U.S. patent application number 12/044666 was filed with the patent office on 2009-01-01 for b-cyclodextrin derivatives and their use against anthrax lethal toxin.
This patent application is currently assigned to Pinnacle Pharmaceuticals, Inc.. Invention is credited to Noureddine Fahmi, Sidney Hecht.
Application Number | 20090005343 12/044666 |
Document ID | / |
Family ID | 35782209 |
Filed Date | 2009-01-01 |
United States Patent
Application |
20090005343 |
Kind Code |
A1 |
Hecht; Sidney ; et
al. |
January 1, 2009 |
B-CYCLODEXTRIN DERIVATIVES AND THEIR USE AGAINST ANTHRAX LETHAL
TOXIN
Abstract
The invention provides low molecular weight compounds that block
the pore formed by protective antigen and inhibit anthrax toxin
action. Structures of the compounds are derivatives of
.beta.-cyclodextrin. Per-substituted alkylamino derivatives
displayed inhibitory activity, and they were protective against
anthrax lethal toxin action at low micromolar concentrations. Also,
the addition of one of the alkylamino derivatives to the bilayer
lipid membrane with multiple PA channels caused a significant
decrease in membrane conductance. Thus, the invention also provides
methods for protection against anthrax toxicity.
Inventors: |
Hecht; Sidney;
(Charlottesville, VA) ; Fahmi; Noureddine;
(Charlottesville, VA) |
Correspondence
Address: |
KEOWN & ZUCCHERO, LLP
500 WEST CUMMINGS PARK, SUITE 1200
WOBURN
MA
01801
US
|
Assignee: |
Pinnacle Pharmaceuticals,
Inc.
|
Family ID: |
35782209 |
Appl. No.: |
12/044666 |
Filed: |
March 7, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11045423 |
Jan 28, 2005 |
|
|
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12044666 |
|
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Current U.S.
Class: |
514/58 |
Current CPC
Class: |
C08B 37/0012 20130101;
A61P 39/02 20180101; A61P 31/04 20180101; A61K 31/724 20130101;
A61P 1/04 20180101; A61P 31/12 20180101 |
Class at
Publication: |
514/58 |
International
Class: |
A61K 31/724 20060101
A61K031/724; A61P 31/04 20060101 A61P031/04; A61P 31/12 20060101
A61P031/12 |
Goverment Interests
[0002] This work was supported by grant IR43AI052894-01 from the
National Institute of Allergy and Infectious Diseases. The
government has certain rights in this invention.
Claims
1. A method for inhibiting the growth of a bacterium or virus,
comprising contacting the bacterium or virus with a compound having
the formula ##STR00006## wherein R.sub.2 is H, OH, OAc, OMe, or
O(CH.sub.2CH.sub.2O).sub.n; R.sub.3 is H, OH, OAc, OMe,
OSO.sub.3Na, or NH.sub.2; and R.sub.6 is H, NH.sub.2,
SCH.sub.2CH.sub.2NH.sub.2, SCH.sub.2CH.sub.2CH.sub.2NH.sub.2,
SCH.sub.2CH.sub.2CH.sub.2CH.sub.2NH.sub.2, I, N.sub.3, SH, lower
alkyl, S-alkylguanidyl, O-alkylguanidyl, S-aminoalkyl,
O-aminoalkyl, aminoalkyl aralkyl, aryl, heterocyclic ring(s), or
OSO.sub.3Na.
2. The method according to claim 1, wherein R.sub.6 is NH.sub.2,
SCH.sub.2CH.sub.2NH.sub.2, SCH.sub.2CH.sub.2CH.sub.2NH.sub.2, or
SCH.sub.2CH.sub.2CH.sub.2CH.sub.2NH.sub.2.
3. A method for inhibiting the growth of a bacterium or virus,
comprising contacting the bacterium or virus with a compound shown
in FIG. 1.
4. A method for treating a bacterial or viral infection, comprising
administering to a mammal with a bacterial infection a compound
having the formula ##STR00007## wherein R.sub.2 is H, OH, OAc, OMe,
or O(CH.sub.2CH.sub.2O).sub.n; R.sub.3 is H, OH, OAc, OMe,
OSO.sub.3Na, or NH.sub.2; and R.sub.6 is H, NH.sub.2,
SCH.sub.2CH.sub.2NH.sub.2, SCH.sub.2CH.sub.2CH.sub.2NH.sub.2,
SCH.sub.2CH.sub.2CH.sub.2CH.sub.2NH.sub.2, I, N.sub.3, SH, lower
alkyl, S-alkylguanidyl, O-alkylguanidyl, S-aminoalkyl,
O-aminoalkyl, aminoalkyl, aralkyl, aryl, heterocyclic ring(s), or
OSO.sub.3Na.
5. The method according to claim 4, wherein R.sub.6 is NH.sub.2,
SCH.sub.2CH.sub.2NH.sub.2, SCH.sub.2CH.sub.2CH.sub.2NH.sub.2, or
SCH.sub.2CH.sub.2CH.sub.2CH.sub.2NH.sub.2.
6. A method for treating a bacterial or viral infection, comprising
administering to a mammal with a bacterial infection a compound
shown in FIG. 1.
7. A method for preventing a bacterial or viral infection,
comprising administering to a mammal susceptible to a bacterial
infection a compound having the formula ##STR00008## wherein
R.sub.2 is H, OH, OAc, OMe, or O(CH.sub.2CH.sub.2O).sub.n; R.sub.3
is H, OH, OAc, OMe, OSO.sub.3Na, or NH.sub.2; and R.sub.6 is H,
NH.sub.2, SCH.sub.2CH.sub.2NH.sub.2,
SCH.sub.2CH.sub.2CH.sub.2NH.sub.2,
SCH.sub.2CH.sub.2CH.sub.2CH.sub.2NH.sub.2, I, N.sub.3, SH, lower
alkyl, S-alkylguanidyl, O-alkylguanidyl, S-aminoalkyl,
O-aminoalkyl, aminoalkyl, aralkyl, aryl, heterocyclic ring(s), or
OSO.sub.3Na.
8. The method according to claim 7, wherein R.sub.6 is NH.sub.2,
SCH.sub.2CH.sub.2NH.sub.2, SCH.sub.2CH.sub.2CH.sub.2NH.sub.2, or
SCH.sub.2CH.sub.2CH.sub.2CH.sub.2NH.sub.2.
9. A method for preventing a bacterial or viral infection,
comprising administering to a mammal susceptible to a bacterial
infection a compound shown in FIG. 1.
Description
[0001] This application claims priority to U.S. patent application
Ser. No. 11/045,423, filed on Jan. 28, 2005, the contents of which
are incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The invention relates to protection against Anthrax-mediated
biotoxicity.
[0005] 2. Summary of the Related Art
[0006] Bacillus anthracis is one of the most dangerous potential
biological weapons. Currently, there is no effective treatment for
inhalational anthrax, beyond the administration of antibiotics
shortly after exposure. Time delay reduces the effectiveness of
antibiotic treatment. Dixon et al., Anthrax. N. Engl. J. Med.: 341,
815-826 (1999) teaches that major factors playing a role in anthrax
infection are the cytotoxic effect of anthrax toxin, and bacteremia
leading to oxygen and nutritional substance deprivation,
accumulation of various bacterial and host toxic products with
eventual organ failure and death.
[0007] Brossier et al., Toxicon. 39: 1747-1755 (2001) teaches that
the two anthrax toxins are formed by three different proteins:
protective antigen (PA) which either combines with lethal factor
(LF) to form lethal toxin (LeTx), or with edema factor (EF) to form
edema toxin (EdTx). LF and EF are enzymes targeting substrates
within the cytosol, and PA facilitates their transport across the
cell membrane forming a heptameric pore. PA assembles into a
ring-shaped heptamer with a negatively charged lumen and exposes a
hydrophobic surface for binding of LF and EF. Petosa et al., Nature
385: 833-838 (1997) teaches the three-dimensional structure of the
PA pore.
[0008] Karginov et al., FEMS Immun. Med. Microb. 40: 71-74 (2004)
teaches that treatment of Bacillus anthracis infected mice with a
combination of the antibiotic ciprofloxacin and partially purified
antibodies against anthrax protective antigen dramatically
increased survival rates in comparison with antibiotic treatment
alone.
[0009] Although promising, antibodies are less attractive as
potential drugs in comparison with low molecular weight compounds,
which offer potentially better penetration through membranes and
are not sensitive to proteases.
[0010] Therefore, there is a need for new safe and efficient
treatments to supplement to traditional antibiotic
intervention.
BRIEF SUMMARY OF THE INVENTION
[0011] The invention provides new safe and efficient treatments to
supplement to traditional antibiotic intervention.
[0012] In a first aspect, the invention provides low molecular
weight compounds designed to block the pore formed by PA, which can
inhibit anthrax toxin action. The high-affinity blockers of PA
according to the invention are derivatives of beta-cyclodextrin
(D-CD), which is a cyclic molecule comprising seven D-glucose units
and having sevenfold symmetry, like the PA pore.
[0013] In a second aspect, the invention provides methods for
inhibiting the toxic effects of Bacillus anthrasis. The methods
according to this aspect of the invention comprise contacting a
cell with a compound according to the first aspect of the
invention.
[0014] In a third aspect, the invention provides novel methods for
making certain derivatives of .beta.-CD.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows embodiments of compounds according to the
invention.
[0016] FIG. 2 shows protection of RAW 264.7 cells from LeTx-induced
cell death by .beta.-CD derivatives.
[0017] FIG. 3 shows inhibition of cytopathic effect of LeTx
expressed as percentage of the LeTx effect induced in cells not
treated with inhibitor.
[0018] FIG. 4 shows typical tracks of ion conductance for PA
channels reconstituted into planar lipid membranes. The downward
arrow indicates the addition of PrAmBC to the cis side of the
membrane.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0019] The invention relates to protection against Anthrax-mediated
biotoxicity. The invention provides new safe and efficient
treatments to supplement to traditional antibiotic intervention.
The references cited herein reflect the level of knowledge in the
field and are hereby incorporated by reference in their entirety.
In the case of a conflict between the teachings of the cited
references and the present specification, any such conflict shall
be resolved in favor of the latter.
[0020] In a first aspect, the invention provides low molecular
weight compounds designed to block the pore formed by PA, which can
inhibit anthrax toxin action. The high-affinity blockers of PA
according to the invention are preferably derivatives of
beta-cyclodextrin (.beta.-CD), which is a cyclic molecule
comprising seven D-glucose units and having sevenfold symmetry,
like the PA pore. Alternatively, molecules similar to .beta.-CD,
cyclic molecules having sevenfold symmetry, like the PA pore may be
used. The outside diameter of .beta.-CD--15.3 .ANG.--is comparable
with the diameter of the PA channel lumen, which is 20-35 .ANG.
according to X-ray analysis data, and about 12 .ANG. at its most
narrow point according to the measurement of current flow through
the channel. Alternative cyclic molecules should be of similar
size.
[0021] Preferred derivatives of .beta.-CD include
hepta-6-alkylamino derivatives of .beta.-cyclodextrin. .beta.-CD
substituted with positively charged groups of various sizes because
the lumen of the PA pore is mostly negatively charged. Also, the
positively charged groups might alter the local pH inside the
lumen, inhibiting the conformational change required for the
formation of the transmembrane channel.
[0022] Preferred compounds have the formula
##STR00001##
[0023] wherein R.sub.2 is H, OH, OAc, OMe, or O(CH.sub.2CH.sub.2O);
R.sub.3 is H, OH, OAc, OMe, OSO.sub.3Na, or NH.sub.2; and R.sub.6
is H, NH.sub.2, SCH.sub.2CH.sub.2NH.sub.2,
SCH.sub.2CH.sub.2CH.sub.2NH.sub.2,
SCH.sub.2CH.sub.2CH.sub.2CH.sub.2NH.sub.2, I, N.sub.3, SH, lower
alkyl, S-alkylguanidyl, O-alkylguanidyl, S-aminoalkyl,
O-aminoalkyl, aminoalkyl, aralkyl, aryl, heterocyclic ring(s), or
OSO.sub.3Na. Most preferably, R.sub.6 is H, NH.sub.2,
SCH.sub.2CH.sub.2NH.sub.2, SCH.sub.2CH.sub.2CH.sub.2NH.sub.2, or
SCH.sub.2CH.sub.2CH.sub.2CH.sub.2NH.sub.2.
[0024] For purposes of the invention, the term "lower alkyl" means
an alkyl group from 1 to 7 carbon atoms. The terms "alkyl" and
"aryl" include alkyl or aryl groups which may be substituted or
unsubstituted. Preferred substitutions include, without limitation,
substitution with nitrogen containing moieties, including amino
groups, which may be mono or disubstituted, preferably with alkyl
or aryl groups. Also, for purposes of the invention the term
"alkyl" includes chains of 1-7 atoms with one or more nitrogen
atoms and the remainder carbon atoms.
[0025] Particularly preferred derivatives of .beta.-CD are shown in
FIG. 1.
[0026] In a second aspect, the invention provides methods for
inhibiting the toxic effects of Bacillus anthrasis. The methods
according to this aspect of the invention comprise contacting a
cell with a compound according to the first aspect of the
invention. Preferably, the cell is in a mammal, most preferably in
a human.
[0027] The four hepta-6-alkylamino derivatives of
.beta.-cyclodextrin suggested by our structure-based evaluation
were synthesized. These and some other .beta.-cyclodextrin
derivatives synthesized by us or obtained elsewhere (FIG. 2) were
tested for their ability to inhibit cytotoxic effect of LeTx on
mouse macrophage-like cells RAW 264.7.
[0028] Surprisingly, only the alkylamino derivatives originally
suggested based on structure-based design, displayed inhibitory
activity, and they were protective against LeTx action at low
micromolar concentrations (FIG. 3). These experiments also showed
that the compounds were not toxic to RAW 264.7 cells up to 25 .mu.M
concentration, while their IC.sub.50 were as low as 4.4 .mu.M (FIG.
3). The rest of the compounds presented in FIG. 1 displayed no
inhibitory activity at concentrations 100 .mu.M and lower.
[0029] One of the alkylamino derivatives--PrAmBC--was tested for
the ability to block ion conductance through PA channels
reconstituted into planar bilayer lipid membranes. It was
demonstrated that the addition of PrAmBC to the bilayer lipid
membrane with multiple PA channels (about 60) caused a significant
step-like decrease in membrane conductance at 3 nM concentration of
the compound (FIG. 4).
[0030] Persubstituted .beta.-cyclodextrin derivatives can
potentially also be utilized for blocking of other toxins that form
heptameric transmembrane channels, such as staphylococcal
.alpha.-hemolysin. Derivatives of hexameric .alpha.-cyclodextrin
may also find utility against targets such as Helicobacter pylori
VacA toxin or hepatitis C virus p7 protein, which form hexameric
channels and are considered to be important virulence factors in
the pathogenesis of peptic ulcer disease and HCV infection,
respectively.
[0031] In a third aspect, the invention provides novel methods for
making certain derivatives of .beta.-CD. The introduction of an
alkylamino group at the primary position of .beta.-cyclodextrins
proved to be a challenge. The direct alkylation of
per-iodo-.beta.-cyclodextrin with an alcolate nucleophile (derived
from an azidoalkanol for example) would pose some problems since
the basic alcolate may induce elimination or intramolecular
substitutions. Nucleophilic displacement of iodide anions from
per-6-iodo-.beta.-cyclodextrins, employing poor nucleophiles or
elevated temperatures favors the intramolecular substitution
reaction, resulting in the formation of 3,6-anhydro-D-glucopyranose
residues within the structure of per-6-iodo-.beta.-cyclodextrin.
Taking advantage of the higher nucleophilicity of a sulfur atom
over an oxygen atom, we utilized the introduction of a sulfur atom
at the primary position of the .beta.-cyclodextrin followed by a
selective alkylation of the mercapto group, with a
halogenopropionitrile to provide directly a precursor of the target
compound 19. In this case, no supplementary protection and
deprotection steps are required.
[0032] Thus the invention provides an improved method for
synthesizing a substituted .beta.-cyclodextrin, wherein the
improvement comprises introducing a sulfur atom at the primary
position of the .beta.-cyclodextrin followed by a selective
alkylation of the mercapto group, with a halogenopropionitrile.
[0033] The following examples are intended to illustrate certain
particularly preferred embodiments of the invention and are not
intended to limit the scope of the invention.
Example 1
Synthesis of .beta.-cyclodextrin Derivatives
[0034] Reagents. .beta.-cyclodextrin derivatives 1-7 listed in
Table 1 were synthesized at Pinnacle Pharmaceuticals, Inc.
(Charlottesville, Va.). Compounds 12 and 13 were purchased from
Cytrea Ltd (Dublin, Ireland). Sulfo derivatives of
.beta.-cyclodextrin 8-11 were kindly provided by Dr. Gyula Vigh
(Texas A&M University, College Station, Tex.).
.beta.-cyclodextrin 14 was purchased from Sigma (St. Louis, Mo.).
Most chemical reagents were purchased from Aldrich Chemicals or
Fisher Scientific and used without further purification.
Acetonitrile and dichloromethane were distilled from CaH.sub.2. DMF
was distilled from CaH.sub.2 under diminished pressure.
Triethylamine was distilled from P205.
[0035] Analysis. .sup.1H NMR and .sup.13C NMR spectra were recorded
on a General Electric QE-300 or a Varian 300 spectrometer. Moisture
sensitive reactions were conducted under argon in oven-dried
glassware. Analytical thin-layer chromatography was performed on
Merk 60F.sub.254 precoated silica gel plates. Visualization was
performed by ultraviolet light and/or by staining with
phosphomolybdic acid or sulfuric acid. Flash chromatography was
performed using (40-60 .mu.m) silica gel.
Synthesis. Cyclodextrins 2, 3, 4 and 5 were prepared according to
standard procedures.
##STR00002##
(2-Phthalimidoethyl)isothiouronium hydrobromide (9). A suspension
of N-(2-bromoethyl)phthalimide (6) (3.0 g, 11.8 mmol) and thiourea
(1.82 g, 23.96 mmol) in absolute EtOH (5.7 mL) was stirred at
reflux for 18 h after which the product crystallized. After cooling
to room temperature the product was collected by filtration,
washing with small amounts of chilled absolute EtOH and dried under
vacuum. Compound 9 (4.0 g, quantitative yield) was obtained as
colorless crystals; .sup.1H NMR (DMSO-d.sub.6) .delta. 9.04 (brs,
2H), 7.91 (m, 2H); 7.11 (brs, 1H); 3.89 (t, J=5.8 Hz, 2H); 3.52 (t,
J=5.8 Hz, 2H). (3-Phthalimidopropyl)isothiouronium hydrobromide
(10). A suspension of N-(3-bromopropyl)phthalimide (7) (3.0 g, 11
mmol) and thiourea (1.7 g, 22.37 mmol) in absolute EtOH (5.3 mL)
was stirred at reflux for 18 h after which the product
crystallized. After cooling to room temperature the product was
collected by filtration, washing with small amounts of chilled
absolute EtOH (2.times.10 mL) and ether (10 mL) and dried under
vacuum. Compound 10 (3.95 g, quantitative yield) was obtained as
colorless crystals. .sup.1H NMR (DMSO-d.sub.6) .delta. 9.01 (brs,
2H), 7.90 (m, 2H); 3.50 (t, J=6.2 Hz, 2H); 3.20 (t, J=6.6 Hz, 2H);
1.75 (m, 2H). (4-Phthalimidobutyl)isothiouronium hydrobromide (11).
A suspension of N-(4-bromobutyl)phthalimide (8) (1.0 g, 3.5 mmol)
and thiourea (540 mg, 7.08 mmol) in absolute EtOH (1.7 mL) was
stirred at reflux for 18 h. The product did not crystallize as
expected. However, upon cooling to room temperature, the syrupy
mixture started crystallizing after a quick shaking and stirring.
Ether (4 mL) was added and the mixture stirred for 15 min. before
collecting the product by filtration, washing with small amounts of
chilled EtOH. Compound 11 (1.22 g, 96%) was obtained as colorless
solid; .sup.1H NMR (DMSO-d.sub.6) .delta. 9.02 (brs, 2H), 7.89 (m,
4H); 7.13 (brs, 1H); 3.60 (t, J=6.4 Hz, 2H); 3.17 (t, J=6.6 Hz,
2H); 1.67 (m, 4H).
##STR00003##
Heptakis (2,3-di-O-acetyl-6-deoxy-6-iodo)cyclomaltoheptaose (14).
(See Baer et al., Carbohydr. Res. 228: 307 1992). To a solution of
per-6-iodo-.beta.-cyclodextrin (2) (1.0 g, 0.52 mmol) in dry
pyridine (5 mL was added Ac.sub.2O (7.5 mL) and a catalytic amount
of DMAP (6.5 mg, 0.05 mmol). The mixture was stirred at room
temperature under argon for 48 h. The reaction was quenched by
addition of MeOH (15 mL) and the solvents evaporated under
diminished pressure. Coevaporation with small amounts of MeOH
(3.times.4 mL) and toluene (3.times.4 mL) gave a brown residue,
which was purified on a silica gel column (20.times.3 cm). Elution
with a gradient Hexane-EtOAc (1:1 to 1:4) gave compound 14 (1.06 g,
81%) as a colorless solid, which crystallized upon trituration with
diethyl ether; .sup.1H NMR (CDCl.sub.3) .delta. 5.33 (brt, J=8.4
Hz, 1H); 5.2 (d, J=3.6 Hz, 1H); 4.83 (dd, J=3.9, 9.9 Hz, 1H);
3.58-3.81 (complex m, 4H); 2.09 (s, 3H); 2.05 (s, 3H); mass
spectrum (MALDI), calcd. for C.sub.70H.sub.91I.sub.7NaO.sub.42 m/z
2514.8 found 2514.9 [M+Na] (100%). Heptakis
[2,3-di-O-acetyl-6-deoxy-6-(2-phthalimidoethyl)-thio]cyclomaltoh-
eptaose (15). To a solution of heptakis
(2,3-di-O-acetyl-6-deoxy-6-iodo)cyclomaltoheptaose (14) (0.5 g, 0.2
mmol) and (2-phthalimidoethyl)isothiouronium hydrobromide (9) (0.99
g, 3.0 mmol) in dry DMF (20 mL) was added Cs.sub.2CO.sub.3 (1.63 g,
5.0 mmol) and the mixture stirred at room temperature under argon
for 48 h. The mixture was poured into ice (40 g) and 0.5 N HCl (200
mL) was added. The aqueous layer was extracted with dichloromethane
(3.times.50 mL). The combined organic phases were washed
successively with 0.5 N HCl (200 mL) and brine (100 mL), dried
(MgSO.sub.4) and evaporated under diminished pressure. The residue
was purified on a silica gel column (21.times.3 cm) eluting with
EtOAc to give compound 15 (145 mg, 23%) as a colorless solid.
Another fraction (165 mg) was obtained in a slightly impure form.
.sup.1H NMR (CDCl.sub.3) .delta. 7.73 (m, 2H); 7.62 (m, 2H); 5.25
(t, 1H, J=8.7 Hz); 5.10 (brs, 1H); 4.80 (m, 1H); 4.15 (m, 1H); 3.87
(t, 1H, J=8.4 Hz); 3.63 (m, 2H); 3.03 (m, 2H); 2.64 (m, 2H); 2.05
(s, 3H); 2.01 (s, 3H). Heptakis
[2,3-di-O-acetyl-6-deoxy-6-(3-phthalimidopropyl)-thio]cyclomaltoheptaose
(16). To a solution of heptakis
(2,3-di-O-acetyl-6-deoxy-6-iodo)cyclomaltoheptaose (14) (250 mg,
0.1 mmol) and (3-phthalimidopropyl) isothiouronium hydrobromide
(10) (472 mg, 1.37 mmol) in dry DMF (10 mL) was added
Cs.sub.2CO.sub.3 (687 mg, 2.11 mmol) and the mixture stirred at
room temperature under argon for 68 h. The mixture was poured into
ice (50 g) and 0.5 N HCl (100 mL) was added. The aqueous layer was
extracted with dichloromethane (3.times.50 mL). The combined
organic phases were washed successively with 0.5 N HCl (100 mL) and
brine (100 mL), dried (MgSO.sub.4) and evaporated under diminished
pressure. The residue was purified on a silica gel column
(14.times.3 cm) eluting with EtOAc to give compound 15 (188 mg,
59%) as a colorless foam; .sup.1H-NMR (300 MHz) .delta. 7.70 (dd,
2H, J=3.0 Hz, J=5.5 Hz); 7.58 (dd, 2H, J=3.1 Hz, J=5.4 Hz); 5.23
(dd, 1H, J=8.3 Hz, J=9.6 Hz); 5.06 (d, 1H, J=3.8 Hz); 4.80 (dd, 1H,
J=3.8 Hz, J=9.7 Hz); 4.12 (m, 1H); 3.84 (t, 1H); 3.66 (t, 2H, J=6.9
Hz); 3.03 (m, 2H); 2.60 (m, 2H, J=5.9 Hz, J=12.9 Hz); 2.05 (s, 3H);
2.02 (s, 3H); 1.91 (m, 2H; mass spectrum (MALDI), calcd. for
C.sub.147H.sub.161N.sub.7NaO.sub.56S.sub.7 m/z 3166.8 found 3166.8
[M+Na] (40%), 3168.8 (100%) and 3167.8 (80%). Heptakis
[2,3-di-O-acetyl-6-deoxy-6-(4-phthalimidobutyl)-thio]cyclomaltoheptaose
(17). To a solution of heptakis
(2,3-di-O-acetyl-6-deoxy-6-iodo)cyclomaltoheptaose (14) (404 mg,
016 mmol) and (4-phthalimidobutyl) isothiouronium hydrobromide (11)
(870 mg, 2.42 mmol) in dry DMF (16 mL) was added Cs.sub.2CO.sub.3
(1.32 g, 4.04 mmol) and the mixture stirred at room temperature
under argon for 48 h. The mixture was poured into ice (50 g) and
0.5 N HCl (200 mL) was added. The aqueous layer was extracted with
dichloromethane (3.times.50 mL). The combined organic phases were
washed successively with 0.5 N HCl (100 mL) and brine (100 mL),
dried (MgSO.sub.4) and evaporated under diminished pressure. The
residue was purified on a silica gel column (18.times.3 cm) eluting
with EtOAc to give compound 17 (125 mg, 24%) as a colorless solid.
Another fraction (132 mg) was obtained in a slightly impure form.
.sup.1H-NMR (CDCl.sub.3) .delta. 7.74 (m, 2H); 7.64 (m, 2H); 5.26
(m, 1H); 5.12 (d, 1H, J=3.6 Hz); 4.80 (dd, 1H, J=3.7 Hz, J=9.8 Hz);
4.15 (m, 1H); 3.88 (m, 1H); 3.63 (m, 2H); 3.03 (m, 2H); 2.65 (m,
2H); 2.06 (s, 3H); 2.02 (s, 3H); 1.73 (m, 2H); 1.61 (m, 2H); mass
spectrum (MALDI), calcd. for
C.sub.154H.sub.175N.sub.7NaO.sub.56S.sub.7 m/z 3267.5 found 3267.3
[M+Na] (40%). Per-6-(2-aminoethylthio)-.beta.-cyclodextrin (18). A
mixture of compound 15 (100 mg, 31.92 .mu.mol) and hydrazine
monohydrate (1.55 mL, 31.92 mmol) in EtOH--H.sub.2O 1:1 (1.5 mL)
was stirred at 60.degree. C. for 18 h. The solvents were evaporated
under diminished pressure to give a solid, which was suspended in
1N HCl (5 mL) and stirred at rt for 8 h. The insoluble material was
filtered and the filtrate diluted with acetone (25 mL) until the
product precipitated. The supernatant was removed by centrifugation
and the product washed with acetone (4.times.25 mL) and dried under
vacuum. The product 18 (46 mg, 89%) was obtained as a colorless
solid. Mass spectrum (MALDI), calcd. for
C.sub.56H.sub.105N.sub.7O.sub.28S.sub.7 m/z 1548.9 found 1548.8 [M]
(100%). Per-6-(3-aminopropylthio)-.alpha.-cyclodextrin (19). A
mixture of compound 16 (100 mg, 31.38 .mu.mol) and hydrazine
monohydrate (1.54 mL, 31.78 mmol) in EtOH--H.sub.2O 1:1 (1.5 mL)
was stirred at 60.degree. C. for 16 h. The solvents were evaporated
under diminished pressure to give a solid, which was suspended in
1N HCl (5 mL) and stirred at rt for 4 h. The insoluble material was
filtered and the filtrate diluted with acetone (25 mL) until the
product precipitated. The supernatant was removed by centrifugation
and the product washed with acetone (4.times.25 mL) and dried under
vacuum. Compound 19 (53 mg, 85% yield) was obtained as a colorless
solid. .sup.13C-NMR (DMSO-d.sub.6) .delta. 102.09, 84.52, 72.48,
72.23, 71.41, 37.79, 33.03, 29.71, 26.85; mass spectrum (MALDI),
calcd. for C.sub.63H.sub.119N.sub.7NaO.sub.28S.sub.7 m/z 1668.60
found 1668.82 [M+Na] (100%).
Per-6-(4-aminobutylthio)-.beta.-cyclodextrin (20). A mixture of
compound 17 (80 mg, 25.31 .mu.mol) and hydrazine monohydrate (1.22
mL, 25.31 mmol) in EtOH--H.sub.2O 1:1 (1.2 mL) was stirred at
60.degree. C. for 24 h. The solvents were evaporated under
diminished pressure to give a solid, which was suspended in 1N HCl
(5 mL) and stirred at rt for 4 h. The insoluble material was
filtered and the filtrate diluted with acetone (25 mL) until the
product precipitated. The supernatant was removed by centrifugation
and the product washed with acetone (4.times.25 mL) and dried under
vacuum. The product 20 (40 mg, 94%) was obtained as a colorless
solid. .sup.13C-NMR (DMSO-d.sub.6) .delta. 102.05, 84.43, 72.47,
72.22, 71.49, 38.40, 32.85, 32.15, 26.12; mass spectrum (MALDI),
calcd. for C.sub.70H.sub.133N.sub.7O.sub.28S.sub.7 m/z 1745.3 found
1745.9 [M+Na] (100%).
##STR00004##
##STR00005##
Example 2
Protection of Cells from Cytotoxicity
[0036] Recombinant B. anthracis lethal factor (rLF), edema factor
(rEF), and protective antigen (rPA) were acquired from List
Biological Laboratories, Inc. (Campbell, Calif.). Murine RAW 264.7
monocyte-macrophage cell line ATCC TIB-71 was obtained from
American Type Culture Collection (Manassas, Va., USA). The cells
were cultured in phenol free Dulbecco's Modification of Eagle's
Medium/Ham's F-12 50/50 Mix (Mediatech, Inc., Herndon, Va., USA)
supplemented with 10% heat-inactivated fetal bovine serum, 100
units/ml: 100 .mu.g/ml penicillin-streptomycin, 0.1 mM
non-essential amino acids, and 0.5 mM 2-mercaptoethanol at
37.degree. C. in 5% CO.sub.2. The cells were harvested using
Cellstripper.TM. from Mediatech, Inc. and then were washed once
with media to remove the non-enzymatic dissociation solution. RAW
264.7 cells were plated in 96-well flat-bottomed tissue culture
plates from Becton Dickinson (San Jose, Calif., USA) at a
concentration of 10.sup.5 cells/well in the DMEM medium mentioned
above and incubated overnight at 37.degree. C. in 5% CO.sub.2. RAW
264.7 cells were pre-incubated with different concentrations of
tested compounds in DMEM medium for 1 hr at 37.degree. C. in a 5%
CO.sub.2 atmosphere. Then DMEM medium or LeTx (LF=32 ng/ml; PA=500
ng/ml) in the media were added, and the plate was incubated under
the same condition for 4 hrs. Cell viability was estimated using a
MTS kit from Promega (Madison, Wis., USA). A .mu. Quant
spectrophotometer from Bio-Tek Instruments, Inc. (Winooski, Vt.,
USA) was used to obtain OD.sub.570 readings.
Example 3
Inhibition of Ion Conductance
[0037] Ion conductance experiments were performed according to
Montal and Mueller [14] with modifications [15,16]. PA channels
were reconstituted into planar lipid membranes formed from DPhPC;
the membrane bathing solution contained 0.1M KCl, 1 mM EDTA at pH
6.6. Ion conductance through PA channels was measured in the
presence of PrAmBC.
* * * * *