JP2021161092A - Novel cdp-ribitol derivative - Google Patents

Abstract

To provide a novel CDP-ribitol derivative effective for treatment of dystroglycanopathy, a pharmaceutical containing the CDP-ribitol derivative, and a dystroglycanopathy treatment agent containing the CDP-ribitol derivative.SOLUTION: A CDP-ribitol derivative is represented by the formula (I) [where R1-R4 independently represent a C1-6 alkyl-carbonyl group or the like, R5 and R6 independently represent H or the like, R7 and R8 independently represent OH or the like].SELECTED DRAWING: None

Description

The present invention relates to a novel CDP-libitol derivative effective for the treatment of dystroglycan abnormality, a drug containing the CDP-libitol derivative, and a therapeutic agent for dystroglycan abnormality containing the CDP-libitol derivative.

Muscular dystrophy is a general term for hereditary diseases in which progressive muscle weakness is observed, and more than 50 causative genes have been elucidated. Of the muscular dystrophy, Fukuyama-type muscular dystrophy is common in Japan, and the cause is known to be an abnormality in the fukutin (FKTN) gene. Fukuyama-type muscular dystrophy-related diseases such as limb-girdle-type muscular dystrophy, congenital muscular dystrophy, and Walker-Warburg syndrome are found all over the world, and are called dystroglycan abnormalities including Fukuyama-type.

Dystroglycan is a membrane protein that binds to the intracellular cytoskeleton via dystrophin and also to the extracellular basal membrane via sugar chains and laminin, resulting in damage to muscle cells associated with muscle contraction. It acts like a dystrophin to prevent. Dystroglycan undergoes various post-translational modifications such as sugar chain formation. (Non-Patent Document 1). Two molecules of ribitol phosphate are contained in the sugar chain of dystroglycan, and these ribotor phosphates are incorporated into the sugar chain from cytidine diphosphate (CDP) -ribitol by fukutin (FKTN) and FKRP.

The present inventors have also clarified that ISPD (IsoPrenoid synchase Domain contouring) is an enzyme that biosynthesizes CDP-libitol from cytidine triphosphate (CTP) and ribitol-5-phosphate (non-patent). Document 1). That is, the disease caused by the genetic abnormality of ISPD, fukutin, and FKRP can be said to be ribitol phosphate deficiency. Therefore, the present inventors have developed a therapeutic agent for a disease associated with dystroglycan sugar modification abnormality containing CDP-ribitol as an active ingredient (Patent Document 1).

International Publication No. 2018/034343 Pamphlet

Kanagawa M et al., Cell Rep. , 2016, 14 (9), 2209-2223

As described above, the present inventors have developed therapeutic agents for diseases associated with dystroglycan sugar modification abnormalities such as dystroglycan abnormalities. However, there is a need for an even better active ingredient for the treatment of dystroglycan disorders.
Therefore, an object of the present invention is to provide a novel CDP-libitol derivative effective for the treatment of dystroglycan dysfunction, a drug containing the CDP-libitol derivative, and a therapeutic agent for dystroglycan dysfunction containing the CDP-libitol derivative. do.

The present inventors have conducted intensive studies to solve the above problems. As a result, they have found that a specific derivative of CDP-ribitol restores the sugar chain formation of dystroglycan in cells and mice in which the CDP-ribitol biosynthetic enzyme (ISPD) gene is knocked down, and completed the present invention.
Hereinafter, the present invention will be shown.

［式中、
Ｒ¹〜Ｒ⁴は、独立して、Ｈ、Ｃ_1-6アルキル−カルボニル基、またはＣ_1-6アルコキシ−カルボニル基を示し、
Ｒ⁵とＲ⁶は、独立して、Ｈ、Ｃ_1-6アルキル基、置換基を有していてもよいＣ_6-12アリール基、Ｃ_6-12アリール基上に置換基を有していてもよいＣ_6-12アリール−アミノ基、またはＣ_6-12アリール基上に置換基を有していてもよいＣ_6-12アリール−メチル基を示し、
Ｃ_6-12アリール基上の置換基は、Ｃ_1-6アルコキシ基、またはＣ_1-6アルキル−カルボニルオキシ基を示し、
Ｒ⁷とＲ⁸は、独立して、ＯＨ、Ｃ_1-6アルキル−カルボニルオキシ基、Ｃ_1-6アルコキシ−カルボニルオキシ基、Ｃ_1-6アルコキシ基、またはハロゲノ基を示し、
但し、Ｒ¹〜Ｒ⁶がＨである場合、Ｒ⁷とＲ⁸は、独立して、Ｃ_1-6アルキル−カルボニルオキシ基、Ｃ_1-6アルコキシ−カルボニルオキシ基、Ｃ_1-6アルコキシ基、またはハロゲノ基を示す。］
［２］ ＣＤＰ−リビトール誘導体が下記式（Ｉ²）で表されるＣＤＰ−リビトール誘導体である上記［１］に記載のＣＤＰ−リビトール誘導体またはその薬学的に許容される塩。

［式中、Ｒ¹〜Ｒ⁸は上記と同義を示す。］
［３］ Ｒ¹〜Ｒ⁴が、独立して、Ｃ_1-4アルキル−カルボニル基である上記［１］または［２］に記載のＣＤＰ−リビトール誘導体またはその薬学的に許容される塩。
［４］ Ｒ⁵とＲ⁶がＨである上記［１］〜［３］のいずれかに記載のＣＤＰ−リビトール誘導体またはその薬学的に許容される塩。
［５］ Ｒ⁵とＲ⁶が、独立して、Ｃ_6-12アリール基上に置換基を有していてもよいＣ_6-12アリール−メチル基である上記［１］〜［３］のいずれかに記載のＣＤＰ−リビトール誘導体またはその薬学的に許容される塩。
［６］ Ｒ⁵とＲ⁶が、独立して、フェニル基上に置換基としてＣ_1-6アルキル−カルボニルオキシ基を有するベンジル基である上記［１］〜［３］のいずれかに記載のＣＤＰ−リビトール誘導体またはその薬学的に許容される塩。
［７］ Ｒ⁷とＲ⁸がＯＨである上記［１］〜［６］のいずれかに記載のＣＤＰ−リビトール誘導体またはその薬学的に許容される塩。
［８］ 上記［１］〜［７］のいずれかに記載のＣＤＰ−リビトール誘導体またはその薬学的に許容される塩を含有することを特徴とする医薬。
［９］ 上記［１］〜［７］のいずれかに記載のＣＤＰ−リビトール誘導体またはその薬学的に許容される塩を有効成分として含有することを特徴とするジストログリカン異常症治療剤。 [1] A CDP-ribitol derivative or a pharmaceutically acceptable salt thereof, which is represented by the following ^{formula (I 1).}

[During the ceremony,
R ^{1 to} R ⁴ independently represent H, C _1-6 alkyl-carbonyl groups, or C _1-6 alkoxy-carbonyl groups.
R ⁵ and R ⁶ are independently, H, C _1-6 alkyl group which may have a substituent C _6-12 aryl group, optionally having a substituent on the C _6-12 aryl group which may be C _6-12 aryl - amino group or C _6-12 aryl group optionally C _6-12 aryl optionally having substituent on, - a methyl group,
Substituents on the C _6-12 aryl group represent a C _1-6 alkoxy group or a C _1-6 alkyl-carbonyloxy group.
R ⁷ and R ⁸ independently represent OH, C _1-6 alkyl-carbonyloxy group, C _1-6 alkoxy-carbonyloxy group, C _1-6 alkoxy group, or halogeno group.
However, when R ^{1 to} R ⁶ are H, R ⁷ and R ⁸ independently have a C _1-6 alkyl-carbonyloxy group, a C _1-6 alkoxy-carbonyloxy group, and a C _1-6 alkoxy group. , Or a halogeno group. ]
[2] The CDP-ribitol derivative according to the above [1] or a pharmaceutically acceptable salt thereof, wherein the CDP-ribitol derivative is a CDP-ribitol derivative represented by the following ^{formula (I 2).}

[In the formula, R ^{1 to} R ⁸ have the same meaning as above. ]
[3] The CDP-libitol derivative or a pharmaceutically acceptable salt thereof according to the above [1] or [2], wherein ^{R 1 to} R ⁴ are independently C _{1-4 alkyl-carbonyl groups.}
[4] The CDP-ribitol derivative according to any one of the above [1] to [3], wherein ^{R 5} and R ^{6 are H, or a pharmaceutically acceptable salt thereof.}
[5] Of the above [1] to [3], ^{where R 5} and R ⁶ _{are C 6-12} aryl-methyl groups which may independently have a substituent on the C _{6-12 aryl group.} The CDP-libitol derivative according to any one or a pharmaceutically acceptable salt thereof.
[6] The above-mentioned [1] to [3], wherein ^{R 5} and R ⁶ _{are independently benzyl groups having a C 1-6} alkyl-carbonyloxy group as a substituent on the phenyl group. CDP-libitol derivative or pharmaceutically acceptable salt thereof.
[7] The CDP-ribitol derivative according to any one of the above [1] to [6] or a pharmaceutically acceptable salt thereof, wherein ^{R 7} and R ^{8 are OH.}
[8] A drug comprising the CDP-ribitol derivative according to any one of the above [1] to [7] or a pharmaceutically acceptable salt thereof.
[9] A therapeutic agent for dystroglycan dysfunction, which comprises the CDP-ribitol derivative according to any one of the above [1] to [7] or a pharmaceutically acceptable salt thereof as an active ingredient.

The novel CDP-ribitol derivative according to the present invention was superior to CDP-ribitol in the recovery activity of sugar chain formation of dystroglycan in cells and mice in which the CDP-ribitol biosynthetic enzyme (ISPD) gene was knocked down. Therefore, the novel CDP-ribitol derivative according to the present invention is effective for the treatment of dystroglycan dysfunction caused by dystroglycan sugar chain dysplasia.

FIG. 1 is a Western blot photograph showing the recovery effect of dystroglycan sugar chain formation of the novel CDP-ribitol derivative according to the present invention. FIG. 2 is a Western blot photograph showing the recovery effect of dystroglycan sugar chain formation of the novel CDP-ribitol derivative according to the present invention. FIG. 3 is a Western blot photograph showing the recovery effect of dystroglycan sugar chain formation of the novel CDP-ribitol derivative according to the present invention. FIG. 4 is an H & E-stained photograph of the muscle tissue of the test mouse and an immunostained photograph using the antibody IIH6 that binds to the sugar chain of dystroglycan (DG). FIG. 5 is a western blot photograph of a muscle tissue sample of a CDP-libitol synthase (ISPD) gene knockout mouse administered with physiological saline, an aqueous solution of CDP-libitol, or a novel CDP-libitol derivative according to the present invention. FIG. 6 is a graph [(1)] showing the average muscle fiber diameter obtained by immunostaining a muscle tissue sample of the test mouse with laminin and a graph [(2)] showing the muscle fiber diameter distribution. FIG. 7 is a graph [(1)] showing the results of immunostaining with F4 / 80 of the muscle tissue sample of the test mouse and a graph [(2)] showing the results of immunostaining with collagen I.

In the CDP-ribitol derivative represented by the formula (I ¹ ) (hereinafter referred to as "CDP-ribitol derivative (I ¹ )"), the reducing sugar moiety is preferably ribitol and the furanose moiety is preferably ribose in terms of absolute structure. That is, the CDP-ribitol derivative (I ¹ ) may be hydrolyzed in vivo to a nucleoside diphosphate-reducing sugar with nuclease resistance and / or activity superior to that of CDP-ribitol, but in vivo. A CDP-ribitol derivative represented by the formula (I ² ) converted to CDP-ribitol (hereinafter referred to as "CDP-ribitol derivative (I ² )") is preferable. Hereinafter, the CDP-ribitol derivative (I ¹ ) and the CDP-ribitol derivative (I ² ) are collectively referred to as a CDP-ribitol derivative (I).

In the CDP-ribitol derivative (I), R ^{1 to} R ⁴ are H constituting the hydroxyl group or a protecting group for the hydroxyl group of the ribitol moiety. When R ^{1 to} R ⁴ are H, it is considered that the hydrophilicity of the CDP-ribitol derivative (I) is relatively high and the toxicity is relatively low. On the other hand, when R ^{1 to} R ⁴ are C _1-6 alkyl-carbonyl group or C _1-6 alkoxy-carbonyl group, the lipophilicity of the CDP-libitol derivative (I) becomes high and it is easily taken up into cells. On the other hand, it is considered that the C _1-6 alkyl-carbonyl group or the C _1-6 alkoxy-carbonyl group is taken up into the cell and then removed by an intracellular hydrolyzing enzyme such as esterase. In particular, the C _1-6 alkoxy-carbonyl group is considered to be easily hydrolyzed due to its carbonic acid ester structure.

The "C _1-6 alkyl group" refers to a linear or branched monovalent saturated aliphatic hydrocarbon group having 1 or more carbon atoms and 6 or less carbon atoms. For example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, n-hexyl and the like.

"C _1-6 alkoxy group" refers to a linear or branched aliphatic hydrocarbon oxy group having 1 or more carbon atoms and 6 or less carbon atoms. For example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, t-butoxy, n-pentoxy, n-hexoxy and the like.

C _1-6 alkyl R ¹ to R ⁴ - carbonyl group or a C _1-6 alkoxy - constitute a carbonyl group C _1-6 alkyl group or a C _1-6 possible toxicity smaller the number of carbon atoms in the alkoxy group is reduced Because of their properties, the carbon number of these groups is preferably 1 or more and 4 or less, more preferably 1 or 2, and even more preferably 1. In particular, the acetyl group and the methoxycarbonyl group can be said to have high biocompatibility. However, as the number of carbon atoms in these groups increases, the lipophilicity increases, and the intracellular uptake amount may increase.

^{When R 5} and R ⁶ are H in the CDP-libitol derivative (I), or when the phosphate group is a pharmaceutically acceptable salt, the hydrophilicity of the CDP-libitol derivative (I) is high. It is easy to formulate into a liquid drug which is an aqueous solution or a suspension, and it is considered that the toxicity is relatively low. On the other hand, at least one of R ⁵ and R ^6, C _1-6 alkyl group which may have a substituent C _6-12 aryl group, optionally having substituent on C _6-12 aryl group which may C _6-12 aryl - fat when it is methyl group, CDP-ribitol derivative (I) - amino or C _6-12 aryl group optionally C _6-12 aryl optionally having substituent on, Increased solubility may increase intracellular uptake.

The "C _6-12 aryl group" refers to a monovalent aromatic hydrocarbon group having 6 or more and 12 or less carbon atoms. For example, phenyl, naphthyl, indenyl, biphenyl and the like, preferably phenyl.

C _1-6 alkyl group which is a protecting group of a phosphate group, which may have a substituent C _6-12 aryl group which may have a substituent on the C _6-12 aryl group C _{6 -12} aryl - amino groups, and C _6-12 aryl optionally having a substituent on a base C _6-12 aryl - among methyl group, have a substituent on the C _6-12 aryl group The C _6-12 aryl-amino group, _{which may have a substituent, and the C 6-12} aryl-methyl group, which may have a substituent on the C _6-12 aryl group, are easily removed intracellularly, and C _{6-12 C 6-12} aryl-methyl groups, which may have substituents on aryl groups, are more likely to be removed intracellularly, and benzyl groups, which may have substituents on phenyl groups, are more intracellular. It is even easier to remove. In particular, when the C _6-12 aryl or phenyl group has a C _1-6 alkoxy group or a C _1-6 alkyl-carbonyloxy group as a substituent, it is easily removed depending on the substitution position. For example, if the p-position and / or o-position of the benzyl group _{is substituted with a C 1-6} alkoxy group or a C _1-6 alkyl-carbonyloxy group, particularly a C _1-6 alkyl-carbonyloxy group, in vivo. First, when these groups are removed to generate a phenolic hydroxyl group, the phenolic hydroxyl group pulls an electron, and as a result, these groups are eliminated from the phosphate group.

As the C _1-6 alkyl group as R ⁵ and R ⁶ _{, a C 1-4} alkyl group is preferable, a C _1-2 alkyl group is more preferable, and methyl is even more preferable. _{The carbon number of the C 1-6} alkoxy group, which is a substituent on the C _6-12 aryl group or the phenyl group, or the C _1-6 alkyl-carbonyloxy group is preferably 1 or more and 4 or less, preferably 1 or 2. Is more preferable, and 1 is even more preferable.

When R ⁷ and R ⁸ are OH, the original function of ribose is exhibited as it is, and it is considered that the toxicity is low. When R ⁷ and R ⁸ are C _1-6 alkyl-carbonyloxy group or C _1-6 alkoxy-carbonyloxy group, they are relatively lipophilic and easily taken up into cells, while being taken up into cells. After being hydrolyzed, it is hydrolyzed to OH, and it is considered that the original function of ribose is exhibited. When R ⁷ and R ⁸ are a C _1-6 alkyl-carbonyloxy group, a C _1-6 alkoxy-carbonyloxy group, a C _1-6 alkoxy group, or a halogeno group, a CDP-libitol derivative (I). ) Is considered to improve nuclease resistance.

The _{number of carbon atoms of the C 1-6} alkyl group and C _1-6 alkoxy contained in ^{R 7} and R ⁸ is preferably 1 or more and 4 or less, more preferably 1 or 2, and even more preferably 1.

In the CDP-ribitol derivative (I), when R ^{1 to} R ⁶ are all H, R ⁷ and R ⁸ are independently C _1-6 alkyl-carbonyloxy group and C _1-6 alkoxy-carbonyloxy. Shows a group, C _1-6 alkoxy group, or halogeno group. That is, the CDP-ribitol derivative (I) does not contain the unsubstituted CDP-ribitol.

The CDP-ribitol derivative (I) can form a salt with a pyrophosphate group, and the salt is a pharmaceutically acceptable salt. The counter cation constituting the pharmaceutically acceptable salt of the CDP-libitol derivative (I) is not particularly limited as long as it is pharmaceutically acceptable, but for example, alkali metal salts such as sodium ion and potassium ion. Examples thereof include Group 2 metal ions such as ions, magnesium ions and calcium ions. At least one of the phosphate groups may be in the -O-P (= O) (O ^- )-O- state, for example, in solution. In addition, the CDP-ribitol derivative (I) is generally basic, and there is a possibility that a salt may be formed by the amino group of cytosine. The salt is not particularly limited as long as it is a pharmaceutically acceptable salt, and for example, hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, perchlorate, phosphate and the like. Inorganic acid salts; oxalate, malonate, maleate, fumarate, lactate, malate, citrate, tartrate, benzoate, trifluoroacetate, acetate, methanesulfonic acid. Examples include salts, p-toluene sulfonates, trifluoromethane sulfonates, glutamates, asparaginates and other organic acid salts.

The CDP-ribitol derivative (I) can be synthesized, for example, by the following scheme.

In the above reaction, the reducing sugar monophosphate (II) and morpholidate (III) are reacted in a solvent. Since the phosphate group of morpholidate (III) is activated by the morpholine group, it is easy to reduce the reducing sugar monophosphorus even when ^{the reducing sugar has a hydroxyl group or R 7} and R ^{8 are hydroxyl groups.} It reacts with the acid ester (II) to form a pyrophosphate group.

As the solvent used in the above reaction, a solvent exhibiting basicity such as dimethylformamide or pyridine is preferable. In addition, tetrazole, benzimidazolium triflate, trichloroacetic acid and the like may be used to promote the reaction.

The reaction conditions may be adjusted as appropriate. For example, it is preferable to use a dehydrated solvent and carry out the reaction in an atmosphere of an inert gas such as nitrogen gas or argon gas in order to suppress the mixing of water and oxygen. The reaction temperature may be room temperature, for example, 0 ° C. or higher and 40 ° C. or lower. The reaction temperature is preferably 10 ° C. or higher or 15 ° C. or higher, more preferably 20 ° C. or higher, more preferably 35 ° C. or lower, and even more preferably 30 ° C. or lower. The reaction time should be determined by a preliminary experiment, until an increase in the amount of the target compound is not observed, or until either the reducing sugar monophosphate (II) or the morpholidate (III) is consumed by chromatography or the like. However, for example, it may be 10 hours or more and 10 days or less.

After completion of the reaction, normal post-treatment may be performed. For example, after distilling off the solvent, the target compound may be purified by chromatography, recrystallization or the like. Further, the hydroxyl group and the phosphoric acid group ^{may be protected or deprotected, and R 1 to} R ⁸ may be changed.

The reducing sugar monophosphate ester (II) can be easily synthesized. For example, as shown in the scheme below, the carboxy group of aldonic acid or its derivative is reduced, and a phosphoric acid group is introduced at the 5-position using _{phosphoryl chloride (POCl 3).} At this time, by protecting the hydroxyl group of aldonic acid, it becomes possible to selectively introduce the phosphoric acid group to the 5-position.

In addition, morpholidate (III) can be synthesized by a conventional method from a 5'-monophosphate form of a nucleoside or a derivative at the 2'and / or 3'position thereof.

The CDP-ribitol derivative (I) according to the present invention or a pharmaceutically acceptable salt thereof is taken up by cells and then hydrolyzed by an intracellular enzyme to be converted to CDP-ribitol. One of the causes of dystroglycan abnormality is that two molecules of ribitol phosphate are not introduced into the sugar chain of α-dystroglycan, and the sugar chain is not completely formed. The introduction of ribitol phosphate into the sugar chain of α-dystroglycan is as follows: first, CDP-ribitol is synthesized from ribitol phosphate and cytidine triphosphate (CTP) by ISPD, which is a CDP-libitol biosynthetic enzyme, and then from CDP-libitol. It is carried out by the action of fukutin (FKTN) and FKRP. Therefore, dystroglycan abnormalities are considered to be caused by abnormalities in the genes of ISPD, FKTN and / or FKRP. On the other hand, by administration of the CDP-ribitol derivative (I) according to the present invention or a pharmaceutically acceptable salt thereof, CDP-ribitol is supplied intracellularly even if there is an abnormality in the ISPD gene, and FKTN and FKTN Patients with dystroglycan dysfunction should have even a small amount of FKTN and FKRP activity because they cannot survive if FKRP is completely deficient, and by supplying a sufficient amount of CDP-ribitol, FKTN and Even if the activity of FKRP is low, sugar chains of α-dystroglycan are formed, and it is considered that the symptoms are alleviated. Therefore, the CDP-ribitol derivative (I) according to the present invention or a pharmaceutically acceptable salt thereof can be used as an active ingredient of a drug, particularly as an active ingredient of a therapeutic agent for dystroglycan abnormality.

Dystroglycan dysfunction is a disease caused by malformation of the sugar chain of α-dystroglycan as described above, and examples thereof include Fukuyama-type muscular dystrophy, limb-girdle-type muscular dystrophy, congenital muscular dystrophy, and Walker-Warburg syndrome.

The form of the drug containing the CDP-ribitol derivative (I) according to the present invention or a pharmaceutically acceptable salt thereof is not particularly limited, and can be an oral preparation or a parenteral preparation. Examples of the oral preparation include tablets, capsules, granules, powders, fine granules, pills, troches, sublingual preparations, emulsions, suspensions, syrups and the like. Parenteral preparations include injections, ointments, suppositories, creams and the like. The injection can be administered intravenously, subcutaneously, intramuscularly, or the like, and the ointment or cream may be applied to the mucous membrane or locally.

The medicament according to the present invention may contain a pharmaceutical additive in addition to the CDP-ribitol derivative (I) or a pharmaceutically acceptable salt thereof. Examples of the pharmaceutical additive include a solvent, a pH adjuster, an excipient, a disintegrant, a binder, a lubricant, a coating agent, a dye, and the like, but are not particularly limited.

As the medicine according to the present invention, an injection is preferable. As the solvent for the injection, in addition to water, a buffer solution or physiological saline may be used. Further, since the injection needs to be isotonic or substantially isotonic, it may contain sodium ion, potassium ion, calcium ion, chloride ion, acetate ion, hydrogen carbonate ion, lactic acid, glucose and the like.

The dose of the CDP-ribitol derivative (I) or a pharmaceutically acceptable salt thereof according to the present invention may be appropriately adjusted according to the patient's symptoms, severity, age, gender, etc., but for example, 1 It can be 0.01 mg or more and 60 g or less per day. The dose is preferably 0.1 mg or more, preferably 24 g or less, and more preferably 6 g or less. Further, the number of administrations per day can be 1 time or more and 5 times or less, preferably 4 times or less or 3 times or less, and more preferably 2 times or less. In addition, the administration interval may be 1 day or more and 2 weeks or less while observing the progress.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited by the following examples as well as the present invention, and appropriate modifications are made to the extent that it can be adapted to the gist of the above and the following. Of course, it is possible to carry out, and all of them are included in the technical scope of the present invention.

シチジン ５’−モノホスフェート（２．５ｇ）を水（１００ｍＬ）に懸濁し、次にトリエチルアミン（２．１５ｍＬ）を加えて溶解し、ｐＨを７〜８に調整した。これに１−アセチルイミダゾール（８．５ｇ）を加えて室温で約３時間攪拌した。次いでトリエチルアミン（２１５μＬ）と１−アセチルイミダゾール（２．０ｇ）を追加した。反応溶液を濾過した後に、逆相系のＨＰＬＣで精製した。目的のフラクションを集め、凍結乾燥により目的化合物を得た（粗収量：３．０ｇ（トリエチルアミンを含む）。
¹Ｈ−ＮＭＲ（４００ＭＨｚ，Ｄ₂Ｏ）：δ７．９５（ｄ，１Ｈ，Ｊ＝７．８Ｈｚ），６．２２（ｄ，１Ｈ，Ｊ＝５．５Ｈｚ），６．１３（ｄ，１Ｈ，Ｊ＝７．３Ｈｚ），５．４７（ｄｄ，１Ｈ，Ｊ＝３．７，５．５Ｈｚ），５．４４（ｄｄ，１Ｈ，Ｊ＝５．５，５．５Ｈｚ），４．５１（ｍ，１Ｈ），４．１６（ｄｄｄ，１Ｈ，Ｊ＝２．７，４．６，１１．９Ｈｚ），４．０８（ｄｄｄ，１Ｈ，Ｊ＝２．７，５．０，１１．９Ｈｚ），２．１８（ｓ，３Ｈ），２．１１（ｓ，３Ｈ） Example 1: Synthesis of CDP-ribitol diacetate (1) Synthesis of 2', 3'-di-O-acetyl-cytidine 5'-monophosphate

Cytidine 5'-monophosphate (2.5 g) was suspended in water (100 mL), then triethylamine (2.15 mL) was added to dissolve it, and the pH was adjusted to 7-8. 1-Acetylimidazole (8.5 g) was added thereto, and the mixture was stirred at room temperature for about 3 hours. Then triethylamine (215 μL) and 1-acetylimidazole (2.0 g) were added. The reaction solution was filtered and then purified by reverse phase HPLC. The desired fraction was collected and lyophilized to give the desired compound (crude yield: 3.0 g (including triethylamine).
^{1 1} H-NMR (400 MHz, D ₂ O): δ7.95 (d, 1H, J = 7.8 Hz), 6.22 (d, 1H, J = 5.5 Hz), 6.13 (d, 1H, J = 7.3Hz), 5.47 (dd, 1H, J = 3.7, 5.5Hz), 5.44 (dd, 1H, J = 5.5, 5.5Hz), 4.51 (m) , 1H), 4.16 (ddd, 1H, J = 2.7, 4.6, 11.9Hz), 4.08 (ddd, 1H, J = 2.7, 5.0, 11.9Hz), 2.18 (s, 3H), 2.11 (s, 3H)

２’，３’−ジ−Ｏ−アセチル−シチジン ５’−モノホスフェート（２．５ｇ）とモルホリン（１．８ｍＬ）をｔ−ブタノール（５０ｍＬ）と水（５０ｍＬ）の混合溶媒に懸濁し、還流した。これにＮ，Ｎ’−ジイソプロピルカルボジイミド（２．７ｍＬ）をｔ−ブタノール（５０ｍＬ）に溶解した溶液を滴下した。室温に戻した後に、減圧濃縮した。得られた残渣に水を加えて凍結乾燥した。凍結乾燥品（５．２ｇ）を逆相系のＨＰＬＣで精製した。目的のフラクションを集め、凍結乾燥により目的化合物を得た（粗収量：２．０ｇ（溶媒や塩を含む））。
¹Ｈ−ＮＭＲ（４００ＭＨｚ，Ｄ₂Ｏ）：δ７．９２（ｄ，１Ｈ，Ｊ＝７．３Ｈｚ），６．２１（ｄ，１Ｈ，Ｊ＝５．０Ｈｚ），６．１１（ｄ，１Ｈ，Ｊ＝７．８Ｈｚ），５．４８−５．４４（ｍ，２Ｈ），４．５２（ｍ，１Ｈ），４．１１（ｄｄｄ，１Ｈ，Ｊ＝２．３，４．１，１１．９Ｈｚ），４．０５（ｄｄｄ，１Ｈ，Ｊ＝３．２，５．０，１１．９Ｈｚ），３．７９（ｂｒ．ｔ，４Ｈ，Ｊ＝４．８Ｈｚ），３．７５（ｍ，２Ｈ），３．６７（ｂｒ．ｔ，４Ｈ，Ｊ＝４．３Ｈｚ），３．４４（ｂｒ．ｔ，４Ｈ，Ｊ＝５．０Ｈｚ），３．０６（ｍ，４Ｈ），２．１９（ｓ，３Ｈ），２．１２（ｓ，３Ｈ），１．２５（ｄ，１２Ｈ，Ｊ＝６．４Ｈｚ） (2) Synthesis of 2', 3'-di-O-acetyl-cytidine 5'-monophosphate morpholidate 4-morpholin-N, N'-diisopropylcarboxyamidine salt

2', 3'-di-O-acetyl-cytidine 5'-monophosphate (2.5 g) and morpholine (1.8 mL) are suspended in a mixed solvent of t-butanol (50 mL) and water (50 mL) and refluxed. bottom. A solution prepared by dissolving N, N'-diisopropylcarbodiimide (2.7 mL) in t-butanol (50 mL) was added dropwise thereto. After returning to room temperature, the mixture was concentrated under reduced pressure. Water was added to the obtained residue and lyophilized. The lyophilized product (5.2 g) was purified by reverse phase HPLC. The desired fraction was collected and lyophilized to give the desired compound (crude yield: 2.0 g (including solvent and salt)).
^{1 1} H-NMR (400 MHz, D ₂ O): δ7.92 (d, 1H, J = 7.3 Hz), 6.21 (d, 1H, J = 5.0 Hz), 6.11 (d, 1H, J = 7.8Hz), 5.48-5.44 (m, 2H), 4.52 (m, 1H), 4.11 (ddd, 1H, J = 2.3, 4.1, 11.9Hz) ), 4.05 (ddd, 1H, J = 3.2,5.0, 11.9Hz), 3.79 (br.t, 4H, J = 4.8Hz), 3.75 (m, 2H) , 3.67 (br.t, 4H, J = 4.3Hz), 3.44 (br.t, 4H, J = 5.0Hz), 3.06 (m, 4H), 2.19 (s, 3H), 2.12 (s, 3H), 1.25 (d, 12H, J = 6.4Hz)

参考文献（Ｊ．Ｍｅｄ．Ｃｈｅｍ．，１９８４，２７，７１７−７２６）の方法でＤ−リボース ５−ホスフェートから得られるＤ−リビトール ５−ホスフェート（９７０ｍｇ）と、トリ−ｎ−オクチルアミン（２．０５ｍＬ）に脱水ＤＭＦ（５０ｍＬ）を加えて減圧濃縮する操作を２回繰り返した。得られた残渣にアルゴン雰囲気下で脱水ＤＭＦ（５０ｍＬ）を加えて溶解した（Ａ液）。別途、２’，３’−ジ−Ｏ−アセチル−シチジン ５’−モノホスフェート モルフォリデート（２．０ｇ）に脱水ＤＭＦ（５０ｍＬ）を加えて減圧濃縮した。得られた残渣にアルゴン雰囲気下で脱水ピリジン（５０ｍＬ）を加えて溶解した（Ｂ液）。Ａ液とＢ液の２つの溶液を混合し、アルゴン雰囲気下で１Ｈ−テトラゾール（２９４ｍｇ）を加えて室温で攪拌した。５日後に反応混合物を減圧濃縮した。得られた残渣を逆相系のＨＰＬＣで精製した。目的のフラクションを集め、凍結乾燥及びイオン交換樹脂に通すことにより、白色粉末であるＣＤＰ−リビトール ジアセテートの２Ｎａ塩を得た（収量：１１３ｍｇ，収率：４．３％）。
¹Ｈ−ＮＭＲスペクトル（４００ＭＨｚ，Ｄ₂Ｏ）：δ７．９４（ｄ，１Ｈ，Ｊ＝７．３Ｈｚ），６．２２（ｄ，１Ｈ，Ｊ＝５．５Ｈｚ），６．１５（ｄ，１Ｈ，Ｊ＝７．８Ｈｚ），５．４８（ｄｄ，１Ｈ，Ｊ＝４．１，５．５Ｈｚ），５．４４（ｄｄ，１Ｈ，Ｊ＝５．５，５．５Ｈｚ），４．５３（ｍ，１Ｈ），４．２９（ｄｄｄ，１Ｈ，Ｊ＝２．３，５．０，１１．９Ｈｚ），４．２１（ｄｄｄ，１Ｈ，Ｊ＝３．２，５．０，１１．９Ｈｚ），４．１５（ｄｄｄ，１Ｈ，Ｊ＝３．０，５．７，１１．０Ｈｚ），４．０６（ｍ，１Ｈ），３．９１（ｍ，１Ｈ），３．８７（ｍ，１Ｈ），３．８０（ｄｄ，１Ｈ，Ｊ＝３．２，１１．９Ｈｚ），３．７６（ｄｄ，１Ｈ，Ｊ＝５．９，６．９Ｈｚ），３．６４（ｄｄ，１Ｈ，Ｊ＝６．９，１１．９Ｈｚ），２．１８（ｓ，３Ｈ），２．１２（ｓ，３Ｈ）
ＥＳＩ−ＭＳスペクトル（ｎｅｇａｔｉｖｅ ｍｏｄｅ） Ｃ₁₈Ｈ₂₈Ｎ₃Ｏ₁₇Ｐ₂：［Ｍ−Ｈ］^-，ｃａｌｃｄ：６２０．０９，ｆｏｕｎｄ：６２０．１ (3) CDP-ribitol diacetate

D-ribitol 5-phosphate (970 mg) obtained from D-ribose 5-phosphate by the method of reference (J. Med. Chem., 1984, 27, 717-726) and tri-n-octylamine (2. The operation of adding dehydrated DMF (50 mL) to (05 mL) and concentrating under reduced pressure was repeated twice. Dehydrated DMF (50 mL) was added to the obtained residue under an argon atmosphere and dissolved (Liquid A). Separately, dehydrated DMF (50 mL) was added to 2', 3'-di-O-acetyl-cytidine 5'-monophosphate morpholidate (2.0 g), and the mixture was concentrated under reduced pressure. Dehydrated pyridine (50 mL) was added to the obtained residue under an argon atmosphere and dissolved (Liquid B). Two solutions, solution A and solution B, were mixed, 1H-tetrazole (294 mg) was added under an argon atmosphere, and the mixture was stirred at room temperature. After 5 days, the reaction mixture was concentrated under reduced pressure. The obtained residue was purified by reverse phase HPLC. The desired fractions were collected, lyophilized and passed through an ion exchange resin to give a 2Na salt of CDP-ribitol diacetate, which is a white powder (yield: 113 mg, yield: 4.3%).
^{1 1} H-NMR spectrum (400 MHz, D ₂ O): δ7.94 (d, 1H, J = 7.3 Hz), 6.22 (d, 1H, J = 5.5 Hz), 6.15 (d, 1H) , J = 7.8Hz), 5.48 (dd, 1H, J = 4.1,5.5Hz), 5.44 (dd, 1H, J = 5.5,5.5Hz), 4.53 ( m, 1H), 4.29 (ddd, 1H, J = 2.3, 5.0, 11.9Hz), 4.21 (ddd, 1H, J = 3.2, 5.0, 11.9Hz) , 4.15 (ddd, 1H, J = 3.0, 5.7, 11.0Hz), 4.06 (m, 1H), 3.91 (m, 1H), 3.87 (m, 1H) , 3.80 (dd, 1H, J = 3.2, 11.9Hz), 3.76 (dd, 1H, J = 5.9, 6.9Hz), 3.64 (dd, 1H, J = 6) 9.9, 11.9Hz), 2.18 (s, 3H), 2.12 (s, 3H)
ESI-MS spectrum (negative mode) C ₁₈ H ₂₈ N ₃ O ₁₇ P ₂ : [MH] ^- , calcd: 620.09, found: 620.1

Ｌ−リボン酸−１，４−ラクトン（２．０ｇ）を水（１０ｍＬ）に溶解し、１Ｍ ＮａＯＨ水溶液（１４．２ｍＬ）を加えた。２０分後に反応溶液を減圧濃縮した（残渣Ａ）。別の容器に無水酢酸（３０ｍＬ）を入れて氷冷した。これに７０％ＨＣｌＯ₄水溶液（１．５ｍＬ）を加えることで、淡黄色溶液が得られた。前記残渣Ａを０℃以下に冷却し、この淡黄色溶液を加えた。反応混合物を４０℃まで昇温した後に、さらに１時間攪拌した。反応溶液を氷冷し、酢酸ナトリウム（２．４ｇ）と水（４０ｍＬ）を順次加えた。減圧濃縮により得られた残渣に水を加えて再度減圧濃縮した。残渣を逆相系のＨＰＬＣで精製し、目的のフラクションを集め、凍結乾燥により白色粉末である目的化合物を得た（収量：３．７８ｇ，収率：８３％）。
¹Ｈ−ＮＭＲ（４００ＭＨｚ，ＣＤＣｌ₃）：δ５．６３（ｄｄ，１Ｈ，Ｊ＝２．７，９．２Ｈｚ），５．４６（ｄ，１Ｈ，Ｊ＝２．３Ｈｚ），５．３６（ｄｄｄ，１Ｈ，Ｊ＝２．５，４．３，９．２Ｈｚ），４．３５（ｄｄ，１Ｈ，Ｊ＝２．５，１２．６Ｈｚ），４．１７（ｄｄ，１Ｈ，Ｊ＝４．３，１２．６Ｈｚ），２．１７（ｓ，３Ｈ），２．１０（ｓ，３Ｈ），２．０７（ｓ，３Ｈ），２．０３（ｓ，３Ｈ） Example 2: Synthesis of CDP-Ribitol Tetra Acetate (1) 2,3,4,5-Tetra-O-Acetyl-L-Ribbon Acid

L-Ribbon acid-1,4-lactone (2.0 g) was dissolved in water (10 mL) and a 1 M aqueous NaOH solution (14.2 mL) was added. After 20 minutes, the reaction solution was concentrated under reduced pressure (residue A). Acetic anhydride (30 mL) was placed in another container and ice-cooled. A pale yellow solution was obtained by adding a 70% HClO ₄ aqueous solution (1.5 mL) to this. The residue A was cooled to 0 ° C. or lower, and this pale yellow solution was added. The reaction mixture was heated to 40 ° C. and then stirred for an additional hour. The reaction solution was ice-cooled, and sodium acetate (2.4 g) and water (40 mL) were sequentially added. Water was added to the residue obtained by concentration under reduced pressure, and the mixture was concentrated under reduced pressure again. The residue was purified by reverse phase HPLC, the desired fraction was collected, and freeze-dried to obtain the desired compound as a white powder (yield: 3.78 g, yield: 83%).
^{1 1} H-NMR (400 MHz, CDCl ₃ ): δ5.63 (dd, 1H, J = 2.7, 9.2 Hz), 5.46 (d, 1H, J = 2.3 Hz), 5.36 (ddd) , 1H, J = 2.5, 4.3, 9.2Hz), 4.35 (dd, 1H, J = 2.5, 12.6Hz), 4.17 (dd, 1H, J = 4.3) , 12.6Hz), 2.17 (s, 3H), 2.10 (s, 3H), 2.07 (s, 3H), 2.03 (s, 3H)

２，３，４，５−テトラ−Ｏ−アセチル−Ｌ−リボン酸（３．７８ｇ）を脱水ＴＨＦ（５０ｍＬ）に溶解し、−１０℃に冷却した。これにＮ−メチルモルホリン（１．３６ｍＬ）とクロロギ酸イソブチル（１．６１ｍＬ）を順次加えて１０分間攪拌した。さらにＮａＢＨ₄（１．２８ｇ）を加えた後に、−５０℃に冷却した。これにメタノール（８０ｍＬ）を１０分間で滴下した。氷浴し、６Ｍ塩酸（２．７ｍＬ）を加えてｐＨを４に調整した。減圧濃縮し、得られた残渣に酢酸エチルと水を加えて抽出した。有機層を合わせ、無水硫酸ナトリウムで乾燥させた。これを濾過し、濾液と洗浄液を合わせて減圧濃縮した。得られた残渣をシリカゲルカラムクロマトグラフィーにより精製した。目的のフラクションを集めて減圧濃縮、乾燥することで白色粉末として目的化合物を得た（収量：２．６６ｇ，収率：７３％）。なお、目的化合物である２，３，４，５−テトラ−Ｏ−アセチル−Ｌ−リビトールは、１，２，３，４−テトラ−Ｏ−アセチル−Ｄ−リビトールともいう。
¹Ｈ−ＮＭＲ（４００ＭＨｚ，ＣＤＣｌ₃）：δ５．３７（ｄｄ，１Ｈ，Ｊ＝４．６，５．９Ｈｚ），５．３３（ｄｄｄ，１Ｈ，Ｊ＝３．２，４．６，６．９Ｈｚ），５．０７（ｄｄｄ，１Ｈ，Ｊ＝３．７，５．５，５．５Ｈｚ），４．３８（ｄｄ，１Ｈ，Ｊ＝３．２，１１．９Ｈｚ），４．１６（ｄｄ，１Ｈ，Ｊ＝６．９，１１．９Ｈｚ），３．８４（ｍ，１Ｈ），３．６９（ｄｄ，１Ｈ，Ｊ＝５．０，１２．８Ｈｚ），２．１４（ｓ，３Ｈ），２．１１（ｓ，３Ｈ），２．１０（ｓ，３Ｈ），２．０５（ｓ，３Ｈ） (2) Synthesis of 1,2,3,4-tetra-O-acetyl-D-ribitol

2,3,4,5-Tetra-O-acetyl-L-ribbon acid (3.78 g) was dissolved in dehydrated THF (50 mL) and cooled to −10 ° C. N-Methylmorpholine (1.36 mL) and isobutyl chloroformate (1.61 mL) were sequentially added thereto, and the mixture was stirred for 10 minutes. After further adding NaBH ₄ (1.28 g), the mixture was cooled to −50 ° C. Methanol (80 mL) was added dropwise thereto for 10 minutes. The pH was adjusted to 4 by bathing in ice and adding 6M hydrochloric acid (2.7 mL). The mixture was concentrated under reduced pressure, and ethyl acetate and water were added to the obtained residue for extraction. The organic layers were combined and dried over anhydrous sodium sulfate. This was filtered, and the filtrate and the washing liquid were combined and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography. The target fraction was collected, concentrated under reduced pressure, and dried to obtain the target compound as a white powder (yield: 2.66 g, yield: 73%). The target compound 2,3,4,5-tetra-O-acetyl-L-ribitol is also referred to as 1,2,3,4-tetra-O-acetyl-D-ribitol.
^{1 1} H-NMR (400 MHz, CDCl ₃ ): δ5.37 (dd, 1H, J = 4.6, 5.9 Hz), 5.33 (ddd, 1H, J = 3.2, 4.6, 6. 9Hz), 5.07 (ddd, 1H, J = 3.7, 5.5, 5.5Hz), 4.38 (dd, 1H, J = 3.2, 11.9Hz), 4.16 (dd) , 1H, J = 6.9, 11.9Hz), 3.84 (m, 1H), 3.69 (dd, 1H, J = 5.0, 12.8Hz), 2.14 (s, 3H) , 2.11 (s, 3H), 2.10 (s, 3H), 2.05 (s, 3H)

１，２，３，４−テトラ−Ｏ−アセチル−Ｄ−リビトール（１．０ｇ）を脱水ＴＨＦ（２０ｍＬ）に溶解し、−１０℃以下に冷却した。これにジイソプロピルエチルアミン（４．２ｍＬ）とＰＯＣｌ₃（２．０ｍＬ）を順次加えて３０分間攪拌した。反応混合物を−７８℃に冷却し、水（２０ｍＬ）を徐々に加えた。室温で飽和炭酸水素ナトリウム水溶液を加えて中和し、減圧濃縮によりＴＨＦを留去した。得られた残渣（水層）に飽和炭酸水素ナトリウム水溶液とクロロホルムを加えて分液した。有機層に水を加えて抽出し、水層を合わせた。減圧濃縮により液量を減じた。６Ｍ塩酸（１．５ｍＬ）を加えてｐＨを１〜２に調整した後、逆相ＨＰＬＣで精製した。目的のフラクションを集めて凍結乾燥し、得られた残渣に水を加えて溶解して再度凍結乾燥し、含水軟性油状物として目的化合物を得た（粗収量：１．１１ｇ（水を含む））。
¹Ｈ−ＮＭＲ（４００ＭＨｚ，Ｄ₂Ｏ）：δ５．４１（ｄｄ，１Ｈ，Ｊ＝５．５，５．５Ｈｚ），５．３５−５．２８（ｍ，２Ｈ），４．４４（ｄｄ，１Ｈ，Ｊ＝３．２，１２．４Ｈｚ），４．３１（ｄｄ，１Ｈ，Ｊ＝５．９，１２．４Ｈｚ），４．１４（ｄｄｄ，１Ｈ，Ｊ＝３．２，５．９，１１．９Ｈｚ），４．０４（ｄｄｄ，１Ｈ，Ｊ＝５．９，６．９，１１．９Ｈｚ），２．１６（ｓ，３Ｈ），２．１５（ｓ，３Ｈ），２．１３（ｓ，３Ｈ），２．０８（ｓ，３Ｈ） (3) Synthesis of 1,2,3,4-tetra-O-acetyl-D-ribitol 5-phosphate

1,2,3,4-tetra-O-acetyl-D-ribitol (1.0 g) was dissolved in dehydrated THF (20 mL) and cooled to −10 ° C. or lower. _{Diisopropylethylamine (4.2 mL) and POCl 3} (2.0 mL) were sequentially added thereto, and the mixture was stirred for 30 minutes. The reaction mixture was cooled to −78 ° C. and water (20 mL) was added slowly. A saturated aqueous sodium hydrogen carbonate solution was added at room temperature for neutralization, and THF was distilled off by concentration under reduced pressure. A saturated aqueous sodium hydrogen carbonate solution and chloroform were added to the obtained residue (aqueous layer) to separate the liquids. Water was added to the organic layer for extraction, and the aqueous layers were combined. The amount of liquid was reduced by concentration under reduced pressure. After adjusting the pH to 1-2 by adding 6M hydrochloric acid (1.5 mL), the mixture was purified by reverse phase HPLC. The target fraction was collected and freeze-dried, water was added to the obtained residue, dissolved, and freeze-dried again to obtain the target compound as a water-containing soft oil (crude yield: 1.11 g (including water)). ..
^{1 1} H-NMR (400 MHz, D ₂ O): δ5.41 (dd, 1H, J = 5.5, 5.5 Hz), 5.35-5.28 (m, 2H), 4.44 (dd, dd, 1H, J = 3.2, 12.4Hz), 4.31 (dd, 1H, J = 5.9, 12.4Hz), 4.14 (ddd, 1H, J = 3.2,5.9, 11.9Hz), 4.04 (ddd, 1H, J = 5.9, 6.9, 11.9Hz), 2.16 (s, 3H), 2.15 (s, 3H), 2.13 ( s, 3H), 2.08 (s, 3H)

１，２，３，４−テトラ−Ｏ−アセチル−Ｄ−リビトール ５−ホスフェート（１．１１ｇ）を脱水ＤＭＦに溶解し、トリエチルアミン（０．６９ｍＬ）を加えて減圧濃縮した。得られた残渣に脱水ＤＭＦを加えて減圧濃縮する操作を２回繰り返した。アルゴン雰囲気下、得られた残渣を脱水ピリジンに溶解し、トリエチルアミン（０．６９ｍＬ）を加えた（Ａ液）。１Ｈ−テトラゾール（０．２７ｇ）と、別途調製したシチジン ５’−モノホスフェート モルフォリデート（定法によりシチジン ５’−モノホスフェートから調製が可能）（１．８９ｇ）をそれぞれ別の容器Ｂと容器Ｃに入れ、脱水ＤＭＦを加えて減圧濃縮する操作を２回繰り返した。それぞれ得られた残渣をアルゴン雰囲気下で脱水ピリジンに溶解した（順にＢ液およびＣ液）。続いてアルゴン雰囲気下でＡ液に対してＢ液とＣ液を順次加えた。溶媒量は合計で約２０ｍＬであった。アルゴン雰囲気下、室温で５日間攪拌した。反応混合物を減圧濃縮した。得られた残渣に脱水ＤＭＦを加えて再度減圧濃縮した。残渣を逆相系のＨＰＬＣで精製した。目的のフラクションを集め、凍結乾燥、およびイオン交換樹脂に通すことにより、白色粉末として目的化合物の２Ｎａ塩を得た（収量：５４８ｍｇ，収率：２８％）。
¹Ｈ−ＮＭＲ（４００ＭＨｚ，Ｄ₂Ｏ）：δ８．００（ｄ，１Ｈ，Ｊ＝７．３Ｈｚ），６．１２（ｄ，１Ｈ，Ｊ＝７．３Ｈｚ），５．９７（ｄ，１Ｈ，Ｊ＝４．１Ｈｚ），５．３６（ｄｄ，１Ｈ，Ｊ＝５．５，５．５Ｈｚ），５．３１−５．２６（ｍ，２Ｈ），４．４０（ｄｄ，１Ｈ，Ｊ＝３．２，１２．４Ｈｚ），４．３５−４．２３（ｍ，５Ｈ），４．２０−４．１５（ｍ，２Ｈ），４．０６（ｍ，１Ｈ），２．１２９（ｓ，３Ｈ），２．１１８（ｓ，３Ｈ），２．１１８（ｓ，３Ｈ），２．０７１（ｓ，３Ｈ）
ＥＳＩ−ＭＳスペクトル（ｎｅｇａｔｉｖｅ ｍｏｄｅ） Ｃ₂₂Ｈ₃₂Ｎ₃Ｏ₁₉Ｐ₂：［Ｍ−Ｈ］^-，ｃａｌｃｄ：７０４．１１，ｆｏｕｎｄ：７０４．０ (4) Synthesis of CDP-ribitol tetraacetate

1,2,3,4-Tetra-O-acetyl-D-ribitol 5-phosphate (1.11 g) was dissolved in dehydrated DMF, triethylamine (0.69 mL) was added, and the mixture was concentrated under reduced pressure. The operation of adding dehydrated DMF to the obtained residue and concentrating under reduced pressure was repeated twice. The obtained residue was dissolved in dehydrated pyridine under an argon atmosphere, and triethylamine (0.69 mL) was added (Liquid A). Separate container B and container C for 1H-tetrazole (0.27 g) and separately prepared cytidine 5'-monophosphate morpholidate (which can be prepared from cytidine 5'-monophosphate by a conventional method) (1.89 g), respectively. The operation of adding dehydrated DMF and concentrating under reduced pressure was repeated twice. The obtained residues were dissolved in dehydrated pyridine under an argon atmosphere (solutions B and C in that order). Subsequently, liquid B and liquid C were sequentially added to liquid A under an argon atmosphere. The total amount of solvent was about 20 mL. The mixture was stirred at room temperature for 5 days under an argon atmosphere. The reaction mixture was concentrated under reduced pressure. Dehydrated DMF was added to the obtained residue, and the mixture was concentrated again under reduced pressure. The residue was purified by reverse phase HPLC. The desired fraction was collected, lyophilized and passed through an ion exchange resin to obtain a 2Na salt of the target compound as a white powder (yield: 548 mg, yield: 28%).
^{1 1} H-NMR (400 MHz, D ₂ O): δ8.00 (d, 1H, J = 7.3 Hz), 6.12 (d, 1H, J = 7.3 Hz), 5.97 (d, 1H, J = 4.1Hz), 5.36 (dd, 1H, J = 5.5, 5.5Hz), 5.31-5.26 (m, 2H), 4.40 (dd, 1H, J = 3) .2,12.4Hz), 4.35-4.23 (m, 5H), 4.20-4.15 (m, 2H), 4.06 (m, 1H), 2.129 (s, 3H) ), 2.118 (s, 3H), 2.118 (s, 3H), 2.071 (s, 3H)
ESI-MS spectrum (negative mode) C ₂₂ H ₃₂ N ₃ O ₁₉ P ₂ : [MH] ^- , calcd: 704.11, found: 704.0

ＣＤＰ−リビトール テトラアセテート（２５０ｍｇ）を水（３．３４ｍＬ）に溶解し、１−アセチルイミダゾール（３６８ｍｇ）を加えて室温で攪拌した。その後、４回に分けて１−アセチルイミダゾール（合計で６２６ｍｇ）を追加した。反応開始から約４時間後に反応混合物を水で希釈して、逆相系のＨＰＬＣで精製した。目的のフラクションを集め、凍結乾燥、およびイオン交換樹脂に通すことにより、白色粉末である目的化合物の２Ｎａ塩を得た（収量：２２８ｍｇ，収率：７５％）。
¹Ｈ−ＮＭＲ（４００ＭＨｚ，Ｄ₂Ｏ）：δ７．９７（ｄ，１Ｈ，Ｊ＝７．３Ｈｚ），６．２２（ｄ，１Ｈ，Ｊ＝５．５Ｈｚ），６．１６（ｄ，１Ｈ，Ｊ＝７．３Ｈｚ），５．４６（ｄｄ，１Ｈ，Ｊ＝４．１，５．５Ｈｚ），５．４１（ｄｄ，１Ｈ，Ｊ＝５．５，５．５Ｈｚ），５．３６（ｄｄ，１Ｈ，Ｊ＝５．５，５．５Ｈｚ），５．３１−５．２６（ｍ，２Ｈ），４．５２（ｍ，１Ｈ），４．４１（ｄｄ，１Ｈ，Ｊ＝３．２，１２．４Ｈｚ），４．３１−４．２６（ｍ，２Ｈ），４．２０−４．１６（ｍ，２Ｈ），４．０７（ｍ，１Ｈ），２．１８（ｓ，３Ｈ），２．１４（ｓ，３Ｈ），２．１３（ｓ，３Ｈ），２．１２０（ｓ，３Ｈ），２．１１８（ｓ，３Ｈ），２．０８（ｓ，３Ｈ）
ＥＳＩ−ＭＳスペクトル（ｎｅｇａｔｉｖｅ ｍｏｄｅ） Ｃ₂₆Ｈ₃₆Ｎ₃Ｏ₂₁Ｐ₂：［Ｍ−Ｈ］^-，ｃａｌｃｄ：７８８．１３，ｆｏｕｎｄ：７８８．１ Example 3: Synthesis of CDP-Ribitol Hexaacetate

CDP-ribitol tetraacetate (250 mg) was dissolved in water (3.34 mL), 1-acetylimidazole (368 mg) was added, and the mixture was stirred at room temperature. Then, 1-acetylimidazole (626 mg in total) was added in 4 portions. Approximately 4 hours after the start of the reaction, the reaction mixture was diluted with water and purified by reverse phase HPLC. The desired fractions were collected, lyophilized and passed through an ion exchange resin to give a 2Na salt of the target compound as a white powder (yield: 228 mg, yield: 75%).
^{1 1} H-NMR (400 MHz, D ₂ O): δ7.97 (d, 1H, J = 7.3 Hz), 6.22 (d, 1H, J = 5.5 Hz), 6.16 (d, 1H, J = 7.3Hz), 5.46 (dd, 1H, J = 4.1,5.5Hz), 5.41 (dd, 1H, J = 5.5,5.5Hz), 5.36 (dd) , 1H, J = 5.5, 5.5Hz), 5.31-5.26 (m, 2H), 4.52 (m, 1H), 4.41 (dd, 1H, J = 3.2, 12.4Hz), 4.31-4.26 (m, 2H), 4.20-4.16 (m, 2H), 4.07 (m, 1H), 2.18 (s, 3H), 2 .14 (s, 3H), 2.13 (s, 3H), 2.120 (s, 3H), 2.118 (s, 3H), 2.08 (s, 3H)
ESI-MS spectrum (negative mode) C ₂₆ H ₃₆ N ₃ O ₂₁ P ₂ : [MH] ^- , calcd: 788.13, found: 788.1

参考文献（ＷＯ２０１６／０４４１６４）の方法などによりＤ−リボースから得られる５−Ｏ−トリチル−Ｄ−リビトール（６．０ｇ）を脱水ピリジン（６０ｍＬ）に溶解し、４−ジメチルアミノピリジン（０．９３ｇ）を加えて氷冷した。これにブタン酸無水物（１４．９ｍＬ）を加え、続いて脱水クロロホルム（５０ｍＬ）と脱水ピリジン（６０ｍＬ）を加えた。反応容器を氷浴からはずして室温で攪拌した。４０分後に減圧濃縮し、得られた残渣にジメチルエーテルと水を加えて分液、抽出した。有機層を合わせ、飽和炭酸水素ナトリウム水、水、および飽和食塩水で順次洗浄した後に、無水硫酸ナトリウムで乾燥させた。これを濾過し、濾液と洗浄液を合わせて減圧濃縮した。得られた残渣をシリカゲルカラムクロマトグラフィーにより精製した。目的のフラクションを集めて減圧濃縮し、得られた残渣にｎ−ヘキサンを加えて減圧濃縮および乾燥することで、無色油状物として目的化合物を得た（粗収量：６．７３ｇ（溶媒及び試薬由来物を含む））。
¹Ｈ−ＮＭＲ（４００ＭＨｚ，ＣＤＣｌ₃）：δ７．４０−７．２０（ｍ，１５Ｈ），５．４２（ｄｄ，１Ｈ，Ｊ＝５．０，５．９Ｈｚ），５．３４（ｄｄｄ，１Ｈ，Ｊ＝３．２，５．９，５．９Ｈｚ），５．２８（ｄｄｄ，１Ｈ，Ｊ＝３．２，５．０，６．９Ｈｚ），４．３２（ｄｄ，１Ｈ，Ｊ＝３．２，１２．４Ｈｚ），４．１０（ｄｄ，１Ｈ，Ｊ＝６．９，１２．４Ｈｚ），３．２９（ｄｄ，１Ｈ，Ｊ＝３．２，１０．５Ｈｚ），３．１６（ｄｄ，１Ｈ，Ｊ＝５．９，１０．５Ｈｚ），２．４１−２．０６（ｍ，８Ｈ），１．７５−１．４６（ｍ，８Ｈ），０．９７６（ｔ，３Ｈ，Ｊ＝７．３Ｈｚ），０．９１０（ｔ，３Ｈ，Ｊ＝７．３Ｈｚ），０．９０５（ｔ，３Ｈ，Ｊ＝７．３Ｈｚ），０．８７３（ｔ，３Ｈ，Ｊ＝７．３Ｈｚ） Example 4: Synthesis of CDP-ribitol tetrabutyrate (1) Synthesis of 1,2,3,4-tetra-O-butyryl-5-O-trityl-D-ribitol

5-O-trityl-D-ribitol (6.0 g) obtained from D-ribose by the method of Reference (WO2016 / 044164) is dissolved in dehydrated pyridine (60 mL), and 4-dimethylaminopyridine (0.93 g) is dissolved. ) Was added and ice-cooled. To this was added butanoic anhydride (14.9 mL), followed by dehydrated chloroform (50 mL) and dehydrated pyridine (60 mL). The reaction vessel was removed from the ice bath and stirred at room temperature. After 40 minutes, the mixture was concentrated under reduced pressure, dimethyl ether and water were added to the obtained residue, and the mixture was separated and extracted. The organic layers were combined, washed successively with saturated aqueous sodium hydrogen carbonate, water, and saturated brine, and dried over anhydrous sodium sulfate. This was filtered, and the filtrate and the washing liquid were combined and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography. The desired fraction was collected and concentrated under reduced pressure, n-hexane was added to the obtained residue, concentrated under reduced pressure and dried to obtain the desired compound as a colorless oil (crude yield: 6.73 g (derived from solvent and reagent). (Including things)).
^{1 1} H-NMR (400 MHz, CDCl ₃ ): δ7.40-7.20 (m, 15H), 5.42 (dd, 1H, J = 5.0, 5.9 Hz), 5.34 (ddd, 1H) , J = 3.2,5.9,5.9Hz), 5.28 (ddd, 1H, J = 3.2,5.0, 6.9Hz), 4.32 (dd, 1H, J = 3) .2,12.4Hz), 4.10 (dd, 1H, J = 6.9, 12.4Hz), 3.29 (dd, 1H, J = 3.2, 10.5Hz), 3.16 ( dd, 1H, J = 5.9, 10.5Hz), 2.41-2.06 (m, 8H), 1.75-1.46 (m, 8H), 0.976 (t, 3H, J) = 7.3Hz), 0.910 (t, 3H, J = 7.3Hz), 0.905 (t, 3H, J = 7.3Hz), 0.873 (t, 3H, J = 7.3Hz)

１，２，３，４−テトラ−Ｏ−ブチリル−５−Ｏ−トリチル−Ｄ−リビトール（３．８４ｇ）を酢酸（４０ｍＬ）に溶解し、５５℃に加温すると共に水（９ｍＬ）を加えた。５５℃で３時間攪拌した後に、反応混合物を減圧濃縮した。得られた残渣にジエチルエーテルを加え、飽和炭酸水素ナトリウム水、水および飽和食塩水で順次洗浄した後に、無水硫酸ナトリウムで乾燥させた。これを濾過し、濾液と洗浄液を合わせて減圧濃縮した。得られた残渣をシリカゲルカラムクロマトグラフィーにより精製した。目的のフラクションを集めて減圧濃縮し、残渣にｎ−ヘキサンを加えて減圧濃縮、乾燥することで、無色油状物である目的化合物を得た（収量：１．５１ｇ，収率：６１％）。
¹Ｈ−ＮＭＲ（４００ＭＨｚ，ＣＤＣｌ₃）：δ５．３９−５．３３（ｍ，２Ｈ），５．０８（ｄｄｄ，１Ｈ，Ｊ＝３．７，５．５，５．５Ｈｚ），４．３９（ｄｄ，１Ｈ，Ｊ＝３．２，１２．４Ｈｚ），４．１５（ｄｄ，１Ｈ，Ｊ＝６．９，１２．４Ｈｚ），３．８３（ｄｄ，１Ｈ，Ｊ＝３．７，１２．４Ｈｚ），３．６８（ｄｄ，１Ｈ，Ｊ＝５．５，１２．４Ｈｚ），２．３９−２．２５（ｍ，８Ｈ），１．７３−１．６０（ｍ，８Ｈ），０．９６８（ｔ，３Ｈ，Ｊ＝７．３Ｈｚ），０．９６７（ｔ，３Ｈ，Ｊ＝７．３Ｈｚ），０．９５４（ｔ，３Ｈ，Ｊ＝７．３Ｈｚ），０．９３５（ｔ，３Ｈ，Ｊ＝７．３Ｈｚ）
ＥＳＩ−ＭＳスペクトル（ｐｏｓｉｔｉｖｅ ｍｏｄｅ） Ｃ₂₁Ｈ₃₆Ｏ₉Ｎａ：［Ｍ＋Ｎａ］⁺，ｃａｌｃｄ：４５５．２３，ｆｏｕｎｄ：４５５．２ (2) Synthesis of 1,2,3,4-tetra-O-butyryl-D-ribitol

Dissolve 1,2,3,4-tetra-O-butyryl-5-O-trityl-D-ribitol (3.84 g) in acetic acid (40 mL), heat to 55 ° C and add water (9 mL). rice field. After stirring at 55 ° C. for 3 hours, the reaction mixture was concentrated under reduced pressure. Diethyl ether was added to the obtained residue, and the mixture was washed successively with saturated aqueous sodium hydrogen carbonate, water and saturated brine, and dried over anhydrous sodium sulfate. This was filtered, and the filtrate and the washing liquid were combined and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography. The target fraction was collected and concentrated under reduced pressure, n-hexane was added to the residue, concentrated under reduced pressure, and dried to obtain the target compound as a colorless oil (yield: 1.51 g, yield: 61%).
^{1 1} H-NMR (400 MHz, CDCl ₃ ): δ5.39-5.33 (m, 2H), 5.08 (ddd, 1H, J = 3.7, 5.5, 5.5 Hz), 4.39 (Dd, 1H, J = 3.2, 12.4Hz), 4.15 (dd, 1H, J = 6.9, 12.4Hz), 3.83 (dd, 1H, J = 3.7, 12) .4Hz), 3.68 (dd, 1H, J = 5.5, 12.4Hz), 2.39-2.25 (m, 8H), 1.73-1.60 (m, 8H), 0 .968 (t, 3H, J = 7.3Hz), 0.967 (t, 3H, J = 7.3Hz), 0.954 (t, 3H, J = 7.3Hz), 0.935 (t, 3H, J = 7.3Hz)
ESI-MS spectrum (positive mode) C ₂₁ H ₃₆ O ₉ Na: [M + Na] ⁺ , calcd: 455.23, found: 455.2

１，２，３，４−テトラ−Ｏ−ブチリル−Ｄ−リビトール（１．５１ｇ）を脱水ＴＨＦ（３０ｍＬ）に溶解し、−１０℃以下に冷却した。これにジイソプロピルエチルアミン（４．８６ｍＬ）とＰＯＣｌ₃（２．２７ｍＬ）を順次加えて４０分間攪拌した。反応混合物を−７８℃に冷却し、水（２０ｍＬ）を徐々に加えた。室温で飽和炭酸水素ナトリウム水溶液を加えて中和後に減圧濃縮により液量を減じ、残渣（水層）をｎ−ヘキサンで２回洗浄し、ジエチルエーテル（１５０ｍＬ）を加えて氷冷、攪拌した。６Ｍ塩酸（１４ｍＬ）を加えてｐＨを１〜２に調整した後に分液した。分離した水層にジエチルエーテルを加えて抽出し、有機層を合わせた。これを水と飽和食塩水で順次洗浄後に、無水硫酸ナトリウムで乾燥させた。これを濾過し、濾液と洗浄液を合わせ、トリエチルアミン（０．４８ｍＬ）を加えて減圧濃縮、乾燥することで、無色油状物である目的化合物を得た（粗収量：１．９５ｇ（トリエチルアミンを含む））。
¹Ｈ−ＮＭＲ（４００ ＭＨｚ，Ｄ₂Ｏ）：δ５．４４（ｄｄ，１Ｈ，Ｊ＝５．５，５．５Ｈｚ），５．３７（ｄｄｄ，１Ｈ，Ｊ＝２．７，５．９，５．９Ｈｚ），５．３１（ｍ，１Ｈ），４．５１（ｄｄ，１Ｈ，Ｊ＝２．７，１２．４Ｈｚ），４．３０（ｄｄ，１Ｈ，Ｊ＝５．９，１２．４Ｈｚ），４．１０（ｄｄｄ，１Ｈ，Ｊ＝３．２，５．５，１１．４Ｈｚ），３．９８（ｄｄｄ，１Ｈ，Ｊ＝６．４，６．４，１１．４Ｈｚ），２．４５−２．３３（ｍ，８Ｈ），１．６９−１．５５（ｍ，８Ｈ），０．９３６（ｔ，３Ｈ，Ｊ＝７．３Ｈｚ），０．９２９（ｔ，３Ｈ，Ｊ＝７．３Ｈｚ），０．９２３（ｔ，３Ｈ，Ｊ＝７．３Ｈｚ），０．９００（ｔ，３Ｈ，Ｊ＝７．３Ｈｚ）
ＥＳＩ−ＭＳスペクトル（ｎｅｇａｔｉｖｅ ｍｏｄｅ） Ｃ₂₁Ｈ₃₆Ｏ₁₂Ｐ：［Ｍ−Ｈ］^-，ｃａｌｃｄ：５１１．１９，ｆｏｕｎｄ：５１１．２ (3) Synthesis of 1,2,3,4-tetra-O-butyryl-D-ribitol 5-phosphate

1,2,3,4-tetra-O-butyryl-D-ribitol (1.51 g) was dissolved in dehydrated THF (30 mL) and cooled to −10 ° C. or lower. Diisopropylethylamine (4.86 mL) and POCl ₃ (2.27 mL) were sequentially added thereto, and the mixture was stirred for 40 minutes. The reaction mixture was cooled to −78 ° C. and water (20 mL) was added slowly. After neutralization by adding saturated aqueous sodium hydrogen carbonate solution at room temperature, the amount of the liquid was reduced by concentration under reduced pressure, the residue (aqueous layer) was washed twice with n-hexane, diethyl ether (150 mL) was added, and the mixture was ice-cooled and stirred. The pH was adjusted to 1 to 2 by adding 6M hydrochloric acid (14 mL), and then the liquid was separated. Diethyl ether was added to the separated aqueous layer for extraction, and the organic layers were combined. This was washed successively with water and saturated brine, and dried over anhydrous sodium sulfate. This was filtered, the filtrate and the washing solution were combined, triethylamine (0.48 mL) was added, concentrated under reduced pressure, and dried to obtain the target compound as a colorless oil (crude yield: 1.95 g (including triethylamine)). ).
^{1 1} H-NMR (400 MHz, D ₂ O): δ5.44 (dd, 1H, J = 5.5, 5.5 Hz), 5.37 (ddd, 1H, J = 2.7, 5.9, 5.9Hz), 5.31 (m, 1H), 4.51 (dd, 1H, J = 2.7, 12.4Hz), 4.30 (dd, 1H, J = 5.9, 12.4Hz) ), 4.10 (ddd, 1H, J = 3.2,5.5,11.4Hz), 3.98 (ddd, 1H, J = 6.4,6.4,11.4Hz), 2. 45-2.33 (m, 8H), 1.69-1.55 (m, 8H), 0.936 (t, 3H, J = 7.3Hz), 0.929 (t, 3H, J = 7) .3Hz), 0.923 (t, 3H, J = 7.3Hz), 0.900 (t, 3H, J = 7.3Hz)
ESI-MS spectrum (negative mode) C ₂₁ H ₃₆ O ₁₂ P: [MH] ^- , calcd: 511.19, found: 511.2

１，２，３，４−テトラ−Ｏ−ブチリル−Ｄ−リビトール ５−ホスフェート（１．９５ｇ）を脱水ＤＭＦに溶解し、減圧濃縮した。得られた残渣に脱水ＤＭＦを加えて減圧濃縮した。得られた残渣をアルゴン雰囲気下で脱水ピリジンに溶解した（Ａ液）。これとは別に、１Ｈ−テトラゾール（０．３８ｇ）、および別途調製したシチジン ５’−モノホスフェート ホルホリン塩（定法によりシチジン ５’−モノホスフェートから調製が可能）（２．６３ｇ）をそれぞれ別の容器Ｂと容器Ｃに入れ、同じく脱水ＤＭＦを加えて減圧濃縮する操作を２回繰り返し、それぞれ得られた残渣をアルゴン雰囲気下で脱水ピリジンに溶解した（順にＢ液とＣ液）。続いてアルゴン雰囲気下でＡ液に対してＢ液とＣ液を順次加えた。更に脱水ＤＭＦ（５ｍＬ）を加え、アルゴン雰囲気下、室温で１９時間攪拌した。脱水ＤＭＦを加えて減圧濃縮する操作を２回繰り返して得られたシチジン ５’−モノホスフェート モルフォリデート（０．４４ｇ）を脱水ＤＭＦ（５ｍＬ）に溶解して反応混合物に加え、アルゴン雰囲気下室温で終夜攪拌した。減圧濃縮により得られた残渣を逆相系のＨＰＬＣで精製した。目的のフラクションを集め、凍結乾燥により得られた１．１２ｇのうちの０．８７ｇをイオン交換樹脂に通すことにより、白色粉末である目的化合物の２Ｎａ塩を得た（収量：７１０ｍｇ，収率：４７％）。
¹Ｈ−ＮＭＲ（４００ＭＨｚ，Ｄ₂Ｏ）：δ８．０２（ｄ，１Ｈ，Ｊ＝７．３Ｈｚ），６．１３（ｄ，１Ｈ，Ｊ＝７．３Ｈｚ），５．９６（ｄ，１Ｈ，Ｊ＝３．７Ｈｚ），５．３９（ｄｄ，１Ｈ，Ｊ＝４．６，５．９Ｈｚ），５．３１（ｍ，２Ｈ），４．４８（ｄｄ，１Ｈ，Ｊ＝２．７，１２．４Ｈｚ），４．３３−４．１５（ｍ，７Ｈ），４．０５（ｄｄｄ，１Ｈ，Ｊ＝６．４，６．４，１１．４Ｈｚ），２．４０−２．３２（ｍ，８Ｈ），１．６５−１．５４（ｍ，８Ｈ），０．９１（ｂｒ．ｔ，３Ｈ＋３Ｈ＋３Ｈ，Ｊ＝７．３Ｈｚ），０．８９（ｔ，３Ｈ，Ｊ＝７．３Ｈｚ）
ＥＳＩ−ＭＳスペクトル（ｎｅｇａｔｉｖｅ ｍｏｄｅ） Ｃ₃₀Ｈ₄₈Ｎ₃Ｏ₁₉Ｐ₂：［Ｍ−Ｈ］^-，ｃａｌｃｄ：８１６．２４，ｆｏｕｎｄ：８１６．２ (4) Synthesis of CDP-ribitol tetrabutyrate

1,2,3,4-tetra-O-butyryl-D-ribitol 5-phosphate (1.95 g) was dissolved in dehydrated DMF and concentrated under reduced pressure. Dehydrated DMF was added to the obtained residue and concentrated under reduced pressure. The obtained residue was dissolved in dehydrated pyridine under an argon atmosphere (Liquid A). Separately, 1H-tetrazole (0.38 g) and separately prepared cytidine 5'-monophosphate forphorin salt (which can be prepared from cytidine 5'-monophosphate by a conventional method) (2.63 g) are placed in separate containers. The operation of placing B and container C in the same manner, adding dehydrated DMF and concentrating under reduced pressure was repeated twice, and the obtained residues were dissolved in dehydrated pyridine under an argon atmosphere (solutions B and C in that order). Subsequently, liquid B and liquid C were sequentially added to liquid A under an argon atmosphere. Further, dehydrated DMF (5 mL) was added, and the mixture was stirred at room temperature for 19 hours under an argon atmosphere. Cytidine 5'-monophosphate morpholidate (0.44 g) obtained by repeating the operation of adding dehydrated DMF and concentrating under reduced pressure twice was dissolved in dehydrated DMF (5 mL), added to the reaction mixture, and added to the reaction mixture at room temperature under an argon atmosphere. Stirred overnight. The residue obtained by concentration under reduced pressure was purified by reverse phase HPLC. The target fraction was collected and 0.87 g of 1.12 g obtained by freeze-drying was passed through an ion exchange resin to obtain a 2Na salt of the target compound as a white powder (yield: 710 mg, yield:). 47%).
^{1 1} H-NMR (400 MHz, D ₂ O): δ8.02 (d, 1H, J = 7.3 Hz), 6.13 (d, 1H, J = 7.3 Hz), 5.96 (d, 1H, J = 3.7Hz), 5.39 (dd, 1H, J = 4.6, 5.9Hz), 5.31 (m, 2H), 4.48 (dd, 1H, J = 2.7, 12) .4Hz), 4.33-4.15 (m, 7H), 4.05 (ddd, 1H, J = 6.4,6.4,11.4Hz), 2.40-2.32 (m, 8H), 1.65-1.54 (m, 8H), 0.91 (br.t, 3H + 3H + 3H, J = 7.3Hz), 0.89 (t, 3H, J = 7.3Hz)
ESI-MS spectrum (negative mode) C ₃₀ H ₄₈ N ₃ O ₁₉ P ₂ : [MH] ^- , calcd: 816.24, found: 816.2

ＣＤＰ−リビトール テトラブチレート（２５７ｍｇ）を水（１５ｍＬ）に溶解し、トリエチルアミン（１０μＬ）を加えてｐＨを７〜８に調整した。これに１−アセチルイミダゾール（２７７ｍｇ）を加えて室温で攪拌した。その後、８回に分けて１−アセチルイミダゾール（合計で２．２２ｇ）を追加した。反応開始から約７時間後に反応混合物が入った容器を約６℃の低温下で終夜（約１６時間）保管した。翌日、反応混合物を逆相系のＨＰＬＣで精製した。目的のフラクションを集め、凍結乾燥、およびイオン交換樹脂に通すことにより、白色粉末である目的化合物の２Ｎａ塩を得た（収量：１２６ｍｇ，収率：５２％）。
¹Ｈ−ＮＭＲ（４００ＭＨｚ，Ｄ₂Ｏ）：δ７．９９（ｄ，１Ｈ，Ｊ＝７．３Ｈｚ），６．２１（ｄ，１Ｈ，Ｊ＝５．５Ｈｚ），６．１６（ｄ，１Ｈ，Ｊ＝７．３Ｈｚ），５．４５（ｄｄ，１Ｈ，Ｊ＝４．１，５．０Ｈｚ），５．４１−５．３７（ｍ，２Ｈ），５．３２−５．２９（ｍ，２Ｈ），４．５１（ｍ，１Ｈ），４．４７（ｄｄ，１Ｈ，Ｊ＝２．３，１２．４Ｈｚ），４．３１−４．１５（ｍ，４Ｈ），４．０５（ｄｄｄ，１Ｈ，Ｊ＝５．９，５．９，１１．９Ｈｚ），２．４１−２．３２（ｍ，８Ｈ），２．１７（ｓ，３Ｈ），２．１２（ｓ，３Ｈ），１．６５−１．５４（ｍ，８Ｈ），０．９１（ｂｒ．ｔ，３Ｈ＋３Ｈ＋３Ｈ，Ｊ＝７．３Ｈｚ），０．８９（ｔ，３Ｈ，Ｊ＝７．３Ｈｚ）
ＥＳＩ−ＭＳスペクトル（ｎｅｇａｔｉｖｅ ｍｏｄｅ） Ｃ₃₄Ｈ₅₂Ｎ₃Ｏ₂₁Ｐ₂：［Ｍ−Ｈ］^-，ｃａｌｃｄ：９００．２６，ｆｏｕｎｄ：９００．３ Example 5: Synthesis of CDP-ribitol tetrabutyrate, diacetate

CDP-ribitol tetrabutyrate (257 mg) was dissolved in water (15 mL) and triethylamine (10 μL) was added to adjust the pH to 7-8. 1-Acetylimidazole (277 mg) was added thereto, and the mixture was stirred at room temperature. Then, 1-acetylimidazole (2.22 g in total) was added in 8 portions. Approximately 7 hours after the start of the reaction, the container containing the reaction mixture was stored overnight (about 16 hours) at a low temperature of about 6 ° C. The next day, the reaction mixture was purified by reverse phase HPLC. The desired fractions were collected, lyophilized and passed through an ion exchange resin to give a 2Na salt of the target compound as a white powder (yield: 126 mg, yield: 52%).
^{1 1} H-NMR (400 MHz, D ₂ O): δ7.99 (d, 1H, J = 7.3 Hz), 6.21 (d, 1H, J = 5.5 Hz), 6.16 (d, 1H, J = 7.3Hz), 5.45 (dd, 1H, J = 4.1,5.0Hz), 5.41-5.37 (m, 2H), 5.32-5.29 (m, 2H) ), 4.51 (m, 1H), 4.47 (dd, 1H, J = 2.3, 12.4Hz), 4.31-4.15 (m, 4H), 4.05 (ddd, 1H) , J = 5.9, 5.9, 11.9Hz), 2.41-2.32 (m, 8H), 2.17 (s, 3H), 2.12 (s, 3H), 1.65 -1.54 (m, 8H), 0.91 (br.t, 3H + 3H + 3H, J = 7.3Hz), 0.89 (t, 3H, J = 7.3Hz)
ESI-MS spectrum (negative mode) C ₃₄ H ₅₂ N ₃ O ₂₁ P ₂ : [MH] ^- , calcd: 900.26, found: 900.3

ＣＤＰ−リビトール テトラブチレートを水（２０ｍＬ）に溶解し、トリエチルアミン（１０μＬ）を加えてｐＨを７〜８に調整した。これに別途調製した１−ブチリルイミダゾール（０．３９ｍＬ）を加えて室温で攪拌した。その後、６回に分けて１−ブチリルイミダゾール（合計で１．３５ｍＬ）を追加した。反応開始から約６時間後に反応混合物が入った容器を約１３℃の低温下で終夜（約１５時間）保管した。翌日、反応混合物を逆相系のＨＰＬＣで精製した。目的のフラクションを集め、凍結乾燥、およびイオン交換樹脂に通すことにより、白色粉末である目的化合物の２Ｎａ塩を得た（収量：８９ｍｇ，収率：３６％）。
¹Ｈ−ＮＭＲ（４００ＭＨｚ，Ｄ₂Ｏ）：δ７．９９（ｄ，１Ｈ，Ｊ＝７．８Ｈｚ），６．２１（ｄ，１Ｈ，Ｊ＝５．９Ｈｚ），６．１６（ｄ，１Ｈ，Ｊ＝７．８Ｈｚ），５．４８（ｍ，１Ｈ），５．４２（ｍ，１Ｈ），５．３８（ｍ，１Ｈ），５．３４−５．２８（ｍ，２Ｈ），４．５０−４．４５（ｍ，２Ｈ），４．３０−４．２４（ｍ，２Ｈ），４．２１−４．１５（ｍ，２Ｈ），４．０５（ｄｄｄ，１Ｈ，Ｊ＝５．９，５．９，１１．９Ｈｚ），２．４５（ｔ，２Ｈ，Ｊ＝７．３Ｈｚ），２．４０−２．３２（ｍ，１０Ｈ），１．７０−１．５４（ｍ，１２Ｈ），０．９７−０．８６（ｍ，１８Ｈ）
ＥＳＩ−ＭＳスペクトル（ｎｅｇａｔｉｖｅ ｍｏｄｅ） Ｃ₃₈Ｈ₆₀Ｎ₃Ｏ₂₁Ｐ₂：［Ｍ−Ｈ］^-，ｃａｌｃｄ：９５６．３２，ｆｏｕｎｄ：９５６．３ Example 6: Synthesis of CDP-Ribitol Hexabutyrate

CDP-ribitol tetrabutyrate was dissolved in water (20 mL) and triethylamine (10 μL) was added to adjust the pH to 7-8. To this, 1-butyryl imidazole (0.39 mL) prepared separately was added, and the mixture was stirred at room temperature. Then, 1-butyrylimidazole (1.35 mL in total) was added in 6 portions. Approximately 6 hours after the start of the reaction, the container containing the reaction mixture was stored overnight (about 15 hours) at a low temperature of about 13 ° C. The next day, the reaction mixture was purified by reverse phase HPLC. The desired fractions were collected, lyophilized and passed through an ion exchange resin to give a 2Na salt of the target compound as a white powder (yield: 89 mg, yield: 36%).
^{1 1} H-NMR (400 MHz, D ₂ O): δ7.99 (d, 1H, J = 7.8 Hz), 6.21 (d, 1H, J = 5.9 Hz), 6.16 (d, 1H, J = 7.8Hz), 5.48 (m, 1H), 5.42 (m, 1H), 5.38 (m, 1H), 5.34-5.28 (m, 2H), 4.50 -4.45 (m, 2H), 4.30-4.24 (m, 2H), 4.21-4.15 (m, 2H), 4.05 (ddd, 1H, J = 5.9, 5.9, 11.9Hz), 2.45 (t, 2H, J = 7.3Hz), 2.40-2.32 (m, 10H), 1.70-1.54 (m, 12H), 0.97-0.86 (m, 18H)
ESI-MS spectrum (negative mode) C ₃₈ H ₆₀ N ₃ O ₂₁ P ₂ : [MH] ^- , calcd: 956.32, found: 956.3

Ｄ−リボースから誘導した５−Ｏ−トリチル−Ｄ−リビトール（１．７６ｇ，４．４６ｍｍｏｌ）を脱水ピリジン（１８ｍＬ）に溶解し、Ｎ，Ｎ−４−ジメチルアミノピリジン（５４５ｍｇ，４．４６ｍｍｏｌ）を加えて氷冷した。これにイソブタン酸無水物（４．４６ｍＬ，２６．８ｍｍｏｌ）を加え、反応容器を氷浴からはずして室温で攪拌した。１時間３０分後にＮ，Ｎ−ジメチル−１，３−プロパンジアミン（１．３９ｍＬ，１１．１５ｍｍｏｌ）を加えて未反応のイソブタン酸無水物を分解させた後に減圧濃縮した。得られた残渣にｎ−ヘキサン−酢酸エチル（２：１）の混合溶媒を加え、水、１Ｍ塩酸、水、飽和炭酸水素ナトリウム水、水、および飽和食塩水で順次洗浄した後に、無水硫酸ナトリウムで乾燥させた。これを濾過し、濾液と洗浄液を合わせて減圧濃縮した。得られた残渣をシリカゲルカラムクロマトグラフィーにより精製した。目的のフラクションを集めて減圧濃縮、乾燥することで、無色油状物である目的化合物を得た（収量：１．８９ｇ，収率：６３％）。
¹Ｈ−ＮＭＲ（４００ＭＨｚ，ＣＤＣｌ₃）：δ７．４０−７．２０（ｍ，１５Ｈ），５．４０（ｄｄ，１Ｈ，Ｊ＝５．５，５．５Ｈｚ），５．３７（ｄｄｄ，１Ｈ，Ｊ＝３．２，５．９，５．９Ｈｚ），５．２６（ｄｄｄ，１Ｈ，Ｊ＝２．８，５．０，７．３Ｈｚ），４．３１（ｄｄ，１Ｈ，Ｊ＝３．２，１１．９Ｈｚ），４．１１（ｄｄ，１Ｈ，Ｊ＝７．３，１１．９Ｈｚ），３．２９（ｄｄ，１Ｈ，Ｊ＝３．２，１０．５Ｈｚ），３．１９（ｄｄ，１Ｈ，Ｊ＝６．４，１０．５Ｈｚ），２．６４（ｑｑ，１Ｈ，Ｊ＝６．９，６．９Ｈｚ），２．５０（ｑｑ，１Ｈ，Ｊ＝６．９，６．９Ｈｚ），２．４４−２．３３（ｍ，２Ｈ），１．２４（ｄ，３Ｈ，Ｊ＝６．９Ｈｚ），１．２２（ｄ，３Ｈ，Ｊ＝６．９Ｈｚ），１．１２（ｄ，３Ｈ，Ｊ＝７．３Ｈｚ），１．１１（ｄ，３Ｈ，Ｊ＝７．３Ｈｚ），１．０９（ｄ，３Ｈ，Ｊ＝７．３Ｈｚ），１．０８（ｄ，３Ｈ，Ｊ＝６．９Ｈｚ），１．０４（ｄ，３Ｈ，Ｊ＝６．９Ｈｚ），１．０１（ｄ，３Ｈ，Ｊ＝７．３Ｈｚ）
ＥＳＩ−ＭＳスペクトル（ｐｏｓｉｔｉｖｅ ｍｏｄｅ） Ｃ₄₀Ｈ₅₀Ｏ₉Ｎａ：［Ｍ＋Ｎａ］⁺，ｃａｌｃｄ：６９７．３４，ｆｏｕｎｄ：６９７．３ Example 7: Synthesis of CDP-ribitol tetraisobutyrate and diacetate (1) Synthesis of 1,2,3,4-tetra-O-isobutyryl-5-O-trityl-D-ribitol

5-O-trityl-D-ribitol (1.76 g, 4.46 mmol) derived from D-ribose was dissolved in dehydrated pyridine (18 mL) and N, N-4-dimethylaminopyridine (545 mg, 4.46 mmol). Was added and ice-cooled. Isobutane anhydride (4.46 mL, 26.8 mmol) was added thereto, and the reaction vessel was removed from the ice bath and stirred at room temperature. After 1 hour and 30 minutes, N, N-dimethyl-1,3-propanediamine (1.39 mL, 11.15 mmol) was added to decompose the unreacted isobutane anhydride, and then the mixture was concentrated under reduced pressure. A mixed solvent of n-hexane-ethyl acetate (2: 1) was added to the obtained residue, and the mixture was washed successively with water, 1M hydrochloric acid, water, saturated aqueous sodium hydrogen carbonate water, water, and saturated brine, and then anhydrous sodium sulfate. It was dried with. This was filtered, and the filtrate and the washing liquid were combined and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography. The desired fraction was collected, concentrated under reduced pressure, and dried to obtain the desired compound as a colorless oil (yield: 1.89 g, yield: 63%).
¹ H-NMR (400MHz, CDCl ₃ ): δ7.40-7.20 (m, 15H), 5.40 (dd, 1H, J = 5.5, 5.5Hz), 5.37 (ddd, 1H) , J = 3.2, 5.9, 5.9Hz), 5.26 (ddd, 1H, J = 2.8, 5.0, 7.3Hz), 4.31 (dd, 1H, J = 3) .2, 11.9Hz), 4.11 (dd, 1H, J = 7.3, 11.9Hz), 3.29 (dd, 1H, J = 3.2, 10.5Hz), 3.19 ( dd, 1H, J = 6.4, 10.5Hz), 2.64 (qq, 1H, J = 6.9, 6.9Hz), 2.50 (qq, 1H, J = 6.9, 6. 9Hz), 2.44-2.33 (m, 2H), 1.24 (d, 3H, J = 6.9Hz), 1.22 (d, 3H, J = 6.9Hz), 1.12 ( d, 3H, J = 7.3Hz), 1.11 (d, 3H, J = 7.3Hz), 1.09 (d, 3H, J = 7.3Hz), 1.08 (d, 3H, J) = 6.9Hz), 1.04 (d, 3H, J = 6.9Hz), 1.01 (d, 3H, J = 7.3Hz)
ESI-MS spectrum (positive mode) C ₄₀ H ₅₀ O ₉ Na: [M + Na] ⁺ , calcd: 697.34, found: 697.3

１，２，３，４−テトラ−Ｏ−イソブチリル−５−Ｏ−トリチル−Ｄ−リビトール（１．４２ｇ，２．１０ｍｍｏｌ）に９０％ギ酸水（１５ｍＬ）を加え、さらにメタノール（２ｍＬ）を加えて均一な溶液として室温で攪拌した。２０分後に反応混合物を減圧濃縮した。得られた残渣をｎ−ヘキサン：酢酸エチル＝１：１の混合溶媒に溶解し、飽和炭酸水素ナトリウム水、水、および飽和食塩水で順次洗浄した後に、無水硫酸ナトリウムで乾燥させた。これを濾過し、濾液と洗浄液を合わせて減圧濃縮した。得られた残渣をシリカゲルカラムクロマトグラフィーにより精製した。目的のフラクションを集めて減圧濃縮、乾燥することで、目的化合物を得た（収量：６０４ｍｇ，収率：６７％）。
¹Ｈ−ＮＭＲ（４００ＭＨｚ，ＣＤＣｌ₃）：δ５．３９−５．３４（ｍ，２Ｈ），５．０７（ｄｄｄ，１Ｈ，Ｊ＝３．７，５．５，５．５Ｈｚ），４．４０（ｄｄ，１Ｈ，Ｊ＝２．７，１２．４Ｈｚ），４．１６（ｄｄ，１Ｈ，Ｊ＝６．９，１２．４Ｈｚ），３．８４（ｄｄｄ，１Ｈ，Ｊ＝３．７，７．３，１２．４Ｈｚ），３．６８（ｄｄｄ，１Ｈ，Ｊ＝５．５，５．５，１２．４Ｈｚ），２．６８−２．４８（ｍ，４Ｈ），２．０７（ｄｄ，１Ｈ，Ｊ＝６．０，７．３Ｈｚ），１．２２−１．１６（ｍ，１８Ｈ），１．１５（ｄ，３Ｈ，Ｊ＝７．３Ｈｚ），１．１４（ｄ，３Ｈ，Ｊ＝６．９Ｈｚ）
ＥＳＩ−ＭＳスペクトル（ｐｏｓｉｔｉｖｅ ｍｏｄｅ） Ｃ₂₁Ｈ₃₆Ｏ₉Ｎａ：［Ｍ＋Ｎａ］⁺，ｃａｌｃｄ：４５５．２３，ｆｏｕｎｄ：４５５．２ (2) Synthesis of 1,2,3,4-tetra-O-isobutyryl-D-ribitol

Add 90% formic acid water (15 mL) to 1,2,3,4-tetra-O-isobutyryl-5-O-trityl-D-ribitol (1.42 g, 2.10 mmol), and then add methanol (2 mL). The mixture was stirred at room temperature as a uniform solution. After 20 minutes, the reaction mixture was concentrated under reduced pressure. The obtained residue was dissolved in a mixed solvent of n-hexane: ethyl acetate = 1: 1 and washed successively with saturated aqueous sodium hydrogen carbonate solution, water and saturated brine, and then dried over anhydrous sodium sulfate. This was filtered, and the filtrate and the washing liquid were combined and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography. The target compound was collected, concentrated under reduced pressure, and dried to obtain the target compound (yield: 604 mg, yield: 67%).
^{1 1} H-NMR (400 MHz, CDCl ₃ ): δ5.39-5.34 (m, 2H), 5.07 (ddd, 1H, J = 3.7, 5.5, 5.5 Hz), 4.40 (Dd, 1H, J = 2.7, 12.4Hz), 4.16 (dd, 1H, J = 6.9, 12.4Hz), 3.84 (ddd, 1H, J = 3.7, 7) .3, 12.4Hz), 3.68 (ddd, 1H, J = 5.5, 5.5, 12.4Hz), 2.68-2.48 (m, 4H), 2.07 (dd, dd, 1H, J = 6.0, 7.3Hz), 1.22-1.16 (m, 18H), 1.15 (d, 3H, J = 7.3Hz), 1.14 (d, 3H, J) = 6.9Hz)
ESI-MS spectrum (positive mode) C ₂₁ H ₃₆ O ₉ Na: [M + Na] ⁺ , calcd: 455.23, found: 455.2

１，２，３，４−テトラ−Ｏ−イソブチリル−Ｄ−リビトール（３００ｍｇ，０．６９４ｍｍｏｌ）を脱水ＴＨＦ（６ｍＬ）に溶解し、−１０℃以下に冷却した。これにジイソプロピルエチルアミン（９４４μＬ，５．５５ｍｍｏｌ）とＰＯＣｌ₃（４５２μＬ，４．８６ｍｍｏｌ）を順次加えて２時間攪拌した。反応混合物を−７８℃に冷却し、水（計４ｍＬ）を加えた。室温で飽和炭酸水素ナトリウム水（計８ｍＬ）を加えて中和し、減圧濃縮によりＴＨＦをほぼ留去する際には、途中で飽和炭酸水素ナトリウム水を加えてｐＨを中性にした。得られた残渣（水層）をｎ−ヘキサンで洗浄した後に、６Ｍ塩酸を加えてｐＨを１〜２に調整した後に、ジエチルエーテルを加えて分液した。分離した水層にジエチルエーテルを加えて抽出し、有機層を合わせた。水および飽和食塩水で順次洗浄後に無水硫酸ナトリウムで乾燥させた。これを濾過し、濾液と洗浄液を合わせ、トリエチルアミンを加えて減圧濃縮、乾燥することで、無色油状物である目的化合物を得た（収量：３２４ｍｇ，収率：９１％）。
¹Ｈ−ＮＭＲ（４００ＭＨｚ，Ｄ₂Ｏ）：δ５．４５（ｄｄ，１Ｈ，Ｊ＝５．０，５．９Ｈｚ），５．３７（ｄｄｄ，１Ｈ，Ｊ＝２．８，６．０，６．０Ｈｚ），５．３０（ｄｄｄ，１Ｈ，Ｊ＝３．７，４．６，６．９Ｈｚ），４．５２（ｄｄ，１Ｈ，Ｊ＝２．８，１２．４Ｈｚ），４．３０（ｄｄ，１Ｈ，Ｊ＝６．０，１２．４Ｈｚ），４．１３（ｄｄｄ，１Ｈ，Ｊ＝３．２，５．５，１１．４Ｈｚ），４．００（ｄｄｄ，１Ｈ，Ｊ＝６．４，６．４，１１．４Ｈｚ），２．７６−２．６２（ｍ，３Ｈ），２．６１（ｑｑ，１Ｈ，Ｊ＝６．９，６．９Ｈｚ），１．１９−１．１５（ｍ，１８Ｈ），１．１３（ｄ，３Ｈ，Ｊ＝７．３Ｈｚ），１．１２（ｄ，３Ｈ，Ｊ＝６．９Ｈｚ）
ＥＳＩ−ＭＳスペクトル（ｎｅｇａｔｉｖｅ ｍｏｄｅ） Ｃ₂₁Ｈ₃₆Ｏ₁₂Ｐ：［Ｍ−Ｈ］^-，ｃａｌｃｄ：５１１．１９，ｆｏｕｎｄ：５１１．２ (3) Synthesis of 1,2,3,4-tetra-O-isobutyryl-D-ribitol 5-phosphate

1,2,3,4-Tetra-O-isobutyryl-D-ribitol (300 mg, 0.694 mmol) was dissolved in dehydrated THF (6 mL) and cooled to −10 ° C. or lower. To this, diisopropylethylamine (944 μL, 5.55 mmol) and POCl ₃ (452 μL, 4.86 mmol) were sequentially added, and the mixture was stirred for 2 hours. The reaction mixture was cooled to −78 ° C. and water (4 mL total) was added. Saturated sodium hydrogen carbonate water (8 mL in total) was added at room temperature for neutralization, and when THF was substantially distilled off by concentration under reduced pressure, saturated sodium hydrogen carbonate water was added on the way to neutralize the pH. The obtained residue (aqueous layer) was washed with n-hexane, and then 6M hydrochloric acid was added to adjust the pH to 1-2, and then diethyl ether was added to separate the liquids. Diethyl ether was added to the separated aqueous layer for extraction, and the organic layers were combined. After washing sequentially with water and saturated brine, the mixture was dried over anhydrous sodium sulfate. This was filtered, the filtrate and the washing liquid were combined, triethylamine was added, concentrated under reduced pressure, and dried to obtain the target compound as a colorless oil (yield: 324 mg, yield: 91%).
^{1 1} H-NMR (400 MHz, D ₂ O): δ5.45 (dd, 1H, J = 5.0, 5.9 Hz), 5.37 (ddd, 1H, J = 2.8, 6.0, 6) .0Hz), 5.30 (ddd, 1H, J = 3.7, 4.6, 6.9Hz), 4.52 (dd, 1H, J = 2.8, 12.4Hz), 4.30 ( dd, 1H, J = 6.0, 12.4Hz), 4.13 (ddd, 1H, J = 3.2,5.5,11.4Hz), 4.00 (ddd, 1H, J = 6. 4,6.4,11.4Hz), 2.76-2.62 (m, 3H), 2.61 (qq, 1H, J = 6.9, 6.9Hz), 1.19-1.15 (M, 18H), 1.13 (d, 3H, J = 7.3Hz), 1.12 (d, 3H, J = 6.9Hz)
ESI-MS spectrum (negative mode) C ₂₁ H ₃₆ O ₁₂ P: [MH] ^- , calcd: 511.19, found: 511.2

１，２，３，４−テトラ−Ｏ−イソブチリル−Ｄ−リビトール ５−ホスフェート（３１９ｍｇ，０．６２２ｍｍｏｌ）を脱水ピリジンに溶解し、減圧濃縮する操作を３回繰り返した。得られた残渣をアルゴン雰囲気下で脱水ピリジンに溶解した（Ａ液）。これとは別に、１Ｈ−テトラゾール（９６．０ｍｇ，１．３７ｍｍｏｌ）と、別途調製した２’，３’−ジ−Ｏ−アセチル−シチジン ５’−モノホスフェート モルフォリデート（定法によりシチジン５’−モノホスフェートから調製が可能）（４４４ｍｇ，０．９３３ｍｍｏｌ）をそれぞれ別の容器Ｂと容器Ｃに入れ、同じく脱水ピリジンを加えて減圧濃縮する操作を３回繰り返し、それぞれ得られた残渣をアルゴン雰囲気下で脱水ピリジンに溶解した（順にＢ液とＣ液）。続いてアルゴン雰囲気下でＡ液に対してＢ液とＣ液を順次加えた。アルゴン雰囲気下、室温で終夜攪拌した。減圧濃縮により得られた残渣に脱水ＤＭＦを加えて減圧濃縮した。得られた残渣を逆相系のＨＰＬＣで精製した。目的のフラクションを集め、凍結乾燥、およびイオン交換樹脂に通すことにより、白色粉末である目的化合物の２Ｎａ塩を得た（収量：２７１ｍｇ，収率：４６％）。
¹Ｈ−ＮＭＲ（４００ＭＨｚ，Ｄ₂Ｏ）：δ８．００（ｄ，１Ｈ，Ｊ＝７．８Ｈｚ），６．２０（ｄ，１Ｈ，Ｊ＝５．５Ｈｚ），６．１６（ｄ，１Ｈ，Ｊ＝７．３Ｈｚ），５．４４（ｄｄ，１Ｈ，Ｊ＝４．６，５．５Ｈｚ），５．４１−５．３７（ｍ，２Ｈ），５．３１−５．２６（ｍ，２Ｈ），４．５２−４．４６（ｍ，２Ｈ），４．３０−４．１３（ｍ，４Ｈ），４．０６（ｄｄｄ，１Ｈ，Ｊ＝６．９，６．９，１１．４Ｈｚ），２．６８−２．５４（ｍ，４Ｈ），２．１６（ｓ，３Ｈ），２．１１（ｓ，３Ｈ），１．１５−１．１２（ｍ，１８Ｈ），１．１１（ｄ，３Ｈ，Ｊ＝６．９Ｈｚ），１．１０（ｄ，３Ｈ，Ｊ＝６．９Ｈｚ）
ＥＳＩ−ＭＳスペクトル（ｎｅｇａｔｉｖｅ ｍｏｄｅ） Ｃ₃₄Ｈ₅₂Ｎ₃Ｏ₂₁Ｐ₂：［Ｍ−Ｈ］^-，ｃａｌｃｄ：９００．２６，ｆｏｕｎｄ：９００．２ (4) Synthesis of CDP-ribitol tetraisobutyrate and diacetate

The operation of dissolving 1,2,3,4-tetra-O-isobutyryl-D-ribitol 5-phosphate (319 mg, 0.622 mmol) in dehydrated pyridine and concentrating under reduced pressure was repeated three times. The obtained residue was dissolved in dehydrated pyridine under an argon atmosphere (Liquid A). Separately, 1H-tetrazole (96.0 mg, 1.37 mmol) and 2', 3'-di-O-acetyl-cytidine 5'-monophosphate morpholidate prepared separately (cytidine 5'-by the conventional method). (Can be prepared from monophosphate) (444 mg, 0.933 mmol) was placed in separate containers B and C, respectively, and the operation of adding dehydrated pyridine and concentrating under reduced pressure was repeated three times, and the obtained residues were subjected to an argon atmosphere. Dissolved in dehydrated pyridine (solutions B and C in that order). Subsequently, liquid B and liquid C were sequentially added to liquid A under an argon atmosphere. The mixture was stirred overnight at room temperature under an argon atmosphere. Dehydrated DMF was added to the residue obtained by concentration under reduced pressure, and the mixture was concentrated under reduced pressure. The obtained residue was purified by reverse phase HPLC. The desired fractions were collected, lyophilized and passed through an ion exchange resin to give a 2Na salt of the target compound as a white powder (yield: 271 mg, yield: 46%).
^{1 1} H-NMR (400 MHz, D ₂ O): δ8.00 (d, 1H, J = 7.8 Hz), 6.20 (d, 1H, J = 5.5 Hz), 6.16 (d, 1H, J = 7.3Hz), 5.44 (dd, 1H, J = 4.6, 5.5Hz), 5.41-5.37 (m, 2H), 5.31-5.26 (m, 2H) ), 4.52-4.46 (m, 2H), 4.30-4.13 (m, 4H), 4.06 (ddd, 1H, J = 6.9, 6.9, 11.4Hz) , 2.68-2.54 (m, 4H), 2.16 (s, 3H), 2.11 (s, 3H), 1.15-1.12 (m, 18H), 1.11 (d) , 3H, J = 6.9Hz), 1.10 (d, 3H, J = 6.9Hz)
ESI-MS spectrum (negative mode) C ₃₄ H ₅₂ N ₃ O ₂₁ P ₂ : [MH] ^- , calcd: 900.26, found: 900.2

Ｄ−リボースから誘導した５−Ｏ−トリチル−Ｄ−リビトール（１．５０ｇ，３．８０ｍｍｏｌ）を脱水クロロホルム（１５ｍＬ）に溶解し、Ｎ，Ｎ−４−ジメチルアミノピリジン（２．７９ｇ，２２．８ｍｍｏｌ）を加えて氷冷した。これにクロロギ酸メチル（１．７５ｍＬ，２２．８ｍｍｏｌ）を加え、反応容器を氷浴からはずして室温で攪拌した。２時間３０分後にクロロギ酸メチル（０．２９２ｍＬ，３．８０ｍｍｏｌ）を追加し、更に１時間室温で攪拌した。１Ｍ塩酸、水および飽和食塩水で順次洗浄した後に、無水硫酸ナトリウムで乾燥させた。これを濾過し、濾液と洗浄液を合わせて減圧濃縮した。得られた残渣をシリカゲルカラムクロマトグラフィーにより精製した。目的のフラクションを集めて減圧濃縮、乾燥することで、無色油状物である目的化合物を得た（収量：１．２２ｇ，収率：５１％）。
¹Ｈ−ＮＭＲ（４００ＭＨｚ，ＣＤＣｌ₃）：δ７．４３−７．２０（ｍ，１５Ｈ），５．３５（ｄｄ，１Ｈ，Ｊ＝４．１，５．９Ｈｚ），５．２２（ｄｄｄ，１Ｈ，Ｊ＝３．２，３．７，７．３Ｈｚ），５．１２（ｄｄｄ，１Ｈ，Ｊ＝３．７，５．５，５．５Ｈｚ），４．４９（ｄｄ，１Ｈ，Ｊ＝３．２，１２．４Ｈｚ），４．２６（ｄｄ，１Ｈ，Ｊ＝６．９，１２．４Ｈｚ），３．８２（ｓ，３Ｈ），３．７７（ｓ，３Ｈ），３．７４（ｓ，３Ｈ），３．７２（ｓ，３Ｈ），３．３８（ｄｄ，１Ｈ，Ｊ＝３．２，１０．５Ｈｚ），３．２６（ｄｄ，１Ｈ，Ｊ＝５．５，１０．５Ｈｚ）
ＥＳＩ−ＭＳスペクトル（ｐｏｓｉｔｉｖｅ ｍｏｄｅ） Ｃ₃₂Ｈ₃₄Ｏ₁₃Ｎａ：［Ｍ＋Ｎａ］⁺，ｃａｌｃｄ：６４９．１９，ｆｏｕｎｄ：６４９．２ Example 8: CDP-Ribitol Tetramethoxycarbonyl, Diacetate (1) Synthesis of 1,2,3,4-tetra-O-methoxycarbonyl-5-O-trityl-D-ribitol

5-O-trityl-D-ribitol (1.50 g, 3.80 mmol) derived from D-ribose was dissolved in dehydrated chloroform (15 mL) and N, N-4-dimethylaminopyridine (2.79 g, 22. 8 mmol) was added and ice-cooled. Methyl chloroformate (1.75 mL, 22.8 mmol) was added thereto, and the reaction vessel was removed from the ice bath and stirred at room temperature. After 2 hours and 30 minutes, methyl chloroformate (0.292 mL, 3.80 mmol) was added, and the mixture was further stirred at room temperature for 1 hour. After washing sequentially with 1M hydrochloric acid, water and saturated brine, the mixture was dried over anhydrous sodium sulfate. This was filtered, and the filtrate and the washing liquid were combined and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography. The target compounds were collected, concentrated under reduced pressure, and dried to obtain the target compound as a colorless oil (yield: 1.22 g, yield: 51%).
^{1 1} H-NMR (400 MHz, CDCl ₃ ): δ7.43-7.20 (m, 15H), 5.35 (dd, 1H, J = 4.1,5.9 Hz), 5.22 (ddd, 1H) , J = 3.2, 3.7, 7.3Hz), 5.12 (ddd, 1H, J = 3.7, 5.5, 5.5Hz), 4.49 (dd, 1H, J = 3) .2,12.4Hz), 4.26 (dd, 1H, J = 6.9, 12.4Hz), 3.82 (s, 3H), 3.77 (s, 3H), 3.74 (s) , 3H), 3.72 (s, 3H), 3.38 (dd, 1H, J = 3.2, 10.5Hz), 3.26 (dd, 1H, J = 5.5, 10.5Hz)
ESI-MS spectrum (positive mode) C ₃₂ H ₃₄ O ₁₃ Na: [M + Na] ⁺ , calcd: 649.19, found: 649.2

１，２，３，４−テトラ−Ｏ−メトキシカルボニル−５−Ｏ−トリチル−Ｄ−リビトール（１．１９ｇ，１．９０ｍｍｏｌ）に８０％ギ酸／メタノール（１５ｍＬ）を加えて室温で攪拌した。３０分後に反応混合物を減圧濃縮した。得られた残渣をｎ−ヘキサン：酢酸エチル＝１：１の混合溶媒に溶解し、飽和炭酸水素ナトリウム水、水および飽和食塩水で順次洗浄した後に無水硫酸ナトリウムで乾燥させた。これを濾過し、濾液と洗浄液を合わせて減圧濃縮した。得られた残渣をシリカゲルカラムクロマトグラフィーにより精製した。目的のフラクションを集めて減圧濃縮、乾燥することで目的化合物を得た（収量：３８８ｍｇ，収率：５３％）。
¹Ｈ−ＮＭＲ（４００ＭＨｚ，ＣＤＣｌ₃）：δ５．２８（ｄｄ，１Ｈ，Ｊ＝４．１，５．０Ｈｚ），５．２５（ｄｄｄ，１Ｈ，Ｊ＝２．８，４．１，６．９Ｈｚ），５．０２（ｄｄｄ，１Ｈ，Ｊ＝４．１，５．５，５．５Ｈｚ），４．５４（ｄｄ，１Ｈ，Ｊ＝２．８，１２．４Ｈｚ），４．３１（ｄｄ，１Ｈ，Ｊ＝６．９，１２．４Ｈｚ），３．９４（ｄｄｄ，１Ｈ，Ｊ＝４．１，６．４，１２．４Ｈｚ），３．８５−３．８０（ｍ，７Ｈ），３．８２（ｓ，３Ｈ），３．７９（ｓ，３Ｈ），２．０９（ｄｄ，１Ｈ，Ｊ＝６．４，６．４Ｈｚ）
ＥＳＩ−ＭＳスペクトル（ｐｏｓｉｔｉｖｅ ｍｏｄｅ） Ｃ₁₃Ｈ₂₀Ｏ₁₃Ｎａ：［Ｍ＋Ｎａ］⁺，ｃａｌｃｄ：４０７．０８，ｆｏｕｎｄ：４０７．１ (2) Synthesis of 1,2,3,4-tetra-O-methoxycarbonyl-D-ribitol

80% formic acid / methanol (15 mL) was added to 1,2,3,4-tetra-O-methoxycarbonyl-5-O-trityl-D-ribitol (1.19 g, 1.90 mmol), and the mixture was stirred at room temperature. After 30 minutes, the reaction mixture was concentrated under reduced pressure. The obtained residue was dissolved in a mixed solvent of n-hexane: ethyl acetate = 1: 1, washed successively with saturated aqueous sodium hydrogen carbonate, water and saturated brine, and dried over anhydrous sodium sulfate. This was filtered, and the filtrate and the washing liquid were combined and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography. The desired fraction was collected, concentrated under reduced pressure, and dried to obtain the desired compound (yield: 388 mg, yield: 53%).
^{1 1} H-NMR (400 MHz, CDCl ₃ ): δ5.28 (dd, 1H, J = 4.1,5.0 Hz), 5.25 (ddd, 1H, J = 2.8, 4.1, 6. 9Hz), 5.02 (ddd, 1H, J = 4.1,5.5,5.5Hz), 4.54 (dd, 1H, J = 2.8,12.4Hz), 4.31 (dd) , 1H, J = 6.9, 12.4Hz), 3.94 (ddd, 1H, J = 4.1,6.4,12.4Hz), 3.85-3.80 (m, 7H), 3.82 (s, 3H), 3.79 (s, 3H), 2.09 (dd, 1H, J = 6.4, 6.4 Hz)
ESI-MS spectrum (positive mode) C ₁₃ H ₂₀ O ₁₃ Na: [M + Na] ⁺ , calcd: 407.08, found: 407.1

１，２，３，４−テトラ− Ｏ−メトキシカルボニル−Ｄ−リビトール（３３５ｍｇ，０．８７２ｍｍｏｌ）を脱水ＴＨＦ（１５ｍＬ）に溶解し、−１０℃以下に冷却した。これにジイソプロピルエチルアミン（１．２１ｍＬ，６．９８ｍｍｏｌ）とＰＯＣｌ₃（５６７μＬ，６．１０ｍｍｏｌ）を順次加えて２時間攪拌した。反応混合物を−７８℃に冷却し、水（計５ｍＬ）を加えた。室温で飽和炭酸水素ナトリウム水（計８ｍＬ）を加えて中和し、減圧濃縮によりＴＨＦをほぼ留去する際には途中で飽和炭酸水素ナトリウム水を加えてｐＨを中性にした。得られた残渣（水層）をジエチルエーテルで洗浄した後に、６Ｍ塩酸を加えてｐＨを１〜２に調整した後に酢酸エチルを加えて分液した。分離した水層に酢酸エチルを加えて抽出し、有機層を合わせた。水と飽和食塩水で順次洗浄後に無水硫酸ナトリウムで乾燥させた。これを濾過し、濾液と洗浄液を合わせ、トリエチルアミンを加えて減圧濃縮、乾燥することで、無色油状物である目的化合物を得た（収量：３０３ｍｇ（¹Ｈ−ＮＭＲの積分値からの補正収量），収率：７４％）。
¹Ｈ−ＮＭＲ（４００ＭＨｚ，ＣＤＣｌ₃）：δ５．２９（ｄｄ，１Ｈ，Ｊ＝４．１，６．４Ｈｚ），５．２３（ｄｄｄ，１Ｈ，Ｊ＝３．２，４．１，６．９Ｈｚ），５．１２（ｍ，１Ｈ），４．５０（ｄｄ，１Ｈ，Ｊ＝３．２，１２．４Ｈｚ），４．３１（ｄｄ，１Ｈ，Ｊ＝６．９，１２．４Ｈｚ），４．２１（ｄｄｄ，１Ｈ，Ｊ＝３．２，５．５，１１．９Ｈｚ），４．０９（ｄｄｄ，１Ｈ，Ｊ＝５．０，６．９，１１．９Ｈｚ），３．８０１（ｓ，３Ｈ），３．７９５（ｓ，３Ｈ），３．７９２（ｓ，３Ｈ），３．７７（ｓ，３Ｈ）
ＥＳＩ−ＭＳスペクトル（ｎｅｇａｔｉｖｅ ｍｏｄｅ） Ｃ₁₃Ｈ₂₀Ｏ₁₆Ｐ：［Ｍ−Ｈ］^-，ｃａｌｃｄ：４６３．０５，ｆｏｕｎｄ： ４６３．０ (3) Synthesis of 1,2,3,4-tetra-O-methoxycarbonyl-D-ribitol 5-phosphate

1,2,3,4-tetra-O-methoxycarbonyl-D-ribitol (335 mg, 0.872 mmol) was dissolved in dehydrated THF (15 mL) and cooled to −10 ° C. or lower. Diisopropylethylamine (1.21 mL, 6.98 mmol) and POCl ₃ (567 μL, 6.10 mmol) were sequentially added thereto, and the mixture was stirred for 2 hours. The reaction mixture was cooled to −78 ° C. and water (5 mL total) was added. Saturated sodium hydrogen carbonate water (8 mL in total) was added at room temperature for neutralization, and when THF was substantially distilled off by concentration under reduced pressure, saturated sodium hydrogen carbonate water was added on the way to neutralize the pH. The obtained residue (aqueous layer) was washed with diethyl ether, and then 6M hydrochloric acid was added to adjust the pH to 1-2, and then ethyl acetate was added to separate the liquids. Ethyl acetate was added to the separated aqueous layer for extraction, and the organic layers were combined. After washing sequentially with water and saturated brine, it was dried over anhydrous sodium sulfate. This was filtered, the filtrate and the washing solution were combined, triethylamine was added, concentrated under reduced pressure, and dried to obtain the target compound as a colorless oil (yield: 303 mg ( ^{corrected yield from 1} H-NMR integrated value). , Yield: 74%).
^{1 1} H-NMR (400 MHz, CDCl ₃ ): δ5.29 (dd, 1H, J = 4.1,6.4 Hz), 5.23 (ddd, 1H, J = 3.2, 4.1, 6. 9Hz), 5.12 (m, 1H), 4.50 (dd, 1H, J = 3.2, 12.4Hz), 4.31 (dd, 1H, J = 6.9, 12.4Hz), 4.21 (ddd, 1H, J = 3.2,5.5,11.9Hz), 4.09 (ddd, 1H, J = 5.0, 6.9, 11.9Hz), 3.801 ( s, 3H), 3.795 (s, 3H), 3.792 (s, 3H), 3.77 (s, 3H)
ESI-MS spectrum (negative mode) C ₁₃ H ₂₀ O ₁₆ P: [MH] ^- , calcd: 463.05, found: 463.0

１，２，３，４−テトラ−Ｏ−メトキシカルボニル−Ｄ−リビトール ５−ホスフェート（３０３ｍｇ，０．６５ｍｍｏｌ）、２’，３’−ジ−Ｏ−アセチル−シチジン ５’−モノホスフェート モルフォリデート（６９７ｍｇ，０．９８ｍｍｏｌ）と１Ｈ−テトラゾール（１００ｍｇ，１．４３ｍｍｏｌ）を脱水ピリジン（７ｍＬ）に溶解し、アルゴン雰囲気下、室温で２日間攪拌した。反応液を減圧濃縮し、残渣に脱水ＤＭＦを加えて減圧濃縮した。得られた残渣を逆相系のＨＰＬＣで精製した。目的のフラクションを集め、凍結乾燥、およびイオン交換樹脂に通すことにより、白色粉末である目的化合物の２Ｎａ塩を得た（収量：３２５ｍｇ，収率：５５％）。
¹Ｈ−ＮＭＲ（４００ＭＨｚ，Ｄ₂Ｏ）：δ７．９８（ｄ，１Ｈ，Ｊ＝７．３Ｈｚ），６．２１（ｄ，１Ｈ，Ｊ＝５．０Ｈｚ），６．１６（ｄ，１Ｈ，Ｊ＝７．８Ｈｚ），５．４４（ｄｄ，１Ｈ，Ｊ＝４．１，５．５Ｈｚ），５．４０（ｄｄ，１Ｈ，Ｊ＝５．５，５．５Ｈｚ），５．２９（ｄｄ，１Ｈ，Ｊ＝５．０，５．０Ｈｚ），５．２３−５．１６（ｍ，２Ｈ），４．５５（ｄｄ，１Ｈ，Ｊ＝３．２，１２．８Ｈｚ），４．５２（ｍ，１Ｈ），４．３８（ｄｄ，１Ｈ，Ｊ＝６．０，１２．４Ｈｚ），４．３０（ｄｄｄ，１Ｈ，Ｊ＝２．３，４．１，１１．９Ｈｚ），４．２３（ｄｄｄ，１Ｈ，Ｊ＝３．２，５．５，１１．９Ｈｚ），４．１８（ｄｄｄ，１Ｈ，Ｊ＝３．２，４．１，１１．９Ｈｚ），４．１１（ｄｄｄ，１Ｈ，Ｊ＝６．０，６．０，１１．９Ｈｚ），３．８２（ｍ，６Ｈ），３．８１（ｓ，３Ｈ），３．７９（ｓ，３Ｈ），２．１７（ｓ，３Ｈ），２．１２（ｓ，３Ｈ）
ＥＳＩ−ＭＳスペクトル（ｎｅｇａｔｉｖｅ ｍｏｄｅ） Ｃ₂₆Ｈ₃₆Ｎ₃Ｏ₂₅Ｐ₂：［Ｍ−Ｈ］^-，ｃａｌｃｄ：８５２．１１，ｆｏｕｎｄ：８５２．１ (4) CDP-ribitol tetramethoxycarbonyl, diacetate

1,2,3,4-tetra-O-methoxycarbonyl-D-libitol 5-phosphate (303 mg, 0.65 mmol), 2', 3'-di-O-acetyl-cytidine 5'-monophosphate morpholidate (697 mg, 0.98 mmol) and 1H-tetrazole (100 mg, 1.43 mmol) were dissolved in dehydrated pyridine (7 mL), and the mixture was stirred at room temperature for 2 days under an argon atmosphere. The reaction mixture was concentrated under reduced pressure, dehydrated DMF was added to the residue, and the mixture was concentrated under reduced pressure. The obtained residue was purified by reverse phase HPLC. The desired fractions were collected, lyophilized and passed through an ion exchange resin to give a 2Na salt of the target compound as a white powder (yield: 325 mg, yield: 55%).
^{1 1} H-NMR (400 MHz, D ₂ O): δ7.98 (d, 1H, J = 7.3 Hz), 6.21 (d, 1H, J = 5.0 Hz), 6.16 (d, 1H, J = 7.8Hz), 5.44 (dd, 1H, J = 4.1,5.5Hz), 5.40 (dd, 1H, J = 5.5,5.5Hz), 5.29 (dd) , 1H, J = 5.0, 5.0Hz), 5.23-5.16 (m, 2H), 4.55 (dd, 1H, J = 3.2, 12.8Hz), 4.52 ( m, 1H), 4.38 (dd, 1H, J = 6.0, 12.4Hz), 4.30 (ddd, 1H, J = 2.3, 4.1, 11.9Hz), 4.23 (Ddd, 1H, J = 3.2,5.5,11.9Hz), 4.18 (ddd, 1H, J = 3.2,4.1,11.9Hz), 4.11 (ddd, 1H) , J = 6.0, 6.0, 11.9Hz), 3.82 (m, 6H), 3.81 (s, 3H), 3.79 (s, 3H), 2.17 (s, 3H) ), 2.12 (s, 3H)
ESI-MS spectrum (negative mode) C ₂₆ H ₃₆ N ₃ O ₂₅ P ₂ : [MH] ^- , calcd: 852.11, found: 852.1

参考文献（Ｅｕｒ．Ｊ．Ｏｒｇ．Ｃｈｅｍ．，２００９，１８，１９６７−１９７５）に記載の方法で得られる２’，３’−ジ−Ｏ−アセチル−シチジン（０．５０ｇ）を脱水ＤＭＦ（２０ｍＬ）に溶解し、アルゴン雰囲気下で氷冷した。これに参考文献（Ｊ．Ｍｅｄ．Ｃｈｅｍ．，２０１５，５８，６１１４−６１３０）に記載の方法で得られるペンタノイルオキシベンジツ ビス（ジイソプロピルアミノ）−ホスホロアミダイト（１．３４ｇ）を加え、更に４，５−ジシアノイミダゾール（０．１８ｇ）の脱水ＤＭＦ（０．８０ｍＬ）溶液をアルゴン雰囲気下で滴下した。アルゴン雰囲気下、室温で３０分間攪拌した後に酢酸エチルを加え、飽和炭酸水素ナトリウム水溶液、水および飽和食塩水で順次洗浄した。無水硫酸ナトリウムで乾燥後に濾過し、濾液と洗浄液を合わせて減圧濃縮により得られた残渣をシリカゲルカラムクロマトグラフィーにより精製した。目的のフラクションを集めて減圧濃縮、乾燥することで、油状物である目的化合物を得た（粗収量：０．４９１ｇ（溶媒等を含む））。
¹Ｈ−ＮＭＲ（４００ＭＨｚ，ＣＤＣｌ₃）：δ７．９０（ｄ，０．５Ｈ，Ｊ＝７．８Ｈｚ），７．７２（ｄ，０．５Ｈ，Ｊ＝７．３Ｈｚ），７．４１（ｄ，１Ｈ，Ｊ＝８．７Ｈｚ），７．３８（ｄ，１Ｈ，Ｊ＝８．７Ｈｚ），７．０４（ｄ，１Ｈ，Ｊ＝８．７Ｈｚ），７．０２（ｄ，１Ｈ，Ｊ＝８．７Ｈｚ），６．４８（ｄ，０．５Ｈ，Ｊ＝７．３Ｈｚ），６．３８（ｄ，０．５Ｈ，Ｊ＝５．５Ｈｚ），５．４２（ｄｄ，０．５Ｈ，Ｊ＝４．１，５．５Ｈｚ），５．３９−５．３６（ｍ，１Ｈ），５．２９（ｄｄ，０．５Ｈ，Ｊ＝５．５，７．３Ｈｚ），５．０１（ｄ，０．５Ｈ，Ｊ＝７．３Ｈｚ），４．７８（ｄｄ，０．５Ｈ，Ｊ＝７．３，１２．４Ｈｚ），４．７４−４．６９（ｍ，１Ｈ），４．５７−４．５２（ｍ，１Ｈ），４．２６（ｍ，１Ｈ），４．０２（ｄｄｄ，０．５Ｈ，Ｊ＝２．３，５．５，１１．０Ｈｚ），３．８３−３．８１（ｍ，１Ｈ），３．７９（ｄｄｄ，０．５Ｈ，Ｊ＝１．４，４．６，１１．０Ｈｚ），３．７５−３．４９（ｍ，２Ｈ），２．５９（ｔ，１Ｈ，Ｊ＝７．３Ｈｚ），２．５７（ｔ，１Ｈ，Ｊ＝７．３Ｈｚ），２．１０１（ｓ，１．５Ｈ），２．０９６（ｓ，１．５Ｈ），２．０８２（ｓ，１．５Ｈ），２．０４５（ｓ，１．５Ｈ），１．７７−１．６９（ｍ，２Ｈ），１．５０−１．４０（ｍ，２Ｈ），１．２９−１．２０（ｍ，１２Ｈ），０．９８０（ｔ，１．５Ｈ，Ｊ＝７．３Ｈｚ），０．９７７（ｔ，１．５Ｈ，Ｊ＝７．３Ｈｚ）
ＥＳＩ−ＭＳスペクトル（ｐｏｓｉｔｉｖｅ ｍｏｄｅ） Ｃ₃₁Ｈ₄₆Ｎ₄Ｏ₁₀Ｐ：［Ｍ＋Ｈ］⁺，ｃａｌｃｄ：６６５．３０，ｆｏｕｎｄ：６６５．３ Example 9: Synthesis of CDP-ribitol hexaacetate-PB1 (1) Synthesis of 2', 3'-di-O-acetyl-cytidine-5'-(4-pentanoyloxybenzyl N, N-diisopsopyl phosphoramidite)

Dehydrated DMF (20 mL) of 2', 3'-di-O-acetyl-cytidine (0.50 g) obtained by the method described in Reference (Eur. J. Org. Chem., 2009, 18, 1967-1975). ), And ice-cooled in an argon atmosphere. To this, pentanoyloxybenzibis (diisopropylamino) -phosphoroamidite (1.34 g) obtained by the method described in Reference (J. Med. Chem., 2015, 58, 6114-6130) is added, and further. A dehydrated DMF (0.80 mL) solution of 4,5-dicyanoimidazole (0.18 g) was added dropwise under an argon atmosphere. After stirring at room temperature for 30 minutes under an argon atmosphere, ethyl acetate was added, and the mixture was washed successively with saturated aqueous sodium hydrogen carbonate solution, water and saturated brine. After drying over anhydrous sodium sulfate, the mixture was filtered, the filtrate and the washing solution were combined, and the residue obtained by concentration under reduced pressure was purified by silica gel column chromatography. The desired fraction was collected, concentrated under reduced pressure, and dried to obtain the target compound as an oil (crude yield: 0.491 g (including solvent and the like)).
¹ H-NMR (400MHz, CDCl ₃ ): δ7.90 (d, 0.5H, J = 7.8Hz), 7.72 (d, 0.5H, J = 7.3Hz), 7.41 (d) , 1H, J = 8.7Hz), 7.38 (d, 1H, J = 8.7Hz), 7.04 (d, 1H, J = 8.7Hz), 7.02 (d, 1H, J = 8.7Hz), 6.48 (d, 0.5H, J = 7.3Hz), 6.38 (d, 0.5H, J = 5.5Hz), 5.42 (dd, 0.5H, J) = 4.1, 5.5 Hz), 5.39-5.36 (m, 1H), 5.29 (dd, 0.5H, J = 5.5, 7.3 Hz), 5.01 (d, 0.5H, J = 7.3Hz), 4.78 (dd, 0.5H, J = 7.3, 12.4Hz), 4.74-4.69 (m, 1H), 4.57-4 .52 (m, 1H), 4.26 (m, 1H), 4.02 (ddd, 0.5H, J = 2.3,5.5,11.0Hz), 3.83-3.81 ( m, 1H), 3.79 (ddd, 0.5H, J = 1.4, 4.6, 11.0Hz), 3.75-3.49 (m, 2H), 2.59 (t, 1H) , J = 7.3Hz), 2.57 (t, 1H, J = 7.3Hz), 2.101 (s, 1.5H), 2.096 (s, 1.5H), 2.082 (s) , 1.5H), 2.045 (s, 1.5H), 1.77-1.69 (m, 2H), 1.50-1.40 (m, 2H), 1.29-1.20 (M, 12H), 0.980 (t, 1.5H, J = 7.3Hz), 0.977 (t, 1.5H, J = 7.3Hz)
ESI-MS spectrum (positive mode) C ₃₁ H ₄₆ N ₄ O ₁₀ P: [M + H] ⁺ , calcd: 665.30, found: 665.3

アルゴン雰囲気下で１，２，３，４−テトラ−Ｏ−アセチル−Ｄ−リビトール ５−ホスフェート（２３２ｍｇ）、トリエチルアミン（５８μＬ）、および２’，３’−ジ−Ｏ−アセチル−シチジン−５’−（４−ペンタノイルオキシベンジル Ｎ，Ｎ−ジイソプロピル ホスホロアミダイト）（４６３ｍｇ）を脱水アセトニトリル（１４ｍＬ）に溶解して室温で攪拌した。これに４，５−ジシアノイミダゾール（８２ｍｇ）を脱水アセトニトリル（１ｍＬ）に溶解したものを少しずつ計７回に分けて加えた。２０分間攪拌した後に、約５．０Ｍ ｔ−ブチルヒドロペルオキシド／デカン溶液（１９０μＬ）を加えた。更に４０分間攪拌した後に、約５．０Ｍ ｔ−ブチルヒドロペルオキシド／デカン溶液（８０μＬ）を追加した。２０分後にｎ−ヘキサンで洗浄した後に、減圧濃縮によりアセトニトリルを留去した。残渣に脱水アセトニトリルを加えて再度減圧濃縮した。得られた残渣を逆相系のＨＰＬＣで精製した。目的のフラクションを集め、凍結乾燥に得られた２２６ｍｇのうちの１４０ｍｇをイオン交換樹脂に通すことにより、白色粉末である目的化合物のＮａ塩を得た（収量：１１９ｍｇ，収率：４１％）。
¹Ｈ−ＮＭＲ（４００ＭＨｚ，Ｄ₂Ｏ）：δ７．６０（ｄ，０．５Ｈ，Ｊ＝７．８Ｈｚ），７．５７（ｄ，０．５Ｈ，Ｊ＝７．３Ｈｚ），７．５６−７．５１（ｍ，２Ｈ），７．１８−７．１２（ｍ，２Ｈ），６．１０−６．０７（ｍ，１Ｈ），５．９７（ｄ，０．５Ｈ， Ｊ＝７．８ Ｈｚ）， ５．９０（ｄ， ０．５Ｈ， Ｊ＝７．３ Ｈｚ）， ５．３６（ｄｄ，０．５Ｈ，Ｊ＝５．５，５．５Ｈｚ），５．３３（ｄｄ，０．５Ｈ，Ｊ＝５．５，５．５Ｈｚ），５．２８−５．２１（ｍ，６Ｈ），４．４４−４．３４（ｍ，３Ｈ），４．３２−４．２２（ｍ，２Ｈ），４．２０−４．１２（ｍ，１Ｈ），４．０９−４．０２（ｍ，１Ｈ），２．６４（ｔ，１Ｈ，Ｊ＝７．３Ｈｚ），２．６３（ｔ，１Ｈ，Ｊ＝７．３Ｈｚ），２．１４４（ｓ，３Ｈ），２．１２２（ｓ，１．５Ｈ），２．１２０（ｓ，１．５Ｈ），２．１０３（ｓ，１．５Ｈ），２．０９８（ｓ，３Ｈ），２．０８８（ｓ，３Ｈ），２．０８０（ｓ，１．５Ｈ），２．０６０（ｓ，１．５Ｈ），２．０５７（ｓ，１．５Ｈ），１．７４−１．６６（ｍ，２Ｈ），１．４６−１．３６（ｍ，２Ｈ），０．９３（ｔ，３Ｈ，Ｊ＝７．３Ｈｚ）
ＥＳＩ−ＭＳスペクトル（ｎｅｇａｔｉｖｅ ｍｏｄｅ） Ｃ₃₈Ｈ₅₀Ｎ₃Ｏ₂₃Ｐ₂：［Ｍ−Ｈ］^-，ｃａｌｃｄ：９７８．２３，ｆｏｕｎｄ：９７８．２ (2) Synthesis of CDP-ribitol hexaacetate-PB1

1,2,3,4-tetra-O-acetyl-D-libitol 5-phosphate (232 mg), triethylamine (58 μL), and 2', 3'-di-O-acetyl-cytidine-5'in an argon atmosphere -(4-Pentanoyloxybenzyl N, N-diisopropylphosphoroamidite) (463 mg) was dissolved in dehydrated acetonitrile (14 mL) and stirred at room temperature. To this, 4,5-dicyanoimidazole (82 mg) dissolved in dehydrated acetonitrile (1 mL) was added little by little in 7 portions in total. After stirring for 20 minutes, about 5.0 M t-butyl hydroperoxide / decane solution (190 μL) was added. After stirring for an additional 40 minutes, about 5.0 M t-butyl hydroperoxide / decane solution (80 μL) was added. After washing with n-hexane after 20 minutes, acetonitrile was distilled off by concentration under reduced pressure. Dehydrated acetonitrile was added to the residue, and the mixture was concentrated again under reduced pressure. The obtained residue was purified by reverse phase HPLC. The target fraction was collected and 140 mg of the 226 mg obtained by freeze-drying was passed through an ion exchange resin to obtain a Na salt of the target compound as a white powder (yield: 119 mg, yield: 41%).
^{1 1} H-NMR (400 MHz, D ₂ O): δ7.60 (d, 0.5H, J = 7.8 Hz), 7.57 (d, 0.5H, J = 7.3 Hz), 7.56- 7.51 (m, 2H), 7.18-7.12 (m, 2H), 6.10-6.07 (m, 1H), 5.97 (d, 0.5H, J = 7.8) Hz), 5.90 (d, 0.5H, J = 7.3 Hz), 5.36 (dd, 0.5H, J = 5.5, 5.5 Hz), 5.33 (dd, 0.35 Hz). 5H, J = 5.5, 5.5Hz), 5.28-5.21 (m, 6H), 4.44-4.34 (m, 3H), 4.32-4.22 (m, 2H) ), 4.20-4.12 (m, 1H), 4.09-4.02 (m, 1H), 2.64 (t, 1H, J = 7.3Hz), 2.63 (t, 1H) , J = 7.3Hz), 2.144 (s, 3H), 2.122 (s, 1.5H), 2.120 (s, 1.5H), 2.103 (s, 1.5H), 2.098 (s, 3H), 2.088 (s, 3H), 2.080 (s, 1.5H), 2.060 (s, 1.5H), 2.057 (s, 1.5H) , 1.74-1.66 (m, 2H), 1.46-1.36 (m, 2H), 0.93 (t, 3H, J = 7.3Hz)
ESI-MS spectrum (negative mode) C ₃₈ H ₅₀ N ₃ O ₂₃ P ₂ : [MH] ^- , calcd: 978.23, found: 978.2

１，２，３，４−テトラ−Ｏ−アセチル−Ｄ−リビトール（３８４ｍｇ，１．２０ｍｍｏｌ）を脱水ジクロロメタン（１５ｍＬ）に溶解し、アルゴン雰囲気下で氷冷した。これに参考文献（Ｊ．Ｍｅｄ．Ｃｈｅｍ．，２０１５，５８，６１１４−６１３０）に記載の方法で得られる４−ペンタノイルオキシベンジル ビス（ジイソプロピルアミノ）−ホスホロアミダイト（１．０５ｇ，２．４０ｍｍｏｌ）を加えた。４，５−ジシアノイミダゾール（１４２ｍｇ，１２０ｍｍｏｌ）／脱水ＤＭＦ（６３０μＬ）の溶液を滴下し、反応容器を氷浴からはずしてアルゴン雰囲気下で攪拌した。３０分後に酢酸エチルで希釈し、飽和炭酸水素ナトリウム水、水および飽和食塩水で順次洗浄した。無水硫酸マグネシウムで乾燥後に濾過し、濾液と洗浄液を合わせて減圧濃縮し、得られた残渣をシリカゲルカラムクロマトグラフィーにより精製した。目的のフラクションを集めて減圧濃縮、乾燥することで目的化合物を得た（粗収量：７８２ｍｇ，粗収率：９９％）。
¹Ｈ−ＮＭＲ（４００ＭＨｚ，ＣＤＣｌ₃）：δ７．３６−７．３２（ｍ，２Ｈ），７．０４−７．０２（ｍ，２Ｈ），５．３９（ｄｄ，０．５Ｈ，Ｊ＝５．０，６．０Ｈｚ），５．３５（ｄｄ，０．５Ｈ，Ｊ＝５．０，５．０Ｈｚ），５．３２（ｍ，１Ｈ），５．２６−５．２０（ｍ，１Ｈ），４．７７−４．５７（ｍ，２Ｈ），４．３４（ｄｄ，０．５Ｈ，Ｊ＝３．２，１１．９Ｈｚ），４．３３（ｄｄ，０．５Ｈ，Ｊ＝３．２，１２．４Ｈｚ），４．１６（ｄｄ，０．５Ｈ，Ｊ＝１．８，１２．４Ｈｚ），４．１５（ｄｄ，０．５Ｈ，Ｊ＝１．８，１２．４Ｈｚ），３．８４（ｄｄｄ，０．５Ｈ，Ｊ＝３．７，６．９，１１．４Ｈｚ），３．７８−３．７４（ｍ，１Ｈ），３．６７（ｍ，０．５Ｈ），３．６６−３．５６（ｍ，２Ｈ），２．５５（ｔ，２Ｈ，Ｊ＝７．３Ｈｚ），２．０８（ｓ，１．５Ｈ），２．０６２（ｓ，１．５Ｈ），２．０５６（ｓ，３Ｈ），２．０４６（ｓ，３Ｈ），２．０３４（ｓ，１．５Ｈ），２．０３０（ｓ，１．５Ｈ），１．７７−１．７０（ｍ，２Ｈ），１．４９−１．４０（ｍ，２Ｈ），１．２１−１．１５（ｍ，１２Ｈ），０．９７（ｔ，３Ｈ，Ｊ＝７．３Ｈｚ）
ＥＳＩ−ＭＳスペクトル（ｐｏｓｉｔｉｖｅ ｍｏｄｅ） Ｃ₃₁Ｈ₄₉ＮＯ₁₂Ｐ：［Ｍ＋Ｈ］⁺，ｃａｌｃｄ：６５８．３０，ｆｏｕｎｄ：６５８．３ Example 10: Synthesis of CDP-ribitol hexaacetate-PB2 (1) 1,2,3,4-tetra-O-acetyl-D-ribitol-5- (4-pentanoyloxybenzyl N, N-diisopropylphosphoro) Synthesis of amidite

1,2,3,4-tetra-O-acetyl-D-ribitol (384 mg, 1.20 mmol) was dissolved in dehydrated dichloromethane (15 mL) and ice-cooled under an argon atmosphere. To this, 4-pentanoyloxybenzylbis (diisopropylamino) -phosphoroamidite (1.05 g, 2.40 mmol) obtained by the method described in Reference (J. Med. Chem., 2015, 58, 6114-6130). ) Was added. A solution of 4,5-dicyanoimidazole (142 mg, 120 mmol) / dehydrated DMF (630 μL) was added dropwise, the reaction vessel was removed from the ice bath, and the mixture was stirred under an argon atmosphere. After 30 minutes, the mixture was diluted with ethyl acetate and washed successively with saturated aqueous sodium hydrogen carbonate solution, water and saturated brine. After drying over anhydrous magnesium sulfate, the mixture was filtered, the filtrate and washing solution were combined and concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography. The desired fraction was collected, concentrated under reduced pressure, and dried to obtain the desired compound (crude yield: 782 mg, crude yield: 99%).
¹ H-NMR (400MHz, CDCl ₃ ): δ7.36-7.32 (m, 2H), 7.04-7.02 (m, 2H), 5.39 (dd, 0.5H, J = 5) .0, 6.0Hz), 5.35 (dd, 0.5H, J = 5.0, 5.0Hz), 5.32 (m, 1H), 5.26-5.20 (m, 1H) , 4.77-4.57 (m, 2H), 4.34 (dd, 0.5H, J = 3.2, 11.9Hz), 4.33 (dd, 0.5H, J = 3.2) , 12.4Hz), 4.16 (dd, 0.5H, J = 1.8, 12.4Hz), 4.15 (dd, 0.5H, J = 1.8, 12.4Hz), 3. 84 (ddd, 0.5H, J = 3.7, 6.9, 11.4Hz), 3.78-3.74 (m, 1H), 3.67 (m, 0.5H), 3.66 -3.56 (m, 2H), 2.55 (t, 2H, J = 7.3Hz), 2.08 (s, 1.5H), 2.062 (s, 1.5H), 2.056 (S, 3H), 2.046 (s, 3H), 2.034 (s, 1.5H), 2.030 (s, 1.5H), 1.77-1.70 (m, 2H), 1.49-1.40 (m, 2H), 1.21-1.15 (m, 12H), 0.97 (t, 3H, J = 7.3Hz)
ESI-MS spectrum (positive mode) C ₃₁ H ₄₉ NO ₁₂ P: [M + H] ⁺ , calcd: 658.30, found: 658.3

ＣＭＰ ジアセテート（２７０ｍｇ，０．６６３ｍｍｏｌ）を脱水ＤＭＦ（２０ｍＬ）に溶解し、減圧濃縮する操作を２回繰り返した。得られた残渣を脱水ＤＭＦ（３０ｍＬ）に溶解し、トリエチルアミン（８３．１μＬ，０．５７７ｍｍｏｌ）を加えて中和した（Ａ液）。これとは別に、１，２，３，４−テトラ−Ｏ−アセチル−Ｄ−リビトール−５−（４−ペンタノイルオキシベンジル Ｎ，Ｎ−ジイソプロピル ホスホロアミダイト）（４７９ｍｇ，０．７２９ｍｍｏｌ）を脱水アセトニトリル（２７ｍＬ）に溶解し、これに前記Ａ液を加えた。更に脱水ＤＭＦ（５ｍＬ）で２回洗浄した。続いて４，５−ジシアノイミダゾール（８６．１ｍｇ，０．７２９ｍｍｏｌ）の脱水ＤＭＦ（１ｍＬ）溶液を室温で加えた。２０分間攪拌した後に、７０％ｔ−ブチルヒドロペルオキシド水溶液（４２７μＬ，３．３２ｍｍｏｌ）を加えた。更に２時間後に７０％ｔ−ブチルヒドロペルオキシド水溶液（１７１μＬ，１．３３ｍｍｏｌ）を追加した。更に１時間３０分間攪拌した後に反応混合物を減圧濃縮し、得られた残渣を逆相系のＨＰＬＣで精製した。目的のフラクションを集め、凍結乾燥、およびイオン交換樹脂に通すことにより、白色粉末である目的化合物のＮａ塩を得た（収量：３２８ｍｇ，収率：５０％）。
¹Ｈ−ＮＭＲ（４００ＭＨｚ，Ｄ₂Ｏ）：δ７．７５（ｄ，０．５Ｈ，Ｊ＝７．８Ｈｚ），７．７３（ｄ，０．５Ｈ，Ｊ＝７．３Ｈｚ），７．５０（ｄ，２Ｈ，Ｊ＝８．７Ｈｚ），７．１５（ｍ，２Ｈ），６．１４（ｍ，１Ｈ），５．９９（ｄ，０．５Ｈ，Ｊ＝７．８Ｈｚ），５．９６（ｄ，０．５Ｈ，Ｊ＝７．３Ｈｚ），５．３９−５．２８（ｍ，３Ｈ），５．２４−５．１７（ｍ，４Ｈ），４．４６（ｍ，１Ｈ），４．３７−４．１３（ｍ，６Ｈ），２．６４（ｔ，２Ｈ，Ｊ＝７．３Ｈｚ），２．１５５（ｓ，１．５Ｈ），２．１５１（ｓ，１．５Ｈ），２．１０９（ｓ，１．５Ｈ），２．１０４（ｓ，１．５Ｈ），２．１００（ｍ，３Ｈ），２．０８４（ｓ，１．５Ｈ），２．０７６（ｍ，４．５Ｈ），２．０６（ｓ，１．５Ｈ），２．０５（ｓ，１．５Ｈ），１．７４−１．６７（ｍ，２Ｈ），１．４６−１．３７（ｍ，２Ｈ），０．９４（ｔ，３Ｈ，Ｊ＝７．３Ｈｚ）
ＥＳＩ−ＭＳスペクトル（ｎｅｇａｔｉｖｅ ｍｏｄｅ） Ｃ₃₈Ｈ₅₀Ｎ₃Ｏ₂₃Ｐ₂：［Ｍ−Ｈ］^-，ｃａｌｃｄ：９７８．２３，ｆｏｕｎｄ：９７８．２ (2) Synthesis of CDP-ribitol hexaacetate-PB2

The operation of dissolving CMP diacetate (270 mg, 0.663 mmol) in dehydrated DMF (20 mL) and concentrating under reduced pressure was repeated twice. The obtained residue was dissolved in dehydrated DMF (30 mL) and neutralized by adding triethylamine (83.1 μL, 0.577 mmol) (Liquid A). Separately, dehydration of 1,2,3,4-tetra-O-acetyl-D-ribitol-5- (4-pentanoyloxybenzyl N, N-diisopropylphosphoroamidite) (479 mg, 0.729 mmol) It was dissolved in acetonitrile (27 mL), and the solution A was added thereto. Further, it was washed twice with dehydrated DMF (5 mL). Subsequently, a dehydrated DMF (1 mL) solution of 4,5-dicyanoimidazole (86.1 mg, 0.729 mmol) was added at room temperature. After stirring for 20 minutes, a 70% aqueous solution of t-butyl hydroperoxide (427 μL, 3.32 mmol) was added. After an additional 2 hours, a 70% aqueous solution of t-butyl hydroperoxide (171 μL, 1.33 mmol) was added. After further stirring for 1 hour and 30 minutes, the reaction mixture was concentrated under reduced pressure, and the obtained residue was purified by reverse phase HPLC. The desired fraction was collected, lyophilized and passed through an ion exchange resin to obtain a Na salt of the target compound as a white powder (yield: 328 mg, yield: 50%).
^{1 1} H-NMR (400 MHz, D ₂ O): δ7.75 (d, 0.5H, J = 7.8 Hz), 7.73 (d, 0.5H, J = 7.3 Hz), 7.50 ( d, 2H, J = 8.7Hz), 7.15 (m, 2H), 6.14 (m, 1H), 5.99 (d, 0.5H, J = 7.8Hz), 5.96 ( d, 0.5H, J = 7.3Hz), 5.39-5.28 (m, 3H), 5.24-5.17 (m, 4H), 4.46 (m, 1H), 4. 37-4.13 (m, 6H), 2.64 (t, 2H, J = 7.3Hz), 2.155 (s, 1.5H), 2.151 (s, 1.5H), 2. 109 (s, 1.5H), 2.104 (s, 1.5H), 2.100 (m, 3H), 2.084 (s, 1.5H), 2.076 (m, 4.5H) , 2.06 (s, 1.5H), 2.05 (s, 1.5H), 1.74-1.67 (m, 2H), 1.46-1.37 (m, 2H), 0 .94 (t, 3H, J = 7.3Hz)
ESI-MS spectrum (negative mode) C ₃₈ H ₅₀ N ₃ O ₂₃ P ₂ : [MH] ^- , calcd: 978.23, found: 978.2

Test Example 1: In vitro analysis Human fetal kidney cells (HEK cells) in which CDP-ribitol synthase (ISPD) was knocked out were prepared according to a known technique (Kanagawa et al, 2016). The prepared ISPD-KO HEK cells were ^{seeded on a 6-} well plate at 1.0 × 10 6 cells / well, and 10% inactivated fetal bovine serum (manufactured by Sigma), 100 U / mL penicillin, and 100 μg / mL streptomycin (manufactured by WAKO). ) Was cultured overnight in a high glucose Dulbecco modified Eagle's medium (manufactured by WAKO) to allow the cells to settle, and then each concentration of the test compound was added. After 24 hours, the cells were washed with PBS and stripped with TBS containing a 1% detergent (Triton X-100) and a protease inhibitor (Nacalai). After stirring at 4 ° C. for 1 hour, 10 μL of Wheat Germ Agglutinin (WGA) -agarose beads (manufactured by Vector Laboratories) was added to the soluble fraction, and the mixture was stirred at 4 ° C. for 16 hours. After washing with TBS containing 0.1% surfactant (Triton X-100), it was eluted with SDS-PAGE loading buffer. The protein was separated by 4 to 15% linear gradient SDS gel (manufactured by Bio-Rad) and transferred to a PVDF membrane (manufactured by Millipore). Blots were labeled with the following antibodies and developed by horseradish peroxidase (HRP) -enhanced chemiluminescence reagent ("Supersignal West Pico PLUS or ECL Prime" developed by GE Healthcare). In addition, for comparison, the same experiment was conducted when the test compound was not used and when normal HEK cells were used. The results are shown in Figures 1-3.
Antibody mouse anti-complete sugar chain modified type-DG ("IIH6" manufactured by Millipore)
Rat anti-DG core (“3D7” Ohtsuka et al., 2015)
Mouse anti-DG ("8D5" manufactured by Novocastra)
In addition, "IIH6" binds to the sugar chain of α-dystroglycan (DG), "3D7" binds to the core of α-DG, and "8D5" binds to β-DG.

(1) Results of CDP-Ribitol Acetate As shown in FIG. 1, CDP-Ribitol (CDP-Rbo) recovered the formation of sugar chains at 500 μM, but CDP-Ribitol Tetraacetate (TetA, Example 2). ) Recovered sugar chain formation at 200 μM, indicating that the sugar chain recovery effect of CDP-ribitol tetraacetate is stronger than that of CDP-ribitol.

(2) Results of CDP-Ribitol Butyrate As shown in Fig. 2, CDP-Ribitol tetraacetate (TetA, Example 2) recovered the formation of sugar chains at 200 μM, whereas CDP-Ribitol tetra. Butyrate (TetB, Example 4) and CDP-ribitol tetrabutyrate, diacetate (TetB / DiA, Example 5) are 100 μM, CDP-ribitol hexabutyrate (HexB, Example 6) is 50 μm and sugar chain. CDP-ribitol butyrate tended to have a stronger sugar chain recovery effect than CDP-ribitol acetate.

(3) Results of CDP-Ribitol PB As shown in the result shown in FIG. 3, CDP-Ribitol hexaacetate-PB1 CDP-Ribitol isobutyrate (BP1, Example 9) in which phosphoric acid on the citidine side is esterified is 10 μM. CDP-ribitol hexaacetate-PB2 (BP2, Example 10) in which the phosphoric acid on the ribitol side was esterified was 50 μM, and the formation of sugar chains was restored. It is considered that the intracellular uptake of CDP-ribitol increased due to the esterification of the negatively charged pyrophosphate group.

Test Example 2: In vivo analysis-Muscle tissue observation (1) Preparation of model mouse Ispd ^{lox / lox} mice (Lee, et al., 2014) in which loxP sequences are inserted into the 5'side and 3'side of the mouse Ispd gene exon2, respectively. ) Was crossed with Cre-expressing knock-in (CreKI) mice having Myf5 specifically expressed in muscle progenitor cells as a promoter (Jackson Lab, B6.129S4-Myf5 ^{tm3 (cre) Sor} / J). ^{Furthermore, Ispd loxP / +} heterozygous mice having Myf5-Cre were ^{crossed with Ispd lox / lox} mice to generate Myf5-Ispd-cKO mice in which CDP-ribitol synthase (ISPD) was knocked out with the expression of Myf5. Hereinafter, [Ispd ^{lox / lox} : Myf5-CreKI (+)] is referred to as cKO, [Ispd ^{lox / +} : Myf5-CreKI (+)] is referred to as HET, and [Ispd ^{lox / lox} : Myf5-CreKI (-)] is referred to as WT. write. Animal experiments were conducted with the permission of the Kobe University Graduate School Animal Experiment Management Committee.
In addition, cKO had a lower weight than WT, a shorter life than WT and HT, and had weaker grip strength and lower muscle function than WT and HT even at 4 weeks of age.
In addition, cKO had a lower muscle weight and a tendency toward muscular atrophy than WT and HET, and had a high creatine kinase (CK) value, which is an index of muscle destruction.
Furthermore, Western blots detected fully sugar-chained dystroglycan (DG) in WT and HET, but not in cKO. The core protein of DG was also observed in WT and HET, but the sugar chain-modified DG band was detected more intensely. On the other hand, in cKO, only the band of the core protein without sugar chain modification was observed. From these results, it was shown that the sugar chain of DG was shed in the muscle tissue of cKO.

(2) Preparation of muscle tissue sample The muscle tissue removed from the mouse was embedded in an OTC compound (manufactured by Sakura Fintek) and frozen in isopentane (manufactured by Nakarai) cooled in liquid nitrogen. Frozen sections of 7 μm were prepared using a cryostat and used for histological analysis and fluorescent immunostaining.

(3) Pathological analysis by H & E staining and immunostaining In H & E staining, hematoxylin was stained for 2 minutes and eosin was stained for 1 minute, and then dehydrated with ethanol and xylene.
In immunostaining with IIH6, the sections were fixed in a mixed solvent of cold ethanol: acetic acid = 1: 1 for 1 minute, and then blocked for 1 hour at room temperature using a MOM blocking reagent (manufactured by Vector Laboratories) containing 5% goat serum. .. The reaction was carried out overnight at 4 ° C. using the primary antibody diluted in MOM dilution.
For H & E staining, it was encapsulated with "Permount" Fisher Scientific as an embedding agent for preparing frozen sections, and for immunostaining, it was encapsulated with "TISSU MOUNT" manufactured by Shiraimatsu Kikai. The sections were observed with a fluorescence microscope (“Leica DMR” manufactured by Leica Microsystems). The results are shown in FIG.
As shown in the results shown in FIG. 4, in the tissue of cKO, the findings characteristic of muscular dystrophy such as the size difference of muscle fibers, the increase of the central core, and the increase of fibrosis were observed as compared with WT and HET. In addition, immunostaining with IIH6, which binds to the sugar chain of α-dystroglycan (DG), showed that the fluorescence signal was decreased, indicating that the sugar chain of DG was shed.

In addition, immunostaining was performed using the following antibodies.
Rat anti-laminin ("4H8-2" manufactured by Alexis Biochemicals)
Rabbit anti-collagen I (manufactured by Bio-Rad)
Rat anti-F4 / 80 (manufactured by BioLegend)
For immunostaining with laminin, F4 / 80 and collagen I, sections were blocked with PBS containing 3% bovine serum albumin (BSA) for 1 hour at room temperature, followed by the primary antibody diluted with PBS containing 1% BSA. The reaction was carried out at 4 ° C. overnight. The next day, the sections were washed with PBS and reacted with a secondary antibody using a dye (“Alexa Fluor 488” or “Alexa Fluor 555” from Molecular Probes) for 30 minutes at room temperature and then washed with PBS.
As a result of laminin staining, it was shown that the muscle fiber size of cKO was relatively large and small as compared with WT and HET, and the muscle fiber size was not uniform and varied widely. Moreover, in the muscle tissue of cKO, the proportion of muscle fibers having a central core was remarkably increased as compared with WT. Furthermore, as a result of staining with antibody F4 / 80, which is widely used as a macrophage marker, macrophage infiltration is stronger in cKO muscle tissue than in WT, and as a result of collagen staining, fibrosis of muscle tissue is progressing. It has been shown. All of these are typical findings of muscular dystrophy, and it can be said that cKO, which is a muscle-specific ISPD-deficient mouse, serves as a model for Fukuyama-type muscular dystrophy-related diseases.

Test Example 3: In vivo analysis-Western blot analysis After anesthetizing 4 of the 7-week-old cKO model mice, CDP-libitol (CDP-Rbo) or CDP-libitol tetraacetate (TetA, Example 2) was saline. 40 μL of a 0.1 M aqueous solution or physiological saline solution dissolved in water was injected into the tibialis anterior muscle and 60 μL into the gastrocnemius muscle, and 3 days later, each aqueous solution was injected after the same anesthesia. Muscle tissue was removed 3 days after the second injection.
After crushing the muscle tissue using a polytron homogenizer in TBS to which a protease inhibitor was added, a surfactant (Triton X-100) having a final concentration of 1% was added, and the mixture was inverted and mixed at 4 ° C. for 1 hour. To the soluble fraction, 10 to 30 μL of Wheat Germ Agglutinin (WGA) -agarose beads (manufactured by Vector Laboratories) was added, and the mixture was stirred at 4 ° C. for 16 hours. After washing with TBS containing 0.1% surfactant (Triton X-100), it was eluted with SDS-PAGE loading buffer. The obtained sample was subjected to Western blotting in the same manner as in Test Example 1. The results are shown in FIG.
As shown in the results shown in FIG. 5, when CDP-ribitol was administered, sugar chain formation of α-dystroglycan (DG) was slightly observed as compared with the case where physiological saline was administered.
On the other hand, when CDP-ribitol tetraacetate (TetA) was administered, DG sugar chain formation was clearly observed, and high sugar chain recovery activity was shown. In addition, the model mice to which CDP-ribitol tetraacetate (TetA) was administered were no different from the model mice to which the same amount of physiological saline was administered at the age of 8 weeks. The toxicity of TetA) is considered to be low.

Test Example 4: In vivo analysis-Immunostaining analysis The same procedure as in Test Example 3 except that the administration was continued twice a week for 3 consecutive weeks, and the tissue was removed 3 days after the final administration, and the tissue was removed with Test Example 2. Pathological analysis by immunostaining with laminin, F4 / 80 and collagen I was performed under similar conditions. The results of immunostaining with laminin are shown in FIG. 6, the results of immunostaining with F4 / 80 are shown in FIG. 7 (1), and the results of immunostaining with collagen I are shown in FIG. 7 (2). In FIG. 6, “*” indicates a case where there is a significant difference at p <0.05 as a result of one-way ANOVA of the mean value by Turkey's multiple comparison test, and “***” indicates p <0. The case where there is a significant difference at .001 is shown. In FIG. 7, “*” indicates a case where there is a significant difference at p <0.05 in Dunn's multiple comparison after the Kruskal-Wallis test.
As shown in the results shown in FIG. 6, the muscle fiber diameter tended to increase slightly even when CDP-ribitol (CDP-Rbo) was administered as compared with the control example (physiological saline), but CDP-ribitol tended to increase. When tetraacetate (TetA) was administered, the muscle fiber diameter was significantly increased as compared with the control example and the case where CDP-ribitol was administered. Looking at the muscle fiber diameter distribution (FIG. 6 (2)), it can be seen that when CDP-ribitol tetraacetate (TetA) was administered, the small diameter muscle fibers decreased and the large diameter muscle fibers increased.
As shown in the result shown in FIG. 7 (1), when CDP-ribitol tetraacetate (TetA) was administered, the infiltration of macrophages into muscle tissue was significantly reduced as compared with the control example.
In addition, as shown in the result shown in FIG. 7 (2), when CDP-ribitol tetraacetate (TetA) was administered, muscle fibrosis was observed as compared with the control example and the case where CDP-ribitol was administered. Can be seen to have decreased significantly.
The above results indicate that TetA is effective in treating Fukuyama muscular dystrophy and related diseases.

Claims (9)

A CDP-ribitol derivative or a pharmaceutically acceptable salt thereof, which is represented by the following formula (I ^1).

[During the ceremony,
R ^{1 to} R ⁴ independently represent H, C _1-6 alkyl-carbonyl groups, or C _1-6 alkoxy-carbonyl groups.
R ⁵ and R ⁶ are independently, H, C _1-6 alkyl group which may have a substituent C _6-12 aryl group, optionally having a substituent on the C _6-12 aryl group which may be C _6-12 aryl - amino group or C _6-12 aryl group optionally C _6-12 aryl optionally having substituent on, - a methyl group,
Substituents on the C _6-12 aryl group represent a C _1-6 alkoxy group or a C _1-6 alkyl-carbonyloxy group.
R ⁷ and R ⁸ independently represent OH, C _1-6 alkyl-carbonyloxy group, C _1-6 alkoxy-carbonyloxy group, C _1-6 alkoxy group, or halogeno group.
However, when R ^{1 to} R ⁶ are H, R ⁷ and R ⁸ independently have a C _1-6 alkyl-carbonyloxy group, a C _1-6 alkoxy-carbonyloxy group, and a C _1-6 alkoxy group. , Or a halogeno group. ] The CDP-ribitol derivative according to claim 1, wherein the CDP-ribitol derivative is a CDP-ribitol derivative represented by the following ^{formula (I 2), or a pharmaceutically acceptable salt thereof.}

[In the formula, R ^{1 to} R ⁸ have the same meaning as above. ] The CDP-ribitol derivative according to claim 1 or 2, wherein R ^{1 to} R ⁴ are independently C _{1-4 alkyl-carbonyl groups, or a pharmaceutically acceptable salt thereof.} The CDP-ribitol derivative according to any one of claims 1 to 3, wherein R ⁵ and R ^{6 are H, or a pharmaceutically acceptable salt thereof.} The CDP according to any one of claims 1 to 3, wherein R ⁵ and R ⁶ _{are C 6-12} aryl-methyl groups which may independently have a substituent on the C _{6-12 aryl group.} -Libitol derivative or pharmaceutically acceptable salt thereof. The CDP-libitol derivative according to any one of claims 1 to 3, wherein R ⁵ and R ⁶ _{are independently benzyl groups having a C 1-6} alkyl-carbonyloxy group as a substituent on the phenyl group, or a CDP-libitol derivative thereof. A pharmaceutically acceptable salt. The CDP-ribitol derivative according to any one of claims 1 to 6, wherein R ⁷ and R ^{8 are OH, or a pharmaceutically acceptable salt thereof.} A medicament containing the CDP-ribitol derivative according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof. A therapeutic agent for dystroglycan dysfunction, which comprises the CDP-ribitol derivative according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof as an active ingredient.

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