U.S. patent application number 14/257938 was filed with the patent office on 2014-09-04 for trehalose compound, method for producing same, and pharmaceutical product containing the compound.
This patent application is currently assigned to GLYTECH, INC.. The applicant listed for this patent is GLYTECH, INC.. Invention is credited to Hiroshi Imagawa, Mugio Nishizawa, Masataka Oda, Jun Sakurai, Hirofumi Yamamoto.
Application Number | 20140248317 14/257938 |
Document ID | / |
Family ID | 42128548 |
Filed Date | 2014-09-04 |
United States Patent
Application |
20140248317 |
Kind Code |
A1 |
Nishizawa; Mugio ; et
al. |
September 4, 2014 |
TREHALOSE COMPOUND, METHOD FOR PRODUCING SAME, AND PHARMACEUTICAL
PRODUCT CONTAINING THE COMPOUND
Abstract
A trehalose compound having high immunopotentiating activity and
low toxicity is represented by formula (1). (In the formula, X and
X' each represents a phenyl, a naphthyl, R.sub.1--CHR.sub.1--
(wherein R.sub.1 and R.sub.2 each represents a C.sub.7-C.sub.21
alkyl group or the like) or the like; and n and n' each
independently represents an integer of 0-3). The compound exhibits
a high activating effect on macrophages and neutrophils.
##STR00001##
Inventors: |
Nishizawa; Mugio;
(Tokushima, JP) ; Imagawa; Hiroshi; (Tokushima,
JP) ; Yamamoto; Hirofumi; (Tokushima, JP) ;
Sakurai; Jun; (Tokushima, JP) ; Oda; Masataka;
(Tokushima, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GLYTECH, INC. |
Kyoto |
|
JP |
|
|
Assignee: |
GLYTECH, INC.
Kyoto
JP
|
Family ID: |
42128548 |
Appl. No.: |
14/257938 |
Filed: |
April 21, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13126842 |
May 25, 2011 |
8741871 |
|
|
PCT/JP2009/005650 |
Oct 27, 2009 |
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14257938 |
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Current U.S.
Class: |
424/278.1 |
Current CPC
Class: |
A61P 37/04 20180101;
A61P 35/00 20180101; Y02A 50/30 20180101; C07H 13/08 20130101; A61P
39/02 20180101; A61P 31/00 20180101; Y02A 50/473 20180101; C07H
13/04 20130101; A61P 31/04 20180101; A61P 43/00 20180101; C07H
13/06 20130101; A61K 31/7024 20130101 |
Class at
Publication: |
424/278.1 |
International
Class: |
C07H 13/06 20060101
C07H013/06 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 31, 2008 |
JP |
2008-282613 |
Feb 27, 2009 |
JP |
2009-046824 |
Claims
1. A method for immunostimulating; activating a macrophage;
activating a neutrophil; activating the phagocytosis of phagocytic
cells; neutralizing a bacterial toxin; or preventing or treating
infectious disease or cancer in a mammal, comprising administering
a therapeutically effective amount of a compound represented by the
following formula (1) to the mammal: ##STR00067## wherein X
represents phenyl, naphthyl, or a group represented by
R.sub.1--CHR.sub.2--, and X' represents phenyl, naphthyl, or a
group represented by R.sub.1'--CHR.sub.2'--, wherein R.sub.1,
R.sub.1', R.sub.2 and R.sub.2' independently represent a hydrogen
atom or a C.sub.1-C.sub.21 alkyl group, and with regard to R.sub.1,
R.sub.1', R.sub.2 and R.sub.2', a hydrogen atom in each alkyl group
may be replaced by an alkoxy group, all or some of alkyl groups may
form a 4- to 8-membered ring, and R.sub.1 and R.sub.2, and R.sub.1'
and R.sub.2' may bind to each other to form a 4- to 8-membered
ring, and wherein n and n' independently represent an integer of 0
to 3, with the proviso that the compound is not the following
compounds: (1) a compound, in which X represents
R.sub.1--CHR.sub.2--, X' represents R.sub.1'--CHR.sub.2'--,
R.sub.1, R.sub.1', R.sub.2 and R.sub.2' independently represent a
hydrogen atom or an unsubstituted linear C.sub.1-C.sub.6 alkyl
group, and n and n' represent 0; (2) a compound, in which X
represents R.sub.1--CHR.sub.2--, X' represents
R.sub.1'--CHR.sub.2'--, any one of R.sub.1, R.sub.1', R.sub.2 and
R.sub.2' represent a C.sub.14 linear alkyl group, and n and n'
represent 0; (3) a compound, in which X represents
R.sub.1--CHR.sub.2--, X' represents R.sub.1'--CHR.sub.2'--, all of
R.sub.1, R.sub.1', R.sub.2 and R.sub.2' represent a hydrogen atom,
and n and n' independently represent 0 to 3; (4) a compound, in
which X represents R.sub.1--CHR.sub.2--, X' represents
R.sub.1'--CHR.sub.2'--, any three of R.sub.1, R.sub.1', R.sub.2 and
R.sub.2', represent a hydrogen atom, the remaining one of R.sub.1,
R.sub.1', R.sub.2 and R.sub.2' represents a C.sub.1-C.sub.21 linear
alkyl group, and n and n' independently represent 0 to 3; (5) a
compound, in which X represents R.sub.1--CHR.sub.2--, X' represents
R.sub.1'--CHR.sub.2'--, any one of R.sub.1 and R.sub.2 represents a
hydrogen atom, any one of R.sub.1' and R.sub.2' represents a
hydrogen atom, and both the remaining one of R.sub.1 and R.sub.2
and the remaining one of R.sub.1' and R.sub.2' independently
represent a C.sub.1-C.sub.21 linear alkyl group, and n and n'
independently represent 0 to 3; and (6) a compound, in which X
represents phenyl, X' represents phenyl, and n and n' represent
0.
2. The method according to claim 1, wherein X represents
R.sub.1--CHR.sub.2-- and X' represents R.sub.1--CHR.sub.2'--.
3. The method according to claim 2, wherein R.sub.1, R.sub.1',
R.sub.2 and R.sub.2' independently represent a linear
C.sub.7-C.sub.21 alkyl group, and n and n' independently represent
0 or 1.
4. The method according to claim 2, wherein R.sub.1, R.sub.1',
R.sub.2 and R.sub.2' independently represent a linear
C.sub.8-C.sub.16 alkyl group, and n and n' independently represent
0.
5. The method according to claim 2, wherein R.sub.1, R.sub.1',
R.sub.2 and R.sub.2' independently represent a linear
C.sub.9-C.sub.14 alkyl group, and n and n' independently represent
1.
6. The method according to claim 1, wherein R.sub.1 is identical to
R.sub.1', R.sub.2 is identical to R.sub.2', and n is identical to
n'.
7. The method according to claim 1, wherein the compound is
selected from the group consisting of:
6,6'-bis-O-(2-octyldecanoyl)-.alpha.,.alpha.'-trehalose,
6,6'-bis-O-(2-nonylundecanoyl)-.alpha.,.alpha.'-trehalose,
6,6'-bis-O-(2-decyldodecanoyl)-.alpha.,.alpha.'-trehalose,
6,6'-bis-O-(2-undecyltridecanoyl)-.alpha.,.alpha.'-trehalose,
6,6'-bis-O-(2-dodecyltetradecanoyl)-.alpha.,.alpha.'-trehalose,
6,6'-bis-O-(2-tridecylpentadecanoyl)-.alpha.,.alpha.'-trehalose,
6,6'-bis-O-(2-pentadecylheptadecanoyl)-.alpha.,.alpha.'-trehalose,
and
6,6'-bis-O-(2-hexadecyloctadecanoyl)-.alpha.,.alpha.'-trehalose.
8. The method according to claim 1, wherein the compound is
selected from the group consisting of:
6,6'-bis-O-(3-nonyldodecanoyl)-.alpha.,.alpha.'-trehalose,
6,6'-bis-O-(3-decyltridecanoyl)-.alpha.,.alpha.'-trehalose,
6,6'-bis-O-(3-undecyltetradecanoyl)-.alpha.,.alpha.'-trehalose,
6,6'-bis-O-(3-dodecylpentadecanoyl)-.alpha.,.alpha.'-trehalose,
6,6'-bis-O-(3-tridecylhexadecanoyl)-.alpha.,.alpha.'-trehalose, and
6,6'-bis-O-(3-tetradecylheptadecanoyl)-.alpha.,.alpha.'-trehalose.
9. The method according to claim 1, wherein the compound is
selected from the group consisting of:
6,6'-bis-O-(2-decyldodecanoyl)-.alpha.,.alpha.'-trehalose,
6,6'-bis-O-(3-nonyldodecanoyl)-.alpha.,.alpha.'-trehalose,
6,6'-bis-O-(3-tridecylhexadecanoyl)-.alpha.,.alpha.'-trehalose, and
6,6'-bis-O-(3-tetradecylheptadecanoyl)-.alpha.,.alpha.'-trehalose.
10. A method for inducing a cytokine, wherein the cytokine is
selected from the group consisting of IL-8, TNF-.alpha., and
IFN-.gamma.; activating a neutrophil; activating the phagocytosis
of phagocytic cells; neutralizing a bacterial toxin; preventing or
treating bacterial infectious disease, wherein the bacterium is
selected from the group consisting of Welch bacillus, Pseudomonas
aeruginosa, and enteropathogenic Escherichia coli; or preventing or
treating cancer, wherein the cancer is infiltrative cancer in a
mammal, comprising administering a therapeutically effective amount
of a compound represented by the following formula (2) to the
mammal: ##STR00068## wherein X represents phenyl, naphthyl, or a
group represented by R.sub.1--CHR.sub.2--, and X' represents
phenyl, naphthyl, or a group represented by R.sub.1'--CHR.sub.2'--,
wherein R.sub.1, R.sub.1', R.sub.2 and R.sub.2' independently
represent a hydrogen atom or a C.sub.1-C.sub.21 alkyl group, and
with regard to R.sub.1, R.sub.1', R.sub.2 and R.sub.2', a hydrogen
atom in each alkyl group may be replaced by a hydroxyl group or an
alkoxy group, all or some of alkyl groups may form a 4- to
8-membered ring, and R.sub.1 and R.sub.2, and R.sub.1' and R.sub.2'
may bind to each other to form a 4- to 8-membered ring, and wherein
n and n' independently represent an integer of 0 to 3, with the
proviso that the compound is not the following compounds: (1) a
compound, in which X represents R.sub.1--CHR.sub.2--, X' represents
R.sub.1'--CHR.sub.2'--, R.sub.1, R.sub.1', R.sub.2 and R.sub.2'
independently represent a hydrogen atom or an unsubstituted linear
C.sub.1-C.sub.6 alkyl group, and n and n' represent 0; and (2) a
compound, in which X represents R.sub.1--CHR.sub.2--, X' represents
R.sub.1'--CHR.sub.2'--, R.sub.1, R.sub.1', R.sub.2 and R.sub.2'
represent a C.sub.14 linear alkyl group, and n and n' represent
0.
11. The method according to claim 10, wherein the method is
characterized for inducing a cytokine, wherein the cytokine is
selected from the group consisting of IL-8, TNF-.alpha., and
IFN-.gamma.; activating a neutrophil; or neutralizing a bacterial
toxin in a mammal.
12. A method for neutralizing a bacterial toxin in a mammal,
comprising administering a therapeutically effective amount of a
compound represented by the following formula (3) to the mammal:
##STR00069## wherein X represents phenyl, naphthyl, or a group
represented by R.sub.1--CHR.sub.2--, and X' represents phenyl,
naphthyl, or a group represented by R.sub.1'--CHR.sub.2'--, wherein
R.sub.1, R.sub.1', R.sub.2 and R.sub.2' independently represent a
hydrogen atom or a C.sub.1-C.sub.21 alkyl group, and with regard to
R.sub.1, R.sub.1', R.sub.2 and R.sub.2', a hydrogen atom in each
alkyl group may be replaced by a hydroxyl group or an alkoxy group,
all or some of alkyl groups may form a 4- to 8-membered ring, and
R.sub.1 and R.sub.2, and R.sub.1' and R.sub.2' may bind to each
other to form a 4- to 8-membered ring, and wherein n and n'
independently represent an integer of 0 to 3.
13. The method according to claim 12, wherein the compound is
selected from the group consisting of:
6,6'-bis-O-(2-decyldodecanoyl)-.alpha.,.alpha.'-trehalose,
6,6'-bis-O-(2-tetradecylhexadecanoyl)-.alpha.,.alpha.'-trehalose,
6,6'-his-O-(3-nonyldodecanoyl)-.alpha.,.alpha.'-trehalose,
6,6'-bis-O-(3-tridecylhexadecanoyl)-.alpha.,.alpha.'-trehalose, and
6,6'-bis-O-(3-tetradecylheptadecanoyl)-.alpha.,.alpha.'-trehalose.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a divisional application of and,
thereby, claims benefit under 35 U.S.C. .sctn.120 to U.S. patent
application Ser. No. 13/126,842 filed on Apr. 29, 2011, titled,
"TREHALOSE COMPOUND, METHOD FOR PRODUCING SAME, AND PHARMACEUTICAL
PRODUCT CONTAINING THE COMPOUND," which is a national stage
application of PCT Application No. PCT/JP2009/005650, filed on Oct.
27, 2009, which claims priority to Japanese Patent Application No.
2009-046824 filed on Feb. 27, 2009 and Japanese Patent Application
No. 2008-282613 filed on Oct. 31, 2008. This application claims the
benefit and priority of all prior applications and incorporates
their disclosures by reference in their entireties.
TECHNICAL FIELD
[0002] The present invention relates to a trehalose compound, a
method for producing the same, and a pharmaceutical product
comprising the same.
BACKGROUND ART
[0003] Various types of infectious diseases, such as those caused
by bacteria, viruses or fungi, have been known. As a therapeutic
method for infectious diseases caused by bacteria, administration
of antibiotics has been carried out. However, such administration
of antibiotics has been problematic in terms of the emergence of
microbes resistant to antibiotics. It has also caused in-hospital
infection of such resistant microbes. In addition, administration
of antibiotics has also been problematic in terms of opportunistic
infection, which is common in immune-compromised patients who are
infected with HIV, who are under treatment with anticancer agents,
and who are elder people or children, etc.
[0004] Moreover, in the case of infection with enteropathogenic
Escherichia coli such as O-157 or Shigella dysenteriae, verotoxin
is generated in vivo, and severe symptoms such as combination with
hemolytic uremic syndrome may occur particularly in the case of
elder people or children with compromised immune systems. To such
infectious diseases, antibiotics may be administered in some cases.
However, it has been pointed out that administration of antibiotics
may cause to kill bacteria, and as a result, toxin existing in the
bacteria may be released to outside of the bacteria at once, so
that the condition may be deteriorated. On the other hand, in the
case of highly contagious infection diseases, such as O-157, which
are transmissible only by ingestion of several hundreds of to
several thousands of bacteria, there may be cases in which
administration of antibiotics should be selected to prevent
secondary infection. With regard to other therapeutic methods,
since approximately 10% of all the O-157 infection cases may
combine with hemolytic uremic syndrome, plasma exchange, dialysis
therapy, and the like are carried out on such O-157 infection
cases. These are all therapeutic methods which may impose an undue
burden on patients.
[0005] Furthermore, as a method for coping with toxin generated by
bacteria, symptomatic therapy is mainly carried out. Other methods
include the use of a toxin adsorbent, the use of an antibody
against such toxin, and the like. Such adsorption methods involving
the use of activated carbon or the like may cause side effects such
as constipation. Further, the use of an antibody is inconvenient in
that an antibody must be developed against each type of toxin.
[0006] Thus, a method for treating infectious disease caused by
such pathogenic bacteria or a method for suppressing the onset of
such infectious disease has been searched for. Recently, it has
been attempted to treat or prevent infectious diseases by enhancing
a patient's own immune system without administering
antibiotics.
[0007] Various compounds have been studied in order to find out
substances for enhancing the patient's own immune system. As
examples of ingredients obtained from natural products, trehalose
dimycolate (TDM) and trehalose dicorynomycolate (TDCM), which are
diester compounds of trehalose, have been known. TDM has been
discovered as a glycolipid existing on the cell cortex of
Mycobacterium tuberculosis, and it has been known to exhibit
immunostimulatory activity and anticancer activity. Moreover, TDCM
has been isolated, as a homolog having fewer carbon atoms than
those of TDM, from related Corynebacterium spp. It has been
revealed that both TDCM and a stereoisomer thereof exhibit
immunostimulatory activity and anticancer activity.
[0008] However, TDM and TDCM are highly toxic, and thus, they
cannot be used as pharmaceutical products. Accordingly, in order to
use TDM and TDCM as pharmaceutical products, it has been necessary
to synthesize compounds with reduced toxicity, while maintaining or
enhancing their activity.
[0009] Hence, a trehalose 6,6'-diester compound, which is a
trehalose fatty acid ester composition, has been synthesized as a
TDM derivative. Various tests, such as a toxicity test and a
macrophage activating test, have been performed on the trehalose
6,6'-diester compound (see Non Patent Literature 1). In this
publication, the presence or absence of a .beta.-hydroxyl group,
compounds having 30, 32 or 48 carbon atoms as a length of the alkyl
portion of a lipid, compounds in which the ester bond between a
sugar and a lipid has been replaced by an amide bond, etc. have
been studied. In terms of toxicity, this publication describes that
an ester bond and a long chain fatty acid greatly contribute to
toxicity. This publication describes a compound having 30 carbon
atoms as a length of a side chain fatty acid portion. However, in
order to obtain a compound having high activity and low toxicity,
the binding manner of a sugar and a side chain, the presence or
absence of a substituent for a side chain fatty acid, the length of
an alkyl group constituting such a side chain fatty acid, and the
like have not been comprehensively studied and have not been
optimized. Tests have been just sporadically carried out on several
compounds as described above. Even with regard to immunostimulatory
action, the contents thereof have not been studied in detail.
[0010] On the other hand, with regard to a TDM derivative, the
present inventors have synthesized a TDM derivative having an ester
bond or an amide bond, in which the .beta.-hydroxyl group has been
replaced by a hydrogen atom or a methoxy group (see Patent
Literature 1). However, the derivative described in this
publication has a comparatively short alkyl portion of fatty acid
(approximately 7 carbon atoms). With regard to its activity, only
activity as an adenosine A3 receptor antagonist was measured.
[0011] The present inventors have succeeded in substituting the
hydroxyl group of TDCM with a hydrogen atom, so that it could not
be an asymmetric carbon atom, as well as in synthesizing an amide
derivative of TDCM, in which the ester bond has been replaced by an
amide bond. The inventors have then confirmed that these amide
derivatives have immunostimulatory action (see Patent Literature
2). However, thereafter, it has been found that these amide
derivatives have action to induce cancer, and as a result, it could
not help giving up the use of the amide derivatives as
pharmaceutical compounds.
[0012] Therefore, a sufficiently effective and highly safe method
for treating various symptoms caused by pathogenic bacteria or
suppressing the onset of such symptoms, using such a TDM or TDCM
derivative, has not yet been established. Thus, it has been desired
to develop an effective and safe method for treating the symptoms
caused by pathogenic bacteria or suppressing the onset of the
symptoms.
CITATION LIST
Patent Literature
[0013] [Patent Literature 1] WO2007/111214 [0014] [Patent
Literature 2] WO2008/093700
Non Patent Literature
[0015] [Non Patent Literature 1] Numata et al., Chem. Pharm. Bull.
(1985), 33(10), 4544-4555
SUMMARY OF INVENTION
Technical Problem
[0016] When TDM and TDCM derivatives are synthesized, since TDM and
TDCM themselves are highly toxic, it is necessary to synthesize
compounds having activity and also having low toxicity. As prior
art techniques, several TDM and TDCM modified bodies have been
known. However, TDM and TDCM are glycolipids, their sugar chain
contains many hydroxyl groups and has high polarity, and it is
difficult to synthesize them. Thus, structure activity correlation,
such as the relationship between type of structure and the type of
activity, has not yet been clarified.
[0017] Under the aforementioned circumstances, it is an object of
the present invention to produce a large number of TDM and TDCM
derivatives and to provide a compound having high activity and low
toxicity and a pharmaceutical product comprising the compound.
[0018] Moreover, in prior art techniques, the use of antibiotics
against pathogenic bacteria has been directed towards inhibition of
the growth of Escherichia coli, the killing of Escherichia coli,
and the like, which resulted in prevention of the release of toxin
from the Escherichia coli. Thus, antibiotics were not able to
detoxify the generated toxin itself. In contrast, it is an object
of the present invention to provide a pharmaceutical product
capable of reducing the toxicity of a toxin, even in a case in
which bacteria have grown to such an extent that they generate the
toxin.
Solution to Problem
[0019] The present inventors have found that a trehalose diester
compound represented by formula (1) exhibits excellent
antibacterial activity on infectious diseases caused by pathogenic
bacteria and has low toxicity. In addition, the inventors have also
found that, among the trehalose diester compounds represented by
the formula (1), a compound, in which X represents
R.sub.1--CHR.sub.2--, X' represents R.sub.1'--CHR.sub.2'--, and as
a branched form of a lipid portion thereof, particularly n and n'
each represent 0 (hereinafter referred to as an .alpha.-branched
compound), and particularly, n and n' each represent 1 (hereinafter
referred to as a .beta.-branched compound), is particularly
excellent. Moreover, the inventors have also found that, among the
trehalose diester compounds represented by the formula (1), a
compound, in which an alkyl chain portion represented by R.sub.1,
R.sub.2, R.sub.1' or R.sub.2' in the formula (1) has a specific
length (the number of carbon atoms is 10, 14 or the like), tends to
have maximum activity. Furthermore, the aforementioned publications
and the like do not describe that the trehalose diester compound is
useful as an antibacterial agent, even when toxin generated by
bacteria is administered. In contrast, the present inventors have
found in mouse in-vivo tests that the present trehalose diester
compound is useful, when not only bacteria, but also toxin
generated by such bacteria is administered to mice.
[0020] The present invention provides a compound represented by the
following formula (1):
##STR00002##
[0021] wherein X represents phenyl, naphthyl, or a group
represented by R.sub.1--CHR.sub.2--, and X' represents phenyl,
naphthyl, or a group represented by R.sub.1'--CHR.sub.2'--,
[0022] wherein R.sub.1, R.sub.1', R.sub.2 and R.sub.2'
independently represent a hydrogen atom or a C.sub.1-C.sub.21
alkoxy group, and with regard to R.sub.1, R.sub.1', R.sub.2 and
R.sub.2', a hydrogen atom in each alkyl group may be replaced by a
hydroxyl group or an alkoxy group, all or some of alkyl groups may
form a 4- to 8-membered ring, and R.sub.1 and R.sub.2, and R.sub.1'
and R.sub.2' may bind to each other to form a 4- to 8-membered
ring, and
[0023] wherein n and n' independently represent an integer of 0 to
3,
[0024] with the proviso that that the compound is not the following
compounds:
(1) a compound, in which X represents R.sub.1--CHR.sub.2--, X'
represents R.sub.1'--CHR.sub.2'--, R.sub.1, R.sub.1', R.sub.2 and
R.sub.2' independently represent a hydrogen atom or an
unsubstituted linear C.sub.1-C.sub.6 alkyl group, and n and n'
represent 0; and (2) a compound, in which X represents
R.sub.1--CHR.sub.2--, X' represents R.sub.1'--CHR.sub.2'--,
R.sub.1, R.sub.1', R.sub.2 and R.sub.2' represent a C.sub.14 linear
alkyl group, and n and n' represent 0.
[0025] In addition, the present invention provides a pharmaceutical
composition comprising the compound represented by the formula (1)
and a pharmaceutically acceptable carrier.
[0026] Moreover, the present invention provides the pharmaceutical
composition comprising the compound represented by the formula (1)
and a pharmaceutically acceptable carrier, wherein the
pharmaceutical composition is used as an immunostimulator, a
macrophage activator, a neutrophil activator, an agent for
activating the phagocytosis of phagocytic cells, an anti-bacterial
infection agent, or a bacterial toxin neutralizer.
[0027] Furthermore, the present invention provides use of the
compound represented by the formula (1) in the manufacture of a
pharmaceutical composition for use as an immunostimulator, a
macrophage activator, a neutrophil activator, an agent for
activating the phagocytosis of phagocytic cells, an anti-bacterial
infection agent, or a bacterial toxin neutralizer.
[0028] Further, the present invention provides a method for
preventing or treating the infectious disease in a mammal including
a human, comprising administering a therapeutically effective
amount of the compound represented by the formula (1) to the
mammal.
[0029] Still further, the present invention provides a bacterial
toxin neutralizer, comprising a compound represented by the
following formula (2):
##STR00003##
[0030] wherein X represents phenyl, naphthyl, or a group
represented by R.sub.1--CHR.sub.2--, and
X' represents phenyl, naphthyl, or a group represented by
R.sub.1'--CHR.sub.2'--,
[0031] wherein R.sub.1, R.sub.1', R.sub.2 and R.sub.2'
independently represent a hydrogen atom or a C.sub.1-C.sub.21
alkoxy group, and with regard to R.sub.1, R.sub.1', R.sub.2 and
R.sub.2', a hydrogen atom in each alkyl group may be replaced by a
hydroxyl group or an alkoxy group, or all or some of alkyl groups
may form a 4- to 8-membered ring, or R.sub.1 and R.sub.2, and
R.sub.1' and R.sub.2' may bind to each other to form a 4- to
8-membered ring, and
[0032] wherein n and n' independently represent an integer of 0 to
3.
Advantageous Effects of Invention
[0033] Since the trehalose compound of the present invention has
high immunostimulatory activity and also has low toxicity, it is
useful for providing an excellent pharmaceutical product, which
highly acts on infectious diseases caused by pathogenic
bacteria.
[0034] The trehalose compound of the present invention has action
to activate cellular immunity. The present trehalose compound
activates neutrophils or macrophages, so as to enhance their
phagocytosis, so as to exhibit antibacterial action. Specifically,
according to the compound of the present invention, since bacteria
themselves are ingested by neutrophils or macrophages, the amount
of toxin released to outside of the bacteria is small. As a result,
the present invention is able to provide a low-risk pharmaceutical
product, which may not cause the release of toxin due to
destruction of Escherichia coli upon administration of
antibiotics.
[0035] Moreover, the trehalose compound of the present invention
exhibits toxicity-reducing action even on toxin itself. Thus, the
present invention is able to provide a pharmaceutical product,
which is effective even in a case in which the degree of infection
in an infectious disease caused by Escherichia coli has progressed
and such Escherichia coli has grown and has released toxin to
outside of the bacteria.
[0036] Furthermore, as a result of activation of neutrophils or
macrophages by administration of the trehalose compound of the
present invention, they can ingest multi-drug-resistant bacteria
generated as a result of administration of antibiotics, as well as
non-resistant bacteria. Accordingly, the present invention is able
to provide a pharmaceutical product having therapeutic effects also
on infectious diseases caused by multi-drug-resistant bacteria.
[0037] Further, the trehalose compound of the present invention
activates cellular immunity, but it does not cause excessive immune
response. Accordingly, the present invention is able to provide a
pharmaceutical product, which hardly causes a risk of such
excessive immune response that an antibody against an antibody
administered as a pharmaceutical product may be generated.
[0038] Still further, the trehalose compound of the present
invention includes compounds that do not contain asymmetric carbon
atoms. That is to say, the trehalose compound according to the
present invention can be efficiently synthesized in a large volume
by a method for producing the trehalose compound of the present
invention, without involving asymmetric synthesis.
BRIEF DESCRIPTION OF DRAWINGS
[0039] FIG. 1 shows the amount of active oxygen released from mouse
intraperitoneal macrophages in a case in which TDCM, a vehicle or
the test compound of the present invention was allowed to act on
the mouse intraperitoneal macrophages.
[0040] FIG. 2 shows the phagocytic ability of mouse intraperitoneal
macrophages in a case in which TDCM, a vehicle or the test compound
of the present invention was allowed to act on the mouse
intraperitoneal macrophages.
[0041] FIG. 3 shows the amount of active oxygen released from
rabbit neutrophils in a case in which TDCM or the test compound of
the present invention was allowed to act on the rabbit
neutrophils.
[0042] FIG. 4 shows the phagocytic ability of rabbit neutrophils in
a case in which TDCM or the test compound of the present invention
was allowed to act on the rabbit neutrophils.
[0043] FIG. 5 shows the amount of IL-8 released from human-derived
THP-1 cells in a case in which TDCM, a vehicle or the test compound
of the present invention was allowed to act on the human-derived
THP-1 cells.
[0044] FIG. 6 shows the concentration of IL-6 in mouse plasma in a
case in which TDCM, a vehicle or the test compound of the present
invention was administered to mice.
[0045] FIG. 7 shows the concentration of IFN-.gamma. in mouse
plasma in a case in which TDCM, a vehicle or the test compound of
the present invention was administered to mice.
[0046] FIG. 8 shows the concentration of TNF-.alpha. in mouse
plasma in a case in which TDCM, a vehicle or the test compound of
the present invention was administered to mice.
[0047] FIGS. 9A and 9B show the amount of MTP-1.beta. and the
amount of TNF-.alpha., respectively, released from human-derived
cells in a case in which TDCM, a vehicle or the test compound of
the present invention was allowed to act on the human-derived
cells. The value was indicated with a mean.+-.S.D. (n=5). The
symbol * indicates the case of P<0.01, compared with the
control.
[0048] FIG. 10 shows the cytotoxicity of TDCM, a vehicle or the
test compound of the present invention in human-derived THP-1
cells. The white bar shows the results obtained by treating the
cells with the test compound for 2 hours, whereas the black bar
shows the results obtained by treating the cells with the test
compound for 24 hours. The value was indicated with a mean.+-.S.D.
(n=5). The symbol * indicates the case of P <0.01, compared with
the control.
[0049] FIGS. 11A and 11B show the measurement results of the
mutagenicity of TDCM, a vehicle or the test compound of the present
invention in the absence or in the present of S9 mix, respectively,
according to an Ames test.
[0050] FIGS. 12A and 12B-D show the number of cells and microscopic
images, respectively, obtained in a case in which a vehicle or the
test compound of the present invention was allowed to act on mouse
intraperitoneal infiltrating cells. The value was indicated with a
mean.+-.S.D. (n=5). The symbol * indicates the case of P<0.01,
compared with the control.
[0051] FIGS. 13A and 13B show the results of Giemsa staining, which
were obtained in a case in which a vehicle or the test compound of
the present invention, respectively, was allowed to act on mouse
intraperitoneal infiltrating cells.
[0052] FIGS. 14A-B and 14C-D show the images of CD-8-positive cells
in mouse intraperitoneal infiltrating cells, on which a vehicle or
the test compound of the present invention, respectively, was
allowed to act, which were observed under a fluorescent
microscope.
[0053] FIGS. 15A and 15B show the measurement results of
CD-8-positive cells in mouse intraperitoneal infiltrating cells, on
which a vehicle or the test compound of the present invention,
respectively, was allowed to act, which were obtained by flow
cytometry.
[0054] FIG. 16A shows the influence of a vehicle or the test
compound of the present invention on the number of surviving mice,
to which each of the above described compounds was administered and
then Welch bacillus was then administered. FIG. 16B shows
experimental schedule. The symbol * indicates the case of
P<0.01, compared with the control. The test was carried out 8
times, and standard results were shown.
[0055] FIG. 17A shows the influence of a vehicle or the test
compound of the present invention on the number of surviving mice,
to which each of the above described compounds was administered and
then Pseudomonas aeruginosa was then administered. FIG. 17B shows
experimental schedule. The symbol * indicates the case of
P<0.01, compared with the control. The test was carried out 8
times, and standard results were shown.
[0056] FIG. 18A shows the influence of a vehicle or the test
compound of the present invention on the number of surviving mice,
to which each of the above described compounds was administered
after completion of the administration of Pseudomonas
aeruginosa.
[0057] FIG. 18B shows experimental schedule. The symbol * indicates
the case of P<0.01, compared with the control. The test was
carried out 8 times, and standard results were shown.
[0058] FIG. 19 shows the number of Pseudomonas aeruginosa cells in
mouse blood, in a case in which Pseudomonas aeruginosa was
administered to mice, to which a vehicle or the test compound of
the present invention had been administered.
[0059] FIGS. 20A and 20B show the influence of a vehicle or the
test compound of the present invention on body weight and cancer
metastasis in breast cancer cell-inoculated mice, respectively.
[0060] FIG. 20C shows experimental schedule.
DETAILED DESCRIPTION
[0061] Hereinafter, preferred embodiments of the present invention
will be described. The compound represented by the formula (1) may
be present in the form of a pharmaceutically acceptable salt or
solvate thereof.
[0062] The term "C.sub.1-C.sub.21 alkyl group" is used in the
present specification to include an alicyclic hydrocarbon group in
which all or some of aliphatic hydrocarbon groups form a 4- to
8-membered ring, as well as a linear or branched aliphatic
hydrocarbon group containing 1 to 21 carbon atoms. In a preferred
aspect, the "C.sub.1-C.sub.21 alkyl group" of the compound
represented by the formula (1) of the present invention is a linear
aliphatic hydrocarbon group. Examples of the "C.sub.1-C.sub.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, an n-heptyl
group, an n-octyl group, an n-nonyl group, an n-decyl group, an
n-undecyl group, an n-dodecyl group, an n-tridecyl group, an
n-tetradecyl group, an n-pentadecyl group, an n-hexadecyl group, an
n-heptadecyl group, an n-octadecyl group, an n-nonadecyl group, an
n-icosyl group, and an n-henicosyl group. Examples of an alicyclic
hydrocarbon group in which all of aliphatic hydrocarbon groups form
a ring include a cyclobutyl group, a cyclopentyl group, a
cyclohexyl group, a cycloheptyl group, and a cyclooctyl group.
Examples of an alicyclic hydrocarbon group in which some of
aliphatic hydrocarbon groups form a ring include a
cyclohexyl-n-octyl group, a cyclohexyl-n-nonyl group, and a
cycloheptyl-n-octyl group. R.sub.1, R.sub.1', R.sub.2 or R.sub.2'
is preferably a linear alkyl group, and more preferably a linear
alkyl group containing 10 to 16 carbon atoms. It is particularly
preferably an n-decyl group which is a linear alkyl group
containing 10 carbon atoms, when n is 0, or it is particularly
preferably an n-nonyl group which is a linear alkyl group
containing 9 carbon atoms, an n-tridecyl group which is a linear
alkyl group containing 13 carbon atoms, or an n-tetradecyl group
which a linear alkyl group containing 14 carbon atoms, when n is 1.
It is most preferably an n-decyl group.
[0063] With regard to R.sub.1, R.sub.1', R.sub.2 and R.sub.2', the
hydrogen atom in each alkyl group may be replaced by a hydroxyl
group or an alkoxy group. The term "alkoxy group" is used herein to
mean a substituent group having a structure in which a linear or
branched aliphatic hydrocarbon group containing 1 to 21 carbon
atoms binds to an oxygen atom. Examples of such an alkoxy group
include a methoxy group, an ethoxy group, a propoxy group, a butoxy
group, a pentyloxy group, a hexyloxy group, and a heptyloxy group.
It is preferably a linear alkoxy group. Examples of the alkyl group
substituted with such an alkoxy group include a methoxy dodecyl
group, an ethoxy undecyl group, a propoxy decyl group, a pentyloxy
nonyl group, a hexyloxy octyl group, a hexyloxy heptyl group, and a
pentyloxy octyl group. When the hydrogen atom in each alkyl group
is replaced by a hydroxyl group or an alkoxy group, the
substitution position may be anywhere in each alkyl group. It is
preferably a compound in which a hydrogen atom binding to the
terminal carbon atom of the alkyl group is replaced by a hydroxyl
group or an alkoxy group. When such an alkoxy group binds to the
terminal carbon atom of the alkyl group, it adopts a linear ether
structure to which a hydrocarbon group binds via an oxygen atom.
With regard to the sum of intervening oxygen atoms and carbon atoms
constituting a hydrocarbon group, as with the aforementioned length
of the alkyl group of a hydrocarbon group, the number of carbon and
oxygen atoms constituting an alkoxyalkyl group is preferably 2 to
21, and more preferably 10 to 16 when n is 0, and 9 to 15 when n is
1.
[0064] R.sub.1 and R.sub.2, and R.sub.1' and R.sub.2' may bind to
each other to form a 4- to 8-membered ring. When X is
R.sub.1--CHR.sub.2--, the carbon atom to which R.sub.1 and R.sub.2
bind, and such R.sub.1 and R.sub.2 all become constituent atoms of
the 4- to 8-membered ring. In addition, alkyl groups constituting
R.sub.1 and R.sub.2 may be branched alkyl groups, and in such a
case, some of the branched alkyl groups may constitute the 4- to
8-membered ring, so that it may adopt such a structure as
cycloalkyl substituted with an alkyl group. Moreover, the cases in
which a ring is formed include the cases of being replaced by the
aforementioned substituents. The same applies not only to X but
also to X'. Examples of such a 4- to 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 compound, the 4- to 8-membered ring is
preferably a cyclohexyl group or a cycloheptyl group.
[0065] R.sub.1 and R.sub.2, and R.sub.1' and R.sub.2' may be
identical to or different from each other. From the viewpoint of
synthetic efficiency, preferably, R.sub.1 is identical to R.sub.1',
and R.sub.2 is identical to R.sub.2'.
[0066] Moreover, with regard to the relationship between R.sub.1
and R.sub.2, R.sub.1 and R.sub.2 may be identical to or different
from each other. In one of the preferred aspects, it is a compound,
in which the number of carbon atoms of R.sub.1 is identical to the
number of carbon atoms of R.sub.2, or the number of carbon atoms of
R.sub.1 is larger (1 or 2 carbon atoms) or smaller (1 or 2 carbon
atoms) than the number of carbon atoms of R.sub.2. For example, it
is a compound, in which R.sub.1 has 14 carbon atoms and R.sub.2 has
16 carbon atoms. Other than the above described compound, preferred
examples also include a compound, in which R.sub.1 is a short chain
alkyl group containing 1 to 5 carbon atoms and R.sub.2 is a long
chain alkyl group containing 10 to 16 carbon atoms, and a compound,
in which R.sub.1 is a long chain alkyl group containing 10 to 16
carbon atoms and R.sub.2 is a short chain alkyl group containing 1
to 5 carbon atoms. The relationship between R.sub.1' and R.sub.2'
is the same as the aforementioned relationship between R.sub.1 and
R.sub.2. Thus, it is possible to read R.sub.1 as R.sub.1', and
R.sub.2 as R.sub.2', in the aforementioned relationship between
R.sub.1 and R.sub.2.
[0067] n and n' may be identical to or different from each other.
From the viewpoint of synthetic efficiency, n is preferably
identical to n'. In addition, from the viewpoint of activity, a
compound in which n or n' is 0, and a compound in which n or n' is
1, are preferable.
[0068] Trehalose has three isomers, namely, an .alpha.,.alpha.'
form, an .alpha.,.beta.' form, and a .beta.,.beta.' form. Of these,
an .alpha.,.alpha.' form is preferable as the trehalose compound of
the present invention.
[0069] The compound represented by the formula (1) and a salt
thereof may be present in the form of a solvate. Such a solvate is
included in the scope of the present invention. Moreover,
radiolabeled compounds of the aforementioned compound represented
by the formula (1), which are useful for biological studies, are
also included in the scope of the present invention.
[0070] The compound of the present invention is preferably the
aforementioned compound represented by the formula (1). Each
substituent in the formula has the following characteristics. The
following characteristics can be independently selected, singly or
in combination, unless they include contradictions.
(A) X is a group represented by R.sub.1--CHR.sub.2--. (B) X' is a
group represented by R.sub.1'--CHR.sub.2'--. (C) R.sub.1 and
R.sub.1' independently represent an unsubstituted C.sub.1-C.sub.21
alkyl group. (D) R.sub.2 and R.sub.2' independently represent a
hydrogen atom or an unsubstituted C.sub.1-C.sub.21 alkyl group. (E)
R.sub.1 and R.sub.1'' independently represent a linear
C.sub.1-C.sub.21 alkyl group. (F) R.sub.2 and R.sub.2'
independently represent a hydrogen atom or a linear
C.sub.1-C.sub.21 alkyl group. (G) R.sub.1 and R.sub.1'
independently represent an unsubstituted and linear
C.sub.1-C.sub.21 alkyl group. (H) R.sub.2 and R.sub.2'
independently represent a hydrogen atom, or an unsubstituted and
linear C.sub.1-C.sub.21 alkyl group. (I) R.sub.1 and R.sub.1'
independently represent an unsubstituted and linear
C.sub.7-C.sub.21 alkyl group. (J) R.sub.2 and R.sub.2'
independently represent an unsubstituted and linear
C.sub.7-C.sub.21 alkyl group. (K) R.sub.1 and R.sub.1' are
identical to each other, and represent an unsubstituted
C.sub.1-C.sub.21 alkyl group. (L) R.sub.2 and R.sub.2' are
identical to each other, and represent a hydrogen atom or an
unsubstituted C.sub.1-C.sub.21 alkyl group. (M) R.sub.1 and
R.sub.1' are identical to each other, and represent a linear
C.sub.1-C.sub.21 alkyl group. (N) R.sub.2 and R.sub.2' are
identical to each other, and represent a hydrogen atom or a linear
C.sub.1-C.sub.21 alkyl group. (O) R.sub.1 and R.sub.1' are
identical to each other, and represent an unsubstituted and linear
C.sub.1-C.sub.21 alkyl group. (P) R.sub.2 and R.sub.2' are
identical to each other, and represent a hydrogen atom, or an
unsubstituted and linear C.sub.1-C.sub.21 alkyl group. (Q) R.sub.1
and R.sub.1' are identical to each other, and represent an
unsubstituted and linear C.sub.7-C.sub.21 alkyl group. (R) R.sub.2
and R.sub.2' are identical to each other, and represent an
unsubstituted and linear C.sub.7-C.sub.21 alkyl group. (S) n and n'
independently represent 0 or 1. (T) n and n' are 0. (U) n and n'
are 1.
[0071] The compound of the present invention is preferably the
compound represented by the formula (1), or a pharmaceutically
acceptable salt or solvate thereof, which has the following
structures.
(V) X is a group represented by R.sub.1--CHR.sub.2--; X' is a group
represented by R.sub.1'--CHR.sub.2'--, wherein R.sub.1, R.sub.1',
R.sub.2 and R.sub.2' independently represent a hydrogen atom or a
C.sub.1-C.sub.21 alkyl group, and with regard to such R.sub.1,
R.sub.1', R.sub.2 and R.sub.2', the hydrogen atom in each alkyl
group may be replaced by a hydroxyl group or an alkoxy group, all
or some of alkyl groups may form a 4- to 8-membered ring, and
R.sub.1 and R.sub.2, and R.sub.1' and R.sub.2' may bind to each
other to form a 4- to 8-membered ring; and n and n' independently
represent an integer of 0 to 3. (W) X is a group represented by
R.sub.1--CHR.sub.2--; X' is a group represented by
R.sub.1'--CHR.sub.2'--, wherein R.sub.1, R.sub.1', R.sub.2 and
R.sub.2' independently represent a linear C.sub.7-C.sub.21 alkyl
group, and with regard to such R.sub.1, R.sub.1', R.sub.2 and
R.sub.2', the hydrogen atom in each alkyl group may be replaced by
a hydroxyl group or an alkoxy group, and all or some of alkyl
groups may form a 4- to 8-membered ring; and n and n' independently
represent an integer of 0 or 1. (X) X is a group represented by
R.sub.1--CHR.sub.2--; X' is a group represented by
R.sub.1'--CHR.sub.2'--; wherein R.sub.1, R.sub.1', R.sub.2 and
R.sub.2' independently represent a C.sub.8-C.sub.16 alkyl group,
and with regard to such R.sub.1, R.sub.1', R.sub.2 and R.sub.2',
the hydrogen atom in each alkyl group may be replaced by a hydroxyl
group or an alkoxy group, and all or some of alkyl groups may form
a 4- to 8-membered ring; and n and n' independently represent 0.
(Y) X is a group represented by R.sub.1--CHR.sub.2--; X' is a group
represented by R.sub.1'--CHR.sub.2'--, wherein R.sub.1, R.sub.1',
R.sub.2 and R.sub.2' independently represent a C.sub.8-C.sub.14
alkyl group, and with regard to such R.sub.1, R.sub.1', R.sub.2 and
R.sub.2', the hydrogen atom in each alkyl group may be replaced by
a hydroxyl group or an alkoxy group, and all or some of alkyl
groups may form a 4- to 8-membered ring; and n and n' independently
represent 1. (Z) X is a group represented by R.sub.1--CHR.sub.2--;
X' is a group represented by R.sub.1'--CHR.sub.2'--; wherein
R.sub.1 and R.sub.1' are identical to each other and represent a
hydrogen atom or a C.sub.1-C.sub.21 alkyl group, and with regard to
such R.sub.1 and R.sub.1', the hydrogen atom in each alkyl group
may be replaced by a hydroxyl group or an alkoxy group, and all or
some of alkyl groups may form a 4- to 8-membered ring; R.sub.2 and
R.sub.2' are identical to each other and represent a hydrogen atom
or a C.sub.1-C.sub.21 alkyl group, and with regard to such R.sub.1
and R.sub.2', the hydrogen atom in each alkyl group may be replaced
by a hydroxyl group or an alkoxy group, and all or some of alkyl
groups may form a 4- to 8-membered ring; R.sub.1 and R.sub.2, and
R.sub.1' and R.sub.2' may bind to each other to form a 4- to
8-membered ring; and n and n' are identical to each other and
represent an integer of 0 to 3. (AA) X is a group represented by
R.sub.1--CHR.sub.2--; X' is a group represented by
R.sub.1'--CHR.sub.2'--; wherein R.sub.1 and R.sub.1' are identical
to each other and represent a C.sub.1-C.sub.21 alkyl group, and the
hydrogen atom in each alkyl group may be replaced by a hydroxyl
group or an alkoxy group; R.sub.2 and R.sub.2' are identical to
each other and represent a C.sub.7-C.sub.21 alkyl group, and the
hydrogen atom in each alkyl group may be replaced by a hydroxyl
group or an alkoxy group; and n and n' are identical to each other
and represent 0 or 1. (BB) X is a group represented by
R.sub.1--CHR.sub.2--; X' is a group represented by
R.sub.1'--CHR.sub.2'--; wherein R.sub.1 and R.sub.1' are identical
to each other and represent a C.sub.7-C.sub.21 alkyl group, and the
hydrogen atom in each alkyl group may be replaced by a hydroxyl
group or an alkoxy group; R.sub.2 and R.sub.2' are identical to
each other and represent a hydrogen atom or a C.sub.1-C.sub.21
alkyl group, and the hydrogen atom in each alkyl group may be
replaced by a hydroxyl group or an alkoxy group; and n and n' are
identical to each other and represent 0 or 1. (CC) X is a group
represented by R.sub.1--CHR.sub.2--; X' is a group represented by
R.sub.1'--CHR.sub.2'--; wherein R.sub.1 and R.sub.1' are identical
to each other and represent a linear C.sub.7-C.sub.21 alkyl group,
and the hydrogen atom in each alkyl group may be replaced by a
hydroxyl group or an alkoxy group; R.sub.2 and R.sub.2' are
identical to each other and represent a linear C.sub.7-C.sub.21
alkyl group, and the hydrogen atom in each alkyl group may be
replaced by a hydroxyl group or an alkoxy group; and n and n' are
identical to each other and represent 0 or 1. (DD) X is a group
represented by R.sub.1--CHR.sub.2--; X' is a group represented by
R.sub.1'--CHR.sub.2'--; wherein R.sub.1 and R.sub.1' are identical
to each other and represent an unsubstituted and linear
C.sub.1-C.sub.21 alkyl group; R.sub.2 and R.sub.2' are identical to
each other, and represent a hydrogen atom, or an unsubstituted and
linear C.sub.7-C.sub.21 alkyl group; and n and n' are identical to
each other and represent 0 or 1. (EE) X is a group represented by
R.sub.1--CHR.sub.2--; X' is a group represented by
R.sub.1'--CHR.sub.2'--; wherein R.sub.1 and R.sub.1' are identical
to each other and represent an unsubstituted and linear
C.sub.7-C.sub.21 alkyl group; R.sub.2 and R.sub.2' are identical to
each other, and represent a hydrogen atom, or an unsubstituted and
linear C.sub.1-C.sub.21 alkyl group; and n and n' are identical to
each other and represent 0 or 1. (FF) X is a group represented by
R.sub.1--CHR.sub.2--; X' is a group represented by
R.sub.1'--CHR.sub.2'--; wherein R.sub.1 and R.sub.1' are identical
to each other and represent an unsubstituted and linear
C.sub.7-C.sub.21 alkyl group; R.sub.2 and R.sub.2' are identical to
each other and represent an unsubstituted and linear
C.sub.7-C.sub.21 alkyl group; and n and n' are identical to each
other and represent 0 or 1. (GG) X is a group represented by
R.sub.1--CHR.sub.2--; X' is a group represented by
R.sub.1'--CHR.sub.2'--; wherein R.sub.1 and R.sub.1' are identical
to each other and represent an unsubstituted and linear
C.sub.8-C.sub.16 alkyl group; R.sub.2 and R.sub.2' are identical to
each other and represent an unsubstituted and linear
C.sub.8-C.sub.16 alkyl group; and n and n' are identical to each
other and represent 0 or 1. (HH) X is a group represented by
R.sub.1--CHR.sub.2--; X' is a group represented by
R.sub.1'--CHR.sub.2'--; wherein R.sub.1 and R.sub.1' are identical
to each other and represent an unsubstituted and linear
C.sub.8-C.sub.16 alkyl group; R.sub.2 and R.sub.2' are identical to
each other and represent an unsubstituted and linear
C.sub.8-C.sub.16 alkyl group; and n and n' are identical to each
other and represent 0. (II) X is a group represented by
R.sub.1--CHR.sub.2--; X' is a group represented by
R.sub.1'--CHR.sub.2'--; wherein R.sub.1 and R.sub.1' are identical
to each other and represent an unsubstituted and linear
C.sub.9-C.sub.14 alkyl group; R.sub.2 and R.sub.2' are identical to
each other and represent an unsubstituted and linear
C.sub.9-C.sub.14 alkyl group; and n and n' are identical to each
other and represent 1. (JJ) X is a group represented by
R.sub.1--CHR.sub.2--; X' is a group represented by
R.sub.1'--CHR.sub.2'--; wherein R.sub.1, R.sub.1', R.sub.2 and
R.sub.2' are identical to one another and represent an
unsubstituted and linear C.sub.10 alkyl group; and n and n'
represent 0. (KK) X is a group represented by R.sub.1--CHR.sub.2--;
X' is a group represented by R.sub.1'--CHR.sub.2'--; wherein
R.sub.1, R.sub.1', R.sub.2 and R.sub.2' are identical to one
another and represent an unsubstituted and linear C.sub.9,
C.sub.13, or C.sub.14 alkyl group; and n and n' represent 1.
[0072] Specific examples of the trehalose compound of the present
invention include the following compounds: [0073]
6,6'-bis-O-(2-octyldecanoyl)-.alpha.,.alpha.'-trehalose, [0074]
6,6'-bis-O-(2-nonylundecanoyl)-.alpha.,.alpha.'-trehalose, [0075]
6,6'-bis-O-(2-decyldodecanoyl)-.alpha.,.alpha.'-trehalose, [0076]
6,6'-bis-O-(2-undecyltridecanoyl)-.alpha.,.alpha.'-trehalose,
[0077]
6,6'-bis-O-(2-dodecyltetradecanoyl)-.alpha.,.alpha.'-trehalose,
[0078]
6,6'-bis-O-(2-tridecylpentadecanoyl)-.alpha.,.alpha.'-trehalose,
[0079]
6,6'-bis-O-(2-pentadecylheptadecanoyl)-.alpha.,.alpha.'-trehalose,
[0080]
6,6'-bis-O-(2-hexadecyloctadecanoyl)-.alpha.,.alpha.'-trehalose,
[0081] 6,6'-bis-O-(3-nonyldodecanoyl)-.alpha.,.alpha.'-trehalose,
[0082] 6,6'-bis-O-(3-decyltridecanoyl)-.alpha.,.alpha.'-trehalose,
[0083]
6,6'-bis-O-(3-undecyltetradecanoyl)-.alpha.,.alpha.'-trehalose,
[0084]
6,6'-bis-O-(3-dodecylpentadecanoyl)-.alpha.,.alpha.'-trehalose,
[0085]
6,6'-bis-O-(3-tridecylhexadecanoyl)-.alpha.,.alpha.'-trehalose,
[0086]
6,6'-bis-O-(3-tetradecylheptadecanoyl)-.alpha.,.alpha.'-trehalose,
[0087] 6,6'-bis-O-(benzoyl)-.alpha.,.alpha.'-trehalose, [0088]
6,6'-bis-O-(2-naphthylcarbonyl)-.alpha.,.alpha.'-trehalose, [0089]
6,6'-bis-O-(cyclohexanecarbonyl)-.alpha.,.alpha.'-trehalose, [0090]
6,6'-bis-O-(cycloheptanecarbonyl)-.alpha.,.alpha.'-trehalose,
[0091]
6,6'-bis-O-(2-tetradecyloctadecanoyl)-.alpha.,.alpha.'-trehalose,
[0092]
6,6'-bis-O-(14-methoxy-2-(12-methoxydodecyl)-tetradecanoyl)-.alpha.,.alph-
a.'-trehalose, and [0093]
6,6'-bis-O-(15-hydroxy-2-(13-hydroxytridecyl)-pentadecanoyl)-.alpha.,.alp-
ha.'-trehalose.
[0094] Preferred examples of the trehalose compound of the present
invention include the following compounds: [0095]
6,6'-bis-O-(2-octyldecanoyl)-.alpha.,.alpha.'-trehalose, [0096]
6,6'-bis-O-(2-nonylundecanoyl)-.alpha.,.alpha.'-trehalose, [0097]
6,6'-bis-O-(2-decyldodecanoyl)-.alpha.,.alpha.'-trehalose, [0098]
6,6'-bis-O-(2-undecyltridecanoyl)-.alpha.,.alpha.'-trehalose,
[0099]
6,6'-bis-O-(2-dodecyltetradecanoyl)-.alpha.,.alpha.'-trehalose,
[0100]
6,6'-bis-O-(2-tridecylpentadecanoyl)-.alpha.,.alpha.'-trehalose,
[0101]
6,6'-bis-O-(2-pentadecylheptadecanoyl)-.alpha.,.alpha.'-trehalose,
and [0102]
6,6'-bis-O-(2-hexadecyloctadecanoyl)-.alpha.,.alpha.'-trehalose.
[0103] Other preferred examples of the trehalose compound of the
present invention include the following compounds: [0104]
6,6'-bis-O-(3-nonyldodecanoyl)-.alpha.,.alpha.'-trehalose, [0105]
6,6'-bis-O-(3-decyltridecanoyl)-.alpha.,.alpha.'-trehalose, [0106]
6,6'-bis-O-(3-undecyltetradecanoyl)-.alpha.,.alpha.'-trehalose,
[0107]
6,6'-bis-O-(3-dodecylpentadecanoyl)-.alpha.,.alpha.'-trehalose,
[0108]
6,6'-bis-O-(3-tridecylhexadecanoyl)-.alpha.,.alpha.'-trehalose, and
[0109]
6,6'-bis-O-(3-tetradecylheptadecanoyl)-.alpha.,.alpha.'-trehalose.
[0110] More preferred examples of the trehalose compound of the
present invention include the following compounds: [0111]
6,6'-bis-O-(2-decyldodecanoyl)-.alpha.,.alpha.'-trehalose, [0112]
6,6'-bis-O-(3-nonyldodecanoyl)-.alpha.,.alpha.'-trehalose, [0113]
6,6'-bis-O-(3-tridecylhexadecanoyl)-.alpha.,.alpha.'-trehalose, and
[0114]
6,6'-bis-O-(3-tetradecylheptadecanoyl)-.alpha.,.alpha.'-trehalose.
[0115] A preferred example of the bacterial toxin neutralizer of
the present invention is a bacterial toxin neutralizer comprising
any one of the following compounds: [0116]
6,6'-bis-O-(2-decyldodecanoyl)-.alpha.,.alpha.'-trehalose, [0117]
6,6'-bis-O-(2-tetradecyldodecanoyl)-.alpha.,.alpha.'-trehalose,
[0118] 6,6'-bis-O-(3-nonyldodecanoyl)-.alpha.,.alpha.'-trehalose,
[0119]
6,6'-bis-O-(3-tridecylhexadecanoyl)-.alpha.,.alpha.'-trehalose, and
[0120]
6,6'-bis-O-(3-tetradecylheptadecanoyl)-.alpha.,.alpha.'-trehalose.
[0121] The pharmaceutical composition and immunostimulator of the
present invention, etc. are characterized in that they comprise the
above described trehalose compound.
[0122] The trehalose compound of the present invention is used as
an immunostimulator having high action to activate macrophages or
neutrophils. Accordingly, the trehalose compound of the present
invention is useful as an agent for preventing or treating diseases
associated with a biological defense mechanism due to immune
system, including infectious diseases such as bacterial infection,
viral infection and fungus infection, opportunistic infectious
disease, and multi-drug-resistant infectious disease.
<Production Method>
[0123] The compound represented by the formula (1) of the present
invention can be synthesized by the following two steps (a) and
(b):
[0124] (a) a step of allowing carbonyl compounds represented by
formula (4) and formula (6) to simultaneously or successively act
on a trehalose compound represented by formula (3), so as to carry
out an esterification reaction; and
[0125] (b) a step of deprotecting a protecting group for the
hydroxyl group of a trehalose compound represented by formula (5)
which is obtained in the above described step (a).
[0126] With regard to the expression "allowing carbonyl compounds
represented by formula (4) and formula (6) to simultaneously or
successively act on . . . " in the step (a), the carbonyl compounds
represented by the formula (4) and the formula (6) may be
successively acted on the trehalose compound, as shown in Synthetic
Scheme 1 below. Otherwise, as shown in Synthetic Scheme 2 below,
the carbonyl compounds represented by the formula (4) and the
formula (6) may be simultaneously acted on the trehalose compound.
Further, when the carbonyl compounds represented by the formula (4)
and the formula (6) are identical to each other, these compounds
may be used as a carbonyl compound represented by the formula (4),
and they may be acted on the trehalose compound simultaneously,
namely, at a single reaction stage, as shown in Synthetic Scheme 3
below. It is to be noted that a compound represented by formula (2)
may also be synthesized as in the case of the compound represented
by the formula (1).
[0127] The compound represented by the formula (1) of the present
invention can be produced by the method shown in the following
Synthetic Scheme 1.
##STR00004##
[0128] In the above synthetic scheme, R.sub.1, R.sub.1', R.sub.2,
R.sub.2', n and n' have the same definitions as those described
above. R.sub.3 and R.sub.3' each represent a protecting group for
the hydroxyl group of sugar. Y and Y' independently represent a
hydroxyl group or a halogen atom. In the above scheme, the
trehalose compound represented by the formula (3) has an
.alpha.,.alpha.' form. However, an .alpha.,.beta.' form and a
.beta.,.beta.' form can also be synthesized in the same manner as
that described above. In the present invention, however, an
.alpha.,.alpha.' form is preferable.
[0129] Herein, as R.sub.3 and R.sub.3', known protecting groups for
hydroxyl group can be used. For example, protecting groups for
hydroxyl group described in Protecting groups in Organic chemistry
(John Wiley & Sons INC., New York 1991, ISBN 0-471-62301-6) can
be used. Specific examples of such a protecting group include:
arylalkyl groups such as a benzyl group, a .epsilon.-methoxybenzyl
group and a biphenylmethyl group; acyl groups such as an acetyl
group; alkoxycarbonyl groups such as a methoxycarbonyl group and a
tart-butoxycarbonyl group; and trialkylsilyl groups such as a
trimethylsilyl group. Of these, a benzyl group is preferable.
R.sub.3 and R.sub.3' may be identical to or different from each
other. Preferably, R.sub.3 and R.sub.3' are identical to each
other.
[0130] Y and Y' independently represent a hydroxyl group or a
halogen atom. Examples of such a halogen atom include a fluorine
atom, a chlorine atom, and an iodine atom. Y and Y' are preferably
hydroxyl groups.
[0131] <Step (a): Esterification Reaction>
[0132] This is a step of allowing the carbonyl compounds
represented by the formula (4) and the formula (6) to successively
act on the trehalose compound represented by the formula (3), so as
to carry out an esterification reaction between the trehalose
compound and the carbonyl compounds.
[0133] The trehalose compound represented by the formula (3) is a
trehalose compound whose hydroxyl groups other than those at
position 6 and at position 6' are protected by protecting groups.
As such a trehalose compound, either a commercially available
product, or a compound synthesized from trehalose or the like
according to a known method, may be used. For example,
2,3,4,2',3',4'-hexabenzoxy-.alpha.,.alpha.'-trehalose, the
protecting group of which is a benzyl group, is commercially
available, and this product can be preferably used. Moreover, an
.alpha.,.alpha.' form of trehalose is naturally present, and thus
it can be easily obtained. In trehalose, hydroxyl groups at
position 6 and at position 6' have reactivity that is different
from that of other hydroxyl groups. Hence, the trehalose compound
represented by the formula (3), wherein hydroxyl groups other than
those at position 6 and at position 6' are protected, can be
relatively easily synthesized by introducing protecting groups for
hydroxyl groups into natural trehalose according to a known
method.
[0134] Furthermore, as carbonyl compounds represented by the
formula (4) and the formula (6), commercially available products,
compounds synthesized in accordance with the after-mentioned
Synthetic Schemes 4, 5 and 6, and compounds synthesized according
to a known method, may be used.
[0135] In the step of allowing the carbonyl compound represented by
the formula (4) to act on the trehalose compound represented by the
formula (3), the hydroxyl groups at position 6 and at position 6'
in the trehalose compound represented by the formula (3) may be
both esterified. A compound, in which both of the hydroxyl groups
at position 6 and at position 6' are esterified, is referred to as
a diester form. An intermediate compound, in which only either one
hydroxyl group is esterified, is referred to as a monoester form.
When the carbonyl compound represented by the formula (4) is used
in an approximately equivalent amount with respect to the trehalose
compound represented by the formula (3), a large amount of
monoester form is generated as a result of steric hindrance. In
order to enhance the reaction yield of a desired compound, a
monoester form, in which the desired hydroxyl group at position 6
has been esterified, may be separated and purified after completion
of the reaction. Alternatively, in order to enhance the reaction
yield, either one hydroxyl group of the hydroxyl groups at position
6 and at position 6' of the trehalose compound represented by the
formula (3) used as a raw material, which is not to be esterified
with the carbonyl compound represented by the formula (4), has been
selectively protected in advance, and after completion of the
esterification reaction of the other hydroxyl group, it may be
selectively deprotected. With regard to the order of esterification
of hydroxyl groups, the hydroxyl group at position 6 may be
previously esterified, or the hydroxyl group at position 6' may
also be previously esterified.
[0136] In this esterification reaction, methods that are commonly
used as general esterification reactions, and methods known to
persons skilled in the art, may be broadly applied.
[0137] When Y or Y' is a hydroxyl group in the carbonyl compound
represented by the formula (4) or the formula (6), a known
esterification reaction between carboxylic acid and alcohol can be
widely applied. For example, a dehydration method (including a
carbodiimide method), a mixed acid anhydride method and an active
esterification method can be applied. These methods can be
preferably carried out using a condensing agent in an inactive
solvent.
[0138] The condensing agent used in the aforementioned methods may
be a dehydrating agent or any other agent commonly used in the
esterification reaction between alcohol and carboxylic acid.
Examples of such a condensing agent include: mineral acids such as
hydrogen chloride, sulfuric acid and hydrochloric acid; organic
acids such as paratoluenesulfonic acid and camphorsulfonic acid;
dehydrating agents including Lewis acid such as boron fluoride
etherate; acid halides such as phosphorous trichloride, phosphorus
tribromide, phosphorus pentachloride, phosphorus oxychloride and
thionyl chloride; mixed acid anhydrides such as ethyl chloroformate
and methanesulfonyl chloride; carbodiimides such as
N,N'-dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide and
1-ethyl-3-dimethylaminopropylcarbodiimide; and other condensing
agents such as N,N-carbonyldiimidazole,
2-ethoxy-N-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) and
triphenylphosphine-carbon tetrachloride (complex). These condensing
agents may be used singly or in combination of two or more types.
The ratio between a raw material compound and a condensing agent
used is not particularly limited, and it can be appropriately
selected from a wide range.
[0139] The esterification reaction can be carried out in a suitable
solvent. The type of such a solvent is not particularly limited, as
long as it is an inactive solvent, which has moderate ability to
dissolve a raw material compound and which does not affect the
esterification reaction. A wide range of known solvents can be
used. Examples of a solvent used in the above described
esterification reaction include: aromatic hydrocarbons such as
benzene, toluene and xylene; halogenated aromatic hydrocarbons such
as chlorobenzene and dichlorobenzene; aliphatic hydrocarbons such
as n-hexane, cyclohexane and petroleum ether; aliphatic halogenated
hydrocarbons such as dichloromethane, 1,2-dichloroethane,
chloroform and carbon tetrachloride; ethers such as diethyl ether,
diisopropyl ether, dioxane, tetrahydrofuran, ethylene glycol
dimethyl ether and ethylene glycol diethyl ether; ketones such as
acetone, 2-butanone and methyl isobutyl ketone; nitriles such as
acetonitrile, propionitrile and benzonitrile; amides such as
N,N-dimethylformamide and hexamethylphosphoric triamide (HMPA); and
sulfoxides such as dimethyl sulfoxide. These solvents may be used
singly or in combination of two or more types.
[0140] Furthermore, in the esterification reaction, a wide range of
known reaction promoters can be used. Examples of such a reaction
promoter include: catalysts such as dimethylformamide,
dimethylamidopyridine and 4-pyrrolidinopyridine; and drying agents
such as anhydrous magnesium sulfate and molecular sieves (4A, 5A).
These reaction promoters may be added into a reaction system.
Further, in order to promote the esterification reaction,
apparatuses such as a Dean-Stark water separation apparatus and a
Soxhlet extractor may also be used. These reaction promoters or
apparatuses may be used singly or in combination of two or more
types. A catalyst may be used in combination with a drying agent.
The ratio between a raw material compound and a reaction promoter
used is not particularly limited, and it can be appropriately
selected from a wide range.
[0141] The amount of a raw material compound used in the present
reaction is not particularly limited, and it can be appropriately
selected from a wide range. When the compound represented by the
formula (4) and the compound represented by the formula (6) are
successively reacted, the carbonyl compound represented by the
formula (4) is used in an amount of generally 0.5 to 1.8 moles, and
preferably 0.8 to 1.2 moles, with respect to 1 mole of the
trehalose compound represented by the formula (3). In addition, the
carbonyl compound represented by the formula (6) is used in an
amount of generally 0.5 to 1.8 moles, and preferably 0.8 to 1.2
moles, with respect to 1 mole of the monoester form represented by
the formula (5).
[0142] Moreover, the reaction temperature applied to the
esterification reaction is not particularly limited, either. In
general, it may be set within the range from -10.degree. C. to the
boiling point of a solvent used. The reaction is carried out
generally at a temperature from 0.degree. C. to 200.degree. C., and
preferably from a room temperature to 100.degree. C.
[0143] The reaction time depends on the type of a raw material
compound and the amount used, and reaction conditions such as a
reaction temperature. In general, the reaction time can be
appropriately adjusted within the range from 1 hour to 1 week, and
it is preferably 1 to 24 hours, and more preferably 3 to 10
hours.
[0144] After completion of the reaction, common treatments such as
separation and elimination of by-products, drying, and distillation
of a solvent, are performed on a reaction mixture. Thereafter, the
reaction product is purified by a common method such as silica gel
column chromatography.
[0145] When Y or Y' is a halogen atom in the carbonyl compound
represented by the formula (4) or the formula (6), the
esterification reaction can be carried out in a suitable solvent,
and as necessary, in the presence of a base.
[0146] As solvents, the same inactive solvents as those used in the
above described esterification reaction can be used.
[0147] Examples of a base include: alkaline metal hydroxides such
as sodium hydroxide and potassium hydroxide; alkaline-earth metal
hydroxides such as calcium hydroxide; alkaline metal carbonates
such as sodium carbonate and potassium carbonate; alkaline metal
hydrogencarbonates such as sodium hydrogencarbonate and potassium
hydrogencarbonate; alkaline metal acetates such as sodium acetate
and potassium acetate; alkaline-earth metal acetates such as
calcium acetate; alkaline metal hydrides such as sodium hydride and
potassium hydride; alkaline-earth metal hydrides such as calcium
hydride; ammonium salts such as ammonium hydroxide, ammonium
carbonate and ammonium acetate; and tertiary amines such as
trimethylamine, triethylamine, N,N-dimethylaniline, pyridine,
4-(dimethylamino)pyridine, diazabicyclooctane (DABCO),
diazabicyclononene (DBN) and diazabicycloundecene (DBU). These
bases may be used singly or in combination of two or more
types.
[0148] The amounts of a raw material compound and a base that are
subjected to the present reaction are not particularly limited. The
amounts can be appropriately selected from a wide range. When the
compound represented by the formula (4) and the compound
represented by the formula (6) are successively reacted, the
carbonyl compound represented by the formula (4) is used in an
amount of generally 0.5 to 1.8 moles, and preferably 0.8 to 1.2
moles, with respect to 1 mole of the trehalose compound represented
by the formula (2). A base is used in an amount of generally 0.5 to
1.8 moles, and preferably 0.8 to 1.2 moles, with respect to 1 mole
of the trehalose compound represented by the formula (2). Moreover,
the carbonyl compound represented by the formula (6) is used in an
amount of generally 0.5 to 1.8 moles, and preferably 0.8 to 1.2
moles, with respect to 1 mole of the monoester form represented by
the formula (5). A base is used in an amount of generally 0.5 to
1.8 moles, and preferably 0.8 to 1.2 moles, with respect to 1 mole
of the monoester form represented by the formula (5).
[0149] As in the case of the aforementioned esterification
reaction, the reaction temperature may be generally set within the
range from -10.degree. C. to the boiling point of a solvent used.
As in the case of the aforementioned esterification reaction, the
reaction time depends on the above described concentration,
temperature, etc. In general, the reaction time can be
appropriately adjusted within the range from 0.1 to 10 hours.
[0150] Even when a carbonyl compound in which X represents a
hydroxyl group and a carbonyl group in which X' represents a
halogen atom are used as the compound represented by the formula
(4) and the compound represented by the formula (6), and also when
a carbonyl compound in which X represents a halogen atom and a
carbonyl group in which X' represents a hydroxyl group are used as
the compound represented by the formula (4) and the compound
represented by the formula (6), the compound represented by the
formula (7) can also be synthesized by appropriately selecting
appropriate reaction conditions from the above described reaction
conditions.
[0151] <Step (b)>
[0152] Deprotection of the hydroxyl groups of sugar is carried out
on the compound represented by the formula (7) in which the
position 6 and position 6' of trehalose have been esterified and
the hydroxyl groups of sugar have been protected, which was
obtained in the above described step (a), so as to obtain a
trehalose diester compound of interest, which is represented by the
formula (1).
[0153] In order to leave protecting groups from the protected
hydroxyl groups in the compound represented by the formula (7), the
deprotection method suitable for the protecting groups, which is
described in the aforementioned publication, can be appropriately
applied, for example.
[0154] In a case in which the protecting group for R.sub.3 is a
benzyl group, for example, a catalytic hydrogenation reaction can
be applied. The catalytic hydrogenation reaction is carried out in
a hydrogen atmosphere in the presence of a catalyst.
[0155] As catalysts, a wide range of known catalysts can be used,
as long as they can be used in such a catalytic hydrogenation
reaction. Examples of such a catalyst include platinum oxide,
platinum carbon, palladium hydroxide, palladium carbon, and Raney
nickel.
[0156] The catalyst is used in an amount of generally about 0.001%
to 50% by weight, and preferably about 0.01% to 10% by weight, with
respect to the weight of the compound represented by the formula
(7).
[0157] The hydrogen pressure is not particularly limited, and it
can be appropriately selected from a wide range. It is generally
about 0.8 to 100 atmospheres, and preferably about 1 to 3
atmospheres.
[0158] This reaction is generally carried out in a suitable
solvent. As solvents, a wide range of solvents can be used, as long
as they are inactive solvents that do not affect the reaction.
Examples of a solvent used herein 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; carboxylic acids such as formic acid and
acetic acid; and the mixed solvents thereof.
[0159] The temperature applied in this reaction is generally about
0.degree. C. to 100.degree. C., and preferably about 10.degree. C.
to 40.degree. C. The reaction time depends on the amount of a
substrate, a temperature, the type of a catalyst, etc. Using the
theoretical amount of hydrogen consumption as a guideline, the
reaction may be terminated. The reaction time is generally about 1
to 50 hours, and preferably 1 to 30 hours.
[0160] After completion of the reaction, common treatments such as
the removal of the catalyst by filtration and distillation of the
solvent are performed, and the reaction product is then purified by
common methods such as solvent extraction and silica gel column
chromatography.
[0161] The compound represented by the formula (1) of the present
invention can also be produced by the methods shown in the
following Synthetic Scheme 2 or Synthetic Scheme 3.
##STR00005##
[0162] In the above synthetic scheme, R.sub.1, R.sub.1', R.sub.2,
R.sub.2', R.sub.3, R.sub.3', n and n' have the same definitions as
those described above.
[0163] Synthetic Scheme 2 is a scheme whereby the carbonyl
compounds represented by the formula (4) and the formula (6) are
allowed to simultaneously act on the trehalose compound represented
by the formula (3) in the step (a) that is an esterification
reaction. The step (b) that is a deprotection reaction is the same
as that in Synthetic Scheme 1.
[0164] The esterification reaction and the deprotection reaction
can be carried out in the same manner as that in Synthetic Scheme 1
with the exception that the carbonyl compounds represented by the
formula (4) and the formula (6) are allowed to simultaneously act
on the trehalose compound represented by the formula (3) in the
step (a).
[0165] When the carbonyl compounds represented by the formula (4)
and the formula (6) are allowed to simultaneously act on the
aforementioned trehalose compound, a compound of interest can be
obtained by separating and purifying it from the generated
product.
[0166] When R.sub.1 and R.sub.1', R.sub.2 and R.sub.2', and n and
n' are identical to each other in the compound represented by the
formula (1), the above described Synthetic Scheme 2 can be
particularly represented by Synthetic Scheme 3 below. When R.sub.1
and R.sub.1', R.sub.2 and R.sub.2', and n and n' are not identical
to each other in the compound represented by the formula (1), from
the viewpoint of the enhancement of a reaction yield, the compound
is preferably synthesized by the method as shown in Synthetic
Scheme 1.
##STR00006##
[0167] In the above synthetic scheme, R.sub.1, R.sub.2, R.sub.3,
R.sub.3' and n have the same definitions as those described
above.
[0168] In Synthetic Scheme 3, only one type of carbonyl compound
(4) is allowed to act on the trehalose compound represented by the
formula (3) in the step (a) that is an esterification reaction.
Thus, this is a scheme whereby the esterification reaction is
carried out simultaneously, namely, at a single reaction stage, and
the step (b) that is a deprotection reaction is the same as that in
Synthetic Scheme 1.
[0169] The esterification reaction and the deprotection reaction in
Synthetic Scheme 3 can be carried out in the same manner as the
reaction in the above described Synthetic Scheme 1 with the
exception that the used amount of the carbonyl compound represented
by the formula (4) is increased. With regard to the used amount,
specifically, with respect to 1 mole of the trehalose compound
represented by the formula (3), the carbonyl compound represented
by the formula (4) is used in an amount of generally 1.8 to 5
moles, and preferably 2 to 3 moles; a condensing agent is used in
an amount of generally 1.8 to 5 moles, and preferably 2 to 4 moles;
and a base is used in an amount of 1.8 to 8 moles, and preferably 2
to 6 moles.
[0170] The compound represented by the formula (7) may be used to
the subsequent reaction without performing isolation and
purification. However, it is preferable to remove reagents used in
the esterification and by-products before subjecting the compound
to the subsequent reaction.
[0171] Carbonyl Compounds Used as Raw Materials>
[0172] As the carbonyl compound represented by the formula (4) or
the formula (6), which is used as a raw material compound in the
above described Synthetic Scheme 1, 2 or 3, a commercially
available product can be used, or a compound can be produced by a
method known to persons skilled in the art. For example, as a
compound wherein, in the formula (4) or the formula (6), X or X'
represents a phenyl group and n or n' represents 0, benzoic acid or
a benzoic acid halide can be used.
[0173] Among the compounds represented by the formula (4), a
compound, wherein X represents R.sub.1--CHR.sub.2-- and n
represents 0, can also be produced by the following Synthetic
Scheme 4 or 5.
[0174] Hereafter, the carbonyl compound represented by the formula
(4) is described as a compound of interest. However, the carbonyl
compound represented by the formula (6) can also be produced in the
same manner.
##STR00007##
[0175] In the above synthetic scheme, R.sub.1 and R.sub.2 have the
same definitions as those described above. R.sub.4 represents an
alkyl group containing 1 to 6 carbon atoms, and Hal represents a
halogen atom.
[0176] Synthetic Scheme 4 is a step of subjecting the compound
represented by the formula (8) to an ordinary alkylation reaction
to obtain the carbonyl compound represented by the formula (4). A
commercially available product can be used as the compound
represented by the formula (8) that is a raw material compound.
When R.sub.1 is a linear alkyl group containing 10 carbon atoms,
for example, ethyl dodecanoate or the like can be used. An acid
ester having a desired length as a side chain alkyl of a compound
of interest may be used.
[0177] Various methods, such as the method described in Creger, J.
Am. Chem. Soc., Vol. 92, pp. 1397-98, 1970, can be used in the
alkylation reaction. More specifically, a strong base may be added
to a solution of the compound represented by the formula (8) to
eliminate the hydrogen atom at position 2, and thereafter, an alkyl
halide may be allowed to react with the compound represented by the
formula (8). As an example, alkylation can be carried out by the
following reaction.
[0178] First, the compound represented by the formula (8) is
allowed to react with a strong base. The type of such a strong base
is not particularly limited, as long as it has action to eliminate
a hydrogen atom. Examples of such a strong base include: alkaline
metal hydroxides such as sodium hydroxide and potassium hydroxide;
and alkaline-earth metal hydroxides such as calcium hydroxide.
These bases may be used singly or in combination of two or more
types. Moreover, lithium diisopropylamide may be used in
combination, so as to carry out a proton-lithium exchange reaction.
The type of a solvent is not particularly limited, as long as it is
an inactive solvent, which has moderate ability to dissolve a raw
material compound and which does not affect the esterification
reaction. A wide range of known solvents can be used. Examples of a
solvent used in the above described alkylation reaction include:
aromatic hydrocarbons such as benzene, toluene and xylene;
aliphatic hydrocarbons such as n-hexane, cyclohexane and petroleum
ether; and ethers such as diethyl ether, diisopropyl ether,
dioxane, tetrahydrofuran, ethylene glycol dimethyl ether, and
ethylene glycol diethyl ether. These solvents may be used singly or
in combination of two or more types.
[0179] In the present reaction, the ratio between the compound
represented by the formula (8) and the strong base can be
appropriately selected from a wide range. In general, the strong
base or the like is used in an amount of approximately 0.9 to 5
times the mole of the compound represented by the formula (8). The
reaction temperature applied in this reaction is generally about
-80.degree. C. to 60.degree. C., and preferably about 0.degree. C.
to 60.degree. C. The reaction time is set at about 5 minutes to 6
hours, and preferably about 5 minutes to 1 hour.
[0180] Subsequently, an alkyl halide is added to the reaction
mixture. As such an alkyl halide, an alkane halide having a desired
length of carbon chain portion, such as 1-iodooctane,
1-iodoheptane, 1-iododecane, 1-iodoundecane, 1-iodododecane or
1-iodotridecane, may be used as an alkyl group on the side chain of
a compound of interest. Halides include a chloride, an iodide, a
bromide, and the like. Of these, an iodide is preferable. In the
present reaction, the ratio between the compound represented by the
formula (8) and the alkyl halide can be appropriately selected from
a wide range. In general, the alkyl halide is used in an amount of
approximately 1.1 to 3 times the mole of the compound represented
by the formula (8). The reaction temperature applied in this
reaction may be generally set around a room temperature. The
reaction time is generally set at about 2 to 12 hours.
[0181] After completion of the reaction, a known isolation and
purification method, such as silica gel column chromatography or
vacuum distillation, is applied to isolate and purify the compound
of interest.
[0182] Among the compounds represented by the formula (4), a
compound, wherein X represents R.sub.1--CHR.sub.2-- and n
represents 0, can also be produced by the following Synthetic
Scheme 5.
##STR00008##
[0183] In the above synthetic scheme, R.sub.1, R.sub.2, R.sub.4 and
Hal have the same definitions as those described above.
[0184] Synthetic Scheme 5 is a step of subjecting the compound
represented by the formula (11) to an ordinary alkylation reaction
to obtain the carbonyl compound represented by the formula (4). A
commercially available product can be used as the compound
represented by the formula (11) that is a raw material compound.
Examples of such a commercially available product include diethyl
malonate, dimethyl malonate, dipropyl malonate, dibutyl malonate,
diisopropyl malonate, di-tert-butyl malonate, dicyclohexyl
malonate, diphenyl malonate, and dibenzyl malonate.
[0185] As described above, the alkylation reaction can be carried
out by allowing a strong base to react with the compound
represented by the formula (11) used as a raw material compound,
and then allowing an alkyl halide to act on the aforementioned
compound.
[0186] In the step of allowing a strong base to react with the
compound represented by the formula (11), the ratio between the
compound represented by the formula (11) and the strong base can be
appropriately selected from a wide range. In general, the strong
base or the like is used in an amount of approximately 0.9 to 5
times the mole of the compound represented by the formula (11). The
reaction temperature applied in this reaction is generally about
-80.degree. C. to 60.degree. C., and preferably about 0.degree. C.
to 60.degree. C. The reaction time is set at about 5 minutes to 6
hours, and preferably about 5 minutes to 1 hour.
[0187] Subsequently, in the step of adding an alkyl halide to the
reaction mixture, the ratio between the compound represented by the
formula (11) and the alkyl halide can be appropriately selected
from a wide range. In general, the alkyl halide represented by the
formula (12) and the alkyl halide represented by the formula (9)
are used in each amount of approximately 0.8 to 1.2 times the mole
of the compound represented by the formula (11). When the compound
of interest is a compound wherein R.sub.1 and R.sub.2 are identical
to each other, only one type of alkyl halide may be used. In
general, such an alkyl halide is used in an amount of approximately
2.2 to 4 times the mole of the compound represented by the formula
(11). On the other hand, when the compound of interest is a
compound wherein R.sub.1 and R.sub.2 are not identical to each
other, as described above, the alkyl halide represented by the
formula (12) and the alkyl halide represented by the formula (9)
are allowed to simultaneously act on the compound, so that only the
compound of interest can be isolated and purified. Otherwise,
different alkyl halides are allowed to successively act on the
compound, namely, after one type of alkyl halide has been allowed
to act on the compound, the other type of alkyl halide is allowed
to act thereon, and thereafter, the compound of interest may be
isolated and purified. As such isolation and purification methods,
known isolation and purification methods, such as silica gel column
chromatography or vacuum distillation, can be applied.
[0188] Among the compounds represented by the formula (4), a
compound, wherein X represents R.sub.1--CHR.sub.2-- and n
represents 1, can also be produced by the following Synthetic
Scheme 6.
[0189] <Synthetic Scheme 6>
[0190] Synthetic Scheme 6 can be described in the following
Synthetic Schemes 6-1 to 6-5.
##STR00009##
[0191] In the above synthetic scheme, R.sub.1 has the same
definitions as those described above.
[0192] Synthetic Scheme 6-1 is a reaction regarding the dehydration
binding of N,O-dimethylhydroxyamine to carboxylic acid used as a
raw material compound.
[0193] A basic condensing agent is allowed to act on the carboxylic
acid represented by the formula (13). A wide range of known basic
condensing agents can be used herein. Examples of such a basic
condensing agent include carbonyldiimidazole,
dimethylaminopyridine, piperidine, pyrrolidine, pyridine,
imidazole, N,N,N',N'-tetramethylurea, bis(pentamethylene)urea, and
1,1-carbonyldipyrrole. A commercially available product can be used
as the carboxylic acid that is a raw material compound. For
example, heptanoic acid, octanoic acid, decanoic acid, and
undecanoic acid are used. An acid having a desired length as a side
chain alkyl of a compound of interest may be used. As solvents, a
wide range of solvents can be used, as long as they are inactive
solvents, which have moderate ability to dissolve a raw material
compound and which do not affect the esterification reaction.
Examples of a solvent used in the above described esterification
reaction include: aromatic hydrocarbons such as benzene, toluene
and xylene; halogenated aromatic hydrocarbons such as chlorobenzene
and dichlorobenzene; aliphatic hydrocarbons such as n-hexane,
cyclohexane and petroleum ether; aliphatic halogenated hydrocarbons
such as dichloromethane, 1,2-dichloroethane, chloroform and carbon
tetrachloride; and ethers such as diethyl ether, diisopropyl ether,
dioxane, tetrahydrofuran, ethylene glycol dimethyl ether and
ethylene glycol diethyl ether. These solvents may be used singly or
in combination of two or more types.
[0194] In the present reaction, the use ratio between the
carboxylic acid represented by the formula (13) and
carbonyldiimidazole or the like can be appropriately selected from
a wide range. In general, it is approximately 0.8 to 2.0 moles. The
reaction temperature applied in this reaction is generally about
-80.degree. C. to 60.degree. C., and preferably about 0.degree. C.
to 60.degree. C. The reaction time is set at about 5 minutes to 6
hours, and preferably about 30 minutes to 3 hours.
[0195] Subsequently, the N,O-dimethylhydroxyamine represented by
the formula (14) is allowed to react with the reaction product.
1-hydroxybenzotriazole or the like may also be used instead of the
N,O-dimethylhydroxyamine. The ratio between the carboxylic acid
represented by the formula (13) and the N,O-dimethylhydroxyamine
can be appropriately selected from a wide range. In general, the
N,O-dimethylhydroxyamine may be used in an amount of approximately
0.8 to 1.5 moles with respect to 1 mole of the carboxylic acid
represented by the formula (13). The reaction temperature is
generally about -80.degree. C. to 60.degree. C., and preferably
about 0.degree. C. to 60.degree. C. The reaction time may be set at
about 10 minutes to 10 hours.
[0196] After completion of the reaction, a known isolation and
purification method, such as silica gel column chromatography or
vacuum distillation, is applied to isolate and purify the compound
of interest.
##STR00010##
[0197] In the above synthetic scheme, R.sub.1, R.sub.2 and Hal have
the same definitions as those described above.
[0198] Synthetic Scheme 6-2 is a reaction of allowing an alkyl
halide to act on the compound represented by the formula (15) to
synthesize a ketone body.
[0199] In the present reaction, the alkyl halide represented by the
formula (9) may be allowed to act on metallic magnesium in an ether
solvent to prepare a Grignard reagent, and the thus prepared
reagent may be used in the reaction. As such a metallic magnesium,
polished shaved magnesium is preferably used. In addition, lithium,
sodium, zinc, indium or the like may also be used.
[0200] As the alkyl halide represented by the formula (9), the same
alkyl halides as those described above can be used.
[0201] The present reaction is carried out in an ether solvent.
Examples of such an ether solvent include diethyl ether,
diisopropyl ether, dioxane, tetrahydrofuran, tetrahydropyran,
ethylene glycol dimethyl ether and ethylene glycol diethyl ether.
These solvent may be used singly or in combination of two or more
types.
[0202] In the present reaction, the ratio between the compound
represented by the formula (15) and the alkyl halide can be
appropriately selected from a wide range. In general, the alkyl
halide is used in an amount of approximately 0.8 to 5 times the
mole of the compound represented by the formula (15). The reaction
temperature applied in this reaction is generally about 0.degree.
C. to 80.degree. C. The reaction time is about 5 minutes to 6
hours.
[0203] After completion of the reaction, a known isolation and
purification method, such as silica gel column chromatography or
vacuum distillation, is applied to isolate and purify the compound
of interest.
##STR00011##
[0204] In the above synthetic scheme, R.sub.1, R.sub.2 and R.sub.4
have the same definitions as those described above. R.sub.5 and
R.sub.6 each represent an alkyl group, an alkoxy group, an aryl
group, or an aryloxy group. These groups may be replaced by a
halogen atom and the like.
[0205] Synthetic Scheme 6-3 is a reaction of allowing a Wittig
reagent or a Horner-Emmons reagent to react with the ketone
compound represented by the formula (16) in the presence of a
strong base, so as to form a carbon-carbon double bond. The
compound represented by the formula (17) in the above described
synthetic scheme is a Horner-Emmons reagent. Instead of the
Horner-Emmons reagent, the Wittig reagent may also be used.
[0206] The type of such a strong base is not particularly limited,
as long as it has action to eliminate a hydrogen atom. Examples of
such a strong base include: alkaline metal hydroxides such as
sodium hydroxide and potassium hydroxide; and alkaline-earth metal
hydroxides such as calcium hydroxide. These bases may be used
singly or in combination of two or more types. The type of a
solvent is not particularly limited, as long as it is an inactive
solvent, which has moderate ability to dissolve a raw material
compound and which does not affect the esterification reaction. A
wide range of known solvents can be used. Examples of a solvent
used in the present reaction include: aromatic hydrocarbons such as
benzene, toluene and xylene; aliphatic hydrocarbons such as
n-hexane, cyclohexane and petroleum ether; and ethers such as
diethyl ether, diisopropyl ether, dioxane, tetrahydrofuran,
ethylene glycol dimethyl ether, and ethylene glycol diethyl ether.
These solvents may be used singly or in combination of two or more
types.
[0207] In the present reaction, the ratio between the compound
represented by the formula (16) and the strong base can be
appropriately selected from a wide range. In general, the strong
base or the like is used in an amount of approximately 1.1 to 8
times the mole of the compound represented by the formula (16). The
reaction temperature applied in this reaction is generally about
-80.degree. C. to 60.degree. C. The reaction time is generally set
at about 5 minutes to 3 hours.
[0208] Subsequently, a Wittig reagent or a Horner-Emmons reagent is
allowed to react with the reaction mixture. As such a Wittig
reagent or a Horner-Emmons reagent, a wide range of known reagents
can be used. The type of such an agent is not particularly limited,
as long as it is able to form a carbon-carbon double bond between
the ketone compound represented by the formula (16) and an acid
ester. Examples of a Wittig reagent include
ethoxycarbonylmethyl(triphenyl)phosphonium bromide and ethyl
(triphenylphosphoranylidene)acetate. Examples of a Horner-Emmons
reagent include ethyl (diaryl)phosphonoacetates such as ethyl
diphenylphosphonoacetate, and ethyl (dialkyl)phosphonoacetates such
as ethyl diethylphosphonoacetate. Of these, ethyl
diethylphosphonoacetate is preferable.
[0209] In the present reaction, the ratio between the compound
represented by the formula (16) and ethyl diethylphosphonoacetate
can be appropriately selected from a wide range. In general, ethyl
diethylphosphonoacetate is used in an amount of approximately 1.1
to 10 times the mole of the compound represented by the formula
(16). The reaction temperature applied in this reaction may be
generally set at a room temperature. The reaction time is generally
set at about 2 to 30 hours.
[0210] After completion of the reaction, a known isolation and
purification method, such as silica gel column chromatography or
vacuum distillation, is applied to isolate and purify the compound
of interest.
##STR00012##
[0211] In the above synthetic scheme, R.sub.1, R.sub.2 and R.sub.4
have the same definitions as those described above.
[0212] Synthetic Scheme 6-4 is a reaction of performing a catalytic
hydrogenation reaction on the carboxylic acid ester having an
unsaturated bond, represented by the formula (18), so as to convert
it to a saturated carboxylic acid ester.
[0213] The catalytic hydrogenation reaction is carried out in a
hydrogen atmosphere in the presence of a catalyst.
[0214] As catalysts, a wide range of known catalysts can be used,
as long as they can be used in a catalytic hydrogenation reaction.
Examples of such a catalyst include platinum oxide, platinum
carbon, palladium hydroxide, palladium carbon, and Raney
nickel.
[0215] The catalyst is used in an amount of generally about 0.001%
to 50% by weight, and preferably about 0.01% to 10% by weight, with
respect to the weight of the compound represented by the formula
(18).
[0216] The hydrogen pressure is not particularly limited, and it
can be appropriately selected from a wide range. It is generally
about 0.8 to 100 atmospheres, and preferably about 1 to 3
atmospheres.
[0217] This reaction is generally carried out in a suitable
solvent. As solvents, a wide range of solvents can be used, as long
as they are inactive solvents that do not affect the reaction.
Examples of a solvent used herein 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; carboxylic acids such as formic acid and
acetic acids; and the mixed solvents thereof.
[0218] The temperature applied in the present reaction is generally
about 0.degree. C. to 100.degree. C., and preferably about
10.degree. C. to 40.degree. C. The reaction time depends on the
amount of a substrate, a temperature, the type of a catalyst, etc.
Using the theoretical amount of hydrogen consumption as a
guideline, the reaction may be terminated. The reaction time is
generally about 1 to 50 hours, and preferably 1 to 30 hours.
[0219] After completion of the reaction, common treatments such as
the removal of the catalyst by filtration and distillation of the
solvent are performed, and the reaction product is then purified by
common methods such as solvent extraction and silica gel column
chromatography.
##STR00013##
[0220] In the above synthetic scheme, R.sub.1, R.sub.2 and R.sub.4
have the same definitions as those described above.
[0221] Synthetic Scheme 6-5 is a reaction of hydrolyzing the
carboxylic acid ester represented by the formula (19) to obtain
desired carboxylic acid.
[0222] As such hydrolytic reactions, various types of known
reactions can be used. Such a hydroxylic reaction is carried out
under acidic conditions or under basic conditions, or it may be
carried out in the form of an enzyme reaction.
[0223] In order to create basic conditions, a base may be added to
a solvent. As such bases, a wide range of substances can be used,
as long as they generate hydride ions. Examples of such a base
include: alkaline metal hydroxides such as sodium hydroxide and
potassium hydroxide; alkaline-earth metal hydroxides such as
calcium hydroxide; alkaline metal carbonates such as sodium
carbonate and potassium carbonate; alkaline metal
hydrogencarbonates such as sodium hydrogencarbonate and potassium
hydrogencarbonate; alkaline metal acetates such as sodium acetate
and potassium acetate; alkaline-earth metal acetates such as
calcium acetate; alkaline metal hydrides such as sodium hydride and
potassium hydride; alkaline-earth metal hydrides such as calcium
hydride; ammonium salts such as ammonium hydroxide, ammonium
carbonate and ammonium acetate; and tertiary amines such as
trimethylamine, triethylamine, N,N-dimethylaniline, pyridine,
4-(dimethylamino)pyridine, diazabicyclooctane (DABCO),
diazabicyclononene (DEN) and diazabicycloundecene (DBU). These
bases may be used singly or in combination of two or more
types.
[0224] The solvent may be an inactive solvent, which has moderate
ability to dissolve a raw material compound and which does not
affect the esterification reaction. A wide range of known solvents
can be used. Examples of a solvent used in the above described
esterification reaction include: aromatic hydrocarbons such as
benzene, toluene and xylene; halogenated aromatic hydrocarbons such
as chlorobenzene and dichlorobenzene; aliphatic hydrocarbons such
as n-hexane, cyclohexane and petroleum ether; aliphatic halogenated
hydrocarbons such as dichloromethane, 1,2-dichloroethane,
chloroform and carbon tetrachloride; ethers such as diethyl ether,
diisopropyl ether, dioxane, tetrahydrofuran, ethylene glycol
dimethyl ether and ethylene glycol diethyl ether; ketones such as
acetone, 2-butanone and methyl isobutyl ketone; nitriles such as
acetonitrile, propionitrile and benzonitrile; amides such as
N,N-dimethylformamide and hexamethylphosphoric triamide (HMPA); and
sulfoxides such as dimethyl sulfoxide. These solvents may be used
singly or in combination of two or more types.
[0225] In the present reaction, the reaction temperature, the
reaction time, and the like can be appropriately selected from a
wide range. The reaction temperature is generally about 0.degree.
C. to 100.degree. C. The reaction time is generally about 30
minutes to 20 hours.
[0226] After completion of the reaction, a known isolation and
purification method, such as silica gel column chromatography or
vacuum distillation, is applied to isolate and purify the compound
of interest.
[0227] Among the compounds represented by the formula (4), a
compound, wherein X represents R.sub.1--CHR.sub.2-- and n
represents 2, can also be produced by the following Synthetic
Scheme 7, for example.
##STR00014##
[0228] In the above synthetic scheme, R.sub.1 and R.sub.2 have the
same definitions as those described above.
[0229] Synthetic Scheme 7 is a reaction in which the diethyl
3-hydroxypropanoylphosphonate represented by the formula (21) is
allowed to react as a Horner-Emmons reagent with the ketone
compound represented by the formula (16) to form a carbon-carbon
double bond, so as to obtain the compound represented by the
formula (22), a catalytic hydrogenation reaction is then performed
thereon to obtain the compound represented by the formula (23), an
oxidation reaction of alcohol is then performed thereon to obtain
the compound represented by the formula (24) as a carbonyl
compound. In the above synthetic scheme, only one of cis-trans
stereoisomers of double bond is described with regard to the
compound represented by the formula (22). However, examples of such
stereoisomers are not limited to the aforementioned
stereoisomer.
[0230] The reaction using a Horner-Emmons reagent and the catalytic
hydrogenation reaction can be carried out in the same manner as
described above, while referring to Synthetic Scheme 6-3 and
Synthetic Scheme 6-4, respectively. In addition, the oxidation
reaction of alcohol can be carried out by oxidizing alcohol with a
strong oxidizer. Such an alcohol oxidation reaction can be carried
out, as appropriate, according to a known method such as oxidation
with chromic acid or Jones oxidation. As an example, oxidation with
chromic acid can be carried out using the salts or complexes of
chromic anhydride, chromic acid, dichromic acid, etc.
[0231] After completion of the reaction, common treatments such as
the removal of the catalyst by filtration and distillation of the
solvent are performed, and the reaction product is then purified by
common methods such as solvent extraction and silica gel column
chromatography.
[0232] In the present invention, the term "immunostimulation" means
the activation of various immune actions such as cellular immunity
or humoral immunity. The immunostimulator may be an agent that
exhibits any of these immunostimulatory actions. The trehalose
compound of the present invention is considered to carry out the
activation of the immune actions of macrophages or neutrophils,
which are referred to as, at least, cellular immunity in the immune
system. A wide range of circumstances, in which the aforementioned
cells release cytokines as a result of the activation of cellular
immunity, and thereby humoral immunity is also activated, are also
included in the immunostimulation.
[0233] Macrophage originally has phagocytosis to foreign matters
entering from the outside. In the present specification, the term
"macrophage activation" means action to enhance the phagocytosis of
macrophage, and as a result, the adhesive property of macrophage to
tissues and the mobility thereof are improved, and it englobes
bacteria invading from the outside and degenerated self-components.
In a state in which macrophages are activated, it has been known
that the release of nitric oxide (NO) and the release of active
oxygen are increased. The released amounts of these free substances
are measured, and the obtained value can be used as an indicator of
macrophage activation. In addition, acceleration in the
phagocytosis itself is measured, and the obtained value can also be
used as an indicator of macrophage activation.
[0234] Neutrophil itself originally has phagocytosis to foreign
matters, as with macrophage. In the present specification, the term
"neutrophil activation" means action to enhance the phagocytosis of
neutrophil, and as a result, the neutrophil englobes bacteria and
the like. Also, in a state in which neutrophils are activated, it
has been known that the release of nitric oxide (NO) and the
release of active oxygen are increased. It has also been known that
the release of physiologically active substances is observed as a
result of degranulation of microgranules and azurophile granules by
neutrophil activation. The released amounts of these free
substances are measured, and the obtained value can be used as an
indicator of neutrophil activation. In addition, acceleration in
the phagocytosis itself is measured, and the obtained value can
also be used as an indicator of neutrophil activation.
[0235] In the present specification, phagocytic cells include
macrophages, monocytes, polymorphonuclear leukocytes, dendritic
cells, and the like. The "phagocytosis" means the action of these
cells as immune-system cells to incorporate foreign matters from
the outside, such as pathogen bacteria, into the vesicles thereof
and to fuse the vesicles with lysosomes in the cells, so as to
digest the foreign matters. Activation of the phagocytosis of
phagocytic cells is not particularly limited, as long as it is
action to activate any of these phagocytic cells so as to enhance
the phagocytosis thereof. Such activation of the phagocytosis of
phagocytic cells is preferably action to accelerate the
phagocytosis of either one or both of macrophage and
neutrophil.
[0236] In the present specification, the type of an anti-bacterial
infection agent is not particularly limited, as long as it is able
to reduce infectious disease caused by bacteria, namely, various
symptoms caused by the presence of bacteria in a body. Examples of
bacteria include Welch bacillus, Pseudomonas aeruginosa, and
enteropathogenic Escherichia coli.
[0237] In the present specification, the bacterial toxin
neutralizer means an agent for reducing the action of toxin
generated by bacteria. Anti-bacterial infection agents include
those that suppress the growth of bacteria or the release of toxin
from bacteria, so as to reduce various symptoms caused by bacteria.
On the other hand, toxin neutralizers include those that reduce the
action of toxin even under circumstances in which only the toxin
acts on a living body. Such action includes action to adsorb toxin,
action to modify toxin to an inactive product, action to
incorporate toxin into phagocytic cells, and further action to
digest the thus incorporated toxin.
[0238] In the present invention, the anticancer agent means an
agent, which has antitumor activity and is used for the prevention
or treatment of cancer. The tumor, on which the anticancer agent of
the present invention acts, includes both primary tumor and
metastatic tumor. Accordingly, the anticancer agent of the present
invention may be used not only for the treatment of primary cancer
and metastatic tumor, but also for prevention of metastatic tumor
at the same time of or after the treatment of primary cancer.
Moreover, in the present invention, examples of the tumor, on which
the anticancer agent acts, include breast cancer, testicular
cancer, orchioncus, pancreatic cancer, phrenic tumor, lung cancer,
ovarian cancer, stomach cancer, gallbladder cancer, kidney cancer,
prostatic cancer, esophageal cancer, liver cancer, oral cancer,
colonic cancer, large bowel cancer, rectal cancer, uterine cancer,
bile duct cancer, islet cell adenoma, adrenal cortical carcinoma,
bladder cancer, thyroid cancer, skin cancer, malignant carcinoid
tumor, melanoma, glioma, osteosarcoma, myeloma, soft tissue
sarcoma, neuroblastoma, malignant lymphoma, and leukemia. Of these,
breast cancer, testicular cancer, pancreatic cancer or pherenic
cancer is preferable.
[0239] In the present specification, the trehalose compound of the
present invention, or a pharmaceutical composition comprising the
aforementioned compound and a pharmaceutically acceptable carrier,
can be used as a pharmaceutical product such as an
immunostimulator, a bacterial toxin neutralizer or an anticancer
agent.
[0240] In the present specification, a pharmaceutically acceptable
carrier is not particularly limited, as long as it is
pharmacologically and pharmaceutically acceptable. Examples of such
a pharmaceutically acceptable carrier include: carriers commonly
used in the production of pharmaceutical preparations, such as an
excipient, a binder, a dispersant, a thickener, a lubricant, a pH
adjuster or a solubilizer; an antibiotic; an antibacterial agent; a
disinfectant; an antiseptic; a builder; a bleaching agent; an
enzyme; a chelating agent; a defoaming agent; a coloring agent (a
dye, a pigment, etc.); a softening agent; a moisturizing agent; a
surfactant; an antioxidant; an aromatic; a corrigent; a flavoring
agent; and a solvent. The pharmaceutically acceptable carrier can
be mixed within the range that does not impair the activity of the
trehalose compound (I) of the present invention. Addition of such a
carrier may have influence on the absorbing property of the
trehalose compound (I) of the present invention or the blood
concentration thereof, and thus it is also possible to change the
disposition of the compound.
[0241] In the present specification, the method for administering
the compound of the present case means a method for administering
the trehalose compound of the present invention itself or a
pharmaceutical composition comprising the present compound to a
human or an animal. The compound of the present case or the
pharmaceutical composition can be formulated in the form of an
ordinary medical preparation. Such a medical preparation can be
prepared, as appropriate, using the aforementioned pharmacological
carrier. The dosage form is not particularly limited, and it is
selected and used, as appropriate, depending on therapeutic
purpose. Representative examples of a dosage form include a tablet,
a pill, a powder, a liquid agent, a suspension, an emulsion, a
granule, a capsule, a suppository, an injection (an liquid agent, a
suspension, an emulsion, etc.) These pharmaceutical preparations
may be produced by a commonly used method.
[0242] The dose of the aforementioned medical preparation may be
selected, as appropriate, depending on direction of use, the age
and sex of a patient, the degree of disease, and other conditions.
In general, the trehalose compound (1) used as an active ingredient
is administered in a dose of 0.01 to 100 mg, and preferably 0.1 to
50 mg per day per kg of body weight, once or divided over several
administrations.
[0243] The aforementioned dose may be fluctuated depending on
various conditions. Thus, there is a case in which a dose smaller
than the aforementioned range is sufficient. Also, there is a case
in which a dose higher than the aforementioned range is
necessary.
[0244] It is to be noted that the terms are used to explain
specific embodiments in the present specification. Thus, they are
not intended to limit the scope of the invention.
[0245] In addition, the term "include" used in the present
specification intends to mean that the described matters (a member,
a step, an element, a number, etc.) are present, and thus, it does
not exclude the presence of other matters (a member, a step, an
element, a number, etc.), with the exception that the term
"include" should be contextually understood in an apparently
different way.
[0246] Unless there are other definitions, all of the terms used
herein (including technical terms and scientific terms) have the
same meanings as those broadly understood by persons skilled in the
technical field, to which the present invention pertains. Unless
other definitions are clearly stated, the terms used herein should
be understood to have meanings that are compatible with the
meanings used in the present specification and the related
technical field, and the terms should not be interpreted to have
ideal meanings or excessively formal meanings.
[0247] There may be a case in which the embodiment of the present
invention is described with reference to a schematic view. When
such a schematic view is used, there may be a case in which
exaggerated descriptions are used to provide clear explanation.
[0248] The terms such as "first" and "second" are used to indicate
various elements. However, it is understood that these elements
should not be restricted by such terms. These terms are used to
distinguish one element from other elements. Thus, it is possible
to indicate, for example, the first element as the second element,
and likewise, to indicate the second element as the first element.
This does not deviate from the scope of the present invention.
[0249] Hereinafter, the present invention will be more specifically
described in the following examples. These examples are not
intended to limit the present invention. It may also be possible to
add modification to the present invention, as appropriate, within
the range suitable for the above-mentioned or after-mentioned
contents. Such modified inventions are also included in the
technical scope of the present invention.
EXAMPLES
Example
Chemical Synthesis of Trehalose Diester Compound
[0250] The trehalose compound represented by the formula (3) of
Synthetic Scheme 1, wherein the hydroxyl group of sugar has been
protected, and a desired carbonyl compound represented by the
formula (4) or the formula (6), were subjected to an esterification
reaction, so as to synthesize the compound represented by the
formula (7). Thereafter, the hydroxyl group of sugar was
deprotected, so as to obtain a desired trehalose diester
compound.
[0251] Several production examples will be described below.
However, these production examples are not intended to limit 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-.alpha.,.alpha.'-
-trehalose
##STR00015##
[0253] The carboxylic acid (2-decyldodecanoic acid) (145 mg, 425
.mu.mol) obtained by the method described in Production Example C-1
and a trehalose derivative
(2,3,4,2',3',4'-hexabenzoxy-.alpha.,.alpha.'-trehalose) (150 mg,
170 .mu.mol) were dissolved in an anhydrous dichloromethane
solution (2 ml). Thereafter, powdered molecular sieves 4A (0.3 g),
4-dimethylaminopyridine (20.8 mg, 170 .mu.mol),
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
[hereinafter abbreviated as EDCI] (97.8 mg, 510 .mu.mol) were
successively added to the solution, and the mixed solution was then
heated to reflux for 4 hours. Thereafter, the reaction solution was
filtrated with Celite-535, and distilled water was then added
thereto, followed by extraction with dichloromethane three times.
Anhydrous magnesium sulfate was added to the organic layer to dry
it, and the resultant was filtrated and was then concentrated. The
residue was purified using column chromatography (hexane:ethyl
acetate=10:1), so as to obtain
6,6'-bis-O-(2-decyldodecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha-
.,.alpha.'-trehalose (242 mg, 93%) as a diester form in the form of
a colorless amorphous solid.
colorless syrup; [.alpha.].sub.D.sup.20 +57.7.degree. (c 0.9
CHCl.sub.3); FT IR (neat) 3088, 3064, 3031, 2941, 2862, 1946, 1869,
1804, 1741 cm.sup.-1; .sup.1H NMR (300 MHz in CDCl.sub.3)
.delta.0.87 (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);
.sup.13C NMR (75 MHz in CDCl.sub.3) .delta.14.13, 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.
Production Example A-2
Synthesis of
6,6'-bis-O-(2-octyldecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha.,.alpha.'-t-
rehalose
##STR00016##
[0255]
6,6'-Bis-O-(2-octyldecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha.,.alp-
ha.'-trehalose was obtained by the same method as that applied in
Production Example A-1, using the 2-octyldecanoic acid obtained by
the method described in Production Example C-2 as carboxylic
acid.
colorless syrup; [.alpha.].sub.D.sup.16 +62.3.degree. (c 1.0
CHCl.sub.3); FT IR (neat) 3088, 3064, 3031, 2927, 2855, 1947, 1868,
1808, 1737 cm.sup.-1; .sup.1H NMR (300 MHz in CDCl.sub.3)
.delta.0.85 (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); .sup.13CNMR (75 MHz in CDCl.sub.3)
.delta.14.12, 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.sup.++Na); HRMS (FAB.sup.+) m/z calcd
for C.sub.90H.sub.126O.sub.13Na (M.sup.++Na) 1437.9096. Found
1437.9126.
Production Example A-3
Synthesis of
6,6'-bis-O-(2-nonylundecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha.,.alpha.'-
-trehalose
##STR00017##
[0257]
6,6'-Bis-O-(2-nonylundecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha.,.a-
lpha.'-trehalose was obtained by the same method as that applied in
Production Example A-1, using the 2-nonylundecanoic acid obtained
by the method described in Production Example C-3 as carboxylic
acid.
colorless syrup; [.alpha.].sub.D.sup.16 +64.8.degree. (c 1.0
CHCl.sub.3); FT IR (neat) 3088, 3064, 3031, 2926, 2854, 1946, 1871,
1806, 1738 cm.sup.-1; .sup.1H NMR (300 MHz in CDCl.sub.3)
.delta.0.86 (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); .sup.13CNMR (75 MHz in CDCl.sub.3)
.delta.14.11, 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.sup.++Na); HRMS
(FAB.sup.+) m/z calcd for C.sub.94H.sub.134O.sub.13Na (M.sup.++Na)
1493.9722. Found 1493.9701.
Production Example A-4
Synthesis of
6,6'-bis-O-(2-undecyltridecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha.,.alph-
a.'-trehalose
##STR00018##
[0259]
6,6'-Bis-O-(2-undecyltridecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha.-
,.alpha.'-trehalose was obtained by the same method as that applied
in Production Example A-1, using the 2-undecyltridecanoic acid
obtained by the method described in Production Example C-4 as
carboxylic acid.
colorless syrup; [.alpha.].sub.D.sup.20 +60.0.degree. (c 0.9
CHCl.sub.3); FT IR (neat) 3088, 3064, 3031, 2940, 2862, 1946, 1869,
1805, 1740 cm.sup.-1; .sup.1H NMR (300 MHz in CDCl.sub.3)
.delta.0.87 (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=12.0 Hz), 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);
.sup.13C NMR (75 MHz in CDCl.sub.3) .delta.14.12, 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.
Production Example A-5
Synthesis of
6,6'-bis-O-(2-dodecyltetradecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha.,.al-
pha.'-trehalose
##STR00019##
[0261]
6,6'-Bis-O-(2-dodecyltetradecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alph-
a.,.alpha.'-trehalose was obtained by the same method as that
applied in Production Example A-1, using the 2-dodecyltetradecanoic
acid obtained by the method described in Production Example
C.sub.1-5 as carboxylic acid.
colorless syrup; [.alpha.].sub.D.sup.20 +60.8.degree. (c 1.0
CHCl.sub.3); FT IR (neat) 3088, 3064, 3031, 2940, 2862, 1946, 1869,
1804, 1739 cm.sup.-1; .sup.1H NMR (300 MHz in CDCl.sub.3)
.delta.0.88 (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);
.sup.13C NMR (75 MHz in CDCl.sub.3) .delta.14.12, 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.
Production Example A-6
Synthesis of
6,6'-bis-O-(2-tridecylpentadecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha.,.a-
lpha.'-trehalose
##STR00020##
[0263]
6,6'-Bis-O-(2-tridecylpentadecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alp-
ha.,.alpha.'-trehalose was obtained by the same method as that
applied in Production Example A-1, using the
2-tridecylpentadecanoic acid obtained by the method described in
Production Example C.sub.1-6 as carboxylic acid.
colorless syrup; [.alpha.].sub.D.sup.20 +50.2.degree. (c 1.1
CHCl.sub.3); FT IR (neat) 3088, 3064, 3031, 2929, 2855, 1945, 1868,
1804, 1739 cm.sup.-1; .sup.1H NMR (300 MHz in CDCl.sub.3)
.delta.0.88 (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);
.sup.13C NMR (75 MHz in CDCl.sub.3) .delta.14.11, 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.
Production Example A-7
Synthesis of
6,6'-bis-O-(2-pentadecylheptadecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha.,-
.alpha.'-trehalose
##STR00021##
[0265]
6,6'-Bis-O-(2-pentadecylheptadecanoyl)-2,3,4,2',3',4'-hexabenzyl-.a-
lpha.,.alpha.'-trehalose was obtained by the same method as that
applied in Production Example A-1, using the
2-pentadecylheptadecanoic acid obtained by the method described in
Production Example C-8 as carboxylic acid.
colorless syrup; [.alpha.].sub.D.sup.20 +44.3.degree. (c 1.0
CHCl.sub.3); FT IR (neat) 3088, 3064, 3031, 2941, 2861, 1945, 1868,
1812, 1739 cm.sup.-1; .sup.1H NMR (300 MHz in CDCl.sub.3)
.delta.0.88 (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=10.8 Hz), 4.99
(2H, d, J=10.5 Hz), 5.18 (2H, d, J=3.6 Hz), 7.22-7.37 (30H, m);
.sup.13C NMR (75 MHz in CDCl.sub.3) .delta.14.14, 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.
Production Example A-8
Synthesis of
6,6'-bis-O-(2-hexadecyloctadecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha.,.a-
lpha.'-trehalose
##STR00022##
[0267]
6,6'-Bis-O-(2-hexadecyloctadecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alp-
ha.,.alpha.'-trehalose was obtained by the same method as that
applied in Production Example A-1, using the
2-hexadecyloctadecanoic acid obtained by the method described in
Production Example C-9 as carboxylic acid.
colorless syrup; [.alpha.].sub.D.sup.20 +48.8.degree. (c 1.0
CHCl.sub.3); FT IR (neat) 3088, 3064, 3031, 2938, 2857, 1944, 1869,
1808, 1739 cm.sup.-1; NMR (300 MHz in CDCl.sub.3) .delta.0.88 (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=10.5 Hz), 4.99 (2H, d, J=10.8 Hz), 5.18
(2H, d, J=3.6 Hz), 7.22-7.37 (30H, m); .sup.13C NMR (75 MHz in
CDCl.sub.3) .delta.14.15, 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.
Example 1
Production Example .alpha.-1
Synthesis of
6,6'-bis-O-(2-decyldodecanoyl)-.alpha.,.alpha.'-trehalose
##STR00023##
[0269] The
6,6'-bis-O-(2-decyldodecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha-
.,.alpha.'-trehalose (200 mg, 131 enol) obtained by the method
described in Production Example A-1 was dissolved in a mixed
solvent (4 ml) of chloroform:methanol:acetic acid (1:1:0.05), and
palladium hydroxide (20 w/w %, 8 mg, 11.4 .mu.mol) was then added
to the obtained solution. The obtained mixture was stirred under 1
atmospheric pressure of hydrogen for 6 hours. Thereafter, the
reaction mixture was filtrated and was then concentrated, and the
residue was purified using column chromatography
(dichloromethane:methanol=10:1), so as to obtain
6,6'-bis-O-(2-decyldodecanoyl)-.alpha.,.alpha.'-trehalose (125 mg,
97%) in the form of a colorless amorphous solid.
colorless syrup; [.alpha.].sub.D.sup.20 +61.8.degree. (c 1.0
CHCl.sub.3); FT IR (neat) 3358, 2940, 2861, 1746 cm.sup.-1; .sup.1H
NMR (300 MHz in C.sub.5D.sub.5N) .delta.0.88 (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); .sup.13C NMR (75 MHz in C.sub.5D.sub.5N)
.delta.14.29, 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.sup.++Na); FIRMS (FAB.sup.+) m/z
calcd for C.sub.56H.sub.106O.sub.13Na (M.sup.++Na) 1009.7532. Found
1009.7498.
Example 2
Production Example .alpha.-2
Synthesis of
6,6'-bis-O-(2-octyldecanoyl)-.alpha.,.alpha.'-trehalose
##STR00024##
[0271] 6,6'-Bis-O-(2-octyldecanoyl)-.alpha.,.alpha.'-trehalose was
obtained by the same method as that applied in Production Example
.alpha.-1, using the
6,6'-bis-O-(2-octyldecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha.,.alpha.'-t-
rehalose obtained by the method described in Production Example A-2
as a raw material compound.
[0272] colorless syrup; [.alpha.].sub.D.sup.14 +70.2.degree. (c 0.6
CHCl.sub.3); FT IR (neat) 3300, 2929, 2856, 1743 cm.sup.-1; .sup.1H
NMR (300 MHz in C.sub.5D.sub.5N) .delta.0.86 (12H, t, J=6.9 Hz),
1.21 (40H, m), 1.46 (8H, m), 1.57 (4H, m), 1.80 (4H, m), 2.56 (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), 4.88 (2H, dd, J=12.0, 4.8 Hz), 5.08 (4H, m), 5.87
(2H, d, J=3.6 Hz); .sup.13C NMR (75 MHz in C.sub.5D.sub.5N)
.delta.13.69, 22.32, 27.12, 27.19, 28.96, 29.11, 29.33, 31.47,
32.23, 45.55, 63.25, 70.94, 71.30, 72.69, 74.18, 95.14, 175.68;
FABMS m/z (%) 898 (M.sup.++Na); HRMS (FAB.sup.+)m/z calcd for
C.sub.48H.sub.90O.sub.13Na (M.sup.++Na) 897.6279. Found
897.6249.
Example 3
Production Example .alpha.-3
Synthesis of
6,6'-bis-O-(2-nonylundecanoyl)-.alpha.,.alpha.'-trehalose
##STR00025##
[0274] 6,6'-Bis-O-(2-nonylundecanoyl)-.alpha.,.alpha.'-trehalose
was obtained by the same method as that applied in Production
Example .alpha.-1, using the
6,6'-bis-O-(2-nonylundecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha.,.alpha.'-
-trehalose obtained by the method described in Production Example
A-3 as a raw material compound.
colorless syrup; [.alpha.].sub.D.sup.14 +64.9.degree. (c 0.6
CHCl.sub.3); FT IR (neat) 3306, 2928, 2855, 1742 cm.sup.-1; .sup.1H
NMR (300 MHz in C.sub.5D.sub.5N) .delta.0.87 (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); .sup.13C NMR (75 MHz in C.sub.5D.sub.5N)
.delta.13.69, 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.sup.++Na); HRMS (FAB.sup.+) m/z calcd
for C.sub.52H.sub.98O.sub.13Na (M.sup.++Na) 953.6905. Found
953.6862.
Example 4
Production Example .alpha.-4
Synthesis of
6,6'-bis-O-(2-undecyltridecanoyl)-.alpha.,.alpha.'-trehalose
##STR00026##
[0276] 6,6'-Bis-O-(2-undecyltridecanoyl)-.alpha.,.alpha.'-trehalose
was obtained by the same method as that applied in Production
Example .alpha.-1, using the
6,6'-bis-O-(2-undecyltridecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha.,.alph-
a.'-trehalose obtained by the method described in Production
Example A-4 as a raw material compound.
colorless syrup; [.alpha.].sub.D.sup.14 +58.5.degree. (c 1.0
CHCl.sub.3); FT IR (neat) 3297, 2934, 2856, 1742 cm.sup.-1; .sup.1H
NMR (300 MHz in C.sub.5D.sub.5N) .delta.0.88 (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); .sup.13C NMR (75 MHz in C.sub.5D.sub.5N)
.delta.14.29, 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.sup.++Na); HRMS (FAB.sup.+) m/z calcd for
C.sub.60H.sub.114O.sub.13Na (M.sup.++Na) 1065.8157. Found
1065.8160.
Example 5
Production Example .alpha.-5
Synthesis of
6,6'-bis-O-(2-dodecyltetradecanoyl)-.alpha.,.alpha.'-trehalose
##STR00027##
[0278]
6,6'-Bis-O-(2-dodecyltetradecanoyl)-.alpha.,.alpha.'-trehalose was
obtained by the same method as that applied in Production Example
.alpha.-1, using the
6,6'-bis-O-(2-dodecyltetradecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha.,.al-
pha.'-trehalose obtained by the method described in Production
Example A-5 as a raw material compound. colorless syrup;
[.alpha.].sub.D.sup.14 +54.6.degree. (c 1.0 CHCl.sub.3); FT IR
(neat) 3310, 2937, 2857, 1742 cm.sup.-1; .sup.1H NMR (300 MHz in
C.sub.5D.sub.5N) .delta.0.88 (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); .sup.13C NMR (75 MHz in C.sub.5D.sub.5N) .delta.14.30, 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.sup.++Na); HRMS (FAB.sup.+) m/z calcd for
C.sub.64H.sub.122O.sub.13Na (M.sup.++Na) 1121.8784. Found
1121.8831.
Example 6
Production Example .alpha.-6
Synthesis of
6,6'-bis-O-(2-tridecylpentadecanoyl)-.alpha.,.alpha.'-trehalose
##STR00028##
[0280]
6,6'-Bis-O-(2-tridecylpentadecanoyl)-.alpha.,.alpha.'-trehalose was
obtained by the same method as that applied in Production Example
.alpha.-1, using the
6,6'-bis-O-(2-tridecylpentadecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha.,.a-
lpha.'-trehalose obtained by the method described in Production
Example A-6 as a raw material compound. colorless syrup;
[.alpha.].sub.D.sup.19 +52.7.degree. (c 0.6 CHCl.sub.3); FT IR
(neat) 3313, 2927, 2854, 1741 cm.sup.-1; .sup.1H NMR (300 MHz in
C.sub.5D.sub.5N) .delta.0.88 (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); .sup.13C NMR (75 MHz in C.sub.5D.sub.5N) .delta.14.28, 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.sup.++Na); HRMS (FAB.sup.+) m/z calcd for
C.sub.68H.sub.130O.sub.13Na (M.sup.++Na) 1177.9409. Found
1177.9404.
Example 7
Production Example .alpha.-7
Synthesis of
6,6'-bis-O-(2-pentadecylheptadecanoyl)-.alpha.,.alpha.'-trehalose
##STR00029##
[0282]
6,6'-Bis-O-(2-pentadecylheptadecanoyl)-.alpha.,.alpha.'-trehalose
was obtained by the same method as that applied in Production
Example .alpha.-1, using the
6,6'-bis-O-(2-pentadecylheptadecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha.,-
.alpha.'-trehalose obtained by the method described in Production
Example A-7 as a raw material compound. colorless syrup;
[.alpha.].sub.D.sup.19 +44.8.degree. (c 0.5 CHCl.sub.3); FT IR
(neat) 3330, 2925, 2853, 1741 cm.sup.-1; .sup.1H NMR (300 MHz in
C.sub.5D.sub.5N) .delta.0.87 (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); .sup.13C NMR (75 MHz in C.sub.5D.sub.5N) .delta.14.23, 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.sup.++Na); HRMS (FAB.sup.+) m/z calcd for
C.sub.76H.sub.146O.sub.13Na (M.sup.++Na) 1290.0661. Found
1290.0677.
Example 8
Production Example .alpha.-8
Synthesis of
6,6'-bis-O-(2-hexadecyloctadecanoyl)-.alpha.,.alpha.'-trehalose
##STR00030##
[0284]
6,6'-Bis-O-(2-hexadecyloctadecanoyl)-.alpha.,.alpha.'-trehalose was
obtained by the same method as that applied in Production Example
.alpha.-1, using the
6,6'-bis-O-(2-hexadecyloctadecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha.,.a-
lpha.'-trehalose obtained by the method described in Production
Example A-8 as a raw material compound. colorless syrup;
[.alpha.].sub.D.sup.17 +45.1.degree. (c 0.5 CHCl.sub.3); FT IR
(neat) 3308, 2937, 2856, 1741 cm.sup.-1; .sup.1H NMR (300 MHz in
C.sub.5D.sub.5N) .delta.0.87 (12H, t, =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); .sup.13C NMR (75 MHz in C.sub.5D.sub.5N) .delta.14.25, 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.sup.++Na); HRMS (FAB.sup.+) m/z calcd for
C.sub.80H.sub.154O.sub.13Na (M.sup.++Na) 1346.1288. Found
1346.1287.
Production Example B-1
Synthesis of
6,6'-bis-O-(3-nonyldodecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha.,.alpha.'-
-trehalose
##STR00031##
[0286] The carboxylic acid (3-nonyldodecanoic acid) (236 mg, 724
.mu.mol) obtained by the method described in Production Example D-1
and a trehalose derivative
(2,3,4,2',3',4'-hexabenzoxy-.alpha.,.alpha.'-trehalose) (256 mg,
290 .mu.mol) were dissolved in an anhydrous dichloromethane
solution (10 ml). Thereafter, powdered molecular sieves 4A (1 g),
4-dimethylaminopyridine (35.4 mg, 290 .mu.mol),
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
[hereinafter abbreviated as EDCI] (222 mg, 1.16 mmol) were
successively added to the solution, and the mixed solution was then
heated to reflux for 10 hours. Thereafter, the reaction solution
was filtrated with Celite-535, and a saturated saline solution was
then added thereto, followed by extraction with dichloromethane two
times. Anhydrous magnesium sulfate was added to the organic layer
to dry it, and the resultant was filtrated and was then
concentrated. The residue was purified using column chromatography
(hexane:ethyl acetate=15:1), so as to obtain
6,6'-bis-O-(3-nonyldodecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha-
.,.alpha.'-trehalose (350 mg, 80%) as a diester form in the form of
a colorless amorphous solid.
colorless syrup; [.alpha.].sub.D.sup.21 +65.3.degree. (c 1.0
CHCl.sub.3); FT IR (neat) 3088, 3063, 3031, 2925, 2853, 1944, 1871,
1806, 1739 cm.sup.-1; .sup.1H NMR (300 MHz in CDCl.sub.3)
.delta.0.87 (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=12.0 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); .sup.13C NMR (75 MHz in CDCl.sub.3)
.delta.14.13, 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.sup.++Na); HRMS (FAB.sup.+) m/z calcd for
C.sub.96H.sub.138O.sub.13Na (M.sup.++Na) 1522.0036. Found
1522.0020.
Production Example B-2
Synthesis of
6,6'-bis-O-(3-octylundecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha.,.alpha.'-
-trehalose
##STR00032##
[0288]
6,6'-Bis-O-(3-octylundecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha.,.a-
lpha.'-trehalose was obtained by the same method as that applied in
Production Example B-1, using the 3-octylundecanoic acid obtained
by the method described in Production Example D-2 as carboxylic
acid.
colorless syrup; [.alpha.].sub.D.sup.20 +41.8.degree. (c 2.0
CHCl.sub.3); FT IR (neat) 3088, 3064, 3031, 2932, 2855, 1947, 1867,
1806, 1739 cm.sup.-1; .sup.1H NMR (300 MHz in CDCl.sub.3)
.delta.0.86 (6H, t, J=6.9 Hz), 80.87 (6H, t, J=6.9 Hz), 1.20 (56H,
m), 1.81 (2H, m), 2.20 (40, d, J=6.9 Hz), 3.54 (20, 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=12.0 Hz), 4.72 (20, d,
J=12.0 Hz), 4.86 (4H, d, J=10.5 Hz), 5.00 (2H, d, J=10.5 Hz), 5.17
(20, d, J=3.6 Hz), 7.23-7.37 (30H, m); .sup.13C NMR (75 MHz in
CDCl.sub.3) .delta.14.14, 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.sup.++Na); HRMS (FAB.sup.+) m/z calcd for
C.sub.92H.sub.130O.sub.13Na (M.sup.++Na) 1465.9409. Found
1465.9392.
Production Example B-3
Synthesis of
6,6'-bis-O-(3-decyltridecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha.,.alpha.-
'-trehalose
##STR00033##
[0290]
6,6'-Bis-O-(3-decyltridecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha.,.-
alpha.'-trehalose was obtained by the same method as that applied
in Production Example B-1, using the 3-decyltridecanoic acid
obtained by the method described in Production Example D-3 as
carboxylic acid.
colorless syrup; [.alpha.].sub.D.sup.20 +53.6.degree. (c 1.0
CHCl.sub.3); FT IR (neat) 3088, 3063, 3030, 2926, 2854, 1946, 1874,
1804, 1739 cm.sup.-1; .sup.1H NMR (300 MHz in CDCl.sub.3)
.delta.0.87 (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=12.0 Hz), 4.86 (4H, d,
J=10.5 Hz), 5.00 (2H, d, J=10.5 Hz), 5.17 (25, d, J=3.6 Hz),
7.23-7.36 (30H, m); .sup.13C NMR (75 MHz in CDCl.sub.3)
.delta.14.14, 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.sup.++H+Na); HRMS (FAB.sup.+) m/z calcd for
C.sub.100H.sub.147O.sub.13Na (M.sup.++H+Na) 1579.0787. Found
1579.0763.
Production Example 5-4
Synthesis of
6,6'-bis-O-(3-undecyltetradecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha.,.al-
pha.'-trehalose
##STR00034##
[0292]
6,6'-Bis-O-(3-undecyltetradecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alph-
a.,.alpha.'-trehalose was obtained by the same method as that
applied in Production Example B-1, using the 3-undecyltetradecanoic
acid obtained by the method described in Production Example D-4 as
carboxylic acid.
colorless syrup; [.alpha.].sub.D.sup.20 +58.1.degree. (c 1.0
CHCl.sub.3); FT IR (neat) 3088, 3064, 3031, 2926, 2854, 1946, 1867,
1806, 1739 cm.sup.-1; .sup.1H NMR (300 MHz in CDCl.sub.3)
.delta.0.88 (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=12.0 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),
7.19-7.36 (30H, m); .sup.13C NMR (75 MHz in CDCl.sub.3)
.delta.14.14, 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.sup.++Na); HRMS (FAB.sup.+) m/z calcd for
C.sub.104H.sub.154O.sub.13Na (M.sup.++Na) 1634.1288. Found
1634.1298.
Production Example B-5
Synthesis of
6,6'-bis-O-(3-dodecylpentadecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha.,.al-
pha.'-trehalose
##STR00035##
[0294]
6,6'-Bis-O-(3-dodecylpentadecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alph-
a.,.alpha.'-trehalose was obtained by the same method as that
applied in Production Example B-1, using the 3-dodecylpentadecanoic
acid obtained by the method described in Production Example D-5 as
carboxylic acid.
colorless syrup; [.alpha.].sub.D.sup.20 +68.9.degree. (c 0.9
CHCl.sub.3); FT IR (neat) 3088, 3063, 3031, 2925, 2853, 1944, 1867,
1806, 1739 cm.sup.-1; .sup.1H NMR (300 MHz in CDCl.sub.3)
.delta.0.88 (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=10.5 Hz), 5.18
(2H, d, J=3.3 Hz), 7.21-7.37 (30H, m); .sup.13C NMR (75 MHz in
CDCl.sub.3) .delta.14.14, 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.sup.++H+Na); HRMS (FAB.sup.+) m/z calcd for
C.sub.108H.sub.163O.sub.13Na (M.sup.++H+Na) 1691.1993. Found
1691.1992.
Production Example B-6
Synthesis of
6,6'-bis-O-(3-tridecylhexadecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha.,.al-
pha.'-trehalose
##STR00036##
[0296]
6,6'-Bis-O-(3-tridecylhexadecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alph-
a.,.alpha.'-trehalose was obtained by the same method as that
applied in Production Example B-1, using commercially available
3-tridecylhexadecanoic acid (manufactured by Wako Pure Chemical
Industries, Ltd.) as carboxylic acid.
colorless syrup; [.alpha.].sub.D.sup.20 +49.5.degree. (c 0.9
CHCl.sub.3); FT IR (neat) 3088, 3064, 3031, 2925, 2853, 1944, 1871,
1806, 1739 cm.sup.-1; .sup.1H NMR (300 MHz in CDCl.sub.3)
.delta.0.88 (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=10.8 Hz), 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); .sup.13C
NMR (75 MHz in CDCl.sub.3) .delta.14.16, 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.sup.++H+Na); HRMS
(FAB.sup.+) m/z calcd for C.sub.112H.sub.171O.sub.13Na
(M.sup.++H+Na) 1747.2619. Found 1747.2618.
Production Example B-7
Synthesis of
6,6'-bis-O-(3-tetradecylheptadecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha.,-
.alpha.'-trehalose
##STR00037##
[0298]
6,6'-Bis-O-(3-tetradecylheptadecanoyl)-2,3,4,2',3',4'-hexabenzyl-.a-
lpha.,.alpha.'-trehalose was obtained by the same method as that
applied in Production Example B-1, using the
3-tetradecylheptadecanoic acid obtained by the method described in
Production Example D-6 as carboxylic acid.
colorless syrup; [.alpha.].sub.D.sup.20 +43.7.degree. (c 1.0
CHCl.sub.3); FT IR (neat) 3087, 3064, 3032, 2924, 2853, 1943, 1871,
1796, 1739 cm.sup.-1; .sup.1H NMR (300 MHz in CDCl.sub.3)
.delta.0.88 (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); .sup.13C
NMR (75 MHz in CDCl.sub.3) .delta.14.16, 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.sup.++Na); HRMS
(FAB.sup.+) m/z calcd for C.sub.116H.sub.178O.sub.13Na (M.sup.++Na)
1802.3185. Found 1802.3175.
Example 9
Production Example .beta.-1
Synthesis of
6,6'-bis-O-(3-nonyldodecanoyl)-.alpha.,.alpha.'-trehalose
##STR00038##
[0300] The
6,6'-bis-O-(3-nonyldodecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha-
.,.alpha.-trehalose (350 mg, 233 .mu.mol) obtained by the method
described in Production Example B-1 was dissolved in a mixed
solvent (5 ml) of chloroform:methanol:acetic acid (5:1:0.5), and
palladium hydroxide (3 w/w %, 13 mg, 18.5 .mu.mol) was then added
to the obtained solution. The obtained mixture was stirred under 1
atmospheric pressure of hydrogen for 27 hours. Thereafter, the
reaction mixture was filtrated and was then concentrated, and the
residue was purified using column chromatography
(dichloromethane:methanol=15:1), so as to obtain
6,6'-bis-O-(3-nonyldodecanoyl)-.alpha.,.alpha.'-trehalose (135 mg,
61%) in the form of a colorless amorphous solid.
colorless syrup; [.alpha.].sub.D.sup.21 +62.8.degree. (c 0.7
CHCl.sub.3); FT IR (neat) 3316, 2926, 2854, 1743 cm.sup.-1; .sup.1H
NMR (300 MHz in C.sub.5D.sub.5N) .delta.0.81 (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); .sup.13C NMR (75 MHz in C.sub.5D.sub.5N)
.delta.14.29, 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.sup.++Na); HRMS (FAB.sup.+) m/z calcd
for C.sub.54H.sub.102O.sub.13Na (M.sup.++Na) 981.7218. Found
981.7198.
Example 15
Production Example .beta.-2
Synthesis of
6,6'-bis-O-(3-octylundecanoyl)-.alpha.,.alpha.-trehalose
##STR00039##
[0302] 6,6'-Bis-O-(3-octylundecanoyl)-.alpha.,.alpha.'-trehalose
was obtained by the same method as that applied in Production
Example .beta.-1, using the
6,6'-bis-O-(3-octylundecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha.,.alpha.'-
-trehalose obtained by the method described in Production Example
B-2 as a raw material compound.
colorless syrup; [.alpha.].sub.D.sup.21 +70.7.degree. (c 0.4
CHCl.sub.3); FT IR (neat) 3275, 2925, 2854, 1742 cm.sup.-1; .sup.1H
NMR (300 MHz in C.sub.5D.sub.5N) 60.86 (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); .sup.13C NMR (75 MHz in C.sub.5D.sub.5N)
.delta.14.28, 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.sup.++Na); HRMS (FAB.sup.+) m/z calcd
for C.sub.50H.sub.94O.sub.13Na (M.sup.++Na) 925.6592. Found
925.6585.
Example 10
Production Example .beta.-3
Synthesis of
6,6'-bis-O-(3-decyltridecanoyl)-.alpha.,.alpha.'-trehalose
##STR00040##
[0304] 6,6'-Bis-O-(3-decyltridecanoyl)-.alpha.,.alpha.'-trehalose
was obtained by the same method as that applied in Production
Example .beta.-1, using the
6,6'-bis-O-(3-decyltridecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha.,.alpha.-
'-trehalose obtained by the method described in Production Example
B-3 as a raw material compound.
colorless syrup; [.alpha.].sub.D.sup.21 +60.9.degree. (c 0.9
CHCl.sub.3); FT IR (neat) 3279, 2924, 2854, 1742 cm.sup.-1; .sup.1H
NMR (300 MHz in C.sub.5D.sub.5N) .delta.0.83 (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); .sup.13C NMR (75 MHz in
C.sub.5D.sub.5N) .delta.14.10, 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.sup.++Na); HRMS (FAB.sup.+)
m/z calcd for C.sub.53H.sub.110O.sub.13Na (M.sup.++Na) 1037.7903.
Found 1037.7874.
Example 11
Production Example .beta.-4
Synthesis of
6,6'-bis-O-(3-undecyltetradecanoyl)-.alpha.,.alpha.'-trehalose
##STR00041##
[0306]
6,6'-Bis-O-(3-undecyltetradecanoyl)-.alpha.,.alpha.'-trehalose was
obtained by the same method as that applied in Production Example
.beta.-1, using the
6,6'-bis-O-(3-undecyltetradecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha.,.al-
pha.'-trehalose obtained by the method described in Production
Example B-4 as a raw material compound. colorless syrup;
[.alpha.].sub.D.sup.21 +60.6.degree. (c 0.5 CHCl.sub.3); FT IR
(neat) 3289, 2925, 2853, 1743 cm.sup.-1; .sup.1H NMR (300 MHz in
C.sub.5D.sub.5N) .delta.0.86 (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); .sup.13C NMR (75 MHz in C.sub.5D.sub.5N)
.delta.14.30, 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.sup.++Na); HRMS (FAB.sup.+) m/z calcd for
C.sub.62H.sub.118O.sub.13Na (M.sup.++Na) 1093.8470. Found
1093.8458.
Example 12
Production Example .beta.-5
Synthesis of
6,6'-bis-O-(3-dodecylpentadecanoyl)-.alpha.,.alpha.'-trehalose
##STR00042##
[0308]
6,6'-Bis-O-(3-dodecylpentadecanoyl)-.alpha.,.alpha.'-trehalose was
obtained by the same method as that applied in Production Example
.beta.-1, using the
6,6'-bis-O-(3-dodecylpentadecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha.,.al-
pha.'-trehalose obtained by the method described in Production
Example B-5 as a raw material compound. colorless syrup;
[.alpha.].sub.D.sup.21 +52.5.degree. (c 0.3 CHCl.sub.3); FT IR
(neat) 3271, 2923, 2853, 1743 cm.sup.-1; .sup.1H NMR (300 MHz in
C.sub.5D.sub.5N) .delta.0.84 (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); .sup.13C NMR (75 MHz in C.sub.5D.sub.5N)
.delta.14.18, 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.sup.++Na); HRMS (FAB.sup.+) m/z calcd for
C.sub.66H.sub.126O.sub.13Na (M.sup.++Na) 1149.9110. Found
1149.9103.
Example 13
Production Example .beta.-6
Synthesis of
6,6'-bis-O-(3-tridecylhexadecanoyl)-.alpha.,.alpha.'-trehalose
##STR00043##
[0310]
6,6'-Bis-O-(3-tridecylhexadecanoyl)-.alpha.,.alpha.'-trehalose was
obtained by the same method as that applied in Production Example
.beta.-1, using the
6,6'-bis-O-(3-tridecylhexadecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha.,.al-
pha.'-trehalose obtained by the method described in Production
Example B-6 as a raw material compound. colorless syrup;
[.alpha.].sub.D.sup.21 +42.3.degree. (c 0.5 CHCl.sub.3); FT IR
(neat) 3321, 2925, 2853, 1742 cm.sup.-1; .sup.1H NMR (300 MHz in
C.sub.5D.sub.5N) 50.83 (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);
.sup.13C NMR (75 MHz in C.sub.5D.sub.5N) .delta.14.30, 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.sup.++Na); HRMS (FAB.sup.+) m/z calcd for
C.sub.70H.sub.134O.sub.13Na (M.sup.++Na) 1205.9770. Found
1205.9746.
Example 14
Production Example .beta.-7
Synthesis of
6,6'-bis-0-(3-tetradecylheptadecanoyl)-.alpha.,.alpha.'-trehalose
##STR00044##
[0312]
6,6'-Bis-O-(3-tetradecylheptadecanoyl)-.alpha.,.alpha.'-trehalose
was obtained by the same method as that applied in Production
Example .beta.-1, using the
6,6'-bis-O-(3-tetradecylheptadecanoyl)-2,3,4,2',3',4'-hexabenzyl-.alpha.,-
.alpha.'-trehalose obtained by the method described in Production
Example B-7 as a raw material compound. colorless syrup;
[.alpha.].sub.D.sup.21 +55.7.degree. (c 1.0 CHCl.sub.3); FT IR
(neat) 3310, 2925, 2853, 1743 cm.sup.-1; .sup.1H NMR (300 MHz in
C.sub.5D.sub.5N) .delta.0.84 (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); .sup.13C NMR (75 MHz in C.sub.5D.sub.5N)
.delta.14.05, 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.sup.++Na); HRMS (FAB.sup.+) m/z calcd
for C.sub.74H.sub.142O.sub.13Na (M.sup.++Na) 1262.0348. Found
1262.0348.
Synthesis of Carboxylic Acid Used as Raw Material
Production Example C-1
Synthesis of 2-decyl dodecanoate
##STR00045##
[0314] Anhydrous THF (11 ml) was added to a dried double-necked
flask, and sodium hydride (60 w/w %, 397 mg, 9.93 mmol) was then
added thereto. The obtained mixture was cooled to 0.degree. C., and
diethyl malonate (530 mg, 3.31 mmol) was then added dropwise
thereto. The obtained mixture was stirred at 0.degree. C. for 10
minutes, and 1-iododecane (2.22 g, 8.28 mmol) was then added
thereto, followed by stirring at a room temperature for 6 hours.
Thereafter, a saturated ammonium chloride aqueous solution was
added to the reaction solution, and the resultant was then
extracted with ether three times. The organic layer was dried over
anhydrous magnesium sulfate, and it was filtrated and was then
concentrated. The obtained residue was dissolved in a mixed solvent
of a 10 N sodium hydroxide aqueous solution (4 ml) and n-butanol (8
ml), and the solution was then heated to reflux for 6 hours.
Thereafter, the reaction solution was cooled to a room temperature,
and 1 N hydrochloric acid was then added thereto, followed by
extraction with ether three times. The organic layer was dried over
anhydrous sodium sulfate, and it was filtrated and was then
concentrated. The obtained residue was dissolved in acetic acid
(3.3 ml), and the mixed solution was then heated to reflux for 18
hours. After cooling the reaction solution, it was concentrated
under a reduced pressure to remove acetic acid. The residue was
purified by silica gel column chromatography (hexane:ethyl
acetate=7:1), so as to obtain carboxylic acid (2-decyl dodecanoate)
(710 mg, 63%) in the form of a white amorphous powder.
white powder; FT IR (neat) 3041, 2943, 2857, 2689, 1714 cm.sup.-1;
.sup.1H NMR (300 MHz in CDCl.sub.3) .delta.0.88 (6H, t, J=7.6 Hz),
1.21 (32H, m), 1.48 (2H, m), 1.61 (2H, m), 2.34 (1H, m); .sup.13C
NMR (75 MHz in CDCl.sub.3) .delta.14.13, 22.72, 27.40, 29.37,
29.50, 29.64, 31.95, 32.19, 45.61, 183.22; CIMS m/z (%) 341
(M.sup.++H); HRMS (CI.sup.+) m/z calcd for
C.sub.22H.sub.45O.sub.2(M.sup.++H) 341.3420. Found 341.3421.
Production Example C-2
Synthesis of 2-octyl decanoate
##STR00046##
[0316] 2-Octyl decanoate was obtained by the same method as that
applied in Production Example C-1, using 1-iodooctane instead of
1-iododecane.
white powder; FT IR (neat) 3032, 2927, 2856, 1707 cm.sup.-1;
.sup.1H NMR (300 MHz in CDCl.sub.3) .delta.0.88 (6H, t, J=6.9 Hz),
1.26 (24H, m), 1.48 (2H, m), 1.63 (2H, m), 2.34 (1H, m); .sup.13C
NMR (75 MHz in CDCl.sub.3) .delta.14.12, 22.70, 27.40, 29.30,
29.45, 29.60, 31.90, 32.20, 45.66, 183.50; CIMS m/z (%) 285
(M.sup.++H); HRMS (CI.sup.+) m/z calcd for
C.sub.18H.sub.37O.sub.2(M.sup.++H) 285.2794. Found 285.2772.
Production Example C-3
Synthesis of 2-nonyl undecanoate
##STR00047##
[0318] 2-Nonyl undecanoate was obtained by the same method as that
applied in Production Example C-1, using 1-iodononane instead of
1-iododecane.
white powder; FT IR (neat) 3019, 2934, 2858, 1712 cm.sup.-1;
.sup.1H NMR (300 MHz in CDCl.sub.3) .delta.0.88 (6H, t, J=6.9 Hz),
1.26 (28H, m), 1.48 (2H, m), 1.61 (2H, m), 2.33 (1H, m); .sup.13C
NMR (75 MHz in CDCl.sub.3) .delta.14.14, 22.72, 27.40, 29.34,
29.50, 29.60, 31.92, 32.20, 45.60, 183.11; CIMS m/z (%) 313
(M.sup.++H); HRMS (CI.sup.+) m/z calcd for
C.sub.20H.sub.41O.sub.2(M.sup.++H) 313.3106. Found 313.3111.
Production Example C-4
Synthesis of 2-undecyl tridecanoate
##STR00048##
[0320] 2-Undecyl tridecanoate was obtained by the same method as
that applied in Production Example C-1, using 1-iodoundecane
instead of 1-iododecane.
white powder; FT IR (neat) 3028, 2941, 2858, 1712 cm.sup.-1;
.sup.1H NMR (300 MHz in CDCl.sub.3) .delta.0.88 (6H, t, J=6.9 Hz),
1.25 (36H, m), 1.48 (2H, m), 1.61 (2H, m), 2.35 (1H, m); .sup.13C
NMR (75 MHz in CDCl.sub.3) .delta.14.17, 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.sup.++H); HRMS (CI.sup.+) m/z calcd for
C.sub.24H.sub.490O.sub.2(M.sup.++H) 369.3732. Found 369.3731.
Production Example C-5
Synthesis of 2-dodecyl tetradecanoate
##STR00049##
[0322] 2-Dodecyl tetradecanoate was obtained by the same method as
that applied in Production Example C-1, using 1-iodododecane
instead of 1-iododecane.
white powder; FT IR (neat) 3028, 2943, 2860, 2691, 1714 cm.sup.-1;
.sup.1H NMR (300 MHz in CDCl.sub.3) .delta.0.88 (6H, t, J=7.1 Hz),
1.25 (40H, m), 1.48 (2H, m), 1.61 (2H, m), 2.34 (1H, m); C NMR (75
MHz in CDCl.sub.3) .delta.14.16, 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.sup.++H); HRMS (CI.sup.+) m/z calcd for
C.sub.26H.sub.53O.sub.2(M.sup.++H) 397.4045. Found 397.4043.
Production Example C-6
Synthesis of 2-tridecyl pentadecanoate
##STR00050##
[0324] 2-Tridecyl pentadecanoate was obtained by the same method as
that applied in Production Example C-1, using 1-iodotridecane
instead of 1-iododecane.
white powder; FT IR (neat) 3032, 2922, 2851, 2691, 1712 cm.sup.-1;
.sup.1H NMR (300 MHz in CDCl.sub.3) .delta.0.88 (6H, t, J=6.9 Hz),
1.25 (44H, m), 1.48 (2H, m), 1.61 (2H, m), 2.35 (1H, m); .sup.13C
NMR (75 MHz in CDCl.sub.3) .delta.14.14, 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.sup.++H); HRMS (CI.sup.+) m/z calcd for
C.sub.28H.sub.57O.sub.2(M.sup.++H) 425.4358. Found 425.4341.
Production Example C-7
Synthesis of 2-tetradecyl hexadecanoate
##STR00051##
[0326] 2-Tetradecyl hexadecanoate was obtained by the same method
as that applied in Production Example C-1, using 1-iodotetradecane
instead of 1-iododecane.
white powder; FT IR (neat) 3028, 2928, 2854, 2684, 1706 cm.sup.-1;
.sup.1H NMR (300 MHz in CDCl.sub.3) .delta.0.88 (6H, t, J=7.2 Hz),
1.25 (48H, m), 1.48 (2H, m), 1.60 (2H, m), 2.34 (1H, m); .sup.13C
NMR (75 MHz in CDCl.sub.3) .delta.14.13, 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.sup.++H); HRMS (CI.sup.+) m/z calcd for
C.sub.30H.sub.61O.sub.2 (M.sup.++H) 453.4671. Found 453.4677.
Production Example C-8
Synthesis of 2-pentadecyl heptadecanoate
##STR00052##
[0328] 2-Pentadecyl heptadecanoate was obtained by the same method
as that applied in Production Example C-1, using 1-iodopentadecane
instead of 1-iododecane.
white powder; FT IR (neat) 3028, 2911, 2848, 2650, 1703 cm.sup.-1;
.sup.1H NMR (300 MHz in CDCl.sub.3) .delta.0.88 (6H, t, J=6.9 Hz),
1.25 (52H, m), 1.48 (2H, m), 1.61 (2H, m), 2.35 (1H, m); .sup.13C
NMR (75 MHz in CDCl.sub.3) .delta.14.05, 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.sup.++H); HRMS (CI.sup.+) m/z calcd for
C.sub.32H.sub.65O.sub.2(M.sup.++H) 481.4984. Found 481.4977.
Production Example C-9
Synthesis of 2-hexadecyl octadecanoate
##STR00053##
[0330] 2-Hexadecyl octadecanoate was obtained by the same method as
that applied in Production Example C-1, using 1-iodohexadecane
instead of 1-iododecane.
white powder; FT IR (neat) 3028, 2914, 2848, 2691, 1705 cm.sup.-1;
.sup.1H NMR (300 MHz in CDCl.sub.3) .delta.0.88 (6H, t, J=6.9 Hz),
1.25 (56H, m), 1.48 (2H, m), 1.61 (2H, m), 2.35 (1H, m); .sup.13C
NMR (75 MHz in CDCl.sub.3) .delta.14.14, 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.sup.++H); HRMS (CI.sup.+) m/z calcd for
C.sub.34H.sub.69O.sub.2(M.sup.++H) 509.5297. Found 509.5298.
Production Example D-1
Synthesis of 3-nonyl dodecanoate
[0331] 3-Nonyl dodecanoate was synthesized by the following
Production Examples D-1-1 to D-1-5.
Production Example D-1-1
Synthesis of N-methoxy-N-methyldecanamide
##STR00054##
[0333] Decanoic acid (4 g, 23.2 mmol) was dissolved in an anhydrous
dichloromethane solution (80 mL), and 1,1-carbonyldiimidazole (4.5
g, 27.9 mmol) was then added to the solution, followed by stirring
for 1.5 hours. Subsequently, N,O-dimethylhydroxyamine hydrochloride
(2.7 g, 27.9 mmol) was added to the reaction solution, and the
obtained mixture was further stirred for 3 hours. After addition of
distilled water, the reaction solution was extracted with
dichloromethane two times. Anhydrous magnesium sulfate was added to
the organic layer to dry it, and the resultant was filtrated and
was then concentrated. The obtained residue was purified using
column chromatography (hexane:ethyl acetate=8:1), so as to obtain
N-methoxy-N-methyldecanamide (4.8 g, 97%) as an amide body in the
form of a colorless transparent liquid.
colorless oil; FT IR (neat) 2927, 2854, 1731 cm.sup.-1; .sup.1H NMR
(300 MHz in CDCl.sub.3) .delta.0.88 (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); .sup.13C NMR (75 MHz in CDCl.sub.3) .delta.14.09, 22.65, 24.65,
29.28, 29.45, 31.86, 61.17, 174.77; CIMS m/z (%) 215 (M.sup.+);
HRMS (CI.sup.+) m/z calcd for C.sub.12H.sub.25NO.sub.2(M.sup.+)
215.1921. Found 215.1903.
Production Example D-1-2
Synthesis of 10-nonadecanone
##STR00055##
[0335] Polished shaved magnesium (3.2 g, 134 mmol) was heated, and
thereafter, an anhydrous THF solution (68 ml) of 1-bromononane
(12.9 ml, 67.5 mmol) was slowly added dropwise thereto. After
heating to reflux for 1.5 hours, the reaction solution was cooled
to a room temperature, and it was then added dropwise to a THF (80
mL) solution of the N-methoxy-N-methyldecanamide (4.8 g, 22.5 mmol)
obtained by the method described in Production Example D-1-1. After
the obtained mixture had been stirred for 30 minutes, 1 N
hydrochloric acid was added to the reaction solution, followed by
extraction with diethyl ether two times. Anhydrous magnesium
sulfate was added to the organic layer to dry it, and the resultant
was filtrated and was then concentrated. The obtained residue was
purified using column chromatography (hexane:dichloromethane=8:1),
so as to obtain 10-nonadecanone (6.0 g, 95%) as a ketone body in
the form of a white amorphous solid.
colorless solid; FT IR (neat) 2953, 2916, 2847, 1698 cm.sup.-1;
.sup.1H NMR (300 MHz in CDCl.sub.3) .delta.0.88 (6H, t, J=6.9 Hz),
1.26 (24H, m), 1.55 (4H, m), 2.38 (4H, t, J=7.5 Hz); .sup.13C NMR
(75 MHz in CDCl.sub.3) .delta.14.03, 22.63, 23.84, 29.24, 29.41,
31.84, 42.74, 211.49; CIMS m/z (%) 282 (M.sup.+); HRMS (CI.sup.+)
m/z calcd for C.sub.19H.sub.38O (M.sup.+) 282.2882. Found
282.2902.
Production Example D-1-3
Synthesis of ethyl 3-nonyl-2-dodecanoate
##STR00056##
[0337] Sodium hydride (60 w/w %, 6 g, 142 mmol) was dissolved in
anhydrous THF (200 ml), and the obtained mixture was then cooled to
0.degree. C. Ethyl diethylphosphonoacetate (34 ml, 171 mmol) was
added dropwise to the reaction solution, and the obtained mixture
was then stirred for 30 minutes. Thereafter, the temperature of the
obtained reaction solution was increased to a room temperature, and
the 10-nonadecanone (6.0 g, 21.3 mmol) obtained by the method
described in Production Example D-1-2 was then added to the
reaction solution. The obtained mixture was further heated to
reflux for 18 hours. Thereafter, distilled water was added to the
reaction solution, followed by extraction with diethyl ether two
times. Anhydrous magnesium sulfate was added to the organic layer
to dry it, and the resultant was filtrated and was then
concentrated. The obtained residue was purified using column
chromatography (hexane:dichloromethane=6:1), so as to obtain ethyl
3-nonyl-2-dodecanoate (7.6 g, 99%) as an ester form in the form of
a colorless transparent liquid.
colorless oil; FT IR (neat) 2928, 2855, 1718 cm.sup.-1; .sup.1H NMR
(300 MHz in CDCl.sub.3) .delta.0.88 (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); .sup.13C NMR (75 MHz in
CDCl.sub.3) .delta.14.14, 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.sup.+); HRMS (CI.sup.+) m/z calcd for
C.sub.23H.sub.44O.sub.2 (M.sup.+) 352.3349. Found 352.3345.
Production Example D-1-4
Synthesis of ethyl 3-nonyldodecanoate
##STR00057##
[0339] The ethyl 3-nonyl-2-dodecanoate (7.6 g, 21.7 mmol) obtained
by the method described in Production Example D-1-3 was dissolved
in a mixed solvent (100 ml) of chloroform:methanol (5:1). A
PtO.sub.2 catalyst (3 w/w %, 223 mg, 983 .mu.mol) was added to the
solution, and the obtained mixture was then stirred under 1
atmospheric pressure of hydrogen for 23 hours. Thereafter, the
PtO.sub.2 catalyst was removed using a filter paper, and the
residue was then concentrated. The obtained residue was purified
using column chromatography (hexane:diethyl ether=50:1), so as to
obtain ethyl 3-nonyldodecanoate (7.1 g, 92%) in the form of a
colorless transparent liquid.
colorless oil; FT IR (neat) 2929, 2855, 1739 cm.sup.-1; .sup.1H NMR
(300 MHz in CDCl.sub.3) .delta.0.88 (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);
.sup.13C NMR (75 MHz in CDCl.sub.3) .delta.14.16, 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; CIMS m/z (%) 354 (M.sup.+); HRMS (CI.sup.+) m/z
calcd for C.sub.23H.sub.46O.sub.2 (M.sup.+) 354.3508. Found
354.3503.
Production Example D-1-5
Synthesis of 3-nonyl dodecanoate
##STR00058##
[0341] The ethyl 3-nonyldodecanoate (6.3 g, 17.7 mmol) obtained by
the method described in Production Example D-1-4 was dissolved in a
water-saturated butanol solution (80 ml), and KOH (10 g, 177 mmol)
was then added to the solution, followed by heating to reflux for
4.5 hours. Thereafter, 1 N hydrochloric acid was added to the
reaction solution, and the obtained mixture was then extracted with
diethyl ether two times. Anhydrous magnesium sulfate was added to
the organic layer to dry it, and the resultant was filtrated and
was then concentrated. The obtained residue was purified using
column chromatography (hexane:ethyl acetate=10:1), so as to obtain
3-nonyl dodecanoate (6.3 g, 99%) in the form of a colorless
transparent liquid.
colorless oil; FT IR (neat) 2925, 2854, 1709 cm.sup.-1; .sup.1H NMR
(200 MHz in CDCl.sub.3) .delta.0.88 (6H, t, J=6.6 Hz), 1.26 (32H,
m), 1.85 (1H, m), 2.27 (2H, d, J=6.9 Hz); .sup.13C NMR (75 MHz in
CDCl.sub.3) .delta.14.16, 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.sup.+); HRMS
(CI.sup.+) m/z calcd for C.sub.21H.sub.42O.sub.2 (M.sup.+)
326.3129. Found 326.3157.
Production Example D-2
Synthesis of 3-octyl undecanoate
##STR00059##
[0343] As starting compounds, using octanoic acid instead of the
decanoic acid described in Production Example D-1-1, also using
1-bromooctane instead of the 1-bromononane described in Production
Example D-1-2, and using the compound produced in each step in the
subsequent step, 3-octyl undecanoate was synthesized by the same
methods as those described in Production Examples D-1-1 to
D-1-5.
colorless oil; FT IR (neat) 2926, 2855, 1712 cm.sup.-1; .sup.1H NMR
(300 MHz in CDCl.sub.3) 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); .sup.13C NMR (75 MHz in
CDCl.sub.3) 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.sup.+); HRMS
(CI.sup.+) m/z calcd for C.sub.19H.sub.38O.sub.2 (M.sup.+)
298.2876. Found 298.2874.
Production Example D-3
Synthesis of 3-decyl tridecanoate
##STR00060##
[0345] As starting compounds, using undecanoic acid instead of the
decanoic acid described in Production Example D-1-1, also using
1-bromodecane instead of the 1-bromononane described in Production
Example D-1-2, and using the compound produced in each step in the
subsequent step, 3-decyl tridecanoate was synthesized by the same
methods as those described in Production Examples D-1-1 to
D-1-5.
colorless oil; FT IR (neat) 2935, 2857, 1711 cm.sup.-1; .sup.1H NMR
(300 MHz in CDCl.sub.3) .delta.0.88 (6H, t, J=6.6 Hz), 1.26 (36H,
m), 1.85 (1H, m), 2.27 (2H, d, J=6.9 Hz); .sup.13C NMR (75 MHz in
CDCl.sub.3) .delta.14.15, 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.sup.+); HRMS
(CI.sup.+) m/z calcd for C.sub.23H.sub.46O.sub.2(M.sup.+) 354.3498.
Found 354.3503.
Production Example D-4
Synthesis of 3-undecyl tetradecanoate
##STR00061##
[0347] As starting compounds, using dodecanoic acid instead of the
decanoic acid described in Production Example D-1-1, also using
1-bromoundecane instead of the 1-bromononane described in
Production Example D-1-2, and using the compound produced in each
step in the subsequent step, 3-undecyl tetradecanoate was
synthesized by the same methods as those described in Production
Examples D-1-1 to D-1-5.
colorless solid; FT IR (neat) 2923, 2853, 1707 cm.sup.-1; .sup.1H
NMR (300 MHz in CDCl.sub.3) .delta.0.88 (6H, t, J=6.9 Hz), 1.26
(40H, m), 1.85 (1H, m), 2.27 (2H, d, J=6.9 Hz); .sup.13C NMR (75
MHz in CDCl.sub.3) .delta.14.17, 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.sup.+); HRMS (CI.sup.+) m/z calcd for
C.sub.25H.sub.50O.sub.2(M.sup.+) 382.3811. Found 382.3816.
Production Example D-5
Synthesis of 3-dodecyl pentadecanoate
##STR00062##
[0349] Using the ethyl 3-dodecyl pentadecanoate obtained by the
method described in Production Example D-5-4 instead of the ethyl
3-nonyl dodecanoate described in Production Example D-1-5,
3-dodecyl pentadecanoate was obtained by the same method as that
described in Production Example D-1-5.
[0350] As starting compounds, using tridecanoic acid instead of the
decanoic acid described in Production Example D-1-1, also using
1-bromododecane instead of the 1-bromononane described in
Production Example D-1-2, and using the compound produced in each
step in the subsequent step, 3-dodecyl pentadecanoate was
synthesized by the same methods as those described in Production
Examples D-1-1 to D-1-5.
colorless solid; FT IR (neat) 2928, 2854, 1709 cm.sup.-1; .sup.1H
NMR (300 MHz in CDCl.sub.3) .delta.0.88 (6H, t, J=6.6 Hz), 1.26
(44H, m), 1.85 (1H, m), 2.27 (2H, d, J=6.9 Hz); .sup.13C NMR (75
MHz in CDCl.sub.3) .delta.14.15, 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.sup.+); HRMS (CI.sup.+) m/z calcd for C.sub.27H.sub.54O.sub.2
(M.sup.+) 410.4066. Found 410.4095.
Production Example D-6
Synthesis of 3-tetradecyl heptadecanoate
##STR00063##
[0352] As starting compounds, using pentadecanoic acid instead of
the decanoic acid described in Production Example D-1-1, also using
1-bromotetradecane instead of the 1-bromononane described in
Production Example D-1-2, and using the compound produced in each
step in the subsequent step, 3-tetradecyl heptadecanoate was
synthesized by the same methods as those described in Production
Examples D-1-1 to D-1-5.
colorless solid; FT IR (neat) 2915, 2849, 1704 cm.sup.-1; .sup.1H
NMR (300 MHz in CDCl.sub.3) .delta.0.88 (6H, t, J=6.9 Hz), 1.26
(52H, m), 1.85 (1H, m), 2.27 (2H, d, J=6.9 Hz); .sup.13C NMR (75
MHz in CDCl.sub.3) .delta.14.15, 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.sup.+);
HRMS (CI.sup.+) m/z calcd for C.sub.31H.sub.62O.sub.2(M.sup.+)
466.4685. Found 466.4717.
Synthesis of Other Compounds
[0353] The compounds of Examples 16 to 22 were obtained by the same
manner as in the above described production examples. These are
compounds wherein, in the formula (1) of the present invention, X,
X', n and n' are as shown in Table 1 below.
##STR00064##
TABLE-US-00001 TABLE 1 X n X' n' Example 16 Phenyl 0 Phenyl 0
Example 17 Naphthyl 0 Naphthyl 0 Example 18 Cyclohexyl 0 Cyclohexyl
0 Example 19 Cycloheptyl 0 Cycloheptyl 0 Example 20
CH(C.sub.14H.sub.29C.sub.16H.sub.33) 0
CH(C.sub.14H.sub.29C.sub.16H.sub.33) 0 Example 21 C.sub.14H.sub.29
0 C.sub.14H.sub.29 0 Example 22 C.sub.16H.sub.33 0 C.sub.16H.sub.33
0 Example 23 CH(C.sub.12H.sub.24OCH.sub.3).sub.2 0
CH(C.sub.12H.sub.24OCH.sub.3).sub.2 0 Example 24
CH(C.sub.13H.sub.26OH).sub.2 0 CH(C.sub.13H.sub.26OH).sub.2 0
[Measurement of Physiological Activity]
[0354] The following test was carried out on the compound of the
present invention, so as to measure its activity.
[0355] With regard to the compounds of the present invention, which
are described in Examples, Example numbers, Production Example
numbers, and chemical structure formulae thereof are shown in Table
2 below. The compounds described in Examples are all the compound
represented by the formula (1) of the present invention, and in the
formula (1), X, X', R.sub.1, R.sub.1', R.sub.2, R.sub.2', n and n'
are as shown in Table 2 below.
##STR00065##
TABLE-US-00002 TABLE 2 X: R.sub.1-CHR.sub.2-- Production X':
R.sub.1'-CHR.sub.2'-- Example Example R.sub.1 and R.sub.1' R.sub.2
and R.sub.2' n and n' 2 .alpha.-2 n-C.sub.8H.sub.17
n-C.sub.8H.sub.17 0 3 .alpha.-3 n-C.sub.9H.sub.19 n-C.sub.9H.sub.19
0 1 .alpha.-1 n-C.sub.10H.sub.21 n-C.sub.10H.sub.21 0 4 .alpha.-4
n-C.sub.11H.sub.23 n-C.sub.11H.sub.23 0 5 .alpha.-5
n-C.sub.12H.sub.25 n-C.sub.12H.sub.25 0 6 .alpha.-6
n-C.sub.13H.sub.27 n-C.sub.13H.sub.27 0 7 .alpha.-7
n-C.sub.15H.sub.31 n-C.sub.15H.sub.31 0 8 .alpha.-8
n-C.sub.16H.sub.33 n-C.sub.16H.sub.33 0 15 .beta.-2
n-C.sub.8H.sub.17 n-C.sub.8H.sub.17 1 9 .beta.-1 n-C.sub.9H.sub.19
n-C.sub.9H.sub.19 1 10 .beta.-3 n-C.sub.10H.sub.21
n-C.sub.10H.sub.21 1 11 .beta.-4 n-C.sub.11H.sub.23
n-C.sub.11H.sub.23 1 12 .beta.-5 n-C.sub.12H.sub.25
n-C.sub.12H.sub.25 1 13 .beta.-6 n-C.sub.13H.sub.27
n-C.sub.13H.sub.27 1 14 .beta.-7 n-C.sub.14H.sub.29
n-C.sub.14H.sub.29 1
[0356] In the test, a known natural compound derived from tubercle
bacillus, TDCM, was used as a positive control. It is to be noted
that TDCM is represented by the following chemical formula.
##STR00066##
Test Example 1
Measurement of Ability to Activate Macrophages
Test Example 1 (1)
Measurement of Active Oxygen Released from Mouse Intraperitoneal
Macrophages Using Fluorescence Intensity Measurement Apparatus
<Preparation of Phosphate Buffer (PBS)>
[0357] 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 (D.W.).
[0358] <Preparation of Mouse Intraperitoneal Macrophages>
[0359] 3 ml of 5% thioglycolate medium (Difco, BD, code. 225640,
Lot. 6192372) was administered into the abdominal cavity of a mouse
(ICR mouse (SPF), 5-week-old, male). Four days after the
administration, the mouse was sacrificed with the use of diethyl
ether. The epidermis in the center of abdomen was partially cut
with scissors, and thereafter, the abdomen was picked up and the
epidermis thereof was then peeled off. Thereafter, using a 10-ml
syringe equipped with a 26G needle, 5 ml of 0.05% EDTA-containing
PBS (-) (EDTA 2Na, nuclease and protease tested, Nacalai Tesque)
was totally injected into the abdominal cavity. Thereafter, the
abdomen was massaged about 40 to 50 times by picking up the side of
the abdomen. Using a 23G injection needle, liquid in the abdominal
cavity was slowly collected into a small centrifuge tube. This
operation was repeatedly carried out two times. The collected
microphages were centrifuged at 1000 rpm for 8 minutes. The
supernatant was discarded, and the precipitate was then suspended
in an RPMI 1640 medium (RPMI-1640 containing L-glutamine and phenol
red, Wako, 189-02025, Lot. WRM8043, which contained 10.61%
inactivated serum (Bio West) and 1% penicillin streptomycin
(GIBCO)). The centrifuge tube was filled with the RPMI 1640 medium,
and it was then centrifuged again at 1000 rpm for 8 minutes. The
supernatant was discarded, and the precipitate was then suspended
in an RPMI 1640 medium. Thereafter, the number of macrophages was
counted with One Cell Counter (Wakenyaku Co., Ltd.). The obtained
macrophages were diluted with an RPMI-1640 medium to have any given
concentration. The thus obtained mouse intraperitoneal macrophages
were used in the subsequent test.
[0360] <Preparation of 40 mM Test Compound Solution>
[0361] A 40 mM test compound solution was prepared as follows.
[0362] 0.7 g of BSA was weighed into a large test tube. Then, 10 ml
of sterilized PBS (-) was added to the test tube, and the obtained
mixture was fully stirred. Thereafter, using an LPS removal column
(Endo Trap (trademark) red 1/1 (proofs)), impurities were removed
from the BSA solution. Thereafter, the thus treated BSA solution
was filtrated through a sterile filter (0.2 .mu.m). Subsequently,
protein was quantified using Nano Drop ND-1000, and it was then
diluted with sterilized PBS (-) so as to result in a final
concentration of 2%. The weighed test compound and 250 .mu.l of 2%
BSA (prepared by dissolving 2% BSA in PBS (-)) were placed in a
homogenizer, and they were then treated with a bath type
ultrasonicator for 150 seconds, while they were placed in the
homogenizer. The thus prepared solution was transferred into an
Eppendorf tube, and was then used in the subsequent test. A
positive control was prepared using TDCM as a test compound in the
same manner as described above. A negative control was prepared
without adding test compounds in the same manner as described
above. Hereinafter, a prepared solution, which contains no test
compounds, is referred to as a "vehicle."
[0363] <Preparation of Hank's Balanced Salt Solution
(HBS)>
[0364] 0.4 g of potassium chloride, 0.06 g of potassium dihydrogen
phosphate, 0.107 g of disodium hydrogen phosphate, and 8 g of
sodium chloride were dissolved in distilled water (D.W.), and the
solution was adjusted to pH 7.4 with 1 N sodium hydroxide, and the
total amount of the solution was adjusted to 1000 ml with distilled
water (D.W.), so as to prepare a Hank's balanced salt solution
(HBS).
[0365] <Preparation of Glucose- and BSA-Containing Hank's
Balanced Salt Solution (HBSG-BSA)>
[0366] 0.1 g of glucose and 0.03 g of BSA (Sigma) were dissolved in
100 ml of the above prepared HBS, so as to prepare a glucose- and
BSA-containing Hank's balanced salt solution (HBSG-BSA) (prepared
when used).
[0367] <Measurement of Active Oxygen Released from
Macrophages>
[0368] Mouse intraperitoneal macrophages, a 40 mM test compound
solution, and a BSA-containing Hank's balanced salt solution
(HBSG-BSA) were prepared in the same manner as described above.
[0369] After the macrophages had been washed with HBSG-BSA, they
were then suspended in approximately 5 mL of HBSG-BSA, and 100
.mu.l of the obtained suspension was then dispensed into each well
of a 96-well Collagen Well (TC-PLATE 96 WELL, STERILE WITH LID, IND
PACKED). Thereafter, it was incubated at 37.degree. C. for 1 hour,
so that the cells were adhered to the well. The supernatant was
removed, and 100 .mu.l of HBSG-BSA was then added to the residue.
Then, 1 .mu.l of 10 mM H.sub.2DCFDA
(2',7'-dichlorodihydrofluorescein diacetate, Invitrogen) was added
to the reaction solution. The reaction solution was incubated at
37.degree. C. for 1 hour. The supernatant was removed, and HBSG-BSA
that contained a test compound or a vehicle used as a negative
control was added to the residue, so that the final concentration
of the test compound could be 50 .mu.M. One hour later, the amount
of active oxygen was measured using Genios fluorescence intensity
measurement apparatus.
[0370] The above-mentioned H.sub.2DCFDA is a fluorescent probe, and
its fluorescence intensity is increased in the presence of hydrogen
peroxide (H.sub.2O.sub.2). Thus, by measuring such fluorescence
intensity, the amount of hydrogen peroxide (H.sub.2O.sub.2)
generated can be measured. Hydrogen peroxide (H.sub.2O.sub.2) is
derived from superoxide (O.sup.2-) generated by macrophages, and
thus, using the amount of hydrogen peroxide (H.sub.2O.sub.2)
generated as an indicator, the activation level of macrophages can
be measured.
[0371] The measurement results are shown in FIG. 1.
[0372] FIG. 1 <Amount of active oxygen from mouse
intraperitoneal macrophages>
[0373] As shown in FIG. 1, all of the test compounds of the present
invention exhibited action to promote the generation of active
oxygen from mouse intraperitoneal macrophages, at a level
equivalent to or higher than that of TDCM. Among the test compounds
of the present invention, in particular, the compound of Example 1
and the compound of Example 9 exhibited high activity that was two
times higher than the activity of TDCM.
Test Example 1 (2)
Measurement of Phagocytic Ability of Mouse Intraperitoneal
Macrophages
[0374] Mouse intraperitoneal macrophages, a 40 mM test compound
solution, and an RPMI1640 medium were prepared in the same manner
as described above.
[0375] In the following experiment, Fluoresbrite (trademark)
Carboxylate Microspheres (2.58% Solids-Latex) YG (Polysciences,
Inc.) were used as fluorescent beads.
[0376] Macrophages were added to a TC-Plate (TC-PLATE 24 WELL,
STERILE WITH LID, IND PACKED, greiner bio-one), so that they became
80% confluent. The macrophages were incubated at 37.degree. C. for
2 hours. Thereafter, the supernatant was discarded, and the cells
were then washed with 500 .mu.l of RPMI 1640 medium two times. Each
composition shown in Table 3 below was added to the TC-Plate, and
it was then incubated at 37.degree. C. for 2 hours. Thereafter, the
supernatant was discarded, and the cells were then washed with 300
.mu.l of sterilized PBS (-). In order to remove fluorescent beads
that had not been incorporated into the cells, the aforementioned
washing operation was repeatedly carried out two times. Macrophages
were peeled off with 200 .mu.l of sterilized PBS (-), and were then
transferred into an Eppendorf tube. The macrophages were
centrifuged at 1,500 rpm for 8 minutes. Thereafter, the supernatant
was removed, and the residue was then fully suspended in 100 .mu.l
of sterilized PBS (-). The suspension was again centrifuged at
1,500 rpm for 8 minutes. Thereafter, the supernatant was removed,
and the residue was then fully suspended in 100 .mu.l of sterilized
PBS (-).
[0377] Using FUJI FILM FLA-2000, the amount of fluorescent beads
incorporated into the cells was measured [fluorescence intensity
(Fluor 473 nm, Y520 Filter)]. For analysis, Image Reader V1.4J was
used.
TABLE-US-00003 TABLE 3 1 2 Test compound (final -- 0.45
concentration: 50 .mu.M) LPS (--) 2% BSA in PBS 0.45 -- Fluorescent
beads (2.0 .times. 10.sup.7 beads/mL) 100 100 RPMI 1640 medium
199.5 199.5
[0378] The measurement results are shown in FIG. 2.
[0379] FIG. 2 <Phagocytic ability of mouse intraperitoneal
macrophages>
[0380] As shown in FIG. 2, all of the test compounds of the present
invention exhibited action to activate the phagocytosis of mouse
intraperitoneal macrophages, at a level equivalent to or higher
than that of TDCM. Among the test compounds of the present
invention, in particular, the compound of Example 1 and the
compound of Example 9 exhibited high activity that was
approximately two times higher than the activity of TDCM.
Test Example 2
Measurement of Ability to Activate Neutrophils
<Preparation of Rabbit Neutrophil Suspension>
[0381] A Hank's balanced salt solution (HBS) and a glucose- and
BSA-containing Hank's balanced salt solution (HBSG-BSA) were
prepared in the same manner as that in Test Example 1 (1).
[0382] <Preparation of Citric Acid-Glucose Solution (ACD
Solution)>
[0383] 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 (D.W.), and the
obtained solution was preserved at 4.degree. C. before use.
[0384] <Preparation of Erythrolysis Solution (Lysis
Solution)>
[0385] 0.037 g of EDTA, 1 g of potassium hydrogen carbonate, and
8.3 g of ammonium chloride were dissolved in 1000 ml of distilled
water (D.W.), and the obtained solution was preserved at 4.degree.
C. before use.
[0386] <Preparation of Phosphate Buffer (PBS)>
[0387] A phosphate buffer was prepared in the same manner as that
in Test Example 1 (1).
[0388] <Preparation of 1.2% Dextran-PBS Solution>
[0389] 1.2 g of dextran T500 (Pharmacia) was dissolved in 100 ml of
distilled water (D.W.), and the obtained solution was then
sterilized in an autoclave (121.degree. C., 20 minutes). The
solution was preserved at 4.degree. C. before use.
[0390] <Method for Preparing Rabbit Neutrophil
Suspension>
[0391] 5 ml of the ACD solution was placed in a 20-ml syringe
(injection needle [Nigro]), so that the inside of the syringe was
entirely rinsed therewith. 20 ml of blood was collected from the
central auricular artery of a rabbit using the syringe, and the
collected blood was gently shaken upside down. Thereafter, the
blood was dispensed into three centrifuge tubes (15 ml type;
Falcon), and each tube was then subjected to centrifugation at
4.degree. C. at 1,500 rpm for 5 minutes. Thereafter, the
supernatant was recovered into a centrifuge tube (50 ml type;
Falcon). A 1.2% dextran-PBS solution was added to the recovered
supernatant in an amount equal the supernatant, and the obtained
mixture was then gently shaken upside down. Thereafter, the mixture
was left at a room temperature for 30 minutes or more. The
emergence of an interface was confirmed, and the supernatant was
placed in a new centrifuge tube (50 ml type; Falcon). A 1.2%
dextran-PBS solution was added to the remaining solution in an
amount equal the remaining solution, and the obtained mixture was
then gently shaken upside down. Thereafter, the mixture was left at
a room temperature for 30 minutes or more. The emergence of an
interface was confirmed, and the supernatant was placed in a new
centrifuge tube (50 ml type; Falcon). The recovered supernatant was
centrifuged at 4.degree. C. at 2,000 rpm for 10 minutes, and the
supernatant was then removed. 15 ml of the Lysis solution was added
to the precipitate, and the obtained mixture was then gently
suspended. Thereafter, 5 ml of the Lysis solution was further added
to the suspension, and the obtained mixture was then gently shaken
upside down. The reaction solution was left in ice for 5 minutes.
Thereafter, HBSG-BSA was added to the reaction solution to a total
amount of 50 ml, and the obtained solution was then centrifuged at
4.degree. C. at 2,000 rpm for 10 minutes, followed by the removal
of the supernatant. The precipitate was suspended in 2 ml of
HBSG-BSA, and the obtained cell suspension was gently laminated on
an upper layer of 2 ml of Lymphoprep [Nycomed, 808068] (centrifuge
tube, 15 ml type). It was then centrifuged at 1200 rpm for 20
minutes (conditions of the centrifugal machine: accel 0.5, break
Off), and the supernatant was then removed using an aspirator. In
order to remove the remaining Lyphoprep, the precipitate
(neutrophils) was suspended in MBSG-BSA, and it was then
centrifuged again at 1,500 rpm for 5 minutes. Then, the supernatant
was removed. The neutrophils were suspended in HBSG-BSA, and the
number of cells was then counted using a cell number counting
machine "Celltac" [Nihon Kohden Corporation].
[0392] <Preparation of Emulsion Solution of Test
Compound>
[0393] An emulsion solution of a test compound and an emulsion
solution of TDCM were prepared in the same manner as that in Test
Example 1 (1).
<Preparation of 10 mM H.sub.2DCFDA
(2',7'-dichlorodihydrofluorescein diacetate (Molecular
Probes)>
[0394] 4.86 mg of H.sub.2DCFDA was dissolved in 1 ml of DMSO.
Test Example 2 (1)
Measurement of Amount of Active Oxygen Released from Rabbit
neutrophils
[0395] The influence of the test compound on the release of active
oxygen from rabbit neutrophils was measured by the following
procedures.
[0396] Neutrophils (1.0.times.10.sup.5 cells/100 .mu.l) were plated
on a 96-well plate (Falcon). To this plate, 1 .mu.l of 10 mM
H.sub.2DCFDA was added, and it was then incubated at 37.degree. C.
for 1 hour. In order to remove residual H.sub.2DCFDA, 300 .mu.l of
HBS was added to the reaction product so as to suspend it, and the
obtained suspension was then centrifuged at 8000 rpm for 5 minutes.
The supernatant was removed, and the residue was then suspended in
HBS. Then, HBS was added to the suspension to a final concentration
of 50 .mu.M. The obtained mixture was incubated at 37.degree. C.
for 2 hours, and the amount of active oxygen released was measured
using a fluorescence measurement apparatus (Ex: 485 nm, Em: 535
nm).
[0397] The results are shown in FIG. 3.
[0398] FIG. 3 <Amount of active oxygen released from rabbit
neutrophils>
[0399] As shown in FIG. 3, almost of the test compounds of the
present invention exhibited action to activate the release of
active oxygen from rabbit neutrophils, at a level equivalent to or
higher than that of TDCM. Among the test compounds of the present
invention, in particular, the compound of Example 1 exhibited the
activity was approximately two times higher than the activity of
TDCM.
Test Example 2 (2)
Measurement of Phagocytic Ability of Rabbit Neutrophils
[0400] (2) Method for Producing Ampicillin-Resistant Escherichia
coli and Opsonized Escherichia coli
<Preparation of L-Broth>
[0401] 10 g of tryptophan, 5 g of NaCl, 5 g of Yeast Extract, and
ml of MgSO.sub.4 were dissolved in 1 L of distilled water
(D.W.).
[0402] <Preparation of Opsonizing Reagent>
[0403] 10 mg of an opsonizing reagent (BioParticles Opsonizing
Reagent (Molecular Probes)) was dissolved in 500 .mu.l of ultrapure
water.
[0404] <Production of Ampicillin-Resistant Escherichia
coli>
[0405] Cells (JM109) used for electroporation were dissolved in
ice, and in order to introduce an ampicillin-resistant gene into
Escherichia coli, 2 .mu.l of an ampicillin-resistant plasmid
(pT7Blue, Novagen, 100 ng/.mu.l) was added to the JM109, followed
by suspension. The obtained suspension was transferred to a cuvette
used for electroporation, and it was then pulsed (2.5 kV, 200
.OMEGA., 25 mF). The pulsed cell mass solution was transferred into
a 5-ml tube containing 1 ml of L-broth medium, and it was then
cultured at 37.degree. C. for 3 hours. The resultant solution was
applied to a L-broth agar medium containing 0.005% ampicillin, and
it was then cultured at 37.degree. C. overnight (wherein, before
the solution was dispersed on the medium, it was centrifuged at
3,500 rpm for 5 minutes, 800 .mu.l of the supernatant was removed,
the precipitate was then suspended, and the suspension was then
dispersed on a petri dish). On the following day, a small amount of
bacteria growing from the petri dish was scraped off, and it was
then dissolved in 2 ml of L-broth medium, followed by a shaking
culture for approximately 4 hours.
[0406] <Method for Producing Opsonized Escherichia coli>
[0407] 100 .mu.l of the above produced ampicillin-resistant
Escherichia coli solution and 100 .mu.l of the dissolved opsonizing
reagent were suspended in an Eppendorf tube. The obtained
suspension was incubated at 37.degree. C. for 1 hour, and it was
then suspended in 300 .mu.l of PBS. The suspension was centrifuged
at 1200 G for 15 minutes, and the supernatant was then removed. The
obtained solution was further suspended in 300 .mu.l of PBS, and
the suspension was then centrifuged at 1200 G for 15 minutes,
followed by the removal of the supernatant. This operation was
repeatedly carried out two times. Thereafter, 1 .mu.l of the cell
solution was added to 100 .mu.l of L-broth, and it was fully
suspended. Then, the suspension was 100-fold diluted. The thus
100-fold diluted cell solution was added to 10-.mu.l OneCell
Counter, and the number of cells was then counted under a
microscope.
[0408] <Measurement of Phagocytic Ability of Rabbit
Neutrophils>
[0409] 1.0.times.10.sup.5 neutrophils prepared by the above
described method were fractionated into an Eppendorf tube, and the
cells were then suspended in FIBS. Thereafter, it was treated with
an emulsion solution of a 50-.mu.M test compound or an emulsion
solution of TDCM for 1 hour. Using a 2% BSA solution as a control,
and the same treatment as described above was performed on the
neutrophils. 300 .mu.l of FIBS was added to the reaction solution
so as to suspend it, and the obtained suspension was then
centrifuged at 1200 G for 10 minutes, followed by the removal of
the supernatant. Further, 300 .mu.l of HBS was added to the
obtained solution so as to suspend it, and the obtained suspension
was then centrifuged at 1200 G for 10 minutes, followed by the
removal of the supernatant. 1.0.times.10.sup.7 cells of opsonized
ampicillin-resistant Escherichia coli were fully suspended in the
treated neutrophils, and the obtained suspension was then incubated
at 37.degree. C. for 1 hour. After the removal of residual
Escherichia coli, 100 .mu.l of 0.5% Triton X-100 (in a normal
saline solution) was added to the residue, and it was then fully
suspended therein. The obtained suspension was incubated at
37.degree. C. for 30 minutes. Subsequently, in order to
comparatively quantify the amount of Escherichia coli incorporated
into the inside of the neutrophils by disintegrating the
neutrophils by a treatment with Triton X-100, the Triton
X-100-treated solution as a whole was dispersed on a 0.005%
ampicillin-containing common agar medium, and it was then spread
with the use of a bacteria spreader. Thereafter, it was incubated
at 37.degree. C. for 24 hours, and the number of colonies of
Escherichia coli incorporated into the neutrophils was counted.
[0410] The results are shown in FIG. 4.
[0411] FIG. 4 <Phagocytic activity of rabbit neutrophils>
[0412] As shown in FIG. 4, all of the compounds of the present
invention exhibited action to activate the phagocytic ability of
rabbit neutrophils. Among the compounds of the present invention,
in particular, the compound of Example 1 exhibited the activity
that was approximately 2 times higher than that of TDCM.
Test Example 3
Measurement of Cytokine Released from Mouse Intraperitoneal
Macrophages by Treatment with Test Compound
[0413] Mouse intraperitoneal macrophages, a 40 mM test compound
solution, and an RPMI 1640 medium were prepared in the same manner
as described above.
[0414] Macrophages were added to a TC-Plate, so that they became
80% confluent. The macrophages were incubated at 37.degree. C. for
2 hours. Thereafter, the supernatant was discarded, and the cells
were then washed with 500 .mu.l of RPMI 1640 medium. This washing
operation was repeatedly carried out two times. An emulsion
solution of the test compound (final concentration: 100 .mu.M) was
allowed to act on the macrophages, and two hours later, the medium
was transferred into another Eppendorf tube. It was centrifuged at
10,000 rpm for 10 minutes, and the supernatant was further
transferred into another Eppendorf tube. This supernatant was used
as a sample, and the released cytokines were analyzed using ELISA
kit (IL-6, TNF-.alpha. Quantikine Immunoassay (R & D System
(trademark)).
[0415] With regard to the release of IL-6 from mouse
intraperitoneal macrophages, the amount of IL-6 released was
approximately 15 pg/ml in the case of using a vehicle as a negative
control. In contrast, the compound of Example 1, which seemed to
have particularly high activity among the compounds of the present
invention, exhibited an activity of approximately 200 pg/ml.
Moreover, with regard to the release of TNF-.alpha. from mouse
intraperitoneal macrophages, the amount of TNF-.alpha. released was
approximately 80 pg/ml in the case of using a vehicle as a negative
control. In contrast, the compound of Example 1 exhibited an
activity of approximately 1000 pg/ml.
Test Example 4
Method for Measuring IL-8 Released from THP-1 Cells, which Involves
Treatment with Test Compound
[0416] An RPMI medium solution was prepared in the same manner as
described above. Using this medium solution, an RPMI medium
solution of the test compound was prepared as follows.
[0417] 1.0 mg of the test compound was dissolved in 25 .mu.l of
DMSO by an ultrasonic treatment for approximately 1 minute. The
thus obtained 40 mM test compound stock solution was added to 100
.mu.l of RPMI medium to a final concentration of 50 .mu.M. The
obtained mixture was treated with ultrasonic wave for 5 seconds. In
the case of preparing the vehicle used as a control, instead of the
40 mM test compound stock solution, only the solvent was added in
the same amount as described above to the RPMI medium, and it was
then treated with ultrasonic wave for 5 seconds.
[0418] THP-1 cells (purchased from Cell Bank, RIKEN BioResource
Center) were added to an RPMI medium to a cell density of
1.0.times.10.sup.6 cells/100 .mu.l, and 100 .mu.l each of the
obtained solution was dispensed into a sterile Eppendorf tube. 100
.mu.l of the RPMI medium solution of the test compound as prepared
above was treated with ultrasonic wave for 5 seconds, and the total
amount of the solution was then added to the Eppendorf tube, into
which the cells had been dispensed. Two hours later, the reacted
Eppendorf tube was centrifuged at 5000 rpm for 5 minutes, and the
amount of IL-8 released into the supernatant was then measured
using ELISA kit (human IL-8 ELISA kit (R & D Systems
(trademark)).
[0419] The test results are shown in FIG. 5.
[0420] Among the compounds of the present invention, the compounds,
which seemed to have particularly high immunostimulatory activity,
were measured. As a result, as shown in FIG. 5, the compound of
Example 1 and the compound of Example 9 exhibited activities that
were approximately 0.6 times and approximately 0.8 times higher
than the activity of TDCM, respectively.
Test Example 5
Measurement of IL-6, INF-.alpha., and IFN-.gamma. Released into
Peripheral Blood of Test Compound Administered Mouse
[0421] An emulsion solution of the test compound was prepared as
follows.
[0422] Hereinafter, the term "test compound" is used to include all
of compounds synthesized in Production Examples .alpha.-1 to -8,
compounds synthesized in Production Examples .beta.-1 to -7, and
TDCM used as a comparative example.
[0423] A test compound was weighed (100 .mu.g/mouse), and using a
micro spatula, the total amount of the test compound was placed at
the bottom of a homogenizer (WEATON U.S.A., 10 ml). A droplet of
mineral oil (Nacalai Tesque) was added dropwise thereto, followed
by homogenization. The obtained mixture was treated with a
bath-type ultrasonic wave generator for 150 seconds. Thereafter,
1.0 ml of normal saline solution containing 1.1% Tween 80
(polyoxyethylene sorbitan monooleate, Nacalai Tesque) and 5.6%
mannitol was further added to the homogenizer. The components were
homogenized several times, so that the mineral oil, in which the
test compound was dissolved, was well mixed with the solvent. The
resultant solution was transferred into an Eppendorf tube, and it
was then subjected to pasteurization at 62.degree. C. for 30
minutes.
[0424] Homogenizers had previously been immersed in ice for 3
minutes. Various test compounds (1.0 mg each) were each placed in
such a homogenizer, mineral oil was then added to the homogenizer,
and the obtained mixture was treated with ultrasonic wave for 150
seconds. After confirming that the oil became sticky, 1.0 ml of
normal saline solution (containing 1.1% Tween and 5.6% mannitol)
was added to the homogenizer, and the obtained mixture was then
homogenized for approximately 1 minute. The sample was transferred
into an Eppendorf tube, and it was then subjected to pasteurization
at 62.degree. C. for 30 minutes.
[0425] The thus prepared emulsion solution of test compound (100
.mu.g/mouse) was administered via an intravenous injection to a
group of two mice. Two hours later, blood collected from the heart
(heparin blood collection). The collected blood was centrifuged at
10,000 rpm for 10 minutes, and using only plasma, the amounts of
various types of cytokines were measured using ELISA kit (IL-6,
INF-.alpha., IFN-.gamma. Quantikine Immunoassay (R & D Systems
(trademark)).
[0426] The measurement results are shown in FIGS. 6, 7 and 8.
[0427] FIG. 6 <IL-6 concentration (pg/ml) in mouse
plasma>
[0428] FIG. 7 <IFN-.gamma. concentration (pg/ml) in mouse
plasma>
[0429] FIG. 8 <TNF-.alpha. concentration (pg/ml) in mouse
plasma>
[0430] As shown in FIG. 6, an increase in the plasma IL-6
concentration was observed in mice, to which the test compounds of
the present invention had been administered. Among the test
compounds of the present invention, in particular, the compound of
Example 1 and the compound of Example 9 exhibited high
IL-6-releasing activities, which were, respectively, approximately
1.2 times and approximately 1.5 times higher than that of TDCM as a
known trehalose diester compound from nature, which was used as a
positive control. The compound of Example 13 and the compound of
Example 14 also exhibited activities, which were, respectively,
approximately a half of and approximately equivalent to that of
TDCM.
[0431] As shown in FIG. 7, an increase in the plasma IFN-.gamma.
concentration was observed in mice, to which the test compounds of
the present invention had been administered. Among the test
compounds of the present invention, in particular, the compound of
Example 1 and the compound of Example 9 each exhibited
IFN-.gamma.-releasing activity, which was approximately 1.5 times
higher than that of TDCM used as a positive control. The compound
of Example 13 and the compound of Example 14 also exhibited
activities, which were, respectively, approximately a half of and
approximately equivalent to that of TDCM.
[0432] As shown in FIG. 8, an increase in the plasma TNF-.alpha.
concentration was observed in mice, to which the test compounds of
the present invention had been administered. However, even the
activity of the compound of Example 1 was the same level as that of
TDCM used as a positive control. Other compounds of the present
invention generally exhibited approximately 1/2 of that of
TDCM.
Test Example 6
Mouse Survival Test Involving Administration of Welch Bacillus
(Compound of Example 1)
[0433] As test compounds, 1 mg each of the compound synthesized by
the method described in Production Example .alpha.-1 and TDCM were
weighed. Emulsion solutions of the test compounds (1 mg/ml) were
prepared in the same manner as described above.
[0434] Welch bacillus (Type A; NTCT8237) was prepared as
follows.
[0435] <Method for Preparing Welch Bacillus>
[0436] 7.4 mg of Brain Heart Infusion (BHI) (Difco) powders were
dissolved in 200 ml of distilled water. 40 ml of the obtained
solution (hereinafter this solution is referred to as a "BHI
medium") was withdrawn with a measuring pipette, and it was then
added to a 200-ml flask, followed by performing a high-pressure
steam sterilization (121.degree. C., 20 minutes). In addition, 5 ml
of the BHI medium was added to two screw-top test tubes, and they
were then subjected to a high-pressure steam sterilization.
Moreover, a glass tube-equipped rubber plug (with a cotton plug)
and a glass tube were also subjected to a high-pressure steam
sterilization. In a clean bench, an appropriate amount of bacteria
for preservation, which had grown on a cooked meat medium, was
collected with a sterilized Pasteur pipette, and it was then
inoculated on the PHI medium in the aforementioned sterilized
screw-top test tube. Subsequently, it was incubated at 37.degree.
C. overnight.
[0437] In a clean bench, bacteria that had grown in the screw-top
test tube (wherein the inside of the clean bench in which bacteria
had grown became clouded, and gas was generated) was transferred
into a 200-ml Erlenmeyer flask, which contained 40 ml of PHI
medium. A glass tube was inserted into the medium, so as to carry
out nitrogen substitution for 10 minutes. Subsequently, the glass
tube-equipped rubber plug (with a cotton plug) was equipped in the
Erlenmeyer flask (since Welch bacillus generates gas, it is
necessary to make an air vent), and incubation was then carried out
at 37.degree. C. for 4 to 5 hours. Thereafter, the cultured Welch
bacillus was transferred into a centrifuge tube, it was then
centrifuged (9000 rpm, 15 minutes), and the supernatant was then
removed. 20 ml of sterilized normal saline solution was added to
the precipitate, so that the bacteria were suspended therein. The
suspension was centrifuged (9000 rpm, 15 minutes), and the
supernatant was then removed. The obtained precipitate was added to
the BHI medium in the sterilized screw-top test tube, and the
number of cells was then counted using OneCell Counter
(manufactured by OneCell Inc.).
[0438] <Method for Conducting Survival Test Involving
Administration of Welch Bacillus>
[0439] Mice (ICR, 6-week-old) were divided into three groups, and
the following test was then carried out.
[0440] An emulsion solution containing the test compound, an
emulsion solution containing TDCM, or an emulsion solution alone,
which was to be used as a control, was intraperitoneally
administered in an amount of 100 .mu.g/mouse to a group of four
mice (100 .mu.l/mouse, in the case of the emulsion solution alone).
Three hours later, Welch bacillus (2.4.times.10.sup.7 cells/mouse)
was intraperitoneally administered to the mice. Thereafter, the
condition of the mice was observed.
[0441] The results are shown in Table 4.
TABLE-US-00004 TABLE 4 Mouse survival rate after administration of
Welch bacillus Number of surviving mice/ number of treated mice
Test 2 hours 6 hours 12 hours 24 hours 48 hours compound later
later later later later Example 1 4/4 4/4 4/4 3/4 3/4 TDCM 4/4 4/4
4/4 4/4 4/4 Control 4/4 3/4 0/4 0/4 0/4
[0442] As shown in Table 4, in Welch bacillus lethal models, three
out of the four mice, to which the compound of Example 1 (the
compound of the present invention) had been administered, escaped
death.
Test Example 7
Mouse Survival Test Involving Administration of Welch Bacillus
Toxin (Compound of Example 1)
[0443] Emulsion solutions of the test compounds were prepared in
the same manner as described above.
[0444] A Welch bacillus toxin was prepared as follows.
[0445] <Method for Preparing Welch Bacillus Toxin>
[0446] A Bacillus subtilis .alpha.-toxin gene transformant was
cultured in L-Broth at 37.degree. C. for 14 hours, while stirring.
Thereafter, the culture was centrifuged at 4.degree. C. at 8,000
rpm for 20 minutes, and while stirring the culture supernatant
under cooling on ice, a small amount of ammonium sulfate (Nacalai
Tesque) was periodically added thereto. Thereafter, the reaction
solution was adjusted to contain saturated ammonium sulfate having
a final concentration of 70% (472 g/L), and it was then left
overnight. Subsequently, the reaction solution was centrifuged at
4.degree. C. at 9,500 rpm for 30 minutes, and the generated
precipitate was dissolved in 0.02 M TB (ph 7.5). It was dialyzed
against the same buffer as described above at 4.degree. C.
overnight. After completion of the dialysis, the reaction solution
was centrifuged at 4.degree. C. at 15,000 rpm for 30 minutes, and
the obtained supernatant was used as a crude toxin (ammonium
sulfate toxin) sample. This crude toxin sample was diluted with 1 M
NaCl-TB (pH 7.5) so as to have a final concentration of 0.5 M NaCl.
Thereafter, the crude toxin sample was applied to a copper chelate
affinity column (1.5.times.9 cm) that had previously been
equilibrated with 0.5 M NaCl-TB (pH 7.5), and then, 100 ml each of
0.5 M NaCl-TB (pH 7.5), 0.5 M NaCl-0.1 M PB (pH 6.5), 0.5 M
NaCl-0.02 M acetate buffer (pH 4.5), and 0.5 M NaCl-0.1 M PB (pH
6.5) were successively applied to the column. Subsequently, toxin
bound into 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 the eluant was
then filtrated with a syringe filter (DISMIC-ADVANTEC). The
filtrate was subjected to centrifugal concentration using an
ultrafiltration filter Amicon (trademark) Ultra-15-30K (Millipore),
and the concentrate was then dialyzed against 0.02 M TB (pH 8.0) at
4.degree. C. overnight. Thereafter, the resultant was centrifuged
at 15,000 rpm for 30 minutes, and it was then; applied to UNO
(trademark) Q-1 R Column (BIO-RAD). Elution was carried out by
linearly changing a sodium chloride concentration in 0.02 M TB (pH
8.0) from 0 M to 0.05 M at a flow rate of 1.0 ml/min. Elution (0.5
ml) fractions, in each of which a preparation line was observed in
an Ouchterlony reaction with respect to an anti-.alpha.-toxin serum
and each of which was found to contain an approximately 43-kDa
single band corresponding to the .alpha.-toxin by SOS-PACE, were
collected, and the gathered fraction was then concentrated to
approximately 1.0 ml. This concentrate was dialyzed against 0.02 M
TB (pH 7.5) at 4.degree. C. overnight, and it was then centrifuged
at 4.degree. C. at 15,000 rpm for 30 minutes. The supernatant (a
recombinant .alpha.-toxin) was fractionated. The presence or
absence of impurities contained in the obtained recombinant
.alpha.-toxin was confirmed by SDS-PACE, and it was then preserved
at -80.degree. C. before use.
[0447] <Survival Test Method Involving Administration of Welch
Bacillus Toxin>
[0448] Mice (ICR, 6-week-old) were divided into three groups, and
the following test was then carried out.
[0449] An emulsion solution containing the test compound, an
emulsion solution containing TDCM, or an emulsion solution alone,
which was to be used as a control, was intraperitoneally
administered in an amount of 100 .mu.g/mouse to a group of four
mice (100 .mu.l/mouse, in the case of the emulsion solution alone).
Three hours later, Welch bacillus toxin (200 ng/mouse) was
intraperitoneally administered to the mice. Thereafter, the
condition of the mice was observed.
[0450] The results are shown in Table 5.
TABLE-US-00005 TABLE 5 Mouse survival rate after administration of
Welch bacillus toxin Number of surviving mice/ number of treated
mice Test 2 hours 6 hours 12 hours 24 hours 48 hours compound later
later later later later Example 1 4/4 4/4 4/4 3/4 3/4 TDCM 4/4 4/4
4/4 2/4 2/4 Control 4/4 3/4 0/4 0/4 0/4
[0451] As shown in Table 5, in Welch bacillus toxin lethal models,
three out of the four mice, to which the compound of Example 1 (the
compound of the present invention) had been administered, escaped
death.
Test Example 8
Mouse Survival Test Involving Administration of Pseudomonas
aeruginosa (Compound of Example 1)
[0452] As a test compound, 1 mg of the compound synthesized by the
method described in Production Example .alpha.-1 was weighed. An
emulsion solution of the test compound was prepared in the same
manner as described above.
[0453] Pseudomonas aeruginosa (Fhu-071115 strain) derived from
patients was used, and it was prepared as follows.
[0454] <Method for preparing Pseudomonas aeruginosa>
[0455] L-broth was withdrawn with a 40-ml measuring pipette and was
then added to a 200-ml flask, and the flask was then sealed with a
sponge plug. At the same time, 5 ml of L-broth was added to each of
two other screw-top test tubes. The thus prepared flask and
screw-top test tubes were subjected to an autoclave at 121.degree.
C. for 20 minutes. After the L-broth medium had been cooled to a
room temperature, Pseudomonas aeruginosa, which had been stored in
an ultra low temperature freezer, was added into 40 ml of the
L-broth the medium in a clean bench. The obtained mixture was
shaken in an incubator overnight. Thereafter, the resultant was
centrifuged at 9000 rpm for 15 minutes, and the supernatant was
then removed. Thereafter, 20 ml of sterilized normal saline
solution was added to the precipitate, the mixture was then blended
with the use of a Vortex mixer, and it was then centrifuged at 9000
rpm for 15 minutes, followed by the removal of the supernatant.
This process was carried out three times. Thereafter, 4.5 ml of
sterilized normal saline solution was added to the resultant, and
the mixture was then blended with the use of a Vortex mixer. The
obtained solution was used as a strain stock solution. Using a
1000-fold diluted strain solution, the number of cells was counted
with OneCell Counter. Thereafter, the strain stock solution was
diluted to a desired concentration, and the thus diluted solution
was then used in the subsequent test.
[0456] <Survival Test Method Involving Administration of
Pseudomonas aeruginosa>
[0457] Mice were divided into two groups, and the following two
types of experiments were carried out.
(A) The aforementioned emulsion solution containing the test
compound (100 .mu.g/mouse) was intraperitoneally administered to a
group of three mice (ICR, 5-week-old). Three hours later,
Pseudomonas aeruginosa (5.0.times.10.sup.7 cells/mouse) was
intraperitoneally administered to the mice. Thereafter, the
condition of the mice was observed. (B) Pseudomonas aeruginosa
(2.0.times.10.sup.7 cells/mouse) was intraperitoneally administered
to a group of three mice (ICR, 5-week-old). Three hours later, the
aforementioned emulsion solution containing the test compound (100
.mu.g/mouse) was intraperitoneally administered to the mice.
Thereafter, the condition of the mice was observed.
[0458] The results are shown in Table 6 and Table 7.
TABLE-US-00006 TABLE 6 (Group A) Mouse survival rate obtained in
the case of pre- administration of test compound and administration
of Pseudomonas aeruginosa Number of surviving mice/ number of
treated mice Test 2 hours 4 hours 15 hours 24 hours 48 hours
compound later later later later later Example 1 3/3 3/3 2/3 2/3
2/3 Control 3/3 3/3 0/3 0/3 0/3
[0459] As shown in Table 6, in group A to which the test compound
had previously been administered before administration of
Pseudomonas aeruginosa, two out of the three mice escaped death
caused by Pseudomonas aeruginosa. At the beginning of infection,
there was no significant difference from the control group.
However, from approximately 8 hours after the infection, the
surviving mice were able to walk.
TABLE-US-00007 TABLE 7 (Group B) Mouse survival rate obtained in
the case of administration of Pseudomonas aeruginosa and
administration of test compound Number of surviving mice/ number of
treated mice Test 2 hours 4 hours 15 hours 24 hours 48 hours
compound later later later later later Example 1 3/3 3/3 3/3 1/3
1/3 Control 3/3 3/3 0/3 0/3 0/3
[0460] As shown in Table 7, in group B to which the test compound
was administered after administration of Pseudomonas aeruginosa,
one out of the three mice escaped death caused by Pseudomonas
aeruginosa.
Test Example 9
Release of Cytokine from THP-1 Cells
[0461] The amounts of various types of cytokines and chemokines
from human monocytic leukemia cell-derived THP-1 cells, which had
been treated with the test compound obtained in Example 9, were
measured by the ELISA method. In addition, the same analysis was
performed also on human lung cancer cell-derived A549 cells and
human colon cancer cell-derived DLD-1 cells.
[0462] <Method for Culturing THP-1 Cells>
(1) Serum Lot Check
[0463] The cultured THP-1 cells were transferred into a 15-ml
centrifuge tube in a clean bench. The cells were centrifuged at
1,000 rpm for 5 minutes at 20.degree. C., and the supernatant was
then removed. The precipitate was suspended in 1 ml of RPMI 1640
medium (containing FBS for use in 10% lot check), and the number of
cells was then counted using a hemocytometer (Kayagaki Irika Kogyo
Co., Ltd.) and a cover glass (Sansyo Co., Ltd.). Thereafter, the
solution was diluted with a medium to a cell density of
2.5.times.10.sup.5 cells/ml. 0.5 ml each of the cell suspension was
dispensed into MULTI WELL PLATE 24 wells (SUMILON) used for
suspension culture. The cell growth and the shape thereof were
observed once a day. Two or three days later, the culture solution
was 100-fold diluted with a medium, and then, the number of cells
in 4 .mu.l of the solution was counted using a counting slide. The
cell growth conditions were compared. Cells, the growth and shape
of which were good, were recovered from the well, and they were
then washed with the same medium. Thereafter, the cell solution was
diluted again to a cell density of 2.5.times.10.sup.5 cells/ml. 1
ml of the cell suspension was dispensed into MULTI WELL PLATE 6
wells (SUMILON) used for suspension culture, and it was then
incubated at 37.degree. C. in a 5% CO.sub.2 atmosphere. The medium
was exchanged with a new one every 24 hours. The plate itself was
centrifuged (TONY) at 1,800 rpm for 5 minutes, the medium was then
slowly removed, and 1 ml of new medium was then added. Such a
medium exchange operation was carried out two times. On the third
day or later, after the medium had been exchanged with a new one,
the cells were 100-fold diluted, and the number of cells was then
counted using a counting slide. The degree of cell growth was
measured. Thereafter, this operation was repeatedly carried out for
several days, and media, the growth and shape of which were good,
were selected.
(2) Inactivation and Dispending of Serum
[0464] 500 ml of fetal bovine serum (BBS) (Biowest) that had been
thawed at 4.degree. C. was incubated for 30 minutes, while stirring
it sometimes at 56.degree. C., so as to inactivate it. Thereafter,
30 ml each of the serum was dispensed in a 50-ml centrifuge tube in
a clean bench without filtrating it, and it was then preserved at
-80.degree. C. The preserved serum was thawed at 4.degree. C. when
used.
(3) RPMI 1640 Medium (10% Inactivated FBS+1% Penicillin
Streptomycin)
[0465] 60 ml of the inactivated FBS and 5.6 ml of penicillin
streptomycin (GIBCO) that had been filtrated with Acrodisc 25 mm
Syringe Filter (Pall Corporation) were added to 500 ml of RPMI 1640
liquid medium (Wako) in a clean bench, and they were then mixed so
that they became homogeneous. The prepared medium was directly used
without filtrating it. The medium was preserved at 4.degree. C.,
and it was returned to a room temperature when used.
(4) Subculture of Cells
[0466] THP-1 cells were cultured in a 75-cm.sup.2 suspension
culture flask (SUMILON) (had grown at 90% or more), and the shape
and growth of the cells were observed. When the cell shape was good
and the cell growth rate was high, the cell suspension was
transferred into another flask, or a new medium was added to the
used flask in an amount of two times the amount of the cell
suspension, so as to carry out subculture. When the cell growth
rate was low, the condition was continuously observed, or a new
medium was added to the cell suspension in an equal amount thereof,
so as to carry out subculture.
(5) Preservation of Cells
[0467] 10 to 15 ml of the cells that had been cultured in the
75-cm.sup.2 suspension culture flask (had grown at 90% or more)
were transferred into a centrifuge tube, and it was then
centrifuged at 1,000 rpm for 5 minutes. Then, the supernatant was
removed. Thereafter, 1 ml of cell cryopreservation solution, Cell
banker (Nippon Zenyaku Kogyo Co., Ltd.) was added to the
precipitate, followed by suspension. The obtained suspension was
dispensed into a serum tube, and it was then preserved at
-80.degree. C.
(6) Re-Culture of Preserved Cells
[0468] The cells that had been frozen at -80.degree. C. were
quickly thawed in a 37.degree. C. water bath. The cell preservation
solution was immediately added to a 15-ml centrifuge tube, to which
9 to 10 ml of RPMI 1640 medium had previously been added, and the
obtained mixture was then blended by shaking it upside down. The
mixture was centrifuged at 1,000 rpm for 5 minutes, and the
supernatant was then removed. The precipitate was resuspended in 10
ml of new medium, and the cell suspension was then transferred into
a 75-cm.sup.2 suspension culture flask. Thereafter, a new medium
was added to the cell suspension to a total amount of 20 to 30 ml,
and the obtained mixture was then cultured at 37.degree. C. in a 5%
CO.sub.2 atmosphere.
[0469] <Preparation of ELISA Kit Reagents>
Preparation of Washing Solution
[0470] A washing solution was 25-fold diluted with sterile
distilled water (S.D.W.), and it was then used at a room
temperature.
[0471] Preparation of Standard Substrate Solution
[0472] 600 .mu.L of diluent solution for standard substrate
solution had previously been added to two microtubes. A standard
substrate was dissolved in 1 mL of diluent solution for standard
substrate solution (2450 pg/mL), 100 .mu.L of the obtained solution
was then transferred into a microtube, and it was then dissolved
therein (350 pg/mL). 100 .mu.L of the solution was further
transferred into another microtube, it was then dissolved therein
(50 pg/mL), followed by dilution. The diluent solution for standard
substrate solution was used as a control (0 pg/mL).
[0473] Preparation of Coloring Solution
[0474] Color reagent A and color reagent B were mixed in equal
amounts. A coloring solution in an amount of 100
.mu.l/well.times.the number of wells to be measured, was added to
the mixture. This solution was prepared within 15 minutes before
use.
[0475] <Measurement of Sample>
[0476] An ELISA kit reagent to be used was returned to a room
temperature. A standard substrate solution having a different
concentration and an assay buffer (50 .mu.L) were added to each
well, and 50 .mu.L of sample was further added thereto. The plate
was gently tapped for 1 minute, and a cover was applied onto the
plate, followed by incubation at a room temperature for 2 hours.
Thereafter, the resultant was washed with a washing solution five
times (wherein an aspirator was usable), and 100 .mu.L of the
prepared conjugate solution was then added to each well. A cover
was applied onto the plate, and it was then incubated at a room
temperature for 2 hours. Thereafter, the resultant was washed with
a washing solution five times, and under light-shielded conditions,
100 .mu.L of mixed solution consisting of color reagent solutions A
and B was added to each well. The plate was incubated at a room
temperature in a dark place for 30 minutes. Finally, 100 .mu.L of
reaction termination solution was added to each well, and within 30
minutes after the addition, absorbance was measured using a
microplate reader (Molecular Devices spectra MAX 340PC) (450 nm-550
nm). From a straight line graph of standard substrate solution, the
amount of cytokine released from each sample was calculated.
[0477] <Experimental Method>
[0478] The cultured THP-1 cells were transferred into a 50-ml
centrifuge tube in a clean bench. The cells were centrifuged at
1,000 rpm for 5 minutes at 20.degree. C., and the supernatant was
then removed. The precipitate was suspended in 1 ml of new
serum-free RPMI 1640 medium (Wako), and 10 .mu.l of the cell
suspension was then added to a sterile Eppendorf tube, to which 990
.mu.l of serum-free RPMI 1640 medium had previously been added, so
that the solution was 100-fold diluted. The number of cells in 4
.mu.l of the 100-fold diluted solution was counted with a blood
cell counter, and the cell suspension was then diluted with
serum-free RPMI 1640 medium to a cell density of 1.times.10.sup.7
cells/ml. 100 .mu.L of RPMI medium was added to an Eppendorf tube,
and a 40 mM test compound (Example 9)/DMSO solution was added
thereto to a concentration of 200 .mu.M. Thereafter, ultrasonic
wave was applied to the mixture for 5 seconds. 100 .mu.l of THP-1
cells were added thereto (final concentration of the test compound:
100 .mu.M). The obtained mixture was incubated at 37.degree. C. for
2 hours, and it was then centrifuged at 5000 rpm for 10 minutes.
The supernatant was transferred into another Eppendorf tube, and
the sample was then measured using the ELISA kit.
[0479] It is to be noted that a test compound,
6,6'-bis-O-(2-tetradecylhexanoyl)-.alpha.,.alpha.'-trehalose
(hereinafter referred to as a "control compound A"), and TDCM were
used as positive controls, and that these compounds were also
measured in the same manner as described above. In addition, a
negative control was prepared by adding no test compounds
(vehicle).
[0480] <Experimental Results>
[0481] It became clear that MIP-1.beta. was released from THP-1
cells that had been treated with the compound of Example 9 at a
level that was approximately 8 to 10 times higher than that of
THP-1 cells that had been treated with the positive control.
Moreover, the release level of TNF-.alpha. from THP-1 cells treated
with the compound of Example 9 was almost equivalent to the release
level of TNF-.alpha. from THP-1 cells treated with the negative
control. The results are shown in FIGS. 9A and 9B. As shown in FIG.
9A, the release level of MIP-1.beta. from THP-1 cells treated with
the compound of Example 1 was higher than the release level of
MIP-1.beta. from THP-1 cells treated with the positive control. On
the other hand, as shown in FIG. 9B, the release level of
TNF-.alpha. from THP-1 cells treated with the compound of Example 1
was almost equivalent to the release level of TNF-.alpha. from
THP-1 cells treated with the negative control (data not shown).
Text Example 10
Analysis of Cytotoxicity to THP-1 Cells and Mutagenicity
Test>
<Analysis of Cytotoxicity to THP-1 Cells Using Trypan
Blue>
Preparation of Reagent
Preparation of 0.3% Trypan Blue/PBS (-)
[0482] 0.3 g of trypan blue (Nacalai) was dissolved in 100 ml of
PBS (-).
[0483] Preparation of THP-1 Cells
[0484] The cultured THP-1 cells were transferred into a 50-ml
centrifuge tube in a clean bench. The cells were then centrifuged
at 1,000 rpm for 5 minutes at 20.degree. C., and the supernatant
was then removed. The precipitate was suspended in 1 ml of new
serum-free RPMI 1640 medium (Wako). 10 .mu.l of this cell
suspension was added to a sterile Eppendorf tube, to which 990
.mu.l of serum-free RPMI 1640 medium had previously been added, so
that the solution was 100-fold diluted. The number of cells in 10
.mu.l of the 100-fold diluted solution was counted with a blood
cell counter, and the cell suspension was diluted with serum-free
RPMI 1640 medium to a cell density of 1.times.10.sup.7
cells/ml.
[0485] <Experimental Method>
[0486] A test compound (Example 9)/DMSO solution, or DMSO was added
to 100 .mu.l of RPMI medium to a concentration of 200 .mu.M.
Thereafter, ultrasonic wave was applied to the mixture for 5
seconds. It is to be noted that a test compound,
6-O-(2-decyldecanoyl)-.alpha.-glucose (hereinafter referred to as a
"control compound B"), and TDCM were used to prepare positive
controls in the same manner as described above. In addition, a
negative control was prepared by adding no test compounds
(vehicle). 100 .mu.l of THP-1 cells, which had been adjusted to a
cell density of 1.times.10.sup.7 cells/ml, were added thereto, and
the obtained mixture was then incubated at 37.degree. C. for 2
hours or 24 hours. Subsequently, 20 .mu.l of 0.3% trypan blue/PBS
(-) was added to the reaction solution so as to suspend the
reaction solution therein, and thereafter, the survival rate of the
cells was immediately analyzed using a cell number measuring
apparatus (CYRORECON).
[0487] Mutagenicity Test (Ames Test)>
Preparation of Reagent
Preparation of 0.1 M Sodium Phosphate Buffer
[0488] 5.68 g of Na.sub.2HPO.sub.4 was dissolved in 200 mL of
distilled water. Thereafter, NaH.sub.2HPO.sub.4.2H.sub.2O dissolved
in 100 ml of distilled water was gradually added to the obtained
solution, so as to adjust the solution to pH 7.4. The solution was
then subjected to high-pressure steam sterilization.
[0489] Preparation of Minimal Glucose Agar Medium
(1) VP medium: 0.4 g of MgSO.sub.4.7H.sub.2O, 4 g of citric acid
H.sub.2O, 20 g of K.sub.2HPO.sub.4, and 7 g of
NaNH.sub.4HPO.sub.4.4H.sub.2O were dissolved in 200 mL of distilled
water, and the obtained solution was then subjected to
high-pressure steam sterilization. (2) 40 g of glucose was
dissolved in 200 mL of distilled water, and the obtained solution
was then subjected to high-pressure steam sterilization. (3) 30 g
of powdered agar was suspended in 1600 mL of distilled water, and
the obtained suspension was then subjected to high-pressure steam
sterilization.
[0490] After the reagent of (3) had been cooled to approximately
60.degree. C., the reagent of (1) was mixed with the reagent of
(2), and the approximately 30 mL each of the obtained mixture was
dispersed on a Petri dish.
[0491] Preparation of Upper-Layer Agar Medium
[0492] 1.2 g of powdered agar and 1 g of NaCl were suspended in 200
mL of water, and the obtained suspension was then subjected to
high-pressure steam sterilization. Thereafter, the resultant was
transferred into a 50-ml tube. Before use, 20 mL of 0.5 mM
histidine/biotin solution was mixed with it, and the obtained
mixture was kept warm at 47.degree. C.
[0493] Preparation of Oxoid Nutrient Broth Medium Used for Culture
Of Salmonella Typhii
[0494] 2.5 g of Oxoid Nutrient Broth medium (Difco) was dissolved
in 100 ml of distilled water, and 5 ml of the obtained solution was
then placed in a screw-top test tube, followed by sterilization.
Thereafter, approximately 10 .mu.L of cell solution of TA98
(Salmonella typhmurium TA98) was inoculated into the test tube, and
the obtained mixture was then subjected to a shaking culture at
37.degree. C. overnight, so as to prepare a cell suspension.
[0495] Preparation of S9 Mix
[0496] 9 ml of Co factor A mix (ORIENTAL YEAST) was added to 1 ml
of S9.
Preparation of Standard Mutagenic Substance
Preparation of 4-NQO/DMSO
[0497] 0.3 mg of 4-Nitroquinoline N-oxide (4-NQO) (Tokyo Chemical
Industry Co., Ltd.) was dissolved in 10 ml of DMSO.
Preparation Of 2-Aminoanthracene/DMSO
[0498] 0.5 mg of 2-aminoanthracene (ALDRICH) was dissolved in 30 ml
of DMSO.
[0499] <Experimental Method>
[0500] 10 .mu.L each of the standard mutagenic substance, the test
compound of Example 9, the control compound B, and TDCM were each
placed in an Eppendorf tube. Two Eppendorf tubes were prepared for
each of the aforementioned compounds. 0.5 mL of S9 mix or 100 mM
phosphate buffer was added to each tube, and 100 .mu.L of the cell
suspension was also added thereto, followed by preincubation
(shaking at 37.degree. C. for 20 minutes). Thereafter, 2 mL of soft
agar containing histidine-biotin (which was incubated at 47.degree.
C.) was added to the reaction solution, followed by moderate
suspension. The obtained solution was dispersed on a minimal
glucose agar medium, and it was then incubated (37.degree. C., 2
days). Thereafter, the number of His.sup.+ colonies was
counted.
[0501] <Experimental Results>
[0502] In order to examine cytotoxicity to THP-1 cells, the
survival rates of THP-1 cells, which had been treated with the test
compound obtained in Example 9 for 2 hours and for 24 hours, were
analyzed by trypan blue staining. As a result, cytotoxicity was
observed in the cells, which had been treated with the control
compound B. In contrast, such cytotoxicity was not observed in the
cells, which had been treated with the compound obtained in Example
9. The results are shown in FIG. 10. Moreover, in the case of the
compound obtained in Example 1 as well, significant results could
be obtained (data not shown).
[0503] Furthermore, an Ames test was carried out on the test
compound obtained in Example 9 in the presence or absence of S9
mix. In both cases, the test compound did not exhibit mutagenicity.
Further, the mutagenicity of the standard substance
(2-aminoanthracene, 4NQO) was positive under the present analytical
conditions (FIGS. 11A and 11B), Still further, in the case of the
compound obtained in Example 1 as well, significant results could
be obtained (data not shown).
Test Example 11
Cell Infiltration into Mouse Abdominal Cavity
<Preparation of PBS (-) Solution>
[0504] 4 g of NaCl, 1.45 g of Na.sub.2HPO.sub.4.12H.sub.2O, 0.1 g
of KH.sub.2PO.sub.4, and 0.1 g of KCl were dissolved in 500 ml of
distilled water, and the obtained solution was then subjected to
high-pressure steam sterilization at 121.degree. C. for 20
minutes.
[0505] Preparation of 0.05% EDTA/PBS (-) Solution>
[0506] 50 mg of EDTA (Nacalai Tesque code. 151-30) was dissolved in
100 ml of PBS (-), and the obtained solution was then subjected to
mechanical sterilization using a 0.2-.mu.m filter.
[0507] <Preparation of 1 Mg/mL Emulsion Solution>
[0508] 0.9 g of NaCl, 1.1 ml of Polyoxyethylene Sorbitan Monooleate
(Tween 80), and 5.6 g of D (-)-Mannitol were dissolved in 100 ml of
distilled water, and the obtained solution was then subjected to
mechanical sterilization using a 0.2-.mu.m filter. 1 mg of the test
compound obtained in Example 9 was placed at the bottom of a
homogenizer (WEATON U.S.A., 10 mL, AS ONE Corp.). A droplet of
mineral oil was added thereto, and the obtained mixture was then
homogenized while applying ultrasonic wave thereto for 2 minutes.
Thereafter 1 mL of 1.1% Tween-5.6% Mannitol Saline was added to the
reaction solution, and the obtained mixture was then homogenized
until it became clouded and homogeneous. The total amount of the
solution of the test compound was transferred into an Eppendorf
test tube, and it was then treated at 62.degree. C. for 30 minutes
for pasteurization. The control compound A and TDCM were used as
test compounds (positive controls). On the other hand, no test
compounds were added to the emulsion solution to prepare a negative
control (vehicle).
[0509] <Recovery of Intraperitoneal Infiltrating Cells>
[0510] A solution containing the 1 mg/mL test compound (Example 9)
was intraperitoneally administered to mice (ICR mice (SPF)
(4-week-old, male, body weight: 20-22 g) to a concentration of 100
.mu.g/mouse.
[0511] The mice, to which the test compound had been
intraperitoneally administered, were sacrificed with the use of
diethyl ether, 2 hours or 24 hours after the administration. The
epidermis in the center of abdomen was partially cut with scissors,
and thereafter, the abdomen was picked up and the epidermis thereof
was then peeled off. The peritoneum was lightly picked up with
tweezers, and 5 mL of 0.05% EDTA in PBS (-) was injected in the
total amount into the abdominal cavity, using a 10-mL syringe
equipped with a 26G needle, while paying attention not to inject
the needle into organs. Thereafter, the abdomen was massaged about
40 to 50 times by picking up the side of the abdomen. Thereafter,
liquid in the abdominal cavity was slowly collected into a small
size centrifuge tube. This operation was carried out again. The
collected cells were centrifuged at 1,000 rpm for 10 minutes. The
supernatant was removed, and the precipitate was then suspended in
an RPMI 1640 medium. The centrifuge tube was filled with an RPMI
1640 medium, and it was then centrifuged at 1,000 rpm for 10
minutes again. The supernatant was discarded, and the precipitate
was then suspended in an RPMI 1640 medium. Thereafter, the number
of cells was counted using a cell counter. The suspension was then
diluted with an RPMI-1640 medium to any given concentration.
[0512] <Giemsa Staining of Cells>
<Preparation of 1/15 M Sodium Phosphate Buffer (pH 6.4)>
[0513] 6.0 g of NaH.sub.2PO.sub.4 and 7.06 g of Na.sub.2HPO.sub.4
were each dissolved in 250 ml of distilled water, and a sodium
dihydrogen phosphate solution was then added to a disodium hydrogen
phosphate solution, while measuring the pH thereof, so as to adjust
the pH value to pH 6.4. Thereafter, the obtained solution was
sterilized in an autoclave at 121.degree. C. for 20 minutes.
[0514] A 1 mg/mL test compound solution was intraperitoneally
administered to mice to a concentration of 100 .mu.g/mouse.
Twenty-four hours later, cells infiltrating into the abdominal
cavity were recovered using EDTA/PBS (-). Thereafter, the cells
were centrifuged at 1000 rpm for 8 minutes, and the supernatant was
then removed. The cells were suspended in 100 .mu.L of phosphate
buffer, and the obtained suspension was placed on a slide glass for
staining. After confirming the evaporation of water content, 10 to
15 droplets of May-Grunwald solution were added dropwise thereto on
a staining vat, and it was then left for 2 to 3 minutes. Without
throwing away the May-Grunwald solution, 10 to 15 droplets of
phosphate buffer were added dropwise thereto, and it was then left
for 2 to 3 minutes. An adequate amount of Giemsa staining solution
was added to the reaction solution, and the obtained mixture was
then left for 30 minutes. Thereafter, the slide glass was turned
over, and water was then supplied thereto. The slide glass was
dried, and it was then observed under a microscope.
[0515] <Analysis of CD8-Positive Cells in Mouse Abdominal Cavity
by Flow Cytometry>
Preparation of Reagents
Preparation of PBS (-) Solution
[0516] A PBS (-) solution was prepared by the same method as that
applied in Test Example 11.
[0517] Preparation of 1 Mg/mL Test Compound (Emulsion Solution)
[0518] An emulsion solution containing a 1 mg/ml, test compound was
prepared by the same method as that applied in Test Example 11.
[0519] Preparation of 0.05% EDTA/PBS (-) Solution
[0520] 50 mg of EDTA 2Na was dissolved in 100 ml of PBS (-), and
the obtained solution was then sterilized in an autoclave at
121.degree. C. for 20 minutes.
[0521] Preparation of 0.5% BSA-0.05% EDTA/PBS (-) Solution
[0522] 50 mg of EDTA 2Na was dissolved in 100 ml of PBS (-), and
the obtained solution was then sterilized in an autoclave at
121.degree. C. for 20 minutes. Thereafter, 0.5% BSA was dissolved
in the reaction solution when used.
[0523] <Experimental Method>
[0524] A 1 mg/mL test compound emulsion solution was
intraperitoneally administered to mice (100 .mu.g/mouse).
Twenty-four hours later, cells infiltrating in the abdominal cavity
were recovered using 0.05% EDTA/PBS (-). Thereafter, the collected
peritoneal cells were centrifuged at 300 g for 10 minutes, and the
supernatant was then removed. The prepared peritoneal cells were
suspended in 1 ml of 0.05% EDTA (dissolved in 0.5% BSA/PBS). The
cell suspension was filtrated with a mesh used for flow cytometry,
and the number of cells was then counted in a 100-fold diluted cell
suspension. Cells in each sample were adjusted to a cell density of
10.sup.7 cells/sample, and they were then centrifuged at 300 g for
10 minutes, followed by the removal of the supernatant. The
precipitate was suspended in 100 .mu.l of 0.00% EDTA (dissolved in
0.5% BSA/PBS), and 10 .mu.l each of CD11b, CD4 and CD8 antibodies
(FITC anti-mouse CD11b/Mac-1 (BECKMAN), FITC anti-mouse CD4
(BECKMAN COULTER) and PE anti-mouse CD8a (BD Pharmingen),
respectively) were added. The obtained mixture was incubated at
2.degree. C. to 8.degree. C. in a dark place for 10 minutes, and
the cells were then suspended in 1 to 2 ml of 0.05% EDTA (dissolved
in 0.5% BSA/PBS). The cell suspension was centrifuged at 300 g for
10 minutes, and the supernatant was then removed. The precipitate
was suspended in 1 ml of 0.05% EDTA (dissolved in 0.5% BSA/PBS),
and the solution was then analyzed by flow cytometry.
[0525] <Experimental Results>
[0526] The cells were suspended in 1 mL of PBS (-). 100 .mu.L of
Hemolynac (hemolytic hemoglobin reagent) was added to the cell
suspension so as to destroy erythrocytes. Thereafter, the number of
cells was counted using a cell measurement apparatus (CYTORECON, GE
Healthcare).
[0527] Moreover, the prepared cells were inoculated on a 24-well
Collagen Well (Greiner), and were incubated for 2 hours.
Thereafter, the supernatant was aspirated, and then, the cells were
repeatedly washed with an RPMI medium two times. 300 .mu.l of RPMI
medium was added to the resultant cells, and the cells were then
observed under an inverted microscope.
[0528] As a result, an increase in infiltrating cells was observed
time-dependently in the abdominal cavity of mice, to which the test
compound obtained in Example 9 had been administered. Twenty-four
hours later, the number of infiltrating cells became 15 to 20 times
higher than that had been treated with a vehicle (FIGS. 12A-D).
[0529] Moreover, in order to analyze infiltrating cells, the shape
of the cells was observed by Giemsa staining, and the cells were
treated with various antibodies against a monocyte and macrophage
antigen (CD11b), a lymphocyte antigen (CD4) and an NH cell antigen
(CD8). The cells were then analyzed by flow cytometry. As a result,
twenty-four hours after administration of the test compound
obtained in Example 9, the total number of CD11b-positive cells,
CD4-positive cells and CD8-positive cells was 15 to 20 times larger
than the number of cells that had been treated with a vehicle.
However, the abundance ratio of the CD11b-positive cells and
CD4-positive cells that had been treated with the test compound of
Example 9 was hardly changed from that in the case of being treated
with a vehicle. On the other hand, the abundance ratio of the
CD8-positive cells became approximately 2 to 3 times higher than
that in the case of being treated with a vehicle (FIGS. 13A-B,
14A-D, and 15A-B).
[0530] As described above, it was found that phagocyte system cells
were accumulated in mouse abdominal cavity by the action of the
test compound of Example 9, and that, in particular, the ratio of
NH cells was high.
Test Example 12
Influence of Test Compound on Welch Bacillus-Infected Mice or
Pseudomonas aeruginosa-Infected Mice (Compound of Example 9)
<Method for Preparing Welch Bacillus>
Preparation of COOKED MEAT Medium (Hereinafter Abbreviated as a "CM
Medium" at Times)
[0531] A CM medium (125 mg/ml in D.W.) was added to a screw-top
test tube, and it was then boiled for 15 minutes, so as to remove
air from the CM medium. High-pressure steam sterilization was
performed in an autoclave (121.degree. C., 20 minutes), and the
resultant was then cooled to a room temperature.
[0532] Preparation of Brain Heart Infusion (Hereinafter Abbreviated
as "BHI") Medium
[0533] 37 mg of BHI medium was dissolved in 100 ml of distilled
water. Thereafter, 4.5 mL of the obtained solution was added to a
screw-top test tube, and 40 mL of the obtained solution was added
to an Erlenmeyer flask. They were sealed with sponge plugs, and
high-pressure steam sterilization was then carried out thereon in
an autoclave (121.degree. C., 20 minutes). Thereafter, the
resultants were cooled to a room temperature.
[0534] Preservation of Cells
[0535] C. perfringens Type-A NCTC8237 (PLC+) was added to the
prepared CM medium, and it was then cultured at 37.degree. C. for 2
days. The obtained culture was preserved as a preserved cell
solution at a room temperature.
[0536] Culture of Cells and Preparation of Cell Solution
[0537] 0.2 mL of cell solution was collected from each preserved
cell solution, and it was then added to 4.5 mL of BHI medium used
for pre-culture, followed by a culture at 37.degree. C. overnight.
The total amount of this culture solution was added to 40 mL of BHI
medium, and nitrogen substitution was then carried out (10
minutes). Thereafter, it was cultured again at 37.degree. C. for 5
hours. Thereafter, the culture solution was added to a 50-mL tube,
and it was then centrifuged (4.degree. C., 9000 rpm, 15 minutes).
The supernatant was removed, and a normal saline solution was added
to the precipitate to fully wash it. Thereafter, the resultant was
centrifuged again (4.degree. C., 9000 rpm, 15 minutes), and cells
were then collected. This washing operation was repeatedly carried
out two times. 4.5 mL of BHI medium was added to the precipitate so
as to suspend it. The obtained suspension was used as a stock
solution. This stock solution was 1000-fold diluted, and the
diluted solution was then subjected to high-pressure steam
sterilization in an autoclave (121.degree. C., 20 minutes).
Thereafter, the number of cells was counted using OneCell Counter.
A cell solution having a cell density of 1.times.10.sup.8 cells/mL
was produced, and was used in the subsequent experiment.
[0538] <Method for Preparing Pseudomonas eruginosa>
Preparation of Luria Bertani Broth Medium (L-Broth)
[0539] 10.0 g of Bacto.TM. Tryptone (Difco), 5.0 g of Bacto.TM.
Yeast Extract (Difco), and 5.0 g of sodium chloride (NaCl) (Nacalai
Tesque) were dissolved in distilled water, and 1.0 mL of 1 M
MgSO.sub.4 was then added to the obtained solution. Then, 1 N NaOH
was added to the solution to adjust it to pH 7.5. Distilled water
was added to the solution to a total amount of 1,000 mL.
Thereafter, the obtained solution was subjected to high-pressure
steam sterilization in an autoclave at 121.degree. C. for 20
minutes, and the reaction solution was then cooled to a room
temperature.
[0540] Preservation of Cells
[0541] 0.2 mL of Pseudomonas aeruginosa (P. Aeruginosa) was added
to 10 mL of L-Broth, and the mixture was then subjected to a
shaking culture at 37.degree. C. overnight. Thereafter, 1 mL of
sterile glycerin was added to the obtained culture solution, and
the obtained mixture was then blended with the use of a Vortex
mixer. 300 .mu.L each of the cell solution was dispensed into a
sterile Eppendorf tube, and it was then preserved at -80.degree.
C.
[0542] 0.2 mL each of the preserved cell solution was added to 40
mL of L-Broth medium, and the mixture was then subjected to a
shaking culture at 37.degree. C. overnight. Thereafter, the culture
was transferred into a 50-mL tube, and it was then centrifuged
(4.degree. C., 9000 rpm, 15 minutes). The supernatant was removed,
and a normal saline solution was added to the precipitate to fully
wash it. Thereafter, the resultant was centrifuged again (4.degree.
C., 9000 rpm, 15 minutes), and cells were then collected. This
washing operation was repeatedly carried out two times. 4.5 mL of
normal saline solution was added to the precipitate so as to
suspend it. The obtained suspension was used as a stock solution.
This stock solution was 10000-fold diluted, and the thus diluted
cell solution was then subjected to high-pressure steam
sterilization in an autoclave (121.degree. C., 20 minutes).
Thereafter, the number of cells was counted using OneCell Counter.
A cell solution having a cell density of 1.times.10.sup.8 cells/mL
was produced, and was used in the subsequent experiment.
[0543] <Experimental Method: Pre-Administration of Test
Compound>
[0544] An emulsion solution containing 100 .mu.g/mouse test
compound (Example 9) was intraperitoneally administered to mice.
Twenty-four hours later, 3.0.times.10.sup.10 CFU/ml, Pseudomonas
aeruginosa or 5.0.times.10.sup.7 CFU/mL Welch bacillus was
intraperitoneally administered to the mice. Thereafter, the mice
were observed every 2 hours.
[0545] Experimental Method: Post-Administration of Test
Compound>
[0546] 3.0.times.10.sup.10 CFU/mL Pseudomonas aeruginosa was
intraperitoneally administered to mice. Three hours later, an
emulsion solution containing 100 .mu.g/mouse test compound (Example
9) was intraperitoneally administered to the mice. Thereafter, the
mice were observed every 2 hours.
[0547] <Experimental Results>
[0548] As shown in FIGS. 16A-B and 17A-B, the lethality of the mice
that had been treated with the test compound of Example 9 was
significantly suppressed. In addition, the test compound of Example
9 was administered to mice, three hours after infection with
Pseudomonas aeruginosa. As a result, the lethality of the mice was
significantly suppressed (FIGS. 18A-B).
Test Example 13
Septicemia Observation
[0549] An emulsion solution containing 100 .mu.g/mouse test
compound (Example 9) was intraperitoneally administered to the
mice. Twenty-four hours later, the mice were infected with
3.0.times.10.sup.10 CFU/mL Pseudomonas aeruginosa. Fifteen hours
after administration of the bacteria, blood was collected from the
heart using an injection with a needle tip containing a small
amount of heparin, and 200 .mu.L of whole blood was then inoculated
on a common agar medium. Thereafter, it was incubated in an
incubator for 16 hours. Subsequently, the number of colonies on the
medium was counted.
[0550] When there were a large number of cells, whole blood was
diluted with a normal saline solution by a factor of 10, 100, 1000
or 10000, and the thus diluted solution was inoculated on a common
agar medium.
[0551] As a result, as shown in FIG. 19, when only the bacteria
were administered, or when the bacteria and a vehicle were
administered, the bacteria were detected in mouse blood. However,
when the test compound of Example 9 was administered, the bacteria
were not detected in mouse blood.
Test Example 14
Antitumor Effects
[0552] The 100 .mu.g/mouse test compound obtained in Example 9
(emulsion solution) was intraperitoneally administered to each
mouse, and twenty-four hours later, breast cancer cells (FM3A
cells) were inoculated into the abdominal cavity thereof. Nineteen
days later, the body weight of each mouse was measured. In
addition, the tissue sections of diaphragm, pancreas and testis
were subjected to HE staining, and they were then observed under a
microscope. As a result, the body weight of the breast cancer
cell-inoculated mouse was increased by approximately 10 g, when
compared with a control mouse, and it was observed that the mouse
contained a large amount of ascites fluid. Moreover, significant
infiltration and metastasis of the cancer cells were observed in
the diaphragm, pancreas and testis of the breast cancer
cell-inoculated mouse. On the other hand, in the case of a mouse,
into which the breast cancer cells were inoculated, after the mouse
had been treated with the test compound of the example, the body
weight of the mouse was equivalent to that of a control mouse.
Moreover, infiltration and metastasis of the cancer cells into
various organs were not observed at all (FIGS. 20A-C).
[0553] The trehalose compound of the present invention has
immunostimulatory action that is superior to or equivalent to that
of TDCM. Moreover, the present trehalose compound has toxicity that
is significantly lower than that of TDCM. Thus, this compound can
be preferably used as a pharmaceutical product. It was found that
the toxicity of the compound of the present application is low, not
only in model mice, but also in human-derived cells. Furthermore,
it was also shown that the compound of the present application has
low mutagenicity.
[0554] Further, a .beta.-hydroxyl group was replaced by a hydrogen
atom, and .alpha.-branched or .beta.-branched fatty acid, wherein
the branched carbon chain (which is a portion represented by
R.sub.1, R.sub.2, R.sub.1' or R.sub.2' in the formula (1)) consists
of approximately 7 to 20 carbon atoms, was used, and these
components were successively synthesized and examined. As a result,
it was found that, among trehalose compounds having various
structures, a .alpha.-branched compound containing 10 carbon atoms
or a .beta.-branched compound containing 9, 13 or 14 carbon atoms,
has maximum activity. Moreover, it was also found that the
cancer-inducing activity of an amide bond can also be suppressed by
substituting the amide bond of the prior art technique with an
ester bond.
[0555] Still further, the trehalose compound of the present
invention was found to be significantly useful even in in vivo
tests, in that the present compound reduced the lethality of mice,
to which Welch bacillus had been intraperitoneally administered.
The point that the present trehalose compound reduces the lethality
of mice, to which toxin generated by Welch bacillus had been
intraperitoneally administered, is revolutionary. Moreover, it was
also found that the excellent antibacterial activity of the
trehalose compound of the present invention reduces the lethality
of mice, to which Pseudomonas aeruginosa had been intraperitoneally
administered.
[0556] Still further, with respect of the action mechanism of the
trehalose compound of the present invention, it was demonstrated
that when this compound was administered to mice, the
H.sub.2O.sub.2 activity of neutrophil, the phagocytic ability of
neutrophil, and the phagocytic ability of macrophage were enhanced.
Thus, it is suggested that the aforementioned anti-infectious
disease actions would be provided as a result of the activation of
the cellular immunity of macrophage or neutrophil.
[0557] These test results suggest that the trehalose compound of
the present invention would provide the activation of the cellular
immunity of macrophage or neutrophil, and thus that it would be
useful for a wide range of infectious diseases caused by bacteria,
viruses, fungi and the like, which would be targets of the
phagocytosis of macrophage or neutrophil. Accordingly, the
trehalose compound of the present invention is more advantageous
than other compounds of the prior art in that it involves a method
that is more simple and reliable than the methods of the prior art,
in which the causal bacterium as a target must be found when an
antibiotic is used, and an antibiotic having an antibacterial
spectrum to the causal bacterium must be appropriately selected,
and also which has a risk regarding the appearance of a
drug-resistant bacterium, and in which such a drug-resistant
bacterium actually appeared, another antibiotic having an
antibacterial spectrum to the drug-resistant bacterium must be
appropriately selected.
[0558] Still further, in the present situation of the prior art
techniques in which antibiotics are not effective for virus and
thus virus infection must be prevented by administration of
vaccine, the trehalose compound of the present invention may be
useful in that it could provide a therapeutic agent for
viruses.
[0559] Among the trehalose diester compounds of the present
invention, a compound having particularly high activity had
activity that was approximately 2 times higher than that of TDCM,
and a compound having further particularly high activity had
activity that was approximately 8 to 10 times higher than that of
TDCM. It was demonstrated that these compounds are particularly
useful in that they also have low toxicity.
[0560] Still further, cytokine response was measured after
administration of the trehalose compound of the present invention.
As a result, the release of IL-6, IFN-.gamma. and TNF-.alpha. all
tended to increase. Moreover, both an in vitro test and an in vivo
test were carried out. As a result, it was found that, among the
compounds of the present invention, a compound having particularly
high activity could bring on a significant increase in the
aforementioned cytokines in the in vitro test, but that it could
not increase the release of TNF-.alpha. so much in in vivo test.
Furthermore, it was also found that, among the compounds of the
present invention, such a compound having particularly high
activity activates the release of IL-8 from human-derived THP-1
cells less strongly than TDCM does. Further, as a result of the
measurement using human-derived THP-1 cells, A549 cells and DLD-1
cells, it was found that such a compound having a particularly high
activity, among the compounds of the present invention, exhibited
significant activity of activating the release of NIP-1.beta. from
THP-1 cells, whereas it did not particularly enhance the releasing
activity of TNF-.alpha..
[0561] Immunostimulation has been traditionally emphasized, and the
induction of cytokine and chemokine is useful for activation of
immunity. On the other hand, it has been known that, if immunity is
excessively activated, it may rather have harmful effects,
including anaphylactic shock and allergy as typical examples. The
trehalose compound of the present invention does not excessively
activate the release of inflammatory TNF-.alpha. or IL-8 known as a
chemokine, which may become a factor of causing the aforementioned
excessive inflammatory response. Although the present trehalose
compound has immunostimulatory action, it is unlikely that immune
response excessively acts to generate side effects such as
inflammation. On the other hand, there is also case in which it is
important that chemokine and the like are released at once to
activate immunocytes in one process of immune response. Thus, there
is also an aspect in which the release of a large amount of
cytokine or chemokine is useful, depending on various conditions
such as an infectious state and the physical conditions of an
infected subject.
[0562] As stated above, because of the properties of cytokine or
chemokine, there are three cases, namely, a case in which the
release of a large amount of cytokine or chemokine is favorable, a
case in which such release is unfavorable, and a case in which such
release does not have particular importance. Thus, there are
various cases depending on situation. When the release of IL-8 or
TNF-.alpha. is desired, or when the release of IL-8 or TNF-.alpha.
is not particularly important, a desired amount of specific type of
the trehalose compound of the present invention can be used to
accelerate the release of IL-8, TNF-.alpha. or the like or can be
used without intending to suppress such release. Such use of the
present trehalose compound is also included in the scope of the
present invention.
[0563] Furthermore, it was found that the trehalose compound of the
present invention significantly suppresses the growth of cancer
cells in breast cancer cell-inoculated mice, to which the trehalose
compound of the present invention has been administered, and that
the present trehalose compound also suppresses infiltration or
metastasis of the cancer cells into other organs. This demonstrates
that the compound of the present application has antitumor
activity.
[0564] Therefore, the usefulness and safety of the trehalose
compound of the present invention as an anti-infectious disease
therapeutic agent and as an anticancer agent can be clearly
demonstrated.
INDUSTRIAL APPLICABILITY
[0565] Since the trehalose compound provided by the present
invention has high immunostimulatory activity and also has low
toxicity, it is useful for the treatment of infectious diseases
caused by pathogenic bacteria. Specifically, using the trehalose
compound of the present invention, it is possible to provide a
pharmaceutical product, which hardly causes a risk of having side
effects such as the release of toxin due to destruction of cell
masses upon administration of antibiotics, and which has action to
reduce the toxicity of toxin owned by pathogenic bacteria. In
addition, using the trehalose compound of the present invention, it
is possible to provide a pharmaceutical product which has
therapeutic effects on infectious diseases caused by
multi-drug-resistant bacteria. Moreover, the compound of the
present invention is also useful for the production of a low-risk
pharmaceutical product, which may not cause a risk of generating
excessive immune response.
[0566] Furthermore, by applying the method for producing a
trehalose compound of the present invention, the trehalose compound
according to the present invention can be efficiently synthesized
in a large volume without involving asymmetric synthesis.
* * * * *