WO2020213794A1 - Novel crystalline form of antiviral agent and preparation method therefor - Google Patents

Abstract

The inventors of the present invention have overcome a hydrolysis problem caused by the moisture absorption of currently commercially available tenofovir alafenamide hemifumarate, and a novel crystalline form of a free base of tenofovir alafenamide, which has water solubility equivalent to that of acid addition salts, has excellent tackiness and uniformity, which are properties that facilitate formulation, and has low electrostatic force, can be used as a therapeutic or preventive pharmaceutical composition that is effective in the treatment of HIV-1 infection and chronic hepatitis B.

Description

Novel crystalline form of antiviral agent and method for manufacturing same

The present invention relates to a novel crystal form of tenofovir alafenamide free base and a method for preparing the same.

Tenofovir alafenamide hemifumarate is a nucleotide analog reverse transcriptase and HBV polymerase inhibitor, developed by Gilead Sciences and sold under the brand name vemlidy. It is a useful drug for the treatment of chronic hepatitis B infection.

Tenofovir alafenamide hemifumarate is described in Korean Patent Publication No. 10-0767432, Korean Patent Publication No. 10-0749160, and Korean Patent Publication No. 10-1612642, and has a chemical name 9-[(R)- The hemifumarate of 2-[[(S)-[[(S)-1-(isopropoxycarbonyl)ethyl]amino]phenoxyphosfinyl]methoxy]propyl]adenine is represented by the following structural formula (Chemical Formula 1) ).

[Formula 1]

Tenofovir alafenamide is hydrolyzed by water in the body and converted into tenofovir form to show medicinal effects. Accordingly, it has been reported that such a form of tenofovir alafenamide has a problem of deteriorating safety due to moisture absorption (Journal of Pharmaceutical and Biomedical Analysis 131 (2016) 146-155).

According to published reports, tenofovir alafenamide hemifumarate is hydrolyzed in the presence of water to generate formaldehyde, and the amine groups of the formed formaldehyde and tenofovir alafenamide cause condensation reaction, which is an impurity, tenofovir. It has been reported to generate a dimer (Tenofovir dimer) (Pharmaceutical Research, 18:234-237(2001); Pharmaceutical Research, 17:1098-1103(2000), Journal of Pharmaceutical and Biomedical Analysis 131 (2016) 146-155 ).

Since this is a crystalline solid in a salt form that is friendly to moisture, it causes a problem in that stability is deteriorated by moisture. Therefore, there is a very need for a new crystalline solid that can improve the stability problem due to moisture.

In the Republic of Korea Patent Publication No. 10-0767432, the Republic of Korea Patent Publication No. 10-0749160, and the Republic of Korea Patent Publication No. 10-1612642, tenofovir alla is not passed through the free base of tenofovir alafenamide, Phenamide fumarate is prepared. The prepared tenofovir alafenamide fumarate contains monofumarate, hemifumarate, and the like, and is purified to finally produce tenofovir alafenamide hemifumarate.

Therefore, in Korean Patent Publication No. 10-0767432, Korean Patent Publication No. 10-0749160, and Korean Patent Publication No. 10-1612642, there is no report on the tenofovir alafenamide free base as a crystalline solid.

In published patent publication US2004/0018150, it is reported that fumarate is produced from a mucous oil that is not a crystalline solid when preparing tenofovir free base.

Tenofovir free base in the form of oil has a problem of inhibiting stability and purity compared to the crystalline solid form. Therefore, there is a high possibility that these oil forms do not meet the purity and stability standards of pharmaceuticals that require strict management standards.

Therefore, as a result of trying to overcome this problem, the present inventors secured a tenofovir alafenamide free base as a novel crystalline solid rather than tenofovir alafenamide free base of the mucous oil-like form.

International Publication No. WO2016/205141 A1 discloses various acid addition salts and co-crystals of tenofovir alafenamide, but there is no data on physicochemical properties and stability thereof.

The present inventors prepared and analyzed tenofovir alafenamide hemifumarate disclosed in Korean Patent Publication No. 10-1612642, and as a result, adhesiveness is strong, grain size distribution of crystals is uneven, and adhesion during tableting is problematic. , It was confirmed that it had a problem of poor stability and hygroscopicity.

Therefore, the present inventors are thermodynamically stable, low hygroscopicity, easy storage at room temperature, non-sticky, solubility equal to acid addition salts, and new crystalline form of tenofovir alafen which is advantageous for formulation even without preparing acid addition salts. To disclose the amide free base.

The present inventors overcome the problem of being hydrolyzed by moisture absorption of tenofovir alafenamide hemifumarate currently on the market, and the water solubility is equivalent to the acid addition salt, and has excellent adhesion and uniformity, which are properties that are easy to formulate. A novel crystalline free base of tenofovir alafenamide with low electrostatic force was developed.

According to another embodiment of the present invention, the present invention in powder X-ray diffraction (PXRD) analysis of 2θ diffraction angles 7.431±0.2, 9.723±0.2, 11.231±0.2, 11.595±0.2, 11.949±0.2, 12.239±0.2, 12.897±0.2 , 13.287±0.2, 14.343±0.2, 14.877±0.2, 15.424±0.2, 15.672±0.2, 16.178±0.2, 17.24±0.2, 17.593±0.2, 18.275±0.2, 19.082±0.2, 19.515±0.2, 19.93±0.2, 20.536 ±0.2, 21.294±0.2, 21.897±0.2, 22.185±0.2, 22.407±0.2, 22.93±0.2, 23.316±0.2, 24.009±0.2, 24.516±0.2, 24.961±0.2, 25.467±0.2, 25.933±0.2, 26.697±0.2 , 26.929±0.2, 27.25±0.2, 27.619±0.2, 28.188±0.2, 28.941±0.2, 29.439±0.2, 29.997±0.2, 31.096±0.2, 31.876±0.2, 32.682±0.2, 33.032±0.2, 34.593±0.2 It provides a tenofovir alafenamide free base represented by the following formula (2), characterized by having a powder X-ray diffraction pattern having a typical peak.

[Formula 2]

For example, intensities and peak positions of powder X-ray diffraction of one embodiment of tenofovir alafenamide free base crystal form according to the present invention may be as shown in Table 1 below.

[Table 1]

[Correction 19.11.2019 under Rule 91]

In addition, the present invention provides a tenofovir alafenamide free base showing an endothermic peak of 121.30°C±3°C and 123.63°C±3°C in temperature differential scanning calorimetry (DSC) analysis using a sealed fan.

In addition, the present invention provides a tenofovir alafenamide free base having a novel crystal structure in an anhydrous crystalline form that does not have a thermogravimetric decrease before 100°C in thermogravimetric (TGA) analysis.

The present inventors sought to obtain a tenofovir alafenamide free base as a novel crystalline solid capable of overcoming the problem of the conventional tenofovir free base in the form of a viscous oil.

The present inventors attempted to obtain a novel crystalline free base of tenofovir alafenamide having excellent purity, excellent physicochemical properties and stability while overcoming the disadvantages of the conventional tenofovir alafenamide hemifumarate.

As a result, the present inventors developed a method and conditions for preparing a new crystal form of tenofovir alafenamide free base having the above-described advantages, and the tenofovir alafenamide free base prepared in this way is thermodynamically stable. And, as such, it was found to be suitable for use as an active ingredient in pharmaceuticals.

In FIG. 2, the crystalline tenofovir alafenamide free base of the present invention represented by Chemical Formula 2 exhibits a different X-ray diffraction pattern than tenofovir alafenamide hemifumarate.

According to one embodiment of the present invention, the crystalline tenofovir alafenamide free base of the present invention has an X-ray diffraction pattern shown in FIG. 1.

According to an embodiment of the present invention, the crystalline tenofovir alafenamide free base of the present invention has the characteristics of the calorimetric curve and thermogravimetric analysis result of the temperature differential scanning (DSC) calorimetry of FIG. 3.

The present invention overcomes the problem of being hydrolyzed by moisture absorption of tenofovir alafenamide hemifumarate, which is currently on the market, and has excellent tackiness and uniformity, which is a property that is easy to formulate and has an aqueous solubility equal to that of an acid addition salt As a novel crystalline form of tenofovir alafenamide with low electrostatic force, it is suitable for use as an active ingredient in pharmaceuticals.

According to another aspect of the present invention, the present invention provides a method for preparing a crystalline tenofovir alafenamide free base comprising the following steps:

(a) dissolving or suspending tenofovir alafenamide acid addition salt in methylene chloride or a mixed solvent of methylene chloride and methanol, and adding sodium carbonate or sodium hydrogen carbonate aqueous solution to adjust the pH, and then separating the organic layer;

(b) adding magnesium sulfate (MgSO ₄ ) or sodium sulfate (Na ₂ SO ₄ ) as a desiccant to the resultant of step (a) and stirring;

(c) filtering the resultant of step (b) and then concentrating and drying under reduced pressure;

(d) acetone, isopropanol, ethyl acetate in the resultant of step (c); Or adding a cosolvent of acetone ethyl acetate, and obtaining a crystalline tenofovir alafenamide free base through vigorous stirring and selective seeding.

Step (a) Tenofovir alafenamide acid addition salt treatment

In the present invention, a step obtained by dissolving or suspending tenofovir alafenamide acid addition salt in methylene chloride or a mixed solvent of methylene chloride and methanol, adjusting the pH by adding sodium carbonate or sodium hydrogen carbonate aqueous solution, and separating the organic layer by extraction Includes.

On the other hand, treatment of an aqueous sodium carbonate or sodium hydrogen carbonate solution and a methylene chloride or a mixed solvent of methylene chloride and methanol added to the tenofovir alafenamide acid addition salt is not restricted to the order.

The tenofovir alafenamide acid addition salt used in the present invention includes various acid addition salts of tenofovir alafenamide, for example, tenofovir alafenamide hemifumarate can be used.

Adjusting the pH by treatment with sodium carbonate or sodium hydrogen carbonate aqueous solution means making the conditions of 9-10.

Steps (b)-(c): Crystalline form by recrystallization Tenofovir Alafenamide Free base purchase

In step (b), magnesium sulfate or sodium sulfate is added as a desiccant and stirred to remove moisture.

It means a step of filtering the resultant in step (c), preferably at 5°C, and concentrating and drying under reduced pressure at 50°C or less.

Step (d): Crystalline form by recrystallization Tenofovir Alafenamide Free base purchase

Ethanol or isopropanol and a co-solvent of the two solvents were added to the resultant of step (c) or the starting material of step (a) (ii), followed by vigorous stirring and selective seeding as needed. Through the crystalline tenofovir alafenamide free base is obtained.

Acetone or ethyl acetate and the mixing ratio of these two solvents can be prepared in various ways, and the amount of the solvent used is specifically 10-50 ml per 1 g of tenofovir alafenamide free base.

It is advantageous to vigorously stir the tenofovir alafenamide free base and the solvent for crystallization, and the stirring time is 1-8 hours, more specifically 1-4 hours.

Upon stirring, the tenofovir alafenamide crystalline free base according to the present invention can be selectively seeded to accelerate the precipitation time of crystals. Seeding is not essential, but it is effective when a crystalline solid does not precipitate even after stirring for several hours.

The effects of the present invention are as follows.

Tenofovir alafenamide free base according to the present invention can minimize the generation of related substances over time compared to tenofovir alafenamide hemifumarate, thereby reducing the amount of impurities produced during the storage process of the product. Can increase.

In addition, the tenofovir alafenamide free base according to the present invention has excellent physicochemical properties equivalent to that of the acid addition salt hemifumarate salt, which has excellent flow chart, uniformity, no electrostatic force, and water solubility. It can be used as a useful active ingredient.

1 shows a powder X-ray diffraction pattern of tenofovir alafenamide free base crystalline solid prepared according to an embodiment of the present invention.

FIG. 2 shows a powder X-ray diffraction pattern of tenofovir alafenamide free base and tenofovir alafenamide hemifumarate prepared according to an embodiment of the present invention.

3 is a thermogravimetric analysis-temperature differential scanning calorimeter (TGA-DSC) results of tenofovir alafenamide free base crystalline solid prepared according to an embodiment of the present invention.

Figure 4 shows the results of the hydrogen nuclear magnetic resonance spectroscopy (H-NMR) of tenofovir alafenamide free base prepared according to an embodiment of the present invention.

Figure 5 shows the results of the carbon nuclear magnetic resonance spectroscopy (C-NMR) of tenofovir alafenamide free base prepared according to an embodiment of the present invention.

6 is a graph showing the results of accelerated stability (40 degrees, RH 75%) of tenofovir alafenamide free base and tenofovir alafenamide hemifumarate prepared according to an embodiment of the present invention.

7 is a graph showing the results of severe stability (60 degrees, RH 75%) of tenofovir alafenamide free base and tenofovir alafenamide hemifumarate prepared according to an embodiment of the present invention.

FIG. 8 is a result of comparing the uniformity and distribution of the particles according to the particle size of tenofovir alafenamide free base and tenofovir alafenamide hemifumarate prepared according to an embodiment of the present invention using a particle size analyzer (PSA). Show.

9 is a comparison of the shape of particles according to tenofovir alafenamide free base and tenofovir alafenamide hemifumarate prepared according to an embodiment of the present invention.

FIG. 10 shows a comparison of powder photographs of tenofovir alafenamide free base and tenofovir alafenamide hemifumarate prepared according to an embodiment of the present invention. This was compared by putting it in a PE bag used as an actual packaging container. Tenofovir alafenamide hemifumarate is difficult to subdivide due to static electricity, and is lumped due to stickiness, whereas tenofovir alla according to the present invention The free base of phenamide does not have static electricity, and it is possible to confirm a homogeneous state with good flowability.

The present invention will be described in more detail through the following examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

[Example 1] Preparation of new crystal form of tenofovir alafenamide free base

At room temperature, 20 g of tenofovir alafenamide hemifumarate was dissolved by adding 500 mL of methylene chloride and 50 mL of methanol. A 10% aqueous sodium carbonate solution was added and stirred for 10 minutes, while the pH was adjusted to about 9-10, and the organic layer was separated. After removing moisture from the organic layer using magnesium sulfate, it was filtered and concentrated under reduced pressure at 45 degrees. Thereafter, 100 mL of ethyl acetate was added, stirred at room temperature for 2 hours, and filtered (washed with 20 mL of ethyl acetate) to obtain 12 g of a new crystal form of tenofovir alafenamide free base.

[Example 2] Preparation of new crystal form of tenofovir alafenamide free base

At room temperature, 20 g of tenofovir alafenamide hemifumarate was dissolved by adding 500 mL of methylene chloride and 50 mL of methanol. A 10% aqueous sodium carbonate solution was added and stirred for 10 minutes, while the pH was adjusted to about 9-10, and the organic layer was separated. After removing moisture from the organic layer using magnesium sulfate, it was filtered and concentrated under reduced pressure at 45 degrees. Thereafter, 100 mL of acetone was added, stirred at room temperature for 2 hours, and filtered (washed with 20 mL of acetone) to obtain 13 g of a new crystalline form of tenofovir alapenamide free base.

[Example 3] Preparation of new crystal form of tenofovir alafenamide free base

At room temperature, 20 g of tenofovir alafenamide hemifumarate was dissolved by adding 500 mL of methylene chloride and 50 mL of methanol. A 10% aqueous sodium carbonate solution was added and stirred for 10 minutes, while the pH was adjusted to about 9-10, and the organic layer was separated. After removing moisture from the organic layer using magnesium sulfate, it was filtered and concentrated under reduced pressure at 45 degrees. Thereafter, 100 mL of isopropyl alcohol was added, stirred at room temperature for 2 hours, and filtered (washed with 20 mL of isopropyl alcohol) to obtain 10.4 g of a new crystal form of tenofovir alafenamide free base.

[Example 4] Preparation of new crystal form of tenofovir alafenamide free base

At room temperature, 20 g of tenofovir alafenamide hemifumarate was dissolved by adding 500 mL of methylene chloride and 50 mL of methanol. A 10% aqueous sodium carbonate solution was added and stirred for 10 minutes, while the pH was adjusted to about 9-10, and the organic layer was separated. After removing moisture from the organic layer using magnesium sulfate, it was filtered and concentrated under reduced pressure at 45 degrees. Thereafter, 50 mL of ethyl acetate and 50 mL of acetone were added, stirred at room temperature for 2 hours, and filtered (washed with 10 mL of ethyl acetate and 10 mL of acetone) to obtain 11 g of a new crystal form of tenofovir alafenamide free base.

[Experimental Example 1] Powder X-ray diffraction (PXRD)

PXRD analysis (see Fig. 1) was performed on an X-ray powder diffractometer (D8 Advance) using Cu Kα radiation. The instrument was equipped with tube power, and the amount of current was set at 45 kV and 40 mA. The divergence and scattering slits were set to 1°, and the light receiving slits were set to 0.2 mm. A θ-2θ continuous scan of 3° minutes (0.4 sec/0.02° interval) from 5 to 35°2θ was used.

[Experimental Example 2] Thermogravimetric Analysis (TGA)

Using a TGA Q50 obtained from TA company, measurement (see Fig. 3) was performed using a TGA fan at a scan rate of 10°C/min from 30°C to 300°C under nitrogen purification.

[Experimental Example 3] Temperature Differential Scanning Calorimetry (DSC)

Using DSC Q20 obtained from TA company, DSC measurement (see Fig. 3) was performed in a closed pan at a scan rate of 10°C/min from 30°C to 300°C under nitrogen purification.

[Experimental Example 4] Evaluation of the solubility of a new crystal form of tenofovir alafenamide free base

First, tenofovir alafenamide hemifumaric acid in order to compare with tenofovir alafenamide hemifumarate by measuring the water solubility and solubility at pH 6.8 of the new crystal form of tenofovir alafenamide free base prepared in the above example. The concentration was set from 10 µg/ml to 50 µg/ml of salt, diluted 10 times, and showed linearity. A reliable value was obtained with R ² =0.993 at the indicated linearity. Using this linearity, tenofovir alafenamide hemifumarate and tenofovir alafenamide free base new crystal forms were added until a solid precipitated in 5 ml of water and a pH 6.8 aqueous solution. After stirring for 1 hour, the mixture was allowed to stand for 1 hour. After sampling 1 ml of the supernatant of the still solution, it was diluted 10 times with methanol, and the diluted solvent was analyzed by HPLC, and the solubility was measured using the area of the tenofovir alafenamide peak. The HPLC chromatogram is shown in Figure 9. The results are summarized in Table 2 below.

[Table 2]

As shown in Table 2, the solubility of the new crystal form of tenofovir alafenamide free base and tenofovir alafenamide hemifumarate showed equivalent results. Therefore, it was confirmed that even free bases other than acid addition salts can exhibit the same solubility as acid addition salts.

[ Experimental example 5] Tenofovir Alafenamide Free base New crystal form and Tenofovir Evaluation of acceleration and severe stability of alafenamide hemifumarate

A new crystal form of tenofovir alafenamide free base prepared by Examples and tenofovir alafenamide hemifumarate were accelerated and stability tests under severe conditions were performed. As a result, in Fig. 6 accelerated stability (40 degrees, 75% relative humidity), the new crystal form of tenofovir alafenamide free base remained stable for 30 days without any effect of purity, but tenofovir alafenamide hemifumarate was It was confirmed that the stability was poor as the purity decreased from the 6th day.

7 shows that even in severe stability (60 degrees, 75% relative humidity), the new crystal form of tenofovir alafenamide free base remained stable for 30 days without any effect of purity, but tenofovir alafenamide hemifumarate was 6 It was confirmed that the purity was lowered after 1 and that the severe stability was poor.

Therefore, it was confirmed that the stability of the new crystal form of tenofovir alafenamide free base was superior to that of tenofovir alafenamide hemifumarate in accelerated and severe conditions.

[ Experimental example 6] Tenofovir Alafenamide Free base New crystal form particle flow chart, uniformity, electrostatic force comparative evaluation

In the case of solid powder, the mixing properties with excipients, flow of particles, and uniformity of particles are very important when tableting. In general, it is known that the particle distribution is symmetrical on the particle size distribution map, and the very uniform shape makes tableting easy. The particle distribution of tenofovir alafenamide free base new crystal form of the present invention and tenofovir alafenamide hemifumarate were compared and analyzed. The particle size was generally measured using a mastersizer 2000 equipment using a solid dispersion method. Fig. 8 shows the particle diagrams of tenofovir alafenamide free base and tenofovir alafenamide hemifumarate of the present invention, respectively.

As shown in FIG. 8, in the case of the crystalline tenofovir alafenamide free base of the present invention, a uniform distribution of symmetrical peaks was shown, whereas tenofovir alafenamide hemifumarate was an asymmetrical having a wide and divergent peaks. It was confirmed that it has a particle distribution diagram.

In addition, in FIG. 9, the crystal shape of the crystal tenofovir alafenamide free base of the present invention is small particles, while the tenofovir alafenamide hemifumarate is uneven and differently agglomerated particles.

In FIG. 10, the adhesiveness of each solid can be confirmed. Tenofovir alafenamide hemifumarate has strong viscosity and can be confirmed that it adheres to the PE bag by electrostatic force, but the tenofovir alafenamide free base of the present invention has no electrostatic force and has an even particle form. It was confirmed to represent. In conclusion, tenofovir alafenamide free base of the present invention is a solid form that is easier to tablet than tenofovir alafenamide hemifumarate, and its flow rate, uniformity, and electrostatic force are greatly improved, making it a very easy solid for pharmaceutical tableting. It was confirmed that it was in shape.

As described above, specific parts of the present invention have been described in detail, and it is obvious that these specific techniques are only preferred embodiments and are not intended to limit the scope of the present invention to those of ordinary skill in the art. Therefore, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (4)

1. Powder X-ray diffraction (PXRD) analysis with characteristic peaks at 2θ diffraction angles including 7.431±0.2, 11.231±0.2, 12.897±0.2, 19.082±0.2, 19.515±0.2, 21.294±0.2 and 22.407±0.2. It has a line diffraction pattern, and shows an endothermic peak of 121.30°C±3°C and 123.63°C±3°C in temperature differential scanning calorimetry (DSC) analysis, and there is no decrease in thermoweight before 100°C in thermogravimetric analysis. Tenofovir alafenamide free base crystalline form represented by the following formula (2), characterized by an anhydrous crystalline form.

   [Formula 2]

   
2. [Correction 19.11.2019 under Rule 91]
   The tenofovir alafenamide free base crystalline form according to claim 1, wherein the tenofovir alafenamide free base crystalline form has a powder X-ray diffraction pattern shown in FIG. 1.
3. [Correction 19.11.2019 under Rule 91]
   The tenofovir alafenamide free base crystalline form according to claim 1, wherein the tenofovir alafenamide free base crystalline form is characterized by a calorie curve of temperature differential scanning calorimetry and an anhydrous crystalline form of thermogravimetric analysis shown in FIG. .
4. A therapeutic or prophylactic pharmaceutical composition useful in the treatment of HIV-1 infection and chronic hepatitis B as a cleotide analog reverse transcriptase and HBV polymerase inhibitor comprising tenofovir alafenamide free base crystalline form according to claim 1.

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