KR101985791B1 - Process for producing di(nucleoside 5'-)polyphosphate - Google Patents

Abstract

(상기 화학식 1에서, B¹ 및 B²는 각각 독립적으로 피리미딘 염기이고, n은 0~4의 자연수이다)A process for producing di (nucleoside 5'-) polyphosphate represented by the following formula (1) is provided. Di (nucleoside 5'-) polyphosphate comprises two substances each independently selected from the group consisting of nucleoside 5'-triphosphate (NTP) and nucleoside 5'-monophosphate (NMP) In the presence of a copper metal salt.
[Chemical Formula 1]

(Wherein B ¹ and B ² are each independently a pyrimidine base and n is a natural number of 0 to 4)

Description

PROCESS FOR PRODUCING DI (NUCLEOSIDE 5 ' -) POLYPHOSPHATE < RTI ID = 0.0 >

The present invention relates to a process for preparing di (nucleoside 5'-) polyphosphate, and more particularly to a process for producing di (nucleoside 5'-) polyphosphate by reacting a nucleoside 5'-triphosphate with a nucleoside 5'-monophosphate in the presence of a copper metal salt (Nucleoside 5 ' -) polyphosphate.

Among the di (nucleoside 5'-) polyphosphates represented by the following formula 1, for example, P ¹ , P ⁴ -di (uridine 5'-) tetraphosphate (Diquafosol, Up4U) And is expected to be developed as an expectorant agent or a therapeutic agent for pneumonia because it has an effect of inducing a bile drainage.

[Chemical Formula 1]

(Wherein B ¹ and B ² are each independently a pyrimidine base and n is a natural number of 0 to 4)

The synthesis of such di (pyrimidine nucleoside 5'-) polyphosphate can be carried out, for example, by using Uridine 5'-monophosphate (UMP) as a starting material in the case of Up4U, diphenylphosphochloridate (DPC ) And a phosphating agent such as pyrophosphate (PPi) have been disclosed in Patent Document 1, etc. However, such a known synthetic method has a drawback that the synthesis yield of the target compound is extremely low, i.e., less than about 10% There was a problem.

W0 1999/005155

A problem to be solved by the present invention is to provide a method for producing di (nucleoside 5'-) polyphosphate with high synthesis efficiency and yield.

The present invention has been made in view of the above problems, and it is an object of the present invention to provide a method of manufacturing the same.

According to an embodiment of the present invention, there is provided a method for producing a di (nucleoside 5'-) polyphosphate represented by the following formula 1, comprising reacting a nucleoside 5'-triphosphate (NTP) and a nucleoside 5'-monophosphate (NMP) in the presence of a copper metal salt.

[Chemical Formula 1]

Wherein B ¹ and B ² are each independently a pyrimidine base and n is a natural number of 2 to 4.

The NTP may be uridine 5'-triphosphate (UTP), and the NMP may be uridine 5'-monophosphate (UMP).

The di (nucleoside 5'-) polyphosphate may be represented by the following formula (2).

(2)

The copper metal salt may include at least one selected from the group consisting of chloride, bromide, nitrate, sulfur oxides, iodides, acetic acid and hydrates thereof.

The copper metal salt may include at least one of copper bromide and copper chloride.

1 to 5 equivalents of the NMP may be used per 1 equivalent of the NTP.

According to another aspect of the present invention, there is provided a process for preparing di (uridine 5'-) tetraphosphate represented by the following general formula (2), comprising reacting uridine 5'-triphosphate (UTP) -Monophosphate (UMP) in the presence of a copper metal salt.

(2)

The copper metal salt may be used in an amount of 1 to 6 equivalents based on 1 equivalent of UTP.

The copper metal salt may include at least one of copper bromide and copper chloride.

The copper metal salt may include copper chloride hydrate.

The UMP may be used in an amount of 1 to 5 equivalents based on 1 equivalent of the UTP.

The details of other embodiments are included in the detailed description and drawings.

According to embodiments of the present invention, di (nucleoside 5'-) polyphosphates, especially P ¹ , P ⁴ -d (uridine 5'-) tetraphosphate (Up4U), can be synthesized with high synthesis efficiency and yield have.

The effects according to the embodiments of the present invention are not limited by the contents exemplified above, and more various effects are included in the specification.

Figures 1 and 2 are HPLC chromatograms after reacting with 3 equivalents of copper bromide in the Experimental Example.
Figures 3 and 4 are HPLC chromatograms after reacting with 2.4 equivalents of copper bromide in the experimental examples.
Figures 5 and 6 are HPLC chromatograms after reacting with 2 equivalents of copper bromide in the experimental examples.
Figures 7 and 8 are HPLC chromatograms after reacting with 1.2 equivalents of copper bromide in the Experimental Example.
9 and 10 are HPLC chromatograms after reacting with 2 equivalents of copper sulfate in Experimental Example.
Fig. 11 is an HPLC chromatogram after reaction with 1.2 equivalents of copper sulfate in Experimental Example.
12 and 13 are HPLC chromatograms after reacting with 2 equivalents of copper chloride in the experimental examples.
14 and 15 are HPLC chromatograms obtained after the reaction was carried out using 3 equivalents of cupric chloride dihydrate and 1.2 equivalents of UMP in Experimental Example.
16 and 17 are HPLC chromatograms obtained after the reaction was carried out using 3 equivalents of cupric chloride dihydrate and 2 equivalents of UMP in Experimental Example.

BRIEF DESCRIPTION OF THE DRAWINGS The advantages and features of the present invention and the manner of achieving them will become apparent with reference to the embodiments described in detail below with reference to the accompanying drawings. The present invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Is provided to fully convey the scope of the invention to those skilled in the art, and the invention is only defined by the scope of the claims.

When an element is referred to as " comprising ", it means that it can include other elements, not excluding other elements unless specifically stated otherwise.

Although the first, second, etc. are used to describe various components, it goes without saying that these components are not limited by these terms. These terms are used only to distinguish one component from another. Therefore, it goes without saying that the first component mentioned below may be the second component within the technical scope of the present invention.

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. Also, preferred methods or samples are described in this specification, but similar or equivalent ones are also included in the scope of the present invention. The contents of all publications referred to herein are incorporated herein by reference.

Hereinafter, embodiments of the present invention will be described in detail.

A method for preparing di (nucleoside 5'-) polyphosphate according to an embodiment of the present invention includes nucleoside 5'-triphosphate (NTP) And nucleoside 5'-monophosphate (NMP), in the presence of a copper metal salt, in the presence of a copper metal salt.

Di (nucleoside 5 '-) polyphosphate) can be represented by the following formula (1).

[Chemical Formula 1]

In the above formula (1), B ¹ and B ² each independently represents a pyrimidine base, and n may be a natural number of 2 to 4.

Specifically, a di (nucleoside 5'-) polyphosphate can be prepared by reacting NTP with NTP using a copper metal salt as a catalyst, reacting NMP with NMP, or reacting NTP with NMP.

In an exemplary embodiment, NTP and NMP can be reacted in the presence of a copper metal salt to produce di (nucleoside 5'-) tetraphosphate, but is not limited thereto.

The copper metal salt may include at least one of chloride, bromide, nitrate, sulfur oxides, iodides, acetates and hydrates thereof of copper. In an exemplary embodiment, the copper metal salt may be copper (I) bromide, copper (II) bromide, copper (I) chloride, copper (II) chloride or a mixture of two or more thereof. In another embodiment, the copper metal salt may comprise copper chloride hydrate. However, the present invention is not limited thereto.

The nucleoside may be a pyrimidine nucleoside comprising a pyrimidine base, and in particular may be uridine. In some embodiments, the NTP may be uridine 5'-triphosphate (UTP) and the NMP may be uridine 5'-monophosphate (UMP). When UTP and UMP are reacted under a copper metal salt, di (uridine 5'-) tetraphosphate (Up4U) as shown in the following formula 2 can be obtained.

(2)

Hereinafter, an example of producing Up4U by reacting UTP and UMP under a copper metal salt will be described in more detail.

The manufacturing method of Up4U may include the following manufacturing steps.

(1) a step of producing a UTP solution containing UTP, a tertiary amine salt, a water-soluble organic solvent and a condensing agent

(2) preparing a UMP solution containing UMP, a tertiary amine salt and a water-soluble organic solvent

(3) reacting the UTP solution with the UMP solution in the presence of a copper metal salt to produce an Up4U tertiary amine salt; and

(4) purifying Up4U tertiary amine salt to prepare Up4U tetrasodium

Specifically, in step (1), the UTP trisodium aqueous solution is passed through a column filled with a cation exchange resin and reacted with a tertiary amine to form a UTP tertiary amine salt. Then, water is removed by concentration under reduced pressure, And dissolving it in an organic solvent to prepare the UTP solution.

The tertiary amine salt may be a salt of a non-reactive tertiary amine. 'No reactivity' means that the reactivity is so low that it may be possible to substantially ignore the reaction mechanism by the tertiary amine in designing the process. Examples of such tertiary amines include, but are not limited to, tri-n-butylamine and diisopropylethylamine.

The tertiary amine may be contained in an amount of 3 to 5 equivalents to 1 equivalent of UTP. When the tertiary amine is contained in an amount less than 3 equivalents relative to 1 equivalent of UTP, the stability of UTP may be lowered, and if it exceeds 5 equivalents, it may be disadvantageous in terms of process efficiency and industrial efficiency.

The water-soluble organic solvent is a solvent capable of dissolving a copper metal salt, for example, dimethylformamide (DMF), dimethylacetamide (DMA), formamide (FA), pyridine, dioxane, dimethylsulfoxide, And mixtures thereof. However, not limited thereto, various other solvents capable of dissolving a copper metal salt may be used.

Condensers include carbodiimides such as diisopropylcarbodiimide (DIPC), dicyclohexylcarbodiimide (DCC), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, (CDI), diphenylphosphochloridate (DPC), and the like, and a sulfonic acid halide such as toluene sulfonyl chloride, and in particular, may include carbodiimides.

The condensing agent may be contained in an amount of 1 to 4 equivalents, preferably 1 to 2 equivalents, per 1 equivalent of UTP. If the condensing agent is contained in an amount less than 1 equivalent based on 1 equivalent of UTP, the reaction may not be completed. When the amount of the condensing agent is more than 4 equivalents, a step of removing the condensing agent is required. It can be disadvantageous from the side.

In the step (2), the UMP solution may be prepared by adding a tertiary amine and a water-soluble organic solvent to a free UMP (UMP-free).

The tertiary amine and the water-soluble organic solvent may be the same as those exemplified above, and may be the same as or different from the tertiary amine and / or the water-soluble organic solvent of the UTP solution.

The tertiary amine may be contained in an amount of 1 to 1.5 equivalents, preferably 1 to 1.2 equivalents to 1 equivalent of UMP. If the amount of the tertiary amine is less than 1 equivalent based on 1 equivalent of UTP, the solubility in the water-soluble organic solvent may be lowered and the reaction may proceed slowly. If the amount of the tertiary amine is more than 1.5 equivalents, the pH is increased and the residue should be removed Which can be disadvantageous in terms of process efficiency.

The step (3) may be a step of adding an UMP solution and a copper metal salt to the UTP solution to prepare an Up4U tertiary amine salt, and the copper metal salt may act as a catalyst for the reaction between UTP and UMP.

The UMP solution may be mixed such that the UMP is 1 to 5 equivalents, preferably 1 to 3 equivalents to 1 equivalent of UTP in the UTP solution. If the UMP is mixed with less than 1 equivalent of UTP per 1 equivalent, the yield may be lowered. If the UMP is mixed in excess of 5 equivalents, it may be disadvantageous in terms of process efficiency and industrial .

The copper metal salt may be a chloride, a bromide, a nitrate, a sulfur oxide, an iodide, an acetic acid or a mixture of two or more thereof, preferably copper (I) bromide, copper (II) , Copper (II) chloride, or a mixture of two or more thereof.

The copper metal salt may be contained in an amount of 1 to 6 equivalents to 1 equivalent of UTP in the UTP solution. When the copper metal salt is mixed with less than 1 equivalent of UTP per 1 equivalent, the reaction between UTP and UMP may be insignificant. As the equivalent amount of the copper metal salt increases, the reaction efficiency between UTP and UMP may be increased. However, The increase in the reaction efficiency is small compared with the increase in the amount of copper metal salt remaining after the reaction, which may be disadvantageous in terms of process efficiency.

The copper metal salt can be added to the UTP solution in the form of a solution dissolved in a water-soluble organic solvent, thereby preventing impurities from being formed when the copper metal salt is added to the solid.

In step (4), the water-soluble organic solvent and dimethylformamide are removed from the Up4U tertiary amine salt by vacuum concentration and anion resin adsorption to obtain Up4U ammonium salt, which is passed through a cation exchange resin, crystallized with ethanol, It may be a step of obtaining sodium.

When an Up4U tertiary amine salt is adsorbed to an anion resin, there is an advantage that it can be removed by eluting dimethylformamide having a high boiling point.

Preparation Example: Preparation of P ¹ , P ⁴ -di (uridine 5'-) tetraphosphate (Up ⁴ U)

(1) Preparation of uridine 5'-triphosphate (UTP) solution

5.5 mL of UTP trisodium (content: 92.2%, manufactured by HANGZHOU MEIYA PHARMACEUTICAL CO. LTD.) Was dissolved in 55 mL of distilled water, passed through a column filled with 55 mL of a cation exchange resin (SCR-BH H type, Samyang Corp.) After washing, it was neutralized with 9.5 mL of tri-n-butylamine. After concentrating under reduced pressure at 40 DEG C, 1,4-dioxane was added to the residue and the filtrate was concentrated under reduced pressure to remove water. To this was added dimethylformamide (50 mL) to prepare a solution of UTP tri-n-butylamine salt in dimethylformamide, 2.35 mL of diisopropylcarbodiimide was added, and the mixture was stirred for 3 hours to obtain a dimethyl formate of UTP tri-n-butylamine Amide diisopropylcarbodiimide solution was prepared.

(2) Preparation of uridine 5'-monophosphate (UMP) solution

55 mL of dimethylformamide was added to 3.84 g of UMP (content 96.6%, manufactured by HANGZHOU MEIYA PHARMACEUTICAL CO. LTD), and 4.99 mL of tri-n-butylamine was added thereto to prepare a dimethylformamide solution of UMP tri-n-butylamine salt Respectively.

(3) Preparation of Up4U tertiary amine salt

To the dimethylformamide diisopropylcarbodiimide solution of the UTP tri-n-butylamine salt prepared in the above (1) was added the dimethylformamide solution of the UMP tri-n-butylamine salt prepared in the above (2) A solution obtained by dissolving various copper metal salts in 40 mL of dimethylformamide was added thereto, and the mixture was allowed to react at room temperature for the time shown in Table 1 below.

Copper metal salt Copper metal salt equivalent UMP equivalent Reaction time Copper bromide (II) 3 equivalents 1.2 equivalent 3 hours Copper bromide (II) 2.4 equivalent 1.2 equivalent 6 hours Copper bromide (II) 2 equivalents 1.2 equivalent 6 hours Copper bromide (II) 1.2 equivalent 1.2 equivalent 6 hours Copper sulfate (II) 1.2 equivalent 1.2 equivalent 6 hours Copper sulfate (II) 2 equivalents 1.2 equivalent 6 hours Copper Chloride (II) 2 equivalents 1.2 equivalent 6 hours Copper chloride < RTI ID = 0.0 > (II) 3 equivalents 1.2 equivalent 3 hours Copper chloride < RTI ID = 0.0 > (II) 3 equivalents 2 equivalents 3 hours

(4-1) Preparation of Up4U tetra sodium (3 equivalents of copper (II) bromide, 1 equivalent of UMP)

In the above reaction (3), 3 equivalents of copper (II) bromide was reacted for 3 hours, and 140 ml of distilled water was added to the reaction solution of Up4U tertiary amine salt. Then, 220 ml of anion exchange resin (Amberlite IRA 67 Cl chloride type) The reaction mixture was washed with a 50% aqueous solution of DMF, distilled water, 0.1 N hydrochloric acid and distilled water, and sequentially eluted with 0.3 molar aqueous solution of ammonium hydrogencarbonate, 0.4 molar aqueous solution and 0.6 molar aqueous solution. Then, the fraction containing Up4U ammonium salt was taken out and concentrated under reduced pressure. And the mixture was concentrated under reduced pressure at 50 ° C. The residue was dissolved in distilled water, passed through 220 mL of cation exchange resin (SCR-BH Na type sodium type, Samyang Corp.), washed with distilled water, and concentrated under reduced pressure. The residue was dissolved in distilled water (10 mL), and 80 mL of ethanol was added. The precipitated crystals were filtered and dried at 40 캜 in vacuo to obtain 5.45 g of Up4U tetrasodium (yield: 62.1%).

(4-2) Preparation of Up4U tetra sodium (3 equivalents of copper (II) chloride dihydrate, 2 equivalents of UMP)

Except that 3 equivalents of the copper (II) chloride dihydrate in (3) above and 2 equivalents of UMP (6.4 g UMP) were used in the preparation of the UMP tri-n-butylamine salt-dimethylformamide solution Proceeding as described in (4-1), 6.93 g of Up 4 U tetrasodium (yield: 78.9%) was obtained.

<Experimental Example: Analysis of Up4 U Synthesis Efficiency by Type and Equivalent of Copper Metal Salt>

In (3) of the above preparation example, the reaction solution was subjected to HPLC (measurement wavelength: 262 nm), reduction of UTP trisodium (retention time: about 6.7 minutes) and UMP (retention time: about 2.5 minutes) 14 minutes) was analyzed as an area percent (%). The results were as shown in Table 2, and each HPLC chromatogram was as shown in Figs.

Copper metal salt equivalent weight Reaction time UTP area% UMP area% Up4U Area% Copper bromide (II) 3 equivalents 3 hours 0.11 9.96 64.62 Copper bromide (II) 2.4 equivalent 6 hours 3.93 19.996 52.96 Copper bromide (II) 2 equivalents 6 hours 2.79 14.298 51.71 Copper bromide (II) 1.2 equivalent 6 hours 0 23.88 34.12 Copper sulfate (II) 1.2 equivalent 6 hours 39.39 30.61 14.98 Copper sulfate (II) 2 equivalents 6 hours 9.12 29.11 31.35 Copper Chloride (II) 2 equivalents 6 hours 2.5 11.07 58.38

As shown in Table 2 above, as the equivalence of the copper metal salt increases, the area% of Up4U increases in the HPLC chromatogram, indicating that the reaction progresses more. It is also understood that copper bromide and copper chloride are more effective in reaction than copper sulfate because copper bromide and copper chloride are more soluble in dimethylformamide, a water-soluble organic solvent than copper sulfate.

Further, when copper (II) chloride was used, the yield was improved as the equivalence of UMP was increased as shown in Table 3 below. Figures 14 and 15 are HPLC chromatograms using 1.2 equivalents of UMP and Figures 16 and 17 are HPLC chromatograms using 2 equivalents of UMP.

Copper metal salts and equivalents UMP equivalent Reaction time Up4U Yield 3 equivalents of copper (II) chloride hydrate 1.2 3 hours 49.3% 3 equivalents of copper (II) chloride hydrate 2.0 3 hours 78.9%

While the present invention has been described in connection with what is presently considered to be practical exemplary embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, You will understand. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (11)

Reacting two substances each independently selected from the group consisting of nucleoside 5'-triphosphate (NTP) and nucleoside 5'-monophosphate (NMP) in the presence of a copper metal salt,
Wherein the copper metal salt is used in an amount of 1 to 6 equivalents based on 1 equivalent of the nucleoside 5'-triphosphate (NTP), and the di (nucleoside 5'-) polyphosphate represented by the following formula (1).
[Chemical Formula 1]

(Wherein B ¹ and B ² are each independently a pyrimidine base and n is a natural number of 2 to 4) The method according to claim 1,
Wherein the NTP is uridine 5'-triphosphate (UTP) and the NMP is uridine 5'-monophosphate (UMP). The method according to claim 1,
Wherein the di (nucleoside 5'-) polyphosphate is di (uridine 5'-) tetraphosphate (Up4U) represented by the following formula (2).
(2)

The method according to claim 1,
Wherein the copper metal salt is at least one selected from the group consisting of copper chloride, bromide, nitrate, sulfur oxides, iodides, acetic acid and hydrates thereof, . 5. The method of claim 4,
Wherein the copper metal salt comprises at least one of copper bromide and copper chloride. The method according to claim 1,
Wherein said di (nucleoside 5'-) polyphosphate is prepared using from 1 to 5 equivalents of said NMP for said NTP equivalents. Reacting uridine 5'-triphosphate (UTP) and uridine 5'-monophosphate (UMP) in the presence of a copper metal salt
The copper metal salt is used in an amount of 1 to 6 equivalents based on 1 equivalent of uridine 5'-triphosphate (UTP), and is a process for producing di (uridine 5'-) tetraphosphate represented by the following formula (2).
(2)

delete 8. The method of claim 7,
Wherein the copper metal salt comprises at least one of copper bromide and copper chloride. 8. The method of claim 7,
Wherein the copper metal salt comprises copper chloride hydrate. &Lt; RTI ID = 0.0 &gt; 5. &lt; / RTI &gt; 8. The method of claim 7,
Wherein the UMP is used in an amount of 1 to 5 equivalents relative to 1 equivalent of the UTP.

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