WO2010050178A1 - トレハロース化合物、その製造方法、及び該化合物を含有する医薬 - Google Patents
トレハロース化合物、その製造方法、及び該化合物を含有する医薬 Download PDFInfo
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- WO2010050178A1 WO2010050178A1 PCT/JP2009/005650 JP2009005650W WO2010050178A1 WO 2010050178 A1 WO2010050178 A1 WO 2010050178A1 JP 2009005650 W JP2009005650 W JP 2009005650W WO 2010050178 A1 WO2010050178 A1 WO 2010050178A1
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- trehalose
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- 0 CCCCCCCCC(*)C(OCC([C@](C(*)C1OCc2ccccc2)OCc2ccccc2)O[C@@]1O[C@@](C(C1OCc2ccccc2)OCc2ccccc2)OC(COC(C(*)CCCCCCCC)=O)[C@]1O)=C Chemical compound CCCCCCCCC(*)C(OCC([C@](C(*)C1OCc2ccccc2)OCc2ccccc2)O[C@@]1O[C@@](C(C1OCc2ccccc2)OCc2ccccc2)OC(COC(C(*)CCCCCCCC)=O)[C@]1O)=C 0.000 description 2
- OYVXVLSZQHSNDK-UHFFFAOYSA-N CC(N(C)OC)=O Chemical compound CC(N(C)OC)=O OYVXVLSZQHSNDK-UHFFFAOYSA-N 0.000 description 1
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7024—Esters of saccharides
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- 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
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
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- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/02—Antidotes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
- C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
- C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
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- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
- C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
- C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
- C07H13/06—Fatty acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
- C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
- C07H13/08—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals directly attached to carbocyclic rings
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
- C07H3/04—Disaccharides
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates to a trehalose compound, a method for producing the same, and a medicine containing the compound.
- verotoxin is produced in the body, especially in elderly people and children with weak resistance, such as hemolytic uremic syndrome. May have unusual symptoms.
- Antibiotics may be administered for such infections, but by administering antibiotics the bacteria are killed, and toxins inside the bacteria are released to the outside of the bacteria at the same time. It has been pointed out that there is a risk of the condition getting worse.
- highly contagious infections such as O-157 that develop only by ingesting hundreds to thousands of bacteria, in order to prevent secondary infection, In some cases, antibiotics have to be chosen.
- hemolytic uremic syndrome may occur in about 10% of cases with O-157, and in this case, plasma exchange or dialysis therapy is performed. It can be said that this is a heavy treatment.
- symptomatic treatment is a priority method for coping with toxins produced by bacteria, and other methods include the use of toxin adsorbents and the use of antibodies against toxins.
- side effects such as constipation, and the use of antibodies is inconvenient in that antibodies must be developed for each toxin.
- TDM trehalose dimycolate
- TDCM trehalose dicorynomycolate
- TDM is found as a glycolipid present on the cell surface of Mycobacterium tuberculosis and is known to exhibit immunostimulatory activity and anticancer activity.
- TDCM a homologue having a shorter carbon number than TDM, was isolated from the cognate Diphtheria bacterium (Corynebacterium spp), and it was revealed that TDCM and its stereoisomer show immunostimulatory activity and anticancer activity, respectively. Has been.
- TDM and TDCM are highly toxic and cannot be used as pharmaceuticals. Therefore, in order to use it as a medicine, it was necessary to synthesize a compound having reduced toxicity while maintaining or enhancing the activity.
- a trehalose 6,6′-diester compound which is an ester of trehalose and a fatty acid
- tests such as a toxicity test and a macrophage activation action were performed (see Non-Patent Document 1).
- the presence or absence of a ⁇ -hydroxyl group, the compound having 30,32,48 carbon atoms in the length of the alkyl part of the lipid, the compound in which the ester bond between the sugar and the lipid is replaced with an amide bond, etc. are examined. In terms, it was considered that ester bonds and long-chain fatty acids made important contributions to toxicity.
- the present inventors have synthesized a derivative or the like having an ester bond or an amide bond and changing the ⁇ -position hydroxyl group to a hydrogen atom or a methoxy group with respect to the TDM derivative (see Patent Document 1).
- the derivatives described in the literature have a relatively short alkyl moiety of fatty acid of about 7 carbon atoms, and the activity is only measured as an adenosine A3 receptor antagonist.
- the present inventors succeeded in synthesizing amide derivatives of TDCM in which the hydroxyl group of TDCM is changed to a hydrogen atom so that it is not an asymmetric carbon and the ester bond is changed to an amide bond.
- the immunostimulatory effect was confirmed (refer patent document 2).
- these amide derivatives were later found to have an effect of inducing cancer, and it was unavoidable to use them as pharmaceutical compounds.
- TDM or TDCM derivative a highly safe treatment or onset suppression method has not yet been established for various symptoms derived from pathogenic bacteria, but an effective and safe treatment or onset suppression method has been established. Is desired.
- TDM and TDCM When synthesizing derivatives of TDM and TDCM, since TDM and TDCM themselves are highly toxic, it is necessary to synthesize a compound having activity and low toxicity. Although some modified TDM or TDCM have been known as the prior art, TDM and TDCM are glycolipids, and sugar chains have many hydroxyl groups and high polarity, which may be difficult to synthesize. The structure-activity relationship of which structure leads to what activity has not yet been clarified.
- an object of the present invention is to produce many derivatives of TDM and TDCM, and to provide a compound having high activity and low toxicity, and a medicament containing the compound.
- the use of antibiotics against pathogenic bacteria as a conventional technique is intended to prevent E. coli from releasing toxins by inhibiting the growth of E. coli and killing E. coli.
- the present invention provides a pharmaceutical that can reduce the toxicity of a toxin even when the bacteria grow to produce a toxin. The purpose is to provide.
- the present inventors have found that the trehalose diester compound represented by the formula (1) exhibits excellent antibacterial activity against infectious diseases caused by pathogenic bacteria and has low toxicity.
- X is R 1 —CHR 2 — and X ′ is R 1 ′ —CHR 2 ′ —
- ⁇ -branched compound a compound in which n and n ′ are particularly 0
- ⁇ -branched compound a compound in which n and n ′ are 1 in particular
- the activity tends to be maximized for a specific length.
- the trehalose diester compound it has not been known so far that it is useful as an antibacterial agent even when the toxin itself produced by the fungus is administered in the above-mentioned literature and the like. In the in vivo test, it was found that it is effective not only when the bacterium is administered but also when the toxin itself produced from the bacterium is administered.
- the present invention provides the following formula (1):
- X is a group represented by phenyl, naphthyl, or R 1 —CHR 2 —;
- X ′ is a group represented by phenyl, naphthyl, or R 1 ′ —CHR 2 ′ —
- R 1 , R 1 ′, R 2 and R 2 ′ are each independently a hydrogen atom or a C 1 -C 21 alkyl group, and with respect to R 1 , R 1 ′, R 2 and R 2 ′, each alkyl group
- the hydrogen atom therein may be substituted by a hydroxyl group or an alkoxy group, and all or part of each alkyl group may form a 4-8 membered ring, and R 1 and R 2 , R 1 'And R 2 ' may be connected to each other to form a 4-8 membered ring
- n and n ′ are each independently an integer of 0 to 3.
- X is R 1 —CHR 2 —
- X ′ is R 1 ′ —CHR 2 ′ —
- R 1 , R 1 ′, R 2 and R 2 ′ are each independently hydrogen A compound that is an atomic or unsubstituted and straight-chain C 1 -C 6 alkyl group, wherein n and n ′ are 0, and
- X is R 1 —CHR 2 —
- X ′ is R 1 ′ —CHR 2 ′ —
- R 1 , R 1 ′, R 2 and R 2 ′ are C 14 linear alkyl groups And excluding compounds in which n and n ′ are 0]
- the compound represented by these is provided.
- the present invention also provides a pharmaceutical composition
- a pharmaceutical composition comprising a compound represented by formula (1) and a pharmacologically acceptable carrier.
- the present invention also relates to a medicament comprising a compound represented by the formula (1) and a pharmacologically acceptable carrier, an immunostimulator, a macrophage activator, a neutrophil activator, a phagocytic phagocytic cell
- a pharmaceutical composition used as an action activator, antibacterial infection agent, or fungus-producing toxin neutralizer.
- the present invention also relates to a pharmaceutical composition used as an immunostimulant, macrophage activator, neutrophil activator, phagocytic phagocytic activator, antibacterial infection agent, or fungus-producing toxin neutralizer.
- a pharmaceutical composition used as an immunostimulant, macrophage activator, neutrophil activator, phagocytic phagocytic activator, antibacterial infection agent, or fungus-producing toxin neutralizer.
- the use of a compound of formula (1) for the manufacture is provided.
- the present invention also relates to a method for preventing or treating infectious diseases in mammals including humans, There is provided a method comprising administering to the mammal a therapeutically effective amount of a compound represented by formula (1).
- X is a group represented by phenyl, naphthyl, or R 1 —CHR 2 —;
- X ′ is a group represented by phenyl, naphthyl, or R 1 ′ —CHR 2 ′ —
- R 1 , R 1 ′, R 2 and R 2 ′ are each independently a hydrogen atom or a C 1 -C 21 alkyl group, and with respect to R 1 , R 1 ′, R 2 and R 2 ′, each alkyl group
- the hydrogen atom therein may be substituted by a hydroxyl group or an alkoxy group, and all or part of each alkyl group may form a 4-8 membered ring, and R 1 and R 2 , R 1 'And R 2 ' may be connected to each other to form a 4-8 membered ring, n and n ′ are each independently an integer of 0 to 3.
- a fungus-producing toxin neutralizing agent comprising a
- the trehalose compound of the present invention has high immunostimulatory activity and low toxicity, it is useful for providing an excellent pharmaceutical against infection caused by pathogenic bacteria.
- the trehalose compound of the present invention has an action of activating cellular immunity, and exhibits antibacterial action by activating neutrophils and macrophages and enhancing their phagocytic action. That is, according to the compound of the present invention, since the bacteria themselves are taken into neutrophils and macrophages, the release of toxins to the outside of the bacteria is small, and the risk of toxin release by destruction of Escherichia coli during antibiotic administration is low. A medicament can be provided.
- the trehalose compound of the present invention exhibits a toxicity reducing action against the toxin itself. Therefore, according to the present invention, it is possible to provide an effective medicine even in the case where the degree of infection progresses in an infectious disease caused by Escherichia coli, etc., resulting in a situation where Escherichia coli and the like proliferate and produce toxins outside the fungus. Can do.
- the activation of neutrophils and macrophages by administration of the trehalose compound of the present invention enables predation of multidrug-resistant bacteria produced by antibiotic administration as well as non-resistant bacteria. Therefore, it is possible to provide a medicament that has a therapeutic effect on infections caused by multidrug-resistant bacteria.
- the trehalose compound of the present invention activates cellular immunity, but an excessive immune response hardly occurs. Therefore, according to the present invention, for example, an antibody against an antibody administered as a medicine in vivo can be produced against an antibody medicine, and a medicine with a low risk that an excessive immune response is generated can be provided.
- the trehalose compound of the present invention includes a compound not containing an asymmetric carbon atom. That is, the trehalose compound according to the present invention can be efficiently synthesized in a large amount by the method for producing a trehalose compound of the present invention without including asymmetric synthesis.
- FIG. 2 shows a CD-8 positive cell image by a fluorescence microscope in mouse peritoneal infiltrating cells treated with TDCM, vehicle or a test compound of the present invention. Shows the measurement results of CD-8 positive cells by flow cytometry in mouse peritoneal infiltrating cells treated with TDCM, vehicle or the test compound of the present invention.
- the compound represented by Formula (1) may exist in the form of the pharmacologically acceptable salt or solvate.
- the “C 1 -C 21 alkyl group” means a linear or branched aliphatic hydrocarbon group having 1 to 21 carbon atoms, or all or part of the aliphatic hydrocarbon group. It also includes an alicyclic hydrocarbon group forming a 4-8 membered ring. In one of the preferred embodiments, the “C 1 -C 21 alkyl group” of the compound represented by formula (1) of the present invention is a linear aliphatic hydrocarbon group.
- C 1 -C 21 alkyl group which is a linear aliphatic hydrocarbon group
- examples of the “C 1 -C 21 alkyl group” which is a linear aliphatic hydrocarbon group include a methyl group, an ethyl group, an n-propyl group, an n-butyl group, an n-pentyl group, an n-hexyl group, n-heptyl group, n-octyl group, n-nonyl group, n-decyl group, n-undecyl group, n-dodecyl group, n-tridecyl group, n-tetradecyl group, n-pentadecyl group, n-hexadecyl group, Examples thereof include n-heptadecyl group, n-octadecyl group, n-nonadecyl group, n-icosyl group
- Examples of the alicyclic hydrocarbon group in which all of the aliphatic hydrocarbon groups form a ring include a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, a cyclooctyl group, and the like.
- Examples of the alicyclic hydrocarbon group in which a part of the aliphatic hydrocarbon group forms a ring include a cyclohexyl-n-octyl group, a cyclohexyl-n-nonyl group, and a cycloheptyl-n-octyl group. Group, etc. can be mentioned.
- R 1 , R 1 ′, R 2 , or R 2 ′ is preferably a linear alkyl group, more preferably a linear alkyl group having 10 to 16 carbon atoms, particularly preferably When n is 0, it is an n-decyl group which is a linear alkyl group having 10 carbon atoms, and when n is 1, an n-nonyl group which is a linear alkyl group having 9 carbon atoms, N-tridecyl group, which is a 13 linear alkyl group, or n-tetradecyl group, which is a linear alkyl group having 14 carbon atoms, and most preferably an n-decyl group.
- the hydrogen atom in each alkyl group may be substituted with a hydroxyl group or an alkoxy group.
- the alkoxy group is a substituent having a structure in which a linear or branched aliphatic hydrocarbon having 1 to 21 carbon atoms is bonded to an oxygen atom, such as a methoxy group, an ethoxy group, a propoxy group, Examples include butoxy group, pentyloxy group, hexyloxy group, heptyloxy group and the like.
- it is a straight-chain alkoxy group, and as an alkyl group substituted by an alkoxy group, for example, a methoxydodecyl group, ethoxyundecyl group, propoxydecyl group, pentyloxynonyl group, hexyloxyoctyl group, hexyloxyheptyl group, And a pentyloxyoctyl group.
- each alkyl group is substituted by a hydroxyl group or an alkoxy group
- the position of substitution may be any in each alkyl group, but preferably the hydrogen atom bonded to the terminal carbon atom of the alkyl group is It is a compound substituted by a hydroxyl group or an alkoxy group.
- the hydrocarbon group is bonded via an oxygen atom to form a linear ether structure, and the intervening oxygen atom and the carbon atom constituting the hydrocarbon group are It is preferable that the number of carbon and oxygen atoms constituting the alkoxyalkyl group is 2 to 21 as the sum of the numbers is the same as the length of the alkyl group of the hydrocarbon group, and more preferably, when n is 0, 10 to 16, and 9 to 15 when n is 1.
- R 1 and R 2 , R 1 ′ and R 2 ′ may be connected to each other to form a 4-8 membered ring, and when X is R 1 —CHR 2 —, R 1 and Both the carbon atom to which R 2 is bonded and R 1 and R 2 are constituent atoms of a 4-8 membered ring.
- the alkyl group constituting R 1 and R 2 may be a branched alkyl group. In this case, a part of the branched alkyl group constitutes a 4-8 membered ring and is substituted with the alkyl group. A structure like cycloalkyl may be used. Further, the case where a ring is formed includes the case where it is substituted with the same substituent as described above.
- Examples of the 4-8 membered ring include a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, and a cyclooctyl group. From the viewpoint of the structural stability of the compound, a cyclohexyl group or a cycloheptyl group is preferable.
- R 1 and R 1 ′, R 2 and R 2 ′ may be the same or different. From the viewpoint of the efficiency of synthesis, R 1 is preferably the same as R 1 ′, and R 2 is R 2. Same as'.
- R 1 and R 2 may be the same or different, as one preferred embodiment, the number of carbon atoms of R 1 is compared with the number of carbon atoms in R 2 The same compound having 1 or 2 carbon atoms or 1 or 2 carbon atoms less than that of R 2 .
- R 1 is a short-chain alkyl group having 1 to 5 carbon atoms
- R 2 is a long-chain alkyl group having 10 to 16 carbon atoms, and vice versa.
- 1 is a long chain alkyl group such as 10 to 16 and R 2 is a short chain alkyl group such as 1 to 5 carbon atoms.
- R 1 ′ and R 2 ′ is the same as the relationship between R 1 and R 2.
- R 1 is R 1 ′ and R 2 is R 2 ′. It can be read as R 2 '.
- N and n ′ may be the same or different. From the viewpoint of the efficiency of synthesis, it is preferable that n and n ′ are the same. From the viewpoint of activity, compounds in which n to n ′ are 0 and compounds in which n to n ′ are 1 are preferable.
- trehalose has three isomers, ⁇ , ⁇ ′, ⁇ , ⁇ ′, and ⁇ , ⁇ ′.
- the trehalose compound of the present invention is preferably an ⁇ , ⁇ ′ form.
- the compound of formula (1) and salts thereof may exist as solvates, which are also within the scope of the present invention.
- the scope of the present invention also includes radiolabeled compounds of the compound of formula (1) useful for biological research.
- a preferred compound of the present invention is a compound represented by the above formula (1), and each substituent in the formula has the following characteristics. The following features can be selected independently, alone or in combination as long as they do not conflict.
- (C) R 1 and R 1 ′ are each independently an unsubstituted C 1 -C 21 alkyl group.
- R 2 and R 2 ′ are each independently a hydrogen atom or an unsubstituted C 1 -C 21 alkyl group.
- R 1 and R 1 ′ are each independently a linear C 1 -C 21 alkyl group.
- R 2 and R 2 ′ are each independently a hydrogen atom or a linear C 1 -C 21 alkyl group.
- R 1 and R 1 ′ are each independently an unsubstituted and linear C 1 -C 21 alkyl group.
- R 2 and R 2 ′ are each independently a hydrogen atom or an unsubstituted and linear C 1 -C 21 alkyl group.
- R 1 and R 1 ′ are each independently an unsubstituted and linear C 7 -C 21 alkyl group.
- R 2 and R 2 ′ are each independently an unsubstituted and linear C 7 -C 21 alkyl group.
- K R 1 and R 1 ′ are identical and are an unsubstituted C 1 -C 21 alkyl group.
- L R 2 and R 2 ′ are the same and are a hydrogen atom or an unsubstituted C 1 -C 21 alkyl group.
- M R 1 and R 1 ′ are the same and are a linear C 1 -C 21 alkyl group.
- N R 2 and R 2 ′ are the same and are a hydrogen atom or a linear C 1 -C 21 alkyl group.
- R 1 and R 1 ′ are the same, unsubstituted and straight-chain C 1 -C 21 alkyl groups.
- P R 2 and R 2 ′ are the same and are a hydrogen atom or an unsubstituted and linear C 1 -C 21 alkyl group.
- Q R 1 and R 1 ′ are the same, unsubstituted and straight-chain C 7 -C 21 alkyl groups.
- R R 2 and R 2 ′ are the same, unsubstituted and straight-chain C 7 -C 21 alkyl groups.
- S n and n ′ are each independently 0 or 1.
- T n and n ′ are 0.
- U n and n ′ are 1.
- a preferred compound of the present invention is a compound represented by the formula (1), or a pharmacologically acceptable salt or solvate thereof, and has the following structure.
- each alkyl group is a hydroxyl group , May be substituted by an alkoxy group, all or part of each alkyl group may form a 4-8 membered ring, and R 1 and R 2 , R 1 ′ and R 2 ′ are Each may be linked to each other to form a 4-8 membered ring; n and n ′ are each independently an integer of 0 to 3.
- X is a group represented by R 1 —CHR 2 —;
- X is a group represented by R 1 —CHR 2 —;
- X ′ is a group represented by R 1 ′ —CHR 2 ′ —, wherein R 1 , R 1 ′, R 2 and R 2 ′ are each independently a linear C 8 -C 16 alkyl group, and with respect to R 1 , R 1 ′, R 2 and R 2 ′, the hydrogen atom in each alkyl group is a hydroxyl group , May be substituted by an alkoxy group, and all or part of each alkyl group may form a 4-8 membered ring; n and n ′ are 0.
- X is a group represented by R 1 —CHR 2 —;
- X ′ is a group represented by R 1 ′ —CHR 2 ′ —, wherein R 1 , R 1 ′, R 2 and R 2 ′ are each independently a linear C 8 -C 14 alkyl group, and with respect to R 1 , R 1 ′, R 2 and R 2 ′, the hydrogen atom in each alkyl group is a hydroxyl group , May be substituted by an alkoxy group, and all or part of each alkyl group may form a 4-8 membered ring; n and n ′ are 1.
- (Z) X is a group represented by R 1 —CHR 2 —;
- X ′ is a group represented by R 1 ′ —CHR 2 ′ —, wherein R 1 and R 1 ′ are The same, a hydrogen atom or a C 1 -C 21 alkyl group, and with respect to R 1 and R 1 ′, the hydrogen atom in each alkyl group may be substituted with a hydroxyl group or an alkoxy group; All or part of may form a 4-8 membered ring;
- R 2 and R 2 ′ are the same and are a hydrogen atom or a C 1 -C 21 alkyl group, and R 2 , R 2 ′
- the hydrogen atom in each alkyl group may be substituted with a hydroxyl group or an alkoxy group, and all or part of each alkyl group may form a 4-8 membered ring; and
- R 1 and R 2, R 1 'and R 2' is to form a 4-8 membere
- X is a group represented by R 1 —CHR 2 —;
- X ′ is a group represented by R 1 ′ —CHR 2 ′ —;
- R 1 and R 1 ′ are the same there is a C 1 -C 21 alkyl group, a hydrogen atom in each alkyl group, a hydroxyl group, it may be substituted by an alkoxy group;
- R 2 and R 2 ' is the same, C 7 - A C 21 alkyl group, the hydrogen atom in each alkyl group may be substituted by a hydroxyl group, an alkoxy group;
- n and n ′ are the same and are 0 or 1.
- X is a group represented by R 1 —CHR 2 —;
- X ′ is a group represented by R 1 ′ —CHR 2 ′ —;
- R 1 and R 1 ′ are the same there are a C 7 -C 21 alkyl group, a hydrogen atom in each alkyl group, a hydroxyl group, it may be substituted by an alkoxy group;
- R 2 and R 2 ' are identical and hydrogen atoms, Alternatively, it is a C 1 -C 21 alkyl group, and the hydrogen atom in each alkyl group may be substituted with a hydroxyl group or an alkoxy group;
- n and n ′ are the same and are 0 or 1.
- X is a group represented by R 1 —CHR 2 —;
- X ′ is a group represented by R 1 ′ —CHR 2 ′ —;
- R 1 and R 1 ′ are the same
- R 2 and R 2 ′ are the same;
- n and n ′ are the same and are 0 or 1 .
- X is a group represented by R 1 —CHR 2 —;
- X ′ is a group represented by R 1 ′ —CHR 2 ′ —;
- R 1 and R 1 ′ are the same
- R 2 and R 2 ′ are the same and are a hydrogen atom or an unsubstituted and straight-chain C 7 -C 21 alkyl group
- N and n ′ are the same and are 0 or 1.
- X is a group represented by R 1 —CHR 2 —;
- X ′ is a group represented by R 1 ′ —CHR 2 ′ —;
- R 1 and R 1 ′ are the same An unsubstituted and straight-chain C 7 -C 21 alkyl group;
- R 2 and R 2 ′ are the same and are a hydrogen atom or an unsubstituted and straight-chain C 1 -C 21 alkyl group
- N and n ′ are the same and are 0 or 1.
- FF FF
- X is a group represented by R 1 —CHR 2 —
- X ′ is a group represented by R 1 ′ —CHR 2 ′ —
- R 1 and R 1 ′ are the same
- R 2 and R 2 ′ are the same, unsubstituted and straight-chain C 7 -C 21 alkyl group
- n and n ′ is the same and is 0 or 1.
- X is a group represented by R 1 —CHR 2 —;
- X ′ is a group represented by R 1 ′ —CHR 2 ′ —;
- R 1 and R 1 ′ are the same An unsubstituted and straight-chain C 8 -C 16 alkyl group;
- R 2 and R 2 ′ are the same, unsubstituted and straight-chain C 8 -C 16 alkyl group;
- n and n ′ is the same and is 0 or 1.
- (HH) X is a group represented by R 1 —CHR 2 —;
- X ′ is a group represented by R 1 ′ —CHR 2 ′ —;
- R 1 and R 1 ′ are the same An unsubstituted and straight-chain C 8 -C 16 alkyl group;
- R 2 and R 2 ′ are the same, unsubstituted and straight-chain C 8 -C 16 alkyl group;
- n and n ′ is 0.
- X is a group represented by R 1 —CHR 2 —;
- X ′ is a group represented by R 1 ′ —CHR 2 ′ —;
- R 1 and R 1 ′ are the same An unsubstituted and straight-chain C 9 -C 14 alkyl group;
- R 2 and R 2 ′ are the same, unsubstituted and straight-chain C 9 -C 14 alkyl group;
- n and n ′ is 1.
- X is a group represented by R 1 —CHR 2 —;
- X ′ is a group represented by R 1 ′ —CHR 2 ′ —;
- R 1 , R 1 ′, R 2 and R 2 ' is the same, unsubstituted and straight-chain C 10 alkyl group;
- n and n' are 0.
- X is a group represented by R 1 —CHR 2 —;
- X ′ is a group represented by R 1 ′ —CHR 2 ′ —;
- R 1 , R 1 ′, R 2 and R 2 ′ is the same, unsubstituted and linear C 9 , C 13 , or C 14 alkyl group;
- n and n ′ are 1.
- trehalose compound of the present invention As specific examples of the trehalose compound of the present invention, the following compounds can be exemplified.
- Examples of compounds suitable as the trehalose compound of the present invention include the following.
- any of the following compounds 6,6′-bis-O- (2-decyldodecanoyl) - ⁇ , ⁇ ′-trehalose, 6,6′-bis-O- (2-tetradecyldodecanoyl) - ⁇ , ⁇ ′-trehalose, 6,6′-bis-O- (3-nonyldodecanoyl) - ⁇ , ⁇ ′-trehalose, 6,6′-bis-O- (3-tridecylhexadecanoyl) - ⁇ , ⁇ ′-trehalose, or
- An example is a fungus-producing toxin neutralizing agent characterized by containing 6,6′-bis-O- (3-tetradecylheptadecanoyl) - ⁇ , ⁇ ′-trehalose.
- the pharmaceutical composition and immunostimulant of the present invention are characterized by containing the trehalose compound.
- the trehalose compound of the present invention is an immunostimulant having a high activation action on macrophages and neutrophils. Therefore, the trehalose compound of the present invention can be used as a prophylactic or therapeutic agent for diseases associated with immune defense such as bacterial infections, viral infections, fungal infections, opportunistic infections, multidrug resistant infections, etc. It is effective as an agent.
- ⁇ Manufacturing method> The compound represented by Formula (1) of this invention is compoundable by the two processes shown by the following (a) and (b).
- (A) A step of performing an esterification reaction by causing the carbonyl compound represented by the formula (4) and the formula (6) to act on the trehalose compound represented by the formula (3) simultaneously or sequentially.
- the compound represented by Formula (2) can also be synthesized in the same manner as the compound represented by Formula (1).
- the compound represented by Formula (1) of this invention can be manufactured by the method represented by the following synthetic scheme 1. ⁇ Synthesis scheme 1>
- R 1 , R 1 ′, R 2 , R 2 ′, n and n ′ are the same as described above.
- R 3 and R 3 ′ represent a protecting group for the hydroxyl group of the sugar.
- Y and Y ′ each independently represent a hydroxyl group or a halogen atom.
- the trehalose compound represented by the formula (3) is an ⁇ , ⁇ ′ form, but an ⁇ , ⁇ ′ form and a ⁇ , ⁇ ′ form can be synthesized in the same manner. However, in the present invention, ⁇ and ⁇ ′ forms are preferred.
- R 3 and R 3 ′ those known as a protecting group for a hydroxyl group can be used.
- a protecting group for a hydroxyl group described in Protecting groups in Organic chemistry John Wiley & Sons INC., New York 1991, ISBN 0-471-62301-6
- arylalkyl groups such as benzyl group, p-methoxybenzyl group and biphenylmethyl group
- acyl groups such as acetyl group
- alkoxycarbonyl groups such as methoxycarbonyl group and tert-butoxycarbonyl group
- Examples thereof include an alkylsilyl group.
- a benzyl group is preferred.
- R 3 and R 3 ′ may be the same or different and are preferably the same.
- Y and Y ′ are each independently a hydroxyl group or a halogen atom, and examples of the halogen atom include a fluorine atom, a chlorine atom, and an iodine atom.
- Y and Y ′ are preferably a hydroxyl group.
- the trehalose compound represented by the formula (3) is allowed to react with the carbonyl compound represented by the formula (4) and the formula (6) in order to carry out an esterification reaction between the trehalose compound and the carbonyl compound.
- the trehalose compound represented by the formula (3) is a compound in which hydroxyl groups other than the 6-position and 6'-position of trehalose are protected with a protecting group, and a commercially available one is used or synthesized from trehalose by a known method. Can be used. For example, 2,3,4,2 ′, 3 ′, 4′-hexabenzoxy- ⁇ , ⁇ ′-trehalose in which the protecting group is a benzyl group is commercially available and can be suitably used. . In addition, ⁇ and ⁇ ′ forms of trehalose exist in nature and are readily available.
- the carbonyl compounds represented by the formulas (4) and (6) are commercially available, or synthesized by a known method in addition to those synthesized by the synthesis scheme 4, 5, or 6 described later. Can be used.
- both the 6-position and 6′-position hydroxyl groups in the trehalose compound represented by the formula (3) are used.
- esterification A compound in which both the 6-position and 6'-position hydroxyl groups are esterified is called a diester, and an intermediate compound in which only one of them is esterified is called a monoester.
- the monoester product in which the desired 6-position hydroxyl group is esterified after the reaction may be separated and purified.
- esterification with the carbonyl compound represented by the formula (4) among the 6-position and 6′-position hydroxyl groups of the trehalose compound represented by the formula (3) as a raw material is performed.
- One undesired hydroxyl group may be selectively protected, and the other hydroxyl group may be selectively deprotected after the esterification reaction.
- the 6'-position hydroxyl group may be esterified first in addition to the case where the 6-position hydroxyl group is esterified first.
- esterification reaction a method commonly used as a general esterification reaction and a method known to those skilled in the art can be widely used.
- condensing agent used in these methods a dehydrating agent is included, and those usually used in the esterification reaction of alcohol and carboxylic acid can be widely used.
- condensing agents include mineral acids such as hydrogen chloride, sulfuric acid and hydrochloric acid; organic acids such as paratoluenesulfonic acid and camphorsulfonic acid; dehydrating agents such as Lewis acids such as boron fluoride etherate; phosphorus trichloride and triodorous Acid halide generators such as phosphorus chloride, phosphorus pentachloride, phosphorus oxychloride and thionyl chloride; mixed acid anhydride generators such as ethyl chloroformate and methanesulfonyl chloride; N, N′-dicyclohexylcarbodiimide (DCC), diisopropyl Carbodiimides such as carbodiimide, 1-ethyl-3-dimethylaminopropylcarbodi
- the esterification reaction can be performed in a suitable solvent.
- Any solvent may be used as long as it is an inert solvent that has appropriate solubility with respect to the raw material compound and does not adversely affect the esterification reaction.
- the solvent used in the esterification reaction include aromatic hydrocarbons such as benzene, toluene and xylene; halogenated aromatic hydrocarbons such as chlorobenzene and dichlorobenzene: aliphatics such as n-hexane, cyclohexane and petroleum ether.
- Hydrocarbons such as dichloromethane, 1,2-dichloroethane, chloroform, carbon tetrachloride; ethers such as diethyl ether, diisopropyl ether, dioxane, tetrahydrofuran, ethylene glycol dimethyl ether, ethylene glycol diethyl ether; acetone, 2 -Ketones such as butanone and methyl isobutyl ketone; nitriles such as acetonitrile, propionitrile and benzonitrile; N, N-dimethylformamide, hexamethylphosphoric triamide (HMPA) Amides and the like; may be mentioned sulfoxide such as dimethyl sulfoxide. These solvents may be used alone or in combination of two or more.
- ethers such as diethyl ether, diisopropyl ether, dioxane, tetrahydrofuran, ethylene glycol dimethyl ether, ethylene
- reaction accelerators can be widely used.
- the reaction accelerator include catalysts such as dimethylformamide, dimethylamide pyridine and 4-pyrrolidinopyridine, and desiccants such as anhydrous magnesium sulfate and molecular sieves (4A, 5A). These reaction accelerators may be added to the reaction system.
- an apparatus such as a Dean-Stark water separator or a Soxhlet extractor may be used.
- reaction accelerators or apparatuses may be used singly or in combination of two or more, and a catalyst and a desiccant may be used in combination.
- the use ratio of the raw material compound and the reaction accelerator is not particularly limited, and can be appropriately selected from a wide range.
- the amount of the raw material compound used for this reaction is not particularly limited and is appropriately selected from a wide range.
- the compound represented by the formula (4) and the compound represented by the formula (6) are sequentially reacted, the compound represented by the formula (4) is represented with respect to 1 mol of the trehalose compound represented by the formula (3).
- the carbonyl compound is usually used in an amount of 0.5 to 1.8 mol, preferably 0.8 to 1.2 mol.
- the carbonyl compound represented by the formula (6) is usually 0.5 to 1.8 moles, preferably 0.8 to 1.2 moles per mole of the monoester represented by the formula (5). Use mol.
- reaction temperature of the esterification reaction is not particularly limited, but it may be usually within a range from ⁇ 10 ° C. to the boiling point temperature of the solvent used. Usually, it is carried out at 0 to 200 ° C., preferably from room temperature to 100 ° C.
- reaction time varies depending on the reaction conditions such as the type of raw material compound and the amount used, reaction temperature, etc., it can usually be appropriately adjusted within the range of 1 hour to 1 week, preferably 1 to 24 hours, more preferably 3 to 10 hours.
- reaction mixture After completion of the reaction, the reaction mixture is subjected to general treatments such as separation and removal of by-products, drying and evaporation of the solvent, and then purified by a general method such as silica gel column chromatography.
- general treatments such as separation and removal of by-products, drying and evaporation of the solvent, and then purified by a general method such as silica gel column chromatography.
- the esterification reaction is carried out in a suitable solvent in the presence of a base as necessary. be able to.
- the same inert solvent used in the esterification reaction can be used.
- Examples of the base include hydroxides of alkali metals such as sodium hydroxide and potassium hydroxide; hydroxides of alkaline earth metals such as calcium hydroxide; carbonates of alkali metals such as sodium carbonate and potassium carbonate; Alkali metal hydrogen carbonates such as sodium hydrogen and potassium hydrogen carbonate; Alkali metal acetates such as sodium acetate and potassium acetate; Alkaline earth metal acetates such as calcium acetate; Alkaline such as sodium hydride and potassium hydride Metal hydrides; hydrides of alkaline earth metals such as calcium hydride; ammonium salts such as ammonium hydroxide, ammonium carbonate and ammonium acetate; trimethylamine, triethylamine, N, N-dimethylaniline, pyridine, 4- (dimethylamino ) Pyridine, diazabicyclooctane (DAB) O), diazabicyclononene (DBN), mention may be made of a ter
- the amount of the raw material compound and base used for this reaction is also not particularly limited and is appropriately selected from a wide range.
- the compound represented by the formula (4) and the compound represented by the formula (6) are sequentially reacted, the compound represented by the formula (4) is represented with respect to 1 mol of the trehalose compound represented by the formula (2).
- the carbonyl compound is usually used in an amount of 0.5 to 1.8 mol, preferably 0.8 to 1.2 mol
- the base is usually used in an amount of 0.5 to 1.8 mol, preferably 0.8 to 1.2 mol. Use mol.
- the carbonyl compound represented by the formula (6) is usually 0.5 to 1.8 moles, preferably 0.8 to 1.2 moles per mole of the monoester represented by the formula (5).
- the base is usually used in an amount of 0.5 to 1.8 mol, preferably 0.8 to 1.2 mol.
- the reaction temperature is usually in the range from ⁇ 10 ° C. to the boiling point temperature of the solvent used, as in the esterification reaction.
- the reaction time varies depending on the concentration, temperature, etc., as in the esterification reaction, but can be appropriately adjusted in the range of usually 0.1 to 10 hours.
- a catalytic hydrogenation reaction can be applied.
- the catalytic hydrogenation reaction is performed in the presence of a catalyst under a hydrogen atmosphere.
- catalysts can be widely used as long as they are used for catalytic hydrogenation reaction, and examples thereof include platinum oxide, platinum carbon, palladium hydroxide, palladium carbon, Raney nickel and the like.
- the amount of the catalyst used is usually about 0.001 to 50% by weight, preferably about 0.01 to 10% by weight, based on the compound represented by the formula (7).
- the hydrogen pressure is not particularly limited and can be appropriately selected from a wide range.
- the hydrogen pressure is usually about 0.8 to 100 atm, preferably about 1 to 3 atm.
- the reaction is usually carried out in a suitable solvent, and any solvent can be used as long as it is inert so as not to adversely affect the reaction.
- the solvent used include aliphatic halogenated hydrocarbons such as dichloromethane, 1,2-dichloroethane, chloroform and carbon tetrachloride; alcohols such as methanol, ethanol and isopropanol; esters such as methyl formate, methyl acetate and ethyl acetate.
- a carboxylic acid such as formic acid or acetic acid, or a mixed solvent thereof.
- the temperature of this reaction is usually about 0 to 100 ° C., preferably about 10 to 40 ° C.
- the reaction time varies depending on the base mass, temperature, type of catalyst, etc., but the reaction may be terminated based on the theoretical amount of hydrogen consumption. Usually, it is about 1 to 50 hours, preferably 1 to 30 hours.
- the catalyst is filtered and the solvent is distilled off, followed by purification by a general method such as solvent extraction or silica gel column chromatography.
- the compound represented by the formula (1) of the present invention can also be produced by the method shown in the following synthesis scheme 2 or 3. ⁇ Synthesis scheme 2>
- R 1 , R 1 ′, R 2 , R 2 ′, R 3 , R 3 ′, n and n ′ are the same as described above.
- Synthesis scheme 2 is a scheme in which the carbonyl compound represented by formula (4) and formula (6) is simultaneously acted on the trehalose compound represented by formula (3) in step (a), which is an esterification reaction.
- Step (b), which is a deprotection reaction, is the same as in Synthesis Scheme 1.
- the esterification reaction and deprotection reaction are synthesized in the step (a) except that the trehalose compound represented by the formula (3) is allowed to simultaneously act on the carbonyl compound represented by the formula (4) and the formula (6).
- the reaction can be performed in the same manner as in Scheme 1.
- the carbonyl compound represented by the formula (4) and the carbonyl compound represented by the formula (6) are allowed to act simultaneously, it can be obtained by separating and purifying the target compound from the product.
- the above synthesis scheme 2 particularly includes the following synthesis scheme 3 It can be expressed as In the case where the compound represented by the formula (1) is not a compound in which R 1 and R 1 ′, R 2 and R 2 ′, n and n ′ are the same, from the viewpoint of increasing the reaction yield. It is preferable to synthesize by the method represented by Synthesis Scheme 1.
- R 1 , R 2 , R 3 , R 3 ′ and n are the same as described above.
- step (a) which is an esterification reaction
- step (b) which is a deprotection reaction
- the esterification reaction and deprotection reaction in Synthesis Scheme 3 can be performed in the same manner as in the above-mentioned Synthesis Scheme 1 except that the amount of the carbonyl compound represented by Formula (4) is increased.
- the carbonyl compound represented by the formula (4) is usually 1.8 to 5 mol, preferably 2 to 3 with respect to 1 mol of the trehalose compound represented by the formula (3).
- the condensing agent is usually 1.8 to 5 mol, preferably 2 to 4 mol
- the base is usually 1.8 to 8 mol, preferably 2 to 6 mol.
- the compound represented by the formula (7) can be used in the next reaction without isolation and purification, but it is preferable to remove the reagent and by-product used in the esterification reaction.
- the carbonyl compound represented by formula (4) or formula (6) which is a raw material compound, can be produced by a method known to those skilled in the art in addition to using a commercially available one. it can.
- benzoic acid or a benzoic acid halide can be used as the compound in which X to X ′ are phenyl groups and n to n ′ are 0.
- a compound in which X is R 1 —CHR 2 — and n is 0 can also be produced by the following synthesis scheme 4 or 5.
- R 1 and R 2 are the same as described above.
- R 4 represents an alkyl group having 1 to 6 carbon atoms, and Hal represents a halogen atom.
- Synthesis scheme 4 is a step in which a compound represented by formula (8) is subjected to a normal alkylation reaction to obtain a carbonyl compound represented by formula (4).
- the compound represented by formula (8) which is a raw material compound a commercially available compound can be used.
- R 1 is a linear alkyl group having 10 carbon atoms
- ethyl dodecanoate or the like is used.
- Can do An ester of an acid having a desired length may be used as the side chain alkyl of the target compound.
- alkylation reaction various methods such as the method described in Creger, J. Am. Chem. Soc., Vol. 92, pages 1397-98, 1970 can be used. More specifically, after adding a strong base to the solution of the compound represented by the formula (8) and extracting a hydrogen atom at the 2-position, an alkyl halide may be reacted. As an example, alkylation can be carried out by the following reaction.
- the strong base is not particularly limited as long as it has an action of extracting a hydrogen atom, and examples thereof include alkali metal hydroxides such as sodium hydroxide and potassium hydroxide; alkaline earth metal hydroxides such as calcium hydroxide and the like. Can be mentioned. These bases may be used individually by 1 type, and may be used in mixture of 2 or more types.
- proton-lithium exchange reaction may be performed in combination with lithium diisopropylamide. Any solvent may be used as long as it is an inert solvent that has appropriate solubility with respect to the raw material compound and does not adversely affect the esterification reaction.
- solvent used in the alkylation reaction examples include aromatic hydrocarbons such as benzene, toluene and xylene; aliphatic hydrocarbons such as n-hexane, cyclohexane and petroleum ether; diethyl ether, diisopropyl ether, dioxane, tetrahydrofuran, Examples include ethers such as ethylene glycol dimethyl ether and ethylene glycol diethyl ether. These solvents may be used alone or in combination of two or more.
- the ratio of the compound represented by the formula (8) and the strong base can be appropriately selected from a wide range, but usually a strong base or the like is added to the compound represented by the formula (8). About 0.9 to 5 times mole is used.
- the reaction temperature at this time is usually about ⁇ 80 to 60 ° C., preferably about 0 ° C. to 60 ° C.
- the reaction time is about 5 minutes to 6 hours, preferably about 5 minutes to 1 hour.
- an alkyl halide is added to the reaction mixture.
- the halogenated alkyl include 1-iodooctane, 1-iodoheptane, 1-iododecane, 1-iodoundecane, 1-iodododecane, 1-iodotridecane, etc.
- An alkane halide having a carbon chain moiety having a desired length may be used.
- the halide include chlorinated products, iodinated products, brominated products, and the like, and preferred are iodinated products.
- the ratio of the compound represented by the formula (8) and the alkyl halide can be appropriately selected from a wide range, but usually the alkyl halide is compared with the compound represented by the formula (8).
- the reaction temperature at this time is usually about room temperature.
- the reaction time is usually about 2 to 12 hours.
- the target compound is isolated and purified by applying known isolation and purification methods such as silica gel column chromatography and vacuum distillation.
- a compound in which X is R 1 —CHR 2 — and n is 0 can also be produced by the following synthesis scheme 5.
- R 1 , R 2 , R 4 and Hal are the same as described above.
- Synthesis scheme 5 is a step of subjecting the compound represented by formula (11) to a normal alkylation reaction to obtain a carbonyl compound represented by formula (4).
- the compound represented by the formula (11) which is a raw material compound commercially available compounds can be used.
- diethyl malonate, dimethyl malonate, dipropyl malonate, didibutyl malonate, diisopropyl malonate, di-malonate examples thereof include tert-butyl, dicyclohexyl malonate, diphenyl malonate, and dibenzyl malonate.
- the alkylation reaction can be carried out in the same manner as described above by reacting the compound represented by the formula (11), which is a raw material compound, with a strong base and then reacting with an alkyl halide.
- the ratio of the compound represented by the formula (11) and the strong base can be appropriately selected from a wide range.
- a strong base or the like is used in an amount of about 0.9 to 5 moles compared to the compound represented by 11).
- the reaction temperature at this time is usually about ⁇ 80 to 60 ° C., preferably about 0 ° C. to 60 ° C.
- the reaction time is about 5 minutes to 6 hours, preferably about 5 minutes to 1 hour.
- the ratio of the compound represented by the formula (11) and the alkyl halide can be appropriately selected from a wide range, but usually the compound represented by the formula (11)
- the alkyl halide represented by the formula (12) and the alkyl halide represented by the formula (9) are each used in an amount of about 0.8 to 1.2 times mol.
- the target compound is a compound in which R 1 and R 2 are the same
- the number of alkyl halides may be one, and usually a halogenated alkyl is 2. with respect to the compound represented by formula (11). Use about 2 to 4 moles.
- the alkyl halide represented by the formula (12) and the alkyl halide represented by the formula (9) are allowed to act simultaneously as described above.
- different alkyl halides are allowed to act in sequence. After acting on one alkyl halide, isolation and purification are performed, and the other alkyl halide is allowed to act.
- the compound may be isolated and purified.
- known isolation and purification methods such as silica gel column chromatography and vacuum distillation can be applied.
- a compound in which X is R 1 —CHR 2 — and n is 1 can also be produced by the following synthesis scheme 6.
- Synthesis scheme 6 can be described as the following synthesis schemes 6-1 to 6-5. ⁇ Synthesis scheme 6-1>
- R 1 is the same as described above.
- Synthesis scheme 6-1 is a reaction in which N, O-dimethylhydroxyamine is dehydrated and bound to a carboxylic acid that is a raw material compound.
- a basic condensing agent is allowed to act on the carboxylic acid represented by the formula (13). Any known basic condensing agent can be widely used. Examples of the basic condensing agent include carbonyldiimidazole, 4-dimethylaminopyridine, piperidine, pyrrolidine, pyridine, imidazole, N, N, N Examples include ', N'-tetramethylurea, bis (pentamethylene) urea, 1,1-carbonyldipyrrole, and the like.
- a commercially available carboxylic acid that is a raw material compound can be used, and examples thereof include heptanoic acid, octanoic acid, decanoic acid, and undecanoic acid.
- An acid having a desired length may be used as the side chain alkyl of the target compound.
- Any solvent may be used as long as it is an inert solvent that has appropriate solubility with respect to the raw material compound and does not adversely affect the esterification reaction.
- the solvent used in the esterification reaction include aromatic hydrocarbons such as benzene, toluene and xylene; halogenated aromatic hydrocarbons such as chlorobenzene and dichlorobenzene: aliphatics such as n-hexane, cyclohexane and petroleum ether.
- Hydrocarbons aliphatic halogenated hydrocarbons such as dichloromethane, 1,2-dichloroethane, chloroform, carbon tetrachloride; ethers such as diethyl ether, diisopropyl ether, dioxane, tetrahydrofuran, ethylene glycol dimethyl ether, ethylene glycol diethyl ether Can do. These solvents may be used alone or in combination of two or more.
- the ratio of the carboxylic acid represented by formula (13) to carbonyldiimidazole can be appropriately selected from a wide range, and is usually about 0.8 to 2.0 mol.
- the reaction temperature at this time is usually about ⁇ 80 to 60 ° C., preferably about 0 ° C. to 60 ° C.
- the reaction time is about 5 minutes to 6 hours, preferably about 30 minutes to 3 hours.
- N, O-dimethylhydroxyamine represented by the formula (14) is reacted.
- N, O-dimethylhydroxyamine, 1-hydroxybenzobenzotriazole and the like can also be used.
- the ratio of the carboxylic acid represented by the formula (13) and N, O-dimethylhydroxyamine can be appropriately selected from a wide range.
- the carboxylic acid represented by the formula (13) is N, About 0.8 to 1.5 moles of O-dimethylhydroxyamine may be used.
- the reaction is usually about ⁇ 80 to 60 ° C., preferably about 0 ° C. to 60 ° C.
- the reaction time may be about 10 minutes to 10 hours.
- Synthesis scheme 6-2 is a reaction in which an alkyl halide is allowed to act on the compound represented by formula (15) to synthesize a ketone body.
- an alkyl halide represented by the formula (9) can be reacted with magnesium metal in an ether solvent to prepare a Grignard reagent and used in the reaction.
- a Grignard reagent As the metallic magnesium, polished ground magnesium is preferably used, and lithium, sodium, zinc, indium and the like can also be used.
- alkyl halide represented by the formula (9) the same ones as described above can be used.
- the reaction is carried out in an ether solvent system
- examples of the ether solvent system include diethyl ether, diisopropyl ether, dioxane, tetrahydrofuran, tetrahydropyran, ethylene glycol dimethyl ether, and ethylene glycol diethyl ether. These solvents may be used alone or in combination of two or more.
- the ratio of the compound represented by the formula (15) and the alkyl halide can be appropriately selected from a wide range, but usually the alkyl halide is compared with the compound represented by the formula (15). About 0.8 to 5 times mol.
- the reaction temperature at this time is usually about 0 ° C. to 80 ° C.
- the reaction time is about 5 minutes to 6 hours.
- R 1 , R 2 and R 4 are the same as described above.
- R 5 and R 6 represent an alkyl group, an alkoxy group, an aryl group or an aryloxy group, and these may be substituted with a halogen atom or the like.
- Synthesis scheme 6-3 is a reaction in which a ketone compound represented by formula (16) is reacted with a Wittig reagent or Horner-Emmons reagent in the presence of a strong base to form a carbon-carbon double bond.
- the compound represented by the formula (17) described in the above synthesis scheme is a Horner-Emmons reagent, but a Wittig reagent may be used instead.
- the strong base is not particularly limited as long as it has an action of extracting a hydrogen atom, and examples thereof include alkali metal hydroxides such as sodium hydroxide and potassium hydroxide; alkaline earth metal hydroxides such as calcium hydroxide and the like. Can be mentioned. These bases may be used individually by 1 type, and may be used in mixture of 2 or more types. Any solvent may be used as long as it is an inert solvent that has appropriate solubility with respect to the raw material compound and does not adversely affect the esterification reaction.
- solvent used in the reaction examples include aromatic hydrocarbons such as benzene, toluene and xylene; aliphatic hydrocarbons such as n-hexane, cyclohexane and petroleum ether; diethyl ether, diisopropyl ether, dioxane, tetrahydrofuran and ethylene glycol. Mention may be made of ethers such as dimethyl ether and ethylene glycol diethyl ether. These solvents may be used alone or in combination of two or more.
- the ratio of the compound represented by the formula (16) and the strong base can be appropriately selected from a wide range, but usually a strong base or the like is added to the compound represented by the formula (16). About 1.1 to 8 moles are used.
- the reaction temperature at this time is usually about ⁇ 80 to 60 ° C.
- the reaction time is usually about 5 minutes to 3 hours.
- the Wittig reagent or Horner-Emmons reagent is reacted with the reaction mixture.
- the Wittig reagent or Horner-Emmons reagent known ones can be widely used.
- any carbon-carbon double bond may be formed between the ketone compound represented by formula (16) and the acetate ester.
- Examples of Wittig reagents include ethoxycarbonylmethyl (trimethyl Examples of phenyl) phosphonium bromide, ethyl (triphenylphosphoranylidene) acetate, Horner-Emmons reagent include ethyl (diaryl) phosphonoacetate such as ethyldiphenylphosphonoacetate, ethyldiethylphosphonoacetate, etc. And ethyl (dialkyl) phosphonoacetate. Ethyl diethyl phosphonoacetate is preferable.
- the ratio of the compound represented by the formula (16) and ethyl diethyl phosphonoacetate can be appropriately selected from a wide range, but is usually ethyl with respect to the compound represented by the formula (16).
- Diethylphosphonoacetate is used in an amount of about 1.1 to 10 times mol.
- the reaction temperature at this time is usually about room temperature.
- the reaction time is usually about 2 to 30 hours.
- R 1 , R 2 and R 4 are the same as described above.
- Synthesis Scheme 6-4 is a reaction in which a catalytic hydrogenation reaction is performed on a carboxylic acid ester having an unsaturated bond represented by Formula (18) to form a saturated carboxylic acid ester.
- the catalytic hydrogenation reaction is performed in a hydrogen atmosphere and in the presence of a catalyst.
- catalysts can be widely used as long as they are used for catalytic hydrogenation reaction, and examples thereof include platinum oxide, platinum carbon, palladium hydroxide, palladium carbon, Raney nickel and the like.
- the amount of the catalyst used is usually about 0.001 to 50% by weight, preferably about 0.01 to 10% by weight, based on the compound represented by the formula (18).
- the hydrogen pressure is not particularly limited and can be appropriately selected from a wide range.
- the hydrogen pressure is usually about 0.8 to 100 atm, preferably about 1 to 3 atm.
- the reaction is usually carried out in an appropriate solvent, and any solvent can be used as long as it is inert so as not to adversely affect the reaction.
- the solvent used include aliphatic halogenated hydrocarbons such as dichloromethane, 1,2-dichloroethane, chloroform and carbon tetrachloride; alcohols such as methanol, ethanol and isopropanol; esters such as methyl formate, methyl acetate and ethyl acetate.
- a carboxylic acid such as formic acid or acetic acid, or a mixed solvent thereof.
- the temperature of this reaction is usually about 0 to 100 ° C., preferably about 10 to 40 ° C.
- the reaction time varies depending on the base mass, temperature, type of catalyst, etc., but the reaction may be terminated based on the theoretical amount of hydrogen consumption. Usually, it is about 1 to 50 hours, preferably about 1 to 30 hours.
- R 1 , R 2 and R 4 are the same as described above.
- Synthesis Scheme 6-5 is a step of obtaining a desired carboxylic acid by hydrolyzing the carboxylic acid ester represented by the formula (19).
- the hydrolysis reaction various known reactions can be used.
- the reaction may be performed under acidic conditions, basic conditions, or as an enzymatic reaction.
- a base may be added to a solvent, and any substance that produces hydride ions as a base can be used widely.
- alkali metal hydroxides such as sodium hydroxide and potassium hydroxide; water Alkali earth metal hydroxides such as calcium oxide; alkali metal carbonates such as sodium carbonate and potassium carbonate; alkali metal hydrogen carbonates such as sodium hydrogen carbonate and potassium hydrogen carbonate; alkalis such as sodium acetate and potassium acetate Metal acetates; alkaline earth metal acetates such as calcium acetate; alkali metal hydrides such as sodium hydride and potassium hydride; alkaline earth metal hydrides such as calcium hydride; ammonium hydroxide and carbonic acid Ammonium salts such as ammonium and ammonium acetate; trimethylamine, triethylamine, N, N Dimethylaniline, pyridine, 4- (dimethylamino) pyridine, diazabicyclooctane (DABCO),
- DABCO
- the solvent may be any inert solvent that has moderate solubility in the raw material compound and does not adversely affect the esterification reaction, and a wide variety of known solvents can be used.
- the solvent used in the esterification reaction include aromatic hydrocarbons such as benzene, toluene and xylene; halogenated aromatic hydrocarbons such as chlorobenzene and dichlorobenzene: aliphatics such as n-hexane, cyclohexane and petroleum ether.
- Hydrocarbons such as dichloromethane, 1,2-dichloroethane, chloroform, carbon tetrachloride; ethers such as diethyl ether, diisopropyl ether, dioxane, tetrahydrofuran, ethylene glycol dimethyl ether, ethylene glycol diethyl ether; acetone, 2 -Ketones such as butanone and methyl isobutyl ketone; nitriles such as acetonitrile, propionitrile and benzonitrile; N, N-dimethylformamide, hexamethylphosphoric triamide (HMPA) Amides and the like; may be mentioned sulfoxide such as dimethyl sulfoxide. These solvents may be used alone or in combination of two or more.
- ethers such as diethyl ether, diisopropyl ether, dioxane, tetrahydrofuran, ethylene glycol dimethyl ether, ethylene
- the reaction temperature, reaction time, etc. can be appropriately selected from a wide range, the reaction temperature is usually about 0 ° C. to 100 ° C., and the reaction time is usually about 30 minutes to 20 hours. Can do.
- the target compound is isolated and purified by applying known isolation and purification methods such as silica gel column chromatography and vacuum distillation.
- R 1 and R 2 are the same as described above.
- a ketone compound represented by the formula (16) is reacted with a diethyl 3-hydroxypropanoyl phosphonate represented by the formula (21) as a Horner-Emmons reagent to produce a carbon-carbon double bond.
- a catalytic hydrogenation reaction is performed to obtain a compound represented by the formula (23)
- an oxidation reaction of alcohol is performed, and the formula (24) as a carbonyl compound is obtained.
- the compound represented by formula (22) only one of the double bond cis-trans stereoisomers is described for the compound represented by formula (22), but the compound is not limited to the stereoisomer. .
- the reaction using the Horner-Emmons reagent and the catalytic hydrogenation reaction can be similarly performed with reference to Synthesis Scheme 6-3 and Synthesis Scheme 6-4, respectively.
- the alcohol oxidation reaction can be performed by oxidizing the alcohol with a strong oxidizing agent, and can be appropriately performed using a known method such as chromic acid oxidation or Jones oxidation.
- chromic acid oxidation can be performed using a salt or complex of chromic anhydride, chromic acid, dichromic acid, or the like.
- the catalyst is filtered and the solvent is distilled off, followed by purification by a general method such as solvent extraction or silica gel column chromatography.
- immunostimulation refers to activating various immunity effects such as cellular immunity and humoral immunity, and the immunostimulant indicates any of these immunity activation effects. Anything is acceptable.
- the trehalose compound of the present invention is assumed to activate at least immune functions such as macrophages and neutrophils called cellular immunity in the immune system. In addition, it includes a wide range of situations in which humoral immunity is further activated by the release of cytokines from these cells.
- macrophage activation means that macrophages originally have phagocytosis against foreign substances from the outside, but act to enhance macrophage phagocytosis, and macrophage adherence to tissues. , And motility is improved, and refers to a state of phagocytosing bacteria that have entered from the outside and denatured self-components. It is known that the release of nitric oxide (NO) and the release of active oxygen increase when macrophages are activated. The release amount of these free substances can be measured as an index of macrophage activation, or the enhancement of phagocytosis itself can be measured and used as an index of macrophage activation.
- NO nitric oxide
- neutrophil activation is a state in which neutrophils originally have phagocytosis against foreign substances similar to macrophages, but enhance phagocytosis of neutrophils and phagocytose bacteria etc. It is. It is known that even when neutrophils are activated, release of nitric oxide (NO) and release of active oxygen increase. In addition, it is also known that the release of physiologically active substances by degranulation of microgranules and azurophilic granules is observed by neutrophil activation. The amount of release of these free substances can be measured and used as an indicator of neutrophil activation, or the enhancement of phagocytosis itself can be measured and used as an indicator of neutrophil activation.
- NO nitric oxide
- phagocytic cells include macrophages and monocytes, polynuclear leukocytes, dendritic cells, etc.
- the phagocytic action refers to immune system cells such as pathogens as foreign substances from the outside. This is the action of digesting the foreign substance by incorporating into the vesicle and fusing the vesicle with the intracellular lysosome.
- the phagocytic activation of a phagocytic cell is not particularly limited as long as it activates any of these phagocytic cells to enhance its phagocytic action. Preferably, it enhances the phagocytic action of one or both of macrophages and neutrophils.
- the antibacterial infectious agent may be any agent that reduces infection caused by bacteria, that is, various symptoms caused by the presence of bacteria in the body.
- bacteria include Pseudomonas aeruginosa and pathogenic Escherichia coli in addition to Clostridium perfringens.
- the fungus-producing toxin neutralizing agent refers to a substance that reduces the action of bacteria-producing toxins.
- antibacterial infectious agents there are those that alleviate the symptoms caused by bacteria by suppressing the growth of bacteria or the release of toxins from bacteria.
- it reduces the action of the toxin, adsorbs the toxin, or modifies the toxin into an inactive one, incorporates the toxin into the phagocytic cell, and further incorporates it It refers to actions such as digesting toxins.
- the anticancer agent means an agent having antitumor activity and used for the prevention or treatment of cancer.
- the tumor to which the anticancer agent acts may be a primary tumor or a metastatic tumor. Therefore, the anticancer agent of the present invention may be used not only for the treatment of primary cancer and metastatic tumor, but also for the prevention of metastatic tumor simultaneously with or after treatment of primary cancer.
- tumors to which the anticancer agent acts in the present invention include, for example, breast cancer, testicular cancer, testicular tumor, pancreatic cancer, diaphragm tumor, lung cancer, ovarian cancer, gastric cancer, gallbladder cancer, kidney cancer, prostate cancer, esophageal cancer, liver cancer, Oral cancer, colon cancer, colon cancer, rectal cancer, uterine cancer, bile duct cancer, islet cell cancer, adrenocortical cancer, bladder cancer, thyroid cancer, skin cancer, malignant carcinoid tumor, melanoma, glioma, osteosarcoma, myeloma, soft part
- tissue sarcoma neuroblastoma, malignant lymphoma, leukemia and the like.
- breast cancer, testicular cancer, pancreatic cancer or diaphragm tumor can be preferably exemplified.
- the trehalose compound of the present invention or a pharmaceutical composition containing the compound and a pharmacologically acceptable carrier is used for pharmaceutical uses such as an immunostimulant, a bacterial toxin neutralizing agent, and an anticancer agent.
- a pharmacologically acceptable carrier such as an immunostimulant, a bacterial toxin neutralizing agent, and an anticancer agent.
- the pharmacologically acceptable carrier is not particularly limited as long as it is pharmacologically and pharmaceutically acceptable.
- excipients for example, excipients, binders, dispersants, thickeners, lubricants, pH adjusters, solubilizers, etc. in addition to carriers generally used in the preparation of preparations, antibiotics, antibacterial agents, bactericides, Preservatives, builders, bleaches, enzymes, chelating agents, antifoaming agents, colorants (dyes, pigments, etc.), softeners, moisturizers, surfactants, antioxidants, fragrances, flavoring agents, flavoring agents, solvents, etc. Is included.
- the pharmacologically acceptable carrier can be blended within a range that does not interfere with the activity of the trehalose compound (1) of the present invention.
- the absorbability and blood of the trehalose compound (1) of the present invention can be increased. It can also affect medium levels and cause changes in pharmacokinetics.
- the method of administering the present compound is a method of administering the trehalose compound of the present invention itself or a pharmaceutical composition containing the compound to a human or an animal, and the present compound or pharmaceutical composition is usually It can be formulated in the form of a medical preparation.
- the medical preparation is appropriately prepared using the pharmacological carrier.
- the dosage form There is no particular limitation on the dosage form, and it is appropriately selected depending on the purpose of treatment. Typical examples thereof include tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, suppositories, injections (solutions, suspensions, emulsions) and the like. These preparations may be produced by a commonly used method.
- the dosage of the above-mentioned medical preparation may be appropriately selected according to usage, patient age, sex, disease severity, and other conditions.
- the active ingredient trehalose compound (1) is 1 kg body weight per day. 0.01 to 100 mg, preferably 0.1 to 50 mg, is administered once to several times.
- a dose smaller than the above range may be sufficient, or a dose exceeding the above range may be necessary.
- Embodiments of the present invention may be described with reference to schematic diagrams, but in the case of schematic diagrams, they may be exaggerated for clarity of explanation.
- terms such as first, second, etc. are used to represent various elements, it is understood that these elements should not be limited by those terms. These terms are only used to distinguish one element from another, for example, the first element is referred to as the second element, and similarly, the second element is the first element. Can be made without departing from the scope of the present invention.
- Production Example A-1 Synthesis of 6,6′-bis-O- (2-decyldodecanoyl) -2,3,4,2 ′, 3 ′, 4′-hexabenzyl- ⁇ , ⁇ ′-trehalose]
- Carboxylic acid (2-decyldodecanoic acid) (145 mg, 425 ⁇ mol) and trehalose derivative (2,3,4,2 ′, 3 ′, 4′-hexabenzoxy- ⁇ ) obtained by the method described in Production Example C-1 , ⁇ ′-trehalose) (150 mg, 170 ⁇ mol) in anhydrous dichloromethane solution (2 ml), powdered molecular sieves 4A (0.3 g), 4-dimethylaminopyridine (20.8 mg, 170 ⁇ mol), 1-ethyl-3- (3-Dimethylaminopropyl) carbodiimide hydrochloride [hereinafter abbreviated as EDCI] (97.8 mg, 510 ⁇ mol) was sequentially added, and the mixture was heated to reflux for 4 hours.
- EDCI 1-ethyl-3- (3-Dimethylaminopropyl carbodiimide hydrochloride
- Example 1 Production Example ⁇ -1 [Synthesis of 6,6′-bis-O- (2-decyldodecanoyl) - ⁇ , ⁇ ′-trehalose]
- Example 2 Production Example ⁇ -2 [Synthesis of 6,6′-bis-O- (2-octyldecanoyl) - ⁇ , ⁇ ′-trehalose]
- Example 3 Production Example ⁇ -3 [Synthesis of 6,6′-bis-O- (2-nonylundecanoyl) - ⁇ , ⁇ ′-trehalose]
- Example 5 Production example ⁇ -5 [Synthesis of 6,6′-bis-O- (2-dodecyltetradecanoyl) - ⁇ , ⁇ ′-trehalose]
- Example 6 Production example ⁇ -6 [Synthesis of 6,6′-bis-O- (2-tridecylpentadecanoyl) - ⁇ , ⁇ ′-trehalose]
- Example 7 Production Example ⁇ -7 [Synthesis of 6,6′-bis-O- (2-pentadecylheptadecanoyl) - ⁇ , ⁇ ′-trehalose]
- Example 8 Production Example ⁇ -8 [Synthesis of 6,6′-bis-O- (2-hexadecyloctadecanoyl) - ⁇ , ⁇ ′-trehalose]
- EDCI 1-ethyl-3- (3 -Dimethylaminopropyl) carbodiimide hydrochloride
- carboxylic acid 3-octylundecanoic acid obtained by the method described in Production Example D-2 was used, and 6,6′-bis-O- (3-octylunound was produced by the same method as in Production Example B-1.
- Decanoyl) -2,3,4,2 ′, 3 ′, 4′-hexabenzyl- ⁇ , ⁇ ′-trehalose was obtained.
- 6,6′-bis-O- (3-decyl) was prepared by the same method as in Production Example B-1 using 3-decyltridecanoic acid obtained by the method described in Production Example D-3.
- Tridecanoyl) -2,3,4,2 ′, 3 ′, 4′-hexabenzyl- ⁇ , ⁇ ′-trehalose was obtained.
- carboxylic acid commercially available 3-tridecylhexadecanoic acid (manufactured by Wako Pure Chemical Industries, Ltd.) was used, and in the same manner as in Production Example B-1, 6,6′-bis-O- (3-tridecylhexa Decanoyl) -2,3,4,2 ′, 3 ′, 4′-hexabenzyl- ⁇ , ⁇ ′-trehalose was obtained.
- Example 9 Production Example ⁇ -1 [Synthesis of 6,6′-bis-O- (3-nonyldodecanoyl) - ⁇ , ⁇ ′-trehalose]
- Example 15 Production Example ⁇ -2 [Synthesis of 6,6′-bis-O- (3-octylundecanoyl) - ⁇ , ⁇ ′-trehalose]
- Example 10 Production Example ⁇ -3 [Synthesis of 6,6′-bis-O- (3-decyltridecanoyl) - ⁇ , ⁇ ′-trehalose]
- Example 11 Production Example ⁇ -4 [Synthesis of 6,6′-bis-O- (3-undecyltetradecanoyl) - ⁇ , ⁇ ′-trehalose]
- Example 12 Production Example ⁇ -5 [Synthesis of 6,6′-bis-O- (3-dodecylpentadecanoyl) - ⁇ , ⁇ ′-trehalose]
- Example 13 Production Example ⁇ -6 [Synthesis of 6,6′-bis-O- (3-tridecylhexadecanoyl) - ⁇ , ⁇ ′-trehalose]
- Example 14 Production Example ⁇ -7 [Synthesis of 6,6′-bis-O- (3-tetradecylheptadecanoyl) - ⁇ , ⁇ ′-trehalose]
- the obtained residue was dissolved in a mixed solvent of 10N aqueous sodium hydroxide solution (4 ml) and n-butanol (8 ml), and heated under reflux for 6 hours. Then, it cooled to room temperature, 1N hydrochloric acid was added, it extracted 3 times with ether, the organic layer was dried using the anhydrous sodium sulfate, and it concentrated after filtering.
- the obtained residue was dissolved in acetic acid (3.3 ml) and heated to reflux for 18 hours. After cooling, concentrated under reduced pressure to remove acetic acid.
- Decanoic acid (4 g, 23.2 mmol) was dissolved in anhydrous dichloromethane solution (80 mL), 1,1-carbonyldiimidazole (4.5 g, 27.9 mmol) was added, and the mixture was stirred for 1.5 hours. Then, N, O-dimethylhydroxyamine hydrochloride (2.7 g, 27.9 mmol) was added, and the mixture was further stirred for 3 hours. After adding distilled water, extraction was performed twice using dichloromethane. The organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated.
- octanoic acid was used instead of decanoic acid described in Production Example D-1-1
- 1-bromooctane was used instead of 1-bromononane described in Production Example D-1-2.
- the produced compound was used in the next step and synthesized in the same manner as described in Production Examples D-1-1 to D-1-5 to obtain 3-octylundecanoic acid.
- pentadecanoic acid was used instead of decanoic acid described in Production Example D-1-1
- 1-bromotetradecane was used instead of 1-bromononane described in Production Example D-1-2.
- the produced compound was used in the next step and synthesized in the same manner as described in Production Examples D-1-1 to D-1-5 to obtain 3-tetradecylheptadecanoic acid.
- Example compounds are compounds represented by the formula (1) of the present invention, in which X, X ′, R 1 , R 1 ′, R 2 , R 2 ′, n and n ′ are the following: Show things.
- TDCM which is a known natural compound derived from Mycobacterium tuberculosis, was used as a positive control. Note that TDCM is represented by the following chemical structural formula.
- Test Example 1 (Measurement of macrophage activation ability) Test Example 1 (1) ⁇ Measurement of active oxygen release from mouse peritoneal macrophages using a fluorescence intensity measuring device> ⁇ Preparation of phosphate buffer (PBS)> 8.0 g of sodium chloride, 0.2 g of potassium chloride, 1.15 g of disodium hydrogen phosphate and 0.2 g of potassium dihydrogen phosphate were dissolved in 1000 ml of distilled water (DW).
- PBS phosphate buffer
- DW distilled water
- mice 3 ml of 5% thioglycolic acid medium (Difco, BD, code. 225640, Lot. 6192372) was administered into the abdominal cavity of mice (ICR mice (SPF), 5 weeks old, male). Four days after administration, the mice were killed using diethyl ether. A cut was made in the epidermis at the center of the abdomen with scissors, the abdomen was pinched and the abdomen epidermis was peeled off.
- 5% thioglycolic acid medium Difco, BD, code. 225640, Lot. 6192372
- a total volume of 5 ml of PBS ( ⁇ ) (EDTA 2Na, nuclease and protease tested, Nacalai Tesque) containing 0.05% EDTA was injected intraperitoneally with a 10 ml syringe equipped with a 26G needle. After that, massage was performed about 40-50 times by pinching the side of the abdomen. The fluid inside the abdominal cavity is slowly collected into a small centrifuge tube with a 23G needle. This operation was repeated twice. The collected macrophages were centrifuged at 1000 rpm for 8 minutes. The supernatant was discarded and RPMI1640 medium (containing RPMI-1640, L-glutamine and phenol red, Wako, 189-02025, Lot.
- RPMI1640 medium containing RPMI-1640, L-glutamine and phenol red, Wako, 189-02025, Lot.
- a 40 mM test compound solution was prepared as follows. 0.7 g BSA was weighed into a large test tube. After adding 10 ml of sterilized PBS ( ⁇ ) and stirring well, the impurities contained in the BSA solution were removed using an LPS removal column (Endo Trap TM red 1/1 (proofs)). Thereafter, the treated BSA solution was filtered through a sterilizing filter (0.2 ⁇ m). Next, the protein was quantified using Nano Drop ND-1000, and diluted with sterile PBS ( ⁇ ) so that the final concentration was 2%.
- the weighed test compound was placed in a homogenizer together with 250 ⁇ l of 2% BSA (2% BSA dissolved in PBS ( ⁇ )) and treated with a bath-type ultrasonic device for 150 seconds while in the homogenizer.
- the solution thus prepared was transferred to an eppen and used for the following tests.
- the positive control was prepared in the same manner using TDCM as the test compound, and the negative control was prepared in the same manner without adding any test compound.
- the preparation solution containing no test compound is referred to as “vehicle”.
- HBS Hanks balanced salt solution
- HBSG-BSA Hanks balanced salt solution containing glucose and BSA> Glucose (0.1 g) and BSA (sigma) (0.03 g) were dissolved in 100 ml of HBS prepared as described above to prepare a Hanks balanced salt solution (HBSG-BSA) containing glucose and BSA (prepared at the time of use).
- HBSG-BSA Hanks balanced salt solution
- HBSG-BSA containing the test compound or vehicle as a negative control was added so that the final concentration of the test compound was 50 ⁇ M, and Genios fluorescence was added after 1 hour. It was measured with an intensity measuring device.
- H 2 DCFDA is the fluorescence probe, in the presence of hydrogen peroxide (H 2 O 2), the fluorescence intensity increases, by measuring the fluorescence intensity, hydrogen peroxide (H 2 O 2 ) Production amount can be measured.
- Hydrogen peroxide (H 2 O 2 ) is derived from superoxide (O 2 ⁇ ) produced by macrophages, and the degree of activation of macrophages is determined using the amount of hydrogen peroxide (H 2 O 2 ) produced as an index. Can be represented.
- FIG. Figure 1 ⁇ Amount of active oxygen released from mouse peritoneal macrophages> As shown in FIG. 1, all of the test compounds of the present invention showed the action of promoting the production of active oxygen from mouse peritoneal macrophages, equivalent to or more than TDCM. In particular, among the compounds of the present invention, the compound of Example 1 and the compound of Example 9 showed a high activity more than twice that of TDCM.
- Test Example 1 (2) ⁇ Measurement of mouse peritoneal macrophage phagocytosis> Mouse peritoneal macrophages, 40 mM test compound solution, and RPMI 1640 medium were prepared as described above.
- Fluoresbrite (trademark) Carboxylate Microspheres (2.58% Solids-Latex) YG (Polysciences, Inc.) was used as the fluorescent beads.
- Macrophages were added to TC-plates (TC-PLATE 24WELL, STERILE WITH LID, IND PACKED, greiner bio-one) to 80% confluent. After incubation at 37 ° C. for 2 hours, the supernatant was discarded and the cells were washed twice with 500 ⁇ l of RPMI 1640 medium.
- the composition shown in Table 3 below was assembled in a TC-plate and incubated at 37 ° C. for 2 hours. After removing the supernatant, the cells were washed with 300 ⁇ l of sterile PBS ( ⁇ ). This washing operation was repeated twice to remove unincorporated fluorescent beads.
- Macrophages were peeled off with 200 ⁇ l of sterile PBS ( ⁇ ), and the macrophages were transferred to an eppen. After centrifugation at 1,500 rpm for 8 minutes, the supernatant was removed and well suspended in 100 ⁇ l of sterile PBS ( ⁇ ). Again, after centrifugation at 1,500 rpm for 8 minutes, the supernatant was removed and well suspended in 100 ⁇ l of sterile PBS ( ⁇ ).
- FUJIFILM FLA-2000 was used to measure the amount of fluorescent beads incorporated into the cells [fluorescence intensity (Fluor 473 nm, Y520 Filter)].
- Fluorescence intensity Fluor 473 nm, Y520 Filter
- Image Reader V1.4J was used for the analysis.
- FIG. Fig. 2 ⁇ Mouse peritoneal macrophage phagocytosis> As shown in FIG. 2, all of the test compounds of the present invention showed an action of activating the phagocytosis of mouse peritoneal macrophages in the same manner or more than TDCM. In particular, among the compounds of the present invention, the compound of Example 1 and the compound of Example 9 exhibited about twice as high activity as TDCM.
- Test Example 2 (Measurement of neutrophil activation ability) ⁇ Preparation of rabbit neutrophil suspension> Hanks balanced salt solution (HBS), glucose and BSA-containing Hanks balanced salt solution (HBSG-BSA) were prepared in the same manner as in Test Example 1 (1).
- HBS Hanks balanced salt solution
- HBSG-BSA BSA-containing Hanks balanced salt solution
- ⁇ Preparation of citric acid-glucose solution > 6.25 g of sodium citrate, 3.125 g of citric acid and 5 g of glucose were dissolved in 250 ml of distilled water (DW) and stored at 4 ° C. until use.
- the precipitate was suspended in 2 ml of HBSG-BSA, and this cell suspension was gently layered on the upper layer of 2 ml of Lymphoprep [Nycomed, 808068] (centrifuge tube, 15 ml type), and centrifuged at 1200 rpm for 20 minutes (centrifuge conditions: accel) 0.5, break Off), and the supernatant was removed with an aspirator.
- the sediment (neutrophils) was suspended in HBSG-BSA, centrifuged again at 1,500 rpm for 5 minutes, and the supernatant was removed. Neutrophils were suspended in HBSG-BSA, and the number of cells was measured with a cell count device “celltac” [Nihon Kohden].
- Test Example 2 (1) ⁇ Measurement of active oxygen release from rabbit neutrophils> The influence on the release of active oxygen from rabbit neutrophils was measured by the following procedure.
- a 96-well plate (Falcon) was seeded with neutrophils (1.0 ⁇ 10 5 cells / 100 ⁇ l). To this, 1 ⁇ l of 10 mM H 2 DCFDA was added and incubated at 37 ° C. for 1 hour. In order to remove excess H 2 DCFDA, 300 ⁇ l of HBS was added and suspended, followed by centrifugation at 8000 rpm for 5 minutes. After removing the supernatant and suspending with HBS, HBS was added to a final concentration of 50 ⁇ M. After incubation at 37 ° C. for 2 hours, the amount of released active oxygen was measured with a fluorescence measuring device (Ex: 485 nm, Em: 535 nm).
- FIG. Figure 3 Reactive oxygen release from rabbit neutrophils>
- the compound of the present invention exhibited an action of activating the release of active oxygen from rabbit neutrophils to the same extent or more than TDCM.
- the compound of Example 1 showed about twice the activity of TDCM.
- Test Example 2 (2) ⁇ Measurement of rabbit neutrophil phagocytic ability> (2) Ampicillin resistant E. coli and opsonized E. coli ⁇ Preparation of L-broth> 10 g of tryptophan, 5 g of NaCl, 5 g of Yeast Extract and 1 ml of MgSO 4 were dissolved in 1 L of distilled water (DW).
- opsonizing agent 10 mg of opsonizing agent (BioParticles Opsonizing Reagent (Molecular Probes) was dissolved in 500 ⁇ l of ultrapure water.
- ⁇ Opsonized Escherichia coli preparation method 100 ⁇ l of the ampicillin-resistant E. coli solution prepared above and 100 ⁇ l of the dissolved opsonizing agent were suspended in an Eppendorf tube. The obtained suspension was incubated at 37 ° C. for 1 hour, and the suspension was suspended in 300 ⁇ l of PBS, and then centrifuged at 1200 G for 15 minutes to remove the supernatant. Further, the obtained liquid was suspended in 300 ⁇ l of PBS and centrifuged at 1200 G for 15 minutes to remove the supernatant. Repeated twice. 1 ⁇ l of the bacterial solution was added to 100 ⁇ l of L-broth, suspended well and diluted 100 times. The bacterial solution diluted 100 times was added to a 10 ⁇ l one-cell counter, and the number of bacteria was counted with a microscope.
- FIG. Figure 4 ⁇ Rabbit neutrophil phagocytic activity> As shown in FIG. 4, all of the compounds of the present invention exhibited an action of activating the phagocytic ability of rabbit neutrophils. In particular, among the compounds of the present invention, the compound of Example 1 showed about twice the activity of TDCM.
- Test example 3 ⁇ Measurement of cytokine release from mouse peritoneal macrophages by test compound treatment> Mouse peritoneal macrophages, 40 mM test compound solution, and RPMI 1640 medium were prepared as described above.
- Macrophages were added to the TC-plate so as to be 80% confluent. After 2 hours incubation at 37 ° C., the supernatant was discarded and the cells were washed with 500 ⁇ l RPMI 1640 medium. This washing operation was repeated twice. An emulsion solution of the test compound (final concentration 100 ⁇ M) was allowed to act on the macrophages, and after 2 hours, the medium was transferred to another eppen.
- the supernatant is further transferred to another eppen, and using the supernatant as a sample, the released cytokine is ELISA kit (IL-6, TNF- ⁇ Quantikine Immunoassay (R & D Systems (trademark)). And analyzed.
- IL-6 TNF- ⁇ Quantikine Immunoassay
- the value of vehicle as a negative control was about 15 pg / ml for the release of IL-6, whereas among the compounds of the present invention, the activity is considered to be particularly high.
- the compound of Example 1 showed an activity of about 200 pg / ml.
- the value of vehicle as a negative control was about 80 pg / ml, whereas the compound of Example 1 showed an activity of about 1000 pg / ml.
- Test example 4 ⁇ Measurement of IL-8 release from THP-1 cells by treatment with test compound>
- the RPMI medium solution was prepared in the same manner as described above, and using this, an RPMI medium solution of the test compound was prepared as follows.
- Test compound 1.0 mg was sonicated in 25 ⁇ l of DMSO for about 1 minute and dissolved.
- the 40 mM test compound stock solution thus obtained was added to 100 ⁇ l of RPMI medium to a concentration of 50 ⁇ M (final concentration) and treated with ultrasound for 5 seconds.
- As a control vehicle the same amount of solvent alone was added to the RPMI medium instead of the 40 mM test compound stock solution, and treated with ultrasound for 5 seconds.
- THP-1 cells purchased from RIKEN cells BANK
- RIKEN cells BANK were prepared in RPMI medium to 1.0 ⁇ 10 6 cells / 100 ⁇ l, and dispensed in 100 ⁇ l sterilized eppenes.
- 100 ⁇ l of the RPMI medium solution of the test compound prepared as described above was sonicated for 5 seconds, and then added to the Eppenes in which the cells were dispensed. After 2 hours, the reaction eppen was centrifuged at 5000 rpm for 5 minutes, and the amount of IL-8 released in the supernatant was measured using an ELISA kit (human IL-8 ELISA kit (R & D Systems TM)).
- test results are shown in FIG. Among the compounds of the present invention, when a compound considered to have particularly high immunostimulatory activity was measured, as shown in FIG. 5, the compound of Example 1 and the compound of Example 9 were about 0.6% of TDCM. The activity was about 0.8 times.
- Test Example 5 ⁇ Measurement of IL-6, TNF- ⁇ and IFN- ⁇ release into peripheral blood in test compound-treated mice> An emulsion solution of the test compound was prepared as follows.
- test compounds refer to the compounds synthesized in Production Examples ⁇ -1 to 8, compounds synthesized in Production Examples ⁇ -1 to 7, and TDCM as a comparative example.
- test compound was weighed (100 ⁇ g / mouse), and the total amount was placed in the bottom of a homogenizer (WEATON USA 10 ml) using a micropartel.
- a homogenizer WEATON USA 10 ml
- One drop of mineral oil Nacalai Tesque
- bath type ultrasonic waves for 150 seconds.
- 1.0 ml of physiological saline containing 1.1% Tween 80 (polyoxyethylene sorbitan monooleate, Nacalai Tesque) and 5.6% mannitol was added to the homogenizer. Homogenized several times and thoroughly mixed the test compound-dissolved mineral oil and solvent.
- the completed solution was transferred to an Eppendorf tube and pasteurized at 62 ° C. for 30 minutes.
- the homogenizer was placed on ice for 3 minutes in advance. 1.0 mg of various test compounds were each taken in a homogenizer, mineral oil was added and treated with ultrasound for 150 seconds. After confirming that the oil was sticky, 1.0 ml of physiological saline (containing 1.1% Tween and 5.6% mannitol) was added thereto and homogenized for about 1 minute. The sample was transferred to an eppen and pasteurized at 62 ° C. for 30 minutes.
- test compound emulsion solution 100 ⁇ g / mouse prepared as described above was administered to 2 mice per group by intravenous injection, and 2 hours later, heart blood (heparin blood collection) was performed. After centrifugation at 10,000 rpm for 10 minutes, various plasma cytokines were measured using ELISA alone (IL-6, TNF- ⁇ , IFN- ⁇ Quantikine Immunoassay (R & D Systems TM)) using plasma alone.
- FIG. 6 ⁇ IL-6 concentration in mouse plasma (pg / ml)> FIG. 7 ⁇ Mouse Plasma IFN- ⁇ Concentration (pg / ml)> Fig. 8 ⁇ TNF- ⁇ concentration in mouse plasma (pg / ml)>
- an increase in plasma IL-6 concentration was observed in the mice administered with the test compound of the present invention.
- the compound of Example 1 and the compound of Example 9 are both about 1.2 times the TDCM, which is a known natural trehalose diester compound as a positive control, respectively.
- the IL-6 releasing activity was about 1.5 times as high.
- the compound of Example 13 and the compound of Example 14 also showed about half the same activity against TDCM, respectively.
- Test Example 6 ⁇ Mouse survival test by administration of Clostridium perfringens (compound of Example 1)> As a test compound, 1 mg each of the compound synthesized by the method described in Production Example ⁇ -1 and TDCM were weighed, and an emulsion solution (1 mg / ml) of the test compound was prepared in the same manner as described above.
- C. perfringens (TypeA NTCT8237) was prepared as follows.
- BHI Brain Heart Infusion
- Bacteria grown in the screw-mouth test tube (turbidity inside the clean bench becomes turbid and gas is generated) are transferred to a 200 ml Erlenmeyer flask containing 40 ml of BHI medium in the clean bench, and a glass tube is inserted into the medium. Then, nitrogen substitution was performed for 10 minutes. Next, a rubber stopper with a glass tube (with a cotton stopper) was attached to the Erlenmeyer flask (Because Clostridium perfringens produces a gas, an air hole is necessary) and incubated at 37 ° C. for 4-5 hours. The cultured C.
- perfringens was transferred to a centrifuge tube, centrifuged (9000 rpm, 15 minutes), and the supernatant was removed.
- 20 ml of sterile physiological saline was added to the sediment to suspend the bacteria, followed by centrifugation (9000 rpm, 15 minutes), and the supernatant was removed.
- BHI medium in a screw-cap test tube in which the obtained sediment was sterilized was added, and the number of bacteria was counted with a one-cell counter (manufactured by One-Cell).
- mice ICR, 6 weeks old
- 100 ⁇ g / mouse 100 ⁇ l / mouse in the case of emulsion solution only
- an emulsion solution of a test compound 100 ⁇ l / mouse in the case of emulsion solution only
- mice 100 ⁇ l / mouse in the case of emulsion solution only
- mice 100 ⁇ l / mouse in the case of emulsion solution only
- mice 100 ⁇ l / mouse (100 ⁇ l / mouse in the case of emulsion solution only) was administered intraperitoneally to each group of 4 mice, an emulsion solution of a test compound, an emulsion solution of TDCM, or an emulsion solution as a control alone.
- C. perfringens 2.4 ⁇ 10 7 cells / mouse
- mice administered with the compound of Example 1 which is a compound of the present invention escaped from lethality in 4 mice in the administration group in a lethal model of Clostridium perfringens.
- Test Example 7 ⁇ Mouse survival test by administration of Clostridium perfringens toxin (compound of Example 1)> A test compound emulsion solution (1 mg / ml) was prepared as described above. C. perfringens toxin was prepared as follows.
- Bacillus subtilis ⁇ -toxin gene transformant is cultured in L-Broth at 37 ° C. for 14 hours, centrifuged at 4 ° C. and 8,000 rpm for 20 minutes, and the culture supernatant is stirred under ice-cooling with ammonium sulfate ( A small amount of ammonium sulfate (Nacalai Tesque) was periodically added to a final concentration of 70% saturated ammonium sulfate (472 g / L) and left overnight.
- a crude toxin preparation was applied to a column (1.5 ⁇ 9 cm), followed by 0.5M NaCl-TB (pH 7.5), 0.5M NaCl-0.1M PB (pH 6.5), 0.5M. NaCl-0.02M acetate buffer (pH 4.5) and 0.5M NaCl-0.1M PB (pH 6.5) were sequentially flowed in 100 ml portions.
- the toxin bound in the column was eluted with 100 ml of 15 mM L-histidine (Nacalai Tesque) -0.5 M NaCl-0.1 M PB (pH 6.5), and this eluate was then filtered with a syringe filter (DISMIC-ADVANTEC).
- mice ICR, 6 weeks old
- mice 100 ⁇ g / mouse (100 ⁇ l / mouse in the case of emulsion solution only) was administered intraperitoneally to each group of 4 mice, an emulsion solution of a test compound, an emulsion solution of TDCM, or an emulsion solution as a control alone.
- C. perfringens toxin 200 ng / mouse was intraperitoneally administered to the mice. Thereafter, follow-up was performed.
- Test Example 8 ⁇ Mouse survival test by administration of Pseudomonas aeruginosa (compound of Example 1)> As a test compound, 1 mg of the compound synthesized by the method described in Production Example ⁇ -1 was weighed, and an emulsion solution of the test compound was prepared in the same manner as described above. Pseudomonas aeruginosa (Fhu-0711115 strain) was derived from a patient and prepared as follows.
- L-broth was taken with a 40 ml measuring pipette, put into one 200 ml flask, and sealed with a sponge. In addition, 5 ml of L-broth was placed in two separate screw-cap test tubes. The flask and screw test tube were autoclaved at 121 ° C. for 20 minutes. After the L-broth medium had cooled to room temperature, Pseudomonas aeruginosa stored in an ultra-low temperature freezer was added to 40 ml of L-broth in a clean bench. Shaking was performed overnight in the culture room.
- Centrifugation was performed at 9000 rpm for 15 minutes, and the supernatant was removed. After adding 20 ml of sterilized physiological saline and mixing with vortex, the step of centrifuging at 9000 rpm for 15 minutes and removing the supernatant was performed three times. 4.5 ml of sterilized physiological saline was added and mixed using a vortex to make a bacterial stock solution. The bacterial solution diluted 1000 times was used to count the number of bacteria with a one-cell counter, and then the bacterial stock solution was diluted to a desired concentration and used for the following tests.
- mice were divided into two groups and the following two experiments were performed.
- A An emulsion solution (100 ⁇ g / mouse) of the above test compound was intraperitoneally administered to 3 mice (ICR, 5 weeks old) per group, and after 3 hours, Pseudomonas aeruginosa (5.0 ⁇ 10 5). 7 cells / mouse) was administered intraperitoneally. Thereafter, follow-up was performed.
- Test Example 9 ⁇ Cytokine release from THP-1 cells> The amount of each cytokine and chemokine released from THP-1 cells derived from human monocytic leukemia cells treated with the test compound obtained in Example 9 was measured by ELISA. In addition, the same analysis was performed using A549 cells derived from human lung cancer cells and DLD-1 cells derived from human colon cancer cells.
- ⁇ Culture method of THP-1 cells (1) Serum lot check The cultured THP-1 cells were transferred to a 15 ml centrifuge tube in a clean bench. The cells were centrifuged at 1,000 rpm for 5 minutes at 20 ° C., and the supernatant was removed. Each sediment was suspended in 1 ml of RPMI 1640 (containing 10% lot check FBS) medium, and the number of cells was counted using a hemocytometer (Higaki Medical Science Co., Ltd.) and a cover glass (Sansho). To 2.5 ⁇ 10 5 cells / ml.
- MULTI WELL PLATE 24wells SUMILON
- cell growth and morphology were observed once a day.
- the culture was diluted 100-fold with the medium, 4 ⁇ l was taken, the number of cells was counted with a counting plate, and the quality of growth was compared.
- Cells with good growth and morphology are recovered from the well, washed with the same medium, diluted again to 2.5 ⁇ 10 5 cells / ml, and 1 ml is added to MULTI WELL PLATE 6 wells (SUMILON) for suspension culture at 37 ° C. It was incubated under the conditions of 5% CO 2.
- the medium was changed every 24 hours, and the whole plate was centrifuged (TOMY) at 1,800 rpm for 5 minutes. Then, the medium was slowly removed, and 1 ml of the medium was newly added. The medium exchange operation was performed twice. After the third day, after changing the medium, the cells were diluted 100 times, counted with a counting plate, and the degree of proliferation was measured. Thereafter, this operation was performed for several days, and a medium with good growth and morphology was selected.
- RPMI 1640 medium (10% non-immobilized FBS + 1% Penicillin Streptomycin)
- a clean bench add 5.6 ml of non-immobilized FBS and Penicillin Streptomycin (GIBCO) 5.6 ml filtered with Acrodisc 25 mm Syringe Filter (Pall Corporation) to 500 ml of RPMI 1640 liquid medium (Wako).
- the prepared medium was used as it was without filtering. Storage was performed at 4 ° C. and the temperature was returned to room temperature before use.
- THP-1 cells were cultured in a 75 cm 2 suspension culture flask (SUMILON) (90% or higher growth), and their morphology and proliferation were observed. If the cells were well shaped and grew quickly, the cell suspension was transferred to a new flask or transplanted with approximately twice the amount of fresh medium added to the flask in use. When the growth was slow, the progress was observed as it was, or an equal amount of fresh medium was added and transplanted.
- SUPILON suspension culture flask
- Standard substrate solution 600 ⁇ L of standard substrate solution dilution is put in two microtubes. Dissolve the standard substrate in 1 mL of standard substrate diluent (2450 pg / mL), transfer it to a 100 ⁇ L microtube, dissolve (350 pg / mL), add 100 ⁇ L to another microtube and dissolve. (50 pg / mL) for dilution. A dilution for standard substrate solution was used as a control (0 pg / mL).
- Color Reagent Preparation Color reagent A and color reagent B were mixed in equal amounts, and 100 ⁇ l / well ⁇ well color solution to be measured was added. This was prepared within 15 minutes before use.
- sample measurement> The reagents of the ELISA kit to be used were returned to room temperature, 50 ⁇ L of various concentrations of standard substrate solution and assay buffer were added to each well, and 50 ⁇ L of the sample was further added. The plate was lightly tapped for 1 minute, the plate was covered and incubated for 2 hours at room temperature. Thereafter, the plate was washed 5 times with a washing solution (aspirator can be used), 100 ⁇ L of the prepared conjugate solution was added, the plate was covered, and incubated at room temperature for 2 hours.
- a washing solution aspirator can be used
- the number of cells was counted with 4 ⁇ l of a 100-fold diluted solution using a hemocytometer, and the cell suspension was diluted to 1 ⁇ 10 7 cells / ml with serum-free RPMI 1640 medium.
- 100 ⁇ L of RPMI medium was taken in an eppen, 40 mM test compound (Example 9) / DMSO solution to 200 ⁇ M was added, and ultrasonic waves were applied for 5 seconds.
- 100 ⁇ l THP-1 cells were added (final concentration of test compound 100 ⁇ M). After incubation at 37 ° C.
- control compound A 6,6′-bis-O- (2-tetradecylhexanoyl) - ⁇ , ⁇ ′-trehalose
- TDCM 6,6′-bis-O- (2-tetradecylhexanoyl) - ⁇ , ⁇ ′-trehalose
- control compound A 6,6′-bis-O- (2-tetradecylhexanoyl) - ⁇ , ⁇ ′-trehalose
- Test Example 10 ⁇ Cytotoxicity study and mutagenicity test for THP-1 cells> ⁇ Study of cytotoxicity of THP-1 cells using trypan blue> Reagent preparation Preparation of 0.3% Trypan Blue / PBS ( ⁇ ) 0.3 g of trypan blue (nacarai) was dissolved in 100 ml of PBS ( ⁇ ).
- THP-1 cells The cultured THP-1 cells were transferred to a 50 ml centrifuge tube in a clean bench, centrifuged at 1,000 rpm for 5 minutes at 20 ° C., and the supernatant was removed. The sediment was suspended in 1 ml of fresh serum-free RPMI 1640 medium (Wako). 10 ⁇ l of this cell suspension was added to a sterile Eppendorf tube to which 990 ⁇ l of serum-free RPMI 1640 medium had been added in advance, and diluted 100 times. 10 ⁇ l of 100-fold diluted solution was counted with a hemocytometer, and the cell suspension was diluted with serum-free RPMI 1640 medium to 1 ⁇ 10 7 cells / ml.
- serum-free RPMI 1640 medium 1 ⁇ 10 7 cells / ml.
- test compound B 6-O- (2-decyldocanoyl) - ⁇ -glucose
- control compound B 6-O- (2-decyldocanoyl) - ⁇ -glucose
- TDCM 6-O- (2-decyldocanoyl) - ⁇ -glucose
- negative control no test compound was added, and the same control was prepared (Vehicle).
- 100 ⁇ l of THP-1 cells prepared to 1.0 ⁇ 10 7 cells / ml were added and incubated at 37 ° C. for 2 hours or 24 hours. Thereafter, 20 ⁇ l of 0.3% trypan blue / PBS ( ⁇ ) was added and suspended, and immediately the cell viability was analyzed with a cell number measuring device (CYRORECON).
- VB medium 0.4 g of MgSO 4 .7H 2 O, 4 g of citrate H 2 O, 20 g of K 2 HPO 4 , 7 g of NaNH 4 HPO 4 .4H 2 O are dissolved in 200 mL of distilled water. And then autoclaved.
- Glucose 40 g was dissolved in distilled water 200 mL and autoclaved.
- agar medium 1.2 g of powder agar and 1 g of NaCl were suspended in 200 mL of water, sterilized by high-pressure steam, and transferred to a 50 mL tube. Before use, 20 mL of 0.5 mM histidine / biotin solution was mixed and kept at 47 ° C.
- Oxoid Nutrient Broth Medium for Salmonella typhi 2.5 g of Oxoid Nutrient Broth (Difco) was dissolved in 100 ml of distilled water, 5 ml of which was placed in a screw test tube and sterilized. Thereafter, about 10 ⁇ L of bacterial solution of TA98 (Salmonella typhimurium TA98) was inoculated and cultured overnight at 37 ° C. with shaking to prepare a bacterial suspension.
- Example 9 ⁇ Experimental result>
- the survival rate of THP-1 cells treated with the test compound obtained in Example 9 for 2 hours and 24 hours was analyzed by trypan blue staining. Although cytotoxicity was observed in the treated cells, no cytotoxicity was observed in the cells treated with the compound obtained in Example 9. The results are shown in FIG. Similarly, significant results were obtained for the compound obtained in Example 1 (data not shown). Furthermore, the Ames test was performed on the test compound obtained in Example 9 in the presence and absence of S9mix, but none of the test compounds showed mutagenicity. Further, the mutagenicity of the standard substance (2-amino anthracene, 4NQO) under the analysis conditions was positive (FIG. 11). Similarly, significant results were obtained for the compound obtained in Example 1 (data not shown).
- Test Example 11 ⁇ Cell infiltration into mouse abdominal cavity> ⁇ Preparation of PBS (-) solution> 4 g of NaCl, 1.45 g of Na 2 HPO 4 ⁇ 12H 2 O, 0.1 g of KH 2 PO 4 and 0.1 g of KCl were dissolved in 500 ml of distilled water and autoclaved at 121 ° C. for 20 minutes.
- test compound solution was transferred to an eppen and treated at 62 ° C. for 30 minutes for pasteurization.
- a test compound was prepared in the same manner using control compound A and TDCM.
- no test compound was added and the same was prepared (vehicle).
- test compound (Example 9) solution was intraperitoneally administered to mice (ICR mice (SPF) (4 weeks old, male, body weight: 20 to 22 g)) to a concentration of 100 ⁇ g / mouse. Mice that received the test compound ip were killed after 2 or 24 hours using diethyl ether. A cut was made in the epidermis at the center of the abdomen with scissors, the abdomen was pinched and the abdomen epidermis was peeled off.
- the peritoneum was gently lifted with tweezers, and 5 mL of 0.05% EDTA in PBS ( ⁇ ) was injected into the abdominal cavity with a 10 mL syringe attached with a 26 G needle so that the needle did not pierce the internal organs. After that, massage was performed about 40-50 times by pinching the side of the abdomen. The fluid inside the abdominal cavity was slowly collected into a small centrifuge tube. This operation was repeated again. The collected cells were centrifuged at 1,000 rpm for 10 minutes. The supernatant was removed and the precipitate was suspended in RPMI 1640 medium. The centrifuge tube was filled with RPMI 1640 medium and centrifuged again at 1,000 rpm for 10 minutes. After discarding the supernatant and suspending in RPMI 1640 medium, the number of cells is counted using a cell counter. RPMI-1640 medium was used to dilute to any concentration.
- the cells were suspended in 100 ⁇ L of phosphate buffer and placed on a glass slide for staining. After confirming that the water had evaporated, 10-15 drops of May-Grünwald liquid were dropped on the staining pad and allowed to stand for 2-3 minutes. Without flowing the May-Grünwald solution, 10 to 15 drops of phosphate buffer were formed and allowed to stand for 2 to 3 minutes. An appropriate amount of Giemsa staining solution was added and left for 30 minutes. After running the water with the slide glass on the back, the slide glass was dried and observed with a microscope.
- test compound 1 mg / mL test compound (emulsion solution)
- the test compound was prepared in the same manner as in Test Example 11.
- ⁇ Experiment method> A 1 mg / mL test compound emulsion solution was intraperitoneally administered to mice (100 ⁇ g / mouse), and cells infiltrated into the peritoneal cavity were collected 24 hours later using 0.05% EDTA / PBS ( ⁇ ). Thereafter, the collected peritoneal cells were centrifuged at 300 g for 10 minutes, and the supernatant was removed. The prepared peritoneal cells were suspended in 1 ml of 0.05% EDTA (dissolved in 0.5% BSA / PBS). The cell suspension was filtered with a mesh for flow site, and the number of cells was measured with the cell suspension diluted 100 times.
- the abundance ratio of CD8 positive cells was increased by about 2 to 3 times compared to the vehicle-treated mice (FIGS. 13 to 15). From the above, it was shown that the test compound of Example 9 accumulates phagocytic cells in the mouse abdominal cavity, and in particular, the ratio of NK cells was shown to be large.
- Test Example 12 ⁇ Effects on C. perfringens or Pseudomonas aeruginosa-infected mice (Compound of Example 9)> ⁇ Method for preparing Clostridium perfringens> Preparation of COOKED MEAT medium (hereinafter sometimes abbreviated as CM medium) CM medium (125 mg / ml in DW) was added to a threaded test tube and boiled for 15 minutes to degas the air in the CM medium. High-pressure steam sterilization with an autoclave (121 ° C., 20 minutes) was performed, and the mixture was cooled to room temperature.
- CM medium COOKED MEAT medium
- BHI Brain Heart Infusion
- C.I. perfringens Type-A NCTC8237 (PLC +) was added, cultured at 37 ° C. for 2 days, and stored as a stock solution at room temperature.
- Bacterial culture and preparation of bacterial solution 0.2 mL of the bacterial solution was taken from each preserved bacterial solution, added to 4.5 mL BHI medium for preculture, and cultured at 37 ° C overnight. The total amount of this culture solution was added to 40 mL BHI medium, and after nitrogen substitution (10 minutes), the cells were again cultured at 37 ° C. for 5 hours. Then, it moved to the culture solution 50mL tube, and centrifuged (4 degreeC, 9000 rpm, 15 minutes). The supernatant was removed, physiological saline was added and washed well, and then the cells were collected by centrifugation (4 ° C., 9000 rpm, 15 minutes). This washing was repeated twice.
- fungus 0.2 mL of P. aeruginosa was added to 10 mL of L-Broth and shaken overnight at 37 ° C. To this culture solution, 1 mL of sterilized glycerin (glycerin) was added and vortexed. 300 ⁇ L of the bacterial solution was dispensed into a sterilized eppen and stored at ⁇ 80 ° C.
- This suspension was used as a stock solution, and a bacterial solution obtained by diluting the stock solution 10,000 times was autoclaved (121 ° C., 20 minutes) using an autoclave, and then the number of bacteria was measured using a one-cell canter.
- a bacterial solution having a bacterial concentration of 1 ⁇ 10 8 cells / mL was prepared and used in the experiment.
- test compound > 100 ⁇ g / mouse test compound (Example 9) emulsion solution was administered intraperitoneally, and 24 hours later, 3.0 ⁇ 10 10 CFU / mL of Pseudomonas aeruginosa or 5.0 ⁇ 10 7 CFU / mL of C. perfringens Administered intraperitoneally, mice were observed every 2 hours.
- mice post-test compound administration> 3.0 ⁇ 10 10 CFU / mL of Pseudomonas aeruginosa was intraperitoneally administered, and 3 hours later, 100 ⁇ g / mouse of the test compound (Example 9) emulsion solution was intraperitoneally administered. Thereafter, the mice were observed every 2 hours.
- Test Example 13 ⁇ Sepsis observation> 100 ⁇ g / mouse of the test compound (Example 9) emulsion solution was intraperitoneally administered and 24 hours later, 3.0 ⁇ 10 10 CFU / mL of Pseudomonas aeruginosa was infected. 15 hours after administration of the bacteria, heart blood was collected with a syringe with a little heparin in the needle tip, 200 ⁇ L of whole blood was seeded on a normal agar medium, and incubated in an incubator for 16 hours. The number of colonies on the medium was counted.
- Test Example 14 ⁇ Anti-tumor effect> 100 ⁇ g / mouse of the test compound (emulsion solution) obtained in Example 9 was intraperitoneally administered to mice, and 24 hours later, breast cancer cells (FM3A cells) were inoculated intraperitoneally. After 19 days, mice were weighed. Further, tissue sections of the diaphragm, pancreas, and testis were observed with a microscope after HE staining. As a result, the breast cancer cell-inoculated mice had an increased body weight of about 10 g compared to the control mice, and a large amount of ascites was observed.
- FM3A cells breast cancer cells
- mice treated with the test compounds of the Examples and inoculated with breast cancer cells had the same body weight as that of the control mice, and further, infiltration of cancer cells into each organ and metastasis were not observed at all (Fig. 20).
- the trehalose compound of the present invention is superior to TDCM or has an immunostimulatory action comparable to that of TDCM, but has a markedly reduced toxicity compared to TDCM and can be suitably used as a pharmaceutical product. It has been found that the toxicity of the present compound is reduced not only in model mice but also in human-derived cells. Moreover, it was shown that this-application compound also has low mutagenicity.
- the ⁇ hydroxyl group is replaced with a hydrogen atom
- the fatty acid is ⁇ -branched or ⁇ -branched
- branched carbon chains R 1 , R 2 in formula (1), R 1 ′ or R 2 ′
- R 1 , R 2 in formula (1), R 1 ′ or R 2 ′ branched carbon chains each having about 7 to 20 carbon atoms are synthesized and tested one by one, so that ⁇ -branched type has 10 carbon atoms and ⁇ -branched type.
- the cancer induction activity which an amide bond has can be suppressed by making an amide bond of a prior art into an ester bond.
- the trehalose compound of the present invention is very useful in an in vivo test in that it reduces lethality in mice administered intraperitoneally with C. perfringens. In addition, it is epoch-making in that the lethality is reduced even in the mouse intraperitoneally administered with the toxin produced by Clostridium perfringens. Furthermore, it has been found that the trehalose compound of the present invention reduces the lethality of the bacterium by excellent antibacterial activity even when administered intraperitoneally with Pseudomonas aeruginosa.
- the trehalose compound of the present invention brings about activation of cellular immunity such as macrophages and neutrophils, and bacteria, viruses, fungi and the like that are subject to phagocytosis of macrophages and neutrophils This suggests that it is useful for a wide range of infections caused by This is because, in the treatment of infectious diseases, when antibiotics are used, the target causative bacteria must be determined, and antibiotics having an antibacterial spectrum must be appropriately selected. When a resistant bacterium emerges, it has the advantage of being a simple and reliable method compared to the conventional method in which another antibiotic having an antibacterial spectrum for the resistant bacterium must be selected appropriately. .
- particularly active compounds have an activity that is about 2 times that of TDCM, particularly about 8 times to 10 times that of a highly active compound, and have low toxicity. It was shown to be particularly useful.
- IL-6, IFN- ⁇ , and TNF- ⁇ release all showed an increasing tendency.
- in vitro tests and in vivo tests are carried out.
- particularly active compounds may cause a significant increase in these cytokines in the in vitro test, but in in vivo tests. Found that it does not increase TNF- ⁇ release significantly.
- IL-8 release activity from human-derived THP-1 cells it has been found that particularly highly active compounds of the present invention are not as strongly activated as TDCM.
- Immunostimulation has long been regarded as important, and induction of cytokines and chemokines is useful for activating immunity.
- immunity is activated too much, it becomes rather harmful as typified by anaphylactic shock and allergy.
- the trehalose compound of the present invention is not excessively activated against the release of inflammatory TNF- ⁇ which can be a factor causing these excessive inflammatory reactions and IL-8 known as chemokine, and has an immunostimulatory effect. This indicates that there is a low possibility of side effects such as inflammation due to an excessive immune reaction.
- cytokines and chemokines are preferably released in a large amount depending on their properties, otherwise they may not be a problem, and may vary depending on the situation.
- the compound should be released or inhibited to release IL-8 or TNF- ⁇ . It is also within the scope of the present invention to use a particular type of trehalose compound of the present invention in the desired amount, without intending to be done.
- the trehalose compound provided by the present invention has high immunostimulatory activity and low toxicity, and is useful for the treatment of infectious diseases caused by pathogenic bacteria. Specifically, by using the trehalose compound of the present invention, there is little risk of causing a side effect such as toxin release due to destruction of bacterial cells upon administration of antibiotics, and a drug having a toxicity-reducing action on the toxin of the pathogenic bacteria itself Can be provided. In addition, by using the trehalose compound of the present invention, it is possible to provide a medicament that has a therapeutic effect on infections caused by multidrug-resistant bacteria. Furthermore, the compounds of the present invention are also useful in the manufacture of pharmaceuticals with a low risk of causing an excessive immune response. Moreover, the trehalose compound according to the present invention can be efficiently synthesized in a large amount by the method for producing a trehalose compound of the present invention without including asymmetric synthesis.
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Abstract
Description
[式中、
Xは、フェニル、ナフチル、または、R1-CHR2-で表される基であり、
X’は、フェニル、ナフチル、または、R1’-CHR2’-で表される基であり、
ここで、
R1、R1’、R2及びR2’は、それぞれ独立に、水素原子またはC1-C21アルキル基であり、R1、R1’、R2、R2’に関し、各アルキル基中の水素原子は、水酸基、アルコキシ基によって置換されていてもよく、各アルキル基の全部または一部は4-8員環を形成していてもよく、また、R1及びR2、R1’及びR2’は、それぞれ互いに連結して4-8員環を形成していてもよく、
n及びn’は、それぞれ独立に、0から3の整数である。
但し、
(1)Xが、R1-CHR2-であり、X’が、R1’-CHR2’-であり、R1、R1’、R2及びR2’が、それぞれ独立に、水素原子または無置換かつ直鎖のC1-C6アルキル基であり、n及びn’が0である化合物、及び、
(2)Xが、R1-CHR2-であり、X’が、R1’-CHR2’-であり、R1、R1’、R2及びR2’がC14直鎖アルキル基であり、n及びn’が0である化合物
を除く]
で表される化合物を提供する。
治療上有効量の式(1)で表される化合物を当該哺乳動物に投与することを含む方法を提供する。
[式中、
Xは、フェニル、ナフチル、または、R1-CHR2-で表される基であり、
X’は、フェニル、ナフチル、または、R1’-CHR2’-で表される基であり、
ここで、
R1、R1’、R2及びR2’は、それぞれ独立に、水素原子またはC1-C21アルキル基であり、R1、R1’、R2、R2’に関し、各アルキル基中の水素原子は、水酸基、アルコキシ基によって置換されていてもよく、各アルキル基の全部または一部は4-8員環を形成していてもよく、また、R1及びR2、R1’及びR2’は、それぞれ互いに連結して4-8員環を形成していてもよく、
n及びn’は、それぞれ独立に、0から3の整数である。]
で表される化合物を含有することを特徴とする菌産生毒素中和剤を提供する。
(A)Xは、R1-CHR2-で表される基である。
(B)X’は、R1’-CHR2’-で表される基である。
(C)R1及びR1’は、それぞれ独立に、無置換のC1-C21アルキル基である。
(D)R2及びR2’は、それぞれ独立に、水素原子または無置換のC1-C21アルキル基である。
(E)R1及びR1’は、それぞれ独立に、直鎖のC1-C21アルキル基である。
(F)R2及びR2’は、それぞれ独立に、水素原子または直鎖のC1-C21アルキル基である。
(G)R1及びR1’は、それぞれ独立に、無置換かつ直鎖のC1-C21アルキル基である。
(H)R2及びR2’は、それぞれ独立に、水素原子または無置換かつ直鎖のC1-C21アルキル基である。
(I)R1及びR1’は、それぞれ独立に、無置換かつ直鎖のC7-C21アルキル基である。
(J)R2及びR2’は、それぞれ独立に、無置換かつ直鎖のC7-C21アルキル基である。
(K)R1及びR1’は、同一であって、無置換のC1-C21アルキル基である。
(L)R2及びR2’は、同一であって、水素原子または無置換のC1-C21アルキル基である。
(M)R1及びR1’は、同一であって、直鎖のC1-C21アルキル基である。
(N)R2及びR2’は、同一であって、水素原子または直鎖のC1-C21アルキル基である。
(O)R1及びR1’は、同一であって、無置換かつ直鎖のC1-C21アルキル基である。
(P)R2及びR2’は、同一であって、水素原子または無置換かつ直鎖のC1-C21アルキル基である。
(Q)R1及びR1’は、同一であって、無置換かつ直鎖のC7-C21アルキル基である。
(R)R2及びR2’は、同一であって、無置換かつ直鎖のC7-C21アルキル基である。
(S)n及びn’は、それぞれ独立に、0または1である。
(T)n及びn’は、0である。
(U)n及びn’は、1である。
(V)Xは、R1-CHR2-で表される基であり;X’は、R1’-CHR2’-で表される基であり、ここで、R1、R1’、R2及びR2’は、それぞれ独立に、水素原子またはC1-C21アルキル基であり、R1、R1’、R2、R2’に関し、各アルキル基中の水素原子は、水酸基、アルコキシ基によって置換されていてもよく、各アルキル基の全部または一部は4-8員環を形成していてもよく、また、R1及びR2、R1’及びR2’は、それぞれ互いに連結して4-8員環を形成していてもよく;n及びn’は、それぞれ独立に、0から3の整数である。
(W)Xは、R1-CHR2-で表される基であり;X’は、R1’-CHR2’-で表される基であり、ここで、R1、R1’、R2及びR2’は、それぞれ独立に、直鎖のC7-C21アルキル基であり、R1、R1’、R2、R2’に関し、各アルキル基中の水素原子は、水酸基、アルコキシ基によって置換されていてもよく、各アルキル基の全部または一部は4-8員環を形成していてもよく;n及びn’は、それぞれ独立に、0または1である。
(X)Xは、R1-CHR2-で表される基であり;X’は、R1’-CHR2’-で表される基であり;ここで、R1、R1’、R2及びR2’は、それぞれ独立に、直鎖のC8-C16アルキル基であり、R1、R1’、R2、R2’に関し、各アルキル基中の水素原子は、水酸基、アルコキシ基によって置換されていてもよく、各アルキル基の全部または一部は4-8員環を形成していてもよく;n及びn’は、0である。
(Y)Xは、R1-CHR2-で表される基であり;X’は、R1’-CHR2’-で表される基であり、ここで、R1、R1’、R2及びR2’は、それぞれ独立に、直鎖のC8-C14アルキル基であり、R1、R1’、R2、R2’に関し、各アルキル基中の水素原子は、水酸基、アルコキシ基によって置換されていてもよく、各アルキル基の全部または一部は4-8員環を形成していてもよく;n及びn’は、1である。
(Z)Xは、R1-CHR2-で表される基であり;X’は、R1’-CHR2’-で表される基であり;ここで、R1及びR1’は同一であって、水素原子またはC1-C21アルキル基であり、R1、R1’に関し、各アルキル基中の水素原子は、水酸基、アルコキシ基によって置換されていてもよく、各アルキル基の全部または一部は4-8員環を形成していてもよく;R2及びR2’は同一であって、水素原子またはC1-C21アルキル基であり、R2、R2’に関し、各アルキル基中の水素原子は、水酸基、アルコキシ基によって置換されていてもよく、各アルキル基の全部または一部は4-8員環を形成していてもよく;また、R1及びR2、R1’及びR2’は、それぞれ互いに連結して4-8員環を形成していてもよく;n及びn’は同一であって、0から3の整数である。
(AA)Xは、R1-CHR2-で表される基であり;X’は、R1’-CHR2’-で表される基であり;R1及びR1’は、同一であって、C1-C21アルキル基であり、各アルキル基中の水素原子は、水酸基、アルコキシ基によって置換されていてもよく;R2及びR2’は、同一であって、C7-C21アルキル基であり、各アルキル基中の水素原子は、水酸基、アルコキシ基によって置換されていてもよく;n及びn’は、同一であって、0または1である。
(BB)Xは、R1-CHR2-で表される基であり;X’は、R1’-CHR2’-で表される基であり;R1及びR1’は、同一であって、C7-C21アルキル基であり、各アルキル基中の水素原子は、水酸基、アルコキシ基によって置換されていてもよく;R2及びR2’は、同一であって、水素原子、または、C1-C21アルキル基であり、各アルキル基中の水素原子は、水酸基、アルコキシ基によって置換されていてもよく;n及びn’は、同一であって、0または1である。
(CC)Xは、R1-CHR2-で表される基であり;X’は、R1’-CHR2’-で表される基であり;R1及びR1’は、同一であって、直鎖のC7-C21アルキル基であり、各アルキル基中の水素原子は、水酸基、アルコキシ基によって置換されていてもよく;R2及びR2’は、同一であって、直鎖のC7-C21アルキル基であり、各アルキル基中の水素原子は、水酸基、アルコキシ基によって置換されていてもよく;n及びn’は、同一であって、0または1である。
(DD)Xは、R1-CHR2-で表される基であり;X’は、R1’-CHR2’-で表される基であり;R1及びR1’は、同一であって、無置換かつ直鎖のC1-C21アルキル基であり;R2及びR2’は、同一であって、水素原子、または、無置換かつ直鎖のC7-C21アルキル基であり;n及びn’は、同一であって、0または1である。(EE)Xは、R1-CHR2-で表される基であり;X’は、R1’-CHR2’-で表される基であり;R1及びR1’は、同一であって、無置換かつ直鎖のC7-C21アルキル基であり;R2及びR2’は、同一であって、水素原子、または、無置換かつ直鎖のC1-C21アルキル基であり;n及びn’は、同一であって、0または1である。
(FF)Xは、R1-CHR2-で表される基であり;X’は、R1’-CHR2’-で表される基であり;R1及びR1’は、同一であって、無置換かつ直鎖のC7-C21アルキル基であり;R2及びR2’は、同一であって、無置換かつ直鎖のC7-C21アルキル基であり;n及びn’は、同一であって、0または1である。
(GG)Xは、R1-CHR2-で表される基であり;X’は、R1’-CHR2’-で表される基であり;R1及びR1’は、同一であって、無置換かつ直鎖のC8-C16アルキル基であり;R2及びR2’は、同一であって、無置換かつ直鎖のC8-C16アルキル基であり;n及びn’は、同一であって、0または1である。
(HH)Xは、R1-CHR2-で表される基であり;X’は、R1’-CHR2’-で表される基であり;R1及びR1’は、同一であって、無置換かつ直鎖のC8-C16アルキル基であり;R2及びR2’は、同一であって、無置換かつ直鎖のC8-C16アルキル基であり;n及びn’は、0である。
(II)Xは、R1-CHR2-で表される基であり;X’は、R1’-CHR2’-で表される基であり;R1及びR1’は、同一であって、無置換かつ直鎖のC9-C14アルキル基であり;R2及びR2’は、同一であって、無置換かつ直鎖のC9-C14アルキル基であり;n及びn’は、1である。
(JJ)Xは、R1-CHR2-で表される基であり;X’は、R1’-CHR2’-で表される基であり;R1、R1’、R2及びR2’は、同一であって、無置換かつ直鎖のC10アルキル基であり;n及びn’は、0である。
(KK)Xは、R1-CHR2-で表される基であり;X’は、R1’-CHR2’-で表される基であり;R1、R1’、R2及びR2’は、同一であって、無置換かつ直鎖のC9、C13、または、C14アルキル基であり;n及びn’は、1である。
6,6’-ビス-O-(2-ノニルウンデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(2-デシルドデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(2-ウンデシルトリデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(2-ドデシルテトラデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(2-トリデシルペンタデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(2-ペンタデシルヘプタデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(2-ヘキサデシルオクタデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(3-ノニルドデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(3-デシルトリデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(3-ウンデシルテトラデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(3-ドデシルペンタデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(3-トリデシルヘキサデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(3-テトラデシルヘプタデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(ベンゾイル)-α,α’-トレハロース、
6,6’-ビス-O-(2-ナフチルカルボニル)-α,α’-トレハロース、
6,6’-ビス-O-(シクロヘキサンカルボニル)-α,α’-トレハロース、
6,6’-ビス-O-(シクロヘプタンカルボニル)-α,α’-トレハロース、
6,6’-ビス-O-(2-テトラデシルオクタデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(14-メトキシ-2-(12-メトキシドデシル)-テトラデカノイル)-α,α’-トレハロース、または、
6,6’-ビス-O-(15-ヒドロキシ-2-(13-ヒドロキシトリデシル)-ペンタデカノイル)-α,α’-トレハロース。
6,6’-ビス-O-(2-ノニルウンデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(2-デシルドデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(2-ウンデシルトリデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(2-ドデシルテトラデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(2-トリデシルペンタデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(2-ペンタデシルヘプタデカノイル)-α,α’-トレハロース、または、
6,6’-ビス-O-(2-ヘキサデシルオクタデカノイル)-α,α’-トレハロース。
6,6’-ビス-O-(3-デシルトリデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(3-ウンデシルテトラデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(3-ドデシルペンタデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(3-トリデシルヘキサデカノイル)-α,α’-トレハロース、または、
6,6’-ビス-O-(3-テトラデシルヘプタデカノイル)-α,α’-トレハロース。
6,6’-ビス-O-(3-ノニルドデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(3-トリデシルヘキサデカノイル)-α,α’-トレハロース、または、
6,6’-ビス-O-(3-テトラデシルヘプタデカノイル)-α,α’-トレハロース。
6,6’-ビス-O-(2-デシルドデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(2-テトラデシルドデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(3-ノニルドデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(3-トリデシルヘキサデカノイル)-α,α’-トレハロース、または、
6,6’-ビス-O-(3-テトラデシルヘプタデカノイル)-α,α’-トレハロースを含有することを特徴とする菌産生毒素中和剤を例示することができる。
<製造方法>
本発明の式(1)で表される化合物は、以下の(a)及び(b)で示される二つの工程により合成することができる。
<合成スキーム1>
式(3)で表されるトレハロース化合物に、式(4)及び式(6)で表されるカルボニル化合物を順次作用させ、トレハロース化合物とカルボニル化合物とのエステル化反応を行う工程である。
上記工程(a)において得られた、トレハロースの6位及び6’位がエステル化され、糖の水酸基が保護された、式(7)で表される化合物に対し、糖の水酸基の脱保護を行うことにより、目的とする、式(1)で表されるトレハロースジエステル化合物を得る。
<合成スキーム2>
上記合成スキーム1、2、または3において、原料化合物である式(4)または式(6)で表されるカルボニル化合物は、市販のものを用いる他、当業者に公知の方法により製造することができる。例えば、式(4)または式(6)において、XないしX’がフェニル基であり、nないしn’が0である化合物としては、安息香酸または安息香酸のハロゲン化物を用いることができる。
<合成スキーム4>
合成スキーム6は、以下の合成スキーム6-1から6-5として説明することができる
。
<合成スキーム6-1>
<合成スキーム6-2>
<合成スキーム6-3>
<合成スキーム6-4>
ることができる。
<合成スキーム6-5>
<合成スキーム7>
ことも可能である。
また、本明細書において用いられる「含む」との用語は、文脈上明らかに異なる理解をすべき場合を除き、記述された事項(部材、ステップ、要素、数字など)が存在することを意図するものであり、それ以外の事項(部材、ステップ、要素、数字など)が存在することを排除しない。
異なる定義が無い限り、ここに用いられるすべての用語(技術用語及び科学用語を含む。)は、本発明が属する技術の当業者によって広く理解されるのと同じ意味を有する。ここに用いられる用語は、異なる定義が明示されていない限り、本明細書及び関連技術分野における意味と整合的な意味を有するものとして解釈されるべきであり、理想化され、又は、過度に形式的な意味において解釈されるべきではない。
本発明の実施態様は模式図を参照しつつ説明される場合があるが、模式図である場合、説明を明確にするために、誇張されて表現されている場合がある。
第一の、第二のなどの用語が種々の要素を表現するために用いられるが、これらの要素はそれらの用語によって限定されるべきではないことが理解される。これらの用語は一つの要素を他の要素と区別するためのみに用いられているのであり、 例えば、第一の要素を第二の要素と記し、同様に、第二の要素は第一の要素と記すことは、本発明の範囲を逸脱することなく可能である。
合成スキーム1で示された式(3)で表される糖の水酸基が保護されたトレハロース化合物と、式(4)または式(6)で表されるそれぞれ所望のカルボニル化合物とのエステル化反応を行い、式(7)で表される化合物を合成した後、糖の水酸基の脱保護を行い、所望のトレハロースジエステル化合物を得た。
製造例A-1
[6,6’-ビス-O-(2-デシルドデカノイル)-2,3,4,2’,3’,4’-ヘキサベンジル-α,α’-トレハロースの合成]
colorless syrup; [α]D 20 +57.7o(c 0.9 CHCl3); FT IR (neat) 3088, 3064, 3031, 2941, 2862, 1946, 1869, 1804, 1741 cm-1 ; 1H NMR (300 MHz in CDCl3) δ0.87 (12H, t, J = 6.9 Hz), 1.14-1.32 (64H, m), 1.43 (4H, m), 1.55 (4H, m), 2.32 (2H, m), 3.54 (2H, dd, J = 9.0, 3.6 Hz), 3.56 (2H, t, J = 9.0 Hz), 4.04 (2H, t, J = 9.0 Hz), 4.10 (2H, m), 4.19 (4H, m), 4.53 (2H, d, J = 10.8 Hz), 4.67 (2H, d, J = 11.7 Hz), 4.72 (2H, d, J = 11.7 Hz), 4.85 (2H, d, J = 10.8 Hz), 4.87 (2H, d, J = 10.8 Hz), 4.99 (2H, d, J = 10.8 Hz), 5.18 (2H, d, J = 3.6 Hz), 7.22-7.37 (30H, m); 13C NMR (75 MHz in CDCl3) δ14.13, 22.69, 27.41, 27.45, 29.33, 29.35, 29.51, 29.53, 29.63, 31.90, 32.32, 45.70, 62.07, 69.16, 73.04, 75.28, 75.71, 77.82, 79.68, 81.55, 93.78, 127.38, 127.61, 127.73, 127.86, 127.92, 128.34, 128.43, 137.79, 137.94, 138.59, 176.16.
[6,6’-ビス-O-(2-オクチルデカノイル)-2,3,4,2’,3’,4’-ヘキサベンジル-α,α’-トレハロースの合成]
colorless syrup; [α]D 16 +62.3o(c 1.0 CHCl3); FT IR (neat) 3088, 3064, 3031, 2927, 2855, 1947, 1868, 1808, 1737 cm-1 ; 1H NMR (300 MHz in CDCl3) δ0.85 (6H, t, J = 6.9 Hz), 0.86(6H, t, J = 6.9 Hz), 1.12-1.34 (48H, m), 1.43 (4H, m), 1.55 (4H, m), 2.32 (2H, m), 3.54 (2H, dd, J = 9.6, 3.6 Hz), 3.57 (2H, t, J = 9.3 Hz), 4.04 (2H, t, J = 9.6 Hz), 4.10 (2H, m), 4.19 (4H, m), 4.53 (2H, d, J = 10.5 Hz),4.67 (2H, d, J = 11.7 Hz), 4.71 (2H, d, J = 11.7 Hz), 4.85 (2H, d, J = 10.5 Hz), 4.87 (2H, d, J = 10.5 Hz), 4.99 (2H, d, J = 10.5 Hz), 5.18 (2H, d, J = 3.6 Hz), 7.22-7.38 (30H, m); 13CNMR (75 MHz in CDCl3) δ14.12, 22.64, 22.67, 27.40, 27.45, 29.27, 29.45, 29.48, 29.62, 31.83, 31.85, 32.34, 45.71, 62.06, 69.15, 73.04, 75.27, 75.71, 77.24, 77.82, 79.68, 81.55, 93.75,127.37, 127.61, 127.72, 127.84, 127.91, 127.93, 128.38, 128.44, 137.80, 137.95, 138.59, 176.15; FABMS m/z (%)1438 (M++Na); HRMS (FAB+) m/z calcd for C90H126O13Na (M++Na) 1437.9096, Found 1437.9126.
[6,6’-ビス-O-(2-ノニルウンデカノイル)-2,3,4,2’,3’,4’-ヘキサベンジル-α,α’-トレハロースの合成]
colorless syrup; [α]D 16 +64.8o(c 1.0 CHCl3); FT IR (neat) 3088, 3064, 3031, 2926, 2854, 1946, 1871, 1806, 1738 cm-1 ; 1H NMR (300 MHz in CDCl3) δ0.86 (6H, t, J = 7.0 Hz), 0.87(6H, t, J = 7.0 Hz), 1.10-1.34 (56H, m), 1.43 (4H, m), 1.55 (4H, m), 2.32 (2H, m), 3.55 (2H, dd, J = 9.6, 3.6 Hz), 3.57 (2H, t, J = 9.3 Hz), 4.04 (2H, t, J = 9.6 Hz), 4.11 (2H, m), 4.19 (4H, m), 4.53 (2H, d, J = 10.2 Hz),4.67 (2H, d, J = 11.7 Hz), 4.72 (2H, d, J = 11.7 Hz), 4.85 (2H, d, J = 10.2 Hz), 4.87 (2H, d, J = 10.5 Hz), 4.99 (2H, d, J = 10.5 Hz), 5.18 (2H, d, J = 3.6 Hz), 7.22-7.37 (30H, m); 13CNMR (75 MHz in CDCl3) δ14.11, 22.67, 27.39, 27.45, 29.28, 29.30, 29.48, 29.51, 29.56, 29.57, 29.61, 31.85, 31.88, 32.33, 45.69, 62.06, 69.15, 73.03, 75.26, 75.70, 77.23, 77.82, 79.68,81.54, 93.75, 127.36, 127.59,127.71, 127.83, 127.90, 127.91, 128.37, 128.42, 137.79, 137.93, 138.58, 176.12;FABMS m/z (%) 1934 (M++Na); HRMS (FAB+) m/z calcd for C94H134O13Na (M++Na) 1493.9722, Found 1493.9701.
[6,6’-ビス-O-(2-ウンデシルトリデカノイル)-2,3,4,2’,3’,4’-ヘキサベンジル-α,α’-トレハロースの合成]
colorless syrup; [α]D 20 +60.0o(c 0.9 CHCl3); FT IR (neat) 3088, 3064, 3031, 2940, 2862, 1946, 1869, 1805, 1740 cm-1 ; 1H NMR (300 MHz in CDCl3) δ0.87 (12H, t, J = 6.9 Hz), 1.14-1.34 (72H, m), 1.43 (4H, m), 1.56 (4H, m), 2.32 (2H, m), 3.54 (2H, dd, J = 10.2, 3.6 Hz), 3.56 (2H, t, J = 8.7 Hz), 4.04 (2H, t, J = 10.2 Hz), 4.12 (2H, m), 4.19 (4H, m), 4.53 (2H, d, J =10.5 Hz), 4.67 (2H, d, J = 12.0Hz), 4.72 (2H, d, J = 12.0 Hz), 4.85 (2H, d, J = 10.8 Hz), 4.88 (2H, d, J = 10.5 Hz), 4.99 (2H, d, J = 10.8 Hz), 5.18 (2H, d, J = 3.6 Hz), 7.22-7.37 (30H, m); 13C NMR (75 MHz in CDCl3)δ14.12, 22.67, 27.40, 27.44, 29.34, 29.50, 29.52, 29.62, 31.90, 32.30, 45.68, 62.05, 69.14,73.03, 75.25, 75.69, 77.80, 79.66, 81.52,93.76, 127.35, 127.58, 127.70, 127.82, 127.90, 128.36, 128.40, 137.76, 137.92, 138.57, 176.12.
[6,6’-ビス-O-(2-ドデシルテトラデカノイル)-2,3,4,2’,3’,4’-ヘキサベンジル-α,α’-トレハロースの合成]
colorless syrup; [α]D 20 +60.8o(c 1.0 CHCl3); FT IR (neat) 3088, 3064, 3031, 2940, 2862, 1946, 1869, 1804, 1739 cm-1 ; 1H NMR (300 MHz in CDCl3) δ0.88 (12H, t, J = 6.9 Hz), 1.14-1.34 (80H, m), 1.43 (4H, m), 1.56 (4H, m), 2.32 (2H, m), 3.54 (2H, dd, J = 9.6, 3.6 Hz), 3.57 (2H, t, J = 8.4 Hz), 4.04 (2H, t, J = 9.6 Hz), 4.12 (2H, m), 4.19 (4H, m), 4.53 (2H, d, J = 10.5 Hz), 4.67 (2H, d, J = 12.0 Hz), 4.72 (2H, d, J = 12.0 Hz), 4.86 (2H, d, J = 10.8 Hz), 4.87 (2H, d, J = 10.5 Hz), 4.99 (2H, d, J = 10.8 Hz), 5.18 (2H, d, J = 3.6 Hz), 7.24-7.37 (30H, m); 13C NMR (75 MHz in CDCl3) δ14.12, 22.69, 27.41, 27.46, 29.36, 29.53, 29.66, 31.92, 32.32, 45.70, 62.09, 69.19, 73.07, 75.27, 75.71, 77.86, 79.72, 81.56, 93.78, 127.71, 127.62, 127.74, 127.86, 127.94, 128.40, 128.45, 137.83, 138.00, 138.64, 176.17.
[6,6’-ビス-O-(2-トリデシルペンタデカノイル)-2,3,4,2’,3’,4’-ヘキサベンジル-α,α’-トレハロースの合成]
colorless syrup; [α]D 20 +50.2o(c 1.1 CHCl3); FT IR (neat) 3088, 3064, 3031, 2929, 2855, 1945, 1868, 1804, 1739 cm-1 ; 1H NMR (300 MHz in CDCl3) δ0.88 (12H, t, J = 6.6 Hz), 1.10-1.34 (88H, m), 1.44 (4H, m), 1.56 (4H, m), 2.32 (2H, m), 3.54 (2H, dd, J = 9.8, 3.6 Hz), 3.58 (2H, t, J = 9.8 Hz), 4.04 (2H, t, J = 9.8 Hz), 4.13 (2H, m), 4.19 (4H, m), 4.53 (2H, d, J = 10.8 Hz), 4.66 (2H, d, J = 12.0 Hz), 4.73 (2H, d, J = 12.0 Hz), 4.85 (2H, d, J = 11.0 Hz), 4.87 (2H, d, J = 10.8 Hz), 4.99 (2H, d, J = 11.0 Hz), 5.18 (2H, d, J = 3.6 Hz), 7.20-7.38 (30H, m); 13C NMR (75 MHz in CDCl3) δ14.11, 22.70, 27.42, 27.46, 29.37, 29.54, 29.66, 31.93, 32.33, 45.72, 62.12, 69.23, 73.10, 75.26, 75.69, 77.23, 77.67, 77.91, 79.77, 81.58, 93.77, 127.42, 127.61, 127.74, 127.85, 127.94, 128.40, 128.44, 128.46, 137.86, 138.05, 138.68, 176.15.
[6,6’-ビス-O-(2-ペンタデシルヘプタデカノイル)-2,3,4,2’,3’,4’-ヘキサベンジル-α,α’-トレハロースの合成]
colorless syrup; [α]D 20 +44.3o(c 1.0 CHCl3); FT IR (neat) 3088, 3064, 3031, 2941, 2861, 1945, 1868, 1812, 1739 cm-1 ; 1H NMR (300 MHz in CDCl3) δ0.88 (12H, t, J = 7.2 Hz), 1.12-1.38 (104H, m), 1.43 (4H, m), 1.56 (4H, m), 2.32 (2H, m), 3.54 (2H, dd, J = 9.6, 3.6 Hz), 3.57 (2H, t, J = 8.7 Hz), 4.04 (2H, t, J = 9.6 Hz), 4.12 (2H, m), 4.20 (4H, m), 4.53 (2H, d, J = 10.5 Hz), 4.67 (2H, d, J = 12.0 Hz), 4.72 (2H, d, J = 12.0 Hz), 4.86 (2H, d, J = 10.8 Hz), 4.87 (2H, d, J = 10.8Hz), 4.99 (2H, d, J = 10.5 Hz), 5.18 (2H, d, J = 3.6 Hz), 7.22-7.37 (30H, m); 13C NMR (75 MHz in CDCl3) δ14.14, 22.70, 27.25, 27.42, 27.45, 29.38, 29.55, 29.68, 29.72, 31.94, 32.32, 45.70, 62.07, 69.16, 73.05, 75.29, 75.72, 77.82, 79.68, 81.55, 93.78, 127.40, 127.62, 127.75,127.95, 128.41, 128.45, 137.81, 137.95, 138.61, 176.19.
[6,6’-ビス-O-(2-ヘキサデシルオクタデカノイル)-2,3,4,2’,3’,4’-ヘキサベンジル-α,α’-トレハロースの合成]
colorless syrup; [α]D 20 +48.8o(c 1.0 CHCl3); FT IR (neat) 3088, 3064, 3031, 2938, 2857, 1944, 1869, 1808, 1739 cm-1 ; 1H NMR (300 MHz in CDCl3) δ0.88 (12H, t, J = 6.9 Hz), 1.10-1.36 (112H, m), 1.43 (4H, m), 1.56 (4H, m), 2.32 (2H, m), 3.55 (2H, dd, J = 9.3, 3.6 Hz), 3.57 (2H, t, J = 9.0 Hz), 4.04 (2H, t, J = 9.3 Hz), 4.11 (2H, m), 4.19 (4H, m), 4.53 (2H, d, J = 10.5 Hz), 4.67 (2H, d, J = 12.0 Hz), 4.72 (2H, d, J = 12.0 Hz), 4.86 (2H, d, J =10.8 Hz), 4.87 (2H, d, J = 10.5Hz), 4.99 (2H, d, J = 10.8 Hz), 5.18 (2H, d, J = 3.6 Hz), 7.22-7.37 (30H, m); 13C NMR (75 MHz in CDCl3) δ14.15, 22.71, 27.43, 29.39, 29.56, 29.69, 29.73, 31.94, 32.31, 45.70, 62.08, 69.17, 73.05, 75.31, 75.74, 77.83, 79.68, 81.56, 93.81, 127.41, 127.64, 127.76, 127.96, 128.42, 128.47, 137.83, 138.00, 138.62, 176.22.
[6,6’-ビス-O-(2-デシルドデカノイル)-α,α’-トレハロースの合成]
colorless syrup; [α]D 20 +61.8o(c 1.0 CHCl3); FT IR (neat) 3358, 2940, 2861, 1746 cm-1 ; 1H NMR (300 MHz in C5D5N) δ0.88 (12H, t, J = 6.9 Hz), 1.25 (56H, m),1.45 (8H, m), 1.58 (4H, m), 1.83 (4H, m), 2.58 (2H, m), 4.18 (2H, t, J = 9.3 Hz), 4.29 (2H, dd, J = 9.3, 3.6 Hz), 4.73 (2H, t, J = 9.3 Hz), 4.88 (2H, dd, J = 11.7, 5.1 Hz), 5.07 (4H, m), 5.87 (2H, d, J = 3.6 Hz); 13C NMR (75 MHz in C5D5N) δ14.29, 22.95, 27.75, 27.80, 29.62, 29.80, 29.90, 29.95, 32.14, 32.82, 46.16, 63.93, 71.54, 71.94, 73.31, 74.81, 95.69, 176.26; FABMS m/z (%) 1010 (M++Na); HRMS (FAB+) m/z calcd for C56H106O13Na (M++Na) 1009.7532, Found 1009.7498.
[6,6’-ビス-O-(2-オクチルデカノイル)-α,α’-トレハロースの合成]
[6,6’-ビス-O-(2-ノニルウンデカノイル)-α,α’-トレハロースの合成]
colorless syrup; [α]D 14+64.9o (c 0.6 CHCl3); FT IR (neat) 3306, 2928, 2855, 1742 cm-1 ; 1H NMR (300 MHz in C5D5N) δ0.87 (12H, t, J = 6.9 Hz), 1.22 (48H, m), 1.42 (8H, m), 1.54 (4H, m), 1.79 (4H, m), 2.56 (2H, m), 4.19 (2H, t, J = 9.0 Hz),4.39 (2H, dd, J = 9.0, 3.6 Hz), 4.74 (2H, t, J = 9.0 Hz), 4.88 (2H, dd, J = 11.7, 4.8 Hz), 5.08 (4H, m), 5.88 (2H, d, J = 3.6 Hz); 13C NMR (75 MHz in C5D5N) δ13.69, 22.32, 27.14, 27.20, 28.97, 29.17, 29.27, 29.34, 31.51, 32.22, 45.55, 63.28, 70.94, 71.31, 72.69, 74.18, 95.13, 175.67; FABMS m/z (%) 954 (M++Na); HRMS (FAB+) m/z calcd for C52H98O13Na (M++Na) 953.6905,Found 953.6862.
実施例4:製造例α-4
[6,6’-ビス-O-(2-ウンデシルトリデカノイル)-α,α’-トレハロースの
合成]
colorless syrup; [α]D 14 +58.5o(c 1.0 CHCl3); FT IR (neat) 3297, 2934, 2856, 1742 cm-1 ; 1H NMR (300 MHz in C5D5N) δ0.88 (12H, t, J = 6.6 Hz), 1.26 (64H, m),1.45 (8H, m), 1.58 (4H, m), 1.82 (4H, m), 2.58 (2H, m), 4.18 (2H, t, J = 9.0 Hz), 4.29 (2H, dd, J = 9.0, 3.6 Hz), 4.73 (2H, t, J = 9.0 Hz), 4.88 (2H, dd, J = 11.7, 5.1 Hz), 5.07 (4H, m), 5.88 (2H, d, J = 3.6 Hz); 13C NMR (75 MHz in C5D5N) δ14.29, 22.94, 27.75, 27.80, 29.63, 29.81, 29.96, 32.14, 32.81, 46.15, 63.93, 71.54, 71.94, 73.31, 74.81, 95.67, 176.25; FABMS m/z (%) 1066 (M++Na); HRMS (FAB+) m/z calcd for C60H114O13Na (M++Na) 1065.8157, Found 1065.8160.
[6,6’-ビス-O-(2-ドデシルテトラデカノイル)-α,α’-トレハロースの合成]
colorless syrup; [α]D 14 +54.6o(c 1.0 CHCl3); FT IR (neat) 3310, 2937, 2857, 1742 cm-1 ; 1H NMR (300 MHz in C5D5N) δ0.88 (12H, t, J = 6.9 Hz), 1.28 (72H, m),1.46 (8H, m), 1.58 (4H, m), 1.82 (4H, m), 2.59 (2H, m), 4.18 (2H, t, J = 9.0 Hz), 4.29 (2H, dd, J = 9.0, 3.6 Hz), 4.73 (2H, t, J = 9.0 Hz), 4.88 (2H, dd, J = 11.7, 5.1 Hz), 5.08 (4H, m), 5.87 (2H, d, J = 3.6 Hz); 13C NMR (75 MHz in C5D5N) δ14.30, 22.97, 27.77, 27.82, 29.66, 29.83, 29.99, 32.17, 32.82, 46.16, 63.94, 71.53, 71.95, 73.31, 74.80, 95.66, 176.24; FABMS m/z (%) 1122 (M++Na); HRMS (FAB+) m/z calcd for C64H122O13Na (M++Na) 1121.8784, Found 1121.8831.
[6,6’-ビス-O-(2-トリデシルペンタデカノイル)-α,α’-トレハロースの合成]
colorless syrup; [α]D 19 +52.7o(c 0.6 CHCl3); FT IR (neat) 3313, 2927, 2854, 1741 cm-1 ; 1H NMR (300 MHz in C5D5N) δ0.88 (12H, t, J = 6.9 Hz), 1.29 (80H, m),1.46 (8H, m), 1.58 (4H, m), 1.83 (4H, m), 2.59 (2H, m), 4.19 (2H, t, J = 9.6 Hz), 4.29 (2H, dd, J = 9.6, 3.6 Hz), 4.74 (2H, t, J = 9.6 Hz), 4.89 (2H, dd, J = 11.7, 5.1 Hz), 5.08 (4H, m), 5.88 (2H, d, J = 3.6 Hz); 13C NMR (75 MHz in C5D5N) δ14.28, 22.94, 27.75, 27.80, 29.63, 29.82, 29.98, 32.14, 32.80, 46.14, 63.92, 71.53, 71.94, 73.30, 74.79, 95.66, 176.21; FABMS m/z (%) 1178 (M++Na); HRMS (FAB+) m/z calcd for C68H130O13Na (M++Na) 1177.9409, Found 1177.9404.
[6,6’-ビス-O-(2-ペンタデシルヘプタデカノイル)-α,α’-トレハロースの合成]
colorless syrup; [α]D 19 +44.8o(c 0.5 CHCl3); FT IR (neat) 3330, 2925, 2853, 1741 cm-1 ; 1H NMR (300 MHz in C5D5N) δ0.87 (12H, t, J = 7.2 Hz), 1.31 (96H, m),1.47 (8H, m), 1.58 (4H, m), 1.83 (4H, m), 2.59 (2H, m), 4.19 (2H, t, J = 9.0 Hz), 4.29 (2H, dd, J = 9.0, 3.6 Hz), 4.74 (2H, t, J = 9.0 Hz), 4.88 (2H, dd, J = 11.4, 4.8 Hz), 5.08 (4H, m), 5.87 (2H, d, J = 3.6 Hz); 13C NMR (75 MHz in C5D5N) δ14.23, 22.89, 27.73, 27.79, 29.58, 29.80, 29.91, 29.97, 32.09, 32.78, 46.13, 63.92, 71.55, 71.95, 73.32, 74.80, 95.67, 176.21; FABMS m/z (%) 1290 (M++Na); HRMS (FAB+) m/z calcd for C76H146O13Na (M++Na) 1290.0661, Found 1290.0677.
[6,6’-ビス-O-(2-ヘキサデシルオクタデカノイル)-α,α’-トレハロースの合成]
colorless syrup; [α]D 17 +45.1o(c 0.5 CHCl3); FT IR (neat) 3308, 2937, 2856, 1741 cm-1 ; 1H NMR (300 MHz in C5D5N) δ0.87 (12H, t, J = 6.9 Hz), 1.31 (104H, m), 1.46 (8H, m), 1.57 (4H, m), 1.80 (4H, m), 2.56 (2H, m), 4.19 (2H, t, J = 9.3 Hz), 4.29 (2H, dd, J = 9.3, 3.6 Hz), 4.74 (2H, t, J = 9.3 Hz), 4.88 (2H, dd, J = 11.4, 4.8 Hz), 5.08 (4H, m), 5.87 (2H, d, J = 3.6 Hz); 13C NMR (75 MHz in C5D5N)δ14.25, 22.90, 27.75, 27.80, 29.60, 29.82, 29.91, 29.99, 32.10, 32.80, 46.14, 63.91, 71.55,71.95, 73.32, 74.80, 95.66, 176.24; FABMS m/z (%) 1346 (M++Na); HRMS (FAB+) m/z calcd for C80H154O13Na (M++Na) 1346.1288, Found 1346.1287.
[6,6’-ビス-O-(3-ノニルドデカノイル)-2,3,4,2’,3’,4’-ヘキサベンジル-α,α’-トレハロースの合成]
colorless syrup; [α]D 21 +65.3o(c 1.0 CHCl3); FT IR (neat) 3088, 3063, 3031, 2925, 2853, 1944, 1871, 1806, 1739 cm-1 ; 1H NMR (300 MHz in CDCl3) δ0.87 (12H, t, J = 5.1 Hz), 1.20 (64H, m), 1.81 (2H, m), 2.20 (4H, d, J = 6.9 Hz), 3.54 (2H,t, J = 9.3 Hz), 3.56 (2H, m), 4.04 (2H, t, J = 9.3 Hz), 4.09 (4H, m), 4.23 (2H,m), 4.51 (2H, d, J = 10.5 Hz), 4.67 (2H, d, J = 12.0 Hz), 4.72 (2H, d, J = 12.0Hz), 4.86 (4H, d, J = 10.5 Hz), 5.00 (2H, d, J = 10.5 Hz), 5.17 (2H, d, J = 3.6Hz), 7.23-7.37 (30H, m); 13C NMR (75 MHz in CDCl3) δ14.13, 22.68, 26.51, 29.33, 29.56, 29.64, 29.92, 31.89, 33.65, 33.76, 34.89, 39.08, 62.35, 69.12, 72.94, 75.30, 75.70, 77.60, 79.38, 81.56, 94.02, 127.44, 127.63, 127.78, 127.92, 128.09,128.41, 128.47, 137.78, 137.84, 138.60, 173.24; FABMS m/z (%) 1522 (M++Na); HRMS (FAB+) m/z calcd for C96H138O13Na (M++Na) 1522.0036, Found 1522.0020.
[6,6’-ビス-O-(3-オクチルウンデカノイル)-2,3,4,2’,3’,4’-ヘキサベンジル-α,α’-トレハロースの合成]
colorless syrup; [α]D 20 +41.8o(c 2.0 CHCl3); FT IR (neat) 3088, 3064, 3031, 2932, 2855, 1947, 1867, 1806, 1739 cm-1 ; 1H NMR (300 MHz in CDCl3) δ0.86 (6H, t, J = 6.9 Hz), δ0.87 (6H, t, J = 6.9 Hz), 1.20 (56H, m), 1.81 (2H, m), 2.20 (4H, d, J = 6.9 Hz), 3.54 (2H, t, J = 8.4 Hz), 3.56 (2H, m), 4.04 (2H, t, J = 9.3 Hz), 4.09 (4H, m), 4.23 (2H, m), 4.51 (2H, d, J =10.5 Hz), 4.67 (2H, d, J = 12.0Hz), 4.72 (2H, d, J = 12.0 Hz), 4.86 (4H, d, J = 10.5 Hz), 5.00 (2H, d, J = 10.5 Hz), 5.17 (2H, d, J = 3.6 Hz), 7.23-7.37 (30H, m); 13C NMR (75 MHz in CDCl3) δ14.14, 22.69, 26.52, 29.32, 29.61, 29.94, 31.89, 33.69, 33.80, 34.91, 39.10, 62.38, 69.14,72.96, 75.29, 75.70, 77.62, 79.40, 81.58, 93.95, 94.04, 127.44, 127.62, 127.77, 127.91, 128.07, 128.41, 128.46, 137.78, 137.85, 138.59, 173.23; FABMS m/z (%) 1466 (M++Na); HRMS (FAB+) m/z calcd for C92H130O13Na (M++Na) 1465.9409, Found 1465.9392.
[6,6’-ビス-O-(3-デシルトリデカノイル)-2,3,4,2’,3’,4’-ヘキサベンジル-α,α’-トレハロースの合成]
colorless syrup; [α]D 20 +53.6o(c 1.0 CHCl3); FT IR (neat) 3088, 3063, 3030, 2926, 2854, 1946, 1874, 1804, 1739 cm-1 ; 1H NMR (300 MHz in CDCl3) δ0.87 (12H, t, J = 6.9 Hz), 1.21 (72H, m), 1.81 (2H, m), 2.19 (4H, d, J = 6.9 Hz), 3.54 (2H,t, J = 8.4 Hz), 3.56 (2H, m), 4.04 (2H, t, J = 8.4 Hz), 4.11 (4H, m), 4.21 (2H,m), 4.51 (2H, d, J = 10.5 Hz), 4.67 (2H, d, J = 12.0 Hz), 4.72 (2H, d, J = 12.0Hz), 4.86 (4H, d, J = 10.5 Hz), 5.00 (2H, d, J = 10.5 Hz), 5.17 (2H, d, J = 3.6Hz), 7.23-7.36 (30H, m); 13C NMR (75 MHz in CDCl3) δ14.14, 22.70, 26.51, 29.36, 29.66, 29.95, 31.93, 33.67, 77.23, 77.62, 79.40, 81.58, 94.04, 127.46, 127.65,127.80, 127.93, 128.10, 128.43, 128.49, 137.80, 137.87, 138.62, 173.27; FABMS m/z (%) 1579 (M++H+Na); HRMS (FAB+) m/z calcd for C100H147O13Na (M++H+Na) 1579.0787, Found 1579.0763.
[6,6’-ビス-O-(3-ウンデシルテトラデカノイル)-2,3,4,2’,3’,4’-ヘキサベンジル-α,α’-トレハロースの合成]
colorless syrup; [α]D 20 +58.1o(c 1.0 CHCl3); FT IR (neat) 3088, 3064, 3031, 2926, 2854, 1946, 1867, 1806, 1739 cm-1 ; 1H NMR (300 MHz in CDCl3) δ0.88 (12H, t, J = 7.2 Hz), 1.21 (80H, m), 1.81 (2H, m), 2.19 (4H, d, J = 6.9 Hz), 3.54 (2H,t, J = 8.4 Hz), 3.55 (2H, m), 4.04 (2H, t, J = 6,9 Hz), 4.10 (4H, m), 4.20 (2H,m), 4.51 (2H, d, J = 10.5 Hz), 4.67 (2H, d, J = 12.0 Hz), 4.71 (2H, d, J = 12.0Hz), 4.86 (4H, d, J = 10.5 Hz), 5.00 (2H, d, J = 10.5 Hz), 5.17 (2H, d, J = 3.6Hz), 7.19-7.36 (30H, m); 13C NMR (75 MHz in CDCl3) δ14.14, 22.71, 26.53, 29.38, 29.67, 29.95, 31.93, 33.68, 33.79, 34.91, 39.10, 62.37, 69.14, 72.96, 75.31, 75.71, 77.63, 79.40, 81.58, 94.04, 127.46, 127.64, 127.80, 127.93, 128.10, 128.43, 128.49, 137.80, 137.87, 138.62, 173.27; FABMS m/z (%) 1635 (M++Na); HRMS (FAB+) m/z calcd for C104H154O13Na (M++Na) 1634.1288, Found 1634.1298.
[6,6’-ビス-O-(3-ドデシルペンタデカノイル)-2,3,4,2’,3’,
4’-ヘキサベンジル-α,α’-トレハロースの合成]
colorless syrup; [α]D 20 +68.9o(c 0.9 CHCl3); FT IR (neat) 3088, 3063, 3031, 2925, 2853, 1944, 1867, 1806, 1739 cm-1 ; 1H NMR (300 MHz in CDCl3) δ0.88 (12H, t, J = 6.9 Hz), 1.21 (88H, m), 1.81 (2H, m), 2.20 (4H, d, J = 6.9 Hz), 3.54 (2H,t, J = 8.7 Hz), 3.57 (4H, m), 4.05 (2H, t, J = 8.7 Hz), 4.05 (2H, t, J = 8.7 Hz), 4.11 (2H, m), 4.21 (2H, m), 4.52 (2H, d, J = 10.5 Hz), 4.67 (2H, d, J = 12.3 Hz), 4.72 (2H, d, J = 12.3 Hz), 4.86 (4H, d, J = 10.5 Hz), 5.01 (2H, d, J = 10.5Hz), 5.18 (2H, d, J = 3.3 Hz), 7.21-7.37 (30H, m); 13C NMR (75 MHz in CDCl3) δ14.14, 22.70,26.53, 29.38, 29.67, 29.95, 31.92, 33.67, 33.78, 34.91, 39.10, 62.37, 69.14, 72.96, 75.30, 75.71, 77.63, 79.40, 81.57, 94.03, 127.45, 127.63, 127.79, 127.93, 128.09, 128.42, 128.48, 137.79, 137.87, 138.62, 173.26; FABMS m/z (%) 1691 (M++H+Na); HRMS (FAB+) m/z calcd for C108H163O13Na (M++H+Na) 1691.1993, Found 1691.1992.
[6,6’-ビス-O-(3-トリデシルヘキサデカノイル)-2,3,4,2’,3’
,4’-ヘキサベンジル-α,α’-トレハロースの合成]
colorless syrup; [α]D 20 +49.5o(c 0.9 CHCl3); FT IR (neat) 3088, 3064, 3031, 2925, 2853, 1944, 1871, 1806, 1739 cm-1 ; 1H NMR (300 MHz in CDCl3) δ0.88 (12H, t, J = 6.9 Hz), 1.23 (96H, m), 1.81 (2H, m), 2.20 (4H, d, J = 6.9 Hz), 3.54 (4H,m), 4.04 (2H, t, J = 9.3 Hz), 4.11 (4H, m), 4.21 (2H, m), 4.51 (2H, d, J = 10.8Hz), 4.67 (2H, d, J = 12.0 Hz), 4.72 (2H, d, J = 12.0 Hz), 4.86 (4H, d, J = 10.8 Hz), 5.00 (2H, d, J = 10.8 Hz), 5.17 (2H, d, J = 3.6 Hz), 7.23-7.37 (30H, m); 13C NMR (75 MHz in CDCl3)δ14.16, 22.72, 26.54, 29.40, 29.69, 29.72, 29.97, 31.95, 33.68, 33.79, 34.92, 39.11, 62.37,69.14, 72.96, 75.33, 75.72, 77.24, 77.62,79.40, 81.59, 94.07, 127.46, 127.66, 127.81, 127.94, 128.11, 128.44, 128.50, 137.80, 137.87,138.63, 173.28; FABMS m/z (%) 1691 (M++H+Na); HRMS (FAB+) m/z calcd for C112H171O13Na (M++H+Na) 1747.2619, Found 1747.2618.
[6,6’-ビス-O-(3-テトラデシルヘプタデカノイル)-2,3,4,2’,3’,4’-ヘキサベンジル-α,α’-トレハロースの合成]
colorless syrup; [α]D 20 +43.7o(c 1.0 CHCl3); FT IR (neat) 3087, 3064, 3032, 2924, 2853, 1943, 1871, 1796, 1739 cm-1 ; 1H NMR (300 MHz in CDCl3) δ0.88 (12H, t, J = 6.9 Hz), 1.23 (104H, m), 1.80 (2H, m), 2.19 (4H, d, J = 6.9 Hz), 3.54 (4H, m), 4.04 (2H, t, J = 9.6 Hz), 4.11(4H, m), 4.21 (2H, m), 4.51 (2H, d, J = 10.5 Hz), 4.67 (2H, d, J = 12.0 Hz), 4.72 (2H, d, J = 12.0 Hz), 4.86 (4H, d, J = 10.5 Hz), 5.00 (2H, d, J = 10.5 Hz), 5.17 (2H, d, J = 3.3 Hz), 7.22-7.37 (30H, m);13C NMR (75 MHz in CDCl3) δ14.16, 22.72, 26.55, 29.39, 29.70, 29.73, 29.97, 31.95, 33.68, 33.79, 34.92, 39.11, 62.38, 69.15, 72.97, 75.32, 75.72, 77.23, 77.63, 79.41, 81.59, 94.06, 127.46, 127.65, 127.81, 127.94, 128.11, 128.44, 128.49, 137.81, 137.88, 138.63, 173.27; FABMS m/z (%) 1802 (M++Na); HRMS (FAB+) m/z calcdfor C116H178O13Na (M++Na) 1802.3185, Found 1802.3175.
[6,6’-ビス-O-(3-ノニルドデカノイル)-α,α’-トレハロースの合成]
colorless syrup; [α]D 21 +62.8o(c 0.7 CHCl3); FT IR (neat) 3316, 2926, 2854, 1743 cm-1 ; 1H NMR (300 MHz in C5D5N) δ0.81 (12H, t, J = 6.9 Hz), 1.23 (64H, m),1.99 (2H, m), 2.33 (4H, d, J = 6.6 Hz), 4.13 (2H, t, J = 9.6 Hz), 4.25 (2H, dd,J = 9.6, 3.9 Hz), 4.74 (2H, t, J = 9.6 Hz), 4.78 (2H, d, J = 12.3 Hz), 4.93 (2H, d, J = 12.3 Hz), 4.98 (2H, m), 5.81 (2H, d, J = 3.9 Hz); 13C NMR (75 MHz in C5D5N) δ14.29, 22.94, 29.84, 29.62, 29.91, 29.94, 30.22, 32.12, 34.10, 35.23, 39.33, 64.26, 71.48, 71.96,73.35, 74.82, 95.82, 173.51; FABMS m/z (%) 982 (M++Na);HRMS (FAB+) m/z calcd for C54H102O13Na (M++Na) 981.7218, Found 981.7198.
[6,6’-ビス-O-(3-オクチルウンデカノイル)-α,α’-トレハロースの合成]
colorless syrup; [α]D 21 +70.7o(c 0.4 CHCl3); FT IR (neat) 3275, 2925, 2854, 1742 cm-1 ; 1H NMR (300 MHz in C5D5N) δ0.86 (12H, t, J = 7.2 Hz), 1.24 (56H, m),2.03 (2H, m), 2.37 (4H, d, J = 6.6 Hz), 4.19 (2H, t, J = 9.3 Hz), 4.31 (2H, dd,J = 9.6, 3.6 Hz), 4.74 (2H, t, J = 9.3 Hz), 4.85 (2H, dd, J = 11.7, 5.7 Hz), 5.01 (2H, d, J = 11.7 Hz), 5.11 (2H, m), 5.89 (2H, d, J = 3.9 Hz); 13C NMR (75 MHzin C5D5N) δ14.28, 22.93, 26.83, 26.87, 29.59, 29.87, 30.21, 32.11, 34.10, 35.23, 39.32, 64.26, 71.48, 71.95, 73.35, 74.83, 95.82, 173.52; FABMS m/z (%) 926 (M++Na); HRMS (FAB+) m/z calcd for C50H94O13Na (M++Na) 925.6592, Found 925.6585.
[6,6’-ビス-O-(3-デシルトリデカノイル)-α,α’-トレハロースの合成]
colorless syrup; [α]D 21 +60.9o(c 0.9 CHCl3); FT IR (neat) 3279, 2924, 2854, 1742 cm-1 ; 1H NMR (300 MHz in C5D5N) δ0.83 (12H, t, J = 6.9 Hz), 1.24 (72H, m),2.00 (2H, m), 2.35 (4H, d, J = 6.6 Hz), 4.14 (2H, t, J = 8.7 Hz), 4.26 (2H, dd,J = 9.6, 3.3 Hz), 4.74 (2H, t, J = 9.0 Hz), 4.78 (2H, dd, J = 11.7, 5.4 Hz), 4.95 (2H, d, J = 12.0 Hz), 5.01 (2H, m), 5.82 (2H, d, J = 3.9 Hz); 13C NMR (75 MHzin C5D5N) δ14.10, 22.74, 26.65, 29.41, 29.73, 30.00, 31.92, 33.87, 35.03, 39.16, 64.10, 71.10, 71.57, 72.92, 74.38, 95.14, 173.52; FABMS m/z (%) 1037 (M++Na);HRMS (FAB+) m/z calcd for C58H110O13Na (M++Na) 1037.7903, Found 1037.7874.
[6,6’-ビス-O-(3-ウンデシルテトラデカノイル)-α,α’-トレハロースの合成]
colorless syrup; [α]D 21 +60.6o(c 0.5 CHCl3); FT IR (neat) 3289, 2925, 2853, 1743 cm-1 ; 1H NMR (300 MHz in C5D5N) δ0.86 (12H, t, J = 6.9 Hz), 1.28 (80H, m),2.04 (2H, m), 2.38 (4H, d, J = 6.6 Hz), 4.19 (2H, t, J = 9.0 Hz), 4.31 (2H, dd,J = 9.0, 3.6 Hz), 4.74 (2H, t, J = 9.0 Hz), 4.85 (2H, dd, J = 11.7, 5.1 Hz), 5.05 (2H, d, J = 11.7 Hz), 5.01 (2H, m), 5.89 (2H, d, J = 3.9 Hz); 13C NMR (75 MHzin C5D5N) δ14.30, 22.96, 26.92, 29.64, 30.00, 30.27, 32.14, 34.14, 35.26, 39.37, 64.30, 71.52, 72.00, 73.40, 74.87, 95.86, 173.53; FABMS m/z (%) 1094 (M++Na);HRMS (FAB+) m/z calcd for C62H118O13Na (M++Na) 1093.8470, Found 1093.8458.
[6,6’-ビス-O-(3-ドデシルペンタデカノイル)-α,α’-トレハロースの
合成]
colorless syrup; [α]D 21+52.5o (c 0.3 CHCl3); FT IR (neat) 3271, 2923, 2853, 1743 cm-1 ; 1H NMR (300 MHz in C5D5N) δ0.84 (12H, t, J = 6.3 Hz), 1.25 (88H, m), 2.02 (2H, m),2.36 (4H, d, J = 6.6 Hz), 4.15 (2H, t, J = 9.0 Hz), 4.27 (2H, dd, J= 9.9, 3.6 Hz), 4.74 (2H, t, J = 9.6 Hz), 4.81 (2H, dd, J = 11.4, 4.8 Hz), 4.97(2H, d, J = 11.4 Hz), 5.02 (2H, m), 5.84 (2H, d, J = 2.4 Hz); 13C NMR (75 MHz in C5D5N) δ14.18, 22.82, 26.73, 29.50, 29.87, 30.12, 32.01, 33.97, 35.12, 39.25,64.18, 71.25, 71.71, 73.06, 74.51, 95.38, 173.52; FABMS m/z (%) 1150 (M++Na); HRMS (FAB+) m/z calcd for C66H126O13Na (M++Na) 1149.9110, Found 1149.9103.
[6,6’-ビス-O-(3-トリデシルヘキサデカノイル)-α,α’-トレハロースの合成]
colorless syrup; [α]D 21 +42.3o(c 0.5 CHCl3); FT IR (neat) 3321, 2925, 2853, 1742 cm-1 ; 1H NMR (300 MHz in C5D5N) δ0.83 (12H, t, J = 6.6 Hz), 1.27 (96H, m),2.02 (2H, m), 2.36 (4H, d, J = 6.9 Hz), 4.15 (2H, t, J = 9.3 Hz), 4.27 (2H, dd,J = 9.3, 3.3 Hz), 4.75 (2H, t, J = 9.3 Hz), 4.80 (2H, dd, J = 12.0, 5.4 Hz), 4.97 (2H, d, J = 12.0 Hz), 5.02 (2H, m), 5.84 (2H, d, J = 3.6 Hz); 13C NMR (75 MHzin C5D5N) δ14.30, 22.96, 26.91, 29.65, 30.03, 30.28, 32.15, 34.12, 35.25, 39.36, 64.28, 71.51, 71.99, 73.38, 74.85, 95.83, 173.51; FABMS m/z (%) 1206 (M++Na);HRMS (FAB+) m/z calcd for C70H134O13Na (M++Na) 1205.9770, Found 1205.9746.
[6,6’-ビス-O-(3-テトラデシルヘプタデカノイル)-α,α’-トレハロー
スの合成]
colorless syrup; [α]D 21 +55.7o(c 1.0 CHCl3); FT IR (neat) 3310, 2925, 2853, 1743 cm-1 ; 1H NMR (300 MHz in C5D5N) δ0.84 (12H, t, J = 6.9 Hz), 1.26 (104H, m), 2.03 (2H, m), 2.37 (4H, d, J = 6.6 Hz), 4.15 (2H, t, J = 9.9 Hz), 4.28 (2H, dd, J = 9.6, 3.9 Hz), 4.74 (2H, t, J =9.9 Hz), 4.81 (2H, dd, J = 11.7, 5.4 Hz), 4.97 (2H, d, J = 11.7 Hz), 5.03 (2H, m), 5.84 (2H,d, J = 3.6 Hz); 13C NMR (75 MHz in C5D5N) δ14.05, 22.67, 26.55, 29.36, 29.67, 29.74, 29.94,31.87, 33.74, 34.95, 39.14, 64.02, 70.91, 71.46, 72.77, 74.20, 94.69, 173.45; FABMS m/z (%) 1262 (M++Na); HRMS (FAB+) m/z calcd for C74H142O13Na (M++Na) 1262.0348, Found 1262.0348.
製造例C-1
[2-デシルドデカン酸の合成]
white powder; FT IR (neat) 3041, 2943, 2857, 2689, 1714 cm-1 ; 1H NMR (300 MHzin CDCl3) δ0.88 (6H, t, J = 7.6Hz), 1.21 (32H, m), 1.48 (2H, m), 1.61 (2H, m),2.34 (1H, m); 13C NMR (75 MHz in CDCl3) δ14.13, 22.72, 27.40, 29.37, 29.50, 29.64, 31.95, 32.19, 45.61, 183.22; CIMS m/z (%) 341 (M++H); HRMS (CI+) m/z calcd for C22H45O2(M++H) 341.3420, Found 341.3421.
[2-オクチルデカン酸の合成]
white powder; FT IR (neat) 3032, 2927, 2856, 1707 cm-1; 1H NMR (300 MHz in CDCl3) δ0.88 (6H, t, J = 6.9Hz), 1.26 (24H, m), 1.48 (2H, m), 1.63 (2H, m), 2.34 (1H, m); 13C NMR (75 MHz in CDCl3) δ14.12, 22.70, 27.40, 29.30, 29.45, 29.60, 31.90, 32.20, 45.66, 183.50; CIMS m/z (%) 285 (M++H); HRMS (CI+) m/z calcd for C18H37O2(M++H) 285.2794, Found 285.2772.
[2-ノニルウンデカン酸の合成]
white powder; FT IR (neat) 3019, 2934, 2858, 1712 cm-1; 1H NMR (300 MHz in CDCl3) δ0.88 (6H, t, J = 6.9Hz), 1.26 (28H, m), 1.48 (2H, m), 1.61 (2H, m), 2.33 (1H, m); 13C NMR (75 MHz in CDCl3) δ14.14, 22.72, 27.40, 29.34, 29.50, 29.60, 31.92, 32.20, 45.60, 183.11; CIMS m/z (%) 313 (M++H); HRMS (CI+) m/z calcd for C20H41O2(M++H) 313.3106, Found 313.3111.
[2-ウンデシルトリデカン酸の合成]
white powder; FT IR (neat) 3028, 2941, 2858, 1712 cm-1 ; 1H NMR (300 MHz in CDCl3) δ0.88 (6H, t, J = 6.9Hz), 1.25 (36H, m), 1.48 (2H, m), 1.61 (2H, m), 2.35 (1H, m); 13C NMR (75 MHzin CDCl3) δ14.17, 22.74, 27.41, 29.40, 29.51, 29.61, 29.65, 29.69, 29.72, 31.97, 32.20, 45.55, 182.81; CIMS m/z (%) 369 (M++H); HRMS (CI+) m/z calcd for C24H49O2(M++H) 369.3732, Found369.3731.
[2-ドデシルテトラデカン酸の合成]
white powder; FT IR (neat) 3028, 2943, 2860, 2691, 1714 cm-1 ; 1H NMR (300 MHzin CDCl3) δ0.88 (6H, t, J = 7.1Hz), 1.25 (40H, m), 1.48 (2H, m), 1.61 (2H, m),2.34 (1H, m); 13C NMR (75 MHz in CDCl3) δ14.16, 22.76, 27.43, 29.43, 29.54, 29.64, 29.68, 29.71, 29.72, 31.99, 32.22, 45.68, 183.46; CIMS m/z (%) 397 (M++H); HRMS (CI+) m/z calcd for C26H53O2(M++H) 397.4045, Found 397.4043.
[2-トリデシルペンタデカン酸の合成]
white powder; FT IR (neat) 3032, 2922, 2851, 2691, 1712 cm-1 ; 1H NMR (300 MHzin CDCl3) δ0.88 (6H, t, J = 6.9Hz), 1.25 (44H, m), 1.48 (2H, m), 1.61 (2H, m),2.35 (1H, m); 13C NMR (75 MHz in CDCl3) δ14.14, 22.72, 27.40, 29.39, 29.50, 29.60, 29.64, 29.69, 31.96, 32.19, 45.54, 182.79; CIMS m/z (%) 425 (M++H); HRMS (CI+) m/z calcd for C28H57O2(M++H) 425.4358, Found 425.4341.
[2-テトラデシルへキサデカン酸の合成]
white powder; FT IR (neat) 3028, 2928, 2854, 2684, 1706 cm-1 ; 1H NMR (300 MHzin CDCl3) δ0.88 (6H, t, J = 7.2Hz), 1.25 (48H, m), 1.48 (2H, m), 1.60 (2H, m),2.34 (1H, m); 13C NMR (75 MHz in CDCl3) δ14.13, 22.72, 27.40, 29.40, 29.50, 29.60, 29.64, 29.73, 31.96, 32.19, 45.57, 182.87; CIMS m/z (%) 453 (100 M++H); HRMS (CI+) m/z calcd for C30H61O2(M++H) 453.4671, Found 453.4677.
[2-ペンタデシルヘプタデカン酸の合成]
white powder; FT IR (neat) 3028, 2911, 2848, 2650, 1703 cm-1 ; 1H NMR (300 MHzin CDCl3) δ0.88 (6H, t, J = 6.9Hz), 1.25 (52H, m), 1.48 (2H, m), 1.61 (2H, m),2.35 (1H, m); 13C NMR (75 MHz in CDCl3) δ14.05, 22.65, 27.33, 29.33, 29.43, 29.53, 29.57, 29.66, 31.90, 32.14, 45.43, 182.41; CIMS m/z (%) 481 (M++H); HRMS (CI+) m/z calcd for C32H65O2(M++H) 481.4984, Found 481.4977.
[2-ヘキサデシルオクタデカン酸の合成]
white powder; FT IR (neat) 3028, 2914, 2848, 2691, 1705 cm-1 ; 1H NMR (300 MHzin CDCl3) δ0.88 (6H, t, J = 6.9Hz), 1.25 (56H, m), 1.48 (2H, m), 1.61 (2H, m),2.35 (1H, m); 13C NMR (75 MHz in CDCl3) δ14.14, 22.72, 27.40, 29.40, 29.50, 29.60, 29.64, 29.73, 31.96, 32.20, 45.51, 182.51; CIMS m/z (%) 509 (M++H); HRMS (CI+) m/z calcd for C34H69O2(M++H) 509.5297, Found 509.5298.
[3-ノニルドデカン酸の合成]
以下の製造例D-1-1からD-1-5に記載の方法により、3-ノニルドデカン酸を合成した。
製造例D-1-1
[N-メトキシ-N-メチルデカンアミドの合成]
colorless oil; FT IR (neat) 2927, 2854, 1731 cm-1 ; 1H NMR (300 MHz in CDCl3) δ0.88 (3H, t, J = 6.9 Hz), 1.27 (12H, m), 1.63 (2H, m), 2.41 (2H, t, J = 7.8 Hz), 3.18 (3H, s), 3.68 (3H, s); 13C NMR (75 MHz in CDCl3) δ14.09, 22.65, 24.65, 29.28, 29.45, 31.86, 61.17, 174.77; CIMS m/z (%) 215 (M+); HRMS (CI+) m/z calcd for C12H25NO2(M+) 215.1921, Found 215.1903.
[10-ノナデカノンの合成]
colorless solid; FT IR (neat) 2953, 2916, 2847, 1698 cm-1 ; 1H NMR (300 MHz inCDCl3) δ0.88 (6H, t, J = 6.9 Hz), 1.26 (24H, m), 1.55 (4H, m), 2.38 (4H, t, J = 7.5 Hz); 13C NMR (75 MHz in CDCl3) δ14.03, 22.63, 23.84, 29.24, 29.41, 31.84,42.74, 211.49; CIMS m/z (%) 282 (M+); HRMS (CI+) m/z calcd for C19H38O (M+) 282.2882, Found 282.2902.
[エチル3-ノニル-2-ドデケノエートの合成]
colorless oil; FT IR (neat) 2928, 2855, 1718 cm-1 ; 1H NMR (300 MHz in CDCl3) δ0.88 (6H, t, J = 6.9 Hz), 1.27 (27H, m), 1.44 (4H, m), 2.12 (2H, t, J = 7.8 Hz), 2.58 (2H, t, J = 8.1 Hz), 4.14 (2H, q, J = 7.2 Hz), 5.61 (1H, br s); 13C NMR (75 MHz in CDCl3) δ14.14, 14.35, 22.70, 27.68, 28.75, 29.34, 29.49, 29.52, 29.60, 30.01, 31.92, 32.19, 38.44, 59.42, 114.98, 165.07, 166.66; CIMS m/z (%) 352 (M+); HRMS (CI+) m/z calcd for C23H44O2 (M+) 352.3349, Found 352.3345.
[エチル3-ノニルドデカノエートの合成]
colorless oil; FT IR (neat) 2929, 2855, 1739 cm-1 ; 1H NMR (300 MHz in CDCl3) δ0.88 (6H, t, J = 6.6 Hz), 1.26 (35H, m), 1.84 (1H, m), 2.21 (2H, d, J = 6.6 Hz), 4.12 (2H, q, J = 7.2 Hz); 13C NMR (75 MHz in CDCl3) δ14.16, 14.32, 22.73, 26.55, 29.38, 29.64, 29.66, 29.93, 31.95, 33.91, 35.10, 39.40, 60.07, 173.73; CIMSm/z (%) 354 (M+); HRMS (CI+) m/z calcd for C23H46O2 (M+) 354.3508, Found 354.3503.
[3-ノニルドデカン酸の合成]
colorless oil; FT IR (neat) 2925, 2854, 1709 cm-1 ; 1H NMR (200 MHz in CDCl3) δ0.88 (6H, t, J = 6.6 Hz), 1.26 (32H, m), 1.85 (1H, m), 2.27 (2H, d, J = 6.9 Hz); 13C NMR (75 MHz in CDCl3) δ14.16, 22.73, 26.52, 29.38, 29.65, 29.89, 31.95, 33.79, 34.87, 39.00, 179.94; CIMS m/z (%) 326 (M+); HRMS (CI+) m/z calcd for C21H42O2(M+) 326.3129, Found 326.3157.
[3-オクチルウンデカン酸の合成]
colorless oil; FT IR (neat) 2926, 2855, 1712 cm-1 ; 1H NMR (300 MHz in CDCl3) d 0.88 (6H, t, J = 6.6 Hz), 1.26 (28H, m), 1.85 (1H, m), 2.27 (2H, d, J = 6.9 Hz); 13C NMR (75 MHz in CDCl3) d 14.16, 22.71, 26.52, 29.34, 29.61, 29.89, 31.93, 33.80, 34.88, 38.94, 179.58; CIMS m/z (%) 298 (M+); HRMS (CI+) m/z calcd for C19H38O2(M+) 298.2876, Found 298.2874.
[3-デシルトリデカン酸の合成]
colorless oil; FT IR (neat) 2935, 2857, 1711 cm-1 ; 1H NMR (300 MHz in CDCl3) δ0.88 (6H, t, J = 6.6 Hz), 1.26 (36H, m), 1.85 (1H, m), 2.27 (2H, d, J = 6.9 Hz); 13C NMR (75 MHz in CDCl3) δ14.15, 22.72, 26.51, 29.38, 29.66, 29.88, 31.94, 33.78, 34.86, 38.95, 179.79; CIMS m/z (%) 354 (M+); HRMS (CI+) m/z calcd for C23H46O2(M+) 354.3498, Found 354.3503.
[3-ウンデシルテトラデカン酸の合成]
colorless solid; FT IR (neat) 2923, 2853, 1707 cm-1 ; 1H NMR (300 MHz in CDCl3) δ0.88 (6H, t, J = 6.9 Hz), 1.26 (40H, m), 1.85 (1H, m), 2.27 (2H, d, J = 6.9 Hz); 13C NMR (75 MHz in CDCl3) δ14.17, 22.74, 26.53, 29.40, 29.69, 29.72, 29.89, 31.96, 33.79, 34.88, 38.98, 179.82; CIMS m/z (%) 382 (M+); HRMS (CI+) m/z calcd for C25H50O2(M+) 382.3811, Found 382.3816.
[3-ドデシルペンタデカン酸の合成]
colorless solid; FT IR (neat) 2928, 2854, 1709 cm-1 ; 1H NMR (300 MHz in CDCl3) δ0.88 (6H, t, J = 6.6 Hz), 1.26 (44H, m), 1.85 (1H, m), 2.27 (2H, d, J = 6.9 Hz); 13C NMR (75 MHz in CDCl3) δ14.15, 22.72, 26.51, 29.39, 29.68, 29.71, 29.88, 31.96, 33.78, 34.86, 39.00, 180.00; CIMS m/z (%) 410 (M+); HRMS (CI+) m/z calcd for C27H54O2(M+) 410.4066, Found 410.4095.
[3-テトラデシルヘプタデカン酸の合成]
colorless solid; FT IR (neat) 2915, 2849, 1704 cm-1 ; 1H NMR (300 MHz in CDCl3) δ0.88 (6H, t, J = 6.9 Hz), 1.26 (52H, m), 1.85 (1H, m), 2.27 (2H, d, J = 6.9 Hz); 13C NMR (75 MHz in CDCl3) δ14.15, 22.73, 26.52, 29.41, 29.73, 29.90, 31.96, 33.78, 34.88, 38.99, 179.72; CIMS m/z (%) 466 (M+); HRMS (CI+) m/z calcd for C31H62O2(M+) 466.4685, Found 466.4717.
上記製造例と同様にして、実施例16から22の化合物を得た。当該化合物は、本発明の式(1)で表される化合物について、式中、X、X’、n及びn’が、以下の表1に表わされるものである。
本発明の化合物につき、以下の試験を行い、活性を測定した。
本発明の実施例化合物について、実施例番号、製造例番号、及び、その化学構造式を、以下の表2に示す。実施例化合物は、本発明の式(1)で表される化合物であり、式中、X、X’、R1、R1’、R2、R2’、n及びn’は、以下のものを示す。
試験例1(1)<蛍光強度測定装置を用いたマウス腹腔マクロファージからの活性酸素遊離測定>
<リン酸バッファー(PBS)の調製>
塩化ナトリウム8.0g、塩化カリウム0.2g、リン酸水素二ナトリウム1.15g、リン酸二水素カリウム0.2gを1000mlの蒸留水(D.W.)で溶解した。
5%チオグリコール酸培地(Difco、BD、code.225640、Lot.6192372)3mlをマウス(ICRマウス(SPF)、5週齢、オス)腹腔に投与した。投与4日後、ジエチルエーテルを用いてマウスを死亡させた。腹部中央の表皮に少しハサミで切れ込みをいれ、腹部をつまみ腹部表皮を剥ぎ取った。26Gの針を取り付けた10mlのシリンジで0.05%EDTA含有PBS(-)(EDTA 2Na、ヌクレアーゼ及びプロテアーゼtested、ナカライテスク)5mlを腹腔内に全量注入した。その後、腹部の横をつまむようにして40~50回程度マッサージした。腹腔内部の液を23G注射針でゆっくりと小の遠沈管に採取する。この操作を2回繰り返した。採取したマクロファージを1000rpmで8分間遠心した。上清を捨て、RPMI1640培地(RPMI-1640、L-グルタミン及びフェノールレッド含有、和光、189-02025、Lot.WRM8043に10.61%非動化血清(Bio West)、1%ペニシリンストレプトマイシン(GIBCO)を含む)で沈殿を懸濁した。遠沈管をRPMI1640培地で満たし、再度1000rpmで8分間遠心した。上清を捨て、RPMI1640培地で懸濁後、One Cell Counter(和研薬)でマクロファージの数を計測した。RPMI-1640培地を用いて、任意の濃度に希釈した。このようにして得られたマウス腹腔マクロファージを以下の試験に用いた。
40mM試験化合物溶液を以下のようにして調製した。
BSA0.7gを大試験管に秤量した。滅菌PBS(-)10mlを加え、よく撹拌後、LPS除去カラム(Endo Trap(商標)red 1/1(proofs))を用い、BSA溶液に含有されている不純物を除いた。その後、処理BSA溶液を滅菌フィルター(0.2μm)で濾過した。次に、Nano Drop ND-1000を用いて蛋白定量し、最終的な濃度が2%となるように滅菌PBS(-)で希釈した。秤量した試験化合物を、2%BSA(PBS(-)中にBSA2%を溶解したもの)250μlと共にホモジナイザーに入れ、ホモジナイザーに入れたままバスタイプ超音波装置で150秒間処理した。このようにして調製した溶液をエッペンに移し、以下の試験に使用した。ポジティブコントロールとしては、試験化合物として、TDCMを用いて同様に調製し、また、ネガティブコントロールとしては、試験化合物を何も加えず、同様に調製した。試験化合物を含まない調製液について、以下、「vehicle」という。
塩化カリウム0.4g、リン酸二水素カリウム0.06g、リン酸水素二ナトリウム0.107g、塩化ナトリウム8gを蒸留水(D.W.)で溶解後、1N水酸化ナトリウムでpH7.4に調整し、蒸留水(D.W.)で全量1000mlとして、ハンクス平衡化塩溶液(HBS)を調製した。
グルコース0.1g、及び、BSA(sigma)0.03gを、上記で調製したHBS100mlに溶解し、グルコース及びBSA含有ハンクス平衡化塩溶液(HBSG-BSA)を調製した(用時調製)。
マウス腹腔マクロファージ、40mM試験化合物溶液、及び、BSA含有ハンクス平衡化塩溶液(HBSG-BSA)は、前記と同様にして調製した。
HBSG-BSAでマクロファージを洗浄後、5mL程度のHBSG-BSAで懸濁し、96ウェルのコラーゲンウェル(TC-PLATE 96WELL,STERILE WITH LID,IND PACKED)に100μlずつ分注し、37℃で1時間インキュベートを行い、細胞をウェルに張り付かせた。上清を除去し、HBSG-BSAを100μl加え、10mMのH2DCFDA(2’,7’-ジクロロジヒドロフルオレセインジアセテート、invitrogen)を1μl添加した。37℃で1時間インキュベートを行い、上清を除去後、試験化合物を含んだHBSG-BSAまたはネガティブコントロールとしてのvehicleを、試験化合物の最終濃度が50μMになるように添加し、1時間後Genios蛍光強度測定装置で測定した。
図1<マウス腹腔マクロファージからの活性酸素遊離量>
図1に示されるように、本発明試験化合物は、いずれも、TDCMと同等もしくはそれ以上に、マウス腹腔マクロファージからの活性酸素の産生を促進する作用を示した。特に、本発明化合物のうち、実施例1の化合物、及び、実施例9の化合物は、TDCMの2倍を超える高活性を示した。
<マウス腹腔マクロファージ貪食能の測定>
マウス腹腔マクロファージ、40mM試験化合物溶液、及び、RPMI1640培地は、前記と同様にして調製した。
図2<マウス腹腔マクロファージ貪食能>
図2に示されるように、本発明試験化合物は、いずれも、TDCMと同等もしくはそれ以上に、マウス腹腔マクロファージの貪食作用を活性化する作用を示した。特に、本発明化合物のうち、実施例1の化合物、及び、実施例9の化合物は、TDCMの約2倍の高活性を示した。
<ウサギ好中球懸濁液の調製>
ハンクス平衡化塩溶液(HBS)、グルコース及びBSA含有ハンクス平衡化塩溶液(HBSG-BSA)は、試験例1(1)と同様にして調製した。
クエン酸ナトリウム6.25g、クエン酸3.125g及びグルコース5gを蒸留水(D.W.)250mlで溶解し、使用するまで4℃で保存した。
EDTA0.037g、炭酸水素カリウム1g及び塩化アンモニウム8.3gを蒸留水(D.W.)1000mlで溶解し、使用するまで4℃で保存した。
試験例1(1)と同様にして調製した。
デキストランT500(ファルマシア)1.2gを蒸留水(D.W.)100mlで溶解し、オートクレーブ(121℃、20分間)で滅菌し、使用するまで4℃で保存した。
5mlのACD液を20mlのシリンジ(注射針[Nipro])に取り、シリンジ内を一様にリンスした。ウサギの耳介中心動脈から血液をシリンジで20ml採取し、静かに転倒混和後、遠沈管(15mlタイプ:Falcon)3本に分注し、4℃、1,500rpmで5分間遠心後、上清を遠沈管(50mlタイプ:Falcon)に回収した。回収した上清と等量の1.2%デキストラン-PBS溶液を加えて緩やかに転倒混和した後、室温で30分間以上放置した。境界面を確認し、上清を新しい遠沈管(50mlタイプ:Falcon)に入れた。残りの溶液に等量の1.2%デキストラン-PBS溶液を加え緩やかに転倒混和した後、室温で30分間以上放置した。境界面を確認し、上清を遠沈管(50mlタイプ:Falcon)に入れた。回収した上清を4℃、2,000rpmで10分間遠心後、上清を除いた。沈渣にLysis液15ml加え、静かに懸濁後、さらに、Lysis液5ml加えて転倒混和し、氷中で5分間放置した。HBSG-BSAで全量を50mlとし、4℃、2,000rpmで10分間遠心後、上清を除いた。沈渣をHBSG-BSA2mlで懸濁し、この細胞懸濁液をLymphoprep[Nycomed,808068]2ml(遠沈管、15mlタイプ)の上層に静かに重層し、1200rpmで20分間遠心後(遠心機の条件:accel 0.5、break Off)、上清をアスピレーターで除いた。残存するLyphoprepを除くため、沈渣(好中球)をHBSG-BSAに懸濁し、再び、1,500rpmで5分間遠心後、上清を除いた。好中球をHBSG-BSAで懸濁し、細胞数計測装置「celltac」[日本光電]で細胞数を測定した。
試験化合物及びTDCMのエマルジョン溶液を試験例1(1)と同様にして調製した。
4.86mgのH2DCFDAを1mlのDMSOで溶解した。
<ウサギ好中球からの活性酸素遊離量の測定>
ウサギ好中球からの活性酸素遊離に対する影響を以下の手順で測定した。
図3<ウサギ好中球からの活性酸素遊離量>
図3に示されるように、本発明化合物は、おおむねTDCMと同程度もしくはそれ以上に、ウサギ好中球からの活性酸素遊離を活性化する作用を示した。特に、本発明化合物のうち、実施例1の化合物は、TDCMの約2倍の活性を示した。
<ウサギ好中球貪食能の測定>
(2)アンピシリン耐性大腸菌及びオプソニン化大腸菌作成法
<L-brothの調製>
蒸留水(D.W.)1Lに対してトリプトファン10g、NaCl5g、Yeast Extract5g及びMgSO41mlを溶解した。
10mgのオプソニン化剤(BioParticles Opsonizing Reagent(Molucular Probes))を500μlの超純水で溶解した。
エレクトロポーレーション用セル(JM109)を氷中で溶解し、アンピシリン耐性の遺伝子を大腸菌に導入するため、JM109に2μlのアンピシリン耐性プラスミド(pT7Blue、Novagen、100ng/μl)を加え懸濁し、その懸濁液をエレクトロポーレーション用キュベットに移しパルス(2.5kV,200Ω,25mF)をかけた。パルスをかけた菌液を、L-broth培地1mlを入れた5mlチューブに移し、37℃で3時間培養した。該溶液を0.005%アンピシリンを含むL-brothの寒天培地に塗布し、37℃で一晩培養した(培地に撒く際に、3,500rpmで5分間遠心を行った後、上清を800μl除き、沈渣を懸濁後、シャーレに撒いた)。翌日シャーレに生えた菌を少量かきとり、2mlのL-broth培地に溶解し、約4時間攪拌培養を行った。
上記で作成したアンピシリン耐性大腸菌溶液100μlと溶解したオプソニン化剤100μlをエッペンドルフチューブ内で懸濁した。得られた懸濁液を37℃で1時間インキュベーションし、該懸濁液を300μlのPBSで懸濁後、1200G、15分間遠心し上清を除去した。さらに、得られた液を300μlのPBSで懸濁後、1200G、15分間遠心し上清を除去した。2回繰り返した。100μlのL-brothに1μlの菌液を加えよく懸濁し100倍希釈した。100倍希釈した菌液を10μlワンセルカウンターに添加し、顕微鏡により菌数をカウントした。
上記方法により調製した好中球1.0×105個をエッペンに分取し、HBSで懸濁後、50μMの試験化合物のエマルジョン溶液またはTDCMのエマルジョン溶液で1時間処理を行った。コントロールは2%BSA溶液を用いて同様に行った。300μlのHBSを加え懸濁し、1200Gで10分間遠心後、上清を除去した。さらに、得られた液に300μlのHBSを加え懸濁し、1200Gで10分間遠心後、上清を除去した。処理好中球に1.0×107cellsのオプソニン化したアンピシリン耐性大腸菌をよく懸濁後、37℃で1時間インキュベーションした。余分な大腸菌を除去後、0.5%TritonX-100(生理食塩水中)を100μl加え、よく懸濁し、37℃で30分間インキュベーションを行った。TritonX-100処理により好中球を破壊することによって、好中球内部に取り込まれていた大腸菌の量を比較定量するため、次に、TritonX-100処理溶液全量を0.005%アンピシリン含有普通寒天培地にまき、コンラージ棒で全体に広げた。24時間、37℃でインキュベーションを行い、好中球内部に取り込まれていた大腸菌のコロニー数をカウントした。
図4<ウサギ好中球貪食活性>
図4に示されるように、本発明化合物は、いずれもウサギ好中球の貪食能を活性化する作用を示した。特に、本発明化合物のうち、実施例1の化合物は、TDCMの約2倍の活性を示した。
<試験化合物処理によるマウス腹腔マクロファージからのサイトカイン遊離測定>
マウス腹腔マクロファージ、40mM試験化合物溶液、及び、RPMI1640培地は、前記と同様にして調製した。
<試験化合物処理によるTHP-1細胞からのIL-8遊離測定法>
RPMI培地溶液は前記と同様にして調製し、これを用いて、試験化合物のRPMI培地溶液を以下のようにして調製した。
本発明化合物のうち、特に免疫賦活活性が高いと考えられる化合物について測定したところ、図5に示されるように、実施例1の化合物、及び、実施例9の化合物は、TDCMの約0.6倍、約0.8倍の活性を示した。
<試験化合物投与マウスにおける抹消血中へのIL-6、TNF-α、IFN-γの遊離測定>
試験化合物のエマルジョン溶液を以下のようにして調製した。
図7<マウス血漿中IFN-γ濃度(pg/ml)>
図8<マウス血漿中TNF-α濃度(pg/ml)>
図6に見られるように、本発明試験化合物を投与したマウスにおいて、血漿中IL-6濃度の増加が見られた。特に、本発明化合物のうち、実施例1の化合物、実施例9の化合物は、いずれも、ポジティブコントロールとした天然由来の公知のトレハロースジエステル化合物であるTDCMに対し、それぞれ、約1.2倍、約1.5倍程度の高いIL-6遊離活性を示した。実施例13の化合物、及び、実施例14の化合物も、TDCMに対し、それぞれ、約半分、同程度の活性を示した。
<ウェルシュ菌投与によるマウス生存試験(実施例1の化合物)>
試験化合物として、製造例α―1に記載の方法により合成した化合物、及び、TDCMをそれぞれ1mg秤量し、上記と同様にして、試験化合物のエマルジョン溶液(1mg/ml)を調製した。
ウェルシュ菌(TypeA NTCT8237)は、以下のように調製した。
ブレインハートインフュージョン(BHI)(Difco)の粉末7.4mgを蒸留水200mlに溶かした)。得られた溶液(以下、この溶液を「BHI培地」という。)40mlをメスピペットでとり、200mlのフラスコに加え、高圧蒸気滅菌(121℃、20分間)を行った。また、ねじ口試験管2本に5mlのBHI培地を入れ、高圧蒸気滅菌を行った。さらにガラス管付きゴム栓(綿栓付き)及びガラス管を高圧蒸気滅菌した。クリーンベンチ内で、クックドミート培地に生えた保存用の菌を滅菌したパスツールピペットで適量採取し、上記滅菌ねじ口試験管内のBHI培地に菌植えした。次いで、37℃で一晩インキュベーションした。
マウス(ICR、6週齢)を3群に分け、以下の試験を行った。
試験化合物のエマルジョン溶液、TDCMのエマルジョン溶液、又は、コントロールとしてのエマルジョン溶液のみ、を1群4匹のマウスに対し100μg/マウスずつ(エマルジョン溶液のみの場合は、100μl/マウスずつ)腹腔内投与した。3時間後、ウェルシュ菌(2.4×107cells/マウス)をマウスに腹腔内投与した。その後、経過観察を行った。
<ウェルシュ菌毒素投与によるマウス生存試験(実施例1の化合物)>
試験化合物のエマルジョン溶液(1mg/ml)を上記と同様にして調製した。
ウェルシュ菌毒素は、以下のようにして調製した。
枯草菌α毒素遺伝子トランスフォーマントをL-Broth中で攪拌しながら37℃、14時間培養後、4℃、8,000rpmで20分間遠心し、培養上清を氷冷下で攪拌しながら、硫酸アンモニウム(硫安)(ナカライテスク)を定期的に少量添加後、終濃度70%飽和硫安(472g/L)とし、一晩放置した。その後、4℃、9,500rpmで30分間遠心し、生じた沈査を0.02M TB(pH7.5)に溶解させ、同緩衝液で4℃、一晩透析した。透析後、4℃、15,000rpmで30分間遠心し、この上清を粗毒素(硫安毒素)標品とした。この粗毒素標品を終濃度0.5M NaClとなるように、1M NaCl-TB(pH7.5)で希釈後、予め、0.5M NaCl-TB(pH7.5)で平衡化した銅キレートアフィニティーカラム(1.5×9cm)に粗毒素標品をアプライし、続いて、0.5M NaCl-TB(pH7.5)、0.5M NaCl-0.1M PB(pH6.5)、0.5M NaCl-0.02M酢酸緩衝液(pH4.5)、そして、0.5M NaCl-0.1M PB(pH6.5)を順次、100mlずつ流した。次に、カラム中に結合した毒素を15mM L-ヒスチジン(ナカライテスク)-0.5M NaCl-0.1M PB(pH6.5)100mlで溶出させた後、この溶出液をシリンジフィルター(DISMIC-ADVANTEC)でろ過後、限外ろ過フィルターAmicon(商標)Ultra-15-3OK(MILLIPORE)で遠心濃縮し、この濃縮液を0.02M TB(pH8.0)で4℃、一晩透析し、15,000rpmで30分間遠心後、UNO(商標)Q-1 R Column(BIO-RAD)にアプライした。溶出は、0.02M TB(pH8.0)中の塩化ナトリウム濃度を0から0.05Mまで直線的に変化させ、流量1.0ml/minで行った。各溶出(0.5ml)画分において、抗α毒素血清に対するオクタロニー反応で沈降線が認められ、かつ、SDS-PAGEで、α毒素に相当する約43kDaの単一バンドを含む画分を集め、約1.0mlまで濃縮した。この濃縮液を0.02M TB(pH7.5)で4℃、一晩透析した後、4℃、15,000rpmで30分間遠心し、その上清(組み換えα毒素)を分取した。得られた組み換えα毒素は、SDS-PAGEで不純物の有無を確認後、使用まで-80℃で保存した。
マウス(ICR、6週齢)を3群に分け、以下の試験を行った。
試験化合物のエマルジョン溶液、TDCMのエマルジョン溶液、又は、コントロールとしてのエマルジョン溶液のみ、を1群4匹のマウスに対し100μg/マウスずつ(エマルジョン溶液のみの場合は、100μl/マウスずつ)腹腔内投与した。3時間後、ウェルシュ菌毒素(200ng/マウス)をマウスに腹腔内投与した。その後、経過観察を行った。
<緑膿菌投与によるマウス生存試験(実施例1の化合物)>
試験化合物として、製造例α―1に記載の方法により合成した化合物を1mg秤量し、上記と同様にして、試験化合物のエマルジョン溶液を調製した。
緑膿菌(Fhu-071115strain)は患者由来のものを用い、以下のようにして調製した。
L-brothを40mlメスピペットでとり、200mlのフラスコ1本に入れ、スポンジ栓をした。また、別のねじ口試験管2本に5mlのL-brothを入れた。当該フラスコ及びねじ口試験管を121℃で20分間オートクレーブにかけた。L-broth培地が室温まで冷えた後、クリーンベンチ内で40mlのL-brothに超低温フリーザーにて保管していた緑膿菌を加えた。培養室において、一晩シェイキングを行った。9000rpmで15分間遠心を行い、上清を除去した。滅菌生理食塩水20mlを加えてボルテックスを用いて混和した後、9000rpmで15分間遠心を行い、上清を除去する、という工程を3回行った。滅菌生理食塩水4.5mlを加え、ボルテックスを用いて混和し、これを菌原液とした。1000倍に希釈した菌液を用い、ワンセルカウンターで菌数を数えた後、所望の濃度に菌原液を希釈して、以下の試験に用いた。
マウスを2群に分け、以下の二通りの実験を行った。
(A)1群3匹のマウス(ICR、5週齢)に対し、上記試験化合物のエマルジョン溶液(100μg/マウス)を腹腔内投与し、3時間経過後、緑膿菌(5.0×107cells/マウス)を腹腔内投与した。その後、経過観察を行った。(B)1群3匹のマウス(ICR、5週齢)に対し、緑膿菌(2.0×107cells/マウス)を腹腔内投与し、上記試験化合物のエマルジョン溶液(100μg/マウス)を腹腔内投与し、3時間経過後、上記試験化合物のエマルジョン溶液(100μg/マウス)を腹腔内投与した。その後、経過観察を行った。
<THP-1細胞からのサイトカイン遊離>
実施例9で得られた試験化合物で処理した、ヒト単球性白血病細胞由来のTHP-1細胞からの各々のサイトカインおよびケモカイン遊離量をELISA法により測定した。また、ヒト肺がん細胞由来A549細胞、及び、ヒト大腸癌細胞由来DLD-1細胞を用い、同様の解析を行った。
(1)血清のロットチェック
培養したTHP-1細胞をクリーンベンチ内で15mlの遠沈管に移した。細胞を1,000rpmで5分間、20℃で遠心後、上清を除去した。その沈渣を、それぞれRPMI 1640(10%ロットチェック用FBSを含む)培地1mlに懸濁し、ヘモサイトメーター(萱垣医理科工業)とカバーグラス(三商)を用いて細胞数を数えた後、培地で2.5×105cells/mlに希釈した。浮遊培養用MULTI WELL PLATE 24wells(SUMILON)に細胞懸濁液を0.5mlずつ分注し、1日1回細胞の増殖や形態を観察した。2~3日後に培養液を培地で100倍希釈し、4μlをとって計数板で細胞数をカウントし、増殖の良し悪しを比較した。増殖および形態が良い細胞をwellから回収し同じ培地で洗浄後、再び2.5×105cells/mlになるように希釈し、浮遊培養用MULTI WELL PLATE 6wells(SUMILON)に1ml入れ、37℃、5%CO2の条件下でインキュベーションした。24時間ごとに培地交換を行い、プレートごと、1,800rpmで5分間遠心(TOMY)した後、ゆっくりと培地を除去し、新しく培地を1ml加えた。培地の交換作業を2回行った。3日目以降は、培地を交換後、細胞を100倍希釈して、計数板で数え、増殖の程度を測定した。その後、数日間この操作を行い、増殖や形態が良い培地を選択した。
4℃で解凍したFetal bovine serum(FBS)(Biowest)500mlを56℃で時々撹拌しながら30分間インキュベーションし、非動化を行った。その後、血清は濾過せずに、クリーンベンチ内で50mlの遠沈管に30mlずつ分注し、-80℃で保存した。使用前は予め4℃で解凍した。
クリーンベンチ内で、RPMI 1640液体培地(Wako)500mlに、非動化したFBS 60mlと、Acrodisc 25mm Syringe Filter(Pall Corporation)で濾過したPenicillin Streptomycin(GIBCO)5.6mlを加え、均一になるように混和した。作製した培地は、濾過せずにそのまま使用した。保存は4℃で行い、使用前は予め室温に戻した。
THP-1細胞を75cm2の浮遊培養用フラスコ(SUMILON)(90%増殖以上)で培養し、形態および増殖を観察した。細胞の形がよく、増殖が早い場合には、細胞懸濁液を新しいフラスコに移すか、または、使用中のフラスコに、新しい培地を、約2倍量添加して植え継ぎを行った。増殖が遅い場合は、そのまま経過を観察するか、新しい培地を等量加えて植え継ぎを行った。
75cm2の浮遊培養用フラスコ(90%増殖以上)で培養した細胞10~15mlを遠沈管に移し、1,000rpm、5分間遠心し、上清を除去した。その沈渣に、細胞凍結保存液Cell banker(日本全薬工業)1mlを加え、懸濁し、セラムチューブに分注し、-80℃で保存した。
-80℃で凍結した細胞を、37℃の水浴で急速に解凍した。直ちに、細胞保存液を予めRPMI 1640培地を9~10ml加えておいた15ml遠沈管に添加し転倒混和した。1,000rpmで5分間遠心後、上清を除去して10mlの新しい培地で再懸濁し、細胞懸濁液を75cm2の浮遊培養用フラスコに移した。その後、新しい培地を加えて全量を20~30mlにし、37℃、5%CO2の条件下で培養した。
洗浄液の調製
洗浄液を超純水(S.D.W.)で25倍に希釈し、室温にて使用した。
2つのマイクロチューブに標準基質液用希釈液を600μL入れておく。スタンダードの基質を標準基質液用希釈液1mLで溶解し(2450pg/mL)、それを100μL、マイクロチューブに移し、溶解し(350pg/mL)、これをさらに、100μLを別のマイクロチューブに加え溶解して(50pg/mL)希釈を行った。標準基質液用希釈液をコントロールとして用いた(0pg/mL)。
color reagent Aとcolor reagent Bを等量ずつ混合し、100μl/well×測定するwellの呈色液を加えた。これは使用前15分以内に調製しておた。
使用するELISAキットの試薬を常温に戻し、各ウェルに、種々の濃度の標準基質液、および、assay bufferを50μL入れ、さらに、サンプルを50μL添加した。プレートを1分間軽くタップさせ、プレートにカバーをかけ、室温で2時間インキュベートした。その後、洗浄液で5回洗浄し(アスピレーターの使用可)、調製したconjugate液を100μLずつ添加し、プレートにカバーをかけ、室温で2時間インキュベートした。その後、洗浄液で5回洗浄後、遮光しながら、color reagent溶液AとBの混合液を100μLずつ添加し、プレートを室温、暗所で30分間インキュベートした。最後に、反応停止液を100μLずつ添加し、30分間以内にマイクロプレートリーダー(Molecular Devices spectra MAX 340PC)で吸光度を測定した。(450nm-550nm)。標準基質液の直線グラフから、各サンプルのサイトカイン遊離量を算出した。
培養したTHP-1細胞をクリーンベンチ内で50mlの遠沈管に移し、1,000rpmで5分間、20℃で遠心後、上清を除去した。その沈渣を、新しい無血清RPMI 1640培地(Wako)1mlに懸濁し、細胞懸濁液を、予め無血清RPMI 1640培地を990μl加えておいた滅菌エッペンドルフチューブに10μl加えて100倍希釈した。100倍希釈液4μlを血球計数板で細胞数を数え、細胞懸濁液を無血清RPMI 1640培地で1×107cells/mlに希釈した。100μLのRPMI培地をエッペンにとり、200μMとなるようにの40mMの試験化合物(実施例9)/DMSO溶液を添加し、超音波に5秒間かけた。100μlのTHP-1細胞を加えた(試験化合物の最終濃度100μM)。37℃、2時間インキュベーション後、5000rpmで10分間遠心し、上清を別のエッペンにとりELISAキットによりサンプル測定を行った。
なお、ポジティブコントロールとしては、試験化合物として、6,6’-ビス-O-(2-テトラデシルヘキサノイル)-α,α’-トレハロース(以下「対照化合物A」という)、およびTDCMを用いて同様に測定を行った。また、ネガティブコントロールとしては、試験化合物を何も加えず、同様に調製した(vehicle)。
ポジティブコントロールと比較し、実施例9の化合物で処理したTHP-1細胞からのMIP-1βの遊離が、約8~10倍高いことが明らかとなった。また、TNF-αの遊離は、ネガティブコントロール細胞と比較してほとんど同等であった。結果を図9に示す。また、実施例1で得られた化合物については、MIP-1βの遊離についてはポジティブコントロールに比べて好適な結果が得られた一方で、TNF-αの遊離はネガティブコントロール細胞と比較してほとんど同等であった(データ示さず)。
<THP-1細胞に対する細胞毒性検討、及び、変異原性試験>
<THP-1細胞のトリパンブルーを用いた細胞毒性の検討>
試薬の調製
0.3%トリパンブルー/PBS(-)の調製
0.3gのトリパンブルー(nacarai)をPBS(-)100mlで溶解した。
培養したTHP-1細胞をクリーンベンチ内で50mlの遠沈管に移し、1,000rpmで5分間、20℃で遠心後、上清を除去した。その沈渣を、新しい無血清RPMI 1640培地(Wako)1mlに懸濁した。この細胞懸濁液を、予め無血清RPMI 1640培地を990μl加えた滅菌エッペンドルフチューブに10μl加え、100倍希釈した。100倍希釈液10μlを血球計数板で細胞数を数え、細胞懸濁液を無血清RPMI 1640培地で1×107cells/mlになるように希釈した。
RPMI培地100μlに200μMとなるように試験化合物(実施例9)/DMSO溶液、または、DMSOを添加し、5秒間超音波を行った。なお、ポジティブコントロールとしては、試験化合物として、6-O-(2-デシルドカノイル)-α-グルコース(以下「対照化合物B」という)、およびTDCMを用いて同様に調製した。また、ネガティブコントロールとしては、試験化合物を何も加えず、同様に調製した(Vehicle)。1.0×107cells/mlに調製したTHP-1細胞100μlを加え37℃で2時間、または、24時間インキュベーションした。その後、0.3% トリパンブルー/PBS(-)を20μl加え懸濁した後、すぐに細胞数測定装置(CYRORECON)で細胞の生存率を解析した。
試薬の調製
0.1 Mリン酸ナトリウム緩衝液の調製
Na2HPO4 5.68gを蒸留水200mLに溶解し、NaH2HPO4・2H2Oを蒸留水100mLに溶解したものを徐々に加えてpH7.4に調整し、高圧蒸気滅菌した。
(1)VB培地:MgSO4・7H2O 0.4g、クエン酸H2O 4g、K2HPO4 20g、NaNH4HPO4・4H2O 7gを蒸留水200mLに溶解し高圧蒸気滅菌した。
(2)グルコース40gを蒸留水200mLに溶解し高圧蒸気滅菌した。
(3)粉末寒天30gを蒸留水1600mLに懸濁し高圧蒸気滅菌した。
(3)の試薬が約60℃に冷えた後、(1)と(2)の試薬を混合し、約30mLずつシャーレにまいた。
粉末寒天1.2gとNaCl 1gを水200mLに懸濁し高圧蒸気滅菌し、50mlのチューブに移した。使用前に0.5mMのヒスチジン/ビオチン溶液を20mL混合して47℃に保温した。
Oxoid Nutrient Broth(Difco)2.5gを蒸留水100mlに溶解し、その内5mlをねじ試験管に入れて滅菌した。その後、TA98(Salmonella typhimurium TA98)の菌液約10μLを接種して37℃で一晩、振とう培養して菌懸濁液を調製した。
S9 1mlにCo factorA mix(ORIENTAL YEAST)9mlを加えた。
4-NQO/DMSOの調製
4-Nitroquinoline N-oxide(4-NQO)(東京化成工業株式会社)0.3mgを10mlのDMSOに溶解した。
2-aminoanthrathen/DMSOの調製
2-aminoanthracen(ALDRICH)0.5mgを30mlのDMSOに溶解した。
エッペンに標準変異原性物質、実施例9の試験化合物、対照化合物B、またはTDCM10μLを各2本ずつ2組用意した。S9mixまたは100mMリン酸緩衝液0.5mLを加えた後、菌懸濁液100μLを加え、プレインキュベーションした(37℃、20分シェイキング)。ヒスチジン-ビオチンを含むソフトアガー(47℃に保温したもの)2mLを加え軽く懸濁し、最小グルコース寒天培地にまき、インキュベーションした(37℃、2日間)。その後、His+のコロニー数を数えた。
<実験結果>
THP-1細胞に対する細胞毒性を検討するため、実施例9で得られた試験化合物で2時間、及び、24時間処理したTHP-1細胞の生存率をトリパンブルー染色により分析した結果、対照化合物B処理細胞では細胞毒性が認められたが、実施例9で得られた化合物で処理した細胞では、細胞毒性が観察されなかった。結果を図10に示す。また実施例1で得られた化合物についても同様に有意な結果が得られた(データ示さず)。
さらに、実施例9で得られた試験化合物に関してS9mix存在下、及び、非存在下においてAmes試験を行ったが、いずれの試験化合物も変異原性を示さなかった。また、本解析条件下における標準物質(2-amino anthracene, 4NQO)の変異原性は、陽性であった(図11)。また実施例1で得られた化合物についても同様に有意な結果が得られた(データ示さず)。
<マウス腹腔内への細胞浸潤>
<PBS(-)溶液の調製>
NaCl 4g、Na2HPO4・12H2O 1.45g、KH2PO4 0.1g、KCl 0.1gを蒸留水500mlに溶解し、121℃で20分間高圧蒸気滅菌を行った。
EDTA (nacalai tesque code.151-30)50mgをPBS(-)100mlに溶解後、0.2μmのフィルターを用い濾過滅菌した。
NaCl 0.9g、Polyoxyethylene Sorbitan Monooleate(Tween80) 1.1ml、D(-)-Mannitol 5.6gを蒸留水 100mlに溶解し、0.2μmのフィルターで滅菌濾過を行った。実施例9で得られた試験化合物1mgをホモジナイザー(WEATON USA 10mL、アズワン)の底部に取り、1滴のMinelal Oilを添加し、2分間超音波に当てながらホモジナイズした。その後、1mLの1.1% Tween-5.6% Mannitol Salineを加え、液が白濁し、均一になるまでホモジナイズした。エッペンに試験化合物の溶液を全量移し、低温殺菌のため62℃で30分間処理した。ポジティブコントロールとしては、試験化合物として、対照化合物AおよびTDCMを用いて同様に調製した。また、ネガティブコントロールとしては、試験化合物を何も加えず、同様に調製した(vehicle)。
1mg/mLの試験化合物(実施例9)溶液を100μg/マウスとなるように、マウス(ICRマウス(SPF)(4週齢、雄、体重:20~22g))に腹腔投与した。
試験化合物を腹腔投与したマウスを、2時間後、または、24時間後、ジエチルエーテルを使用して死亡させた。腹部中央の表皮に少しハサミで切れ込みを入れ、腹部をつまみ腹部表皮を剥ぎ取った。ピンセットで腹膜を軽く持ち上げ、内臓に針が刺さらないように26Gの針を取り付けた10mLのシリンジで0.05% EDTA in PBS(-) 5mLを腹腔内に全量注入した。その後、腹部の横をつまむようにして40~50回程度マッサージした。腹腔内部の液をゆっくりと小サイズの遠沈管に採取した。この操作を再度繰り返した。採取した細胞を1,000rpmで10分間遠心した。上清を除去し、RPMI1640培地で沈殿を懸濁した。遠沈管をRPMI1640培地で満たし、再度1,000rpmで10分間遠心した。上清を捨て、RPMI1640培地で懸濁後、細胞計数盤を用いて細胞数を計測する。RPMI-1640培地を用いて、任意の濃度に希釈した。
<1/15 M リン酸ナトリウムバッファー(pH6.4)の調製>
NaH2PO4 6.0g、Na2HPO4 7.06gをそれぞれ250mlの蒸留水で溶解し、リン酸水素二ナトリウム溶液にpHを測定しながらリン酸二水素ナトリウム溶液を添加してpH6.4に合わせ、オートクレーブ121℃ 20分間で滅菌した。
1mg/mLの試験化合物溶液を100μg/マウスになるようにマウスに腹腔投与し、24時間後に0.05% EDTA/PBS(-)を用いて腹腔内に浸潤した細胞を回収した。その後、1000rpmで8分間遠心を行い、上清を除去した。細胞を100μLのリン酸緩衝液で懸濁し、染色用スライドガラスの上に載せた。水分が蒸発したのを確認し、染色用バッドの上でメイグリュンワルド液を10~15滴、滴化し、2~3分間放置した。メイグリュンワルド液を流さずに、リン酸緩衝液を10~15滴、滴化し、2~3分間放置した。適当な量のギムザ染色液を添加し、30分間放置した。スライドガラスを裏面にして流水後、スライドガラスを乾燥させ、顕微鏡により観察した。
試薬の調製
試験例11と同じ方法で調製した。
試験例11と同じ方法で調製した。
EDTA2Na 50mgを100mlのPBS(-)に溶解し、オートクレーブで121℃、20分間滅菌を行った。
EDTA2Na 50mgを100mlのPBS(-)に溶解し、オートクレーブで121℃、20分間滅菌を行う。その後、用事調整で0.5%のBSAを溶解した。
1mg/mLの試験化合物エマルション溶液をマウス(100μg/マウス)に腹腔投与し、24時間後に0.05%EDTA/PBS(-)を用いて腹腔内に浸潤した細胞を回収した。その後、採取した腹腔細胞を300gで10分間遠心後、上清を除去した。調製した腹腔細胞を1mlの0.05%EDTA(0.5%BSA/PBSで溶解)で懸濁した。細胞懸濁液をフローサイト用メッシュでろ過を行い、100倍希釈した細胞懸濁液で細胞数を測定した。各々のサンプルの細胞を107cells/sampleに調整後、300gで10分間遠心を行い、上清を除去した。100μlの0.05%EDTA(0.5%BSA/PBSで溶解)で懸濁し、10μlのCD11b、CD4、CD8の抗体(それぞれ、FITC anti-mouse CD11b/Mac-1(BECKMAN)、FITC anti-mouse CD4(BECKMAN COULTER)、PE anti-mouse CD8a(BD Pharmingen))を添加した。2-8℃、暗所、10分間インキュベート後、1-2mlの0.05%EDTA(0.5%BSA/PBSで溶解)で細胞を懸濁し、300gで10分間遠心後、上清を除去した。1mlの0.05%EDTA(0.5%BSA/PBSで溶解)で懸濁後、フローサイトメトリーで解析した。
PBS(-) 1mLで細胞を懸濁した。ヘモライナック(溶血ヘモグロビン試薬)100μLを細胞懸濁液に加え赤血球を破壊した。その後、細胞測定装置(CYTORECON、GE healthcare)により細胞数を測定した。
また、調製した細胞を24wellのコラーゲンウェル(greiner)に播種し、2時間インキュベーションした。その後、上清を吸引し、RPMI培地による細胞の洗浄を2回繰り返した。RPMI培地 300μl加え倒立顕微鏡により細胞を観察した。
その結果、実施例9で得られた試験化合物を投与したマウス腹腔内では、時間依存的に浸潤細胞の増加が認められ、24時間処理後には、Vehicle処理と比較して15~20倍増加していた(図12)。
以上より、実施例9の試験化合物により、マウス腹腔内で食細胞系の細胞が集積することが示され、中でもNK細胞の割合が大きいことが示された。
<ウェルシュ菌感染マウスまたは緑膿菌感染マウスへの影響(実施例9の化合物)>
<ウェルシュ菌調製方法>
COOKED MEAT培地の調製 (以下CM培地と省略することもある)
CM培地 (125mg/ml in D.W.)をネジ付試験管に加え、15分間煮沸してCM培地内の空気を脱気した。オートクレーブ(121℃,20分)による高圧蒸気滅菌を行い、室温まで冷却した。
37mgのBHI培地を100mlの蒸留水に溶解し、ネジ付試験管に4.5mL、三角フラスコに40mL加えてスポンジ栓をし、オートクレーブ(121℃,20分)で高圧蒸気滅菌を行ったあと、室温まで冷却した。
作製したCM培地にC. perfringens Type-A NCTC8237(PLC+)を添加し、37 ℃で2日間培養し、保存菌液として室温で保管した。
各保存菌液から菌液を0.2mLとり、前培養用4.5mL BHI培地内に添加し、37℃で、一晩培養した。この培養液全量を40mLBHI培地内に添加し、窒素置換(10分間)を行ったあと、再び37℃で、5時間培養した。その後、培養液50mLチューブに移し、遠心分離(4℃,9000 rpm,15分)を行った。上清を除去し、生理食塩水を加えてよく洗浄した後、再び遠心分離(4℃,9000rpm,15分)を行い集菌した。この洗浄を2回繰り返した。その沈殿にBHI培地を4.5mL加えて懸濁した。この懸濁液を原液とした。原液を1000倍に希釈して、オートクレーブ(121℃,20分)により高圧蒸気滅菌を行った後、ワンセルカンターを使用し、菌数を計測した。菌濃度が1×108cells/mLの菌液を作製し、実験に使用した。
Luria‐Bertani Broth培地 (L‐Broth)の調製
BactoTM Tryptone(Difco)10.0g、BactoTM Yeast Extract(Difco)5.0g、及び、塩化ナトリウム(NaCl)(nacalai tesque)5.0gを蒸留水に溶解し、1M MgSO4を1.0mLを加え、1N NaOHでpH7.5に調整し、蒸留水で全量を1,000mLにした後、オートクレーブで、121℃、20分間、高圧蒸気滅菌し、室温まで冷却した。
緑膿菌(P.aeruginosa)0.2mLをL‐Broth 10mLに添加し、37℃で、一晩シェイキング培養した。この培養液に滅菌グリセリン1mL(グリセリン)を加え、ボルテックスを行った。菌液を300μLずつ滅菌エッペンに分注し、-80℃で保存した。
100μg/マウスの試験化合物(実施例9)エマルション溶液を腹腔投与し、24時間後、3.0×1010CFU/mLの緑膿菌または、5.0×107CFU/mLのウェルシュ菌を腹腔に投与し、マウスを2時間おきに観察した。
3.0×1010CFU/mLの緑膿菌を腹腔に投与し3時間後、100μg/マウスの試験化合物(実施例9)エマルション溶液を腹腔投与した。その後、マウスを2時間おきに観察した。
図16および17に示すように、実施例9の試験化合物で処理マウスの致死は、著しく抑制された。また、緑膿菌感染3時間後に実施例9の試験化合物を投与した結果、マウスの致死は、有意に抑制された(図18)。
<敗血症観察>
100μg/マウスの試験化合物(実施例9)エマルション溶液を腹腔投与し、24時間後、3.0×1010CFU/mLの緑膿菌を感染させた。菌投与15時間後にへパリンを針先に少し入れた注射器で心臓採血を行い、全血200μLを普通寒天培地上に播種し、16時間、培養器でインキュベーションした。培地上のコロニー数をカウントした。
菌数が多い場合は、全血を生理食塩水で10倍、100倍、1000倍、10000倍に希釈し、普通寒天培地に播種した。
その結果、図19に示すように、菌のみを投与した場合、または、菌およびVehicleを投与した場にはマウス血液内に菌が検出されたが、実施例9の試験化合物を投与した場合には、血液内で菌が検出されなかった。
<抗腫瘍効果>
実施例9で得られた試験化合物(エマルション溶液)100μg/マウスをマウスに腹腔内へ投与し、24時間後に乳癌細胞(FM3A細胞)を腹腔内に接種した。19日後にマウスの体重測定を行った。また、横隔膜、膵臓、そして、精巣の組織切片をHE染色後、顕微鏡により観察した。その結果、乳癌細胞接種マウスは、コントロールマウスと比較して体重が約10g増加しており、多量の腹水が認められた。また、乳癌細胞接種マウスの横隔膜、膵臓、そして、精巣に著しい癌細胞の浸潤、及び、転移が観察された。一方、実施例の試験化合物で処理した後に乳癌細胞を接種したマウスは、コントロールマウスと同様の体重であり、さらに、各臓器への癌細胞の浸潤、及び、転移は全く認められなかった(図20)。
以上のように、サイトカインやケモカインはその性質により、多量に遊離されることが好ましい場合、そうでない場合、特に問題にならない場合とがあり、状況に応じて様々ではありうるが、本発明のトレハロース化合物を、IL-8やTNF-αの遊離が所望される場合や、特に問題とならない場合には、IL-8やTNF-αなどの遊離を亢進させるように、または、これらの遊離が抑制されることを意図することなく、特定の種類の本発明のトレハロース化合物を所望の量で用いることも、本発明の範囲内である。
また、本発明のトレハロース化合物の製造方法により、不斉合成を含まずに、本発明に係るトレハロース化合物を大量に効率よく合成することができる。
Claims (15)
- 以下の式(1):
[式中、
Xは、フェニル、ナフチル、または、R1-CHR2-で表される基であり、
X’は、フェニル、ナフチル、または、R1’-CHR2’-で表される基であり、
ここで、
R1、R1’、R2及びR2’は、それぞれ独立に、水素原子またはC1-C21アルキル基であり、R1、R1’、R2、R2’に関し、各アルキル基中の水素原子は、水酸基、アルコキシ基によって置換されていてもよく、各アルキル基の全部または一部は4-8員環を形成していてもよく、また、R1及びR2、R1’及びR2’は、それぞれ互いに連結して4-8員環を形成していてもよく、
n及びn’は、それぞれ独立に、0から3の整数である。
但し、
(1)Xが、R1-CHR2-であり、X’が、R1’-CHR2’-であり、R1、R1’、R2及びR2’が、それぞれ独立に、水素原子または無置換かつ直鎖のC1-C6アルキル基であり、n及びn’が0である化合物、及び、
(2)Xが、R1-CHR2-であり、X’が、R1’-CHR2’-であり、R1、R1’、R2及びR2’がC14直鎖アルキル基であり、n及びn’が0である化合物
を除く]
で表される化合物。 - 請求項1に記載の化合物であって、
Xが、R1-CHR2-であり、X’が、R1’-CHR2’-である
化合物。 - 請求項2に記載の化合物であって、
R1、R1’、R2及びR2’は、それぞれ独立に、直鎖C7-C21アルキル基であり、
n及びn’は、それぞれ独立に0または1である
化合物。 - 請求項2に記載の化合物であって、
R1、R1’、R2及びR2’は、それぞれ独立に、直鎖C8-C16アルキル基であり、
n及びn’が0である
化合物。 - 請求項2に記載の化合物であって、
R1、R1’、R2及びR2’は、それぞれ独立に、直鎖C9-C14アルキル基であ
り、
n及びn’は、1である
化合物。 - 請求項1に記載の化合物であって、
R1がR1’と同一であり、R2がR2’と同一であり、nがn’と同一である、
化合物。 - 請求項1に記載の化合物であって、以下のいずれかの化合物:
6,6’-ビス-O-(2-オクチルデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(2-ノニルウンデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(2-デシルドデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(2-ウンデシルトリデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(2-ドデシルテトラデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(2-トリデシルペンタデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(2-ペンタデシルヘプタデカノイル)-α,α’-トレハロース、又は、
6,6’-ビス-O-(2-ヘキサデシルオクタデカノイル)-α,α’-トレハロース。 - 請求項1に記載の化合物であって、以下のいずれかの化合物:
6,6’-ビス-O-(3-ノニルドデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(3-デシルトリデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(3-ウンデシルテトラデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(3-ドデシルペンタデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(3-トリデシルヘキサデカノイル)-α,α’-トレハロース、又は、
6,6’-ビス-O-(3-テトラデシルヘプタデカノイル)-α,α’-トレハロース。 - 請求項1に記載の化合物であって、以下のいずれかの化合物:
6,6’-ビス-O-(2-デシルドデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(3-ノニルドデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(3-トリデシルヘキサデカノイル)-α,α’-トレハロース、又は、
6,6’-ビス-O-(3-テトラデシルヘプタデカノイル)-α,α’-トレハロース。 - 請求項1から9のいずれかに記載の化合物及び薬理学的に許容できるキャリアを含有する医薬組成物。
- 請求項10に記載の医薬であって、免疫賦活剤、マクロファージ賦活剤、好中球賦活剤、貪食細胞の食菌作用賦活剤、抗細菌感染症剤、菌産生毒素中和剤、または、抗癌剤として用いられる医薬組成物。
- 免疫賦活剤、マクロファージ賦活剤、好中球賦活剤、貪食細胞の食菌作用賦活剤、抗細菌感染症剤、菌産生毒素中和剤、または、抗癌剤として用いられる医薬組成物の製造のための、請求項1から9のいずれかに記載の化合物の使用。
- ヒトを含む哺乳動物の感染症、または、癌の予防方法又は治療方法であって、
治療上有効量の請求項1から9のいずれかに記載の化合物を当該哺乳動物に投与することを含む方法。 - 以下の式(2):
[式中、
Xは、フェニル、ナフチル、または、R1-CHR2-で表される基であり、
X’は、フェニル、ナフチル、または、R1’-CHR2’-で表される基であり、
ここで、
R1、R1’、R2及びR2’は、それぞれ独立に、水素原子またはC1-C21アルキル基であり、R1、R1’、R2、R2’に関し、各アルキル基中の水素原子は、水酸基、アルコキシ基によって置換されていてもよく、各アルキル基の全部または一部は4-8員環を形成していてもよく、また、R1及びR2、R1’及びR2’は、それぞれ互いに連結して4-8員環を形成していてもよく、
n及びn’は、それぞれ独立に、0から3の整数である。]
で表される化合物を含有することを特徴とする菌産生毒素中和剤。 - 以下のいずれかの化合物:
6,6’-ビス-O-(2-デシルドデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(2-テトラデシルヘキサデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(3-ノニルドデカノイル)-α,α’-トレハロース、
6,6’-ビス-O-(3-トリデシルヘキサデカノイル)-α,α’-トレハロース、又は、
6,6’-ビス-O-(3-テトラデシルヘプタデカノイル)-α,α’-トレハロースを含有することを特徴とする菌産生毒素中和剤。
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CN2009801436265A CN102203110A (zh) | 2008-10-31 | 2009-10-27 | 海藻糖化合物、其制造方法以及含有该化合物的药品 |
EP09823291A EP2351764A4 (en) | 2008-10-31 | 2009-10-27 | TREHALOSE COMPOUND, PROCESS FOR PRODUCING THE SAME, AND PHARMACEUTICAL PRODUCT CONTAINING THE COMPOUND |
JP2010535655A JP5552056B2 (ja) | 2008-10-31 | 2009-10-27 | トレハロース化合物、その製造方法、及び該化合物を含有する医薬 |
US13/126,842 US8741871B2 (en) | 2008-10-31 | 2009-10-27 | Trehalose compound, method for producing same, and pharmaceutical product containing the compound |
US14/257,938 US20140248317A1 (en) | 2008-10-31 | 2014-04-21 | Trehalose compound, method for producing same, and pharmaceutical product containing the compound |
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JP2008282613 | 2008-10-31 | ||
JP2008-282613 | 2008-10-31 | ||
JP2009046824 | 2009-02-27 | ||
JP2009-046824 | 2009-02-27 |
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US13/126,842 A-371-Of-International US8741871B2 (en) | 2008-10-31 | 2009-10-27 | Trehalose compound, method for producing same, and pharmaceutical product containing the compound |
US14/257,938 Division US20140248317A1 (en) | 2008-10-31 | 2014-04-21 | Trehalose compound, method for producing same, and pharmaceutical product containing the compound |
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WO2010050178A1 true WO2010050178A1 (ja) | 2010-05-06 |
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PCT/JP2009/005650 WO2010050178A1 (ja) | 2008-10-31 | 2009-10-27 | トレハロース化合物、その製造方法、及び該化合物を含有する医薬 |
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US (2) | US8741871B2 (ja) |
EP (2) | EP2351764A4 (ja) |
JP (1) | JP5552056B2 (ja) |
KR (1) | KR20110082584A (ja) |
CN (1) | CN102203110A (ja) |
TW (1) | TW201016221A (ja) |
WO (1) | WO2010050178A1 (ja) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2012016367A1 (zh) * | 2010-08-06 | 2012-02-09 | 山东大学 | 海藻糖衍生物及其制备方法与应用 |
JP2014227404A (ja) * | 2013-05-27 | 2014-12-08 | 公益財団法人微生物化学研究会 | 新規化合物レンツトレハロース、その製造方法、及びその用途、並びに、新規微生物 |
JP2021501791A (ja) * | 2017-11-02 | 2021-01-21 | ヴィクトリア リンク リミテッド | ブラルテミシン類似体 |
JP2021514000A (ja) * | 2018-02-21 | 2021-06-03 | ザ ユニバーシティー オブ モンタナ | ジアリールトレハロース化合物及びその使用 |
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WO2014081969A1 (en) | 2012-11-21 | 2014-05-30 | University Of Louisville Research Foundation, Inc | Compositions and methods for reducing oxidative damage |
US9084720B2 (en) | 2013-05-07 | 2015-07-21 | BioBlast Pharma Ltd. | Compositions and methods for treating oculopharyngeal muscular dystrophy |
KR20160009617A (ko) | 2013-05-07 | 2016-01-26 | 바이오 블라스트 파마 리미티드 | 트레할로스의 비경구 투여에 의한 단백질 응집 근병증 및 신경퇴행성 질환의 치료 |
US11406591B2 (en) | 2015-02-09 | 2022-08-09 | University Of Louisville Research Foundation, Inc. | Ophthalmic compositions and methods for reducing oxidative damage to an eye lens |
CZ309423B6 (cs) * | 2016-06-06 | 2022-12-28 | Apigenex S.R.O. | Lipofilní deriváty trehalózy, jejich příprava a farmaceutická využitelnost |
WO2019197595A2 (en) | 2018-04-13 | 2019-10-17 | Glaxosmithkline Biologicals Sa | Compounds and uses |
CN110501483B (zh) * | 2019-07-25 | 2020-12-29 | 同济大学 | 一种中草药活性的检测方法 |
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- 2009-10-27 WO PCT/JP2009/005650 patent/WO2010050178A1/ja active Application Filing
- 2009-10-27 CN CN2009801436265A patent/CN102203110A/zh active Pending
- 2009-10-27 JP JP2010535655A patent/JP5552056B2/ja not_active Expired - Fee Related
- 2009-10-27 EP EP09823291A patent/EP2351764A4/en not_active Withdrawn
- 2009-10-27 KR KR1020117011968A patent/KR20110082584A/ko not_active Application Discontinuation
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Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012016367A1 (zh) * | 2010-08-06 | 2012-02-09 | 山东大学 | 海藻糖衍生物及其制备方法与应用 |
US8889651B2 (en) | 2010-08-06 | 2014-11-18 | Joyochem Co., Ltd. | Trehalose derivatives, preparation method and uses thereof |
JP2014227404A (ja) * | 2013-05-27 | 2014-12-08 | 公益財団法人微生物化学研究会 | 新規化合物レンツトレハロース、その製造方法、及びその用途、並びに、新規微生物 |
JP2021501791A (ja) * | 2017-11-02 | 2021-01-21 | ヴィクトリア リンク リミテッド | ブラルテミシン類似体 |
JP7360385B2 (ja) | 2017-11-02 | 2023-10-12 | ヴィクトリア リンク リミテッド | ブラルテミシン類似体 |
JP2021514000A (ja) * | 2018-02-21 | 2021-06-03 | ザ ユニバーシティー オブ モンタナ | ジアリールトレハロース化合物及びその使用 |
JP7448954B2 (ja) | 2018-02-21 | 2024-03-13 | ザ ユニバーシティー オブ モンタナ | ジアリールトレハロース化合物及びその使用 |
Also Published As
Publication number | Publication date |
---|---|
TW201016221A (en) | 2010-05-01 |
EP2351764A4 (en) | 2012-09-05 |
JP5552056B2 (ja) | 2014-07-16 |
US8741871B2 (en) | 2014-06-03 |
US20110218171A1 (en) | 2011-09-08 |
KR20110082584A (ko) | 2011-07-19 |
CN102203110A (zh) | 2011-09-28 |
EP2351764A1 (en) | 2011-08-03 |
EP2567963A1 (en) | 2013-03-13 |
EP2351764A8 (en) | 2011-09-21 |
US20140248317A1 (en) | 2014-09-04 |
JPWO2010050178A1 (ja) | 2012-03-29 |
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