WO2007081031A1 - Glycosyltransferase inhibitor - Google Patents

Glycosyltransferase inhibitor Download PDF

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WO2007081031A1
WO2007081031A1 PCT/JP2007/050534 JP2007050534W WO2007081031A1 WO 2007081031 A1 WO2007081031 A1 WO 2007081031A1 JP 2007050534 W JP2007050534 W JP 2007050534W WO 2007081031 A1 WO2007081031 A1 WO 2007081031A1
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transferase
compound
group
reaction
chemical
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PCT/JP2007/050534
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French (fr)
Japanese (ja)
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Shin-Ichiro Nishimura
Hirosato Kondo
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Hokkaido University
Shionogi & Co., Ltd.
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Publication of WO2007081031A1 publication Critical patent/WO2007081031A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A61K31/7072—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid having two oxo groups directly attached to the pyrimidine ring, e.g. uridine, uridylic acid, thymidine, zidovudine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A61K31/708—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid having oxo groups directly attached to the purine ring system, e.g. guanosine, guanylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C07H19/207—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide

Definitions

  • the present invention relates generally to the field of enzymology. More particularly, the present invention relates to an inhibitor of glycosyltransferase and use thereof.
  • glycosyltransferases are generally composed of simple sugars such as UDP, GDP, and CMP.
  • V transfer sugars one by one to sugar acceptors with various structures using sugar nucleotides with attached nucleotidyl groups as sugar donors. Controlling the synthesis of sugar chains with complex expression, timing and activity of these various glycosyltransferases, it is thought to have an extremely important influence on the phenotype of cells.
  • Non-Patent Document 1 Palci c, MM, ⁇ , 3 ⁇ 4. (2001; Assays for Glycosyltransf erases, Trends m Glycoscience and Glycotechnology, 13, 361-370).
  • Non-patent Document 2 Fluorescence Resonance Energ y Transfer: also referred to as FRET in the present specification
  • This method overcomes the drawbacks of the conventional method as described above, and allows the observation of the state of continuous enzyme reaction, which is safe, simple, and highly sensitive, and was impossible in principle by the conventional method.
  • Non-Patent Document 8 Edmund D. Matayoshi, Gary T. Wang, Gran t A. Krafft, John Erickson (1990)
  • Non-Patent Document 9 Science, 247, 954—957, Gary T. Wang, et. al. (1993), Anal. Biochem. 210, 351—359
  • non-specific Permit 10 Linda LM, Clark W. Smith, Zhong— See Yin Zhang (1992), J. Med.
  • non-patent document ll Kimito Washiya, Tetsuya Furuike, Fumio Nakajima, Yuan J. Lee, Shin-Ichiro Nishimura (2000), Anal. Biochem. 283, 39-48).
  • This report demonstrates the usefulness of the method for ⁇ 2,6 sialyltransferases. The reason why this has become possible is that the importance of the enzyme has been recognized, and that the knowledge of the mode of binding between the substrate and the enzyme active site has become available. That's what I mean.
  • Patent Document 1 WO2004Z069855.
  • a design focusing on galactose transferase tributophan was proposed.
  • Patent Document 1 International Publication No. 2004Z069855 Pamphlet
  • Non-patent literature l Palcic, MM, Keiko, S. (2001) Assays for Glycosyltransf erases, Trends in Glycoscience and Glycotechnology, 13, 361—370
  • Non-patent literature 2 Nathalie, P., Sylvain, C., Hugues. D (1995), Angew. Chem. Int. Eds. Engl., 34, 1239—1241.
  • Non-Patent Document 3 Kaoru Omichi, Tokuji Ikenaka (1986), J. Biochem. 99, 291-294.
  • Non-Patent Document 4 Soumitra. Et.al. (1994), Nucleic Acids Research, 22, 315 5--3159.
  • Non-Patent Document 5 Lee, S, P. et. Al. (1995), Anal. Biochem. 227, 295- 301.
  • Patent Document 6 Kyung, B. Lee, Koji Matsuoka, Shin— Ichiro Nishimura, Y uan. C. Lee. (1995), Anal. Biochem. 230, 31— 36.
  • Non-Patent Document 7 Koji Matsuoka, Shin -Ichiro Nishimura, Yuan C. Lee. (1 995), Carbohydrate Research, 276, 31-42.
  • Non-Patent Document 8 Edmund D. Matayoshi, Gary T. Wang, Grant A. Krafft, John Erickson (1990), Science, 247, 954—957.
  • Non-Patent Document 9 Gary T. Wang, et. Al. (1993), Anal. Biochem. 210, 351—3 59.
  • Non-Patent Document 10 Linda L. M., Clark W. Smith, Zhong— Yin Zhang (1992), J. Med. Chem. 35, 3727-3730.
  • Non-Patent Document 11 Kimito Washiya, Tetsuya Furuike, Fumio Nakajima, Yuan C. Lee, Shin-Ichiro Nishimura (2000), Anal. Biochem. 283, 39-48 Disclosure of the Invention
  • an object of the present invention is to provide a method for designing a novel mechanism of transglycosylase activity regulation (for example, an inhibitor) different from conventional methods.
  • the above-mentioned problem is focused on conformational change due to the substrate of the enzyme, and by applying the relationship between the change and catalytic activity to the design of an enzyme activity regulator (for example, inhibitor), Specifically, the problem was solved by providing a novel compound in which a bulky group was introduced into a modified nucleotide and sugar via a triazole group, an oxime group, a hydrazone group, or the like.
  • the present invention provides the following.
  • R is Anolequinore
  • B is a sugar component
  • C is a nucleotidyl group
  • sugar component is GlcNAc
  • A is an azide
  • the above C is a phosphate of adenosine, deoxyadenosine, guanosine, deoxyguanosine, cytidine, deoxythymidine, thymidine (ribothymidine), deoxythymidine or uridine, or a modified form thereof.
  • Compound or its salt is a phosphate of adenosine, deoxyadenosine, guanosine, deoxyguanosine, cytidine, deoxythymidine, thymidine (ribothymidine), deoxythymidine or uridine, or a modified form thereof.
  • the above B is D-galactose, L-fucose, sialic acid, D-GlcNAc, D-GaIN Ac, D-ManNAc, D-mannose, or D-glucose or a derivative thereof.
  • B is a sugar component
  • C is a nucleotidyl group
  • X is a bulky group
  • C is a phosphate of adenosine, deoxyadenosine, guanosine, deoxyguanosine, cytidine, deoxythymidine, thymidine (ribothymidine), deoxythymidine or uridine or a variant thereof. Or its salt.
  • the above B is D-galactose, L-fucose, sialic acid, D-GlcNAc, D-GalNAc, D-ManNAc, D-mannose, or D-gnolecose or its derivatives, items 7, 7 ', The compound or its salt in any one of 8-10.
  • a glycosyltransferase inhibitor comprising the compound according to any one of items 7 to 13 or 7 ′.
  • glycosyltransferases are ⁇ , 4 galactose transferase, ⁇ 1, 3 galactose transferase, ⁇ 81, 4-galactose transferase,
  • glycosyltransferase inhibitor according to Item 14 selected from the group consisting of 1,6 xylosetransferase, ⁇ -xylosetransferase, j81,3 glucuronyltransferase and hyaluronic acid synthase.
  • glycosyltransferases are al, 4 galactose transferase, ⁇ 1, 3 galactose transferase, ⁇ 81, 4-galactose transferase,
  • composition for treating or preventing a condition, disorder or disease caused by an abnormal activity of glycosyltransferase comprising the compound according to any one of items 7 to 13.
  • a method for screening for an inhibitor of glycosyltransferase comprising the following steps:
  • glycosyltransferase is fucose transferase V;
  • the acceptor is a compound having the acceptor and the acceptor.
  • the conditions under which the glycosyltransferase reaction proceeds are as follows: 50 mM HEPES (pH 7.2), 10 mM manganese chloride, ImM acceptor, 200 ⁇ 2. donor, 2.5 mU fucose transferase (FucT) V, 30 M inhibitor candidate compound 23.
  • glycosyltransferase is ⁇ ⁇ , 6-fucose transferase Vin,
  • the acceptor is a compound having the acceptor and the acceptor.
  • the conditions under which the glycosyltransferase reaction proceeds are as follows: 50 mM force codylate buffer (pH 7.5), 10 mM manganese chloride, 100 acceptor, 50 ⁇ donor, 80 ⁇ ⁇ ] fucose transferase VIII, 50 ⁇ 25.
  • glycosyltransferase is a sialyltransferase
  • the acceptor is a compound having the acceptor and the acceptor.
  • the conditions under which the glycosyltransferase reaction proceeds are 40 mM cacodylate-HCl (pH 7.5), 0.5 g / ml BSA, 0.1% surfactant, ImM acceptor, 200 M donor, 1 mU a 2-6 sial. 27.
  • the conditions under which the glycosyltransferase reaction proceeds are 40 mM cacodylate-HCl (pH 7.5), 0.5 g / ml BSA, 0.1% surfactant, ImM acceptor, 200 M donor, 1 mU ⁇ 2-3 sial.
  • glycosyltransferase is N-acetylgalatatosamine transferase, and the acceptor is N-acetylgalatatosamine transferase
  • Item 22 The method according to Item 21, wherein the donor is UDP-GalNAc.
  • the conditions under which the glycosyltransferase reaction proceeds are as follows: 50 mM imidazole-HC1 buffer solution (containing 0.1% poly (oxyethylene), pH 7.2), 500 ⁇ acceptor, 100 ⁇ donor, 100 ⁇ inhibitor candidate 29.
  • the time for reacting the acceptor and the donor is set based on the initial speed of glycosyltransferase, wherein the initial speed is a reaction of mixing the acceptor, the glycosyltransferase and the donor, Item 22.
  • the above reaction is carried out under conditions of 50 mM force codylate buffer (pH 7.5), 10 mM manganese chloride, 100 M acceptor, 50 ⁇ donor, 80 ⁇ fucose transferase VIII The method according to 32.
  • reaction was carried out using 40 mM cacodylate-HCl (pH 7.5), 0.5 g / ml BSA, 0.1% surfactant, ImM acceptor, 200 M donor, ⁇ 2-6 sialyltransferase (SEQ ID NO: 6 (amino acid Sequence)) According to item 32, which is performed under the condition of lmU Law.
  • the reaction was carried out using 40 mM cacodylate-HCl (pH 7.5), 0.5 g / ml BSA, 0.1% surfactant, ImM acceptor, 200 M donor, ⁇ 2-3 sialyltransferase (SEQ ID NO: 7 (amino acid Sequence))
  • the method according to item 32 which is carried out under the condition of lmU.
  • the reaction consists of 50 mM imidazole-HC1 buffer solution (containing 0.1% poly (oxyethylene), ⁇ 7.2), 500 ⁇ ⁇ acceptor, 100 ⁇ donor, 10 mM saline, manganese, lng / ⁇ 1 ⁇ — 33.
  • reaction rate is measured by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS).
  • Item 33 The method according to Item 32, wherein the initial velocity is calculated at a reaction rate of 10 to 20%.
  • Item 33 The method according to Item 32, wherein the initial speed is 60 minutes in the case of fucose transferase VIII. (Item 41)
  • the acceptor, the donor, the glycosyltransferase, and the substance used for the click reaction are mixed and reacted, and the influence of the substance used for the click reaction on the enzyme reaction is confirmed. Process for avoiding the influence
  • the glycosyltransferase is fucose transferase (FucT) V, and the material used for the click reaction is 6 azido GDP fucose, acetylene compound, copper sulfate, tris [(1-benzyl-1H-1, 2, 3- 42.
  • the glycosyltransferase is fucose transferase ⁇ , and the substance used for the click reaction is 6 azide GDP fucose, acetylene compound, copper sulfate, tris [(1 benzyl-1H— 1, 2, 3 triazole-4 yl) 42.
  • the glycosyltransferase is ⁇ 2,3 sialyltransferase, and the substance used in the click reaction 1S alkyne compound, copper sulfate, ascorbic acid, tris [(1 benzyl 1 1H— 1, 2, 3 -triazole— 4 42.
  • the glycosyltransferase is ⁇ 2,6 sialyltransferase, and the substance used in the click reaction 1S alkyne compound, copper sulfate, ascorbic acid, tris [(1 benzyl 1 1H— 1, 2, 3 -triazole-4 42.
  • the glycosyltransferase is N-acetylgalatatosamine transferase, and the substance used for the click reaction is UDP—N3—GalNAc, acetylene compound, tris [(1 benzyl— 1H-1, 2, 3 triazole— 4 44.
  • a method for calculating the inhibition constant of an inhibitor of glycosyltransferase comprising the following steps:
  • glycosyltransferase an acceptor of the glycosyltransferase, a donor of the glycosyltransferase, and the inhibition Mixing with a candidate drug and reacting under conditions and time for the glycosyltransferase reaction to proceed;
  • step II measuring the reaction product of the acceptor and the donor in the reaction product after the reaction of step I);
  • step III) The step of calculating the value force measurement constant measured in step i)
  • the conditions under which the glycosyltransferase reaction proceeds are 50 mM HEPES (pH 7.2), 10 mM manganese chloride, ImM acceptor, 50 or 200 donors, 2.5 mU fucose transferase (FucT) V, 0 to: LOO ⁇ 49.
  • glycosyltransferase reaction proceeds are as follows: 50 mM force codylate buffer (pH 7.5), 10 mM sodium chloride manganese, 100 acceptor, 12.5-75 ⁇ M donor, 8
  • glycosyltransferase reaction proceeds were 40 mM cacodylate-HCl (pH 7.5), 0.5 g / ml BSA, 0.1% surfactant, ImM acceptor, 200 M donor, 1 mU ⁇ 2-6 sial. 49.
  • a method according to item 48 which is an acid transferase (SEQ ID NO: 6 (amino acid sequence)), 0 to 100 ⁇ inhibitor candidate compound mixture.
  • glycosyltransferase reaction proceeds are 40 mM cacodylate-HCl (pH 7.5), 0.5 g / ml BSA, 0.1% surfactant, ImM acceptor, 200 M donor, 1 mU a 2-3 sial 49.
  • the conditions under which the glycosyltransferase reaction proceeds are 50 mM imidazole-HC1 buffer solution ( 0.1% containing poly (oxyethylene), pH 7.2), 500 ⁇ acceptor, 100 ⁇ donor, 10 mM manganese chloride, lng / / X 1 N-acetyl galatatosamine transferase ppGalNAcT-2, 0- 49.
  • the present invention provides a glycosyltransferase inhibitor having a completely novel structure and a screening method thereof.
  • Fig. 1 shows a spectrum in which only the labeled chitotriose has power in the synthesis of the glycosyl acceptor of Example 1 of the present invention (top) and a spectrum in which only the labeled galactosylchitotriose has only power (bottom). .
  • FIG. 2 shows the result of confirming the presence of the product by electrospray Z mass spectrometry (ESI-MS) in Example 1.
  • FIG. 3 shows the results showing that the compound of the present invention has the ability to inhibit fucose transferase.
  • the amount of the product of the enzyme reaction is shown. The smaller the value, the stronger the inhibitory activity.
  • Fig. 4 shows the reaction over time with inhibitor activity when the reaction was carried out in a total of 2 reactions (+) with the inhibitor mixture (200 ⁇ ) added to the reaction conditions (-). It is a result which shows that it measures.
  • Figure 5 shows the donor concentrations measured at 50 ⁇ , 200 ⁇ , and the inhibitor concentrations at 100 ⁇ , 50 ⁇ , ⁇ , 12.5 / ⁇ ⁇ , ⁇ / ⁇ ⁇ ⁇ From the results of changes over time, the result of Dickson plot when the enzyme reaction time is 10 minutes, which is the measurement range of the initial rate, is shown.
  • FIG. 6B shows the click reaction yields measured by ESI-MS for acetylene compounds (acetylene library A1 to A36, L2N and L3N).
  • the yield of the graph is ES
  • the relative ratio of peak intensity ratio in the IMS negative mode is shown.
  • FIG. 7A shows a mixture of inhibitor candidate compounds (200) in addition to the reaction conditions (control).
  • Fig. 7B shows the addition of a mixture of candidate inhibitor compounds (30 ⁇ () (acetylene compounds: acetylene compounds A1 to A36, L2N and L3N, N3GDP-Fuc (+)) and their inhibitors
  • acetylene compounds acetylene compounds A1 to A36, L2N and L3N, N3GDP-Fuc (+)
  • acetylene derivatives A1 to A36 and L2N added with only the acetylene derivative corresponding to the candidate compound is shown.
  • FIG. 8A shows a reaction mixture (control) further added with a naphthalene-containing sugar nucleotide mixture (13b, 200 M), or an inhibitor mixture (13e, 200 having a phosphate with known Ki). The results of a total of three reactions with addition of ⁇ ) are shown.
  • Fig. 8 ⁇ shows the results for A16 in the acetylene library, with donor concentrations measured at 37.5 ⁇ , 20 ⁇ , 5 ⁇ and inhibitor concentrations at 0 ⁇ , 0.5 ⁇ , and 1 ⁇ . From the results of changes over time, Dickson plots are shown when the enzyme reaction time is 10 minutes, which is the measurement range of the initial rate.
  • Figure 8C shows the donor concentration for L3N in the acetylene library at 5% and 25%.
  • FIG. 8D shows the result of MALDI-TOFMS of the glycopeptide.
  • FIG. 8 (b) shows the time course of the reaction rate of a newly synthesized glycopeptide which is an acceptor of fucose transferase.
  • FIG. 8F shows the effect of the click reaction residue (Cu 2+ , GDP-6-N3 fucose) on the enzyme reaction. The amount of the product of the enzyme reaction is shown. The smaller the value, the stronger the inhibitory activity.
  • FIG. 8G shows the results of an enzyme activity inhibition experiment in a compound in which acetylene compound strength described in an acetylene library was also synthesized.
  • Figure 8H shows the Dickson plot for GDP-6-N3-Fucose.
  • FIG. 81 shows inhibitory concentrations for GDP-6-N3 fucose and acetylene libraries A2, A3 and A34.
  • FIG. 9 shows the details of the reaction product over time by direct mass analysis with MALDI-TOFMS (Bruker, UltraFLEX) using deuterium aoWR-labeled compound as an internal standard. .
  • the amount of the enzyme reaction product is shown. The smaller the value, the stronger the inhibitory activity.
  • FIG. 10 shows how enzyme activity is traced in Example 2. Circles indicate sugars transferred by enzyme activity. It is understood that this causes the spectrum to shift.
  • FIG. 11 shows the results of an enzyme activity inhibition experiment in Example 2.
  • FIG. 12A shows an ESI mass spectrum of an inhibitor preparation containing compound 31.
  • FIG. 12B shows the ionization ratio according to ESI mass spectrum of an inhibitor preparation containing a compound synthesized by a click reaction of an acetylene compound and a 5-position azide CMP sialic acid derivative.
  • the yield of the graph shows the relative ratio of peak intensity ratio in ESI-MS negative mode.
  • FIG. 12C shows an ionization ratio by ESI mass spectrum of an inhibitor preparation containing a compound synthesized by a click reaction of an acetylene compound and a 9-position azide CMP sialic acid derivative.
  • the yield of the graph shows the relative ratio of peak intensity ratio in ESI-MS negative mode.
  • FIG. 13 shows the inhibitory effect of inhibitor 31 on a-2,6-sialyltransferase and ⁇ -2,3-sialyltransferase.
  • the graph shows 0.26, indicating that the force was also suppressed to 26%, that is, the inhibition rate was 74%. Yes. Shows the amount of the product of the enzyme reaction. The smaller the value, the stronger the inhibitory activity.
  • FIG. 14 is a schematic diagram of a compound synthesized by a click reaction of an acetylene compound ( ⁇ 1 to ⁇ 36 of the acetylene library) and a 5-position azide CMP sialic acid derivative. The result of the inhibitory activity measurement about a transferase is shown.
  • FIG. 15A shows compounds synthesized by a click reaction of an acetylene compound (A1 to A36 of the acetylene library) and a 5-position azide CMP sialic acid derivative.
  • FIG. 15B shows the synthesis of a compound synthesized by a click reaction of an acetylene compound (A37 to A41 in the acetylene library) and a 5-position azide CMP sialic acid derivative.
  • Fig. 15C shows ⁇ -2,3-sialic acid transfer of acetylene compounds (A37, A39, A40 in the acetylene library) and compounds synthesized by click reaction of 5-position azide CMP sialic acid derivatives. The result of the inhibitory activity measurement about an enzyme is shown.
  • FIG. 16 shows acetylene compounds (acetylene library A1 to A36, L2N, L2A,
  • FIG. 17 shows a compound synthesized by a click reaction of an acetylene compound (acetylene library A1 to A36, L2N, L2A, L2C, L3N, L3A, L3C) and a 9-position azide CMP sialic acid derivative. -Shows the results of measuring the inhibitory activity of 2,3 sialyltransferases.
  • FIG. 18 shows UDP—N3—GalNAc, acetylene compound, tris [(1-1benzyl-1H-1,2,2,3 triazol-4-yl) methyl] amine (TBTA), and copper sulfate. And the yield of click reaction in the case of mixing and reacting sodium ascorbate. The yield in the graph shows the relative ratio of peak intensity ratio in ESI-MS negative mode.
  • Figure 19 shows the measurement of inhibitory activity of ppGalNAcT 2 for compounds synthesized by the click reaction of acetylene compounds (acetylene library Al—A36, L2N, L3N) and UDP—N3—GalNAc. The results are shown.
  • the “sugar chain” refers to a compound formed by one or more unit sugars (monosaccharide and Z or a derivative thereof).
  • the hydroxyl group and amino group of this unit sugar may be protected with an appropriate protecting group.
  • the protecting group include acetyl, benzyl, benzoyl, t-butoxycarbonyl, t-butyldimethylsilyl group and the like.
  • sugar chains examples include polysaccharides (darcose, galactose, mannose, fucose, xylose, N-acetyl dalcosamine, N-acetyl galatatosamine, sialic acid, and complexes thereof contained in the living body. And derivatives), as well as complex polysaccharides such as degraded polysaccharides, glycoproteins, proteodaricans, glycosaminodarlicans, glycolipids, etc. Not. Therefore, in the present specification, the sugar chain is defined as “polysaccharide” or “carbohydrate”. , "Carbohydrate” and “sugar” can be used interchangeably.
  • the “sugar chain” in the present specification may include both a sugar chain and a sugar chain-containing substance.
  • ⁇ ⁇ , 2-—, ⁇ ⁇ , 3-—, ⁇ ⁇ , 4-—, ⁇ ⁇ , 6 1, ⁇ ⁇ , 2-—, etc. depending on the position of unit sugars, and their indications are combined Indicate the position of the carbon in the unit sugar, and the anomer ( ⁇ , j8) for the bond.
  • the information on glycan binding is complex and forces that are difficult to simplify, such as polypeptides and polynucleotides. [Column is Trends in Glycoscience and Glycotechnology 14, 127-137 (2002). It has been proposed to represent sugar chains, and such notation is used in this specification as needed.
  • dice They are called dice, tritose, tetrose, pentose, hexose, heptose, otatose, nonose and decourse, respectively.
  • it corresponds to aldehydes or ketones of chain polyhydric alcohols.
  • the former is called aldose and the latter is called ketose.
  • a "monosaccharide derivative” refers to a substance in which one or more hydroxyl groups on a monosaccharide are substituted with another substituent and the resulting substance is not within the range of a monosaccharide.
  • Examples of such monosaccharide derivatives include saccharides having a carboxyl group (for example, aldonic acids converted to carboxylic acids by acidification at the C 1 position (for example, D— (Darconic acid), uronic acid with terminal C atom converted to carboxylic acid (D-D-glucuronic acid with oxidized glucose), sugar with amino group or amino derivative (for example, acetylated amino group)
  • carboxyl group for example, aldonic acids converted to carboxylic acids by acidification at the C 1 position (for example, D— (Darconic acid), uronic acid with terminal C atom converted to carboxylic acid (D-D-glucuronic acid with oxidized glucose), sugar with amino group or amino derivative (for example, acetylated amino group)
  • N-acetyl-D-darcosamine, N-acetyl-D-galactosamine, etc. sugars having both amino and carboxyl groups (eg, N-acetylethyl neuraminic
  • Gal is an abbreviation for galactose.
  • Gal derivative or “galatose derivative” refers to a substance in which one or more hydrogen atoms on Gal are substituted with another substituent and are not within the range of the resulting material force SGal. Examples of such substituents include those listed in the following (Organic Chemistry) section, UDP or UDP derivatives, and the like.
  • sugar chain-containing substance refers to a substance containing a sugar chain and substances other than sugar chains. Many of such sugar chain-containing substances are found in the living body. For example, in addition to polysaccharides contained in the living body, complex polysaccharides such as degraded polysaccharides, glycoproteins, proteodalycans, glycosaminoglycans, glycolipids, etc. Examples of the biomolecular force include, but are not limited to, a wide range of sugar chains that are decomposed or derived.
  • a substance eg, an enzyme
  • a moiety eg, a moiety that interacts with a substrate on a certain enzyme or a specific amino acid (eg, tryptophan) on a certain enzyme
  • a “specific” “Can interact with” refers to the ability to interact with the substance or part thereof with a specificity that is higher than the specificity for the substance or part other than the part.
  • the substance or part other than the substance or part thereof may comprise a part other than the intended part of a similar substance or certain enzyme.
  • Such an ability is a specific interaction with at least a certain amount of the substance or part thereof when exposed to conditions that dissociate non-specific interactions with the substance or part other than the substance or part thereof.
  • interaction means that when two objects are referred to, the two objects exert a force on each other.
  • interactions include, but are not limited to, covalent bonds, hydrogen bonds, van der Waals forces, ionic interactions, nonionic interactions, hydrophobic interactions, electrostatic interactions, etc.
  • the interaction is a hydrogen bond, a hydrophobic interaction, or the like.
  • shared bond is used in the ordinary sense in the field, and an electron pair is shared by two atoms. It is a chemical bond that is formed.
  • hydrogen bond is used in a normal sense in the field, and the hydrogen nucleus is exposed by attracting an extra-nuclear electron of only one hydrogen atom with a high electronegativity, This is a bond that is created by attracting another atom with high electronegativity, for example, between a hydrogen atom and an atom with high electronegativity (fluorine, oxygen, nitrogen, etc.).
  • the "level” such as an interaction is a degree indicating the strength of the interaction,,, and “strength” both with another substance (or part) of a certain substance (or part). ) Can be used to determine the strength of the interaction.
  • the level of such interaction is, for example, measured values such as fluorescence intensity in FRET, refractive index observation by the SPR (surface plasmon resonance) method used for interactions such as lectins, enzyme enzyme activity measurement ( Radiolabeling, biochemical measurement by ELISA, etc.) can be used.
  • FRET, SPR, and enzyme activity measurement are well known in the art and are described in detail elsewhere in this specification.
  • falmakophore model refers to a model used for forming a desired interaction or the like for a certain configuration. Such a model is used to design a compound with a certain action. For the design of such compounds, for example, ADAM & EVE (Pharmaceutical Molecular Design Laboratory, Tokyo) can be used. In the present invention, it is understood that further novel compounds can be provided using such a pharmacophore model based on the compounds specifically provided in the present invention.
  • a condition, disorder or disease caused by abnormal glycosyltransferase activity means that the level of glycosyltransferase activity is higher or lower than the normal level or such level.
  • a biological state, disorder or disease associated with Such conditions, disorders or diseases include, for example, rheumatoid arthritis, immune disease, lupus erythematosus, cancer or cancer metastasis, bacterial infection, diabetes, metabolic disease, hormonal function, stress, osteoporosis However, it is not limited to them.
  • a state, disorder or disease caused by an abnormal activity of j8 1, 4 galactosyltransferase means that the activity of j8 1, 4-galactose transferase is higher or lower than a normal level. Force caused by or the condition of the body with such a level, disorder Or disease.
  • Such conditions, disorders or diseases include, for example, rheumatoid arthritis, immune disease, lupus erythematosus, cancer or cancer metastasis, bacterial infection, diabetes, metabolic disease, hormonal function, stress, osteoporosis, etc. Is not limited to them.
  • a condition, disorder, or disease caused by abnormal activity of a course transferase includes a force generated by the activity of fucose transferase being higher or lower than a normal level, or such a level. It refers to the condition, disorder or disease associated with the living body.
  • Diseases associated with fucose transferase include immune diseases, cancer or cancer metastasis, bacterial infection, atherosclerosis, antibody-dependent cell injury, and signal transduction.
  • the OC 1, 3 fucose transferase family is highly expressed by canceration. More specifically, the disease associated with fucose transferase VIII may include, for example, emphysema. In cancer patients, knockout suppresses proliferation, ⁇ 1, 6 Fucosyl ⁇ habutoglobin is found at a high rate in spleen cancer patients, and this enzyme decreases in emphysema! It becomes clear that! /
  • a state, disorder or disease caused by an abnormal activity of 2,3-sialyltransferase means that the activity of a 2,3-sialyltransferase is higher than the normal level.
  • Diseases associated with a 2,3-sialyltransferase include immune disorders, cancers such as colorectal / rectal cancer, breast cancer, leukemia, or cancer metastasis.
  • a state, disorder or disease caused by abnormal activity of 2,6-sialyltransferase means that the activity of a 2,6-sialyltransferase is higher than the normal level.
  • Diseases associated with a 2,6-sialyltransferase include immune disorders, cancers such as colorectal / rectal cancer, breast cancer, and leukemia, or cancer metastasis.
  • FRET indicates a direct excitation energy transfer between two fluorescent materials when the fluorescence emission wavelength from one overlaps the absorption wavelength of the other fluorescent material. The phenomenon was discovered by Perin in the old days, and by Fenorester. (Forster, Th. (1948), Ann. Phys. 2: 55-75 .;).
  • the fluorescent material absorbs light of a certain wavelength and is excited by the absorbed light energy.
  • the fluorescent substance emits fluorescence when returning from the excited state to the ground state.
  • Excited phosphors can be thought of as vibrationally excited dipoles, and an electric field depending on the distance R from the dipoles appears (Clegg, RM (1996), XF Wang and B. Herman (eds.), John Wiley & Sons, New York, pp. 179-252 .;).
  • FRET has the property that it will not be observed suddenly at distances exceeding the R value (distance
  • the enzyme substrate is labeled with two fluorescent substances at the position where the degradation site is sandwiched, and an enzyme reaction is performed.
  • the distance between the fluorescent materials is close, and FRET occurs. Therefore, the light emitted from the fluorescent donor is weak and the light emitted from the fluorescent acceptor is observed strongly.
  • the hydrolysis reaction proceeds, the distance between the fluorescent materials increases, and FRET is no longer observed. Then, the light emission from the fluorescent donor is recovered, and the light emission from the fluorescent acceptor is weakened.
  • the reaction of the two-substrate system is the reverse of the one-substrate system described above, and the two enzyme substrates are fluorescently labeled to cause the enzyme reaction.
  • a fluorescently labeled sugar donor and sugar acceptor are prepared.
  • the separation of excitation and observation wavelengths is good, and the ability to estimate the distance between fluorescent substrates when a product is produced by an enzymatic reaction.
  • the enzyme reaction is actually performed. Since it is a fluorescence measurement, it is very sensitive and the reaction is carried out at a fairly dilute substrate concentration. This has the advantage of requiring less substrate mass, and also has the disadvantage that the concentration of one substrate cannot be increased, especially in the enzyme activity measurement of a two-substrate system. Therefore, first fix the concentration of one substrate, first determine the app and v app values, and then change the fixed substrate concentration step by step.
  • the terms “protein”, “polypeptide”, “oligopeptide” and “peptide” are used interchangeably herein and refer to a polymer of amino acids of any length.
  • the polymer may be linear or branched or cyclic.
  • the amino acid may be a modified amino acid, which may be natural or non-natural.
  • the term can also encompass those assembled into a complex of multiple polypeptide chains.
  • the term also encompasses natural or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification (eg, Conjugation with labeling component).
  • This definition also includes, for example, polypeptides (eg, including unnatural amino acids, etc.), peptide-like compounds (eg, peptoids) containing one or more amino acids, and known in the art. Other modifications are included. Therefore, when the enzyme targeted by the screening method of the present invention is a polypeptide, such a variant may be used.
  • polypeptides eg, including unnatural amino acids, etc.
  • peptide-like compounds eg, peptoids
  • polynucleotide As used herein, the terms “polynucleotide”, “oligonucleotide” and “nucleic acid” are used interchangeably herein and refer to a nucleotide polymer of any length.
  • nucleic acid molecule is also used herein interchangeably with nucleic acids, oligonucleotides, and polynucleotides and includes cDNA, mRNA, genomic DNA, and the like. As used herein, nucleic acids and nucleic acid molecules can be included in the concept of the term “gene”.
  • gene refers to a factor that defines a genetic trait. Usually arranged in a certain order on the chromosome. A structural gene that regulates the primary structure of a protein is called a regulatory gene. As used herein, “gene” may refer to “polynucleotide”, “oligonucleotide” and “nucleic acid” and Z or “protein” “polypeptide”, “oligopeptide” and “peptide”.
  • gene product also refers to “polynucleotide”, “oligonucleotide” and “nucleic acid” expressed by a gene, and Z or “protein” “polypeptide”, “oligopeptide” and Refers to “peptide”.
  • homology of genes (for example, nucleic acid sequences, amino acid sequences, etc.) refers to the degree of identity of two or more gene sequences to each other. Therefore, the higher the homology between two genes, the higher the sequence identity or similarity. Whether two genes have homology can be determined by direct sequence comparison or, in the case of nucleic acids, hybridization methods under stringent conditions.
  • the DNA sequence between the gene sequences is typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90% If they are 95%, 96%, 97%, 98% or 99% identical, the genes have homology.
  • “similarity” of a gene eg, nucleic acid sequence, amino acid sequence, etc. refers to the above homology.
  • the degree of identity of two or more gene sequences relative to each other when existing substitutions are considered positive (identical). Thus, if there is a conservative substitution, identity and similarity differ depending on the presence of the conservative substitution. In the absence of conservative substitutions, identity and similarity indicate the same number.
  • identity value in this specification usually refers to the value when aligned using the above BLAST and default conditions. However, if a higher value is obtained by changing the parameter, the highest value is the identity value. When identity is evaluated in multiple areas, the highest value is used as the identity value.
  • the term "foreign gene” refers to a gene that does not naturally exist in an organism. Such a foreign gene may be a gene that is naturally occurring in the organism, or may be a gene that is naturally present in another organism, or an artificially synthesized gene. May be. An organism containing such a foreign gene can express a gene product that is not naturally expressed.
  • amino acid may be natural or non-natural.
  • “Derivatives “Amino acid” or “amino acid analog” refers to an amino acid that is different from a naturally occurring amino acid but has the same function as the original amino acid. Such derivative amino acids and amino acid analogs are well known in the art.
  • the term “natural amino acid” refers to the L-isomer of natural amino acids.
  • Natural amino acids are glycine, alanine, parin, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, ⁇ -carboxyglutamic acid , Arginine, orthine, and lysine. Unless otherwise indicated, all amino acids referred to in this specification are L-forms, and forms using D-form amino acids are also within the scope of the present invention.
  • the term “unnatural amino acid” means an amino acid that is not normally found naturally in proteins.
  • non-natural amino acids include norleucine, para-nitrophenenolanine, homophenenolanine, para-fluororeorophyllaranine, 3-amino-2-benzylpropionic acid, homoarginine D-form or L-form and D-fe-lualanin Is mentioned.
  • Amino acid analog refers to a molecule that is similar to the physical properties and defects or functions of amino acids that are not amino acids. Examples of amino acid analogs include ethionine, canavanine, 2-methylglutamine and the like.
  • Amino acid mimetics refers to compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • Amino acids may be referred to herein by either their commonly known three letter symbol power or by the one letter symbol recommended by the IUPAC — IUB Biochemica 1 Nomenclature Commission. Nucleotides can also be referred to by the generally accepted single letter code.
  • nucleotide or amino acid is the same as a given nucleotide or amino acid in a polynucleotide or polypeptide as a reference for comparison with respect to a certain polynucleotide molecule or polypeptide molecule.
  • it refers to an amino acid that contributes to such a change.
  • a "corresponding" gene for example, an enzyme
  • the corresponding gene of a gene can be an ortholog of that gene.
  • the gene corresponding to human j8 1,4-galactosyltransferase may be an enzyme (or its gene) responsible for similar activities of other species.
  • nucleotide refers to a nucleoside wherein the sugar moiety is a phosphate ester. Nucleotides usually encode amino acids. Glycosyltransferase substrates usually take the form of sugar nucleotides. Nucleic acids are polymers (polynucleotides) of nucleotides (pyrimidine nucleotides and purine nucleotides) whose bases are pyrimidine bases or purine bases. A sugar moiety with D-ribose is called a ribonucleotide and is obtained by RNA hydrolysis.
  • a sugar moiety with D-2-deoxyribose is called deoxyribonucleotide and is obtained by enzymatic degradation of DNA.
  • Creotide is usually called adenylic acid or deoxydelic acid, but it is bound to the sugar moiety of the nucleoside to indicate the position of phosphoric acid and the number of phosphates, and adenosine-5 '-phosphate (English name is abbreviated as 5'—AMP. The same applies below).
  • a derivative derived from a ribonucleoside is referred to as a ribonucleotide, and a derivative derived from a deoxyribonucleoside is referred to as a deoxyribonucleotide.
  • Deoxyribonucleoside 3'-phosphate or deoxyribonucleoside 5'-phosphate is obtained by enzymatic degradation of DNA.
  • nucleotides exist in a free state, and are located at the 5 'position of the nucleoside.
  • diphosphate there are nucleoside diphosphates and nucleoside triphosphates with an additional phosphate group.
  • nucleotide is a group, it is called a nucleotidyl group.
  • nucleoside refers to a compound in which a base and a sugar form an N-glycoside bond.
  • Sugars with D-ribose are ribonucleosides, and those containing purine bases are called purine ribonucleosides (or purine ribosides, generally purine nucleosides) and are obtained by RNA degradation.
  • a sugar with D-ribose is called a ribonucleoside
  • a sugar with D 2'-deoxy xyribose is called a de xyribonucleoside.
  • fragment refers to a polypeptide or polynucleotide having a sequence length of 1 to n-1 with respect to a full-length polypeptide or polynucleotide (length n). Say Chido.
  • the length of the fragment can be changed as appropriate according to its purpose. For example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10 in the case of a polypeptide. , 15, 2, 0, 25, 30, 40, 50 and more, and lengths expressed in integers not specifically listed here (for example, 11 etc.) are also suitable as lower limits. It can be.
  • polypeptides and polynucleotides examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides. !, NA! /, An integer length (eg 11) may also be appropriate as a lower limit.
  • the lengths of polypeptides and polynucleotides can be represented by the number of amino acids or nucleic acids, respectively, as described above, but the above numbers are not absolute, as long as they have the same function. It is intended that the above-mentioned number as the upper limit or subtraction includes the upper and lower numbers (or, for example, 10% above and below) of that number. In order to express such intention, in this specification, “about” may be added before the number. However, it should be understood herein that the presence or absence of “about” does not affect the interpretation of the value.
  • biological activity refers to an activity that can be possessed in a living body by a certain factor (eg, a polypeptide or a protein), and an activity that exhibits various functions. Is included.
  • a factor eg, an enzyme
  • the biological activity includes the enzyme activity.
  • an agent is a ligand
  • it includes binding to the corresponding receptor.
  • biological activity can be measured by techniques well known in the art.
  • a method for producing a polypeptide for example, primary cultured cells or established cells that produce the polypeptide are cultured and isolated or purified from the culture supernatant or the like.
  • the method of obtaining the polypeptide is mentioned.
  • a gene encoding the polypeptide is incorporated into an appropriate expression vector, and an expression host is transformed using the gene, and the recombinant cell is transformed from the culture supernatant of the transformed cell.
  • Peptides can be obtained.
  • the host cell is not particularly limited as long as it expresses a polypeptide that retains physiological activity, and various host cells conventionally used in gene manipulation (for example, E. coli, yeast, animal cells, etc.) Can be used.
  • the polypeptide derived from the cells thus obtained is a natural-type polypeptide.
  • one or more amino acids in the amino acid sequence may be substituted, added and Z or deleted, and the sugar chain may be substituted, added and Z or deleted. Also good.
  • Certain amino acids can be substituted for other amino acids in protein structures, such as force thionic regions or substrate molecule binding sites, without an apparent reduction or loss of interaction binding capacity. It is the ability and nature of the protein to define the biological function of a protein. Thus, specific amino acid substitutions can be made in the amino acid sequence or at the level of its DNA coding sequence, resulting in a protein that still retains its original properties after substitution. Thus, various modifications can be made to the peptide disclosed herein or the corresponding DNA encoding this peptide without any apparent loss of biological utility.
  • hydrophobicity index of amino acids can be taken into account.
  • the importance of the hydrophobic amino acid index in conferring interactive biological functions in proteins is generally recognized in the art (Kyte. J and Doolittle, RFJ Mol. Biol. 157 (1): 105-132, 1982).
  • the hydrophobic nature of amino acids contributes to the secondary structure of the protein produced, and then defines the interaction of the protein with other molecules (eg, enzymes, substrates, receptors, DNA, antibodies, antigens, etc.).
  • Each amino acid is assigned a hydrophobicity index based on their hydrophobicity and charge properties.
  • the hydrophobicity index is preferably within ⁇ 2 and more preferably within ⁇ 1. Even more preferably within ⁇ 0.5. It is understood in the art that such substitution of amino acids based on hydrophobicity is efficient. As described in US Pat. No. 4,554,101, the following hydrophilicity indices are assigned to amino acid residues!
  • an amino acid can be substituted with another that has a similar hydrophilicity index and can still provide a biological equivalent.
  • the hydrophilicity index is preferably within ⁇ 2, more preferably within ⁇ 1, and even more preferably within ⁇ 0.5.
  • conservative substitution is similar to the amino acid substitution, as described above, in the hydrophilicity index or Z and hydrophobicity index with the amino acid to be replaced with the original amino acid. This refers to substitution. Examples of conservative substitutions include those having a hydrophilicity index or hydrophobicity index of 2 or less, preferably ⁇ 1 or less, more preferably ⁇ 0.5 or less. But not limited to them.
  • conservative substitutions are well known to those skilled in the art, for example, substitutions within the following groups: arginine and lysine; dartamic acid and aspartic acid; serine and threonine; glutamine and asparagine; Examples include, but are not limited to, leucine and isoleucine.
  • the "variant” refers to a substance in which a part of the original substance such as a polypeptide or polynucleotide is changed. Such variants include substitutional variants, addition variants, deletion variants, truncated variants, allelic variants, and the like. The substitution need not be a conservative substitution as described above.
  • the substituted amino acid may be a natural amino acid or a non-natural amino acid. Alternatively, the substituted amino acid may be an amino acid analog. Alleles are genetic variants that belong to the same locus and are distinguished from each other. Therefore, "Alleles" A “child variant” refers to a variant that has an allelic relationship to a gene.
  • allelic variants usually have a sequence that is identical or very similar to its corresponding allele and usually has a rarely different biological activity with nearly the same biological activity. May have.
  • “Species homologue or homolog” means homology (preferably at least 60% homology, more preferably at least 80%, at a certain amino acid level or nucleotide level within a certain species. 85% or more, 90% or more, 95% or more homology). The method for obtaining such species homologues will be apparent from the description herein.
  • “Ortholog”, also called orthologous gene refers to a gene derived from speciation from a common ancestor with two genes.
  • human and mouse ⁇ -hemoglobin genes are orthologs.
  • Human ⁇ -hemoglobin genes and j8 hemoglobin genes are paralogs (genes generated by gene duplication). is there.
  • Orthologs are useful for estimating molecular phylogenetic trees. Since orthologs usually have the same function as V and the original species in another species, for example, orthologs of glycosyltransferases are also present in the present invention. May be useful.
  • enzyme refers to a biocatalytic molecule that is a biomolecule such as a protein that acts in living cells.
  • substrate refers to a substance that, when used in a reaction with an active substance such as an enzyme, causes a reaction under the action of an activating substance (for example, an enzyme).
  • an activating substance for example, an enzyme
  • starch is a substrate for amylase
  • hydrogen peroxide is a substrate for force tarase.
  • the product is also a substrate for the reverse reaction.
  • Coenzymes are often one of the enzyme substrates. In the case of transferase, both donor and acceptor molecules are substrates.
  • transferase is a general term for enzymes that catalyze a group transfer reaction.
  • transferase may be used interchangeably with “transferase”.
  • the group transfer reaction is represented by the following equation (6):
  • the group Y is transferred from one compound (donor) to another (acceptor) Done in the form.
  • the "donor” in the group transfer reaction of the present specification means the compound X—Y (donor) of the above formula (6) and can be used interchangeably with the “donor substrate”.
  • the “acceptor” in the group transfer reaction of the present specification means the compound Z—H (acceptor) of the above formula (6), and can be used interchangeably with “acceptor monosubstrate”.
  • glycosyltransferase refers to a sugar (corresponding to group Y in the above formula (6); unit sugar or sugar chain) corresponding to compound X—Y in the above formula (6). ) To another place (equivalent to the compound Z—H of the above formula (6)).
  • glycosyltransferase include galactose transferase, glucose transferase, sialic acid transferase, mannose transferase, fucose transferase, xylose transferase, N-acetylylcosamine transferase, and N-acetyl galatatosamine transferase. However, it is not limited to them.
  • glycosyltransferases examples include ⁇ , 4-galactose transferase, ⁇ 1, 3-galactose transferase, ⁇ ⁇ , 4-galactose transferase, ⁇ ⁇ , 3-galactose transferase, ⁇ ⁇ , 6-galactose transferase, 2,6 sialyltransferase, 1,4 galactose transferase, ceramide galactose transferase, ⁇ , 2 fucose transferase, ⁇ 1, 3 fucose transferase, ⁇ 1, 4 fucose transferase, 1, 1, 6 fucose transferase, 1, 1, 3— ⁇ acetyl galatatosamine transferase, ⁇ , 6— ⁇ acetyl galatatosamine transferase, ⁇ ⁇ , 4— ⁇ Acetylgallatatosamine transferase, polypeptide ⁇ Acetylgalata
  • GalT galactose transferase
  • Gal galactose
  • An enzyme that strictly transfers ⁇ -type or j8-type to the sugar acceptor The ⁇ type has ⁇ , 2—, ⁇ , 3—, ⁇ 4, 41, and ⁇ 1, 6 bond types, and the j8 type has j81, 3—, ⁇ ⁇ , 4 bond types.
  • ⁇ 1-ceramide bond type involved in the synthesis of galatose ceramide.
  • GalT In eukaryotes, GalT, with some exceptions, exists in the Golgi in a mouth-colorized manner, producing a variety of sugar chains. J81, 4 GalT is known not only to play a role in sugar chain synthesis but also to exist on the cell surface, and to act as a cell adhesion molecule throughout the embryogenesis process (DA Hinton). , BD Shur, (1994), Trends Glycosci. Glycotech., 6, 375-385. 4-1, ⁇ 1,3—, ⁇ 1,4-linked galactose enzyme (Christelle B., Emmanuel B., David HJ, Roberto AG an d Anne I. (1998), J. Biochem. 123, 1000-1009., T.
  • the structure and function of which are well examined are mammals such as ushi, pig, and human.
  • the substrate specificity is particularly investigated for ⁇ ⁇ , 4-linked enzymes. is there.
  • This enzyme is relatively easy to isolate and purify, and since it was quickly put on the market, a lot of knowledge about substrate recognition ability and substrate analogue inhibition has already been obtained (Philippe C , Olilier RM, (2001), Bioorg. Med. Chem. 9, 30 77-3092.; Hironobu H., Tsuyoshi E. and Yasuhiro K., (1997), J. Org. Chem.
  • GalT It is GalT that has been most well studied to date. Seven types of this enzyme have been discovered in humans due to its sequence homology. What is generally marketed is called U8, human-derived (substantially the same sequence) j8 1, 4— GalTl. From UDP- Gal to Gal-N-acetyl-darcosamine (hereinafter GlcNAc) To be transferred. It is also deeply involved in the production of ratatose in breast milk, in which case it interacts with ⁇ -lactalbumin and recognizes glucose as a sugar acceptor. Some of these enzymes are also present on the cell surface and are not only used for sugar chain synthesis but also as a cell adhesion molecule.
  • GlcNAc Gal-N-acetyl-darcosamine
  • This enzyme transfers Gal from UDp-Gal to the non-reducing terminal Gal of ⁇ -acetyllactosamine (La cNAc) in ⁇ 1,3-linked form.
  • a human blood group antigen synthase and an ⁇ -gal antigen called Galili epitope such as swine and sushi, and those that synthesize isoglobo-type glycolipids (T. Hennet, (2002), Cell Mol. Life Sci. 59, 1081— 1095.)
  • a method for producing a Galili epitope synthase by genetic recombination in E. coli has already been established and is commercially available.
  • Fucose transferase translocates Fuc to various acceptors using GDP-Fuc as a sugar donor.
  • ⁇ ⁇ , 2-—, ⁇ ⁇ , 3-—, ⁇ 1, 4 1, ⁇ 1, 6 1 are known, and different subtypes of fucose transferase are catalyzed.
  • NMR, X-ray crystal structures, etc. no reports on physical three-dimensional structure analysis (NMR, X-ray crystal structures, etc.) have been made for any species or subtype. Some subtypes show high amino acid sequence homology (Breton, C., Oriol, R., Imberty, A. (1998) Glycobiology 8, 87— 94.), in general, the acceptor specificity is very different.
  • This enzyme transfers Fuc from GDP-Fuc to ⁇ 1,2-linked non-reducing galactose.
  • Known to synthesize human blood group antigens and related sugar chains (Kelly, RJ, Rouquier, 3 ⁇ 4., U orgi, D., Lennon,.., And Lowe, JB (1 995) J Biol. Chem. 270, 4640—4649., Larsen, RD, Ernst, LK, Nair, RP, and Lowe, JB (1990) Proc. Natl. Acad. Sci. USA. 87, 6 674- 6678 .;) .
  • This enzyme transfers Fuc from GDP-Fuc to GlcNAc of N-acetyllactosamine in a 1,3-linked form.
  • This enzyme is known to be involved in biological reactions such as immune response and cancer metastasis by creating a sugar chain called Lewis type antigen (Kaneko, M., Kudo, T., Iwasaki, ⁇ ., Ikehara, , Nishihara, S., Nakagawa, S., Sasaki, ⁇ ., Shiina, ⁇ ., Inoko, ⁇ ., Saitou, ⁇ ., And Narimatsu, ⁇ . (1999) FEBS Lett., 452, 237-242 Natsuka, S., and Lowe, JB (1994) Curr. Opin. Struct. Biol., 4, 683—691., Kannagi, R. (2002) Curr. Opin. Str uct. Biol. 12. 599- 608 .;).
  • This enzyme transfers Fuc from GDP-Fuc to the asparagine-linked GlcNAc present at the reducing end of the N-type sugar chain in a 1,6-linked form (Miyoshi, E., Noda, K., Yamaguchi, Y , Inoue, S., Ikeda, Y., Wang, W., Ko, J. ⁇ ., Uozumi, ⁇ ., Li, W., and Taniguchi, ⁇ . (1999) Biochim. Biophys. Acta.
  • This fucosyl mushroom is found in many glycoprotein sugar chains and is known as the best tumor in the development of liver cancer (Taketa, K., Endo, Y., Sekiya, C., Tanikawa, K., , Taga, ⁇ ., Satomura, S., Matsuura, S., Kawai, ⁇ ., And Hirai, ⁇ . (1993) Cancer Res. 53, 5419— 5423., Hutchinson, WL, Du, M. Q., Johnson, PJ, and Williams, R.
  • a 1,3 fucose transferase family 1 is highly expressed by canceration. More specifically, the disease associated with fucose transferase VIII may include, for example, emphysema.
  • the disease associated with fucose transferase VIII may include, for example, emphysema.
  • knockout results in suppression of growth sera in patients with spleen cancer show a high rate of a1,6Fucosyl ⁇ haptoglobin, and emphysema has reduced levels of this enzyme It is summer.
  • This enzyme transfers sialic acid from CMP sialic acid to ⁇ 2,3 linked form to the galactose residue located at the non-reducing terminus of glycoprotein mucin-type sugar chain, asparagine and glycosphingolipid.
  • This enzyme is located on the non-reducing terminus of the glycoprotein mucin-type glycosylation chain rasparagine and glycosphingolipid on the Golgi body, mainly on the terminal galactose residue of ratatothose N-acetyl latatosamine. Transfers sialic acid in ⁇ 2,6 bond form using acid as sugar donor.
  • the compound of the present invention can be represented by a schematic diagram of ( ⁇ )-( ⁇ )-(C).
  • (B) can include a sugar component (that is, a sugar or a derivative thereof).
  • the target glycosyltransferase is advantageously a sugar as a substrate or an equivalent thereof.
  • (C) may include nucleotides or their equivalents.
  • (C) and (B) may contain a spacer or may be directly coupled.
  • the spacer can be expressed as (s), and is a part having an action of coupling the part (A) and the part (B).
  • (S) may not be present when the (A) part and the (B) part are directly bonded.
  • one or preferably both (A) part and (B) part and a part that does not inhibit their intended function are used.
  • spacer refers to a chemical moiety that is introduced to provide an appropriate distance between two or more characteristic parts.
  • Examples of such a moiety include, but are not limited to, an alkylene group, an ether group, ethylene glycol, a peptide, and a lipid.
  • a person skilled in the art can select and synthesize an appropriate spacer portion according to the above-mentioned characteristic portion.
  • sucrose component refers to a group in a form in which hydrogen is removed so that it can be combined with other moieties.
  • a monovalent form in which one hydrogen is removed from a sugar and a divalent group in which two hydrogens are removed from a sugar.
  • the sugar component include darcos, galactose, mannose, fucose, xylose, N-acetylyldarcosamine, N-acetylgalatatosamine, and sialic acid having two hydrogen atoms.
  • the sugar component may be a monovalent group in which one hydrogen is removed from a sugar chain of two or more sugars, or a divalent group in which two hydrogens are removed from a sugar chain of two or more sugars.
  • the group represented by is preferred as the “sugar component”.
  • C may be acil (preferably acetyl).
  • C may be nucleoside phosphate or nucleoside diphosphate.
  • C may be a pyrimidine nucleoside phosphate or a purine nucleoside phosphate.
  • C can also be a phosphate of adenosine, deoxyadenosine, guanosine, deoxyguanosine, cytidine, deoxythymidine, thymidine (ribothymidine), deoxythymidine or uridine or variants thereof.
  • this modification can mention arbitrary modifications.
  • such variants include adenosine, deoxyadenosine, guanosine, deoxyguanosine, cytidine, deoxythymidine, thymidine (ribothymidine), deoxythymidine or uridine, or other Examples include, but are not limited to, those in which any substituents listed in the above are bonded.
  • “C” has the following formula:
  • B is D-galactose, L-fucose, sialic acid, D-GlcNAc, D-GalNAc ⁇
  • glycosyltransferase inhibitors synthesized by click chemistry specifically inhibit a 2-3 sialyltransferases.
  • phosphate refers to a substance in which the phosphate power is also removed from hydrogen, and exists as a phosphate ester, phosphate or ion.
  • the phosphate may exist as a single or multiple phosphate group, for example, including monophosphate, diphosphate, triphosphate, specifically adenosine, deoxyadenosine, guanosine, deoxy. Examples include xyguanosine, cytidine, deoxythymidine, thymidine (ribothymidine), deoxythymidine or uridine, or phosphates of these modifications.
  • Adenosine diphosphate is also called ADP
  • adenosine triphosphate is also called ATP.
  • the compound of the present invention can be represented by a schematic diagram of (X)-(Y)-(B)-(C) (also expressed as X-Y-B-C).
  • B can be a sugar component
  • C can be a nucleotide
  • X is a bulky group
  • “bulky group” refers to any group having the ability to cause steric hindrance. Whether or not a steric hindrance has occurred can be estimated by computer modeling. Nucleic acid configuration is calculated by molecular configuration analysis methods and precise measurement of interatomic distances, etc. Based on these results, the base chemical structure that can cause steric hindrance is designed. can do. Such steric hindrance can be performed if the target enzyme is identified.
  • the initial structure is a structure obtained by an experiment such as X-ray crystal structure analysis or NMR analysis (including a PDB registered structure) or a predicted structure obtained by calculation through molecular modeling. To date, most of the structure has been obtained by X-ray crystallography except for small domains.
  • the predicted structure is prepared by molecular modeling such as homology modeling.
  • the structure obtained in this way is not used as it is, but the dynamic structure and keyhole information of the expected binding site are analyzed by MD calculation. It is possible to predict the binding state of a drug to a target protein and to search for further binding sites for structure conversion.
  • the bulky group may have a substituent! /, May have an alkyl, or have a substituent! /, May have! /, Alkenyl, an alkyl which may have a substituent, a carbocyclic group which may have a substituent, or a heterocyclic group which may have a substituent.
  • aromatic (functional) group or “aromatic ring (functional) group” refers to a ring compound having an aromatic characteristic or a portion thereof, and generally ⁇ electrons are 4 ⁇ + It has a stable structure including two cyclic conjugated systems.
  • aromatic functional groups can also interact with other aromatic functional groups.
  • the aromatic functional group include, but are not limited to, a benzene ring, naphthalene, anthracene, furan, pyridine, azulene, cyclooctatetradianion and the like.
  • the aromatic functional group may be a carbocyclic ring or a heterocyclic ring. Further, the aromatic functional group may be substituted with a substituent R, and the “substituted aromatic functional group” means an aromatic functional group substituted with a substituent selected below. .
  • alcohol refers to an organic compound in which one or more hydrogen atoms of an aliphatic hydrocarbon are substituted with a hydroxyl group. In this specification, it is also expressed as ROH.
  • R is an alkyl group. Preferably R is C1 C6 alkyl obtain.
  • examples of the alcohol include, but are not limited to, methanol, ethanol, 1-propanol, 2-propanol V-, and the like.
  • alkyl refers to a monovalent group produced by loss of one hydrogen atom in an aliphatic hydrocarbon (alkane) force such as methane, ethane, or propane.
  • n 2n + l is represented by one (where n is a positive integer).
  • Alkyl can be linear or branched.
  • substituted alkyl refers to an alkyl in which H of the alkyl is substituted by the substituent specified below.
  • C1-C2 alkyl C1-C3 alkyl, C1-C4 alkyl, C1-C5 alkyl, C1-C6 alkyl, C1-C7 alkyl, C1-C8 alkyl, C1-C9 alkyl, C1-C10 alkyl Cl-C11 alkyl or C1-C12 alkyl, C1-C2 substituted alkyl, C1-C3 substituted alkyl, C1-C4 substituted alkyl, C1-C5 substituted alkyl, C1-C6 substituted Alkyl, C1-C7 substituted alkyl, C1-C8 substituted alkyl, C1-C9 substituted alkyl, C1-C10 substituted alkyl, C1-C11 substituted alkyl or C1-C12 substituted Or alkyl.
  • C 1 -C 10 alkyl means a linear or branched alkyl having 1 to 10 carbon atoms, such as methyl (CH—), ethyl (CH 1), n-propyl (CH CH CH—), iso
  • An alkyl refers to a C1-C10 alkyl having one or more hydrogen atoms replaced by a substituent.
  • R C1-C6 alkyl is preferred, and C1-C6 alkyl is particularly preferred! /.
  • cycloalkyl refers to alkyl having a cyclic structure.
  • substituted cycloalkyl refers to cycloalkyl in which H of cycloalkyl is substituted by a substituent specified below.
  • C3-C4 cycloalkyl C3-C5 Cycloalkyl, C3-C6 cycloalkyl, C3-C7 cycloalkyl, C3-C8 cycloalkyl, C3-C9 cycloalkyl, C3-C10 cycloalkyl, C3-C11 cycloalkyl, C3-C12 cycloalkyl, C3-C4 substituted Cycloalkyl, C3-C5 substituted cycloalkyl, C3-C6 substituted cycloalkyl, C3-C7 substituted cycloalkyl, C3-C8 substituted cycloalkyl, C3-C9 substituted cycloalkyl, It can be a C3-C10 substituted cycloalkyl, a C3-C11 substituted cycloalkyl, or a C3-C12 substituted cycloalkyl.
  • cycloalkyl is
  • alkell is a monovalent group such as ethylene or propylene that is generated by losing one hydrogen atom of an aliphatic hydrocarbon having one double bond in the molecule. , Generally expressed as CH 1 (where n is a positive integer greater than or equal to 2). "Replaced
  • Alkal formed refers to a alkal in which the H of the alkal is substituted by the substituent specified below.
  • Specific examples include C2 to C3, C2 to C4, C2 to C5 alkenyl, C2 to C6, C2 to C7, C2 to C8, C2 to C9. , C2 to C10, 2 to 11 or 2 to 12, C2 to C3 substituted, C2 to C4 substituted, C2 to C5 Substituted alkell, C2-C6 substituted alkal, C2-C7 substituted alkal, C2-C8 substituted alkal, C2-C9 substituted alkal, C2 -C There can be 10 substituted alks, 2 to 11 substituted arks, or 2 to 12 substituted arks.
  • the 10-substituted alkenyl is a C2 to C10 alkal, in which one or more hydrogen atoms are substituted with a substituent.
  • cycloalkenyl refers to an alkke having a cyclic structure.
  • “Substituted cycloalkenyl” refers to a cycloalkenyl in which H of the cycloalkenyl is substituted by the substituent specified below. Specific examples include C3-C4 cycloalkenyl, C3-C5 cycloalkell, C3-C6 cycloalkenyl, C3-C7 cycloalkenyl.
  • preferred cycloalkenyls include 1-cyclopental, 2-cyclohexyl and the like.
  • alkynyl refers to a monovalent group formed by losing one hydrogen atom from an aliphatic hydrocarbon having one triple bond in the molecule, such as acetylene. Generally expressed as CH 1 (where n is a positive integer greater than or equal to 2). "Substituted al n 2n_3
  • alkyl refers to an alkyl in which H of the alkyl is substituted by the substituent specified below.
  • Specific examples include C2-C3 alkyl, C2-C4 alkyl, C2-C5 alkyl, C2-C6 alkyl, C2-C7 alkyl, C2-C8 alkyl, C2- C9 alkyl, C2 to C10 alkyl, C2 to C11 alkyl, C2 to C12 alkynyl, C2 to C3 substituted alkyl, C2 to C4 substituted alkyl, C2 to C5 substituted Alkyls, C2-C6 substituted alkyls, C2-C7 substituted alkyls, C2-C8 substituted alkyls, C2-C9 substituted alkyls, C2-C10 substituted Alkyl, C2-C11 substituted alkyl or C2-C12 substituted alkyl.
  • C2 to C10 alkyl means, for example, a linear or branched alkyl containing 2 to 10 carbon atoms, such as ethur (CH ⁇ C—), 1 propynyl ( CH C ⁇ C) and the like are exemplified. Also, for example, C2-C10 substituted alkyl
  • Nyl refers to a C2 to C10 alkyl having one or more hydrogen atoms replaced by a substituent.
  • alkoxy refers to a monovalent group formed by loss of a hydrogen atom of a hydroxy group of an alcohol, and is generally represented by C H O (where n is 1 or more).
  • Substituted alkoxy refers to alkoxy in which H of alkoxy is substituted by the substituent specified below. Specific examples include C1-C2 alkoxy, C1 -C3 alkoxy, C1-C4 alkoxy, C1-C5 alkoxy, C1-C6 alkoxy, C1-C7 alkoxy, C1-C8 alkoxy, C1-C9 alkoxy, C1-C10 alkoxy, C1-C11 alkoxy, C1-C12 alkoxy, C1-C2 substituted alkoxy, C1-C3 substituted alkoxy, C1-C4 substituted alkoxy, C1-C5 substituted alkoxy, C1-C6 substituted alkoxy, C1-C7 substituted alkoxy, C1 It can be -C8 substituted alkoxy, C1-C9 substituted alkoxy, C1-C10 substituted alkoxy, C1-C11 substituted alkoxy or
  • Mouth poxy (CH CH CH O—) and the like are exemplified.
  • Carbocyclic group means a group containing a cyclic structure containing only carbon, and includes the above-mentioned “cycloalkyl”, “substituted cycloalkyl”, “cycloalkenyl”. And a group other than “substituted cycloalkenyl”.
  • Carbocyclic groups can be aromatic or non-aromatic and can be monocyclic or polycyclic.
  • the “substituted carbocyclic group” refers to a carbocyclic group in which H of the carbocyclic group is substituted by the substituent specified below.
  • C3-C4 carbocyclic group C3-C5 carbocyclic group, C3-C6 carbocyclic group, C3-C7 carbocyclic group, C3-C8 carbocyclic group, C3-C9 carbocyclic group, C3-C10.
  • Carbocyclic group C3-C11 carbocyclic group, C3-C12 carbocyclic group, C3-C4-substituted carbocyclic group, C3-C5-substituted carbocyclic group, C3-C6-substituted carbocyclic group, C3- C7 substituted carbocyclic group, C3-C8 substituted carbocyclic group, C3-C9 substituted carbocyclic group, C3-C10 substituted carbocyclic group, C3-C11 substituted carbocyclic group or C3 It can be a C12 substituted carbocyclic group.
  • the carbocyclic group can also be a C4-C7 carbocyclic group or a C4-C7 substituted carbocyclic group.
  • Examples of the carbon ring group include those in which one phenyl hydrogen atom is deleted.
  • the hydrogen deletion position may be any position chemically possible, whether on an aromatic ring or on a non-aromatic ring.
  • heterocyclic group refers to a group having a cyclic structure including carbon and heteroatoms.
  • the heteroatoms are selected from the group consisting of 0, S and N forces, and may be the same or different, and may be contained in one or more than one.
  • Hetero Cyclic groups can be aromatic or non-aromatic and can be monocyclic or polycyclic.
  • “Substituted hetero ring group” means a hetero ring group in which H of the hetero ring group is substituted by the substituent specified below.
  • C3-C4 carbocyclic group C3-C5 carbocyclic group, C3-C6 carbocyclic group, C3-C7 carbocyclic group, C3-C8 carbocyclic group, C3-C9 carbocyclic group, C3-C10.
  • Carbocyclic group C3-C11 carbocyclic group, C3-C12 carbocyclic group, C3-C4-substituted carbocyclic group, C3-C5-substituted carbocyclic group, C3-C6-substituted carbocyclic group, C3 -C7 substituted carbocyclic group, C3-C8 substituted carbocyclic group, C3-C9 substituted carbocyclic group, C3-C10 substituted carbocyclic group, 3-3-111 substituted carbocycle
  • One or more carbon atoms of the group or the same 3- to C12-substituted carbocyclic group may be substituted with a heteroatom.
  • a heterocyclic group can also be one in which one or more heteroatoms are substituted for the carbon atoms of a C4-C7 carbocyclic group or a C4-C7 substituted carbocyclic group.
  • the heterocyclic group include a cetyl group, a pyrrolyl group, a furyl group, an imidazolyl group, and a pyridyl group.
  • the hydrogen deletion position may be any position chemically possible, and may be on an aromatic ring or a non-aromatic ring.
  • a carbocyclic group or a heterocyclic group may be substituted with a divalent substituent in addition to being substituted with a monovalent substituent as defined below.
  • halogen refers to a monovalent group of an element such as fluorine (F), chlorine (C1), bromine (Br), iodine (I) belonging to Group 7B of the periodic table.
  • hydroxy refers to a group represented by OH.
  • substituted hydroxy refers to a hydroxy in which H is substituted with a substituent as defined below.
  • thiol is a group in which an oxygen atom of a hydroxy group is substituted with a sulfur atom (mercapto group), and is represented by —SH.
  • substituted thiol refers to a group in which H of mercapto is substituted with a substituent as defined below.
  • cyan refers to a group represented by —CN.
  • Niro means -NO
  • Carboxy refers to a group represented by —COOH. "Replaced ⁇ Carboxy '' means a carboxy H substituted with a substituent as defined below.
  • acyl refers to a monovalent group formed by removing OH from a carboxylic acid.
  • Representative examples of the acyl group include acetyl (CH 2 CO 3), benzoyl (C 3 H 2 CO 3), etc.
  • Substituted acyl refers to a hydrogen substituted with the substituent defined below.
  • amide refers to a group obtained by substituting hydrogen of ammonia with an acid group (acyl group), preferably —CONH
  • Substituted amide refers to a substituted amide.
  • aldehyde refers to a group represented by “one CHO”. This group reacts with an amino group to form an oxime group.
  • thiocarbol is a group in which an oxygen atom in carbonyl is substituted with a sulfur atom, and includes a characteristic group — (C ⁇ S) —.
  • Thiocarbol includes thioketones and thioaldehydes.
  • Substituted thiocarbol means thiocarbonyl substituted with a substituent selected as described below.
  • sulfol is a generic term for a substance containing SO which is a characteristic group.
  • Substituted sulfol means sulfol substituted with a substituent selected below.
  • sulfier refers to a generic term for a substance containing SO— which is a characteristic group.
  • Substituted sulfiel means a sulfiel that is substituted with a substituent selected below!
  • aryl refers to a group formed by leaving one hydrogen atom bonded to an aromatic hydrocarbon ring, and is included in the carbocyclic group in the present specification.
  • substitution refers to replacement of one or more hydrogen atoms in an organic compound or substituent with another atom or atomic group.
  • One hydrogen atom can be removed and substituted with a monovalent substituent, and two hydrogen atoms can be removed and substituted with a divalent substituent.
  • substituent R may be present in one or more, each independently hydrogen, alkyl, cycloalkyl , Alcohol, cycloalkenyl, alkyl, alkoxy, carbocyclic group, heterocyclic group, halogen, hydroxy, thiol, silane-containing nitro, ami-containing carboxy, acyl, thiocarboxy, amide, substituted amide , Substituted carbo- yl, substituted thiocarbol, substituted sulfol, and substituted sulfiel.
  • the substituent R may be independently selected from the group consisting of hydrogen, alkyl, and substituted alkyl force, when there are multiple substituents R. More preferably, independently, when there are a plurality, R may be independently selected from the group consisting of hydrogen and C1-C6 alkyl. All of R may have a substituent other than hydrogen, but may preferably have at least one hydrogen, more preferably 2 to n (where n is the number of R). It may be preferred that the number of hydrogens in the substituent is large.
  • a large substituent or a polar substituent is a force that may impair the effects of the present invention (particularly, interaction with an aldehyde group).
  • the substituent other than hydrogen may preferably be C1-C6 alkyl, C1-C5 alkyl, C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, methyl and the like.
  • the effect of the present invention may be enhanced, it may be preferable to have a large substituent. Even more preferably, all of R may be hydrogen.
  • optical isomer refers to one or a pair of a pair of compounds that have a mirror-image relationship with a crystal or molecule and cannot be superimposed. It is a form of stereoisomer, and the other properties are the same, but only the optical rotation is different.
  • substitution refers to replacement of one or more hydrogen atoms in an organic compound or substituent with another atom or atomic group.
  • One hydrogen atom can be removed and substituted with a monovalent substituent, and two hydrogen atoms can be removed and substituted with a divalent substituent.
  • Substituents include alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkell, substituted alkalk, cycloalkenyl, substituted cycloalkyl, alkyl- , Substituted alkyl, alkoxy, substituted alkoxy, carbocyclic group, substituted carbocyclic group, heterocyclic group, substituted heterocyclic group, halogen, hydroxy, substituted hydroxy, thiol , Substituted thiol, thio-substituted nitro, ami-substituted ami-substituted carboxy, strength rubamoyl, substituted carboxy, acyl, silylamino, substituted acil, thiocarboxy, substituted thiocarboxy, amide, substituted Amides, substituted carbols, substituted thiocarbols, substituted sulfols or substituted s
  • substituent for the further substitution examples include alkyl, cycloalkyl, alkyl, cycloalkenyl, alkyl, alkoxy, halogen , Hydroxy, thiol, nitro-containing nitro, carboxy-amino, rubamoyl, acyl, acylamino, thiocarboxy and amide.
  • the “protection reaction” refers to a reaction in which a protective group such as Boc (t-butyloxycarboxyl) is added to a functional group desired to be protected.
  • a protective group such as Boc (t-butyloxycarboxyl)
  • deprotection reaction refers to a reaction that removes a protecting group such as Boc.
  • Examples of the deprotection reaction include a reaction such as a reduction reaction using PdZC.
  • the target product is contaminated from the reaction solution (unreacted weight loss). , By-products, solvents, etc.) after removal by methods commonly used in the art (eg extraction, distillation, washing, concentration, precipitation, filtration, drying, etc.) For example, it can be isolated by a combination of adsorption, elution, distillation, precipitation, precipitation, chromatography, etc.).
  • the azido sugar of the present specification refers to the target sugar as Christian Vogel, Stephan Bergemann, An drej—Jakob Ott, Thisbe K. Lindhorst, Joacnim Theim, Wilhelm V. Dahlhoff, Chris terHallgren, Monica M. Palcic and Ole Hindsgaul. , Liebigs Ann. (1997) 6 (Refer to 612-612 etc. to introduce t-butyldimethylsilyl (TBDMS) group and tosyl group, and tosyl group reacts with NaN in nonpolar organic solvents such as DMF.
  • TDMS t-butyldimethylsilyl
  • the TBDMS group can be eliminated and cyclized. If necessary, the azide sugar can be acetylated.
  • the azido sugar is obtained, it is protected by acetylation, and then halolated, and a phosphate group is introduced into the halogenated portion.
  • Acetylation is performed by covering acetic anhydride in pyridine. Is called.
  • the vaporization can be performed, for example, by adding halogen titanium (eg, TiBr) in an organic solvent (eg, methylene chloride-ethyl acetate 10: 1).
  • halogen titanium eg, TiBr
  • organic solvent eg, methylene chloride-ethyl acetate 10: 1).
  • organic solvent toluene-methyl cyanide, etc.
  • the target compound of the present invention can be obtained by adding GMP morpholidate and 1H tetrazole in pyridine to the azido sugar phosphate thus obtained and purifying it with Dowex50 X8 or the like. it can.
  • the example scheme is listed below.
  • screening refers to selecting a large number of candidates for a target substance or organism having a certain specific property by a specific operation and Z or evaluation method.
  • screening can be performed by using components such as enzymes, acceptors, donors, and systems, sugar chain arrays, and the like.
  • a library generated using an in silico (system using a computer) system using a real substance such as in vitro or in vivo may be used.
  • compounds obtained by screening having a desired activity are also included within the scope of the present invention.
  • the present invention also contemplates providing a drug by means of computer modeling based on the disclosure of the present invention.
  • copinatorial chemistry refers to a technology for simultaneously synthesizing a large number and variety of compounds by decomposing and combining synthetic reactions into elementary reactions.
  • US Merrif ield's
  • Furka's Split Synthesis Method
  • a reaction is believed by bonding a functional group that becomes a scaffold for organic synthesis called a linker on a solid support such as a bead. To do. Unit structures called building blocks are linked to this linker in a chain. Before each reaction, divide the beads into several equal parts (split), combine the different building blocks, and place them back in one container. By repeating Z splits and pooling n times, a library with ZX n kinds of compounds can be obtained. By performing the reaction for each individual split, the difference in the reactivity of the building blocks can be adjusted, and a uniform library can be obtained. By this method, it is possible to obtain a library having as many kinds of compound powers as possible.
  • liquid phase synthesis refers to a method of preparing a mixture library by liquid phase synthesis using a liquid phase instead of solid phase synthesis.
  • Liquid phase synthesis includes, for example, a mixture synthesis method in which a reaction is performed using a mixture of building blocks that react under the same reaction conditions.
  • the LPCS method in which split synthetic beads are replaced with soluble polymers, can also be used.
  • a library can be prepared in the same manner as split synthesis by using a capture agent for recovery from a solvent.
  • parallel synthesis refers to a method of performing a reaction on a defined register (well, pin, chip, or small section on a membrane). In this method, normal organic synthesis takes place in each individual register. In order to obtain the type of compound required, it is necessary to prepare a considerable number of registers. In solid phase synthesis, pins are usually used, but chip-type synthesis methods have been developed to synthesize more compounds in the same way. As with the split method, many types of compounds can be synthesized by combining normal synthesis. Normalel synthesis is excellent in terms of library reproducibility and structure confirmation, because a library with a single compound strength can be obtained.
  • an "automatic synthesizer (synthetic bot)" can be used for preparing a combinatorial library.
  • the automatic synthesizer performs automatic synthesis by integrating normal synthesis. This overcomes the disadvantage that the number of compounds produced simultaneously is limited compared to split synthesis.
  • the present invention enables the efficient identification, selection and design of enzyme activity modulators (including inhibitory compounds) through the use of molecular design techniques. Therefore, in the present invention Based on the disclosure of the present invention, it is also contemplated that a drug by computer modeling is provided.
  • Computer programs for performing computer modeling can also be used in the process of designing or selecting chemicals. The following are examples of such programs.
  • MCSS Multiple Copy Simultaneous Search Method Proteins: Structure, Function and Genetics, 11, 29-34 (1991)).
  • MCSS is available from Molecular Simulations, San Diego, CA.
  • AUTODOCK DS Goodsell et al., “Automated Docking of Substrates to Proteins by Simulated Annealing J, Proteins: Structure, Function, and Genetics, 8, 195—202 (1990)).
  • AUTODOCK is a Scripps Research Institute. , Available from La Jolla, CA.
  • DOCK is available from the University of California, San Francisco, CA.
  • each fragment's relevance can be performed on a 3D image displayed on the computer screen in relation to the structural coordinates of the target enzyme. This can be followed by model building in a manual using software such as Quanta or Sybyl [Tripos Associates, St. Louis, MO].
  • Useful programs that can be used in linking individual compounds or portions thereof include: [0168] 1. CAVEAT (PA Bartlett et al., "CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active Molecules J (Molecular Recognition in Chemical and Biological Problems, Special Pu b., Royal Chem. Soc., 78 , 182-196 (1989)); G, Lauri and PA Bartlett, "CAVEAT: a Program to Facilitate the Design of Organic Moleculesj, J. Comput. Aided Mol. Des., 8, 51-66 (1994). CAVEAT is available from the University of California, Berkeley, CA.
  • HOOK (MB Eisen et al., "HOOK: A Program for Finding Novel Molecular Architectures that Satisfy the Chemical and Steric Requirements of a Macromolecule Binding Site", Proteins: Struct., Funct., Genet., 19, 199-221 (1994)). HOOK is available from Molecular Simulations, San Diego, CA.
  • an inhibitory or other enzyme binding compound is Compounds can be designed as whole molecules or entirely new, such as by the ability to use empty binding sites or, where necessary, include portions of several known inhibitors. Many novel ligand design methods are known in the art as follows.
  • LUDI H. — J. Bohm, “The Computer Program LUDI: A New Met hod for the De Novo Design of Enzyme InhibitorsJ, J. Comp. Aid.
  • LUDI is available from Molecular Simulations Incorporated, San Diego, CA.
  • an effective enzyme interaction site inhibitor should preferably exhibit a relatively small energy difference (ie, a small deformation energy of binding) between its bound and free states.
  • the most efficient enzyme inhibitors should preferably be designed with a deformation energy of binding of about lOkcalZ mol or less (more preferably 7 kcalZ mol or less). Enzyme inhibitors can interact with the binding pocket in two or more conformations that are similar in total binding energy. In these cases, the deformation energy of binding is thought to be the difference between the energy of the free substance and the average energy of these conformations observed when the inhibitor binds to the protein.
  • Substances designed or selected to bind to the enzyme are preferably calculated so that there is no electrostatic repulsive interaction with the target enzyme and surrounding water molecules in its bound state. Can be further optimized. Such non-complementary electrostatic interactions include charge-charge repulsion interactions, dipole-dipole repulsion interactions and charge-dipole repulsion interactions.
  • Another approach possible with the present invention is the computational screening of small molecule databases for compounds or parts thereof that can bind in whole or in part to the enzyme.
  • the quality of such a substance's fit to the binding site can be determined by either geometric complementarity or an estimated interaction energy deviation [EC Meng et al., J. Comp. Chem., 16, 505-524 (1992)].
  • the present invention uses a computerized quantitative structure activity relationship (QSAR) moirelay technique as a tool for screening efficacy for modulatory activity for compounds of the present invention. Includes the resulting compounds.
  • the computer technology includes the production of a substrate model, a pharmacophore, and a homologous model of the active site of the present invention, which are created by several converters.
  • the method for modeling the normal characteristic group of an interactant for a substance from data obtained in vitro is CAT ALYST pharmacophore method (Ekins et al., Pharmacogenetics, 9: 477-4 89, 1999; Ekins et al., Pharmacol. & Exp.
  • Fitting a compound to the active site can be performed using any of a variety of computer modeling techniques known in the art. Visual inspection and manual manipulation of compounds to active sites are described in QUANTA (Molecular Simulations, Burlington, MA, 1992), SYBYL (Molecular Modeling Software, Tripos Associates, Inc., St. Louis, MO, 1992), AMBER (Weiner et a 1., J. Am. Chem. Soc., 106: 765—784, 1984), CHARMM (Brooks et a 1., J. Comp. Chem., 4: 187-217, 1983), etc. You can use a program like this. In addition, energy can be minimized by using a standard force field such as CHARMM, AMBER, etc.
  • the present invention relates to pharmaceuticals (eg, pharmaceuticals such as vaccines, health foods, proteins or lipids having reduced antigenicity), and cosmetic compositions.
  • the pharmaceutical and cosmetic composition may further comprise a pharmaceutically acceptable carrier and the like.
  • the pharmaceutically acceptable carrier contained in the medicament of the present invention include any substance known in the art.
  • Such suitable formulation materials or pharmaceutically acceptable carriers include antioxidants, preservatives, colorants, flavors, and diluents, emulsifiers, suspending agents, solvents, fillers, fillers. These include, but are not limited to, dosages, buffers, delivery vehicles, diluents, excipients and / or pharmaceutical adjuvants.
  • the medicament of the present invention is administered in the form of a composition comprising the compound, or a variant or derivative thereof, together with one or more physiologically acceptable carriers, excipients or diluents.
  • a suitable vehicle can be water for injection, physiological solution, or artificial cerebrospinal fluid, which can be supplemented with other materials common to compositions for parenteral delivery. is there.
  • Acceptable carriers, excipients or stabilizers used herein are non-toxic to the recipient and are preferably inert at the dosages and concentrations used. And the following: phosphates, citrates, or other organic acids; ascorbic acid, a tocopherol; low molecular weight polypeptides; proteins (eg, serum albumin, gelatin or immunoglobulin); hydrophilic Polymers (eg, polybulurpyrrolidone); amino acids (eg, glycine, glutamine, asparagine, arginine or lysine); monosaccharides, disaccharides and other carbohydrates (including glucose, mannose, or dextrin); chelating agents (eg, EDTA); sugar alcohols (eg mannitol or sorbitol); Salt-forming counterions (eg sodium); and Z or non-ionic surfactants (eg Tween, pluronic or polyethylene glycol (PEG)).
  • phosphates, citrates, or other organic acids
  • Exemplary suitable carriers include neutral buffered saline or saline mixed with serum albumin.
  • the product is a suitable excipient (e.g. For example, it is formulated as a lyophilizing agent using sucrose.
  • Other standard carriers, diluents and excipients may be included as desired.
  • Other exemplary compositions include Tris buffer at pH 7.0—8.5 or acetate buffer at pH 4.0—5.5, which are sardine, sorbitol, or suitable substitutes thereof. Can be included.
  • the medicament of the present invention may be administered orally or parenterally.
  • the medicament of the present invention can be administered intravenously or subcutaneously.
  • the medicament used in the present invention may be in the form of a pharmaceutically acceptable aqueous solution free of pyrogens.
  • a pharmaceutically acceptable composition can be easily prepared by those skilled in the art by considering pH, isotonicity, stability, and the like.
  • the administration method includes oral administration, parenteral administration (e.g., intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, rectal administration, intravaginal administration, local administration to the affected area, Skin administration, etc.).
  • Formulations for such administration can be provided in any dosage form. Examples of such a preparation form include liquids, injections, and sustained release agents.
  • the medicament of the present invention may be a physiologically acceptable carrier, excipient, or stabilizer as necessary (Japanese Pharmacopoeia 14th edition or its latest edition, Remington's Pharmaceutical sciences, 18th Edition, AR Gennaro, ed., MacK Publishing Company, 1990, etc.) and a composition with the desired degree of purity and prepared and stored in the form of a lyophilized cake or aqueous solution Can be done.
  • the amount of the composition used in the treatment method of the present invention depends on the purpose of use, the target disease (type, severity, etc.), the patient's age, weight, sex, medical history, cell morphology or type, etc. In view of this, it can be easily determined by those skilled in the art.
  • the frequency with which the treatment method of the present invention is applied to a subject (or patient) also depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, gender, medical history, treatment course, etc. In view of this, it can be easily determined by those skilled in the art. Examples of the frequency include administration once a few months every day (for example, once a week, once a month). It is preferable to administer once a week-once a month with the progress of the test.
  • the cosmetic can be prepared while complying with the regulations stipulated by the authorities. [0191] (Pesticides)
  • composition of the present invention can also be used as an agrochemical component.
  • agrochemical component When processed as an agrochemical composition, it may contain agronomically acceptable carriers, excipients, stabilizers and the like, if necessary.
  • composition of the present invention is used as an agrochemical, herbicide (such as virazolate), insecticide 'acaricide (such as diazinon), fungicide (such as probenazole), plant growth regulator (such as E.g., noclobutrazole), nematicides (e.g., benomyl), synergists (e.g., pipeto-rubbutoxide), attractants (e.g., eugenol), repellents (e.g., Creosote, etc.), pigments (eg, Food Blue No. 1), fertilizers (eg, urea, etc.) can also be mixed if necessary.
  • herbicide such as virazolate
  • insecticide 'acaricide such as diazinon
  • fungicide such as probenazole
  • plant growth regulator such as E.g., noclobutrazole
  • nematicides e.g., benomyl
  • synergists e.g.,
  • the present invention can also be used in the health 'food field.
  • the points to be noted when used as an oral medicine should be considered as necessary.
  • it is used as a functional food such as specified health foods or 'health foods', it is preferable to treat them in accordance with pharmaceuticals.
  • the present invention can be applied not only to medical treatment but also to food inspection, quarantine, pharmaceutical inspection, forensic medicine, agriculture, livestock, fishery, forestry, etc. that require biomolecule inspection. is there .
  • the present invention also contemplates use for food safety purposes (eg, BSE testing).
  • the site of interaction of the enzyme with the substrate of the enzyme can be identified by analyzing the complex of the enzyme and the substrate by a physical method such as X-ray structural analysis. Alternatively, known data may be used for such interaction sites.
  • a physical method such as X-ray structural analysis.
  • known data may be used for such interaction sites.
  • Asp252, Asp254, Gly292, Trp314, Gly315, Glu317, Asp318, Met344, His347, and the like may be interaction sites.
  • This identification is preferably a more accurate identification, but as explained below, it is possible to identify the A part and thereby that part or If it is possible to produce a compound containing that moiety, it may not necessarily be necessary to identify it at all, and it may not need to be identified at all.
  • structural change necessary for catalytic reaction of an enzyme is required for performing a catalytic reaction that the enzyme plays in an enzyme whose conformation changes due to interaction with a substrate.
  • a structural change For example, ushi j8 1, 4 galactose transferase (SEQ ID NO: 1 (nucleic acid sequence) and 2 (amino acid sequence)), human
  • the sequence ()) is the enzyme, the conformational change caused by the binding of the donor substrate UDP Gal to the above enzyme is such a structural change. Due to this structural change, an acceptor (GlcNAc) binding site appears.
  • Identification of the structural change site responsible for the structural change necessary for the catalytic reaction of the enzyme can be performed using a physical method well known in the art (for example, X-ray structural analysis). Once the site responsible for the structural change is identified, the behavior of the site can be analyzed to help identify the following interacting parts.
  • the A moiety that interacts with the interaction site can be identified using techniques well known in the art.
  • the B moiety that interacts with the structural change site can also be identified using techniques well known in the art.
  • Such techniques include, but are not limited to, computer modeling, biochemical techniques, combinatorial chemistry, screening using genetic algorithms, and the like. Such techniques are for example
  • an enzyme that is a target of the screening method of the present invention may preferably be one that requires two or more molecules for the catalytic reaction.
  • examples of such enzymes include transferases (eg, acetyltransferases, glycosyltransferases, aminotransferases, phosphotransferases, dartamyl transferases, C1 compound transferases, ketone group transferases).
  • transferases eg, acetyltransferases, glycosyltransferases, aminotransferases, phosphotransferases, dartamyl transferases, C1 compound transferases, ketone group transferases.
  • aldehyde transferase acyl transferase, alkyl transferase, nitrogen-containing residue transferase, thiotransferase, peptide transferase, etc.
  • lyase removal addition enzyme
  • enzymes that require two or more molecules include, but are not limited to, enzymes that require a coenzyme (eg, NADPH, NADP, CoQ, etc.) for catalytic reactions.
  • a coenzyme eg, NADPH, NADP, CoQ, etc.
  • the enzyme targeted by the screening method of the present invention is a glycosyltransferase.
  • Glycosyltransferases usually require a donor substrate and an acceptor substrate for the catalytic reaction, and the binding of the donor substrate to the enzyme often causes a conformational change and promotes the catalytic reaction. By using such a mechanism, the screening method of the present invention can be applied.
  • the substrate in the screening method of the present invention, includes a donor and an acceptor, and an enzyme that catalyzes a reaction between the donor and the acceptor is used.
  • the structural change of the enzyme is caused by the donor, but is not limited thereto.
  • the A moiety interacts with the donor, and the B moiety may be a moiety responsible for entry of the acceptor into the catalytic site, but is not limited thereto.
  • the part responsible for the entry of the acceptor to the catalytic site can be easily determined by those skilled in the art by physical methods well known in the art.
  • known data can be used as the part responsible for the entry of the acceptor into the catalytic site.
  • such a conformational change is used for designing an activity regulator (eg, inhibitor).
  • examples of glycosyltransferases include ⁇ , 4-galactosyltransferase, ⁇ 1,3-galactosetransferase, ⁇ , 4-galactosyltransferase , ⁇ ⁇ , 3-galactose transferase, ⁇ ⁇ , 6-galactose transferase, 2, 6 sialyltransferase, 3,4 galactose transferase, ceramide galactose transferase, ⁇ ⁇ , 2 fucose transferase Enzymes, ⁇ 1,3 fucose transferase, ⁇ 1,4 fucose transferase, -1,6 fucose transferase, -1,3--acetyl galatatosamine transferase, ⁇ ⁇ , 6- ⁇ acetyl galata Tosamine transferase, ⁇ ⁇ , 4—
  • the enzyme targeted by the screening method of the present invention is ushi and human j81,4 galactosyltransferase shown in SEQ ID NOs: 2 and 4
  • the enzyme substrate comprises UDP galactose
  • the structural change site is a site containing tryptophan at amino acid position 314 of the polypeptide sequence shown in SEQ ID NO: 2 or 4.
  • similar moieties can be utilized.
  • the corresponding part can be utilized.
  • the enzyme used in the screening method of the present invention may be a wild-type enzyme, but for convenience of design, a variant (for example, a conservative variant, an allelic variant, etc.) Alternatively, fragments can be used. Alternatively, an enzyme variant containing an unnatural amino acid or amino acid analog can be used depending on the purpose of the design. Such variants and fragments can be produced using techniques well known in the art, and those skilled in the art can apply the present invention to the present invention by combining these techniques appropriately.
  • the present invention provides a method for screening for an inhibitor of glycosyltransferase.
  • This method comprises the following steps: I) a glycosyltransferase and an acceptor of the glycosyltransferase , A donor of the glycosyltransferase, and a candidate for the inhibitor, and the reaction is performed under conditions and time under which the glycosyltransferase reaction proceeds; and ii) a reaction product after the reaction in the step I) Measuring the reaction product of the acceptor with the donor.
  • the screening method of the present invention comprises, as necessary, 1) measuring the time course of the glycosyltransferase reaction, calculating the initial speed, and designing a screening based on the initial speed. 2) If necessary, confirm the effect of the click reaction residue on the enzyme reaction and take measures to avoid the effect, 3) Screen the inhibitor candidate compound, 4) Screening Can be performed by calculating the inhibition constant of the hit compound
  • an inhibitor candidate compound that can be screened in the screening method of the present invention can be produced by a click reaction (click chemistry).
  • the click reaction refers to a reaction in which triazole is synthesized from an acetylene compound and an azide compound by [3 + 2] cycloaddition efficiently in an aqueous system.
  • the yield of the inhibitor candidate substance produced by the click reaction can be determined, for example, by electrospray Z mass spectrometry (ESI-MS). The concentration of this inhibitor can be adjusted as necessary.
  • examples of the glycosyltransferase that can be used in the screening method of the present invention include el, 4 galactose transferase, ⁇ 1, 3 galactose transferase, ⁇ ⁇ , 4-galactose transferase.
  • the glycosyltransferase may be fucose transferase V, al, 6 fucose transferase VIII, «2,3 sialyltransferase, ⁇ 2,6 sialyltransferase.
  • the glycosyltransferase used by the screening method of the present invention is fucose transferase V
  • the acceptor is fucose transferase V
  • the conditions under which this glycosyltransferase reaction proceeds are, for example, 50 mM HEPES (pH 7.2), 10 mM manganese chloride, ImM acceptor, 200 M donor, 2.5 mU fucose transferase (FucT) V, 30 / z M inhibitor candidate compound mixture.
  • glycosyltransferase used in the screening of the present invention is ⁇ ⁇ , 6 fucose transferase VIII, and the acceptor is
  • the conditions under which this glycosyltransferase reaction proceeds are, for example, 50 mM force codylate buffer ( ⁇ 7.5), 10 mM salt-manganese, 100 ⁇ M Putter, 50 donors, 80 ⁇ ⁇ ] fucose transferase VIII, 50 GDP—6 ⁇
  • the glycosyltransferase that can be used in the screening of the present invention is a sialyltransferase, and the acceptor is
  • the conditions under which this glycosyltransferase reaction proceeds are, for example, 40 mM cacodylate-HCl (pH 7.5), 0.5 g / ml BSA, 0.1% surfactant. , Lm M acceptor, 200 M donor, lmU ⁇ 2-6 sialyltransferase (SEQ ID NO: 6 (amino acid sequence)).
  • the conditions under which the glycosyltransferase reaction proceeds are 40 mM cacodylate-HCl (pH 7.5), 0.5 g / ml BSA, 0.1% surfactant. Agent, ImM acceptor, 200 M donor, lmU ⁇ 2-3 sialyltransferase (SEQ ID NO: 7 (amino acid sequence)).
  • the glycosyltransferase that can be used in the screening method of the present invention is N-acetylethylgalatatosamine transferase, and the acceptor is
  • esoso / Loo ⁇ df / i3d III TCOTSO / LOOZ OAV
  • the donor can be UDP-GalNAc.
  • the conditions under which the glycosyltransferase reaction proceeds are, for example, 50 mM imidazole-HC1 buffer solution (containing 0.1% Triton X-100, pH 7.2), 500 ⁇ acceptor, 100 ⁇ donor, 100 ⁇ inhibitor candidate compound mixture, 10 mM manganese chloride, IngZ 1 N-acetylethyl latatosaminyltransferase ppGal NAcT-2.
  • the conditions under which the glycosyltransferase reaction proceeds can be acetylene candy, but are not limited thereto.
  • an acceptor that can be used in the screening methods of the present invention can be labeled with an isotope (eg, deuterium, light hydrogen, etc.).
  • an isotope eg, deuterium, light hydrogen, etc.
  • deuterium label can be used as an internal standard
  • light hydrogen label can be used for inhibitor activity evaluation.
  • initial rate of glycosyltransferase means that a glycosyltransferase, a glycosyltransferase acceptor, and a glycosyltransferase donor are mixed and reacted to produce a reaction product of the acceptor and the donor. It can be calculated by measuring the reaction rate over time.
  • the time for reacting the acceptor and the donor may be set based on the initial speed.
  • the initial speed may be a point in time when 1 to 20% of the reaction proceeds. For example, if the 10% time point is 30 minutes and the 20% time point is 90 minutes, the time corresponding to the initial speed of 10-20% is 60 minutes.
  • the reaction is, for example, 50 mM HEPES (pH 7.2), 10 mM manganese chloride, ImM acceptor, 200 ⁇ donor, 2.5 mU fucose transferase (FucT) V It can be carried out under conditions.
  • reaction is performed, for example, with 50 mM force codylate buffer (pH
  • reaction is performed, for example, with 40 mM cacodylate—HCl (pH 7
  • 0.5g / ml BSA, 0.1% Triton CF—54, ImM acceptor, 200 ⁇ M donor, ⁇ 2-6 sialyltransferase (SEQ ID NO: 6 (amino acid sequence)) can be performed under the condition of lmU.
  • the reaction comprises, for example, 40 mM cacodylate-HCl (pH 7.5), 0.5 g / ml BSA, 0.1% Triton CF-54, ImM acceptor, 200 ⁇ donor, ⁇ 2-3 sialyltransferase (SEQ ID NO: 7 (amino acid sequence)) can be performed under the condition of lmU.
  • the reaction may be performed, for example, with 50 mM imidazole-HC1 buffer solution (containing 0.1% Triton X-100, pH 7.2), 500 ⁇ acceptor, 100 ⁇ donor, 10 mM chloride.
  • Manganese, IngZ 1 N-Acetylgalatatosamine transferase ppGalNAcT-2 can be performed under the conditions.
  • reaction rate can be measured by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), but is not limited thereto.
  • MALDI-TOF-MS matrix-assisted laser desorption ionization time-of-flight mass spectrometry
  • the initial speed can be calculated in terms of a reaction rate of 10 to 20%. In the case of fucose transferase VIII, the initial rate can be 60 minutes.
  • the screening method of the present invention comprises reacting the acceptor, the donor, the glycosyltransferase, and the substance used for the click reaction by mixing them, and depending on the substance used for the click reaction.
  • the method may further include a step of confirming the influence on the enzyme reaction and avoiding the influence if there is such an influence.
  • the glycosyltransferase that can be used in the screening method of the present invention is fucose transferase (FucT) V
  • the substance used in the click reaction is 6-adducted GDP fucose, acetylene compound , Copper sulfate, tris [(1-benzyl-1- 1H-1,2,3-triazol-4-yl) methyl] amine (TBTA), or sodium ascorbate.
  • the glycosyltransferase that can be used in the screening method of the present invention is fucose transferase VIII
  • the substance used for the click reaction is 6azide GDP fucose, acetylene compound, copper sulfate , Tris [(1—Benzyl 1H— 1, 2, 3— Riazol-4-yl) methyl] amine (TBTA), or sodium ascorbate.
  • the glycosyltransferase that can be used in the screening method of the present invention is a 2,3 sialyltransferase
  • the substance used in the click reaction is an alkyne compound, copper sulfate.
  • Ascorbic acid tris [(1-benzyl-1H-1,2,3-triazole-4-yl) methyl] amine (TBTA), t-BuOH or CMP-azidosialic acid.
  • the glycosyltransferase that can be used in the screening method of the present invention is a 2,6-sialyltransferase
  • the substance used in the click reaction is an alkyne compound, copper sulfate, or the like.
  • Ascorbic acid tris [(1-benzyl-1H-1,2,3-triazole-4-yl) methyl] amine (TBTA), t-BuOH or CMP-azidosialic acid.
  • the glycosyltransferase that can be used in the screening method of the present invention is N-acetylethylgalatatosamine transferase
  • the substance used in the click reaction is UDP-N- GalNAc, acetylene compound, tris [(1—benzyl— 1H— 1, 2
  • the screening method of the present invention subtracts the inhibitory activity of a substance that affects the enzyme reaction from the inhibitory activity of the inhibitor candidate compound mixture. The influence on the reaction can be avoided.
  • a workaround that may be used in the present invention may be EDTA, for example in the case of Cu 2+.
  • the substances that affect the enzyme reaction are Cu2 + and GDP-6-N fucose.
  • Steps are avoided, for example, by EDTA, avoiding the impact of GDP- 6-N fucose
  • the means to do can be subtraction.
  • the present invention provides a method for calculating the inhibition constant of an inhibitor of glycosyltransferase.
  • This method comprises the following steps: I) Glycosyltransferase and the glycosyltransferase A step of mixing a scepter, a donor of the glycosyltransferase, and a candidate for the inhibitor and reacting them under conditions and time for which the glycosyltransferase reaction proceeds; II) a reaction product after the reaction in the step I) Measuring the reaction product of the acceptor with the donor; and
  • the inhibition constant can be calculated, for example, by determining Ki by a Dixon plot or by calculating IC50, but is not limited thereto.
  • the conditions under which the glycosyltransferase reaction proceeds are, for example, 50 mM HEPES (pH 7.2), 10 mM manganese chloride, ImM acceptor, 50 or 200 ⁇ M donor 2.5 mU fucose transferase (FucT) V, 0 to: LOO ⁇ inhibitor Can be a candidate compound mixture.
  • the conditions under which the glycosyltransferase reaction proceeds are, for example, 50 mM force codylate buffer (pH 7.5), 10 mM manganese chloride, 100 M acceptor, 12.5. ⁇ 75 donor, 80 ⁇ U fucose transferase VIII, 0-100 ⁇ ⁇ inhibitor inhibitor compound mixture.
  • the conditions under which the glycosyltransferase reaction proceeds are, for example, 40 mM cacodylate-HCl (pH 7.5), 0.5 g / ml BSA, 0.1% Triton CF. — 54, ImM acceptor, 200 M donor, lmU ⁇ 2-6 sialyltransferase (SEQ ID NO: 6 (amino acid sequence)), 0 to: L00 ⁇ ⁇ inhibitor inhibitor compound mixture.
  • the conditions under which the glycosyltransferase reaction proceeds are, for example, 40 mM cacodylate-HCl (pH 7.5), 0.5 g / ml BSA, 0.1% Triton CF. — 54, ImM acceptor, 200 M donor, lmU ⁇ 2-3 sialyltransferase (SEQ ID NO: 7 (amino acid sequence)), 0 to: L00 ⁇ ⁇ inhibitor inhibitor compound mixture.
  • the conditions under which the glycosyltransferase reaction proceeds are, for example, 50 mM imidazole-HC1 buffer solution (containing 0.1% Triton X-100, pH 7.2, 4 1), 500 ⁇ ⁇ acceptor, 100 ⁇ ⁇ donor, 10 mM manganese chloride, IngZ ⁇ ⁇ ⁇ ⁇ -acetyl galatatosamine transferase ppGalNAcT-2, 0-100 / ⁇ ⁇ ⁇ inhibitor Can be a candidate compound mixture.
  • the inhibition constant of the glycosyltransferase inhibitor of the present invention is calculated.
  • an internal standard may be mixed in addition to the inhibitor candidate compound mixture.
  • the present invention provides a glycosyltransferase acceptor.
  • This acceptor has the following structural formula:
  • ZER is the MS-sensitized moiety
  • NY is the natural mimic moiety
  • K is the modifiable moiety
  • the ZER of the glycosyltransferase acceptor of the present invention is represented by the following formula:
  • the NY of the glycosyltransferase acceptor of the present invention is:
  • the kappa of the glycosyltransferase acceptor of the present invention is:
  • glycosyltransferase acceptor of the present invention is, for example,
  • the present invention provides a method for producing a factor that modulates the activity of an enzyme.
  • a scheme for producing such an enzyme activity regulator is a scheme using conformational change, which is not conceivable from the conventional concept.
  • the target compound can be produced by appropriately combining the disclosure of this specification and techniques well known in the art. it can.
  • the step of preparing a molecule containing the A portion and the B portion is also performed by using or combining methods known in the art.
  • Such molecules can be prepared using various techniques such as chemical synthesis, biochemical or molecular biological (genetic engineering) production techniques, combinatorial chemistry, genetic algorithms, computer modeling, fitting, etc. .
  • a technique for example, techniques described in Susumu Okazaki, Hiroyuki Okamoto, biological computer simulation, chemical doujin; B. Fraserreid, K. Tatsuta, J. Thiem, Glycoscience, Springer can be used.
  • the present invention provides a composition comprising a compound provided in the present invention.
  • Such compounds usually have glycosyltransferase inhibitory activity.
  • Such a composition can be used in various fields such as pharmaceuticals, cosmetics, agricultural chemicals, foods (eg, health foods), biochemical research, and polymer production.
  • the composition of the present invention needs to conform to the standards and regulations applicable to pharmaceuticals, and if there are similar standards and regulations in other fields, such standards And must comply with regulations.
  • a regulator can be an inhibitor but can also be an active factor.
  • the yarn composition of the present invention can be used for the prevention, treatment, diagnosis, prognosis and the like of conditions, disorders and diseases involving ⁇ 1,4 galactosyltransferase.
  • Conditions, disorders, and diseases involving ⁇ 1,4 galactose transferase include, for example, cancer or cancer metastasis, Examples include, but are not limited to, cancer drug resistance, infectious diseases, chronic diseases (eg, arthritis, rheumatoid arthritis), diabetes, and cardiovascular diseases such as arteriosclerosis.
  • the composition of the present invention can be used for prevention, treatment, diagnosis, prognosis and the like of conditions, disorders and diseases involving fucose transferase.
  • Conditions, disorders or diseases involving fucose transferase include immune disease, cancer or cancer metastasis, bacterial infection, atherosclerosis, antibody-dependent cytotoxicity, signal transduction. It is not limited.
  • the composition of the present invention can be used for the prevention, treatment, diagnosis, prognosis and the like of conditions, disorders and diseases involving a 2,3-sialyltransferase.
  • «2,3-Sialyltransferase-related diseases include, but are not limited to, immune diseases, cancers such as colorectal cancer, breast cancer, leukemia, or cancer metastasis.
  • composition of the present invention can be used for prevention, treatment, diagnosis, prognosis and the like of conditions, disorders and diseases involving a 2,6-sialyltransferase.
  • a 2,6-Sialyltransferase-related diseases include, but are not limited to, immune diseases, cancers such as colon, rectal cancer, breast cancer, leukemia, or cancer metastasis.
  • the composition of the present invention comprises the above prevention, treatment, or regulation by regulating the activity of ⁇ ,, 4 galactose transferase, fucose transferase, glycan 2,3 sialyltransferase or oc 2,6 sialyltransferase. It can be used for diagnosis and prognosis.
  • the modulating activity may preferably be an inhibitory activity, but may also be an active activity. Inhibitory activity is preferred.
  • ⁇ ,, 4-galactose transferase-mediated galactose transfer plays a critical role in the development of a disorder or disease (eg, certain cancer metastasis or drug resistance), fucose transferase Disorders or diseases in which fucose transfer plays a significant role in the onset of the disorder or disease, ⁇ Disorders in which sialic acid transfer carried by sialyltransferase plays a significant role in the development of the disorder or disease
  • Prevention, treatment (treatment) or prognosis of a subject suffering from a disorder or disease in which the transfer of sialic acid carried by the 2,6 sialyltransferase plays a critical role in the onset of the disorder or disease By administering an appropriate dose, the prevention, treatment, prognosis and the like can be performed.
  • Such administration may be oral or parenteral.
  • parenteral administration include intravenous injection, intramuscular injection, subcutaneous injection, rectal administration, intravaginal administration, and direct administration to the affected area. But not limited to them.
  • the compositions of the present invention may contain suitable additives. The selection and addition of such additives are within the technical scope of the field, and can be easily performed by those skilled in the art, for example, by referring to the Japanese Pharmacopoeia 14th Amendment and its supplements. .
  • the fraction was concentrated under reduced pressure to remove the solvent, the residue was dissolved in methanol (5 ml), and a 60% aqueous acetic acid solution (10 ml) was stirred at 60 ° C. for 4 hours.
  • the reaction solution was removed by concentration under reduced pressure, and water was completely removed by azeotropic distillation with toluene several times.
  • the obtained compound was dissolved in pyridine (30 ml), and acetic anhydride (15 ml) was stirred at room temperature under ice cooling.
  • the solution was extracted with chloroform, washed with 1N sulfuric acid aqueous solution, saturated aqueous sodium hydrogen carbonate solution and brine in this order, and the organic layer was collected and dried over magnesium sulfate.
  • the labeled compound was obtained by reacting at 90 ° C. for 1 hour. After cooling, the reaction mixture was mixed 100 times with a matrix solution (2,5-dihydroxybenzoic acid, lOmgZmL 30% acetonitrile solution), and labeled directly by MALDI-TOF MS (Bruker, UltraFLEX). A spectrum with only chitotriose power was obtained.
  • the internal standard was a deuterium label, and the inhibitor activity was evaluated with a light hydrogen label.
  • reaction solution 50 mM HEPES buffer containing lOmM manganese chloride, 25 1
  • the calorie was adjusted to ⁇ ⁇ and 200 ⁇ ⁇ .
  • fucose transferase 2.5 mU, 51
  • reaction was diluted 10-fold with acetonitrile to inactivate the enzyme.
  • reaction solution was further diluted 10-fold with water, mixed 10-fold with a matrix solution (2,5-dihydroxybenzoic acid, 10 mg ZmL 30% acetonitrile solution), and MALDI-TOF MS (Bruker, UltraFLEX). Direct mass spectrometry was performed.
  • Buffer 50 mM HEPES, pH 7.2, lOmM MnC12 Donor: 200
  • the compounds of the present invention were found to have the ability to inhibit fucose transferase.
  • the relative ratio of the product of the enzyme reaction is shown. The smaller the value, the stronger the inhibitory activity.
  • the invented compound was found to have inhibitory activity, and changes with time were observed.
  • the reaction solution (50 mM HEPES buffer containing 10 mM manganese chloride, 25 1) was added with GDP fucose, aoWR-labeled galactosylchitotriose, sodium ascorbate, and copper sulfate, respectively, with a final concentration of 200 ⁇ , ImM, 400 We added it so that it became ⁇ ⁇ , 200 ⁇ .
  • Fucose transferase (2.5 mU, 51) was added, and the reaction was performed at a reaction temperature of 37 ° C.
  • the enzyme was inactivated by taking a part of the reaction solution every 10 minutes, 20 minutes, 30 minutes, 60 minutes, and 80 minutes and diluting the reaction solution 10-fold with acetonitrile.
  • reaction solution was further diluted 10-fold with water, mixed 10-fold with a matrix solution (2,5-dihydroxybenzoic acid, lOmgZmL 30% acetonitrile solution), and directly with MALDI-TOF MS (Bruker, UltraFLEX). Mass analyzed.
  • reaction condition (-) the reaction condition (+) plus the inhibitor mixture (200 M) (+).
  • the reaction conditions are as follows.
  • Donor concentrations were measured at 50 ⁇ , 200 ⁇ , and inhibitor concentrations at 100, 50, 25 ⁇ , 12. ⁇ , and 0 / ⁇ ⁇ ⁇ ⁇ . Based on the results of the change over time, the enzyme reaction time was set to 10 minutes, which is the initial velocity measurement range.
  • the reaction conditions are as follows.
  • Buffer 50 mM HEPES, pH 7.2, 10 mM MnC12
  • Donor 50 young ⁇ MA 200 / z M
  • R is:
  • reaction solution 50 mM HEPES buffer containing 10 mM manganese chloride, 25 1
  • GDP fucose, aoWR-labeled galactosylchitotriose, sodium ascorbate, and copper sulfate respectively
  • a final concentration of 200 ⁇ , ImM, 400 We added it so that it became ⁇ ⁇ , 200 ⁇ .
  • Fucose transferase (2.5 mU, 51) was added, and the reaction was performed at a reaction temperature of 37 ° C. After reacting for 40 minutes, the reaction solution was diluted 10-fold with acetonitrile to inactivate the enzyme.
  • reaction solution was further diluted 10-fold with water, mixed 10-fold with a matrix solution (2,5-dihydroxybenzoic acid, 10 mg / mL 30% acetonitrile solution), and MALDI-TOF MS (manufactured by Bruker, Direct mass spectrometry with UltraFLEX).
  • Buffer 50 mM HEPES, pH 7.2, 10 mM MnC12
  • Inhibitor candidate compound mixed solution 13a-13e, N3GDP—Fuc (+)
  • acetylene derivative (12a-12e, N3GDPFuc (—)
  • FIGS. 7A and 7B The results are shown in FIGS. 7A and 7B. As shown in FIG. 7A, it was revealed that there was a stronger inhibitory activity when the acetylene derivative was bound to the compound 11 by the click reaction (N 3GDPFuc (+)) than the free acetylene derivative. It was also demonstrated that naphthalene-containing sugar nucleotides (13b) have very strong activity compared to the other four types. FIG. 7A shows the relative ratio of the products of the enzyme reaction, and the smaller the value, the stronger the inhibitory activity. Furthermore, as shown in FIG. 7B, A16, A31, A34. L2N and L3N (same compounds as 13b) of the acetylene library showed an inhibition rate of 60% or more.
  • reaction solution 50 mM HEPES buffer containing 10 mM manganese chloride, 25 1
  • Fucose transferase 2.5 mU, 51
  • a portion of the reaction solution was taken every 10 minutes, 20 minutes, 30 minutes, 60 minutes, 80 minutes, and 160 minutes, and the reaction solution was diluted 10-fold with acetonitrile and inactivated the enzyme.
  • reaction solution was further diluted 10-fold with water, then mixed 10-fold with a matrix solution (2,5-dihydroxybenzoic acid, 10 mg / mL 30% acetonitrile solution), and MALDI-TOF MS (Bruker, UltraFLEX) was mixed. ) And mass analysis directly.
  • Buffer 50 mM HEPES, pH 7.2, 10 mM MnC12
  • the inhibition activity was measured by the following four methods.
  • the reaction conditions are as follows.
  • Buffer 50 mM HEPES, pH 7.2, 10 mM MnC12
  • A16 of the acetylene library was measured at donor concentrations of 37.5 / ⁇ ⁇ , 20 / ⁇ ⁇ , and inhibitor concentrations of 0 / z M, 0.5 M, and 1 M.
  • the donor concentration was measured at 5 M, 25 M, 50 ⁇
  • the inhibitor concentration was measured at 0.1 ⁇ , 0.5 ⁇ , 1 ⁇ , ⁇ , ⁇ .
  • the enzyme reaction time was set to 10 minutes, which is the initial velocity measurement range.
  • the reaction conditions are as follows.
  • Buffer 50 mM HEPES, pH 7.2, 10 mM MnC12
  • Donor 50 young ⁇ MA 200 / z M
  • Fmoc amino acid was reacted with MSNT and N-methylimidazole in dichloromethane and immobilized on Fmoc HMPA-PEGA resin.
  • the Fmoc protecting group was deprotected with 20% piperidine in DMF solvent.
  • Fmoc amino acid coupling prior to the introduction of sugar aspartate is: O-benzotriazole 1-yl N, N, ⁇ ', ⁇ , monotetramethyl-mu-tetrafluoroborate (TBTU), ⁇ -methylmorpholine ( ⁇ ), 1-hydroxy
  • TBTU monotetramethyl-mu-tetrafluoroborate
  • ⁇ ⁇ -methylmorpholine
  • HOBt benzotriazole
  • Fmoc sugar gas formic acid
  • DMF 3- (diethoxyphosphoryloxy) -1,2,3 benzotriazi-4- (3H) one (DEPBT), diisopropylethylamine (DIEA). It was cut out. This reaction was repeated to extend the peptide. Finally, the peptide was excised from the resin in water using trifluoroacetic acid (TFA) and triisoprovir silyl compounds (TIPs), and freeze-dried in a 0.1% TFA aqueous solution.
  • TFA trifluoroacetic acid
  • TIPs triisoprovir silyl compounds
  • the glycopeptide was purified using HPLC.
  • Column is Supelco Discovery HS C18 (25cm x 10mm), 0.1% TFA aqueous solution and acetonitrile solvent, gradient of acetononitrile from 10% to 60% in 60 minutes, flow rate 2.8 ⁇ / t Separation and purification were performed under conditions.

Abstract

An object of the invention is to provide various inhibitors of various glycosyltransferases. The object was achieved in the invention by focusing on the conformation change of the enzyme induced by the substrate and applying the correlation between the change and the catalytic activity to a design of a regulatory factor (such as an inhibitor) of an enzymatic activity, more specifically, by providing a novel compound obtained by introducing a bulky group into a modified nucleotide or sugar via a triazole group, an oxime group, a hydrazone group or the like.

Description

明 細 書  Specification
糖転移酵素阻害剤  Glycosyltransferase inhibitor
技術分野  Technical field
[0001] 本発明は、概して、酵素学の分野に関する。より詳細には、本発明は、糖転移酵素 の阻害剤およびその利用に関する。  [0001] The present invention relates generally to the field of enzymology. More particularly, the present invention relates to an inhibitor of glycosyltransferase and use thereof.
背景技術  Background art
[0002] 糖鎖は、生体内において、多用で重要な役割を果たしていることが知られている。  [0002] It is known that sugar chains play many and important roles in vivo.
細胞表面に存在する種々のタンパク質および脂質などは、突き出した糖鎖をもって おり、それら糖鎖は、各種細胞、ホルモン、あるいは感性性の細菌、ウィルス、毒素と Various proteins and lipids present on the cell surface have protruding sugar chains, and these sugar chains are linked to various cells, hormones, or sensitive bacteria, viruses, and toxins.
V、つた細胞外の環境との接着部位として働 ヽて 、る。 V, acting as a site of adhesion with the extracellular environment.
[0003] それら糖タンパク質や糖脂質は、その前駆体が細胞内の小胞体、ゴルジ体を通過 する際に、糖転移酵素と呼ばれる一群の糖鎖伸長酵素によって複雑な糖鎖修飾を 受け、機能を発現する。糖転移酵素は一般的に、単糖に UDP、 GDP、 CMPなどと [0003] When these precursors pass through the endoplasmic reticulum and Golgi apparatus in the cell, these glycoproteins and glycolipids undergo complex sugar chain modification by a group of sugar chain extenders called glycosyltransferases. Is expressed. Glycosyltransferases are generally composed of simple sugars such as UDP, GDP, and CMP.
V、つたヌクレオチジル基が結合した糖ヌクレオチドを糖ドナーとして、多様な構造を持 っ糖ァクセプターへ糖を一つずつ転移させる。これら多種多様の糖転移酵素の発現 の場所、タイミング、活性が複雑な糖鎖の合成をコントロールし、細胞の表現形に極 めて重要な影響を与えて ヽると考えられる。 V, transfer sugars one by one to sugar acceptors with various structures using sugar nucleotides with attached nucleotidyl groups as sugar donors. Controlling the synthesis of sugar chains with complex expression, timing and activity of these various glycosyltransferases, it is thought to have an extremely important influence on the phenotype of cells.
[0004] 生体内において糖鎖の持つ重要性が認識されるにつれて、糖鎖合成酵素である 糖転移酵素の生体内での働きを知ることが、生物の発生、分化、維持の理解のため に重要であることが近年わ力つてきた。現在までにその由来も含め、数十種にも及ぶ 糖転移酵素が発見され、多くの研究者が各々の必要とする情報を得るために、様々 な手法を用いて糖転移酵素の活性測定を行ってきた (例えば、非特許文献 1 = Palci c, M. M. , ΚΘΪΚΟ, ¾ . (2001; Assays for Glycosyltransf erases, Trends m Glycoscience and Glycotechnology, 13, 361— 370参照)。  [0004] As the importance of sugar chains in the living body is recognized, knowing the action of glycosyltransferase, a sugar chain synthase, in vivo is important for understanding the development, differentiation and maintenance of living organisms. In recent years it has become more important to be important. To date, dozens of glycosyltransferases, including their origin, have been discovered, and many researchers have used various methods to measure glycosyltransferase activity in order to obtain the information they need. (See, for example, Non-Patent Document 1 = Palci c, MM, ΚΘΪΚΟ, ¾. (2001; Assays for Glycosyltransf erases, Trends m Glycoscience and Glycotechnology, 13, 361-370).
[0005] 現在では酵素活性測定の手段として、基質反応量をラジオアイソトープを用いて検 出する手法、特定の糖鎖を認識して接着する抗体ゃレクチンに蛍光標識を施すこと で検出する手法、などが広く用いられている。これらの手法はその感度、検体使用料 の少なさにおいて非常に優秀であり、今後も広く採用されてゆくと思われる。しかしな 力 これらの手法を用いるためには、ラジオアイソト一プを用いる危険性、それに伴う 専門の施設の必要性、特殊な抗体ゃレクチンの入手困難さ、高度な専門知識を必 要とするなど、多くの欠点を伴っていることも事実である。こういった背景から、より安 全、簡便で、高感度な糖転移酵素活性測定法の確立が強く望まれていた。 [0005] Currently, as a means of measuring enzyme activity, a method of detecting the amount of substrate reaction using a radioisotope, a method of detecting an antibody that recognizes and adheres to a specific sugar chain by applying a fluorescent label, Are widely used. These methods are sensitive, specimen usage fees It is very excellent in that there are few, and it will be widely adopted in the future. However, in order to use these methods, the risk of using radioisotopes, the need for specialized facilities, the difficulty of obtaining special antibodies and lectins, and the need for advanced expertise, etc. It is also true that it has many drawbacks. Against this background, the establishment of a safer, simpler, and more sensitive method for measuring glycosyltransferase activity has been strongly desired.
近年、蛍光エネルギー移動（非特許文献 2= Fluorescence Resonance Energ y Transfer:本明細書において FRETともいう）という原理に基づいた酵素活性測 定法が注目されている。この手法では上記のような従来法の欠点を克服し、安全、簡 便、高感度、さらに従来法では原理的に不可能であった、連続的な酵素反応の様子 を観察することができる。しカゝしながら、現在までにこの手法を用いた酵素活性測定 は、 1基質反応を行う酵素（アミラーゼ (例えば、非特許文献 2 = Nathalie, P. , Sylv ain, C. , Hugues. D. (1995) , Angew. Chem. Int. Eds. Engl. , 34, 1239— 1241、および非特許文献 3=Kaoru Omichi, Tokuji Ikenaka (1986) , J. Bio chem. 99, 291—294参照）、ヌクレァーゼ（例ぇば、非特許文献4 = 3011111辻 . et . al. (1994) , Nucleic Acids Research, 22, 3155— 3159、および非特許文 献 5=Lee, S, P. et. al. (1995) , Anal. Biochem. 227, 295— 301参照）、セラ ミドグリカナーゼ（例えば、非特許文献 6=Kyung, B. Lee, Koji Matsuoka, Shi n- Ichiro Nishimura, Yuan. C. Lee. (1995) , Anal. Biochem. 230, 31— 3 6、および非特許文献 7=Koji Matsuoka, Shin -Ichiro Nishimura, Yuan C . Lee. (1995) , Carbohydrate Research, 276, 31— 42参照），ぺプチダ—ゼ（ 例えば、非特許文献 8=Edmund D. Matayoshi, Gary T. Wang, Grant A. Krafft, John Erickson(1990) ,非特許文献 9 = Science, 247, 954— 957、 G ary T. Wang, et. al. (1993) , Anal. Biochem. 210, 351— 359、および非特 許文献 10=Linda L. M. , Clark W. Smith, Zhong— Yin Zhang (1992) , J . Med. Chem. 35, 3727— 3730参照））等【こつ!ヽてのみ行われてきた。これらの 例では、酵素の認識部位力 外れた位置に蛍光エネルギー移動を行う 2種の蛍光物 質を導入することにより、酵素反応によって回復する蛍光ドナー力 の蛍光強度の変 化を観察することでその活性を測定しており、この手法の有用性が示されてきた。し 力しながら、 2基質系酵素である糖転移酵素の活性測定については、酵素の重要性 に関する認識不足や、基質標識困難さ、標識による酵素活性への影響を懸念して、 最近まで行われていなかった。し力しながら当研究室において、最近初めて糖転移 酵素につ 1ヽて FRET法を用いた酵素活性測定例が報告された (例えば、非特許文 献 l l =Kimito Washiya, Tetsuya Furuike, Fumio Nakajima, Yuan じ. L ee, Shin -Ichiro Nishimura (2000) , Anal. Biochem. 283, 39— 48参照）。 この報告では、 α 2, 6シアル酸転移酵素について、その手法の有用性が示されてい る。こういったことが可能になってきた背景には、その酵素の重要性が認知されてきた ことに加え、基質と酵素活性部位との結合様式にっ 、ての知見が得られるようになつ てさたこと〖こよる。 In recent years, an enzyme activity measurement method based on the principle of fluorescence energy transfer (Non-patent Document 2 = Fluorescence Resonance Energ y Transfer: also referred to as FRET in the present specification) has attracted attention. This method overcomes the drawbacks of the conventional method as described above, and allows the observation of the state of continuous enzyme reaction, which is safe, simple, and highly sensitive, and was impossible in principle by the conventional method. However, until now, enzyme activity measurement using this method has been as follows: (1) enzyme that performs a substrate reaction (eg, amylase (eg, Non-Patent Document 2 = Nathalie, P., Sylvain, C., Hugues. D. (1995), Angew. Chem. Int. Eds. Engl., 34, 1239—1241, and non-patent literature 3 = Kaoru Omichi, Tokuji Ikenaka (1986), J. Bio chem. 99, 291—294), nuclease. (For example, Non-Patent Document 4 = 3 0 11111 辻. Et. Al. (1994), Nucleic Acids Research, 22, 3155-3159, and Non-Patent Document 5 = Lee, S, P. et. Al. (1995), Anal. Biochem. 227, 295—301), ceramide glycanase (for example, Non-Patent Document 6 = Kyung, B. Lee, Koji Matsuoka, Shin-Ichiro Nishimura, Yuan. C. Lee. (1995) , Anal. Biochem. 230, 31—36, and non-patent document 7 = Koji Matsuoka, Shin-Ichiro Nishimura, Yuan C. Lee. (1995), Carbohydrate Research, 276, 31—42), peptidase (For example, Non-Patent Document 8 = Edmund D. Matayoshi, Gary T. Wang, Gran t A. Krafft, John Erickson (1990), Non-Patent Document 9 = Science, 247, 954—957, Gary T. Wang, et. al. (1993), Anal. Biochem. 210, 351—359, and non-specific Permit 10 = Linda LM, Clark W. Smith, Zhong— See Yin Zhang (1992), J. Med. Chem. 35, 3727-3730)) etc. In these examples, by introducing two types of fluorescent substances that transfer fluorescence energy to a position that deviates from the recognition site of the enzyme, the change in the fluorescence intensity of the fluorescence donor force recovered by the enzyme reaction is observed. Its activity has been measured and the utility of this technique has been demonstrated. Shi However, the measurement of the activity of glycosyltransferase, a two-substrate enzyme, has been carried out until recently due to lack of recognition regarding the importance of the enzyme, difficulty in labeling the substrate, and the effect of labeling on enzyme activity. There wasn't. However, our laboratory recently reported an example of enzyme activity measurement using the FRET method for the first time as a glycosyltransferase (for example, non-patent document ll = Kimito Washiya, Tetsuya Furuike, Fumio Nakajima, Yuan J. Lee, Shin-Ichiro Nishimura (2000), Anal. Biochem. 283, 39-48). This report demonstrates the usefulness of the method for α 2,6 sialyltransferases. The reason why this has become possible is that the importance of the enzyme has been recognized, and that the knowledge of the mode of binding between the substrate and the enzyme active site has become available. That's what I mean.
[0007] 本研究では、 α 2, 6シアル酸転移酵素同様、その基質特異性について研究が進 んで 、るガラクトース転移酵素にっ 、て FRET法を利用して活性測定を行 、、この手 法の汎用性を広めることを目的とした。この手法が確立することにより、細胞抽出画分 中の糖転移酵素の活性量を容易に測定できるようになるだけでなぐプレート上で酵 素反応を行うことでハイスループットな酵素阻害剤の評価が可能になると期待される  [0007] In this study, as with α2,6-sialyltransferase, research on substrate specificity has progressed, and galactose transferase has been used to measure activity using the FRET method. The purpose was to spread the versatility of. By establishing this method, it is possible to easily measure the amount of glycosyltransferase activity in cell extract fractions. By performing an enzyme reaction on a plate, high-throughput enzyme inhibitors can be evaluated. Expected to be possible
[0008] 従来の阻害剤設計の研究は、酵素と基質との間の相互作用に基づき、基質との競 合作用を探索することによって行われてきた。しかし、このような競合作用による相互 作用の干渉では、いくらか基質が酵素と結合するということが避けられず、効率よい 阻害剤の設計には限度がある。そこで、新たなメカニズムに基づく阻害剤の新たな設 計方法に対する要望がある。 [0008] Conventional inhibitor design research has been performed by searching for a competitive action with a substrate based on the interaction between the enzyme and the substrate. However, such interference of interactions due to competitive action unavoidably causes some substrate binding to the enzyme, and there is a limit to the design of efficient inhibitors. Therefore, there is a demand for a new method for designing inhibitors based on a new mechanism.
[0009] このような状況下で、本発明者らは、新たな糖転移阻害剤の設計'製造法を創出し た (特許文献 l =WO2004Z069855)。この特許文献では、ガラクトース転移酵素 のトリブトファンと着目した設計を提唱した。 [0009] Under these circumstances, the present inventors have created a new design and production method for glycosyl transfer inhibitors (Patent Document 1 = WO2004Z069855). In this patent document, a design focusing on galactose transferase tributophan was proposed.
[0010] しかし、このような組み合わせ以外のものについては、具体的な物を提唱していな い。 [0010] However, there is no specific proposal for anything other than such combinations.
[0011] 従って、新たな構造骨格を有する糖転移酵素を提供することにおいて未だに需要 は存在する。 特許文献 1：国際公開第 2004Z069855号パンフレット [0011] Accordingly, there is still a need in providing glycosyltransferases having a new structural skeleton. Patent Document 1: International Publication No. 2004Z069855 Pamphlet
非特許文献 l : Palcic, M. M. , Keiko, S. (2001) Assays for Glycosyltransf erases, Trends in Glycoscience and Glycotechnology, 13, 361— 370 非特許文献 2 : Nathalie, P. , Sylvain, C. , Hugues. D. (1995) , Angew. Che m. Int. Eds. Engl. , 34, 1239— 1241.  Non-patent literature l: Palcic, MM, Keiko, S. (2001) Assays for Glycosyltransf erases, Trends in Glycoscience and Glycotechnology, 13, 361—370 Non-patent literature 2: Nathalie, P., Sylvain, C., Hugues. D (1995), Angew. Chem. Int. Eds. Engl., 34, 1239—1241.
非特許文献 3 :Kaoru Omichi, Tokuji Ikenaka (1986) , J. Biochem. 99, 291 - 294.  Non-Patent Document 3: Kaoru Omichi, Tokuji Ikenaka (1986), J. Biochem. 99, 291-294.
非特許文献 4: Soumitra. et. al. (1994) , Nucleic Acids Research, 22, 315 5- 3159.  Non-Patent Document 4: Soumitra. Et.al. (1994), Nucleic Acids Research, 22, 315 5--3159.
非特許文献 5 :Lee, S, P. et. al. (1995) , Anal. Biochem. 227, 295- 301. ^^特許文献 6 :Kyung, B. Lee, Koji Matsuoka, Shin— Ichiro Nishimura, Y uan. C. Lee. (1995) , Anal. Biochem. 230, 31— 36.  Non-Patent Document 5: Lee, S, P. et. Al. (1995), Anal. Biochem. 227, 295- 301. ^^ Patent Document 6: Kyung, B. Lee, Koji Matsuoka, Shin— Ichiro Nishimura, Y uan. C. Lee. (1995), Anal. Biochem. 230, 31— 36.
非特許文献 7 :Koji Matsuoka, Shin -Ichiro Nishimura, Yuan C. Lee. (1 995) , Carbohydrate Research, 276, 31—42.  Non-Patent Document 7: Koji Matsuoka, Shin -Ichiro Nishimura, Yuan C. Lee. (1 995), Carbohydrate Research, 276, 31-42.
非特許文献 8 : Edmund D. Matayoshi, Gary T. Wang, Grant A. Krafft, Jo hn Erickson (1990) , Science, 247, 954— 957.  Non-Patent Document 8: Edmund D. Matayoshi, Gary T. Wang, Grant A. Krafft, John Erickson (1990), Science, 247, 954—957.
非特許文献 9 : Gary T. Wang, et. al. (1993) , Anal. Biochem. 210, 351— 3 59.  Non-Patent Document 9: Gary T. Wang, et. Al. (1993), Anal. Biochem. 210, 351—3 59.
非特許文献 10 : Linda L. M. , Clark W. Smith, Zhong— Yin Zhang (1992) , J. Med. Chem. 35, 3727- 3730.  Non-Patent Document 10: Linda L. M., Clark W. Smith, Zhong— Yin Zhang (1992), J. Med. Chem. 35, 3727-3730.
非特許文献 11 :Kimito Washiya, Tetsuya Furuike, Fumio Nakajima, Yua n C. Lee, Shin -Ichiro Nishimura (2000) , Anal. Biochem. 283, 39—48 発明の開示  Non-Patent Document 11: Kimito Washiya, Tetsuya Furuike, Fumio Nakajima, Yuan C. Lee, Shin-Ichiro Nishimura (2000), Anal. Biochem. 283, 39-48 Disclosure of the Invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0012] 従って、種々の糖転移酵素の種々の阻害剤を提供することが必要である。 [0012] Therefore, it is necessary to provide various inhibitors of various glycosyltransferases.
[0013] このような状況の下、本発明は、従来の方法とは異なる新規のメカニズム糖転移酵 素の活性調節因子 (例えば、阻害因子)を設計する方法を提供することを課題とする 課題を解決するための手段 [0013] Under such circumstances, an object of the present invention is to provide a method for designing a novel mechanism of transglycosylase activity regulation (for example, an inhibitor) different from conventional methods. Means for solving the problem
[0014] 上記課題は、本発明において、酵素の基質によるコンフオメーシヨン変化に着目し 、その変化と触媒活性との関連を酵素活性調節因子 (例えば、阻害因子)の設計に 応用することによって、具体的には、ヌクレオチドおよび糖の改変体に、トリァゾール 基、ォキシム基、ヒドラゾン基等を介して嵩高基を導入した新規ィ匕合物を提供すること によって解決された。  [0014] In the present invention, the above-mentioned problem is focused on conformational change due to the substrate of the enzyme, and by applying the relationship between the change and catalytic activity to the design of an enzyme activity regulator (for example, inhibitor), Specifically, the problem was solved by providing a novel compound in which a bulky group was introduced into a modified nucleotide and sugar via a triazole group, an oxime group, a hydrazone group, or the like.
[0015] 本発明は、以下を提供する。  [0015] The present invention provides the following.
(項目 1)以下の構造式：  (Item 1) The following structural formula:
A-B-C  A-B-C
を有する、化合物またはその塩であって、  A compound or a salt thereof,
式中  In the formula
Aは、アジド（― N )、アルデヒド置換基（― C ( = 0)—R)またはアルデヒド（— CHO)  A is azide (—N), aldehyde substituent (—C (= 0) —R) or aldehyde (—CHO)
3  Three
であり、 Rはァノレキノレであり、  R is Anolequinore,
Bは、糖成分であり、  B is a sugar component,
Cは、ヌクレオチジル基である、  C is a nucleotidyl group,
ただし、糖成分が GlcNAcである場合は、 Aはアジドである、  However, when the sugar component is GlcNAc, A is an azide.
化合物またはその塩。  Compound or salt thereof.
(項目 2)上記 Cは、ヌクレオシドーリン酸、ヌクレオシドニリン酸またはヌクレオシド三リ ン酸である、項目 1に記載の化合物またはその塩。  (Item 2) The compound or a salt thereof according to item 1, wherein C is nucleoside phosphoric acid, nucleoside diphosphate or nucleoside triphosphate.
(項目 3)上記 Cは、ピリミジンヌクレオシドリン酸またはプリンヌクレオシドリン酸である 、項目 1に記載の化合物またはその塩。  (Item 3) The compound or a salt thereof according to Item 1, wherein C is pyrimidine nucleoside phosphate or purine nucleoside phosphate.
(項目 4)上記 Cは、アデノシン、デォキシアデノシン、グアノシン，デォキシグアノシン 、シチジン、デォキシチミジン、チミジン（リボチミジン）、デォキシチミジンまたはゥリジ ンあるいはそれらの改変体のホスフェートである、項目 1に記載の化合物またはその 塩。  (Item 4) The above C is a phosphate of adenosine, deoxyadenosine, guanosine, deoxyguanosine, cytidine, deoxythymidine, thymidine (ribothymidine), deoxythymidine or uridine, or a modified form thereof. Compound or its salt.
(項目 5)上記 Bは、 D—ガラクトース、 L—フコース、シアル酸、 D— GlcNAc、 D— Ga IN Ac, D— ManNAc、 D—マンノース、もしくは D—グルコースまたはその誘導体で ある、項目 1〜4のいずれかに記載の化合物またはその塩。 (Item 5) The above B is D-galactose, L-fucose, sialic acid, D-GlcNAc, D-GaIN Ac, D-ManNAc, D-mannose, or D-glucose or a derivative thereof. The compound or its salt in any one of the items 1-4 which are.
(項目 5A)上記 Aがアジドである、項目 1〜5のいずれかに記載の化合物またはその 塩。  (Item 5A) The compound or salt thereof according to any one of Items 1 to 5, wherein A is azide.
(項目 5B)上記 Aがアルデヒド置換基である、項目 1〜5のいずれかに記載の化合物 またはその塩。  (Item 5B) The compound or salt thereof according to any one of Items 1 to 5, wherein A is an aldehyde substituent.
(項目 5C)上記 Aがアルデヒドである、項目 1〜5のいずれかに記載の化合物または その塩。  (Item 5C) The compound or salt thereof according to any one of Items 1 to 5, wherein A is an aldehyde.
(項目 6)以下の式：  (Item 6) The following formula:
[化 21] [Chemical 21]
Figure imgf000007_0001
Figure imgf000007_0001
[化 23]
Figure imgf000008_0001
[化 27] [Chemical 23]
Figure imgf000008_0001
[Chemical 27]
Figure imgf000009_0001
Figure imgf000009_0001
[化 28] [Chemical 28]
Figure imgf000009_0002
Figure imgf000009_0002
または Or
[化 28- 1]  [Chemical 28-1]
Figure imgf000009_0003
Figure imgf000009_0003
に示す化合物。 The compound shown in.
(項目 7)以下の構造式 (Item 7) The following structural formula
X-Y-B-C X-Y-B-C
を有する化合物であって、 式中 A compound having In the formula
Bは、糖成分であり、  B is a sugar component,
Cは、ヌクレオチジル基であり、  C is a nucleotidyl group,
Xは、嵩高基であり、 Yは、— O— N =基または、— NH— N =基または 1, ァゾール基  X is a bulky group, Y is —O—N = group or —NH—N = group or 1, azole group
[化 29] [Chemical 29]
Figure imgf000010_0001
である、
Figure imgf000010_0001
Is,
化合物またはその塩。 Compound or salt thereof.
(項目 7' )上記 Cは、ァシル (好ましくはァセチル)である項目 7に記載の化合物また はその塩。  (Item 7 ′) The compound or a salt thereof according to item 7, wherein C is acil (preferably acetyl).
(項目 8)上記 Cは、ヌクレオシドーリン酸、ヌクレオシドニリン酸またはヌクレオシド三リ ン酸である、項目 7に記載の化合物またはその塩。  (Item 8) The compound or a salt thereof according to item 7, wherein C is nucleoside phosphoric acid, nucleoside diphosphate or nucleoside triphosphate.
(項目 9)上記 Cは、ピリミジンヌクレオシドリン酸またはプリンヌクレオシドリン酸である 、項目 7に記載の化合物またはその塩。  (Item 9) The compound or a salt thereof according to Item 7, wherein C is pyrimidine nucleoside phosphate or purine nucleoside phosphate.
(項目 10)上記 Cは、アデノシン、デォキシアデノシン、グアノシン，デォキシグアノシ ン、シチジン、デォキシチミジン、チミジン（リボチミジン）、デォキシチミジンまたはゥリ ジンあるいはそれらの改変体のホスフェートである、項目 7に記載の化合物またはそ の塩。  (Item 10) The compound according to item 7, wherein C is a phosphate of adenosine, deoxyadenosine, guanosine, deoxyguanosine, cytidine, deoxythymidine, thymidine (ribothymidine), deoxythymidine or uridine or a variant thereof. Or its salt.
(項目 11)上記 Bは、 D—ガラクトース、 L—フコース、シアル酸、 D— GlcNAc、 D— GalNAc、 D— ManNAc、 D—マンノース、もしくは D—グノレコースまたはその誘導 体である、項目 7、 7'、 8〜10のいずれかに記載の化合物またはその塩。  (Item 11) The above B is D-galactose, L-fucose, sialic acid, D-GlcNAc, D-GalNAc, D-ManNAc, D-mannose, or D-gnolecose or its derivatives, items 7, 7 ', The compound or its salt in any one of 8-10.
(項目 12) Xは、以下： (Item 12) X is as follows:
[化 30A]
Figure imgf000011_0001
[Chemical 30A]
Figure imgf000011_0001
Η3Ν 0^ ^ 0Η 3 Ν 0 ^ ^ 0
+Η3Ν' '。^\。/\/0 、 + ^
Figure imgf000011_0002
+ Η 3 Ν ''. ^ \. / \ / 0 , + ^
Figure imgf000011_0002
[化 30Β] [Chemical 30Β]
Figure imgf000012_0001
Figure imgf000012_0001
[化 30C] [Chemical 30C]
Figure imgf000013_0001
Figure imgf000013_0001
[化 30D] [Chemical 30D]
Figure imgf000014_0001
Figure imgf000014_0001
[化 30E] [Chemical 30E]
Figure imgf000015_0001
Figure imgf000015_0001
[化 30G] [Chemical 30G]
Figure imgf000016_0001
Figure imgf000016_0001
[化 30H] [Chemical 30H]
Figure imgf000017_0001
Figure imgf000017_0002
Figure imgf000017_0003
Figure imgf000017_0001
Figure imgf000017_0002
Figure imgf000017_0003
Figure imgf000017_0004
Figure imgf000017_0005
Figure imgf000017_0004
Figure imgf000017_0005
[化 301] O〜リ [Chemical 301] O ~ Li
。〜o 、 0〜o ^-- 0〜o、. -. ~o, 0 ~o ^ - 0 ~o ,. -
、〇■〜, ○ ■ ~
■ 0_^0〜O .ハ0〜. 一 1- N ■ 0_ ^ 0- O. C 0- . 1 1- N
O  O
Figure imgf000018_0001
または
Figure imgf000018_0001
Or
のいずれかから選択され、上記 Xが 2つの結合部位を有する基である場合は、 C B — Y— X— Y— B— Cを形成する、項目 7、 7，、 8〜： 11のいずれかに記載の化合物。 (項目 13) Any one of the above, and when X is a group having two binding sites, CB — Y— X— Y— B— C is formed, any of items 7, 7, 8 to 11: Compound described in 1. (Item 13)
[化 31] [Chemical 31]
81031 18 81031 18
または
Figure imgf000019_0001
Or
Figure imgf000019_0001
Figure imgf000019_0002
Figure imgf000019_0002
[化 3ト 1] [Chemical 3rd 1]
Figure imgf000020_0001
Figure imgf000020_0001
[化 3ト 2] [Chemical 3rd 2]
Figure imgf000021_0001
Figure imgf000021_0002
Figure imgf000021_0001
Figure imgf000021_0002
Figure imgf000021_0003
Figure imgf000021_0003
Figure imgf000021_0004
である、化合物。
Figure imgf000021_0004
A compound.
(項目 14)項目 7〜 13または 7'のいずれか 1項に記載の化合物を含む、糖転移酵素 阻害剤。  (Item 14) A glycosyltransferase inhibitor comprising the compound according to any one of items 7 to 13 or 7 ′.
(項目 15)上記糖転移酵素は、 αΐ, 4 ガラクトース転移酵素、 《1, 3 ガラクトー ス転移酵素， ι81, 4—ガラクトース転移酵素， |81, 3—ガラクトース転移酵素， θΐ, 6 ガラクトース転移酵素、《2, 6 シアル酸転移酵素、《1, 4 ガラクトース転移 酵素、セラミドガラタトース転移酵素、 αΐ, 2 フコース転移酵素、ひ1, 3 フコース 転移酵素、 al, 4ーフコース転移酵素、 αΐ, 6 フコース転移酵素、 α 1, 3— Ν— ァセチルガラタトサミン転移酵素、《1, 6— Ν ァセチルガラタトサミン転移酵素、 /3 1, 4 Ν ァセチルガラタトサミン転移酵素、ポリペプチド Ν ァセチルガラタトサミン 転移酵素、 /31, 4 Νァセチルダルコサミン転移酵素、 131, 2— Νァセチルダルコ サミン転移酵素、 β ΐ, 3— Νァセチルダルコサミン転移酵素、 β ΐ, 6—Νァセチルダ ルコサミン転移酵素、 αΐ, 4—Νァセチルダルコサミン転移酵素、 β ΐ, 4 マンノー ス転移酵素、 αΐ, 2 マンノース転移酵素、《1, 3 マンノース転移酵素、《1, 4 マンノース転移酵素、《1, 6 マンノース転移酵素、《1, 2 グルコース転移酵 素、 αΐ, 3 グルコース転移酵素、 α2, 3 シアル酸転移酵素、 α 2, 6 シアル酸 転移酵素、 αΐ, 6—ダルコサミン転移酵素、《1, 6 キシロース転移酵素、 βキシロ ース転移酵素、 j81, 3 グルクロン酸転移酵素およびヒアルロン酸合成酵素からな る群より選択される、項目 14に記載の糖転移酵素阻害剤。 (Item 15) The above glycosyltransferases are αΐ, 4 galactose transferase, << 1, 3 galactose transferase, ι81, 4-galactose transferase, | 81, 3-galactose transferase, θΐ, 6 galactose transferase, << 2, 6 sialyltransferase, << 1, 4 galactose transferase, ceramide galactose transferase, αΐ, 2 fucose transferase, 1,3 fucose transferase, al, 4-fucose transferase, αΐ, 6 fucose transferase, α 1, 3— Ν-acetyl galatatosamine transferase, << 1, 6- ァ acetyl galatatosamine transferase, / 3 1, 4 ァ acetyl galatatosamine transferase, Polypeptide Ν Acetyl galatatosamine transferase, / 31, 4 Ν Acetyl darcosamine transferase, 131, 2— Ν Acetyl darcosamine transferase, β ΐ, 3— Ν acetyl dalcosamine transferase, β ΐ, 6 —Νacetylyl lucosamine transferase, α ΐ, 4—Νacetylyl darcosamine transferase, β ΐ, 4 mannose transferase, αΐ, 2 mannose transferase, << 1, 3 mannose transferase, << 1, 4 mannose transferase Enzyme, << 1, 6 Mannose transferase, << 1, 2 Glucosetransferase, αΐ, 3 Glucosetransferase, α2,3 Sialyltransferase, α2,6 Sialyltransferase, αΐ, 6-Darcosaminyltransferase Item 15. The glycosyltransferase inhibitor according to Item 14, selected from the group consisting of 1,6 xylosetransferase, β-xylosetransferase, j81,3 glucuronyltransferase and hyaluronic acid synthase.
(項目 16)上記糖転移酵素は、 al, 4 ガラクトース転移酵素、《1, 3 ガラクトー ス転移酵素， ι81, 4—ガラクトース転移酵素， |81, 3—ガラクトース転移酵素， β ΐ, 6 ガラクトース転移酵素、ひ1, 2 フコース転移酵素、ひ1, 3 フコース転移酵素 、 al, 4ーフコース転移酵素、 al, 6 フコース転移酵素および α 2, 3 シアル酸 転移酵素、 α2, 6 シアル酸転移酵素、からなる群より選択される、項目 14に記載 の糖転移酵素阻害剤。  (Item 16) The above glycosyltransferases are al, 4 galactose transferase, << 1, 3 galactose transferase, ι81, 4-galactose transferase, | 81, 3-galactose transferase, β ΐ, 6 galactose transferase , 1, 2 fucose transferase, 1, 3, 3 fucose transferase, al, 4-fucose transferase, al, 6 fucose transferase, α 2,3 sialyltransferase, α2, 6 sialyltransferase, Item 15. The glycosyltransferase inhibitor according to Item 14, selected from the group.
(項目 17)項目 7〜 13のいずれか 1項に記載の化合物を含む、糖転移酵素の活性の 異常に起因する状態、障害または疾患を処置または予防するための組成物。  (Item 17) A composition for treating or preventing a condition, disorder or disease caused by an abnormal activity of glycosyltransferase, comprising the compound according to any one of items 7 to 13.
(項目 18)項目 7〜 13のいずれか 1項に記載の化合物を含む組成物を投与する工程 を包含する、糖転移酵素の活性の異常に起因する状態、障害または疾患を処置また は予防するための方法。 (Item 18) Treating or preventing a condition, disorder or disease caused by an abnormal activity of glycosyltransferase, comprising the step of administering a composition comprising the compound according to any one of items 7 to 13 Way for.
(項目 19)項目 1〜13のいずれか 1項に記載の化合物の、糖転移酵素の活性の異 常に起因する状態、障害または疾患を処置または予防するための医薬の製造のた めの使用。 (項目 20)項目 1〜6または 5A〜5Cのいずれ力 1項に記載の化合物と、アルキレン 基、アミノ基、またはアミノォキシ基を有する以下の化合物： (Item 19) Use of the compound according to any one of Items 1 to 13 for the manufacture of a medicament for treating or preventing a condition, disorder or disease caused by abnormal glycosyltransferase activity. (Item 20) Any one of Items 1 to 6 or 5A to 5C, and the following compound having an alkylene group, an amino group, or an aminooxy group:
[化 32] [Chemical 32]
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000025_0001
[化 35] [Chemical 35]
Figure imgf000026_0001
Figure imgf000026_0001
t£S0S0/L00Zd£/13d 93
Figure imgf000027_0001
t £ S0S0 / L00Zd £ / 13d 93
Figure imgf000027_0001
cr" 、ι\τ
Figure imgf000027_0002
cr ", ι \ τ
Figure imgf000027_0002
[化 35- 1]
Figure imgf000028_0001
[Chemical 35-1]
Figure imgf000028_0001
[化 35A]
Figure imgf000028_0002
[Chemical 35A]
Figure imgf000028_0002
Figure imgf000028_0003
Figure imgf000028_0003
Figure imgf000029_0001
Figure imgf000029_0001
[化 35- 3] [Chemical 35-3]
Figure imgf000030_0001
Figure imgf000030_0001
[化 35- 4] [Chemical 35-4]
Figure imgf000031_0001
Figure imgf000031_0001
[化 35- 5]
Figure imgf000032_0001
[Chemical 35-5]
Figure imgf000032_0001
Figure imgf000032_0002
または
Figure imgf000032_0002
Or
とを混合して、アルキレン基とアジド基が反応する力 またはアルデヒド基とアミノ基も しくはァミノォキシ基が反応する条件下で、項目 7〜 13のいずれかに記載の化合物 を生成させる工程を包含する、項目 7〜 13のいずれかに記載の化合物の製造方法。 (項目 21) And a process for producing a compound according to any one of Items 7 to 13 under a condition in which an alkylene group and an azide group react or a condition in which an aldehyde group and an amino group or an aminooxy group react. The method for producing a compound according to any one of Items 7 to 13. (Item 21)
糖転移酵素の阻害剤をスクリーニングするための方法であって、該方法は、以下の 工程： A method for screening for an inhibitor of glycosyltransferase comprising the following steps:
I)糖転移酵素と、該糖転移酵素のァクセプターと、該糖転移酵素のドナーと、該阻害 剤の候補とを混合し、該糖転移酵素反応が進行する条件および時間で反応させるェ 程;ならびに  I) a process in which a glycosyltransferase, an acceptor of the glycosyltransferase, a donor of the glycosyltransferase, and a candidate for the inhibitor are mixed and reacted under conditions and a time at which the glycosyltransferase reaction proceeds; And
II)該 I)工程の反応後の反応物中の該ァクセプターと該ドナーとの反応生成物を測 定する工程、  II) a step of measuring a reaction product of the acceptor and the donor in the reaction product after the reaction of the step I),
を包含する、方法。 Including the method.
(項目 22) (Item 22)
前記糖転移酵素が、フコース転移酵素 Vであり、 The glycosyltransferase is fucose transferase V;
前記ァクセプターが、 The acceptor is
[化 35- 6]
Figure imgf000033_0001
であり、そして [Chemical 35-6]
Figure imgf000033_0001
And
前記ドナーが、 The donor
[化 35- 7][Chemical 35- 7]
Figure imgf000033_0002
である、項目 21に記載の方法。
Figure imgf000033_0002
The method according to item 21, wherein
(項目 23) (Item 23)
前記糖転移酵素反応が進行する条件が、 50mM HEPES (pH 7. 2)、 10mM 塩化マンガン、 ImM ァクセプター、 200 μ Μ ドナー、 2. 5mU フコース転移酵 素 (FucT) V、 30 M 阻害剤候補化合物混合物である、項目 22に記載の方法。 (項目 24) The conditions under which the glycosyltransferase reaction proceeds are as follows: 50 mM HEPES (pH 7.2), 10 mM manganese chloride, ImM acceptor, 200 μ 2. donor, 2.5 mU fucose transferase (FucT) V, 30 M inhibitor candidate compound 23. A method according to item 22, which is a mixture. (Item 24)
前記糖転移酵素が、 α ΐ, 6フコース転移酵素 Vinであり、 The glycosyltransferase is α ΐ, 6-fucose transferase Vin,
前記ァクセプターが、 The acceptor is
[化 35- 8]
Figure imgf000034_0001
であり、そして [Chemical 35-8]
Figure imgf000034_0001
And
前記ドナー力 S、 The donor force S ,
[化 35B] H2
Figure imgf000034_0002
である、項目 21に記載の方法。 [Chemical 35B] H 2
Figure imgf000034_0002
The method according to item 21, wherein
(項目 25) (Item 25)
前記糖転移酵素反応が進行する条件が、 50 mM 力コジル酸緩衝液 (pH 7. 5) 、10 mM 塩化マンガン、 100 ァクセプター、 50 μ Μ ドナー、 80 μ \] フコース転移酵素 VIII、 50 μ Μ GDP— 6N3フコース、 50 μ Μ 阻害剤候補化合 物混合物である、項目 24に記載の方法。 The conditions under which the glycosyltransferase reaction proceeds are as follows: 50 mM force codylate buffer (pH 7.5), 10 mM manganese chloride, 100 acceptor, 50 μΜ donor, 80 μ \] fucose transferase VIII, 50 μΜ 25. The method of item 24, which is a mixture of GDP—6N3 fucose, 50 μ μ inhibitor candidate compound.
(項目 26) 前記糖転移酵素が、シアル酸転移酵素であり、 (Item 26) The glycosyltransferase is a sialyltransferase,
前記ァクセプターが、 The acceptor is
[化 35- 9]  [Chem. 35-9]
Figure imgf000035_0001
であり、そして
Figure imgf000035_0001
And
前記ドナーが、 The donor
[化 35- 10] [Chemical 35-10]
Figure imgf000035_0002
Figure imgf000035_0002
である、項目 21に記載の方法。 The method according to item 21, wherein
(項目 27) (Item 27)
前記糖転移酵素反応が進行する条件が、 40mM cacodylate-HCl (pH7. 5)、 0 . 5g/ml BSA、0. 1% 界面活性剤、 ImM ァクセプター、 200 M ドナー、 1 mU a 2- 6シアル酸転移酵素（配列番号 6 (アミノ酸配列））である、項目 26に記載 の方法。 The conditions under which the glycosyltransferase reaction proceeds are 40 mM cacodylate-HCl (pH 7.5), 0.5 g / ml BSA, 0.1% surfactant, ImM acceptor, 200 M donor, 1 mU a 2-6 sial. 27. The method according to item 26, which is an acid transferase (SEQ ID NO: 6 (amino acid sequence)).
(項目 28) (Item 28)
前記糖転移酵素反応が進行する条件が、 40mM cacodylate-HCl (pH7. 5)、 0 . 5g/ml BSA、0. 1% 界面活性剤、 ImM ァクセプター、 200 M ドナー、 1 mU α 2- 3シアル酸転移酵素（配列番号 7 (アミノ酸配列））である、項目 26に記載 の方法。 The conditions under which the glycosyltransferase reaction proceeds are 40 mM cacodylate-HCl (pH 7.5), 0.5 g / ml BSA, 0.1% surfactant, ImM acceptor, 200 M donor, 1 mU α 2-3 sial. Item 26, which is an acid transferase (SEQ ID NO: 7 (amino acid sequence)). the method of.
(項目 29)  (Item 29)
前記糖転移酵素が、 N—ァセチルガラタトサミン転移酵素であり、 前記ァクセプターが、 The glycosyltransferase is N-acetylgalatatosamine transferase, and the acceptor is
[化 35- 11] [Chemical 35- 11]
Figure imgf000037_0001
干
Figure imgf000037_0001
Dried
Figure imgf000037_0002
Figure imgf000037_0002
Figure imgf000037_0003
Figure imgf000037_0003
Figure imgf000037_0004
Figure imgf000037_0004
esoso/Loo∑df/i3d 9ε TCOTSO/LOOZ: OAV であり、そして esoso / Loo∑df / i3d 9ε TCOTSO / LOOZ: OAV And
前記ドナーは、 UDP— GalNAcである、項目 21に記載の方法。 Item 22. The method according to Item 21, wherein the donor is UDP-GalNAc.
(項目 30) (Item 30)
前記糖転移酵素反応が進行する条件が、 50 mM イミダゾールー HC1緩衝溶液（ 0. 1% ポリ（ォキシエチレン）を含む、 pH7. 2)、 500 μ Μ ァクセプター、 100 μ Μ ドナー、 100 μ Μ 阻害剤候補化合物混合物、 10 mM 塩化マンガン、 In g/ μ ΐ N—ァセチルガラタトサミン転移酵素 ppGalNAcT— 2である、項目 29に記 載の方法。 The conditions under which the glycosyltransferase reaction proceeds are as follows: 50 mM imidazole-HC1 buffer solution (containing 0.1% poly (oxyethylene), pH 7.2), 500 μΜ acceptor, 100 μΜ donor, 100 μΜ inhibitor candidate 29. The method according to item 29, which is a compound mixture, 10 mM manganese chloride, In g / μΐ N-acetylylgalatatosamine transferase ppGalNAcT-2.
(項目 31) (Item 31)
前記糖転移酵素反応が進行する条件が、ァセチル化である、項目 21に記載の方法 (項目 32) The method according to item 21, wherein the condition under which the glycosyltransferase reaction proceeds is acetylation (item 32)
前記ァクセプターと前記ドナーとを反応させる時間は、糖転移酵素の初速に基づい て設定され、ここで、該初速は、前記ァクセプターと、前記糖転移酵素と、前記ドナー とを混合して反応させ、該ァクセプターと該ドナーとの反応生成物の反応率を経時的 に測定することにより算出される、項目 21に記載の方法。 The time for reacting the acceptor and the donor is set based on the initial speed of glycosyltransferase, wherein the initial speed is a reaction of mixing the acceptor, the glycosyltransferase and the donor, Item 22. The method according to Item 21, which is calculated by measuring a reaction rate of a reaction product between the acceptor and the donor over time.
(項目 33) (Item 33)
前記反応が、 50mM HEPES (pH 7. 2)、 10mM 塩化マンガン、 ImM ァクセ プター、 200 M ドナー、 2. 5mU フコース転移酵素（FucT) Vである条件下で実 施される、項目 32に記載の方法。 33. The item according to item 32, wherein the reaction is carried out under conditions of 50 mM HEPES (pH 7.2), 10 mM manganese chloride, ImM acceptor, 200 M donor, 2.5 mU fucose transferase (FucT) V. Method.
(項目 34) (Item 34)
前記反応が、 50 mM 力コジル酸緩衝液（pH 7. 5)、 10 mM 塩化マンガン、 1 00 M ァクセプター、 50 μ Μ ドナー、 80 μ フコース転移酵素 VIIIであ る条件下で実施される、項目 32に記載の方法。 The above reaction is carried out under conditions of 50 mM force codylate buffer (pH 7.5), 10 mM manganese chloride, 100 M acceptor, 50 μΜ donor, 80 μ fucose transferase VIII The method according to 32.
(項目 35) (Item 35)
前記反応が、 40mM cacodylate-HCl (pH7. 5)、 0. 5g/ml BSA、 0. 1% 界面活性剤、 ImM ァクセプター、 200 M ドナー、 α 2— 6シアル酸転移酵素（ 配列番号 6 (アミノ酸配列）） lmUである条件下で実施される、項目 32に記載の方 法。 The reaction was carried out using 40 mM cacodylate-HCl (pH 7.5), 0.5 g / ml BSA, 0.1% surfactant, ImM acceptor, 200 M donor, α 2-6 sialyltransferase (SEQ ID NO: 6 (amino acid Sequence)) According to item 32, which is performed under the condition of lmU Law.
(項目 36)  (Item 36)
前記反応が、 40mM cacodylate-HCl (pH7. 5)、0. 5g/ml BSA、 0. 1% 界面活性剤、 ImM ァクセプター、 200 M ドナー、 α 2— 3シアル酸転移酵素（ 配列番号 7 (アミノ酸配列）） lmUである条件下で実施される、項目 32に記載の方 法。 The reaction was carried out using 40 mM cacodylate-HCl (pH 7.5), 0.5 g / ml BSA, 0.1% surfactant, ImM acceptor, 200 M donor, α2-3 sialyltransferase (SEQ ID NO: 7 (amino acid Sequence)) The method according to item 32, which is carried out under the condition of lmU.
(項目 37)  (Item 37)
前記反応が、 50mM イミダゾールー HC1緩衝溶液 (0. 1% ポリ（ォキシエチレン） を含む、 ρΗ7. 2)、 500 μ Μ ァクセプター、 100 μ Μ ドナー、 10 mM 塩ィ匕 マンガン、 lng/ μ 1 Ν—ァセチルガラタトサミン転移酵素 ppGalNAcT— 2である条 件下で実施される、項目 32に記載の方法。 The reaction consists of 50 mM imidazole-HC1 buffer solution (containing 0.1% poly (oxyethylene), ρΗ7.2), 500 μ ク acceptor, 100 μΜ donor, 10 mM saline, manganese, lng / μ1Ν— 33. A method according to item 32, which is performed under the condition of cetylgalatatosamine transferase ppGalNAcT-2.
(項目 38) (Item 38)
前記反応率が、マトリックス支援レーザー脱離イオンィ匕 飛行時間型質量分析 (MA LDI-TOF-MS)により測定される、項目 32に記載の方法。 33. The method according to item 32, wherein the reaction rate is measured by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS).
(項目 39) (Item 39)
前記初速が、反応率 10〜20%の点で算出される、項目 32に記載の方法。 Item 33. The method according to Item 32, wherein the initial velocity is calculated at a reaction rate of 10 to 20%.
(項目 40) (Item 40)
前記初速が、フコース転移酵素 VIIIの場合、 60分である、項目 32に記載の方法。 (項目 41) Item 33. The method according to Item 32, wherein the initial speed is 60 minutes in the case of fucose transferase VIII. (Item 41)
前記ァクセプターと、前記ドナーと、前記糖転移酵素と、クリック反応に用いた物質と を混合して反応させ、該クリック反応に用いた物質による酵素反応への影響を確認し 、該影響があれば該影響を回避する工程 The acceptor, the donor, the glycosyltransferase, and the substance used for the click reaction are mixed and reacted, and the influence of the substance used for the click reaction on the enzyme reaction is confirmed. Process for avoiding the influence
をさらに包含する、項目 21に記載の方法。 The method of item 21, further comprising:
(項目 42) (Item 42)
前記糖転移酵素がフコース転移酵素 (FucT) Vであり、前記クリック反応に用いた物 質が 6アジド GDPフコース、アセチレン化合物、硫酸銅、トリス [ (1—ベンジル— 1H - 1, 2, 3—トリァゾール— 4—ィル)メチル]ァミン (TBTA)、またはァスコルビン酸ナ トリウムである、項目 41に記載の方法。 (項目 43) The glycosyltransferase is fucose transferase (FucT) V, and the material used for the click reaction is 6 azido GDP fucose, acetylene compound, copper sulfate, tris [(1-benzyl-1H-1, 2, 3- 42. The method according to item 41, which is triazol-4-yl) methyl] amine (TBTA) or sodium ascorbate. (Item 43)
前記糖転移酵素がフコース転移酵素 νιπであり、前記クリック反応に用いた物質が、 6アジド GDPフコース、アセチレン化合物、硫酸銅、トリス [ (1 ベンジル— 1H— 1, 2, 3 トリァゾールー 4 ィル)メチル]ァミン (ΤΒΤΑ)、またはァスコルビン酸ナトリウ ムである、項目 41に記載の方法。 The glycosyltransferase is fucose transferase νιπ, and the substance used for the click reaction is 6 azide GDP fucose, acetylene compound, copper sulfate, tris [(1 benzyl-1H— 1, 2, 3 triazole-4 yl) 42. The method according to Item 41, which is methyl] amine (ΤΒΤΑ) or sodium ascorbate.
(項目 44) (Item 44)
前記糖転移酵素が α 2, 3シアル酸転移酵素であり、前記クリック反応に用いた物質 1S アルキン化合物、硫酸銅、ァスコルビン酸、トリス [ (1 ベンジル一 1H— 1, 2, 3 —トリァゾール— 4—ィル)メチル]ァミン (TBTA)、 t— BuOHまたは CMP アジドシ アル酸である、項目 41に記載の方法。 The glycosyltransferase is α 2,3 sialyltransferase, and the substance used in the click reaction 1S alkyne compound, copper sulfate, ascorbic acid, tris [(1 benzyl 1 1H— 1, 2, 3 -triazole— 4 42. A method according to item 41, which is —yl) methyl] amine (TBTA), t—BuOH or CMP azidosialic acid.
(項目 45) (Item 45)
前記糖転移酵素が α 2, 6シアル酸転移酵素であり、前記クリック反応に用いた物質 1S アルキン化合物、硫酸銅、ァスコルビン酸、トリス [ (1 ベンジル一 1H— 1, 2, 3 —トリァゾール— 4—ィル)メチル]ァミン (TBTA)、 t— BuOHまたは CMP アジドシ アル酸である、項目 41に記載の方法。 The glycosyltransferase is α 2,6 sialyltransferase, and the substance used in the click reaction 1S alkyne compound, copper sulfate, ascorbic acid, tris [(1 benzyl 1 1H— 1, 2, 3 -triazole-4 42. A method according to item 41, which is —yl) methyl] amine (TBTA), t—BuOH or CMP azidosialic acid.
(項目 46) (Item 46)
前記糖転移酵素が N ァセチルガラタトサミン転移酵素であり、前記クリック反応に用 いた物質が、 UDP— N3— GalNAc、アセチレン化合物、トリス [ (1 ベンジル— 1H - 1, 2, 3 トリァゾール— 4—ィル)メチル]ァミン (TBTA)、硫酸銅、ァスコルビン酸 ナトリウムである、項目 41に記載の方法。 The glycosyltransferase is N-acetylgalatatosamine transferase, and the substance used for the click reaction is UDP—N3—GalNAc, acetylene compound, tris [(1 benzyl— 1H-1, 2, 3 triazole— 4 44. The method of item 41, wherein the compound is —yl) methyl] amine (TBTA), copper sulfate, sodium ascorbate.
(項目 47) (Item 47)
前記阻害剤候補ィ匕合物混合物の阻害活性から、該酵素反応に影響する物質の阻 害活性を減算することにより、前記酵素反応への影響を回避する、項目 41に記載の 方法。 42. The method according to item 41, wherein the inhibitory activity of the substance that affects the enzyme reaction is subtracted from the inhibitory activity of the inhibitor candidate mixture mixture to avoid the influence on the enzyme reaction.
(項目 48) (Item 48)
糖転移酵素の阻害剤の阻害定数を算出するための方法であって、該方法は、以下 の工程： A method for calculating the inhibition constant of an inhibitor of glycosyltransferase, the method comprising the following steps:
I)糖転移酵素と、該糖転移酵素のァクセプターと、該糖転移酵素のドナーと、該阻害 剤の候補とを混合し、該糖転移酵素反応が進行する条件および時間で反応させるェ 程； I) glycosyltransferase, an acceptor of the glycosyltransferase, a donor of the glycosyltransferase, and the inhibition Mixing with a candidate drug and reacting under conditions and time for the glycosyltransferase reaction to proceed;
II)該 I)工程の反応後の反応物中の該ァクセプターと該ドナーとの反応生成物を測 定する工程;ならびに  II) measuring the reaction product of the acceptor and the donor in the reaction product after the reaction of step I); and
III)該 Π)工程において測定した値力 測定定数を算出する工程、  III) The step of calculating the value force measurement constant measured in step i)
を包含する、方法。 Including the method.
(項目 49)  (Item 49)
前記糖転移酵素反応が進行する条件が、 50mM HEPES (pH 7. 2)、 10mM 塩化マンガン、 ImM ァクセプター、 50若しくは 200 のドナー、 2. 5mU フコース 転移酵素 (FucT) V、 0〜： LOO μ Μ 阻害剤候補化合物混合物である、項目 48に記 載の方法。 The conditions under which the glycosyltransferase reaction proceeds are 50 mM HEPES (pH 7.2), 10 mM manganese chloride, ImM acceptor, 50 or 200 donors, 2.5 mU fucose transferase (FucT) V, 0 to: LOO μΜ 49. The method according to item 48, which is a mixture of candidate inhibitor compounds.
(項目 50) (Item 50)
前記糖転移酵素反応が進行する条件が、 50 mM 力コジル酸緩衝液 (pH 7. 5) 、 10 mM 塩ィ匕マンガン、 100 ァクセプター、 12. 5〜75 ^ M ドナー、 8The conditions under which the glycosyltransferase reaction proceeds are as follows: 50 mM force codylate buffer (pH 7.5), 10 mM sodium chloride manganese, 100 acceptor, 12.5-75 ^ M donor, 8
0 U フコース転移酵素 νΐΠ、 0〜： LOO /z M 阻害剤候補化合物混合物である、 項目 48に記載の方法。 49. The method according to Item 48, wherein 0 U fucose transferase νΐΠ, 0˜: LOO / z M inhibitor candidate compound mixture.
(項目 51) (Item 51)
前記糖転移酵素反応が進行する条件が、 40mM cacodylate -HCl (pH7. 5)、 0 . 5g/ml BSA、 0. 1 % 界面活性剤、 ImM ァクセプター、 200 M ドナー、 1 mU α 2— 6シアル酸転移酵素（配列番号 6 (アミノ酸配列））、 0〜 100 Μ 阻害 剤候補化合物混合物である、項目 48に記載の方法)。 The conditions under which the glycosyltransferase reaction proceeds were 40 mM cacodylate-HCl (pH 7.5), 0.5 g / ml BSA, 0.1% surfactant, ImM acceptor, 200 M donor, 1 mU α 2-6 sial. 49. A method according to item 48, which is an acid transferase (SEQ ID NO: 6 (amino acid sequence)), 0 to 100Μ inhibitor candidate compound mixture.
(項目 52) (Item 52)
前記糖転移酵素反応が進行する条件が、 40mM cacodylate -HCl (pH7. 5)、 0 . 5g/ml BSA、 0. 1 % 界面活性剤、 ImM ァクセプター、 200 M ドナー、 1 mU a 2— 3シアル酸転移酵素（配列番号 7 (アミノ酸配列））、 0〜100 M 阻害 剤候補化合物混合物である、項目 48に記載の方法。 Conditions under which the glycosyltransferase reaction proceeds are 40 mM cacodylate-HCl (pH 7.5), 0.5 g / ml BSA, 0.1% surfactant, ImM acceptor, 200 M donor, 1 mU a 2-3 sial 49. The method according to item 48, wherein the method is an acid transferase (SEQ ID NO: 7 (amino acid sequence)), 0-100 M inhibitor candidate compound mixture.
(項目 53) (Item 53)
前記糖転移酵素反応が進行する条件が、 50 mM イミダゾールー HC1緩衝溶液（ 0. 1% ポリ（ォキシエチレン）を含む、 pH7. 2)、 500 μ Μ ァクセプター、 100 Μ ドナー、 10 mM 塩化マンガン、 lng/ /X 1 N—ァセチルガラタトサミン転移 酵素 ppGalNAcT—2、 0-100 μ Μ 阻害剤候補化合物混合物である、項目 48に 記載の方法。 The conditions under which the glycosyltransferase reaction proceeds are 50 mM imidazole-HC1 buffer solution ( 0.1% containing poly (oxyethylene), pH 7.2), 500 μΜ acceptor, 100 Μ donor, 10 mM manganese chloride, lng / / X 1 N-acetyl galatatosamine transferase ppGalNAcT-2, 0- 49. A method according to item 48, which is a mixture of 100 μ μ inhibitor candidate compounds.
(項目 54) (Item 54)
前記糖転移酵素反応において、阻害剤候補化合物混合物に加えて内部標準が混 合される、項目 48に記載の方法。 49. The method according to Item 48, wherein in the glycosyltransferase reaction, an internal standard is mixed in addition to the inhibitor candidate compound mixture.
(項目 55) (Item 55)
[化 35- 15][Chemical 35- 15]
Figure imgf000042_0001
Figure imgf000042_0001
または Or
[化 35- 17] = [Chemical 35- 17] =
=  =
Figure imgf000043_0001
Figure imgf000043_0001
Q - - <Π>  Q--<Π>
ェ ェ =F Yeah = F
=。 =.
~ ,固固 μ一 ~, Solid
~ - c~ - <~>  ~-c ~-<~>
o  o
o二 ◦  o Two ◦
S = o  S = o
◦ ◦
Figure imgf000043_0002
Figure imgf000043_0002
l7CS0S0/.00Zdf/X3d zv IC0180/.00Z OAV である、糖転移酵素のァクセプター。 l7CS0S0 / .00Zdf / X3d zv IC0180 / .00Z OAV A glycosyltransferase acceptor.
[0016] 従って、本発明のこれらおよび他の利点は、以下の詳細な説明を読みかつ理解す れば、当業者には明白になることが理解される。  [0016] Accordingly, it is understood that these and other advantages of the present invention will become apparent to those of ordinary skill in the art upon reading and understanding the following detailed description.
発明の効果  The invention's effect
[0017] 本発明は、全く新規な構造の糖転移酵素阻害剤およびそのスクリーニング方法を 提供する。  [0017] The present invention provides a glycosyltransferase inhibitor having a completely novel structure and a screening method thereof.
図面の簡単な説明  Brief Description of Drawings
[0018] [図 1]図 1は、本発明の実施例 1のグリコシルァクセプターの合成における標識キトトリ オースのみ力もなるスペクトル (上）および標識ガラクトシルキトトリオースのみ力もなる スペクトル（下）を示す。  [0018] [Fig. 1] Fig. 1 shows a spectrum in which only the labeled chitotriose has power in the synthesis of the glycosyl acceptor of Example 1 of the present invention (top) and a spectrum in which only the labeled galactosylchitotriose has only power (bottom). .
[図 2]図 2は、実施例 1にお 、て生成物の存在をエレクトロスプレイ Z質量分析 (ESI -MS)によって確認した結果を示す。  FIG. 2 shows the result of confirming the presence of the product by electrospray Z mass spectrometry (ESI-MS) in Example 1.
[図 3]図 3は、本発明の化合物が、フコース転移酵素の阻害能力を持つことを示す結 果を示す。酵素反応の生成物の量を示しており、小さいほど阻害活性が強いことを 示す。  FIG. 3 shows the results showing that the compound of the present invention has the ability to inhibit fucose transferase. The amount of the product of the enzyme reaction is shown. The smaller the value, the stronger the inhibitory activity.
[図 4]図 4は、反応条件（―）に更に阻害剤混合物（200 μ Μ)を加えたもの（+ )の計 2つの反応を行い、阻害剤を加えた場合の阻害活性を経時的に測定することを示す 結果である。  [Fig. 4] Fig. 4 shows the reaction over time with inhibitor activity when the reaction was carried out in a total of 2 reactions (+) with the inhibitor mixture (200 μΜ) added to the reaction conditions (-). It is a result which shows that it measures.
[図 5]図 5は、ドナーの濃度を 50 μ Μ、 200 μ Μおよび阻害剤の濃度を 100 Μ、 5 0 Μ、 Μ、 12. 5 /ζ Μ、 Ο /ζ Μで測定し、上記の経時変化の結果より、酵素反 応時間は初速度の測定範囲である 10分とした場合のディクソンプロットをした結果を 示す。  [Figure 5] Figure 5 shows the donor concentrations measured at 50 μΜ, 200 μΜ, and the inhibitor concentrations at 100 Μ, 50 Μ, Μ, 12.5 / ζ Μ, Ο / ζ 上 記From the results of changes over time, the result of Dickson plot when the enzyme reaction time is 10 minutes, which is the measurement range of the initial rate, is shown.
[図 6Α]図 6Αは、化合物 l l (10mM水溶液）を 2 /z L、アセチレン誘導体（12a〜12e 、 10mM)を 2 レァスコルビン酸ナトリウムと硫酸銅を混ぜた水溶液 (ァスコルビン 酸ナトリウム Z硫酸銅 = 20mMZl0mM) 2/z Lを混合して室温で 12時間反応させ 、生成物の存在を ESI - MSによって確認したときの結果を示す。  [Fig. 6Α] Fig. 6Α shows that the compound ll (10 mM aqueous solution) is 2 / z L, the acetylene derivatives (12a-12e, 10 mM) are 2 aqueous solutions of sodium lascorbate and copper sulfate (sodium corsolate Z copper sulfate = 20 mMZl0 mM) ) 2 / z L is mixed and reacted at room temperature for 12 hours, and the result when the presence of the product is confirmed by ESI-MS is shown.
[図 6B]図 6Bは、アセチレン化合物（アセチレンライブラリの A1〜A36、 L2Nおよび L 3N)について、 ESI— MSで測定したクリック反応の収率を示す。グラフの収率は ES I MSネガティブモードでのピーク強度比の相対比を示す。 FIG. 6B shows the click reaction yields measured by ESI-MS for acetylene compounds (acetylene library A1 to A36, L2N and L3N). The yield of the graph is ES The relative ratio of peak intensity ratio in the IMS negative mode is shown.
[図 7A]図 7Aは、反応条件 (コントロール)に更に阻害剤候補ィ匕合物混合物 (200 [FIG. 7A] FIG. 7A shows a mixture of inhibitor candidate compounds (200) in addition to the reaction conditions (control).
M)を加えたもの（13a〜13e、 N3GDP— Fuc ( + ) )、アセチレン誘導体のみを添カロ したもの（12a〜12e、 N3GDPFuc (—））の計 11種の反応を行った結果を示す。酵 素反応の生成物の量を示しており、小さいほど阻害活性が強いことを示す。 The results of a total of 11 reactions, including those with M) added (13a-13e, N3GDP—Fuc (+)) and those with only acetylene derivatives added (12a-12e, N3GDPFuc (—)) are shown. The amount of the product of the enzyme reaction is shown, and the smaller the value, the stronger the inhibitory activity.
[図 7B]図 7Bは、阻害剤候補化合物混合物（30 μ Μ)を加えたもの（アセチレン化合 物：アセチレンライブラリの A1〜A36、 L2Nおよび L3N、 N3GDP—Fuc ( + ) )およ びその阻害剤候補ィ匕合物に対応するアセチレン誘導体のみを添加したもの（ァセチ レンライブラリの A1〜A36、 L2Nの反応を行った結果を示す。  [Fig. 7B] Fig. 7B shows the addition of a mixture of candidate inhibitor compounds (30 μ （) (acetylene compounds: acetylene compounds A1 to A36, L2N and L3N, N3GDP-Fuc (+)) and their inhibitors The result of the reaction of acetylene derivatives A1 to A36 and L2N added with only the acetylene derivative corresponding to the candidate compound is shown.
[図 8A]図 8Aは、反応条件 (コントロール）に更にナフタレン含有糖ヌクレオチド混合 物（13b、 200 M)を加えたもの、若しくは Kiが判明しているリン酸を有する阻害剤 混合物（13e、 200 μ Μ)を加えたものの計 3種類の反応を行った結果を示す。 [FIG. 8A] FIG. 8A shows a reaction mixture (control) further added with a naphthalene-containing sugar nucleotide mixture (13b, 200 M), or an inhibitor mixture (13e, 200 having a phosphate with known Ki). The results of a total of three reactions with addition of μΜ) are shown.
[図 8Β]図 8Βは、アセチレンライブラリの A16について、ドナーの濃度を 37. 5 Μ、 2 0 Μ、 5 Μおよび阻害剤の濃度を 0 Μ、 0. 5 Μ、 1 Μで測定し、上記の経 時変化の結果より、酵素反応時間は初速度の測定範囲である 10分とした場合のディ クソンプロットをした結果を示す。 [Fig. 8Β] Fig. 8Β shows the results for A16 in the acetylene library, with donor concentrations measured at 37.5 Μ, 20 Μ, 5 Μ and inhibitor concentrations at 0 Μ, 0.5 Μ, and 1 、. From the results of changes over time, Dickson plots are shown when the enzyme reaction time is 10 minutes, which is the measurement range of the initial rate.
[図 8C]図 8Cは、アセチレンライブラリの L3Nについて、ドナーの濃度を 5 Μ、 25 [Figure 8C] Figure 8C shows the donor concentration for L3N in the acetylene library at 5% and 25%.
Μ、 50 μ Μおよび阻害剤の濃度を 0.： L Μ、 0. 5 Μ、： L Μ、 5 Μ、 10 Μで 測定し、上記の経時変化の結果より、酵素反応時間は初速度の測定範囲である 10 分とした場合のディクソンプロットをした結果を示す。 Μ, 50 μΜ and inhibitor concentrations measured at 0 .: L Μ, 0.5 Μ,: L Μ, 5 Μ, 10 Μ. From the results of the above changes over time, the enzyme reaction time is The result of Dickson plot when the measurement range is 10 minutes is shown.
[図 8D]図 8Dは、糖ペプチドの MALDI— TOFMSの結果を示す。  FIG. 8D shows the result of MALDI-TOFMS of the glycopeptide.
[図 8Ε]図 8Εは、新規に合成したフコース転移酵素のァクセプターである糖ペプチド について、その反応率の経時変化を示す。  [FIG. 8 (b)] FIG. 8 (b) shows the time course of the reaction rate of a newly synthesized glycopeptide which is an acceptor of fucose transferase.
[図 8F]図 8Fは、クリック反応残留物（Cu2+、 GDP— 6— N3フコース）による酵素反応 への影響を示す。酵素反応の生成物の量を示しており、小さいほど阻害活性が強い ことを示す。 [FIG. 8F] FIG. 8F shows the effect of the click reaction residue (Cu 2+ , GDP-6-N3 fucose) on the enzyme reaction. The amount of the product of the enzyme reaction is shown. The smaller the value, the stronger the inhibitory activity.
[図 8G]図 8Gは、アセチレンライブラリに記載のアセチレンィ匕合物力も合成した化合 物における酵素活性阻害実験の結果を示す。 [図 8H]図 8Hは、 GDP— 6—N3—フコースについてのディクソンプロットを示す。 [FIG. 8G] FIG. 8G shows the results of an enzyme activity inhibition experiment in a compound in which acetylene compound strength described in an acetylene library was also synthesized. [Figure 8H] Figure 8H shows the Dickson plot for GDP-6-N3-Fucose.
[図 81]図 81は、 GDP— 6—N3 フコースおよびアセチレンライブラリの A2, A3, A3 4につ 、ての阻害濃度を示す。 FIG. 81 shows inhibitory concentrations for GDP-6-N3 fucose and acetylene libraries A2, A3 and A34.
[図 9]図 9は、重水素 aoWR標識ィ匕合物を内部標準として用いて MALDI— TOFMS (Bruker社製、 UltraFLEX)で直接質量分析することによって、反応生成物の経時 変化について詳細を示す。酵素反応の生成物の量を示しており、小さいほど阻害活 性が強いことを示す。  [FIG. 9] FIG. 9 shows the details of the reaction product over time by direct mass analysis with MALDI-TOFMS (Bruker, UltraFLEX) using deuterium aoWR-labeled compound as an internal standard. . The amount of the enzyme reaction product is shown. The smaller the value, the stronger the inhibitory activity.
[図 10]図 10は、実施例 2における酵素活性の追跡の様子を示す。丸は、酵素活性に よって転移された糖を示す。これによりスペクトルが転移することが理解される。  FIG. 10 shows how enzyme activity is traced in Example 2. Circles indicate sugars transferred by enzyme activity. It is understood that this causes the spectrum to shift.
[図 11]図 11は、実施例 2における酵素活性阻害実験の結果を示す。 FIG. 11 shows the results of an enzyme activity inhibition experiment in Example 2.
[図 12A]図 12Aは、化合物 31を含む阻害剤調製液の ESIマススペクトルを示す。 FIG. 12A shows an ESI mass spectrum of an inhibitor preparation containing compound 31.
[図 12B]図 12Bは、アセチレン化合物と 5位アジド体 CMPシアル酸誘導体のクリック 反応により合成されたィ匕合物を含む阻害剤調製液の ESIマススペクトルによるイオン 化比率を示す。グラフの収率は ESI— MSネガティブモードでのピーク強度比の相対 比を示す。 FIG. 12B shows the ionization ratio according to ESI mass spectrum of an inhibitor preparation containing a compound synthesized by a click reaction of an acetylene compound and a 5-position azide CMP sialic acid derivative. The yield of the graph shows the relative ratio of peak intensity ratio in ESI-MS negative mode.
[図 12C]図 12Cは、アセチレン化合物と 9位アジド体 CMPシアル酸誘導体のクリック 反応により合成されたィ匕合物を含む阻害剤調製液の ESIマススペクトルによるイオン 化比率を示す。グラフの収率は ESI— MSネガティブモードでのピーク強度比の相対 比を示す。  [FIG. 12C] FIG. 12C shows an ionization ratio by ESI mass spectrum of an inhibitor preparation containing a compound synthesized by a click reaction of an acetylene compound and a 9-position azide CMP sialic acid derivative. The yield of the graph shows the relative ratio of peak intensity ratio in ESI-MS negative mode.
[図 13]図 13は、阻害剤 31の、 a— 2, 6シアル酸転移酵素および α—2, 3シアル酸 転移酵素に対する阻害効果を示す。縦軸は、阻害剤を含まない実験をコントロール（ = 1)とし、阻害剤を加えた場合の転移生成物の量を相対比で表している。例えば、 化合物 4の α 2, 3シアル酸転移酵素における棒グラフにおいて、グラフは 0. 26を示 していること力も転移が 26%に抑制された、すなわち阻害率は 74%であることを示し ている。酵素反応の生成物の量を示しており、小さいほど阻害活性が強いことを示す  FIG. 13 shows the inhibitory effect of inhibitor 31 on a-2,6-sialyltransferase and α-2,3-sialyltransferase. The vertical axis represents the amount of transfer product in the case of adding an inhibitor as a relative ratio, with the experiment containing no inhibitor as a control (= 1). For example, in the bar graph of α4,3 sialyltransferase of compound 4, the graph shows 0.26, indicating that the force was also suppressed to 26%, that is, the inhibition rate was 74%. Yes. Shows the amount of the product of the enzyme reaction. The smaller the value, the stronger the inhibitory activity.
[図 14]図 14は、アセチレン化合物（アセチレンライブラリの Α1〜Α36)と 5位アジド体 CMPシアル酸誘導体のクリック反応により合成されたィ匕合物について、ひ 2, 6シ アル酸転移酵素についての阻害活性測定の結果を示す。 [FIG. 14] FIG. 14 is a schematic diagram of a compound synthesized by a click reaction of an acetylene compound (Α1 to Α36 of the acetylene library) and a 5-position azide CMP sialic acid derivative. The result of the inhibitory activity measurement about a transferase is shown.
[図 15A]図 15Aは、アセチレン化合物（アセチレンライブラリの A1〜A36)と 5位アジ ド体 CMPシアル酸誘導体のクリック反応により合成された化合物について、 a— 2, [FIG. 15A] FIG. 15A shows compounds synthesized by a click reaction of an acetylene compound (A1 to A36 of the acetylene library) and a 5-position azide CMP sialic acid derivative.
3シアル酸転移酵素についての阻害活性測定の結果を示す。 The result of the inhibitory activity measurement about 3 sialyltransferase is shown.
[図 15B]図 15Bは、アセチレン化合物（アセチレンライブラリの A37〜A41)と 5位アジ ド体 CMPシアル酸誘導体のクリック反応により合成された化合物について、 a— 2, [FIG. 15B] FIG. 15B shows the synthesis of a compound synthesized by a click reaction of an acetylene compound (A37 to A41 in the acetylene library) and a 5-position azide CMP sialic acid derivative.
3シアル酸転移酵素についての阻害活性測定の結果を示す。 The result of the inhibitory activity measurement about 3 sialyltransferase is shown.
[図 15C]図 15Cは、アセチレン化合物（アセチレンライブラリの A37、 A39、 A40)と 5 位アジド体 CMPシアル酸誘導体のクリック反応により合成されたィ匕合物について、 α - 2, 3シアル酸転移酵素についての阻害活性測定の結果を示す。  [Fig. 15C] Fig. 15C shows α-2,3-sialic acid transfer of acetylene compounds (A37, A39, A40 in the acetylene library) and compounds synthesized by click reaction of 5-position azide CMP sialic acid derivatives. The result of the inhibitory activity measurement about an enzyme is shown.
[図 16]図 16は、アセチレン化合物（アセチレンライブラリの A1〜A36、 L2N、 L2A、 FIG. 16 shows acetylene compounds (acetylene library A1 to A36, L2N, L2A,
L2C、 L3N、 L3A、 L3C)と 9位アジド体 CMPシアル酸誘導体のクリック反応により 合成された化合物について、 α - 2, 6シアル酸転移酵素についての阻害活性測定 の結果を示す。 L2C, L3N, L3A, L3C) and 9-position azide CMP sialic acid derivative synthesized compounds by click reaction, α-2, 6 sialyltransferase inhibitory activity measurement results are shown.
[図 17]図 17は、アセチレン化合物（アセチレンライブラリの A1〜A36、 L2N、 L2A、 L2C、 L3N、 L3A、 L3C)と 9位アジド体 CMPシアル酸誘導体のクリック反応により 合成された化合物について、 α - 2, 3シアル酸転移酵素についての阻害活性測定 の結果を示す。  [FIG. 17] FIG. 17 shows a compound synthesized by a click reaction of an acetylene compound (acetylene library A1 to A36, L2N, L2A, L2C, L3N, L3A, L3C) and a 9-position azide CMP sialic acid derivative. -Shows the results of measuring the inhibitory activity of 2,3 sialyltransferases.
[図 18]図 18は、 UDP—N3— GalNAcと、アセチレン化合物と、トリス [ ( 1一べンジル - 1H - 1 , 2, 3 トリァゾールー 4—ィル)メチル]ァミン (TBTA)と、硫酸銅と、ァス コルビン酸ナトリウムとを混合して反応させた場合のクリック反応の収率を示す。グラフ の収率は ESI— MSネガティブモードでのピーク強度比の相対比を示す。  [FIG. 18] FIG. 18 shows UDP—N3—GalNAc, acetylene compound, tris [(1-1benzyl-1H-1,2,2,3 triazol-4-yl) methyl] amine (TBTA), and copper sulfate. And the yield of click reaction in the case of mixing and reacting sodium ascorbate. The yield in the graph shows the relative ratio of peak intensity ratio in ESI-MS negative mode.
[図 19]図 19はアセチレン化合物（アセチレンライブラリの Al—A36、 L2N, L3N)と UDP— N3— GalNAcのクリック反応にょり合成された化合物にっぃてppGalNAcT 2につ 、ての阻害活性測定の結果を示す。 [Figure 19] Figure 19 shows the measurement of inhibitory activity of ppGalNAcT 2 for compounds synthesized by the click reaction of acetylene compounds (acetylene library Al—A36, L2N, L3N) and UDP—N3—GalNAc. The results are shown.
配列表フリーテキスト Sequence listing free text
(配列表の説明）  (Explanation of sequence listing)
(配列番号 1)ゥシ ι8 1 , 4 ガラクトース転移酵素核酸配列 (配列番号 2)ゥシ |8 1, 4 ガラクトース転移酵素アミノ酸配列 (SEQ ID NO: 1) Usi ι8 1, 4 Galactose transferase nucleic acid sequence (SEQ ID NO: 2) Ushi | 8 1, 4 Galactosyltransferase amino acid sequence
(配列番号 3)ヒト 1, 4 ガラクトース転移酵素核酸配列  (SEQ ID NO: 3) human 1, 4 galactosyltransferase nucleic acid sequence
(配列番号 4)ヒト |8 1, 4 ガラクトース転移酵素アミノ酸配列  (SEQ ID NO: 4) Human | 8 1, 4 Galactosyltransferase amino acid sequence
(配列番号 5)アミノ酸配列 HGVTSAPDTR  (SEQ ID NO: 5) Amino acid sequence HGVTSAPDTR
(配列番号 6)ラット a 2, 3 シアル酸転移酵素アミノ酸配列  (SEQ ID NO: 6) Amino acid sequence of rat a 2, 3 sialyltransferase
(配列番号 7)ラット a 2, 6 シアル酸転移酵素アミノ酸配列  (SEQ ID NO: 7) Rat a 2, 6 Sialyltransferase amino acid sequence
(配列番号 8)フコース転移酵素 Vアミノ酸配列  (SEQ ID NO: 8) Fucosyltransferase V amino acid sequence
(配列番号 9)フコース転移酵素 VIIIアミノ酸配列  (SEQ ID NO: 9) Fucosyltransferase VIII amino acid sequence
(配列番号 10)アミノ酸配列 WRGTTPSPVPTTSTTSAP  (SEQ ID NO: 10) Amino acid sequence WRGTTPSPVPTTSTTSAP
(配列番号 11) N—ァセチルガラタトサミン転移酵素 ppGalNAcT— 2アミノ酸配列 発明を実施するための最良の形態  (SEQ ID NO: 11) N-Acetylgalatatosamine transferase ppGalNAcT-2 amino acid sequence BEST MODE FOR CARRYING OUT THE INVENTION
[0020] 以下、本発明を説明する。本明細書の全体にわたり、単数形の表現は、特に言及 しない限り、その複数形の概念をも含むことが理解されるべきである。また、本明細書 において使用される用語は、特に言及しない限り、当該分野で通常用いられる意味 で用いられることが理解されるべきである。  [0020] Hereinafter, the present invention will be described. Throughout this specification, it should be understood that expression in the singular also includes the concept of the plural unless specifically stated otherwise. In addition, it is to be understood that the terms used in this specification are used in the meaning normally used in the art unless otherwise specified.
[0021] (用語）  [0021] (Terminology)
以下に本明細書において特に使用される用語の定義を列挙する。  Listed below are definitions of terms particularly used in the present specification.
[0022] 本明細書において「糖鎖」とは、単位糖 (単糖および Zまたはその誘導体)が 1っ以 上連なってできたィ匕合物をいう。この単位糖の水酸基およびアミノ基は、適切な保護 基で保護されていてもよい。この保護基としては、ァセチル、ベンジル、ベンゾィル、 t ブトキシカルボニル、 tーブチルジメチルシリル基などが挙げられる。単位糖が 2つ 以上連なる場合は、各々の単位糖同士の間は、グリコシド結合による脱水縮合によつ て結合する。このような糖鎖としては、例えば、生体中に含有される多糖類 (ダルコ ス、ガラクトース、マンノース、フコース、キシロース、 N ァセチルダルコサミン、 N— ァセチルガラタトサミン、シアル酸ならびにそれらの複合体および誘導体)の他、分解 された多糖、糖タンパク質、プロテオダリカン、グリコサミノダリカン、糖脂質などの複合 生体分子力 分解または誘導された糖鎖など広範囲なものが挙げられるがそれらに 限定されない。したがって、本明細書では、糖鎖は、「多糖 (ポリサッカリド)」、「糖質」 、「炭水化物」および「糖」と互換可能に使用され得る。また、特に言及しない場合、本 明細書において「糖鎖」は、糖鎖および糖鎖含有物質の両方を包含することがある。 単位糖同士の結合は、その位置によって、 α ΐ, 2—、 α ΐ, 3—、 α ΐ, 4—、 α ΐ , 6 一、 β ΐ , 2—などがあり、それらの表示は結合する単位糖における炭素の位置を併 記すること、および通常その結合に関するァノマー（ α、 j8 )を記載する。糖鎖の結合 に関する情報は複雑であり、ポリペプチド、ポリヌクレオチドのように簡素化することが 困難である力 [列 は、 Trends in Glycoscience and Glycotechnology 14 , 127— 137 (2002)では、リニアコードで糖鎖を表すことを提唱しており、必要に応 じて、本明細書においてもそのような表示法を利用する。 In the present specification, the “sugar chain” refers to a compound formed by one or more unit sugars (monosaccharide and Z or a derivative thereof). The hydroxyl group and amino group of this unit sugar may be protected with an appropriate protecting group. Examples of the protecting group include acetyl, benzyl, benzoyl, t-butoxycarbonyl, t-butyldimethylsilyl group and the like. When two or more unit sugars are connected, each unit sugar is bonded by dehydration condensation using a glycosidic bond. Examples of such sugar chains include polysaccharides (darcose, galactose, mannose, fucose, xylose, N-acetyl dalcosamine, N-acetyl galatatosamine, sialic acid, and complexes thereof contained in the living body. And derivatives), as well as complex polysaccharides such as degraded polysaccharides, glycoproteins, proteodaricans, glycosaminodarlicans, glycolipids, etc. Not. Therefore, in the present specification, the sugar chain is defined as “polysaccharide” or “carbohydrate”. , "Carbohydrate" and "sugar" can be used interchangeably. Further, unless otherwise specified, the “sugar chain” in the present specification may include both a sugar chain and a sugar chain-containing substance. There are α ΐ, 2-—, α ΐ, 3-—, α ΐ, 4-—, α ΐ, 6 1, β ΐ, 2-—, etc., depending on the position of unit sugars, and their indications are combined Indicate the position of the carbon in the unit sugar, and the anomer (α, j8) for the bond. The information on glycan binding is complex and forces that are difficult to simplify, such as polypeptides and polynucleotides. [Column is Trends in Glycoscience and Glycotechnology 14, 127-137 (2002). It has been proposed to represent sugar chains, and such notation is used in this specification as needed.
[0023] 本明細書において「単糖」とは、これより簡単な分子に加水分解されず、一般式 C H Oで表される化合物をいう。ここで、 n= 2、 3、 4、 5、 6、 7、 8、 9および 10であるIn the present specification, the “monosaccharide” refers to a compound that is not hydrolyzed to a simpler molecule and is represented by the general formula C 3 H 2 O. Where n = 2, 3, 4, 5, 6, 7, 8, 9 and 10.
2n n 2n n
ものを、それぞれジ才ース、トリ才ース、テトロース、ペントース、へキソース、ヘプトー ス、オタトース、ノノースおよびデコースという。一般に鎖式多価アルコールのアルデヒ ドまたはケトンに相当するもので、前者をアルド—ス，後者をケト—スという。  They are called dice, tritose, tetrose, pentose, hexose, heptose, otatose, nonose and decourse, respectively. Generally, it corresponds to aldehydes or ketones of chain polyhydric alcohols. The former is called aldose and the latter is called ketose.
[0024] 本明細書にぉ 、て「単糖の誘導体」とは、単糖上の一つ以上の水酸基が別の置換 基に置換され、結果生じる物質が単糖の範囲内にないものをいう。そのような単糖の 誘導体としては、カルボキシル基を有する糖 (例えば、 C 1位が酸ィ匕されてカルボン 酸となったアルドン酸 (例えば、 D—ダルコ一スが酸ィ匕された D—ダルコン酸）、末端 の C原子がカルボン酸となったゥロン酸（D—グルコースが酸化された D グルクロン 酸）、アミノ基またはァミノ基の誘導体 (例えば、ァセチルイ匕されたァミノ基)を有する 糖 (例えば、 N ァセチル— D—ダルコサミン、 N ァセチル— D ガラクトサミンなど )、アミノ基およびカルボキシル基を両方とも有する糖 (例えば、 N ァセチルノイラミ ン酸 (シアル酸）、 N ァセチルムラミン酸など）、デォキシィ匕された糖 (例えば、 2— デォキシー D リボース）、硫酸基を含む硫酸化糖、リン酸基を含むリン酸化糖、 UD P誘導体 Gal誘導体などがあるがそれらに限定されない。あるいは、へミアセタール 構造を形成した糖にぉ 、て、アルコールと反応してァセタール構造のグリコシドもまた 、単糖の誘導体の範囲内にある。  [0024] As used herein, a "monosaccharide derivative" refers to a substance in which one or more hydroxyl groups on a monosaccharide are substituted with another substituent and the resulting substance is not within the range of a monosaccharide. Say. Examples of such monosaccharide derivatives include saccharides having a carboxyl group (for example, aldonic acids converted to carboxylic acids by acidification at the C 1 position (for example, D— (Darconic acid), uronic acid with terminal C atom converted to carboxylic acid (D-D-glucuronic acid with oxidized glucose), sugar with amino group or amino derivative (for example, acetylated amino group) For example, N-acetyl-D-darcosamine, N-acetyl-D-galactosamine, etc., sugars having both amino and carboxyl groups (eg, N-acetylethyl neuraminic acid (sialic acid), N-acetyl muramic acid, etc.), deoxygenated sugars (For example, 2-deoxy D-ribose), sulfated sugars containing sulfate groups, phosphorylated sugars containing phosphate groups, UDP derivative Gal derivatives, etc. Not constant. Alternatively, the per cent sugar forming the Miasetaru structure Te, glycosides also of Asetaru structure by reacting with alcohol, are within the scope of the monosaccharide derivatives.
[0025] 本明細書中において「Gal」とは、ガラクトースの略号である。本明細書中において「 Gal誘導体」または「ガラタトース誘導体」とは、 Gal上の一つ以上の水素原子が別の 置換基に置換され、結果生じる物質力 SGalの範囲内にないものをいう。そのような置 換基としては、下記の（有機化学)の項で列挙される置換基または UDPもしくは UDP 誘導体などが挙げられる。 [0025] In this specification, "Gal" is an abbreviation for galactose. In this specification, “ “Gal derivative” or “galatose derivative” refers to a substance in which one or more hydrogen atoms on Gal are substituted with another substituent and are not within the range of the resulting material force SGal. Examples of such substituents include those listed in the following (Organic Chemistry) section, UDP or UDP derivatives, and the like.
[0026] 本明細書にぉ 、て「糖鎖含有物質」とは、糖鎖および糖鎖以外の物質を含む物質 をいう。このような糖鎖含有物質は、生体内に多く見出され、例えば、生体中に含有 される多糖類の他、分解された多糖、糖タンパク質、プロテオダリカン、グリコサミノグ リカン、糖脂質などの複合生体分子力も分解または誘導された糖鎖など広範囲なも のが挙げられるがそれらに限定されない。  In the present specification, the term “sugar chain-containing substance” refers to a substance containing a sugar chain and substances other than sugar chains. Many of such sugar chain-containing substances are found in the living body. For example, in addition to polysaccharides contained in the living body, complex polysaccharides such as degraded polysaccharides, glycoproteins, proteodalycans, glycosaminoglycans, glycolipids, etc. Examples of the biomolecular force include, but are not limited to, a wide range of sugar chains that are decomposed or derived.
[0027] 本明細書において、ある物質 (例えば、酵素）またはある部分 (例えば、ある酵素上 の基質と相互作用する部分あるいはある酵素上の特定のアミノ酸 (例えば、トリプトフ アン)）と「特異的に相互作用し得る」とは、その物質またはその部分に対して、その物 質またはその部分以外に対する特異性よりも高い特異性をもって相互作用すること ができる能力をいう。好ましくは、その物質またはその部分以外の物質または部分は 、類似の物質またはあるいはある酵素の意図される部分以外の部分を含み得る。そ のような能力は、その物質またはその部分以外の物質または部分との非特異的相互 作用を解離させる条件下に曝されるとき、少なくとも一定量のその物質またはその部 分との特異的相互作用が残存することを判定することによって確認することができる。 具体的には、例えば、そのような能力は、ある酵素上の特定の部分に対する特異的 な相互作用をいう場合は、その特定の部分に対する相互作用が他の部分よりも測定 可能な程度に有意に多いことによって確認することができる。そのような相互作用は、 物理学的な測定手法 (例えば、 NMRなど）によって判定することができる。  [0027] As used herein, a substance (eg, an enzyme) or a moiety (eg, a moiety that interacts with a substrate on a certain enzyme or a specific amino acid (eg, tryptophan) on a certain enzyme) and a “specific” “Can interact with” refers to the ability to interact with the substance or part thereof with a specificity that is higher than the specificity for the substance or part other than the part. Preferably, the substance or part other than the substance or part thereof may comprise a part other than the intended part of a similar substance or certain enzyme. Such an ability is a specific interaction with at least a certain amount of the substance or part thereof when exposed to conditions that dissociate non-specific interactions with the substance or part other than the substance or part thereof. This can be confirmed by determining that the action remains. Specifically, for example, when such an ability refers to a specific interaction with a specific part on an enzyme, the interaction with that specific part is more significant than the other part. It can be confirmed by the fact that it is too much. Such interactions can be determined by physical measurement techniques (eg NMR).
[0028] 本明細書において「相互作用」とは、 2つの物体について言及するとき、その 2つの 物体が相互に力を及ぼしあうことをいう。そのような相互作用としては、例えば、共有 結合、水素結合、ファンデルワールス力、イオン性相互作用、非イオン性相互作用、 疎水性相互作用、静電的相互作用などが挙げられるがそれらに限定されない。好ま しくは、相互作用は、水素結合、疎水性相互作用などである。本明細書において「共 有結合」とは、当該分野における通常の意味で用いられ、電子対が 2つの原子に共 有されて形成する化学結合をいう。本明細書において「水素結合」とは、当該分野に おける通常の意味で用いられ、電気的陰性度の高い原子に一つしかない水素原子 の核外電子が引き寄せられて水素原子核が露出し、これが別の電気的陰性度の高 い原子を引き寄せて生じる結合をいい、例えば、水素原子と電気的陰性度の高い（ フッ素、酸素、窒素などの）原子との間にできる。 [0028] In this specification, "interaction" means that when two objects are referred to, the two objects exert a force on each other. Examples of such interactions include, but are not limited to, covalent bonds, hydrogen bonds, van der Waals forces, ionic interactions, nonionic interactions, hydrophobic interactions, electrostatic interactions, etc. Not. Preferably, the interaction is a hydrogen bond, a hydrophobic interaction, or the like. In this specification, the term “shared bond” is used in the ordinary sense in the field, and an electron pair is shared by two atoms. It is a chemical bond that is formed. In the present specification, the term “hydrogen bond” is used in a normal sense in the field, and the hydrogen nucleus is exposed by attracting an extra-nuclear electron of only one hydrogen atom with a high electronegativity, This is a bond that is created by attracting another atom with high electronegativity, for example, between a hydrogen atom and an atom with high electronegativity (fluorine, oxygen, nitrogen, etc.).
[0029] 本明細書において相互作用などの「レベル」とは、その相互作用など強さを示す程 度を 、 、、「強度」とも 、、ある物質 (または部分)の別の物質 (または部分）に対す る相互作用の強さを判断するために使用され得る。そのような相互作用のレベルは、 例えば、 FRETにおいて蛍光の強さなどの測定値、レクチンなどの相互作用に関し て用いられる SPR (表面プラズモン共鳴)法による屈折率観測、酵素の酵素活性測 定 (放射性標識、 ELISAなどによる生化学的測定)を用いることができる。 FRET, S PR法、酵素活性測定は、当該分野において周知であり、本明細書において他の場 所に詳細に説明されている。  [0029] In this specification, the "level" such as an interaction is a degree indicating the strength of the interaction,,, and "strength" both with another substance (or part) of a certain substance (or part). ) Can be used to determine the strength of the interaction. The level of such interaction is, for example, measured values such as fluorescence intensity in FRET, refractive index observation by the SPR (surface plasmon resonance) method used for interactions such as lectins, enzyme enzyme activity measurement ( Radiolabeling, biochemical measurement by ELISA, etc.) can be used. FRET, SPR, and enzyme activity measurement are well known in the art and are described in detail elsewhere in this specification.
[0030] 本明細書において「フアルマコフォアモデル」とは、ある立体配位について、目的と する相互作用などが形成されるために用いられるモデルをいう。このようなモデルは、 ある作用を有する化合物を設計するために用いられる。そのような化合物の設計に は、例えば、 ADAM &EVE (医薬分子設計研究所、東京)などを利用することがで きる。本発明では、本発明において具体的に提供された化合物をもとにこのようなフ アルマコフォアモデルを利用してさらなる新規ィ匕合物を提供できることが理解される。  [0030] In this specification, "falmakophore model" refers to a model used for forming a desired interaction or the like for a certain configuration. Such a model is used to design a compound with a certain action. For the design of such compounds, for example, ADAM & EVE (Pharmaceutical Molecular Design Laboratory, Tokyo) can be used. In the present invention, it is understood that further novel compounds can be provided using such a pharmacophore model based on the compounds specifically provided in the present invention.
[0031] 本明細書において「糖転移酵素の活性の異常に起因する状態、障害または疾患」 とは、糖転移酵素の活性が通常のレベルより高いまたは低いことによって生じるかま たはそのようなレベルが伴う生体の状態、障害または疾患をいう。そのような状態、障 害または疾患には、例えば、慢性関節リウマチ、免疫性疾患、エリテマトーデス、がん またはがんの転移、細菌感染、糖尿病、代謝性疾患、ホルモン機能、ストレス、骨粗 鬆症などが挙げられるがそれらに限定されない。  [0031] As used herein, "a condition, disorder or disease caused by abnormal glycosyltransferase activity" means that the level of glycosyltransferase activity is higher or lower than the normal level or such level. A biological state, disorder or disease associated with. Such conditions, disorders or diseases include, for example, rheumatoid arthritis, immune disease, lupus erythematosus, cancer or cancer metastasis, bacterial infection, diabetes, metabolic disease, hormonal function, stress, osteoporosis However, it is not limited to them.
[0032] 本明細書において「 j8 1, 4 ガラクトース転移酵素の活性の異常に起因する状態 、障害または疾患」とは、 j8 1, 4—ガラクトース転移酵素の活性が通常のレベルより 高いまたは低いことによって生じる力またはそのようなレベルが伴う生体の状態、障害 または疾患をいう。そのような状態、障害または疾患には、例えば、慢性関節リウマチ 、免疫性疾患、エリテマトーデス、がんまたはがんの転移、細菌感染、糖尿病、代謝 性疾患、ホルモン機能、ストレス、骨粗鬆症などが挙げられるがそれらに限定されな い。 [0032] In the present specification, "a state, disorder or disease caused by an abnormal activity of j8 1, 4 galactosyltransferase" means that the activity of j8 1, 4-galactose transferase is higher or lower than a normal level. Force caused by or the condition of the body with such a level, disorder Or disease. Such conditions, disorders or diseases include, for example, rheumatoid arthritis, immune disease, lupus erythematosus, cancer or cancer metastasis, bacterial infection, diabetes, metabolic disease, hormonal function, stress, osteoporosis, etc. Is not limited to them.
[0033] 本明細書においてけコース転移酵素の活性の異常に起因する状態、障害または 疾患」とは、フコース転移酵素の活性が通常のレベルより高いまたは低いことによって 生じる力またはそのようなレベルが伴う生体の状態、障害または疾患をいう。フコース 転移酵素に関連する疾患としては、免疫性疾患、がんまたはがんの転移、細菌感染 、ァテローム性動脈硬化症、抗体依存性細胞傷害、シグナル伝達が挙げられる。 OC 1 , 3フコース転移酵素ファミリ一は癌化により高発現している。より詳細には、フコース 転移酵素 VIIIに関連する疾患としては、例えば、肺気腫が挙げられ得る。がん患者 において、ノックアウトすると増殖が抑制されること、脾がん患者における血清では α 1 , 6Fucosylィ匕ハブトグロビンが高率で発見されること、肺気腫ではこの酵素が減つ て！、ることが明らかになって！/、る。  [0033] As used herein, a condition, disorder, or disease caused by abnormal activity of a course transferase includes a force generated by the activity of fucose transferase being higher or lower than a normal level, or such a level. It refers to the condition, disorder or disease associated with the living body. Diseases associated with fucose transferase include immune diseases, cancer or cancer metastasis, bacterial infection, atherosclerosis, antibody-dependent cell injury, and signal transduction. The OC 1, 3 fucose transferase family is highly expressed by canceration. More specifically, the disease associated with fucose transferase VIII may include, for example, emphysema. In cancer patients, knockout suppresses proliferation, α 1, 6 Fucosyl 匕 habutoglobin is found at a high rate in spleen cancer patients, and this enzyme decreases in emphysema! It becomes clear that! /
[0034] 本明細書において「ひ 2, 3—シアル酸転移酵素の活性の異常に起因する状態、障 害または疾患」とは、 a 2, 3—シアル酸転移酵素の活性が通常のレベルより高いま たは低いことによって生じる力またはそのようなレベルが伴う生体の状態、障害または 疾患をいう。 a 2, 3—シアル酸転移酵素に関連する疾患としては、免疫性疾患、結 腸 ·直腸がん、乳がん、白血病などのがんまたはがんの転移が挙げられる。  [0034] In the present specification, "a state, disorder or disease caused by an abnormal activity of 2,3-sialyltransferase" means that the activity of a 2,3-sialyltransferase is higher than the normal level. A force, or a condition, disorder or disease associated with a force or such level resulting from being high or low. Diseases associated with a 2,3-sialyltransferase include immune disorders, cancers such as colorectal / rectal cancer, breast cancer, leukemia, or cancer metastasis.
[0035] 本明細書において「ひ 2, 6—シアル酸転移酵素の活性の異常に起因する状態、障 害または疾患」とは、 a 2, 6—シアル酸転移酵素の活性が通常のレベルより高いま たは低いことによって生じる力またはそのようなレベルが伴う生体の状態、障害または 疾患をいう。 a 2, 6—シアル酸転移酵素に関連する疾患としては、免疫性疾患、結 腸 ·直腸がん、乳がん、白血病などのがんまたはがんの転移が挙げられる。  [0035] In the present specification, "a state, disorder or disease caused by abnormal activity of 2,6-sialyltransferase" means that the activity of a 2,6-sialyltransferase is higher than the normal level. A force, or a condition, disorder or disease associated with a force or such level resulting from being high or low. Diseases associated with a 2,6-sialyltransferase include immune disorders, cancers such as colorectal / rectal cancer, breast cancer, and leukemia, or cancer metastasis.
[0036] (蛍光エネルギー移動（FRET)法）  [0036] (Fluorescence energy transfer (FRET) method)
FRETは、 2つの蛍光物質間で、一方からの蛍光発光波長がもう一方の蛍光物質 の吸収波長に重なった時に生じる、直接的な励起エネルギーの移動のことを示す。 その現象の発見は、古くはペリンによってなされ、さらにフエノレスターによってその原 理が示された（Forster, Th. (1948) , Ann. Phys. 2 : 55— 75.；)。 FRET indicates a direct excitation energy transfer between two fluorescent materials when the fluorescence emission wavelength from one overlaps the absorption wavelength of the other fluorescent material. The phenomenon was discovered by Perin in the old days, and by Fenorester. (Forster, Th. (1948), Ann. Phys. 2: 55-75 .;).
[0037] 蛍光物質はある波長の光を吸収し、吸収した光エネルギーによって励起する。蛍光 物質は励起状態から基底状態に戻るときに蛍光を発する。励起した蛍光物質は振動 励起双極子として考えることができ、その双極子からの距離 Rに依存する電場が現れ る（Clegg, R. M. (1996) , X. F. Wang and B. Herman (eds. ) , John Wiley & Sons, New York, pp. 179— 252.；)。双極子の極めて近傍（R< <励起光 波長）の電場においては電場の強度は 1ZR3によって支配されており、この双極子 近接電場の中に双極子相互作用によってその励起エネルギーを吸収する物質 (蛍 光ァクセプター）が存在すると、蛍光発光を行うことなく励起エネルギーが直接蛍光ァ クセプターに移動する。この現象を FRETと呼び、蛍光ァクセプターが蛍光ドナーの 電場エネルギーを吸収する確立力 S、蛍光ドナーの電場強度の 2乗に比例するため、 FRETが起こる確立は距離の 6乗に反比例する（Clegg、前出、 Selvin, P. R. (199 5) , Methods Enzymol. 246, 300— 334.；)。 [0037] The fluorescent material absorbs light of a certain wavelength and is excited by the absorbed light energy. The fluorescent substance emits fluorescence when returning from the excited state to the ground state. Excited phosphors can be thought of as vibrationally excited dipoles, and an electric field depending on the distance R from the dipoles appears (Clegg, RM (1996), XF Wang and B. Herman (eds.), John Wiley & Sons, New York, pp. 179-252 .;). In an electric field very close to the dipole (R <<excitation light wavelength), the electric field strength is dominated by 1ZR 3 , and the substance that absorbs the excitation energy by dipole interaction in this dipole near electric field ( In the presence of a fluorescent acceptor), the excitation energy moves directly to the fluorescent acceptor without emitting fluorescence. This phenomenon is called FRET, and the probability that the fluorescent acceptor absorbs the electric field energy of the fluorescent donor S is proportional to the square of the electric field strength of the fluorescent donor, so the probability that FRET occurs is inversely proportional to the sixth power of the distance (Clegg, Supra, Selvin, PR (199 5), Methods Enzymol. 246, 300—334 .;).
[0038] FRETは R値を越える距離にお!ヽては急激に観測されなくなる性質をもっため（距 [0038] FRET has the property that it will not be observed suddenly at distances exceeding the R value (distance
0  0
離の 6乗に反比例することによる）、分子が結合、あるいは相互作用することを観測す るセンサ一としての役割を担うことができる。  It can play a role as a sensor that observes the binding or interaction of molecules (by being inversely proportional to the sixth power of separation).
[0039] 例えば加水分解酵素の活性測定に関しては、その酵素基質について分解部位を 挟んだ所で 2つの蛍光物質で標識し、酵素反応を行う。加水分解が起こる前では蛍 光物質間の距離が近く FRETが起こっているため、蛍光ドナー力もの発光は弱く蛍 光ァクセプターからの発光が強く観測される。加水分解反応が進むことによって蛍光 物質間の距離が広がり、 FRETは観測されなくなる。すると蛍光ドナーからの発光が 回復し、蛍光ァクセプターからの発光は弱められる。この酵素反応を分光器のセル内 で行うことで、連続的に酵素反応を観察することが可能になる。  [0039] For example, for measuring the activity of hydrolase, the enzyme substrate is labeled with two fluorescent substances at the position where the degradation site is sandwiched, and an enzyme reaction is performed. Before the hydrolysis occurs, the distance between the fluorescent materials is close, and FRET occurs. Therefore, the light emitted from the fluorescent donor is weak and the light emitted from the fluorescent acceptor is observed strongly. As the hydrolysis reaction proceeds, the distance between the fluorescent materials increases, and FRET is no longer observed. Then, the light emission from the fluorescent donor is recovered, and the light emission from the fluorescent acceptor is weakened. By performing this enzyme reaction in the spectroscope cell, it is possible to observe the enzyme reaction continuously.
[0040] 2基質系の反応においては上記の 1基質系の逆になり、 2つの酵素基質について それぞれ蛍光標識を行い、酵素反応を行わせる。 2つの基質が反応し結合すること によって蛍光物質間の距離が縮まり、 FRETが観測されるようになるヒト |8 1, 4—ガラ クトース転移酵素核酸配列。  [0040] The reaction of the two-substrate system is the reverse of the one-substrate system described above, and the two enzyme substrates are fluorescently labeled to cause the enzyme reaction. A human | 8 1,4-galactosyltransferase nucleic acid sequence in which FRET is observed when the distance between the fluorescent substances decreases as the two substrates react and bind.
[0041] 次に FRETを用いた糖転移酵素の活性量、および合成した基質に対する速度定 数測定法について簡単に説明する。 [0041] Next, the amount of glycosyltransferase activity using FRET and the rate of the synthesized substrate were determined. The number measurement method will be briefly described.
[0042] 1.まず蛍光標識した糖ドナーおよび糖ァクセプターを調製する。蛍光物質ペアの 選択については、より大きなスペクトルの重なりを持ち、励起、観測波長の分離が良く 、酵素反応によって生成物となったときの蛍光基質間距離の推定力 適切な R  [0042] 1. First, a fluorescently labeled sugar donor and sugar acceptor are prepared. For the selection of fluorescent substance pairs, there is a larger spectral overlap, the separation of excitation and observation wavelengths is good, and the ability to estimate the distance between fluorescent substrates when a product is produced by an enzymatic reaction.
0値を 持ち、また酵素反応を妨げない、といったことを考慮すべきである。  It should be taken into account that it has a zero value and does not interfere with the enzymatic reaction.
[0043] 2.酵素反応の経過を蛍光ドナー発光の減少、あるいは蛍光ァクセプター発光の増 加から観察する。分光器力 得られる情報はあくまで相対強度に過ぎないが、既知 活性の酵素を基準に検量線を作成することでこの情報だけでも活性量測定は可能 である。酵素速度定数 (V 値)の決定をするためには相対強度を絶対反応量へ変 max  [0043] 2. Observe the progress of the enzyme reaction from a decrease in fluorescence donor emission or an increase in fluorescence acceptor emission. Spectroscopic force Information obtained is only relative intensity, but it is possible to measure the amount of activity using this information alone by creating a calibration curve based on an enzyme with known activity. To determine the enzyme rate constant (V value), change the relative intensity to the absolute reaction amount max
換する必要がある (κ値のみ決定する場合は必ずしも必要ではない)。そのため酵 素反応初期の生成物のない状態と、酵素反応が完全に終了した状態の間で検量線 を作成し、絶対反応量を算出する。  (It is not always necessary if only the κ value is determined). Therefore, a calibration curve is created between the state where there is no product in the initial stage of the enzyme reaction and the state where the enzyme reaction is completely completed, and the absolute reaction amount is calculated.
[0044] 3.実際に酵素反応を行なう。蛍光測定であるので非常に感度がよいため、かなり希 薄な基質濃度で反応を行なうことになる。これは必要な基質量が少なくて済む利点と 同時に、特に 2基質系の酵素活性測定においては一方の基質の濃度を高濃度にで きない欠点を合わせ持つ。そのためまず一方の基質濃度を固定し、見力けの app、 v app値をまず求め、次に固定していた基質濃度を段階的に変化させ、それぞれの max [0044] 3. The enzyme reaction is actually performed. Since it is a fluorescence measurement, it is very sensitive and the reaction is carried out at a fairly dilute substrate concentration. This has the advantage of requiring less substrate mass, and also has the disadvantage that the concentration of one substrate cannot be increased, especially in the enzyme activity measurement of a two-substrate system. Therefore, first fix the concentration of one substrate, first determine the app and v app values, and then change the fixed substrate concentration step by step.
濃度について κ app、 V appを求める。算出したいくつかの V appについて、固定し m max max Obtain κ app and V app for the concentration. For some calculated V apps , fix m max max
た基質濃度に対して 2次プロットを行い、真の K 、V 値を算出する。  Perform a quadratic plot against the substrate concentration and calculate the true K and V values.
m max  m max
[0045] (分子生物学）  [0045] (Molecular biology)
本明細書において使用される用語「タンパク質」「ポリペプチド」、「オリゴペプチド」 および「ペプチド」は、本明細書において同じ意味で使用され、任意の長さのアミノ酸 のポリマーをいう。このポリマーは、直鎖であっても分岐していてもよぐ環状であって もよい。アミノ酸は、天然のものであっても非天然のものであってもよぐ改変されたァ ミノ酸であってもよい。この用語はまた、複数のポリペプチド鎖の複合体へとァセンブ ルされたものも包含し得る。この用語はまた、天然または人工的に改変されたアミノ酸 ポリマーも包含する。そのような改変としては、例えば、ジスルフイド結合形成、グリコ シル化、脂質化、ァセチル化、リン酸ィ匕または任意の他の操作もしくは改変（例えば、 標識成分との結合体化)。この定義にはまた、例えば、アミノ酸の 1または 2以上のァ ナログを含むポリペプチド (例えば、非天然のアミノ酸などを含む）、ペプチド様ィ匕合 物（例えば、ぺプトイド）および当該分野において公知の他の改変が包含される。従 つて、本発明のスクリーニング方法が目的とする酵素がポリペプチドである場合、この ような改変体を使用してもよ 、。 As used herein, the terms “protein”, “polypeptide”, “oligopeptide” and “peptide” are used interchangeably herein and refer to a polymer of amino acids of any length. The polymer may be linear or branched or cyclic. The amino acid may be a modified amino acid, which may be natural or non-natural. The term can also encompass those assembled into a complex of multiple polypeptide chains. The term also encompasses natural or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification (eg, Conjugation with labeling component). This definition also includes, for example, polypeptides (eg, including unnatural amino acids, etc.), peptide-like compounds (eg, peptoids) containing one or more amino acids, and known in the art. Other modifications are included. Therefore, when the enzyme targeted by the screening method of the present invention is a polypeptide, such a variant may be used.
[0046] 本明細書において使用される用語「ポリヌクレオチド」、「オリゴヌクレオチド」および「 核酸」は、本明細書において同じ意味で使用され、任意の長さのヌクレオチドのポリ マーをいう。 [0046] As used herein, the terms "polynucleotide", "oligonucleotide" and "nucleic acid" are used interchangeably herein and refer to a nucleotide polymer of any length.
[0047] 用語「核酸分子」はまた、本明細書において、核酸、オリゴヌクレオチド、およびポリ ヌクレオチドと互換可能に使用され、 cDNA、 mRNA、ゲノム DNAなどを含む。本明 細書では、核酸および核酸分子は、用語「遺伝子」の概念に含まれ得る。  [0047] The term "nucleic acid molecule" is also used herein interchangeably with nucleic acids, oligonucleotides, and polynucleotides and includes cDNA, mRNA, genomic DNA, and the like. As used herein, nucleic acids and nucleic acid molecules can be included in the concept of the term “gene”.
[0048] 本明細書において、「遺伝子」とは、遺伝形質を規定する因子をいう。通常染色体 上に一定の順序に配列している。タンパク質の一次構造を規定する構造遺伝子とい い、その発現を左右するものを調節遺伝子という。本明細書では、「遺伝子」は、「ポリ ヌクレオチド」、「オリゴヌクレオチド」および「核酸」ならびに Zまたは「タンパク質」「ポ リペプチド」、「オリゴペプチド」および「ペプチド」をさすことがある。本明細書におい てはまた、「遺伝子産物」とは、遺伝子によって発現された「ポリヌクレオチド」、「オリゴ ヌクレオチド」および「核酸」ならびに Zまたは「タンパク質」「ポリペプチド」、「オリゴぺ プチド」および「ペプチド」をさす。本明細書において遺伝子 (例えば、核酸配列、アミ ノ酸配列など）の「相同性」とは、 2以上の遺伝子配列の、互いに対する同一性の程 度をいう。従って、ある 2つの遺伝子の相同性が高いほど、それらの配列の同一性ま たは類似性は高い。 2種類の遺伝子が相同性を有する力否かは、配列の直接の比 較、または核酸の場合ストリンジェントな条件下でのハイブリダィゼ—シヨン法によって 調べられ得る。 2つの遺伝子配列を直接比較する場合、その遺伝子配列間で DNA 配列が、代表的には少なくとも 50%同一である場合、好ましくは少なくとも 70%同一 である場合、より好ましくは少なくとも 80%、 90%、 95%、 96%、 97%、 98%または 99%同一である場合、それらの遺伝子は相同性を有する。本明細書において、遺伝 子 (例えば、核酸配列、アミノ酸配列など）の「類似性」とは、上記相同性において、保 存的置換をポジティブ（同一）とみなした場合の、 2以上の遺伝子配列の、互いに対 する同一性の程度をいう。従って、保存的置換がある場合は、その保存的置換の存 在に応じて同一性と類似性とは異なる。また、保存的置換がない場合は、同一性と類 似性とは同じ数値を示す。 [0048] As used herein, "gene" refers to a factor that defines a genetic trait. Usually arranged in a certain order on the chromosome. A structural gene that regulates the primary structure of a protein is called a regulatory gene. As used herein, “gene” may refer to “polynucleotide”, “oligonucleotide” and “nucleic acid” and Z or “protein” “polypeptide”, “oligopeptide” and “peptide”. As used herein, “gene product” also refers to “polynucleotide”, “oligonucleotide” and “nucleic acid” expressed by a gene, and Z or “protein” “polypeptide”, “oligopeptide” and Refers to “peptide”. As used herein, “homology” of genes (for example, nucleic acid sequences, amino acid sequences, etc.) refers to the degree of identity of two or more gene sequences to each other. Therefore, the higher the homology between two genes, the higher the sequence identity or similarity. Whether two genes have homology can be determined by direct sequence comparison or, in the case of nucleic acids, hybridization methods under stringent conditions. When directly comparing two gene sequences, the DNA sequence between the gene sequences is typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90% If they are 95%, 96%, 97%, 98% or 99% identical, the genes have homology. In the present specification, “similarity” of a gene (eg, nucleic acid sequence, amino acid sequence, etc.) refers to the above homology. The degree of identity of two or more gene sequences relative to each other when existing substitutions are considered positive (identical). Thus, if there is a conservative substitution, identity and similarity differ depending on the presence of the conservative substitution. In the absence of conservative substitutions, identity and similarity indicate the same number.
[0049] 本明細書では塩基配列の類似性、同一性および相同性の比較は、配列分析用ッ —ルである BLASTを用いてデフォルトパラメータを用いて算出される。同一性の検 索は ί列えば、 NCBIの BLAST 2. 2. 9 (2004. 5. 12 発行）を用いて行うこと力 S できる。本明細書における同一性の値は通常は上記 BLASTを用い、デフォルトの 条件でァラインした際の値をいう。ただし、パラメーターの変更により、より高い値が出 る場合は、最も高い値を同一性の値とする。複数の領域で同一性が評価される場合 はそのうちの最も高い値を同一性の値とする。  [0049] In the present specification, comparison of base sequence similarity, identity and homology is calculated using BLAST, a sequence analysis tool, using default parameters. Searching for identity can be done using NCBI's BLAST 2.2.9 (issued 12 May 2004). The identity value in this specification usually refers to the value when aligned using the above BLAST and default conditions. However, if a higher value is obtained by changing the parameter, the highest value is the identity value. When identity is evaluated in multiple areas, the highest value is used as the identity value.
[0050] 本明細書において「外来遺伝子」とは、ある生物において、その生物には天然には 存在しない遺伝子をいう。そのような外来遺伝子は、その生物に天然に存在する遺 伝子を改変したものであってもよぐ天然において他の生物に存在する遺伝子であつ てもよく、人工的に合成した遺伝子であってもよい。そのような外来遺伝子を含む生 物は、天然では発現しない遺伝子産物を発現し得る。  [0050] As used herein, the term "foreign gene" refers to a gene that does not naturally exist in an organism. Such a foreign gene may be a gene that is naturally occurring in the organism, or may be a gene that is naturally present in another organism, or an artificially synthesized gene. May be. An organism containing such a foreign gene can express a gene product that is not naturally expressed.
[0051] 人工的に合成した遺伝子を作製するための DNA合成技術および核酸ィ匕学にっ ヽ ては、例えば、 Gait, M. J. (1985) . Oligonucleotide Synthesis : A Practical Approach, IRLPress ; Gait, M. J. (1990) . Oligonucleotide Synthesis : A Practical Approach, IRL Press ; Eckstein, F. 、上 991) . Oligonucleotides and Analogues : A Practical Approac, IRL Press ; Adams, R. L. etal. ( 1992) . The Biochemistry of the Nucleic Acids, Chapman&Hall; Sha barova, Z. et al. (1994) . Advanced Organic Chemistry of Nucleic A cids, Weinheim ; Blackburn, G. M. et al. (1996) . Nucleic Acids in Che mistry and Biology, Oxford University Press； Hermanson, G. T. (1996 ) . Bioconjugate Techniques, Academic Pressなどに， d載されており、これら は本明細書において関連する部分が参考として援用される。  [0051] Regarding DNA synthesis technology and nucleic acid science for producing artificially synthesized genes, see, for example, Gait, MJ (1985). Oligonucleotide Synthesis: A Practical Approach, IRLPress; Gait, MJ ( 1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F., 991). Oligonucleotides and Analogues: A Practical Approac, IRL Press; Adams, RL etal. (1992). Sha barova, Z. et al. (1994). Advanced Organic Chemistry of Nucleic A cids, Weinheim; Blackburn, GM et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, GT (1996 ). Bioconjugate Techniques, Academic Press, etc., which are incorporated herein by reference in their entirety.
[0052] 本明細書にぉ 、て、「アミノ酸」は、天然のものでも非天然のものでもよ 、。「誘導体 アミノ酸」または「アミノ酸アナログ」とは、天然に存在するアミノ酸とは異なるがもとの アミノ酸と同様の機能を有するものをいう。そのような誘導体アミノ酸およびアミノ酸ァ ナログは、当該分野において周知である。用語「天然のアミノ酸」とは、天然のァミノ 酸の L—異性体を意味する。天然のアミノ酸は、グリシン、ァラニン、パリン、ロイシン、 イソロイシン、セリン、メチォニン、トレオニン、フエ二ルァラニン、チロシン、トリプトファ ン、システィン、プロリン、ヒスチジン、ァスパラギン酸、ァスパラギン、グルタミン酸、グ ルタミン、 γ —カルボキシグルタミン酸、アルギニン、オル-チン、およびリジンである 。特に示されない限り、本明細書でいう全てのアミノ酸は L体である力 D体のアミノ酸 を用いた形態もまた本発明の範囲内にある。用語「非天然アミノ酸」とは、タンパク質 中で通常は天然に見出されないアミノ酸を意味する。非天然アミノ酸の例として、ノル ロイシン、パラ一ニトロフエニノレアラニン、ホモフエニノレアラニン、パラ一フノレオロフェニ ルァラニン、 3—アミノー 2—ベンジルプロピオン酸、ホモアルギニンの D体または L体 および D—フエ-ルァラニンが挙げられる。「アミノ酸アナログ」とは、アミノ酸ではない 力 アミノ酸の物性および Ζまたは機能に類似する分子をいう。アミノ酸アナログとし ては、例えば、ェチォニン、カナバニン、 2—メチルグルタミンなどが挙げられる。アミ ノ酸模倣物とは、アミノ酸の一般的な化学構造とは異なる構造を有するが、天然に存 在するアミノ酸と同様な様式で機能する化合物をいう。 [0052] As used herein, "amino acid" may be natural or non-natural. "Derivatives “Amino acid” or “amino acid analog” refers to an amino acid that is different from a naturally occurring amino acid but has the same function as the original amino acid. Such derivative amino acids and amino acid analogs are well known in the art. The term “natural amino acid” refers to the L-isomer of natural amino acids. Natural amino acids are glycine, alanine, parin, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, γ-carboxyglutamic acid , Arginine, orthine, and lysine. Unless otherwise indicated, all amino acids referred to in this specification are L-forms, and forms using D-form amino acids are also within the scope of the present invention. The term “unnatural amino acid” means an amino acid that is not normally found naturally in proteins. Examples of non-natural amino acids include norleucine, para-nitrophenenolanine, homophenenolanine, para-fluororeorophyllaranine, 3-amino-2-benzylpropionic acid, homoarginine D-form or L-form and D-fe-lualanin Is mentioned. “Amino acid analog” refers to a molecule that is similar to the physical properties and defects or functions of amino acids that are not amino acids. Examples of amino acid analogs include ethionine, canavanine, 2-methylglutamine and the like. Amino acid mimetics refers to compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
[0053] アミノ酸は、その一般に公知の 3文字記号力、または IUPAC— IUB Biochemica 1 Nomenclature Commissionにより推奨される 1文字記号のいずれかにより、本 明細書中で言及され得る。ヌクレオチドも同様に、一般に受け入れられた 1文字コー ドにより言及され得る。 [0053] Amino acids may be referred to herein by either their commonly known three letter symbol power or by the one letter symbol recommended by the IUPAC — IUB Biochemica 1 Nomenclature Commission. Nucleotides can also be referred to by the generally accepted single letter code.
[0054] 本明細書において、「対応する」ヌクレオチドまたはアミノ酸とは、あるポリヌクレオチ ド分子またはポリペプチド分子にぉ 、て、比較の基準となるポリヌクレオチドまたはポ リペプチドにおける所定のヌクレオチドまたはアミノ酸と同様の作用を有する力、また は有することが予測されるヌクレオチドまたはアミノ酸を 、 、、特に酵素分子にあって は、活性部位中の同様の位置に存在し触媒活性に同様の寄与をするアミノ酸をいう 。あるいは、酵素分子のコンフオメーシヨン変化に寄与する場合、そのような変化に寄 与するアミノ酸をいう。 [0055] 本明細書において、「対応する」遺伝子 (例えば、酵素）とは、ある種において、比 較の基準となる種における所定の遺伝子と同様の作用を有するか、または有すること が予測される遺伝子をいい、そのような作用を有する遺伝子が複数存在する場合、 進化学的に同じ起源を有するものをいう。従って、ある遺伝子の対応する遺伝子は、 その遺伝子のオルソログであり得る。従って、ヒト j8 1, 4—ガラクトース転移酵素に対 応する遺伝子は、他の種の同様の活性を担う酵素（またはその遺伝子)であり得る。 [0054] In the present specification, the "corresponding" nucleotide or amino acid is the same as a given nucleotide or amino acid in a polynucleotide or polypeptide as a reference for comparison with respect to a certain polynucleotide molecule or polypeptide molecule. A nucleotide or amino acid that has the action of, or is predicted to have, in the case of an enzyme molecule, an amino acid that is present at a similar position in the active site and contributes similarly to catalytic activity. . Alternatively, when it contributes to a change in the conformation of an enzyme molecule, it refers to an amino acid that contributes to such a change. [0055] In the present specification, a "corresponding" gene (for example, an enzyme) has, or is predicted to have, in a certain species, the same action as that of a predetermined gene in a species serving as a reference for comparison. When there are multiple genes having such an action, they have the same origin in terms of evolutionary chemistry. Thus, the corresponding gene of a gene can be an ortholog of that gene. Thus, the gene corresponding to human j8 1,4-galactosyltransferase may be an enzyme (or its gene) responsible for similar activities of other species.
[0056] 本明細書にぉ 、て「ヌクレオチド」は、糖部分が燐酸エステルになって 、るヌクレオ シドをいう。ヌクレオチドは通常アミノ酸をコードする。糖転移酵素の基質は通常糖ヌ クレオチドの形態をとる。核酸は塩基がピリミジン塩基またはプリン塩基のヌクレオチド (ピリミジンヌクレオチドおよびプリンヌクレオチド）の重合体 (ポリヌクレオチド)である。 糖部分が D—リボースのものをリボヌクレオチドといい， RNAの加水分解によって得 られる。糖部分が D— 2—デォキシリボースのものをデォキシリボヌクレオチドといい， DNAの酵素分解によって得られる。クレオチドは通常アデ-ル酸，デォキシアデ- ル酸とよんで 、るが，正確にはヌクレオシドの糖部分に結合して 、る燐酸の位置と燐 酸の数を示して，アデノシン— 5'——燐酸 (英文名を略して 5 '— AMPとよぶ.以下 同様)。リボヌクレオシドから誘導されたものをリボヌクレオチド、デォキシリボヌクレオ シドから誘導されたものをデォキシリボヌクレオチドと 、う。 DNAの酵素分解によりデ ォキシリボヌクレオシド 3' -リン酸あるいはデォキシリボヌクレオシド 5' -リン酸が得られ る.核酸以外にもヌクレオチドは遊離の状態で存在し，ヌクレオシドの 5'位で二リン酸 と結合して 、るヌクレオシドニリン酸，リン酸基がさらに 1つ延びたヌクレオシド三リン 酸がある。ヌクレオチドが基となる場合は、ヌクレオチジル基という。  [0056] As used herein, "nucleotide" refers to a nucleoside wherein the sugar moiety is a phosphate ester. Nucleotides usually encode amino acids. Glycosyltransferase substrates usually take the form of sugar nucleotides. Nucleic acids are polymers (polynucleotides) of nucleotides (pyrimidine nucleotides and purine nucleotides) whose bases are pyrimidine bases or purine bases. A sugar moiety with D-ribose is called a ribonucleotide and is obtained by RNA hydrolysis. A sugar moiety with D-2-deoxyribose is called deoxyribonucleotide and is obtained by enzymatic degradation of DNA. Creotide is usually called adenylic acid or deoxydelic acid, but it is bound to the sugar moiety of the nucleoside to indicate the position of phosphoric acid and the number of phosphates, and adenosine-5 '-phosphate (English name is abbreviated as 5'—AMP. The same applies below). A derivative derived from a ribonucleoside is referred to as a ribonucleotide, and a derivative derived from a deoxyribonucleoside is referred to as a deoxyribonucleotide. Deoxyribonucleoside 3'-phosphate or deoxyribonucleoside 5'-phosphate is obtained by enzymatic degradation of DNA. In addition to nucleic acids, nucleotides exist in a free state, and are located at the 5 'position of the nucleoside. In combination with diphosphate, there are nucleoside diphosphates and nucleoside triphosphates with an additional phosphate group. When a nucleotide is a group, it is called a nucleotidyl group.
[0057] 本明細書において「ヌクレオシド」とは、塩基と糖とが N—グリコシド結合をした化合 物をいう。糖が D—リボースのものがリボヌクレオシドで，そのうちプリン塩基を含むも のはプリンリボヌクレオシド (またはプリンリボシド，一般にはプリンヌクレオシド）とよば れ， RNAの分解によって得られる。糖が D—リボースのものをリボヌクレオシド， D 2' -デ才キシリボースのものをデ才キシリボヌクレオシドという。  [0057] As used herein, "nucleoside" refers to a compound in which a base and a sugar form an N-glycoside bond. Sugars with D-ribose are ribonucleosides, and those containing purine bases are called purine ribonucleosides (or purine ribosides, generally purine nucleosides) and are obtained by RNA degradation. A sugar with D-ribose is called a ribonucleoside, and a sugar with D 2'-deoxy xyribose is called a de xyribonucleoside.
[0058] 本明細書にぉ 、て、「フラグメント」とは、全長のポリペプチドまたはポリヌクレオチド（ 長さが n)に対して、 l〜n— 1までの配列長さを有するポリペプチドまたはポリヌクレオ チドをいう。フラグメントの長さは、その目的に応じて、適宜変更することができ、例え ば、その長さの下限としては、ポリペプチドの場合、 3、 4、 5、 6、 7、 8、 9、 10、 15, 2 0、 25、 30、 40、 50およびそれ以上のアミノ酸が挙げられ、ここの具体的に列挙して いない整数で表される長さ (例えば、 11など)もまた、下限として適切であり得る。また 、ポリヌクレオチドの場合、 5、 6、 7、 8、 9、 10、 15, 20、 25、 30、 40、 50、 75、 100 およびそれ以上のヌクレオチドが挙げられ、ここの具体的に列挙して!/、な!/、整数で表 される長さ（例えば、 11など)もまた、下限として適切であり得る。本明細書において、 ポリペプチドおよびポリヌクレオチドの長さは、上述のようにそれぞれアミノ酸または核 酸の個数で表すことができるが、上述の個数は絶対的なものではなぐ同じ機能を有 する限り、上限または加減としての上述の個数は、その個数の上下数個（または例え ば上下 10%)のものも含むことが意図される。そのような意図を表現するために、本 明細書では、個数の前に「約」を付けて表現することがある。しかし、本明細書では、「 約」のあるなしはその数値の解釈に影響を与えな ヽことが理解されるべきである。 [0058] As used herein, "fragment" refers to a polypeptide or polynucleotide having a sequence length of 1 to n-1 with respect to a full-length polypeptide or polynucleotide (length n). Say Chido. The length of the fragment can be changed as appropriate according to its purpose. For example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10 in the case of a polypeptide. , 15, 2, 0, 25, 30, 40, 50 and more, and lengths expressed in integers not specifically listed here (for example, 11 etc.) are also suitable as lower limits. It can be. In the case of polynucleotides, examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides. !!, NA! /, An integer length (eg 11) may also be appropriate as a lower limit. In the present specification, the lengths of polypeptides and polynucleotides can be represented by the number of amino acids or nucleic acids, respectively, as described above, but the above numbers are not absolute, as long as they have the same function. It is intended that the above-mentioned number as the upper limit or subtraction includes the upper and lower numbers (or, for example, 10% above and below) of that number. In order to express such intention, in this specification, “about” may be added before the number. However, it should be understood herein that the presence or absence of “about” does not affect the interpretation of the value.
[0059] 本明細書において「生物学的活性」とは、ある因子 (例えば、ポリペプチドまたはタ ンパク質）力 生体内において有し得る活性のことをいい、種々の機能を発揮する活 性が包含される。例えば、ある因子が酵素である場合、その生物学的活性は、その 酵素活性を包含する。別の例では、ある因子がリガンドである場合、そのリガンドが対 応するレセプターへの結合を包含する。そのような生物学的活性は、当該分野にお V、て周知の技術によって測定することができる。  [0059] In this specification, "biological activity" refers to an activity that can be possessed in a living body by a certain factor (eg, a polypeptide or a protein), and an activity that exhibits various functions. Is included. For example, when a factor is an enzyme, the biological activity includes the enzyme activity. In another example, when an agent is a ligand, it includes binding to the corresponding receptor. Such biological activity can be measured by techniques well known in the art.
[0060] 本明細書において、ポリペプチドを生産する方法としては、例えば、そのポリべプチ ドを産生する初代培養細胞または株化細胞を培養し、培養上清などから単離または 精製することによりそのポリペプチドを得る方法が挙げられる。あるいは、遺伝子操作 手法を利用して、そのポリペプチドをコードする遺伝子を適切な発現ベクターに組み 込み、これを用いて発現宿主を形質転換し、この形質転換細胞の培養上清から組換 えポリペプチドを得ることができる。上記宿主細胞は、生理活性を保持するポリぺプ チドを発現するものであれば、特に限定されず、従来力 遺伝子操作において利用 される各種の宿主細胞 (例えば、大腸菌、酵母、動物細胞など)を用いることが可能 である。このようにして得られた細胞に由来するポリペプチドは、天然型のポリべプチ ドと実質的に同一の作用を有する限り、アミノ酸配列中の 1以上のアミノ酸が置換、付 加および Zまたは欠失していてもよぐ糖鎖が置換、付加および Zまたは欠失してい てもよい。 [0060] In the present specification, as a method for producing a polypeptide, for example, primary cultured cells or established cells that produce the polypeptide are cultured and isolated or purified from the culture supernatant or the like. The method of obtaining the polypeptide is mentioned. Alternatively, using a gene manipulation technique, a gene encoding the polypeptide is incorporated into an appropriate expression vector, and an expression host is transformed using the gene, and the recombinant cell is transformed from the culture supernatant of the transformed cell. Peptides can be obtained. The host cell is not particularly limited as long as it expresses a polypeptide that retains physiological activity, and various host cells conventionally used in gene manipulation (for example, E. coli, yeast, animal cells, etc.) Can be used. The polypeptide derived from the cells thus obtained is a natural-type polypeptide. As long as it has substantially the same action as that of the amino acid sequence, one or more amino acids in the amino acid sequence may be substituted, added and Z or deleted, and the sugar chain may be substituted, added and Z or deleted. Also good.
[0061] あるアミノ酸は、相互作用結合能力の明らかな低下または消失なしに、例えば、力 チオン性領域または基質分子の結合部位のようなタンパク質構造において他のアミ ノ酸に置換され得る。あるタンパク質の生物学的機能を規定するのは、タンパク質の 相互作用能力および性質である。従って、特定のアミノ酸の置換がアミノ酸配列にお いて、またはその DNAコード配列のレベルにおいて行われ得、置換後もなお、もとの 性質を維持するタンパク質が生じ得る。従って、生物学的有用性の明らかな損失なし に、種々の改変が、本明細書において開示されたペプチドまたはこのペプチドをコー ドする対応する DNAにお 、て行われ得る。  [0061] Certain amino acids can be substituted for other amino acids in protein structures, such as force thionic regions or substrate molecule binding sites, without an apparent reduction or loss of interaction binding capacity. It is the ability and nature of the protein to define the biological function of a protein. Thus, specific amino acid substitutions can be made in the amino acid sequence or at the level of its DNA coding sequence, resulting in a protein that still retains its original properties after substitution. Thus, various modifications can be made to the peptide disclosed herein or the corresponding DNA encoding this peptide without any apparent loss of biological utility.
[0062] 上記のような改変を設計する際に、アミノ酸の疎水性指数が考慮され得る。タンパク 質における相互作用的な生物学的機能を与える際の疎水性アミノ酸指数の重要性 は、一般に当該分野で認められている（Kyte. Jおよび Doolittle, R. F. J. Mol. Bi ol. 157 (1) : 105- 132, 1982)。アミノ酸の疎水的性質は、生成したタンパク質の 二次構造に寄与し、次いでそのタンパク質と他の分子 (例えば、酵素、基質、レセプ ター、 DNA、抗体、抗原など）との相互作用を規定する。各アミノ酸は、それらの疎水 性および電荷の性質に基づく疎水性指数を割り当てられる。それらは:イソロイシン（ +4. 5)；バリン（+4. 2)；ロイシン（ + 3. 8)；フエ-ルァラニン（ + 2. 8)；システィン Zシスチン（ + 2. 5)；メチォニン（ + 1. 9)；ァラニン（ + 1. 8)；グリシン（一0. 4)；スレ ォニン（一 0. 7) ;セリン（一0. 8)；トリプトファン（一0. 9) ;チロシン（一1. 3) ;プロリン (— 1. 6) ;ヒスチジン（一3. 2)；グノレタミン酸（一 3. 5)；グノレタミン（一3. 5) ;ァスパラ ギン酸（一3. 5)；ァスパラギン（一3. 5) ;リジン（一3. 9)；およびアルギニン（一4. 5) )である。  [0062] In designing such modifications, the hydrophobicity index of amino acids can be taken into account. The importance of the hydrophobic amino acid index in conferring interactive biological functions in proteins is generally recognized in the art (Kyte. J and Doolittle, RFJ Mol. Biol. 157 (1): 105-132, 1982). The hydrophobic nature of amino acids contributes to the secondary structure of the protein produced, and then defines the interaction of the protein with other molecules (eg, enzymes, substrates, receptors, DNA, antibodies, antigens, etc.). Each amino acid is assigned a hydrophobicity index based on their hydrophobicity and charge properties. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); ferulalanin (+2.8); cystine Z cystine (+2.5); methionine (+ 1. 9); alanine (+1. 8); glycine (one 0.4); threonine (one 0.7); serine (one 0.8); tryptophan (one 0.9); tyrosine (one 1 3); Proline (— 1. 6); Histidine (1 3.2); Gnoretamic acid (1 3.5); Gnoretamine (1 3.5); Aspartic acid (1 3.5); Asparagine (1) 3.5); lysine (one 3.9); and arginine (one 4.5)).
[0063] あるアミノ酸を、同様の疎水性指数を有する他のアミノ酸により置換して、そして依 然として同様の生物学的機能を有するタンパク質 (例えば、酵素活性において等価 なタンパク質)を生じさせ得ることが当該分野で周知である。このようなアミノ酸置換に おいて、疎水性指数が ± 2以内であることが好ましぐ ± 1以内であることがより好まし ぐおよび ±0. 5以内であることがさらにより好ましい。疎水性に基づくこのようなァミノ 酸の置換は効率的であることが当該分野において理解される。米国特許第 4、 554、 101号に記載されるように、以下の親水性指数がアミノ酸残基に割り当てられて!/、る： アルギニン（ + 3. 0)；リジン（ + 3. 0)；ァスパラギン酸（ + 3. 0± 1)；グルタミン酸（+ 3. 0± 1)；セリン（ + 0. 3)；ァスパラギン（ + 0. 2)；グルタミン（ + 0. 2)；グリシン（0)； スレオニン（一0. 4)；プロリン（一0. 5 ± 1)；ァラニン（一0. 5)；ヒスチジン（一0. 5)； システィン（一1. 0)；メチォニン（一1. 3)；バリン（一 1. 5)；ロイシン（一1. 8)；ィソロ イシン（一 1. 8) ;チロシン（一2. 3)；フエ-ルァラニン（一2. 5)；およびトリプトファン（ 3. 4)。アミノ酸が同様の親水性指数を有しかつ依然として生物学的等価体を与え 得る別のものに置換され得ることが理解される。このようなアミノ酸置換において、親 水性指数が ± 2以内であることが好ましぐ ± 1以内であることがより好ましぐおよび ±0. 5以内であることがさらにより好ましい。 [0063] The ability to substitute one amino acid with another amino acid having a similar hydrophobicity index and still produce a protein with a similar biological function (eg, a protein equivalent in enzyme activity) Are well known in the art. In such amino acid substitutions, the hydrophobicity index is preferably within ± 2 and more preferably within ± 1. Even more preferably within ± 0.5. It is understood in the art that such substitution of amino acids based on hydrophobicity is efficient. As described in US Pat. No. 4,554,101, the following hydrophilicity indices are assigned to amino acid residues! /, Arginine (+3.0); lysine (+3.0); Aspartic acid (+ 3.0 ± 1); glutamic acid (+ 3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); Threonine (one 0.4); proline (one 0.5 ± 1); alanine (one 0.5); histidine (one 0.5); cystine (one 1.0); methionine (one 1.3); Valine (one 1.5); leucine (one 1.8); isoloicin (one 1.8); tyrosine (one 2.3); ferrolanine (one 2.5); and tryptophan (3.4) . It is understood that an amino acid can be substituted with another that has a similar hydrophilicity index and can still provide a biological equivalent. In such an amino acid substitution, the hydrophilicity index is preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.
[0064] 本発明にお 、て、「保存的置換」とは、アミノ酸置換にぉ 、て、元のアミノ酸と置換さ れるアミノ酸との親水性指数または Zおよび疎水性指数が上記のように類似して 、る 置換をいう。保存的置換の例としては、例えば、親水性指数または疎水性指数が、士 2以内のもの同士、好ましくは ± 1以内のもの同士、より好ましくは ±0. 5以内のもの 同士のものが挙げられるがそれらに限定されない。従って、保存的置換の例は、当業 者に周知であり、例えば、次の各グループ内での置換:アルギニンおよびリジン；ダル タミン酸およびァスパラギン酸；セリンおよびスレオニン；グルタミンおよびァスパラギン ；ならびにパリン、ロイシン、およびイソロイシン、などが挙げられるがこれらに限定され ない。 [0064] In the present invention, "conservative substitution" is similar to the amino acid substitution, as described above, in the hydrophilicity index or Z and hydrophobicity index with the amino acid to be replaced with the original amino acid. This refers to substitution. Examples of conservative substitutions include those having a hydrophilicity index or hydrophobicity index of 2 or less, preferably ± 1 or less, more preferably ± 0.5 or less. But not limited to them. Thus, examples of conservative substitutions are well known to those skilled in the art, for example, substitutions within the following groups: arginine and lysine; dartamic acid and aspartic acid; serine and threonine; glutamine and asparagine; Examples include, but are not limited to, leucine and isoleucine.
[0065] 本明細書において、「改変体」とは、もとのポリペプチドまたはポリヌクレオチドなどの 物質に対して、一部が変更されているものをいう。そのような改変体としては、置換改 変体、付加改変体、欠失改変体、短縮 (truncated)改変体、対立遺伝子変異体な どが挙げられる。置換は、上述の保存的置換でもよぐそうでなくてもよい。置換され るアミノ酸は、天然のアミノ酸であってもよぐ非天然のアミノ酸であってもよい。あるい は、置換されるアミノ酸は、アミノ酸アナログでもよい。対立遺伝子 (allele)とは、同一 遺伝子座に属し、互いに区別される遺伝的改変体のことをいう。従って、「対立遺伝 子変異体」とは、ある遺伝子に対して、対立遺伝子の関係にある改変体をいう。その ような対立遺伝子変異体は、通常その対応する対立遺伝子と同一または非常に類似 性の高い配列を有し、通常はほぼ同一の生物学的活性を有する力 まれに異なる生 物学的活性を有することもある。「種相同体またはホモログ (homolog)」とは、ある種 の中で、ある遺伝子とアミノ酸レベルまたはヌクレオチドレベルで、相同性 (好ましくは 、 60%以上の相同性、より好ましくは、 80%以上、 85%以上、 90%以上、 95%以上 の相同性)を有するものをいう。そのような種相同体を取得する方法は、本明細書の 記載から明らかである。「オルソログ（ortholog)」とは、オルソロガス遺伝子（ortholo gous gene)ともいい、二つの遺伝子がある共通祖先からの種分化に由来する遺伝 子をいう。例えば、多重遺伝子構造をもつヘモグロビン遺伝子ファミリーを例にとると 、ヒトおよびマウスの αヘモグロビン遺伝子はオルソログである力 ヒトの αへモグロビ ン遺伝子および j8ヘモグロビン遺伝子はパラログ (遺伝子重複で生じた遺伝子)であ る。オルソログは、分子系統樹の推定に有用である。オルソログは、通常別の種にお V、てもとの種と同様の機能を果たして 、ることがあり得ることから、本発明にお 、て、 例えば、糖転移酵素のオルソログもまた、本発明において有用であり得る。 [0065] In the present specification, the "variant" refers to a substance in which a part of the original substance such as a polypeptide or polynucleotide is changed. Such variants include substitutional variants, addition variants, deletion variants, truncated variants, allelic variants, and the like. The substitution need not be a conservative substitution as described above. The substituted amino acid may be a natural amino acid or a non-natural amino acid. Alternatively, the substituted amino acid may be an amino acid analog. Alleles are genetic variants that belong to the same locus and are distinguished from each other. Therefore, "Alleles" A “child variant” refers to a variant that has an allelic relationship to a gene. Such allelic variants usually have a sequence that is identical or very similar to its corresponding allele and usually has a rarely different biological activity with nearly the same biological activity. May have. “Species homologue or homolog” means homology (preferably at least 60% homology, more preferably at least 80%, at a certain amino acid level or nucleotide level within a certain species. 85% or more, 90% or more, 95% or more homology). The method for obtaining such species homologues will be apparent from the description herein. “Ortholog”, also called orthologous gene, refers to a gene derived from speciation from a common ancestor with two genes. For example, taking the hemoglobin gene family with multigene structure as an example, human and mouse α-hemoglobin genes are orthologs. Human α-hemoglobin genes and j8 hemoglobin genes are paralogs (genes generated by gene duplication). is there. Orthologs are useful for estimating molecular phylogenetic trees. Since orthologs usually have the same function as V and the original species in another species, for example, orthologs of glycosyltransferases are also present in the present invention. May be useful.
[0066] 本明細書において「酵素」とは、生細胞内で作用するタンパク質などの生体分子に よる生体触媒分子をいう。  [0066] As used herein, the term "enzyme" refers to a biocatalytic molecule that is a biomolecule such as a protein that acts in living cells.
[0067] 本明細書において「基質」とは、酵素などの活性ィ匕物質との反応において用いられ る場合、ある活性化物質 (例えば、酵素)の作用を受けて反応を起す物質をいう。特 に酵素反応において、例えば澱粉はアミラーゼの基質であり、過酸化水素は力タラ 一ゼの基質である。酵素反応が可逆的なときは，生成物も逆反応の基質である。また 補酵素が酵素の基質の一つであることも多い。転移酵素の場合、ドナー分子および ァクセプター分子の両方が基質である。  [0067] In this specification, "substrate" refers to a substance that, when used in a reaction with an active substance such as an enzyme, causes a reaction under the action of an activating substance (for example, an enzyme). Particularly in enzymatic reactions, for example, starch is a substrate for amylase, and hydrogen peroxide is a substrate for force tarase. When the enzyme reaction is reversible, the product is also a substrate for the reverse reaction. Coenzymes are often one of the enzyme substrates. In the case of transferase, both donor and acceptor molecules are substrates.
[0068] 本明細書にぉ 、て「転移酵素」とは、基転移反応を触媒する酵素の総称を 、う。本 明細書において、「転移酵素」は「トランスフェラーゼ」と互換可能に使用され得る。基 転移反応は、以下の式 (6) :  In the present specification, the term “transferase” is a general term for enzymes that catalyze a group transfer reaction. In the present specification, “transferase” may be used interchangeably with “transferase”. The group transfer reaction is represented by the following equation (6):
X-Y+Z-H X-H + Z-Y (6)  X-Y + Z-H X-H + Z-Y (6)
に示すように、一つの化合物 (供与体)から基 Yが他の化合物 (受容体）に転移する 形で行われる。 As shown, the group Y is transferred from one compound (donor) to another (acceptor) Done in the form.
[0069] 本明細書の基転移反応における「ドナー」とは、上記式 (6)の化合物 X— Y (供与体 )を意味し、「ドナー基質」と互換可能に使用され得る。本明細書の基転移反応にお ける「ァクセプター」とは、上記式 (6)の化合物 Z— H (受容体)を意味し、「ァクセプタ 一基質」と互換可能に使用され得る。  [0069] The "donor" in the group transfer reaction of the present specification means the compound X—Y (donor) of the above formula (6) and can be used interchangeably with the “donor substrate”. The “acceptor” in the group transfer reaction of the present specification means the compound Z—H (acceptor) of the above formula (6), and can be used interchangeably with “acceptor monosubstrate”.
[0070] 本明細書において「糖転移酵素」とは、糖 (上記式 (6)の基 Yに相当；単位糖または 糖鎖)をある場所 (上記式 (6)の化合物 X— Yに相当）から別の場所 (上記式 (6)の化 合物 Z— Hに相当）へと転移させるよう触媒する作用を有する酵素をいう。糖転移酵 素としては、例えば、ガラクトース転移酵素、グルコース転移酵素、シアル酸転移酵 素、マンノース転移酵素、フコース転移酵素、キシロース転移酵素、 N ァセチルダ ルコサミン転移酵素、および N ァセチルガラタトサミン転移酵素などが挙げられるが それらに限定されない。そのような糖転移酵素としては、例えば、糖転移酵素の例と しては、 αΐ, 4—ガラクトース転移酵素、 《1, 3—ガラクトース転移酵素， β ΐ, 4— ガラクトース転移酵素， β ΐ, 3—ガラクトース転移酵素， β ΐ, 6—ガラクトース転移酵 素、 ひ 2, 6 シアル酸転移酵素、 ひ1, 4 ガラクトース転移酵素、セラミドガラタトー ス転移酵素、 αΐ, 2 フコース転移酵素、 《1, 3 フコース転移酵素、 《1, 4 フ コース転移酵素、 ひ1, 6 フコース転移酵素、 ひ1, 3— Ν ァセチルガラタトサミン 転移酵素、 αΐ, 6— Ν ァセチルガラタトサミン転移酵素、 β ΐ, 4— Ν ァセチルガ ラタトサミン転移酵素、ポリペプチド Ν ァセチルガラタトサミン転移酵素、 /31, 4-Ν ァセチルダルコサミン転移酵素、 131, 2— Νァセチルダルコサミン転移酵素、 β ΐ, 3 —Νァセチルダルコサミン転移酵素、 β ΐ, 6— Νァセチルダルコサミン転移酵素、 a 1, 4—Nァセチルダルコサミン転移酵素、 β ΐ, 4 マンノース転移酵素、 αΐ, 2— マンノース転移酵素、 《1, 3 マンノース転移酵素、 《1, 4 マンノース転移酵素、 αΐ, 6 マンノース転移酵素、 αΐ, 2 グルコース転移酵素、 α 1, 3 グルコース 転移酵素、 α2, 3 シアル酸転移酵素、 《2, 6 シアル酸転移酵素、 《1, 6 グ ルコサミン転移酵素、 αΐ, 6—キシロース転移酵素、 j8キシロース転移酵素（プロテ ォグリカンコア構造合成酵素）、 β ΐ, 3—グルクロン酸転移酵素、ヒアルロン酸合成 酵素、他の糖ヌクレオチドを糖ドナーとして用いる糖転移酵素およびドルコールリン 酸型糖ドナーを用いる糖転移酵素などが挙げられるがそれらに限定されない。 [0070] As used herein, "glycosyltransferase" refers to a sugar (corresponding to group Y in the above formula (6); unit sugar or sugar chain) corresponding to compound X—Y in the above formula (6). ) To another place (equivalent to the compound Z—H of the above formula (6)). Examples of the glycosyltransferase include galactose transferase, glucose transferase, sialic acid transferase, mannose transferase, fucose transferase, xylose transferase, N-acetylylcosamine transferase, and N-acetyl galatatosamine transferase. However, it is not limited to them. Examples of such glycosyltransferases include αΐ, 4-galactose transferase, << 1, 3-galactose transferase, β ΐ, 4-galactose transferase, β ΐ, 3-galactose transferase, β ΐ, 6-galactose transferase, 2,6 sialyltransferase, 1,4 galactose transferase, ceramide galactose transferase, αΐ, 2 fucose transferase, << 1, 3 fucose transferase, 《1, 4 fucose transferase, 1, 1, 6 fucose transferase, 1, 1, 3—Ν acetyl galatatosamine transferase, αΐ, 6—Ν acetyl galatatosamine transferase, β ΐ, 4— Ν Acetylgallatatosamine transferase, polypeptide ァ Acetylgalatatosamine transferase, / 31, 4-Ν Acetyl darcosamine transferase, 131, 2- Ν Acetyl darcosamine transferase, β ΐ, 3 —Ν Saminyltransferase, β ΐ, 6-Νacetylyldarcosaminetransferase, a 1, 4-N-acetylyldarcosaminetransferase, βΐ, 4 mannose transferase, αΐ, 2-mannosetransferase, << 1, 3 Mannose transferase, 《1, 4 Mannose transferase, αΐ, 6 Mannose transferase, αΐ, 2 Glucose transferase, α 1, 3 Glucose transferase, α2, 3 Sialyltransferase, 《2, 6 Sialyltransferase , 《1, 6 glucosamine transferase, α ΐ, 6-xylose transferase, j8 xylose transferase (proteoglycan core synthase), β ΐ, 3-glucuronic acid transferase, hyaluronic acid synthase, other sugar nucleotides Glucosyltransferase and dolcorrin using as a sugar donor Examples include, but are not limited to, glycosyltransferases using acid-type sugar donors.
[0071] 本明細書にぉ 、て「ガラタトース転移酵素」（本明細書にぉ 、て GalTとも表記する） は、ガラクトース（以下 Gal)を UDP— Galから N ァセチル—ダルコサミンを末端に 持つ多様な糖ァクセプターへ、ァノマー位の立体選択制について厳格に α型あるい は j8型で転移させる酵素をいう。 α型では αΐ, 2—、 αΐ, 3—、 αΐ, 4一、 α 1, 6 結合型が存在し、 j8型では j81, 3—、 β ΐ, 4 結合型が存在する。このほか、ガ ラタトースセラミド合成に関わる β 1—セラミド結合型が存在する。真核生物において 、 GalTは一部の例外を除きゴルジ体内に口—カラィズして存在し、多様な糖鎖を生 み出している。また j81, 4 GalTは、糖鎖合成の役割のみならず、細胞表層に存在 し、授精ゃ胚発生の過程にお!ヽて細胞接着分子としての働きを持つことが知られて いる（D. A. Hinton, B. D. Shur, (1994), Trends Glycosci. Glycotech. , 6 , 375-385. )o現在までに、哺乳動物においては 19の異なった GalTが発見され ており、これらは αΐ, 3—、 αΐ, 4一、 β 1, 3—、 β 1, 4 結合型のガラクトース酵 素である（Christelle B. , Emmanuel B. , David H.J. , Roberto A. G. an d Anne I. (1998), J. Biochem. 123, 1000—1009. 、 T. Hennet, (2002) , Cell. Mol. Life Sci. 59, 1081— 1095.；)。これらは j81—セラミド型酵素を除き 、全てタイプ II膜貫通型で、短い N末端細胞質側領域と膜貫通領域、幹領域を持ち 酵素活性部位を含む大きな C末端活性ドメインをゴルジ内腔に向けている。  [0071] In the present specification, "galatose transferase" (hereinafter also referred to as GalT) is a variety of galactose (hereinafter referred to as Gal) having a terminal from UDP-Gal to N-acetyl-darcosamine. An enzyme that strictly transfers α-type or j8-type to the sugar acceptor. The α type has αΐ, 2—, αΐ, 3—, α 4, 41, and α 1, 6 bond types, and the j8 type has j81, 3—, β ΐ, 4 bond types. In addition, there is a β 1-ceramide bond type involved in the synthesis of galatose ceramide. In eukaryotes, GalT, with some exceptions, exists in the Golgi in a mouth-colorized manner, producing a variety of sugar chains. J81, 4 GalT is known not only to play a role in sugar chain synthesis but also to exist on the cell surface, and to act as a cell adhesion molecule throughout the embryogenesis process (DA Hinton). , BD Shur, (1994), Trends Glycosci. Glycotech., 6, 375-385. 4-1, β 1,3—, β 1,4-linked galactose enzyme (Christelle B., Emmanuel B., David HJ, Roberto AG an d Anne I. (1998), J. Biochem. 123, 1000-1009., T. Hennet, (2002), Cell. Mol. Life Sci. 59, 1081-1095 .;). These are all type II transmembrane types, except for the j81-ceramide type enzyme, with a short N-terminal cytoplasmic region, transmembrane region, and stem region, and a large C-terminal active domain containing the enzyme active site toward the Golgi lumen Yes.
[0072] その構造、機能についてよく調べられているのはゥシ、ブタ、ヒトなど哺乳類由来の ものが多ぐ中でも基質特異性について特に調べられているのは、 β ΐ, 4 結合型 酵素である。この酵素は比較的単離、精製が容易であり、早くに市販されるようになつ たことから、すでに多くの基質認識能、基質類似物阻害作用についての知見が得ら れている（Philippe C. , Olilier R. M. , (2001), Bioorg. Med. Chem. 9, 30 77-3092. ； Hironobu H. , Tsuyoshi E. and Yasuhiro K. , (1997), J. Org. Chem. 62, 1914—1915. ； Shuichi T. , Sang J. C. , Yasuhiro I. , Y oshitaka I. , Armin S. , Robert I. L. , Jiangyue W. , Takashi H. , Gary S. and Chi-Huey W. , (1999), Bioorg. Med. Chem. 7, 401—409. ； T akashi H. , Brion W. M. , Ruo W. and Chi-Huey W. , (1997), Bioor g. Med. Chem. 5, 497— 500. ； Sang J. C. , Shuichi T. and Chi-Huey W. , ( 1998) , Bioorg. Med. Chem. Let. 8, 3359— 3364. ; 01e H. , Kanw al J. Kaur. , Geeta S. , Magdalena B. , Suzanne C. C. , Louis D. H. a nd Monica M. P. , ( 1991) , J. Biol. Chem. 266, 17858— 17862.；)。また、 ブタの臓器をヒトに移植する際の拒絶反応に関わる ex Gal抗原を合成するひ 1 , 3— GalTにつ!/、ても多くの知見が得られて!/、る。以下に上記 2系列の GalTにつ!/、て簡 単にその特徴を述べる。 [0072] The structure and function of which are well examined are mammals such as ushi, pig, and human. Among them, the substrate specificity is particularly investigated for β ΐ, 4-linked enzymes. is there. This enzyme is relatively easy to isolate and purify, and since it was quickly put on the market, a lot of knowledge about substrate recognition ability and substrate analogue inhibition has already been obtained (Philippe C , Olilier RM, (2001), Bioorg. Med. Chem. 9, 30 77-3092.; Hironobu H., Tsuyoshi E. and Yasuhiro K., (1997), J. Org. Chem. 62, 1914—1915 ; Shuichi T., Sang JC, Yasuhiro I., Y oshitaka I., Armin S., Robert IL, Jiangyue W., Takashi H., Gary S. and Chi-Huey W., (1999), Bioorg. Med Chem. 7, 401—409. Takashi H., Brion WM, Ruo W. and Chi-Huey W., (1997), Bioor. g. Med. Chem. 5, 497- 500.; Sang JC, Shuichi T. and Chi-Huey W., (1998), Bioorg. Med. Chem. Let. 8, 3359— 3364.; 01e H., Kanw al J. Kaur., Geeta S., Magdalena B., Suzanne CC, Louis DH and Monica MP, (1991), J. Biol. Chem. 266, 17858-17862 .;). In addition, much knowledge has been obtained about 1,3-GalT, which synthesizes ex Gal antigens involved in rejection when transplanting porcine organs to humans! Below, the characteristics of the two series of GalTs! / Are briefly described.
( 1) β 1 , 4 - GalT  (1) β 1, 4-GalT
現在までにもっともよく研究されている GalTである。この酵素はヒトにおいてそのシ —クエンス相同性から、 7種類のものが発見されている。一般に市販されているもの は、ゥシ、ヒト由来（ほぼ同一の配列を有する）の j8 1 , 4— GalTlと呼ばれるもので、 UDP— Galから Galを N—ァセチルーダルコサミン（以下 GlcNAc)に転移させる。母 乳中のラタトースの生成にも深く関わり、この場合は αラクトアルブミンと相互作用して 、グルコースを糖ァクセプターとして認識する。一部のこの酵素は細胞表層にも存在 し、糖鎖合成のみならず、細胞接着分子としての働きを持つことがわ力つている。早く 力 市販され容易に入手可能であるという事情から、多くの研究者が基質特異性に ついて検証しており、糖ドナー（Thomas B. , Thomas S. , Darius -Jean N. , Ricardo G. G. , Henrik C. and Lother E. , (2001) , ChemBioChem. 2 , 884— 894. ； Yasuhiro K. , Tsuyoshi E. , Hiroyuki O. , Hisashi K. Hir onobu H. , ( 1995) , Carbo. Res. 269, 273— 294. ； Geeta S. , Ole H. an d Monica M. P. , ( 1993) , Carbo. Res. 245, 137—144. ； Elizabeth E. B . , Petety V. B. and Pradman K. Q. , ( 1995) , Glycocon. J. 12, 865— 8 78. ； Lawrence J. B. and Robert D. R. , ( 1982) , Biochemistry 21 , 634 0— 6343. ； Tsuyoshi E. , Yasuhiro K. , Hisashi K. and Hironobu H. , ( 1996) , Bioorg. Med. Chem. 4, 1939— 1948. ； Wong C. H. , Whiteside It is GalT that has been most well studied to date. Seven types of this enzyme have been discovered in humans due to its sequence homology. What is generally marketed is called U8, human-derived (substantially the same sequence) j8 1, 4— GalTl. From UDP- Gal to Gal-N-acetyl-darcosamine (hereinafter GlcNAc) To be transferred. It is also deeply involved in the production of ratatose in breast milk, in which case it interacts with α-lactalbumin and recognizes glucose as a sugar acceptor. Some of these enzymes are also present on the cell surface and are not only used for sugar chain synthesis but also as a cell adhesion molecule. Due to the fact that it is commercially available and easily available, many researchers have verified the substrate specificity, and sugar donors (Thomas B., Thomas S., Darius-Jean N., Ricardo GG, Henrik C. and Lother E., (2001), ChemBioChem. 2, 884— 894.; Yasuhiro K., Tsuyoshi E., Hiroyuki O., Hisashi K. Hir onobu H., (1995), Carbo. Res. 269, 273-294 .; Geeta S., Ole H. an d Monica MP, (1993), Carbo. Res. 245, 137-144 .; Elizabeth E. B., Petety VB and Pradman KQ, (1995), Glycocon. Jr. 12, 865— 8 78.; Lawrence JB and Robert DR, (1982), Biochemistry 21, 634 0— 6343.; Tsuyoshi E., Yasuhiro K., Hisashi K. and Hironobu H., (1996), Bioorg Med. Chem. 4, 1939— 1948.; Wong CH, Whiteside
G. M. , ( 1994) , 「Enzymes in synthetic organic chemistryj , pp264— 269.；)、糖ァクセプター（Yoshihiro N. , Hideaki T. , Kazukiyo Κ. and Joa chim T. , (2001) , Org. let. 3, 1— 3. ； Torsten W. , Yoshihiro N. , Volk er S . and Joachim T. , ( 1994) , J. Org. Chem. 59, 6744— 6747. ； Yoshi hiro N. , Torsten W. , Volker S. and Joachim T. , ( 1993) , J. Am. Che m. Soc. 115, 2536— 2537. ； Yasuniro K. , Hisashi K. , Tsuyosni E. , H ironobu H. , ( 1998) , Carbo. Res. 306, 361— 378. ； Monica M. P. , ( 19 87) , Carbo. Res. 159, 315— 324. ； Yasuhiro K. , Hironobu H. and His ashi K. , ( 1992) , Carbo. Res. 229, C5— C9. ； Yasuhiro K. , Hironobu H. , Seiichiro O. , (2000) , Carbo. Res. 323, 44—48. ； Chi-Huey W. , Yoshitaka I. , Thomas K. , Christine G. N. , David P. D. and Gary C . L. , ( 1991) , J. Am. Chem. Soc. 113, 8137— 8145.；)の両方【こつ ヽて、ネィ ティブな基質でなくともある程度まで基質修飾に対する寛容性があることが知られて きている。さらに最近になって X線結晶構造解析の結果が報告され (Louis N. G. , Christian C. and Yves B. , ( 1999) , EMBO. J. 18, 3546— 3557. , ； B. R amakrishnan and Pradman K. Q. , (2001) , J. Mol. Biol. 310, 205— 21 8. )、この酵素は逐次（sequential order)型の反応機構で基質と結合し（Boopat hy R. , Elizabeth Β. and Pradman K. Q. , (2002) , Biochem. Biophys. Res. Comm. 291 , 11 13— 1118. )、糖ドナーの結合によって、大きな構造変化を 起こすことが確認された（Boopathyら、前出； Ramakrishnan, P. V. Balajji and Pradman K. Q. , (2002) , J. Mol. Biol. 318, 491— 502.；)。 GM, (1994), "Enzymes in synthetic organic chemistry, pp264—269 .;), sugar acceptors (Yoshihiro N., Hideaki T., Kazukiyo Κ. And Joa chim T., (2001), Org. Let. 3, 1— 3. ； Torsten W., Yoshihiro N., Volk er S. and Joachim T., (1994), J. Org. Chem. 59, 6744— 6747 .; Yoshi hiro N., Torsten W., Volker S. and Joachim T., (1993), J. Am. Chem. Soc. 115, 2536— 2537. Yasuniro K., Hisashi K., Tsuyosni E., H ironobu H., (1998), Carbo. Res. 306, 361— 378.; Monica MP, (19 87 ), Carbo. Res. 159, 315— 324.; Yasuhiro K., Hironobu H. and His ashi K., (1992), Carbo. Res. 229, C5— C9.; Yasuhiro K., Hironobu H., Seiichiro O., (2000), Carbo. Res. 323, 44—48 .; Chi-Huey W., Yoshitaka I., Thomas K., Christine GN, David PD and Gary C. L., (1991), J. Am. Chem. Soc. 113, 8137-8145 .;) Both have been known to be tolerant of substrate modification to some extent even if not a native substrate. More recently, the results of X-ray crystal structure analysis have been reported (Louis NG, Christian C. and Yves B., (1999), EMBO. J. 18, 3546— 3557.,; B. R amakrishnan and Pradman KQ , (2001), J. Mol. Biol. 310, 205—21 8.), this enzyme binds to the substrate in a sequential order type reaction mechanism (Boopat hy R., Elizabeth Β. And Pradman KQ, (2002), Biochem. Biophys. Res. Comm. 291, 11 13— 1118.), it was confirmed that a large structural change was caused by the binding of a sugar donor (Boopathy et al., Supra; Ramakrishnan, PV Balajji and Pradman KQ, (2002), J. Mol. Biol. 318, 491-502 .;).
他の 6種類の |8 1 , 4 GalTについては未検証な部分が多い。アミノ酸配列全体の 相同性は低いものの、 β ΐ , 4— GalTlにおいて触媒活性に関わる特徴的な配列に ついてはよく保存されている（Christelle B. , Emmanuel B. , David Η. J. , R oberto A. G. and Anne I. ( 1998) , J. Biochem. 123, 1000—1009.、 ( 15 ) N. Lo, J. H. Shaper, J. Pevsner, N. L. Shaper, ( 1998) , Glycobiology, 8 , 517 - 526. )。現在までに考えられていることには、糖ドナーである UDP— Galの 結合については全ての 13 1 , 4 GalTについてほぼ同様で、糖ァクセプターの基質 特異性について大きな差があるものと考えられている（糖脂質を好むもの、ポリラクト サミンの合成に絡むもの、ラタトシルセラミドの合成を行うもの、プロテオダリカン糖鎖 のコア構造の合成に関わるもの等)。 [0075] (2) a l, 3-GalT The other six types | 8 1, 4 GalT have many unverified parts. Although the homology of the whole amino acid sequence is low, the characteristic sequences involved in catalytic activity in β ΐ, 4-GalTl are well conserved (Christelle B., Emmanuel B., David Η. J., R oberto AG and Anne I. (1998), J. Biochem. 123, 1000—1009., (15) N. Lo, JH Shaper, J. Pevsner, NL Shaper, (1998), Glycobiology, 8, 517-526.). What has been considered to date is that the binding of the sugar donor UDP-Gal is almost the same for all 13 1, 4 GalT and there is a large difference in the substrate specificity of the sugar acceptor. (Such as those that prefer glycolipids, those involved in the synthesis of polylactosamine, those that synthesize latatosylceramide, those involved in the synthesis of the core structure of the proteodalycan sugar chain). [0075] (2) al, 3-GalT
この酵素は、 UDp— Galから Galを α 1, 3—結合型で Ν—ァセチルラクトサミン (La cNAc)の非還元末端 Galに転移させる。ヒトの血液型抗原の合成酵素と、ブタおよび ゥシなどの Galili epitope と呼ばれる α Gal抗原を作るもの、イソグロボ系糖脂質 合成を行うものが知られている（T. Hennet, (2002) , Cell. Mol. Life Sci. 59, 1081— 1095. ) o Galili epitopeの合成酵素についてはすでに大腸菌の遺伝子 組み換えで生成する手法が確立されており、市販されている。基質類似物の転移活 性についての報告では、糖ドナーについてはおよそ |8 1, 4—結合型と類似した特異 性を持つと思われる（Keiko S. , Taketo U. , Ole Η. , Nina Ο. L. S. , War ren W. W. and Monica M. P. , (2000) , J. Am. Chem. Soc. 122, 1261 - 1269. )。これら a l, 3—結合型酵素はその立体選択性が |8 1, 4—結合型と異 なるが、酵素反応に重要な特徴的なアミノ酸配列は ι8 1— 4結合型酵素とよく類似し ており、立体構造は類似していると思われる（Christelle B.前出）。 X線結晶解析 の結果（Louis N. G. , Christophe B. , Anup Κ. Μ. , Ole Η. , Joel Η. S . and David H. J. , (2001) , ΕΜΒΟ J. 20, 638— 649. ； Ester B. , G. J. Swaminathan, Yingnan Z. , Ramanathan N. , Keith B. and K. R. Ach arya, (2001) , J. Biol. Chem. 276, 48608—48614. )力、らも j8 1, 4—型酵素と 同様、基質の結合によって大きな構造変化を起こすことが確認されている (Ester B . , Yingnan Z. , G. J. Swaminathan, Yingnan Z. , Keith B. and K. R. Acharya. , (2002) , J. Biol. Chem. 277, 28310— 28318.；)。  This enzyme transfers Gal from UDp-Gal to the non-reducing terminal Gal of α-acetyllactosamine (La cNAc) in α 1,3-linked form. It is known that a human blood group antigen synthase and an α-gal antigen called Galili epitope such as swine and sushi, and those that synthesize isoglobo-type glycolipids (T. Hennet, (2002), Cell Mol. Life Sci. 59, 1081— 1095.) o A method for producing a Galili epitope synthase by genetic recombination in E. coli has already been established and is commercially available. In the report on the transfer activity of substrate analogues, sugar donors appear to have a specificity approximately similar to | 8 1, 4—linked (Keiko S., Taketo U., Ole le., Nina Ο). LS, Warren WW and Monica MP, (2000), J. Am. Chem. Soc. 122, 1261-1269.). These al, 3-linked enzymes differ in stereoselectivity from the | 8 1,4-linked type, but the characteristic amino acid sequence important for the enzymatic reaction is very similar to the ι8 1-4 linked enzyme. The three-dimensional structure seems to be similar (Christelle B. supra). Results of X-ray crystallography (Louis NG, Christophe B., Anup Κ. Μ., Ole Η., Joel Η. S. and David HJ, (2001), ΕΜΒΟ J. 20, 638—649 .; Ester B. , GJ Swaminathan, Yingnan Z., Ramanathan N., Keith B. and KR Ach arya, (2001), J. Biol. Chem. 276, 48608—48614.) It has been confirmed that a large structural change is caused by the binding of the substrate (Ester B., Yingnan Z., GJ Swaminathan, Yingnan Z., Keith B. and KR Acharya., (2002), J. Biol. Chem. 277, 28310—28318 .;).
[0076] (3)フコース転移酵素  [0076] (3) Fucose transferase
フコース転移酵素は GDP— Fucを糖ドナーとして Fucを様々なァクセプターへ転 移する。その結合様式は以下に示すような α ΐ, 2—、 α ΐ, 3—、 α 1, 4一、 α 1, 6 一の結合型が知られ、それぞれ異なるサブタイプのフコース転移酵素が触媒反応を 行う（Becker, D. J. , Lowe, J. B. (2003) Glycobiology 13, 41R- 53R.；)。現 在までに物理学的な立体構造解析に関する報告 (NMR、 X線結晶構造など）はいか なる生物種、サブタイプについてもなされていない。サブタイプの中には高いアミノ酸 シークェンス相同性を示すものがある一方（Breton, C. , Oriol, R. , Imberty, A. (1998) Glycobiology 8, 87— 94. )、一般にァクセプター特異性は大きく 異なる。 Fucose transferase translocates Fuc to various acceptors using GDP-Fuc as a sugar donor. As shown below, α ΐ, 2-—, α ΐ, 3-—, α 1, 4 1, α 1, 6 1 are known, and different subtypes of fucose transferase are catalyzed. (Becker, DJ, Lowe, JB (2003) Glycobiology 13, 41R-53R .;). To date, no reports on physical three-dimensional structure analysis (NMR, X-ray crystal structures, etc.) have been made for any species or subtype. Some subtypes show high amino acid sequence homology (Breton, C., Oriol, R., Imberty, A. (1998) Glycobiology 8, 87— 94.), in general, the acceptor specificity is very different.
[0077] (4) a l, 2— FucT  [0077] (4) a l, 2— FucT
この酵素は GDP— Fucから Fucを α 1, 2—結合型で非還元末端のガラクトースに 転移させる。ヒトの血液型抗原及びその類縁糖鎖を合成することで知られている (Kel ly, R. J. , Rouquier, ¾. , U orgi, D. , Lennon, . . , and Lowe, J. B. (1 995) J. Biol. Chem. 270, 4640—4649.、 Larsen, R. D. , Ernst, L. K. , Nair, R. P. , and Lowe, J. B. (1990) Proc. Natl. Acad. Sci. USA. 87, 6 674- 6678.；)。  This enzyme transfers Fuc from GDP-Fuc to α1,2-linked non-reducing galactose. Known to synthesize human blood group antigens and related sugar chains (Kelly, RJ, Rouquier, ¾., U orgi, D., Lennon,.., And Lowe, JB (1 995) J Biol. Chem. 270, 4640—4649., Larsen, RD, Ernst, LK, Nair, RP, and Lowe, JB (1990) Proc. Natl. Acad. Sci. USA. 87, 6 674- 6678 .;) .
[0078] (5) a l, 3 -FucT 1, 4— FucT)  [0078] (5) a l, 3 -FucT 1, 4— FucT)
この酵素は GDP— Fucから Fucを a 1, 3—結合型で N—ァセチルラクトサミンの Gl cNAcに転移させる。この酵素は Lewis型抗原と呼ばれる糖鎖を作ることで、免疫反 応やがんの転移といった生体反応に関わることで知られる（Kaneko, M. , Kudo, T. , Iwasaki, Η. , Ikehara, Υ. , Nishihara, S. , Nakagawa, S. , Sasaki, Κ. , Shiina, Τ. , Inoko, Η. , Saitou, Ν. , and Narimatsu, Η. (1999) FEBS Lett. , 452, 237- 242.、 Natsuka, S. , and Lowe, J. B. (1994) Curr. Opin. Struct. Biol. , 4, 683— 691.、 Kannagi, R. (2002) Curr. Opin. Str uct. Biol. 12. 599 - 608.；)。  This enzyme transfers Fuc from GDP-Fuc to GlcNAc of N-acetyllactosamine in a 1,3-linked form. This enzyme is known to be involved in biological reactions such as immune response and cancer metastasis by creating a sugar chain called Lewis type antigen (Kaneko, M., Kudo, T., Iwasaki, Η., Ikehara, , Nishihara, S., Nakagawa, S., Sasaki, Κ., Shiina, Τ., Inoko, Η., Saitou, Ν., And Narimatsu, Η. (1999) FEBS Lett., 452, 237-242 Natsuka, S., and Lowe, JB (1994) Curr. Opin. Struct. Biol., 4, 683—691., Kannagi, R. (2002) Curr. Opin. Str uct. Biol. 12. 599- 608 .;).
[0079] (6) a l, 6— FucT  [0079] (6) a l, 6— FucT
この酵素は GDP— Fucから Fucを a 1, 6—結合型で N—型糖鎖の還元末端に存 在するァスパラギン結合 GlcNAcに転移させる（Miyoshi, E. , Noda, K. , Yamag uchi, Y. , Inoue, S. , Ikeda, Y. , Wang, W. , Ko, J. Η. , Uozumi, Ν. , Li, W. , and Taniguchi, Ν. (1999) Biochim. Biophys. Acta. 1473, 9— 20. ) 。このフコシルイ匕は多くの糖タンパク質糖鎖に見られ、肝臓癌の発生時などの腫瘍マ 一力一として知られる（Taketa, K. , Endo, Y. , Sekiya, C. , Tanikawa, K. , Κ oji, Τ. , Taga, Η. , Satomura, S. , Matsuura, S. , Kawai, Τ. , and Hirai, Η. (1993) Cancer Res. 53, 5419— 5423. , Hutchinson, W. L. , Du, M . Q. , Johnson, P. J. , and Williams, R. (1991) Hepatology 13, 683— 6 88. )0最近の研究では、このフコシルイ匕の増加が IgGlの抗体依存性細胞傷害活 性を抑制すること（Shields, R. L. , Lai, J. , Keck, R. , O ' Connell, L. Y. , Ho ng, K. , Meng, Y. G. , Weikert, S. H. , and Presta, L. G. (2002) J. Biol . Chem. 277, 26733— 26740.、 Shinkawa, T. , Nakamura, K. , Yamane, N. , Shoji— Hosaka, E. , Kanda, Y. , Sakurada, M. , Uchida, K. , Anaza wa, H. , Satoh, M. , Yamasaki, M. , Hanai, N. , and Shitara, K. (2003) J. Biol. Chem. 278, 3466— 3473. )、また上皮細胞増殖因子受容体による細 胞内シグナル伝達にも関与することが報告されている（Wang, X. , Ihara, H. , Mi yoshi, E. , Honke, K. , Taniguchi, N. , and Gu, J. (2005) J. Biol. Chem . Nov 29 ; [Epub ahead of print])。 a 1, 3フコース転移酵素ファミリ一は癌 化により高発現している。より詳細には、フコース転移酵素 VIIIに関連する疾患として は、例えば、肺気腫が挙げられ得る。がん患者において、ノックアウトすると増殖が抑 制されること、脾がん患者における血清では a 1, 6Fucosylィ匕ハプトグロビンが高率 で発見されること、肺気腫ではこの酵素が減っていることが明らかになつている。 This enzyme transfers Fuc from GDP-Fuc to the asparagine-linked GlcNAc present at the reducing end of the N-type sugar chain in a 1,6-linked form (Miyoshi, E., Noda, K., Yamaguchi, Y , Inoue, S., Ikeda, Y., Wang, W., Ko, J. Η., Uozumi, Ν., Li, W., and Taniguchi, Ν. (1999) Biochim. Biophys. Acta. 1473, 9— 20.) This fucosyl mushroom is found in many glycoprotein sugar chains and is known as the best tumor in the development of liver cancer (Taketa, K., Endo, Y., Sekiya, C., Tanikawa, K., , Taga, Η., Satomura, S., Matsuura, S., Kawai, Τ., And Hirai, Η. (1993) Cancer Res. 53, 5419— 5423., Hutchinson, WL, Du, M. Q., Johnson, PJ, and Williams, R. (1991) Hepatology 13, 683— 6 88.) 0 In a recent study, this increase in fucosyl moth suppresses antibody-dependent cytotoxic activity of IgGl (Shields, RL, Lai, J., Keck, R., O 'Connell, LY, Ho ng, K., Meng, YG, Weikert, SH, and Presta, LG (2002) J. Biol. Chem. 277, 26733— 26740., Shinkawa, T., Nakamura, K., Yamane, N., Shoji— Hosaka, E., Kanda, Y., Sakurada, M., Uchida, K., Anaza wa, H., Satoh, M., Yamasaki, M., Hanai, N., and Shitara, K. (2003) J Biol. Chem. 278, 3466— 3473.) and also reported to be involved in intracellular signaling by epidermal growth factor receptors (Wang, X., Ihara, H., Mi yoshi, E., Honke, K., Taniguchi, N., and Gu, J. (2005) J. Biol. Chem. Nov 29; [Epub ahead of print]). a 1,3 fucose transferase family 1 is highly expressed by canceration. More specifically, the disease associated with fucose transferase VIII may include, for example, emphysema. In cancer patients, knockout results in suppression of growth, sera in patients with spleen cancer show a high rate of a1,6Fucosyl 匕 haptoglobin, and emphysema has reduced levels of this enzyme It is summer.
[0080] (7) a 2, 3 - SiaT  [0080] (7) a 2, 3-SiaT
この酵素は、糖タンパク質のムチン型糖鎖ゃァスパラギン結合糖鎖およびスフイン ゴ糖脂質の非還元末端に位置するガラクトース残基に CMP シアル酸から α 2, 3 結合型でシアル酸を転移する。  This enzyme transfers sialic acid from CMP sialic acid to α2,3 linked form to the galactose residue located at the non-reducing terminus of glycoprotein mucin-type sugar chain, asparagine and glycosphingolipid.
[0081] (8) a 2, 6 - SiaT  [0081] (8) a 2, 6-SiaT
この酵素は、ゴルジ体にぉ ヽて糖タンパク質のムチン型糖鎖ゃァスパラギン結合糖 鎖、およびスフインゴ糖脂質の非還元末端に位置する、主にラタトースゃ Nァセチル ラタトサミンの末端ガラクトース残基に CMP シアル酸を糖ドナーとして α 2, 6 結 合型でシアル酸を転移する。  This enzyme is located on the non-reducing terminus of the glycoprotein mucin-type glycosylation chain rasparagine and glycosphingolipid on the Golgi body, mainly on the terminal galactose residue of ratatothose N-acetyl latatosamine. Transfers sialic acid in α 2,6 bond form using acid as sugar donor.
[0082] (有機化学）  [0082] (Organic Chemistry)
1つの局面では、本発明の化合物は、 (Α) - (Β) - (C)という模式図で表すことが できる。  In one aspect, the compound of the present invention can be represented by a schematic diagram of (Α)-(Β)-(C).
[0083] (Α)は、トリァゾールまたはォキシムを形成する能力を有する基 (例えば、アジド（― Ν )、アルデヒド置換基（ C ( = Ο)— R)またはアルデヒド（― CHO) )である。 [0084] (B)は、糖成分 (すなわち、糖またはその誘導体)を挙げることができる。好ましくは 、ターゲットとする糖転移酵素が基質とする糖またはその等価物であることが有利で ある。 [0083] (Α) is a group capable of forming triazole or oxime (for example, azide (—Ν), aldehyde substituent (C (= C) —R) or aldehyde (—CHO)). [0084] (B) can include a sugar component (that is, a sugar or a derivative thereof). Preferably, the target glycosyltransferase is advantageously a sugar as a substrate or an equivalent thereof.
[0085] (C)は、ヌクレオチドまたはその等価物を挙げることができる。  [0085] (C) may include nucleotides or their equivalents.
[0086] ここで（C)と（B)とは、スぺーサ一が入っていても直接結合していても良い。ここで、 スぺーサ一は (s)と表すことができ、 (A)部分と (B)部分とを結合する作用を有する 部分である。（S)は、（A)部分と (B)部分とが直接結合する場合はなくてもよい。好ま しくは、（S)としては、（A)部分および (B)部分のいずれかまたは好ましくは両方と、 それらの意図される機能を阻害しないような部分が用いられる。ここで、「スぺーサー（ 部分)」とは、 2つ以上の特徴的部分の間に適切な距離をもうけさせるために導入され る化学的部分をいう。そのような部分としては、例えば、アルキレン基、エーテル基、 エチレングリコール、ペプチド、脂質などが挙げられるがそれらに限定されない。当業 者は、上記特徴的部分に応じて、適切なスぺーサ一部分を選択および合成すること ができる。 [0086] Here, (C) and (B) may contain a spacer or may be directly coupled. Here, the spacer can be expressed as (s), and is a part having an action of coupling the part (A) and the part (B). (S) may not be present when the (A) part and the (B) part are directly bonded. Preferably, as (S), one or preferably both (A) part and (B) part and a part that does not inhibit their intended function are used. As used herein, “spacer” refers to a chemical moiety that is introduced to provide an appropriate distance between two or more characteristic parts. Examples of such a moiety include, but are not limited to, an alkylene group, an ether group, ethylene glycol, a peptide, and a lipid. A person skilled in the art can select and synthesize an appropriate spacer portion according to the above-mentioned characteristic portion.
[0087] 本明細書にぉ 、て「糖成分」とは、他の部分と結合し得るように水素が取れた形式 の基をいい、例えば、糖から 1つの水素が取れた形式の一価の基、糖から 2つの水素 が取れた形式の二価の基などを挙げることができる。糖成分としては、例えば、ダルコ —ス、ガラクトース、マンノース、フコース、キシロース、 N—ァセチルダルコサミン、 N —ァセチルガラタトサミン、シアル酸力も水素が二個取れたものを挙げることができる 。あるいは、糖成分は、二糖以上の糖鎖から 1つの水素が取れた形式の一価の基、 二糖以上の糖鎖から 2つの水素が取れた形式の二価の基などであってもよ 、。好ま しくは、以下の式：  [0087] As used herein, the term "sugar component" refers to a group in a form in which hydrogen is removed so that it can be combined with other moieties. For example, a monovalent form in which one hydrogen is removed from a sugar. And a divalent group in which two hydrogens are removed from a sugar. Examples of the sugar component include darcos, galactose, mannose, fucose, xylose, N-acetylyldarcosamine, N-acetylgalatatosamine, and sialic acid having two hydrogen atoms. Alternatively, the sugar component may be a monovalent group in which one hydrogen is removed from a sugar chain of two or more sugars, or a divalent group in which two hydrogens are removed from a sugar chain of two or more sugars. Yo ... Preferably, the following formula:
[化 41]
Figure imgf000071_0001
[Chemical 41]
Figure imgf000071_0001
で表される基が「糖成分」として好まし ヽ。 The group represented by is preferred as the “sugar component”.
好ましくは、 Cは、ァシル (好ましくはァセチル）であり得る。  Preferably C may be acil (preferably acetyl).
[0088] 好ましくは、 Cは、ヌクレオシドーリン酸またはヌクレオシドニリン酸であり得る。 [0088] Preferably, C may be nucleoside phosphate or nucleoside diphosphate.
[0089] 好ましくは、 Cは、ピリミジンヌクレオシドリン酸またはプリンヌクレオシドリン酸であり 得る。 [0089] Preferably, C may be a pyrimidine nucleoside phosphate or a purine nucleoside phosphate.
[0090] 好ましくはまた、 Cは、アデノシン、デォキシアデノシン、グアノシン，デォキシグアノ シン、シチジン、デォキシチミジン、チミジン（リボチミジン）、デォキシチミジンまたはゥ リジンあるいはそれらの改変体のホスフェートであり得る。ここで、この改変体は、機能 が阻害されない限り、任意の改変体を挙げることができる。例えば、そのような改変体 としては、アデノシン、デォキシアデノシン、グアノシン，デォキシグアノシン、シチジン 、デォキシチミジン、チミジン（リボチミジン）、デォキシチミジンまたはゥリジンにアルキ ル基が結合したもの、あるいは、他の以下に掲げる任意の置換基が結合したものを 挙げることができるがそれらに限定されない。「C」としては以下の式：  [0090] Preferably, C can also be a phosphate of adenosine, deoxyadenosine, guanosine, deoxyguanosine, cytidine, deoxythymidine, thymidine (ribothymidine), deoxythymidine or uridine or variants thereof. Here, as long as this function is not inhibited, this modification can mention arbitrary modifications. For example, such variants include adenosine, deoxyadenosine, guanosine, deoxyguanosine, cytidine, deoxythymidine, thymidine (ribothymidine), deoxythymidine or uridine, or other Examples include, but are not limited to, those in which any substituents listed in the above are bonded. “C” has the following formula:
[化 42]  [Chemical 42]
Figure imgf000071_0002
で示される基が好ましい。
Figure imgf000071_0002
Is preferred.
[0091] 好ましくは、 Bは、 D -ガラクトース、 L -フコース、シアル酸、 D- GlcNAc、 D-GalNAcゝ[0091] Preferably, B is D-galactose, L-fucose, sialic acid, D-GlcNAc, D-GalNAc ゝ
D-ManNAc, D-マンノース、もしくは D-グルコースまたはその誘導体であり得る。（B)It can be D-ManNAc, D-mannose, or D-glucose or a derivative thereof. (B)
- (C)の好ましい組み合わせとしては、 (a) - (y)、 (b) - (y)、 (c) - (y)、 (d) - (z)-Preferred combinations of (C) include (a)-(y), (b)-(y), (c)-(y), (d)-(z)
、 (e) - (y)、 (a) - (y)、および (f) - (x)が挙げられる。 , (E)-(y), (a)-(y), and (f)-(x).
[0092] さらに好ましくは、 (A) - (B) - (C)は、 [0092] More preferably, (A)-(B)-(C) is
[0093] [化 101] [0093] [Chemical 101]
Figure imgf000072_0001
Figure imgf000072_0001
[0094] [化 102]  [0094] [Chemical 102]
Figure imgf000072_0002
Figure imgf000072_0002
[0095] [化 103]  [0095] [Chemical 103]
Figure imgf000072_0003
Figure imgf000072_0003
[0096] [化 104]
Figure imgf000073_0001
Figure imgf000073_0002
Figure imgf000073_0003
Figure imgf000073_0004
[0096] [Chemical 104]
Figure imgf000073_0001
Figure imgf000073_0002
Figure imgf000073_0003
Figure imgf000073_0004
[0100] または [0100] or
[0101] [化 108] [0101] [Chemical 108]
Figure imgf000073_0005
Figure imgf000073_0005
[0102] あるいは [化 108- 1] [0102] or [Chemical 108-1]
Figure imgf000074_0001
Figure imgf000074_0001
に示すィ匕合物であり得る。 The compound shown in FIG.
特に、化合物  In particular, the compound
[化 108- 2] [Chemical 108-2]
Figure imgf000074_0002
Figure imgf000074_0002
とクリックケミストリーで合成された糖転移酵素阻害剤は、 a 2— 3シアル酸転移酵素 を特異的に阻害する。 And glycosyltransferase inhibitors synthesized by click chemistry specifically inhibit a 2-3 sialyltransferases.
本明細書において「ホスフェート」とは、リン酸力も水素がとれたものをさし、リン酸ェ ステル、リン酸塩またはイオンとして存在する。ホスフェートは、ホスフェート基が単数 または複数結合したものとして存在し得、例えば、一リン酸、二リン酸、三リン酸を含 み、具体的には、アデノシン、デォキシアデノシン、グアノシン，デォキシグアノシン、 シチジン、デォキシチミジン、チミジン（リボチミジン）、デォキシチミジンまたはゥリジン あるいはそれらの改変体のホスフェートなどを挙げることができる。アデノシンの二リン 酸は ADP、アデノシンの三リン酸は ATPとも称される。 [0104] 別の局面において、本発明の化合物は、（X) - (Y) - (B) - (C)という模式図 (X —Y—B— Cとも表す)で表すことができる。ここで、式中 Bは、糖成分であり得、 Cは、 ヌクレオチドであり得、 Xは、嵩高基であり、 Yは、— O— N =基または、— NH— N = 基または 1, 2, 3—トリアゾール基であり得る。 In the present specification, “phosphate” refers to a substance in which the phosphate power is also removed from hydrogen, and exists as a phosphate ester, phosphate or ion. The phosphate may exist as a single or multiple phosphate group, for example, including monophosphate, diphosphate, triphosphate, specifically adenosine, deoxyadenosine, guanosine, deoxy. Examples include xyguanosine, cytidine, deoxythymidine, thymidine (ribothymidine), deoxythymidine or uridine, or phosphates of these modifications. Adenosine diphosphate is also called ADP, and adenosine triphosphate is also called ATP. [0104] In another aspect, the compound of the present invention can be represented by a schematic diagram of (X)-(Y)-(B)-(C) (also expressed as X-Y-B-C). Wherein B can be a sugar component, C can be a nucleotide, X is a bulky group, Y is —O—N = group or —NH—N = group or 1, It can be a 2,3-triazole group.
[0105] ここで、 A、 Bおよび Cは、上記 (A) - (B) - (C)と同様の基を用いることができる。 Here, as A, B and C, the same groups as in the above (A)-(B)-(C) can be used.
[0106] 本明細書において「嵩高基」とは、立体障害を起こし得る能力を有する任意の基を いう。立体障害を起こしているかどうかは、コンピュータモデリングにより推測すること ができる。分子の立体配置の解析方法や原子間距離の精密な測定方法などにより、 核酸の立体配置が計算されるので、これらの結果に基づ!/、て立体障害を起こし得る 塩基の化学構造をデザインすることができる。このような立体障害は、ターゲットとなる 酵素が特定されれば、行うことができる。 X線結晶構造解析または NMR解析といった 実験により得られた構造 (PDB登録構造を含む）、または分子モデリングにより計算 から求めた予測構造を初期構 造とする。現在までは、小さなドメインを除き構造のほ とんどが X線結晶構造解析により得られて ヽる。構造解析できな ヽ膜存在型タンパク や、配列相同性が高 いファミリータンパクについてはホモロジ一モデリング法などの 分子モデリングにより予想構造を作製する。このようにして得られた構造はそのまま使 わず、 MD計算により動的な構造 や予想結合部位の鍵穴情報を分析する。薬物の 標的タンパクへの結合状態を予想し、構造変換のための更なる結合部位を探索する ことができる。 As used herein, “bulky group” refers to any group having the ability to cause steric hindrance. Whether or not a steric hindrance has occurred can be estimated by computer modeling. Nucleic acid configuration is calculated by molecular configuration analysis methods and precise measurement of interatomic distances, etc. Based on these results, the base chemical structure that can cause steric hindrance is designed. can do. Such steric hindrance can be performed if the target enzyme is identified. The initial structure is a structure obtained by an experiment such as X-ray crystal structure analysis or NMR analysis (including a PDB registered structure) or a predicted structure obtained by calculation through molecular modeling. To date, most of the structure has been obtained by X-ray crystallography except for small domains. For capsular proteins that cannot be structurally analyzed or for family proteins with high sequence homology, the predicted structure is prepared by molecular modeling such as homology modeling. The structure obtained in this way is not used as it is, but the dynamic structure and keyhole information of the expected binding site are analyzed by MD calculation. It is possible to predict the binding state of a drug to a target protein and to search for further binding sites for structure conversion.
[0107] 嵩高基としては、置換基を有して!/、てもよ 、アルキル、置換基を有して!/、てもよ！/、ァ ルケニル、置換基を有していてもよいアルキ -ル、置換基を有していてもよい炭素環 基、または置換基を有していてもよい複素環基を包含する。  [0107] The bulky group may have a substituent! /, May have an alkyl, or have a substituent! /, May have! /, Alkenyl, an alkyl which may have a substituent, a carbocyclic group which may have a substituent, or a heterocyclic group which may have a substituent.
[0108] このような嵩高基としては、例えば、  [0108] Examples of such bulky groups include:
[0109] [化 109A]
Figure imgf000076_0001
[0109] [Chemical 109A]
Figure imgf000076_0001
Η3Ν 0^ ^ 0Η 3 Ν 0 ^ ^ 0
+Η3Ν' '。^\。/\/0 、 + ^
Figure imgf000076_0002
+ Η 3 Ν ''. ^ \. / \ / 0 , + ^
Figure imgf000076_0002
[化 109B] [Chemical 109B]
Figure imgf000077_0001
Figure imgf000077_0001
[化 109C] [Chemical 109C]
Figure imgf000078_0001
Figure imgf000078_0001
[化 109D] [Chemical 109D]
Figure imgf000079_0001
Figure imgf000079_0001
[化 109E] [Chemical 109E]
Figure imgf000080_0001
Figure imgf000080_0001
[化 109G] [Chemical 109G]
Figure imgf000081_0001
Figure imgf000081_0001
[化 109H] [Chemical 109H]
Figure imgf000082_0001
Figure imgf000082_0002
Figure imgf000082_0003
Figure imgf000082_0001
Figure imgf000082_0002
Figure imgf000082_0003
Figure imgf000082_0004
Figure imgf000082_0005
Figure imgf000082_0004
Figure imgf000082_0005
[化 1091] [Chemical 1091]
Figure imgf000083_0001
Figure imgf000083_0001
ο〜0 0〜0--^0〜0^- - または ο ~ 0 0 ~ 0- ^ 0 ~ 0 ^--or
[0110] を挙げることができるがそれらに限定されない。 [0110] can be mentioned, but is not limited thereto.
[0111] 本明細書において、「芳香族 (官能)基」または「芳香環 (官能)基」とは、芳香族の 特性を有する環系化合物またはその部分を 、 、、一般に π電子が 4η+ 2個ある環 状共役系を含む安定な構造を有する。このような芳香族官能基は、他の芳香族官能 基とも相互作用することができる。芳香族官能基の例としては、例えば、ベンゼン環、 ナフタレン、アントラセン、フラン、ピリジン、ァズレン、シクロォクタテトラエンジァニォ ンなどが挙げられるがそれらに限定されない。芳香族官能基は、炭素環であってもよ くへテロ環であってもよい。また、芳香族官能基は、置換基 Rで置換されていてもよく 、「置換された芳香族官能基」とは、下記において選択される置換基で置換されてい る芳香族官能基を意味する。  [0111] In the present specification, "aromatic (functional) group" or "aromatic ring (functional) group" refers to a ring compound having an aromatic characteristic or a portion thereof, and generally π electrons are 4η + It has a stable structure including two cyclic conjugated systems. Such aromatic functional groups can also interact with other aromatic functional groups. Examples of the aromatic functional group include, but are not limited to, a benzene ring, naphthalene, anthracene, furan, pyridine, azulene, cyclooctatetradianion and the like. The aromatic functional group may be a carbocyclic ring or a heterocyclic ring. Further, the aromatic functional group may be substituted with a substituent R, and the “substituted aromatic functional group” means an aromatic functional group substituted with a substituent selected below. .
[0112] 本明細書において「アルコール」とは、脂肪族炭化水素の 1または 2以上の水素原 子をヒドロキシル基で置換した有機化合物をいう。本明細書においては、 ROHとも表 記される。ここで、 Rは、アルキル基である。好ましくは、 Rは、 C1 C6アルキルであり 得る。アルコールとしては、例えば、メタノール、エタノール、 1—プロパノール、 2—プ ロノ V—ルなどが挙げられるがそれらに限定されない。 In the present specification, “alcohol” refers to an organic compound in which one or more hydrogen atoms of an aliphatic hydrocarbon are substituted with a hydroxyl group. In this specification, it is also expressed as ROH. Here, R is an alkyl group. Preferably R is C1 C6 alkyl obtain. Examples of the alcohol include, but are not limited to, methanol, ethanol, 1-propanol, 2-propanol V-, and the like.
[0113] 本明細書において「アルキル」とは、メタン、ェタン、プロパンのような脂肪族炭化水 素（アルカン)力も水素原子が一つ失われて生ずる 1価の基をいい、一般に C H [0113] As used herein, "alkyl" refers to a monovalent group produced by loss of one hydrogen atom in an aliphatic hydrocarbon (alkane) force such as methane, ethane, or propane.
n 2n+ l 一で表される（ここで、 nは正の整数である)。アルキルは、直鎖または分枝鎖であり得 る。本明細書において「置換されたアルキル」とは、以下に規定する置換基によって アルキルの Hが置換されたアルキルをいう。これらの具体例は、 C1〜C2アルキル、 C1〜C3アルキル、 C1〜C4アルキル、 C1〜C5アルキル、 C1〜C6アルキル、 C1 〜C7アルキル、 C1〜C8アルキル、 C1〜C9アルキル、 C1〜C10アルキル、 Cl〜 C11アルキルまたは C1〜C12アルキル、 C1〜C2置換されたアルキル、 C1〜C3置 換されたアルキル、 C1〜C4置換されたアルキル、 C1〜C5置換されたアルキル、 C 1〜C6置換されたアルキル、 C1〜C7置換されたアルキル、 C1〜C8置換されたァ ルキル、 C1〜C9置換されたアルキル、 C1〜C10置換されたアルキル、 C1〜C11 置換されたアルキルまたは C1〜C 12置換されたアルキルであり得る。ここで、例えば C 1〜C 10アルキルとは、炭素原子を 1〜 10個有する直鎖または分枝状のアルキル を意味し、メチル（CH—）、ェチル（C H一）、 n—プロピル（CH CH CH—）、イソ  n 2n + l is represented by one (where n is a positive integer). Alkyl can be linear or branched. In the present specification, the “substituted alkyl” refers to an alkyl in which H of the alkyl is substituted by the substituent specified below. Specific examples of these include C1-C2 alkyl, C1-C3 alkyl, C1-C4 alkyl, C1-C5 alkyl, C1-C6 alkyl, C1-C7 alkyl, C1-C8 alkyl, C1-C9 alkyl, C1-C10 alkyl Cl-C11 alkyl or C1-C12 alkyl, C1-C2 substituted alkyl, C1-C3 substituted alkyl, C1-C4 substituted alkyl, C1-C5 substituted alkyl, C1-C6 substituted Alkyl, C1-C7 substituted alkyl, C1-C8 substituted alkyl, C1-C9 substituted alkyl, C1-C10 substituted alkyl, C1-C11 substituted alkyl or C1-C12 substituted Or alkyl. Here, for example, C 1 -C 10 alkyl means a linear or branched alkyl having 1 to 10 carbon atoms, such as methyl (CH—), ethyl (CH 1), n-propyl (CH CH CH—), iso
3 2 5 3 2 2 プロピル（（CH ) CH―)、 n—ブチル（CH CH CH CH―)、 n—ペンチル（CH C  3 2 5 3 2 2 Propyl ((CH) CH—), n-butyl (CH CH CH CH—), n-pentyl (CH C
3 2 3 2 2 2 3 3 2 3 2 2 2 3
H CH CH CH一）、 n—へキシル（CH CH CH CH CH CH一）、 n—ヘプチルH CH CH CH 1), n-hexyl (CH CH CH CH CH CH 1), n-heptyl
2 2 2 2 3 2 2 2 2 2 2 2 2 2 3 2 2 2 2 2
(CH CH CH CH CH CH CH― )、 n—ォクチル（CH CH CH CH CH CH C (CH CH CH CH CH CH CH—), n-octyl (CH CH CH CH CH CH C
3 2 2 2 2 2 2 3 2 2 2 2 23 2 2 2 2 2 2 3 2 2 2 2 2
H CH ―)、 n—ノニル（CH CH CH CH CH CH CH CH CH ―)、 n—デシルH CH ―), n-nonyl (CH CH CH CH CH CH CH CH CH ―), n-decyl
2 2 3 2 2 2 2 2 2 2 2 2 2 3 2 2 2 2 2 2 2 2
(CH CH CH CH CH CH CH CH CH CH一）、 C (CH ) CH CH CH (C (CH CH CH CH CH CH CH CH CH CH 1), C (CH) CH CH CH (C
3 2 2 2 2 2 2 2 2 2 3 2 2 23 2 2 2 2 2 2 2 2 2 3 2 2 2
H ) 、— CH CH (CH ) などが例示される。また、例えば、 C1〜C10置換されたァH), —CH 2 CH 2 (CH 2) and the like are exemplified. Also, for example, C1-C10 substituted key
3 2 2 3 2 3 2 2 3 2
ルキルとは、 C1〜C10アルキルであって、そのうち 1または複数の水素原子が置換 基により置換されているものをいう。 Rとしては、 C1〜C6アルキルが好ましぐ特に C 1〜C6アルキルが好まし!/、。  An alkyl refers to a C1-C10 alkyl having one or more hydrogen atoms replaced by a substituent. As R, C1-C6 alkyl is preferred, and C1-C6 alkyl is particularly preferred! /.
[0114] 本明細書において「シクロアルキル」とは、環式構造を有するアルキルをいう。「置換 されたシクロアルキル」とは、以下に規定する置換基によってシクロアルキルの Hが置 換されたシクロアルキルをいう。具体例としては、 C3〜C4シクロアルキル、 C3〜C5 シクロアルキル、 C3〜C6シクロアルキル、 C3〜C7シクロアルキル、 C3〜C8シクロ アルキル、 C3〜C9シクロアルキル、 C3〜C10シクロアルキル、 C3〜C11シクロアル キル、 C3〜C12シクロアルキル、 C3〜C4置換されたシクロアルキル、 C3〜C5置換 されたシクロアルキル、 C3〜C6置換されたシクロアルキル、 C3〜C7置換されたシク 口アルキル、 C3〜C8置換されたシクロアルキル、 C3〜C9置換されたシクロアルキ ル、 C3〜C10置換されたシクロアルキル、 C3〜C11置換されたシクロアルキルまた は C3〜C12置換されたシクロアルキルであり得る。例えば、シクロアルキルとしては、 シクロプロピル、シクロへキシルなどが例示される。 In this specification, “cycloalkyl” refers to alkyl having a cyclic structure. “Substituted cycloalkyl” refers to cycloalkyl in which H of cycloalkyl is substituted by a substituent specified below. Specific examples include C3-C4 cycloalkyl, C3-C5 Cycloalkyl, C3-C6 cycloalkyl, C3-C7 cycloalkyl, C3-C8 cycloalkyl, C3-C9 cycloalkyl, C3-C10 cycloalkyl, C3-C11 cycloalkyl, C3-C12 cycloalkyl, C3-C4 substituted Cycloalkyl, C3-C5 substituted cycloalkyl, C3-C6 substituted cycloalkyl, C3-C7 substituted cycloalkyl, C3-C8 substituted cycloalkyl, C3-C9 substituted cycloalkyl, It can be a C3-C10 substituted cycloalkyl, a C3-C11 substituted cycloalkyl, or a C3-C12 substituted cycloalkyl. For example, cycloalkyl is exemplified by cyclopropyl, cyclohexyl and the like.
[0115] 本明細書において「ァルケ-ル」とは、エチレン、プロピレンのような、分子内に二重 結合を一つ有する脂肪族炭化水素力 水素原子が一つ失われて生ずる 1価の基を いい、一般に C H 一で表される（ここで、 nは 2以上の正の整数である）。「置換さ [0115] In the present specification, "alkell" is a monovalent group such as ethylene or propylene that is generated by losing one hydrogen atom of an aliphatic hydrocarbon having one double bond in the molecule. , Generally expressed as CH 1 (where n is a positive integer greater than or equal to 2). "Replaced
n 2n_ l  n 2n_ l
れたァルケ-ル」とは、以下に規定する置換基によってァルケ-ルの Hが置換された ァルケ-ルをいう。具体例としては、 C2〜C3アルケ-ル、 C2〜C4アルケ-ル、 C2 〜C5アルケニル、 C2〜C6ァルケ-ル、 C2〜C7ァルケ-ル、 C2〜C8アルケ -ル、 C2〜C9ァルケ-ル、 C2〜C10ァルケ-ル、じ2〜じ11ァルケ-ルまたはじ2〜じ12 ァルケ-ル、 C2〜C3置換されたァルケ-ル、 C2〜C4置換されたァルケ-ル、 C2 〜C5置換されたァルケ-ル、 C2〜C6置換されたァルケ-ル、 C2〜C7置換された ァルケ-ル、 C2〜C8置換されたァルケ-ル、 C2〜C9置換されたァルケ-ル、 C2 〜C 10置換されたァルケ-ル、じ2〜じ11置換されたァルケ-ルまたはじ2〜じ12置 換されたァルケ-ルであり得る。ここで、例えば C2〜C10アルキルとは、炭素原子を 2〜： L0個含む直鎖または分枝状のァルケ-ルを意味し、ビュル (CH =CH— )、ァリ  “Alkal formed” refers to a alkal in which the H of the alkal is substituted by the substituent specified below. Specific examples include C2 to C3, C2 to C4, C2 to C5 alkenyl, C2 to C6, C2 to C7, C2 to C8, C2 to C9. , C2 to C10, 2 to 11 or 2 to 12, C2 to C3 substituted, C2 to C4 substituted, C2 to C5 Substituted alkell, C2-C6 substituted alkal, C2-C7 substituted alkal, C2-C8 substituted alkal, C2-C9 substituted alkal, C2 -C There can be 10 substituted alks, 2 to 11 substituted arks, or 2 to 12 substituted arks. Here, for example, C2 to C10 alkyl means a linear or branched alkaryl containing 2 to: L0 carbon atoms, such as bull (CH = CH—), ally
2  2
ル（CH =CHCH ―)、 CH CH = CH—などが例示される。また、例えば、 C2〜C  (CH 2 = CHCH —), CH 2 CH = CH— and the like. For example, C2 ~ C
2 2 3  2 2 3
10置換されたァルケ-ルとは、 C2〜C10ァルケ-ルであって、そのうち 1または複数 の水素原子が置換基により置換されて 、るものを 、う。  The 10-substituted alkenyl is a C2 to C10 alkal, in which one or more hydrogen atoms are substituted with a substituent.
[0116] 本明細書において「シクロアルケ-ル」とは、環式構造を有するァルケ-ルをいう。「 置換されたシクロアルケ-ル」とは、以下に規定する置換基によってシクロアルケ-ル の Hが置換されたシクロアルケ-ルをいう。具体例としては、 C3〜C4シクロアルケ- ル、 C3〜C5シクロアルケ-ル、 C3〜C6シクロアルケ-ル、 C3〜C7シクロアルケ- ル、 C3〜C8シクロアルケ-ル、 C3〜C9シクロアルケ-ル、 C3〜C10シクロアルケ -ル、 C3〜C11シクロアルケ-ル、 C3〜C12シクロアルケ-ル、 C3〜C4置換され たシクロアルケ-ル、 C3〜C5置換されたシクロアルケ-ル、 C3〜C6置換されたシク ロアルケ-ル、 C3〜C7置換されたシクロアルケ-ル、 C3〜C8置換されたシクロアル ケ -ル、 C3〜C9置換されたシクロアルケ-ル、 C3〜C10置換されたシクロアルケ- ル、じ3〜じ11置換されたシクロァルケ-ルまたはじ3〜じ12置換されたシクロァルケ -ルであり得る。例えば、好ましいシクロアルケ-ルとしては、 1—シクロペンテ-ル、 2—シクロへキセ-ルなどが例示される。 As used herein, “cycloalkenyl” refers to an alkke having a cyclic structure. “Substituted cycloalkenyl” refers to a cycloalkenyl in which H of the cycloalkenyl is substituted by the substituent specified below. Specific examples include C3-C4 cycloalkenyl, C3-C5 cycloalkell, C3-C6 cycloalkenyl, C3-C7 cycloalkenyl. C3 to C8 cycloalkenyl, C3 to C9 cycloalkenyl, C3 to C10 cycloalkenyl, C3 to C11 cycloalkenyl, C3 to C12 cycloalkenyl, C3 to C4 substituted cycloalkenyl, C3 to C5 Substituted cycloalkenyl, C3-C6 substituted cycloalkenyl, C3-C7 substituted cycloalkenyl, C3-C8 substituted cycloalkenyl, C3-C9 substituted cycloalkenyl, C3 It can be a C10 substituted cycloalkenyl, 3 to 11 substituted cycloalkyl or 3 to 12 substituted cycloalkyl. For example, preferred cycloalkenyls include 1-cyclopental, 2-cyclohexyl and the like.
[0117] 本明細書において「アルキニル」とは、アセチレンのような、分子内に三重結合を一 つ有する脂肪族炭化水素から水素原子が一つ失われて生ずる 1価の基を 、い、一 般に C H 一で表される（ここで、 nは 2以上の正の整数である）。「置換されたアル n 2n_3 [0117] In the present specification, "alkynyl" refers to a monovalent group formed by losing one hydrogen atom from an aliphatic hydrocarbon having one triple bond in the molecule, such as acetylene. Generally expressed as CH 1 (where n is a positive integer greater than or equal to 2). "Substituted al n 2n_3
キ -ル」とは、以下に規定する置換基によってアルキ-ルの Hが置換されたアルキ- ルをいう。具体例としては、 C2〜C3アルキ-ル、 C2〜C4アルキ-ル、 C2〜C5アル キ -ル、 C2〜C6アルキ-ル、 C2〜C7アルキ-ル、 C2〜C8アルキ -ル、 C2〜C9 アルキ -ル、 C2〜C10アルキ-ル、 C2〜C11アルキ-ル、 C2〜C12アルキニル、 C2〜C3置換されたアルキ-ル、 C2〜C4置換されたアルキ-ル、 C2〜C5置換され たアルキ-ル、 C2〜C6置換されたアルキ-ル、 C2〜C7置換されたアルキ-ル、 C 2〜C8置換されたアルキ-ル、 C2〜C9置換されたアルキ-ル、 C2〜C10置換され たアルキ-ル、 C2〜C11置換されたアルキ-ルまたは C2〜C12置換されたアルキ -ルであり得る。ここで、例えば、 C2〜C10アルキ-ルとは、例えば炭素原子を 2〜1 0個含む直鎖または分枝状のアルキ-ルを意味し、ェチュル（CH≡C—）、 1 プロ ピニル（CH C≡C )などが例示される。また、例えば、 C2〜C10置換されたアルキ  The term “alkyl” refers to an alkyl in which H of the alkyl is substituted by the substituent specified below. Specific examples include C2-C3 alkyl, C2-C4 alkyl, C2-C5 alkyl, C2-C6 alkyl, C2-C7 alkyl, C2-C8 alkyl, C2- C9 alkyl, C2 to C10 alkyl, C2 to C11 alkyl, C2 to C12 alkynyl, C2 to C3 substituted alkyl, C2 to C4 substituted alkyl, C2 to C5 substituted Alkyls, C2-C6 substituted alkyls, C2-C7 substituted alkyls, C2-C8 substituted alkyls, C2-C9 substituted alkyls, C2-C10 substituted Alkyl, C2-C11 substituted alkyl or C2-C12 substituted alkyl. Here, for example, C2 to C10 alkyl means, for example, a linear or branched alkyl containing 2 to 10 carbon atoms, such as ethur (CH≡C—), 1 propynyl ( CH C≡C) and the like are exemplified. Also, for example, C2-C10 substituted alkyl
3  Three
ニルとは、 C2〜C10アルキ-ルであって、そのうち 1または複数の水素原子が置換 基により置換されて 、るものを 、う。  Nyl refers to a C2 to C10 alkyl having one or more hydrogen atoms replaced by a substituent.
[0118] 本明細書において「アルコキシ」とは、アルコール類のヒドロキシ基の水素原子が失 われて生ずる 1価の基をいい、一般に C H O で表される（ここで、 nは 1以上の [0118] As used herein, "alkoxy" refers to a monovalent group formed by loss of a hydrogen atom of a hydroxy group of an alcohol, and is generally represented by C H O (where n is 1 or more).
n 2n+ l  n 2n + l
整数である)。「置換されたアルコキシ」とは、以下に規定する置換基によってアルコ キシの Hが置換されたアルコキシをいう。具体例としては、 C1〜C2アルコキシ、 C1 〜C3アルコキシ、 C1〜C4アルコキシ、 C1〜C5アルコキシ、 C1〜C6アルコキシ、 C 1〜C7アルコキシ、 C1〜C8アルコキシ、 C1〜C9アルコキシ、 C1〜C10アルコキシ 、 C1〜C11アルコキシ、 C1〜C12アルコキシ、 C1〜C2置換されたアルコキシ、 C1 〜C3置換されたアルコキシ、 C1〜C4置換されたアルコキシ、 C1〜C5置換されたァ ルコキシ、 C1〜C6置換されたアルコキシ、 C1〜C7置換されたアルコキシ、 C1〜C 8置換されたアルコキシ、 C1〜C9置換されたアルコキシ、 C1〜C10置換されたアル コキシ、 C1〜C11置換されたアルコキシまたは C 1〜C 12置換されたアルコキシであ り得る。ここで、例えば、 C1〜C10アルコキシとは、炭素原子を 1〜： L0個含む直鎖ま たは分枝状のアルコキシを意味し、メトキシ（CH O— )、エトキシ（C H O— )、 n—プ Is an integer). “Substituted alkoxy” refers to alkoxy in which H of alkoxy is substituted by the substituent specified below. Specific examples include C1-C2 alkoxy, C1 -C3 alkoxy, C1-C4 alkoxy, C1-C5 alkoxy, C1-C6 alkoxy, C1-C7 alkoxy, C1-C8 alkoxy, C1-C9 alkoxy, C1-C10 alkoxy, C1-C11 alkoxy, C1-C12 alkoxy, C1-C2 substituted alkoxy, C1-C3 substituted alkoxy, C1-C4 substituted alkoxy, C1-C5 substituted alkoxy, C1-C6 substituted alkoxy, C1-C7 substituted alkoxy, C1 It can be -C8 substituted alkoxy, C1-C9 substituted alkoxy, C1-C10 substituted alkoxy, C1-C11 substituted alkoxy or C1-C12 substituted alkoxy. Here, for example, C1-C10 alkoxy means linear or branched alkoxy containing 1 to L0 carbon atoms, and includes methoxy (CH 2 O—), ethoxy (CHO—), n— The
3 2 5  3 2 5
口ポキシ（CH CH CH O—）などが例示される。  Mouth poxy (CH CH CH O—) and the like are exemplified.
3 2 2  3 2 2
[0119] 本明細書にぉ 、て「炭素環基」とは、炭素のみを含む環状構造を含む基であって、 上記の「シクロアルキル」、「置換されたシクロアルキル」、 「シクロアルケ-ル」、「置換 されたシクロアルケ-ル」以外の基を 、う。炭素環基は芳香族系または非芳香族系で あり得、そして単環式または多環式であり得る。「置換された炭素環基」とは、以下に 規定する置換基によって炭素環基の Hが置換された炭素環基を 、う。具体例として は、 C3〜C4炭素環基、 C3〜C5炭素環基、 C3〜C6炭素環基、 C3〜C7炭素環基 、 C3〜C8炭素環基、 C3〜C9炭素環基、 C3〜C10炭素環基、 C3〜C11炭素環基 、 C3〜C12炭素環基、 C3〜C4置換された炭素環基、 C3〜C5置換された炭素環 基、 C3〜C6置換された炭素環基、 C3〜C7置換された炭素環基、 C3〜C8置換さ れた炭素環基、 C3〜C9置換された炭素環基、 C3〜C10置換された炭素環基、 C3 〜C11置換された炭素環基または C3〜C12置換された炭素環基であり得る。炭素 環基はまた、 C4〜C7炭素環基または C4〜C7置換された炭素環基であり得る。炭 素環基としては、フエニル基力 水素原子が 1個欠失したものが例示される。ここで、 水素の欠失位置は、化学的に可能な任意の位置であり得、芳香環上であってもよぐ 非芳香環上であってもよ 、。  In the present specification, the term “carbocyclic group” means a group containing a cyclic structure containing only carbon, and includes the above-mentioned “cycloalkyl”, “substituted cycloalkyl”, “cycloalkenyl”. And a group other than “substituted cycloalkenyl”. Carbocyclic groups can be aromatic or non-aromatic and can be monocyclic or polycyclic. The “substituted carbocyclic group” refers to a carbocyclic group in which H of the carbocyclic group is substituted by the substituent specified below. Specific examples include C3-C4 carbocyclic group, C3-C5 carbocyclic group, C3-C6 carbocyclic group, C3-C7 carbocyclic group, C3-C8 carbocyclic group, C3-C9 carbocyclic group, C3-C10. Carbocyclic group, C3-C11 carbocyclic group, C3-C12 carbocyclic group, C3-C4-substituted carbocyclic group, C3-C5-substituted carbocyclic group, C3-C6-substituted carbocyclic group, C3- C7 substituted carbocyclic group, C3-C8 substituted carbocyclic group, C3-C9 substituted carbocyclic group, C3-C10 substituted carbocyclic group, C3-C11 substituted carbocyclic group or C3 It can be a C12 substituted carbocyclic group. The carbocyclic group can also be a C4-C7 carbocyclic group or a C4-C7 substituted carbocyclic group. Examples of the carbon ring group include those in which one phenyl hydrogen atom is deleted. Here, the hydrogen deletion position may be any position chemically possible, whether on an aromatic ring or on a non-aromatic ring.
[0120] 本明細書にぉ 、て「ヘテロ環基」とは、炭素およびへテロ原子をも含む環状構造を 有する基をいう。ここで，ヘテロ原子は、 0、 Sおよび N力もなる群より選択され、同一 であっても異なっていてもよく、 1つ含まれていても 2以上含まれていてもよい。ヘテロ 環基は、芳香族系または非芳香族系であり得、そして単環式または多環式であり得 る。「置換されたへテロ環基」とは、以下に規定する置換基によってへテロ環基の Hが 置換されたへテロ環基をいう。具体例としては、 C3〜C4炭素環基、 C3〜C5炭素環 基、 C3〜C6炭素環基、 C3〜C7炭素環基、 C3〜C8炭素環基、 C3〜C9炭素環基 、 C3〜C10炭素環基、 C3〜C11炭素環基、 C3〜C12炭素環基、 C3〜C4置換さ れた炭素環基、 C3〜C5置換された炭素環基、 C3〜C6置換された炭素環基、 C3 〜C7置換された炭素環基、 C3〜C8置換された炭素環基、 C3〜C9置換された炭 素環基、 C3〜C10置換された炭素環基、じ3〜じ11置換された炭素環基またはじ3 〜C 12置換された炭素環基の 1つ以上の炭素原子をへテロ原子で置換したものであ り得る。ヘテロ環基はまた、 C4〜C7炭素環基または C4〜C7置換された炭素環基 の炭素原子を 1つ以上へテロ原子で置換したものであり得る。ヘテロ環基としては、 チェニル基、ピロリル基、フリル基、イミダゾリル基、ピリジル基などが例示される。水 素の欠失位置は、化学的に可能な任意の位置であり得、芳香環上であってもよぐ非 芳香環上であってもよい。 In the present specification, the term “heterocyclic group” refers to a group having a cyclic structure including carbon and heteroatoms. Here, the heteroatoms are selected from the group consisting of 0, S and N forces, and may be the same or different, and may be contained in one or more than one. Hetero Cyclic groups can be aromatic or non-aromatic and can be monocyclic or polycyclic. “Substituted hetero ring group” means a hetero ring group in which H of the hetero ring group is substituted by the substituent specified below. Specific examples include C3-C4 carbocyclic group, C3-C5 carbocyclic group, C3-C6 carbocyclic group, C3-C7 carbocyclic group, C3-C8 carbocyclic group, C3-C9 carbocyclic group, C3-C10. Carbocyclic group, C3-C11 carbocyclic group, C3-C12 carbocyclic group, C3-C4-substituted carbocyclic group, C3-C5-substituted carbocyclic group, C3-C6-substituted carbocyclic group, C3 -C7 substituted carbocyclic group, C3-C8 substituted carbocyclic group, C3-C9 substituted carbocyclic group, C3-C10 substituted carbocyclic group, 3-3-111 substituted carbocycle One or more carbon atoms of the group or the same 3- to C12-substituted carbocyclic group may be substituted with a heteroatom. A heterocyclic group can also be one in which one or more heteroatoms are substituted for the carbon atoms of a C4-C7 carbocyclic group or a C4-C7 substituted carbocyclic group. Examples of the heterocyclic group include a cetyl group, a pyrrolyl group, a furyl group, an imidazolyl group, and a pyridyl group. The hydrogen deletion position may be any position chemically possible, and may be on an aromatic ring or a non-aromatic ring.
[0121] 本明細書において、炭素環基またはへテロ環基は、下記に定義されるように 1価の 置換基で置換され得ることに加えて、 2価の置換基で置換され得る。そのような二価 の置換は、ォキソ置換 ( = O)またはチォキソ置換（ = S)であり得る。  [0121] In the present specification, a carbocyclic group or a heterocyclic group may be substituted with a divalent substituent in addition to being substituted with a monovalent substituent as defined below. Such divalent substitutions can be oxo substitution (= O) or thixo substitution (= S).
[0122] 本明細書にぉ 、て「ハロゲン」とは、周期表 7B族に属するフッ素 (F)、塩素（C1)、 臭素（Br)、ヨウ素（I)などの元素の 1価の基をいう。  [0122] As used herein, the term "halogen" refers to a monovalent group of an element such as fluorine (F), chlorine (C1), bromine (Br), iodine (I) belonging to Group 7B of the periodic table. Say.
[0123] 本明細書において「ヒドロキシ」とは、 OHで表される基をいう。「置換されたヒドロ キシ」とは、ヒドロキシの Hが下記で定義される置換基で置換されて 、るものを 、う。  As used herein, “hydroxy” refers to a group represented by OH. “Substituted hydroxy” refers to a hydroxy in which H is substituted with a substituent as defined below.
[0124] 本明細書において「チオール」とは、ヒドロキシ基の酸素原子を硫黄原子で置換し た基 (メルカプト基)であり、—SHで表される。「置換されたチオール」とは、メルカプト の Hが下記で定義される置換基で置換されて 、る基を 、う。  In this specification, “thiol” is a group in which an oxygen atom of a hydroxy group is substituted with a sulfur atom (mercapto group), and is represented by —SH. The term “substituted thiol” refers to a group in which H of mercapto is substituted with a substituent as defined below.
[0125] 本明細書において「シァノ」とは、—CNで表される基をいう。「ニトロ」とは、 -NO  In the present specification, “cyan” refers to a group represented by —CN. "Nitro" means -NO
2 で表される基をいう。「ァミノ」とは、 -NHで表される基をいう。「置換されたァミノ」と  The group represented by 2. “Amino” refers to a group represented by —NH. With "substituted amino"
2  2
は、ァミノの Hが以下で定義される置換基で置換されたものを 、う。  In which the amino H is substituted with a substituent as defined below.
[0126] 本明細書において「カルボキシ」とは、—COOHで表される基をいう。「置換された カルボキシ」とは、カルボキシの Hが以下に定義される置換基で置換されたものをいう In the present specification, “carboxy” refers to a group represented by —COOH. "Replaced `` Carboxy '' means a carboxy H substituted with a substituent as defined below.
[0127] 本明細書にぉ 、て「チォカルボキシ」とは、カルボキシ基の酸素原子を硫黄原子で 置換した基をいい、 C ( = S) OH、— C ( = 0) SHまたは— CSSHで表され得る。「 置換されたチォカルボキシ」とは、チォカルボキシの Hが以下に定義される置換基で 置換されたものをいう。 As used herein, “thiocarboxy” refers to a group in which the oxygen atom of a carboxy group is replaced by a sulfur atom, and C (═S) OH, —C (= 0) SH, or —SSH Can be represented. “Substituted thiocarboxy” refers to thiocarboxy H substituted with the substituents defined below.
[0128] 本明細書において「ァシル」とは、カルボン酸から OHを除いてできる 1価の基をいう 。ァシル基の代表例としては、ァセチル（CH CO )、ベンゾィル（C H CO )など  In the present specification, “acyl” refers to a monovalent group formed by removing OH from a carboxylic acid. Representative examples of the acyl group include acetyl (CH 2 CO 3), benzoyl (C 3 H 2 CO 3), etc.
3 6 5  3 6 5
が挙げられる。「置換されたァシル」とは、ァシルの水素を以下に定義される置換基で 置換したものをいう。  Is mentioned. “Substituted acyl” refers to a hydrogen substituted with the substituent defined below.
[0129] 本明細書にぉ 、て「アミド」とは、アンモニアの水素を酸基 (ァシル基)で置換した基 であり、好ましくは、 -CONH  In the present specification, the term “amide” refers to a group obtained by substituting hydrogen of ammonia with an acid group (acyl group), preferably —CONH
2で表される。「置換されたアミド」とは、アミドが置換され たものをいう。  Represented by 2. “Substituted amide” refers to a substituted amide.
[0130] 本明細書において「カルボ-ル」とは、アルデヒドおよびケトンの特性基である一（C  [0130] As used herein, "carbol" is a characteristic group of aldehydes and ketones (C
=o) を含むものを総称したものをいう。「置換されたカルボ-ル」は、下記におい て選択される置換基で置換されているカルボ二ル基を意味する。  = o) is a generic term for things including “Substituted carbonyl” means a carbonyl group substituted with a substituent selected as described below.
[0131] 本明細書において「アルデヒド」とは、「一 CHO」で表される基をいう。この基は、アミ ノ基と反応してォキシム基を形成する。本明細書にお!ヽて「置換されたアルデヒド基」 とは、一般に「一 CRO」（一 C ( = 0)—R)で表される基をいう。 Rとしては、下記にお いて選択される置換基を挙げることができる。この基もまた、通常アミノ基と反応して ォキシム基を形成する。  In the present specification, “aldehyde” refers to a group represented by “one CHO”. This group reacts with an amino group to form an oxime group. In the present specification, a “substituted aldehyde group” generally refers to a group represented by “one CRO” (one C (= 0) —R). Examples of R include substituents selected in the following. This group also usually reacts with an amino group to form an oxime group.
[0132] 本明細書にぉ 、て「チォカルボ-ル」とは、カルボニルにおける酸素原子を硫黄原 子に置換した基であり、特性基—（C = S)—を含む。チォカルボ-ルには、チオケト ンおよびチォアルデヒドが含まれる。「置換されたチォカルボ-ル」とは、下記におい て選択される置換基で置換されたチォカルボニルを意味する。  In the present specification, “thiocarbol” is a group in which an oxygen atom in carbonyl is substituted with a sulfur atom, and includes a characteristic group — (C═S) —. Thiocarbol includes thioketones and thioaldehydes. “Substituted thiocarbol” means thiocarbonyl substituted with a substituent selected as described below.
[0133] 本明細書において「スルホ -ル」とは、特性基である SO を含むものを総称し  [0133] As used herein, "sulfol" is a generic term for a substance containing SO which is a characteristic group.
2  2
たものをいう。「置換されたスルホ -ル」とは、下記において選択される置換基で置換 されたスルホ -ルを意味する。 [0134] 本明細書において「スルフィエル」とは、特性基である SO—を含むものを総称し たものをいう。「置換されたスルフィエル」とは、下記において選択される置換基で置 換されて!/、るスルフィエルを意味する。 Say something. “Substituted sulfol” means sulfol substituted with a substituent selected below. In this specification, “sulfier” refers to a generic term for a substance containing SO— which is a characteristic group. “Substituted sulfiel” means a sulfiel that is substituted with a substituent selected below!
[0135] 本明細書において「ァリ—ル」とは、芳香族炭化水素の環に結合する水素原子が 1 個離脱して生ずる基をいい、本明細書において、炭素環基に包含される。  In the present specification, the “aryl” refers to a group formed by leaving one hydrogen atom bonded to an aromatic hydrocarbon ring, and is included in the carbocyclic group in the present specification. .
[0136] 本明細書においては、特に言及がない限り、置換は、ある有機化合物または置換 基中の 1または 2以上の水素原子を他の原子または原子団で置き換えることをいう。 水素原子を 1つ除去して 1価の置換基に置換することも可能であり、そして水素原子 を 2つ除去して 2価の置換基に置換することも可能である。  [0136] In the present specification, unless otherwise specified, substitution refers to replacement of one or more hydrogen atoms in an organic compound or substituent with another atom or atomic group. One hydrogen atom can be removed and substituted with a monovalent substituent, and two hydrogen atoms can be removed and substituted with a divalent substituent.
[0137] 本発明の物質または上で定義した官能基が置換基 Rによって置換されている場合 、そのような置換基 Rは、単数または複数存在し、それぞれ独立して、水素、アルキル 、シクロアルキル、ァルケ-ル、シクロアルケ-ル、アルキ -ル、アルコキシ、炭素環 基、ヘテロ環基、ハロゲン、ヒドロキシ、チォ—ル、シァ入ニトロ、アミ入カルボキシ、 ァシル、チォカルボキシ、アミド、置換されたアミド、置換されたカルボ-ル、置換され たチォカルボ-ル、置換されたスルホ-ルおよび置換されたスルフィエルからなる群 より選択される。好ましくは、置換基 Rは、複数存在する場合それぞれ独立して、水素 、アルキルおよび置換されたアルキル力もなる群より選択され得る。より好ましくは、独 立して、 Rは、複数存在する場合それぞれ独立して、水素および C1〜C6アルキルか らなる群より選択され得る。 Rは、すべてが水素以外の置換基を有していても良いが、 好ましくは、少なくとも 1つの水素、より好ましくは、 2〜n (ここで nは Rの個数）の水素 を有し得る。置換基のうち水素の数が多いことが好ましくあり得る。大きな置換基また は極性のある置換基は本発明の効果 (特に、アルデヒド基との相互作用）に障害を有 し得る力らである。従って、水素以外の置換基としては、好ましくは、 C1〜C6アルキ ル、 C1〜C5アルキル、 C1〜C4アルキル、 C1〜C3アルキル、 C1〜C2アルキル、 メチルなどであり得る。ただし、本発明の効果を増強し得ることもあることから、大きな 置換基を有することもまた好ましくあり得る。さらにより好ましくは Rのすべてが水素で あり得る。  [0137] When the substance of the present invention or the functional group defined above is substituted by a substituent R, such substituent R may be present in one or more, each independently hydrogen, alkyl, cycloalkyl , Alcohol, cycloalkenyl, alkyl, alkoxy, carbocyclic group, heterocyclic group, halogen, hydroxy, thiol, silane-containing nitro, ami-containing carboxy, acyl, thiocarboxy, amide, substituted amide , Substituted carbo- yl, substituted thiocarbol, substituted sulfol, and substituted sulfiel. Preferably, the substituent R may be independently selected from the group consisting of hydrogen, alkyl, and substituted alkyl force, when there are multiple substituents R. More preferably, independently, when there are a plurality, R may be independently selected from the group consisting of hydrogen and C1-C6 alkyl. All of R may have a substituent other than hydrogen, but may preferably have at least one hydrogen, more preferably 2 to n (where n is the number of R). It may be preferred that the number of hydrogens in the substituent is large. A large substituent or a polar substituent is a force that may impair the effects of the present invention (particularly, interaction with an aldehyde group). Accordingly, the substituent other than hydrogen may preferably be C1-C6 alkyl, C1-C5 alkyl, C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, methyl and the like. However, since the effect of the present invention may be enhanced, it may be preferable to have a large substituent. Even more preferably, all of R may be hydrogen.
[0138] 本明細書において、 Cl、 C2、、、 Cnは、炭素数を表す。従って、 C1は炭素数 1個 の置換基を表すために使用される。 [0138] In the present specification, Cl, C2, ..., Cn represent the number of carbon atoms. Therefore, C1 has 1 carbon Used to represent the substituents of
[0139] 本明細書において、「光学異性体」とは、結晶または分子の構造が鏡像関係にあつ て、重ねあわせることのできない一対の化合物の一方またはその組をいう。立体異性 体の一形態であり、他の性質は同じであるにもかかわらず、旋光性のみが異なる。  [0139] In this specification, "optical isomer" refers to one or a pair of a pair of compounds that have a mirror-image relationship with a crystal or molecule and cannot be superimposed. It is a form of stereoisomer, and the other properties are the same, but only the optical rotation is different.
[0140] 本明細書においては、特に言及がない限り、置換は、ある有機化合物または置換 基中の 1または 2以上の水素原子を他の原子または原子団で置き換えることをいう。 水素原子を 1つ除去して 1価の置換基に置換することも可能であり、そして水素原子 を 2つ除去して 2価の置換基に置換することも可能である。  [0140] In this specification, unless otherwise specified, substitution refers to replacement of one or more hydrogen atoms in an organic compound or substituent with another atom or atomic group. One hydrogen atom can be removed and substituted with a monovalent substituent, and two hydrogen atoms can be removed and substituted with a divalent substituent.
[0141] 置換基としては、アルキル、置換されたアルキル、シクロアルキル、置換されたシク 口アルキル、ァルケ-ル、置換されたァルケ-ル、シクロアルケ-ル、置換されたシク ロアルケ-ル、アルキ -ル、置換されたアルキ -ル、アルコキシ、置換されたアルコキ シ、炭素環基、置換された炭素環基、ヘテロ環基、置換されたへテロ環基、ハロゲン 、ヒドロキシ、置換されたヒドロキシ、チオール、置換されたチオール、シァ入ニトロ、 アミ入置換されたアミ入カルボキシ、力ルバモイル、置換されたカルボキシ、ァシル 、ァシルァミノ、置換されたァシル、チォカルボキシ、置換されたチォカルボキシ、アミ ド、置換されたアミド、置換されたカルボ-ル、置換されたチォカルボ-ル、置換され たスルホ-ルまたは置換されたスルフィエルが挙げられるがそれらに限定されない。  [0141] Substituents include alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkell, substituted alkalk, cycloalkenyl, substituted cycloalkyl, alkyl- , Substituted alkyl, alkoxy, substituted alkoxy, carbocyclic group, substituted carbocyclic group, heterocyclic group, substituted heterocyclic group, halogen, hydroxy, substituted hydroxy, thiol , Substituted thiol, thio-substituted nitro, ami-substituted ami-substituted carboxy, strength rubamoyl, substituted carboxy, acyl, silylamino, substituted acil, thiocarboxy, substituted thiocarboxy, amide, substituted Amides, substituted carbols, substituted thiocarbols, substituted sulfols or substituted sulfiers. Be, but are not limited to them.
[0142] 上記の置換基が置換された基である場合は、そのさらなる置換のための置換基とし ては、アルキル、シクロアルキル、ァルケ-ル、シクロアルケ-ル、アルキ -ル、アルコ キシ、ハロゲン、ヒドロキシ、チオール、シァ入ニトロ、アミ入カルボキシ、力ルバモイ ル、ァシル、ァシルァミノ、チォカルボキシおよびアミドが挙げられる。  [0142] When the above substituent is a substituted group, examples of the substituent for the further substitution include alkyl, cycloalkyl, alkyl, cycloalkenyl, alkyl, alkoxy, halogen , Hydroxy, thiol, nitro-containing nitro, carboxy-amino, rubamoyl, acyl, acylamino, thiocarboxy and amide.
[0143] 本明細書にぉ 、て「保護反応」とは、 Boc (t—ブチルォキシカルボ-ル）のような保 護基を、保護が所望される官能基に付加する反応をいう。保護基により官能基を保 護することによって、より反応性の高い官能基の反応を抑制し、より反応性の低い官 能基のみを反応させることができる。  In the present specification, the “protection reaction” refers to a reaction in which a protective group such as Boc (t-butyloxycarboxyl) is added to a functional group desired to be protected. By protecting the functional group with a protecting group, the reaction of the functional group with higher reactivity can be suppressed, and only the functional group with lower reactivity can be reacted.
[0144] 本明細書にぉ ヽて「脱保護反応」とは、 Bocのような保護基を脱離させる反応を ヽぅ [0144] Throughout this specification, "deprotection reaction" refers to a reaction that removes a protecting group such as Boc.
。脱保護反応としては、 PdZCを用いる還元反応のような反応が挙げられる。 . Examples of the deprotection reaction include a reaction such as a reduction reaction using PdZC.
[0145] 本発明の各方法において、目的とする生成物は、反応液から夾雑物 (未反応減量 、副生成物、溶媒など)を、当該分野で慣用される方法 (例えば、抽出、蒸留、洗浄、 濃縮、沈澱、濾過、乾燥など）によって除去した後に、当該分野で慣用される後処理 方法 (例えば、吸着、溶離、蒸留、沈澱、析出、クロマトグラフィーなど)を組み合わせ て処理して単離し得る。 [0145] In each method of the present invention, the target product is contaminated from the reaction solution (unreacted weight loss). , By-products, solvents, etc.) after removal by methods commonly used in the art (eg extraction, distillation, washing, concentration, precipitation, filtration, drying, etc.) For example, it can be isolated by a combination of adsorption, elution, distillation, precipitation, precipitation, chromatography, etc.).
[0146] 本明細書のアジド糖は、目的とする糖を、 Christian Vogel, Claudia Bergemann, An drej— Jakob Ott, ThisbeK. Lindhorst, Joacnim Theim, Wilhelm V. Dahlhoff, Chris terHallgren, MonicaM. Palcic and Ole Hindsgaul, Liebigs Ann. (1997) 6(Π -612など を参照して、 t-ブチルジメチルシリル (TBDMS)基およびトシル基を導入し、トシル基 を DMFなどの非極性有機溶媒中で NaNと反応することによって、アジド基導入し、  [0146] The azido sugar of the present specification refers to the target sugar as Christian Vogel, Claudia Bergemann, An drej—Jakob Ott, Thisbe K. Lindhorst, Joacnim Theim, Wilhelm V. Dahlhoff, Chris terHallgren, Monica M. Palcic and Ole Hindsgaul. , Liebigs Ann. (1997) 6 (Refer to 612-612 etc. to introduce t-butyldimethylsilyl (TBDMS) group and tosyl group, and tosyl group reacts with NaN in nonpolar organic solvents such as DMF. By introducing an azido group,
3  Three
その後、 TBDMS基を脱離させ環化させて、作製することができる。必要に応じてァ ジド糖は、ァセチル化させることができる。  Thereafter, the TBDMS group can be eliminated and cyclized. If necessary, the azide sugar can be acetylated.
[0147] 模式的なアジド糖合成例としてアジドフコースの例を以下に記載する。 [0147] An example of azidofucose is described below as a schematic example of azido sugar synthesis.
[0148] [化 110] [0148] [Chem 110]
Figure imgf000092_0001
Figure imgf000092_0001
[0149] ー且アジド糖が得られたら、これをァセチル化保護し、ハロ化して、ハロ化した部分 にリン酸基を導入する。ァセチル化は、ピリジン中で無水酢酸をカ卩えることによって行 われる。ノ、口化は、例えば、有機溶媒 (例えば、塩化メチレン—酢酸ェチル 10 : 1)中 でハロゲンィ匕チタン (例えば、 TiBr )をカ卩えて行うことができる。リン酸基の導入は、 [0149] Once the azido sugar is obtained, it is protected by acetylation, and then halolated, and a phosphate group is introduced into the halogenated portion. Acetylation is performed by covering acetic anhydride in pyridine. Is called. The vaporization can be performed, for example, by adding halogen titanium (eg, TiBr) in an organic solvent (eg, methylene chloride-ethyl acetate 10: 1). The introduction of phosphate groups
4  Four
有機溶媒（トルエン—シアン化メチルなど）中でテトラプチルアンモ -ゥムホスフェート を加え、その後メタノール中などで水酸ィ匕アンモ-ゥムをカ卩えることによって達成する ことができる。  This can be achieved by adding tetraptylammonium phosphate in an organic solvent (toluene-methyl cyanide, etc.) and then covering the ammonium hydroxide in methanol or the like.
[0150] このようにして得られたアジド糖ホスフェートに、 GMPモルホリデートおよび 1Hテト ラゾールを、ピリジン中で加え、 Dowex50 X 8などによって精製することによって、 目 的とする本発明の化合物を得ることができる。以下にその例示スキームを掲げる。  [0150] The target compound of the present invention can be obtained by adding GMP morpholidate and 1H tetrazole in pyridine to the azido sugar phosphate thus obtained and purifying it with Dowex50 X8 or the like. it can. The example scheme is listed below.
[0151] [化 111]  [0151] [Chem 111]
tctra -butyl ammoni tctra -butyl ammoni
Toluene-  Toluene-
1) GMP-mo 1) GMP-mo
1H -tetr  1H -tetr
I  I
2) Dowex 2) Dowex
Figure imgf000093_0001
Figure imgf000093_0001
[0152] さらに、本発明のアジド化合物またはアルデヒドもしくはアルデヒド誘導体ィ匕合物を 用いて、糖転移酵素阻害活性を有する本発明の化合物を作製するには、ァスコルビ ン酸、硫酸銅の存在下で水中で、アルキレン基またはアミノ基を有する嵩高基を提供 する化合物またはその前駆体を加えることによって達成することができる。以下にそ の例示スキームを掲げる。 [0153] [化 112] [0152] Furthermore, in order to produce the compound of the present invention having glycosyltransferase inhibitory activity using the azide compound or the aldehyde or aldehyde derivative compound of the present invention, in the presence of ascorbic acid and copper sulfate. This can be accomplished by adding a compound or precursor thereof that provides a bulky group with an alkylene or amino group in water. Below is an example scheme. [0153] [Chem 112]
Figure imgf000094_0001
Figure imgf000094_0001
Inihibitor  Inihibitor
[0154] (スクリーニング）  [0154] (Screening)
本明細書において、「スクリーニング」とは、目的とするある特定の性質をもつ物質ま たは生物などを、特定の操作および Zまたは評価方法で多数の候補力 選抜するこ とをいう。本明細書では、酵素、ァクセプター、ドナー等の成分、およびシステム、糖 鎖アレイなどを用いることによって、スクリーニングを行うことができる。スクリーニング は、インビトロ、インビボなど実在物質を用いた系を使用してもよぐインシリコ（コンビ ユータを用いた系）の系を用いて生成されたライブラリーを用いてもよい。本発明では 、所望の活性を有するスクリーニングによって得られた化合物もまた、本発明の範囲 内に包含されることが理解される。また本発明では、本発明の開示をもとに、コンビュ ータモデリングによる薬物が提供されることも企図される。  In this specification, “screening” refers to selecting a large number of candidates for a target substance or organism having a certain specific property by a specific operation and Z or evaluation method. In the present specification, screening can be performed by using components such as enzymes, acceptors, donors, and systems, sugar chain arrays, and the like. For the screening, a library generated using an in silico (system using a computer) system using a real substance such as in vitro or in vivo may be used. In the present invention, it is understood that compounds obtained by screening having a desired activity are also included within the scope of the present invention. The present invention also contemplates providing a drug by means of computer modeling based on the disclosure of the present invention.
[0155] (コンビナトリアルケミストリ）  [0155] (Combinatorial chemistry)
本明細書において「コピナトリアルケミストリ」とは、合成反応を素反応に分解して組 み合わせることにより、同時に多数かつ多様な化合物を合成する技術をいう。 Merrif ield (米国）の「ペプチド固相合成法」（1963年)を基礎とした自動合成化技術の進展 と、 Furka (ノヽンガリー）らの「スプリット合成法」（1991年)の技術などに端を発し、種 々の技術が周知となっており、当業者は、適宜本発明に適用することができる。  As used herein, “copinatorial chemistry” refers to a technology for simultaneously synthesizing a large number and variety of compounds by decomposing and combining synthetic reactions into elementary reactions. The development of automatic synthesis technology based on Merrif ield's (US) "Peptide Solid Phase Synthesis Method" (1963) and Furka's "Split Synthesis Method" (1991). Various techniques are well known, and those skilled in the art can appropriately apply the present invention.
[0156] 本明細書において使用される「スプリット法」では、ビーズなどの固体支持体上にリ ンカーと呼ばれる有機合成の足場となる官能基が結合されることによって反応が信仰 する。このリンカ一には、ビルディングブロックという単位構造が連鎖的に結合される。 各反応の前にビーズを数等分 (スプリット）し、異なるビルディングブロックを結合させ た後，再び 1つの容器に戻す。 Z等分のスプリットとプールを n回繰り返すことにより、 Z X n種類の化合物力 なるライブラリーが得られる。反応を個々のスプリットごとに行う ことでビルディングブロックの反応性の違 ヽを調節し、均一なライブラリーを得ることが できる。この方法により、できるだけ多くの種類の化合物力もなるライブラリーを得るこ とがでさる。 [0156] In the "split method" used in the present specification, a reaction is believed by bonding a functional group that becomes a scaffold for organic synthesis called a linker on a solid support such as a bead. To do. Unit structures called building blocks are linked to this linker in a chain. Before each reaction, divide the beads into several equal parts (split), combine the different building blocks, and place them back in one container. By repeating Z splits and pooling n times, a library with ZX n kinds of compounds can be obtained. By performing the reaction for each individual split, the difference in the reactivity of the building blocks can be adjusted, and a uniform library can be obtained. By this method, it is possible to obtain a library having as many kinds of compound powers as possible.
[0157] 本明細書において「液相合成」は、固相合成の代わりに液相を用いて混合物ライブ ラリーを液相合成で作成する方法をいう。液相合成には、例えば、同じ反応条件で反 応するビルディングブロックの混合物を利用して反応を行う混合物合成法がある。あ るいは、スプリット合成のビーズを可溶性ポリマーに置き換えた、 LPCS法もまた使用 可能である。この方法は、溶媒からの回収に捕捉剤を利用することで、スプリット合成 と同じようにライブラリーを作製することができる。  In this specification, “liquid phase synthesis” refers to a method of preparing a mixture library by liquid phase synthesis using a liquid phase instead of solid phase synthesis. Liquid phase synthesis includes, for example, a mixture synthesis method in which a reaction is performed using a mixture of building blocks that react under the same reaction conditions. Alternatively, the LPCS method, in which split synthetic beads are replaced with soluble polymers, can also be used. In this method, a library can be prepared in the same manner as split synthesis by using a capture agent for recovery from a solvent.
[0158] 本明細書にぉ 、て「パラレル合成」とは、位置を定めたレジスター（ゥエルやピン、チ ップまたは膜上の小区画)上で反応を行う方法をいう。この方法では、個々のレジスタ 一で通常の有機合成が行われる。必要な化合物の種類を得るためには相当数のレ ジスターを調製する必要がある。固相合成では通常ピンを用いることが多いが、より 多くの化合物を同じように合成するために、チップ型の合成法が開発されている。ノ ラレル合成の組合せにより、スプリット法同様多くの種類の化合物が合成可能である 。 ノラレル合成は、単一化合物力もなるライブラリーが得られるため、ライブラリーの再 現性や構造確認などの面で優れて ヽる。  [0158] As used herein, "parallel synthesis" refers to a method of performing a reaction on a defined register (well, pin, chip, or small section on a membrane). In this method, normal organic synthesis takes place in each individual register. In order to obtain the type of compound required, it is necessary to prepare a considerable number of registers. In solid phase synthesis, pins are usually used, but chip-type synthesis methods have been developed to synthesize more compounds in the same way. As with the split method, many types of compounds can be synthesized by combining normal synthesis. Normalel synthesis is excellent in terms of library reproducibility and structure confirmation, because a library with a single compound strength can be obtained.
[0159] 本明細書においてコンビナトリアルライブラリの調製には、「自動合成装置 (合成口 ボット）」を用いることができる。 自動合成装置は、ノ ラレル合成を集積ィ匕することによ つて、 自動合成を行う。このことにより、スプリット合成に較べ同時に作られる化合物数 が限られるという欠点を克服することができる。  [0159] In the present specification, an "automatic synthesizer (synthetic bot)" can be used for preparing a combinatorial library. The automatic synthesizer performs automatic synthesis by integrating normal synthesis. This overcomes the disadvantage that the number of compounds produced simultaneously is limited compared to split synthesis.
[0160] (化合物設計）  [0160] (Compound design)
従って、本発明は、分子設計技術の使用により、酵素活性調節因子 (阻害化合物 を含む)を効率よく同定、選択、および設計することを可能にする。従って、本発明で は、本発明の開示をもとに、コンピュータモデリングによる薬物が提供されることも企 図される。 Thus, the present invention enables the efficient identification, selection and design of enzyme activity modulators (including inhibitory compounds) through the use of molecular design techniques. Therefore, in the present invention Based on the disclosure of the present invention, it is also contemplated that a drug by computer modeling is provided.
[0161] コンピュータモデリングを行うためのコンピュータープログラムもまた、化学物質を設 計または選択するプロセスにおいて使用され得る。このようなプログラムとしては、以 下が挙げられる。  [0161] Computer programs for performing computer modeling can also be used in the process of designing or selecting chemicals. The following are examples of such programs.
[0162] 1. GRID (P. J. Goodford, 「A Computational Procedure for Determini ng Energetically Favoraole Binding Sites on Biologically Important [0162] 1. GRID (P. J. Goodford, "A Computational Procedure for Determining Energetically Favoraole Binding Sites on Biologically Important
MacromoleculesJ , J. Med. Chem. , 28, 849— 857頁（1985) )。 GRIDは、 Oxford University, Oxford, UKから入手可能である。 Macromolecules J, J. Med. Chem., 28, 849—page 857 (1985)). GRID is available from Oxford University, Oxford, UK.
[0163] 2. MCSS (A. Mirankerら， 「Functionality Maps of Binding Sites : A [0163] 2. MCSS (A. Miranker et al., “Functionality Maps of Binding Sites: A
Multiple Copy Simultaneous Search MethodJ Proteins : Structure, F unction and Genetics, 11, 29— 34頁（1991) )。 MCSSは、 Molecular Sim ulations, San Diego, CAから入手可能である。 Multiple Copy Simultaneous Search Method Proteins: Structure, Function and Genetics, 11, 29-34 (1991)). MCSS is available from Molecular Simulations, San Diego, CA.
[0164] 3. AUTODOCK(D. S. Goodsellら， 「Automated Docking of Substrate s to Proteins by Simulated AnnealingJ , Proteins : Structure, Function , and Genetics, 8, 195— 202頁（1990) )。 AUTODOCKは、 Scripps Resea rch Institute, La Jolla, CAから入手可能である。  [0164] 3. AUTODOCK (DS Goodsell et al., “Automated Docking of Substrates to Proteins by Simulated Annealing J, Proteins: Structure, Function, and Genetics, 8, 195—202 (1990)). AUTODOCK is a Scripps Research Institute. , Available from La Jolla, CA.
[0165] 4. DOCK (I. D. Kuntzら， 「A Geometric Approach to Macromolecule  [0165] 4. DOCK (I. D. Kuntz et al., “A Geometric Approach to Macromolecule
-Ligand InteractionsJ , J. Mol. Biol. , 161, 269— 288頁（1982) )。 DOCK は、 University of California, San Francisco, CA力ら入手可會である。  -Ligand Interactions J, J. Mol. Biol., 161, 269—288 (1982)). DOCK is available from the University of California, San Francisco, CA.
[0166] 一旦、適切な化合物が選択されると、それらは、単一化合物または複合体にァセン プリすることができる。構築に先だって、対象となる酵素の構造座標に関連してコンビ ユーター画面上に表示される 3次元イメージ上で、互いのフラグメントの関連性の視 覚的検査が行われ得る。これに続き、 Quantaまたは Sybyl [Tripos Associates, St. Louis, MO]のようなソフトウェアを用いるマ-ユアルでのモデル構築が行われ 得る。  [0166] Once the appropriate compounds are selected, they can be assembled into a single compound or complex. Prior to construction, a visual inspection of each fragment's relevance can be performed on a 3D image displayed on the computer screen in relation to the structural coordinates of the target enzyme. This can be followed by model building in a manual using software such as Quanta or Sybyl [Tripos Associates, St. Louis, MO].
[0167] 個々の化合物またはその部分を連結させる際に使用され得る有用なプログラムは、 以下を含む。 [0168] 1. CAVEAT (P. A. Bartlettら、「CAVEAT:A Program to Facilitate th e Structure― Derived Design of Biologically Active MoleculesJ (Mol ecular Recognition in Chemical and Biological Problems, Special Pu b. , Royal Chem. Soc. , 78, 182— 196頁（1989) )； G, Lauriおよび P. A. Ba rtlett, 「CAVEAT: a Program to Facilitate the Design of Organic M oleculesj , J. Comput. Aided Mol. Des. , 8, 51— 66頁（1994) )。 CAVEAT は、 University of California, Berkeley, CAから入手可能である。 [0167] Useful programs that can be used in linking individual compounds or portions thereof include: [0168] 1. CAVEAT (PA Bartlett et al., "CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active Molecules J (Molecular Recognition in Chemical and Biological Problems, Special Pu b., Royal Chem. Soc., 78 , 182-196 (1989)); G, Lauri and PA Bartlett, "CAVEAT: a Program to Facilitate the Design of Organic Moleculesj, J. Comput. Aided Mol. Des., 8, 51-66 (1994). CAVEAT is available from the University of California, Berkeley, CA.
[0169] 2. ISIS (MDL Information Systems, San Leandro, CA)のような 3Dデー タベースシステム 。この分野は、 Y. C. Martin, 「3D Database Searching in Drug Design」，J. Med. Chem. , 35, 2145— 2154頁（1992)【こお!/、て概説 される。  [0169] 2. 3D database systems such as ISIS (MDL Information Systems, San Leandro, CA). This field is reviewed by Y. C. Martin, “3D Database Searching in Drug Design”, J. Med. Chem., 35, 2145–2154 (1992).
[0170] 3. HOOK(M. B. Eisenら， 「HOOK:A Program for Finding Novel Mo lecular Architectures that Satisfy the Chemical and Steric Require ments of a Macromolecule Binding Site」, Proteins : Struct. , Funct. , Genet. , 19, 199— 221頁（1994) )。 HOOKは、 Molecular Simulations, Sa n Diego, CAから入手可能である。  [0170] 3. HOOK (MB Eisen et al., "HOOK: A Program for Finding Novel Molecular Architectures that Satisfy the Chemical and Steric Requirements of a Macromolecule Binding Site", Proteins: Struct., Funct., Genet., 19, 199-221 (1994)). HOOK is available from Molecular Simulations, San Diego, CA.
[0171] 上記のように一度に 1つの化合物またはその部分を段階的様式で対象となる酵素 の阻害因子などとして構築することを進める代わりに、阻害性または他の酵素結合ィ匕 合物は、空の結合部位を使用する力、または必要に応じていくつかの既知の阻害因 子の部分を含める方法などにより、分子全体あるいは全く新規に化合物を設計するこ とができる。当該分野において、以下のような多くの新規リガンド設計方法が知られて いる。  [0171] Instead of proceeding to construct one compound or part thereof at a time as an inhibitor of the target enzyme, etc. in a stepwise manner as described above, an inhibitory or other enzyme binding compound is Compounds can be designed as whole molecules or entirely new, such as by the ability to use empty binding sites or, where necessary, include portions of several known inhibitors. Many novel ligand design methods are known in the art as follows.
[0172] 1. LUDI (H. — J. Bohm, 「The Computer Program LUDI :A New Met hod for the De Novo Design of Enzyme InhibitorsJ , J. Comp. Aid. [0172] 1. LUDI (H. — J. Bohm, “The Computer Program LUDI: A New Met hod for the De Novo Design of Enzyme InhibitorsJ, J. Comp. Aid.
Molec. Design, 6 61— 78頁（1992) )。 LUDIは、 Molecular Simulations I ncorporated, San Diego, CA力ら入手可會である。 Molec. Design, 6 61—78 (1992)). LUDI is available from Molecular Simulations Incorporated, San Diego, CA.
[0173] 2. LEGEND (Y. Nishibataら， Tetrahedron, 47, 8985頁（1991) )。 LEGEN[0173] 2. LEGEND (Y. Nishibata et al., Tetrahedron, 47, 8985 (1991)). LEGEN
Dは、 Molecular Simulations Incorporated, San Diego, CA力ら入手可會 である。 D is available from Molecular Simulations Incorporated, San Diego, CA It is.
[0174] 3. LeapFrog (Tripos Associates, St. Louis, MOから入手可能である）。  [0174] 3. LeapFrog (available from Tripos Associates, St. Louis, MO).
[0175] 4. SPROUT (V. Gilletら， 「SPROUT:A Program for Structure Genera tion」， J. Comput. Aided Mol. Design, 7, 127— 153頁（1993) )。 SPROUT は、 University of Leeds, UKから入手可能である。  [0175] 4. SPROUT (V. Gillet et al., “SPROUT: A Program for Structure Generation”, J. Comput. Aided Mol. Design, 7, 127-153 (1993)). SPROUT is available from the University of Leeds, UK.
[0176] 他の分子モデリング技術もまた、本発明に従って使用され得る [例えば、 N. C. Co henら, 「Molecular Modeling Software and Metnods for Medicinal C hemistryj , J. Med. Chem. , 33, 883— 894頁（1990)を参照のこと； M. A. Na viaおよび M. A. Murcko, 「The Use of Structural Information in Drug DesignJ , Current Opinions in Structural Biology, 2, 202— 210頁 (19 92)もまた参照のこと； L. M. Balbesら， 「A Perspective of Modern Method s in Computer― Aided Drug DesignJ , (Reviews in Computational C hemistry, vol. 5, K. B. Lipkowitzおよび D. B. Boyd編， VCH, New York, 3 37— 380頁（1994) ) ;W. C. Guida, 「Software For Structure -Based Dm g DesignJ , Curr. Opin. Struct. Biology. , 4, 777— 781頁（1994) ]。  [0176] Other molecular modeling techniques can also be used in accordance with the present invention [see, eg, NC Cohen et al., “Molecular Modeling Software and Metnods for Medicinal Chemistryj, J. Med. Chem., 33, 883-894 ( 1990); see MA Na via and MA Murcko, “The Use of Structural Information in Drug Design J, Current Opinions in Structural Biology, 2, 202—210 (1992); LM Balbes et al., "A Perspective of Modern Methods in Computer-Aided Drug DesignJ, (Reviews in Computational Chemistry, vol. 5, KB Lipkowitz and DB Boyd, VCH, New York, 3 37-380 (1994)); WC Guida, "Software For Structure -Based Dm Design J, Curr. Opin. Struct. Biology., 4, 777-781 (1994)].
[0177] 一旦上記の方法によりィ匕合物が設計される力または選択されると、その物質が酵素 相互作用部位に結合し得る効率が、計算による評価により試験され、そして最適化さ れ得る。例えば、有効な酵素相互作用部位阻害因子は、好ましくは、その結合状態 と遊離状態との間に相対的に小さなエネルギー差 (すなわち、結合の小さな変形ェ ネルギー）を示さなければならない。従って、最も効率的な酵素阻害因子は、好ましく は、約 lOkcalZモル以下（より好ましくは、 7kcalZモル以下）結合の変形エネルギ 一を用いて設計されるべきである。酵素阻害因子は、全結合エネルギーにおいて類 似する 2つ以上のコンフオメーシヨンで結合ポケットと相互作用し得る。これらの場合、 結合の変形エネルギーは、遊離物質のエネルギーと阻害因子がタンパク質に結合 するとき観察されるこれらのコンフオメーシヨンの平均エネルギーとの間の差であると 考えられる。  [0177] Once the compound is designed or selected by the above method, the efficiency with which the substance can bind to the enzyme interaction site can be tested and optimized by computational evaluation. . For example, an effective enzyme interaction site inhibitor should preferably exhibit a relatively small energy difference (ie, a small deformation energy of binding) between its bound and free states. Thus, the most efficient enzyme inhibitors should preferably be designed with a deformation energy of binding of about lOkcalZ mol or less (more preferably 7 kcalZ mol or less). Enzyme inhibitors can interact with the binding pocket in two or more conformations that are similar in total binding energy. In these cases, the deformation energy of binding is thought to be the difference between the energy of the free substance and the average energy of these conformations observed when the inhibitor binds to the protein.
[0178] 酵素に結合するように設計または選択される物質は、その結合状態において、好ま しくは、標的酵素および周囲の水分子との静電的斥力相互作用がないように、計算 によりさらに最適化され得る。このような非相補的静電的相互作用は、電荷—電荷斥 力相互作用、双極子一双極子斥力相互作用および電荷一双極子斥力相互作用を 含む。 [0178] Substances designed or selected to bind to the enzyme are preferably calculated so that there is no electrostatic repulsive interaction with the target enzyme and surrounding water molecules in its bound state. Can be further optimized. Such non-complementary electrostatic interactions include charge-charge repulsion interactions, dipole-dipole repulsion interactions and charge-dipole repulsion interactions.
[0179] 特定のコンピューターソフトウェアは、化合物変形エネルギーおよび静電的相互作 用を評価する分野において入手可能である。このような使用のために設計されたプロ グラムの例として、以下が挙げられる： Gaussian 94, revision C (M. J. Frisch, Gaussian, Inc. , Pittsburgh, PA 1995)； AMBER, version 4. 1 (P. A. Kol lman, University of California at San Francisco, 1995)； QUANTA/ CHARMM (Molecular Simulations, Inc. , San Diego, C A 1995) ;Insigh t Il/Discover, (Molecular Simulations, Inc. , San Diego, CA 1995)； DelPhi (Molecular Simulations, Inc. , San Diego, CA 1995) ;および AM SOL (Quantum Chemistry Program Exchange, Indiana University)。こ れらのプログラムは、例えば、 Silicon Graphicsワークステーション（例えば、「IMP ACT」グラフィクスを備える Indigo2)を用いて実行され得る。他のハードウェアシステ ムおよびソフトウェアパッケージもまた、当業者に公知である。 [0179] Specific computer software is available in the field of assessing compound deformation energy and electrostatic interaction. Examples of programs designed for such use include: Gaussian 94, revision C (MJ Frisch, Gaussian, Inc., Pittsburgh, PA 1995); AMBER, version 4.1 (PA Kol lman, University of California at San Francisco, 1995); QUANTA / CHARMM (Molecular Simulations, Inc., San Diego, CA 1995); Insigh t Il / Discover, (Molecular Simulations, Inc., San Diego, CA 1995); DelPhi (Molecular Simulations, Inc., San Diego, CA 1995); and AM SOL (Quantum Chemistry Program Exchange, Indiana University). These programs may be run using, for example, a Silicon Graphics workstation (eg, Indigo 2 with “IMP ACT” graphics). Other hardware systems and software packages are also known to those skilled in the art.
[0180] 本発明により可能な別のアプローチは、酵素に全体または部分的に結合し得る化 合物またはその部分についての低分子データベースの計算上のスクリーニングであ る。このスクリーニングにおいて、結合部位へのこのような物質の適合の質は、形状的 相補性または見積もられた相互作用エネルギーの ヽずれかにより判定され得る [E. C. Mengら， J. Comp. Chem. , 16, 505— 524頁（1992) ]。  [0180] Another approach possible with the present invention is the computational screening of small molecule databases for compounds or parts thereof that can bind in whole or in part to the enzyme. In this screening, the quality of such a substance's fit to the binding site can be determined by either geometric complementarity or an estimated interaction energy deviation [EC Meng et al., J. Comp. Chem., 16, 505-524 (1992)].
[0181] (ファノレマコフォア）  [0181] (Fanoremacophore)
本発明は、他の実施形態において、本発明の化合物に対する調節活性について の有効性のスクリーニングの道具として、コンピュータによる定量的構造活性相関（q uantitative structure activity relationship = QSAR)モアノレィ匕技術を使用 して得られる化合物を包含する。ここで、コンピューター技術は、いくつかのコンビュ —タによって作成した基質铸型、フアルマコフォア、ならびに本発明の活性部位の相 同モデルの作製などを包含する。一般に、インビトロで得られたデ―タから、ある物質 に対する相互作用物質の通常の特性基をモデル化することに対する方法は、 CAT ALYST フアルマコフォア法（Ekins et al. 、 Pharmacogenetics, 9 : 477〜4 89, 1999 ; Ekins et al.、 Pharmacol. & Exp. Ther. , 288 : 21〜29, 199 9 ; Ekins et al.、 Pharmacol. & Exp. Ther. , 290 : 429〜438, 1999 ;Ek ins et al.、 Pharmacol. & Exp. Ther. , 291 : 424〜433, 1999)および比 較分十電界分析 (comparative molecular field analysis ; CoMF A) (Jones e t al. , Drug Metabolism & Disposition, 24 : 1〜6, 1996)などを使用して 行うことができる。本発明において、コンビュ一タモデリングは、分子モデル化ソフトゥ エア（例えば、 CATALYST™バージョン 4 (Molecular Simulations, Inc. , San Diego, CA)など）を使用して行われ得る。 The present invention, in another embodiment, uses a computerized quantitative structure activity relationship (QSAR) moirelay technique as a tool for screening efficacy for modulatory activity for compounds of the present invention. Includes the resulting compounds. Here, the computer technology includes the production of a substrate model, a pharmacophore, and a homologous model of the active site of the present invention, which are created by several converters. In general, the method for modeling the normal characteristic group of an interactant for a substance from data obtained in vitro is CAT ALYST pharmacophore method (Ekins et al., Pharmacogenetics, 9: 477-4 89, 1999; Ekins et al., Pharmacol. & Exp. Ther., 288: 21-29, 199 9; Ekins et al., Pharmacol. & Exp. Ther., 290: 429-438, 1999; Ekins et al., Pharmacol. & Exp. Ther., 291: 424-433, 1999) and comparative molecular field analysis; CoMF A ) (Jones et al., Drug Metabolism & Disposition, 24: 1-6, 1996). In the present invention, computer modeling can be performed using molecular modeling software such as CATALYST ™ version 4 (Molecular Simulations, Inc., San Diego, Calif.).
活性部位に対する化合物のフィッティングは、当該分野で公知の種々のコンビユー タモデリング技術のいずれかを使用してで行うことができる。視覚による検査および活 性部位に対する化合物のマニュアルによる操作は、 QUANTA (Molecular Simul ations, Burlington, MA, 1992)、 SYBYL (Molecular Modeling Software , Tripos Associates, Inc. , St. Louis, MO, 1992)、 AMBER (Weiner et a 1. , J. Am. Chem. Soc. , 106 : 765— 784, 1984)、 CHARMM (Brooks et a 1. 、J. Comp. Chem. , 4 : 187〜217, 1983)などのようなプログラムを使用して行 うことができる。これにカロえ、 CHARMM, AMBERなどのような標準的な力の場を使 用してエネルギーの最小化を行うこともできる。他のさらに特殊ィ匕されたコンビュ一タ モデリングは、 GRID (Goodford et al. 、J. Med. Chem. , 28 : 849〜857, 198 5) , MCSS (Miranker and Karplus, Function and Genetics, 11 : 29〜34 , 1991)、 AUTODOCK (Goodsell and Olsen, Proteins : S tructure, Func tion and Genetics, 8 : 195〜202, 1990)、 DOCK (Kuntz et al. , J. Mol. Biol. , 161 : 269-288, (1982) )などを含む。さらなる構造の化合物は、空白の活 性部位、既知の低分子化合物における活性部位などに、 LUDKBohm, J. Comp. Aid. Molec. Design, 6 : 61〜78, 1992)、 LEGEND (Nishibata and Itai, T etrahedron, 47 : 8985, 1991)、 Leap Frog (Tripos Associates, St. Louis, MO)などのようなコンピュ一タ一プログラムを使用して新規に構築することもできる。 このようなモデリングは、当該分野において周知慣用されており、当業者は、本明細 書の開示に従って、適宜本発明の範囲に入る化合物を設計することができる。 Fitting a compound to the active site can be performed using any of a variety of computer modeling techniques known in the art. Visual inspection and manual manipulation of compounds to active sites are described in QUANTA (Molecular Simulations, Burlington, MA, 1992), SYBYL (Molecular Modeling Software, Tripos Associates, Inc., St. Louis, MO, 1992), AMBER (Weiner et a 1., J. Am. Chem. Soc., 106: 765—784, 1984), CHARMM (Brooks et a 1., J. Comp. Chem., 4: 187-217, 1983), etc. You can use a program like this. In addition, energy can be minimized by using a standard force field such as CHARMM, AMBER, etc. Other more specialized computer modeling is GRID (Goodford et al., J. Med. Chem., 28: 849-857, 198 5), MCSS (Miranker and Karplus, Function and Genetics, 11: 29-34, 1991), AUTODOCK (Goodsell and Olsen, Proteins: Structure, Function, Genetics, 8: 195-202, 1990), DOCK (Kuntz et al., J. Mol. Biol., 161: 269- 288, (1982)). Additional structural compounds include blank active sites, active sites in known low molecular weight compounds, etc., LUDKBohm, J. Comp. Aid. Molec.Design, 6: 61-78, 1992), LEGEND (Nishibata and Itai, Tetrahedron, 47: 8985, 1991), Leap Frog (Tripos Associates, St. Louis, MO). Such modeling is well known and commonly used in the art, and those skilled in the art According to the disclosure of the description, compounds that fall within the scope of the present invention can be designed as appropriate.
[0183] (医薬'ィ匕粧品など、およびそれを用いる治療、予防など）  [0183] (Pharmaceuticals, cosmetics, etc., and treatment and prevention using the same)
別の局面において、本発明は、医薬 (例えば、ワクチン等の医薬品、健康食品、タ ンパク質または脂質は抗原性を低減した医薬品）およびィ匕粧用組成物に関する。こ の医薬およびィ匕粧用組成物は、薬学的に受容可能なキャリアなどをさらに含み得る。 本発明の医薬に含まれる薬学的に受容可能なキャリアとしては、当該分野において 公知の任意の物質が挙げられる。  In another aspect, the present invention relates to pharmaceuticals (eg, pharmaceuticals such as vaccines, health foods, proteins or lipids having reduced antigenicity), and cosmetic compositions. The pharmaceutical and cosmetic composition may further comprise a pharmaceutically acceptable carrier and the like. Examples of the pharmaceutically acceptable carrier contained in the medicament of the present invention include any substance known in the art.
[0184] そのような適切な処方材料または薬学的に受容可能なキャリアとしては、抗酸化剤 、保存剤、着色料、風味料、および希釈剤、乳化剤、懸濁化剤、溶媒、フィラー、増 量剤、緩衝剤、送達ビヒクル、希釈剤、賦形剤および/または薬学的アジュバント挙 げられるがそれらに限定されない。代表的には、本発明の医薬は、化合物、またはそ の改変体もしくは誘導体を、 1つ以上の生理的に受容可能なキャリア、賦形剤または 希釈剤とともに含む組成物の形態で投与される。例えば、適切なビヒクルは、注射用 水、生理的溶液、または人工脳脊髄液であり得、これらには、非経口送達のための 組成物に一般的な他の物質を補充することが可能である。  [0184] Such suitable formulation materials or pharmaceutically acceptable carriers include antioxidants, preservatives, colorants, flavors, and diluents, emulsifiers, suspending agents, solvents, fillers, fillers. These include, but are not limited to, dosages, buffers, delivery vehicles, diluents, excipients and / or pharmaceutical adjuvants. Typically, the medicament of the present invention is administered in the form of a composition comprising the compound, or a variant or derivative thereof, together with one or more physiologically acceptable carriers, excipients or diluents. . For example, a suitable vehicle can be water for injection, physiological solution, or artificial cerebrospinal fluid, which can be supplemented with other materials common to compositions for parenteral delivery. is there.
[0185] 本明細書で使用される受容可能なキャリア、賦形剤または安定化剤は、レシピエン トに対して非毒性であり、そして好ましくは、使用される投薬量および濃度において不 活性であり、そして以下が挙げられる：リン酸塩、クェン酸塩、または他の有機酸;ァス コルビン酸、 a トコフエロール;低分子量ポリペプチド；タンパク質（例えば、血清ァ ルブミン、ゼラチンまたは免疫グロブリン）；親水性ポリマー（例えば、ポリビュルピロリ ドン）；アミノ酸 (例えば、グリシン、グルタミン、ァスパラギン、アルギニンまたはリジン） ；モノサッカリド、ジサッカリドおよび他の炭水化物（グルコース、マンノース、またはデ キストリンを含む）；キレート剤（例えば、 EDTA)；糖アルコール (例えば、マン-トー ルまたはソルビトール）；塩形成対イオン (例えば、ナトリウム）；ならびに Zあるいは非 イオン性表面活性化剤（例えば、 Tween、プル口ニック (pluronic)またはポリエチレ ングリコール（PEG) )。  [0185] Acceptable carriers, excipients or stabilizers used herein are non-toxic to the recipient and are preferably inert at the dosages and concentrations used. And the following: phosphates, citrates, or other organic acids; ascorbic acid, a tocopherol; low molecular weight polypeptides; proteins (eg, serum albumin, gelatin or immunoglobulin); hydrophilic Polymers (eg, polybulurpyrrolidone); amino acids (eg, glycine, glutamine, asparagine, arginine or lysine); monosaccharides, disaccharides and other carbohydrates (including glucose, mannose, or dextrin); chelating agents (eg, EDTA); sugar alcohols (eg mannitol or sorbitol); Salt-forming counterions (eg sodium); and Z or non-ionic surfactants (eg Tween, pluronic or polyethylene glycol (PEG)).
[0186] 例示の適切なキャリアとしては、中性緩衝化生理食塩水、または血清アルブミンと 混合された生理食塩水が挙げられる。好ましくは、その生成物は、適切な賦形剤 (例 えば、スクロース）を用いて凍結乾燥剤として処方される。他の標準的なキャリア、希 釈剤および賦形剤は所望に応じて含まれ得る。他の例示的な組成物は、 pH7. 0— 8. 5の Tris緩衝剤または pH4. 0— 5. 5の酢酸緩衝剤を含み、これらは、さら〖こ、ソ ルビトールまたはその適切な代替物を含み得る。 [0186] Exemplary suitable carriers include neutral buffered saline or saline mixed with serum albumin. Preferably, the product is a suitable excipient (e.g. For example, it is formulated as a lyophilizing agent using sucrose. Other standard carriers, diluents and excipients may be included as desired. Other exemplary compositions include Tris buffer at pH 7.0—8.5 or acetate buffer at pH 4.0—5.5, which are sardine, sorbitol, or suitable substitutes thereof. Can be included.
[0187] 本発明の医薬は、経口的または非経口的に投与され得る。あるいは、本発明の医 薬は、静脈内または皮下で投与され得る。全身投与されるとき、本発明において使用 される医薬は、発熱物質を含まない、薬学的に受容可能な水溶液の形態であり得る 。そのような薬学的に受容可能な組成物の調製は、 pH、等張性、安定性などを考慮 することにより、当業者は、容易に行うことができる。本明細書において、投与方法は 、経口投与、非経口投与 (例えば、静脈内投与、筋肉内投与、皮下投与、皮内投与 、粘膜投与、直腸内投与、膣内投与、患部への局所投与、皮膚投与など)であり得る 。そのような投与のための処方物は、任意の製剤形態で提供され得る。そのような製 剤形態としては、例えば、液剤、注射剤、徐放剤が挙げられる。  [0187] The medicament of the present invention may be administered orally or parenterally. Alternatively, the medicament of the present invention can be administered intravenously or subcutaneously. When administered systemically, the medicament used in the present invention may be in the form of a pharmaceutically acceptable aqueous solution free of pyrogens. Such a pharmaceutically acceptable composition can be easily prepared by those skilled in the art by considering pH, isotonicity, stability, and the like. In this specification, the administration method includes oral administration, parenteral administration (e.g., intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, rectal administration, intravaginal administration, local administration to the affected area, Skin administration, etc.). Formulations for such administration can be provided in any dosage form. Examples of such a preparation form include liquids, injections, and sustained release agents.
[0188] 本発明の医薬は、必要に応じて生理学的に受容可能なキャリア、賦型剤または安 定化剤 (日本薬局方第 14版またはその最新版、 Remington' s Pharmaceutical sciences, 18th Edition, A. R. Gennaro, ed. , MacK Publishing Compan y, 1990などを参照）と、所望の程度の純度を有する組成物とを混合することによつ て、凍結乾燥されたケーキまたは水溶液の形態で調製され保存され得る。  [0188] The medicament of the present invention may be a physiologically acceptable carrier, excipient, or stabilizer as necessary (Japanese Pharmacopoeia 14th edition or its latest edition, Remington's Pharmaceutical sciences, 18th Edition, AR Gennaro, ed., MacK Publishing Company, 1990, etc.) and a composition with the desired degree of purity and prepared and stored in the form of a lyophilized cake or aqueous solution Can be done.
[0189] 本発明の処置方法において使用される組成物の量は、使用目的、対象疾患 (種類 、重篤度など)、患者の年齢、体重、性別、既往歴、細胞の形態または種類などを考 慮して、当業者が容易に決定することができる。本発明の処置方法を被検体 (または 患者)に対して施す頻度もまた、使用目的、対象疾患 (種類、重篤度など)、患者の 年齢、体重、性別、既往歴、および治療経過などを考慮して、当業者が容易に決定 することができる。頻度としては、例えば、毎日 数ケ月に 1回（例えば、 1週間に 1回 1ヶ月に 1回）の投与が挙げられる。 1週間ー1ヶ月に 1回の投与を、経過を見なが ら施すことが好ましい。  [0189] The amount of the composition used in the treatment method of the present invention depends on the purpose of use, the target disease (type, severity, etc.), the patient's age, weight, sex, medical history, cell morphology or type, etc. In view of this, it can be easily determined by those skilled in the art. The frequency with which the treatment method of the present invention is applied to a subject (or patient) also depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, gender, medical history, treatment course, etc. In view of this, it can be easily determined by those skilled in the art. Examples of the frequency include administration once a few months every day (for example, once a week, once a month). It is preferable to administer once a week-once a month with the progress of the test.
[0190] 本発明が化粧品として使用されるときもまた、当局の規定する規制を遵守しながら 化粧品を調製することができる。 [0191] (農薬） [0190] When the present invention is used as a cosmetic, the cosmetic can be prepared while complying with the regulations stipulated by the authorities. [0191] (Pesticides)
本発明の組成物は、農薬の成分としても用いることができる。農薬組成物として処 方される場合、必要に応じて、農学的に受容可能なキャリア、賦型剤または安定化剤 などを含み得る。  The composition of the present invention can also be used as an agrochemical component. When processed as an agrochemical composition, it may contain agronomically acceptable carriers, excipients, stabilizers and the like, if necessary.
[0192] 本発明の組成物が、農薬として使用される場合は、除草剤 (ビラゾレートなど)、殺 虫'殺ダニ剤 (ダイアジノンなど)、殺菌剤 (プロべナゾールなど)、植物成長調整剤 ( 例、ノクロブトラゾールなど）、殺線虫剤（例、べノミルなど）、共力剤（例、ピぺ口-ル ブトキサイドなど）、誘引剤 (例、オイゲノールなど）、忌避剤 (例、クレオソートなど）、 色素 (例、食用青色 1号など)、肥料 (例、尿素など)などもまた必要に応じて混合され 得る。  [0192] When the composition of the present invention is used as an agrochemical, herbicide (such as virazolate), insecticide 'acaricide (such as diazinon), fungicide (such as probenazole), plant growth regulator (such as E.g., noclobutrazole), nematicides (e.g., benomyl), synergists (e.g., pipeto-rubbutoxide), attractants (e.g., eugenol), repellents (e.g., Creosote, etc.), pigments (eg, Food Blue No. 1), fertilizers (eg, urea, etc.) can also be mixed if necessary.
[0193] (保健'食品）  [0193] (Health 'food)
本発明はまた、保健'食品分野においても利用することができる。このような場合、 上述の経口医薬として用いられる場合の留意点を必要に応じて考慮すべきである。 特に、特定保健食品のような機能性食品'健康食品などとして使用される場合には、 医薬に準じた扱 、を行うことが好ま 、。  The present invention can also be used in the health 'food field. In such a case, the points to be noted when used as an oral medicine should be considered as necessary. In particular, when it is used as a functional food such as specified health foods or 'health foods', it is preferable to treat them in accordance with pharmaceuticals.
[0194] 本発明は上記のように、医療以外にも、食品検査、検疫、医薬品検査、法医学、農 業、畜産、漁業、林業などで、生体分子の検査が必要なものに全て適応可能である 。本発明においては特に、食料の安全目的のための（たとえば、 BSE検査)使用も企 図される。  [0194] As described above, the present invention can be applied not only to medical treatment but also to food inspection, quarantine, pharmaceutical inspection, forensic medicine, agriculture, livestock, fishery, forestry, etc. that require biomolecule inspection. is there . In particular, the present invention also contemplates use for food safety purposes (eg, BSE testing).
[0195] (相互作用部位;構造変化）  [0195] (Interaction site; structural change)
酵素におけるその酵素の基質との相互作用部位は、酵素と基質との複合体を X線 構造解析などの物理学的手法により分析することによって同定することができる。ある いは、そのような相互作用部位は既知のデータを用いてもよい。例えば、ヒト j8 1, 4 —ガラタトース組成物転移酵素の場合、 J. Mol. Biol 318, 491— 502 (2002)に 記載されるように、ゥシ配列番号 2および対応するヒトの配列（配列番号 4)において A sp252、 Asp254、 Gly292、 Trp314, Gly315、 Glu317、 Asp318、 Met344、 Hi s347などが相互作用部位であり得る。この同定は、より精確に同定することが好まし いが、以下に説明するように、 A部分を同定することができ、それによりその部分また はその部分を含む化合物を製造することができれば、必ずしも、精確である必要はな ぐ同定する必要が全くない場合もあり得る。 The site of interaction of the enzyme with the substrate of the enzyme can be identified by analyzing the complex of the enzyme and the substrate by a physical method such as X-ray structural analysis. Alternatively, known data may be used for such interaction sites. For example, in the case of human j8 1,4-galatose composition transferase, as described in J. Mol. Biol 318, 491-502 (2002), Usi SEQ ID NO: 2 and the corresponding human sequence (SEQ ID NO: In 4), Asp252, Asp254, Gly292, Trp314, Gly315, Glu317, Asp318, Met344, His347, and the like may be interaction sites. This identification is preferably a more accurate identification, but as explained below, it is possible to identify the A part and thereby that part or If it is possible to produce a compound containing that moiety, it may not necessarily be necessary to identify it at all, and it may not need to be identified at all.
[0196] 本明細書において酵素の「触媒反応に必要な構造変化」とは、基質との相互作用 によりコンフオメーシヨンが変化する酵素において、その酵素が担う触媒反応を行うの に必要とされる構造上の変化をいう。例えば、ゥシ j8 1, 4 ガラクトース転移酵素（配 列番号 1 (核酸配列）および 2 (アミノ酸配列)）、ヒト |8 1, 4 ガラクトース転移酵素 (配 列番号 3 (核酸配列)および 4 (アミノ酸配列)）がその酵素である場合、ドナー基質で ある UDP Galの上記酵素への結合により弓 Iき起こされるコンフオメ シヨン変化が そのような構造変化にあたる。この構造変化により、ァクセプター（GlcNAc)結合部 位が出現する。  [0196] In the present specification, "structural change necessary for catalytic reaction" of an enzyme is required for performing a catalytic reaction that the enzyme plays in an enzyme whose conformation changes due to interaction with a substrate. A structural change. For example, ushi j8 1, 4 galactose transferase (SEQ ID NO: 1 (nucleic acid sequence) and 2 (amino acid sequence)), human | 8 1, 4 galactose transferase (SEQ ID NO: 3 (nucleic acid sequence) and 4 (amino acid sequence) When the sequence ()) is the enzyme, the conformational change caused by the binding of the donor substrate UDP Gal to the above enzyme is such a structural change. Due to this structural change, an acceptor (GlcNAc) binding site appears.
[0197] このような酵素の触媒反応に必要な構造変化を担う構造変化部位の同定は、当該 分野において周知の物理学的手法 (例えば、 X線構造解析)を用いて行うことができ る。いったん構造変化を担う部位が同定されると、その部位の挙動を解析することで、 以下の相互作用する部分の同定の助けとすることができる。  [0197] Identification of the structural change site responsible for the structural change necessary for the catalytic reaction of the enzyme can be performed using a physical method well known in the art (for example, X-ray structural analysis). Once the site responsible for the structural change is identified, the behavior of the site can be analyzed to help identify the following interacting parts.
[0198] 本発明のスクリーニング方法において、相互作用部位と相互作用する A部分は、当 該分野において周知の技法を用いて同定することができる。本発明のスクリーニング 方法において、構造変化部位と相互作用する B部分もまた、当該分野において周知 の技法を用いて同定することができる。そのような技法としては、例えば、コンビュ一タ モデリング、生化学的手法、コンビナトリアルケミストリ、遺伝的アルゴリズムを用いた スクリーニングなどが挙げられるがそれらに限定されない。そのような技法は、例えば [0198] In the screening method of the present invention, the A moiety that interacts with the interaction site can be identified using techniques well known in the art. In the screening method of the present invention, the B moiety that interacts with the structural change site can also be identified using techniques well known in the art. Such techniques include, but are not limited to, computer modeling, biochemical techniques, combinatorial chemistry, screening using genetic algorithms, and the like. Such techniques are for example
、本明細書において引用する文献または最新創薬ィ匕学 (上 Z下) Wermuth編、長 瀬博監訳、テクノミック（1997)などの一般的な教科書に記載されており、当業者は それら技法を適宜使用または組み合わせることによって本発明のスクリーニング方法 に応用することができる。 , Literatures cited in this specification or the latest drug discovery science (upper Z lower) Wermuth, edited by Hiroshi Nagase, technomic (1997), etc. Can be applied to the screening method of the present invention by appropriately using or combining them.
[0199] 1つの実施形態において、本発明のスクリーニング方法が対象とする酵素は、好ま しくは、触媒反応に 2以上の分子が必要であるものが使用され得る。そのような酵素と しては、例えば、転移酵素 (例えば、ァセチル転移酵素、糖転移酵素、アミノ基転移 酵素、リン酸転移酵素、ダルタミル転移酵素、 C1化合物転移酵素、ケトン基転移酵 素、アルデヒド基転移酵素、ァシル基転移酵素、アルキル基転移酵素、含窒素残基 転移酵素、チォ—ル基転移酵素、ペプチド転移酵素など)、リア—ゼ (除去付加酵素[0199] In one embodiment, an enzyme that is a target of the screening method of the present invention may preferably be one that requires two or more molecules for the catalytic reaction. Examples of such enzymes include transferases (eg, acetyltransferases, glycosyltransferases, aminotransferases, phosphotransferases, dartamyl transferases, C1 compound transferases, ketone group transferases). Element, aldehyde transferase, acyl transferase, alkyl transferase, nitrogen-containing residue transferase, thiotransferase, peptide transferase, etc.), lyase (removal addition enzyme)
)、シンターゼ (合成酵素、リガーゼ)などが挙げられる。あるいは、そのような 2つ以上 の分子を必要とする酵素としては、補酵素（例えば， NADPH、 NADP、 CoQなど） などを触媒反応に必要とする酵素が挙げられるがそれらに限定されない。 ), Synthase (synthetic enzyme, ligase) and the like. Alternatively, enzymes that require two or more molecules include, but are not limited to, enzymes that require a coenzyme (eg, NADPH, NADP, CoQ, etc.) for catalytic reactions.
[0200] 好ましい実施形態において、本発明のスクリーニング方法が対象とする酵素は、糖 転移酵素である。糖転移酵素は、通常ドナー基質およびァクセプター基質をその触 媒反応に必要とし、ドナー基質の酵素への結合により、コンフオメーシヨン変化を起こ し、触媒反応が促進されることが多い。このようなメカニズムを用いて、本発明のスクリ 一二ング方法を適用することができる。 [0200] In a preferred embodiment, the enzyme targeted by the screening method of the present invention is a glycosyltransferase. Glycosyltransferases usually require a donor substrate and an acceptor substrate for the catalytic reaction, and the binding of the donor substrate to the enzyme often causes a conformational change and promotes the catalytic reaction. By using such a mechanism, the screening method of the present invention can be applied.
[0201] 1つの好ましい実施形態において、本発明のスクリーニング方法では、上記基質は、 ドナーおよびァクセプターを含み、酵素は、このドナーとこのァクセプターとの間の反 応を触媒するものが使用される。通常、酵素の構造変化は、該ドナーにより生じるが 、これらに限定されない。この好ましい実施形態において、上記 A部分はドナーと相 互作用し、上記 B部分はこのァクセプターの触媒部位への進入を担う部分であっても よいが、それらに限定されない。ここで、ァクセプターの触媒部位への進入を担う部 分は、当該分野において周知の物理学的手法などによって当業者は容易に決定す ることができる。あるいは、ァクセプターの触媒部位への進入を担う部分は既知のデ ータを用いることもできる。重要なのは、本発明において、このようなコンフオメーショ ン変化を活性調節因子 (例えば、阻害因子)の設計に用いることである。  [0201] In one preferred embodiment, in the screening method of the present invention, the substrate includes a donor and an acceptor, and an enzyme that catalyzes a reaction between the donor and the acceptor is used. Usually, the structural change of the enzyme is caused by the donor, but is not limited thereto. In this preferred embodiment, the A moiety interacts with the donor, and the B moiety may be a moiety responsible for entry of the acceptor into the catalytic site, but is not limited thereto. Here, the part responsible for the entry of the acceptor to the catalytic site can be easily determined by those skilled in the art by physical methods well known in the art. Alternatively, known data can be used as the part responsible for the entry of the acceptor into the catalytic site. Importantly, in the present invention, such a conformational change is used for designing an activity regulator (eg, inhibitor).
[0202] 本発明のスクリーニング方法の 1つの好ましい実施形態において、糖転移酵素の例 としては、 β ΐ , 4—ガラクトース転移酵素、 《1 , 3—ガラクトース転移酵素， β ΐ , 4— ガラクトース転移酵素， β ΐ , 3—ガラクトース転移酵素， β ΐ , 6—ガラクトース転移酵 素、 ひ 2, 6 シアル酸転移酵素、 ひ 1 , 4 ガラクトース転移酵素、セラミドガラタトー ス転移酵素、 α ΐ , 2 フコース転移酵素、 《1 , 3 フコース転移酵素、 《1 , 4 フ コース転移酵素、 ひ 1 , 6 フコース転移酵素、 ひ 1 , 3— Ν ァセチルガラタトサミン 転移酵素、 α ΐ , 6— Ν ァセチルガラタトサミン転移酵素、 β ΐ , 4— Ν ァセチルガ ラタトサミン転移酵素、ポリペプチド Ν ァセチルガラタトサミン転移酵素、 13 1 , 4 -N ァセチルダルコサミン転移酵素、 13 1 , 2— Nァセチルダルコサミン転移酵素、 j8 1 , 3 —Nァセチルダルコサミン転移酵素、 β ΐ , 6— Νァセチルダルコサミン転移酵素、 a 1 , 4—Nァセチルダルコサミン転移酵素、 β ΐ , 4 マンノース転移酵素、 α ΐ , 2— マンノース転移酵素、《1 , 3 マンノース転移酵素、《1 , 4 マンノース転移酵素、 α ΐ , 6 マンノース転移酵素、 α ΐ , 2 グルコース転移酵素、 α 1 , 3 グルコース 転移酵素、 α 2, 3 シアル酸転移酵素、《2, 6 シアル酸転移酵素、《1 , 6 グ ルコサミン転移酵素、 α ΐ , 6—キシロース転移酵素、 j8キシロース転移酵素（プロテ ォグリカンコア構造合成酵素）、 β ΐ , 3—グルクロン酸転移酵素、ヒアルロン酸合成 酵素、他の糖ヌクレオチドを糖ドナーとして用いる糖転移酵素およびドルコールリン 酸型糖ドナーを用いる糖転移酵素などが挙げられるがこれらに限定されない。もっと も好ましくは、本発明のスクリーニング方法が対象とする酵素の一つは、 β ΐ , 4ーガ ラタトース転移酵素である。 [0202] In one preferred embodiment of the screening method of the present invention, examples of glycosyltransferases include βΐ, 4-galactosyltransferase, << 1,3-galactosetransferase, βΐ, 4-galactosyltransferase , Β ΐ, 3-galactose transferase, β ΐ, 6-galactose transferase, 2, 6 sialyltransferase, 3,4 galactose transferase, ceramide galactose transferase, α ΐ, 2 fucose transferase Enzymes, << 1,3 fucose transferase, << 1,4 fucose transferase, -1,6 fucose transferase, -1,3--acetyl galatatosamine transferase, α ΐ, 6- ァ acetyl galata Tosamine transferase, β ΐ, 4— Ν Acetylgalatatosamine transferase, polypeptide ァ Acetylgalatatosamine transferase, 13 1, 4 -N Acetyl darcosamine transferase, 13 1, 2— N-acetyl darcosamine transferase, j8 1, 3 — N-acetyl darcosamine transferase, β ΐ, 6— acetyl dalcosamine transferase, a 1, 4 —N-acetylylcolosamine transferase, β,, 4 mannose transferase, α ΐ, 2— mannose transferase, << 1, 3 mannose transferase, << 1, 4 mannose transferase, α ΐ, 6 mannose transferase, α ,, 2 Glucosetransferase, α1,3 Glucosetransferase, α2,3 Sialyltransferase, << 2,6 Sialyltransferase, << 1,6 Glucosaminetransferase, α酵素, 6-Xylose Transfer Enzymes, j8 xylose transferase (proteoglycan core structure synthase), β ΐ, 3-glucuronic acid transferase, hyaluronic acid synthase, glycosyltransferases using other sugar nucleotides as sugar donors and dolcorrin Although such glycosyltransferase using the mold sugar donor are not limited to. More preferably, one of the enzymes targeted by the screening method of the present invention is βΐ, 4-galactose transferase.
[0203] 本発明のスクリーニング方法が対象とする酵素が配列番号 2および 4に示されるゥ シおよびヒト j8 1 , 4 ガラクトース転移酵素である好ましい実施形態において、その 酵素の基質は、 UDP ガラクトースを含み、上記構造変化部位は、配列番号 2また は 4に示されるポリペプチド配列のアミノ酸 314位のトリプトファンを含む部位である。 当然、類似の酵素の場合、同様の部分が利用され得る。あるいは、対応する酵素の 場合、対応する部分が利用され得る。  [0203] In a preferred embodiment in which the enzyme targeted by the screening method of the present invention is ushi and human j81,4 galactosyltransferase shown in SEQ ID NOs: 2 and 4, the enzyme substrate comprises UDP galactose The structural change site is a site containing tryptophan at amino acid position 314 of the polypeptide sequence shown in SEQ ID NO: 2 or 4. Of course, in the case of similar enzymes, similar moieties can be utilized. Alternatively, in the case of a corresponding enzyme, the corresponding part can be utilized.
[0204] 本発明のスクリーニング方法で利用される酵素は、野生型の酵素であってもよいが、 その設計の便宜のために、改変体 (例えば、保存的改変体、対立遺伝子改変体など )あるいはフラグメントを用いることもできる。あるいは、その設計の目的に応じて、非 天然のアミノ酸またはアミノ酸アナログを含む酵素改変体を用いることもできる。このよ うな改変体およびフラグメントの生産は、当該分野において周知の技法を用いて行う ことができ、当業者はそれら周知技術を適宜用いる力組み合わせて本発明に適用す ることがでさる。  [0204] The enzyme used in the screening method of the present invention may be a wild-type enzyme, but for convenience of design, a variant (for example, a conservative variant, an allelic variant, etc.) Alternatively, fragments can be used. Alternatively, an enzyme variant containing an unnatural amino acid or amino acid analog can be used depending on the purpose of the design. Such variants and fragments can be produced using techniques well known in the art, and those skilled in the art can apply the present invention to the present invention by combining these techniques appropriately.
[0205] (ドナー、ァクセプターを用いたスクリーニングの方法）  [0205] (Screening method using donor and acceptor)
別の局面において、本発明は糖転移酵素の阻害剤をスクリーニングするための方 法を提供する。この方法は、以下の工程: I)糖転移酵素と、該糖転移酵素のァクセプ ターと、該糖転移酵素のドナーと、該阻害剤の候補とを混合し、該糖転移酵素反応 が進行する条件および時間で反応させる工程；ならびに Π)該 I)工程の反応後の反 応物中の該ァクセプターと該ドナーとの反応生成物を測定する工程、を包含する。 In another aspect, the present invention provides a method for screening for an inhibitor of glycosyltransferase. This method comprises the following steps: I) a glycosyltransferase and an acceptor of the glycosyltransferase , A donor of the glycosyltransferase, and a candidate for the inhibitor, and the reaction is performed under conditions and time under which the glycosyltransferase reaction proceeds; and ii) a reaction product after the reaction in the step I) Measuring the reaction product of the acceptor with the donor.
[0206] 1つの好ましい実施形態において、本発明のスクリーニング方法は、必要に応じて、 1)糖転移反応の経時変化を測定し、初速の算出し、その初速に基づくスクリーニン グを設計すること、 2)必要に応じて、クリック反応の残留物による酵素反応への影響 を確認し、その影響を回避する手段を講じること、 3)阻害剤候補化合物をスクリー- ングすること、 4)スクリーニングによりヒットしたィ匕合物の阻害定数を算出することによ り実施され得る c  [0206] In one preferred embodiment, the screening method of the present invention comprises, as necessary, 1) measuring the time course of the glycosyltransferase reaction, calculating the initial speed, and designing a screening based on the initial speed. 2) If necessary, confirm the effect of the click reaction residue on the enzyme reaction and take measures to avoid the effect, 3) Screen the inhibitor candidate compound, 4) Screening Can be performed by calculating the inhibition constant of the hit compound
[0207] 1つの実施形態において、本発明のスクリーニング方法においてスクリーニングされ 得る阻害剤候補化合物は、クリック反応 (クリックケミストリー)で製造され得る。本明細 書において、クリック反応とは、クリック反応とはアセチレンィ匕合物とアジドィ匕合物から 水系で効率よく [3 + 2]付加環化によってトリァゾールを合成する反応をいう。クリック 反応より製造された阻害剤候補物質の収率は、例えば、エレクトロスプレイ Z質量分 析 (ESI— MS)により求めることができる。この阻害剤の濃度は、必要に応じて調整 することができる。  [0207] In one embodiment, an inhibitor candidate compound that can be screened in the screening method of the present invention can be produced by a click reaction (click chemistry). In this specification, the click reaction refers to a reaction in which triazole is synthesized from an acetylene compound and an azide compound by [3 + 2] cycloaddition efficiently in an aqueous system. The yield of the inhibitor candidate substance produced by the click reaction can be determined, for example, by electrospray Z mass spectrometry (ESI-MS). The concentration of this inhibitor can be adjusted as necessary.
[0208] 1つの実施形態において、本発明のスクリーニング方法において使用され得る糖転 移酵素の例としては、 e l, 4 ガラクトース転移酵素、 《1, 3 ガラクトース転移酵 素， β ΐ, 4—ガラクトース転移酵素， β ΐ, 3—ガラクトース転移酵素， β ΐ, 6—ガラク トース転移酵素、 ひ 2, 6 シアル酸転移酵素、 ひ 1, 4 ガラクトース転移酵素、セラ ミドガラタトース転移酵素、 α ΐ, 2 フコース転移酵素、 《1, 3 フコース転移酵素、 α ΐ, 4ーフコース転移酵素、 α ΐ, 6 フコース転移酵素、 α 1, 3— Ν ァセチルガ ラタトサミン転移酵素、 α ΐ, 6— Ν ァセチルガラタトサミン転移酵素、 β ΐ, 4-Ν- ァセチルガラタトサミン転移酵素、ポリペプチド Ν ァセチルガラタトサミン転移酵素（ 例えば、 Ν—ァセチルガラタトサミン転移酵素 ppGalNAcT— 2)、 β 1, 4— Νァセチ ルダルコサミン転移酵素、 β ΐ, 2— Νァセチルダルコサミン転移酵素、 13 1 , 3— Νァ セチルダルコサミン転移酵素、 13 1 , 6— Νァセチルダルコサミン転移酵素、 α ΐ, 4— Νァセチルダルコサミン転移酵素、 /3 1, 4 マンノース転移酵素、 α ΐ, 2 マンノー ス転移酵素、 α ΐ, 3 マンノース転移酵素、《1, 4 マンノース転移酵素、《1, 6 マンノース転移酵素、《1, 2 グルコース転移酵素、《1, 3 グルコース転移酵 素、 « 2, 3 シアル酸転移酵素、 « 2, 6 シアル酸転移酵素、 α ΐ, 6 ダルコサミ ン転移酵素、 α ΐ, 6—キシロース転移酵素、 j8キシロース転移酵素（プロテオグリカ ンコア構造合成酵素）、 β ΐ, 3—グルクロン酸転移酵素、ヒアルロン酸合成酵素、他 の糖ヌクレオチドを糖ドナーとして用いる糖転移酵素およびドルコールリン酸型糖ド ナーを用いる糖転移酵素などが挙げられるがこれらに限定されない。好ましくは、糖 転移酵素は、フコース転移酵素 V、 a l, 6フコース転移酵素 VIII、 « 2, 3 シアル 酸転移酵素、 α 2, 6 シアル酸転移酵素であり得る。 [0208] In one embodiment, examples of the glycosyltransferase that can be used in the screening method of the present invention include el, 4 galactose transferase, << 1, 3 galactose transferase, β ガ, 4-galactose transferase. Enzyme, β ΐ, 3-galactose transferase, β ΐ, 6-galactose transferase, 2, 2, 6 sialyltransferase, 1, 1, 4 galactose transferase, ceramide galatatosyl transferase, α ΐ, 2 fucose transferase , 《1, 3 fucose transferase, α ΐ, 4-fucose transferase, α ΐ, 6 fucose transferase, α 1, 3— チ ル acetylcetone ratatosamine transferase, α ΐ, 6- ァ acetyl galatatosamine transferase, β ΐ, 4-Ν- acetyl galatatosamine transferase, polypeptide ァ acetyl galatatosamine transferase (eg, Ν-acetyl galatatosamine transferase ppGalNAcT-2), β 1, 4— — Cetyldarcosaminetransferase, β ΐ, 2— Νacetylyldarcosaminetransferase, 13 1, 3— cetyldarcosamine transferase, 13 1, 6— cetyldarcosamine transferase, α ΐ, 4— Acetyldarcosamine transferase, / 3 1, 4 Mannose transferase, α ΐ, 2 Manno Transferase, α ΐ, 3 mannose transferase, << 1, 4 mannose transferase, << 1, 6 mannose transferase, << 1, 2 glucose transferase, << 1, 3 glucose transferase, «2, 3 sial Acid transferase, «2, 6 sialyltransferase, α ΐ, 6 darcosamine transferase, α ΐ, 6-xylose transferase, j8 xylose transferase (proteoglycan core structure synthase), β ΐ, 3-glucuron Examples include, but are not limited to, acid transferases, hyaluronic acid synthases, glycosyltransferases using other sugar nucleotides as sugar donors, and glycosyltransferases using dolcor phosphate-type sugar donors. Preferably, the glycosyltransferase may be fucose transferase V, al, 6 fucose transferase VIII, «2,3 sialyltransferase, α 2,6 sialyltransferase.
[0209] 1つの実施形態において、本発明のスクリーニング方法により使用される糖転移酵 素は、フコース転移酵素 Vであり、ァクセプターは、  [0209] In one embodiment, the glycosyltransferase used by the screening method of the present invention is fucose transferase V, and the acceptor is
[0210] [化 300]  [0210] [Chemical 300]
Figure imgf000108_0001
であり、そしてドナーは、
Figure imgf000108_0001
And the donor is
[0211] [化 301] [0211] [Chemical 301]
Figure imgf000108_0002
である。
Figure imgf000108_0002
It is.
好ましい実施形態において、この糖転移酵素反応が進行する条件は、例えば、 50 mM HEPES (pH 7. 2)、 10mM 塩化マンガン、 ImM ァクセプター、 200 M ドナー、 2. 5mU フコース転移酵素 (FucT)V、 30 /z M 阻害剤候補化合物混合 物であり得る。 In a preferred embodiment, the conditions under which this glycosyltransferase reaction proceeds are, for example, 50 mM HEPES (pH 7.2), 10 mM manganese chloride, ImM acceptor, 200 M donor, 2.5 mU fucose transferase (FucT) V, 30 / z M inhibitor candidate compound mixture.
別の実施形態において、本発明のスクリーニングにおいて使用される糖転移酵素 は、 α ΐ, 6フコース転移酵素 VIIIであり、ァクセプターは、  In another embodiment, the glycosyltransferase used in the screening of the present invention is α ΐ, 6 fucose transferase VIII, and the acceptor is
[化 302]  [Chem 302]
OOPMI  OOPMI
一 N o a  One N o a
OOPMI  OOPMI
一 N  One N
Figure imgf000109_0001
Figure imgf000109_0001
であり、そしてドナーは、  And the donor is
[0213] [化 303]  [0213] [Chem 303]
H2 H 2
HO OH
Figure imgf000109_0002
HO OH
Figure imgf000109_0002
[0214] 好ま 、実施形態にぉ 、て、この糖転移酵素反応が進行する条件は、例えば、 50 mM 力コジル酸緩衝液（ρΗ 7. 5)、 10 mM 塩ィ匕マンガン、 100 ^ M ァクセ プター、 50 ドナー、 80 μ \] フコース転移酵素 VIII、 50 GDP— 6Ν[0214] Preferably, according to the embodiment, the conditions under which this glycosyltransferase reaction proceeds are, for example, 50 mM force codylate buffer (ρΗ7.5), 10 mM salt-manganese, 100 ^ M Putter, 50 donors, 80 μ \] fucose transferase VIII, 50 GDP—6Ν
3フコース、 50 M 阻害剤候補化合物混合物であり得る。 Can be 3 fucose, 50 M inhibitor candidate compound mixture.
[0215] 1つ実施形態において、本発明のスクリーニングにおいて使用され得る糖転移酵素 は、シアル酸転移酵素であり、ァクセプターは、 [0215] In one embodiment, the glycosyltransferase that can be used in the screening of the present invention is a sialyltransferase, and the acceptor is
[0216] [化 304] [0216] [Chem 304]
Figure imgf000110_0001
であり、そしてドナーは、
Figure imgf000110_0001
And the donor is
[0217] [化 305]  [0217] [Chem 305]
Figure imgf000110_0002
Figure imgf000110_0002
であり得る。  It can be.
[0218] 好ま 、実施形態にぉ 、て、この糖転移酵素反応が進行する条件は、例えば、 40 mM cacodylate-HCl (pH7. 5)、0. 5g/ml BSA、 0. 1% 界面活性剤、 lm M ァクセプター、 200 M ドナー、 lmU α 2— 6シアル酸転移酵素（配列番号 6 (アミノ酸配列））であり得る。  [0218] Preferably, according to the embodiment, the conditions under which this glycosyltransferase reaction proceeds are, for example, 40 mM cacodylate-HCl (pH 7.5), 0.5 g / ml BSA, 0.1% surfactant. , Lm M acceptor, 200 M donor, lmU α 2-6 sialyltransferase (SEQ ID NO: 6 (amino acid sequence)).
[0219] 別の好ま 、実施形態にぉ 、て、例えば、この糖転移酵素反応が進行する条件は 、40mM cacodylate-HCl (pH7. 5)、0. 5g/ml BSA、 0. 1% 界面活性剤、 ImM ァクセプター、 200 M ドナー、 lmU α 2— 3シアル酸転移酵素（配列番 号 7 (アミノ酸配列））であり得る。 [0220] 1つの実施形態において、本発明のスクリーニング方法において使用され得る糖転 移酵素は、 N—ァセチルガラタトサミン転移酵素であり、ァクセプターは、 [0219] In another preferred embodiment, for example, the conditions under which the glycosyltransferase reaction proceeds are 40 mM cacodylate-HCl (pH 7.5), 0.5 g / ml BSA, 0.1% surfactant. Agent, ImM acceptor, 200 M donor, lmU α2-3 sialyltransferase (SEQ ID NO: 7 (amino acid sequence)). [0220] In one embodiment, the glycosyltransferase that can be used in the screening method of the present invention is N-acetylethylgalatatosamine transferase, and the acceptor is
[0221] [化 306] [0221] [Chemical 306]
Figure imgf000112_0001
干
Figure imgf000112_0001
Dried
Figure imgf000112_0002
Figure imgf000112_0002
Figure imgf000112_0003
Figure imgf000112_0003
Figure imgf000112_0004
Figure imgf000112_0004
esoso/Loo∑df/i3d III TCOTSO/LOOZ: OAV であり、そしてドナーは、 UDP— GalNAcであり得る。 esoso / Loo∑df / i3d III TCOTSO / LOOZ: OAV And the donor can be UDP-GalNAc.
[0222] 好ま 、実施形態にぉ 、て、この糖転移酵素反応が進行する条件は、例えば、 50 mM イミダゾール— HC1緩衝溶液（0. 1 % Triton X— 100を含む、 pH7. 2)、 500 μ Μ ァクセプター、 100 μ Μ ドナー、 100 μ Μ 阻害剤候補化合物混合 物、 10 mM 塩化マンガン、 IngZ 1 N—ァセチルガラタトサミン転移酵素 ppGal NAcT— 2であり得る。 [0222] Preferably, in the embodiment, the conditions under which the glycosyltransferase reaction proceeds are, for example, 50 mM imidazole-HC1 buffer solution (containing 0.1% Triton X-100, pH 7.2), 500 μΜ acceptor, 100 μΜ donor, 100 μΜ inhibitor candidate compound mixture, 10 mM manganese chloride, IngZ 1 N-acetylethyl latatosaminyltransferase ppGal NAcT-2.
[0223] 1つの実施形態において、この糖転移酵素反応が進行する条件は、ァセチルイ匕で あり得るが、これに限定されない。  [0223] In one embodiment, the conditions under which the glycosyltransferase reaction proceeds can be acetylene candy, but are not limited thereto.
[0224] 1つの実施形態において、本発明のスクリーニング方法において使用され得るァク セプターは、同位体 (例えば、重水素、軽水素など）により標識され得る。例えば、重 水素標識体は内部標準として、軽水素標識体は阻害剤の活性評価に用いられ得る  [0224] In one embodiment, an acceptor that can be used in the screening methods of the present invention can be labeled with an isotope (eg, deuterium, light hydrogen, etc.). For example, deuterium label can be used as an internal standard, and light hydrogen label can be used for inhibitor activity evaluation.
[0225] (糖転移酵素の初速の算出） [0225] (Calculation of initial speed of glycosyltransferase)
本明細書において糖転移酵素の「初速」は、糖転移酵素と、糖転移酵素のァクセプ ターと、糖転移酵素のドナーとを混合して反応させ、該ァクセプターと該ドナーとの反 応生成物の反応率を経時的に測定することにより算出することができる。  In the present specification, “initial rate” of glycosyltransferase means that a glycosyltransferase, a glycosyltransferase acceptor, and a glycosyltransferase donor are mixed and reacted to produce a reaction product of the acceptor and the donor. It can be calculated by measuring the reaction rate over time.
[0226] 他の実施形態において、前記ァクセプターと前記ドナーとを反応させる時間は、該 初速に基づいて設定され得る。ここで、例えば、初速は、 1〜20%の反応が進行する 時点であり得る。例えば、 10%の時点が 30分であり、 20%の時点が 90分であるとす ると、 10〜20%の初速に対応する時間は 60分である。 [0226] In another embodiment, the time for reacting the acceptor and the donor may be set based on the initial speed. Here, for example, the initial speed may be a point in time when 1 to 20% of the reaction proceeds. For example, if the 10% time point is 30 minutes and the 20% time point is 90 minutes, the time corresponding to the initial speed of 10-20% is 60 minutes.
[0227] 他の実施形態において、前記反応は、例えば、 50mM HEPES (pH 7. 2)、 10 mM 塩化マンガン、 ImM ァクセプター、 200 μ Μ ドナー、 2. 5mU フコース転 移酵素 (FucT) Vである条件下で実施され得る。 [0227] In another embodiment, the reaction is, for example, 50 mM HEPES (pH 7.2), 10 mM manganese chloride, ImM acceptor, 200 μΜ donor, 2.5 mU fucose transferase (FucT) V It can be carried out under conditions.
[0228] 別の実施形態において、前記反応は、例えば、 50 mM 力コジル酸緩衝液 (pH[0228] In another embodiment, the reaction is performed, for example, with 50 mM force codylate buffer (pH
7. 5)、 10 mM 塩ィ匕マンガン、 100 μ Μ ァクセプター、 50 ^ Μ ドナー、 80 μ \] フコース転移酵素 VIIIである条件下で実施され得る。 7.5), 10 mM salt, manganese, 100 μΜ acceptor, 50 ^ Μ donor, 80 μ \] fucose transferase VIII.
[0229] 別の実施形態において、前記反応は、例えば、 40mM cacodylate— HCl (pH7[0229] In another embodiment, the reaction is performed, for example, with 40 mM cacodylate—HCl (pH 7
. 5)、0. 5g/ml BSA、0. 1 % Triton CF— 54、 ImM ァクセプター、 200 μ M ドナー、 α 2— 6シアル酸転移酵素（配列番号 6 (アミノ酸配列）） lmUである条 件下で実施され得る。 5), 0.5g / ml BSA, 0.1% Triton CF—54, ImM acceptor, 200μ M donor, α 2-6 sialyltransferase (SEQ ID NO: 6 (amino acid sequence)) can be performed under the condition of lmU.
[0230] 別の実施形態において、前記反応は、例えば、 40mM cacodylate -HCl (pH 7 . 5)、0. 5g/ml BSA、0. 1% Triton CF— 54、 ImM ァクセプター、 200 μ Μ ドナー、 α 2— 3シアル酸転移酵素（配列番号 7 (アミノ酸配列）） lmUである条 件下で実施され得る。  [0230] In another embodiment, the reaction comprises, for example, 40 mM cacodylate-HCl (pH 7.5), 0.5 g / ml BSA, 0.1% Triton CF-54, ImM acceptor, 200 μΜ donor, α2-3 sialyltransferase (SEQ ID NO: 7 (amino acid sequence)) can be performed under the condition of lmU.
[0231] 別の実施形態において、前記反応は、例えば、 50mM イミダゾールー HC1緩衝 溶液（0. 1% Triton X— 100を含む、 pH7. 2)、 500 μ Μ ァクセプター、 100 μ Μ ドナー、 10 mM 塩化マンガン、 IngZ 1 N—ァセチルガラタトサミン転 移酵素 ppGalNAcT— 2である条件下で実施され得る。  [0231] In another embodiment, the reaction may be performed, for example, with 50 mM imidazole-HC1 buffer solution (containing 0.1% Triton X-100, pH 7.2), 500 μΜ acceptor, 100 μΜ donor, 10 mM chloride. Manganese, IngZ 1 N-Acetylgalatatosamine transferase ppGalNAcT-2 can be performed under the conditions.
[0232] 1つの実施形態において、前記反応率は、マトリックス支援レーザー脱離イオンィ匕 飛行時間型質量分析 (MALDI— TOF— MS)により測定され得るが、これに限定さ れない。 [0232] In one embodiment, the reaction rate can be measured by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), but is not limited thereto.
[0233] 他の実施形態において、好ましくは、前記初速は、反応率 10〜20%の点で算出さ れ得る。フコース転移酵素 VIIIの場合、初速は 60分であり得る。  [0233] In another embodiment, preferably, the initial speed can be calculated in terms of a reaction rate of 10 to 20%. In the case of fucose transferase VIII, the initial rate can be 60 minutes.
[0234] (クリック反応の残留物による酵素反応への影響）  [0234] (Effects of enzyme reaction by residue of click reaction)
1つの実施形態において、本発明のスクリーニング方法は、前記ァクセプターと、前 記ドナーと、前記糖転移酵素と、クリック反応に用いた物質とを混合して反応させ、該 クリック反応に用いた物質による酵素反応への影響を確認し、該影響があれば該影 響を回避する工程をさらに包含し得る。  In one embodiment, the screening method of the present invention comprises reacting the acceptor, the donor, the glycosyltransferase, and the substance used for the click reaction by mixing them, and depending on the substance used for the click reaction. The method may further include a step of confirming the influence on the enzyme reaction and avoiding the influence if there is such an influence.
[0235] 別の実施形態において、本発明のスクリーニング方法において使用され得る糖転 移酵素は、フコース転移酵素 (FucT) Vであり、前記クリック反応に用いた物質は 6ァ ジド GDPフコース、アセチレン化合物、硫酸銅、トリス [ (1—ベンジル一 1H— 1, 2, 3 —トリァゾールー 4—ィル)メチル]ァミン (TBTA)、またはァスコルビン酸ナトリウムで あり得る。  [0235] In another embodiment, the glycosyltransferase that can be used in the screening method of the present invention is fucose transferase (FucT) V, and the substance used in the click reaction is 6-adducted GDP fucose, acetylene compound , Copper sulfate, tris [(1-benzyl-1- 1H-1,2,3-triazol-4-yl) methyl] amine (TBTA), or sodium ascorbate.
[0236] 別の実施形態において、本発明のスクリーニング方法において使用され得る糖転 移酵素は、フコース転移酵素 VIIIであり、前記クリック反応に用いた物質は、 6アジド GDPフコース、アセチレン化合物、硫酸銅、トリス [ (1—ベンジル一 1H— 1, 2, 3—ト リアゾール—4—ィル)メチル]ァミン (TBTA)、またはァスコルビン酸ナトリウムであり 得る。 [0236] In another embodiment, the glycosyltransferase that can be used in the screening method of the present invention is fucose transferase VIII, and the substance used for the click reaction is 6azide GDP fucose, acetylene compound, copper sulfate , Tris [(1—Benzyl 1H— 1, 2, 3— Riazol-4-yl) methyl] amine (TBTA), or sodium ascorbate.
[0237] 別の実施形態において、本発明のスクリーニング方法において使用され得る糖転 移酵素は、 a 2, 3シアル酸転移酵素であり、前記クリック反応に用いた物質は、アル キン化合物、硫酸銅、ァスコルビン酸、トリス [ ( 1—ベンジル— 1H— 1 , 2, 3—トリァゾ ール— 4—ィル)メチル]ァミン（TBTA)、 t— BuOHまたは CMP—アジドシアル酸で あり得る。  [0237] In another embodiment, the glycosyltransferase that can be used in the screening method of the present invention is a 2,3 sialyltransferase, and the substance used in the click reaction is an alkyne compound, copper sulfate. , Ascorbic acid, tris [(1-benzyl-1H-1,2,3-triazole-4-yl) methyl] amine (TBTA), t-BuOH or CMP-azidosialic acid.
[0238] 別の実施形態において、本発明のスクリーニング方法において使用され得る糖転 移酵素は、 a 2, 6シアル酸転移酵素であり、前記クリック反応に用いた物質は、アル キン化合物、硫酸銅、ァスコルビン酸、トリス [ ( 1—ベンジル— 1H— 1 , 2, 3—トリァゾ ール— 4—ィル)メチル]ァミン（TBTA)、 t— BuOHまたは CMP—アジドシアル酸で あり得る。  [0238] In another embodiment, the glycosyltransferase that can be used in the screening method of the present invention is a 2,6-sialyltransferase, and the substance used in the click reaction is an alkyne compound, copper sulfate, or the like. , Ascorbic acid, tris [(1-benzyl-1H-1,2,3-triazole-4-yl) methyl] amine (TBTA), t-BuOH or CMP-azidosialic acid.
[0239] 別の実施形態において、本発明のスクリーニング方法において使用され得る糖転 移酵素は、 N—ァセチルガラタトサミン転移酵素であり、前記クリック反応に用いた物 質は、 UDP— N — GalNAc、アセチレン化合物、トリス [ ( 1—ベンジル— 1H— 1 , 2  [0239] In another embodiment, the glycosyltransferase that can be used in the screening method of the present invention is N-acetylethylgalatatosamine transferase, and the substance used in the click reaction is UDP-N- GalNAc, acetylene compound, tris [(1—benzyl— 1H— 1, 2
3  Three
, 3—トリァゾール— 4—ィル)メチル]ァミン (TBTA)、硫酸銅、ァスコルビン酸ナトリ ゥムであり得る。  , 3-triazole-4-yl) methyl] amine (TBTA), copper sulfate, sodium ascorbate.
[0240] 他の実施形態にお!、て、本発明のスクリーニング方法では、前記阻害剤候補化合 物混合物の阻害活性から、該酵素反応に影響する物質の阻害活性を減算すること により、前記酵素反応への影響を回避することができる。  [0240] In another embodiment, the screening method of the present invention subtracts the inhibitory activity of a substance that affects the enzyme reaction from the inhibitory activity of the inhibitor candidate compound mixture. The influence on the reaction can be avoided.
[0241] 1つの実施形態において、本発明において使用され得る回避手段は、例えば、 Cu 2+の場合、 EDTAであり得る。フコース転移酵素 VIIIの場合、酵素反応に影響する 物質は、 Cu2+および GDP— 6— Nフコースであり、 Cu2+による影響を回避する手  [0241] In one embodiment, a workaround that may be used in the present invention may be EDTA, for example in the case of Cu 2+. In the case of fucose transferase VIII, the substances that affect the enzyme reaction are Cu2 + and GDP-6-N fucose.
3  Three
段は、例えば、 EDTAによって回避され、 GDP— 6— Nフコースによる影響を回避  Steps are avoided, for example, by EDTA, avoiding the impact of GDP- 6-N fucose
3  Three
する手段は、減算であり得る。  The means to do can be subtraction.
[0242] (阻害剤候補化合物の阻害定数の算出）  [0242] (Calculation of inhibition constant of candidate inhibitor compound)
1つの局面において、本発明は、糖転移酵素の阻害剤の阻害定数を算出するため の方法を提供する。この方法は、以下の工程: I)糖転移酵素と、該糖転移酵素のァク セプターと、該糖転移酵素のドナーと、該阻害剤の候補とを混合し、該糖転移酵素 反応が進行する条件および時間で反応させる工程; II)該 I)工程の反応後の反応物 中の該ァクセプターと該ドナーとの反応生成物を測定する工程;ならびに In one aspect, the present invention provides a method for calculating the inhibition constant of an inhibitor of glycosyltransferase. This method comprises the following steps: I) Glycosyltransferase and the glycosyltransferase A step of mixing a scepter, a donor of the glycosyltransferase, and a candidate for the inhibitor and reacting them under conditions and time for which the glycosyltransferase reaction proceeds; II) a reaction product after the reaction in the step I) Measuring the reaction product of the acceptor with the donor; and
III)該 Π)工程において測定した値カゝら測定定数を算出する工程、を包含する。  III) a step of calculating a measurement constant based on the value measured in the step i).
[0243] 1つの実施形態において、阻害定数は、例えば、ディクソンプロットにより Kiを求め ること、 IC50を求めることにより算出することができるが、これらに限定されない。  [0243] In one embodiment, the inhibition constant can be calculated, for example, by determining Ki by a Dixon plot or by calculating IC50, but is not limited thereto.
[0244] 1つの実施形態にお!、て、前記糖転移酵素反応が進行する条件は、例えば、 50m M HEPES (pH 7. 2)、 10mM 塩化マンガン、 ImM ァクセプター、 50若しくは 200 μの Μドナー、 2. 5mU フコース転移酵素（FucT) V、 0〜： LOO μ Μ 阻害剤 候補化合物混合物であり得る。  [0244] In one embodiment, the conditions under which the glycosyltransferase reaction proceeds are, for example, 50 mM HEPES (pH 7.2), 10 mM manganese chloride, ImM acceptor, 50 or 200 μM donor 2.5 mU fucose transferase (FucT) V, 0 to: LOO μΜ inhibitor Can be a candidate compound mixture.
[0245] 他の実施形態において、前記糖転移酵素反応が進行する条件は、例えば、 50 m M 力コジル酸緩衝液（pH 7. 5)、 10 mM 塩化マンガン、 100 M ァクセプ ター、 12. 5〜75 ドナー、 80 ^ U フコース転移酵素 VIII, 0~100 ^ Μ 阻害剤候補化合物混合物であり得る。  [0245] In another embodiment, the conditions under which the glycosyltransferase reaction proceeds are, for example, 50 mM force codylate buffer (pH 7.5), 10 mM manganese chloride, 100 M acceptor, 12.5. ~ 75 donor, 80 ^ U fucose transferase VIII, 0-100 ^ Μ inhibitor inhibitor compound mixture.
[0246] 他の実施形態にお!、て、前記糖転移酵素反応が進行する条件は、例えば、 40m M cacodylate-HCl (pH7. 5)、 0. 5g/ml BSA、 0. 1% Triton CF— 54、 ImM ァクセプター、 200 M ドナー、 lmU α 2— 6シアル酸転移酵素（配列番 号 6 (アミノ酸配列））、 0〜： L00 Μ 阻害剤候補化合物混合物であり得る。  [0246] In another embodiment, the conditions under which the glycosyltransferase reaction proceeds are, for example, 40 mM cacodylate-HCl (pH 7.5), 0.5 g / ml BSA, 0.1% Triton CF. — 54, ImM acceptor, 200 M donor, lmU α 2-6 sialyltransferase (SEQ ID NO: 6 (amino acid sequence)), 0 to: L00 得 る inhibitor inhibitor compound mixture.
[0247] 別の実施形態にお!、て、前記糖転移酵素反応が進行する条件は、例えば、 40m M cacodylate-HCl (pH7. 5)、 0. 5g/ml BSA、 0. 1% Triton CF— 54、 ImM ァクセプター、 200 M ドナー、 lmU α 2— 3シアル酸転移酵素（配列番 号 7 (アミノ酸配列））、 0〜： L00 Μ 阻害剤候補化合物混合物であり得る。  [0247] In another embodiment, the conditions under which the glycosyltransferase reaction proceeds are, for example, 40 mM cacodylate-HCl (pH 7.5), 0.5 g / ml BSA, 0.1% Triton CF. — 54, ImM acceptor, 200 M donor, lmU α2-3 sialyltransferase (SEQ ID NO: 7 (amino acid sequence)), 0 to: L00 得 る inhibitor inhibitor compound mixture.
[0248] 別の実施形態において、前記糖転移酵素反応が進行する条件は、例えば、 50 m M イミダゾールー HC1緩衝溶液（0. 1% Triton X— 100を含む、 pH7. 2、 4 1)、 500 μ Μ ァクセプター、 100 μ Μ ドナー、 10 mM 塩化マンガン、 IngZ μ \ Ν—ァセチルガラタトサミン転移酵素 ppGalNAcT—2、 0〜100 /ζ Μ 阻害剤 候補化合物混合物であり得る。  [0248] In another embodiment, the conditions under which the glycosyltransferase reaction proceeds are, for example, 50 mM imidazole-HC1 buffer solution (containing 0.1% Triton X-100, pH 7.2, 4 1), 500 μ ク acceptor, 100 μ Μ donor, 10 mM manganese chloride, IngZ μ \ チ ル -acetyl galatatosamine transferase ppGalNAcT-2, 0-100 / ζ 阻 害 inhibitor Can be a candidate compound mixture.
[0249] 好ましい実施形態において、本発明の糖転移酵素の阻害剤の阻害定数を算出す るための方法では、前記糖転移酵素反応において、阻害剤候補化合物混合物に加 えて内部標準が混合され得る。 [0249] In a preferred embodiment, the inhibition constant of the glycosyltransferase inhibitor of the present invention is calculated. In the glycosyltransferase reaction, an internal standard may be mixed in addition to the inhibitor candidate compound mixture.
[0250] 別の局面において、本発明は、糖転移酵素のァクセプターを提供する。このァクセ プタ一は、以下の構造式：  [0250] In another aspect, the present invention provides a glycosyltransferase acceptor. This acceptor has the following structural formula:
ZER-NY-K  ZER-NY-K
を有し、式中、 ZERは MS高感度化部分であり、 NYは天然ミミック部分であり、 Kは 修飾可能部分である。  Where ZER is the MS-sensitized moiety, NY is the natural mimic moiety, and K is the modifiable moiety.
[0251] 1つの実施形態において、本発明の糖転移酵素のァクセプターの前記 ZERは、 [化 307]  [0251] In one embodiment, the ZER of the glycosyltransferase acceptor of the present invention is represented by the following formula:
Figure imgf000117_0001
Figure imgf000117_0001
[化 601] [Chem 601]
Figure imgf000118_0001
Figure imgf000118_0001
であり得る力 これらに限定されない。 The force that can be: Not limited to these.
他の実施形態において、本発明の糖転移酵素のァクセプターの前記 NYは、 [化 308]  In another embodiment, the NY of the glycosyltransferase acceptor of the present invention is:
Figure imgf000118_0002
Figure imgf000118_0002
[化 602]  [Chem 602]
Figure imgf000118_0003
Figure imgf000118_0003
[化 603] o- [Chem 603] o-
申 ¥0 N C ΗΛ N C T I - I - I
Figure imgf000119_0001
N ¥ 0 NC ΗΛ NCTI-I-I
Figure imgf000119_0001
o- ■ o- o: ： o o- ■ o- o:: o
Figure imgf000119_0002
Figure imgf000119_0002
esoso/Loordf/i3d 811 TCOTSO/LOOZ: OAV であり得る力 これらに限定されない。 esoso / Loordf / i3d 811 TCOTSO / LOOZ: OAV The force that can be: Not limited to these.
他の実施形態において、本発明の糖転移酵素のァクセプターの前記 κは、 In another embodiment, the kappa of the glycosyltransferase acceptor of the present invention is:
[化 309] [Chemical 309]
Figure imgf000120_0001
Figure imgf000120_0001
-NH、 -CHまたは CDであり得る力 これらに限定されない。  Forces that can be -NH, -CH or CD, but are not limited to these.
2 3 3  2 3 3
好ましくは、本発明の糖転移酵素のァクセプターは、例えば、  Preferably, the glycosyltransferase acceptor of the present invention is, for example,
[化 310] [Chem 310]
Figure imgf000120_0002
Figure imgf000120_0002
[化 311] [Chemical 311]
Figure imgf000121_0001
または
Figure imgf000121_0001
Or
[化 312] [Chem 312]
Figure imgf000122_0001
干
Figure imgf000122_0001
Dried
Figure imgf000122_0002
Figure imgf000122_0002
Figure imgf000122_0003
Figure imgf000122_0003
Figure imgf000122_0004
Figure imgf000122_0004
esoso/Loo∑df/i3d TCOTSO/LOOZ: OAV であり得る力 これらに限定されない。 esoso / Loo∑df / i3d TCOTSO / LOOZ: OAV The force that can be: Not limited to these.
[0255] (因子の産生）  [0255] (Production of factors)
別の局面において、本発明は、酵素の活性を調節する因子を生産する方法を提供 する。このような酵素の活性調節因子 (例えば、阻害因子)を生産するスキームは、従 来のコンセプトからは想到し得るようなものではない、コンフオメ一シヨン変化を利用し たスキームである。ただし、いったん目的となる因子のモデルまたはその分子自体の 高次構造が明らかになれば、本明細書の開示および当該分野において周知の技法 を適宜組み合わせることによって、目的とする化合物を生産することができる。  In another aspect, the present invention provides a method for producing a factor that modulates the activity of an enzyme. A scheme for producing such an enzyme activity regulator (for example, an inhibitor) is a scheme using conformational change, which is not conceivable from the conventional concept. However, once the model of the target factor or the higher-order structure of the molecule itself is clarified, the target compound can be produced by appropriately combining the disclosure of this specification and techniques well known in the art. it can.
[0256] 本発明の酵素の活性を調節する因子を生産する方法において、 A部分および B部 分を含む分子を調製する工程もまた、当該分野において公知の方法を用いるか、あ るいは組み合わせることによって、実施することができる。そのような分子の調製は、 化学合成、生化学的もしくは分子生物学的 (遺伝子工学的)産生手法、コンビナトリ アルケミストリ、遺伝アルゴリズム、コンピュータモデリング、フィッティングなどの種々 の技法を用いて行うことができる。そのような技法は、例えば、岡崎進、岡本裕幸編、 生体系のコンピュータシミュレーション、化学同人； B. Fraserreid、 K. Tatsuta, J. Thiem, Glycoscience, Springerに記載される技術を用いることができる。  [0256] In the method for producing a factor that regulates the activity of the enzyme of the present invention, the step of preparing a molecule containing the A portion and the B portion is also performed by using or combining methods known in the art. Can be implemented. Such molecules can be prepared using various techniques such as chemical synthesis, biochemical or molecular biological (genetic engineering) production techniques, combinatorial chemistry, genetic algorithms, computer modeling, fitting, etc. . As such a technique, for example, techniques described in Susumu Okazaki, Hiroyuki Okamoto, biological computer simulation, chemical doujin; B. Fraserreid, K. Tatsuta, J. Thiem, Glycoscience, Springer can be used.
[0257] 別の好ましい局面において、本発明は、本発明において提供される化合物を含む 、組成物を提供する。このような化合物は、通常、糖転移酵素の阻害活性を有する。 そのような組成物は、医薬、化粧品、農薬、食品 (例えば、保健食品）、生化学研究、 高分子生産など種々の分野において利用することができる。医薬であれば、本発明 の組成物は、医薬に対して適用される基準および規制に適合している必要があり、 他の分野においても同様の基準および規制が存在する場合、そのような基準および 規制に適合している必要がある。好ましくは、このような調節因子は、阻害因子であり 得るが、活性ィ匕因子であってもよい。  [0257] In another preferred aspect, the present invention provides a composition comprising a compound provided in the present invention. Such compounds usually have glycosyltransferase inhibitory activity. Such a composition can be used in various fields such as pharmaceuticals, cosmetics, agricultural chemicals, foods (eg, health foods), biochemical research, and polymer production. In the case of pharmaceuticals, the composition of the present invention needs to conform to the standards and regulations applicable to pharmaceuticals, and if there are similar standards and regulations in other fields, such standards And must comply with regulations. Preferably, such a regulator can be an inhibitor but can also be an active factor.
[0258] 本発明の糸且成物は、 β 1, 4 ガラクトース転移酵素が関与する状態、障害および 疾患の予防、処置、診断、予後などに使用することができる。 β 1, 4 ガラクトース転 移酵素が関与する状態、障害および疾患としては、例えば、がんまたはがんの転移、 がんの薬剤耐性、感染疾患、慢性疾患 (例えば、関節炎、慢性関節リウマチ）、糖尿 病、動脈硬化のような循環器系の疾患などが挙げられるがそれらに限定されない。ま た、本発明の組成物は、フコース転移酵素が関与する状態、障害および疾患の予防 、処置、診断、予後などに使用することができる。フコース転移酵素が関与する状態、 障害または疾患としては、免疫性疾患、がんまたはがんの転移、細菌感染、ァテロー ム性動脈硬化症、抗体依存性細胞傷害、シグナル伝達が挙げられるがそれらに限 定されない。さらに、本発明の組成物は、 a 2, 3—シアル酸転移酵素が関与する状 態、障害および疾患の予防、処置、診断、予後などに使用することができる。 « 2, 3 ーシアル酸転移酵素に関連する疾患としては、免疫性疾患、結腸 *直腸がん、乳が ん、白血病などのがんまたはがんの転移が挙げられるがそれらに限定されない。さら に、本発明の組成物は、 a 2, 6—シアル酸転移酵素が関与する状態、障害および 疾患の予防、処置、診断、予後などに使用することができる。 a 2, 6—シアル酸転移 酵素に関連する疾患としては、免疫性疾患、結腸，直腸がん、乳がん、白血病などの がんまたはがんの転移が挙げられるがそれらに限定されない。本発明の組成物は、 β ΐ , 4 ガラクトース転移酵素、フコース転移酵素、ひ 2, 3 シアル酸転移酵素また は oc 2, 6 シアル酸転移酵素の活性を調節することによって、上記予防、処置、診 断、予後などに用いることができる。調節活性は、好ましくは阻害活性であり得るが、 活性ィ匕活性であってもよい。阻害活性であることが好ましい。 β ΐ , 4—ガラクトース転 移酵素が担うガラクトースの転移が障害または疾患の発症に重大な役割を果たして いる障害または疾患 (例えば、ある種のがんの転移または薬剤耐性）、フコース転移 酵素が担うフコースの転移が障害または疾患の発症に重大な役割を果たしている障 害または疾患、《2, 3 シアル酸転移酵素が担うシアル酸の転移が障害または疾 患の発症に重大な役割を果たしている障害または疾患、あるいは《2, 6 シアル酸 転移酵素が担うシアル酸の転移が障害または疾患の発症に重大な役割を果たして V、る障害または疾患に罹患する被検体に予防、処置 (治療)または予後に適切な投 与量を投与することによって、予防、処置、予後などを行うことができる。そのような投 与は、経口投与であっても非経口投与であってもよい。非経口投与の場合、静脈注 射、筋肉注射、皮下注射、直腸内投与、膣内投与、患部への直接投与などが挙げら れるがそれらに限定されない。投与の経路に応じて、本発明の組成物は、適切な添 加物を含むことができる。そのような添加物の選択および添カ卩は、当該分野の技術範 囲内にあり、例えば、日本薬局方第 14改正およびその追補などを参照することによ つて当業者は容易に行うことができる。 [0258] The yarn composition of the present invention can be used for the prevention, treatment, diagnosis, prognosis and the like of conditions, disorders and diseases involving β 1,4 galactosyltransferase. Conditions, disorders, and diseases involving β 1,4 galactose transferase include, for example, cancer or cancer metastasis, Examples include, but are not limited to, cancer drug resistance, infectious diseases, chronic diseases (eg, arthritis, rheumatoid arthritis), diabetes, and cardiovascular diseases such as arteriosclerosis. In addition, the composition of the present invention can be used for prevention, treatment, diagnosis, prognosis and the like of conditions, disorders and diseases involving fucose transferase. Conditions, disorders or diseases involving fucose transferase include immune disease, cancer or cancer metastasis, bacterial infection, atherosclerosis, antibody-dependent cytotoxicity, signal transduction. It is not limited. Furthermore, the composition of the present invention can be used for the prevention, treatment, diagnosis, prognosis and the like of conditions, disorders and diseases involving a 2,3-sialyltransferase. «2,3-Sialyltransferase-related diseases include, but are not limited to, immune diseases, cancers such as colorectal cancer, breast cancer, leukemia, or cancer metastasis. Furthermore, the composition of the present invention can be used for prevention, treatment, diagnosis, prognosis and the like of conditions, disorders and diseases involving a 2,6-sialyltransferase. a 2,6-Sialyltransferase-related diseases include, but are not limited to, immune diseases, cancers such as colon, rectal cancer, breast cancer, leukemia, or cancer metastasis. The composition of the present invention comprises the above prevention, treatment, or regulation by regulating the activity of β ,, 4 galactose transferase, fucose transferase, glycan 2,3 sialyltransferase or oc 2,6 sialyltransferase. It can be used for diagnosis and prognosis. The modulating activity may preferably be an inhibitory activity, but may also be an active activity. Inhibitory activity is preferred. β,, 4-galactose transferase-mediated galactose transfer plays a critical role in the development of a disorder or disease (eg, certain cancer metastasis or drug resistance), fucose transferase Disorders or diseases in which fucose transfer plays a significant role in the onset of the disorder or disease, 《Disorders in which sialic acid transfer carried by sialyltransferase plays a significant role in the development of the disorder or disease Prevention, treatment (treatment) or prognosis of a subject suffering from a disorder or disease in which the transfer of sialic acid carried by the 2,6 sialyltransferase plays a critical role in the onset of the disorder or disease By administering an appropriate dose, the prevention, treatment, prognosis and the like can be performed. Such administration may be oral or parenteral. Examples of parenteral administration include intravenous injection, intramuscular injection, subcutaneous injection, rectal administration, intravaginal administration, and direct administration to the affected area. But not limited to them. Depending on the route of administration, the compositions of the present invention may contain suitable additives. The selection and addition of such additives are within the technical scope of the field, and can be easily performed by those skilled in the art, for example, by referring to the Japanese Pharmacopoeia 14th Amendment and its supplements. .
[0259] 以上のように、本発明の好ましい実施形態を用いて本発明を例示してきた力 本発 明は、この実施形態に限定して解釈されるべきものではない。本発明は、特許請求 の範囲によってのみその範囲が解釈されるべきであることが理解される。当業者は、 本発明の具体的な好ましい実施形態の記載から、本発明の記載および技術常識に 基づいて等価な範囲を実施することができることが理解される。本明細書において引 用した特許、特許出願および文献は、その内容自体が具体的に本明細書に記載さ れているのと同様にその内容が本明細書に対する参考として援用されるべきであるこ とが理解される。 [0259] As described above, the power of the present invention exemplified by the preferred embodiment of the present invention. The present invention should not be construed as being limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications, and literature references cited in this specification should be incorporated by reference as if the contents themselves were specifically described in the present specification. Is understood.
実施例  Example
[0260] 以下に、実施例に基づいて本発明を説明するが、以下の実施例は、例示の目的の みに提供される。従って、本発明の範囲は、上記発明の詳細な説明にも下記実施例 にも限定されるものではなぐ特許請求の範囲によってのみ限定される。  [0260] Hereinafter, the present invention will be described based on examples. However, the following examples are provided for illustrative purposes only. Accordingly, the scope of the present invention is limited only by the appended claims, which are not limited to the above detailed description of the invention nor the following examples.
[0261] 以下の化合物設計の説明では、これらの特定の化合物および糖転移酵素 (例えば 、フコース転移酵素）を例示として用いる力 これら以外の酵素および化合物であつ ても、本発明の範囲内に属する限り、同様の設計が可能であることに留意するべきで ある。  [0261] In the following description of compound design, the ability to use these specific compounds and glycosyltransferases (eg, fucose transferase) as examples. Other enzymes and compounds are also within the scope of the present invention. It should be noted that as long as a similar design is possible.
[0262] [化 113]  [0262] [Chem 113]
Figure imgf000125_0001
Figure imgf000125_0001
1 [0263] [化 114] 1 [0263] [Chem 114]
Figure imgf000126_0001
Figure imgf000126_0001
2  2
[0264] [化 115]  [0264] [Chemical 115]
Figure imgf000126_0002
Figure imgf000126_0002
3
Figure imgf000126_0003
MS Three
Figure imgf000126_0003
MS
4 [0266] [化 117] Four [0266] [Chemical 117]
Figure imgf000127_0001
MS
Figure imgf000127_0001
MS
5  Five
[0267] 出発物質である D—ガラクトース力 化合物 5までは、過去に報告されている論文に 沿って合成 行った。 (Christian Vogel, Claudia Bergemann, Andrej— Jako b Ott, Thisbe K. Lindhorst, Joachim Theim, Wilhelm V. Dahlhoff, Ch rister Hallgren, Monica M. Palcic and Ole Hindsgaul, Liebigs Ann. ( 1997) 601 -612)  [0267] The starting material, D-galactose compound up to compound 5, was synthesized according to previously reported papers. (Christian Vogel, Claudia Bergemann, Andrej— Jako b Ott, Thisbe K. Lindhorst, Joachim Theim, Wilhelm V. Dahlhoff, Christer Hallgren, Monica M. Palcic and Ole Hindsgaul, Liebigs Ann. (1997) 601 -612)
[0268] [化 118]  [0268] [Chemical 118]
Figure imgf000127_0002
Figure imgf000127_0002
CH2OTBDMS CH 2 OTBDMS
6  6
[0269] [化 119] [0269] [Chemical 119]
L〇H2〇TBDMS L ○ H 2 ○ TBDMS
[0270] [化 120] [0270] [Chemical 120]
Figure imgf000128_0001
Figure imgf000128_0001
8  8
[0271] [化 121]  [0271] [Chemical 121]
Figure imgf000128_0002
Figure imgf000128_0002
9 9
[0272] (化合物 6 (6— O tーブチルジメチルシリル 2, 3 ;4, 5 ジー O イソプロピリジ ンー 1 O トシル D—ガラクチトーノレ)の合成）  [0272] (Synthesis of Compound 6 (6-O t-Butyldimethylsilyl 2, 3; 4,5 G O Isopropylidin 1 O Tosyl D—Galactotonole))
化合物 5 (2g, 5. 3 lmmol)をジクロロメタン（2ml)、ピリジン（10ml)に溶解し氷冷 下、塩ィ匕トシル（1. 5eq, 7. 97mmol, 1. 52g)、 4 ジメチルァミノピリジン（2. Oeq , 10. 62mmol, 1. 30g)をカ卩え、室温に戻して 15時間撹拌した。溶液をクロ口ホル ムで抽出し、 1N硫酸水溶液、飽和炭酸水素ナトリウム水溶液、食塩水の順に洗浄し 、クロ口ホルム層を回収後、硫酸マグネシウム上で乾燥させた。セライト濾過により乾 燥剤を濾過後、減圧濃縮によりクロ口ホルムを除去した後、残留物をシリカゲルクロマ トグラフィー (展開溶媒:へキサン Z酢酸ェチル =8Zlで精製し、分画を濃縮した。 得られた残留物をベンゼンで凍結乾燥することにより白色非晶質 6 (2. 73g, 96. 8 %)を得た。： R 0. 64(トルエン Z酢酸ェチル =3Zl) ;1H— NMR δ (CDC1 )；Compound 5 (2g, 5.3 lmmol) was dissolved in dichloromethane (2ml) and pyridine (10ml), and ice-cooled, salted tosyl (1.5eq, 7.97mmol, 1.52g), 4dimethylaminopyridine (2. Oeq, 10.62mmol, 1.30g) was added, and the mixture was warmed to room temperature and stirred for 15 hours. Extract the solution with chloroform and wash with 1N sulfuric acid aqueous solution, saturated aqueous sodium hydrogen carbonate solution and brine in this order. The black-form layer was collected and dried over magnesium sulfate. After the desiccant was filtered through Celite filtration, the chloroform was removed by vacuum concentration, and the residue was purified by silica gel chromatography (developing solvent: hexane Z ethyl acetate = 8 Zl, and the fraction was concentrated. The obtained residue was freeze-dried with benzene to obtain white amorphous 6 (2. 73 g, 96.8%): R 0.64 (toluene Z ethyl acetate = 3 Zl); 1H—NMR δ ( CDC1);
0. 09 (m, 6H, — Si— (CH ) ), 3. 89— 3. 96 (m, 9H, t— Bu), 1. 34, 1. 35,0. 09 (m, 6H, — Si— (CH)), 3. 89— 3. 96 (m, 9H, t— Bu), 1. 34, 1. 35,
I. 36. 1. 39 (each s, 12H, isopropyl) , 2.48 (s, 3H, — CH ), 3. 73 (dd, 1I. 36. 1. 39 (each s, 12H, isopropyl), 2.48 (s, 3H, — CH), 3. 73 (dd, 1
H, J 1. 3 Hz, J 4. 0 Hz, H— 6 a), 3. 80(t, 1H, J 7. 7 Hz, H—H, J 1. 3 Hz, J 4.0 Hz, H— 6 a), 3. 80 (t, 1H, J 7. 7 Hz, H—
2), 3. 87 (d, 1H, H-3), 3. 89 (dd, 1H, J 2. 8 Hz, H— 6/3), 3. 99 (m2), 3. 87 (d, 1H, H-3), 3. 89 (dd, 1H, J 2. 8 Hz, H— 6/3), 3. 99 (m
, 1H, H-4), 4. 13 (t, 1H, J 5. 1 Hz, H~lj3), 4. 15—4. 17(m, 1H,, 1H, H-4), 4.13 (t, 1H, J 5.1 Hz, H ~ lj3), 4.15—4.17 (m, 1H,
H-5), 4. 34 (dd, 1H, H— 1), 7. 37, 7. 84 (each d, 4H, aromatic) ; 13C- NMR δ (CDC1 ) ;0. 59, 0. 76(— Si— (CH ) ), 24. 44(t— Bu), 27. 65 (—H-5), 4.34 (dd, 1H, H-1), 7.37, 7.84 (each d, 4H, aromatic); 13C-NMR δ (CDC1); 0.59, 0.76 ( — Si— (CH)), 24. 44 (t— Bu), 27. 65 (—
CH ), 31. 96, 32. 81, 33. 03, 33. 11 (isopropyl) , 68. 94(C— 6), 75. 38 (CH), 31. 96, 32. 81, 33. 03, 33. 11 (isopropyl), 68. 94 (C-6), 75. 38 (
C-l), 83.40(C-3), 83. 84 (C- 2), 84. 20(C— 5), 87. 65(C— 4), 134.C-l), 83.40 (C-3), 83. 84 (C- 2), 84. 20 (C— 5), 87. 65 (C— 4), 134.
II, 135. 79 (aromatic)； Anal. Calcd. for C H O S ： C, 56. 57;H, 7. 98II, 135. 79 (aromatic); Anal. Calcd. For C H O S: C, 56. 57; H, 7. 98
;S, 6. 04. Found :C, 56.43;H, 7. 93;S 6. 24; FAB -MS calcd 531. 2 449(M+H+), found 531. 2422。 ; S, 6. 04. Found: C, 56.43; H, 7.93; S 6.24; FAB-MS calcd 531. 2 449 (M + H + ), found 531. 2422.
(化合物 7(1 アジドー 6— O—t—ブチルジメチルシリル—2, 3;4, 5 ジ—O— イソプロピリジン D—ガラクチトール）の合成）  (Synthesis of Compound 7 (1 Azido 6-O-t-butyldimethylsilyl-2, 3; 4, 5 di-O-isopropylidine D-galactitol)
化合物 6 (2. 73g, 5. 14mmol)をジメチルホルムアミド（20ml)に溶解し、アジ化 ナトリウム（4. Oeq, 20. 56mmol, 1. 34g)をカ卩ぇ 60°Cでー晚撹拌した。溶液を酢 酸ェチルで抽出後、食塩水で洗浄し、有機層を回収した。回収した有機層を硫酸マ グネシゥム上で乾燥後、乾燥剤をセライト濾過し、濾液を減圧濃縮する事により溶媒 を除去した。得られた残查をシリカゲルカラムクロマトグラフィー (展開溶媒:へキサン Z酢酸ェチル =45Zl)によりで精製した。分画を集めて濃縮した後、ベンゼンで凍 結乾燥し、 目的とする白色非晶質 7(1. 80g, 87. 3%)を得た。： R 0. 68 (トルエン Compound 6 (2.73 g, 5.14 mmol) was dissolved in dimethylformamide (20 ml), and sodium azide (4. Oeq, 20.56 mmol, 1.34 g) was stirred at 60 ° C. under agitation. The solution was extracted with ethyl acetate and then washed with brine to recover the organic layer. The collected organic layer was dried over magnesium sulfate, the desiccant was filtered through Celite, and the filtrate was concentrated under reduced pressure to remove the solvent. The resulting residue was purified by silica gel column chromatography (developing solvent: hexane Z ethyl acetate = 45 Zl). The fractions were collected and concentrated, and then freeze-dried with benzene to obtain the desired white amorphous 7 (1.80 g, 87.3%). : R 0. 68 (Toluene
Z酢酸ェチル =7Zl) ;1H—NMR δ (CDC1 ) ;0. 08 (m, 6H, —Si—(CH ) )Zethyl acetate = 7Zl); 1H-NMR δ (CDC1); 0.08 (m, 6H, —Si— (CH))
， 0. 91 (m, 9H， t-Bu), 1. 34， 1. 35， 1. 36， 1.42 (each s， 12H, isopropy 1), 3. 31 (dd, 1H, J 5. 9 Hz, J 14. 1 Hz, H— 6a), 3. 60(dd, 1H, J , 0.91 (m, 9H, t-Bu), 1.34, 1.35, 1.36, 1.42 (each s, 12H, isopropy 1), 3.31 (dd, 1H, J 5.9 Hz, J 14.1 Hz, H— 6a), 3.60 (dd, 1H, J
5, 6α βα, ββ  5, 6α βα, ββ
2. 8 Hz, H— 6 3), 3. 72 (dd, 1H, J 9. 5, 11. 3 Hz, H— 1 3), 3. 84— 2. 8 Hz, H— 6 3), 3. 72 (dd, 1H, J 9. 5, 11. 3 Hz, H— 1 3), 3. 84—
5, 6β 5, 6β
3. 90 (m, 3H, H— lj8, H— 3, H— 4), 4. 00 (m, 1H, H— 2), 4. 13 (m, 1H, H - 5) ; 13C - NMR™ (CDC1 ) ;1. 21, 1. 34(— Si— (CH ) ), 32. 85(t— Bu  3. 90 (m, 3H, H— lj8, H— 3, H— 4), 4.00 (m, 1H, H— 2), 4. 13 (m, 1H, H-5); 13C-NMR ™ (CDC1); 1. 21, 1. 34 (— Si— (CH)), 32. 85 (t— Bu
3 3 2  3 3 2
), 33. 65-33. 83(isopropyl), 59. 5(C— 6), 70. 69(C— 1), 85. 11 (C— 3) , 86. 28(C-4), 87. 73(C— 5), 89. 52(C~2)； Anal. Calcd. for C H N  ), 33. 65-33. 83 (isopropyl), 59.5 (C— 6), 70. 69 (C— 1), 85. 11 (C— 3), 86. 28 (C-4), 87 73 (C—5), 89. 52 (C ~ 2); Anal. Calcd. For CHN
18 35 3 18 35 3
O Si:C, 53. 84;H, 8. 78;N, 10.46. Found :C, 53. 75;H, 8. 74 ;N, 10. 3O Si: C, 53. 84; H, 8. 78; N, 10.46.Found: C, 53. 75; H, 8. 74; N, 10. 3
5 Five
9;FAB— MS calcd 402. 2425 (M+H+), found 402. 2444。 9; FAB— MS calcd 402. 2425 (M + H + ), found 402. 2444.
[0274] (化合物 8(1 アジドー 2, 3;4, 5 ジ O イソプロピリジン D—ガラクチトール )の合成） [0274] (Synthesis of Compound 8 (1 Azido 2, 3; 4, 5 di-O isopropylidine D-galactitol)
化合物 7(1. 80g, 4.48mmol)をテトラヒドロフラン（10ml)に溶かし、氷冷下テトラ ブチルアンモ -ゥムフルオライド（1M THF溶液， 1. 2eq, 5. 28mmol, 6. 17ml) を加え室温で 1時間撹拌した。減圧濃縮により反応溶媒を除去した後、残留物をシリ 力ゲルクロマトグラフィ（展開溶媒:へキサン Z酢酸ェチル =4Zl)で精製した。分画 を回収し減圧濃縮により溶媒を除去し、残留物をエタノールにより再結晶することで 白色粉末 8 (1. 23g, 95. 3%)を得た。： R 0. 21 (トルエン Z酢酸ェチル =7/1 ) ;1H— NMR δ (CDC1 ) ;1. 34, 1. 36, 1. 38, 1.42 (each s, 12H, isop  Compound 7 (1.80 g, 4.48 mmol) was dissolved in tetrahydrofuran (10 ml), and tetrabutylammonium fluoride (1M THF solution, 1.2 eq, 5.28 mmol, 6.17 ml) was added under ice-cooling and stirred at room temperature for 1 hour. . After removing the reaction solvent by concentration under reduced pressure, the residue was purified by silica gel chromatography (developing solvent: hexane Z ethyl acetate = 4Zl). The fractions were collected, the solvent was removed by concentration under reduced pressure, and the residue was recrystallized from ethanol to obtain white powder 8 (1.23 g, 95.3%). : R 0. 21 (Toluene Z ethyl acetate = 7/1); 1H— NMR δ (CDC1); 1. 34, 1. 36, 1. 38, 1.42 (each s, 12H, isop
3  Three
ropyl) , 3. 32 (dd, 1H, J 13. 2 Hz, J 5. 1 Hz, H— 6a), 3. 60 ( βα, ββ 5, 6α  ropyl), 3.32 (dd, 1H, J 13.2 Hz, J 5.1 Hz, H— 6a), 3.60 (βα, ββ 5, 6α
dd, 1H, J 12. 0 Hz, J 5. 6 Hz, H— 1 a ) , 3. 61 (dd, 1H, J 2 la, 1β la, 2 5, 6 β dd, 1H, J 12.0 Hz, J 5. 6 Hz, H— 1 a), 3. 61 (dd, 1H, J 2 la, 1β la, 2 5, 6 β
. 8 Hz, H-6j8), 3. 77 (t, 1H, J 7. 5 Hz, H— 3), 3. 79 (dd, 1H, J 8 Hz, H-6j8), 3. 77 (t, 1H, J 7.5 Hz, H— 3), 3. 79 (dd, 1H, J
3, 4 1 3, 4 1
2.4 Hz, H-lj8), 3. 85 (t, 1H, J 8. 0 Hz, H— 4) , 4. 03 (m, 1H β, 2 4, 5 2.4 Hz, H-lj8), 3.85 (t, 1H, J 8.0 Hz, H— 4), 4.03 (m, 1H β, 2 4, 5
, H-2), 4. 13 (m, 1H, H— 5) 3C— NMR δ (CDC1 ) ;28. 90— 29. 80  , H-2), 4.13 (m, 1H, H— 5) 3C— NMR δ (CDC1); 28. 90— 29. 80
3  Three
(isopropyl), 54. 76 (C— 6), 64. 04 (C— 1), 80. 81 (C— 3), 81. 50 ( C-4), 83. 09 (C-5), 84. 82 (C-2)； Anal. Calcd. for C H N O ： C,  (isopropyl), 54. 76 (C— 6), 64. 04 (C— 1), 80. 81 (C— 3), 81. 50 (C-4), 83. 09 (C-5), 84 82 (C-2); Anal. Calcd. For CHNO: C,
12 21 3 5 12 21 3 5
50. 16;H, 7. 37;N, 14. 63. Found :C, 50. 30;H, 7. 18;N, 14. 52; FAB -MS calcd 288. 1560 (M+H+), found 288. 1556。 50. 16; H, 7. 37; N, 14. 63.Found: C, 50. 30; H, 7. 18; N, 14. 52; FAB -MS calcd 288. 1560 (M + H + ), found 288. 1556.
[0275] (化合物 9(1, 2, 3, 4 O ァセチルー 6 アジドー L ガラクトース）の合成） 二口ナスフラスコに入れたジクロロメタン（5ml)を一 78°Cに冷却して窒素置換した。 塩ィ匕ォキサリル（2. Oeq, 8. 35mmol, 0. 73ml)、ジメチルスルホキシド（4. Oeq, 1 6. 7mmol, 1. 19ml)を滴下後、—78。Cのまま 5分撹拌した。ィ匕合物 8 (1. 20g, 4 . 18mmol)をジクロロメタン（3ml)に溶かしてシリンジでフラスコ内に加えた。 78°C のまま 15分撹拌し、トリエチルァミン（5. Oeq, 20. 88mmol, 2. 91ml)を加え徐々 に 0°Cに戻しながら 2時間撹拌した。溶液をクロ口ホルムで抽出し、食塩水で洗浄した 。有機層を回収し硫酸マグネシウム上で乾燥させた。セライト濾過により乾燥剤を除 去した後、減圧濃縮し得られた残留物をシリカゲルカラムクロマトグラフィー (展開溶 媒:へキサン Z酢酸ェチル =8Zl)で精製した。分画を減圧濃縮し溶媒を除去した 後、残留物をメタノール（5ml)に溶かし、 60%酢酸水溶液（10ml)をカ卩ぇ 60°Cで 4 時間撹拌した。減圧濃縮により反応溶液を除去し、さらにトルエンと数回共沸させるこ とにより水分を完全に除去した。得られた化合物をピリジン (30ml)に溶解し、氷冷下 、無水酢酸（15ml)をカ卩ぇ室温でー晚撹拌した。溶液をクロ口ホルムで抽出し、 1N 硫酸水溶液、飽和炭酸水素ナトリウム水溶液、食塩水の順に洗浄し、有機層を回収 後、硫酸マグネシウム上で乾燥させた。セライト濾過により乾燥剤を除去し、濾液を減 圧濃縮した。濃縮した残留物をシリカゲルカラムクロマトグラフィー (展開溶媒:へキサ ン Z酢酸ェチル =5Zl)で精製し分画を減圧濃縮し、既知化合物 9 (1. lg、 73%) をネ守た。 (Geeta ¾trivastava, Kanwal J. Kaur, Ole Hmasgaul, Monica M . Palcic, J. B. C. , 267 (1992) 22356- 22361) 0 (Synthesis of Compound 9 (1, 2, 3, 4 O Acetyl-6 Azido L Galactose)) Dichloromethane (5 ml) placed in a two-necked eggplant flask was cooled to 78 ° C. and purged with nitrogen. After adding salt oxalyl (2. Oeq, 8. 35 mmol, 0.73 ml) and dimethyl sulfoxide (4. Oeq, 1 6.7 mmol, 1. 19 ml) dropwise, -78. C was stirred for 5 minutes. Compound 8 (1.20 g, 4.18 mmol) was dissolved in dichloromethane (3 ml) and added to the flask with a syringe. The mixture was stirred at 78 ° C for 15 minutes, triethylamine (5. Oeq, 20. 88mmol, 2.91ml) was added, and the mixture was stirred for 2 hours while gradually returning to 0 ° C. The solution was extracted with black mouth form and washed with brine. The organic layer was collected and dried over magnesium sulfate. After removing the desiccant by Celite filtration, the residue obtained by concentration under reduced pressure was purified by silica gel column chromatography (developing solvent: hexane Z ethyl acetate = 8 Zl). The fraction was concentrated under reduced pressure to remove the solvent, the residue was dissolved in methanol (5 ml), and a 60% aqueous acetic acid solution (10 ml) was stirred at 60 ° C. for 4 hours. The reaction solution was removed by concentration under reduced pressure, and water was completely removed by azeotropic distillation with toluene several times. The obtained compound was dissolved in pyridine (30 ml), and acetic anhydride (15 ml) was stirred at room temperature under ice cooling. The solution was extracted with chloroform, washed with 1N sulfuric acid aqueous solution, saturated aqueous sodium hydrogen carbonate solution and brine in this order, and the organic layer was collected and dried over magnesium sulfate. The desiccant was removed by celite filtration, and the filtrate was concentrated under reduced pressure. The concentrated residue was purified by silica gel column chromatography (developing solvent: hexane Z ethyl acetate = 5 Zl), and the fraction was concentrated under reduced pressure to protect the known compound 9 (1. lg, 73%). (Geeta ¾trivastava, Kanwal J. Kaur, Ole Hmasgaul, Monica M. Palcic, JBC, 267 (1992) 22356-22361) 0
[0276] [化 122]  [0276] [Chemical 122]
〇Ac  ○ Ac
Figure imgf000131_0001
Figure imgf000131_0001
〇Ac  ○ Ac
[0277] [化 123]
Figure imgf000132_0001
[0277] [Chemical 123]
Figure imgf000132_0001
Figure imgf000132_0002
Figure imgf000132_0002
[0278] [化 124]  [0278] [Chemical 124]
Figure imgf000132_0003
Figure imgf000132_0003
11  11
[0279] 化合物 9を出発物質として、報告例に沿って化合物 10を合成した。 (Geeta Striv astava, Kanwal J. Kaur, Ole Hindsgaul, Monica M. Palcic, J. B. C. , 26 7 (1992) 22356- 22361) [0279] Compound 10 was synthesized according to the reported examples using compound 9 as a starting material. (Geeta Striv astava, Kanwal J. Kaur, Ole Hindsgaul, Monica M. Palcic, J. B.C., 26 7 (1992) 22356-22361)
化合物 10 (210mg, 0. 54mmol)をピリジンと 3回程共沸させることにより、水を除 去した。その後、化合物 10をピリジン（3ml)に溶解し、 GMPモルフオリデート（2. Oe q, 791. 10mg)、および 1H—テトラゾル（4. Oeq, 151. 31mg)を加えて、窒素置 換し、 2日間攪拌した。  Water was removed by azeotroping compound 10 (210 mg, 0.54 mmol) with pyridine three times. Then, compound 10 is dissolved in pyridine (3 ml), GMP morpholidate (2. Oe q, 791. 10 mg) and 1H-tetrazole (4. Oeq, 151. 31 mg) are added, and the nitrogen is replaced. Stir for 2 days.
[0280] 反応終了後、ピリジンを減圧濃縮により除去した。得られた残留物を水に溶解し、ジ ェチルエーテルを加え洗净後、水層を回収した。得られた水層を減圧濃縮し、逆相 シリカゲルカラムクロマトグラフィー（展開溶媒:水）、 DEAEイオン交換カラムクロマト グラフィー（展開溶媒: 0. 06mM 炭酸水素アンモ-ゥム水溶液）、ゲル濾過カラムク 口マトグラフィ（展開溶媒：水）の順に精製し、最後に Dowex50X8イオン交換クロマト グラフィ (展開溶媒:水）により、リン酸をナトリウム塩に変換することにより、化合物 11 を収率 11%で得た。 [0280] After completion of the reaction, pyridine was removed by concentration under reduced pressure. The obtained residue was dissolved in water, washed with diethyl ether, and the aqueous layer was recovered. The obtained aqueous layer was concentrated under reduced pressure, reversed-phase silica gel column chromatography (developing solvent: water), DEAE ion exchange column chromatography (developing solvent: 0.06 mM aqueous ammonium hydrogen carbonate solution), gel filtration column chromatography. Purification was performed in the order of mouth matography (developing solvent: water), and finally, phosphoric acid was converted to a sodium salt by Dowex50X8 ion exchange chromatography (developing solvent: water), whereby Compound 11 was obtained in a yield of 11%.
[0281] 1H-NMR;8.52(1H, H-8), 4.96(1H, t, J =J =7.8 Hz, H，，一 1)  [0281] 1H-NMR; 8.52 (1H, H-8), 4.96 (1H, t, J = J = 7.8 Hz, H, 1)
1, 2 1, P  1, 2 1, P
, 3.88(1H, d, J =3.2 Hz , H，，一 4), 3.79(1H, m, H，，一 5), 3.67(1  , 3.88 (1H, d, J = 3.2 Hz, H, 1), 3.79 (1H, m, H, 1), 3.67 (1
3, 4  3, 4
H, dd, J =9.6Hz, H，，一 3), 3.61— 3.59 (2H, m, H，，一 2, H，，一 6a),  H, dd, J = 9.6Hz, H, 1 3), 3.61—3.59 (2H, m, H, 1 2, H, 1 6a),
2, 3  twenty three
3.46 (1H, dd, J =13.0 Hz, J =5.5 Hz, H，，一 6 j8 )。  3.46 (1H, dd, J = 13.0 Hz, J = 5.5 Hz, H, 1 6 j8).
5, 6  5, 6
[0282] [化 125] [0282] [Chemical 125]
〔〕S02832 [] S02832
Figure imgf000134_0001
Figure imgf000134_0001
H H
I
Figure imgf000135_0001
I
Figure imgf000135_0001
[0284] (グリコシルァクセプターの合成（軽水素化合物にっ 、て））  [0284] (Glycosyl acceptor synthesis (light hydrogen compounds))
キトトリオース（7. 4mg, 11. 8 mol)を反応溶液（lOOmM 酢酸緩衝液 200 1 )に溶解し、アミノォキシ一 Trp— Arg— OMe試薬（aoWR (H) , 13mg, 25 μ mol) を加え、 90°Cで一時間反応する事により標識化合物を得た。冷却後、反応液をマトリ タス溶液（2, 5—ジヒドロキシ安息香酸、 lOmgZmL 30%ァセトニトリル溶液）で 10 0倍に混合し、 MALDI—TOF MS (Bruker社製、 UltraFLEX)で直接質量分析 することによって、標識キトトリオースのみ力もなるスペクトルを得た。 （スペクトルは図 1上）  Dissolve chitotriose (7.4 mg, 11.8 mol) in the reaction solution (lOOmM acetate buffer 200 1), add aminooxy-trp-Arg-OMe reagent (aoWR (H), 13 mg, 25 μmol), and add 90 By reacting at ° C for 1 hour, a labeled compound was obtained. After cooling, the reaction solution is mixed 100 times with Matritas solution (2,5-dihydroxybenzoic acid, lOmgZmL 30% acetonitrile solution) and directly subjected to mass spectrometry with MALDI-TOF MS (Bruker, UltraFLEX). A spectrum with only the power of labeled chitotriose was obtained. (Spectrum above figure 1)
(グリコシルァクセプターの合成 (重水素化合物にっ 、て） )  (Synthesis of glycosyl acceptors (deuterium compounds))
キトトリオース（7. 4mg, 11. 8 mol)を反応溶液（lOOmM 酢酸緩衝液 200 1 )に溶解し、 d3—アミノォキシ一 Trp— Arg— OMe試薬（aoWR (D) , 13mg, 25 μ mol)を加え、 90°Cで一時間反応する事により標識化合物を得た。冷却後、反応液を マトリクス溶液（2, 5—ジヒドロキシ安息香酸、 lOmgZmL 30%ァセトニトリル溶液） で 100倍に混合し、 MALDI— TOF MS (Bruker社製、 UltraFLEX)で直接質量 分析することによって、標識キトトリオースのみ力もなるスペクトルを得た。  Dissolve chitotriose (7.4 mg, 11.8 mol) in the reaction solution (lOOmM acetate buffer 200 1), and add d3-aminoxy Trp-Arg-OMe reagent (aoWR (D), 13 mg, 25 μmol). The labeled compound was obtained by reacting at 90 ° C. for 1 hour. After cooling, the reaction mixture was mixed 100 times with a matrix solution (2,5-dihydroxybenzoic acid, lOmgZmL 30% acetonitrile solution), and labeled directly by MALDI-TOF MS (Bruker, UltraFLEX). A spectrum with only chitotriose power was obtained.
[0285] (aoWR標識ガラクトシルキトトリオースの合成） [0285] (Synthesis of aoWR-labeled galactosylchitotriose)
軽および重水素 aoWR標識キトトリオース（11. 8 μ mol)に 1M重炭酸緩衝液（600 1)を加え pHを 7. 0とした後、 UDP—ガラクトース（1. 5eq, 9. 2mg,ャマサ醤油 株式会社)、およびガラクトース転移酵素（30mU,東洋紡社製)を添加後、さらに最 終濃度が 2mMとなるように塩ィ匕マンガンをカ卩え、 2日間、ガラクトース糖転移反応を 行った。反応液を一部とり、マトリクス溶液（2, 5—ジヒドロキシ安息香酸、 lOmgZm L 30%ァセトニトリル溶液）で 100倍に混合し、 MALDI— TOF MS (Bruker社製 、 UltraFLEX)で直接質量分析することによって、標識ガラクトシルキトトリオースの み力もなるスペクトルを得た。（スペクトルは図 1下)。 Light and deuterium After adding 1M bicarbonate buffer (600 1) to aoWR labeled chitotriose (11.8 μmol) to pH 7.0, UDP-galactose (1.5 eq, 9.2 mg, yamasa soy sauce Co., Ltd.) and galactose transferase (30 mU, manufactured by Toyobo Co., Ltd.) were added, and further, salt and manganese were prepared so that the final concentration was 2 mM, and galactose sugar transfer reaction was performed for 2 days. Take a part of the reaction solution, mix 100 times with a matrix solution (2,5-dihydroxybenzoic acid, lOmgZm L 30% acetonitrile solution), and perform direct mass analysis with MALDI-TOF MS (Bruker, UltraFLEX) In addition, a spectrum having only the power of labeled galactosylchitotriose was obtained. (The spectrum is the bottom of Figure 1).
[0286] 軽および重水素 aoWR標識ガラクトシルキトトリオースをァクセプターとしてフコース 糖転移反応を行った。 [0286] Light and deuterium fucose transglycosylation was performed using aoWR-labeled galactosylchitotriose as an acceptor.
[0287] 内部標準は重水素標識体、阻害剤活性評価は軽水素標識体で行った。 [0287] The internal standard was a deuterium label, and the inhibitor activity was evaluated with a light hydrogen label.
[0288] 化合物 11 (lOmM水溶液） 2 μ リン酸アセチレン誘導体（lOmM) 2 μ ァスコ ルビン酸ナトリウム（20mM) 1 μ Lおよび硫酸銅（lOmM) 1 μ Lを混合して室温で 12 時間反応させ、生成物の存在を ESI— MSによって確認した。 [0288] Compound 11 (lOmM aqueous solution) 2 μ Acetylene phosphate derivative (lOmM) 2 μ Sodium alcoholate (20 mM) 1 μL and copper sulfate (lOmM) 1 μL were mixed and reacted at room temperature for 12 hours. The presence of product was confirmed by ESI—MS.
[0289] 結果を図 2に示す。示されるように、生成物が二価イオンで検出された。 [0289] The results are shown in FIG. As shown, the product was detected with divalent ions.
[0290] 反応溶液（lOmM塩化マンガンを含む 50mM HEPES緩衝液， 25 1)に GDPフ コース、 aoWR標識ガラクトシルキトトリオース、ァスコルビン酸ナトリウム、および硫酸 銅をそれぞれ最終濃度力 200 μ Μ, ImM, 400 μ Μ, 200 μ Μ、となるようにカロえ 調整した。フコース転移酵素（2. 5mU, 5 1)を添加し、反応温度 37°Cで 40分間反 応した後、反応溶液をァセトニトリルで 10倍に希釈し、酵素を失活させた。反応液を さらに水で 10倍に希釈した後、マトリクス溶液（2, 5—ジヒドロキシ安息香酸、 10mg ZmL 30%ァセトニトリル溶液）で 10倍に混合し、 MALDI—TOF MS (Bruker 社製、 UltraFLEX)で直接質量分析した。 [0290] The reaction solution (50 mM HEPES buffer containing lOmM manganese chloride, 25 1) was added with GDP fucose, aoWR-labeled galactosyl chitotriose, sodium ascorbate, and copper sulfate, respectively, with a final concentration of 200 μΜ, ImM, 400 The calorie was adjusted to μ Μ and 200 μ μ. After addition of fucose transferase (2.5 mU, 51) and reaction at a reaction temperature of 37 ° C for 40 minutes, the reaction solution was diluted 10-fold with acetonitrile to inactivate the enzyme. The reaction solution was further diluted 10-fold with water, mixed 10-fold with a matrix solution (2,5-dihydroxybenzoic acid, 10 mg ZmL 30% acetonitrile solution), and MALDI-TOF MS (Bruker, UltraFLEX). Direct mass spectrometry was performed.
[0291] 前記の反応条件（1)に更に阻害剤（200 M)をカ卩えたもの（2)、また、コントロー ルとして 6アジド GDPフコース（200 μ Μ)を加えたもの（3)およびアセチレン化合物（ 200 μ Μ)を加えたもの（4)の計 4つの反応を行った。反応条件は以下の通りである [0291] The above reaction conditions (1) with an additional inhibitor (200 M) (2), and 6-azide GDP fucose (200 μΜ) as a control (3) and acetylene A total of four reactions were carried out (4) with the addition of the compound (200 μΜ). The reaction conditions are as follows:
[0292] 条件 [0292] Conditions
(1)  (1)
緩衝液： 50mM HEPES, pH 7. 2、 lOmM MnC12 ドナー： 200 Buffer: 50 mM HEPES, pH 7.2, lOmM MnC12 Donor: 200
ァクセプター： ImM  Acceptor: ImM
酵素： フコース転移酵素（FucT)V 2. 5mU  Enzyme: Fucose transferase (FucT) V 2.5 mU
総量； 25 ;z L  Total amount; 25; z L
(共通）  (Common)
それとプラスして  Plus that
(2)阻害剤混合溶液 200 M  (2) Inhibitor mixed solution 200 M
(3)アジド GDP—フコース 200 Μ  (3) Azide GDP—Fucose 200 Μ
(4)アセチレン化合物 200 /ζ Μ  (4) Acetylene compound 200 / ζ Μ
をそれぞれ（1)にカ卩えた。  Was placed in (1).
[0293] その結果を図 3に示す。示されるように、本発明の化合物が、フコース転移酵素の 阻害能力を持つことがわ力つた。酵素反応の生成物の相対比を示しており、小さい ほど阻害活性が強 、ことを示す。  The results are shown in FIG. As shown, the compounds of the present invention were found to have the ability to inhibit fucose transferase. The relative ratio of the product of the enzyme reaction is shown. The smaller the value, the stronger the inhibitory activity.
[0294] 発明したィ匕合物に阻害活性が有することが明らかになつたので、経時変化の観測 を行った。  [0294] The invented compound was found to have inhibitory activity, and changes with time were observed.
[0295] 反応溶液（10mM塩化マンガンを含む 50mM HEPES緩衝液， 25 1)に GDPフ コース、 aoWR標識ガラクトシルキトトリオース、ァスコルビン酸ナトリウム、および硫酸 銅をそれぞれ最終濃度力 200 μ Μ, ImM, 400 μ Μ, 200 μ Μ、となるように添カロし た。フコース転移酵素（2. 5mU, 5 1)を添加し、反応温度 37°Cで反応した。 10分 、 20分、 30分、 60分、 80分経過ごとに反応液を一部とり反応溶液をァセトニトリルで 10倍に希釈することで、酵素を失活した。反応液をさらに水で 10倍に希釈した後、 マトリクス溶液（2, 5—ジヒドロキシ安息香酸、 lOmgZmL 30%ァセトニトリル溶液） で 10倍に混合し、 MALDI-TOF MS (Bruker社製、 UltraFLEX)で直接質量 分析した。  [0295] The reaction solution (50 mM HEPES buffer containing 10 mM manganese chloride, 25 1) was added with GDP fucose, aoWR-labeled galactosylchitotriose, sodium ascorbate, and copper sulfate, respectively, with a final concentration of 200 μΜ, ImM, 400 We added it so that it became μ Μ, 200 μΜ. Fucose transferase (2.5 mU, 51) was added, and the reaction was performed at a reaction temperature of 37 ° C. The enzyme was inactivated by taking a part of the reaction solution every 10 minutes, 20 minutes, 30 minutes, 60 minutes, and 80 minutes and diluting the reaction solution 10-fold with acetonitrile. The reaction solution was further diluted 10-fold with water, mixed 10-fold with a matrix solution (2,5-dihydroxybenzoic acid, lOmgZmL 30% acetonitrile solution), and directly with MALDI-TOF MS (Bruker, UltraFLEX). Mass analyzed.
[0296] 前記の反応条件（―）に更に阻害剤混合物（200 M)を加えたもの（+ )の計 2つ の反応を行った。反応条件は以下の通りである。  [0296] A total of two reactions were carried out: the reaction condition (-) plus the inhibitor mixture (200 M) (+). The reaction conditions are as follows.
[0297] 条件 [0297] Conditions
(一） 緩衝液： 50mM HEPES, pH 7. 2、 lOmM MnC12 (one) Buffer: 50 mM HEPES, pH 7.2, lOmM MnC12
ドナー： 200  Donor: 200
ァクセプター： ImM  Acceptor: ImM
酵素： フコース転移酵素（FucT)V 2. 5mU  Enzyme: Fucose transferase (FucT) V 2.5 mU
総量； 25 ;z L  Total amount; 25; z L
それとプラスして  Plus that
(+ )阻害剤混合溶液 200 M  (+) Inhibitor mixed solution 200 M
を加えた。  Was added.
[0298] その結果を図 4に示す。示されるように、阻害剤を加えた場合の阻害活性を経時的 に測定することが出来た。  The results are shown in FIG. As shown, the inhibitory activity when an inhibitor was added could be measured over time.
[0299] 阻害定数 Kiを算出するために、ディクソンプロットの作製を行った。  [0299] In order to calculate the inhibition constant Ki, a Dixon plot was prepared.
[0300] ドナーの濃度を 50 μ Μ、 200 μ Μおよび阻害剤の濃度を 100 Μ、 50 Μ、 25 μ Μ、 12. Μ、 0 /ζ Μで測定した。上記の経時変化の結果より、酵素反応時間は 初速度の測定範囲である 10分とした。反応条件は以下の通りである。  [0300] Donor concentrations were measured at 50 μΜ, 200 μΜ, and inhibitor concentrations at 100, 50, 25 μΜ, 12.Μ, and 0 / ζ ド ナ ー. Based on the results of the change over time, the enzyme reaction time was set to 10 minutes, which is the initial velocity measurement range. The reaction conditions are as follows.
[0301] 条件  [0301] Conditions
緩衝液： 50mM HEPES, pH 7. 2、 10mM MnC12  Buffer: 50 mM HEPES, pH 7.2, 10 mM MnC12
ドナー： 50若しく ίま 200 /z M  Donor: 50 young ίMA 200 / z M
ァクセプター： ImM  Acceptor: ImM
阻害剤混合溶液： 0〜： LOO M  Inhibitor mixed solution: 0 ~: LOO M
酵素： フコース転移酵素（FucT)V 2. 5mU  Enzyme: Fucose transferase (FucT) V 2.5 mU
総量； 25 ;z L  Total amount; 25; z L
[0302] 以上の結果より、ディクソンプロット（図 5)を作製した。図 5に示すような直線が得ら れ、 Kiは 19. 6 μ Μと算出できた。  [0302] Based on the above results, a Dixon plot (Fig. 5) was prepared. A straight line as shown in Fig. 5 was obtained, and Ki was calculated to be 19.6 μΜ.
[0303] [化 126A] [0303] [Chemical 126A]
Figure imgf000139_0001
Figure imgf000139_0001
[化 126B] [Chemical 126B]
Figure imgf000140_0001
Figure imgf000140_0001
Exact Mass: 82.0783
Figure imgf000140_0002
Exact Mass: 110.1096
Figure imgf000140_0003
Exact Mass: 82.0783
Figure imgf000140_0002
Exact Mass: 110.1096
Figure imgf000140_0003
Exact Mass: 208.2191 126C] Exact Mass: 208.2191 126C]
45
Figure imgf000141_0001
45
Figure imgf000141_0001
Exact Mass: 124.0888  Exact Mass: 124.0888
Figure imgf000141_0002
Figure imgf000141_0002
L3N ^γγ^ο〜ο Ο ο^%L3N ^ γγ ^ ο 〜 ο Ο ο ^%
^¾¾^J ^J Exact Mass: 328.17 ^ ¾¾ ^ J ^ J Exact Mass : 328.17
] ]
Figure imgf000142_0001
Figure imgf000142_0001
[0307] [化 126E]
Figure imgf000143_0001
[0307] [Chemical 126E]
Figure imgf000143_0001
[0308] [化 126F] [0308] [Chemical 126F]
Figure imgf000143_0002
Figure imgf000143_0002
であって、 Rが以下である:  Where R is:
[0309] [化 126G] [0309] [Chemical 126G]
Figure imgf000144_0001
Figure imgf000144_0001
[0310] [化 126H] [0310] [Chemical 126H]
Figure imgf000145_0001
化合物 ll(10mM水溶液）を 2/zL、アセチレン誘導体（12a〜12e、アセチレンラ イブラリの A1〜A36、 L2Nおよび L3N、 10mM)を 2/zL、ァスコルビン酸ナトリウム と硫酸銅を混ぜた水溶液 (ァスコルビン酸ナトリウム Z硫酸銅 = 20mMZl0mM) 2 μ Lを混合して室温で 12時間反応させ、生成物の存在を ESI— MSによって確認し た。
Figure imgf000145_0001
Compound ll (10 mM aqueous solution) 2 / zL, acetylene derivatives (12a-12e, acetylene library A1-A36, L2N and L3N, 10 mM) 2 / zL, sodium ascorbate 2 μL of an aqueous solution containing sodium sulfate and copper sulfate (sodium ascorbate Z copper sulfate = 20 mM Zl0 mM) was mixed and reacted at room temperature for 12 hours, and the presence of the product was confirmed by ESI-MS.
[0312] 結果を図 6Aに示す。示されるように、化合物 13a〜eの生成を確認した。ァセチレ ン化合物（アセチレンライブラリの A1〜A36、 L2Nおよび L3N)について、クリック反 応の収率を ESI— MSで測定した。その結果を図 6Bに示す。収率はほぼ 100%であ つた。グラフの収率は ESI— MSネガティブモードでのピーク強度比の相対比を示す  [0312] The results are shown in Figure 6A. As shown, formation of compounds 13a-e was confirmed. For the acetylene compounds (acetylene library A1-A36, L2N and L3N), the click reaction yield was measured by ESI-MS. The results are shown in Figure 6B. The yield was almost 100%. Graph yield shows relative ratio of peak intensity ratio in ESI-MS negative mode
[0313] 反応溶液（10mM塩化マンガンを含む 50mM HEPES緩衝液， 25 1)に GDPフ コース、 aoWR標識ガラクトシルキトトリオース、ァスコルビン酸ナトリウム、および硫酸 銅をそれぞれ最終濃度力 200 μ Μ, ImM, 400 μ Μ, 200 μ Μ、となるように添カロし た。フコース転移酵素（2. 5mU, 5 1)を添加し、反応温度 37°Cで反応した。 40分 反応させた後、反応溶液をァセトニトリルで 10倍に希釈し、酵素を失活させた。さらに 反応液を水で 10倍に希釈した後、マトリクス溶液（2, 5—ジヒドロキシ安息香酸、 10 mg/mL 30%ァセトニトリル溶液）で 10倍に混合し、 MALDI—TOF MS (Bruk er社製、 UltraFLEX)で直接質量分析した。 [0313] The reaction solution (50 mM HEPES buffer containing 10 mM manganese chloride, 25 1) was added with GDP fucose, aoWR-labeled galactosylchitotriose, sodium ascorbate, and copper sulfate, respectively, with a final concentration of 200 μΜ, ImM, 400 We added it so that it became μ Μ, 200 μΜ. Fucose transferase (2.5 mU, 51) was added, and the reaction was performed at a reaction temperature of 37 ° C. After reacting for 40 minutes, the reaction solution was diluted 10-fold with acetonitrile to inactivate the enzyme. The reaction solution was further diluted 10-fold with water, mixed 10-fold with a matrix solution (2,5-dihydroxybenzoic acid, 10 mg / mL 30% acetonitrile solution), and MALDI-TOF MS (manufactured by Bruker, Direct mass spectrometry with UltraFLEX).
[0314] 前記の反応条件 (コントロール）に更に阻害剤候補化合物混合物（200 μ Μ)をカロ えたもの（ 13a〜 13e、 N3GDP— Fuc ( + ) )およびその阻害剤候補ィ匕合物に対応す るアセチレン誘導体のみを添カ卩したもの（ 12a〜 12e、 N3GDPFuc (―) )の計 11種 、ならびに阻害剤候補ィ匕合物混合物（30 μ Μ)を加えたもの（アセチレンィ匕合物：ァ セチレンライブラリの A1〜A36、 L2Nおよび L3N、 N3GDP—Fuc ( + ) )およびそ の阻害剤候補ィ匕合物に対応するアセチレン誘導体のみを添加したもの（アセチレン ライブラリの A1〜A36、 L2Nおよび L3N、 N3GDPFuc (—））の計 76種の反応を行 つた。反応条件は以下の通りである。  [0314] Corresponding to the mixture of inhibitor candidate compounds (200 μΜ) (13a to 13e, N3GDP—Fuc (+)) and the inhibitor candidate compounds in addition to the above reaction conditions (control) 11 types (12a to 12e, N3GDPFuc (-)) with a mixture of only acetylene derivatives, and a mixture of inhibitor candidate compounds (30 μ μ) (acetylene compounds: A1 to A36 of the acetylene library, L2N and L3N, N3GDP—Fuc (+)) and acetylene derivatives corresponding to the candidate inhibitor compounds were added (A1 to A36 of the acetylene library, L2N and A total of 76 reactions (L3N, N3GDPFuc (—)) were performed. The reaction conditions are as follows.
[0315] 条件  [0315] Conditions
(コントロール）  (Control)
緩衝液： 50mM HEPES, pH 7. 2、 10mM MnC12  Buffer: 50 mM HEPES, pH 7.2, 10 mM MnC12
ドナー： 200 ァクセプター： ImM Donor: 200 Acceptor: ImM
酵素： フコース転移酵素（FucT)V 2. 5mU  Enzyme: Fucose transferase (FucT) V 2.5 mU
総量； 25 ;z L  Total amount; 25; z L
これらは共通。  These are common.
それとプラスして  Plus that
阻害剤候補化合物混合溶液（13a〜13e、 N3GDP— Fuc ( + ) ) 200 M若しくは アセチレン誘導体（12a〜12e、 N3GDPFuc (—）） 200 Mを加えた。  Inhibitor candidate compound mixed solution (13a-13e, N3GDP—Fuc (+)) 200 M or acetylene derivative (12a-12e, N3GDPFuc (—)) 200 M was added.
[0316] その結果を図 7Aおよび図 7Bに示す。図 7Aに示されたように、遊離のアセチレン 誘導体よりも、クリック反応によって化合物 11にアセチレン誘導体が結合した場合 (N 3GDPFuc ( + ) )に、より強い阻害活性があることが明らかになった。また、ナフタレン 含有糖ヌクレオチド（13b)は他の 4種類と比較して、非常に強い活性が有ることもわ 力つた。図 7Aでは酵素反応の生成物の相対比を示しており、小さいほど阻害活性が 強いことを示す。さらに、図 7Bに示されたように、アセチレンライブラリの A16、 A31、 A34. L2Nおよび L3N (13bと同一の化合物）が 60%以上の阻害率を示した。  The results are shown in FIGS. 7A and 7B. As shown in FIG. 7A, it was revealed that there was a stronger inhibitory activity when the acetylene derivative was bound to the compound 11 by the click reaction (N 3GDPFuc (+)) than the free acetylene derivative. It was also demonstrated that naphthalene-containing sugar nucleotides (13b) have very strong activity compared to the other four types. FIG. 7A shows the relative ratio of the products of the enzyme reaction, and the smaller the value, the stronger the inhibitory activity. Furthermore, as shown in FIG. 7B, A16, A31, A34. L2N and L3N (same compounds as 13b) of the acetylene library showed an inhibition rate of 60% or more.
[0317] 5種類の阻害剤候補ィ匕合物において、もっとも強い阻害活性を持つナフタレン含有 糖ヌクレオチド（13b)について、経時変化の観測を行った。  [0317] Of the five candidate inhibitor compounds, the time course of the naphthalene-containing sugar nucleotide (13b) having the strongest inhibitory activity was observed.
[0318] 反応溶液（10mM塩化マンガンを含む 50mM HEPES緩衝液， 25 1)に GDPフ コース、 aoWR標識ガラクトシルキトトリオース、ァスコルビン酸ナトリウム、および硫酸 銅をそれぞれ最終濃度力 200 μ Μ, ImM, 400 μ Μ, 200 μ Μ、となるように添カロし た。フコース転移酵素（2. 5mU, 5 1)を添加し、反応温度 37°Cで反応した。 10分 、 20分、 30分、 60分、 80分、 160分経過ごとに反応液を一部とり反応溶液をァセト 二トリルで 10倍に希釈し、酵素を失活させた。反応液をさらに水で 10倍に希釈した 後、マトリクス溶液（2, 5—ジヒドロキシ安息香酸、 10mg/mL 30%ァセトニトリル溶 液）で 10倍に混合し、 MALDI-TOF MS (Bruker社製、 UltraFLEX)で直接質 量分析した。  [0318] The reaction solution (50 mM HEPES buffer containing 10 mM manganese chloride, 25 1) was added with GDP fucose, aoWR-labeled galactosylchitotriose, sodium ascorbate, and copper sulfate, respectively, with a final concentration of 200 μΜ, ImM, 400 We added it so that it became μ Μ, 200 μΜ. Fucose transferase (2.5 mU, 51) was added, and the reaction was performed at a reaction temperature of 37 ° C. A portion of the reaction solution was taken every 10 minutes, 20 minutes, 30 minutes, 60 minutes, 80 minutes, and 160 minutes, and the reaction solution was diluted 10-fold with acetonitrile and inactivated the enzyme. The reaction solution was further diluted 10-fold with water, then mixed 10-fold with a matrix solution (2,5-dihydroxybenzoic acid, 10 mg / mL 30% acetonitrile solution), and MALDI-TOF MS (Bruker, UltraFLEX) was mixed. ) And mass analysis directly.
[0319] 前記の反応条件 (コントロール）に更にナフタレン含有糖ヌクレオチド混合物（13b、 200 M)を加えたもの、若しくは Kiが判明しているリン酸を有する阻害剤混合物（1 3e、 200 M)を加えたものの計 3種類の反応を行った。反応条件は以下の通りであ る。 [0319] A mixture of the above-mentioned reaction conditions (control) with a naphthalene-containing sugar nucleotide mixture (13b, 200 M), or an inhibitor mixture (13 e, 200 M) having a phosphate with known Ki. A total of three types of reactions were performed. The reaction conditions are as follows: The
[0320] 条件  [0320] Conditions
(コントロール）  (Control)
緩衝液： 50mM HEPES, pH 7. 2、 10mM MnC12  Buffer: 50 mM HEPES, pH 7.2, 10 mM MnC12
ドナー： 200  Donor: 200
ァクセプター： ImM  Acceptor: ImM
酵素： フコース転移酵素（FucT) V 2. 5mU  Enzyme: Fucose transferase (FucT) V 2.5 mU
総量； 25 ;z L  Total amount; 25; z L
それとプラスして  Plus that
(13b)ナフタレンを有する阻害剤混合溶液 200 μ Μ  (13b) Naphthalene-containing inhibitor mixed solution 200 μΜ
若しくは  Or
(13e)リン酸を有する阻害剤混合溶液 200 μ Μ  (13e) Phosphate-containing inhibitor mixed solution 200 μΜ
を加えた。  Was added.
[0321] その結果を図 8Αに示す。示されるように、阻害剤を加えた場合の阻害活性を経時 的に測定することが出来た。また、 Kiが 19. 6 Mと算出されているリン酸誘導体と比 較しても、ナフタレン誘導体は非常に強い阻害活性を有していることが明らかになつ た。  [0321] The results are shown in Fig. 8 (b). As shown, the inhibitory activity when an inhibitor was added could be measured over time. In addition, it became clear that the naphthalene derivative has a very strong inhibitory activity even when compared with the phosphate derivative whose Ki is calculated to be 19.6 M.
[0322] (阻害実験）  [0322] (Inhibition experiment)
以下の 4つにっ 、て阻害活¾を測定した。  The inhibition activity was measured by the following four methods.
(1)ドナーのみのもの  (1) Donor only
(2)ドナー +阻害剤混合溶液のもの  (2) Donor + inhibitor mixed solution
(3)クリックの際に残っていた N3GDPFucoseを混ぜたもの  (3) A mixture of N3GDPFucose left over when clicking
(4)クリックの際に残っていたリン酸アルキンを混ぜたもの  (4) A mixture of alkyne phosphates remaining at the time of click
反応条件は以下の通りである。  The reaction conditions are as follows.
条件  Condition
緩衝液： 50mM HEPES, pH 7. 2, 10mM MnC12  Buffer: 50 mM HEPES, pH 7.2, 10 mM MnC12
ドナー 200  Donor 200
ァクセプター ImM 酵素：フコース転移酵素（FucT)V, lmU/ lO ^ L Acceptor ImM Enzyme: Fucose transferase (FucT) V, lmU / lO ^ L
総量：25 ;z L Total amount: 25; z L
これらは共通。 These are common.
それとプラスして Plus that
(2)リン酸アルキン 2mM、 (3) N3GDPFuc 2mM、または（4)阻害剤混合溶液 200 Mを加えた。重水素 aoWR標識化合物を内部標準として用いて MALDI— T OFMS (Bruker社製、 UltraFLEX)で直接質量分析することによって、反応生成物 の経時変化を測定した。その結果を図 9に示す。図 9は、酵素反応の生成物の量を 示しており、小さ!/、ほど阻害活性が強 、ことを示す。  (2) Alkyne phosphate 2 mM, (3) N3GDPFuc 2 mM, or (4) inhibitor mixed solution 200 M was added. The time course of the reaction product was measured by direct mass spectrometry using MALDI-T OFMS (Bruker, UltraFLEX) using the deuterium aoWR labeled compound as an internal standard. The results are shown in Fig. 9. FIG. 9 shows the amount of the product of the enzyme reaction, and the smaller! /, The stronger the inhibitory activity.
(アセチレンライブラリの Ki算出）  (Calculation of acetylene library Ki)
さらに、アセチレンライブラリの A16および L3Nについての阻害定数 Kiを算出する ために、ディクソンプロットの作成を行った。  In addition, a Dixon plot was created to calculate the inhibition constant Ki for A16 and L3N in the acetylene library.
アセチレンライブラリの A16【こつ!/、て、ドナーの濃度を 37. 5 /ζ Μ、 20 /ζ Μ、 および阻害剤の濃度を 0 /z M、 0. 5 M、 1 Mで測定した。 L3Nについては、ドナ 一の濃度を 5 M、 25 M、 50 μ Μおよび阻害剤の濃度を 0. 1 Μ、 0. 5 Μ、 1 Μ、 Μ、 Μで測定した。上記の経時変化の結果より、酵素反応時間は初 速度の測定範囲である 10分とした。反応条件は以下の通りである。  A16 of the acetylene library was measured at donor concentrations of 37.5 / ζ Μ, 20 / ζ Μ, and inhibitor concentrations of 0 / z M, 0.5 M, and 1 M. For L3N, the donor concentration was measured at 5 M, 25 M, 50 μΜ, and the inhibitor concentration was measured at 0.1 Μ, 0.5 Μ, 1 Μ, Μ, Μ. Based on the results of the change over time, the enzyme reaction time was set to 10 minutes, which is the initial velocity measurement range. The reaction conditions are as follows.
条件 Condition
緩衝液： 50mM HEPES, pH 7. 2、 10mM MnC12 Buffer: 50 mM HEPES, pH 7.2, 10 mM MnC12
ドナー： 50若しく ίま 200 /z M Donor: 50 young ίMA 200 / z M
ァクセプター： ImM Acceptor: ImM
阻害剤混合溶液： 0〜： L00 M Inhibitor mixed solution: 0 ~: L00 M
酵素： フコース転移酵素（FucT)V 2. 5mU Enzyme: Fucose transferase (FucT) V 2.5 mU
総量； 25 ;z L Total amount; 25; z L
以上の結果より、ディクソンプロットを作成した。図 8Bおよび図 8Cに示すような直線 が得られた。アセチレンライブラリの A16についての Kiは 563 μ Μと算出できた（図 8 B)。 L3Nについての Kiは 1. O /z Mと算出できた（図 8C)。  Based on the above results, a Dixon plot was created. A straight line as shown in FIGS. 8B and 8C was obtained. Ki for A16 of the acetylene library was calculated to be 563 μΜ (Fig. 8B). Ki for L3N was calculated as 1. O / z M (Fig. 8C).
(フコース転移酵素のァクセプターである糖ペプチドの調製） Fmocアミノ酸を MSNT、 Nメチルイミダゾールとともにジクロロメタン中で反応させ F moc HMPA— PEGAレジンに固定させた。 Fmoc保護基を DMF溶媒中で 20%ピ ペリジンにより脱保護した。糖ァスパラギン酸導入前の Fmocアミノ酸カップリングは O —ベンゾトリアゾール 1—ィル一 N, N, Ν' , Ν， 一テトラメチルゥ口-ゥムテトラフル ォロボレート（TBTU)、 Ν—メチルモルホリン（ΝΜΜ) , 1—ヒドロキシベンゾトリァゾ ール (HOBt)を使用して DMF中で行!、、糖ァスパラギン酸導入後は（2—クロロェチ ル）—ジイソプロピルアミン（DIC)、 HOBtを使って DMF中で行った。 Fmoc糖ァス パラギン酸を、 3— (ジエトキシホスホリルォキシ）一1, 2, 3 ベンゾトリアジ一 4— (3 H)オン（DEPBT) ,ジイソプロピルェチルァミン（DIEA)を使って DMF中でカツプリ ングさせた。この反応を繰り返してペプチドを伸張させた。最後にトリフルォロ酢酸 (T FA) ,トリイソプロビルシリル化合物（TIPs)を使用して水中でペプチドをレジンから切 り出し、 0. 1%TFA水溶液で凍結乾燥した。 (Preparation of glycopeptide which is an acceptor of fucose transferase) Fmoc amino acid was reacted with MSNT and N-methylimidazole in dichloromethane and immobilized on Fmoc HMPA-PEGA resin. The Fmoc protecting group was deprotected with 20% piperidine in DMF solvent. Fmoc amino acid coupling prior to the introduction of sugar aspartate is: O-benzotriazole 1-yl N, N, Ν ', Ν, monotetramethyl-mu-tetrafluoroborate (TBTU), Ν-methylmorpholine (ΝΜΜ), 1-hydroxy This was carried out in DMF using benzotriazole (HOBt) !, and after introduction of sugar aspartate, it was carried out in DMF using (2-chloroethyl) -diisopropylamine (DIC) and HOBt. Fmoc sugar gas, formic acid, was added in DMF using 3- (diethoxyphosphoryloxy) -1,2,3 benzotriazi-4- (3H) one (DEPBT), diisopropylethylamine (DIEA). It was cut out. This reaction was repeated to extend the peptide. Finally, the peptide was excised from the resin in water using trifluoroacetic acid (TFA) and triisoprovir silyl compounds (TIPs), and freeze-dried in a 0.1% TFA aqueous solution.
糖ペプチドの精製は HPLCを使用して行った。カラムは Supelco Discovery H S C18 (25cm x 10mm)を使用して、 0. 1%TFA水溶液とァセトニトリルの混 合溶媒でァセトニトリル濃度が 60分間で 10%から 60%になるグラジェント、流速 2. 8 \/ t ゝぅ条件で分離精製した。  The glycopeptide was purified using HPLC. Column is Supelco Discovery HS C18 (25cm x 10mm), 0.1% TFA aqueous solution and acetonitrile solvent, gradient of acetononitrile from 10% to 60% in 60 minutes, flow rate 2.8 \ / t Separation and purification were performed under conditions.
生成物を MALDI— TOFMSを使用して確認した。その結果を図 8Dに示す。 (1.経時変化の測定）  The product was confirmed using MALDI—TOFMS. The result is shown in FIG. 8D. (1. Measurement of changes over time)
経時変化を測定し、初速を算出した。以下の反応条件を用いた。  The change with time was measured, and the initial speed was calculated. The following reaction conditions were used.
反応条件 Reaction conditions
50 mM 力コジル酸緩衝液（pH 7. 5)  50 mM force codylate buffer (pH 7.5)
10 mM 塩化マンガン 10 mM manganese chloride
100 ァクセプター 100 acceptors
50 M ドナー  50 M donor
80 U フコース転移酵素 VIII  80 U fucose transferase VIII
トータルの液量を 20 μ Lとし、 37。Cで、 10分、 20分、 30分、 45分、 60分、 90分で サンプリングした。反応を、 2 Lの反応溶液を 18 Lのァセトニトリルに加えることで 停止させた。この反応溶液を、 DHBで 10倍に希釈して MALDI—TOF— MSにより 測定した。評価はァクセプターとプロダクトの比により行った。その結果、新規に合成 された糖ペプチドが糖転移反応のァクセプターとなることが確認された。反応率が 10 〜20%の点。つまり 60分を初速と設定した（図 8E)。 37. Make the total volume 20 μL. At C, sampling was performed at 10, 20, 30, 45, 60, and 90 minutes. The reaction was stopped by adding 2 L of reaction solution to 18 L of acetonitrile. This reaction solution was diluted 10-fold with DHB and purified by MALDI-TOF-MS. It was measured. The evaluation was performed based on the ratio between the acceptor and the product. As a result, it was confirmed that the newly synthesized glycopeptide is an acceptor for the transglycosylation reaction. The reaction rate is 10 to 20%. In other words, 60 minutes was set as the initial speed (Fig. 8E).
(2.クリック反応残留物の影響）  (2. Effect of click reaction residue)
クリック反応残留物（Cu2+、 GDP- 6 -Nフコース）による酵素反応への影響の確 Confirmation of the influence of click reaction residue (Cu 2+ , GDP-6 -N fucose) on enzyme reaction
3  Three
認した。以下の反応条件を用いた。 I confirmed. The following reaction conditions were used.
反応条件 Reaction conditions
50 mM 力コジル酸緩衝液（pH 7. 5)  50 mM force codylate buffer (pH 7.5)
10 mM 塩化マンガン 10 mM manganese chloride
100 ァクセプター 100 acceptors
50 M ドナー  50 M donor
80 U フコース転移酵素 VIII  80 U fucose transferase VIII
この溶液に、  In this solution,
(1) 50 ^ Μ 硫酸銅  (1) 50 ^ 銅 Copper sulfate
(2) 200 ^ Μ EDTA  (2) 200 ^ Μ EDTA
(3) 50 ^ Μ 硫酸銅 + 200 Μ EDTA  (3) 50 ^ 銅 Copper sulfate + 200 Μ EDTA
(4) 50 μ Μ GDP— 6Nフコース  (4) 50 μΜ GDP— 6N fucose
3  Three
をそれぞれ加えて、トータル液量を 20 Lとし、 37°Cで、 60分間で反応させた。 その結果を、図 8Fに示す。酵素反応の生成物の相対比を示しており、小さいほど 阻害活性が強いことを示す。 FucT VIIIに関して、 Cu2+による酵素反応の阻害が 生じたが、 4倍量の EDTAを加えることで改善した。 GDP- 6N—フコースが阻害剤 Each was added to make a total volume of 20 L and reacted at 37 ° C for 60 minutes. The result is shown in FIG. 8F. The relative ratio of the product of the enzyme reaction is shown. The smaller the value, the stronger the inhibitory activity. For FucT VIII, inhibition of enzyme reaction by Cu 2+ occurred, but it was improved by adding 4 times the amount of EDTA. GDP-6N—fucose is an inhibitor
3  Three
として機能することが確認された。従って、スクリーニングの際には、あら力じめ 4倍量 の EDTAを加えて反応を行った。 Was confirmed to function as. Therefore, at the time of screening, the reaction was performed by adding 4 times the amount of EDTA.
(3.スクリーニング）  (3. Screening)
化合物として、上記アセチレンィ匕合物力も合成した化合物を用いた。上記と同様に 4 倍量の EDTAを加えたものをライブラリ化合物としてスクリーニングに使用した。以下 の反応条件を用いた。 As the compound, a compound in which the above acetylene compound strength was also synthesized was used. As described above, 4 times the amount of EDTA was added as a library compound for screening. The following reaction conditions were used.
(同位体ラベル化） 反応液 15 /z Lに飽和重曹水 30 /z Lを加え、さらに 1. 5 Lの無水酢酸（重曹水の 体積の 5%)をカ卩えて 4時間放置してァセチルイ匕した。内部標準となる重水素ラベル を、無水酢酸の代わりに重無水酢酸をカ卩えて重ァセチルイ匕することにより調製した。 反応条件 (Isotope labeling) Saturated aqueous sodium bicarbonate 30 / z L was added to the reaction solution 15 / z L, and 1.5 L of acetic anhydride (5% of the volume of sodium bicarbonate water) was further added and left for 4 hours for acetylation. An internal standard deuterium label was prepared by deuterating acetic anhydride instead of acetic anhydride. Reaction conditions
50 mM 力コジル酸緩衝液（pH 7. 5)  50 mM force codylate buffer (pH 7.5)
10 mM 塩化マンガン  10 mM manganese chloride
100 ァクセプター  100 acceptors
50 M ドナー  50 M donor
80 /z U フコース転移酵素 VIII  80 / z U fucose transferase VIII
50 GDP— 6Nフコース  50 GDP— 6N fucose
3  Three
50 M クリック反応物  50 M click reactant
トータル液量を 20 Lにし、 37°Cで、 60分間で反応させた。前述と同様の方法を 用いてァセチルイ匕し、内部標準を混ぜて測定を行った。その結果を図 8Gに示す。ァ センプリライブラリーの 1、 2、 3、 34が強い阻害活性を示した。  The total liquid volume was adjusted to 20 L and reacted at 37 ° C for 60 minutes. Using the same method as described above, acetylation was performed, and the internal standard was mixed for measurement. The result is shown in FIG. 8G. The assembly library 1, 2, 3, and 34 showed strong inhibitory activity.
[化 1261] [Chemical 1261]
Figure imgf000153_0001
Figure imgf000153_0002
Figure imgf000153_0003
Figure imgf000153_0001
Figure imgf000153_0002
Figure imgf000153_0003
Figure imgf000153_0004
Figure imgf000153_0004
(1.阻害定数の算出） (1. Calculation of inhibition constant)
スクリーニングによってヒットした阻害剤候補ィ匕合物 3種類と阻害機能があった GDP 6— N—フコース (クリック反応に使用する鍵中間体）について阻害定数を算出し Inhibitory constants were calculated for 3 types of inhibitor candidate compounds hit by screening and GDP 6—N-fucose (a key intermediate used for click reaction) that had an inhibitory function.
3 Three
た。 1と 2とは構造が似ているので 1は除外した。 It was. Since 1 and 2 are similar in structure, 1 was excluded.
GDP-6-Nーフコースについては精製品なので、ディクソンプロットにより Kiを算  The GDP-6-N course is a refined product, so Ki is calculated using the Dixon plot.
3  Three
出した。アセチレンライブラリの A2, A3, A34については未精製なので、 IC50を算 出した。 I put it out. Since A2, A3, and A34 of the acetylene library are not purified, IC50 is calculated. I put it out.
(GDP— 6— N —フコースの Ki算出）  (GDP— 6— N — Fucose Ki calculation)
3  Three
反応条件 Reaction conditions
50 mM 力コジル酸緩衝液（pH 7. 5)  50 mM force codylate buffer (pH 7.5)
10 mM 塩化マンガン  10 mM manganese chloride
100 ァクセプター  100 acceptors
12. 5、 25、 50、 75 M ド、ナー  12. 5, 25, 50, 75 M
80 U フコース転移酵素 VIII  80 U fucose transferase VIII
0、 12. 5、 25、 50、 75、 100 /z M GDP— 6Νフコース  0, 12. 5, 25, 50, 75, 100 / z M GDP—6Ν
3  Three
トータル液量を 20 /z Lとし、 37°Cで、 60分間反応させた。ァセチルイ匕後、内部標準 と混合し MALDIにより測定した。  The total liquid volume was 20 / z L, and the reaction was performed at 37 ° C for 60 minutes. After acetylation, it was mixed with an internal standard and measured by MALDI.
結果を図 8Hに示す。この結果より、 Kiは 23 Mと算出された。  The results are shown in Fig. 8H. From this result, Ki was calculated to be 23 M.
(IC50の算出）  (IC50 calculation)
ドナー： 50 Μ Donor: 50 Μ
ァクセプター： 100 Acceptor: 100
緩衝液： 50 mM 力コジル酸緩衝液 (pH 7. 5) Buffer: 50 mM force codylate buffer (pH 7.5)
塩化マンガン： 10 mM Manganese chloride: 10 mM
フコース転移酵素 VIII： 80 /z U Fucose transferase VIII: 80 / z U
阻害剤： 12. 5, 25, 50, 76, 100 μ Μ (ィ匕合物 2、 3、 34、 GDP— 6Ν Inhibitors: 12. 5, 25, 50, 76, 100 μΜ (compounds 2, 3, 34, GDP— 6Ν
3 ーフコース）  3 course)
トータル液量を 20 Lとし、 37°Cで 60分間反応させた。ァセチル化後、内部標準と 混合し MALDIにより測定した。 The total liquid volume was adjusted to 20 L and reacted at 37 ° C for 60 minutes. After acetylation, it was mixed with an internal standard and measured by MALDI.
結果を図 81に示す。このグラフ力もそれぞれの IC50を求めると、以下のとおりであ つ 7こ。  The results are shown in FIG. This graph power is also as follows when each IC50 is calculated.
IC50 (ji M) IC50 (ji M)
GDP— 6— N3フコース 30.  GDP— 6— N3 fucose 30.
2 12. 7 25. 5 2 12. 7 25. 5
11. 2 この結果より、 2と 34、 GDP-6 -Nーフコースに阻害作用があることがわかった。  11. 2 This result shows that 2 and 34, GDP-6 -N-fucose has an inhibitory effect.
3  Three
測定方法により Kは異なるが N3フコースの結果より数値は妥当な値だと考えられる  Although K differs depending on the measurement method, the numerical value is considered to be reasonable from the results of N3 fucose.
[0324] (実施例 2：ォキシムを含有する糖転移酵素阻害剤の合成) [0324] (Example 2: Synthesis of glycosyltransferase inhibitor containing oxime)
[0325] [化 127] [0325] [Chemical 127]
Figure imgf000155_0001
Figure imgf000155_0001
2  2
Figure imgf000155_0002
Figure imgf000155_0002
22  twenty two
[0327] [化 129] [0327] [Chemical 129]
Figure imgf000156_0001
Figure imgf000156_0001
23  twenty three
[0328] [化 130]
Figure imgf000156_0002
[0328] [Chemical 130]
Figure imgf000156_0002
24 twenty four
[0329] [化 131]
Figure imgf000156_0003
[0329] [Chemical 131]
Figure imgf000156_0003
25  twenty five
[0330] [化 132]
Figure imgf000156_0004
[0330] [Chemical 132]
Figure imgf000156_0004
26  26
(化合物 23の合成）  (Synthesis of Compound 23)
既知物質であるゥリジン 5， - (2—ァセトアミドー 2—デォキシ一 (X—D—ガラクトビ ラノシル）ジホスフェート 1 (10 mg, 16 /z mole)を緩衝液（1. 5 mL ; 50 mM ホ スフェート— Na+, pH 6. 0, 0. 5 mM CuSO )に溶解し、報告例（Buelter. T.  The known substance uridine 5,-(2-acetamido-2-deoxy- (X-D-galactobilanosyl) diphosphate 1 (10 mg, 16 / z mole) was added to the buffer (1.5 mL; 50 mM phosphate) Na +, pH 6.0, 0.5 mM CuSO 4) and reported cases (Buelter. T.
4  Four
et al. Chemibiochem 2001, 2, 884— 894.；)に?¾つてィ匕合物 2へと誘導した。 化合物 26 (27 mole)の水溶液 (200 L)を加え室温で 15分攪拌した後、反応 溶液を逆層カラムクロマトグラフィー (WakoGel (登録商標） C— 18：溶媒は水）を用 いて精製し、化合物 23を 7. 3 mg、収率 51 %で得た。反応スキームを以下に示す et al. Chemibiochem 2001, 2, 884—894 .;) Add an aqueous solution (200 L) of compound 26 (27 mole) and stir at room temperature for 15 minutes, then react. The solution was purified using reverse layer column chromatography (WakoGel (registered trademark) C-18: solvent is water) to obtain Compound 23 (7.3 mg, yield 51%). The reaction scheme is shown below.
[化 133][Chemical 133]
Figure imgf000157_0001
Figure imgf000157_0001
(化合物 26の合成）  (Synthesis of Compound 26)
既知化合物である化合物 24 (500 mg, 1. 72mmole)を THF (lOmL)に溶解し 、 PPh (677 mg, 2. 58 mmole)および N—ヒドロキシ一フタルイミド（421 mg Compound 24 (500 mg, 1.72 mmole), a known compound, is dissolved in THF (lOmL), and PPh (677 mg, 2.58 mmole) and N-hydroxymonophthalimide (421 mg) are dissolved.
3 Three
, 2. 58 mmole)を添カ卩した。反応溶液を 0°Cに冷却した後、ジイソプロピルァゾカ ルボキシレート（508 L, 2. 58 mmole)をゆっくり添カ卩し 0°Cで 6時間攪拌した。 反応液を減圧濃縮した後 CHC1を加え、飽和重曹水および飽和食塩水で洗浄処理  , 2. 58 mmole). After the reaction solution was cooled to 0 ° C, diisopropylazocarboxylate (508 L, 2.58 mmole) was slowly added thereto and stirred at 0 ° C for 6 hours. Concentrate the reaction solution under reduced pressure, add CHC1, and wash with saturated aqueous sodium bicarbonate and brine.
3  Three
し CHC1層を分液後、減圧濃縮した。得られた残渣をシリカゲルカラムクロマトグラフThe CHC1 layer was separated and concentrated under reduced pressure. Silica gel column chromatograph
3 Three
ィー（トルエン:ェチルアセテート = 5 : 1)を用いて精製し、中間体ィ匕合物 5 (1. 2g )を副生物との混合物として得た。得られた中間体化合物混合物（337 mg)を MeO H (5mL)に溶解した後、 NH NH— H 0 (112 2. 2 mmole)を添カ卩し、室温 (Toluene: ethyl acetate = 5: 1) to obtain intermediate compound 5 (1.2 g) as a mixture with by-products. The obtained intermediate compound mixture (337 mg) was dissolved in MeO H (5 mL), NH NH—H 0 (112 2.2 mmole) was added, and room temperature was added.
2 2 2  2 2 2
で 18時間攪拌した。沈殿物をセライトろ過した後減圧濃縮し、シリカゲルカラムクロマ トグラフィー（CHC13 ： MeOH= 100 ： 1)を用いて精製することで、化合物 26 を 137 mg収率 93 %で得た。得られた化合物 26に H O (2. 8 mL)および AcO And stirred for 18 hours. The precipitate was filtered through celite, concentrated under reduced pressure, and purified by silica gel column chromatography (CHC13: MeOH = 100: 1). Was obtained with a 137 mg yield of 93%. The resulting compound 26 was mixed with HO (2.8 mL) and AcO
2  2
H ( 13 L, 250 μ mole)を添加し、約 50 mgZmLの水溶液とした。  H (13 L, 250 μmol) was added to make an aqueous solution of about 50 mgZmL.
[0334] (グリコシルァクセプターの合成） [0334] (Synthesis of glycosyl acceptor)
[0335] [化 134] [0335] [Chemical 134]
Figure imgf000158_0001
既知条件（Matsushita, T. et al, Org. Lett. 2005, 7, 877— 880)により調整 したペプチド（アミノ酸配列 HGVTSAPDTR (配列番号 5) ) (5 mg, 4. 8 μ mole) を H 0 (2mL)に溶解し、レブリン酸 N—ヒドロキシスクシンイミドエステル（5. Ίβ μ ηι
Figure imgf000158_0001
Peptide (amino acid sequence HGVTSAPDTR (SEQ ID NO: 5)) (5 mg, 4.8 μmole) prepared under known conditions (Matsushita, T. et al, Org. Lett. 2005, 7, 877—880) was converted to H 0 ( 2 mL) and levulinic acid N-hydroxysuccinimide ester (5. Ίβ μ ηι
2 2
ole (ジォキサン中） 115 L)およびジイソプロピルェチルァミン（16 μ mole (ジォキ サン中） 320 L)を室温で添カ卩した。室温で 12時間反応を行った後、 HPLC (2 0 X 250 mm C—18, YMC Co. , Ltd. )を用いて精製を行い（220 nm U V検出； A液： 50 mM NH +HCO _B液： CH CN 0〜40min A : B = 95 : 5  ole (in dioxane) 115 L) and diisopropylethylamine (16 μmole in dioxane 320 L) were added at room temperature. After reaction at room temperature for 12 hours, purification was performed using HPLC (20 x 250 mm C-18, YMC Co., Ltd.) (220 nm UV detection; solution A: 50 mM NH + HCO _B solution) : CH CN 0 ~ 40min A: B = 95: 5
4 3 3  4 3 3
〜 60 :40 25分で溶出； 5 mLZmin)、化合物 27を 3mg収率 54 %で得た。  ~ 60:40 eluted at 25 min; 5 mLZmin), compound 27 was obtained in 3 mg yield 54%.
[0336] (ポリペプチド N -ァセチルガラタトサミン転移酵素 (ppGalNAcT)の調製） [0336] (Preparation of Polypeptide N-Acetylgalatatosamine Transferase (ppGalNAcT))
(阻害作用の検出）  (Detection of inhibitory effect)
酵素反応溶液を以下のように調整した。  The enzyme reaction solution was prepared as follows.
1 Control実験  1 Control experiment
200 UDP - GalNAc, 1 mM 化合物 7、緩衝液（イミダゾール 50mM p 200 UDP-GalNAc, 1 mM compound 7, buffer (imidazole 50 mM p
H 7. 2, MnCl 10 mM, Triton X— 100 0. 5%)、 ppGalNAcT溶液 20 H 7.2, MnCl 10 mM, Triton X—100 0.5%), ppGalNAcT solution 20
2  2
μレ反 、容直 50 μ L0 μ-reverse, straight 50 μL 0
[0337] 2 化合物 3添加実験 [0337] 2 Compound 3 addition experiment
200 UDP— GalNAc, 200 Μィ匕合物 3、 1 mM 化合物 7、緩衝液 （ィ ミダゾール 50 mM pH 7. 2, MnCl 10mM, Triton X— 100 0. 5%)、ppG  200 UDP— GalNAc, 200 Μ Compound 3, 1 mM Compound 7, Buffer (Imidazole 50 mM pH 7.2, MnCl 10 mM, Triton X—100 0.5%), ppG
2  2
alNAcT溶液 20 L、反応容量 50 L。 [0338] 酵素反応開始後、 10分、 20分、 40分、 80分に 5 μ Lずつ取り、軽水素 aoWR溶 液（5/zl 10mM aoWR(MeOH-H O 1:1中） +CH CN 30/zL+AcOH 1 alNAcT solution 20 L, reaction volume 50 L. [0338] Take 5 μL at 10 min, 20 min, 40 min, and 80 min after the start of the enzyme reaction, and add light hydrogen aoWR solution (5 / zl 10 mM aoWR in MeOH-H O 1: 1) + CH CN 30 / zL + AcOH 1
2 3  twenty three
0/z L;合計 45μ L)に加え反応を終了した。各溶液から 20 μ Lずつ別のエツベンド ルフチューブへ移し、 60°Cで 2時間、常温で 12時間反応を行い、ペプチドを aoWR 標識した。 Matrix溶液（10 1; 10 mg/mL DHB)と標識ペプチド溶液 0. 5μ Lを混合し MALDI TOF MS分析に用いた。  (0 / z L; 45 μL in total) to complete the reaction. 20 μL of each solution was transferred to a separate Ebbendorf tube and reacted at 60 ° C for 2 hours and at room temperature for 12 hours to label the peptide with aoWR. Matrix solution (10 1; 10 mg / mL DHB) and labeled peptide solution 0.5 μL were mixed and used for MALDI TOF MS analysis.
[0339] 反応模式図を以下に示す。  [0339] A reaction schematic diagram is shown below.
[0340] [化 135- 1] [0340] [Chemical 135-1]
rin rin
Figure imgf000160_0001
Figure imgf000160_0001
l7CS0S0/.00Zdf/X3d 691- IC0180/.00Z OAV l7CS0S0 / .00Zdf / X3d 691- IC0180 / .00Z OAV
sl aowR sl aowR
Figure imgf000161_0001
Figure imgf000161_0001
l7CS0S0/.00Zdf/X3d 091· ΐεθΐ80/.ΟΟΖ OAV [0341] 酵素活性の追跡の様子を図 10に示す。酵素反応の生成物の相対比を示しておりl7CS0S0 / .00Zdf / X3d 091 · ΐθΐ80 / .ΟΟΖ OAV [0341] Figure 10 shows the state of enzyme activity tracking. Shows the relative ratio of the products of the enzyme reaction
、小さいほど阻害活性が強いことを示す。 The smaller the value, the stronger the inhibitory activity.
[0342] 結果を図 11に示す。ひし形： Control実験 正方形： 化合物 23添加実験を示すThe results are shown in FIG. Diamond: Control experiment Square: Compound 23 addition experiment
。化合物 23による阻害効果が示された。 . An inhibitory effect by compound 23 was shown.
[0343] このように、ォキシム型化合物でも、アジド型化合物と同様に、糖転移酵素の阻害 活性を観察することができた。 [0343] As described above, the inhibitory activity of glycosyltransferase could be observed with the oxime type compound as well as the azide type compound.
[0344] (実施例 3 :コンピュータシミュレーション） [0344] (Example 3: computer simulation)
全ての構造解析 (合成した基質誘導体の安定構造、 β ΐ , 4 GalT結晶構造との 結合)シミュレーションは、 Sybyl 6. 8ソフトウェアを使用した。基質誘導体の反応後 の安定構造の解析は 300K、溶媒誘電率 78. 36 (水中を想定)を使用し、分子動力 学（Molecular Dynamics)により Insの間自由運動させ、 lfsごとの蛍光基間距離 を測定した。  Sybyl 6.8 software was used for all structural analyzes (stable structure of synthesized substrate derivatives, binding to β,, 4 GalT crystal structure). Analysis of the stable structure of the substrate derivative after the reaction was performed using 300K, solvent permittivity of 78.36 (assuming water), and freely moving during Ins by molecular dynamics, and distance between fluorescent groups for each lfs. Was measured.
[0345] (実施例 4：蛍光エネルギー移動法による酵素活性測定）  [0345] (Example 4: Enzyme activity measurement by fluorescence energy transfer method)
(4^ 調製）  (4 ^ Preparation)
蛍光測定用石英セル (総容量: 600 L)に最終濃度で以下のように基質類似物、 酵素、緩衝液を調製した。  Substrate analogues, enzymes, and buffers were prepared in a quartz cell for fluorescence measurement (total volume: 600 L) at the final concentration as follows.
[0346] 実施例 1および 2にお!/、て製造した化合物： 10 μ Μまたは 50 μ Μ [0346] Compounds prepared in Examples 1 and 2! /, 10 μΜ or 50 μΜ
GalT: 100 ,u U  GalT: 100, u U
緩衝液： 50mM Hepes緩衝剤， pH 6. 5, 10mM MnCl、0. 25mg/mL B  Buffer: 50 mM Hepes buffer, pH 6.5, 10 mM MnCl, 0.25 mg / mL B
2  2
SA.  SA.
酵素をカロえる瞬間を時間 0とし、 2, 4, 8, 16, 32, 64分にスキャンスピード 700nm Z分で測定する。  The time when the enzyme is burned is set to time 0, and the measurement is performed at 2, 4, 8, 16, 32, 64 minutes at a scan speed of 700 nm Z minutes.
[0347] (4^ 2 基質の転移活性の確認） [0347] (Confirmation of transfer activity of 4 ^ 2 substrate)
1. 5mLエツペンドルフチューブ中で以下の条件において酵素反応を行い、生成 物を HPLC (ダンシル基蛍光検出）、 MALDI— TOF— MASSにて確認する。  1. Perform enzyme reaction in a 5 mL Eppendorf tube under the following conditions, and confirm the product with HPLC (dansyl fluorescence detection) and MALDI-TOF-MASS.
[0348] く β ΐ , 4- GalT > [0348] <β ΐ, 4- GalT>
実施例 1および 2において製造した化合物： 200 Mまたは 50 M  Compounds prepared in Examples 1 and 2: 200 M or 50 M
β 1, 4— Gam O /z U 緩衝液： lOOmM Hepes緩衝剤、 pH 6. 5, lOmM MnCl、 0. 25mg/mL β 1, 4— Gam O / z U Buffer: lOOmM Hepes buffer, pH 6.5, lOmM MnCl, 0.25 mg / mL
2  2
BSA.  BSA.
総容量 ^O/zL  Total capacity ^ O / zL
37°C、 1時間インキュベーションした後、続けて 100°C3分の熱処理、  After incubation at 37 ° C for 1 hour, heat treatment at 100 ° C for 3 minutes,
15000rpmZlO分で遠心処理をし、 HPLCオートインジェクターにより、上澄み 20 μ Lをインジエタ卜する。  Centrifuge at 15000 rpm ZlO min, and inject 20 μL of supernatant with HPLC autoinjector.
[0349] 溶出条件： 0分 MeOH 50% :H O 50%から 25分 MeOH 55% :H O 45%に  [0349] Elution condition: 0 min MeOH 50%: H 2 O 50% to 25 min MeOH 55%: H 2 O 45%
2 2 なる線形勾配。流速 0. 5mLZmin、カラム温度 40°C。溶出 14〜15分。  A linear gradient of 2 2. Flow rate 0.5mLZmin, column temperature 40 ° C. Elution 14-15 minutes.
[0350] < al, 3-GalT> [0350] <al, 3-GalT>
実施例 1および 2において製造した化合物： 200 Mまたは 100 M  Compounds prepared in Examples 1 and 2: 200 M or 100 M
a 1, 3-Ο 1Τ:500^υ  a 1, 3-Ο 1Τ: 500 ^ υ
緩衝液： lOOmM Hepes緩衝剤， pH 6. 5, 10mM MnCl、0. 25mg/mL  Buffer: lOOmM Hepes buffer, pH 6.5, 10 mM MnCl, 0.25 mg / mL
2  2
BSA.  BSA.
総容量 ^O/zL  Total capacity ^ O / zL
他の条件は j81, 4— GalTのものと同様にした。  Other conditions were the same as those for j81, 4-GalT.
[0351] 溶出条件： 0分 CH CN 25% :H O 75%から 20分 CH CN 35% :H O 65% [0351] Elution condition: 0 min CH CN 25%: H O 75% to 20 min CH CN 35%: H O 65%
3 2 3 2 になる線形勾配。流速 0. 5mLZmin、カラム温度 40°C。溶出 14〜15分。  A linear gradient that becomes 3 2 3 2. Flow rate 0.5mLZmin, column temperature 40 ° C. Elution 14-15 minutes.
[0352] 溶出ピ一クフラクシヨンを集めて濃縮し、 MALDI— TOF— MASSにより生成物確 認を行う。 [0352] Collect and concentrate the eluted fractions and confirm the product by MALDI-TOF-MASS.
[0353] (4^3 酵素阻害定数の測定）  [0353] (Measurement of 4 ^ 3 enzyme inhibition constant)
1. 5mLのエツペンドルフチューブ中で以下の条件にて酵素反応を行い、 Dixonプ ロットにより阻害定数を算出した。 HPLC蛍光分析において、ピ―ク面積とサンプル 量は検量線により 3000pmoほでは比例関係にあることを確認し、酵素反応量をピ— ク面積から算出した。その他の条件は上述の蛍光エネルギー移動法による酵素活性 測定と同様であった。  1. Enzyme reaction was performed in a 5 mL Eppendorf tube under the following conditions, and the inhibition constant was calculated using the Dixon plot. In HPLC fluorescence analysis, the peak area and the sample amount were confirmed to be proportional to 3000 pmo by a calibration curve, and the amount of enzyme reaction was calculated from the peak area. Other conditions were the same as in the enzyme activity measurement by the fluorescence energy transfer method described above.
[0354] く β ΐ, 4-GalT> [0354] <β ΐ, 4-GalT>
UDP-GahlOO^M  UDP-GahlOO ^ M
実施例 1および 2にお 、て製造したィ匕合物： 0〜200 μ Μ β 1 , 4— GalT ^OO /z U In Examples 1 and 2, the compound produced in 0 to 200 μΜ β 1, 4— GalT ^ OO / z U
緩衝液： 50mM Hepes緩衝剤， pH 6. 5, lOmM MnCl、 0. 2  Buffer: 50 mM Hepes buffer, pH 6.5, lOmM MnCl, 0.2
2 2
Figure imgf000164_0001
Figure imgf000164_0001
総容量 ^O /z L  Total capacity ^ O / z L
インキュベーション時間： 10分。  Incubation time: 10 minutes.
[0355] (4^ 4 結果および考察） [0355] (4 ^ 4 results and discussion)
本実施例により糖転移酵素の新規阻害剤を構築することができることが確認される  This example confirms that a novel inhibitor of glycosyltransferase can be constructed.
[0356] (実施例 5：化合物 29〜34のシアル酸転移酵素阻害活性） [0356] (Example 5: Sialyltransferase inhibitory activity of compounds 29 to 34)
実施例 5では、化合物 28とクリックケミストリーで合成された以下の化合物 29〜34、 ならびにアセチレンィ匕合物と 5位アジド体 CMPシアル酸誘導体のクリック反応により 合成された化合物および 9位アジド体 CMPシアル酸誘導体力 クリック反応により合 成された化合物のシアル酸転移酵素阻害活性を検討した。  In Example 5, the following compounds 29 to 34 synthesized by click chemistry with compound 28, and compounds synthesized by click reaction of acetylene compound and 5-position azide CMP sialic acid derivative and 9-position azide CMP Sialic Acid Derivative Strength We examined the sialyltransferase inhibitory activity of compounds synthesized by click reaction.
[化 136]  [Chemical 136]
Figure imgf000164_0002
Figure imgf000164_0002
[化 137]
Figure imgf000165_0001
Figure imgf000165_0002
Figure imgf000165_0003
[Chemical 137]
Figure imgf000165_0001
Figure imgf000165_0002
Figure imgf000165_0003
[化 138A] [Chemical 138A]
Figure imgf000166_0001
Figure imgf000166_0001
Figure imgf000166_0002
Figure imgf000166_0002
Figure imgf000166_0003
Figure imgf000166_0003
ならびに、 And
アセチレンィ匕合物と 5位アジド体 CMPシアル酸誘導体のクリック反応により合成され た化合物 Compound synthesized by click reaction of acetylene compound and 5-position azide CMP sialic acid derivative
[化 138B] [Chemical 138B]
Figure imgf000166_0004
Figure imgf000166_0004
であって、式中の Rは以下: Where R is:
[化 138C] [Chemical 138C]
Figure imgf000167_0001
Figure imgf000167_0001
[化 138D] [Chemical 138D]
Figure imgf000168_0001
Figure imgf000168_0001
Figure imgf000168_0002
Figure imgf000168_0002
[化 138E]
Figure imgf000169_0001
Figure imgf000169_0002
[Chemical 138E]
Figure imgf000169_0001
Figure imgf000169_0002
Figure imgf000169_0003
Figure imgf000169_0003
41  41
、..0 ^--。^_-0 。 0 ^^Q 0  , ..0 ^-. ^ _- 0. 0 ^^ Q 0
およびアセチレン化合物と 9位アジド体 CMPシアル酸誘導体からクリック反応により 合成された化合物 And acetylene compounds and 9-position azide compounds synthesized by click reaction from CMP sialic acid derivatives
[化 138F] [Chemical 138F]
Figure imgf000169_0004
であって、式中の Rは以下:
Figure imgf000169_0004
Where R is:
[化 138G] [Chemical 138G]
Figure imgf000170_0001
Figure imgf000170_0001
Figure imgf000170_0002
Figure imgf000170_0002
2020
[化 138H] [Chemical 138H]
Figure imgf000171_0001
Figure imgf000171_0001
Figure imgf000171_0002
Figure imgf000171_0002
[化 1381]
Figure imgf000172_0001
[Chemical 1381]
Figure imgf000172_0001
L3C L3C
L2C L2C
(アジド糖ヌクレオチドの合成法) (Synthesis of azido sugar nucleotides)
[化 139] [Chem 139]
Figure imgf000173_0001
Figure imgf000173_0001
(化合物 35の合成） (Synthesis of Compound 35)
アジド酢酸メチノレ（1. Oeq、 0. 733mmol、 84. Omg)を 99%エタノーノレ（3ml)に 溶解し、室温で IN NaOH水溶液（1. Oeq、 0. 733mmol、 0. 733ml)を加え、 1 時間室温で攪拌した。その後、化合物 9 (300mg、 0. 733mmol)を 95%エタノール (2. 73ml)に溶力しておいた溶液を、反応液にカ卩え、室温で 10時間攪拌した。その 後、 DMFで 3回共沸することにより水を除いた。その後、 DMF (4ml)に溶解した後、 トリェチルァミン（1. Oeq、0. 733mmol、0. 102ml)を加えた後にジフエ-ルフォス フォリルアジド（1. Oeq、 0. 733mmol、 0. 158ml)を加え、 18時間室温で攪拌した 。溶液をシリカゲルクロマトグラフィー（展開溶媒:クロ口ホルム Zメタノール =20Zl) で精製し、分画を減圧濃縮することにより黄色非晶質 35 (230mg、 44%)を得た。 R  Methylol azidoacetate (1. Oeq, 0.733 mmol, 84. Omg) was dissolved in 99% ethanol (3 ml), and IN NaOH aqueous solution (1. Oeq, 0.733 mmol, 0.733 ml) was added at room temperature for 1 hour. Stir at room temperature. Thereafter, a solution prepared by dissolving Compound 9 (300 mg, 0.733 mmol) in 95% ethanol (2.73 ml) was added to the reaction solution and stirred at room temperature for 10 hours. After that, water was removed by azeotropic distillation with DMF three times. Then, after dissolving in DMF (4 ml), triethylamine (1. Oeq, 0.733 mmol, 0.102 ml) was added, followed by diphenylphosphoryl azide (1. Oeq, 0.733 mmol, 0.158 ml). Stir for hours at room temperature. The solution was purified by silica gel chromatography (developing solvent: Kuroguchi Form Z methanol = 20 Zl), and the fraction was concentrated under reduced pressure to obtain yellow amorphous 35 (230 mg, 44%). R
f f
=0. 6 (クロ口ホルム Zメタノール =3Zl) lH— NMR δ (CH OD) ; 1. 96 (t、 1H = 0. 6 (black mouth form Z methanol = 3Zl) lH— NMR δ (CH OD); 1.96 (t, 1H
3  Three
、 3eq)、 2. 68 (dd、 1H、 3ax)、 3. 55 (d、 1H、 H— 7)、 3. 67 (q、 1H、 H— 9)、 3. 77 (m、 1H、 H— 8)、 3. 81 (dd、 1H、 H— 9)、 3. 94 (m、 3H、 H— 5、 N—CH—  3eq), 2.68 (dd, 1H, 3ax), 3.55 (d, 1H, H—7), 3.67 (q, 1H, H—9), 3.77 (m, 1H, H — 8), 3.81 (dd, 1H, H—9), 3.94 (m, 3H, H—5, N—CH—
3 2 3 2
CO) , 4. 14 (m、 1H、 H— 4)、 4. 58 (d、 1H、 H— 6)、 7. 33— 7. 38 (m、 3H、 aro matic) , 7. 58 (d、 2H、 aromatic) ESI— MS calcd 457. 139、 found 457. 2 92 CO), 4.14 (m, 1H, H—4), 4.58 (d, 1H, H—6), 7.33— 7.38 (m, 3H, aro matic), 7.58 (d , 2H, aromatic) ESI— MS calcd 457. 139, found 457. 2 92
(化合物 36の合成）  (Synthesis of Compound 36)
化合物 35 (56. 3mg、 123mmol)を水とアセトンの混合溶媒 (溶媒:水 Zァ セトン = 24/1 、 2ml)に溶解し、 N—ブロモスクシミド（4. 0eq、 452mmol、 88mg )を氷冷下で溶液に加えた。反応液を室温に戻し、 1時間攪拌した。その後、シリカゲ ルクロマトグラフィー（展開溶媒:クロ口ホルム Zメタノール = 10Z1)で分画を集め、 減圧濃縮することで既知化合物 36 (16mg、 36%)を得た。 （SarahJ. Luchansky,Compound 35 (56.3 mg, 123 mmol) was dissolved in a mixed solvent of water and acetone (solvent: water Zasetone = 24/1, 2 ml), and N-bromosuccinimide (4.0 eq, 452 mmol, 88 mg) was cooled with ice. To the solution. The reaction solution was returned to room temperature and stirred for 1 hour. Thereafter, the fractions were collected by silica gel chromatography (developing solvent: Kuroguchi Form Z methanol = 10Z1) and concentrated under reduced pressure to obtain a known compound 36 (16 mg, 36%). (SarahJ. Luchansky,
Scarlett Goon, Carolyn R. Bertozzi, Chem. Bio. Chem. , 5 (2004) 3 71 - 374) Scarlett Goon, Carolyn R. Bertozzi, Chem. Bio. Chem., 5 (2004) 3 71-374)
(化合物 37の合成）  (Synthesis of Compound 37)
ィ匕合物 36 (16mg、 45. 7 μ mol)を水 (500 μ 1)に溶解させ、さらに 1N水酸ィ匕ナト リウム水溶液（1. leq、 50 /z l)を添加し、室温で 1時間反応させた。反応液を Dowex 50WX 8 (H+)により中和することで既知化合物 37 (15mg、 97%)を得た。 Compound 36 (16 mg, 45.7 μmol) was dissolved in water (500 μ1), and 1N aqueous sodium hydroxide solution (1. leq, 50 / zl) was added. Reacted for hours. Dowex the reaction solution Neutralization with 50WX 8 (H + ) gave the known compound 37 (15 mg, 97%).
(化合物 38の合成）  (Synthesis of Compound 38)
ィ匕合物 37 (10mg、 28. 6mmol)を Tris— HC1緩衝液（100mM、 3ml、 pH9. 0、 MgC12 20mMを含む）に溶かし、シチジントリフォスフェイト—ニナトリウム（0. 9eq 、 13. 6mg)を加え、続いて CMPシアル酸合成酵素（1U)をカ卩え、 37°Cで 1. 75時 間反応させた。続けて反応液に Tris—HCl緩衝液（900mM、 pH9. 0、 3ml)を加え 、 Alkaline Phosphatase (50U)をカ卩え、 37°Cで 2時間反応させた。不溶物を綿栓 でろ過し、ゲルろ過（G15、溶媒:水）により画分を得て、凍結乾燥させた。続いて、化 合物を水に溶解し、陰イオン交換カラム (DEAE) (溶出溶媒 : 0. 1M NH HCO水  Compound 37 (10 mg, 28.6 mmol) is dissolved in Tris—HC1 buffer (100 mM, 3 ml, pH 9.0, containing 20 mM MgC12), and cytidine triphosphate—sodium disodium (0.9 eq, 13.6 mg) Then, CMP sialic acid synthase (1U) was added and reacted at 37 ° C for 1.75 hours. Subsequently, Tris-HCl buffer solution (900 mM, pH 9.0, 3 ml) was added to the reaction solution, and Alkaline Phosphatase (50 U) was added thereto, followed by reaction at 37 ° C. for 2 hours. Insoluble matter was filtered through a cotton plug, and fractions were obtained by gel filtration (G15, solvent: water) and lyophilized. Subsequently, the compound is dissolved in water and an anion exchange column (DEAE) (elution solvent: 0.1M NH HCO water).
4 3 溶液）により画分を得て、凍結乾燥することにより既知化合物 38 (9. 87mg、 51%)を 得た。  The fraction was obtained from 4 3 solution) and freeze-dried to obtain known compound 38 (9. 87 mg, 51%).
(クリック反応）  (Click reaction)
(化合物 28)  (Compound 28)
20mM 1¾5—11じ1緩衝溶液 117. 6、 5 1)に化合物 28、アルキン化合物、硫 酸銅、ァスコルビン酸、 TBTA、 t—BuOHをそれぞれ最終濃度が 2mM、 2mM、 1 mM、 10mM、 lmM、 40%になるように調製した。室温で 5時間反応させた後、 3時 間スピードバックし、 2mM EDTAを添加することで ImMの阻害剤混合溶液を調製 し、 ESI— MSにより質量分析した。質量分析の結果を図 12Aに示す。  20mM 1¾5-11 1 buffer solution 117.6, 5 1) and compound 28, alkyne compound, copper sulfate, ascorbic acid, TBTA, t-BuOH, respectively, with final concentrations of 2 mM, 2 mM, 1 mM, 10 mM, lmM, Prepared to be 40%. After reacting at room temperature for 5 hours, the reaction mixture was speeded back for 3 hours, 2 mM EDTA was added to prepare an ImM inhibitor mixed solution, and mass spectrometry was performed by ESI-MS. The result of mass spectrometry is shown in FIG. 12A.
(共通）  (Common)
上記反応条件に基づいて、 CMPシアル酸誘導体の 5位アジド体から化合物 29、 3 0、 31を、 9位 ジド体力らィ匕合物 32、 33、 34を合成した。  Based on the above reaction conditions, compounds 29, 30 and 31 were synthesized from the 5-position azide of the CMP sialic acid derivative, and 9-position zide strength compounds 32, 33 and 34 were synthesized.
以下に一例として化合物 31の合成を示しておく。  The synthesis of compound 31 is shown as an example below.
[化 140A] [Chemical 140A]
Figure imgf000176_0001
Figure imgf000176_0001
以下のアセチレン化合物（アセチレンライブラリの A1〜A41)につ!/、ても、上記と同 様に、 5位アジド体シアル酸にクリック反応を施した。 For the following acetylene compounds (A1-A41 in the acetylene library), the click reaction was applied to the 5-position azide sialic acid as described above.
[化 1棚] [1 shelf]
6
Figure imgf000178_0001
6
Figure imgf000178_0001
Exact Mass: 167.1674 Exact Mass: 167.1674
Exact Mass: 172.1252
Figure imgf000178_0002
Exact Mass: 172.1252
Figure imgf000178_0002
Exact Mass: 120.0375 Exact Mass: 103.0422
Figure imgf000178_0003
422Exact Mass: 120.0375 Exact Mass: 103.0422
Figure imgf000178_0003
422
Exact Mass: 70.0055
Figure imgf000178_0004
Exact Mass: 70.0055
Figure imgf000178_0004
Exact Mass: 180.0245 Exact Mass: 180.0245
Exact Mass: 98.0368 Exact Mass: 98.0368
Exact Mass: 126.0681 Exact Mass: 126.0681
〇、\ .OH  〇, \ .OH
Exact Mass: 182.1307 Exact Mass: 182.1307
Exact Mass: 182.1307 Exact Mass: 182.1307
Exact Mass: 70.0419Exact Mass: 70.0419
Figure imgf000178_0005
Figure imgf000178_0005
10 「〇H 19  10 「〇H 19
Exact Mass: 82.0783 Exact Mass: 82.0783
Exact Mass: 70.0419
Figure imgf000178_0006
Exact Mass: 70.0419
Figure imgf000178_0006
[化 140C] Exact Mass: 138.1409
Figure imgf000179_0001
[Chemical 140C] Exact Mass: 138.1409
Figure imgf000179_0001
Figure imgf000179_0002
Figure imgf000179_0002
Exact Mass: 168.1514 Exact Mass: 224.0507
Figure imgf000179_0003
Exact Mass: 168.1514 Exact Mass: 224.0507
Figure imgf000179_0003
Exact Mass: 84.0575
Figure imgf000179_0004
Exact Mass: 84.0575
Figure imgf000179_0004
Exact Mass: 1 12.0888  Exact Mass: 1 12.0888
[化 140D] [Chemical 140D]
Figure imgf000180_0001
Figure imgf000180_0001
Figure imgf000180_0002
Figure imgf000180_0002
上記アセチレン化合物と 5位アジド体 CMPシアル酸誘導体のクリック反応により合 成された化合物を以下に示す。 The compounds synthesized by the click reaction of the above acetylene compound and the 5-position azide CMP sialic acid derivative are shown below.
[化 140E]  [Chemical 140E]
Figure imgf000180_0003
Figure imgf000180_0003
式中の Rは、以下である: R in the formula is:
[化 140F] [Chemical 140F]
Figure imgf000181_0001
Figure imgf000181_0001
[化 140G] [Chemical 140G]
Figure imgf000182_0001
Figure imgf000182_0001
Figure imgf000182_0002
Figure imgf000182_0002
[化 140H] 3 [Chemical 140H] Three
3 8 3 8
Figure imgf000183_0001
Figure imgf000183_0001
Figure imgf000183_0002
Figure imgf000183_0002
41  41
Click反応の収率は ESI— MS (negative mode)で、原料と目的化合物のピーク強 度比により見積もった。結果を図 12Bに示す。アセチレンライブラリの A37〜A41に っ 、ては、原料のピークが消えて 、ることを確認した。 The yield of the Click reaction was ESI—MS (negative mode), and was estimated from the peak intensity ratio of the raw material and the target compound. The results are shown in FIG. 12B. It was confirmed that the peaks of the raw materials disappeared in A37 to A41 of the acetylene library.
以下のアセチレン化合物（アセチレンライブラリの A1〜A36、 L2N、 L2A、 L2C、 L3N、 L3A、 L3C)についても、上記と同様に、 9位アジド体シアル酸にクリック反応 を施した。  For the following acetylene compounds (acetylene library A1 to A36, L2N, L2A, L2C, L3N, L3A, L3C), click reaction was performed on the 9-position azide sialic acid in the same manner as described above.
[化 1则 Cl、[Chemical 1 Cl,
Figure imgf000184_0001
Figure imgf000184_0001
Exact Mass: 102.0236 Exact Mass: 102.0236
Exact Mass: 102.047
Figure imgf000184_0002
Exact Mass: 102.047
Figure imgf000184_0002
Exact Mass: 167.1674 Exact Mass: 167.1674
Exact M ass: 172.1252Exact M ass: 172.1252
Figure imgf000184_0003
Figure imgf000184_0003
Exact M ass: 120.0375 Exact Mass: 103.0422  Exact M ass: 120.0375 Exact Mass: 103.0422
Figure imgf000184_0004
Figure imgf000184_0004
[化 140J] 58
Figure imgf000185_0001
[Chemical 140J] 58
Figure imgf000185_0001
Exact Mass: 126.1045 Exact Mass: 126.1045
23 twenty three
Exact Mass: 208.2191
Figure imgf000185_0002
Exact Mass: 208.2191
Figure imgf000185_0002
Exact Mass: 124.0888  Exact Mass: 124.0888
Figure imgf000185_0003
Figure imgf000185_0003
Exact Mass: 168.1514 Exact Mass: 224.0507
Figure imgf000185_0004
Exact Mass: 168.1514 Exact Mass: 224.0507
Figure imgf000185_0004
Exact Mass: 84.0575  Exact Mass: 84.0575
Figure imgf000185_0005
Figure imgf000185_0005
Exact Mass: 112.0888  Exact Mass: 112.0888
[化 140K]
Figure imgf000186_0001
[Chemical 140K]
Figure imgf000186_0001
L3C  L3C
L2C 上記アセチレン化合物と 9位アジド体 CMPシアル酸誘導体のクリック反応により合 成された化合物を以下に示す。  The compounds synthesized by the click reaction of L2C acetylene compound and 9-position azide CMP sialic acid derivative are shown below.
[化 140L] [Chemical 140L]
Figure imgf000186_0002
式中の Rは、以下である:
Figure imgf000186_0002
R in the formula is:
[ィ匕 140M] [I 匕 140M]
[腸 [Intestines
03 03
Figure imgf000187_0001
Figure imgf000187_0001
l7CS0S0/.00Zdf/X3d 981· ΐεθΐ80/.ΟΟΖ OAV l7CS0S0 / .00Zdf / X3d 981 · εθΐ80 / .ΟΟΖ OAV
Figure imgf000188_0001
Figure imgf000188_0001
Figure imgf000188_0002
Figure imgf000188_0002
[化 140O] [Chemical 140O]
Figure imgf000189_0001
Figure imgf000189_0001
Figure imgf000189_0002
Figure imgf000189_0002
Click反応の収率は ESI— MS (negative mode)で、原料と目的化合物のピーク強 度比により見積もった。結果を図 12Cに示す。 The yield of the Click reaction was ESI—MS (negative mode), and was estimated from the peak intensity ratio of the raw material and the target compound. The results are shown in FIG. 12C.
(0. 5g/ml BSA、 0. 1 % Triton CF— 54 を含む 40mMcacodylate緩衝 溶液、 pH7. 5、 5 /z 1)に CMPシアル酸、 aoWR標識ガラクトシルキトトリオース、阻害 剤をそれぞれ最終濃度が 200 μ M、 lmM、 200 μ Μになるように調整した。シアル 酸転移酵素（以下に示される、 Calbiochemより入手可能な a 2— 6シアル酸転移酵 素または α 2— 3シアル酸転移酵素）（lmU、 5 ^ 1)を添カ卩し、全量 25uLで反応温度 37°Cで 2時間反応した後、反応溶液をァセトニトリルで 5倍に希釈することにより酵素 を失活させた。反応液をさらに水で 2倍に希釈した後、マトリクス溶液（2, 5—ジヒドロ キシ安息香酸、 lOmgZmL 30%ァセトニトリル溶液）で 20倍に混合し、 MALDI— TOF MS (Bruker社製、 UltraFLEX)で直接質量分析した。  (40 mM cacodylate buffer solution containing 0.5 g / ml BSA, 0.1% Triton CF—54, pH 7.5, 5 / z 1) with final concentrations of CMP sialic acid, aoWR-labeled galactosylchitotriose, and inhibitor. Adjustments were made to 200 μM, lmM, and 200 μM. Add sialyltransferase (a2-6 sialyltransferase or α2-3 sialyltransferase available from Calbiochem as shown below) (lmU, 5 ^ 1) to a total volume of 25 uL. After reacting at a reaction temperature of 37 ° C for 2 hours, the enzyme was inactivated by diluting the reaction solution 5-fold with acetonitrile. The reaction solution was further diluted 2 times with water, mixed 20 times with a matrix solution (2,5-dihydroxybenzoic acid, lOmgZmL 30% acetonitrile solution), and MALDI-TOF MS (Bruker, UltraFLEX). Direct mass spectrometry was performed.
[化 141A] [Chemical 141A]
Figure imgf000190_0001
Figure imgf000190_0002
Figure imgf000190_0001
Figure imgf000190_0002
インヒビ夕一Inhibin Yuichi
Figure imgf000190_0003
Figure imgf000190_0003
およびアセチレンィ匕合物と 9位アジド体 CMPシアル酸誘導体のクリック反応により合 成された化合物 And acetylene compound and 9-position azide compound synthesized by click reaction of CMP sialic acid derivative
[化 141B] [Chemical 141B]
Figure imgf000191_0001
式中の Rは以下:
Figure imgf000191_0001
Where R is:
[化 141C] [Chemical 141C]
Figure imgf000192_0001
Figure imgf000192_0001
Figure imgf000192_0002
Figure imgf000192_0002
2020
[化 141D] [Chemical 141D]
Figure imgf000193_0001
Figure imgf000193_0001
Figure imgf000193_0002
Figure imgf000193_0002
[化 141E] [Chemical 141E]
Figure imgf000194_0001
Figure imgf000194_0001
Figure imgf000194_0002
前記の反応条件に置いて、阻害剤 29〜34を、それぞれ α—2, 6シアル酸転移酵 素、 α— 2, 3シアル酸転移酵素について、合計 12の反応を行った。反応条件は以 下の通りである。
Figure imgf000194_0002
Under the reaction conditions described above, the inhibitors 29 to 34 were subjected to a total of 12 reactions for α-2,6-sialyltransferase and α-2,3-sialyltransferase, respectively. The reaction conditions are as follows.
(条件） (Condition)
( a 2— 6シアル酸転移酵素の場合）  (a 2-6 sialyltransferase)
( 1) (1)
緩衝液： 40mM cacodylate -HCl (pH7. 5) , 0. 5g/ml BSA、 0. 1 % Trit on CF- 54 Buffer: 40 mM cacodylate -HCl (pH 7.5), 0.5 g / ml BSA, 0.1% Trit on CF- 54
ドナー： 200 μ M Donor: 200 μM
ァクセプター： ImM Acceptor: ImM
酵素： α 2— 6シアル酸転移酵素（配列番号 6 (アミノ酸配列） ) lmU (Calbiochem より入手） Enzyme: α 2-6 sialyltransferase (SEQ ID NO: 6 (amino acid sequence)) lmU (obtained from Calbiochem)
総量 20 μ 1 Total volume 20 μ 1
(共通） (Common)
それに加えて、 In addition to it,
(2)阻害剤混合溶液 29 200 Μ  (2) Inhibitor mixed solution 29 200 Μ
(3)阻害剤混合溶液 30 200 Μ  (3) Inhibitor mixed solution 30 200 Μ
(4)阻害剤混合溶液 31 200 Μ  (4) Inhibitor mixed solution 31 200 Μ
(5)阻害剤混合溶液 32 200 Μ  (5) Inhibitor mixed solution 32 200 Μ
(6)阻害剤混合溶液 33 200 Μ (7)阻害剤混合溶液 34 200 M (6) Inhibitor mixed solution 33 200 Μ (7) Inhibitor mixed solution 34 200 M
をそれぞれ加えた。 Was added respectively.
( a 2— 3シアル酸転移酵素の場合）  (a 2-3 sialyltransferase)
( 1)  (1)
緩衝液： 40mM cacodylate -HCl (pH7. 5) , 0. 5g/ml BSA、 0. 1 % Trit on CF- 54 Buffer: 40 mM cacodylate -HCl (pH 7.5), 0.5 g / ml BSA, 0.1% Trit on CF- 54
ドナー： 200 μ M Donor: 200 μM
ァクセプター： ImM Acceptor: ImM
酵素： α 2— 3シアル酸転移酵素 （配列番号 7 (アミノ酸配列）） lmU (Calbioche mより入手） Enzyme: α 2-3 sialyltransferase (SEQ ID NO: 7 (amino acid sequence)) lmU (obtained from Calbiochem)
総量 20 μ 1 Total volume 20 μ 1
(共通） (Common)
それに加えて、 In addition to it,
(2)阻害剤混合溶液 29 200 Μ  (2) Inhibitor mixed solution 29 200 Μ
(3)阻害剤混合溶液 30 200 Μ  (3) Inhibitor mixed solution 30 200 Μ
(4)阻害剤混合溶液 31 200 Μ  (4) Inhibitor mixed solution 31 200 Μ
(5)阻害剤混合溶液 32 200 Μ  (5) Inhibitor mixed solution 32 200 Μ
(6)阻害剤混合溶液 33 200 Μ  (6) Inhibitor mixed solution 33 200 Μ
(7)阻害剤混合溶液 34 200 Μ  (7) Inhibitor mixed solution 34 200 Μ
をそれぞれ加えた。重水素標識されたァクセプターを用いてシアル酸転移反応を行 い、内部標準を調製した。阻害剤なし (コントロール)および阻害剤を加えた実験は、 軽水素標識ァクセプターを用いて上記と同様に行った。反応後、内部標準を加え Μ ALDI— TOFにより解析を行った。その結果を図 13に示す。図 13のグラフは酵素反 応の生成物の相対比を示しており、小さいほど阻害活性が強いことを示す。示された ように、クリック反応によって合成した化合物のなかでィ匕合物 31が a 2— 3シアル酸 転移酵素に強い阻害活性が、また《2— 6シアル酸転移酵素 2, 6siaTでは阻害活 性を示さず、基質として認識され転移反応が起こることが明らかとなった。化合物 31 と類似の構造を有する化合物 34は阻害活性を示さないことからクリック反応を行う位 置により阻害活性が大きく異なることが推測される。 Was added respectively. A sialic acid transfer reaction was performed using a deuterium labeled acceptor to prepare an internal standard. Experiments without inhibitor (control) and with inhibitors were performed in the same manner as described above using a light hydrogen labeled acceptor. After the reaction, an internal standard was added and analysis was performed by ALDI-TOF. The results are shown in FIG. The graph of FIG. 13 shows the relative ratio of the enzyme reaction products, and the smaller the value, the stronger the inhibitory activity. As shown, compound 31 has a strong inhibitory activity on a2-3 sialyltransferase, and also on << 2-6 sialyltransferase 2, 6siaT. It was clarified that the transfer reaction was recognized as a substrate. Compound 34, which has a structure similar to Compound 31, does not show inhibitory activity, so it is in the position where click reaction is performed. It is estimated that the inhibitory activity varies greatly depending on the position.
[0360] 上記アセチレン化合物（アセチレンライブラリの A1〜A36)と 5位アジド体 CMPシァ ル酸誘導体のクリック反応により合成された阻害剤についても、上記のひ—2, 6シァ ル酸転移酵素について同様の阻害活性測定を行った。ただし、阻害剤の濃度を 30 μ Μで測定した。その結果を図 14に示す。示されたように、アセチレンライブラリの Α23が、 100%の阻害率を示した。ただし、化合物 4以外すべてに関して転移生成 物が観測された。  [0360] The above acetylene compounds (A1 to A36 in the acetylene library) and the inhibitors synthesized by the click reaction of 5-position azide CMP sialic acid derivatives are also the same as for the above 2-2,6-sialyltransferases. Inhibitory activity was measured. However, the inhibitor concentration was measured at 30 μΜ. The result is shown in FIG. As shown, Α23 of the acetylene library showed 100% inhibition. However, transfer products were observed for all but Compound 4.
[0361] 上記アセチレン化合物（アセチレンライブラリの Α1〜Α41)と 5位アジド体 CMPシァ ル酸誘導体のクリック反応により合成された阻害剤についても、上記のひ—2, 3シァ ル酸転移酵素について同様の阻害活性測定を行った。ただし、アセチレンライブラリ の A1〜A36につ!/ヽて ίま、阻害剤の濃度を 30 μ Μで柳』定し、 37、 38、 40、 41に関 しては阻害剤の濃度を 200 Μで測定した。その結果を図 15Aおよび Βに示す。 化合物 37、 40については阻害活性が強力つたため、阻害剤の濃度を 20 Μにして さらに実験を行った。化合物 39についても同様に測定した。その結果を図 15Cに示 す。化合物 40は 200 Μで阻害活性が 100%であった。また 20 Μでは 38% であった。化合物 39は 20 μ Μの濃度でしか測定を行っていないが 50%と化合物 4 0よりも強い阻害活性を示した。よって 200 Μで活性測定を行った場合は 100% 阻害すると考えられる。これらの結果力も化合物 39が最も強い阻害活性を示した。た だ、し、ィ匕合物 1、 2、 3、 5〜9、 11、 13、 15、 19、 20、 25、 26、 29、 32〜34、 36に関 しては転移生成物が観測された。  [0361] The same applies to the above-mentioned 2-2,3-sialyltransferases for the inhibitors synthesized by the click reaction of the above acetylene compounds (A1 to Α41 of the acetylene library) and the 5-position azide CMP sialic acid derivative. Inhibitory activity was measured. However, for inhibitors A1 to A36 in the acetylene library, the concentration of the inhibitor is determined to be 30 μΜ, and for 37, 38, 40, and 41, the inhibitor concentration is 200 Μ. Measured with The results are shown in Fig. 15A and IV. Since compounds 37 and 40 had strong inhibitory activity, further experiments were carried out at an inhibitor concentration of 20%. Compound 39 was measured in the same manner. The result is shown in Fig. 15C. Compound 40 had an inhibitory activity of 100% at 200 mg. At 20 km, it was 38%. Compound 39, which was measured only at a concentration of 20 μΜ, showed 50% and a stronger inhibitory activity than compound 40. Therefore, if the activity is measured at 200 mg, it is considered to inhibit 100%. As a result, Compound 39 showed the strongest inhibitory activity. However, transition products were observed for compounds 1, 2, 3, 5-9, 11, 13, 15, 19, 20, 25, 26, 29, 32-34, 36. It was done.
[0362] 上記アセチレン化合物（アセチレンライブラリの A1〜A36、 L2N、 L2A、 L2C、 L3 N、 L3A、 L3C)と 9位アジド体 CMPシアル酸誘導体のクリック反応により合成された 阻害剤についても、上記の α—2, 6シアル酸転移酵素、 α—2, 3シアル酸転移酵 素について同様の阻害活性測定を行った。ただし、阻害剤の濃度を 30 μ Μで測 定した。その結果を図 16および図 17に示す。示されたように、アセチレンライブラリの Α23が、 a— 2, 6シアル酸転移酵素では 60%以上の阻害率を示し、 a— 2, 3シァ ル酸転移酵素では 80%以上の阻害率を示した。ただし、 a 2, 6SiaTについて化合 物 1 , 2, 3, 5~ 10, 13, 15〜19, 21 , 22, 24〜29, 34〜36, L2N, L3N, L2C, L3Cに関しては転移生成物が観測され、 oc 2, 3SiaTについて化合物 1〜11、 13、 15、 16、 19〜22、 24〜27、 29〜32、 34、 35、 L2N、 L3N、 L2C, L3Cに関して は転移生成物が観測された。 [0362] The above acetylene compounds (acetylene library A1-A36, L2N, L2A, L2C, L3N, L3A, L3C) and the 9-position azide CMP sialic acid derivatives are also synthesized by the click reaction described above. The same inhibitory activity was measured for α-2,6-sialyltransferase and α-2,3-sialyltransferase. However, the inhibitor concentration was measured at 30 μ 30. The results are shown in FIG. 16 and FIG. As shown, Α23 of the acetylene library showed an inhibition rate of 60% or more for a—2, 6 sialyltransferase, and an inhibition rate of 80% or more for a—2, 3 sialyltransferase. It was. However, for a 2, 6SiaT, compounds 1, 2, 3, 5 to 10, 13, 15 to 19, 21, 22, 24 to 29, 34 to 36, L2N, L3N, L2C, Transfer products are observed for L3C, compounds 1-11, 13, 15, 16, 19-22, 24-27, 29-32, 34, 35 for oc 2, 3SiaT, for L2N, L3N, L2C, L3C A transfer product was observed.
(N—ァセチルガラタトサミン転移酵素のスクリ一ユング）  (N-acetyl galatatosaminyltransferase screen)
(ァクセプターペプチドの合成 (軽水素化合物））  (Synthesis of acceptor peptide (light hydrogen compound))
ペプチド自動合成機（Applied Biosystems社製、 433A Peptide Synthesiz er)により、以下の WR標識 MUC5AC (WRGTTPSPVPTTSTTSAP)を合成した 。無水酢酸 0. 95 ml、N, N—ジイソプロピルェチルァミン 0. 45 ml、 1—ヒドロキシ ベンゾトリアゾール 0. 04 g、 DMF18. 6 mlをカ卩えることにより、 N末端をァセチル 化した。トリフルォロ酢酸 Zトリイソプロビルシラン Z水 = 93Z2Z5の混合溶液を添 加することによりレジンを切断し、アミノ酸の保護基の脱保護を行った。高速液体クロ マトグラフィによって精製し、 MALDI— TOF MS (Bruker社製、 UlyraFLEX)で 直接質量分析することによって、 WR標識 MUC5ACのみカゝらなるスペクトルを得た。 重水素化合物については、重無水酢酸を用いることによって同様に合成した。  The following WR-labeled MUC5AC (WRGTTPSPVPTTSTTSAP) was synthesized by an automatic peptide synthesizer (Applied Biosystems, 433A Peptide Synthesizer). The N-terminal was acetylated by preparing 0.95 ml of acetic anhydride, 0.45 ml of N, N-diisopropylethylamine, 0.04 g of 1-hydroxybenzotriazole, and 18.6 ml of DMF. The resin was cleaved by adding a mixed solution of trifluoroacetic acid Z triisoprovirsilane Z water = 93Z2Z5 to deprotect the amino acid protecting group. By purification by high-performance liquid chromatography and direct mass spectrometry using MALDI-TOF MS (manufactured by Bruker, UlyraFLEX), a spectrum containing only WR-labeled MUC5AC was obtained. The deuterium compound was similarly synthesized by using deuterated acetic anhydride.
[化 142] [Chemical 142]
Figure imgf000198_0001
干
Figure imgf000198_0001
Dried
Figure imgf000198_0002
Figure imgf000198_0002
Figure imgf000198_0003
Figure imgf000198_0003
Figure imgf000198_0004
Figure imgf000198_0004
esoso/Loo∑df/i3d 161 TCOTSO/LOOZ: OAV クリック反応に用いたアセチレン化合物（アセチレンライブラリの A1〜A36、 L2Nおよ び L3N)を以下に示す。 esoso / Loo∑df / i3d 161 TCOTSO / LOOZ: OAV The acetylene compounds (A1 to A36, L2N and L3N in the acetylene library) used for the click reaction are shown below.
[化 143]  [Chemical 143]
.0419.0419
Figure imgf000199_0001
10 广 OH
Figure imgf000199_0001
10 广 OH
Exact Mass: 172.1252  Exact Mass: 172.1252
Exact Mass: 70.0419 Exact Mass: 70.0419
Figure imgf000199_0002
Cl、
Figure imgf000199_0002
Cl,
Exact Mass: 120.0375  Exact Mass: 120.0375
Figure imgf000199_0003
Figure imgf000199_0003
[化 144]
Figure imgf000200_0001
[Chemical 144]
Figure imgf000200_0001
Exact Mass: 310.1933
Figure imgf000200_0002
Exact Mass: 310.1933
Figure imgf000200_0002
Exact Mass: 208.2191 Exact Mass: 208.2191
[化 145]
Figure imgf000201_0001
[Chemical 145]
Figure imgf000201_0001
Exact Mass: 126.1045 Exact Mass: 126.1045
Figure imgf000201_0002
Figure imgf000201_0002
Exact Mass: 124.0888
Figure imgf000201_0003
Exact Mass: 124.0888
Figure imgf000201_0003
Exact Mass: 206.0732 Exact Mass: 206.0732
Figure imgf000201_0004
Figure imgf000201_0004
Exact Mass: 100.016 Exact Mass: 100.016
Figure imgf000201_0005
Figure imgf000201_0005
Exact Mass: 224.0507
Figure imgf000201_0006
上記アセチレン化合物力 クリック反応により合成されたィ匕合物を以下に示す, [化 146]
Figure imgf000202_0001
式中の Rは以下: [化 147] Exact Mass: 224.0507
Figure imgf000201_0006
The above-mentioned compound synthesized by the click reaction is shown below, [Chemical Formula 146]
Figure imgf000202_0001
Where R is: [Chemical 147]
Figure imgf000203_0001
Figure imgf000203_0001
[化 148] [Chemical 148]
Figure imgf000204_0001
Figure imgf000204_0001
[化 149] [Chemical 149]
Figure imgf000205_0001
Figure imgf000205_0001
Figure imgf000205_0002
Figure imgf000205_0002
Figure imgf000205_0003
Figure imgf000205_0003
クリック反応条件： UDP— N3— GalNAc(10 mM水溶液）を 5 1、アセチレン化 合物（10 mMメタノール溶液）を 5 1、トリス [ (1 ベンジル一 1H— 1, 2, 3 トリ ァゾール— 4—ィル)メチル]ァミン (TBTA) (5 mM DMSO溶液）を 5 1、硫酸 銅（5 mM 水溶液）を 5 1、ァスコルビン酸ナトリウム（50 mM水溶液）を 5 μ \ を混合して室温で 3時間反応させ、 ESI - MS (solvent： MeOH)により、阻害剤候 補ィ匕合物の生成を確認した。クリック反応の収率は ESI— MSで測定した。その結果 を図 18に示す。グラフの収率は ESI— MSネガティブモードでのピーク強度比の相 対比を示す。 Click reaction conditions: UDP—N3—GalNAc (10 mM aqueous solution) 51, acetylation 5 1 in the compound (10 mM methanol solution), 51 in Tris [(1 benzyl 1 1H— 1, 2, 3 triazole — 4-yl) methyl] amine (TBTA) (5 mM DMSO solution), sulfuric acid Copper (5 mM aqueous solution) 5 1 and sodium ascorbate (50 mM aqueous solution) 5 μ \ were mixed and reacted at room temperature for 3 hours, and then the inhibitor was supplemented by ESI-MS (solvent: MeOH). Formation of the product was confirmed. The yield of click reaction was measured by ESI-MS. Figure 18 shows the result. The yield of the graph shows the contrast of peak intensity ratio in ESI-MS negative mode.
[0364] (ライブラリーによる阻害剤のスクリーニング）  [0364] (Inhibitor screening by library)
酵素反応は、最終濃度が imidazole— HC1緩衝溶液 (0. 1% Triton X— 100を 含む、 pH7. 2) 50 mM、 UDP— GalNAc 100 M、軽水素 WR標識 MUC5A C 500 M、阻害剤候補化合物 100 M、塩化マンガン 10 mMで行った。 pp GalNAcT- 2 (10 ngZ μ 1)を添加し、全量 40 μ 1、反応温度 37°Cで 40分間反 応した。コントロールとして、阻害剤候補ィ匕合物の代わりにクリック反応試薬を同量カロ えた反応も行った。その後、反応溶液から 2 μ 1とってァセトニトリルで 10倍に希釈す る事により酵素を失活させた。反応溶液を更に水で 2倍に希釈し、マトリクス溶液（2, 5 ジヒドロキシ安息香酸、 10 mgZmL 30%ァセトニトリル溶液)で 10倍に希釈し 、重水素 WR標識化合物を内部標準として加えて MALDI— TOF MS (Bruker社 製、 UlyraFLEX)で直接質量分析することによって、阻害活性を測定した。結果を 図 19に示す。  The final concentration of the enzyme reaction is imidazole—HC1 buffer solution (containing 0.1% Triton X-100, pH 7.2) 50 mM, UDP—GalNAc 100 M, light hydrogen WR-labeled MUC5A C 500 M, candidate inhibitor compound 100 M, manganese chloride 10 mM. pp GalNAcT-2 (10 ngZ μ 1) was added and reacted for 40 minutes at a total temperature of 40 μ 1 and a reaction temperature of 37 ° C. As a control, a reaction in which the same amount of the click reaction reagent was added instead of the inhibitor candidate compound was also performed. Thereafter, 2 μl was taken from the reaction solution and diluted 10-fold with acetonitrile to inactivate the enzyme. The reaction solution is further diluted 2 times with water, diluted 10 times with a matrix solution (2,5 dihydroxybenzoic acid, 10 mgZmL 30% acetonitrile solution), deuterium WR labeled compound is added as an internal standard, and MALDI-TOF Inhibitory activity was measured by direct mass spectrometry using MS (Bruker, UlyraFLEX). The results are shown in Fig. 19.
[0365] (実施例 6 :製剤例）  [Example 6: Formulation example]
実施例 1および 2において製造されたィ匕合物にグルタミン酸ソーダ 5% (重量）、可 溶性澱粉 5% (重量）、ショ糖 5% (重量)および硫酸マグネシウム 7水和物 1% (重量） を含む分散媒と同量混合し、 pH7. 0に修正後、 40°C以下で凍結してから凍結乾 燥を行う。得られた凍結乾燥粉末を 60メッシュの締で粉末化して乾燥粉末を調製す る。これを 2mgと乳糖（日局） 61mg、澱粉（日局） 116, 2mg、結合剤としてポリビ- ルピロリドン K25 (日局） 20mg、滑沢剤としてステアリン酸マグネシウム（日局） 0. 8m gを加えて均一に混合し、打鍵機で圧縮成型し 1錠当たり 200mgの素錠を作り、さら に、ヒドロキシプロピルセルロースを用いてフィルムコーティングを施して白色のフィル ムコーティングされた錠剤を製造する。 The compounds prepared in Examples 1 and 2 were mixed with sodium glutamate 5% (by weight), soluble starch 5% (by weight), sucrose 5% (by weight) and magnesium sulfate heptahydrate 1% (by weight). Mix the same amount with a dispersion medium containing, and adjust to pH 7.0, freeze at 40 ° C or lower, and then freeze-dry. The resulting freeze-dried powder is pulverized by tightening with 60 mesh to prepare a dry powder. 2 mg of this, lactose (JP) 61 mg, starch (JP) 116, 2 mg, polyvinylpyrrolidone K25 (JP) 20 mg as binder, magnesium stearate (JP) 0.8 mg as lubricant In addition, mix evenly and compress with a key press to make uncoated tablets of 200 mg per tablet, and then apply a film coating with hydroxypropylcellulose to form a white film. To produce coated tablets.
[0366] このような錠剤は、健康食品または医薬として投与することができる。  [0366] Such a tablet can be administered as a health food or a medicine.
産業上の利用可能性  Industrial applicability
[0367] 本発明により、新規ィ匕合物および糖転移酵素阻害剤ならびにその利用が提供され た。本発明によって、従来に存在しな力つた強力な阻害因子が提供された。 [0367] The present invention provides novel compounds, glycosyltransferase inhibitors, and uses thereof. According to the present invention, a powerful inhibitory force that has not existed in the past has been provided.

Claims

請求の範囲 The scope of the claims
[1] 以下の構造式：  [1] The following structural formula:
A-B-C  A-B-C
を有する、化合物またはその塩であって、  A compound or a salt thereof,
式中  In the formula
Aは、アジド（― N )、アルデヒド置換基（― C ( = 0)—R)またはアルデヒド（— CHO)  A is azide (—N), aldehyde substituent (—C (= 0) —R) or aldehyde (—CHO)
3  Three
であり、 Rはァノレキノレであり、  R is Anolequinore,
Bは、糖成分であり、  B is a sugar component,
Cは、ヌクレオチジル基である、  C is a nucleotidyl group,
ただし、糖成分が GlcNAcである場合は、 Aはアジドである、  However, when the sugar component is GlcNAc, A is an azide.
化合物またはその塩。  Compound or salt thereof.
[2] 前記 Cは、ヌクレオシドーリン酸、ヌクレオシドニリン酸またはヌクレオシド三リン酸であ る、請求項 1に記載の化合物またはその塩。  [2] The compound or a salt thereof according to claim 1, wherein C is nucleoside-phosphate, nucleoside diphosphate, or nucleoside triphosphate.
[3] 前記 Cは、ピリミジンヌクレオシドリン酸またはプリンヌクレオシドリン酸である、請求項[3] The C is pyrimidine nucleoside phosphate or purine nucleoside phosphate.
1に記載の化合物またはその塩。 1. The compound or a salt thereof according to 1.
[4] 前記 Cは、アデノシン、デォキシアデノシン、グアノシン，デォキシグアノシン、シチジ ン、デォキシチミジン、チミジン（リボチミジン）、デォキシチミジンまたはゥリジンあるい はそれらの改変体のホスフェートである、請求項 1に記載の化合物またはその塩。 [4] The claim 1, wherein C is a phosphate of adenosine, deoxyadenosine, guanosine, deoxyguanosine, cytidine, deoxythymidine, thymidine (ribothymidine), deoxythymidine or uridine, or a variant thereof. Or a salt thereof.
[5] 前記 Bは、 D—ガラクトース、 L—フコース、シアル酸、 D— GlcNAc、 D— GalNAc、[5] The B is D-galactose, L-fucose, sialic acid, D-GlcNAc, D-GalNAc,
D— ManNAc、 D—マンノース、もしくは D—グルコースまたはその誘導体である、請 求項 1に記載の化合物またはその塩。 The compound or a salt thereof according to claim 1, which is D-ManNAc, D-mannose, or D-glucose or a derivative thereof.
[6] 以下の式： [6] The following formula:
Figure imgf000208_0001
Figure imgf000209_0001
Figure imgf000208_0001
Figure imgf000209_0001
剛
Figure imgf000209_0002
Tsuyoshi
Figure imgf000209_0002
剛
Figure imgf000209_0003
Tsuyoshi
Figure imgf000209_0003
t£S0S0/L00Zd£/∑Jd 803 ΐεθΐ80/.ΟΟΖ OAV 寧〔 t £ S0S0 / L00Zd £ / ∑Jd 803 ΐεθΐ80 / .ΟΟΖ OAV Ning
Figure imgf000210_0001
Figure imgf000210_0001
Figure imgf000211_0001
Figure imgf000211_0001
に示すィ匕合物またはその塩。 Or a salt thereof.
以下の構造式  The following structural formula
X-Y-B-C  X-Y-B-C
を有する化合物であって、  A compound having
式中  In the formula
Bは、糖成分であり、  B is a sugar component,
Cは、ヌクレオチジル基であり、  C is a nucleotidyl group,
Xは、嵩高基であり、 Yは、— O— N =基または、— NH— N =基または 1, ァゾール基  X is a bulky group, Y is —O—N = group or —NH—N = group or 1, azole group
Figure imgf000211_0002
である、
Figure imgf000211_0002
Is,
化合物。  Compound.
[8] 前記 Cは、ヌクレオシドーリン酸、ヌクレオシドニリン酸またはヌクレオシド三リン酸であ る、請求項 7に記載の化合物またはその塩。  [8] The compound or a salt thereof according to claim 7, wherein the C is nucleoside phosphoric acid, nucleoside diphosphate or nucleoside triphosphate.
[9] 前記 Cは、ピリミジンヌクレオシドリン酸またはプリンヌクレオシドリン酸である、請求項[9] The C is a pyrimidine nucleoside phosphate or a purine nucleoside phosphate.
7に記載の化合物またはその塩。 7. The compound according to 7, or a salt thereof.
[10] 前記 Cは、アデノシン、デォキシアデノシン、グアノシン，デォキシグアノシン、シチジ ン、デォキシチミジン、チミジン（リボチミジン）、デォキシチミジンまたはゥリジンあるい はそれらの改変体のホスフェートである、請求項 7に記載の化合物またはその塩。 [10] The C is adenosine, deoxyadenosine, guanosine, deoxyguanosine, cytidine, deoxythymidine, thymidine (ribothymidine), deoxythymidine or uridine. 8. The compound according to claim 7 or a salt thereof, wherein is a phosphate of a variant thereof.
[11] 前記 Bは、 D—ガラクトース、 L—フコース、シアル酸、 D— GlcNAc、 D— GalNAc、[11] The B is D-galactose, L-fucose, sialic acid, D-GlcNAc, D-GalNAc,
D— ManNAc、 D—マンノース、もしくは D—グルコースまたはその誘導体である、請 求項 7に記載の化合物またはその塩。 The compound or a salt thereof according to claim 7, which is D-ManNAc, D-mannose, or D-glucose or a derivative thereof.
[12] Xは、以下： [12] X is:
[化 10A]  [Chemical 10A]
Figure imgf000212_0001
[化應]
Figure imgf000212_0001
[Chemical]
Figure imgf000213_0001
Figure imgf000213_0001
[化 10C] [Chemical 10C]
Figure imgf000214_0001
Figure imgf000214_0001
[化 10D] [Chemical 10D]
Figure imgf000215_0001
Figure imgf000215_0001
[化 10E] [Chemical 10E]
Figure imgf000216_0001
Figure imgf000216_0001
Figure imgf000216_0002
Figure imgf000216_0002
[化 10G] [Chemical 10G]
Figure imgf000217_0001
Figure imgf000217_0001
Figure imgf000217_0002
Figure imgf000217_0003
Figure imgf000217_0004
Figure imgf000217_0002
Figure imgf000217_0003
Figure imgf000217_0004
[化蘭] [Bakeran]
Figure imgf000218_0001
Figure imgf000218_0002
Figure imgf000218_0003
Figure imgf000218_0001
Figure imgf000218_0002
Figure imgf000218_0003
Figure imgf000218_0004
Figure imgf000218_0005
Figure imgf000218_0004
Figure imgf000218_0005
[化 101] ■0〜ο 0〜o 0〜O ^ [Chemical 101] ■ 0 to ο 0 to o 0 to O ^
Figure imgf000219_0001
Figure imgf000219_0001
ま は
Figure imgf000219_0002
Or
Figure imgf000219_0002
の！ヽずれかから選択される、請求項 7に記載の化合物。 of! 8. A compound according to claim 7, selected from any one of them.
[13] [ィ匕 11] [13] [Yi 11]
Figure imgf000220_0001
Figure imgf000220_0002
Figure imgf000220_0003
Figure imgf000220_0001
Figure imgf000220_0002
Figure imgf000220_0003
[化 11- 1] [Chem. 11-1]
Figure imgf000221_0001
Figure imgf000221_0001
[化 11— 2] [Chemical 11—2]
Figure imgf000222_0001
Figure imgf000222_0001
Figure imgf000222_0002
Figure imgf000222_0002
Figure imgf000222_0003
Figure imgf000222_0003
Figure imgf000222_0004
である、化合物。
Figure imgf000222_0004
A compound.
[14] 請求項 7〜： 13のいずれか 1項に記載の化合物を含む、糖転移酵素阻害剤。  [14] A glycosyltransferase inhibitor comprising the compound according to any one of claims 7 to 13:
[15] 前記糖転移酵素は、 (X 1, 4 ガラクトース転移酵素、 ex 1, 3 ガラクトース転移酵素 , j8 1, 4—ガラクトース転移酵素， /3 1， 3—ガラクトース転移酵素， j8 1, 6—ガラタト ース転移酵素、 α 2， 6 シアル酸転移酵素、 ひ1, 4 ガラクトース転移酵素、セラミ ドガラタトース転移酵素、 αΐ, 2 フコース転移酵素、ひ1, 3 フコース転移酵素、 αΐ, 4ーフコース転移酵素、 αΐ, 6 フコース転移酵素、 α 1, 3— Ν ァセチルガ ラタトサミン転移酵素、 αΐ, 6— Ν ァセチルガラタトサミン転移酵素、 β ΐ, 4-Ν- ァセチルガラタトサミン転移酵素、ポリペプチド Ν ァセチルガラタトサミン転移酵素、 β 1, 4—Νァセチルダルコサミン転移酵素、 131, 2— Νァセチルダルコサミン転移酵 素、 β ΐ, 3— Νァセチルダルコサミン転移酵素、 β ΐ, 6—Νァセチルダルコサミン転 移酵素、 αΐ, 4—Νァセチルダルコサミン転移酵素、 β ΐ, 4 マンノース転移酵素、 αΐ, 2 マンノース転移酵素、 αΐ, 3 マンノース転移酵素、 α 1, 4 マンノース 転移酵素、 αΐ, 6 マンノース転移酵素、《1, 2 グルコース転移酵素、《1, 3— グルコース転移酵素、 α2, 3 シアル酸転移酵素、《2, 6 シアル酸転移酵素、 a 1, 6—ダルコサミン転移酵素、ひ1, 6 キシロース転移酵素、 j8キシロース転移酵 素、 j81, 3 グルクロン酸転移酵素およびヒアルロン酸合成酵素力 なる群より選択 される、請求項 14に記載の糖転移酵素阻害剤。 [15] The glycosyltransferases are (X 1,4 galactosyltransferase, ex 1,3 galactose transferase, j8 1,4-galactose transferase, / 3 1,3-galactose transferase, j8 1,6— Galatatosyltransferase, α 2,6 sialyltransferase, 1,4 galactose transferase, ceramic Degaratatosyltransferase, αΐ, 2 fucose transferase, 1,3 fucose transferase, αΐ, 4-fucose transferase, αΐ, 6 fucose transferase, α 1, 3—Ν acetylacetyl ratatosamine transferase, αΐ, 6—Ν Cetylgalatatosamine transferase, β ΐ, 4-Ν-Acetyl galatatosamine transferase, polypeptide Ν Acetyl galatatosamine transferase, β 1, 4—Νacetyltilcosamine transferase, 131, 2— Νacetylyl darcosamine transferase, β ΐ, 3-- acetyl dalcosamine transferase, β ΐ, 6- acetyl darcosamine transferase, α ΐ, 4--acetyl darcosamine transferase, β ΐ , 4 mannose transferase, αΐ, 2 mannose transferase, αΐ, 3 mannose transferase, α 1, 4 mannose transferase, αΐ, 6 mannose transferase, << 1, 2 glucose transferase, << 1, 3-glucose transfer Enzymes, alpha 2, 3 sialyltransferase, "2, 6 sialyltransferase, a 1, 6- Darukosamin transferase, shed 1, 6 xylose transferase, j8 xylose transfer enzyme, J81, 3 glucuronosyltransferase 15. The glycosyltransferase inhibitor according to claim 14, wherein the glycosyltransferase inhibitor is selected from the group consisting of hyaluronic acid synthase.
[16] 前記糖転移酵素は、 oc 1, 4 ガラクトース転移酵素、 oc 1, 3 ガラクトース転移酵素 , j81, 4—ガラクトース転移酵素， β ΐ, 3—ガラクトース転移酵素， β ΐ, 6—ガラタト ース転移酵素、 αΐ, 2 フコース転移酵素、《1, 3 フコース転移酵素、《1, 4— フコース転移酵素、《1, 6 フコース転移酵素および a 2, 3 シアル酸転移酵素、 a 2, 6 シアル酸転移酵素からなる群より選択される、請求項 14に記載の糖転移酵 素阻害剤。 [16] The glycosyltransferases are oc 1,4 galactose transferase, oc 1,3 galactose transferase, j81, 4-galactose transferase, β ΐ, 3-galactose transferase, β ΐ, 6-galactose Transferase, αΐ, 2 fucose transferase, << 1, 3 fucose transferase, << 1, 4— fucose transferase, << 1, 6 fucose transferase and a 2, 3 sialyltransferase, a 2, 6 sialic acid 15. The glycosyltransferase inhibitor according to claim 14, which is selected from the group consisting of transferases.
[17] 請求項 7〜13のいずれか 1項に記載の化合物を含む、糖転移酵素の活性の異常に 起因する状態、障害または疾患を処置または予防するための糸且成物。  [17] A thread and a composition for treating or preventing a condition, disorder or disease caused by an abnormal activity of glycosyltransferase, comprising the compound according to any one of claims 7 to 13.
[18] 請求項 7〜 13のいずれか 1項に記載の化合物を含む組成物を投与する工程を包含 する、糖転移酵素の活性の異常に起因する状態、障害または疾患を処置または予 防するための方法。  [18] To treat or prevent a condition, disorder or disease caused by abnormal glycosyltransferase activity, comprising the step of administering a composition comprising the compound according to any one of claims 7 to 13. Way for.
[19] 請求項 1〜13のいずれか 1項に記載の化合物の、糖転移酵素の活性の異常に起因 する状態、障害または疾患を処置または予防するための医薬の製造のための使用。  [19] Use of the compound according to any one of claims 1 to 13 for the manufacture of a medicament for treating or preventing a condition, disorder or disease caused by abnormal glycosyltransferase activity.
[20] 請求項 1〜6のいずれか 1項に記載の化合物と、アルキレン基、アミノ基、またはァミノ ォキシ基を有する以下の化合物： [化 12] [20] The compound according to any one of claims 1 to 6 and the following compound having an alkylene group, an amino group, or an aminooxy group: [Chemical 12]
Figure imgf000224_0001
Figure imgf000224_0001
[化 13] [Chemical 13]
Figure imgf000225_0001
Figure imgf000225_0002
Figure imgf000225_0003
Figure imgf000225_0001
Figure imgf000225_0002
Figure imgf000225_0003
[化 14] [Chemical 14]
Figure imgf000226_0001
Figure imgf000226_0001
[化 15A] [Chemical 15A]
Figure imgf000227_0001
Figure imgf000227_0001
^CS0S0/.00Zdf/X3d 9SS ΐε0180/.00Ζ OAV
Figure imgf000228_0001
^ CS0S0 / .00Zdf / X3d 9SS ΐε0180 / .00Ζ OAV
Figure imgf000228_0001
Cに 、NT
Figure imgf000228_0002
C to NT
Figure imgf000228_0002
[化 15C]
Figure imgf000229_0001
[Chemical 15C]
Figure imgf000229_0001
Figure imgf000230_0001
Figure imgf000230_0002
Figure imgf000230_0003
Figure imgf000230_0004
Figure imgf000230_0005
Figure imgf000230_0001
Figure imgf000230_0002
Figure imgf000230_0003
Figure imgf000230_0004
Figure imgf000230_0005
Figure imgf000231_0001
Figure imgf000231_0002
Figure imgf000231_0003
Figure imgf000231_0001
Figure imgf000231_0002
Figure imgf000231_0003
Figure imgf000231_0004
Figure imgf000231_0004
[化 15G] [Chemical 15G]
Figure imgf000232_0001
Figure imgf000232_0001
[化 15H]
Figure imgf000233_0001
[Chemical 15H]
Figure imgf000233_0001
Figure imgf000233_0002
または
Figure imgf000233_0002
Or
とを混合して、アルキレン基とアジド基が反応する力 またはアルデヒド基とアミノ基も しくはァミノォキシ基が反応する条件下で、請求項 7〜 13の 、ずれかに記載の化合 物を生成させる工程を包含する、請求項 7〜13のいずれかに記載の化合物の製造 方法。 To produce a compound according to any one of claims 7 to 13 under the condition that an alkylene group and an azide group react or a condition where an aldehyde group and an amino group or an aminooxy group react. The method for producing a compound according to any one of claims 7 to 13, comprising a step.
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011519567A (en) * 2008-05-02 2011-07-14 シアトル ジェネティクス，インコーポレーテッド Methods and compositions for preparing antibodies and antibody derivatives having low core fucosylation
US20120035115A1 (en) * 2008-09-23 2012-02-09 Alnylam Pharmaceuticals, Inc. Chemical modifications of monomers and oligonucleotides with cycloaddition
JP2013541503A (en) * 2010-08-05 2013-11-14 シアトル ジェネティクス，インコーポレーテッド Methods for inhibiting protein fucosylation in vivo using fucose analogs
WO2015112013A1 (en) * 2014-01-24 2015-07-30 Synaffix B.V. Process for the attachment of a galnac moiety comprising a (hetero)aryl group to a glcnac moiety, and product obtained thereby
JP2017538446A (en) * 2014-12-01 2017-12-28 アムジエン・インコーポレーテツド Process for manipulating the level of glycan content of glycoproteins
US10266502B2 (en) 2014-01-24 2019-04-23 Synaffix B.V. Process for the cycloaddition of a halogenated 1,3-dipole compound with a (hetero)cycloalkyne
US10350228B2 (en) 2012-08-23 2019-07-16 Seattle Genetics, Inc. Treatment of sickle cell disease and inflammatory conditions
CN112805292A (en) * 2019-08-14 2021-05-14 斯微(上海) 生物科技有限公司 Modified nucleoside and synthetic method thereof
US11168085B2 (en) 2014-01-24 2021-11-09 Synaffix B.V. Process for the cycloaddition of a hetero(aryl) 1,3-dipole compound with a (hetero)cycloalkyne

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004099231A2 (en) * 2003-04-09 2004-11-18 Neose Technologies, Inc. Glycopegylation methods and proteins/peptides produced by the methods
JP2005314382A (en) * 2004-03-30 2005-11-10 Yamasa Shoyu Co Ltd Azidized aminosugar nucleotide and its application
WO2006035057A1 (en) * 2004-09-29 2006-04-06 Novo Nordisk Health Care Ag Modified proteins

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004099231A2 (en) * 2003-04-09 2004-11-18 Neose Technologies, Inc. Glycopegylation methods and proteins/peptides produced by the methods
JP2005314382A (en) * 2004-03-30 2005-11-10 Yamasa Shoyu Co Ltd Azidized aminosugar nucleotide and its application
WO2006035057A1 (en) * 2004-09-29 2006-04-06 Novo Nordisk Health Care Ag Modified proteins

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHEMIBIOCHEM., vol. 2, no. 12, 2001, pages 884 - 894 *
DATABASE CAPLUS [online] BULTER T. ET AL.: "Chemoenzymatic synthesis of biotinylated nucleotide sugars as substrates for glycosyltransferases", XP003016032, accession no. STN Database accession no. (2001:920512) *
TAKAYA K. ET AL.: "Rational Design, Synthesis, and Characterization of Novel Inhibitors for Human beta1,4-Galactosyltransferase", JOURNAL OF MEDICINAL CHEMISTRY, vol. 48, no. 19, 2005, pages 6054 - 6065, XP003016031 *

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