WO2011081083A1 - Neuraminic acid derivative, sialidase activity inhibitor and antiinfluenza drug - Google Patents

Neuraminic acid derivative, sialidase activity inhibitor and antiinfluenza drug Download PDF

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WO2011081083A1
WO2011081083A1 PCT/JP2010/073284 JP2010073284W WO2011081083A1 WO 2011081083 A1 WO2011081083 A1 WO 2011081083A1 JP 2010073284 W JP2010073284 W JP 2010073284W WO 2011081083 A1 WO2011081083 A1 WO 2011081083A1
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acid derivative
neuraminic acid
sialidase
sialidase activity
activity
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PCT/JP2010/073284
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French (fr)
Japanese (ja)
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真 木曽
秀治 石田
マゲッシュ サダゴパン
康夫 鈴木
妙子 宮城
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国立大学法人岐阜大学
学校法人中部大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
    • C07D309/16—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • C07D309/28—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

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  • the present invention relates to neuraminic acid derivatives and sialidase activity inhibitors and anti-influenza drugs containing them as active ingredients.
  • Influenza virus infects birds and the like, is extremely susceptible to mutation during transmission, and develops resistance to existing drugs such as Tamiflu. If a new virus occurs in this way, it may become a worldwide pandemic, causing tremendous social and economic damage. Vaccines are effective for viral infections, but viruses that are highly mutated, such as influenza viruses and HIV, may not be able to cope with vaccines alone. It is an urgent need.
  • hemagglutinin hemagglutinin
  • NA neuraminidase
  • neuraminidase catalyzes a reaction that hydrolyzes sialic acid (I) (see Structural Formula 1 below) of the glycoprotein terminal that binds to hemagglutinin on the surface of the viral molecule (this is called sialidase activity), thereby causing host cells to Accelerate the virus release process from
  • sialic acid I
  • sialidase activity this is called sialidase activity
  • neuraminidase promotes the movement of viral molecules through respiratory mucus, prevents the aggregation of viral molecules, and promotes budding (germination) and release of viruses. For this reason, drugs that inhibit sialidase activity are considered to be effective in preventing and treating influenza.
  • oseltamivir (2) (registered trademark name: Tamiflu) represented by the following structural formula (Chemical Formula 1)
  • zanamivir (3) registered trademark name: Relenza
  • the present invention has been made in view of the above-mentioned conventional circumstances, and selectively inhibits the activity of viral neuraminidase and hardly inhibits human sialidase activity, neuraminic acid derivatives, sialidase activity inhibitors and anti-influenza drugs It is an issue to be solved to provide.
  • the present inventor synthesized a compound in which the terminal of the glycerol part of zanamivir (3) was replaced with an amide, and investigated its sialidase activity. As a result, it was found that the sialidase activity of the virus was selectively inhibited and the human sialidase activity was hardly inhibited, and the present invention was completed.
  • the neuraminic acid derivative of the present invention has the following chemical formula (1) (wherein R 1 and R 2 are hydrogen, an alkyl group having 1 to 8 carbon atoms which may be branched, or a branch having 1 to 8 carbon atoms). Or R 3 is hydrogen or fluorine) or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof.
  • the neuraminic acid derivative of the present invention can be used as a sialidase activity inhibitor. That is, the sialidase activity inhibitor of the present invention contains the neuraminic acid derivative of the present invention as an active ingredient.
  • the anti-influenza drug containing the neuraminic acid derivative of the present invention as an active ingredient has a high sialidase activity inhibitory effect and selectively inhibits viral sialidase activity, and thus has the advantage of fewer side effects.
  • Example 1 In Example 1, neuraminic acid derivative (11) was synthesized according to the synthesis route of Chemical Formula 4 below.
  • Step 1 5-Acetamido-4-azido-7,8,9-tri-O-acetyl-2,3,4,5-tetradeoxy-D-glycero-D-galacto- Synthesis of non-2-enopyranosonic acid methyl ester (4)
  • the starting compound (4) was prepared according to the method of Malcolm Chandler et al. (see J. Chem. Soc. Perkin Trans. 1, 1173-1180 (1995)) The synthesis was performed by the following synthesis route.
  • Step 2 5-Acetamido-4- (N-tert-butoxycarbonyl) amino-7,8,9-tri-O-acetyl-2,3,4,5-tetradeoxy-D-glycero-D-galacto-non-2-enopyranosonic Synthesis of acid methyl ester (5)
  • Compound (4) (7.95 g, 15 mmol) synthesized in Step 1 is dissolved in ethyl acetate (250 ml), Lindra catalyst (4 g), di-tert-butyl dicarbonate (20 ml) Were added successively, and the mixture was stirred in a hydrogen atmosphere, 1 atm, and 50 ° C. for 2 days.
  • Step 3 Synthesis of 5-Acetamido-4- (N-tert-butoxycarbonyl) amino-2,3,4,5-tetradeoxy-D-glycero-D-galacto-non-2-enopyranosonic acid methyl ester (6)
  • the compound (5) (5.3 g, 10 mmol) was dissolved in methanol (50 ml), a catalytic amount of sodium methoxide was added, and the mixture was stirred overnight at room temperature. After the reaction was completed, PH7 was prepared using Dowex-50 ⁇ 8 (H + ). The reaction mixture was filtered and the solvent was distilled off with an evaporator.
  • Step 4 5-Acetamido-4- (N-tert-butoxycarbonyl) amino-2,3,4,5-tetradeoxy-9-O- (p-toluene sulphonyl) -D-glycero-D-galacto-non-2-enopyranosonic acid Synthesis of methyl ester (7)
  • Compound (6) synthesized in Step 3 (2.23 g, 4 mmol) and p-toluenesulfonyl chloride (1.15 g, 18 mmol) are placed in a bifurcated flask in a desiccator containing silica gel. It was dried overnight under reduced pressure.
  • Step 5 5-Acetamido-9-azido-4- (N-tert-butoxycarbonyl) amino-2,3,4,5,9-pentadeoxy-)-D-glycero-D-galacto-non-2-enopyranosonic acid methyl ester
  • Step 5 Compound (7) (1.11 g, 2 mmol) synthesized in Step 4 was dissolved in DMF (10 ml), sodium azide (650 mg, 10 mmol) and 18-crown-6-ether (105 mg, 0.4 mmol). And stirred at 50 ° C. overnight.
  • Step 6 5-Acetamido-4- (N-tert-butoxycarbonyl) amino-2,3,4,5,9-pentadeoxy-9-pentanamido-D-glycero-D-galacto-non-2-enopyranosonic acid methyl ester (9)
  • the azide compound (8) synthesized in Step 5 (243 mg, 0.5 mmol) and N-hydroxysuccinimidyl valeryl ester (Valeryl NHS ester) (200 mg, 1 mmol) were dissolved in dehydrated tetrahydrofuran (3 ml) and trimethyl Phosphine (1M in THF, 1 mL, 1 mmol) was added dropwise at room temperature until nitrogen was evolved and then stirred overnight.
  • Step 7 5-Acetamido-4- (N, N'-bis-tert-butoxycarbonyl) guanidino-2,3,4,5,9-pentadeoxy-9-pentanamido-D-glycero-D-galacto-non-2-enopyranosonic acid Synthesis of methyl ester (10) Compound (9) (195 mg, 0.4 mmol) synthesized in Step 6 was dissolved in dichloromethane (2 ml), and trifluoroacetic acid (2 mL) was added at room temperature. After confirming disappearance of the starting material, the solvent was distilled off with an evaporator.
  • Step 8 Synthesis of 5-Acetamido-4-guanidino-2,3,4,5,9-pentadeoxy-9-pentanamido-D-glycero-D-galacto-non-2-enopyranosonic acid Trifluoroacetic Acid Salt (11)
  • the compound (10) (100 mg, 0.16 mmol) was dissolved in 2 ml of a methanol / THF mixed solvent (5/1), and an alkaline solution in which LiOH (25 mg) was dissolved in 1 ml of water was added to the reaction solution cooled to 0 ° C. Stir for 12 hours.
  • the reaction mixture was passed through an ion exchange resin (Dowex-50 ⁇ 8 (H + )) to pH 7, and then the reaction mixture was filtered and evaporated to obtain a yellow solid. This was dissolved in dichloromethane (2 mL) and trifluoroacetic acid (2 mL) at room temperature. After confirming the disappearance of the starting material, it was dried with an evaporator, and the crude product thus obtained was subjected to reverse phase column chromatography by 0-20% aqueous methanol solution (containing 0.1% trifluoroacetic acid). Purified. The purified fraction was dried and freeze-dried to obtain neuraminic acid derivative (11) as a white solid (64 mg, 76%). 1 H-NMR data and HR-MS data of the obtained neuraminic acid derivative (11) are shown below.
  • Example 2 neuraminic acid derivative (14) was synthesized according to the synthesis route of Chemical Formula 4 below.
  • Step 1 5-Acetamido-4- (N-tert-butoxycarbonyl) amino-2,3,4,5,9-pentadeoxy-9-cyclopropane-carboxamido-D-glycero-D-galacto-non-2-enopyranosonic acid methyl ester
  • Step 12 Synthesis of Azide Compound (8) (243 mg, 0.5 mmol) which is an intermediate compound in Example 1 above and N-hydroxysuccinic acid imidyl ester of cyclopropanecarboxylic acid (cyclopropyl NHS ester) (185 mg, 1 mmol) were dried.
  • Step 2 5-Acetamido-4- (N, N'-bis-tert-butoxycarbonyl) guanidino-2,3,4,5,9-pentadeoxy-9-cyclopropanecarboxamido D-glycero-D-galacto-non-2-enopyranosonic acid methyl
  • ester (13) Compound (12) (188 mg, 0.4 mmol) obtained as described above was added to a mixture of dichloromethane (2 mL) and trifluoroacetic acid (2 mL) at room temperature to confirm the disappearance of the starting material. After that, the reaction solution was evaporated and dried.
  • reaction mixture was passed through an ion exchange resin (Dowex-50 ⁇ 8 (H + )) to adjust the pH to 7, and then the reaction mixture was filtered and evaporated to obtain a yellowish white solid. This was dissolved in dichloromethane (2 mL) and trifluoroacetic acid (2 mL) at room temperature. After confirming the disappearance of the starting material, it was dried with an evaporator, and the crude product thus obtained was subjected to reverse phase column chromatography by 0-20% aqueous methanol solution (containing 0.1% trifluoroacetic acid). Purified.
  • ion exchange resin Dowex-50 ⁇ 8 (H + )
  • 15 ⁇ l of 20 mM sodium acetate buffer (pH 6.0), a predetermined amount of neuraminic acid derivative adjusted to 5 concentrations, and 5 ⁇ l of virus containing 2 units of sialidase were mixed and incubated at 37 ° C. for 15 minutes. Went.
  • one unit of viral sialidase is defined as the amount of enzyme required to produce 1 nmol of MU per minute at 37 ° C. at pH 6.0.
  • 5 ⁇ l of MUNA was added and further incubated at 37 ° C. for 15 minutes.
  • the activity measurement with respect to the sialidase (A / Narita / 1/2009) of influenza virus was measured with the fluorescence measurement using 4-methylumbelliferyl * N-acetylneuraminic acid (MUNA). Evaluated by law.
  • the neuraminic acid derivative (11) was found to have a high activity inhibitory action on the sialidases of A / PR / 8/34 (H1N1), H3N2 and H5N3 viruses. .
  • neuraminic acid derivative (14) is more effective than neuraminic acid derivative (11) against sialidases of A / PR / 8/34 (H1N1), H3N2 and H5N3 viruses. It showed a high activity inhibitory action and was similar to zanamivir (3) and oseltamivir (2). Furthermore, as shown in Table 1, the effect of inhibiting the activity of neuraminic acid derivative (14) on the sialidase of A / Narita / 1/2009 (H1N1) is similar to that of zanamivir (3) and oseltamivir (2). there were.
  • neuraminic acid derivative (11) and neuraminic acid derivative (14) have sufficient effects as sialidase activity inhibitors and antiviral agents.
  • a 500 mM sodium acetate buffer solution pH 4.6 for NEU1 and NEU4, pH 5.5 for NEU2
  • a predetermined amount of neuraminic acid derivative adjusted to 5 concentrations 50 ⁇ g of calf-derived albumin, 20 nmol of MUNA
  • a predetermined amount of recombinant human-sialidase were mixed and incubated at 37 ° C. for 30 minutes.
  • the amount of MU equal to the amount of neuraminic acid released was measured by the same method as in the case of measuring activity inhibition against viral sialidase.
  • the IC 50 value was calculated using a non-linear regression analysis method by Microsoft Excel.
  • the activity inhibition of zanamivir (3) registered trademark name: Relenza
  • neuraminic acid derivative (11) and neuraminic acid derivative (14) synthesized as described above were evaluated for activity inhibition against human-sialidase NEU3 by a fluorescence measurement method by high performance liquid chromatography using malononitrile.
  • neuraminic acid derivative (11) inhibits human-sialidase activity (IC 50 ) against any of four types of sialidases derived from humans (NEU1, NEU2, NEU3 and NEU4). was almost unacceptable.
  • the neuraminic acid derivative (14) was also inhibited against human-sialidase activity against any of the four types of sialidases derived from humans (NEU1, NEU2, NEU3 and NEU4). IC 50 ) was hardly recognized.
  • the neuraminic acid derivative and sialidase activity inhibitor of the present invention can be used as biological tools for examining the function of sialidase.
  • the neuraminic acid derivative of the present invention selectively inhibits viral sialidase activity and hardly inhibits human sialidase activity, and thus can be used as an anti-influenza drug having few side effects.

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Abstract

Provided are a neuraminic acid derivative, which selectively inhibits virus sialidase activity but little inhibits human sialidase activity, a sialidase activity inhibitor, and an antiinfluenza drug. The aforesaid neuraminic acid derivative is characterized by comprising a compound represented by chemical formula (1), a pharmaceutically acceptable salt thereof, a hydrate of the same, a solvate of the same or a prodrug of the same. In chemical formula (1), R1 and R2 represent hydrogen, an optionally branched C1-8 alkyl group or an optionally branched C1-8 cycloalkyl group; and R3 represents hydrogen or fluorine.

Description

ノイラミン酸誘導体、シアリダーゼ活性阻害剤及び抗インフルエンザ薬Neuraminic acid derivatives, sialidase activity inhibitors and anti-influenza drugs
 本発明は、ノイラミン酸誘導体、並びにそれを有効成分として含有するシアリダーゼ活性阻害剤及び抗インフルエンザ薬に関する。 The present invention relates to neuraminic acid derivatives and sialidase activity inhibitors and anti-influenza drugs containing them as active ingredients.
 インフルエンザウイルスは鳥類等が感染し、伝播の過程できわめて変異しやすく、タミフルのような既存の薬剤に対する耐性が発現する。こうして新型ウイルスが発生すると、世界同時流行（パンデミック）となる場合もあり、社会的、経済的に甚大な被害をもたらす。ウイルス感染にはワクチンが有効であるが、インフルエンザウイルスやＨＩＶのような、きわめて変異しやすいウイルスにおいては、ワクチンのみでは対応できない場合もあり、それを補うものとして、抗ウイルス薬の開発が社会的急務となっている。 Influenza virus infects birds and the like, is extremely susceptible to mutation during transmission, and develops resistance to existing drugs such as Tamiflu. If a new virus occurs in this way, it may become a worldwide pandemic, causing tremendous social and economic damage. Vaccines are effective for viral infections, but viruses that are highly mutated, such as influenza viruses and HIV, may not be able to cope with vaccines alone. It is an urgent need.
 ウイルスの表面には、ヘマグルチニン（ＨＡ）及びノイラミニダーゼ（ＮＡ）（シアリダーゼとも呼ばれるが、ウイルス学の分野ではノイラミニダーゼと呼ばれる場合が多い。）という２つの重要な糖タンパク質スパイクが存在しており、ウイルスの侵入や複製に重要な役割をしている。すなわち、ヘマグルチニンは糖複合体の残基に結合するシアル酸の末端を認識し、結合する。これにより、宿主細胞に付着したり、侵入したりするのを媒介する。
 一方、ノイラミニダーゼは、ウイルス分子表面のヘマグルチニンに結合する糖タンパク質端末のシアル酸（Ｉ）（下記構造式化１参照）を加水分解する反応を触媒し（これをシアリダーゼ活性という）、これにより宿主細胞からのウイルスの放出過程を促進する。さらに、ノイラミニダーゼは呼吸器系の粘液を通じてウイルス分子の移動を促し、ウイルス分子の凝集を防ぎ、ウイルスの出芽（発芽）・遊離を促進する。このため、シアリダーゼ活性を阻害する薬剤は、インフルエンザの治療予防に効果があると考えられている。 There are two important glycoprotein spikes on the surface of the virus: hemagglutinin (HA) and neuraminidase (NA) (also called sialidase but often called neuraminidase in the field of virology). It plays an important role in intrusion and replication. That is, hemagglutinin recognizes and binds to the end of sialic acid that binds to the residue of the glycoconjugate. This mediates attachment to and entry of host cells.
On the other hand, neuraminidase catalyzes a reaction that hydrolyzes sialic acid (I) (see Structural Formula 1 below) of the glycoprotein terminal that binds to hemagglutinin on the surface of the viral molecule (this is called sialidase activity), thereby causing host cells to Accelerate the virus release process from In addition, neuraminidase promotes the movement of viral molecules through respiratory mucus, prevents the aggregation of viral molecules, and promotes budding (germination) and release of viruses. For this reason, drugs that inhibit sialidase activity are considered to be effective in preventing and treating influenza.
Figure JPOXMLDOC01-appb-C000002
Figure JPOXMLDOC01-appb-C000002
 現在まで、シアリダーゼ活性を阻害してインフルエンザを予防する薬剤として、下記構造式（化１）に示すオセルタミビル（２）(登録商標名：タミフル)や、ザナミビル（３）（登録商標名：リレンザ)が開発され、さらには、それらの様々な誘導体が開発されている（特許文献１及び２参照）。 To date, oseltamivir (2) (registered trademark name: Tamiflu) represented by the following structural formula (Chemical Formula 1) and zanamivir (3) (registered trademark name: Relenza) are drugs that inhibit sialidase activity to prevent influenza. Further, various derivatives thereof have been developed (see Patent Documents 1 and 2).
Figure JPOXMLDOC01-appb-C000003
Figure JPOXMLDOC01-appb-C000003
特開２００１－３５４６３５JP 2001-354635 A 特開２００８－２９７３２０JP 2008-297320 A
 しかしながら、従来のインフルエンザを予防薬剤では、精神神経疾患、高血糖症、出血性疾患、皮膚疾患、肝臓と腎臓疾患、突然死等の症例と関連する、重篤な副作用の症例が報告されており、問題となっていた。そして、それらの副作用の原因として、上記従来のシアリダーゼ活性阻害剤が、ウイルスのノイラミニダーゼのみならず、ヒトのシアリダーゼに対しても活性阻害を有することに起因するのではないかと考えられている。なぜならば、ヒトのシアリダーゼとウイルスのノイラミニダーゼとは、三次元構造及び活性部位の構造において顕著な類似性を示すからである。このため、ウイルスのシアリダーゼ活性を選択的に阻害し、ヒトのシアリダーゼ活性を阻害しない薬剤の開発が望まれていた。 However, in the case of conventional preventive drugs for influenza, cases of serious side effects related to cases such as neuropsychiatric diseases, hyperglycemia, hemorrhagic diseases, skin diseases, liver and kidney diseases, sudden death have been reported. Was a problem. It is thought that the cause of these side effects may be that the above conventional sialidase activity inhibitors have activity inhibition not only against viral neuraminidase but also against human sialidase. This is because human sialidase and viral neuraminidase show significant similarities in three-dimensional structure and active site structure. Therefore, it has been desired to develop a drug that selectively inhibits viral sialidase activity and does not inhibit human sialidase activity.
 本発明は、上記従来の実情に鑑みてなされたものであり、ウイルスのノイラミニダーゼの活性を選択的に阻害し、ヒトのシアリダーゼ活性を阻害し難い、ノイラミン酸誘導体、シアリダーゼ活性阻害剤及び抗インフルエンザ薬を提供することを解決すべき課題としている。 The present invention has been made in view of the above-mentioned conventional circumstances, and selectively inhibits the activity of viral neuraminidase and hardly inhibits human sialidase activity, neuraminic acid derivatives, sialidase activity inhibitors and anti-influenza drugs It is an issue to be solved to provide.
 本発明者は、上記課題を解決すべく、ザナミビル（３）のグリセロール部分の末端をアミドに替えた化合物を合成し、そのシアリダーゼ活性について調べた。その結果、ウイルスのシアリダーゼ活性を選択的に阻害し、ヒトのシアリダーゼ活性はほとんど阻害しないことを見出し、本発明を完成するに至った。 In order to solve the above problems, the present inventor synthesized a compound in which the terminal of the glycerol part of zanamivir (3) was replaced with an amide, and investigated its sialidase activity. As a result, it was found that the sialidase activity of the virus was selectively inhibited and the human sialidase activity was hardly inhibited, and the present invention was completed.
 すなわち、本発明のノイラミン酸誘導体は、下記化学式（１）（ただし、Ｒ１及びＲ２は、水素又は炭素数１～８の分岐してもよいアルキル基若しくは炭素数１～８の分岐してもよいシクロアルキル基を示し、Ｒ３は水素又はフッ素を示す）又はその薬学上許容される塩、水和物、溶媒和物若しくはプロドラッグからなることを特徴とする。 That is, the neuraminic acid derivative of the present invention has the following chemical formula (1) (wherein R 1 and R 2 are hydrogen, an alkyl group having 1 to 8 carbon atoms which may be branched, or a branch having 1 to 8 carbon atoms). Or R 3 is hydrogen or fluorine) or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof.
Figure JPOXMLDOC01-appb-C000004
Figure JPOXMLDOC01-appb-C000004
 本発明者らは、上記化学式（１）におけるＲ１＝ｎ－ブチル基、Ｒ２＝メチル基、Ｒ３＝水素であるノイラミン酸誘導体や、Ｒ１＝シクロペンチル基、Ｒ２＝メチル基、Ｒ３＝水素であるノイラミン酸誘導体が、ウイルスのシアリダーゼ活性を確実に阻害し、ヒトのシアリダーゼ活性をほとんど阻害しないことを確認している。 The present inventors have used neuraminic acid derivatives in which R 1 = n-butyl group, R 2 = methyl group, R 3 = hydrogen in the chemical formula (1), R 1 = cyclopentyl group, R 2 = methyl group, R It has been confirmed that the neuraminic acid derivative in which 3 = hydrogen reliably inhibits viral sialidase activity and hardly inhibits human sialidase activity.
 以上の結果から、本発明のノイラミン酸誘導体をシアリダーゼ活性阻害剤に利用することができる。すなわち、本発明のシアリダーゼ活性阻害剤は、本発明のノイラミン酸誘導体を有効成分として含有することを特徴とする。 From the above results, the neuraminic acid derivative of the present invention can be used as a sialidase activity inhibitor. That is, the sialidase activity inhibitor of the present invention contains the neuraminic acid derivative of the present invention as an active ingredient.
 さらには、本発明のノイラミン酸誘導体を有効成分とする抗インフルエンザ薬は、シアリダーゼ活性阻害効果が高く、ウイルスのシアリダーゼ活性を選択的に阻害することから、副作用が少ないという利点を有する。 Furthermore, the anti-influenza drug containing the neuraminic acid derivative of the present invention as an active ingredient has a high sialidase activity inhibitory effect and selectively inhibits viral sialidase activity, and thus has the advantage of fewer side effects.
（実施例１）
 実施例１では、下記化４の合成経路に従ってノイラミン酸誘導体（１１）を合成した。 Example 1
In Example 1, neuraminic acid derivative (11) was synthesized according to the synthesis route of Chemical Formula 4 below.
Figure JPOXMLDOC01-appb-C000005
Figure JPOXMLDOC01-appb-C000005
（ステップ１）
 5-Acetamido-4-azido-7,8,9-tri-O-acetyl-2,3,4,5-tetradeoxy-D-glycero-D-galacto-
non-2-enopyranosonic acid methyl ester (4)の合成
 出発物質となる化合物（４）はMalcolm Chandlerらの方法（J. Chem. Soc. Perkin Trans. 1, 1173-1180 (1995)参照）に従い、下記の合成経路で合成を行った。 (Step 1)
5-Acetamido-4-azido-7,8,9-tri-O-acetyl-2,3,4,5-tetradeoxy-D-glycero-D-galacto-
Synthesis of non-2-enopyranosonic acid methyl ester (4) The starting compound (4) was prepared according to the method of Malcolm Chandler et al. (see J. Chem. Soc. Perkin Trans. 1, 1173-1180 (1995)) The synthesis was performed by the following synthesis route.
Figure JPOXMLDOC01-appb-C000006
Figure JPOXMLDOC01-appb-C000006
（ステップ２）
 5-Acetamido-4-(N-tert-butoxycarbonyl)amino-7,8,9-tri-O-acetyl-2,3,4,5-tetradeoxy-D-glycero-D-galacto-non-2-enopyranosonic acid methyl ester (5)の合成
 ステップ１で合成した化合物（４）（7.95g, 15mmol）を酢酸エチル（250ml）に溶解させ、リンドラ―触媒（4g）、二炭酸ジ-tert-ブチル（20ml）を順次加え、水素雰囲気化、１気圧、５０℃で２日間撹拌した。反応が完了した後、反応混合物をセライトろ過し、メタノールで洗浄した。続いてエバポレータで溶媒留去し、残渣をシリカゲルフラッシュカラムクロマトグラフィー(酢酸エチル１００％)で精製し、化合物（５）(9.1 g (98%))を白色泡状物として得た。得られた化合物（５）の1H-NMRデータ及び、HR-MSデータを以下に示す。 (Step 2)
5-Acetamido-4- (N-tert-butoxycarbonyl) amino-7,8,9-tri-O-acetyl-2,3,4,5-tetradeoxy-D-glycero-D-galacto-non-2-enopyranosonic Synthesis of acid methyl ester (5) Compound (4) (7.95 g, 15 mmol) synthesized in Step 1 is dissolved in ethyl acetate (250 ml), Lindra catalyst (4 g), di-tert-butyl dicarbonate (20 ml) Were added successively, and the mixture was stirred in a hydrogen atmosphere, 1 atm, and 50 ° C. for 2 days. After the reaction was completed, the reaction mixture was filtered through celite and washed with methanol. Subsequently, the solvent was distilled off by an evaporator, and the residue was purified by silica gel flash column chromatography (ethyl acetate 100%) to obtain Compound (5) (9.1 g (98%)) as a white foam. 1 H-NMR data and HR-MS data of the obtained compound (5) are shown below.
1H NMR (CDCl3)δ 1.41 (9H, s, NHCOC(CH3)3), 1.92, 2.05, 2.07, 2.10 (12H, 4 × s, 3 × OCOCH3, 1 ×NHCOCH3), 3.78 (3H, s, COOCH3 ), 4.13 to 4.23 (3H, m, H-5, H-6, H-9a), 4.46 (1H, td, J 4,NH  9.6 , J 4,3  2.1, J 4,5  9.6, H-4), 4.66 to 4.70 (2H, m, NH4, H-9b),  5.30 (1H, ddd, J 8,7  7.5, J 8,9a  5.5, J 8,9b  2.8, H-8), 5.46 (1H, dd, J 7,8  4.8, J 7,6  1.3, H-7), 5.54 (1H, d, J NH,5  8.9, NH5), 6.90 (1H, d, J 3,4 2.1,  H-3)
HR-MS (Positive): Calcd. for C23H34N2O12 (M+Na)+, 553.2009, Found 553.2012. 1 H NMR (CDCl 3 ) δ 1.41 (9H, s, NHCOC (CH 3 ) 3 ), 1.92, 2.05, 2.07, 2.10 (12H, 4 × s, 3 × OCOCH 3 , 1 × NHCOCH 3 ), 3.78 (3H , s, COOCH 3 ), 4.13 to 4.23 (3H, m, H-5, H-6, H-9a), 4.46 (1H, td, J 4, NH 9.6, J 4,3 2.1, J 4,5 9.6, H-4), 4.66 to 4.70 (2H, m, NH 4, H-9b), 5.30 (1H, ddd, J 8,7 7.5, J 8,9a 5.5, J 8,9b 2.8, H-8 ), 5.46 (1H, dd, J 7,8 4.8, J 7,6 1.3, H-7), 5.54 (1H, d, J NH, 5 8.9, NH 5 ), 6.90 (1H, d, J 3, 4 2.1,   H-3)
HR-MS (Positive): Calcd.for C 23 H 34 N 2 O 12 (M + Na) + , 553.2009, Found 553.2012.
（ステップ３）
 5-Acetamido-4-(N-tert-butoxycarbonyl)amino-2,3,4,5-tetradeoxy-D-glycero-D-galacto-non-2-enopyranosonic acid methyl ester (6)の合成
 ステップ２で合成した化合物（５）（5.3g，10mmol）をメタノール（50ml）に溶解させ、触媒量のナトリウムメトキシド加え、室温で一晩撹拌した。反応が完了した後、Dowex-50×8(H+)を用いて、ＰＨ７になるよう調製した。反応混合物をろ過した後エバポレータで溶媒留去し、残渣をシリカゲルフラッシュカラムクロマトグラフィー(クロロホルム：メタノール＝１：２０)で精製し、化合物（６）(3.03g，75%)を白色固体として得た。得られた化合物（６）の1H-NMRデータ及び、HR-MSデータを以下に示す。 (Step 3)
Synthesis of 5-Acetamido-4- (N-tert-butoxycarbonyl) amino-2,3,4,5-tetradeoxy-D-glycero-D-galacto-non-2-enopyranosonic acid methyl ester (6) The compound (5) (5.3 g, 10 mmol) was dissolved in methanol (50 ml), a catalytic amount of sodium methoxide was added, and the mixture was stirred overnight at room temperature. After the reaction was completed, PH7 was prepared using Dowex-50 × 8 (H + ). The reaction mixture was filtered and the solvent was distilled off with an evaporator. The residue was purified by silica gel flash column chromatography (chloroform: methanol = 1: 20) to obtain compound (6) (3.03 g, 75%) as a white solid. . 1 H-NMR data and HR-MS data of the obtained compound (6) are shown below.
1H NMR (CD3OD)δ 1.44 (9H, s, NHCOC(CH3)3), 1.98 (3H, s, NHCOCH3), 3.57 to 3.67 (2H, m, H-7, H-9a), 3.76 (3H, s, COOCH3 ), 3.81 (1H, dd, J 9b,9a  11.2, J 9b,8  3.1, H-9b), 3.87 (1H, ddd, J 8,7  11.2, J 8,9a  4.5, J 8,9b  2.2, H-8), 4.05 (1H, dd, J 5,4  8.7, J 5,6  9.8, H-5), 4.22 (1H, dd, J 6,5  9.8, J 6,7  <1, H-6), 4.46 (1H, dd, J 4,5  8.7, J 4,3  2.2, H-4), 5.82 (1H, d, J 3,4 2.2,  H-3); 
HR-MS (Positive): Calcd. for C17H28N2O9 (M+Na)+, 427.1693, Found 427.1673. 1 H NMR (CD 3 OD) δ 1.44 (9H, s, NHCOC (CH 3 ) 3 ), 1.98 (3H, s, NHCOCH 3 ), 3.57 to 3.67 (2H, m, H-7, H-9a), 3.76 (3H, s, COOCH 3 ), 3.81 (1H, dd, J 9b, 9a 11.2, J 9b, 8 3.1, H-9b), 3.87 (1H, ddd, J 8,7 11.2, J 8,9a 4.5 , J 8,9b 2.2, H-8), 4.05 (1H, dd, J 5,4 8.7, J 5,6 9.8, H-5), 4.22 (1H, dd, J 6,5 9.8, J 6, 7 <1, H-6), 4.46 (1H, dd, J 4,5 8.7, J 4,3 2.2, H-4), 5.82 (1H, d, J 3,4 2.2,   H-3);
HR-MS (Positive): Calcd.for C 17 H 28 N 2 O 9 (M + Na) + , 427.1693, Found 427.1673.
（ステップ４）
 5-Acetamido-4-(N-tert-butoxycarbonyl)amino-2,3,4,5-tetradeoxy-9-O-(p-toluene sulphonyl)-D-glycero-D-galacto-non-2-enopyranosonic acid methyl ester (7)の合成
 ステップ３で合成した化合物（６）（2.23g，4mmol）と、p-トルエンスルホニルクロリド（1.15g, 18 mmol）を二股フラスコに入れ、シリカゲルが入っているデシゲータ中で減圧下、一晩乾燥させた。化合物（６）を脱水ピリジン（15 mL）に溶解させ、p-トルエンスルホニルクロリドを、－１０℃で１２時間以上かけて加え、０℃にて一晩撹拌した。反応混合物に脱水メタノールを加えて反応を停止させ、３０分撹拌した後、エバポレータで溶媒留去し、残渣をシリカゲルフラッシュカラムクロマトグラフィー(メタノール／クロロホルム＝0、1/20の順)で精製し、化合物（７）(2.49g ，81%)を白色固体として得た。得られた化合物（６）の1H-NMRデータ及び、HR-MSデータを以下に示す。 (Step 4)
5-Acetamido-4- (N-tert-butoxycarbonyl) amino-2,3,4,5-tetradeoxy-9-O- (p-toluene sulphonyl) -D-glycero-D-galacto-non-2-enopyranosonic acid Synthesis of methyl ester (7) Compound (6) synthesized in Step 3 (2.23 g, 4 mmol) and p-toluenesulfonyl chloride (1.15 g, 18 mmol) are placed in a bifurcated flask in a desiccator containing silica gel. It was dried overnight under reduced pressure. Compound (6) was dissolved in dehydrated pyridine (15 mL), and p-toluenesulfonyl chloride was added at −10 ° C. over 12 hours and stirred at 0 ° C. overnight. The reaction mixture was added with dehydrated methanol to stop the reaction, stirred for 30 minutes, evaporated with an evaporator, and the residue was purified by silica gel flash column chromatography (methanol / chloroform = 0, 1/20 in this order) Compound (7) (2.49 g, 81%) was obtained as a white solid. 1 H-NMR data and HR-MS data of the obtained compound (6) are shown below.
1H NMR (CD3OD)δ 1.43 (9H, s, NHCOC(CH3)3), 1.98 (3H, s, NHCOCH3), 2.45 (3H, s, Ar-CH3 ), 3.51 (1H, dd, J 7,6  <1, J 7,8  9.1, H-7), 3.76 (3H, s, COOCH3 ), 3.96 to 4.16 (4H, m, H-5, H-6, H-8, H-9a), 4.29 (1H, dd, J 9b,9a  9.6, J 9b,8  1.3, H-9b), 4.44 (1H, dd, J 4,3  2.1, J 4,5  9.0, H-4),  5.81 (1H, d, J 3,4 2.1,  H-3), 7.43 (2H, d, H-2', H-6'), 7.80 (2H, d, H-3', H-5')
HR-MS (Positive): Calcd. for C24H34N2O11S (M+Na)+, 581.1781, Found 581.1791. 1 H NMR (CD 3 OD) δ 1.43 (9H, s, NHCOC (CH 3 ) 3 ), 1.98 (3H, s, NHCOCH 3 ), 2.45 (3H, s, Ar-CH 3 ), 3.51 (1H, dd , J 7,6 <1, J 7,8 9.1, H-7), 3.76 (3H, s, COOCH 3 ), 3.96 to 4.16 (4H, m, H-5, H-6, H-8, H -9a), 4.29 (1H, dd, J 9b, 9a 9.6, J 9b, 8 1.3, H-9b), 4.44 (1H, dd, J 4,3 2.1, J 4,5 9.0, H-4), 5.81 (1H, d, J 3,4 2.1,   H-3), 7.43 (2H, d, H-2 ', H-6'), 7.80 (2H, d, H-3 ', H-5')
HR-MS (Positive): Calcd.for C 24 H 34 N 2 O 11 S (M + Na) + , 581.1781, Found 581.1791.
（ステップ５）
 5-Acetamido-9-azido-4-(N-tert-butoxycarbonyl)amino-2,3,4,5,9-pentadeoxy-)-D-glycero-D-galacto-non-2-enopyranosonic acid methyl ester (8)の合成
 ステップ４で合成した化合物（７）（1.11g，2mmol）をＤＭＦ（10ml）に溶解させ、アジ化ナトリウム（650mg, 10mmol）及び１８-クラウン-６-エーテル（105mg, 0.4 mmol）を加えて５０℃で一晩撹拌した。その後、室温まで冷却し、エバポレータで溶媒留去し、残渣をシリカゲルフラッシュカラムクロマトグラフィー(メタノール／クロロホルム＝0、1/20の順)で精製し、化合物（８）(770mg，92%)を白色泡状物として得た。得られた化合物（８）の1H-NMRデータ及び、HR-MSデータを以下に示す。 (Step 5)
5-Acetamido-9-azido-4- (N-tert-butoxycarbonyl) amino-2,3,4,5,9-pentadeoxy-)-D-glycero-D-galacto-non-2-enopyranosonic acid methyl ester ( Synthesis of 8) Compound (7) (1.11 g, 2 mmol) synthesized in Step 4 was dissolved in DMF (10 ml), sodium azide (650 mg, 10 mmol) and 18-crown-6-ether (105 mg, 0.4 mmol). And stirred at 50 ° C. overnight. Thereafter, the mixture was cooled to room temperature, the solvent was distilled off with an evaporator, and the residue was purified by silica gel flash column chromatography (methanol / chloroform = 0, 1/20 in this order) to obtain compound (8) (770 mg, 92%) in white Obtained as a foam. 1 H-NMR data and HR-MS data of the obtained compound (8) are shown below.
1H NMR (CD3OD)δ 1.44 (9H, s, NHCOC(CH3)3), 1.99 (3H, s, NHCOCH3), 3.38 (1H, dd, J 9a,9b  12.8, J 9a,8  6, H-9a), 3.52 to 3.56 (2H, m, H-7, H-9b), 3.77 (3H, s, COOCH3 ), 4.01 to 4.06 (2H, m, H-5, H-8), 4.22 (1H, dd, J 6,7  <1, J 6,5 11.0, H-6), 4.46 (1H, dd, J 4,3  2.3, J 4,5  9.6, H-4),  5.81 (1H, d, J 3,4 2.3,  H-3)
HR-MS (Positive): Calcd. for C17H27N5O8 (M+Na)+, 452.1757, Found 452.1756. 1 H NMR (CD 3 OD) δ 1.44 (9H, s, NHCOC (CH 3 ) 3 ), 1.99 (3H, s, NHCOCH 3 ), 3.38 (1H, dd, J 9a, 9b 12.8, J 9a, 8 6 , H-9a), 3.52 to 3.56 (2H, m, H-7, H-9b), 3.77 (3H, s, COOCH 3 ), 4.01 to 4.06 (2H, m, H-5, H-8), 4.22 (1H, dd, J 6,7 <1, J 6,5 11.0, H-6), 4.46 (1H, dd, J 4,3 2.3, J 4,5 9.6, H-4), 5.81 (1H , d, J 3,4 2.3,   H-3)
HR-MS (Positive): Calcd.for C 17 H 27 N 5 O 8 (M + Na) + , 452.1757, Found 452.1756.
（ステップ６）
 5-Acetamido-4-(N-tert-butoxycarbonyl)amino-2,3,4,5,9-pentadeoxy-9-pentanamido-D-glycero-D-galacto-non-2-enopyranosonic acid methyl ester (9)の合成
 ステップ５で合成したアジド化合物（８）（243mg, 0.5 mmol）とＮ-ヒドロキシスクシンイミジルバレリルエステル(Valeryl NHS ester )（200mg, 1mmol）とを脱水テトラヒドロフラン（3ml）に溶解させ、トリメチルホスフィン（1M in THF, 1mL, 1mmol）を、窒素が発生するまで室温で滴下した後、一晩撹拌した。そして、エバポレータで溶媒留去し、残渣に水（20ml）を加えて、酢酸エチル（20 ml）で抽出した。水層を酢酸エチル（10 ml）で再度抽出し、先に抽出した酢酸エチル層と合わせ、飽和炭酸水素ナトリウム溶液、水（20mL）で順次洗浄し、硫酸ナトリウムを加えて乾燥させた。ろ過した後、エバポレータで溶媒留去し、残渣をシリカゲルフラッシュカラムクロマトグラフィー(メタノール／クロロホルム＝0、1/20の順)で精製し、化合物（９）(204mg，74%)を白色固体として得た。得られた化合物（９）の1H-NMRデータ及び、HR-MSデータを以下に示す。 (Step 6)
5-Acetamido-4- (N-tert-butoxycarbonyl) amino-2,3,4,5,9-pentadeoxy-9-pentanamido-D-glycero-D-galacto-non-2-enopyranosonic acid methyl ester (9) The azide compound (8) synthesized in Step 5 (243 mg, 0.5 mmol) and N-hydroxysuccinimidyl valeryl ester (Valeryl NHS ester) (200 mg, 1 mmol) were dissolved in dehydrated tetrahydrofuran (3 ml) and trimethyl Phosphine (1M in THF, 1 mL, 1 mmol) was added dropwise at room temperature until nitrogen was evolved and then stirred overnight. Then, the solvent was distilled off with an evaporator, water (20 ml) was added to the residue, and the mixture was extracted with ethyl acetate (20 ml). The aqueous layer was extracted again with ethyl acetate (10 ml), combined with the previously extracted ethyl acetate layer, washed successively with saturated sodium bicarbonate solution and water (20 mL), dried by adding sodium sulfate. After filtration, the solvent was distilled off with an evaporator, and the residue was purified by silica gel flash column chromatography (methanol / chloroform = 0, 1/20 in this order) to obtain compound (9) (204 mg, 74%) as a white solid. It was. 1 H-NMR data and HR-MS data of the obtained compound (9) are shown below.
1H NMR (CD3OD)δ 0.93 (3H, t, H-4'), 1.36 (2H, sex, , H-3'), 1.43 (9H, s, NHCOC(CH3)3), 1.59 (2H, p, H-2'), 1.97 (3H, s, NHCOCH3), 2.23 (2H, t, H-1'), 3.32 (1H, H-9a, mixed with CD3OD peak), 3.43 (1H, dd, J 7,6 <1, J 7,8 8.9, H-7), 3.56 (1H, dd, J 9b,9a  13.7, J 9b,8  2.8, H-9b), 3.76 (3H, s, COOCH3 ), 3.92 (1H, ddd, J 8,7 9.6, J 8,9a 6.2, J 8,9b 2.8, H-8), 4.04 (1H, dd, J 5,4 =  J 5,6 10.3, H-5), 4.25 (1H, dd, J 6,5 11.0, J 6,7 <1, H-6), 4.47 (1H, dd, J 4,5 8.9 , J 4,3 2.0, H-4),  5.82 (1H, d, J 3,4 2.0,  H-3)
HR-MS (Positive): Calcd. for C22H37N3O9 (M+Na)+, 510.2427, Found 510.2482. 1 H NMR (CD 3 OD) δ 0.93 (3H, t, H-4 '), 1.36 (2H, sex,, H-3'), 1.43 (9H, s, NHCOC (CH 3) 3), 1.59 ( 2H, p, H-2 '), 1.97 (3H, s, NHCOCH 3 ), 2.23 (2H, t, H-1'), 3.32 (1H, H-9a, mixed with CD3OD peak), 3.43 (1H, dd, J 7,6 <1, J 7,8 8.9, H-7), 3.56 (1H, dd, J 9b, 9a 13.7, J 9b, 8 2.8, H-9b), 3.76 (3H, s, COOCH 3), 3.92 (1H, ddd , J 8,7 9.6, J 8,9a 6.2, J 8,9b 2.8, H-8), 4.04 (1H, dd, J 5,4 =    J 5,6 10.3, H-5), 4.25 (1H, dd, J 6,5 11.0, J 6,7 <1, H-6), 4.47 (1H, dd, J 4,5 8.9   , J 4,3 2.0, H-4), 5.82 (1H, d, J 3,4 2.0,   H-3)
HR-MS (Positive): Calcd.for C 22 H 37 N 3 O 9 (M + Na) + , 510.2427, Found 510.2482.
（ステップ７）
 5-Acetamido-4-(N,N'-bis-tert-butoxycarbonyl)guanidino-2,3,4,5,9-pentadeoxy-9-pentanamido-D-glycero-D-galacto-non-2-enopyranosonic acid methyl ester (10)の合成
 ステップ６で合成した化合物（９）（195mg，0.4mmol）をジクロロメタン（２ml）に溶解させ、トリフルオロ酢酸（2mL）を室温にて加えた。出発原料の消失を確認した後、エバポレータで溶媒留去した。残渣をＮ，Ｎ'－ジメチルホルムアミド（2ml）に溶解させ、トリエチルアミン（165μl, 1.2mmol）を加えた。N,N’-bis-(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine (Bis-BocPCH)(185mg, 0.6mmol)をジメチルホルムアミド（２ｍｌ）に溶解させた溶液を、反応溶液に滴下し、５０℃にて１６時間攪拌した。エバポレータで溶媒留去し、残渣をシリカゲルフラッシュカラムクロマトグラフィー(メタノール／クロロホルム＝4/10、9/10の順)で精製し、化合物（１０）(141mg，56%) を白色固体として得た。得られた化合物（１０）の1H-NMRデータ及び、HR-MSデータを以下に示す。 (Step 7)
5-Acetamido-4- (N, N'-bis-tert-butoxycarbonyl) guanidino-2,3,4,5,9-pentadeoxy-9-pentanamido-D-glycero-D-galacto-non-2-enopyranosonic acid Synthesis of methyl ester (10) Compound (9) (195 mg, 0.4 mmol) synthesized in Step 6 was dissolved in dichloromethane (2 ml), and trifluoroacetic acid (2 mL) was added at room temperature. After confirming disappearance of the starting material, the solvent was distilled off with an evaporator. The residue was dissolved in N, N′-dimethylformamide (2 ml) and triethylamine (165 μl, 1.2 mmol) was added. A solution of N, N'-bis- (tert-butoxycarbonyl) -1H-pyrazole-1-carboxamidine (Bis-BocPCH) (185 mg, 0.6 mmol) dissolved in dimethylformamide (2 ml) was added dropwise to the reaction solution, Stir at 50 ° C. for 16 hours. The solvent was distilled off by an evaporator, and the residue was purified by silica gel flash column chromatography (in order of methanol / chloroform = 4/10, 9/10) to obtain Compound (10) (141 mg, 56%) as a white solid. 1 H-NMR data and HR-MS data of the obtained compound (10) are shown below.
1H NMR (CD3OD)δ 0.93 (3H, t, H-4'), 1.36 (2H, sex, , H-3'), 1.47, 1.52 (18H, 2 × s, 2 ×, NHCOC(CH3)3), 1.60 (2H, p, H-2'), 1.95 (3H, s, NHCOCH3), 2.23 (2H, t, H-1'), 3.34 (1H, dd, J 9a,9b  13.7, J 9a,8  6.9, H-9a), 3.45 (1H, dd, J 7,6 <1, J 7,8 8.4, H-7), 3.59 (1H, dd, J 9b,9a  13.7, J 9b,8  2.8, H-9b), 3.78 (3H, s, COOCH3 ), 3.93 (1H, ddd, J 8,7 8.4, J 8,9a 6.9, J 8,9b 2.8, H-8), 4.21 (1H, dd, J 5,4 =  J 5,6 10.3, H-5), 4.30 (1H, dd, J 6,5 10.3, J 6,7 <1, H-6), 5.06 (1H, dd, J 4,5 9.6 , J 4,3 2.0, H-4), 5.89 (1H, d, J 3,4 2.0, H-3); HR-MS (Positive): Calcd. for C28H47N5O11 (M+Na)+, 652.3170, Found 652.3232. 1 H NMR (CD 3 OD) δ 0.93 (3H, t, H-4 '), 1.36 (2H, sex,, H-3'), 1.47, 1.52 (18H, 2 × s, 2 ×, NHCOC (CH 3 ) 3 ), 1.60 (2H, p, H-2 '), 1.95 (3H, s, NHCOCH 3 ), 2.23 (2H, t, H-1'), 3.34 (1H, dd, J 9a, 9b 13.7 , J 9a, 8 6.9, H-9a), 3.45 (1H, dd, J 7,6 <1, J 7,8 8.4, H-7), 3.59 (1H, dd, J 9b, 9a 13.7, J 9b , 8 2.8, H-9b) , 3.78 (3H, s, COOCH 3), 3.93 (1H, ddd, J 8,7 8.4, J 8,9a 6.9, J 8,9b 2.8, H-8), 4.21 ( 1H, dd, J 5,4 =    J 5,6 10.3, H-5), 4.30 (1H, dd, J 6,5 10.3, J 6,7 <1, H-6), 5.06 (1H, dd, J 4,5 9.6   , J 4,3 2.0, H-4), 5.89 (1H, d, J 3,4 2.0, H-3); HR-MS (Positive): Calcd.for C 28 H 47 N 5 O 11 (M + Na) + , 652.3170, Found 652.3232.
（ステップ８）
 5-Acetamido-4-guanidino-2,3,4,5,9-pentadeoxy-9-pentanamido-D-glycero-D-galacto-non-2-enopyranosonic acid Trifluoroacetic Acid Salt (11)の合成
 ステップ７で合成した化合物(10) （100mg, 0.16mmol）を、メタノール／ＴＨＦ混合溶媒(5/1)2mlに溶かし、ＬｉＯＨ(25mg)を水1mlに溶かしたアルカリ溶液を、０℃に冷却した反応液に加え、１２時間撹拌を行う。反応終了後、反応混合物をイオン交換樹脂（Dowex-50×8 (H+)）に通してpH7とした後、反応混合物をろ過し、エバポレートして、黄色固体として得た。これをジクロロメタン(2mL)とトリフルオロ酢酸(2mL)に室温下で溶かした。出発物質が消滅していることを確認した後、エバポレータで乾燥させ、こうして得られた粗成物は逆相カラムクロマトグラフィーにより、0～20% メタノール水溶液（0.1% のトリフルオロ酢酸を含む）によって精製した。精製したフラクションを乾燥させ、凍結乾燥し、白色固体としてノイラミン酸誘導体（１１）を(64mg，76%) で得た。得られたノイラミン酸誘導体（１１）の1H-NMRデータ及び、HR-MSデータを以下に示す。 (Step 8)
Synthesis of 5-Acetamido-4-guanidino-2,3,4,5,9-pentadeoxy-9-pentanamido-D-glycero-D-galacto-non-2-enopyranosonic acid Trifluoroacetic Acid Salt (11) The compound (10) (100 mg, 0.16 mmol) was dissolved in 2 ml of a methanol / THF mixed solvent (5/1), and an alkaline solution in which LiOH (25 mg) was dissolved in 1 ml of water was added to the reaction solution cooled to 0 ° C. Stir for 12 hours. After completion of the reaction, the reaction mixture was passed through an ion exchange resin (Dowex-50 × 8 (H + )) to pH 7, and then the reaction mixture was filtered and evaporated to obtain a yellow solid. This was dissolved in dichloromethane (2 mL) and trifluoroacetic acid (2 mL) at room temperature. After confirming the disappearance of the starting material, it was dried with an evaporator, and the crude product thus obtained was subjected to reverse phase column chromatography by 0-20% aqueous methanol solution (containing 0.1% trifluoroacetic acid). Purified. The purified fraction was dried and freeze-dried to obtain neuraminic acid derivative (11) as a white solid (64 mg, 76%). 1 H-NMR data and HR-MS data of the obtained neuraminic acid derivative (11) are shown below.
1H NMR (D2O)δ 0.90 (3H, t, H-4'), 1.32 (2H, sex, , H-3'), 1.57 (2H, p, H-2'), 2.03 (3H, s, NHCOCH3), 2.29 (2H, t, H-1'), 3.36 (1H, dd, J 9a,9b  14.4, J 9a,8  6.8, H-9a), 3.56 to 3.58 (2H, m, H-7, H-9b), 3.98 (1H, ddd, J 8,7 12.3, J 8,9a 10.3, J 8,9b 3.4, H-8), 4.22 (1H, dd, J 5,4 =  J 5,6 10.3, H-5), 4.38 (1H, dd, J 6,5 10.3, J 6,7 <1, H-6), 4.44 (1H, dd, J 4,5 9.6 , J 4,3 2.7, H-4),  5.64 (1H, d, J 3,4 2.1, H-3); HR-MS (Negative): Calcd. For C19H29F3N5O9 (M-H)-, 528.1923, Found 528.1783. 1 H NMR (D 2 O) δ 0.90 (3H, t, H-4 '), 1.32 (2H, sex,, H-3'), 1.57 (2H, p, H-2 '), 2.03 (3H, s, NHCOCH 3 ), 2.29 (2H, t, H-1 '), 3.36 (1H, dd, J 9a, 9b 14.4, J 9a, 8 6.8, H-9a), 3.56 to 3.58 (2H, m, H -7, H-9b), 3.98 (1H, ddd, J 8,7 12.3, J 8,9a 10.3, J 8,9b 3.4, H-8), 4.22 (1H, dd, J 5,4 =    J 5,6 10.3, H-5), 4.38 (1H, dd, J 6,5 10.3, J 6,7 <1, H-6), 4.44 (1H, dd, J 4,5 9.6   , J 4,3 2.7, H-4), 5.64 (1H, d, J 3,4 2.1, H-3); HR-MS (Negative): Calcd. For C 19 H 29 F 3 N 5 O 9 ( MH) - , 528.1923, Found 528.1783.
（実施例２）
 実施例２では、下記化４の合成経路に従ってノイラミン酸誘導体（１４）を合成した。 (Example 2)
In Example 2, neuraminic acid derivative (14) was synthesized according to the synthesis route of Chemical Formula 4 below.
Figure JPOXMLDOC01-appb-C000007
Figure JPOXMLDOC01-appb-C000007
（ステップ１）
5-Acetamido-4-(N-tert-butoxycarbonyl)amino-2,3,4,5,9-pentadeoxy-9-cyclopropane-carboxamido-D-glycero-D-galacto-non-2-enopyranosonic acid methyl ester (12) の合成
 上記実施例１における中間化合物であるアジド化合物(8) (243mg, 0.5 mmol)とシクロプロパンカルボンのN-ヒドロキシコハク酸イミジルエステル(シクロプロピルNHS エステル) (185mg, 1mmol)とを乾燥ＴＨＦ(3ml)に溶かし、撹拌しながらトリメチルホスフィン (1M in THF, 1mL, 1mmol) を室温下で窒素の発生が完了するまで滴下した。そして、反応混合物を１昼夜撹拌し他後、揮発成分を減圧下で留去させ、残渣を酢酸エチル(20 ml)と水 (20 ml)とで抽出した。水相を分離し、さらに酢酸エチル(10ml)で抽出した。そして、酢酸エチル抽出物は飽和NaHCO3及び水（20mL）で洗浄し、無水Na2SO4を加えて乾燥し、濾過し、減圧下で濃縮した。こうして得られた粗成物をシリカゲルカラムクロマトグラフィー(0～5% メタノール／酢酸エチル)で精製し、化合物(12)190mg (収率81%)を白色粉体として得た。得られた化合物（１２）の1H-NMRデータ及び、HR-MSデータを以下に示す。 (Step 1)
5-Acetamido-4- (N-tert-butoxycarbonyl) amino-2,3,4,5,9-pentadeoxy-9-cyclopropane-carboxamido-D-glycero-D-galacto-non-2-enopyranosonic acid methyl ester ( 12) Synthesis of Azide Compound (8) (243 mg, 0.5 mmol) which is an intermediate compound in Example 1 above and N-hydroxysuccinic acid imidyl ester of cyclopropanecarboxylic acid (cyclopropyl NHS ester) (185 mg, 1 mmol) were dried. It was dissolved in THF (3 ml), and trimethylphosphine (1M in THF, 1 mL, 1 mmol) was added dropwise with stirring until nitrogen generation was completed at room temperature. The reaction mixture was stirred for one day and night, and then the volatile components were distilled off under reduced pressure. The residue was extracted with ethyl acetate (20 ml) and water (20 ml). The aqueous phase was separated and further extracted with ethyl acetate (10 ml). The ethyl acetate extract was then washed with saturated NaHCO 3 and water (20 mL), dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduced pressure. The crude product thus obtained was purified by silica gel column chromatography (0-5% methanol / ethyl acetate) to obtain 190 mg (yield 81%) of Compound (12) as a white powder. 1 H-NMR data and HR-MS data of the obtained compound (12) are shown below.
1H NMR (CD3OD)δ 0.76 to 0.90 (4H, 2 × m, H-2', H-3'), 1.45 (9H, s, NHCOC(CH3)3), 1.57 to 1.61 (1H, m, H-1'), 2.01 (3H, s, NHCOCH3), 3.39 to 3.44 (2H, m, H-7, H-9b), 3.61 (1H, dd, J 9b,9a  13.7, J 9b,8  3.4, H-9b), 3.78 (3H, s, COOCH3 ), 3.94 (1H, ddd, J 8,7 9.6, J 8,9a 6.2, J 8,9b 3.4, H-8), 4.05 (1H, dd, J 5,4 =  J 5,6 10.3, H-5), 4.23 (1H, dd, J 6,5 10.3, J 6,7 <1, H-6), 4.46 (1H, dd, J 4,5 10.3, J 4,3 2.7, H-4),  5.82 (1H, d, J 3,4 2.0,  H-3); HR-MS (Positive): Calcd. for C21H33N3O9 (M+Na)+, 494.2114, Found 494.2149. 1 H NMR (CD 3 OD) δ 0.76 to 0.90 (4H, 2 × m, H-2 ', H-3'), 1.45 (9H, s, NHCOC (CH 3 ) 3 ), 1.57 to 1.61 (1H, m, H-1 '), 2.01 (3H, s, NHCOCH 3 ), 3.39 to 3.44 (2H, m, H-7, H-9b), 3.61 (1H, dd, J 9b, 9a 13.7, J 9b, 8 3.4, H-9b), 3.78 (3H, s, COOCH 3), 3.94 (1H, ddd, J 8,7 9.6, J 8,9a 6.2, J 8,9b 3.4, H-8), 4.05 (1H , dd, J 5,4 =    J 5,6 10.3, H-5), 4.23 (1H, dd, J 6,5 10.3, J 6,7 <1, H-6), 4.46 (1H, dd, J 4,5 10.3, J 4, 3 2.7, H-4), 5.82 (1H, d, J 3,4 2.0,   H-3); HR-MS (Positive): Calcd.for C 21 H 33 N 3 O 9 (M + Na) + , 494.2114, Found 494.2149.
（ステップ２）
5-Acetamido-4-(N,N'-bis-tert-butoxycarbonyl)guanidino-2,3,4,5,9-pentadeoxy-9-cyclopropanecarboxamido D-glycero-D-galacto-non-2-enopyranosonic acid methyl ester (13)の合成
 上記のようにして得られた化合物（１２）（188mg，0.4mmol）をジクロルメタン(2mL)とトリフルオロ酢酸(2mL)の混合物に室温下で加え、出発物質の消失を確認した後、反応液をエバポレートして、乾燥した。残渣をジクロルメタン (2ml)とトリエチルアミン (165μl,1.2mmol)の混合液に加えた。N,N'-bis-(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine (Bis-BocPCH)(185mg, 0.6mmol) のジクロルメタン (2ml)溶液を反応液に滴下し、５０℃で１６時間撹拌を行なった後、真空下で溶媒を留去し、シリカゲルカラムクロマトグラフィー(40～90% 酢酸エチル／ヘキサン)で精製し、化合物（１３）146mg (収率61%)を白色粉体として得た。得られた化合物（１３）の1H-NMRデータ及び、HR-MSデータを以下に示す。 (Step 2)
5-Acetamido-4- (N, N'-bis-tert-butoxycarbonyl) guanidino-2,3,4,5,9-pentadeoxy-9-cyclopropanecarboxamido D-glycero-D-galacto-non-2-enopyranosonic acid methyl Synthesis of ester (13) Compound (12) (188 mg, 0.4 mmol) obtained as described above was added to a mixture of dichloromethane (2 mL) and trifluoroacetic acid (2 mL) at room temperature to confirm the disappearance of the starting material. After that, the reaction solution was evaporated and dried. The residue was added to a mixture of dichloromethane (2 ml) and triethylamine (165 μl, 1.2 mmol). A solution of N, N'-bis- (tert-butoxycarbonyl) -1H-pyrazole-1-carboxamidine (Bis-BocPCH) (185 mg, 0.6 mmol) in dichloromethane (2 ml) was added dropwise to the reaction solution and stirred at 50 ° C. for 16 hours. The solvent was distilled off under vacuum, and the residue was purified by silica gel column chromatography (40-90% ethyl acetate / hexane) to obtain 146 mg (yield 61%) of compound (13) as a white powder. . 1 H-NMR data and HR-MS data of the obtained compound (13) are shown below.
1H NMR (CD3OD)δ 0.74 to 0.86 (4H, 2 × m, H-2', H-3'), 1.47, 1.52 (18H, 2 × s, 2 ×, NHCOC(CH3)3), 1.61 to 1.65  (2H, m, H-1'), 1.95 (3H, s, NHCOCH3), 3.37 (1H, dd, J 9a,9b  13.6, J 9a,8  6.2, H-9a), 3.45 (1H, dd, J 7,6 <1, J 7,8 8.9, H-7), 3.58 to 3.61 (1H, m, H-9b), 3.77 (3H, s, COOCH3 ), 3.93 (1H, ddd, J 8,7 9.6, J 8,9a 6.8, J 8,9b 3.4, H-8), 4.21 (1H, dd, J 5,4 =  J 5,6 10.3, H-5), 4.31 (1H, dd, J 6,5 11.0, J 6,7 <1, H-6), 5.04 (1H, dd, J 4,5 10.3 , J 4,3 2.7, H-4), 5.89 (1H, d, J 3,4 2.1, H-3); HR-MS (Positive): Calcd. for C27H43N5O11 (M+Na)+, 636.2857, Found 636.2884. 1 H NMR (CD 3 OD) δ 0.74 to 0.86 (4H, 2 × m, H-2 ', H-3'), 1.47, 1.52 (18H, 2 × s, 2 ×, NHCOC (CH 3 ) 3 ) , 1.61 to 1.65 (2H, m, H-1 '), 1.95 (3H, s, NHCOCH 3 ), 3.37 (1H, dd, J 9a, 9b 13.6, J 9a, 8 6.2, H-9a), 3.45 ( 1H, dd, J 7,6 <1, J 7,8 8.9, H-7), 3.58 to 3.61 (1H, m, H-9b), 3.77 (3H, s, COOCH 3 ), 3.93 (1H, ddd , J 8,7 9.6, J 8,9a 6.8 , J 8,9b 3.4, H-8), 4.21 (1H, dd, J 5,4 =    J 5,6 10.3, H-5), 4.31 (1H, dd, J 6,5 11.0, J 6,7 <1, H-6), 5.04 (1H, dd, J 4,5 10.3   , J 4,3 2.7, H-4), 5.89 (1H, d, J 3,4 2.1, H-3); HR-MS (Positive): Calcd.for C 27 H 43 N 5 O 11 (M + Na) + , 636.2857, Found 636.2884.
（ステップ３） (Step 3)
5-Acetamido-4-guanidino-2,3,4,5,9-pentadeoxy-9-cyclopropanecarboxamido-D-glycero-D-galacto-non-2-enopyranosonic acid Trifluoroacetic Acid Salt (14)の合成
 上記のようにして得られた化合物（１３）(100mg, 0.16mmol)をメタノール/THF(5/1)混合溶媒2mlに溶かし、０℃に冷却下、LiOH (25mg)／H2O(1ml)のアルカリ液を滴下し、12時間撹拌する。反応終了後、反応混合物をイオン交換樹脂（Dowex-50×8 (H+)）に通してpH7とした後、反応混合物をろ過し、エバポレートして、黄白色固体として得た。これをジクロロメタン(2mL)とトリフルオロ酢酸(2mL)に室温下で溶かした。出発物質が消滅していることを確認した後、エバポレータで乾燥させ、こうして得られた粗成物は逆相カラムクロマトグラフィーにより、0～20% メタノール水溶液（0.1% のトリフルオロ酢酸を含む）によって精製した。精製したフラクションを乾燥させ、凍結乾燥し、白色固体としてノイラミン酸誘導体（１４）を67mg (収率83%)で得た。得られたノイラミン酸誘導体（１４）の1H-NMRデータ及び、HR-MSデータを以下に示す。 Synthesis of 5-Acetamido-4-guanidino-2,3,4,5,9-pentadeoxy-9-cyclopropanecarboxamido-D-glycero-D-galacto-non-2-enopyranosonic acid Trifluoroacetic Acid Salt (14) The compound (13) (100 mg, 0.16 mmol) obtained in this manner was dissolved in 2 ml of a methanol / THF (5/1) mixed solvent, and cooled to 0 ° C., an alkaline solution of LiOH (25 mg) / H 2 O (1 ml) was added. Add dropwise and stir for 12 hours. After completion of the reaction, the reaction mixture was passed through an ion exchange resin (Dowex-50 × 8 (H + )) to adjust the pH to 7, and then the reaction mixture was filtered and evaporated to obtain a yellowish white solid. This was dissolved in dichloromethane (2 mL) and trifluoroacetic acid (2 mL) at room temperature. After confirming the disappearance of the starting material, it was dried with an evaporator, and the crude product thus obtained was subjected to reverse phase column chromatography by 0-20% aqueous methanol solution (containing 0.1% trifluoroacetic acid). Purified. The purified fraction was dried and freeze-dried to obtain 67 mg (yield 83%) of neuraminic acid derivative (14) as a white solid. 1 H-NMR data and HR-MS data of the obtained neuraminic acid derivative (14) are shown below.
1H NMR (D2O)δ 0.83 to 0.84 (4H, 2 × m, H-2', H-3'), 1.63 to 1.66 (2H, m, H-1'), 2.02 (3H, s, NHCOCH3), 3.34 (1H, dd, J 9a,9b  13.7, J 9a,8  7.6, H-9a), 3.55 (1H, dd, J 7,8 8.9 , J 7,6 1.4, H-7), 3.59 (1H, dd, J 9b,9a  13.7, J 9b,8  2.8, H-9b), 3.97 (1H, ddd, J 8,7 12.3, J 8,9a 6.9, J 8,9b 3.4, H-8), 4.20 (1H, dd, J 5,4 =  J 5,6 10.3, H-5), 4.36 (1H, dd, J 6,5 10.3, J 6,7 1.4, H-6), 4.42 (1H, dd, J 4,5 9.6 , J 4,3 2.7, H-4),  5.61 (1H, d, J 3,4 2.0, H-3); HR-MS (Negative): Calcd. For C18H25F3N5O9 (M-H)-, 512.1610, Found 512.1729. 1 H NMR (D 2 O) δ 0.83 to 0.84 (4H, 2 × m, H-2 ', H-3'), 1.63 to 1.66 (2H, m, H-1 '), 2.02 (3H, s, NHCOCH 3 ), 3.34 (1H, dd, J 9a, 9b 13.7, J 9a, 8 7.6, H-9a), 3.55 (1H, dd, J 7,8 8.9   , J 7,6 1.4, H-7), 3.59 (1H, dd, J 9b, 9a 13.7, J 9b, 8 2.8, H-9b), 3.97 (1H, ddd, J 8,7 12.3, J 8, 9a 6.9, J 8,9b 3.4, H-8), 4.20 (1H, dd, J 5,4 =    J 5,6 10.3, H-5), 4.36 (1H, dd, J 6,5 10.3, J 6,7 1.4, H-6), 4.42 (1H, dd, J 4,5 9.6   , J 4,3 2.7, H-4), 5.61 (1H, d, J 3,4 2.0, H-3); HR-MS (Negative): Calcd. For C 18 H 25 F 3 N 5 O 9 ( MH) - , 512.1610, Found 512.1729.
＜評 価＞
（ウイルスシアリダーゼに対する活性阻害の測定）
 上記のようにして合成したノイラミン酸誘導体（１１）及びノイラミン酸誘導体（１４）について、インフルエンザＡ型ウイルスのシアリダーゼ（A/PR/8/34、A/Memphis/1/71及びA/Duck/313/4/78）に対する活性阻害を、4-methylumbelliferyl N-acetylneuraminic酸（ＭＵＮＡ）を用いた蛍光測定法によって評価した。 <Evaluation>
(Measurement of activity inhibition against viral sialidase)
About neuraminic acid derivative (11) and neuraminic acid derivative (14) synthesized as described above, influenza A virus sialidase (A / PR / 8/34, A / Memphis / 1/71 and A / Duck / 313) / 4/78) was evaluated by fluorescence measurement using 4-methylumbelliferyl N-acetylneuraminic acid (MUNA).
 すなわち、２０ｍＭの酢酸ナトリウム緩衝液（ｐＨ６．０）１５μｌと、５段階の濃度に調整した所定量のノイラミン酸誘導体と、２ユニットのシアリダーゼを含むウイルス５μｌとを混合し、３７℃で１５分インキュベーションを行った。ここで、ウイルスシアリダーゼの１ユニットとは、ｐＨ６．０で３７℃で１分につき１ｎｍｏｌのＭＵの生成をするために必要な酵素量と定義される。
 次に、５μｌのＭＵＮＡを加え、さらに３７℃で１５分インキュベーションを行った。 That is, 15 μl of 20 mM sodium acetate buffer (pH 6.0), a predetermined amount of neuraminic acid derivative adjusted to 5 concentrations, and 5 μl of virus containing 2 units of sialidase were mixed and incubated at 37 ° C. for 15 minutes. Went. Here, one unit of viral sialidase is defined as the amount of enzyme required to produce 1 nmol of MU per minute at 37 ° C. at pH 6.0.
Next, 5 μl of MUNA was added and further incubated at 37 ° C. for 15 minutes.
 最後に、２００ｍＭ炭酸ナトリウム－重炭酸塩緩衝液（ｐＨ１０．６）２００μｌを加えて反応を終了させた。リリースされるノイラミン酸と等しい量のＭＵは、プレートリーダー（ミトラＬＢ９４０、バートルドＴｅｃｈｎｏｌｏｇｉｅｓ、Ｐｆｏｒｚｈｅｉｍ、ドイツ）によって、３５５ｎｍの励起と４６０ｎｍの蛍光によって検出された。ＩＣ５０値は、ＧｒａｐｈＰａｄ Ｐｒｉｓｍソフトウェア（サンディエゴ、ＣＡ、ＵＳＡ）で非線形回帰分析法を使用して計算した。また、比較のため、オセルタミビル（２）（登録商標名：タミフル）及びザナミビル（３）（登録商標名：リレンザ）の活性阻害についても、同様に測定した。結果を表１並びに図３及び図４に示す。 Finally, 200 μl of 200 mM sodium carbonate-bicarbonate buffer (pH 10.6) was added to terminate the reaction. An amount of MU equal to the released neuraminic acid was detected by a plate reader (Mitra LB940, Barthold Technologies, Pforzheim, Germany) with excitation at 355 nm and fluorescence at 460 nm. IC 50 values were calculated using non-linear regression analysis with GraphPad Prism software (San Diego, CA, USA). For comparison, the activity inhibition of oseltamivir (2) (registered trademark name: Tamiflu) and zanamivir (3) (registered trademark name: Relenza) was also measured in the same manner. The results are shown in Table 1 and FIGS.
 また、上記のようにして合成したノイラミン酸誘導体（１４）について、インフルエンザウイルスのシアリダーゼ（A/Narita/1/2009）に対する活性阻害を、4-methylumbelliferyl N-acetylneuraminic酸（ＭＵＮＡ）を用いた蛍光測定法によって評価した。 Moreover, about the neuraminic acid derivative (14) synthesize | combined as mentioned above, the activity measurement with respect to the sialidase (A / Narita / 1/2009) of influenza virus was measured with the fluorescence measurement using 4-methylumbelliferyl * N-acetylneuraminic acid (MUNA). Evaluated by law.
 すなわち、プラスチック製９６穴ウエルに試料を溶かした酢酸緩衝液（２０ｍM, pH6.0)5 uLを加え、次いでインフルエンザウイルス（シアリダーゼ活性として2単位）液を５uLを加え、３７℃, 15分間、pre-incubateした。続いて、4-MU-Neu5Ac (4-メチルウンベリフェリル-シアル酸）を溶解した基質液を5μLを各ウエルに加え３７℃で15分間保温した後、100 mM Sodium carbonate buffer （100 mM 炭酸緩衝液, pH10.6）を加え反応を停止した。シアリダーゼ活性により遊離した4-MUの蛍光 (Ex:355nm, Em:460 nm) を測定し、IC50を求めた。結果を表１に示す。 That is, 5 μL of acetate buffer (20 mM, pH 6.0) dissolved in a 96-well plastic well was added, and then 5 μL of influenza virus (2 units as sialidase activity) was added, followed by pretreatment at 37 ° C. for 15 minutes. -Incubate. Subsequently, 5 μL of a substrate solution in which 4-MU-Neu5Ac (4-methylumbelliferyl-sialic acid) was dissolved was added to each well and incubated at 37 ° C. for 15 minutes, and then 100 μmM sodium carbonate buffer buffer (100 μmM carbonate buffer) Liquid, pH 10.6) was added to stop the reaction. IC50 was determined by measuring the fluorescence (Ex: 355 nm, Em: 460 nm) of 4-MU released by sialidase activity. The results are shown in Table 1.
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
 図１及び表1に示すように、ノイラミン酸誘導体（１１）は、A/PR/8/34(H1N1)、H3N2及びH5N3のウイルスのシアリダーゼに対して、高い活性阻害作用を奏することが分かった。 As shown in FIG. 1 and Table 1, the neuraminic acid derivative (11) was found to have a high activity inhibitory action on the sialidases of A / PR / 8/34 (H1N1), H3N2 and H5N3 viruses. .
 また、図２及び表１に示すように、ノイラミン酸誘導体（１４）は、A/PR/8/34(H1N1)、H3N2及びH5N3のウイルスのシアリダーゼに対して、ノイラミン酸誘導体（１１）よりも高い活性阻害作用を示し、ザナミビル（３）やオセルタミビル（２）と同程度であった。さらに、表１に示すように、ノイラミン酸誘導体（１４）のA/Narita/1/2009(H1N1) のウイルスのシアリダーゼに対する活性阻害の効果は、ザナミビル（３）やオセルタミビル（２）と同程度であった。 In addition, as shown in FIG. 2 and Table 1, neuraminic acid derivative (14) is more effective than neuraminic acid derivative (11) against sialidases of A / PR / 8/34 (H1N1), H3N2 and H5N3 viruses. It showed a high activity inhibitory action and was similar to zanamivir (3) and oseltamivir (2). Furthermore, as shown in Table 1, the effect of inhibiting the activity of neuraminic acid derivative (14) on the sialidase of A / Narita / 1/2009 (H1N1) is similar to that of zanamivir (3) and oseltamivir (2). there were.
 以上の結果は、ノイラミン酸誘導体（１１）及びノイラミン酸誘導体（１４）がシアリダーゼ活性阻害剤及び抗ウイルス薬として充分な効果を有することを明らかにしている。 The above results reveal that neuraminic acid derivative (11) and neuraminic acid derivative (14) have sufficient effects as sialidase activity inhibitors and antiviral agents.
（ヒト－シアリダーゼ（ＮＥＵ１、ＮＥＵ２及びＮＥＵ４）に対する活性阻害の測定）
 上記のようにして合成したノイラミン酸誘導体（１１）及びノイラミン酸誘導体（１４）について、３種類（ＮＥＵ１、ＮＥＵ２、及びＮＥＵ４）のヒト－シアリダーゼに対する活性阻害を、4-methylumbelliferyl N-acetylneuraminic酸（ＭＵＮＡ）を用いた蛍光測定法によって評価した。 (Measurement of activity inhibition against human-sialidase (NEU1, NEU2 and NEU4))
For the neuraminic acid derivative (11) and neuraminic acid derivative (14) synthesized as described above, activity inhibition against three types of human-sialidase (NEU1, NEU2, and NEU4) was inhibited by 4-methylumbelliferyl N-acetylneuraminic acid (MUNA). ) Was evaluated by a fluorescence measurement method.
 すなわち、５０μＬの反応液において、５００ｍＭの酢酸ナトリウム緩衝液（ＮＥＵ１及びＮＥＵ４に対してはｐＨ４．６、ＮＥＵ２に対してはｐＨ５．５）１０μＬ、５段階の濃度に調整した所定量のノイラミン酸誘導体、５０μｇの子牛由来のアルブミン、２０ｎmolのＭＵＮＡ、所定量の組み換え型ヒト－シアリダーゼを混合し、３７℃で３０分間インキュベーションを行った。そしてリリースされるノイラミン酸と等しい量のＭＵを、ウイルスシアリダーゼに対する活性阻害の測定の場合と同様の方法で測定した。
 なお、ＩＣ５０値は、マイクロソフトエクセルによる非線形回帰分析法を用いて計算した。
 また、比較のため、ザナミビル（３）（登録商標名：リレンザ)の活性阻害についても同様に測定した。 That is, in a 50 μL reaction solution, a 500 mM sodium acetate buffer solution (pH 4.6 for NEU1 and NEU4, pH 5.5 for NEU2) 10 μL, a predetermined amount of neuraminic acid derivative adjusted to 5 concentrations 50 μg of calf-derived albumin, 20 nmol of MUNA, and a predetermined amount of recombinant human-sialidase were mixed and incubated at 37 ° C. for 30 minutes. The amount of MU equal to the amount of neuraminic acid released was measured by the same method as in the case of measuring activity inhibition against viral sialidase.
The IC 50 value was calculated using a non-linear regression analysis method by Microsoft Excel.
For comparison, the activity inhibition of zanamivir (3) (registered trademark name: Relenza) was also measured in the same manner.
（ヒト－シアリダーゼ（ＮＥＵ３）に対する活性阻害の測定）
 上記のようにして合成したノイラミン酸誘導体（１１）及びノイラミン酸誘導体（１４）について、ヒト－シアリダーゼＮＥＵ３に対する活性阻害を、malononitrileを用いた高速液体クロマトグラフィーによる蛍光測定法によって評価した。 (Measurement of activity inhibition against human-sialidase (NEU3))
The neuraminic acid derivative (11) and neuraminic acid derivative (14) synthesized as described above were evaluated for activity inhibition against human-sialidase NEU3 by a fluorescence measurement method by high performance liquid chromatography using malononitrile.
 すなわち、５μＬの反応液において、５００ｍＭの酢酸ナトリウム緩衝液（ｐＨ４．５）１０μＬ、５段階の濃度に調整した所定量のノイラミン酸誘導体、５０μｇの子牛由来のアルブミン、５ｎmolのガングリオシドＧＭ３、５０μｇのトリトンＸ－１００、所定量の組み換え型ヒト－シアリダーゼを混合し、３７℃で３０分間インキュベーションを行った。 That is, in a 5 μL reaction solution, 10 μL of 500 mM sodium acetate buffer (pH 4.5), a predetermined amount of neuraminic acid derivative adjusted to 5 concentrations, 50 μg of calf-derived albumin, 5 nmol of ganglioside GM3, 50 μg Triton X-100 and a predetermined amount of recombinant human-sialidase were mixed and incubated at 37 ° C. for 30 minutes.
 最後に、２００ｍＭ炭酸ナトリウム－重炭酸塩緩衝液（ｐＨ１０．６）２００μｌを加えて反応を終了させた。そしてＧＭ３からリリースされたシアル酸をmalononitrileを用いた高速液体クロマトグラフィーによる蛍光測定法によって測定した。
 なお、ＩＣ５０値は、マイクロソフトエクセルによる非線形回帰分析法を用いて計算した。
 また、比較のため、ザナミビル（３）（登録商標名：リレンザ)の活性阻害についても同様に測定した。 Finally, 200 μl of 200 mM sodium carbonate-bicarbonate buffer (pH 10.6) was added to terminate the reaction. And the sialic acid released from GM3 was measured by the fluorescence measuring method by the high performance liquid chromatography using malononitrile.
The IC 50 value was calculated using a non-linear regression analysis method by Microsoft Excel.
For comparison, the activity inhibition of zanamivir (3) (registered trademark name: Relenza) was also measured in the same manner.
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
 結果を表２並びに図３及び図４に示す。
 表２及び図３に示すように、ノイラミン酸誘導体（１１）は、ヒト由来の４種類のシアリダーゼ（ＮＥＵ１、ＮＥＵ２、ＮＥＵ３及びＮＥＵ４）のいずれに対しても、ヒト－シアリダーゼ活性阻害（ＩＣ５０）はほとんど認められない程度であった。 The results are shown in Table 2 and FIGS.
As shown in Table 2 and FIG. 3, neuraminic acid derivative (11) inhibits human-sialidase activity (IC 50 ) against any of four types of sialidases derived from humans (NEU1, NEU2, NEU3 and NEU4). Was almost unacceptable.
 また、表２及び図４に示すように、ノイラミン酸誘導体（１４）についても、ヒト由来の４種類のシアリダーゼ（ＮＥＵ１、ＮＥＵ２、ＮＥＵ３及びＮＥＵ４）のいずれに対しても、ヒト－シアリダーゼ活性阻害（ＩＣ５０）はほとんど認められない程度であった。 Further, as shown in Table 2 and FIG. 4, the neuraminic acid derivative (14) was also inhibited against human-sialidase activity against any of the four types of sialidases derived from humans (NEU1, NEU2, NEU3 and NEU4). IC 50 ) was hardly recognized.
 以上の結果は、ノイラミン酸誘導体（１１）及びノイラミン酸誘導体（１４）を抗ウイルス薬として用いた場合においても、副作用の可能性が極めて少ないことを示している。 The above results show that even when neuraminic acid derivative (11) and neuraminic acid derivative (14) are used as antiviral drugs, the possibility of side effects is extremely low.
 この発明は上記発明の実施の態様及び実施例の説明に何ら限定されるものではない。特許請求の範囲を逸脱せず、当業者が容易に想到できる範囲で種々の変形態様もこの発明に含まれる。 This invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications are also included in the present invention as long as those skilled in the art can easily conceive without departing from the scope of the claims.
ノイラミン酸誘導体（１１）のウイルス（Ｈ１Ｎ１、Ｈ３Ｎ２及びＨ５Ｎ３）シアリダーゼに対する活性阻害の測定結果を示すグラフである。It is a graph which shows the measurement result of the activity inhibition with respect to the virus (H1N1, H3N2, and H5N3) sialidase of a neuraminic acid derivative (11). ノイラミン酸誘導体（１４）のウイルス（Ｈ１Ｎ１、Ｈ３Ｎ２及びＨ５Ｎ３）シアリダーゼに対する活性阻害の測定結果を示すグラフである。It is a graph which shows the measurement result of the activity inhibition with respect to the virus (H1N1, H3N2, and H5N3) sialidase of a neuraminic acid derivative (14). ノイラミン酸誘導体（１１）のヒト－シアリダーゼ（ＮＥＵ１、ＮＥＵ２、ＮＥＵ３及びＮＥＵ４）に対する活性阻害の測定結果を示すグラフである。It is a graph which shows the measurement result of the activity inhibition with respect to the human sialidase (NEU1, NEU2, NEU3, and NEU4) of neuraminic acid derivative (11). ノイラミン酸誘導体（１４）のヒト－シアリダーゼ（ＮＥＵ１、ＮＥＵ２、ＮＥＵ３及びＮＥＵ４）に対する活性阻害の測定結果を示すグラフである。It is a graph which shows the measurement result of the activity inhibition with respect to the human sialidase (NEU1, NEU2, NEU3, and NEU4) of a neuraminic acid derivative (14).
 本発明のノイラミン酸誘導体及びシアリダーゼ活性阻害剤は、シアリダーゼの機能を調べる生物学的ツールとして利用することができる。また、本発明のノイラミン酸誘導体は、ウイルスのシアリダーゼ活性を選択的に阻害し、ヒトのシアリダーゼ活性をほとんど阻害しないことから、副作用の少ない抗インフルエンザ薬等に利用できる。 The neuraminic acid derivative and sialidase activity inhibitor of the present invention can be used as biological tools for examining the function of sialidase. In addition, the neuraminic acid derivative of the present invention selectively inhibits viral sialidase activity and hardly inhibits human sialidase activity, and thus can be used as an anti-influenza drug having few side effects.

Claims (5)

  1.  下記化学式（１）（ただし、Ｒ１及びＲ２は、水素又は炭素数１～８の分岐してもよいアルキル基若しくは炭素数１～８の分岐してもよいシクロアルキル基を示し、Ｒ３は水素又はフッ素を示す）又はその薬学上許容される塩、水和物、溶媒和物若しくはプロドラッグからなることを特徴とするノイラミン酸誘導体。
    Figure JPOXMLDOC01-appb-C000001
         The following chemical formula (1) (wherein R 1 and R 2 represent hydrogen, an alkyl group having 1 to 8 carbon atoms which may be branched or a cycloalkyl group having 1 to 8 carbon atoms which may be branched, and R 3 Represents hydrogen or fluorine) or a neuraminic acid derivative comprising a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof.
    Figure JPOXMLDOC01-appb-C000001
  2.  Ｒ１はｎ－ブチル基であり、Ｒ２はメチル基であり、Ｒ３は水素であることを特徴とする請求項１記載のノイラミン酸誘導体。 The neuraminic acid derivative according to claim 1, wherein R 1 is an n-butyl group, R 2 is a methyl group, and R 3 is hydrogen.
  3.  Ｒ１はシクロペンチル基であり、Ｒ２はメチル基であり、Ｒ３は水素であることを特徴とする請求項１記載のノイラミン酸誘導体。 The neuraminic acid derivative according to claim 1, wherein R 1 is a cyclopentyl group, R 2 is a methyl group, and R 3 is hydrogen.
  4.  請求項１乃至３のいずれか1項に記載のノイラミン酸誘導体を有効成分として含有することを特徴とするシアリダーゼ活性阻害剤。 A sialidase activity inhibitor comprising the neuraminic acid derivative according to any one of claims 1 to 3 as an active ingredient.
  5.  請求項１乃至３のいずれか1項に記載のノイラミン酸誘導体を有効成分として含有することを特徴とする抗インフルエンザ薬。 An anti-influenza drug comprising the neuraminic acid derivative according to any one of claims 1 to 3 as an active ingredient.
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