U.S. patent application number 13/003414 was filed with the patent office on 2011-09-15 for inositol and trehalose derivatives and pharmaceutical compositions for treating neurodegenerative diseases comprising the same.
Invention is credited to Sung-Kee Chung, Subhash C. Ghosh, Jungkyun Im, Boram Kim, Woo Sirl Lee.
Application Number | 20110224423 13/003414 |
Document ID | / |
Family ID | 41570452 |
Filed Date | 2011-09-15 |
United States Patent
Application |
20110224423 |
Kind Code |
A1 |
Chung; Sung-Kee ; et
al. |
September 15, 2011 |
INOSITOL AND TREHALOSE DERIVATIVES AND PHARMACEUTICAL COMPOSITIONS
FOR TREATING NEURODEGENERATIVE DISEASES COMPRISING THE SAME
Abstract
The invented inositol and trehalose derivatives, prepared by
introducing multiple units of the guanidine group to the backbone
molecules, show excellent blood-brain barrier permeability, and
accordingly, it can be easily transported to the brain tissues and
utilized for the treatment of neurodegenerative diseases such as
Alzheimer's disease and Huntington's disease.
Inventors: |
Chung; Sung-Kee; (Pohang-si,
KR) ; Lee; Woo Sirl; (Guri-si, KR) ; Kim;
Boram; (Gwangju-si, KR) ; Im; Jungkyun;
(Seoul, KR) ; Ghosh; Subhash C.; (Pohang-si,
KR) |
Family ID: |
41570452 |
Appl. No.: |
13/003414 |
Filed: |
March 23, 2009 |
PCT Filed: |
March 23, 2009 |
PCT NO: |
PCT/KR2009/001470 |
371 Date: |
January 10, 2011 |
Current U.S.
Class: |
536/55.1 ;
536/55; 560/168 |
Current CPC
Class: |
A61P 25/14 20180101;
A61P 25/00 20180101; C07H 15/04 20130101; C07H 3/04 20130101; A61P
25/28 20180101 |
Class at
Publication: |
536/55.1 ;
560/168; 536/55 |
International
Class: |
C07H 15/02 20060101
C07H015/02; C07C 279/12 20060101 C07C279/12 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 22, 2008 |
KR |
10-2008-0071383 |
Claims
1. Inositol derivatives of Formula (1) or salts thereof:
##STR00073## wherein, R is ##STR00074## n being an integer in the
range of 1 to 12; and R.sub.1 is hydrogen, alkyl or --COR', R'
being hydrogen, alkyl, aminoalkyl, arylalkyl, cycloalkyl or
heteroalkyl.
2. The inositol derivatives of claim 1, wherein the stereochemistry
of the compound of Formula (1) corresponds to the scyllo-, myo-,
epi-, chino-, allo-, muco-, neo- or cis-inositol isomer, or salts
thereof.
3. Inositol derivatives of Formula (2) or salts thereof:
##STR00075## wherein, R is ##STR00076## n being an integer in the
range of 1 to 12; and R.sub.1 and R.sub.2 are each independently
hydrogen, alkyl or --COR', R' being hydrogen, alkyl, aminoalkyl,
arylalkyl, cycloalkyl or heteroalkyl.
4. The inositol derivatives of claim 3, wherein the structural
framework of the compound of Formula (2) corresponds to the
scyllo-, myo-, epi-, or chino-inositol isomer, or salts
thereof.
5. Trehalose derivatives of Formula (3) or salts thereof:
##STR00077## wherein, R is ##STR00078## n being an integer in the
range of 1 to 12; and R.sub.1 is hydrogen, R, alkyl or --COR', R'
being hydrogen, alkyl, aminoalkyl, arylalkyl, cycloalkyl,
heteroalkyl or a phosphor.
6. Monosaccharide derivatives of Formula (4) or salts thereof;
##STR00079## wherein, R is ##STR00080## n being an integer in the
range of 1 to 12; R.sub.1 is hydrogen, R, alkyl, arylalkyl,
cycloalkyl or heteroalkyl; and R.sub.2 is hydrogen, R or
trityl.
7. The monosaccharide derivatives of claim 6, wherein the compound
of Formula (4) has the structural framework of D-glucose,
D-mannose, D-allose or D-galactose, or salts thereof.
8. Pharmaceutical compositions for treating neurodegenerative
diseases, containing the compound according to claim 1 as an
effective component.
9. The pharmaceutical compositions of claim 8, wherein the
neurodegenerative diseases include Alzheimer's disease,
Huntington's disease, and other closely related diseases.
10. Pharmaceutical compositions for treating neurodegenerative
diseases, containing the compound according to claim 2 as an
effective component.
11. Pharmaceutical compositions for treating neurodegenerative
diseases, containing the compound according to claim 3 as an
effective component.
12. Pharmaceutical compositions for treating neurodegenerative
diseases, containing the compound according to claim 4 as an
effective component.
13. Pharmaceutical compositions for treating neurodegenerative
diseases, containing the compound according to claim 5 as an
effective component.
14. Pharmaceutical compositions for treating neurodegenerative
diseases, containing the compound according to claim 6 as an
effective component.
15. Pharmaceutical compositions for treating neurodegenerative
diseases, containing the compound according to claim 7 as an
effective component.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to inositol and trehalose
derivatives having excellent blood-brain barrier permeability,
which are prepared by introducing several guanidine groups to the
backbone of inositol and trehalose, and to pharmaceutical
compositions for treating neurodegenerative diseases comprising the
same.
BACKGROUND OF THE INVENTION
[0002] Approximately 25 million people worldwide are currently
suffering from Alzheimer's disease, and that number is expected to
double every five years. Studies of the brain tissue of Alzheimer's
disease patients have revealed the presence of aggregated peptides,
particularly senile plaques and neurofibrillary tangles, which have
been reported to be formed from mutated beta-amyloids and Tau
peptides (M. Goedert, M. G Spillantini, Science, 314: 777-784
(2006)). Such aggregated peptides induce cell damage and apoptosis,
resulting in neurodegenerative diseases. The presently available
methods of symptomatic therapy include the use of
acetylcholinesterase inhibitors and NMDA acceptor antagonists, but
such agents do not cure the disease.
[0003] It has been reported that some inositol stereoisomers
prevent beta-amyloids from forming aggregates in in vitro
experiments, and also that they are capable of inhibiting the
aggregation of beta-amyloids in mouse models of Alzheimer's disease
to reduce or alleviate the symptoms of Alzheimer's disease (J.
McLaurin et al., Nature Medicine, 12: 801-808 (2006); J. McLaurin
et al., J. Biol. Chem. 275: 18495-18502 (2000). However, inositol
is known to have difficulty in passing through the blood-brain
barrier (BBB), and the amount thereof transferred to the nervous
system of the brain is insignificantly small (L. M. Lewin et al.,
Biochem. Journal, 156: 375-380 (1976); M. Uldry et al., EMBO
Journal, 20: 4467-4477 (2000). Hence, in order to apply inositol to
the treatment of Alzheimer's disease, a method to enhance the
passage of inositol through the BBB must be developed.
[0004] Huntington's disease, a neurodegenerative disease inducing
dementia, afflicts approximately 30,000 Americans. It has been
shown that most Huntington's disease patients have a mutation in
the CAG (glutamine codon) repeats of chromosome No. 4, which leads
to the production of mutant huntingtin proteins (H. Y. Zoghbi, H.
T. Orr, Annu. Rev. Neuroscie., 23: 217-247 (2000). The mutant
huntingtin proteins are reported to induce mitochondrial
dysfunction in specific nerve cells of the brain, causing the
apoptosis of the nerve cells. No therapeutically effective agent
for Huntington's disease has been developed.
[0005] Trehalose has been reported to efficiently inhibit the
polyglutamine-induced aggregate formation in in vivo mouse model
experiments, alleviating the symptoms of Huntington's disease (M.
Tanaka et al., Nature Medicine, 10: 148-154 (2004). However, it is
also known that trehalose cannot pass through the BBB to reach the
brain (www.huntingtonproject.org/portals/0/trehalose). Thus, in
order to use trehalose as an effective therapeutic agent for
Huntington's disease, a method that enables trehalose to pass
through the BBB must be developed.
[0006] Therefore, the present inventors have endeavored to develop
inositol and trehalose derivatives that can pass through the BBB
and reach the brain for effective treatment of Alzheimer's disease
and Huntington's disease, respectively.
SUMMARY OF THE INVENTION
[0007] Accordingly, it is the object of the present invention to
provide inositol derivatives, trehalose derivatives, and
monosaccharide derivatives, which have improved BBB
permeability.
[0008] It is another object of the present invention to provide
pharmaceutical compositions for the treatment of neurodegenerative
diseases, comprising the inositol derivatives, the trehalose
derivatives, and the monosaccharide derivatives.
[0009] In accordance with one aspect of the present invention, are
provided inositol derivatives of Formula (1) or salts thereof:
##STR00001##
[0010] wherein,
[0011] R is
##STR00002##
n being an integer in the range of 1 to 12; and
[0012] R.sub.1 is hydrogen, R, alkyl or --COR', R' being hydrogen,
alkyl, aminoalkyl, arylalkyl, cycloalkyl or heteroalkyl.
[0013] In accordance with another aspect of the present invention,
are provided inositol derivatives of Formula (2) or salts
thereof:
##STR00003##
[0014] wherein,
[0015] R is
##STR00004##
n being an integer in the range of 1 to 12; and
[0016] R.sub.1 and R.sub.2 are each independently hydrogen, alkyl
or --COR', R' being hydrogen, alkyl, aminoalkyl, arylalkyl,
cycloalkyl or heteroalkyl.
[0017] In accordance with a further aspect of the present
invention, are provided trehalose derivatives of Formula (3) or
salts thereof:
##STR00005##
[0018] wherein,
[0019] R is
##STR00006##
n being an integer in the range of 1 to 12; and
[0020] R.sub.1 is hydrogen, R, alkyl or --COR', R' being hydrogen,
alkyl, aminoalkyl, arylalkyl, cycloalkyl, heteroalkyl or a
phosphor.
[0021] In accordance with a further aspect of the present
invention, are provided monosaccharide derivatives of Formula (4)
or salts thereof:
##STR00007##
[0022] wherein,
[0023] R is
##STR00008##
n being an integer in the range of 1 to 12;
[0024] R.sub.1 is hydrogen, R, alkyl, arylalkyl, cycloalkyl or
heteroalkyl; and
[0025] R.sub.2 is hydrogen, R or trityl.
[0026] In addition, in accordance with a still further aspect of
the present invention, are provided pharmaceutical compositions for
treating neurodegenerative diseases, comprising any one compound
selected from the compounds of Formulas 1 to 4.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] The above and other objects and features of the present
invention will become apparent from the following description of
the invention, when taken in conjunction with the accompanying FIG.
1, which shows the abilities of the compounds according to the
present invention to permeate into the brain through the BBB.
DETAILED DESCRIPTION OF THE INVENTION
[0028] Unless otherwise defined, the technical and scientific terms
used in the present invention have the same meanings generally used
in the relevant technical field. In addition, the aforementioned
literatures are incorporated into the description of the invention
by reference.
[0029] Herein, the term "alkyl" refers to a linear or branched
C.sub.1-12 alkyl, e.g., methyl, ethyl, propyl, hexyl, and
octyl.
[0030] The term "aryl" refers to a monocyclic aromatic group or a
bicyclic group having one or more aromatic rings, and the term
"arylalkyl" refers to a C.sub.1-3 alkyl having 1 to 3 aryl
substituents, including but not limited to benzyl, and trityl.
[0031] The term "cycloalkyl" refers to a saturated monocyclic
C.sub.3-8 hydrocarbon group, including but not limited to
cyclopropyl, cyclobutyl, and cyclohexyl.
[0032] Further, the term "heteroalkyl" refers to a C.sub.1-10 alkyl
having one or more heteroatoms, the heteroatom being oxygen,
sulfur, or nitrogen.
[0033] Hereinafter, a detailed description of the present invention
is given.
[0034] The present invention provides inositol derivatives of
Formula (1) or salts thereof:
##STR00009##
[0035] wherein,
[0036] R is
##STR00010##
n being an integer in the range of 1 to 12; and
[0037] R.sub.1 is hydrogen, R, alkyl or --COR', R' being hydrogen,
alkyl, aminoalkyl, arylalkyl, cycloalkyl or heteroalkyl.
[0038] The stereochemistry of the compound of Formula (1)
corresponds to the scyllo-, myo-, epi-, chino-, allo-, muco-, neo-
or cis-inositol isomer.
[0039] Also, the present invention provides inositol derivatives of
Formula (2) or salts thereof:
##STR00011##
[0040] wherein,
[0041] R is
##STR00012##
n being an integer in the range of 1 to 12; and
[0042] R.sub.1 and R.sub.2 are each independently hydrogen, alkyl
or --COR', R' being hydrogen, alkyl, aminoalkyl, arylalkyl,
cycloalkyl or heteroalkyl.
[0043] The structural framework of the compound of Formula (2)
corresponds to the scyllo-, myo-, epi-, or chino-inositol
isomer
[0044] Also, the present invention provides trehalose derivatives
of Formula (3) or salts thereof:
##STR00013##
[0045] wherein,
[0046] R is
##STR00014##
n being an integer in the range of 1 to 12; and
[0047] R.sub.1 is hydrogen, R, alkyl or --COR', R' being hydrogen,
alkyl, aminoalkyl, arylalkyl, cycloalkyl, heteroalkyl or a
fluorescence probe.
[0048] Also, the present invention provides a monosaccharide
derivative of Formula (4) or a salt thereof:
##STR00015##
[0049] wherein,
[0050] R is
##STR00016##
n being an integer in the range of 1 to 12;
[0051] R.sub.1 is hydrogen, R, alkyl, arylalkyl, cycloalkyl or
heteroalkyl; and
[0052] R.sub.2 is hydrogen, R or trityl.
[0053] The compound of Formula (4) has the structural framework of
D-glucose, D-mannose, D-allose or D-galactose.
[0054] The inositol derivatives of Formulas (1) and (2) according
to the present invention may be prepared by a method comprising the
steps of: 1) introducing amino acid side chains to the hydroxyl
groups of a protected inositol intermediate by acylation to obtain
an intermediate; 2) introducing protected guanidine groups to the
terminal amino groups of the amino acid side chains of the compound
obtained in step 1); and 3) removing the protecting group from the
guanidine group of the compound obtained in step 2) to provide the
inositol derivative of Formulas (1) or (2).
[0055] The trehalose derivatives of Formula (3) according to the
present invention may be prepared by a method comprising the steps
of: 1) introducing amino acid side chains to the hydroxyl groups of
a protected trehalose intermediate by acylation to obtain an
intermediate; 2) introducing protected guanidine groups to the
terminal amino groups of the amino acid side chains of the compound
obtained in step 1); and 3) removing the protecting group from the
guanidine group of the compound obtained in step 2) to provide the
trehalose derivative of Formula (3).
[0056] The monosaccharide derivatives of Formula (4) according to
the present invention may be prepared by a method comprising the
steps of: 1) introducing amino acid side chains to the hydroxyl
groups of a protected monosaccharide intermediate by acylation to
obtain an intermediate; 2) introducing protected guanidine groups
to the terminal amino groups of the amino acid side chains of the
compound obtained in step 1); and 3) removing the protecting group
from the guanidine group of the compound obtained in step 2) to
provide the monosaccharide derivative of Formula (4).
[0057] Specifically, the intermediate for preparing the compound of
Formula (1) can be prepared according to the procedure of Scheme
(1) using a scyllo-inositol derivative, in which the
stereochemistry of the 2-OH group of myo-inositol is inverted, as a
starting material.
##STR00017##
[0058] wherein, Bz is benzoyl, TBS is t-butyldimethylsilyl, and Cbz
is carbobenzoxy.
[0059] Scheme (2) illustrates the procedure for preparing the
compound of Formula (1) from the intermediate obtained in Scheme
(1).
##STR00018##
[0060] wherein, Boc is butyloxycarbonyl, TFA is trifluoroacetic
acid, and Cbz and TBS are the same as defined above.
[0061] The compound of Formula (2) which is a typical example of
the inositol derivative having eight guanidine groups, can be
prepared from 1-O-benzoyl-2,3,4,5-tetrabenzyl-scyllo-inositol
(Korean Patent No. 578732) according to the procedure of Scheme
(3).
##STR00019## ##STR00020## ##STR00021##
[0062] wherein, Bn is benzyl, and Bz, Boc, Cbz and TFA are the same
as defined above.
[0063] Schemes (4) and (5) respectively illustrate the procedures
for preparing the compounds of Formula (3) from trehalose.
##STR00022##
[0064] wherein, Boc and TFA are the same as defined above.
##STR00023## ##STR00024##
[0065] wherein, Tr is trityl, FITC is fluorescein isothiocyanate,
and Boc, Cbz and TFA are the same as defined above.
[0066] The key intermediate for preparing the compound of Formula
(4) can be prepared according to the procedure of Scheme (6) using
D-glucose as a starting material.
##STR00025##
[0067] wherein, Bz and Cbz are the same as defined above.
[0068] Scheme (7) illustrates the procedures for preparing the
compound of Formula (4) from the intermediate obtained in Scheme
(6).
##STR00026##
[0069] wherein, Cbz, Boc and TFA are the same as defined above.
[0070] The present invention provides pharmaceutical compositions
for treating neurodegenerative diseases, comprising any one
compound selected from the compounds of Formulas 1 to 4 and a
pharmaceutically acceptable carrier. The neurodegenerative diseases
may include Alzheimer's disease, Huntington's disease, and closely
related diseases.
[0071] The pharmaceutical composition according to the present
invention may further include an excipient, a distintigrator, a
sweetening agent, a lubricating agent, and a flavoring agent, which
are typically used. Also, the pharmaceutical composition may be
formulated into pharmaceutical preparations in single- or
multiple-dose forms, such as tablets, capsules, powders, granules,
liquids such as suspensions, emulsions or syrups, or parenteral
administration preparations, by typical methods.
[0072] Also, the pharmaceutical composition according to the
present invention may be parenterally or orally administered
depending on the needs, and daily dose of the compound of the
present invention may be 0.01 to 100 mg per 1 kg of adult's body
weight and may be administered at once or in several parts. The
dose for a specific patient may vary depending on body weight, age,
gender, health condition, diet, administration time, administration
method, and disease severity.
[0073] The following Examples are intended to further illustrate
the present invention without limiting its scope.
EXAMPLE
Preparative Example 1
Preparation of Scyllo-Inositol Having Carbobenzoxy (Cbz)-Protected
Linker
<1-1> Introduction of Acetonide Protecting Group
##STR00027##
[0075] 1-O-Bz-2,3,4,5,6-pentahydroxyl-scyllo-inositol (Korean
Patent No. 578732; 9.96 g, 35.05 mmol) was dissolved in
N,N-dimethylformamide (110 ml), and para-toluene sulfonic acid
(3.33 g) was added dropwise thereto to obtain a mixture.
2-Methoxypropene (33.5 ml) was added to the mixture and the mixture
was stirred at room temperature for 16 hours.
[0076] After the completion of the reaction, the reaction mixture
was extracted with ethyl acetate, and the extract was washed
several times with saturated aqueous NaHCO.sub.3 and water. The
organic layer thus obtained was dried over Na.sub.2SO.sub.4,
concentrated under a reduced pressure, and then purified by column
chromatography (ethyl acetate:hexane=1:2) to obtain the title
compound having two isopropylidene groups introduced to the
backbone thereof, as a white foamy solid (6.03 g).
[0077] .sup.1H NMR (CDCl.sub.3): .delta. 1.44-1.55 (m, 12H),
3.67-3.95 (m, 4H), 4.10-4.16 (m, 1H), 5.43 (dd, J=7.9, 10.4 Hz, 1H,
for 3a), 5.60 (t, J=9.2 Hz, 1H, for 3b), 7.42-7.49 (m, 2H),
7.54-7.62 (m, 1H), 8.07-8.10 (m, 2H).
<1-2> Introduction of t-Butyldimethylsilane (TBS) Protecting
Group
##STR00028##
[0078] The compound obtained in Preparative Example <1-1>
(646 mg, 1.77 mmol) was dissolved in N,N-dimethylformamide (3 ml),
and imidazole (534 mg, 3.54 mmol) and t-butyldimethylsilyl chloride
(534 mg, 3.54 mmol) were added thereto at 0.degree. C. The mixture
thus obtained, was warmed to room temperature, and then stirred for
10 hours.
[0079] After the completion of the reaction, the reaction mixture
was extracted with ethyl acetate, and the extract was washed
several times with water and saturated aqueous NaHCO.sub.3. The
organic layer thus obtained was dried over Na.sub.2SO.sub.4,
concentrated under a reduced pressure, and then purified by column
chromatography (ethyl acetate:hexane=1:3) to obtain the title
compound as a white solid (750 mg).
[0080] .sup.1H NMR (CDCl.sub.3): .delta. 0.07, 0.06 (2s, 6H, for
4a), 0.14 (s, 6H, for 4b), 0.73 (s, 9H, for 4a), 0.92 (s, 9H, for
4b), 1.41-1.46 (m, 12H), 3.60-4.10 (m, 5H), 5.47-5.56 (m, 1H),
7.41-7.46 (m, 2H), 7.54-7.56 (m, 1H), 8.00-8.09 (m, 2H).
<1-3> Removal of Benzoyl (Bz) Protecting Group
##STR00029##
[0082] The compound obtained in Preparative Example <1-2>
(700 mg, 1.46 mmol) was dissolved in a mixture of methanol and
dichloromethane (1:4) (2.5 ml), a NaOMe solution (134 .mu.l, 0.58
mmol, 25 wt %) was added thereto, and the mixture thus obtained was
refluxed for 3 hours.
[0083] Thereafter, the mixture was cooled to room temperature,
diluted with dichloromethane, filtered through silica gel, and
concentrated. The concentrate thus obtained was washed with a
mixture of hexane and ethyl acetate (19:1), and then dried in a
vacuum to obtain the title compound as a white solid (547 mg).
[0084] .sup.1H NMR (CDCl.sub.3): .delta. 0.07-0.13 (m, 6H),
0.85-0.90 (m, 9H), 1.40-1.46 (m, 12H), 3.43-3.78 (m, 6H).
<1-4> Introduction of Carbobenzoxy (Cbz)-Protected Linker
##STR00030##
[0086] The compound obtained in Preparative Example <1-3>
(360 mg, 0.960 mmol), 6-benzoyloxycarbonylaminohexanoic acid (290
mg, 1.15 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (221
mg, 1.15 mmol), and 4-dimethylaminopyridine (35 mg, 0.288 mmol)
were dissolved in dichloromethane (3 ml) and stirred at room
temperature for 24 hours.
[0087] After the completion of the reaction, the reaction mixture
was extracted with ethyl acetate, and the extract was washed
several times with saturated aqueous NaHCO.sub.3 and water. The
organic layer thus obtained was dried over Na.sub.2SO.sub.4,
concentrated under a reduced pressure, and then purified by column
chromatography (ethyl acetate:hexane=1:3) to obtain the title
compound as a sticky liquid (485 mg).
[0088] .sup.1H NMR (CDCl.sub.3): .delta. 0.05 (s, 3H, for 6a), 0.08
(s, 3H, for 6a), 0.11 (s, 6H, for 6b), 0.84 (s, 9H, for 6a), 0.90
(s, 9H, for 6b), 1.37-1.53 (m, 16H), 1.63-1.68 (m, 2H), 2.32-2.39
(m, 2H), 3.15-3.19 (m, 2H), 3.50-3.62 (m, 2H), 3.63-3.68 (m, 1H),
3.71-3.79 (m, 1H), 3.89 (dd, J=7.8, 10.0 Hz, 1H), 4.81 (brs, 1H),
5.08 (s, 2H), 5.22 (dd, J=7.8, 10.7 Hz, 1H), 7.30-7.36 (m, 5H).
<1-5> Removal of Acetonide Protecting Group and
t-Butyldimethylsilane Protecting Group
##STR00031##
[0089] The compound obtained in Preparative Example <1-4>
(310 mg, 0.5 mmol) was dissolved in a mixture of dichloromethane
and methanol (1:4, 2 ml), a HCl gas-saturated ethyl acetate
solution (1 ml) was added thereto, and the mixture thus obtained
was stirred at room temperature for 30 min.
[0090] After the completion of the reaction, the reaction mixture
was concentrated under a reduced pressure to completely remove the
solvent, washed with hexane and ether, and dried in a vacuum to
obtain the title compound as a white solid (225 mg).
[0091] .sup.1H NMR (D.sub.2O): .delta. 1.33-1.37 (m, 2H), 1.48-1.53
(m, 2H), 1.62-1.67 (m, 2H), 2.46 (t, J=7.1 Hz, 2H), 3.12 (t, J=6.0
Hz, 2H), 3.34-3.50 (m, 6H), 5.12 (s, 2H), 7.42-7.44 (m, 5H).
Preparative Example 2
Preparation of scyllo-Inositol Having Benzoyl (Bz)-Protected OH
Group and Carbobenzoxy (Cbz)-Protected Linker
<2-1> Introduction of Carbobenzoxy (Cbz)-Protected Linker
##STR00032##
[0093] 1-O-benzoyl-2,3,4,5-tetrabenzyl-scyllo-inositol (Korean
Patent No. 578732; 2.5 g, 3.88 mmol),
6-benzoyloxycarbonylaminohexanoic acid (1.5 mg, 5.65 mmol),
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (1.1 mg, 5.65 mmol)
and 4-dimethylaminopyridine (140 mg, 1.14 mmol) were dissolved in
dichloromethane (20 ml) and stirred at room temperature for 24
hours.
[0094] After the completion of the reaction, the reaction mixture
was extracted with ethyl acetate, and the extract was washed
several times with saturated aqueous NaHCO.sub.3 and water. The
organic layer thus obtained was dried over Na.sub.2SO.sub.4,
concentrated under a reduced pressure, and then purified by column
chromatography (ethyl acetate:hexane=1:3) to obtain the title
compound as a white solid (3.38 g).
[0095] .sup.1H NMR (CDCl.sub.3): .delta. 0.91-1.29 (m, 6H), 2.01
(t, J=7.1 Hz, 2H), 2.88 (br. s, 2H), 3.66-3.70 (m, 4H), 4.56-4.88
(m, 8H), 5.08 (s, 2H), 5.27-5.37 (m, 2H), 7.03-7.53 (m, 28H), 7.94
(d, J=7.2 Hz, 2H).
<2-2> Removal of Benzyl (Bn) Protecting Group and
Carbobenzoxy (Cbz) Protecting Group
##STR00033##
[0097] The compound obtained in Preparative Example <2-1>
(3.38 g, 3.79 mmol) was dissolved in a mixture of dichloromethane
and ethanol (1:1.5, 50 ml), and Pd(OH).sub.2/C (20 wt %) (1.7 g)
was added thereto. The mixture thus obtained was stirred at room
temperature under H.sub.2 gas (4 atm) for one day and filtered
through celite to remove Pd(OH).sub.2/C. The filtrate thus obtained
was concentrated under a reduced pressure to obtain the title
compound as a white sticky solid (1.4 g).
[0098] .sup.1H NMR (CD.sub.3OD): .delta. 1.09-1.20 (m, 2H),
1.37-1.50 (m, 4H), 2.14-2.35 (m, 2H), 2.67 (t, J=7.7 Hz, 2H),
3.38-3.45 (m, 2H), 3.54-3.67 (m, 2H), 5.12-5.25 (m, 2H), 7.47-7.52
(m, 2H), 7.61-7.63 (m, 1H), 8.00-8.03 (m, 2H).
<2-3> Re-Introduction of Carbobenzoxy (Cbz) Protecting
Group
##STR00034##
[0100] The compound obtained in Preparative Example <2-2>
(1.3 g, 3.52 mmol) was dissolved in a mixture of 1,4-dioxane and
water (1:1, 20 ml). Triethylamine (594 .mu.l, 4.22 mmol) was added
thereto, and carbobenzoxy chloride (Cbz-Cl) (980 .mu.l, 7.04 mmol)
was added dropwise at 0.degree. C. for 30 min. The obtained mixture
was stirred at room temperature for 12 hours, concentrated under a
reduced pressure. The water (20 ml) was added to the obtained
concentrates and extracted with ethyl acetate. The organic layer
thus obtained was dried over Na.sub.2SO.sub.4, concentrated under a
reduced pressure, and then purified by column chromatography
(dichloromethane:methanol=12:1) to obtain the title compound as a
white foamy solid (1.2 g).
[0101] .sup.1H NMR (CD.sub.3OD): .delta. 1.04-1.36 (m, 6H),
2.14-2.23 (m, 2H), 2.86-2.89 (m, 2H), 3.38-3.41 (m, 2H), 3.52-3.62
(m, 2H), 5.05 (s, 2H), 5.10-5.20 (m, 2H), 7.35-7.57 (m, 8H),
7.99-8.01 (m, 2H).
Preparative Example 3
Preparation of scyllo-Inositol Having the Same Two Neighboring
Protecting Groups
<3-1> Introduction of Benzoyl (Bz) Protecting Group
##STR00035##
[0103] 1-O-benzoyl-2,3,4,5-tetrabenzyl-scyllo-inositol (Korean
Patent No. 578732; 150 mg, 0.233 mmol) was dissolved in pyridine (3
ml), benzoyl chloride (35 .mu.l, 0.302 mmol) was added dropwise
thereto, and the mixture thus obtained was stirred at 60.degree. C.
for 24 hours.
[0104] After the completion of the reaction, water (5 ml) was added
to the reaction mixture, stirred for 30 min, diluted with ethyl
acetate, and the extract was washed with HCl (1M), NaHCO.sub.3 and
Na.sub.2SO.sub.4. The organic layer thus obtained was dried over
Na.sub.2SO.sub.4, concentrated under a reduced pressure, and then
purified by column chromatography (ethyl acetate:hexane=1:2) to
obtain the title compound having two benzoyl groups as a white
solid (174 mg).
[0105] .sup.1H NMR (CDCl.sub.3): .delta. 3.8-3.82 (m, 4H),
4.63-4.80 (dd, 4H), 4.93 (s, 4H), 5.55-5.57 (m, 2H), 7.05-7.90 (m,
30H).
<3-2> Removal of Benzyl (Bn) Protecting Group
##STR00036##
[0107] The compound obtained in Preparative Example <3-1>
(160 mg, 0.214 mmol) was dissolved in a mixture of dichloromethane
and ethanol (1:2, 12 ml) and Pd(OH).sub.2/C (20 wt %, 75 mg) was
added thereto. The mixture thus obtained was stirred at room
temperature under H.sub.2 gas (4 atm) for one day, and filtered
through celite to remove Pd(OH).sub.2/C. The filtrate thus obtained
was concentrated under a reduced pressure to obtain the title
compound as a white solid compound (94 mg).
[0108] .sup.1H NMR (CD.sub.3OD): .delta. 3.45-3.48 (m, 2H),
3.68-3.74 (m, 2H), 5.34-5.39 (m, 2H), 7.33-7.90 (m, 10H).
Preparative Example 4
Preparation of Trehalose Having the Same Two Protecting Groups
<4-1> Introduction of Trityl (Tr) Protecting Group
##STR00037##
[0110] Trehalose hydrate (2.2 g, 5.82 mmol) and trityl chloride
(7.7 g, 27.21 mmol) were dissolved in pyridine (40 ml) at room
temperature and stirred for 5 hours while being slowly heated to
60.degree. C.
[0111] After the completion of the reaction, the reaction mixture
was diluted with ethyl acetate and the extract was washed with
aqueous hydrochloric acid (1N) and saturated aqueous NaHCO.sub.3.
Thereafter, aqueous NaCl was added dropwise thereto to obtain a
suspension. The suspension was filtered using a Buchner funnel. The
filtrate thus obtained was washed with diethylether, and dried
under a reduced pressure to obtain a white compound (4.37 g).
[0112] m.p. 275-276.degree. C. (dec.)
[0113] .sup.1H NMR (in MeOD, .delta.) 3.37 (app. t, 6H, J=3.8 Hz),
3.57 (dd, 2H, J=9.7 and 3.8 Hz), 3.79 (app. t, 2H, J=9.3 Hz),
4.02-4.09 (m, 2H), 5.36 (d, 2H, J=3.8 Hz, anomeric protons),
7.14-7.25 (m, 18H, trityl protons), 7.41-7.52 (m, 12H, trityl
protons).
Preparative Example 5
Preparation of Glucose Derivative Having Carbobenzoxy
(Cbz)-Protected Linker
<5-1> Preparation of penta-O-Benzoyl-D-Glucose
##STR00038##
[0115] D-glucose (2.0 g, 11 mmol) was dissolved in anhydrous
pyridine (24 ml), and acetic anhydride (8 ml, 67 mmol) was added
thereto, and the mixture thus obtained was stirred at
60.about.65.degree. C. for 3 hours.
[0116] After the completion of the reaction, the reaction mixture
was extracted with dichloromethane, and the extract was washed with
1N HCl, saturated aqueous NaHCO.sub.3 and aqueous NaCl. The product
thus obtained was dried over Na.sub.2SO.sub.4, concentrated under a
reduced pressure, and then recrystallized from methanol/acetone to
obtain the title compound as a white solid (5.7 g).
[0117] .sup.1H NMR (CDCl.sub.3): .delta. 4.40-4.46 (m, 1H, H-5),
4.49 (dd, J=12.23 Hz, 4.70 Hz, 1H, H-6b), 4.65 (dd, J=12.31 Hz,
2.82 Hz, 1H, H-6a), 5.82-5.91 (m, 2H, H-2, H-4), 6.07 (t, J=9.4 Hz,
1H, H-3), 6.31 (d, J=7.99 Hz, 1H, H-1), 7.24-8.05 (m, 25H,
arom).
<5-2> Preparation of 2,3,4,6-tetra-O-Benzoyl-D-Glucose
##STR00039##
[0119] The compound obtained Preparation Example <5-1> (2.0
g, 2.8 mmol) was dissolved in dichloromethane (10 ml), and
hydrobromic acid was added thereto at 0.degree. C., and the mixture
thus obtained was stirred at room temperature for 7 hours.
[0120] After the reaction was completed using ice water, the
reaction mixture was extracted with dichloromethane, and the
extract was washed with saturated aqueous NaHCO.sub.3 and aqueous
NaCl. The product thus obtained was dried over Na.sub.2SO.sub.4 and
concentrated under a reduced pressure. The concentrate thus
obtained was dissolved in acetone/water (6 ml/0.3 ml).
Ag.sub.2CO.sub.3 was added thereto, and the mixture thus obtained
was stirred at room temperature for 3 hours.
[0121] After the completion of the reaction, the reaction mixture
was filtered through celite to remove Ag.sub.2CO.sub.3. The
filtrate thus obtained was concentrated under a reduced pressure,
to obtain the title compound as a white sticky solid (1.7 g).
[0122] .sup.1H NMR (CDCl.sub.3): .delta. 4.16-4.20 (m, 1H, H-5),
4.46 (dd, J=12.24 Hz, 4.99 Hz, 1H, H-6b), 4.61 (dd, J=12.16 Hz,
2.91 Hz, 1H, H-6a), 5.05 (d, J=7.93 Hz, 1H, H-1), 5.32 (t, J=8.0
Hz, 1H, H-2), 5.67 (t, J=9.7 Hz, 1H, H-4), 5.93 (t, J=9.7 Hz, 1H,
H-3), 7.24-8.03 (m, 20H, arom).
<5-3> Preparation of 2,3,4,6-tetra-O-Benzoyl-D-Glucose
Acetimidate
##STR00040##
[0124] The compound obtained in Preparation Example <5-2>
(1.5 g, 2.6 mmol) was dissolved in dichloromethane (10 ml).
1,8-diazabicyclo[5,4,0]undec-7-ene (DBU) (20 ml, 0.13 mmol) and
trichloroacetonitrile (2.6 ml, 26.1 mmol) was added thereto at
0.degree. C., and the mixture was stirred for 4 hours.
[0125] After the completion of the reaction, the reaction mixture
was concentrated under a reduced pressure, and then purified by
column chromatography (ethyl acetate:hexane=1:3) to obtain the
title compound as a white solid (1.2 g).
[0126] .sup.1H NMR (CDCl.sub.3): .delta. 4.46 (dd, J=12.9 Hz, 5.4
Hz, 1H, H-6b), 4.16-4.20 (m, 2H, H-5, H-6a), 5.60 (dd, J=10.2 Hz,
3.7 Hz, 1H, H-2), 5.83 (t, J=9.8 Hz, 1H, H-4), 6.28 (t, J=10.0 Hz,
1H, H-3), 6.84 (d, J=3.7 Hz, 1H, H-1), 5.93 (t, J=9.7 Hz, 1H, H-3),
7.26-8.05 (m, 20H, arom), 8.64 (s, 1H, NH).
<5-4> Preparation of
1-O--(N-Carbobenzoxy-6-Aminopentyl)-2,3,4,6-tetra-O-Benzoyl-D-Glucopyrano-
side
##STR00041##
[0128] N-carbobenzoxy-6-aminopentanol (0.32 g, 1.35 mmol) and
trimethylsilyl trifluoromethanesulfonate (0.25 ml, 1.35 mmol) were
dissolved in dichloromethane (10 ml) and cooled to 0.degree. C. To
this solution, was added a solution obtained by dissolving the
compound obtained in Preparative Example <5-3> (1 g, 1.4
mmol) in dichloromethane (25 ml), and the mixture thus obtained was
stirred for 2 hours.
[0129] After the completion of the reaction, the reaction mixture
was filtered through celite. The filtrate thus obtained was washed
with saturated aqueous NaHCO.sub.3 and aqueous NaCl. The product
thus obtained was dried over Na.sub.2SO.sub.4, concentrated under a
reduced pressure, and then purified by column chromatography (ethyl
acetate:hexane=1:2) to obtain the title compound as a transparent
sticky oil (0.8 g).
[0130] .sup.1H NMR (CDCl.sub.3): .delta. 1.20-1.54 (m, 6H),
2.93-2.95 (d, J=6.1 Hz, 2H), 3.51-3.54 (m, 1H), 3.88-3.95 (m, 1H),
4.10-4.17 (m, 1H, H-5), 4.47 (dd, J=12.2 Hz, 5.0 Hz, 1H, H-6b),
4.63 (d, J=11.9 Hz, 2H, H-6b, NH), 4.81 (d, J=7.7 Hz, 1H, H-1),
5.08 (s, 1H, benzyl), 5.52 (t, J=9.4 Hz, 1H, H-2), 5.68 (t, J=9.7
Hz, 1H, H-4), 5.91 (t, J=9.6 Hz, 1H, H-3), 7.26-8.03 (m, 25H,
arom).
<5-5> Preparation of
1-O--(N-Carbobenzoxy-6-Aminopentyl)-D-Glucopyranose
##STR00042##
[0132] The compound obtained in Preparative Example <5-4>
(0.8 g, 1.0 mmol) was dissolved in methanol (30 ml), a solution (30
.mu.l, 0.1 mmol) of sodium methoxide in methanol was added thereto,
and the mixture thus obtained was stirred at 70.degree. C. for 3
hours.
[0133] After the completion of the reaction, the reaction mixture
was neutralized with acidic cation exchange resin (Dowex
50WX8-100), and then concentrated under a reduced pressure to
obtain the title compound as a colorless sticky solid (369 mg).
[0134] .sup.1H-NMR (CD.sub.3OD): .delta. 1.35-1.58 (m, 6H),
3.03-3.27 (m, J=6.1 Hz 5H), 3.44-3.49 (m, 1H), 3.59-3.64 (m, 1H),
3.80-3.85 (m, 3H), 4.18 (d, J=7.7 Hz, 1H), 5.00 (s, 1H, benzyl),
5.52 (t, J=9.4 Hz, 1H, H-2), 5.68 (t, J=9.7 Hz, 1H, H-4), 5.91 (t,
J=9.6 Hz, 1H, H-3), 7.29 (brs, 5H, arom).
Example 1
Preparation of Scyllo-Inositol Derivative Having Ten Guanidine
Groups
[0135] <1-1> Introduction of Side Chain to scyllo-Inositol by
Acylation
##STR00043##
[0136] The compound having five OH groups obtained in Preparative
Example <1-5> (34 mg, 0.079 mmol), a branched linker having
two guanidine groups (Korean Patent No. 699279; 464 mg, 0.636
mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (151 mg, 0.79
mmol) and 4-dimethylaminopyridine (24 mg, 0.2 mmol) were dissolved
in N,N-dimethylformamide (2 ml), and stirred at room temperature
for two days.
[0137] After the completion of the reaction, the reaction mixture
was washed several times with water and aqueous NaHCO.sub.3. The
organic layer thus obtained was dried over Na.sub.2SO.sub.4,
concentrated under a reduced pressure, and then purified by column
chromatography (dichloromethane:methanol=10:1) to obtain the title
compound having five acyl side chains, as a sticky solid (228
mg).
[0138] .sup.1H NMR (CDCl.sub.3): .delta. 1.25-1.69 (m, 236H),
2.16-2.25 (m, 12H), 2.30-2.46 (m, 30H), 3.14 (m, 2H), 3.43-3.44 (m,
18H), 5.07 (s, 2H), 5.23 (s, 6H), 7.33 (m, 5H), 8.50 (m, 10H),
11.49 (s, 10H).
<1-2> Removal of Carbobenzoxy (Cbz) Protecting Group from
Terminal Amine Group of Linker
##STR00044##
[0139] The compound obtained in Example <1-1> (100 mg, 0.025
mmol) was dissolved in a mixture of methanol and dichloromethane
(9:1) (3 ml), and Pd/C (10 wt %, 50 mg) was added thereto. The
mixture thus obtained was stirred at room temperature under H.sub.2
gas (1 atm) for one day, and filtered through celite to remove
Pd/C. The filtrate thus obtained was concentrated under a reduced
pressure to obtain the title compound as a white sticky solid (95
mg).
[0140] .sup.1H NMR (CDCl.sub.3): .delta. 1.21-2.24 (m, 248H),
2.88-3.70 (m, 50H), 5.23-5.27 (m, 6H), 8.47 (s, 10H), 11.43 (m,
10H).
<1-3> Introduction of Fluorescence Probe (FITC)
##STR00045##
[0142] The compound obtained in Example <1-2> (85 mg, 0.022
mmol) was dissolved in a mixture of tetrahydrofuran and ethanol
(3:2) (0.675 ml), fluorescein-5-isocyanate (10.3 mg, 0.026 mmol)
and triethylamine (9.1 .mu.l, 0.067 mmol) were added thereto, and
the mixture thus obtained was stirred at room temperature for one
day.
[0143] After the completion of the reaction, the reaction mixture
was concentrated under a reduced pressure, and then purified by
column chromatography (dichloromethane:methanol=10:1) to obtain a
dark yellow sticky compound (100 mg).
[0144] .sup.1H NMR (CDCl.sub.3): .delta. 1.14-1.89 (m, 236H),
2.14-3.05 (m, 42H), 3.39-3.42 (m, 20H), 5.22 (s, 6H), 6.58-6.94 (m,
6H), 7.80-8.05 (m, 3H), 8.45 (s, 10H), 11.38 (s, 10H).
<1-4> Removal of N-Boc Protecting Group from
N,N'-di-Butyloxycarbonyl (Boc)-Guanidine Group
##STR00046##
[0145] The compound obtained in Example <1-3> (100 mg) was
dissolved in ethyl acetate (1 ml), HCl gas-saturated ethyl acetate
(2 ml) was added dropwise thereto, and the mixture thus obtained
stirred at room temperature for one day.
[0146] After the completion of the reaction, the reaction mixture
was concentrated under a reduced pressure to remove the solvent,
and washed with ethyl acetate to remove non-polar impurities.
Thereafter, purification by MPLC chromatography (0.1%
trifluoroacetic acid-containing water:acetonitrile=1:1 to 1:2) was
performed to obtain a light green compound (35 mg).
[0147] .sup.1H NMR (CD.sub.3OD): .delta. 1.28-2.40 (m, 72H),
3.30-3.42 (m, 48H), 5.48 (s, 6H), 7.14-8.56 (m, 9H).
Example 2
Preparation of Scyllo-Inositol Derivative Having Five Guanidine
Groups
[0148] <2-1> Introduction of Side Chain to scyllo-Inositol by
Acylation
##STR00047##
[0149] The compound having five OH groups obtained in Preparative
Example <1-5> (85.8 mg, 0.2 mmol), a linear linker having a
single guanidine group (600 mg, 1.6 mmol),
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (383.42 mg, 2 mmol)
and 4-dimethylaminopyridine (61 mg, 0.5 mmol) were dissolved in
N,N-dimethylformamide (2 ml) and stirred at room temperature for
two days.
[0150] After the completion of the reaction, the reaction mixture
was washed several times with water and aqueous NaHCO.sub.3. The
organic layer thus obtained was dried over Na.sub.2SO.sub.4,
concentrated under a reduced pressure, and then purified by column
chromatography (dichloromethane:methanol=10:1) to obtain the title
compound having five acyl side chains, as a sticky solid (402
mg).
[0151] .sup.1H NMR (CDCl.sub.3, 500 MHz): .delta. 1.28-1.38 (m,
12H), 1.47-1.62 (m, 114H), 2.22-2.42 (m, 12H), 3.18-3.21 (m, 2H),
3.37-3.43 (m, 10H), 5.10 (s, 2H), 5.11-5.13 (m, 1H), 5.25 (s, 5H),
7.29-7.36 (m, 5H), 8.29-8.32 (t, J=5.0 Hz, 5H), 11.51 (s, 5H).
<2-2> Removal of Carbobenzoxy (Cbz) Protecting Group from
Terminal Amine Group of the Linker
##STR00048##
[0152] The compound obtained in Example <2-1> (100 mg, 0.025
mmol) was dissolved in a mixture of methanol and dichloromethane
(9:1) (3 ml) and Pd/C (10 wt %, 50 mg) was added thereto. The
mixture thus obtained was stirred at room temperature under H.sub.2
gas (1 atm) for one day and filtered through celite to remove Pd/C.
The filtrate thus obtained was concentrated under a reduced
pressure to obtain the title compound as a white sticky solid (87
mg).
[0153] .sup.1H NMR (CDCl.sub.3): .delta. 1.25-1.56 (m, 126H), 2.21
(t, J=6.8 Hz, 12H), 2.92-2.99 (m, 2H), 3.36-3.48 (m, 10H), 5.23 (s,
6H), 8.29-8.36 (m, 5H), 11.5 (m, 5H).
<2-3> Introduction of Fluorescence Probe (FITC)
##STR00049##
[0155] The compound obtained in Example <2-2> (85 mg, 0.04
mmol) was dissolved in a mixture of tetrahydrofuran and ethanol
(3:2) (0.675 ml), and fluorescein-5-isocyanate (19.1 mg, 0.049
mmol) and triethylamine (17.1 .mu.l, 0.123 mmol) were added
thereto, and stirred at room temperature for one day.
[0156] After the completion of the reaction, the reaction mixture
was concentrated under a reduced pressure, and then purified by
column chromatography (dichloromethane:methanol=10:1) to obtain a
dark yellow sticky compound (104 mg).
[0157] .sup.1H NMR (CDCl.sub.3): .delta. 1.23-1.49 (m, 126H),
2.11-2.25 (m, 12H), 3.05-3.75 (m, 12H), 5.24 (s, 6H), 6.55-7.11 (m,
6H), 7.95-8.17 (m, 3H), 8.35-8.41 (m, 5H), 11.5 (m, 5H).
<2-4> Removal of N-Boc Protecting Group from
N,N'-di-Butyloxycarbonyl (Boc)-Guanidine Group
##STR00050##
[0158] The compound obtained in Example <2-3> (104 mg) was
dissolved in ethyl acetate (1 ml), HCl gas-saturated ethyl acetate
(2 ml) was added dropwise thereto, and the mixture thus obtained
was stirred at room temperature for one day.
[0159] After the completion of the reaction, the reaction mixture
was concentrated under a reduced pressure to remove the solvent,
and washed with ethyl acetate to remove non-polar impurities.
Subsequently, purification by MPLC chromatography (0.1%
trifluoroacetic acid-containing water:acetonitrile=1:1.about.1:2)
was performed to obtain a light green compound (42 mg).
[0160] .sup.1H NMR (CD.sub.3OD): .delta. 1.23-1.38 (m, 12H),
1.58-1.72 (m, 24H), 2.23-2.32 (m, 12H), 3.06-3.15 (m, 10H), 3.61
(m, 1H), 3.94 (m, 1H), 5.43 (s, 6H), 6.54-6.74 (m, 6H), 7.15-7.18
(m, 1H), 7.70-7.73 (m, 1H), 8.01-8.25 (m, 1H).
Example 3
Preparation of Scyllo-Inositol Derivative Having Eight Guanidine
Groups and FITC
[0161] <3-1> Introduction of Side Chain to scyllo-Inositol by
Acylation
##STR00051##
[0162] The compound obtained in Preparative Example <2-3>
(300 mg, 0.59 mmol), a branched linker having two guanidine groups
(Korean Patent No. 699279; 2.61 g, 3.57 mmol),
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (684 mg, 3.57 mmol)
and 4-dimethylaminopyridine (87 mg, 0.71 mmol) were dissolved in
N,N-dimethylformamide (3 ml), and stirred at room temperature for
three days.
[0163] After the completion of the reaction, the reaction mixture
was washed several times with water and aqueous NaHCO.sub.3. The
organic layer thus obtained was dried over Na.sub.2SO.sub.4,
concentrated under a reduced pressure, and then purified by column
chromatography (dichloromethane:methanol=10:1) to obtain the title
compound as a sticky solid (1.85 g).
[0164] .sup.1H NMR (CDCl.sub.3): .delta. 0.91-1.69 (m, 190H),
2.10-2.46 (m, 32H), 2.90-2.95 (m, 4H), 3.38-4.22 (m, 16H),
4.88-5.51 (m, 6H), 5.07 (s, 2H), 7.30-7.36 (m, 5H), 7.39-7.45 (m,
2H), 7.54-7.60 (m, 1H), 7.91-7.95 (m, 2H), 8.49 (brs, 8H), 11.49
(brs, 8H).
<3-2> Removal of Carbobenzoxy (Cbz) Protecting Group from
Terminal Amine Group of the Linker
##STR00052##
[0165] The compound obtained in Example <3-1> (1 g, 0.296
mmol) was dissolved in a mixture of methanol and dichloromethane
(9:1) (25 ml) and Pd/C (10 wt %, 500 mg) was added thereto. The
mixture thus obtained was stirred at room temperature under H.sub.2
gas (4 atm) for one day, and filtered through celite to remove
Pd/C. The filtrate thus obtained was concentrated under a reduced
pressure to obtain the title compound as a white sticky solid (960
mg).
[0166] .sup.1H NMR (CDCl.sub.3): .delta. 1.06-2.26 (m, 202H),
2.88-3.70 (m, 40H), 5.31-5.56 (m, 6H), 7.45-7.50 (m, 2H), 7.61-7.64
(m, 1H), 7.93-7.95 (m, 2H), 8.47 (s, 8H), 11.43 (m, 8H).
<3-3> Introduction of Fluorescence Probe (FITC)
##STR00053##
[0168] The compound obtained in Example <3-2> (879 mg, 0.27
mmol) was dissolved in a mixture of tetrahydrofuran and ethanol
(3:1) (5 ml), fluorescein-5-isocyanate (127 mg, 0.325 mmol),
triethylamine (113 .mu.l, 0.81 mmol) were added thereto, and the
mixture thus obtained was stirred at room temperature for one
day.
[0169] After the completion of the reaction, the reaction mixture
was concentrated under a reduced pressure, and then purified by
column chromatography (dichloromethane:methanol=10:1) to obtain a
dark yellow sticky compound (888 mg).
[0170] .sup.1H NMR (CDCl.sub.3): .delta. 1.02-2.88 (m, 226H),
3.32-3.51 (m, 16H), 3.62-4.12 (m, 2H), 5.05-5.61 (m, 6H), 6.56-7.90
(m, 14H), 8.50 (brs, 8H), 11.44 (brs, 8H).
<3-4> Removal of N-Boc Protecting Group from
N,N'-di-Butyloxycarbonyl (Boc)-Guanidine Group
##STR00054##
[0171] HCl gas-saturated ethyl acetate (10 ml) was added dropwise
to the compound obtained in Example <3-3> (888 mg), and the
solution thus obtained was stirred at room temperature for one
day.
[0172] After the completion of the reaction, the reaction mixture
was concentrated under a reduced pressure, and washed with ethyl
acetate to remove non-polar impurities. Subsequently, purification
by MPLC chromatography (0.1% trifluoroacetic acid-containing
water:acetonitrile=1:1.about.1:2) was performed to obtain a light
green compound (412 mg).
[0173] .sup.1H NMR (CD.sub.3OD): .delta. 1.15-2.38 (m, 80H),
2.88-3.66 (m, 16H), 5.56-5.72 (m, 6H), 7.20-7.70 (m, 10H),
7.91-8.59 (m, 4H).
Example 4
Preparation of Scyllo-Inositol Derivative Having Eight Guanidine
Groups
[0174] <4-1> Introduction of Side Chain to scyllo-Inositol by
Acylation
##STR00055##
[0175] The compound obtained in Preparative Example <3-2> (30
mg, 0.0772 mmol), a branched linker having two guanidine groups
(Korean Patent No. 699279; 451 mg, 0.618 mmol),
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (133 mg, 0.695 mmol),
and 4-dimethylaminopyridine (19 mg, 0.154 mmol) were dissolved in
dichloromethane (3 ml), and stirred at room temperature for three
days.
[0176] After the completion of the reaction, the reaction mixture
was washed several times with water and aqueous NaHCO.sub.3. The
organic layer thus obtained was dried over Na.sub.2SO.sub.4,
concentrated under a reduced pressure, and then purified by column
chromatography (dichloromethane:methanol=10:1) to obtain the title
compound having four acyl side chains as a sticky solid (180
mg).
[0177] .sup.1H NMR (CDCl.sub.3): .delta. 1.33-1.69 (m, 184H),
2.11-2.13 (m, 8H), 2.22-2.47 (m, 24H), 3.41-3.44 (m, 16H),
5.37-5.39 (m, 2H), 5.47-5.52 (m, 2H), 5.64-5.66 (m, 2H), 7.33-7.36
(m, 4H), 7.47-7.52 (m, 2H), 7.83-7.84 (m, 4H), 8.24-8.46 (m, 8H),
11.47 (brs, 8H);
[0178] MS (MALDI-TOF): m/z 3233.606 (M+).
<4-2> Removal of N-Boc Protecting Group from
N,N'-di-Butyloxycarbonyl (Boc)-Guanidine Group
##STR00056##
[0179] The compound obtained in Example <4-1> (80 mg, 0.0247
mmol) was dissolved in ethyl acetate (1 ml), HCl gas-saturated
ethyl acetate (2 ml) was added dropwise thereto, and the mixture
thus obtained was stirred at room temperature for one day.
[0180] After the completion of the reaction, the reaction mixture
was concentrated under a reduced pressure to remove the solvent,
and washed with ethyl acetate to remove non-polar impurities.
Subsequently, purification by MPLC chromatography (0.1%
trifluoroacetic acid-containing water:acetonitrile=70:30) was
performed to obtain a light green compound (45 mg).
[0181] .sup.1H NMR (CD.sub.3OD): .delta. 1.25-2.45 (m, 48H),
3.07-3.49 (m, 40H), 5.70-5.72 (m, 2H), 5.80-5.87 (m, 2H), 5.96-5.98
(m, 2H), 7.49 (t, 4H, J=4.5 Hz), 7.65 (t, 2H, J=4.35 Hz), 7.92 (d,
4H, J=4.5 Hz);
[0182] MS (MALDI-TOF): m/z 1633.085 (M+).
Example 5
Preparation of Trehalose Derivative Having Six Guanidine Groups and
FITC
<5-1> Introduction of Side Chain to Trehalose by
Acylation
##STR00057##
[0184] The compound obtained in Preparative Example <4-1>
(2.19 g, 2.65 mmol), N-butyloxycarbonyl (Boc)-6-aminohexanoic acid
(5.52 g, 23.87 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(4.58 g, 23.86 mmol), and 4-dimethylaminopyridine (0.97 g, 7.96
mmol) were dissolved in N,N-dimethylformamide (60 ml), and stirred
at room temperature for 48 hours.
[0185] After the completion of the reaction, the reaction mixture
was subjected to azeotropic distillation along with toluene at
60.degree. C., diluted with aqueous lithium chloride (5%),
extracted three times with ethyl acetate, and the extract was
washed with saturated aqueous NaHCO.sub.3. The organic layer thus
obtained was dried over Na.sub.2SO.sub.4, concentrated under a
reduced pressure, and then purified by column chromatography (ethyl
acetate:hexane=1:1) to obtain the title compound as a yellow liquid
(3.05 g).
[0186] .sup.1H NMR (CDCl.sub.3) .delta. 1.08-1.75 (m, 90H, CH.sub.3
and NCH.sub.2CH.sub.2CH.sub.2CH.sub.2), 1.85-2.30 (m, 12H,
CO--CH.sub.2), 2.97-3.03 (m, 4H), 3.05-3.14 (m, 12H, NCH.sub.2),
4.08-4.16 (m, 2H), 4.73-4.89 (m, 6H, NH), 5.10 (app. t, 2H, J=10.1
Hz), 5.23 (dd, 2H, J=10.2 and 3.8 Hz), 5.43-5.50 (m, 4H), 7.20-7.42
(m, 30H, trityl protons).
<5-2> Removal of N-Boc Protecting Group from Terminal Amino
Group of the Side Chain
##STR00058##
[0187] The compound obtained in Example <5-1> (625 mg, 0.30
.mu.mol) was dissolved in HCl gas-saturated ethyl acetate (17 ml)
and stirred at room temperature for two days.
[0188] After the completion of the reaction, the reaction mixture
was concentrated under a reduced pressure to remove the solvent,
and washed with ethyl acetate to remove non-polar impurities to
obtain a white sticky compound (368 mg).
[0189] .sup.1H NMR (MeOD) .delta. 1.39-1.72 (m, 36H,
NCH.sub.2CH.sub.2CH.sub.2CH.sub.2), 2.29-2.49 (m, 12H,
CO--CH.sub.2), 2.91-3.11 (m, 12H, NCH.sub.2), 3.56-3.69 (m, 3H),
3.80-3.95 (m, 2H), 4.16-4.42 (m, 2H), 4.90-5.10 (m, 3H), 5.24-5.54
(m, 4H).
<5-3> Conversion of Amino Group into N,N'-di-Boc-Guanidine
Group
##STR00059##
[0190] The compound obtained in Example <5-2> (358 mg, 0.29
mmol) was dissolved in a mixture of 1,4-dioxane and water (5:1, 5
ml), triethylamine (0.8 ml, 4.22 mmol) and
N,N-di-Boc-N'-trifluoromethanesulfonylguanidine (1.63 g, 4.16 mmol)
were added dropwise thereto, and the mixture was stirred at room
temperature for three days.
[0191] After the completion of the reaction, the reaction mixture
was concentrated under a reduced pressure, diluted with ethyl
acetate, and the extract was washed with water and saturated
aqueous NaCl. The product thus obtained was dried over
Na.sub.2SO.sub.4, concentrated under a reduced pressure, and then
purified by column chromatography (ethyl acetate:hexane=2:3) to
obtain the title compound as a white solid (564 mg).
[0192] .sup.1H NMR (CDCl.sub.3) .delta. 1.36-1.75 (m, 144H,
CH.sub.3 and NCH.sub.2CH.sub.2CH.sub.2CH.sub.2), 2.25-2.38 (m, 12H,
CO--CH.sub.2), 3.30-3.45 (m, 12H, NCH.sub.2), 3.62-3.70 (m, 4H),
4.25 (dd, 2H, J=11.8 and 5.0 Hz), 4.38-4.44 (m, 2H), 4.88 (dd, 2H,
J=10.1 and 3.7 Hz), 5.31 (d, 2H, J=3.7 Hz, anomeric protons), 5.39
(app. t, 2H, J=9.9 Hz), 8.31 (br s, 6H), 11.49 (br s, 6H).
<5-4> Introduction of Carbobenzoxy (Cbz)-Protected Linker
##STR00060##
[0194] The compound obtained in Example <5-3> (397 mg, 0.16
mmol), 6-benzyloxycarbonylaminohexanoic acid (512 mg, 1.92 mmol),
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (370 mg, 1.93 mmol)
and 4-dimethylaminopyridine (47 mg, 0.38 mmol) were dissolved in
N,N-dimethylformamide (10 ml) and stirred at room temperature for
48 hours.
[0195] After the completion of the reaction, the reaction mixture
was diluted with aqueous lithium chloride (5%), and extracted three
times with ethyl acetate. The product thus obtained was dried over
Na.sub.2SO.sub.4, concentrated under a reduced pressure, and then
purified by column chromatography (dichloromethane:methanol=50:1)
to obtain a colorless sticky compound (313 mg).
[0196] .sup.1H NMR (CDCl.sub.3) .delta. 1.23-1.73 (m, 156H,
CH.sub.3 and NCH.sub.2CH.sub.2CH.sub.2CH.sub.2), 2.21-2.39 (m, 16H,
CO--CH.sub.2), 3.10-3.24 (m, 4H, CH.sub.2NH--CO), 3.35-3.45 (m,
12H, CH.sub.2NHCN), 3.90-4.05 (m, 4H), 4.17-4.29 (m, 2H), 4.97-5.18
(m, 8H), 5.24-5.37 (m, 4H), 5.47 (app. t, 2H, J=9.7 Hz), 7.28-7.39
(m, 10H, benzene protons), 8.30 (br s, 6H), 11.50 (br s, 6H).
<5-5> Removal of Carbobenzoxy (Cbz) Protecting Group from
Terminal Amine Group of the Linker
##STR00061##
[0197] The compound obtained in Example <5-4> (159 mg, 54
.mu.mol) was dissolved in a mixture of methanol and dichloromethane
(9:1) (19 ml), Pd/C (10 wt %, 120 mg) was added thereto. The
mixture thus obtained was stirred at room temperature under H.sub.2
gas (50 psi) for 16 hours, and filtered through celite to remove
Pd/C. The filtrate thus obtained was concentrated under a reduced
pressure to obtain the title compound as a white sticky solid (130
mg).
[0198] .sup.1H NMR (MeOD) .delta. 1.39-1.78 (m, 156H, CH.sub.3 and
NCH.sub.2CH.sub.2CH.sub.2CH.sub.2), 2.25-2.45 (m, 16H,
CO--CH.sub.2), 2.85-2.99 (m, 4H, CH.sub.2NH.sub.2), 3.32-3.39 (m,
12H, CH.sub.2NH), 3.94-4.11 (m, 2H), 4.09-4.28 (m, 4H), 5.05-5.14
(m, 4H), 5.36 (d, 2H, J=3.6 Hz, anomeric protons), 5.53 (app. t,
2H, J=9.7 Hz).
<5-6> Introduction of Fluorescence Probe (FITC)
##STR00062##
[0200] The compound obtained in Example <5-5> (129 mg, 47.83
.mu.mol) was dissolved in a mixture of tetrahydrofuran, ethanol and
methanol (6:4:1) (4 ml), fluorescein-5-isocyanate (55 mg, 0.13
mmol) and triethylamine (65 .mu.l, 0.34 mmol) were added thereto,
and the mixture thus obtained was stirred at room temperature for
one day.
[0201] After the completion of the reaction, the reaction mixture
was concentrated under a reduced pressure, and then purified by
column chromatography (dichloromethane:methanol=10:1) to obtain a
dark yellow sticky compound (95 mg).
[0202] .sup.1H NMR (CDCl.sub.3) .delta. 1.23-1.66 (m, 156H,
CH.sub.3 and NCH.sub.2CH.sub.2CH.sub.2CH.sub.2), 2.11-2.30 (m, 12H,
CO--CH.sub.2), 3.30-3.48 (m, 12H, CH.sub.2NHC.dbd.N), 3.51-3.69 (m,
4H, CH.sub.2NHC.dbd.S), 3.91-4.01 (m, 4H), 4.10-4.20 (m, 2H),
4.92-5.11 (m, 4H), 5.21-5.31 (m, 2H), 5.49 (app. t, 2H, J=9.3 Hz),
6.57-6.79 (m, 10H), 7.10-7.18 (m, 2H), 7.91-8.11 (m, 2H), 8.31-8.49
(m, 4H), 9.04 (br s, 2H), 11.49 (br s, 6H).
<5-7> Removal of N-Boc Protecting Group from
N,N'-di-Boc-Guanidine Group
##STR00063##
[0203] The compound (92 mg, 26.52 .mu.mol) obtained in Example
<5-6> was dissolved in HCl gas-saturated ethyl acetate (3.5
ml), and stirred at room temperature for two days.
[0204] After the completion of the reaction, the reaction mixture
was concentrated under a reduced pressure to remove the solvent,
washed with ethyl acetate to remove non-polar impurities, and then
purified by MPLC to obtain a yellow sticky compound (42 mg). For
analysis, a small amount of the compound was further purified by
prep-HPLC (water:acetonitrile=75:25, 220 nm).
[0205] .sup.1H NMR (MeOD) .delta. 1.30-1.75 (m, 48H,
NCH.sub.2CH.sub.2CH.sub.2CH.sub.2), 2.24-2.49 (m, 16H,
CO--CH.sub.2), 3.11-3.28 (m, 12H, CH.sub.2NHC.dbd.N), 3.59-3.66 (m,
4H, CH.sub.2NHC.dbd.S), 4.00-4.09 (m, 2H), 4.10-4.28 (m, 4H),
5.04-5.18 (m, 4H), 5.35 (d, 2H, J=3.4 Hz, anomeric protons), 5.54
(app. t, 2H, J=9.8 Hz), 6.55-6.73 (m, 12H), 7.15-7.18 (m, 2H),
7.73-7.80 (m, 2H), 8.32 (br s, 2H).
Example 6
Preparation of Trehalose Derivative Having Six Guanidine Groups
##STR00064##
[0207] The compound obtained in Example <5-3> (26 mg, 10.59
.mu.mol) was dissolved in HCl gas-saturated ethyl acetate (2 ml),
and stirred at room temperature for two days.
[0208] After the completion of the reaction, the reaction mixture
was concentrated under a reduced pressure to remove the solvent,
washed with ethyl acetate to remove non-polar impurities, and then
purified by MPLC, to obtain a white sticky compound (13.7 mg). For
analysis, a small amount of the compound was further purified by
prep-HPLC (water:acetonitrile=75:25, 220 nm).
[0209] .sup.1H NMR (D.sub.2O) .delta. 1.31-1.73 (m, 36H,
NCH.sub.2CH.sub.2CH.sub.2CH.sub.2), 2.39-2.62 (m, 12H,
CO--CH.sub.2), 3.11-3.28 (m, 12H, NCH.sub.2), 3.81 (app. t, 1H,
J=9.12 Hz), 3.95-4.11 (m, 2H), 4.15-4.52 (m, 5H), 4.89-5.12 (m,
2H), 5.19-5.55 (m, 4H).
Example 7
Preparation of Trehalose Derivative Having Eight Guanidine
Groups
<7-1> Introduction of Side Chain to Trehalose by
Acylation
##STR00065##
[0211] Trehalose hydrate (0.87 g, 2.30 mmol), N-Boc-6-aminohexanoic
acid (6.37 g, 27.55 mmol),
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (5.81 g, 30.30 mmol)
and 4-dimethylaminopyridine (0.84 g, 6.89 mmol) were dissolved in
N,N-dimethylformamide (24 ml) and stirred at room temperature for
48 hours.
[0212] After the completion of the reaction, the reaction mixture
was subjected to azeotropic distillation along with toluene at
60.degree. C., diluted with ethyl acetate, and the extract was
washed several times with saturated aqueous NaHCO.sub.3 and water.
The organic layer thus obtained was dried over Na.sub.2SO.sub.4,
concentrated under a reduced pressure, and then purified by column
chromatography (ethyl acetate:hexane=1:1) to obtain the title
compound as a white solid (4.03 g).
[0213] .sup.1H NMR (CDCl.sub.3) .delta. 1.24-1.66 (m, 120H,
CH.sub.3 and NCH.sub.2CH.sub.2CH.sub.2CH.sub.2), 2.20-2.37 (m, 16H,
CO--CH.sub.2), 3.08-3.21 (m, 16H, NCH.sub.2), 3.83-4.03 (m, 4H),
4.20-4.25 (m, 2H), 4.65-5.00 (m, 7H, NH), 5.01-5.09 (m, 4H), 5.29
(d, 2H, J=3.7 Hz, anomeric protons), 5.48 (app. t, 2H, J=9.7
Hz).
<7-2> Removal of N-Boc Protecting Group from Terminal Amino
Group of the Side Chain
##STR00066##
[0214] The compound (1.94 g, 0.95 mmol) obtained in Example
<7-1> was dissolved in a mixture of trifluoroacetic acid
(TFA) and dichloromethane (1:1, 20 ml), and stirred at room
temperature for 6 hours.
[0215] After the completion of the reaction, the reaction mixture
was concentrated under a reduced pressure, and the extract was
washed with a mixture of diethylether and methanol (20:1). The
product thus obtained was dissolved in water and lyophilized to
obtain a brown sticky compound (3.17 g).
[0216] .sup.1H NMR (MeOD) .delta. 1.25-1.39 (m, 16H,
NCH.sub.2CH.sub.2CH.sub.2), 1.42-1.66 (m, 32H,
NCH.sub.2CH.sub.2CH.sub.2CH.sub.2), 2.10-2.35 (m, 16H,
CO--CH.sub.2), 2.71-2.89 (m, 16H, NCH.sub.2), 3.81-3.93 (m, 2H),
4.00-4.15 (m, 4H), 4.91-5.03 (m, 4H), 5.23 (d, 2H, J=3.6 Hz,
anomeric protons), 5.35 (app. t, 2H, J=9.7 Hz).
<7-3> Conversion of Amino Group into N,N'-di-Boc-Guanidine
Group
##STR00067##
[0217] The compound (86 mg, 39.59 .mu.mol) obtained in Example
<7-2> was dissolved in a mixture of 1,4-dioxane and water
(5:1, 2 ml), triethylamine (0.5 ml, 2.64 mmol),
N,N-di-Boc-N'-trifluoromethanesulfonylguanidine (187 mg, 0.48 mmol)
were added dropwise thereto, and the mixture was stirred at room
temperature for three days.
[0218] After the completion of the reaction, the reaction mixture
was concentrated under a reduced pressure, diluted with ethyl
acetate, and washed with water and saturated aqueous NaCl. The
product thus obtained was dried over Na.sub.2SO.sub.4, concentrated
under a reduced pressure, and then purified by column
chromatography (ethyl acetate:hexane=1:2) to obtain the title
compound as a white solid (81 mg).
[0219] .sup.1H NMR (CDCl.sub.3) .delta. 1.28-1.71 (m, 192H,
CH.sub.3 and NCH.sub.2CH.sub.2CH.sub.2CH.sub.2), 2.20-2.39 (m, 16H,
CO--CH.sub.2), 3.33-3.46 (m, 16H, NCH.sub.2), 3.94-4.01 (m, 4H),
4.16-4.24 (m, 2H), 4.97-5.10 (m, 4H), 5.27 (d, 2H, J=3.7 Hz,
anomeric protons), 5.45 (app. t, 2H, J=9.7 Hz), 8.31 (br s, 8H),
11.50 (br s, 8H).
<7-4> Conversion of Amino Group into N,N'-di-Boc-Guanidine
Group
##STR00068##
[0220] The compound obtained in Example <7-3> (20 mg, 6.28
.mu.mol) was dissolved in HCl gas-saturated ethyl acetate (3 ml),
and stirred at room temperature for two days.
[0221] After the completion of the reaction, the reaction mixture
was concentrated under a reduced pressure to remove the solvent,
washed with ethyl acetate to remove non-polar impurities, and then
purified by MPLC to obtain a white sticky compound (13.7 mg). For
analysis, a small amount of the compound was further purified by
prep-HPLC (water:acetonitrile=75:25, 220 nm).
[0222] .sup.1H NMR (D.sub.2O) .delta. 1.29-1.73 (m, 48H,
NCH.sub.2CH.sub.2CH.sub.2CH.sub.2), 2.31-2.49 (m, 16H,
CO--CH.sub.2), 3.12-3.22 (m, 16H, NCH.sub.2), 4.02-4.13 (m, 2H),
4.21-4.40 (m, 4H), 5.15-5.27 (m, 4H), 5.45 (d, 2H, J=3.5 Hz,
anomeric protons), 5.58 (app. t, 2H, J=9.7 Hz).
Example 8
Preparation of Glucose Derivative Having Eight Guanidine Groups
<8-1> Introduction of Side Chain to Glucose by Acylation
##STR00069##
[0224] The compound obtained in Preparative Example <5-5> (50
mg, 0.125 mmol), a branched linker having two guanidine groups (757
mg, 1.000 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (192
mg, 1.000 mmol) and 4-dimethylaminopyridine (23 mg, 0.188 mmol)
were dissolved in N,N-dimethylformamide (2 ml) and stirred at room
temperature for three days.
[0225] After the completion of the reaction, the reaction mixture
was washed several times with water and aqueous NaHCO.sub.3. The
organic layer thus obtained was dried over Na.sub.2SO.sub.4,
concentrated under a reduced pressure, and then purified by column
chromatography (dichloromethane:methanol=10:1) to obtain the title
compound as a sticky solid (306 mg).
[0226] .sup.1H NMR (CDCl.sub.3): .delta. 1.27-1.75 (m, 206H),
2.04-2.53 (m, 32H), 3.15-3.18 (m, 2H), 3.46 (brs, 16H), 3.64-3.81
(m, 2H), 4.16 (brs, 2H), 4.46 (d, J=7.8 Hz, 1H), 4.94-5.24 (m, 5H),
7.34 (s, 5H), 8.52 (s, 8H), 11.49 (s, 8H).
<8-2> Removal of Carbobenzoxy (Cbz) Protecting Group from
Terminal Amine Group of the Linker
##STR00070##
[0227] The compound obtained in Example <8-1> (105 mg, 0.028
mmol) was dissolved in ethanol (8 ml), Pd/C (10 wt %, 100 mg) was
added thereto. This mixture thus obtained was stirred at room
temperature under H.sub.2 gas (1 atm) for one day, and filtered
through celite to remove Pd/C. The filtrate thus obtained was
concentrated under a reduced pressure to obtain the title compound
as a white sticky solid (78 mg).
[0228] .sup.1H NMR (CDCl.sub.3): .delta. 1.25-1.86 (m, 206H),
2.18-2.26 (m, 32H), 3.15-3.18 (m, 2H), 3.46 (brs, 18H), 3.63-4.16
(m, 6H), 4.11-4.16 (m, 3H), 4.52 (brs, 1H), 4.94-5.09 (m, 2H), 5.21
(t, J=9.0 Hz, 1H), 8.60 (s, 8H), 11.41 (s, 8H).
<8-3> Introduction of Fluorescence Probe (FITC)
##STR00071##
[0230] The compound obtained in Example <8-2> (75 mg, 0.020
mmol) was dissolved in a mixture of tetrahydrofuran and ethanol
(5:2) (3.5 ml), fluorescein-5-isocyanate (10 mg, 0.024 mmol) and
triethylamine (24 .mu.l, 0.163 mmol) were added thereto, and the
mixture was stirred at room temperature for one day.
[0231] After the completion of the reaction, the reaction mixture
was concentrated under a reduced pressure, and then purified by
column chromatography (dichloromethane:methanol=8:1) to obtain a
dark yellow sticky compound (29 mg).
[0232] .sup.1H NMR (CDCl.sub.3): .delta. 1.18-1.61 (m, 206H),
2.15-2.95 (m, 34H), 3.39 (brs, 18H), 3.56-3.80 (m, 6H), 4.00-4.16
(m, 3H), 4.43 (brs, 1H), 4.81-5.27 (m, 3H), 6.42-6.99 (m, 6H), 7.27
(brs, 1H), 7.74-7.78 (m, 1H), 7.89 (s, 1H), 8.43 (s, 8H), 11.37 (s,
8H).
<8-4> Removal of N-Boc Protecting Group from
N,N'-di-Boc-Guanidine Group
##STR00072##
[0233] The compound obtained in Example <8-3> (29 mg) was
dissolved in ethyl acetate (1 ml), HCl gas-saturated ethyl acetate
(3 ml) was added dropwise thereto, and the mixture was stirred at
room temperature for one day.
[0234] After the completion of the reaction, the reaction mixture
was concentrated under a reduced pressure to remove the solvent,
washed with ethyl acetate to remove non-polar impurities, and then
purified by MPLC chromatography (0.1% trifluoroacetic
acid-containing water:acetonitrile=1:1.about.1:2) to obtain a light
green compound (3.5 mg).
[0235] .sup.1H NMR (CD.sub.3OD): .delta. 1.36-1.71 (m, 62H),
2.01-2.42 (m, 34H), 3.29-3.42 (m, 18H), 3.52-3.89 (m, 6H),
4.11-4.30 (m, 3H), 4.59-4.71 (m, 1H), 4.81-5.26 (m, 3H), 6.52-6.68
(m, 6H), 7.15 (d, J=8.3 Hz, 1H), 7.67 (d, J=7.1 Hz, 1H), 8.37 (s,
1H).
Test Example 1
Measurement of Permeation into Mouse Brain and Distribution
Therein
[0236] The compounds obtained in Example 1 (94.5 mg/kg), Example 2
(59.5 mg/kg), Example 3 (84.5 mg/kg), Example 5 (90.9 mg/kg) and
Example 8 (83.1 mg/kg) were each dissolved in distilled water, and
intraperitoneally injected into eight-week old C57BL/6 mice
(available from Hyochang Science Inc., Korea). After 20 min, the
injected mice were treated with a solution (4% para-formaldehyde in
PBS (pH 7.4)), the brain of each mouse was collected and stored in
a 0.5 M sucrose PBS solution for one day.
[0237] In a cryostat, the brain was sliced into 15 .mu.m thick
slices, and each slice was placed on a slide glass, dried at
37.degree. C., washed with PBS, treated with 0.3% triton X-100 at
room temperature for 15 min, and then observed with a confocal
microscope. The results are shown in FIG. 1.
[0238] In FIG. 1, (A) shows the result obtained for the mice
injected with only distilled water as a control, (B) to (F) show
the results obtained from the mice injected with the compound of
Examples 1 to 5, respectively.
[0239] As shown in FIG. 1, the inositol, the trehalose and the
monosaccharide derivatives prepared according to the present
invention readily pass through the blood-brain barrier to permeate
into the brain tissue.
[0240] As described above, in accordance with the present
invention, the inositol and the trehalose derivatives have high BBB
permeability and can easily be delivered to the brain tissues, and
thus can be effectively used for the treatment of neurodegenerative
diseases such as Alzheimer's disease, Huntington's disease, and
other related diseases.
[0241] While the invention has been described with respect to the
above specific embodiments, it should be recognized that various
modifications and changes may be made to the invention by those
skilled in the art, which also fall within the scope of the
invention as defined by the appended claims.
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