β-Cyclodextrin derivatives that inhibit anthrax lethal toxin

Article abstract of DOI:10.1016/j.bmc.2005.07.054

Recently, we demonstrated that simultaneous blocking of bacterial growth by antibiotics and inhibition of anthrax toxin action with antibodies against protective antigen were beneficial for the treatment of anthrax. The present study examined the hypothesis that blocking the pore formed by protective antigen can inhibit the action of anthrax toxin. The potential inhibitors were chosen by a structure-based design using β-cyclodextrin as the starting molecule. Several β-cyclodextrin derivatives were evaluated for their ability to protect RAW 264.7 cells from the action of anthrax lethal toxin. Per-substituted aminoalkyl derivatives displayed inhibitory activity and were protective against anthrax lethal toxin action at low micromolar concentrations. These results provide the basis for a structure-based drug discovery program, with the goal of identifying new drug candidates for anthrax treatment.

Full text of DOI:10.1016/j.bmc.2005.07.054

Bioorganic & Medicinal Chemistry 14 (2006) 33–40
b-Cyclodextrin derivatives that inhibit anthrax lethal toxin
a,
Vladimir A. Karginov, Adiamseged Yohannes, Tanisha M. Robinson,
a
a
*
b
Nour Eddine Fahmi, Kenneth Alibek and Sidney M. Hecht
c
d
a
Innovative Biologics, Inc., 10900 University Blvd., Manassas, VA 20110, USA
Pinnacle Pharmaceuticals, Inc., Emerging Technology Center One, 1670 Discovery Dr., Charlottesville, VA 22911, USA
b
c
George Mason University, 10900 University Blvd., Manassas, VA, USA
Departments of Chemistry and Biology, University of Virginia, Charlottesville, VA 22901, USA
d
Received 29 June 2005; revised 27 July 2005; accepted 27 July 2005
Available online 19 September 2005
Abstract—Recently, we demonstrated that simultaneous blocking of bacterial growth by antibiotics and inhibition of anthrax toxin
action with antibodies against protective antigen were beneficial for the treatment of anthrax. The present study examined the
hypothesis that blocking the pore formed by protective antigen can inhibit the action of anthrax toxin. The potential inhibitors were
chosen by a structure-based design using b-cyclodextrin as the starting molecule. Several b-cyclodextrin derivatives were evaluated
for their ability to protect RAW 264.7 cells from the action of anthrax lethal toxin. Per-substituted aminoalkyl derivatives displayed
inhibitory activity and were protective against anthrax lethal toxin action at low micromolar concentrations. These results provide
the basis for a structure-based drug discovery program, with the goal of identifying new drug candidates for anthrax treatment.
ꢀ
2005 Elsevier Ltd. All rights reserved.
1
. Introduction
substrates within the cytosol, and PA facilitates their
transport across the cell membrane forming a heptamer-
6
–8
Bacillus anthracis is one of the most dangerous potential
biological weapons. Currently, there is no effective treat-
ment for inhalational anthrax, beyond the administra-
ic pore. Recently, we demonstrated that treatment of
mice infected with B. anthracis with a combination of
the antibiotic ciprofloxacin and partially purified anti-
bodies against anthrax protective antigen drastically
increased survival rates in comparison with antibiotic
1
tion of antibiotics shortly after exposure. Time delay
reduces the effectiveness of antibiotic treatment. There-
fore, there is need for new, safe, and efficient treatment
to supplement intervention with antibiotics. Major fac-
tors playing a role in anthrax infection are the cytotoxic
effect of anthrax toxin, and bacteremia leading to oxy-
gen and nutritional substance deprivation, as well as
the accumulation of various bacterial and host toxic
5
treatment alone. Although promising, antibodies are
less attractive as potential drugs in comparison with
low molecular weight compounds, which offer potential-
ly better penetration through membranes and are not
sensitive to proteases.
2
products, leading to eventual organ failure and death.
The present study examined the hypothesis that com-
pounds designed to block the pore formed by PA can
inhibit the action of anthrax toxin. PA assembles into
This research was based on the premise that simulta-
neous blocking of bacterial growth by antibiotics and
inhibition of anthrax toxin action would be beneficial
for the treatment of anthrax. The two anthrax toxins
are formed by three proteins: protective antigen (PA)
that 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
a ring-shaped heptamer with a mostly negatively
9,10
3
–5
charged lumen.
The information available on the
three-dimensional structure of the PA pore permits a
structure-based drug design. As a starting point for the
development of high affinity ligands for the PA pore,
we employed b-cyclodextrin (b-CD), a cyclic heptamer
of D-glucose having a hydrophobic cavity. In common
with the PA-pore, b-cyclodextrin has a sevenfold
1
1
˚
symmetry. The outside diameter of b-CD—15.3 A —is
Keywords: Anthrax treatment; Lethal toxin inhibitors; b-Cyclodextrin
derivatives.
of the same order as the diameter of the PA channel
9
˚
lumen, which is 20–35 A, according to X-ray data,
*
Corresponding author. Tel.: +1 703 993 8544; fax: +1 703 993
982; e-mail: vak@innovbio.com
˚
and about 12 A at its narrowest point, according to
8
0
968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.07.054

34
V. A. Karginov et al. / Bioorg. Med. Chem. 14 (2006) 33–40
1
2
the measurement of current flow through the channel.
It was also demonstrated that b-cyclodextrin can par-
tially block ion conductance through a similar hepta-
and C-3) hydroxyl groups may be used as points of
1
functionalization.
5
1
3
meric pore formed by staphylococcal a-toxin.
Monofacially functionalized b-CD derivatives substitut-
ed on the face containing the primary alcohol with seven
positively charged pendant groups represent potentially
interesting molecules to block the function of anthrax
lethal toxin.
Cyclodextrins are widely used as pharmaceutical agents
to enhance solubility, bioavailability, and stability of
drug molecules because they can encapsulate organic
molecules. Methods for selective modifications of
cyclodextrins have been developed and offer excellent
opportunities for the synthesis of appropriate deriva-
1
4
Based on the considerations described above, hepta-6-
aminoalkyl derivatives of b-cyclodextrin were chosen
for modification. b-CDs substituted with positively
charged groups seemed promising because the lumen
of the PA pore is negatively charged. Conceivably, the
positively charged groups might also alter the effective
local pH inside the lumen, inhibiting the conformational
change required for the formation of the functional
1
5
tives. Presently, several derivatives of b-cyclodextrin
are being prepared and evaluated for anti-toxin activity.
2
. Results and discussion
The cyclodextrins a-CD, b-CD, and c-CD are naturally
occurring cyclomaltooligosaccharides containing six,
1
7
transmembrane channel.
1
6
seven, or eight a-(1,4)-D-glucopyranosyl rings. The
cyclic array of saccharides produces a hydrophobic cav-
ity, generally described as a truncated cone. The primary
alcohols are located around the narrow rim and the sec-
ondary alcohols around the wider rim, as shown in Fig-
ure 1. The use of CD derivatives as molecular scaffolds is
of interest since the primary (C-6) and secondary (C-2
2.1. Chemistry
An important reaction reported by Defaye and
co-workers enables the symmetric derivatization of
cyclodextrins on the face containing the primary
1
8
OH groups.
This reaction allows the selective
6
OH
O
O
HO
OH
O
HO
OH
O
OH
primary
face
R6
O
OH
OH
O
OHO
O
3
2
HO
R3O
O
7
O
R2O
O
OH
HO
O
β-cyclodextrin
HO
secondary face
HO
OH
OH
OH
O
OH
O
HO
OH
O
HO
O
O
HO
R2
R3
R6
1
H
H
H
H
H
H
SCH CH NH (AmEtβCD)
2 2 2
2
3
SCH CH CH NH (AmPrβCD)
2 2 2 2
SCH CH CH CH NH (AmBuβCD)
2
2
2
2
2
4
5
6
H
H
H
H
H
H
NH (AmβCD)
I
N3
SH
2
7
8
9
1
1
1
1
1
H
H
H
H
Ac
OSO Na
3
Ac
Me
Me
OSO Na
3
0
1
2
3
4
Me
SO3Na
H
OSO Na
3
OSO Na
3
(CH2 CH O) n (n =2 ,3)
NH2
OH
H
2
(CH2 CH O) n (n =2 ,3)
H
H
H
2
Figure 1. Cyclodextrins employed for study.

V. A. Karginov et al. / Bioorg. Med. Chem. 14 (2006) 33–40
35
replacement of all primary hydroxyl groups of a
cyclodextrin by iodine atoms. Thus, treatment of b-cy-
clodextrin with I and PPh in DMF at 70 ꢁC afforded
O
O
NH.HBr
H N S
2
thiourea
N
n
N
n
2
3
Br
the heptaiodide 5 in 76% yield (Scheme 1). The reac-
tion was performed using a modification to the origi-
nal protocol, as reported by Stoddart and co-
O
O
1
1
1
5
6
7
n = 1
n = 2
n = 3
18 n = 1
19 n = 2
20 n = 3
1
9
workers. The heptaiodide 5 was treated with NaN3
in DMF to give the heptaazide 6 in quantitative yield.
Heptaazide 6 was subjected to reduction using PPh3,
followed by treatment with ammonia to give the
per-6-amino-b-cyclodextrin 4.
NH.HBr
CN
thiourea
CN
Br
H2N
S
2
1
22
However, the introduction of an alkylamino group at
the primary position of b-cyclodextrins proved to be a
challenge. The direct alkylation of per-iodo-b-cyclodex-
trin with an alcoholate nucleophile (derived from an
azidoalkanol, for example) would pose some problems
since the basic alcoholate may induce elimination or
intramolecular substitutions. Some studies have shown
that nucleophilic displacement of iodide anions from
per-6-iodo-b-cyclodextrins, employing poor nucleo-
philes or elevated temperatures, favors the intramolecu-
lar substitution reaction, resulting in the formation of
Scheme 2.
phthalimide and the acetate groups using a large excess
of hydrazine monohydrate in aqueous EtOH gave the
desired products 18–20, isolated as the hydrochlorides.
The structures and symmetrical substitution have been
confirmed by mass spectrometry and C NMR spec-
troscopy (Scheme 4).
1
3
3
,6-anhydro-D-glucopyranose residues within the struc-
2
2.2. Cytotoxicity tests
0
ture of per-6-iodo-b-cyclodextrin.
The four hepta-6-aminoalkyl derivatives of b-cyclodex-
trin, suggested by our structure-based design (1–4), were
synthesized. These and several other b-cyclodextrin
derivatives (Fig. 1) were tested for their ability to inhibit
the cytotoxic effect of LeTx on mouse macrophage-like
RAW 264.7 cells. Strikingly, only the four aminoalkyl
derivatives envisaged as inhibitors of LeTx action actu-
ally displayed LeTx inhibitory activity, and they were
protective against LeTx action at low micromolar con-
centrations (Fig. 2). These experiments also showed that
the compounds were not toxic to RAW 264.7 cells up to
50 lM concentration, while they had IC50 values as low
as 3.3 lM. The rest of the compounds shown in Figure 1
To facilitate the isolation of pure compounds by chro-
matography and to assure that the products obtained
were symmetrically substituted, per-6-iodo-b-cyclodex-
trin 5 was acetylated using Ac O and pyridine in the
presence of a catalytic amount of DMAP to give the
2
1
8b
acylated product 23 in 81% yield (Scheme 2).
Com-
pound 23 reacted smoothly in DMF in the presence of
Cs CO with thioureido derivatives 18, 19, or 20 (ob-
tained from the corresponding N-phthalimido-bro-
2
3
moalkyl precursors by treatment with thiourea in
EtOH (Scheme 3)) to give the desired products 24,
21
2
5, and 26. Finally, simultaneous removal of the
OH
I
N3
O
PPh , I2
O
O
3
NaN3
OH
O
OH
O
OH
O
OH
OH
OH
7
7
7
5
6
thiourea
NaOH
PPh3, NH4OH
SH
NH2
O
O
OH
O
OH
O
OH
OH
7
7
7
4
Scheme 1.

36
V. A. Karginov et al. / Bioorg. Med. Chem. 14 (2006) 33–40
I
I
Ac O
pyridine
2
O
O
9
, 10 or 11
OH
O
OAc
O
cat. DMAP
Cs CO
2
3
DMF
OH
OAc
7
7
5
23
O
N
O
O
NH
2
n
n
S
S
O
2 4 2
N H .H O
OAc
O
OH
O
EtOH
OAc
OH
7
7
24
25
26
n = 1
n = 2
n = 3
1
2
3
n = 1
n = 2
n = 3
Scheme 3.
NH HCl
2.
CN
O
I
S
S
O
O
1
3
BH3.SMe2
OAc
O
OAc
O
OH
O
Cs CO
2
3
DMF
OAc
OAc
OH
7
7
7
2
3
27
28
Scheme 4.
displayed no inhibitory activity at concentrations up to
1
derivatives can potentially also be used to block other
toxins that form heptameric transmembrane channels,
such as staphylococcal a-hemolysin. Derivatives of
hexameric a-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. More generally, this can
be considered to be a general approach for the discov-
ery of new drugs by blocking pores with molecules
having the same dimensions and symmetry as the
pores.
00 lM.
2
.3. Ion conductance studies
One of the aminoalkyl derivatives—AmPrbCD—was
tested for its ability to block ion conductance through
PA channels reconstituted in planar bilayer lipid mem-
branes. It was demonstrated that the addition of
AmPrbCD to the bilayer lipid membrane with multiple
PA channels caused a significant step-like decrease in
membrane conductance at 3 nM AmPrbCD concentra-
tion (E. Nestorovich and S. Bezrukov, personal
communication).
4. Experimental
3. Conclusions
4
.1. Chemistry
The data obtained support the hypothesis that com-
pounds designed to block the pore formed by PA
can inhibit the action of anthrax toxin. The results
presented provide the basis for a structure-based drug
discovery program to identify new drug candidates
for anthrax treatment. Per-substituted b-cyclodextrin
b-Cyclodextrin derivatives 1–7 listed in Figure 1 were
synthesized at Pinnacle Pharmaceuticals, Inc. Thio b-cy-
clodextrin derivatives 8–11 were kindly provided by
Dr. Gyula Vigh (Texas A&M University, College
Station, TX). Compounds 12 and 13 were purchased

V. A. Karginov et al. / Bioorg. Med. Chem. 14 (2006) 33–40
Media LeTx
37
Media
LeTx
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00
1.20
.00
0.80
1
0.60
0.40
0.20
0.00
0
0.39 0.78 1.56 3.12 6.25 12.5 25
50 100
0
0.39 0.78 1.56 3.12 6.25 12.5 25
50
100
AmβCD Concentration (µM)
AmPrβCD Concentration (µM)
Media
LeTx
Media
Le Tx
1.20
1.00
0.80
0.60
0.40
0.20
0.00
1.20
1.00
0.80
0.60
0.40
0.20
0.00
0
0.39 0.78 1.56 3.12 6.25 12.5 25
50
100
0
0.39 0.78 1.56 3.12 6.25 12.5 25
50 100
Am Etβ CD Concentration (µM)
Am Buβ CD Concentration (µM)
IC50
:
AmβCD
12.1 + 3.5 µM
–
AmEtβCD
AmPrβCD
AmBuβCD
9.3 + 0.8 µM
–
3.3 + 1.0 µM
–
6.1 + 2.3 µM
–
Figure 2. Protection of RAW 264.7 cells from LeTx-induced cell death by b-CD derivatives. RAW 264.7 cells were incubated with different
concentrations of the b-CD derivatives with or without LeTx. Each experimental condition was performed in triplicate. Cell viability was determined
by MTS colorimetric assay. Error bars represent standard deviations. IC50 values are means of three to four independent experiments.
from Cytrea (Dublin, Ireland). b-Cyclodextrin (14) was
purchased from Sigma Chemicals (St. Louis, MO).
4.1.2. (2-Phthalimidoethyl)isothiouronium hydrobromide
3
2
(18).
A suspension of 3.0 g (11.8 mmol) of N-(2-
bromoethyl)phthalimide (15) and 1.82 g (23.96 mmol)
of thiourea in 5.7 mL of absolute EtOH was stirred at
reflux for 18 h after which the product had crystallized.
The cooled product was collected by filtration, washed
with small amounts of cold abs EtOH, and dried in vac-
uo. Compound 18 was obtained as colorless crystals:
1
13
4
.1.1. General methods. H NMR and C NMR spectra
were recorded on a General Electric QE-300 or a Varian
00 spectrometer. Moisture sensitive reactions were con-
3
ducted under argon in oven-dried glassware. All chemi-
cal reagents were purchased from Aldrich Chemicals or
Fisher Scientific and used without further purification.
Acetonitrile and dichloromethane were distilled from
CaH . DMF was distilled from CaH under diminished
1
yield 4.0 g (100%); mp 243–245 ꢁC; H NMR (DMSO-
d ) d 3.53 (t, 2H, J = 5.9 Hz), 3.91 (t, 2H, J = 5.9 Hz),
6
7.93 (m, 4H), and 9.07 (br s, 3H).
2
2
pressure. Triethylamine was distilled from P O . Analyt-
5
2
ical thin-layer chromatography was performed on
Merck 60F254 precoated silica gel plates. Visualization
was performed by ultraviolet light or by staining with
phosphomolybdic acid or sulfuric acid. Flash chroma-
tography was performed using (40–60 lm) silica gel.
Melting points were taken with a Mel-Temp melting
point apparatus and are uncorrected. Cyclodextrins 4,
4.1.3. (3-Phthalimidopropyl)isothiouronium hydrobro-
2
4
mide (19). A suspension of 3.0 g (11 mmol) of N-(3-
bromopropyl)phthalimide (16) and 1.7 g (22.4 mmol)
of thiourea in 5.3 mL of abs EtOH was stirred at reflux
for 18 h at which time the product had crystallized. The
cooled product was collected by filtration, washed suc-
cessively with two 10-mL portions of cold abs EtOH
and two 10-mL portions of ether, and then dried in vac-
uo. Compound 19 was obtained as colorless crystals:
5
procedures.
, 6 and 7 were prepared according to literature
8,19,22
1

38
V. A. Karginov et al. / Bioorg. Med. Chem. 14 (2006) 33–40
2
5
yield 3.95 g (100%); mp 236–238 ꢁC (lit. 236–237 ꢁC);
4.1.7. Heptakis [2,3-di-O-acetyl-6-deoxy-6-(3-phthalimi-
dopropyl)-thio]cyclomaltoheptaose (25). To a solution of
250 mg (0.1 mmol) of heptakis (2,3-di-O-acetyl-6-deoxy-
6-iodo)cyclomaltoheptaose (23) and 472 mg (1.37 mmol)
of (3-phthalimidopropyl)isothiouronium hydrobromide
(19) in 10 mL of dry DMF was added 687 mg
(2.11 mmol) Cs CO and the reaction mixture was stir-
red at 23 ꢁC under argon for 68 h. The reaction mixture
was poured onto 50 g of ice and 100 mL of 0.5 N HCl
was added. The aqueous layer was extracted with three
50-mL portions of dichloromethane. The combined
organic phase was washed successively with 100 mL of
1
H NMR (DMSO-d ) d 1.97 (m, 2H), 3.22 (t, 2H,
J = 6.6 Hz), 3.71 (t, 2H, J = 6.2 Hz), 7.88 (m, 4H), and
9
6
.07 (br s, 3H).
4
.1.4. (4-Phthalimidobutyl)isothiouronium hydrobromide
5
2
(
butyl)phthalimide (17) and 0.54 g (7.08 mmol) of thio-
20). A suspension of 1.0 g (3.5 mmol) of N-(4-bromo-
2
3
urea in 1.7 mL of abs EtOH was stirred at reflux for
1
8 h. Upon cooling to room temperature, the syrupy
mixture began crystallizing and was treated with 4 mL
ether. The mixture was stirred for 15 min and the prod-
uct was collected by filtration and washed with a small
amount of cold EtOH. Compound 20 was obtained as
a colorless solid: yield 1.22 g (96%); mp 171–172 ꢁC;
H NMR (DMSO-d ) d 1.70 (m, 4H), 3.18 (t, 2H,
0.5 N HCl and 100 mL brine, dried (MgSO ), and con-
4
centrated under diminished pressure. The residue was
purified on a silica gel column (14 · 3 cm); elution with
EtOAc gave compound 25 as a colorless foam: yield
1
6
1
J = 6.6 Hz) 3.63 (t, 2H, J = 6.1 Hz), 7.89 (m, 4H), and
9
188 mg (59%); H NMR (CDCl ) d 1.91 (m, 2H), 2.02
3
.00 (br s, 3H).
(s, 3H), 2.05 (s, 3H), 2.60 (m, 2H, J = 12.9, 5.9 Hz),
3
.03 (m, 2H), 3.66 (t, 2H, J = 6.9 Hz), 3.84 (t, 1H),
4
altoheptaose (23).
of per-6-iodo-b-cyclodextrin (5) in 5 mL of dry pyri-
.1.5. Heptakis (2,3-di-O-acetyl-6-deoxy-6-iodo)cyclom-
To a solution of 1.0 g (0.52 mmol)
4.12 (m, 1H), 4.80 (dd, 1H, J = 9.7, 3.8 Hz), 5.06 (d,
1H, J = 3.8 Hz), 5.23 (dd, 1H, J = 9.6, 8.3 Hz), 7.58
(dd, 2H, J = 5.4, 3.1 Hz) and 7.70 (dd, 2H, J = 5.5,
3.0 Hz); mass spectrum (MALDI), m/z 3166.8
18b
dine was added 7.5 mL of Ac O and 6.5 mg
(
2
+
0.05 mmol) of 4,4-dimethylaminopyridine. The reac-
[M+Na] , theoretical 3166.8.
tion mixture was stirred at 23 ꢁC under argon for
4
1
8 h. The reaction was quenched by the addition of
5 mL MeOH and the solvent was concentrated under
4.1.8.
Heptakis
[2,3-di-O-acetyl-6-deoxy-6-(4-
phthalimidobutyl)-thio]cyclomaltoheptaose (26). To a
solution of 404 mg (0.16 mmol) of heptakis (2,3-di-O-
acetyl-6-deoxy-6-iodo)cyclomaltoheptaose (23) and
0.87 g (2.4 mmol) of (4-phthalimidobutyl)isothiouroni-
um hydrobromide (20) in 16 mL of dry DMF was added
1.32 g (4.04 mmol) Cs CO and the reaction mixture was
stirred at 23 ꢁC under argon for 48 h. The reaction mix-
ture was poured onto 50 g ice and 200 mL of 0.5 N HCl
was added. The aqueous layer was extracted with three
50-mL portions of dichloromethane. The combined
organic phase was washed successively with 100 mL of
diminished pressure. Co-evaporation with three 4-mL
portions of MeOH and three 4-mL portions of toluene
gave a brown residue, which was purified on a silica gel
column (20 · 3 cm). Elution with a step gradient of
1
:1 ! 1:4 hexane–EtOAc gave compound 23 as a col-
2
3
orless foam, which crystallized upon trituration with
172–
1
8b
ether: yield 1.06 g (81%): mp 180–182 ꢁC (lit.
1
1
3
3
77 ꢁC); H NMR (CDCl ) d 2.05 (s, 3H), 2.09 (s,
3
H), 3.58–3.81 (m, 4H), 4.83 (dd, 1H, J = 9.9,
.9 Hz), 5.20 (d, 1H, J = 3.6 Hz), and 5.33 (br t, 1H,
J = 8.4 Hz); mass spectrum (MALDI), m/z 2514.9
[
0.5 N HCl and 100 mL brine, dried (MgSO ), and then
4
+
M+Na] , theoretical m/z 2514.8.
concentrated under reduced pressure. The residue was
purified on a silica gel column (18 · 3 cm); elution with
EtOAc gave compound 26 as a colorless solid: yield
4.1.6. Heptakis [2,3-di-O-acetyl-6-deoxy-6-(2-phthalimi-
doethyl)-thio]cyclomaltoheptaose (24). To a solution of
125 mg (24%). An additional 132 mg was obtained in a
1
0
6
.5 g (0.2 mmol) of heptakis (2,3-di-O-acetyl-6-deoxy-
-iodo)cyclomaltoheptaose (23) and 0.99 g (3.0 mmol)
slightly impure form; H NMR (CDCl ) d 1.61 (m,
3
2H), 1.73 (m, 2H), 2.02 (s, 3H), 2.06 (s, 3H), 2.65 (m,
2H), 3.03 (m, 2H), 3.63 (m, 2H), 3.88 (m, 1H), 4.15
(m, 1H), 4.80 (dd, 1H, J = 9.8, 3.7 Hz), 5.12 (d, 1H,
J = 3.6 Hz), 5.26 (m, 1H), 7.64 (m, 2H), and 7.74 (m,
of (2-phthalimidoethyl)isothiouronium hydrobromide
18) in 20 mL of dry DMF was added 1.63 g (5.0 mmol)
of Cs CO and the reaction mixture was stirred at 23 ꢁC
(
2
3
+
under argon for 48 h. The reaction mixture was poured
onto 40 g ice and 200 mL of 0.5 N HCl was added. The
aqueous layer was extracted with three 50-mL portions
of dichloromethane. The combined organic phase was
washed successively with 200 mL of 0.5 N HCl and
2H); mass spectrum (MALDI), m/z 3267.3 [M+Na] ,
theoretical 3267.5.
4.1.9. Per-6-(2-aminoethylthio)-b-cyclodextrin (1).
A
mixture of 100 mg (31.9 lmol) of compound 24 and
1.55 mL (31.9 mmol) of hydrazine monohydrate in
1
00 mL of brine, dried (MgSO ), and concentrated un-
4
der diminished pressure. The residue was purified on a
silica gel column (21 · 3 cm); elution with EtOAc affor-
ded compound 24 as a colorless solid: yield 145 mg
1.5 mL of 1:1 EtOH–H O was stirred at 60 ꢁC for
2
18 h. The solvent was concentrated under diminished
pressure to give a solid, that was suspended in 5 mL of
1 N HCl and stirred at 23 ꢁC for 8 h. The insoluble
material was filtered and the filtrate was diluted with
25 mL acetone, causing the product to precipitate. The
supernatant was removed by centrifugation and the
product was washed with four 25-mL portions of ace-
tone and dried in vacuo. The product (1) was obtained
as a colorless solid: yield 46 mg (89%); mp 180–182 ꢁC
(
23%). An additional 165 mg of 24 was obtained in a
1
slightly impure form; H NMR (CDCl ) d 2.01 (s,
3
3
2
1
2
3
H), 2.05 (s, 3H), 2.64 (m, 2H), 3.03 (m, 2H), 3.63 (m,
H), 3.87 (t, 1H, J = 8.4 Hz), 4.15 (m, 1H), 4.80 (m,
H), 5.10 (br s, 1H), 5.25 (t, 1H, J = 8.7 Hz), 7.62 (m,
H) and 7.73 (m, 2H); mass spectrum (MALDI), m/z
+
068.8 [M+Na] , theoretical 3068.7.

V. A. Karginov et al. / Bioorg. Med. Chem. 14 (2006) 33–40
39
1
3
(
dec); C NMR (DMSO-d ) d 102.07, 84.49, 72.51,
PA = 500 ng/mL) in the media was added, and the plate
was incubated under the same conditions for 4 h. Cell
viability was estimated using a MTS kit from Promega
6
72.18, 71.28, 32.70, 29.60; mass spectrum (MALDI),
m/z 1548.8 [M] , theoretical 1548.9.
+
(Madison, WI). A l Quant spectrophotometer from
4
.1.10. Per-6-(3-aminopropylthio)-b-cyclodextrin (2). A
Bio-Tek Instruments (Winooski, VT), was used to make
OD570 readings.
mixture of 100 mg (31.4 lmol) of compound 25 and
1
1
.54 mL (31.78 mmol) of hydrazine monohydrate in
.5 mL of 1:1 EtOH–H O was stirred at 60 ꢁC for 16 h.
4.3. Ion conductance studies
2
The solvent was concentrated under diminished pressure
to give a solid, which was suspended in 5 mL of 1 N HCl
and stirred at 23 ꢁC for 4 h. The insoluble material was
filtered and the filtrate was diluted with 25 mL acetone,
causing the product to precipitate. The supernatant
was removed by centrifugation and the product was
washed with four 25-mL portions of acetone and dried
in vacuo. Compound 2 was obtained as a colorless solid:
Ion conductance experiments were performed according
27,28
2
6
to Montal and Mueller with modifications.
PA
channels were reconstituted in planar lipid membranes
formed from DPhPC; the membrane bathing solution
contained 0.1 M KCl and 1 mM EDTA at pH 6.6. Ion
conductance through PA channels was measured in
the presence of AmPrbCD.
1
3
yield 53 mg (85%); mp 161–163 ꢁC (dec); C NMR
(
DMSO-d ) d 26.85, 29.71, 33.03, 37.79, 71.41, 72.23,
6
Acknowledgments
7
1
2.48, 84.52 and 102.09; mass spectrum (MALDI), m/z
668.8 [M+Na] , theoretical m/z 1668.6.
+
The authors thank S. Bezrukov and E. Nestorovich for
the ion conductance data and G. Vigh for the sulfo b-cy-
clodextrin derivatives. This work was supported by
Grant 1R43AI052894-01 from the National Institute
of Allergy and Infectious Diseases, National Institutes
of Health.
4
.1.11. Per-6-(4-aminobutylthio)-b-cyclodextrin (3). A
mixture of 80 mg (25.3 lmol) of compound 26 and
1
.22 mL (25.3 mmol) of hydrazine monohydrate in
1
.2 mL of 1:1 EtOH–H O was stirred at 60 ꢁC for 24 h.
2
The solvent was concentrated under diminished pressure
to give a solid, which was suspended in 5 mL of 1 N HCl
and stirred at 23 ꢁC for 4 h. The insoluble material was
filtered and the filtrate was diluted with 25 mL acetone,
causing the product to precipitate. The supernatant
was removed by centrifugation and the product was
washed with four 25-mL portions of acetone and dried
in vacuo. The product was obtained as a colorless solid:
References and notes
1
. Bartlett, J. G.; Inglesby, T. V.; Borio, L. Clin. Infect. Dis.
002, 35, 851.
2
2. Dixon, T. C.; Meselson, M.; Guillemin, J.; Hanna, P. C.
N. Engl. J. Med. 1999, 341, 815.
3. Rainey, G. J.; Young, J. A. Nat. Rev. Microbiol. 2004, 2,
1
3
yield 40 mg (94%); mp 172–174 ꢁC (dec); C NMR
7
. Friedlander, A. M. Nature 2001, 414, 160.
21.
(
DMSO-d ) d 26.12, 32.15, 32.85, 38.40, 71.49, 72.22,
6
4
7
1
2.47, 84.43 and 102.05; mass spectrum (MALDI), m/z
745.9 [M+Na] , theoretical m/z 1745.3.
+
5. Karginov, V. A.; Robinson, T.; Riemenschneider, J.;
Golding, B.; Kennedy, M.; Shiloach, J.; Alibek, K. FEMS
Immunol. Med. Microb. 2004, 40, 71.
4
.2. Cell-based assays
6. Brossier, F.; Mock, M. Toxicon 2001, 39, 1747.
7. Collier, R. J.; Young, J. A. Annu. Rev. Cell Dev. Biol.
4
.2.1. Cells and cell culture. Recombinant B. anthracis
2
003, 19, 45.
lethal factor (rLF) and protective antigen (rPA) were ac-
quired from List Biological Laboratories (Campbell,
CA). 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-red free DulbeccoÕs modified
EagleÕs medium (DMEM, Mediatech, Herndon, VA,
USA) supplemented with 10% heat-inactivated fetal bo-
vine serum, 100 U/ml:100 lg/ml penicillin–streptomycin,
8. Moayeri, M.; Leppla, S. H. Curr. Opin. Microbiol. 2004, 7,
19.
9. Petosa, C.; Collier, R. J.; Klimpel, K. R.; Leppla, S. H.;
Liddington, R. C. Nature 1997, 385, 833.
0. Benson, E. L.; Huynh, P. D.; Finkelstein, A.; Collier, R. J.
Biochemistry 1998, 37, 3941.
1
11. Szejtli, J. Chem. Rev. 1998, 98, 1743.
12. Finkelstein, A. Toxicology 1994, 87, 29.
13. Gu, L. Q.; Braha, O.; Conlan, S.; Cheley, S.; Bayley, H.
Nature 1999, 398, 686.
0
2
.1 mM non-essential amino acids, and 0.5 mM of
1
4. Uekama, K.; Hirayama, F.; Irie, T. Chem. Rev. 1998, 98,
2045.
-mercaptoethanol at 37 ꢁC in a 5% CO atmosphere.
2
The cells were harvested by gentle scraping with a cell
scraper and were then washed once with media. RAW
15. Khan, A. R.; Forgo, P.; Stine, K. J.; DÕSouza, V. T. Chem.
Rev. 1998, 98, 1977.
1
6. (a) Szetli, J. Cyclodextrins and their Inclusion Complexes;
Akademiai Kiado: Budapest, 1982; (b) Wenz, G. Angew.
Chem. Int. Ed. Engl. 1994, 33, 803.
2
64.7 cells were plated in 96-well flat-bottomed tissue
culture plates from Becton–Dickinson (San Jose, CA)
5
at a concentration of 10 cells/well in DMEM mentioned
1
1
7. Friedlander, A. M. J. Biol. Chem. 1986, 261, 7123.
8. (a) Gadelle, A.; Defaye, J. Angew. Chem. Int. Ed. Engl.
above and incubated overnight at 37 ꢁC in 5% CO2.
1
991, 30, 78; (b) Baer, H. H.; Berenguel, A. V.; Shu, Y. Y.;
Defaye, J.; Gadelle, A.; Gonz a´ lez, F. S. Carbohydr. Res.
992, 228, 307.
4.2.2. Cytotoxicity neutralization assay. RAW 264.7 cells
were pre-incubated with different concentrations of test-
1
ed compounds in DMEM for 1 h at 37 ꢁC in a 5% CO
atmosphere. Then DMEM or LeTx (LF = 32 ng/mL;
19. Ashton, P. R.; K o¨ niger, R.; Stoddart, J. F. J. Org. Chem.
1996, 61, 903.
2

40
V. A. Karginov et al. / Bioorg. Med. Chem. 14 (2006) 33–40
2
0. (a) Fujita, K.; Yamamura, H.; Imoto, T.; Fujioka, T.;
24. Edwards, M. L.; Prakash, N. J.; Stemerick, D. M.;
Sunkara, S. P.; Bitonti, A. J.; Davis, G. F.; Dumont, J.
A.; Bey, P. J. Med. Chem. 1990, 33, 1369.
25. (a) Kuhnert, N.; Holst, B.; Williamson, G. J. Labelled
Compd. Radiopharm. 2001, 44, 347; (b) Regan, B. M.;
Galysh, F. T.; Morris, R. N. J. Med. Chem 1967, 10, 649.
26. Montal, M.; Mueller, P. Proc. Natl. Acad. Sci. U.S.A.
1972, 69, 3561.
27. Bezrukov, S. M.; Vodyanoy, I. Biophys. J. 1993, 64,
16.
28. Rostovtseva, T. K.; Nestorovich, E. M.; Bezrukov, S. M.
Biophys. J. 2002, 82, 160.
Mihashi, K. J. Org. Chem. 1988, 53, 1943; (b) Fujita, K.;
Tahara, T.; Yamamura, H.; Imoto, T.; Koga, T.; Fujioka,
T.; Mihashi, K. J. Org. Chem. 1990, 55, 877; (c) Ashton, P.
R.; Ellwood, P.; Staton, I.; Stoddart, J. F. J. Org. Chem.
1
991, 56, 7274.
1. Atwell, G. J.; Cain, B. F.; Denny, W. A. J. Med. Chem.
977, 20, 1128.
2
2
2
1
2. Rojas, M. T.; K o¨ niger, R.; Stoddart, J. F.; Kaifer, A. E.
J. Am. Chem. Soc. 1995, 117, 336.
3. Duzhak, V. G. Fiziologicheski Aktivnye Veshchestva 1973,
5, 38.