U.S. patent application number 11/664437 was filed with the patent office on 2009-02-26 for proliferation inhibitor of helicobacter pylori including alpha-n-acetyl-glucosaminyl bond-containing monosaccharide derivatives.
This patent application is currently assigned to SHINSHU UNIVERSITY. Invention is credited to Masaya Fujita, Jun Nakayama, Takashi Shirai, Takashi Yamanoi.
Application Number | 20090054355 11/664437 |
Document ID | / |
Family ID | 39608462 |
Filed Date | 2009-02-26 |
United States Patent
Application |
20090054355 |
Kind Code |
A1 |
Nakayama; Jun ; et
al. |
February 26, 2009 |
Proliferation Inhibitor Of Helicobacter Pylori Including
Alpha-N-Acetyl-Glucosaminyl Bond-Containing Monosaccharide
Derivatives
Abstract
The present invention herein provides a proliferation inhibitor
of Helicobacter pylori comprising a compound that can simply be
mass-produced, can specifically inhibit the proliferation of H.
pylori, which has high safety and never generates any resistant
bacteria, as well as a diet of a food or beverage, and a
pharmaceutical preparation containing the proliferation inhibitor
of Helicobacter pylori. The proliferation inhibitor of Helicobacter
pylori comprises an alpha-N-acetyl-glucosaminyl bond-containing
monosaccharide derivative represented by the following chemical
formula (1) GlcNAc1-alpha-O-Y (1) (in the formula (1), Y is a
straight-, branched- or cyclic-aliphatic hydrocarbon group having 1
to 27 carbon atoms or a straight-, branched- or cyclic-acyl group
having 1 to 27 carbon atoms). The diet of the food, the beverage or
the pharmaceutical preparation comprises the proliferation
inhibitor of Helicobacter pylori.
Inventors: |
Nakayama; Jun;
(Matsumoto-shi, JP) ; Yamanoi; Takashi; (Tokyo,
JP) ; Fujita; Masaya; (Tokyo, JP) ; Shirai;
Takashi; (Tokyo, JP) |
Correspondence
Address: |
OLIFF & BERRIDGE, PLC
P.O. BOX 320850
ALEXANDRIA
VA
22320-4850
US
|
Assignee: |
SHINSHU UNIVERSITY
Matsumoto-Shi, Nagano
JP
THE NOGUCHI INSTITUTE
Tokyo
JP
|
Family ID: |
39608462 |
Appl. No.: |
11/664437 |
Filed: |
January 12, 2007 |
PCT Filed: |
January 12, 2007 |
PCT NO: |
PCT/JP2007/050741 |
371 Date: |
June 20, 2007 |
Current U.S.
Class: |
514/26 ;
536/5 |
Current CPC
Class: |
A61P 35/00 20180101;
C07H 5/04 20130101; A61P 1/04 20180101; A61P 31/04 20180101; A23L
33/10 20160801; A61P 31/00 20180101; A61K 31/7028 20130101 |
Class at
Publication: |
514/26 ;
536/5 |
International
Class: |
A61K 31/7004 20060101
A61K031/7004; C07G 3/00 20060101 C07G003/00; A61P 31/00 20060101
A61P031/00 |
Claims
1. A proliferation inhibitor of Helicobacter pylori comprising an
alpha-N-acetyl-glucosaminyl bond-containing monosaccharide
derivative represented by the following chemical formula (1)
GlcNAc1-alpha-O-Y (1) (in the formula (1), Y is a straight-,
branched- or cyclic-aliphatic hydrocarbon group having 1 to 27
carbon atoms; or a straight-, branched- or cyclic-acyl group having
1 to 27 carbon atoms).
2. A diet of a food or a beverage comprising a proliferation
inhibitor of Helicobacter pylori comprising an
alpha-N-acetyl-glucosaminyl bond-containing monosaccharide
derivative represented by the following chemical formula (1)
GlcNAc1-alpha-O-Y (1) (in the formula (1), Y is a straight-,
branched- or cyclic-aliphatic hydrocarbon group having 1 to 27
carbon atoms or a straight-, branched- or cyclic-acyl group having
1 to 27 carbon atoms).
3. A pharmaceutical preparation comprising a proliferation
inhibitor of Helicobacter pylori comprising an
alpha-N-acetyl-glucosaminyl bond-containing monosaccharide
derivative represented by the following chemical formula (1)
GlcNAc1-alpha-O-Y (1) (in the formula (1), Y is a straight-,
branched- or cyclic-aliphatic hydrocarbon group having 1 to 27
carbon atoms or a straight-, branched- or or cyclic-acyl group
having 1 to 27 carbon atoms).
Description
TECHNICAL FIELD
[0001] The present invention relates to a proliferation inhibitor
comprising an alpha-N-acetyl-glucosaminyl bond-containing
monosaccharide derivative, which can inhibit the proliferation of
Helicobacter pylori (H. pylori) as a causative microorganism for
diseases such as peptic ulcers and gastric cancers.
BACKGROUND ART
[0002] Helicobacter pylori (H. pylori) is a gram-negative bacterium
belonging to the genus Spirillum isolated from the gastric mucous
membrane of a patient suffering from chronic gastritis and
cultivated (Marshall B. J., Warren J. R., Lancet,
1984,1:1311-1315). It has already been elucidated that such H.
pylori is closely related not only to the crises of chronic
gastritis and peptic ulcers, but also to the crises of serious or
advanced gastric disease such as gastric cancers and gastric
malignant lymphoma (Peek R. M. Jr., Blaser M. J., Nature. Rev.
Cancer, 2002, 2:28-37).
[0003] It has been said that the total number of the persons
infected with H. pylori would reach almost a half of the total
population in all of the world, but these gastric diseases are not
always advanced even to serious conditions thereof in all of the
infected persons. This fact clearly indicates that the gastric
mucous membrane in itself would be provided with the mechanism of
protecting the same from the infection of the bacterium, H.
pylori.
[0004] H. pylori inhabits in the superficial mucus secreted from
the surface layer of the gastric mucous, but never inhabits in the
mucous and the glandular mucus secreted from the mucous deep layer.
This glandular mucus inherently contains a sugar chain derived from
O-glycan having an alpha-N-acetyl-glucosaminyl residue (alpha
GlcNAc residue) at the terminal thereof. For this reason, the
foregoing fact would suggest that the sugar chain may protect the
gastric mucous from the infection with H. pylori.
[0005] An article of Kawakubo M. et al., Science, 2004,
305:1003-1006 discloses the effect of alpha GlcNAc residue on the
proliferation of H. pylori. This article likewise discloses that
glycoproteins which are each linked with a core binary-branched
O-glycan having an alpha GlcNAc bond at the un-reduced terminal
(GlcNAc alpha 1-4Gal beta 1-4GlcNAc beta 1-6(GlcNAc alpha 1-4Gal
beta 1-3)GalNAc-R) can substantially inhibit the proliferation and
mobility of H. pylori and can likewise induce considerable changes
such as the elongation of bacterial cells and the formation of
asymmetric contours and the fragmentation thereof. A series of
these changes are not observed at all in case of the O-glycan free
of any alpha GlcNAc residue. In addition, this article also states
that the glycosyl cholesterol components (CGL) which exist in the
surface layer of the bacterial cells are significantly reduced, as
has been deduced from the results obtained by the morphological
observation of H. pylori in the presence of sugar chains each
carrying the foregoing alpha GlcNAc residue.
[0006] H. pylori essentially requires CGL for the survival thereof,
but it cannot synthesize the same by itself (Hirai Y. et al., J.
Bacteriol., 1995, 177:5327-5333). Accordingly, it would be believed
that H. pylon takes in cholesterols from the external world and
adds glucose to the region in the proximity to the membrane of the
bacterial cell to thus construct the cell wall. In this respect, it
would thus be estimated that the foregoing sugar chain carrying the
alpha GlcNAc residue has an ability to inhibit the construction of
such a cell wall. However, it requires the use of multiple steps
and great expense to chemically or enzymatically synthesize the
foregoing core binary-branched O-glycan having an alpha GlcNAc bond
at the un-reduced terminal and therefore, the chemical or enzymatic
synthesis of the O-glycan is not considered to be practicable.
[0007] Moreover, Japanese Patent Provisional Publication No.
2003-517015 discloses substances having an ability of being linked
with H. pylori, which have sugar chains each including smaller Gal
beta 3GlcNAc or Gal beta 3GalNAc, but a method for the preparation
of these substances is quite complicated and therefore, the method
never permits the mass production of the same.
[0008] On the other hand, the presently used methods for treating
patients infected with H. pylori are not ones which make use of
these sugar chains, but they mainly comprise the step of
exterminating bacterial cells through the simultaneous use of the
following three kinds of drugs: a kind of proton pump-inhibitor and
two kinds of antibiotics. In the medical treatment with which the
three kinds of drugs are combined, problems further arise such that
this treatment may induce the generation of resistant bacteria to
thus cause the recurrence of the infectious disease and that they
may cause side effects.
[0009] For this reason, there has been desired for the development
of a proliferation inhibitor of H. pylori comprising alpha GlcNAc
residue-containing sugar chain derivative, which never exerts any
adverse effect on the human body such as a side effect, can be
produced in commercial quantity and can easily be prepared.
SUMMARY OF THE INVENTION
[0010] The present invention has been developed to solve the
foregoing problems. It is an object of the present invention to
provide a proliferation inhibitor of H. pylori comprising a
compound that can simply be mass-produced, can specifically inhibit
the proliferation of H. pylori, which has high safety and never
generates any resistant bacteria, as well as a diet of a food or
beverage and a pharmaceutical preparation containing the
proliferation inhibitor.
[0011] According to the present invention, the proliferation
inhibitor of H. pylori developed for the achievement of the
foregoing object, comprises an alpha-N-acetyl-glucosaminyl
bond-containing monosaccharide derivative represented by the
following chemical formula (1):
GlcNAc1-alpha-O-Y 1)
(In Formula (1), Y is a straight-, branched- or cyclic-aliphatic
hydrocarbon group having 1 to 27 carbon atoms; or a straight-,
branched- or cyclic-acyl group having 1 to 27 carbon atoms).
[0012] In addition, the diet of the food or the beverage developed
for the achievement of the foregoing object, comprises the
foregoing proliferation inhibitor of H. pylori.
[0013] Moreover, the pharmaceutical preparation developed for the
achievement of the foregoing object, comprises the foregoing
proliferation inhibitor of H. pylori.
[0014] The alpha-N-acetyl-glucosaminyl bond-containing
monosaccharide derivative possesses an anti-bacterial effect to
thus inhibit the proliferation of H. pylori. When using this sugar
derivative, there is not any possibility of generating any
resistant bacteria unlike the administration of the antibiotics.
The sugar derivative can simply be produced and is quite suitable
for the industrial production in commercial quantity.
[0015] According to the sugar derivative-containing pharmaceutical
preparation, the sugar derivative inhibits the construction of the
cell wall of H. pylori to thus specifically inhibit the
proliferation of the bacteria and therefore, it can show a drug
efficacy against H. pylori. In addition, the proliferation
inhibitor of H. pylori can be used alone or in combination with
other drugs such as antibiotics to thus completely remove H. pylori
from the stomach and to prevent the recurrence of gastric diseases
such as chronic gastritis, peptic ulcers, gastric cancers and/or
gastric malignant lymphoma. Furthermore, in the structure of the
sugar derivative, the aglycon site thereof is composed of non-toxic
groups such as an aliphatic hydrocarbon group typical of a lower or
higher saturated or unsaturated alkyl group or a cholestanyl group,
or an acyl group, and accordingly, the proliferation inhibitor of
H. pylori would have quite high safety to the human body.
[0016] The diet of the food or the beverage each containing the
proliferation inhibitor of H. pylori is useful for alleviating the
symptoms of gastric diseases and/or the prevention of such
diseases. This sugar derivative shows a strong action of inhibiting
the proliferation of H. pylori. Consequently, if the sugar
derivative is simply incorporated into the diet of these foods and
beverages in a small quantity, the resulting diet of the foods and
the beverages can show excellent H. pylori-resistant effects.
[0017] Moreover, the pharmaceutical preparation comprising the
proliferation inhibitor of H. pylori is effective for curing
gastric diseases caused by the H. pylori such as chronic gastritis
and gastric ulcers. The sugar derivative shows a strong effect of
inhibiting the proliferation of H. pylori and therefore, an
excellent H. pylori-resistant effect can be anticipated simply by
taking a small amount of this pharmaceutical preparation and
accordingly, the latter is useful for the medical treatment of
gastric diseases, the alleviation of the symptoms thereof and/or
the prevention of the same.
BRIEF EXPLANATION OF THE DRAWINGS
[0018] FIG. 1 is a graph showing the H. pylori-resistant effect of
a proliferation inhibitor of H. pylori comprising
alpha-N-acetyl-glucosaminyl bond-containing monosaccharide
derivative (Compound 1) to which the present invention is
applied.
[0019] FIG. 2 is a graph showing the H. pylori-resistant effect of
an proliferation inhibitor of H. pylori comprising
alpha-N-acetyl-glucosaminyl bond-containing monosaccharide
derivative (Compound 2) to which the present invention is
applied.
MODES FOR CARRYING OUT THE INVENTION
[0020] Embodiments according to the present invention will
hereunder be described in detail, but the present invention is, by
no means, limited to these specific embodiments at all.
[0021] The alpha-N-acetyl-glucosaminyl bond-containing
monosaccharide derivative included in the proliferation inhibitor
of H. pylori of the present invention is represented by the
foregoing chemical formula (1), or GlcNAc1-alpha-O-Y, wherein Y is
an aliphatic hydrocarbon group such as an alkyl group. More
specifically, the derivative has such a structure that an
N-acetyl-glucosaminyl group (GlcNAc) is linked at the
alpha-position.
[0022] The alpha-N-acetyl-glucosaminyl bond-containing
monosaccharide derivative has an excellent effect of inhibiting the
proliferation of H. pylori. For instance, when a culture medium
containing a monosaccharide derivative carrying an ethyl group
(GlcNAc-alpha-O-Et) in a concentration of not less than 1.8 mM is
coexistent with H. pylori, the monosaccharide derivative can
control the proliferation ability of H. pylori to a level of not
more than 50%. In particular, such a culture medium containing the
monosaccharide derivative in a concentration of not less than 7.2
mM can completely inhibit the proliferation of H. pylori. This
monosaccharide derivative is never decomposed within not only the
culture mediums, but also the stomach. This
alpha-N-acetyl-glucosaminyl bond-containing monosaccharide
derivative (GlcNAc-alpha-O-Et) is not only a monosaccharide, but
also a compound simply prepared through a single-step synthetic
method and accordingly, it can be produced in commercial quantity
or scale. In addition, this monosaccharide derivative is quite
stable since it is an aliphatic hydrocarbon group-substituted
derivative of N-acetylglucosamine and the substituent is linked to
the glucosamine through an ether bond. Moreover, the aliphatic
hydrocarbon group such as an alkyl group is a quite stable residue.
This monosaccharide derivative is completely free of any residue
harmful to the human body such as an aromatic residue. Therefore,
the safety thereof is considerably high and the monosaccharide
derivative can thus be incorporated into the diet of the foods and
the beverages as well as pharmaceutical preparations.
[0023] In this connection, the substituent Y of the
alpha-N-acetyl-glucosaminyl bond-containing monosaccharide
derivative appearing in the chemical formula (1) may be a group
having 1 to 27 carbon atoms and specific examples thereof include
aliphatic hydrocarbon groups, for instance, straight or branched
alkyl groups such as ethyl groups; and alicyclic alkyl groups
represented by those derived from sterols, or steroid
ring-containing groups such as cholestanyl groups. Similarly, the
substituent Y may be a group having 1 to 27 carbon atoms and
specific examples thereof having 3 carbon atoms include, for
instance, straight acyl groups; branched acyl groups; and alicyclic
acyl groups, for instance, sterol-derived group-containing acyl
groups and steroid ring-containing acyl groups such as
cholestanyl-containing acyl groups.
[0024] The cholestanyl group-containing monosaccharide derivative
(GlcNAc-alpha-cholestanol) shows an H. pylori-proliferation
inhibitory effect on the order of 40 to 50% at a concentration of
not more than 180 .mu.M in the experiments similar to those
described above. This monosaccharide derivative also possesses
characteristic properties similar to those observed for the
derivative: GlcNAc-alpha-O-Et and it would be expected that this
derivative likewise has safety although it is more or less lower
than that observed for GlcNAc-alpha-O-Et.
[0025] These alpha-N-acetyl-glucosaminyl bond-containing
monosaccharide derivatives are used as proliferation inhibitors of
H. pylori. These saccharide derivatives may be used alone, a
plurality of them may be used in combination or they may likewise
be used in combination with a kind of proton pump-inhibitor such as
Lansoprazole or Omeprazole and two kinds of antibiotics such as
Amoxicillin and Clarithromycin.
[0026] This proliferation inhibitor of H. pylori may likewise be
used as an additive for the diet of the foods and the beverages. In
this respect, examples of such diet of the foods and the beverages
may be foods, for instance, dairy products such as yoghurt; and
beverages such as drink water, cocoa and juices. In this
connection, it is preferred to incorporate the proliferation
inhibitor of H. pylori into the diet of these foods and beverages
in an amount ranging from 0.02 to 0.2%. It is more preferred that
the diet of these foods and beverages are those continuously
ingested. This is because the H. pylori-proliferation inhibitory
effect may further be improved and the continuous ingestion thereof
would thus inhibit the peptic diseases, for instance, gastric
diseases such as chronic gastritis.
[0027] This proliferation inhibitor of H. pylori is used as an
effective component to be incorporated into a pharmaceutical
preparation. Such a pharmaceutical preparation may be in any form
such as a tablet, a capsule, a granule, a pill, an emulsion, a
powder, a syrup, a liquid preparation, or an injection. Such a
pharmaceutical preparation may further comprise components for
preparing each pharmaceutical preparation such as excipients,
distilled water and physiological saline; and/or other medical
components. It is more preferred that these pharmaceutical
preparations should be taken once or continuously to thus improve
the H. pylori-proliferation inhibitory effect and accordingly, the
ingestion thereof would permit the curing or alleviation of the
peptic diseases, for instance, gastric diseases such as chronic
gastritis.
[0028] The following are the description of examples which relate
to the preparation of alpha-N-acetyl-glucosaminyl bond-containing
monosaccharide derivatives and the preparation of the proliferation
inhibitor of H. pylori of the present invention.
PREPARATION EXAMPLE 1
[0029] Preparation of GlcNAc alpha-O-Et (1) Through Chemical
Synthesis
[0030] Ethoxy 2-acetamide-2-deoxy-N-acetyl-alpha-D-glucosaminide
(GIcNAc alpha-O-Et (1)) as an example of the
alpha-N-acetyl-glucosaminyl bond-containing monosaccharide
derivative represented by the foregoing chemical formula (1) to
which the present invention is applied is detailed in this
Preparation Example 1. This derivative can be synthesized according
to the following chemical reaction scheme.
##STR00001##
[0031] Specifically, 3.2864 g (14.86 mmol) of
N-acetyl-D-glucosamine was added to a 200 ml volume eggplant-shaped
flask containing HCl gas-bubbled EtOH (50.0 ml) to thus dissolve
the compound into the latter, a tube packed with calcium chloride
was attached to the flask and the resulting solution was then
stirred at room temperature. Whether the reaction was completed or
not was confirmed by the thin layer chromatography (TLC) technique
(developer solvent: chloroform/methanol (3:1)). After the elapse of
85 hours, the reaction system was concentrated and as a result,
pink-colored crystals were precipitated. A small amount of the
resulting crystals was subjected to the thin layer chromatography
(developer solvent: chloroform/methanol (3:1)), followed by the
color development with iodine for 12 hours. The crystals were
purified according to the column chromatography (developer solvent:
chloroform/methanol (3:1)) to thus give a white crystalline product
(alpha-derivative) in a yield of 75%. The identification of the
resulting product was carried out according to the nuclear magnetic
resonance (NMR) spectrometry technique (600 MHz).
.sup.1H-NMR (600 MHz, CDCl.sub.3): .delta.(ppm): 1.06(3H, t, J=7.6
Hz, --CH.sub.2CH.sub.3), 1.90(3H, s, CH.sub.3CO), 3.34(1 H, t,
J=9.6 Hz, H-4), 3.35-3.41 (1H, m, H-5), 3.57-3.66(4H, m, H-2, H-3,
Ha-6,-CHaCHbH.sub.3), 3.72(1H, dd, J=1.4 Hz, J=8.3 Hz,
--CHaCHbH.sub.3), 3.77(1H, dd, J=2.5 Hz, J=10.3 Hz, Hb-6), 4.73(1H,
d, J=4.2 Hz, H-1) .sup.13C-NMR (150 MHz, CDCl.sub.3): .delta.(ppm):
14.67(--CH2CH.sub.3), 22.51(CH.sub.3CO), 54.29(C-2), 61.21
(--CH.sub.2CH.sub.3), 64.58(C-6), 70.69(C-5), 71.77(C-3),
72.39(C-4), 97.29(C-1), 175.08(CH3CO).
[0032] These spectroscopic data clearly support the fact that the
resulting product is GIcNAc alpha-O-Et (1).
Confirmation of H. pylori-Proliferation Inhibitory Effect of
Compound 1 (GlcNAc alpha-O-Et (1))
[0033] The effect of GlcNAc alpha-O-Et on H. pylori was confirmed
according to the following procedures. Bacterial cells of H. pylori
(ATCC 43504) stored in a brucella broth culture medium frozen at
-80.degree. C. were cultured in the same culture medium (3 mL)
supplemented with 10% equine serum at 35.degree. C. in the presence
of 15% CO.sub.2 for 40 hours according to the shaking culture
technique, the movement of bacterial cells was observed under a
microscope and non-coccoid type bacterial cells of H. pylori were
recovered. The culture medium was inspected for the OD values at
600 nm, followed by the dilution thereof with Muller-Hinton culture
medium supplemented with 5.5% equine serum such that the number of
bacterial cells present therein was equal to 4.times.10.sup.7, and
3 mL thereof in total was cultured at 35.degree. C. in the presence
of 15% CO.sub.2 for 24 hours according to the shaking culture
technique, followed by the confirmation by a microscope to thus
give an H. pylori-containing culture medium (bacterial cell
density: 2.times.10.sup.7/mL) used in the test for the confirmation
of the effect of the foregoing compound. On the other hand, there
were prepared Muller-Hinton culture mediums (free of any bacterial
cell of H. pylori) each containing 902.6 .mu.M to 14.4 mM of the
foregoing GlcNAc alpha-O-Et and supplemented with 5% equine serum,
each of the resulting culture mediums was added to the foregoing H.
pylori-containing culture medium in a mixing ratio of 1:1 (by
volume) (total volume: 100 .mu.L each; on a 96-well plate),
followed by the mixing thereof and the subsequent cultivation at
35.degree. C. in the presence of 15% CO.sub.2 for 96 hours. After
the cultivation thereof over a predetermined period of time, the
bacterial cell density thus proliferated was determined by the
determination of the OD values at 600 nm, followed by the
comparison of the result observed for the sample to which the
candidate compound was added with that observed for the negative
control free of any candidate compound (the control depicted in
FIG. 1) to thus evaluate the proliferatiori inhibitory effect of
the compound. In this respect, 1 U herein means 2.9 .mu.mol/mL.
[0034] The results obtained using GlcNAc alpha-O-Et are plotted on
FIG. 1.
[0035] As is clear from the data shown in FIG. 1, when adding
GIcNAc alpha-O-Et in an amount of not less than 625 mU/mL (1.8 mM),
it is confirmed that not less than 50% of the H. pylori
proliferation is inhibited by the action of the compound.
PREPARATION EXAMPLE 2
[0036] Preparation of GLcNAc alpha-Cholestanol (2) Through Chemical
Synthesis)
[0037] A compound:
3-beta-cholestanyl-2-acetamide-3,4,6-tri-O-benzyl-2-deoxy-alpha-D-glucosa-
minide (GLcNAc alpha-cholestanol (2)) as an example of the
alpha-N-acetyl-glucosaminyl bond-containing monosaccharide
derivative represented by the foregoing chemical formula (1) to
which the present invention is applied is detailed in this
Preparation Example 2. This derivative can be synthesized according
to the following chemical reaction scheme.
##STR00002##
[0038] To a 20 ml volume 2-necked flask, Yb(OTf).sub.3 (129 mg,
0.2080 mmol) dried under reduced pressure for 6 hours in an oil
bath maintained at 185.degree. C. was added and then 0.5 ml of
CH.sub.2Cl.sub.2 was added thereto in an Ar gas atmosphere. To the
resulting mixture, a solution of glycosyl acetate (4) (133.2 mg,
0.2496 mmol) and 3-beta-cholestanol (80.8 mg, 0.2080 mmol)
dissolved in CH.sub.2Cl.sub.2 was added, and 0.1M solution of
BF.sub.3Et.sub.2O (62 .mu.l, 6.24 .mu.mol) prepared in a messflask
was then added to the mixture. The resulting mixture was stirred at
room temperature for 21 hours, the progress of the reaction was
confirmed according to the thin layer chromatography technique
(developer solvent: hexane/AcOEt=1:2) and then the reaction was
stopped by the addition of a saturated NaHCO.sub.3 solution. The
reaction system was extracted with a CH.sub.2Cl.sub.2/AcOEt mixed
solvent, the resulting organic phase was washed with a common salt
solution and then dried over Na.sub.2SO.sub.4. Subsequently, the
organic phase was filtered and then concentrated to thus give
unpurified crystals. The unpurified product was purified according
to the preparative thin layer chromatography technique (developer
solvent: benzene/AcOEt=5:1) to thus obtain a glycoside derivative
mixture in a yield of 70% (alpha/beta ratio=23/77; yield of
alpha-derivative (3): 16.1%).
[0039] The following are the spectrometric data of the
alpha-derivative thus isolated obtained by the use of .sup.1H-NMR
and .sup.13C-NMR spectrometers (available from JEOL Ltd.), and
these data clearly indicate that the derivative has the structure
specified above as that for the compound (3).
.sup.1H-NMR (600 MHz, CDCl.sub.3): .delta.(ppm): 1.86(3H, s, H-8),
0.57-1.97(47H, m, 3-beta-cholestanyl), 3.50(1 H, tt, J=5.4 Hz,
J=10.9 Hz, H-1'), 3.68(1H, t, J=9.5 Hz, H-3), 3.67(1H, dd, J=1.9
Hz, J=10.9 Hz, H-6.sub.b), 3.72(1H, t, J=9.3 Hz, H-4), 3.76(1H, dd,
J=4.3 Hz, J=8.8 Hz, H-6.sub.b), 3.89(1H, ddd, J=1.9 Hz, J=4.1 Hz,
J=9.4 Hz, H-5), 4.24(1H, td, J=3.8 Hz, J=9.8 Hz, H-2), 4.91(1H, d,
J=3.8 Hz, H-1), 5.32(1H, d, J=9.3 Hz, -NHAc), 4.50-4.85(6H, m,
--OCH.sub.2Ph), 7.16-7.35(15H, m, --OCH.sub.2Ph); .sup.13C-NMR (150
MHz, CDCl.sub.3): .delta.(ppm): 23.48(C-8),
12.07-56.44(3-beta-cholestanyl), 52.54(C-2), 68.74(C-6),
70.93(C-5), 76.98(C-1'), 78.50(C-4), 80.82(C-3), 96.16(C-1'),
169.62(C-7), 70.40-75.03(--OCH.sub.2Ph), 127.59-128.51
(--OCH.sub.2C(CHCH).sub.2CH), 138.10-138.57(--OCH2C(CHCH).sub.2CH)
2-2. Synthesis of
3-beta-Cholestanyl-2-Acetamide-2-Deoxy-N-Acetyl-alpha-D-Glucosaminide
(GlcNAc alpha-Cholestanol (2))
[0040] To a 20 ml volume 2-necked, eggplant-shaped flask containing
3 ml of THF, 32.1 mg (0.037 mM) of the alpha-D-glucosaminide (3)
prepared above was added and dissolved to THF, followed by the
addition of 54.7 mg (0.39 mmol) of palladium hydroxide and the
stirring of the resulting mixture at room temperature while
bubbling the mixture with hydrogen gas. The progress of the
reaction was confirmed according to the thin layer chromatography
technique (developer solvent: chloroform/methanol (5:1)). After the
elapse of 15 hours, the palladium hydroxide was filtered off from
the reaction mixture, the resulting filtrate was purified according
to the silica gel column chromatography technique (developer
solvent: chloroform/methanol (5:1)) to thus obtain a white
crystalline product. The resulting product was analyzed by the
.sup.1H-NMR spectrometry and accordingly, the results thus obtained
clearly supported the fact that the product had the foregoing
structure represented by the compound 2.
.sup.1H-NMR (600 MHz, CDCl.sub.3): .delta.(ppm):
0.68-1.85(3-beta-cholestanyl), 2.15(3-beta-cholestanyl),
3.52-3.57(3-beta-cholestanyl), 1.97(3H, s, CH3CO), 3.63-3.68(4H, m,
H-3, H-4, H-5, Ha-6), 3.78-3.84(2H, m, H-2, Hb-6), 4.93(1H, d,
J=3.4 Hz, H-1). Confirmation of H. pylori-Proliferation Inhibitory
Effect of Compound 2 (GlcNAc alpha-Cholestanol (2))
[0041] The effect of GIcNAc alpha-cholestanol on H. pylori was
confirmed according to the following procedures. Bacterial cells of
H. pylori (ATCC 43504) stored in a brucella broth culture medium
frozen at -80.degree. C. were cultured in the same culture medium
(3 mL) supplemented with 10% equine serum at 35.degree. C. in the
presence of 15% CO.sub.2 for 40 hours according to the shaking
culture technique, the movement or behavior of bacterial cells was
observed under a microscope and non-coccoid type bacterial cells of
H. pylori were recovered. The culture medium was inspected for the
OD values at 600 nm, followed by the dilution thereof with
Muller-Hinton culture medium supplemented with 5.5% equine serum
such that the number of bacterial cells present therein was equal
to 4.times.10.sup.7, and 3 mL thereof in total was cultured at
35.degree. C. in the presence of 15% CO.sub.2 for 24 hours
according to the shaking culture technique, followed by the
confirmation of the movement or behavior of bacterial cells by a
microscope to thus give an H. pylori-containing culture medium
(bacterial cell density: 2.times.10.sup.7/mL) used in the test for
the confirmation of the effect of the foregoing compound. On the
other hand, there were prepared Muller-Hinton culture mediums (free
of any bacterial cell of H. pylori) each containing 45 .mu.M to 360
.mu.M of the foregoing GIcNAc alpha-cholestanol and supplemented
with 5% equine serum, each of the resulting culture mediums was
added to the foregoing H. pylori-containing culture medium in a
mixing ratio of 1:1 (by volume) (total volume: 100 .mu.L each; on a
96-well plate), followed by the mixing thereof and the subsequent
cultivation at 35.degree. C. in the presence of 15% CO.sub.2 for 96
hours. After the cultivation thereof over a predetermined period of
time, the bacterial cell density thus proliferated was determined
by the measurement of the OD values at 600 nm, followed by the
comparison of the result observed for the sample to which the
candidate compound was added with that observed for the negative
control free of any candidate compound (the control depicted in
FIG. 2) to thus evaluate the proliferation inhibitory effect of the
compound. In this respect, 1 U herein means 2.9 .mu.mol/mL. In this
connection, the foregoing GlcNAc alpha-cholestanol is never
dissolved simply in the culture medium in such a high
concentration. Accordingly, the insoluble fraction included in the
resulting solution was removed, in advance, through filtration. For
this reason, the foregoing concentrations are all expressed in
terms of the concentrations of the compounds while assuming the
complete dissolution thereof in the culture medium and therefore,
the practical concentrations are lower than those indicated or
specified above.
[0042] The results obtained using GIcNAc alpha-cholestanol are
plotted on FIG. 2. The concentration of a solution obtained by
dissolving the compound in an amount as large as possible was
referred to as the concentration A. As a result, it was confirmed
that the use of the compound in an amount of at least 62.5 mU/mL
(180 .mu.M) permitted the inhibition of about 40 to 50% of the
proliferation of H. pylori (within 1.5 to 2 days).
INDUSTRIAL APPLICABILITY
[0043] The alpha-N-acetyl-glucosaminyl bond-containing
monosaccharide derivative shows its antibacterial effect against H.
pylori in such a mechanism that it can inhibit all kinds of
proliferative activities essential or indispensable to the growth
of the bacterial cells, completely unlike the conventional
antibiotics and therefore, it is quite useful as an effective
component of an H. pylori-resistant agent.
[0044] The proliferation inhibitors of H. pylori comprising these
sugar derivatives are effective components for pharmaceutical
preparations as well as additives for supplements, the diet of the
foods and the beverages.
[0045] The diet of the foods and the beverages each comprising the
proliferation inhibitors of H. pylori are useful as functional
foods and beverages as well as health foods and beverages. The
pharmaceutical preparation comprising the proliferation inhibitors
of H. pylori is accordingly useful for curing, alleviation and/or
prevention of gastric diseases such as chronic gastritis and
gastric ulcers, and peptic diseases, which are caused by the
bacterial cells of H. pylori.
* * * * *