JPWO2010150791A1 - Process for producing adenosine tetraphosphate compounds - Google Patents

Process for producing adenosine tetraphosphate compounds Download PDF

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JPWO2010150791A1
JPWO2010150791A1 JP2011519910A JP2011519910A JPWO2010150791A1 JP WO2010150791 A1 JPWO2010150791 A1 JP WO2010150791A1 JP 2011519910 A JP2011519910 A JP 2011519910A JP 2011519910 A JP2011519910 A JP 2011519910A JP WO2010150791 A1 JPWO2010150791 A1 JP WO2010150791A1
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向後　悟
悟 向後
和臣 大沢
和臣 大沢
早川　弘之
弘之 早川
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Abstract

［要約］［課題］アデノシンテトラホスフェートの合成法において、従来の製法に比べて高収率かつより簡易な方法で合成することが可能となる新規な製造法を確立する。［解決手段］式（ＸＩ）化合物から式（Ｉ）化合物を合成する工程において、式（ＸＩ）化合物を、カルボジイミド類を縮合剤として用いて対応する環状トリリン酸体（ＸＩＶ）へ変換し、続けてマグネシウム、マンガンまたは鉄から選択された金属塩の存在下、得られた環状トリリン酸と式（ＶＩＩＩ）化合物とを反応させる。［選択図］（式中、Ａｒはアリール基、Ｘは硫黄原子もしくは酸素原子またはイミノ基のいずれかより選ばれる基、Ｒ１とＲ２は水素原子、アルキルまたはアラルキル基のいずれかより選ばれる基、Ｍは水素原子またはイオンを示す。）[Summary] [Problem] In a method for synthesizing adenosine tetraphosphate, a novel production method capable of being synthesized by a simpler method with a higher yield than conventional methods is established. [Solution] In the step of synthesizing the compound of formula (I) from the compound of formula (XI), the compound of formula (XI) is converted into the corresponding cyclic triphosphate (XIV) using carbodiimides as a condensing agent, and In the presence of a metal salt selected from magnesium, manganese or iron, the obtained cyclic triphosphoric acid is reacted with the compound of formula (VIII). [Selection Figure] (In the formula, Ar is an aryl group, X is a group selected from a sulfur atom, an oxygen atom or an imino group, R1 and R2 are groups selected from a hydrogen atom, an alkyl or an aralkyl group, M represents a hydrogen atom or an ion.)

Description

本発明は、アデノシンテトラホスフェート化合物の効率的な製造法に関する。   The present invention relates to an efficient method for producing an adenosine tetraphosphate compound.

下記式（１）で表されるアデノシンテトラホスフェート化合物またはその塩は、Ｐ２Ｙプリン受容体のアゴニストであり、嚢胞性線維症、慢性閉塞性肺疾患（ＣＯＰＤ）などの治療薬としての開発が期待されている化合物である。   The adenosine tetraphosphate compound represented by the following formula (1) or a salt thereof is an agonist of the P2Y purine receptor and is expected to be developed as a therapeutic agent for cystic fibrosis, chronic obstructive pulmonary disease (COPD) and the like. It is a compound.

Figure 2010150791
（式中、Ｍは水素原子またはＮａ+などのイオンを示す。）
Figure 2010150791
 (In the formula, M represents a hydrogen atom or an ion such as Na + .)

このようなアデノシンテトラホスフェート化合物の合成法としては、従来、例えば下記［化２］に示したように、式（３）化合物を２工程のリン酸化反応により式（５）で表されるトリリン酸誘導体とし、これを１−メチルアデノシン−５’−リン酸イミダゾリデート（７）と反応させることで目的の式（１）化合物とする方法（従来法１）や、式（４）化合物とアデノシン５’−トリリン酸イミダゾリデート、または式（５）化合物とアデノシン５’−モノリン酸イミダゾリデートから合成される式（１’）化合物をメチル化する方法（従来法２）が報告されている（特許文献１）。   As a method for synthesizing such an adenosine tetraphosphate compound, conventionally, for example, as shown in the following [Chemical Formula 2], triphosphoric acid represented by the formula (5) is obtained by subjecting the compound of the formula (3) to a two-step phosphorylation reaction. A derivative, which is reacted with 1-methyladenosine-5′-phosphate imidazolidate (7) to obtain the desired compound of formula (1) (conventional method 1), or a compound of formula (4) and adenosine 5′-triphosphate imidazolidate, or a method of methylating a compound of formula (1 ′) synthesized from a compound of formula (5) and adenosine 5′-monophosphate imidazolidate (conventional method 2) has been reported. (Patent Document 1).

Figure 2010150791
（式中、Ｍは水素原子またはＮａ+などのイオンを示す。）
Figure 2010150791
 (In the formula, M represents a hydrogen atom or an ion such as Na + .)

ＷＯ２００８／０６０６３２WO2008 / 060632 ＷＯ２００８／０１２９４９WO2008 / 012949

Ｏｒｇａｎｉｃ Ｌｅｔｔｅｒｓ，８，２０７５−２０７７（２００６）Organic Letters, 8, 2075-2077 (2006)

しかしながら、これらの従来の合成法は、各工程での合成収率が低いために、式（２）化合物から式（１）化合物を得るまでの通算の単離収率は従来法１で０．３％、従来法２で０．０３〜０．０８％程度と、到底実用的な方法とは言えなかった。   However, since these conventional synthesis methods have a low synthesis yield in each step, the total isolation yield until the compound of the formula (1) is obtained from the compound of the formula (2) is 0. 3%, 0.03 to 0.08% in the conventional method 2, and could not be said to be a practical method at all.

また、従来法１および２とも、工業的に不利とされる、シリカゲルやイオン交換樹脂を用いたカラムクロマトグラフィーによる精製方法が多用される、立体異性体の混合物として生成する式（３）化合物を分離するための工程が必要である、さらに、式（１）化合物を高純度で得るために分取用ＨＰＬＣのような大量合成には不利な装置が必要になる等、工業的生産に適用するには重大な問題点を多数有していた。 Further, in both conventional methods 1 and 2, the compound of the formula (3) produced as a mixture of stereoisomers, which is frequently used in column chromatography using silica gel or ion exchange resin, which is industrially disadvantageous. A process for separation is required. Furthermore, in order to obtain the compound of the formula (1) with high purity, it is necessary to use a disadvantageous apparatus for mass synthesis such as preparative HPLC. Had many serious problems.

より詳細に問題点を分析すれば、式（１）化合物の従来の合成法には、以下（Ａ）〜（Ｃ）に挙げるような大きな問題点があった。   If the problems are analyzed in more detail, the conventional synthesis method of the compound of formula (1) has the following major problems as listed in (A) to (C) below.

（Ａ）目的の式（１）化合物の生産に用いる出発原料の式（３）化合物は、後述の実施例中に示すようにα，β−立体異性体混合物として調製されるため、これらを分離して目的のβ体のみを得るためには、分取用ＨＰＬＣのような大掛かりな精製装置の使用が必須であり、しかも２工程を経た単離収率は２３％と低い。 (A) Since the compound of the formula (3) as a starting material used for the production of the target compound of the formula (1) is prepared as an α, β-stereoisomer mixture as shown in the examples described later, these are separated. Thus, in order to obtain only the target β-form, it is essential to use a large purification apparatus such as preparative HPLC, and the isolation yield after two steps is as low as 23%.

（Ｂ）式（３）化合物を式（４）化合物に、さらに式（４）化合物を式（５）化合物とする工程では、いずれの工程も単離収率が低い上、精製に手間のかかるイオン交換カラムクロマトグラフィーを用いており、収率、精製の簡便さの点で問題がある。 (B) In the step of using the compound of formula (3) as the compound of formula (4) and further using the compound of formula (4) as the compound of formula (5), the isolation yield is low in each step, and the purification takes time. Since ion exchange column chromatography is used, there are problems in terms of yield and ease of purification.

（Ｃ）式（５）化合物を式（１）化合物とする工程では、従来、式（５）化合物を水中、塩化マンガンの存在下、１−メチルアデノシン−５’−リン酸イミダゾリデート（式（７）化合物）と反応させていた。しかしながら、ここで用いられている１−メチルアデノシン−５’−リン酸イミダゾリデートの合成には、アデノシン−５’−リン酸（式（６）化合物）から２工程の操作を必要とする上、１−メチルアデノシン−５’−リン酸イミダゾリデートの単離収率も１８％と低収率でしか得られない。 (C) In the step of converting the compound of formula (5) into the compound of formula (1), conventionally, the compound of formula (5) is converted to 1-methyladenosine-5′-phosphate imidazolidate (formula in the presence of manganese chloride in water). (7) compound). However, the synthesis of 1-methyladenosine-5′-phosphate imidazolidate used here requires two-step operation from adenosine-5′-phosphate (compound of formula (6)). The isolated yield of 1-methyladenosine-5′-phosphate imidazolidate can be obtained only at a low yield of 18%.

本発明者らは鋭意研究を重ねた結果、下記（Ｘ）〜（Ｚ）のような知見を得ると共に、当該方法を用いることで、従来の合成法における手間の掛かる精製操作を大幅に簡略化し、式（１）化合物の収率を公知の方法と比較して格段に向上させることができることを見出した。   As a result of intensive studies, the present inventors obtained the following knowledge (X) to (Z), and greatly simplified the time-consuming purification operation in the conventional synthesis method by using the method. The inventors have found that the yield of the compound of formula (1) can be remarkably improved as compared with known methods.

（Ｘ）式（３）化合物の調製において、後述のように、従来法では１：３．５（α：β）の比で不要なα体が生成する。この問題に対して発明者らは、（ａ）反応系にモレキュラーシーブスを共存させ、ルイス酸を種々検討することにより、目的のβ体の生成比を１：２４〜１：２７にまで改善できること、（ｂ）出発原料に、従来の式（２）化合物に代えて式（８）化合物を用いることで、中間体である式（９）化合物が結晶となるため、この段階で結晶化によって目的とするβ体のみを優先的に得られることを見出した。こうして得られた式（９）化合物を脱保護することにより、式（３）化合物が単一の異性体として得られ、従来法と異なり、工業的に不利なシリカゲルカラムクロマトグラフィーや分取用ＨＰＬＣのような装置を用いることなく、結晶化のみで式（３）化合物を２工程で６９％と高収率で得られることが明らかとなった。 (X) In the preparation of the compound of formula (3), as described later, the conventional method produces an unnecessary α-form at a ratio of 1: 3.5 (α: β). For this problem, the inventors can improve the production ratio of the target β-form from 1:24 to 1:27 by coexisting molecular sieves in the reaction system and examining various Lewis acids. (B) By using the compound of the formula (8) instead of the conventional compound of the formula (2) as the starting material, the compound of the formula (9), which is an intermediate, becomes a crystal. It was found that only the β-form can be obtained preferentially. By deprotecting the compound of formula (9) thus obtained, the compound of formula (3) is obtained as a single isomer, and unlike conventional methods, it is industrially disadvantageous in silica gel column chromatography and preparative HPLC. It was revealed that the compound of the formula (3) can be obtained in a high yield of 69% in two steps only by crystallization without using an apparatus such as

Figure 2010150791
（式中、Ｍｅはメチル基、Ｂｚはベンゾイル基、Ａｃはアセチル基を示す。）
Figure 2010150791
 (In the formula, Me represents a methyl group, Bz represents a benzoyl group, and Ac represents an acetyl group.)

（Ｙ）式（４）化合物の調製において、新たに２，３位の水酸基に保護基を導入した式（１０）化合物を中間体として、塩基の存在下でリン酸化反応を行い、また、式（４）化合物をＮ−メチルイミダゾリデート体を中間体として経由させることによって、収率を大きく改善でき、さらに精製も簡便に実施できる活性炭カラムクロマトグラフィーにより行うことによって、式（５）化合物の単離収率、精製の手間を大幅に改善できることを見出した。 (Y) In the preparation of the compound of the formula (4), a phosphorylation reaction is carried out in the presence of a base using the compound of the formula (10) in which a protective group is newly introduced into the hydroxyl groups at the 2- and 3-positions as an intermediate. (4) By passing the compound through an N-methylimidazolidate as an intermediate, the yield can be greatly improved, and further purification can be carried out easily by activated carbon column chromatography. It was found that the isolation yield and the labor of purification can be greatly improved.

Figure 2010150791
（式中、Ｍｅはメチル基、Ｂｚはベンゾイル基を示す。）
Figure 2010150791
 (In the formula, Me represents a methyl group, and Bz represents a benzoyl group.)

（Ｚ）式（５）化合物を式（１）化合物とする工程に関連する反応としては、従来、アデノシン５’−環状トリリン酸とアデノシン５’−モノリン酸との反応において、亜鉛塩の存在下反応させる方法（非特許文献１）が知られていた。しかしながら、後述の検討のように、亜鉛塩を用いる方法では、高収率を達成するには保護されたアデノシンヌクレオチド原料が必要であり、本発明のような無保護の原料を用いた場合には、合成収率３０．８％と低いものであった。 (Z) As a reaction related to the step of converting the compound of formula (5) into the compound of formula (1), conventionally, in the reaction of adenosine 5′-cyclic triphosphate and adenosine 5′-monophosphate, in the presence of a zinc salt A method of reacting (Non-Patent Document 1) has been known. However, as described later, in the method using a zinc salt, a protected adenosine nucleotide raw material is required to achieve a high yield, and when an unprotected raw material such as the present invention is used, The synthesis yield was as low as 30.8%.

そこで本発明者らは、当該反応に最適な条件を鋭意検討した結果、特定の金属塩、とくにマグネシウムの塩を存在させるのが、最も好適な条件であることを見出した。 Thus, as a result of intensive studies on the optimum conditions for the reaction, the present inventors have found that the presence of a specific metal salt, particularly a magnesium salt, is the most suitable condition.

従来、マグネシウムの塩の存在下、ジ（ピリミジンヌクレオシド５’−）ポリホスフェートを合成する方法に関してすでに報告されている（特許文献２）。しかし、本発明化合物とより近い化合物の反応であるアデノシン５’−環状トリリン酸とアデノシン５’−モノリン酸との反応においては、マグネシウム塩は亜鉛塩より数段劣り、高収率を達成できないことが報告されており（非特許文献１）、アデノシンテトラホスフェートの合成に亜鉛塩に代えてマグネシウム塩を用いたとしても高収率で目的化合物を得ることができるとは到底考えられず、全くの予想外のことであった。 Conventionally, a method for synthesizing di (pyrimidine nucleoside 5'-) polyphosphate in the presence of a magnesium salt has already been reported (Patent Document 2). However, in the reaction of adenosine 5′-cyclic triphosphoric acid and adenosine 5′-monophosphoric acid, which is a reaction of a compound closer to the compound of the present invention, magnesium salt is inferior to zinc salt by several steps, and high yield cannot be achieved. (Non-Patent Document 1), it is not considered that the target compound can be obtained in high yield even if a magnesium salt is used instead of a zinc salt in the synthesis of adenosine tetraphosphate. It was unexpected.

Figure 2010150791
Figure 2010150791

したがって、本発明は以下の通りである。
［１］ 式（ＸＩ）化合物を縮合剤を用いて式（ＸＩＶ）化合物に変換後（工程Ａ）、マグネシウム、マンガンまたは鉄のいずれかより選択された金属塩の存在下、式（ＸＩＩＩ）と反応させることにより式（Ｉ）化合物を得る（工程Ｂ）ことを特徴とするアデノシンテトラホスフェート化合物の製造法。Therefore, the present invention is as follows.
[1] After converting the compound of formula (XI) to the compound of formula (XIV) using a condensing agent (step A), in the presence of a metal salt selected from any of magnesium, manganese or iron, A process for producing an adenosine tetraphosphate compound, characterized in that a compound of formula (I) is obtained by reaction (step B).

Figure 2010150791
Figure 2010150791

（式中、Ａｒはアリール基、Ｘは硫黄原子もしくは酸素原子またはイミノ基のいずれかより選ばれる基、Ｒ１とＲ２は水素原子、アルキルまたはアラルキル基のいずれかより選ばれる基、Ｍは水素原子またはイオンを示す。）(In the formula, Ar is an aryl group, X is a group selected from a sulfur atom, an oxygen atom or an imino group, R 1 and R 2 are groups selected from a hydrogen atom, an alkyl or an aralkyl group, and M is Indicates hydrogen atom or ion.)

［２］Ｒ１がアルキル、Ｒ２が水素原子、Ａｒがアルコキシフェニル、Ｘは酸素原子で表される化合物である、［１］に記載の製造法。[2] The production method according to [1], wherein R 1 is alkyl, R 2 is a hydrogen atom, Ar is alkoxyphenyl, and X is an oxygen atom.

［３］Ｒ１がメチル、Ｒ２が水素原子、Ａｒがメトキシフェニル、Ｘは酸素原子で表される化合物である、［１］に記載の製造法。[3] The production method according to [1], wherein R 1 is methyl, R 2 is a hydrogen atom, Ar is methoxyphenyl, and X is an oxygen atom.

［４］縮合剤がカルボジイミド類である、［１］に記載の製造法。
［５］金属塩がマグネシウム塩である、［１］に記載の製造法。
［６］工程Ａと工程Ｂを連続して行う、［１］に記載の製造法。[4] The production method according to [1], wherein the condensing agent is a carbodiimide.
[5] The production method according to [1], wherein the metal salt is a magnesium salt.
[6] The production method according to [1], wherein the step A and the step B are continuously performed.

本発明の合成法は、従来法と比較して、式（Ｉ）化合物（出発原料（式（ＸＩ）化合物を含め）に至る各工程の収率が高く、また、精製手段として結晶化を多用することにより、精製が著しく容易になっていることが、最大の特長となっている。   Compared with the conventional method, the synthesis method of the present invention has a higher yield in each step leading to the compound of formula (I) (including the starting material (including the compound of formula (XI)), and crystallization is frequently used as a purification means. Thus, the greatest feature is that refining is remarkably facilitated.

すなわち、従来法によると、目的とする式（１）化合物の通算収率は０．０３〜０．３％程度と極めて低いものであったのに対し、本発明では当該化合物を、出発原料（式（ＸＩ）化合物）の原料化合物である式（ＩＩ）化合物から通算した場合であっても、単離収率が約１４％と、きわめて効率よく合成することができる。   That is, according to the conventional method, the total yield of the target compound of the formula (1) was as extremely low as about 0.03 to 0.3%. Even when the total amount is from the compound of formula (II), which is the starting compound of formula (XI) compound), the isolation yield is about 14%, and it can be synthesized very efficiently.

また、従来法では、工業的に不利なクロマトグラフィー操作を頻繁に行っていたのに対し、本発明では、ほとんどの工程で、精製手段として結晶化を用いており、クロマトグラフィーによる精製は、活性炭カラムクロマトグラフィー、陰イオン交換クロマトグラフィーをそれぞれ１回行うのみで済むため、従来法と比較して大幅に精製法が簡略化されている。   In addition, in the conventional method, chromatography operation which is industrially disadvantageous is frequently performed, whereas in the present invention, crystallization is used as a purification means in most steps, and purification by chromatography is performed using activated carbon. Since column chromatography and anion exchange chromatography need only be performed once, the purification method is greatly simplified as compared with the conventional method.

従って、本発明は、高収率かつより簡易な方法でアデノシンテトラホスフェート化合物を合成することが可能であり、工業的な大量合成に好適な方法である。 Therefore, the present invention can synthesize an adenosine tetraphosphate compound with a high yield and a simpler method, and is a suitable method for industrial mass synthesis.

本発明は、下記式（ＸＩ）化合物を縮合剤を用いて式（ＸＩＶ）化合物に変換後（工程Ａ）、金属塩の存在下、式（ＸＩＩＩ）化合物と反応させることにより式（Ｉ）化合物を得る（工程Ｂ）２つの工程からなることを特徴とするアデノシンテトラホスフェート化合物の製造法に関するものである。   In the present invention, a compound of formula (I) is converted into a compound of formula (XIV) using a condensing agent (step A) and then reacted with the compound of formula (XIII) in the presence of a metal salt. (Step B) The present invention relates to a method for producing an adenosine tetraphosphate compound characterized by comprising two steps.

Figure 2010150791
（式中、Ａｒはアリール基、Ｘは硫黄原子もしくは酸素原子またはイミノ基のいずれかより選ばれる基、Ｒ１とＲ２は水素原子、アルキルまたはアラルキル基のいずれかより選ばれる基、Ｍは水素原子、Ｎａ＋などの金属イオン又は置換アンモニウムイオンを示す。）
Figure 2010150791
 (In the formula, Ar is an aryl group, X is a group selected from a sulfur atom, an oxygen atom or an imino group, R 1 and R 2 are groups selected from a hydrogen atom, an alkyl or an aralkyl group, and M is It represents a hydrogen atom, a metal ion such as Na + or a substituted ammonium ion.)

工程Ａにおいて、式（ＸＩ）化合物を式（ＸＩＶ）化合物へ変換するための縮合剤としては、ジシクロヘキシルカルボジイミド、水溶性カルボジイミド、ジイソプロピルカルボジイミドといったカルボジイミド類、ジフェニルホスホロクロリデート等のリン酸ハロゲン化物、塩化トルエンスルホニル等のスルホン酸ハロゲン化物、カルボニルジイミダゾール、等の公知の縮合剤を使用することができ、特にカルボジイミド類が好適である。   In step A, as a condensing agent for converting the compound of formula (XI) to the compound of formula (XIV), carbodiimides such as dicyclohexylcarbodiimide, water-soluble carbodiimide, diisopropylcarbodiimide, phosphoric acid halides such as diphenylphosphorochloridate, Known condensing agents such as sulfonic acid halides such as toluenesulfonyl chloride and carbonyldiimidazole can be used, and carbodiimides are particularly preferred.

反応は、使用する縮合剤により異なるもの、たとえば、ジメチルホルムアミド、ジメチルアセトアミド、ホルムアミド、ジメチルスルホキシド等の単独又は混合溶媒中、式（ＸＩ）化合物１モルに対して縮合剤１〜５モル使用し、０℃〜５０℃、好ましくは１５〜３０℃で１〜２４時間程度反応させることにより実施できる。   The reaction varies depending on the condensing agent used, for example, 1 to 5 mol of condensing agent is used per 1 mol of the compound of formula (XI) in a single or mixed solvent such as dimethylformamide, dimethylacetamide, formamide, dimethyl sulfoxide and the like. The reaction can be carried out at 0 to 50 ° C., preferably 15 to 30 ° C. for about 1 to 24 hours.

より具体的に、縮合剤としてジイソプロピルカルボジイミドを使用する場合、ジメチルホルムアミド中、式（ＸＩ）化合物１モルに対して、ジイソプロピルカルボジイミドを１〜５モル、好ましくは１．２〜２モルを用い、０〜５０℃、好ましくは２０〜３０℃で１〜３時間程度反応させることにより実施できる。   More specifically, when diisopropylcarbodiimide is used as the condensing agent, 1 to 5 mol, preferably 1.2 to 2 mol of diisopropylcarbodiimide is used in 1 mol of the compound of formula (XI) in dimethylformamide. It can be carried out by reacting at -50 ° C, preferably 20-30 ° C for about 1-3 hours.

このようにして得られた式（ＸＩＶ）化合物は、単離することなく連続して次の工程Ｂに利用できる。工程Ｂでは、金属塩の存在下、式（ＸＩＩＩ）化合物と反応させて式（Ｉ）化合物を合成する。   The compound of the formula (XIV) thus obtained can be used continuously in the next step B without isolation. In Step B, the compound of formula (I) is synthesized by reacting with the compound of formula (XIII) in the presence of a metal salt.

反応に共存させる金属塩としては、マグネシウム、マンガン及び鉄のいずれかより選択された金属の塩であれば、特に制限されない。具体的には、フッ化マグネシウム、塩化マグネシウム、臭化マグネシウム、ヨウ化マグネシウム、塩化マンガン、塩化第二鉄等の金属ハロゲン化物、マグネシウム、マンガン、鉄の硫酸、硝酸、リン酸、過塩素酸、テトラフルオロホウ酸塩等といった金属無機酸塩、マグネシウム、マンガン、鉄のトリフルオロメタンスルホン酸、メタンスルホン酸、トルエンスルホン酸、酢酸、トリフルオロ酢酸、ステアリン酸、クエン酸塩等を例示することができる。   The metal salt coexisting in the reaction is not particularly limited as long as it is a metal salt selected from any of magnesium, manganese and iron. Specifically, magnesium fluoride, magnesium chloride, magnesium bromide, magnesium iodide, manganese chloride, ferric chloride and other metal halides, magnesium, manganese, iron sulfate, nitric acid, phosphoric acid, perchloric acid, Examples thereof include metal inorganic acid salts such as tetrafluoroborate, magnesium, manganese, and iron trifluoromethanesulfonic acid, methanesulfonic acid, toluenesulfonic acid, acetic acid, trifluoroacetic acid, stearic acid, and citrate. .

そのような金属塩の中でも、合成収率及び取扱い易さの点でマグネシウム塩が好ましく、特に塩化マグネシウムなどのマグネシウムのハロゲン化物を好適なものとして例示できる。なお、用いる金属塩は、無水物であっても水和物であっても構わない。   Among such metal salts, a magnesium salt is preferable from the viewpoint of synthesis yield and ease of handling, and a magnesium halide such as magnesium chloride is particularly preferable. The metal salt used may be an anhydride or a hydrate.

反応は、式（ＸＩＶ）化合物１モルに対し、式（ＸＩＩＩ）化合物を１〜５モル、好ましくは１〜２モル、及び金属塩１〜５モル、好ましくは１〜２モルをそれぞれ添加し、０〜１００℃、好ましくは１５〜３０℃で、１〜２４時間程度反応させることにより実施できる。   In the reaction, 1 to 5 mol, preferably 1 to 2 mol, and 1 to 5 mol, preferably 1 to 2 mol of a metal salt are added to 1 mol of the compound of formula (XIV), The reaction can be carried out at 0 to 100 ° C., preferably 15 to 30 ° C. for about 1 to 24 hours.

反応後、合成目的の式（Ｉ）化合物は、一般のヌクレオチドの単離精製に使用されている方法（例えば、再結晶法、イオン交換カラムクロマトグラフィー、吸着カラムクロマトグラフィー、活性炭カラムクロマトグラフィーなど）を適宜組み合せて分離精製することができ、必要に応じて塩型とすることもできる。   After the reaction, the compound of formula (I) for the purpose of synthesis is a method used for isolation and purification of general nucleotides (for example, recrystallization method, ion exchange column chromatography, adsorption column chromatography, activated carbon column chromatography, etc.) Can be separated and purified by combining them as appropriate, and can be converted into a salt form as necessary.

次に、原料化合物である上記式（ＸＩ）で表されるリボーストリリン酸誘導体と式（ＸＩＩＩ）で表されるアデノシン５’−モノリン酸誘導体の調製法に関し、詳細に説明する。最初には、式（ＸＩ）化合物は、好ましくは、以下に説明する３つの工程より調製する。 Next, a method for preparing the ribose triphosphate derivative represented by the above formula (XI) and the adenosine 5'-monophosphate derivative represented by the formula (XIII), which are raw material compounds, will be described in detail. Initially, the compound of formula (XI) is preferably prepared from the three steps described below.

第１工程；
式（ＩＩ）化合物を、ルイス酸およびモレキュラーシーブスの存在下、フェノール化合物と反応させ、立体異性体混合物である式（ＩＩＩ）化合物を得、得られた式（ＩＩＩ）化合物から結晶化によりβ体（式（ＩＶ）化合物）のみを分離し、該化合物をアルカリ性条件下、脱保護して式（Ｖ）化合物を得る工程。First step;
The compound of formula (II) is reacted with a phenol compound in the presence of Lewis acid and molecular sieves to obtain a compound of formula (III) which is a stereoisomer mixture, and β-form is obtained by crystallization from the obtained compound of formula (III). A step of separating only (compound of formula (IV)) and deprotecting the compound under alkaline conditions to obtain a compound of formula (V).

Figure 2010150791
（式中、Ａｒはアリール基、Ｘは硫黄原子もしくは酸素原子またはイミノ基のいずれかより選ばれる基、Ｒ３は保護基、Ｒ４はアシル基を示す。）
Figure 2010150791
 (In the formula, Ar represents an aryl group, X represents a group selected from a sulfur atom, an oxygen atom or an imino group, R 3 represents a protecting group, and R 4 represents an acyl group.)

第２工程；
式（Ｖ）化合物の５位水酸基を選択的に保護し、２，３位水酸基を５位保護基とは除去法の異なる保護基で保護後、５位保護基を選択的に除去することにより、式（ＶＩＩ）化合物を得、該化合物を塩基の存在下、リン酸化して式（ＶＩＩＩ）化合物とし、２，３位の保護基を除去して式（ＩＸ）化合物を得る工程。Second step;
By selectively protecting the hydroxyl group at the 5-position of the compound of formula (V) and protecting the hydroxyl group at the 2- and 3-positions with a protecting group having a different removal method from the protecting group at the 5-position, by selectively removing the 5-position protecting group A step of obtaining a compound of formula (VII), phosphorylating the compound in the presence of a base to give a compound of formula (VIII), and removing a protecting group at positions 2 and 3 to obtain a compound of formula (IX).

Figure 2010150791
（式中、Ａｒはアリール基、Ｘは硫黄原子もしくは酸素原子またはイミノ基のいずれかより選ばれる基、Ｒ５とＲ６は保護基、Ｍは水素原子またはイオンを示す。）
Figure 2010150791
 (In the formula, Ar represents an aryl group, X represents a group selected from a sulfur atom, an oxygen atom or an imino group, R 5 and R 6 represent a protecting group, and M represents a hydrogen atom or an ion.)

第３工程；
式（ＩＸ）化合物のリン酸基を式（Ｘ）化合物に変換後、ピロリン酸塩と反応させることにより式（ＸＩ）化合物を得る工程。Third step;
A step of obtaining a compound of formula (XI) by converting a phosphate group of the compound of formula (IX) into a compound of formula (X) and then reacting with a pyrophosphate.

Figure 2010150791
（ＩＸ） （Ｘ） （ＸＩ）
（式中、Ａｒはアリール基、Ｘは硫黄原子もしくは酸素原子またはイミノ基のいずれかより選ばれる基、Ｒ７はイミダゾリル基またはＮ−メチルイミダゾリル基、Ｍは水素原子またはイオンを示す。）
Figure 2010150791
 (IX) (X) (XI)
(In the formula, Ar represents an aryl group, X represents a group selected from a sulfur atom, an oxygen atom or an imino group, R 7 represents an imidazolyl group or an N-methylimidazolyl group, and M represents a hydrogen atom or an ion.)

第１工程は、式（ＩＩ）化合物を、ルイス酸およびモレキュラーシーブスの存在下、フェノール化合物と反応させ、立体異性体混合物である式（ＩＩＩ）化合物を得、得られた式（ＩＩＩ）化合物から結晶化によりβ体（式（ＩＶ）化合物）を分離し、該化合物をアルカリ性条件下、脱保護して式（Ｖ）化合物を得る工程である。 In the first step, the compound of formula (II) is reacted with a phenol compound in the presence of Lewis acid and molecular sieves to obtain a compound of formula (III) which is a stereoisomer mixture, and from the obtained compound of formula (III) In this step, the β-form (compound of formula (IV)) is separated by crystallization, and the compound is deprotected under alkaline conditions to obtain the compound of formula (V).

Ｒ３で表される水酸基の保護基としては、水酸基の保護基として常用されているものであればよく、たとえばエーテル系保護基、アシル系保護基、シリル系保護基、アセタール系保護基などを例示することができる。より具体的には、エーテル系保護基としては、メチルエーテル、第３級ブチルエーテル、ベンジルエーテル、メトキシベンジルエーテル、トリチルエーテルなどを、アシル系保護基としてはアセチル、ベンゾイル、ｐ−クロロベンゾイル、ピバロイルなどを、シリル系保護基としてはｔ−ブチルジメチルシリル、ｔ−ブチルジフェニルシリル、トリメチルシリル、トリエチルシリルなどを、アセタール系保護基としてはイソプロピリデン、エチリデン、メチリデン、ベンジリデン、テトラヒドロピラニル、メトキシメチルなどをそれぞれ使用することができる。これらの中でも、結晶性の良い、ベンゾイル基等のアシル系保護基が好適である。The hydroxyl-protecting group represented by R 3 may be any one commonly used as a hydroxyl-protecting group, such as an ether-based protecting group, an acyl-based protecting group, a silyl-based protecting group, an acetal-based protecting group, etc. It can be illustrated. More specifically, examples of the ether protecting group include methyl ether, tertiary butyl ether, benzyl ether, methoxybenzyl ether, and trityl ether. Examples of the acyl protecting group include acetyl, benzoyl, p-chlorobenzoyl, and pivaloyl. Silyl protecting groups such as t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triethylsilyl, etc., and acetal protecting groups such as isopropylidene, ethylidene, methylidene, benzylidene, tetrahydropyranyl, methoxymethyl, etc. Each can be used. Of these, acyl-based protecting groups such as benzoyl groups having good crystallinity are preferable.

Ｒ４は、アシル基であればよく、アセチル、ベンゾイル、ピバロイル基などを例示することができる。R 4 may be an acyl group, and examples thereof include acetyl, benzoyl, and pivaloyl groups.

このような原料化合物は、市販品を用いるか、公知の方法（Ｈｅｌｖｅｔｉｃａ Ｃｈｉｍｉｃａ Ａｃｔａ，４２，１１７１−１１７３（１９５９）など）により調製することができる。また、公知の方法で調製された式（ＩＩ）を精製することなく、反応に供することもできる。 Such a raw material compound may be a commercially available product or may be prepared by a known method (such as Helvetica Chimica Acta, 42, 1171-1173 (1959)). Moreover, it can also use for reaction, without refine | purifying Formula (II) prepared by the well-known method.

式（ＩＩ）化合物から式（ＩＩＩ）化合物への反応は、式（ＩＩ）化合物を、ジクロロメタン、１，２−ジクロロエタン、トルエン等の有機溶媒中、式（ＩＩ）化合物１モルに対して０．０１〜２モルのルイス酸、式（ＩＩ）化合物１ｇに対して２ｇ以上のモレキュラーシーブスの存在下、１〜１０モル、好ましくは１．２当量のフェノール化合物と０〜５０℃、好ましくは２０〜３０℃で１〜２４時間反応させることにより実施できる。 The reaction from the compound of the formula (II) to the compound of the formula (III) is performed by reacting the compound of the formula (II) in an organic solvent such as dichloromethane, 1,2-dichloroethane, toluene, etc. with respect to 1 mol of the compound of the formula (II). 01 to 2 mol of Lewis acid, 1 to 10 mol, preferably 1.2 equivalents of phenolic compound and 0 to 50 ° C., preferably 20 to 20 mol in the presence of 2 g or more of molecular sieves per 1 g of the compound of formula (II) It can implement by making it react at 30 degreeC for 1 to 24 hours.

当該反応に使用するルイス酸としては、トリフルオロメタンスルホン酸トリメチルシリル、三フッ化ホウ素エーテル錯体、四塩化すず、塩化亜鉛、ヨウ化亜鉛、無水塩化アルミニウム等が例示されるが、トリフルオロメタンスルホン酸トリメチルシリルが最も好適である。 Examples of the Lewis acid used in the reaction include trimethylsilyl trifluoromethanesulfonate, boron trifluoride ether complex, tin tetrachloride, zinc chloride, zinc iodide, anhydrous aluminum chloride, and the like. Trimethylsilyl trifluoromethanesulfonate is exemplified. Most preferred.

また、使用するモレキュラーシーブスは、市販のものを適宜用いることができる。中でもモレキュラーシーブ４Ａがとくに好適である。   Moreover, the molecular sieve to be used can use a commercially available thing suitably. Of these, the molecular sieve 4A is particularly suitable.

フェノール化合物としては、フェノール、チオフェノール、および、これらの２位、４位等をＣ１−４アルキル、Ｃ１−４アルコキシ、Ｃ１−４アルキルチオ、Ｃ１−４アルキルアミノ、Ｃ１−４ジアルキルアミノ、Ｃ１−４アルキルスルホニル、ハロゲノ、ニトロ、シアノ、アミノ、ヒドロキシル等の置換基で置換したものが例示される。Examples of the phenol compound include phenol, thiophenol, and C 1-4 alkyl, C 1-4 alkoxy, C 1-4 alkylthio, C 1-4 alkylamino, C 1-4 at the 2-position and 4-position thereof. Examples thereof include those substituted with a substituent such as dialkylamino, C 1-4 alkylsulfonyl, halogeno, nitro, cyano, amino, hydroxyl and the like.

式（ＩＩＩ）化合物の異性体混合物を結晶化により単一の異性体（ＩＶ）化合物とするには、得られた式（ＩＩＩ）化合物を、使用した式（ＩＩ）化合物に対してアセトン、２−ブタノン等のケトン系有機溶媒および／またはジクロロメタン、クロロホルム等のハロゲン系溶媒に溶解させ、これにメタノール、エタノール、イソプロパノールを貧溶媒として加え、晶析させればよい。 In order to crystallize a mixture of isomers of the compound of formula (III) into a single isomer (IV) compound by crystallization, the compound of formula (III) obtained is acetone, -It may be dissolved in a ketone-based organic solvent such as butanone and / or a halogen-based solvent such as dichloromethane or chloroform, and methanol, ethanol or isopropanol may be added as a poor solvent to cause crystallization.

式（ＩＶ）化合物の保護基を除去し、式（Ｖ）化合物とする反応では、使用した保護基に応じ、酸性加水分解、アルカリ性加水分解、フッ化テトラブチルアンモニウム処理、接触還元などの通常の処理方法から適宜選択して行えばよい。 In the reaction in which the protecting group of the compound of formula (IV) is removed to obtain the compound of formula (V), depending on the protecting group used, ordinary hydrolysis, acid hydrolysis, tetrabutylammonium fluoride treatment, catalytic reduction, etc. What is necessary is just to select suitably from a processing method.

第２工程は、式（Ｖ）化合物の５位水酸基を選択的に保護し、２，３位水酸基を５位保護基とは除去法の異なる保護基で保護後、５位保護基を選択的に除去することにより、式（ＶＩＩ）化合物を得、該化合物を塩基の存在下、リン酸化して式（ＶＩＩＩ）化合物とし、２，３位の保護基を除去して式（ＩＸ）化合物を得る工程である。 In the second step, the 5-position hydroxyl group of the compound of formula (V) is selectively protected, the 2-position hydroxyl group is protected with a protecting group having a different removal method from the 5-position protecting group, and then the 5-position protecting group is selectively treated. To obtain a compound of formula (VII), which is phosphorylated in the presence of a base to give a compound of formula (VIII), and the protecting groups at positions 2 and 3 are removed to give a compound of formula (IX). It is a process to obtain.

Ｒ５で表される５位水酸基の保護基は、１級水酸基の選択的な保護基として常用されているものであればよく、具体的には、ジメトキシトリチル、メトキシトリチル、トリチル、ｔｅｒｔ−ブチルジメチルシリル、ｔｅｒｔ−ブチルジフェニルシリル基などが例示される。The protecting group for the 5-position hydroxyl group represented by R 5 may be any one commonly used as a selective protecting group for the primary hydroxyl group. Specifically, dimethoxytrityl, methoxytrityl, trityl, tert-butyl Examples thereof include dimethylsilyl and tert-butyldiphenylsilyl groups.

また、Ｒ６で表される２，３位水酸基の保護基としては、水酸基の保護などで通常使用されるものであればよく、たとえばアシル系保護基、エーテル系保護基、シリル系保護基、アセタール系保護基などを例示することができる。Further, the protective group for the hydroxyl group at the 2- and 3-positions represented by R 6 may be any one that is usually used for protecting a hydroxyl group, such as an acyl-based protective group, an ether-based protective group, a silyl-based protective group, An acetal type protective group etc. can be illustrated.

より具体的には、エーテル系保護基としては、メチルエーテル、第３級ブチルエーテル、ベンジルエーテル、メトキシベンジルエーテル、トリチルエーテルなどを、アシル系保護基としてはアセチル、ベンゾイル、ｐ−クロロベンゾイル、ピバロイルなどを、シリル系保護基としてはｔｅｒｔ−ブチルジメチルシリル、ｔｅｒｔ−ブチルジフェニルシリル、トリメチルシリル、トリエチルシリルなどを、アセタール系保護基としてはイソプロピリデン、エチリデン、メチリデン、ベンジリデン、テトラヒドロピラニル、メトキシメチルなどをそれぞれ使用することができる。   More specifically, examples of the ether protecting group include methyl ether, tertiary butyl ether, benzyl ether, methoxybenzyl ether, and trityl ether. Examples of the acyl protecting group include acetyl, benzoyl, p-chlorobenzoyl, and pivaloyl. Silyl protecting groups such as tert-butyldimethylsilyl, tert-butyldiphenylsilyl, trimethylsilyl, triethylsilyl, etc., and acetal protecting groups such as isopropylidene, ethylidene, methylidene, benzylidene, tetrahydropyranyl, methoxymethyl and the like. Each can be used.

５位水酸基の保護基の除去は、使用した保護基に応じ、酸性加水分解、アルカリ性加水分解、フッ化テトラブチルアンモニウム処理、接触還元などの通常の処理方法から適宜選択して行えばよい。   The removal of the protecting group at the 5-position hydroxyl group may be appropriately selected from ordinary treatment methods such as acidic hydrolysis, alkaline hydrolysis, tetrabutylammonium fluoride treatment, and catalytic reduction depending on the protecting group used.

得られた式（ＶＩＩ）化合物は、精製することなく次の工程に用いることができる。また、式（ＶＩＩ）化合物を結晶として得たい場合は、Ｒ６で表される保護基としては結晶性の高いｐ−クロロベンゾイル基のようなアシル系保護基を選択し、式（ＶＩＩ）化合物の粗生成物をエタノールといった有機溶媒から結晶化させればよい。The obtained compound of formula (VII) can be used in the next step without purification. When the compound of formula (VII) is desired to be obtained as a crystal, an acyl protecting group such as p-chlorobenzoyl group having high crystallinity is selected as the protecting group represented by R 6 , and the compound of formula (VII) The crude product may be crystallized from an organic solvent such as ethanol.

式（ＩＸ）化合物は、式（ＶＩＩ）化合物の５位水酸基をリン酸化後、２，３位の保護基を除去することにより得られる。   The compound of formula (IX) can be obtained by phosphorylating the 5-position hydroxyl group of the formula (VII) compound and then removing the protective groups at the 2- and 3-positions.

式（ＶＩＩ）化合物を式（ＶＩＩＩ）化合物とするには、ピリジン、アセトニトリル、トルエン、リン酸トリアルキル等の有機溶媒中、式（ＶＩＩ）化合物を、式（ＶＩＩ）化合物１モルに対して１〜１０モル、好ましくは１．５モルの塩基の存在下、１〜５モル、好ましくは１．２当量のリン酸化剤と−３０〜３０℃で１〜２４時間反応させればよい。   In order to convert the compound of formula (VII) into the compound of formula (VIII), the compound of formula (VII) is used in an organic solvent such as pyridine, acetonitrile, toluene, trialkyl phosphate, etc. with respect to 1 mol of the compound of formula (VII). In the presence of 10 to 10 mol, preferably 1.5 mol of base, 1 to 5 mol, preferably 1.2 equivalents of phosphorylating agent may be reacted at -30 to 30 ° C for 1 to 24 hours.

用いる塩基としては、トリエチルアミン、トリブチルアミン等のトリアルキルアミン、ピリジン、ジメチルアミノピリジン、Ｎ−メチルイミダゾール等の複素環アミンを例示することができ、リン酸化剤として、オキシ塩化リン、テトラクロロピロリン酸等や各種アミダイト試薬を例示することができる。 Examples of the base to be used include trialkylamines such as triethylamine and tributylamine, and heterocyclic amines such as pyridine, dimethylaminopyridine and N-methylimidazole. Phosphorus oxychloride and tetrachloropyrophosphate as phosphorylating agents And various amidite reagents.

得られた式（ＶＩＩＩ）化合物を引き続き、脱保護することにより式（ＩＸ）化合物が得られる。 The resulting compound of formula (VIII) is subsequently deprotected to give compound of formula (IX).

保護基の除去は、使用した保護基に応じ、酸性加水分解、アルカリ性加水分解、フッ化テトラブチルアンモニウム処理、接触還元などの通常の処理方法から適宜選択して行えばよい。 The removal of the protecting group may be appropriately selected from ordinary treatment methods such as acidic hydrolysis, alkaline hydrolysis, tetrabutylammonium fluoride treatment and catalytic reduction according to the used protecting group.

例えば、２，３位の保護基としてベンゾイル基などのアシル系保護基を用いた場合、エタノール中、式（ＶＩＩＩ）化合物を、濃アンモニア水と０℃〜室温で１〜３日処理すればよい。この場合、反応の進行に伴い、生成した式（ＩＸ）化合物が析出するので、これを濾取、乾燥するのみで式（ＩＸ）化合物が得られる。 For example, when an acyl protecting group such as a benzoyl group is used as the protecting group at the 2- and 3-positions, the compound of formula (VIII) may be treated with concentrated aqueous ammonia at 0 ° C. to room temperature for 1 to 3 days. . In this case, as the reaction proceeds, the produced compound of formula (IX) precipitates, and the compound of formula (IX) can be obtained simply by filtering and drying.

第３工程は、式（ＩＸ）化合物のリン酸基を式（Ｘ）化合物に変換後、ピロリン酸塩と反応させることにより式（ＸＩ）化合物を得る工程である。 The third step is a step of obtaining the compound of formula (XI) by reacting with a pyrophosphate after converting the phosphate group of the compound of formula (IX) to the compound of formula (X).

式（ＩＸ）化合物のリン酸基を活性化して式（Ｘ）化合物とするには、Ｎ−メチルイミダゾリデート法（Ｒｕｓｓｉａｎ Ｊｏｕｎａｌ ｏｆ Ｂｉｏｏｒｇａｎｉｃ Ｃｈｅｍｉｓｔｒｙ， ２２， ５９９−６０４（１９９６））やイミダゾリデート法（Ｊｏｕｎａｌ ｏｆ Ａｍｅｒｉｃａｎ Ｃｈｅｍｉｃａｌ Ｓｏｃｉｅｔｙ， ８７， １７８５（１９６５））といった公知の方法を利用することができる。 In order to activate the phosphate group of the compound of formula (IX) to form the compound of formula (X), the N-methylimidazolidate method (Russian Journal of Bioorganic Chemistry, 22, 599-604 (1996)) or imidazolidate A known method such as the method (Jonal of American Chemical Society, 87, 1785 (1965)) can be used.

例えば、活性化の方法にＮ−メチルイミダゾリデート法を用いる場合、アセトニトリル等の有機溶媒中、式（ＩＸ）化合物１モルに対して１〜１０モルのジメチルアニリンおよび１〜１０モルのトリエチルアミン存在下、式（ＩＸ）化合物を１〜１０モルのトリフルオロ酢酸無水物と−２０〜３０℃で１０〜６０分間、１〜１０モルのＮ−メチルイミダゾールと１０〜６０分間反応させることによって活性化させることができる。 For example, when the N-methylimidazolidate method is used as the activation method, 1 to 10 mol of dimethylaniline and 1 to 10 mol of triethylamine are present in 1 mol of the compound of formula (IX) in an organic solvent such as acetonitrile. Below, the compound of formula (IX) is activated by reacting with 1 to 10 mol of trifluoroacetic anhydride at -20 to 30 ° C. for 10 to 60 min and with 1 to 10 mol of N-methylimidazole for 10 to 60 min. Can be made.

また、イミダゾリデート法による活性化は、ジメチルホルムアミド、ジメチルアセトアミド等の有機溶媒中、式（ＩＸ）化合物を、式（ＩＸ）化合物１モルに対して１〜５モル、好ましくは３モルのカルボニルジイミダゾールと、０〜３０℃で１〜２４時間反応させることで達成できる。得られた式（Ｘ）化合物は、単離することなく次の工程に利用することが可能である。 In addition, the activation by the imidazolidate method is carried out by using a compound of formula (IX) in an organic solvent such as dimethylformamide or dimethylacetamide in an amount of 1 to 5 mol, preferably 3 mol, of carbonyl with respect to 1 mol of the compound of formula (IX). This can be achieved by reacting with diimidazole at 0 to 30 ° C. for 1 to 24 hours. The obtained compound of the formula (X) can be used for the next step without isolation.

式（Ｘ）化合物は、式（Ｘ）化合物１モルに対して１〜１０モルのピロリン酸塩と反応させることにより、式（ＸＩ）化合物へと変換できる。たとえば、Ｎ−メチルイミダゾリデート法により活性化した場合には、２モル程度のピロリン酸塩と０℃〜室温で１０〜６０分間、イミダゾリデート化では５モル程度のピロリン酸塩と室温で１〜３日間反応させるのが好ましい。また、ピロリン酸はトリエチルアンモニウム、トリブチルアンモニウム等の３級アンモニウム、テトラエチルアンモニウム、テトラブチルアンモニウム、ベンジルトリエチルアンモニウム等の４級アンモニウム塩として使用することができる。 A compound of formula (X) can be converted to a compound of formula (XI) by reacting 1 to 10 mol of pyrophosphate with respect to 1 mol of compound of formula (X). For example, when activated by the N-methylimidazolidate method, about 2 moles of pyrophosphate and 0 ° C. to room temperature for 10 to 60 minutes, and imidazolidation at about 5 moles of pyrophosphate and room temperature. The reaction is preferably performed for 1 to 3 days. In addition, pyrophosphoric acid can be used as a tertiary ammonium such as triethylammonium and tributylammonium, and as a quaternary ammonium salt such as tetraethylammonium, tetrabutylammonium and benzyltriethylammonium.

次に、もう１つの原料化合物である式（ＸＩＩＩ）で表されるアデノシン５’−モノリン酸酸誘導体は、記式（ＸＩＩ）化合物をアルキル化あるいはアラルキル化することによって調製する。   Next, another raw material compound, an adenosine 5'-monophosphate derivative represented by the formula (XIII), is prepared by alkylating or aralkylating the compound of the formula (XII).

Figure 2010150791
（ＸＩＩ） （ＸＩＩＩ）
（式中、Ｒ１、Ｒ２は水素原子、アルキルまたはアラルキルのいずれかより選ばれる基、Ｍは水素原子またはイオンを示す。）
Figure 2010150791
 (XII) (XIII)
(In the formula, R 1 and R 2 are a hydrogen atom, a group selected from alkyl or aralkyl, and M represents a hydrogen atom or an ion.)

式（ＸＩＩ）化合物を式（ＸＩＩＩ）化合物とするには、式（ＸＩＩ）化合物の水酸基を適当な保護基を用いて保護した後、アルキル化あるいはアラルキル化し、引き続き保護基を除去すればよい。   In order to convert the compound of formula (XII) into the compound of formula (XIII), the hydroxyl group of the compound of formula (XII) is protected with an appropriate protecting group, then alkylated or aralkylated, and then the protecting group is removed.

保護基としては、水酸基の保護で通常使用されるものであればよく、たとえばアシル系保護基、エーテル系保護基、シリル系保護基、アセタール系保護基などを例示することができる。   The protecting group is not particularly limited as long as it is usually used for protecting a hydroxyl group, and examples thereof include an acyl protecting group, an ether protecting group, a silyl protecting group, and an acetal protecting group.

より具体的には、エーテル系保護基としては、メチルエーテル、第３級ブチルエーテル、ベンジルエーテル、メトキシベンジルエーテル、トリチルエーテルなどを、アシル系保護基としてはアセチル、ベンゾイル、ｐ−クロロベンゾイル、ピバロイルなどを、シリル系保護基としてはｔｅｒｔ−ブチルジメチルシリル、ｔｅｒｔ−ブチルジフェニルシリル、トリメチルシリル、トリエチルシリルなどを、アセタール系保護基としてはイソプロピリデン、エチリデン、メチリデン、ベンジリデン、テトラヒドロピラニル、メトキシメチルなどをそれぞれ使用することができる。   More specifically, examples of the ether protecting group include methyl ether, tertiary butyl ether, benzyl ether, methoxybenzyl ether, and trityl ether. Examples of the acyl protecting group include acetyl, benzoyl, p-chlorobenzoyl, and pivaloyl. Silyl protecting groups such as tert-butyldimethylsilyl, tert-butyldiphenylsilyl, trimethylsilyl, triethylsilyl, etc., and acetal protecting groups such as isopropylidene, ethylidene, methylidene, benzylidene, tetrahydropyranyl, methoxymethyl and the like. Each can be used.

保護基にて水酸基を保護した式（ＸＩＩ）化合物は、引き続き、アルキル化剤あるいはアラルキル化剤と反応させる。たとえば、上記反応液に対し、１０〜１００モルのヨウ化メチル、硫酸ジメチル、メタンスルホン酸メチル、ジアゾメタン、トリメチルシリルジアゾメタン等のメチル化剤を加え、０〜５０℃で１〜７日間反応させればよい。 The compound of formula (XII) in which the hydroxyl group is protected with a protecting group is subsequently reacted with an alkylating agent or an aralkylating agent. For example, 10 to 100 moles of methyl iodide such as methyl iodide, dimethyl sulfate, methyl methanesulfonate, diazomethane, trimethylsilyldiazomethane, etc. are added to the above reaction solution and reacted at 0-50 ° C. for 1-7 days. Good.

水酸基の保護基の除去は、使用した保護基に応じ、酸性加水分解、アルカリ性加水分解、フッ化テトラブチルアンモニウム処理、接触還元などの通常の処理方法から適宜選択して行えばよい。   The removal of the protective group for the hydroxyl group may be appropriately selected from ordinary treatment methods such as acidic hydrolysis, alkaline hydrolysis, tetrabutylammonium fluoride treatment, and catalytic reduction depending on the protective group used.

このようにして得られた式（ＸＩ）化合物および式（ＸＩＩＩ）化合物は、必要により遊離体から塩型（たとえば、ナトリウム塩などの金属塩、テトラブチルアンモニウム塩などの置換アンモニウム塩など）に変換して、原料化合物として使用すればよい。   The thus obtained formula (XI) compound and formula (XIII) compound are converted from a free form into a salt form (for example, metal salt such as sodium salt, substituted ammonium salt such as tetrabutylammonium salt, etc.) as necessary. Thus, it may be used as a raw material compound.

以下、本発明を実験例及び実施例をあげて具体的に説明するが、本発明はこれらによって何ら限定されるものではない。   Hereinafter, the present invention will be specifically described with reference to experimental examples and examples, but the present invention is not limited thereto.

＜アデノシンテトラホスフェート化合物の合成例＞
Ｐ１−［４−メトキシフェニルβ−Ｄ−リボフラノシド］５−］Ｐ４−（１−メチルアデノシン５’−）テトラリン酸（１）の合成<Synthesis example of adenosine tetraphosphate compound>
P 1 - [4-methoxyphenyl beta-D-ribofuranoside] 5-] P 4 - (1- methyl-adenosine 5') Synthesis of tetralin acid (1)

Figure 2010150791
Figure 2010150791

化合物５（４．０５ｍｍｏｌ）の水溶液（２５０ｍＬ）を、陽イオン交換樹脂（ＰＫ２１６（三菱化学社製）、プロトン型、３０ｍＬ）に通過させ、通過後の樹脂を脱イオン水で洗浄した。通過液、洗浄液に１Ｎ水酸化テトラｎ−ブチルアンモニウム（８．１０ｍＬ、８．１０ｍｍｏｌ）を加え中和後、濃縮した。残渣をアセトニトリル（２０ｍＬ×３）で共沸し、淡黄色アメ状の化合物５をテトラｎ−ブチルアンモニウム塩として得た。   An aqueous solution (250 mL) of compound 5 (4.05 mmol) was passed through a cation exchange resin (PK216 (manufactured by Mitsubishi Chemical Corporation), proton type, 30 mL), and the resin after passing was washed with deionized water. 1N Tetra-n-butylammonium hydroxide (8.10 mL, 8.10 mmol) was added to the passing liquid and the washing liquid, neutralized, and concentrated. The residue was azeotroped with acetonitrile (20 mL × 3) to obtain pale yellow candy-like compound 5 as a tetra n-butylammonium salt.

この残渣をジメチルホルムアミド（２０ｍＬ）に溶解し、ジイソプロピルカルボジイミド（９４０μＬ、６．０７ｍｍｏｌ）を加え、室温で３時間撹拌した。ここに、化合物１２のジメチルホルムアミド溶液、塩化マグネシウムのジメチルホルムアミド溶液を加え、室温で１８時間撹拌した。なお、化合物１２のジメチルホルムアミド溶液は、化合物１２（２．６３ｇ、７．２８ｍｍｏｌ）を脱イオン水（５０ｍＬ）に溶解し、氷冷下、１０％水酸化テトラｎ−ブチルアンモニウムでｐＨを８．０としたものを、４０℃で濃縮、残渣をジメチルホルムアミド２０ｍＬで３回共沸した後、ジメチルホルムアミド１０ｍＬに溶解して調製した。また、塩化マグネシウムのジメチルホルムアミド溶液は、塩化マグネシウム６水和物（１．４８ｇ、７．２８ｍｍｏｌ）をジメチルホルムアミド２０ｍＬで３回共沸した残渣をジメチルホルムアミド１０ｍＬに溶解して調製した。 This residue was dissolved in dimethylformamide (20 mL), diisopropylcarbodiimide (940 μL, 6.07 mmol) was added, and the mixture was stirred at room temperature for 3 hours. A dimethylformamide solution of compound 12 and a dimethylformamide solution of magnesium chloride were added thereto, and the mixture was stirred at room temperature for 18 hours. The dimethylformamide solution of Compound 12 was prepared by dissolving Compound 12 (2.63 g, 7.28 mmol) in deionized water (50 mL) and adjusting the pH to 8. with 10% tetra-n-butylammonium hydroxide under ice cooling. 0 was concentrated at 40 ° C., and the residue was azeotroped three times with 20 mL of dimethylformamide and then dissolved in 10 mL of dimethylformamide. A dimethylformamide solution of magnesium chloride was prepared by dissolving a residue obtained by azeotroping magnesium chloride hexahydrate (1.48 g, 7.28 mmol) three times with 20 mL of dimethylformamide in 10 mL of dimethylformamide.

反応液に冷脱イオン水（３０ｍＬ）を加え撹拌した後、脱イオン水で５００ｍＬとした。ＨＰＬＣによる定量の結果、化合物１の合成収率は７５．６％であった。 Cold deionized water (30 mL) was added to the reaction solution and stirred, and then made up to 500 mL with deionized water. As a result of quantification by HPLC, the synthesis yield of Compound 1 was 75.6%.

１Ｎ水酸化ナトリウム水溶液でｐＨ７．０とした後、ＤＥＡＥセファデックスカラムクロマトグラフィー（ＤＥＡＥセファデックス１５０ｍＬ、０．１０〜０．１５Ｍ塩化リチウムにより溶出）により精製を行った。目的物を含む画分を濃縮後、残渣をメタノール（４０ｍＬ）に溶解し、アセトン（１６０ｍＬ）を加え、生成した沈殿をろ取、乾燥した。得られたリチウム塩を脱イオン水に溶解し、陽イオン交換樹脂カラム（ＰＫ２１６（三菱化学社製）、ナトリウム型、２０ｍＬ）に通過させ、通過後の樹脂を脱イオン水で洗浄した。通過液、洗浄液を合わせ濃縮後、残渣を脱イオン水に溶解、予備凍結後、終夜、凍結乾燥した。白色粉末状の化合物１を得た。 After adjusting the pH to 7.0 with 1N aqueous sodium hydroxide solution, purification was performed by DEAE Sephadex column chromatography (DEAE Sephadex 150 mL, eluted with 0.10 to 0.15 M lithium chloride). After concentrating the fraction containing the desired product, the residue was dissolved in methanol (40 mL), acetone (160 mL) was added, and the resulting precipitate was collected by filtration and dried. The obtained lithium salt was dissolved in deionized water, passed through a cation exchange resin column (PK216 (manufactured by Mitsubishi Chemical), sodium type, 20 mL), and the resin after passing was washed with deionized water. The combined flow-through and washings were concentrated and the residue was dissolved in deionized water, pre-frozen and then lyophilized overnight. A white powdery compound 1 was obtained.

１Ｈ−ＮＭＲ（Ｄ２Ｏ）：δ８．６５（１Ｈ，ｓ，ａｒｏｍａｔｉｃ），８．４６（１Ｈ，ｓ，ａｒｏｍａｔｉｃ），６．９５（２Ｈ，ｄ，ａｒｏｍａｔｉｃ，Ｊ＝９．５），６．８７（２Ｈ，ｄ，ａｒｏｍａｔｉｃ，Ｊ＝９．５），６．１３（１Ｈ，ｄ，Ｈ−１’，Ｊ＝５．５），５．４５（１Ｈ，ｄ，Ｈ−１，Ｊ＝２．０），４．８４（１Ｈ，ｔ，Ｈ−２’ ，Ｊ＝５．０），４．６３（１Ｈ，ｄｄ，Ｈ−３’ ，Ｊ＝３．５，５．０），４．７４（１Ｈ，ｔ，Ｈ−３，Ｊ＝５．５），４．４１（１Ｈ，ｍ，Ｈ−４’），４．３２（１Ｈ，ｄｄ，Ｈ−２，Ｊ＝２．０，５．０），４．２６（１Ｈ，ｑ，Ｈ−４，Ｊ＝５．０），４．２９（１Ｈ，ｍ，Ｈ−５’），４．１９（１Ｈ，ｍ，Ｈ−５ａ），４．１１（１Ｈ，ｍ，Ｈ−５ｂ），３．７８（３Ｈ，ｓ，ｍｅｔｈｙｌ），３．８６（３Ｈ，ｓ，ｍｅｔｈｙｌ）
３１Ｐ−ＮＭＲ（Ｄ２Ｏ）：δ−１１．２０（ｍ），−２３．００（ｍ）
ＭＳ（ＥＳＩ）：ｍ／ｚ ８３８［Ｍ−Ｈ］−，８６０［Ｍ１Ｎａ−Ｈ］−，８８２［Ｍ２Ｎａ−Ｈ］−，９０５［Ｍ３Ｎａ−Ｈ］−，９２６［Ｍ４Ｎａ−Ｈ］−． 1 H-NMR (D 2 O): δ 8.65 (1H, s, aromatic), 8.46 (1H, s, aromatic), 6.95 (2H, d, aromatic, J = 9.5), 6 .87 (2H, d, aromatic, J = 9.5), 6.13 (1H, d, H-1 ′, J = 5.5), 5.45 (1H, d, H−1, J = 2.0), 4.84 (1H, t, H-2 ′, J = 5.0), 4.63 (1H, dd, H-3 ′, J = 3.5, 5.0), 4 .74 (1H, t, H-3, J = 5.5), 4.41 (1H, m, H-4 ′), 4.32 (1H, dd, H-2, J = 2.0, 5.0), 4.26 (1H, q, H-4, J = 5.0), 4.29 (1H, m, H-5 ′), 4.19 (1H, m, H-5a) , 4.11 (1H, m, H-5b), 3.78 (3 , S, methyl), 3.86 (3H, s, methyl)
31 P-NMR (D 2 O): δ-11.20 (m), −23.00 (m)
MS (ESI): m / z 838 [M-H] − , 860 [M1Na—H] − , 882 [M2Na—H] − , 905 [M3Na—H] − , 926 [M4Na—H] − .

実験例１：Ｐ１−［４−メトキシフェニルβ−Ｄ−リボフラノシド］５−］Ｐ４−（１−メチルアデノシン５’−）テトラリン酸（１）合成における金属塩の効果の検討Experimental Example 1: P 1 - [4-methoxyphenyl beta-D-ribofuranoside] 5-] P 4 - (1- methyl-adenosine 5') tetraphosphate (1) Examination of effect of the metal salt in the synthesis

Figure 2010150791
Figure 2010150791

化合物５（１．００ｍｍｏｌ）にトリエチルアミン（４１８μＬ、３．００ｍｍｏｌ）を加え、ジメチルホルムアミド（１０ｍＬ）で３回共沸した。残渣をジメチルホルムアミド（５ｍＬ）に溶解し、ジイソプロピルカルボジイミド（２３２μＬ、１．５０ｍｍｏｌ）を加え、室温で３時間撹拌した。ここに、化合物１２のジメチルホルムアミド溶液、塩化マグネシウム、または塩化亜鉛のジメチルホルムアミド溶液を加え、室温で２４時間撹拌した。反応液を脱イオン水で２５０ｍＬとし、５μＬをＨＰＬＣ（２６０ｎｍ）で分析し、化合物１の合成収率を算出した。なお、化合物１２のジメチルホルムアミド溶液は、化合物１２（５９２ｍｇ、１．６４ｍｍｏｌ）を脱イオン水（２０ｍＬ）に溶解し、氷冷下、１０％水酸化テトラブチルアンモニウムでｐＨを８．０としたものを、４０℃で濃縮、残渣をジメチルホルムアミド１０ｍＬで３回共沸した後、ジメチルホルムアミド２．５ｍＬに溶解して調製した。また、塩化亜鉛のジメチルホルムアミド溶液は、塩化マグネシウム６水和物（３０５ｍｇ、１．５０ｍｍｏｌ）、または塩化亜鉛（２０４ｍｇ、１．５０ｍｍｏｌ）をジメチルホルムアミド１０ｍＬで３回共沸した残渣をジメチルホルムアミド２．５ｍＬに溶解して調製した。その結果を表１に示す。   Triethylamine (418 μL, 3.00 mmol) was added to compound 5 (1.00 mmol), and azeotroped three times with dimethylformamide (10 mL). The residue was dissolved in dimethylformamide (5 mL), diisopropylcarbodiimide (232 μL, 1.50 mmol) was added, and the mixture was stirred at room temperature for 3 hr. To this, a dimethylformamide solution of compound 12, magnesium chloride, or a dimethylformamide solution of zinc chloride was added and stirred at room temperature for 24 hours. The reaction solution was adjusted to 250 mL with deionized water, 5 μL was analyzed by HPLC (260 nm), and the synthesis yield of Compound 1 was calculated. The dimethylformamide solution of Compound 12 was prepared by dissolving Compound 12 (592 mg, 1.64 mmol) in deionized water (20 mL) and adjusting the pH to 8.0 with 10% tetrabutylammonium hydroxide under ice cooling. Was concentrated at 40 ° C., the residue was azeotroped three times with 10 mL of dimethylformamide, and then dissolved in 2.5 mL of dimethylformamide. In addition, a solution of zinc chloride in dimethylformamide was obtained by subjecting magnesium chloride hexahydrate (305 mg, 1.50 mmol) or zinc chloride (204 mg, 1.50 mmol) to dimethylformamide 2. Prepared by dissolving in 5 mL. The results are shown in Table 1.

Figure 2010150791
Figure 2010150791

表１の結果から、金属塩が存在しないときの化合物１の合成収率は４．５９％と極めて低いのに対し、塩化マグネシウムを共存させると合成収率は５３．０％と大きく改善した。一方、塩化亜鉛を用いた場合は合成収率３０．８％と塩化マグネシウムを用いた結果と比較して半分弱で大きく減弱することがわかった。   From the results shown in Table 1, the synthesis yield of Compound 1 when no metal salt is present is extremely low, 4.59%, whereas the synthesis yield is greatly improved to 53.0% when magnesium chloride is present. On the other hand, it was found that when zinc chloride was used, the synthesis yield was 30.8%, which was a little less than half compared with the result using magnesium chloride.

＜原料化合物の合成例１＞
１）２，３、５−トリ−Ｏ−ベンゾイル−１−Ｏ−ｐ−メトキシフェニル−β−Ｄ−リボ−ペントフラノース（９）の合成（方法１：化合物８を出発原料とする方法）<Synthesis Example 1 of Raw Material Compound>
1) Synthesis of 2,3,5-tri-O-benzoyl-1-Op-methoxyphenyl-β-D-ribo-pentofuranose (9) (Method 1: Method using Compound 8 as a starting material)

Figure 2010150791
（式中、Ｂｚはベンゾイル、Ａｃはアセチル、Ｍｅはメチルを示す）
Figure 2010150791
 (In the formula, Bz represents benzoyl, Ac represents acetyl, and Me represents methyl)

化合物（８）（３０．０ｇ、５９．５ｍｍｏｌ）、４−メトキシフェノール（８．８５ｇ、７１．３ｍｍｏｌ）をトルエン（３００ｍＬ）に溶解し、モレキュラーシーブス４Ａ（Ｗａｋｏ、６０ｇ）を加え、室温で１時間撹拌した後、トリフルオロメタンスルホン酸トリメチルシリル（１．２ｍＬ、６．６ｍｍｏｌ）を加え、室温で２４時間撹拌した。反応液にトリエチルアミン（６ｍＬ）を加えてろ過した後、ろ液を飽和重曹水（３００ｍＬ）で洗浄し、無水硫酸マグネシウム上で乾燥、濃縮した。結晶状の残渣を２−ブタノン（６０ｍＬ）に加熱溶解後、エタノール（３００ｍＬ）を添加し、析出した結晶をろ取した。得られた粗結晶（２７．２ｇ）を２−ブタノン（６０ｍＬ）に加熱溶解し、エタノール（３００ｍＬ）を加え再結晶を行い、白色粉末状の化合物９（収量２５．５ｇ、４４．８ｍｍｏｌ、単離収率７５．３％）を得た。   Compound (8) (30.0 g, 59.5 mmol), 4-methoxyphenol (8.85 g, 71.3 mmol) was dissolved in toluene (300 mL), molecular sieves 4A (Wako, 60 g) was added, and 1 at room temperature was added. After stirring for hours, trimethylsilyl trifluoromethanesulfonate (1.2 mL, 6.6 mmol) was added and stirred at room temperature for 24 hours. After adding triethylamine (6 mL) to the reaction solution and filtering, the filtrate was washed with saturated aqueous sodium hydrogen carbonate (300 mL), dried over anhydrous magnesium sulfate and concentrated. The crystalline residue was dissolved in 2-butanone (60 mL) with heating, ethanol (300 mL) was added, and the precipitated crystals were collected by filtration. The obtained crude crystals (27.2 g) were dissolved in 2-butanone (60 mL) with heating, ethanol (300 mL) was added and recrystallized to obtain white powdered compound 9 (yield 25.5 g, 44.8 mmol, single A separation yield of 75.3% was obtained.

１Ｈ−ＮＭＲ（ＣＤＣｌ３）：δ８．０４−７．３２（１５Ｈ，ｍ，ａｒｏｍａｔｉｃ），６．９８（２Ｈ，ｄ，ａｒｏｍａｔｉｃ，Ｊ＝９．０），６．７９（２Ｈ，ｄ，ａｒｏｍａｔｉｃ，Ｊ＝９．０），６．０２（１Ｈ，ｄｄ，Ｈ−３，Ｊ＝５．０，７．０）５．９０（１Ｈ，ｄ，Ｈ−２，Ｊ＝５．０），５．８０（１Ｈ，ｓ，Ｈ−１）４．８２（１Ｈ，ｍ，Ｈ−４），４．７３（１Ｈ，ｄｄ，Ｈ−５ａ，Ｊ＝４．５，１２．０），４．５５（１Ｈ，ｄｄ，Ｈ−５ｂ，Ｊ＝４．５，１２．０），３．７６（３Ｈ，ｓ，ｍｅｔｈｙｌ）． 1 H-NMR (CDCl 3 ): δ 8.04-7.32 (15H, m, aromatic), 6.98 (2H, d, aromatic, J = 9.0), 6.79 (2H, d, aromatic) , J = 9.0), 6.02 (1H, dd, H-3, J = 5.0, 7.0) 5.90 (1H, d, H-2, J = 5.0), 5 .80 (1H, s, H-1) 4.82 (1H, m, H-4), 4.73 (1H, dd, H-5a, J = 4.5, 12.0), 4.55 (1H, dd, H-5b, J = 4.5, 12.0), 3.76 (3H, s, methyl).

２）２，３、５−トリ−Ｏ−ベンゾイル−１−Ｏ−ｐ−メトキシフェニル−β−Ｄ−リボ−ペントフラノース（９）の合成（方法２：Ｄ−リボースを出発原料とする方法） 2) Synthesis of 2,3,5-tri-O-benzoyl-1-Op-methoxyphenyl-β-D-ribo-pentofuranose (9) (Method 2: Method using D-ribose as a starting material)

Figure 2010150791
（式中、Ｂｚはベンゾイル、Ａｃはアセチル、Ｍｅはメチルを示す）
Figure 2010150791
 (In the formula, Bz represents benzoyl, Ac represents acetyl, and Me represents methyl)

Ｄ−リボース（７．５０ｇ、５０．０ｍｍｏｌ）をメタノール（３５ｍＬ）に懸濁後、濃硫酸（１２５μＬ）を滴下し、室温で２４時間撹拌した。反応液にピリジン（３６０μＬ）を加え濃縮後、残渣をジオキサン（２０ｍＬ）で２回共沸し、粗メチルリボシドを得た。   D-ribose (7.50 g, 50.0 mmol) was suspended in methanol (35 mL), concentrated sulfuric acid (125 μL) was added dropwise, and the mixture was stirred at room temperature for 24 hours. After adding pyridine (360 μL) to the reaction solution and concentrating, the residue was azeotroped twice with dioxane (20 mL) to obtain crude methylriboside.

粗メチルリボシドをピリジン（１５ｍＬ）、トルエン（２５ｍＬ）に溶解後、氷冷下、塩化ベンゾイル（２０．０ｍＬ、１７２ｍｍｏｌ）を滴下し、５０℃で１８時間加熱撹拌した。反応液に脱イオン水（１０ｍＬ）を加え５０℃で１時間撹拌後、濃縮した。残渣に酢酸エチル（３００ｍＬ）を加え、脱イオン水（３００ｍＬ）、次いで飽和重曹水（３００ｍＬ）で洗浄し、無水硫酸マグネシウム上で乾燥、濃縮した。残渣をジオキサン（２０ｍＬ）で２回共沸し、粗ベンゾイルエステルとした。   The crude methylriboside was dissolved in pyridine (15 mL) and toluene (25 mL), and then benzoyl chloride (20.0 mL, 172 mmol) was added dropwise under ice cooling, followed by heating and stirring at 50 ° C. for 18 hours. Deionized water (10 mL) was added to the reaction mixture, and the mixture was stirred at 50 ° C. for 1 hr and concentrated. Ethyl acetate (300 mL) was added to the residue, washed with deionized water (300 mL), then saturated aqueous sodium hydrogen carbonate (300 mL), dried over anhydrous magnesium sulfate, and concentrated. The residue was azeotroped twice with dioxane (20 mL) to give the crude benzoyl ester.

粗ベンゾイルエステルをトルエン（２５ｍＬ）に溶解し、無水酢酸（５．７０ｍＬ、６０．３ｍｍｏｌ）を加えた。濃硫酸（１．５ｍＬ）を滴下後、室温で３０分間撹拌した。酢酸ナトリウム（２．５ｇ）、脱イオン水（１０ｍＬ）を加え、室温で３０分間撹拌後、酢酸エチル（３００ｍＬ）を加え、脱イオン水（３００ｍＬ）、ついで飽和重曹水（３００ｍＬ）で洗浄し、無水硫酸マグネシウム上で乾燥、濃縮した。残渣をジオキサン（２０ｍＬ）で２回共沸し、粗化合物８（粗収量２５．８ｇ、精製することなく次の反応に使用）とした。   The crude benzoyl ester was dissolved in toluene (25 mL) and acetic anhydride (5.70 mL, 60.3 mmol) was added. Concentrated sulfuric acid (1.5 mL) was added dropwise, followed by stirring at room temperature for 30 minutes. Sodium acetate (2.5 g) and deionized water (10 mL) were added, and after stirring at room temperature for 30 minutes, ethyl acetate (300 mL) was added, washed with deionized water (300 mL), and then saturated aqueous sodium hydrogen carbonate (300 mL). Dried over anhydrous magnesium sulfate and concentrated. The residue was azeotroped twice with dioxane (20 mL) to give crude compound 8 (crude yield 25.8 g, used for next reaction without purification).

粗化合物８（２５．８ｇ）、４−メトキシフェノール（７．４４ｇ、５９．９ｍｍｏｌ）をトルエン（２５０ｍＬ）に溶解し、モレキュラーシーブス４Ａ（５０ｇ）を加え、室温で１時間撹拌した後、トリフルオロメタンスルホン酸トリメチルシリル（１．０ｍＬ）を加え、室温で２６時間撹拌した。反応液にトリエチルアミン（６ｍＬ）を加えてろ過した後、ろ液を飽和重曹水（３００ｍＬ）で洗浄し、無水硫酸マグネシウム上で乾燥、濃縮した。アメ状の残渣をエタノール（２５０ｍＬ）に溶解、撹拌し、析出した結晶をろ取した。結晶をエタノール（５０ｍＬ）で洗浄後、真空乾燥し、褐色粉末状の化合物９（収量１８．７ｇ、３２．９ｍｍｏｌ、Ｄ−リボースからの単離収率６５．８％）を得た。   Crude compound 8 (25.8 g) and 4-methoxyphenol (7.44 g, 59.9 mmol) were dissolved in toluene (250 mL), molecular sieve 4A (50 g) was added, and the mixture was stirred at room temperature for 1 hour, and then trifluoromethane. Trimethylsilyl sulfonate (1.0 mL) was added, and the mixture was stirred at room temperature for 26 hours. After adding triethylamine (6 mL) to the reaction solution and filtering, the filtrate was washed with saturated aqueous sodium hydrogen carbonate (300 mL), dried over anhydrous magnesium sulfate and concentrated. The candy-like residue was dissolved in ethanol (250 mL) and stirred, and the precipitated crystals were collected by filtration. The crystals were washed with ethanol (50 mL) and then vacuum-dried to obtain brown powdery compound 9 (yield 18.7 g, 32.9 mmol, isolated yield from D-ribose 65.8%).

得られた結晶（１８．７ｇ）を２−ブタノン（４０ｍＬ）に加熱溶解し、エタノール（２００ｍＬ）を加え再結晶（収量１７．０ｇ、２９．９ｍｍｏｌ、再結晶化回収率９０．９％、Ｄ−リボースからの単離収率５９．８％）。 The obtained crystals (18.7 g) were dissolved by heating in 2-butanone (40 mL), ethanol (200 mL) was added, and recrystallization (yield 17.0 g, 29.9 mmol, recrystallization recovery rate 90.9%, D -Isolated yield from ribose 59.8%).

３）１−Ｏ−ｐ−メトキシフェニル−β−Ｄ−リボ−ペントフラノース（３）の合成 3) Synthesis of 1-Op-methoxyphenyl-β-D-ribo-pentofuranose (3)

Figure 2010150791
（式中、Ｂｚはベンゾイル、Ｍｅはメチルを示す）
Figure 2010150791
 (In the formula, Bz represents benzoyl and Me represents methyl)

化合物９（２５．０ｇ、４４．０ｍｍｏｌ）をメタノール（２５０ｍＬ）に懸濁し、ナトリウムメトキシド（１．１９ｇ、２２．０ｍｍｏｌ）を加え、 室温で２２時間撹拌した。反応液に陽イオン交換樹脂（Ｄｏｗｅｘ ５０ＷＸ８−１００、プロトン型）を加えて中和後、ろ過により樹脂を除去し、ろ液を濃縮した。結晶状の残渣をエタノール（１０ｍＬ）に加熱溶解し、イソプロピルエーテル（２００ｍＬ）を加え、析出した結晶をろ取した。結晶をイソプロピルエーテル（５０ｍＬ）で洗浄後、真空乾燥し、白色粉末状の化合物３（１０．３ｇ、４０．２ｍｍｏｌ、収率９１．４％）を得た。   Compound 9 (25.0 g, 44.0 mmol) was suspended in methanol (250 mL), sodium methoxide (1.19 g, 22.0 mmol) was added, and the mixture was stirred at room temperature for 22 hours. A cation exchange resin (Dowex 50WX8-100, proton type) was added to the reaction solution for neutralization, the resin was removed by filtration, and the filtrate was concentrated. The crystalline residue was dissolved in ethanol (10 mL) with heating, isopropyl ether (200 mL) was added, and the precipitated crystals were collected by filtration. The crystals were washed with isopropyl ether (50 mL) and then vacuum dried to obtain white powdered compound 3 (10.3 g, 40.2 mmol, yield 91.4%).

１Ｈ−ＮＭＲ（ＤＭＳＯ−ｄ６）：δ６．９１（２Ｈ，ｄ，ａｒｏｍａｔｉｃ，Ｊ＝９．５），６．８４（２Ｈ，ｄ，ａｒｏｍａｔｉｃ，Ｊ＝１０．０），５．３１（１Ｈ，ｄ，Ｈ−１，Ｊ＝１．０），５．２５（１Ｈ，ｄ，ＯＨ，Ｊ＝５．０），４．９６（１Ｈ，ｄ，ＯＨ，Ｊ＝６．０），４．６６（１Ｈ，ｔ，５−ＯＨ，Ｊ＝５．５），３．９７（２Ｈ，ｍ，Ｈ−２＆３）３．８６（１Ｈ，ｍ，Ｈ−４），４．８２（１Ｈ，ｍ，Ｈ−４），３．５３（１Ｈ，ｄｄｄ，Ｈ−５ａ，Ｊ＝４．０，５．５，１１．５），３．３７（１Ｈ，ｄｔ，Ｈ−５ｂ，Ｊ＝５．５，１１．５），３．６９（３Ｈ，ｓ，ｍｅｔｈｙｌ）． 1 H-NMR (DMSO-d 6 ): δ 6.91 (2H, d, aromatic, J = 9.5), 6.84 (2H, d, aromatic, J = 10.0), 5.31 (1H , D, H-1, J = 1.0), 5.25 (1H, d, OH, J = 5.0), 4.96 (1H, d, OH, J = 6.0), 4. 66 (1H, t, 5-OH, J = 5.5), 3.97 (2H, m, H-2 & 3) 3.86 (1H, m, H-4), 4.82 (1H, m, H-4), 3.53 (1H, ddd, H-5a, J = 4.0, 5.5, 11.5), 3.37 (1H, dt, H-5b, J = 5.5) 11.5), 3.69 (3H, s, methyl).

４）１−Ｏ−ｐ−メトキシフェニル−β−Ｄ−リボ−ペントフラノース５−モノリン酸（４）の合成（方法１：２，３位の保護基としてベンゾイル基を使用する方法） 4) Synthesis of 1-Op-methoxyphenyl-β-D-ribo-pentofuranose 5-monophosphate (4) (Method 1: Method using benzoyl group as protecting group at positions 2 and 3)

Figure 2010150791
（式中、Ｂｚはベンゾイル、Ｍｅはメチルを示す）
Figure 2010150791
 (In the formula, Bz represents benzoyl and Me represents methyl)

化合物３（１０．４ｇ、４０．６ｍｍｏｌ）をジメチルホルムアミド（１００ｍＬ）に溶解し、イミダゾール（６．０８ｇ、８９．３ｍｍｏｌ）、ｔｅｒｔ−ブチルクロロジメチルシラン（６．７３ｇ、４４．５ｍｍｏｌ）を加え、室温で２０時間撹拌した。反応液にメタノール（１０ｍＬ）を加えて濃縮した後、残渣を酢酸エチル（２００ｍＬ）に溶解し、脱イオン水（２００ｍＬ）で洗浄した。有機層を無水硫酸マグネシウム上で乾燥、濃縮後、残渣をピリジンで共沸（２０ｍＬ×２）し、粗シリルエーテルを得た。   Compound 3 (10.4 g, 40.6 mmol) was dissolved in dimethylformamide (100 mL), imidazole (6.08 g, 89.3 mmol), tert-butylchlorodimethylsilane (6.73 g, 44.5 mmol) was added, Stir at room temperature for 20 hours. After adding methanol (10 mL) to the reaction solution and concentrating, the residue was dissolved in ethyl acetate (200 mL) and washed with deionized water (200 mL). The organic layer was dried over anhydrous magnesium sulfate and concentrated, and then the residue was azeotroped with pyridine (20 mL × 2) to obtain a crude silyl ether.

粗シリルエーテルをピリジン（５０ｍＬ）に溶解し、氷冷下、塩化ベンゾイル（１３．０ｍＬ、１１２ｍｍｏｌ）、ジメチルアミノピリジン（２４８ｍｇ、２．０３ｍｍｏｌ）を加え、室温で２４時間撹拌した。反応液に脱イオン水（２０ｍＬ）を加え、室温で１時間撹拌した後、濃縮し、残渣を酢酸エチル（３００ｍＬ）に溶解した。有機層を水（３００ｍＬ）、飽和重曹水（２００ｍＬ×２）で洗浄後、有機層を無水硫酸マグネシウム上で乾燥、濃縮した。残渣をトルエンで共沸（５０ｍＬ×３）し、粗ベンゾイルエステルを得た。   The crude silyl ether was dissolved in pyridine (50 mL), benzoyl chloride (13.0 mL, 112 mmol) and dimethylaminopyridine (248 mg, 2.03 mmol) were added under ice cooling, and the mixture was stirred at room temperature for 24 hours. Deionized water (20 mL) was added to the reaction solution, and the mixture was stirred at room temperature for 1 hour and then concentrated. The residue was dissolved in ethyl acetate (300 mL). The organic layer was washed with water (300 mL) and saturated aqueous sodium bicarbonate (200 mL × 2), and then the organic layer was dried over anhydrous magnesium sulfate and concentrated. The residue was azeotroped with toluene (50 mL × 3) to obtain a crude benzoyl ester.

粗ベンゾイルエステルをメタノール（１６０ｍＬ）に溶解し、酸性フッ化アンモニウム（６．４７ｇ、１１３ｍｍｏｌ）を加え、５０℃で２４時間撹拌した。反応液を濃縮後、残渣を酢酸エチル（３００ｍＬ）に溶解し、飽和重曹水（３００ｍＬ）で洗浄後、有機層を無水硫酸マグネシウム上で乾燥、濃縮した。淡黄色アメ状の粗化合物１０（精製せず、次の反応に使用）を得た。 The crude benzoyl ester was dissolved in methanol (160 mL), acid ammonium fluoride (6.47 g, 113 mmol) was added, and the mixture was stirred at 50 ° C. for 24 hours. The reaction mixture was concentrated, the residue was dissolved in ethyl acetate (300 mL), washed with saturated aqueous sodium hydrogen carbonate (300 mL), and the organic layer was dried over anhydrous magnesium sulfate and concentrated. A pale yellow crude crude compound 10 (not purified, used in the next reaction) was obtained.

粗化合物１０をジオキサン（５０ｍＬ×２）で共沸後、リン酸トリエチル（７７ｍＬ）に溶解した。アルゴン雰囲気下、−１０℃でトリブチルアミン（１３．１ｍＬ、５５．０ｍｍｏｌ）、オキシ塩化リン（４．０６ｍＬ、４３．６ｍｍｏｌ）を加え、同温度で３時間撹拌した。冷５０％アセトニトリル（１８０ｍＬ）、トリエチルアミン（３６．５ｍＬ、２６２ｍｍｏｌ）を加え、−１０℃で３０分間、室温で２時間撹拌した後、クロロホルム（３００ｍＬ）、脱イオン水（３００ｍＬ）を加え撹拌した。水層より、クロロホルム（３００ｍＬ×１０）で抽出し、有機層を合わせて濃縮した。残渣をエタノール（３６０ｍＬ）に溶解し、２８％アンモニア水（１８０ｍＬ）を加え、室温で２日間撹拌した。析出した沈殿をろ取し、エタノールで洗浄後、乾燥し、白色粉末状の化合物４（１１．６ｇ、ジアンモニウム塩として３１．３ｍｍｏｌ、化合物３から収率７７．１％）を得た。   Crude compound 10 was azeotroped with dioxane (50 mL × 2) and then dissolved in triethyl phosphate (77 mL). Under an argon atmosphere, tributylamine (13.1 mL, 55.0 mmol) and phosphorus oxychloride (4.06 mL, 43.6 mmol) were added at −10 ° C., and the mixture was stirred at the same temperature for 3 hours. Cold 50% acetonitrile (180 mL) and triethylamine (36.5 mL, 262 mmol) were added, and the mixture was stirred at −10 ° C. for 30 minutes and at room temperature for 2 hours, and then chloroform (300 mL) and deionized water (300 mL) were added and stirred. The aqueous layer was extracted with chloroform (300 mL × 10), and the organic layers were combined and concentrated. The residue was dissolved in ethanol (360 mL), 28% aqueous ammonia (180 mL) was added, and the mixture was stirred at room temperature for 2 days. The deposited precipitate was collected by filtration, washed with ethanol, and dried to obtain white powdered compound 4 (11.6 g, 31.3 mmol as a diammonium salt, yield 77.1% from compound 3).

１Ｈ−ＮＭＲ（Ｄ２Ｏ）：δ７．１０（２Ｈ，ｄ，ａｒｏｍａｔｉｃ，Ｊ＝９．０），７．００（２Ｈ，ｄ，ａｒｏｍａｔｉｃ，Ｊ＝９．０），５．５７（１Ｈ，ｄ，Ｈ−１，Ｊ＝２．０），４．４４（１Ｈ，ｔ，Ｈ−３，Ｊ＝６．０），４．３６（１Ｈ，ｄｄ，Ｈ−２，Ｊ＝２．０，４．５），４．２２（１Ｈ，ｑ，Ｈ−４，Ｊ＝５．０），３．９５（１Ｈ，ｄｔ，Ｈ−５ａ，Ｊ＝５．０，１１．０），３．８８（１Ｈ，ｄｔ，Ｈ−５ｂ，Ｊ＝５．５，１１．０），３．８２（３Ｈ，ｓ，ｍｅｔｈｙｌ）；３１Ｐ−ＮＭＲ（Ｄ２Ｏ）：δ３．６６（ｓ）． 1 H-NMR (D 2 O): δ 7.10 (2H, d, aromatic, J = 9.0), 7.00 (2H, d, aromatic, J = 9.0), 5.57 (1H, d, H-1, J = 2.0), 4.44 (1H, t, H-3, J = 6.0), 4.36 (1H, dd, H-2, J = 2.0, 4.5), 4.22 (1H, q, H-4, J = 5.0), 3.95 (1H, dt, H-5a, J = 5.0, 11.0), 3.88 (1H, dt, H-5b, J = 5.5, 11.0), 3.82 (3H, s, methyl); 31 P-NMR (D 2 O): δ 3.66 (s).

５）１−Ｏ−ｐ−メトキシフェニル−β−Ｄ−リボ−ペントフラノース５−モノリン酸（４）の合成（方法２：２，３位の保護基としてｐ−クロロベンゾイル基を使用する方法） 5) Synthesis of 1-Op-methoxyphenyl-β-D-ribo-pentofuranose 5-monophosphate (4) (Method 2: Method using p-chlorobenzoyl group as the protecting group at the 2- and 3-positions)

Figure 2010150791
（式中、ｐ-Ｃｌ-Ｂｚはｐ−クロロベンゾイル、Ｍｅはメチルを示す）
Figure 2010150791
 (In the formula, p-Cl-Bz represents p-chlorobenzoyl and Me represents methyl)

化合物３（９．００ｇ、３５．１ｍｍｏｌ）をピリジン（５７ｍＬ）に溶解し、氷冷下、ジメチルアミノピリジン（８６ｍｇ）、ｔｅｒｔ−ブチルクロロジメチルシラン（６．３６ｇ、４２．２ｍｍｏｌ）を加え、同温度で１０分間、室温で２０時間撹拌した。氷冷下、塩化ｐ−クロロベンゾイル（１０．８ｍＬ、８４．２ｍｍｏｌ）を滴下後、４０℃で２４時間撹拌した。反応液に脱イオン水（１０ｍＬ）を加え、４０℃で１時間撹拌後、濃縮し、残渣を酢酸エチル（２００ｍＬ）に溶解した。有機層を脱イオン水（２００ｍＬ）、飽和炭酸水素ナトリウム水溶液（２００ｍＬ×２）で洗浄し、有機層を無水硫酸マグネシウム上で乾燥後、濃縮した。残渣をトルエンで共沸（２０ｍＬ×３）し、粗ｐ−クロロベンゾイルエステルを得た。粗ｐ−クロロベンゾイルエステルをメタノール（３０ｍＬ）、アセトニトリル（３０ｍＬ）に溶解し、ｐ−トルエンスルホン酸（２．０ｇ）を加え、室温で５時間撹拌した。反応液にピリジン（１ｍＬ）を加え中和後、濃縮し、残渣を酢酸エチル（２００ｍＬ）に溶解した。有機層を飽和炭酸水素ナトリウム水溶液（２００ｍＬ）で洗浄し、有機層を無水硫酸マグネシウム上で乾燥後、濃縮した。残渣をジオキサンで共沸（２０ｍＬ×２）し、粗化合物１０’を得た。   Compound 3 (9.00 g, 35.1 mmol) was dissolved in pyridine (57 mL), and dimethylaminopyridine (86 mg) and tert-butylchlorodimethylsilane (6.36 g, 42.2 mmol) were added under ice cooling. Stir at temperature for 10 minutes and at room temperature for 20 hours. Under ice cooling, p-chlorobenzoyl chloride (10.8 mL, 84.2 mmol) was added dropwise, followed by stirring at 40 ° C. for 24 hours. Deionized water (10 mL) was added to the reaction mixture, and the mixture was stirred at 40 ° C. for 1 hr, concentrated, and the residue was dissolved in ethyl acetate (200 mL). The organic layer was washed with deionized water (200 mL) and saturated aqueous sodium hydrogen carbonate solution (200 mL × 2), and the organic layer was dried over anhydrous magnesium sulfate and concentrated. The residue was azeotroped with toluene (20 mL × 3) to obtain crude p-chlorobenzoyl ester. The crude p-chlorobenzoyl ester was dissolved in methanol (30 mL) and acetonitrile (30 mL), p-toluenesulfonic acid (2.0 g) was added, and the mixture was stirred at room temperature for 5 hours. To the reaction solution was added pyridine (1 mL), neutralized and concentrated, and the residue was dissolved in ethyl acetate (200 mL). The organic layer was washed with a saturated aqueous sodium hydrogen carbonate solution (200 mL), and the organic layer was dried over anhydrous magnesium sulfate and concentrated. The residue was azeotroped with dioxane (20 mL × 2) to obtain crude compound 10 ′.

粗化合物１０’（２３．７ｇ）をリン酸トリエチル（６６ｍＬ）に溶解し、０℃でトリブチルアミン（１１．３ｍＬ、４７．４ｍｍｏｌ）、オキシ塩化リン（３．５１ｍＬ、３７．７ｍｍｏｌ）を加え、同温度で３時間撹拌した。反応液に冷５０％アセトニトリル（１５８ｍＬ）、トリエチルアミン（３１．５ｍＬ、２２６ｍｍｏｌ）を加え、０℃で１０分間、室温で４時間撹拌した後、ｎ−ブタノール：酢酸ブチル（１：１、３００ｍＬ）、脱イオン水（３００ｍＬ）を加え撹拌した。水層より、ｎ−ブタノール：酢酸ブチル（１：１、３００ｍＬ×３）で抽出し、有機層を合わせて濃縮した。残渣をエタノール（２４０ｍＬ）に溶解し、２８％アンモニア水（１２０ｍＬ）を加え、室温で２２時間撹拌した。析出した沈殿をろ取し、エタノール（３０ｍＬ×２）で洗浄後、乾燥し、白色粉末状の化合物４（８．６６ｇ、ジアンモニウム塩として２３．４ｍｍｏｌ、化合物３から６６．７％）を得た。 Crude compound 10 ′ (23.7 g) was dissolved in triethyl phosphate (66 mL), tributylamine (11.3 mL, 47.4 mmol) and phosphorus oxychloride (3.51 mL, 37.7 mmol) were added at 0 ° C. Stir at the same temperature for 3 hours. Cold 50% acetonitrile (158 mL) and triethylamine (31.5 mL, 226 mmol) were added to the reaction solution, followed by stirring at 0 ° C. for 10 minutes and at room temperature for 4 hours, and then n-butanol: butyl acetate (1: 1, 300 mL), Deionized water (300 mL) was added and stirred. The aqueous layer was extracted with n-butanol: butyl acetate (1: 1, 300 mL × 3), and the organic layers were combined and concentrated. The residue was dissolved in ethanol (240 mL), 28% aqueous ammonia (120 mL) was added, and the mixture was stirred at room temperature for 22 hr. The deposited precipitate was collected by filtration, washed with ethanol (30 mL × 2) and dried to obtain Compound 4 (8.66 g, 23.4 mmol as a diammonium salt, 66.7% from Compound 3) as a white powder. It was.

６）１−Ｏ−ｐ−メトキシフェニル−β−Ｄ−リボ−ペントフラノース５−トリリン酸（５）の合成（方法１：イミダゾリデート法） 6) Synthesis of 1-Op-methoxyphenyl-β-D-ribo-pentofuranose 5-triphosphate (5) (method 1: imidazolidate method)

Figure 2010150791
（式中、Ｍｅはメチルを示す）
Figure 2010150791
 (In the formula, Me represents methyl)

化合物４（１３．１ｇ、ジアンモニウム塩として３５．４ｍｍｏｌ）を脱イオン水（８００ｍＬ）に溶解し、陽イオン交換樹脂（ＰＫ２１６（三菱化学社製）、プロトン型、２００ｍＬ）に通過させ、通過後の樹脂を脱イオン水で洗浄した。通過液、洗浄液をトリブチルアミン（１８．５ｍＬ、７７．７ｍｍｏｌ）に受けて中和後、濃縮し、残渣をジメチルホルムアミド（１００ｍＬ×３）で共沸した。   Compound 4 (13.1 g, 35.4 mmol as diammonium salt) is dissolved in deionized water (800 mL), passed through a cation exchange resin (PK216 (Mitsubishi Chemical Corporation), proton type, 200 mL), and after passing The resin was washed with deionized water. The passing liquid and the washing liquid were neutralized by receiving tributylamine (18.5 mL, 77.7 mmol), and concentrated, and the residue was azeotroped with dimethylformamide (100 mL × 3).

得られた化合物４のトリブチルアミン塩をジメチルホルムアミド（３５０ｍＬ）に溶解し、カルボニルジイミダゾール（１７．３ｇ、１０７ｍｍｏｌ）を加え、アルゴン雰囲気下、室温で３時間撹拌した。反応液にメタノール（２．９ｍＬ）を加えて１０分間撹拌後、ピロリン酸の３トリブチルアミン塩（５当量）のジメチルホルムアミド溶液（７５０ｍＬ）を加え、室温で１５時間撹拌した。反応液をろ過後、ろ液を濃縮した。残渣を脱イオン水に溶解し、全量を８００ｍＬとし、１Ｎ塩酸でｐＨを１．５とした。これを活性炭カラム（１Ｌ）に吸着し、水洗（５Ｌ）の後、０．２Ｎアンモニア水、２０％エタノール−０．２Ｎアンモニア水で溶出した。溶出液を濃縮後、残渣を０．２Ｎアンモニア水に溶解し、室温で終夜処理した。反応液を濃縮後、水を加えて全量を８００ｍＬとし、ＤＥＡＥセファデックスカラム（５００ｃｃ、５０〜７５〜１００〜２００〜３００ｍＭ炭酸水素アンモニウムで溶出）により精製した。化合物５を含む画分を濃縮後、残渣を水（１００ｍＬ×５）で共沸した。残渣に水を加えて全量を６００ｍＬとし、陽イオン交換樹脂カラム（ＰＫ２１６（三菱化学社製）、プロトン型、２００ｍＬ）に通過させ、通過後の樹脂を脱イオン水で洗浄した。通過液、洗浄液をトリエチルアミン（２６．０ｍＬ、１０９ｍｍｏｌ）に受けて中和後、濃縮した。残渣をジオキサン（１００ｍＬ×３）で共沸し、淡黄色アメ状の化合物５（２８３ｎｍでの紫外線吸収より、収量２５．９ｍｍｏｌ、収率７３．２％）をトリエチルアミン塩として得た。   The tributylamine salt of the obtained compound 4 was dissolved in dimethylformamide (350 mL), carbonyldiimidazole (17.3 g, 107 mmol) was added, and the mixture was stirred at room temperature for 3 hours under an argon atmosphere. Methanol (2.9 mL) was added to the reaction solution, and the mixture was stirred for 10 minutes, and then a dimethylformamide solution (750 mL) of 3 tributylamine salt of pyrophosphate (5 equivalents) was added, and the mixture was stirred at room temperature for 15 hours. After filtration of the reaction solution, the filtrate was concentrated. The residue was dissolved in deionized water to a total volume of 800 mL and the pH was adjusted to 1.5 with 1N hydrochloric acid. This was adsorbed on an activated carbon column (1 L), washed with water (5 L), and eluted with 0.2 N aqueous ammonia and 20% ethanol-0.2 N aqueous ammonia. After concentrating the eluate, the residue was dissolved in 0.2N aqueous ammonia and treated overnight at room temperature. After the reaction solution was concentrated, water was added to make a total volume of 800 mL, and the mixture was purified by a DEAE Sephadex column (eluted with 500 cc, 50 to 75 to 100 to 200 to 300 mM ammonium bicarbonate). After concentrating the fraction containing Compound 5, the residue was azeotroped with water (100 mL × 5). Water was added to the residue to make a total volume of 600 mL, and the solution was passed through a cation exchange resin column (PK216 (manufactured by Mitsubishi Chemical), proton type, 200 mL), and the resin after passing was washed with deionized water. The passing solution and the washing solution were received by triethylamine (26.0 mL, 109 mmol), neutralized, and concentrated. The residue was azeotroped with dioxane (100 mL × 3) to obtain pale yellow candy-like compound 5 (yield 25.9 mmol, yield 73.2% from UV absorption at 283 nm) as a triethylamine salt.

１Ｈ−ＮＭＲ（Ｄ２Ｏ）：δ７．１１（２Ｈ，ｄ，ａｒｏｍａｔｉｃ，Ｊ＝９．０），６．９９（２Ｈ，ｄ，ａｒｏｍａｔｉｃ，Ｊ＝９．０），５．５６（１Ｈ，ｄ，Ｈ−１，Ｊ＝２．０），４．５１（１Ｈ，ｄｄ，Ｈ−３，Ｊ＝５．０，６．０），４．３５（１Ｈ，ｄｄ，Ｈ−２，Ｊ＝１．５，５．０），４．２６（１Ｈ，ｑ，Ｈ−４，Ｊ＝５．０），４．１８（１Ｈ，ｄｔ，Ｈ−５ａ，Ｊ＝４．０，１１．５），４．１１（１Ｈ，ｄｔ，Ｈ−５ｂ，Ｊ＝６．０，１２．０），３．８２（３Ｈ，ｓ，ｍｅｔｈｙｌ），３．１９（Ｈ，ｑ，ＴＥＡ，Ｊ＝７．５），１．２７（Ｈ，ｔ，ＴＥＡ，Ｊ＝７．５）；３１Ｐ−ＮＭＲ（Ｄ２Ｏ）：δ−１０．８２（ｄ），−１１．２１（ｄ），−２３．１７（ｔ）． 1 H-NMR (D 2 O): δ 7.11 (2H, d, aromatic, J = 9.0), 6.99 (2H, d, aromatic, J = 9.0), 5.56 (1H, d, H-1, J = 2.0), 4.51 (1H, dd, H-3, J = 5.0, 6.0), 4.35 (1H, dd, H-2, J = 1.5, 5.0), 4.26 (1H, q, H-4, J = 5.0), 4.18 (1H, dt, H-5a, J = 4.0, 11.5) 4.11 (1H, dt, H-5b, J = 6.0, 12.0), 3.82 (3H, s, methyl), 3.19 (H, q, TEA, J = 7.5 ), 1.27 (H, t, TEA, J = 7.5); 31 P-NMR (D 2 O): δ-10.82 (d), -11.21 (d), -23.17 (T).

＜原料合物の合成例２＞
１−Ｏ−ｐ−メトキシフェニル−β−Ｄ−リボ−ペントフラノース５−トリリン酸（５）の合成（方法２：Ｎ−メチルイミダゾリデート法）<Synthesis example 2 of raw material compound>
Synthesis of 1-Op-methoxyphenyl-β-D-ribo-pentofuranose 5-triphosphate (5) (Method 2: N-methylimidazolidate method)

Figure 2010150791
（式中、Ｍｅはメチルを示す）
Figure 2010150791
 (In the formula, Me represents methyl)

上記で調製した化合物４（１．８５ｇ、ジアンモニウム塩として５．００ｍｍｏｌ）を脱イオン水（５０ｍＬ）に溶解し、陽イオン交換樹脂（ＰＫ２１６（三菱化学社製）、プロトン型、１０ｍＬ）に通過させ、通過後の樹脂を脱イオン水で洗浄した。通過液、洗浄液をトリエチルアミン（１．４０ｍＬ、１０．０ｍｍｏｌ）に受けて中和後、濃縮し、残渣をアセトニトリル（２０ｍＬ×３）で共沸した。   Compound 4 (1.85 g, 5.00 mmol as diammonium salt) prepared above is dissolved in deionized water (50 mL) and passed through a cation exchange resin (PK216 (manufactured by Mitsubishi Chemical Corporation), proton type, 10 mL). The resin after passing was washed with deionized water. The passing liquid and the washing liquid were neutralized by receiving triethylamine (1.40 mL, 10.0 mmol), and concentrated, and the residue was azeotroped with acetonitrile (20 mL × 3).

得られた化合物４のトリエチルアミン塩をアセトニトリル（２０ｍＬ）に溶解し、ジメチルアニリン（２．５５ｍＬ）、トリエチルアミン（０．７０ｍＬ、５．０２ｍｍｏｌ）を加え０℃に冷却した。トリフルオロ酢酸無水物（３．５５ｍＬ）のアセトニトリル溶液（５ｍＬ）を５分間かけて滴下し、０℃で５分間、室温で１０分間撹拌した。反応液を減圧下、３０℃で約１５ｍＬを留去後、Ｎ−メチルイミダゾール（１．２０ｍＬ）、トリエチルアミン（３．５０ｍＬ）のアセトニトリル溶液（５ｍＬ）を加え、０℃で１時間撹拌した。ピロリン酸の３トリブチルアミン塩（２当量）のアセトニトリル溶液（１０ｍＬ）を加え、０℃で５分間、室温で１０分間撹拌した。反応液に脱イオン水（１００ｍＬ）を加え撹拌後、７．５Ｎ水酸化ナトリウム水溶液でｐＨ１０．０に合わせ、クロロホルム（５０ｍＬ）で３回抽出した。水層を１Ｎ塩酸でｐＨ７．０に合わせ濃縮後、脱イオン水で５００ｍＬとした（ＨＰＬＣによる分析では合成収率８１．６％）。この溶液を活性炭カラム（二村化学工業タイコー活性炭３００ｃｃ）に吸着させ、水洗後、０．２Ｎアンモニア水で溶出した。目的物を含む画分を２５０ｍＬに濃縮し、陽イオン交換樹脂（ＰＫ２１６（三菱化学社製）、プロトン型、１０ｍＬ）に通過させ、通過後の樹脂を脱イオン水で洗浄した。通過液、洗浄液をトリエチルアミン（５ｍＬ）に受けて中和後、濃縮した。残渣をアセトニトリル（２０ｍＬ×３）で共沸し、淡黄色アメ状の化合物５（ＨＰＬＣによる定量により、収量３．８６ｍｍｏｌ、収率７７．２％）をトリエチルアミン塩として得た。   The triethylamine salt of the obtained compound 4 was dissolved in acetonitrile (20 mL), dimethylaniline (2.55 mL) and triethylamine (0.70 mL, 5.02 mmol) were added, and the mixture was cooled to 0 ° C. A solution of trifluoroacetic anhydride (3.55 mL) in acetonitrile (5 mL) was added dropwise over 5 minutes, and the mixture was stirred at 0 ° C. for 5 minutes and at room temperature for 10 minutes. About 15 mL of the reaction solution was distilled off at 30 ° C. under reduced pressure, and then an acetonitrile solution (5 mL) of N-methylimidazole (1.20 mL) and triethylamine (3.50 mL) was added, followed by stirring at 0 ° C. for 1 hour. A solution of tritrimethylamine pyrophosphate (2 equivalents) in acetonitrile (10 mL) was added, and the mixture was stirred at 0 ° C. for 5 minutes and at room temperature for 10 minutes. Deionized water (100 mL) was added to the reaction mixture, and the mixture was stirred, adjusted to pH 10.0 with 7.5N aqueous sodium hydroxide solution, and extracted three times with chloroform (50 mL). The aqueous layer was adjusted to pH 7.0 with 1N hydrochloric acid and concentrated, and then made up to 500 mL with deionized water (synthesis yield 81.6% in the analysis by HPLC). This solution was adsorbed on an activated carbon column (Nimura activated carbon 300cc), washed with water, and eluted with 0.2N aqueous ammonia. The fraction containing the target product was concentrated to 250 mL, passed through a cation exchange resin (PK216 (manufactured by Mitsubishi Chemical), proton type, 10 mL), and the resin after passing was washed with deionized water. The passing liquid and the washing liquid were neutralized with triethylamine (5 mL) and concentrated. The residue was azeotroped with acetonitrile (20 mL × 3) to obtain pale yellow candy-like compound 5 (quantity by HPLC: yield 3.86 mmol, yield 77.2%) as a triethylamine salt.

＜原料化合物の合成例３＞
１−メチルアデノシン５’−モノリン酸（１２）の合成<Synthesis Example 3 of Raw Material Compound>
Synthesis of 1-methyladenosine 5′-monophosphate (12)

Figure 2010150791
Figure 2010150791

化合物６（２０．０ｇ、５７．６ｍｍｏｌ）をジメチルアセトアミド（２００ｍＬ）に懸濁し、Ｎ，Ｏ−ビス（トリメチルシリル）アセトアミド（７０ｍＬ、２８６ｍｍｏｌ）を加え、室温で１時間撹拌後、さらにヨウ化メチル（７２ｍＬ、１．１６ｍｏｌ）を加え、室温で４３時間撹拌した。反応液を濃縮後、残渣をエタノール（２９０ｍＬ）に溶解し、トリフルオロ酢酸（２９０μＬ）を加え、氷冷下、３時間撹拌した。生じた沈殿をろ取し、冷エタノール（５０ｍＬ）で洗浄、乾燥した。得られた沈殿を水（２５００ｍＬ）に溶解し、陰イオン交換樹脂カラム（ＩＲＡ６７（ローム＆ハース社製）、ギ酸型、２００ｍＬ）に通過させ、通過後の樹脂を脱イオン水で洗浄した。通過液、洗浄液を合わせて濃縮後、エタノール（３００ｍＬ）を加え、生じた沈殿をろ取、冷エタノール（５０ｍＬ）で洗浄した。真空乾燥後、白色粉末状の化合物１２（１５．４ｇ、遊離酸として４２．６ｍｍｏｌ、収率７４．０％）を得た。 Compound 6 (20.0 g, 57.6 mmol) was suspended in dimethylacetamide (200 mL), N, O-bis (trimethylsilyl) acetamide (70 mL, 286 mmol) was added, and the mixture was stirred at room temperature for 1 hour, and further methyl iodide ( 72 mL, 1.16 mol) was added and stirred at room temperature for 43 hours. After the reaction solution was concentrated, the residue was dissolved in ethanol (290 mL), trifluoroacetic acid (290 μL) was added, and the mixture was stirred for 3 hours under ice cooling. The resulting precipitate was collected by filtration, washed with cold ethanol (50 mL), and dried. The obtained precipitate was dissolved in water (2500 mL) and passed through an anion exchange resin column (IRA67 (Rohm & Haas), formic acid type, 200 mL), and the resin after passing was washed with deionized water. The combined flow-through solution and washing solution were concentrated, ethanol (300 mL) was added, and the resulting precipitate was collected by filtration and washed with cold ethanol (50 mL). After vacuum drying, white powdered compound 12 (15.4 g, 42.6 mmol as a free acid, yield 74.0%) was obtained.

１Ｈ−ＮＭＲ（Ｄ２Ｏ）：δ８．６６（１Ｈ，ｓ，ａｒｏｍａｔｉｃ），８．５３（１Ｈ，ｓ，ａｒｏｍａｔｉｃ），６．１９（１Ｈ，ｄ，Ｈ−１’，Ｊ＝５．５），４．５３（１Ｈ，ｔ，Ｈ−３’，Ｊ＝４．５），４．４１（１Ｈ，ｂｒ．ｔ，Ｈ−４’），４．１４（２Ｈ，ｍ，Ｈ−５’），３．９４（３Ｈ，ｓ，ｍｅｔｈｙｌ）；３１Ｐ−ＮＭＲ（Ｄ２Ｏ）：δ０．５９（ｓ）． 1 H-NMR (D 2 O): δ 8.66 (1H, s, aromatic), 8.53 (1H, s, aromatic), 6.19 (1H, d, H-1 ′, J = 5.5) ), 4.53 (1H, t, H-3 ′, J = 4.5), 4.41 (1H, br. T, H-4 ′), 4.14 (2H, m, H-5 ′). ), 3.94 (3H, s, methyl); 31 P-NMR (D 2 O): δ 0.59 (s).

実験例２：１−Ｏ−ｐ−メトキシフェニル−Ｄ−リボ−ペントフラノース保護体を得る際の反応条件の検討 Experimental Example 2: Examination of reaction conditions for obtaining 1-Op-methoxyphenyl-D-ribo-pentofuranose protector

Figure 2010150791
Figure 2010150791

ルイス酸およびモレキュラーシーブスの存在下、あるいは非存在下、１−Ｏ−アセチル−β−Ｄ−リボ−ペントフラノース保護体とフェノール化合物と反応させ、立体異性体混合物である１−Ｏ−ｐ−メトキシフェニル−Ｄ−リボ−ペントフラノース保護体を合成し、α、β立体異性体それぞれの存在比を検討した。
エントリー１：
１−Ｏ−アセチル−β−Ｄ−リボ−ペントフラノース保護体としては、Ｒがアセチルの化合物２を使用し、公知文献（ＷＯ０８／０６０６３２）に記載の方法に従って反応を行った。反応液を希釈した後、ＨＰＬＣにより分析し、α、β立体異性体それぞれの存在比、α：β比を算出した。In the presence or absence of Lewis acid and molecular sieves, 1-O-acetyl-β-D-ribo-pentofuranose protected with a phenol compound is reacted with 1-Op-methoxy which is a stereoisomer mixture. A phenyl-D-ribo-pentofuranose protector was synthesized, and the abundance ratios of α and β stereoisomers were examined.
Entry 1:
As the 1-O-acetyl-β-D-ribo-pentofuranose protector, compound 2 in which R is acetyl was used, and the reaction was carried out according to the method described in the known literature (WO08 / 060632). The reaction solution was diluted and then analyzed by HPLC, and the abundance ratio and α: β ratio of each of the α and β stereoisomers were calculated.

エントリー２〜５：
１−Ｏ−アセチル−β−Ｄ−リボ−ペントフラノース保護体としては、Ｒがベンゾイルの化合物８を使用し、化合物８および４−メトキシフェノール（１．２当量）を有機溶媒（１０ｍＬ／１ｇの化合物８）に溶解し、モレキュラーシーブス４Ａ（Ｗａｋｏ社、２ｇ／１ｇの化合物８、または非存在下）を加え、室温で１時間撹拌した後、トリフルオロメタンスルホン酸トリメチルシリル（０．４ｍＬ／１ｇの化合物８）を加え、室温で２４時間撹拌した。反応液を希釈後、ＨＰＬＣにより分析し、エントリー１と同様にα：β比を算出した。Entries 2-5:
As the 1-O-acetyl-β-D-ribo-pentofuranose protector, compound 8 in which R is benzoyl was used, and compound 8 and 4-methoxyphenol (1.2 equivalents) were added to an organic solvent (10 mL / 1 g Dissolve in Compound 8), add Molecular Sieves 4A (Wako, 2 g / 1 g of Compound 8 or in the absence), stir at room temperature for 1 hour, and then trimethylsilyl trifluoromethanesulfonate (0.4 mL / 1 g of compound). 8) was added and stirred at room temperature for 24 hours. The reaction solution was diluted and then analyzed by HPLC, and the α: β ratio was calculated in the same manner as in entry 1.

それぞれの条件でのα：β比を表１に示す。なお、表中の「ＴＭＳＯＴｆ」はトリフルオロメタンスルホン酸トリメチルシリルを、「ＭＳ４Ａ」はモレキュラーシーブス４Ａを示す。 Table 1 shows the α: β ratio under each condition. In the table, “TMSOTf” represents trimethylsilyl trifluoromethanesulfonate, and “MS4A” represents molecular sieves 4A.

Figure 2010150791
Figure 2010150791

従来法（特許文献１：ＷＯ０８／０６０６３２）では、α：β比は１：３．５と、目的としていないα体が大量に生成していたのに対し（エントリー１）、モレキュラーシーブス４Ａの存在下、トリフルオロメタンスルホン酸トリメチルシリルをルイス酸として用いると、その比は１：２４〜１：２７に著しく改善した（エントリー３〜５）。一方、モレキュラーシーブス非存在下では１：２．４に低下したことから（エントリー２）、この反応ではモレキュラーシーブスの存在が重要であることがわかった。   In the conventional method (Patent Document 1: WO08 / 060632), the α: β ratio is 1: 3.5, which is a large amount of non-targeted α isomers (entry 1), while molecular sieves 4A exists. Below, when trimethylsilyl trifluoromethanesulfonate was used as the Lewis acid, the ratio was significantly improved from 1:24 to 1:27 (entries 3-5). On the other hand, since it decreased to 1: 2.4 in the absence of molecular sieves (entry 2), the presence of molecular sieves was found to be important in this reaction.

Claims (6)

式（ＸＩ）化合物を縮合剤を用いて式（ＸＩＶ）化合物に変換後（工程Ａ）、マグネシウム、マンガンまたは鉄のいずれかより選択された金属塩の存在下、式（ＸＩＩＩ）と反応させることにより式（Ｉ）化合物を得る（工程Ｂ）ことを特徴とするアデノシンテトラホスフェート化合物の製造法。
Figure 2010150791
 （式中、Ａｒはアリール基、Ｘは硫黄原子もしくは酸素原子またはイミノ基のいずれかより選ばれる基、Ｒ１とＲ２は水素原子、アルキルまたはアラルキル基のいずれかより選ばれる基、Ｍは水素原子またはイオンを示す。）After converting the compound of formula (XI) to the compound of formula (XIV) using a condensing agent (step A), reacting with the compound of formula (XIII) in the presence of a metal salt selected from any of magnesium, manganese or iron A process for producing an adenosine tetraphosphate compound, characterized in that a compound of formula (I) is obtained (step B).
Figure 2010150791
 (In the formula, Ar is an aryl group, X is a group selected from a sulfur atom, an oxygen atom or an imino group, R 1 and R 2 are groups selected from a hydrogen atom, an alkyl or an aralkyl group, and M is Indicates hydrogen atom or ion.) Ｒ１がアルキル、Ｒ２が水素原子、Ａｒがアルコキシフェニル、Ｘは酸素原子で表される化合物である、請求項１に記載の製造法。The production method according to claim 1, wherein R 1 is an alkyl, R 2 is a hydrogen atom, Ar is an alkoxyphenyl, and X is an oxygen atom. Ｒ１がメチル、Ｒ２が水素原子、Ａｒがメトキシフェニル、Ｘは酸素原子で表される化合物である、請求項１に記載の製造法。The production method according to claim 1, wherein R 1 is methyl, R 2 is a hydrogen atom, Ar is methoxyphenyl, and X is an oxygen atom. 縮合剤がカルボジイミド類である、請求項１に記載の製造法。 The production method according to claim 1, wherein the condensing agent is a carbodiimide. 金属塩がマグネシウム塩である、請求項１に記載の製造法。 The production method according to claim 1, wherein the metal salt is a magnesium salt. 工程Ａと工程Ｂを連続して行う、請求項１に記載の製造法。 The manufacturing method of Claim 1 which performs the process A and the process B continuously.
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