KR20210110055A - Preparation Method of P1, P4-di(uridine 5'-)tetraphosphate sodium salt tetrahydrates crystal form A - Google Patents

Abstract

The present invention relates to a method for preparing a crystalline form A of P^1, P^4-di(uridine 5'-)tetraphosphate sodium salt tetrahydrate and a crystalline form A of P^1, P^4-di(uridine 5'-)tetraphosphate sodium salt tetrahydrate prepared thereby, wherein the crystalline form A of P^1, P^4-di(uridine 5'-)tetraphosphate sodium salt tetrahydrate maintains a moisture content of about 5.0 to 11.0 wt%, and thus has been proved to be a novel crystalline form different from conventional crystalline forms. According to the method of the present invention, the crystalline form A of P^1, P^4-di(uridine 5'-)tetraphosphate sodium salt tetrahydrate can be conveniently and stably prepared without the use of a process unsuitable for mass production and a separate apparatus, and thus the crystalline form A can be mass-produced more efficiently.

Description

Preparation Method of P1, P4-di(uridine 5'-)tetraphosphate sodium salt tetrahydrates crystal form A {Preparation Method of P1, P4-di(uridine 5'-)tetraphosphate sodium salt tetrahydrates crystal form A}

The present invention ^{relates to a process for preparing P 1} , P ⁴ -di(uridine 5′-)tetraphosphate sodium salt tetrahydrate crystalline Form A.

A nucleoside is a glycoside compound in which a nucleobase and a pentose are N-glycosidically bonded. When a phosphate group is bonded to the nucleoside by a phosphorylation enzyme, it becomes a nucleotide, a basic component of a DNA chain.

It is known that dinucleoside polyphosphate or a salt thereof, which is a kind of nucleotide, is stable in the body and has an effect on the treatment of diseases because it is similar to a substance in a living body.

In particular, to P ^1, represented by the general formula P ⁴ - di (uridine 5 '), tetra-phosphate ^{(P 1, P 4 -di (} uridine 5 '-) tetraphosphate; hereinafter, "UP ₄ U") or a salt thereof is used as a therapeutic agent for keratoconjunctival epithelial disorders accompanying dry eye syndrome or dry tear syndrome, and is a compound expected to be developed as an expectorant or a therapeutic agent for pneumonia because it has an effect of inducing sputum discharge.

[Formula]

P ¹ , P ⁴ -di(uridine 5'-)tetraphosphate (UP ₄ U) is uridine 5'-triphosphate (UTP), uridine 5'-diphosphate (uridine 5') -diphosphate; UDP) or uridine 5'-monophosphate (UMP) can be synthesized through dehydration condensation reaction.

_{However, UP 4} U obtained by the above method is uridine 5'-tetraphosphate (UP ₄ ), uridine 5'-triphosphate, uridine 5'-diphosphate, uridine 5'-monophosphate, etc. cleoside 5'-(poly)phosphates; And P ^1, P ⁴ - di (uridine 5 ') triphosphate ^{_{(UP 3 U), P 1}} , P 4 - di (uridine 5 '), diphosphate (UP ₂ U), such as the nucleoside poly dinyu Since by-products such as phosphates were mixed, there was a problem that the purity was usually low, around 90%.

Low-purity freeze-dried products have the drawback of being easy to absorb moisture, so _{when formulating UP 4} U as a pharmaceutical, a special manufacturing device is required to exclude moisture, and even after manufacturing, strict packaging is required or the shelf life is prolonged due to low stability. very short

In this regard, ^{a method for preparing an amorphous compound of P 1} , P ⁴ -di(uridine 5′-)tetraphosphate.sodium salt ^{using a freeze dryer (see International Publication No. 1999-05155) and P 1} , P ^{A method for preparing 4} -di(uridine 5'-)tetraphosphate amorphous in high yield (refer to Korean Publication No. 2018-0091689) has been reported.

However, although these amorphous compounds can also be usefully used pharmaceutically, there is a problem in that a separate large-scale investment is required in the manufacturing method, and there is a problem in that it is not efficient due to the disadvantages of large consumption of energy and time.

Accordingly, there is a demand for the development of a manufacturing method that can more efficiently mass-produce high-purity and stable UP _{4 U crystals.}

Domestic Publication No. 2018-0091689

The present inventors tried to develop a method for preparing a crystalline form of ^{P 1} , P ⁴ -di(uridine 5'-)traphosphate.sodium salt hydrate suitable for mass production and efficient. As a result, by confirming that a novel crystalline form can be prepared by dropwise addition of alcohol to the compound in the form of an aqueous solution under certain conditions, the present invention has been completed.

Accordingly, an object of the present invention is to provide a method for preparing crystalline form A of compound tetrahydrate represented by the following formula (1):

[Formula 1]

Another object of the present invention is to provide a crystalline form A of compound tetrahydrate represented by the following formula (1):

[Formula 1]

The present inventors tried to develop a method for preparing a crystalline form of ^{P 1} , P ⁴ -di(uridine 5'-)traphosphate.sodium salt hydrate suitable for mass production and efficient. As a result, it was found that a novel crystalline form can be prepared by dropwise addition of alcohol to a compound in the form of an aqueous solution under certain conditions.

Hereinafter, the present invention will be described in more detail.

One aspect of the present invention P ¹ , P ⁴ -di(uridine 5'-) tetraphosphate sodium salt or a solution containing a hydrate thereof comprising the steps of adding alcohol dropwise; And vacuum drying for 12 hours or more; relates to a method for preparing the crystalline form A of the compound tetrahydrate represented by the following formula (1), including.

[Formula 1]

The compound represented by Formula 1 is P ¹ , P ⁴ -di(uridine 5′-)tetraphosphate.tetrasodium salt (hereinafter, “UP ₄ U.4Na”), which is known as a treatment for dry eye syndrome.

The compound is an antagonist acting on the P2Y2 receptor, and promotes the secretion of the aqueous component as well as the mucous component of tears. This effect is used as a therapeutic agent for each conjunctival epithelial disorder accompanying dry eye syndrome or dry tear syndrome, and since it has a sputum discharge inducing action, it is a compound expected to be developed as an expectorant or a therapeutic agent for pneumonia.

In the present invention, the term "hydrate" refers to a compound of a type formed by recombining water with another compound, and hydrates of salts are also called hydrous salts.

In the present invention, "crystal" or "crystal form" refers to those identified by the characteristics of the appearing X-ray diffraction pattern. Those skilled in the art can understand that the physicochemical properties mentioned in the present invention are specific, and the experimental error depends on the conditions of the instrument, the preparation of the sample, and the purity of the sample.

In particular, it is well known to those skilled in the art that the X-ray diffraction pattern generally varies depending on the conditions of the device, and since the relative intensity of the X-ray diffraction pattern may also vary depending on the experimental conditions, the order of the peak intensities is the only or decisive factor. It's not something you should think about.

In addition, the experimental error of the peak angle is usually 5% or less, the error of these angles must also be considered, and an error of ±0.2° is usually allowed. There is a possibility that an overall offset of the peak angle may occur due to experimental factors such as the height of the sample, and a certain amount of offset is usually allowed.

Accordingly, it can be understood by those skilled in the art that the X-ray diffraction pattern of the crystal or crystalline form of the present invention may not necessarily match the X-ray diffraction pattern of the examples mentioned herein. Any crystalline form having the same or similar pattern to the characteristic peaks of these spectra is within the scope of the present invention. A person skilled in the art can determine whether the crystalline form represented by each spectrum is the same crystalline form or a different crystalline form by comparing the spectrum of the present invention with the spectrum of the unknown crystalline form.

On the other hand, the numerical values and numerical ranges of the present invention are not to be understood as being limited to the numerical values or numerical ranges themselves. It can be understood by those skilled in the art that, depending on the specific technical environment, there may be slight variations in the vicinity of the specific values within the scope not departing from the spirit and purpose of the present invention. In the present invention, the range of variation that can be predicted by a person skilled in the art is often expressed through the term “about”.

In an embodiment of the present invention, crystalline form A of ^{P 1} , P ⁴ -di(uridine 5'-)tetraphosphate.tetrasodium salt (UP ₄ U.4Na) tetrahydrate represented by Formula 1 of the present invention is prepared and X-ray diffraction peaks were identified using an X-ray diffraction analyzer (see Example).

Hereinafter, each step of the method for preparing the crystalline form A of the compound tetrahydrate represented by Formula 1 of the present invention will be described in detail.

P ^One , P ⁴ - Dropwise adding alcohol to a solution containing di(uridine 5'-)tetraphosphate sodium salt or a hydrate thereof

This step is a process of forming crystals by dropwise adding alcohol (crystallization solvent) to a solution (concentrate) containing ^{P 1} , P ^{4 -di(uridine 5′-)tetraphosphate sodium salt or a hydrate thereof.}

The P ¹ , P ⁴ -di(uridine 5′-)tetraphosphate sodium salt or a hydrate thereof and the alcohol have a volume ratio of 1:2 to 10, 1:3 to 10, 1:4 to 10, 1:5 to 10, 1:1 to 5, 1:1 to 6, 1:1 to 7, 1:1 to 8, 1:1 to 9 or 1:1 to 10.

When the weight ratio of alcohol to the solution is less than the above range, there may be a problem in that the yield of the prepared crystalline Form A is significantly lowered to less than 70%.

The alcohol may be methanol.

On the other hand, when ethanol is used as the alcohol, there may be a problem in that a crystalline form such as an octahydrate is generated in a tetrahydrate rather than a crystalline form A.

drying step

This step is a process for generating a hydrate crystalline form. ^{By this process, P 1} , P ⁴ -di(uridine 5'-)tetraphosphate.tetrasodium salt tetrahydrate crystalline Form A having a water content of 5.0 to 11.0% by weight can be prepared.

Accordingly, this step can be carried out for a time sufficient for the moisture content to reach 5.0 to 11.0% by weight, for example at least 12 hours.

When the moisture content exceeds the range of 5.0 to 11.0 wt % ^{, there is a problem in that tetrahydrate crystalline Form A of P 1} , P ⁴ -di(uridine 5′-)tetraphosphate sodium salt cannot be formed.

The method for preparing crystalline Form A of the compound tetrahydrate represented by Formula 1 of the present invention may further include, before the drying step, stirring at 0 to 30° C. for 0.5 to 2.5 hours.

This step is a process of generating a crystalline solid by stirring the resulting crystals at room temperature.

The temperature range for performing this step may be 0 to 30° C., but as long as it is a reaction temperature at which solidification of general pharmaceutical raw materials is possible, it may be carried out without limitation.

The execution time range of this step may also be 0.5 to 2.5 hours, but as long as the reaction time for solidification of general pharmaceutical raw materials is possible, it may be carried out without limitation.

^{P 1} , P ⁴ -di(uridine 5′-)tetraphosphate represented by Formula 1 prepared by the above preparation method. The crystalline form A of tetrasodium salt tetrahydrate may have a water content of 5.0 to 11.0% by weight. .

^{The crystalline form A of P 1} , P ⁴ -di(uridine 5'-)tetraphosphate.tetrasodium salt tetrahydrate represented by Chemical Formula 1 of the present invention has a water content in the above range, so that it is different from the conventional crystalline form It was found to be a novel crystalline form.

One aspect of the present invention relates to crystalline form A of compound tetrahydrate represented by the following formula (1).

[Formula 1]

^{The crystalline form A of P 1} , P ⁴ -di(uridine 5'-)tetraphosphate.tetrasodium salt tetrahydrate represented by Formula 1 of the present invention has diffraction angles of 6.2, 12.2, 13.3, 16.5, and 21.9° ( 2θ) has an X-ray diffraction peak.

In addition, the ^{crystalline form A of P 1} , P ⁴ -di(uridine 5'-)tetraphosphate.tetrasodium salt tetrahydrate represented by Formula 1 is 12.5, 14.1, 14.7, 17.0, 18.8, 19.4, 20.2, 20.9, It has X-ray diffraction peaks with weak intensity at diffraction angles (2θ) of 23.4, 25.2, 26.2, 26.8, 27.2, 27.6, 28.6, 28.9, 30.2, 31.1, 31.6, 32.8, 32.9, and 35.1°.

^{Furthermore, the crystalline form A of P 1} , P ⁴ -di(uridine 5′-)tetraphosphate.tetrasodium salt tetrahydrate represented by Formula 1 may have a water content of 5 to 11% by weight.

^{P 1} , P ⁴ -di(uridine 5′-)tetraphosphate.tetrasodium salt tetrahydrate, which is represented by Chemical Formula 1, and overlaps between the crystalline form A and a method for preparing the same will be omitted in consideration of the complexity of the present specification.

The present invention relates to a ^{method for preparing crystalline form A of P 1} , P ⁴ -di(uridine 5′-)tetraphosphate.sodium salt tetrahydrate and P ¹ , P ⁴ -di(uridine 5′-)tetra prepared thereby Phosphate. It relates to crystalline Form A of sodium salt tetrahydrate, wherein the crystalline Form A of P ¹ , P ⁴ -di(uridine 5'-)tetraphosphate.sodium salt tetrahydrate of the present invention has a water content of about 5.0 to 11.0% by weight. It was found to be a new crystalline form different from the conventional crystalline form by maintaining the ^{In addition, according to the method of the present invention, P 1} , P ⁴ -di(uridine 5′-)tetraphosphate.Crystal form A of sodium salt tetrahydrate simply and stably without using a separate apparatus and a process unsuitable for mass production can be produced, so that the crystalline Form A can be mass-produced more efficiently.

1 is an X-ray powder spectrum result of _{tetrahydrate (4H 2} O) crystalline form A of the compound represented by Formula 1 according to an embodiment of the present invention.
2 is an X-ray powder spectrum result of an _{octahydrate (8H 2} O) crystalline form of the compound represented by Formula 1 according to Comparative Example of the present invention.

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

manufacturing example. Preparation of Diquafosol sodium

This preparation illustrates a representative synthesis example of crude diquafosol sodium. The crude diquafosol sodium used in the recrystallization of the present invention may be used as long as it is crude diquafosol sodium prepared by a conventional method in the art in addition to the method of Preparation Example.

1) Preparation of crude diquafosol sodium

After 0.3L of purified water was added dropwise to UDP.2Na (100 g, 0.223 mol), it was dissolved and stirred at 10° C. for 30 minutes. Then, EDC.HCl (42.8 g, 0.223 mol) and CaCl.2H ₂ O (32.8 g, 0.223 mol) were sequentially added dropwise, followed by stirring at 10° C. for 5 hours.

After completion of the reaction, 0.3 L of purified water and 1.2 L of ethanol were injected to solidify, and stirred at room temperature for about 1 hour. The resulting solid was filtered and _{dried with N 2} to obtain 150 g of crude diquafosol sodium compound (purity about 93%, amorphous).

2) Purification of Diquafosol Sodium

150 g of the obtained crude diquafosol sodium compound was dissolved in deionized water and adsorbed to a weakly basic anion exchange resin (Amberlitye IRA series). Then, after sequentially eluting with deionized water, hydrochloric acid solution and sodium chloride solution, R/O concentration was performed, and the concentrated solution was adsorbed onto a strongly acidic cation exchange resin. Finally, it was eluted with deionized water and concentrated to about 14% solution of sodium diquafosol by R/O concentration.

Example. Diquafosol Sodium Salt (UP ₄ U.4Na) Preparation of crystalline form A of tetrahydrate

5L of methanol was slowly added dropwise to 1L of the concentrate obtained in Preparation Example (14% sodium diquafosol solution) at room temperature to form crystals. The resulting crystals were stirred at room temperature for about 2 hours, and then the crystallized solid was filtered and washed with methanol. The obtained solid was vacuum-dried (up to a moisture content of 5.0 to 11.0%) at 35° C. for 12 hours to prepare a crystalline form A of diquafosol sodium salt tetrahydrate (yield 90.3%, purity 99.9%, moisture content: 8.3% ).

comparative example. Diquafosol Sodium Salt (UP ₄ U.4Na) Preparation of crystalline form of octahydrate

A crystalline form of diquafosol sodium salt octahydrate was prepared by using the same method as in the above Example, except that the crystallized (obtained) solid was vacuum dried for 6 hours (moisture content up to 13.0 to 17.0%) (yield) 96.7%, purity 99.9%, moisture content: 13.9%).

experimental example. Diquafosol Sodium Salt (UP ₄ U.4Na) X-ray diffraction measurement of crystalline form

Bruker Co. X- ray diffractometer D8 ADVANCE using ECO, to measure the X- ray diffraction of U ₂ P ₄ .4Na crystal prepared in the following measurement conditions. The measured X-ray spectra of the crystalline form A of UP4U.4Na tetrahydrate (Example) and the crystalline form of UP4U.4Na octahydrate (Comparative Example) are shown in FIGS. 1 and 2, and the peak data thereof are shown in Table 1 and 2 is shown.

[Measuring conditions]

Source: Cu-Kα, λ = 1.5406 A

Generator setting: 40 kV/25 mA

Goniometer radius: 280 mm

Scan axis: 2theta/theta

Axial Soller slits Primary/Secondary = 2.5°/2.5°

Primary optic: Motorized slit

Secondary optic: Motorized slit

Detector: LynxEye SSD 160

Peak No. 2 θ (°) relative strength One 6.288 100.0% 2 12.242 32.0% 3 12.570 16.0% 4 13.334 61.0% 5 14.151 10.5% 6 14.737 5.6% 7 16.451 63.7% 8 16.660 94.5% 9 17.031 24.1% 10 17.581 30.4% 11 18.877 23.4% 12 19.490 14.8% 13 20.256 6.2% 14 20.962 11.3% 15 21.416 38.3% 16 21.931 58.9% 17 23.449 12.4% 18 25.291 9.3% 19 26.207 8.0% 20 26.879 7.6% 21 27.292 10.4% 22 27.656 4.2% 23 28.902 13.2%

Peak No. 2 θ (°) relative strength One 5.991 100 2 9.250 3.9 3 10.189 11.7 4 11.175 11.9 5 11.675 24.4 6 12.451 34.0 7 13.821 7.6 8 14.218 4.6 9 15.450 17.8 10 16.037 8.6 11 17.303 16.5 12 18.114 11.7 13 19.906 26.4 14 20.737 30.7 15 21.472 20.5 16 22.184 10.2 17 22.930 10.7 18 24.362 4.3 19 25.357 9.4 20 27.662 7.5 21 28.287 7.6

Claims (8)

Dropwise adding alcohol to a solution containing P ¹ , P ⁴ -di(uridine 5′-)tetraphosphate sodium salt or a hydrate thereof; and
drying;
A method for preparing crystalline form A of compound tetrahydrate represented by the following formula (1), comprising:
[Formula 1]

The method according to claim 1, wherein ^{the volume ratio of the alcohol and the solution containing the P 1} , P ⁴ -di(uridine 5′-)tetraphosphate sodium salt or hydrate thereof is 1:1 to 10, the method. The method of claim 1 , wherein the alcohol is methanol. The method according to claim 1, wherein the moisture content of the crystalline Form A prepared by the method is 5.0 to 11.0 wt%. Crystalline form A of compound tetrahydrate represented by the following formula (1):
[Formula 1]

6. The crystalline Form A of claim 5, wherein Form A has X-ray diffraction peaks at diffraction angles (2θ) of 6.2, 12.2, 13.3, 16.5, and 21.9°. 6. The compound of claim 5, wherein Form A is 12.5, 14.1, 14.7, 17.0, 18.8, 19.4, 20.2, 20.9, 23.4, 25.2, 26.2, 26.8, 27.2, 27.6, 28.6, 28.9, 30.2, 31.1, 31.6, 32.8, 32.9, and having an X-ray diffraction peak with weak intensity at a diffraction angle (2θ) of 35.1°. The crystalline Form A according to claim 5, wherein the crystalline Form A has a moisture content of 5.0 to 11.0 wt%.

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