Search for cyclodextrin-based inhibitors of anthrax toxins: Synthesis, structural features, and relative activities

Article abstract of DOI:10.1128/AAC.00693-06

Recently, using structure-inspired drug design, we demonstrated that aminoalkyl derivatives of β-cyclodextrin inhibited anthrax lethal toxin action by blocking the transmembrane pore formed by the protective antigen (PA) subunit of the toxin. In the prese

Full text of DOI:10.1128/AAC.00693-06

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 2006, p. 3740–3753
0066-4804/06/$08.00ϩ0 doi:10.1128/AAC.00693-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 50, No. 11
Search for Cyclodextrin-Based Inhibitors of Anthrax Toxins: Synthesis,
Structural Features, and Relative Activitiesᰔ
Vladimir A. Karginov,1* Ekaterina M. Nestorovich,2 Adiamseged Yohannes,1 Tanisha M. Robinson,1
Nour Eddine Fahmi,3 Frank Schmidtmann,3 Sidney M. Hecht,4 and Sergey M. Bezrukov2
Innovative Biologics, Inc., 10900 University Blvd., Manassas, Virginia 201101; Laboratory of Physical and Structural Biology, NICHD,
National Institutes of Health, Bethesda, Maryland 209822; Pinnacle Pharmaceuticals, Inc., Emerging Technology Center One,
1670 Discovery Dr., Charlottesville, Virginia 229113; and Departments of Chemistry and Biology, University of
Virginia, Charlottesville, Virginia 229014
Received 5 June 2006/Returned for modification 1 August 2006/Accepted 27 August 2006
Recently, using structure-inspired drug design, we demonstrated that aminoalkyl derivatives of ␤-cyclodex-
trin inhibited anthrax lethal toxin action by blocking the transmembrane pore formed by the protective antigen
(PA) subunit of the toxin. In the present study, we evaluate a series of new ␤-cyclodextrin derivatives with the
goal of identifying potent inhibitors of anthrax toxins. Newly synthesized hepta-6-thioaminoalkyl and hepta-
6-thioguanidinoalkyl derivatives of ␤-cyclodextrin with alkyl spacers of various lengths were tested for the
ability to inhibit cytotoxicity of lethal toxin in cells as well as to block ion conductance through PA channels
reconstituted in planar bilayer lipid membranes. Most of the tested derivatives were protective against anthrax
lethal toxin action at low or submicromolar concentrations. They also blocked ion conductance through PA
channels at concentrations as low as 0.1 nM. The activities of the derivatives in both cell protection and
channel blocking were found to depend on the length and chemical nature of the substituent groups. One of
the compounds was also shown to block the edema toxin activity. It is hoped that these results will help to
identify a new class of drugs for anthrax treatment, i.e., drugs that block the pathway for toxin translocation
into the cytosol, the PA channel.
Anthrax is a deadly disease, and its causative agent, Bacillus
anthracis, is considered one of the most dangerous biological
weapons. The absence of an effective treatment for postexpo-
sure inhalational anthrax (19) is mostly due to the fact that
antibiotics alone are not always helpful at this stage because of
the accumulation of toxins. For this reason, an effective ther-
apeutic approach would include the simultaneous blocking of
bacterial growth by antibiotics and inhibition of anthrax toxin
action with antitoxins (11, 14, 26).
The two toxins playing an essential role in anthrax patho-
genesis are formed by three polypeptides secreted by Bacillus
anthracis: protective antigen (PA) combines either with lethal
factor (LF) to form lethal toxin (LeTx) or with edema factor
(EF) to form edema toxin (EdTx). LF and EF are enzymes that
target substrates within the cytosol; PA, after being cleaved to
PA63 by a furin-like protease of the host cell, provides a hepta-
meric pore, (PA63)7, to facilitate LF and EF transport into the
cytosol (6, 8, 20). Recently, we demonstrated that persubsti-
tuted 6-aminoalkyl (per-6-aminoalkyl) derivatives of ␤-cyclo-
dextrin (␤-CD) inhibited anthrax LeTx action in vitro and in
vivo by blocking the PA63 pore (13, 15). The rationale behind
this modification was that the positively charged cyclic mole-
cules having sevenfold symmetry would likely block the PA
channel, which has the same sevenfold symmetry and a pre-
dominantly negatively charged lumen (4). The outside diame-
ter of the ␤-CD molecule, 15.3 Å (28), is comparable to the PA
prepore internal diameter of 20 to 35 Å (25) and the diameter
of the transmembrane pore, which is at least 11 Å at its nar-
rowest part (5, 10) (Fig. 1). Cyclodextrins are widely used as
pharmaceutical agents to enhance the solubility, bioavailabil-
ity, and stability of drug molecules because they can encapsu-
late organic molecules (7, 30). Methods for selective modifi-
cation of cyclodextrins have been developed previously and
offer excellent opportunities for the synthesis of appropriate
derivatives (16). In the present study, we further investigated
the effects of the positively charged pendant groups and the
length of the alkyl spacers on the activity of ␤-cyclodextrin
derivatives, with the goal of identifying more-potent inhibitors
of anthrax toxin. Using hexameric ␣-cyclodextrin derivatives,
we also explored the role of sevenfold symmetry in the cyclo-
dextrin-PA channel interaction.
MATERIALS AND METHODS
Reagents. Recombinant B. anthracis lethal factor, edema factor, and protective
antigen (in PA83 and PA63 forms) were acquired from List Biological Labora-
tories, Inc. (Campbell, CA). The following chemical reagents were used: KCl,
KOH, and HCl; EDTA; “purum” hexadecane (Fluka, Buchs, Switzerland); di-
phytanoyl phosphatidylcholine (Avanti Polar Lipids, Inc., Alabaster, AL); pen-
tane (Burdick and Jackson, Muskegon, MI); and agarose (Bethesda Research
Laboratory, Gaithersburg, MD). Doubly distilled and deionized water was used
to prepare solutions. All solutions were purified by filtration through a 0.45-␮m
filter.
Chemistry. 1H nuclear magnetic resonance (NMR) and 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 glass-
ware. All chemical reagents were purchased from Aldrich Chemicals or Fisher
Scientific and used without further purification. Dimethylformamide (DMF) was
distilled from CaH2 under diminished pressure. Analytical thin-layer chroma-
tography was performed on Merck 60F254 precoated silica gel plates. Visualiza-
tion was performed by UV light or by staining with phosphomolybdic acid or
* Corresponding author. Mailing address: Innovative Biologics, Inc.,
10900 University Blvd., MSN 1A8, Manassas, VA 20110. Phone: (703)
993-8544. Fax: (703) 993-8982. E-mail: vak@innovbio.com.
ᰔ Published ahead of print on 18 September 2006.
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CYCLODEXTRIN-BASED INHIBITORS FOR ANTHRAX
3741
FIG. 1. Schematic illustration of a heptameric ␤-cyclodextrin (left) in comparison with the PA channel (right). The arrows show hydroxyl groups
at positions 2, 3, and 6.
sulfuric acid. Flash chromatography was performed using (40- to 60-␮m) silica
gel. Melting points were taken with a Mel-Temp melting point apparatus and are
uncorrected. N-Bromoalkylphthalimides 1a to 1i were either commercially avail-
able or prepared according to procedures outlined in the literature (1, 15).
Peracetylated 6-iodo-␤-CD 3 and peracetylated 6-iodo-␣-CD 7 were prepared
according to the methods of Baer et al. (3) and Takeo et al. (29). Per-6-amino-
␤-CD and -␣-CD 15 and 16 (2, 12) and cyclodextrins 17 and 18 (31) were
prepared according to procedures outlined in the literature.
5-Phthalimidopentyl isothiuronium bromide (compound 2d). A mixture of
5.0 g (16.8 mmol) of N-(5-bromopentyl)-phthalimide (compound 1d) and 1.41 g
(18.5 mmol) of thiourea was heated at reflux in 10 ml of absolute ethanol (EtOH)
for 18 h. The mixture was cooled to room temperature, and the product was
collected by filtration and then washed with two 10-ml portions of chilled EtOH
and four 15-ml portions of acetone and dried under vacuum. Compound 2d was
obtained as a colorless solid: yield 5.62 g (90%); mp 188 to 190°C; 1H NMR
(dimethyl sulfoxide [DMSO]-d6) ␦ 1.42 (m, 2H), 1.65 (m, 4H), 3.15 (t, 2H, J ϭ
7.2 Hz), 3.61 (t, 2H, J ϭ 6.9 Hz), 7.90 (m, 4H), and 8.97 (br s, 4H).
6-Phthalimidohexyl isothiuronium bromide (compound 2e). A mixture of 6.0 g
(19.3 mmol) of 6-bromohexylphthalimide (compound 1e) and 1.4 g (18.4 mmol)
of thiourea in 20 ml of absolute EtOH was stirred at reflux for 18 h. The solvent
was concentrated under diminished pressure to give a residue which was tritu-
rated with 20 ml of acetone and filtered. The product was washed with three
10-ml portions of acetone and dried under vacuum. Compound 2e was obtained
as a colorless solid: yield 5.95 g (79%); mp 137 to 139°C; 1H NMR (DMSO-d6)
␦ 1.33 to 1.42 (m, 4H), 1.62 (m, 4H), 3.15 (t, 2H, J ϭ 7.5 Hz), 3.60 (t, 2H, J ϭ 7.0
Hz), 7.89 (m, 4H), and 8.99 (br s, 3H).
7-Phthalimidoheptyl isothiuronium bromide (compound 2f). A mixture of
3.9 g (12.0 mmol) of 7-bromoheptylphthalimide (compound 1f) and 1.00 g (13.2
mmol) of thiourea in 15 ml of absolute EtOH was stirred at reflux for 18 h. The
solvent was concentrated under diminished pressure to give a residue which was
triturated with 15 ml of acetone and filtered. The product was washed with three
10-ml portions of acetone and dried under vacuum. Compound 2f was obtained
as a colorless solid: yield 3.76 g (78%); mp 150 to 152°C; 1H NMR (DMSO-d6)
␦ 1.31 (m, 6H), 1.58 (m, 4H), 3.13 (m, 2H, J ϭ 7.2 Hz), 3.57 (t, 2H, J ϭ 7.1 Hz),
7.86 (m, 4H), and 8.99 (br s, 4H).
was concentrated under diminished pressure to give a brown syrup which was
triturated with 90 ml of diethylether (Et2O) and stirred for 18 h. The precipitated
product was filtered, washed with three 15-ml portions of Et2O, and dried under
vacuum. Compound 2g was obtained as a colorless solid: yield 5.42 g (96%); 1
H
NMR (DMSO-d6) ␦ 1.24 to 1.42 (m, 8H), 1.61 (m, 4H), 3.15 (t, 2H, J ϭ 7.3 Hz),
3.59 (t, 2H, J ϭ 7.0 Hz), 7.88 (m, 4H), and 9.03 (br s, 3H).
9-Phthalimidononyl isothiuronium bromide (compound 2h). A mixture of
3.0 g (8.5 mmol) of 9-bromononylphthalimide (compound 1h) and 618 mg (8.11
mmol) of thiourea in 16 ml of EtOH was stirred at reflux for 3 h. The solvent was
concentrated under diminished pressure, and the residue was triturated with 25
ml of acetone. The product was filtered, washed with two 15-ml portions of
acetone, and dried under vacuum. Compound 2h was obtained as a colorless
solid: yield 2.78 g (80%); mp 135 to 137°C; 1H NMR (DMSO-d6) ␦ 1.29 (m,
10H), 1.60 (m, 4H), 3.15 (t, 2H, J ϭ 7.5 Hz), 3.58 (t, 2H, J ϭ 7.2 Hz), 7.88 (m,
4H), 8.95 (br s, 1H), and 9.06 (br s, 1H).
10-Phthalimidodecyl isothiuronium bromide (compound 2i). A mixture of
3.3 g (9.0 mmol) of 10-bromodecylphthalimide (compound 1i) and 654 mg (8.58
mmol) of thiourea in 10 ml of EtOH was stirred at reflux for 3 h. The solvent was
concentrated under diminished pressure, and the residue was triturated with 25
ml of Et2O. The product was filtered, washed with two 15-ml portions of Et2O,
and dried under vacuum. Compound 2i was obtained as a colorless solid: yield
3.57 g (94%); mp 127 to 129°C; 1H NMR (DMSO-d6) ␦ 1.25 (m, 12H), 1.58 (m,
4H), 3.13 (t, 2H, J ϭ 7.5 Hz), 3.56 (t, 2H, J ϭ 7.1 Hz), 7.86 (m, 4H), and 8.98 (br
s, 4H).
Heptakis[2,3-di-O-acetyl-6-deoxy-6-(5-phthalimidopentyl)thio]cyclomalto-
heptaose (compound 4d). To a mixture of 1.5 g (0.60 mmol) of peracetylated
6-iodo-␤-CD 3 and 4.7 g (12.6 mmol) of the thiuronium salt 2d in 60 ml of
anhydrous DMF was added 4.9 g (15.0 mmol) of Cs2CO3. The reaction mixture
was stirred at 23°C under argon for 44 h. The insoluble material was filtered
through Celite, and the solvent was concentrated under diminished pressure.
The residue was partitioned between ethylacetate (EtOAc) (75 ml) and water (75
ml). The organic layer was separated, dried (MgSO4), and evaporated under
diminished pressure. The crude product was dissolved in 6 ml of pyridine and 9
ml of aceticanhydride (Ac2O), and then 8.5 mg of 4-dimethylaminopyridine was
added and the reaction mixture was stirred at 23°C under argon for 3 days. The
reaction was quenched by slow addition of 40 ml of methanol (MeOH), and the
solvent was concentrated under diminished pressure. The residue was parti-
tioned between water (75 ml) and EtOAc (65 ml). The organic layer was dried
8-Phthalimidooctyl isothiuronium bromide (compound 2g). A mixture of
5.25 g (15.5 mmol) of 8-bromooctylphthalimide (compound 1g) and 1.04 g (13.7
mmol) of thiourea in 16 ml of EtOH was stirred at reflux for 18 h. The solvent

3742
KARGINOV ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
(MgSO4) and concentrated under diminished pressure. The crude product was
purified on a silica gel column (17 by 3 cm), eluting with EtOAc. Compound 4d
was obtained as a slightly yellow foam: yield 1.31 g (65%); 13C NMR (acetone-d6)
␦ 20.99, 21.05, 26.79, 28.97, 34.16, 34.42, 38.39, 71.55, 72.63, 79.68, 97.72, 123.70,
133.18, 134.85, 168.76, 170.11, and 171.00.
mg of 4-dimethylaminopyridine was added and the reaction mixture was stirred
at 23°C under argon for 48 h. The reaction was quenched by slow addition of 35
ml of MeOH, and the solvent was concentrated under diminished pressure. The
residue was partitioned between water (75 ml) and EtOAc (65 ml). The organic
layer was dried (MgSO4) and concentrated under diminished pressure. The
crude product was purified on a silica gel column (18 by 4 cm), eluting with 1:4
hexane-EtOAc and then with EtOAc. Compound 4h was obtained as a yellow
foam: yield 0.9 g (75%); 13C NMR (acetone-d6) ␦ 20.36, 20.45, 27.05, 29.13,
30.14, 33.89, 37.86, 71.10, 72.09, 79.00, 97.03, 123.05, 132.55, 134.25, 168.11,
169.46, and 170.37; mass spectrum (ESI), m/z 3,760.2 [MϩNa]ϩ (theoretical
3,758.3).
Heptakis [2,3-di-O-acetyl-6-deoxy-6-(6-phthalimidohexyl)thio]cyclomalto-
heptaose (compound 4e). To a mixture of 1.38 g (0.55 mmol) of peracetylated
6-iodo-␤-CD 3 and 4.5 g (11.6 mmol) of the thiuronium salt 2e in 55 ml of
anhydrous DMF was added 4.51 g (13.9 mmol) of Cs2CO3. The reaction mixture
was stirred at 23°C under argon for 4 days. The insoluble material was filtered
through Celite, and the solvent was concentrated under diminished pressure.
The residue was partitioned between EtOAc (50 ml) and water (50 ml). The
organic layer was separated, dried (MgSO4), and concentrated under diminished
pressure. The crude product was dissolved in 5 ml of pyridine and 7.5 ml of
Ac2O, and then 6.5 mg of 4-dimethylaminopyridine was added and the reaction
mixture was stirred at 23°C under argon for 48 h. The reaction was quenched by
slow addition of 50 ml of MeOH, and the solvent was concentrated under
diminished pressure. The residue was partitioned between water (75 ml) and
EtOAc (65 ml). The organic layer was dried (MgSO4) and concentrated under
diminished pressure. The crude product was purified on a silica gel column (15
by 3 cm), eluting with EtOAc. Compound 4e was obtained as a colorless foam:
yield 1.28 g (67%); 13C NMR (acetone-d6) ␦ 20.96, 21.04, 27.32, 30.79, 34.27,
34.93, 38.46, 71.50, 72.81, 79.58, 97.55, 123.71, 133.21, 134.89, 168.79, 170.11, and
170.96; mass spectrum (matrix-assisted laser desorption ionization [MALDI]),
m/z 3,464.6 [MϩNa]ϩ (theoretical 3,463.9).
Heptakis[2,3-di-O-acetyl-6-deoxy-6-(7-phthalimidoheptyl)thio]cyclomalto-
heptaose (compound 4f). To a mixture of 1.07 g (0.43 mmol) of peracetylated
6-iodo-␤-CD 3 and 3.62 g (9.04 mmol) of the thiuronium salt 2f in 50 ml of
anhydrous DMF was added 3.62 g (9.04 mmol) of Cs2CO3. The reaction mixture
was stirred at 23°C under argon for 48 h. The insoluble material was filtered
through Celite, and the solvent was concentrated under diminished pressure.
The residue was partitioned between EtOAc (50 ml) and water (50 ml). The
organic layer was dried (MgSO4) and concentrated under diminished pressure.
The crude product was dissolved in 36 ml of pyridine and 48 ml of Ac2O, and
then 20 mg of 4-dimethylaminopyridine was added and the reaction mixture was
stirred at room temperature under argon for 64 h. The reaction was quenched by
slow addition of 50 ml of MeOH, and the solvent was concentrated under
diminished pressure. The residue was partitioned between water (75 ml) and
EtOAc (65 ml). The organic layer was dried (MgSO4) and concentrated under
diminished pressure. The crude product was purified on a silica gel column (20
by 4 cm), eluting with 1:4 hexane-EtOAc and then with EtOAc. Compound 4f
was obtained as a colorless foam: yield 0.94 g (62%); 13C NMR (acetone-d6) ␦
20.96, 21.04, 27.58, 34.38, 38.46, 71.45, 72.82, 79.69, 97.61, 123.70, 133.21, 134.89,
168.78, 170.12, and 170.95.
Heptakis [2,3-di-O-acetyl-6-deoxy-6-(8-phthalimidooctyl)thio]cyclomalto-
heptaose (compound 4g). To a mixture of 0.80 g (0.32 mmol) of peracetylated
6-iodo-␤-CD 3 and 2.78 g (6.72 mmol) of the thiuronium salt 2g in 30 ml of
anhydrous DMF was added 2.6 g (8.0 mmol) of Cs2CO3. The reaction mixture
was stirred at 23°C under argon for 64 h. The insoluble material was filtered, and
the solvent was concentrated under diminished pressure. The residue was par-
titioned between EtOAc (50 ml) and water (50 ml). The organic layer was
separated, dried (MgSO4), and concentrated under diminished pressure. The
crude product was dissolved in 24 ml of pyridine and 36 ml of Ac2O, and then 20
mg of 4-dimethylaminopyrididine was added and the reaction mixture was stirred
at 23°C under argon for 48 h. The reaction was quenched by slow addition of 35
ml of MeOH, and the solvent was concentrated under diminished pressure. The
residue was partitioned between water (75 ml) and EtOAc (65 ml). The organic
layer was dried (MgSO4) and concentrated under diminished pressure. The
crude product was purified on a silica gel column (20 by 4 cm), eluting with 1:4
hexane-EtOAc and then with EtOAc. Compound 4g was obtained as a colorless
foam: yield 0.85 g (73%); 13C NMR (acetone-d6) ␦ 20.35, 20.44, 27.05, 30.35,
33.87, 37.85, 71.06, 72.08, 79.00, 97.02, 123.04, 132.55, 134.24, 168.11, 169.46, and
170.37; mass spectrum (electrospray ionization [ESI]), m/z 3,665.0 [MϩNa]ϩ
(theoretical 3,660.2).
Heptakis [2,3-di-O-acetyl-6-deoxy-6-(9-phthalimidononyl)thio]cyclomalto-
heptaose (compound 4h). To a mixture of 0.80 g (0.32 mmol) of peracetylated
6-iodo-␤-CD 3 and 2.70 g (6.27 mmol) of the thiuronium salt 2h in 30 ml of
anhydrous DMF was added 2.6 g (8.0 mmol) of Cs2CO3. The reaction mixture
was stirred at 23°C under argon for 64 h. The insoluble material was filtered, and
the solvent was concentrated under diminished pressure. The residue was par-
titioned between EtOAc (50 ml) and water (50 ml). The organic layer was
separated, dried (MgSO4), and concentrated under diminished pressure. The
crude product was dissolved in 24 ml of pyridine and 36 ml of Ac2O, and then 20
Heptakis[2,3-di-O-acetyl-6-deoxy-6-(10-phthalimidodecyl)thio]cyclomalto-
heptaose (compound 4i). To a mixture of 0.80 g (0.32 mmol) of peracetylated
6-iodo-␤-CD 3 and 2.97 g (6.72 mmol) of the thiuronium salt 2i in 30 ml of
anhydrous DMF was added 2.6 g (8.0 mmol) of Cs2CO3. The reaction mixture
was stirred at 23°C under argon for 64 h. The insoluble material was filtered, and
the solvent was concentrated under diminished pressure. The residue was par-
titioned between EtOAc (50 ml) and water (50 ml). The organic layer was dried
(MgSO4) and concentrated under diminished pressure. The crude product was
dissolved in 24 ml of pyridine and 36 ml of Ac2O, and then 20 mg of 4-dimethyl-
aminopyridine was added and the reaction mixture was stirred at 23°C under
argon for 48 h. The reaction was quenched by slow addition of 35 ml of MeOH,
and the solvent was concentrated under diminished pressure. The residue was
partitioned between water (75 ml) and EtOAc (65 ml). The organic layer was
dried (MgSO4) and concentrated under diminished pressure. The crude product
was purified on a silica gel column (20 by 4 cm), eluting with 1:4 hexane-EtOAc
and then with EtOAc. Compound 4i was obtained as a yellow foam: yield 0.85 g
(69%); 13C NMR (acetone-d6) ␦ 20.35, 20.44, 27.02, 29.38, 30.12, 33.88, 37.85,
70.95, 72.11, 79.03, 96.98, 123.05, 132.56, 134.27, 168.13, 169.48, and 170.35.
Per-6-(5-aminopentylthio)-␤-cyclodextrin (cyclodextrin 5d). A suspension of
1.19 g (0.35 mmol) of cyclodextrin 4d in 20 ml of 1:1 H2O-EtOH was treated with
20 ml of hydrazine monohydrate and heated at 70°C for 18 h. The cooled
reaction mixture was concentrated under diminished pressure. The residue was
triturated with 40 ml of 1 N HCl, and the mixture was stirred at 23°C for 2 h. The
insoluble material was removed by centrifugation, and the supernatant was
treated with acetone until the product precipitated. The product was collected,
washed with three 20-ml portions of acetone, and dried under vacuum. Cyclo-
dextrin 5d was obtained as a colorless solid: yield 395 mg (53%); mp 192 to 194°C
(decomparition [decomp.]); 13C NMR (DMSO-d6) ␦ 26.12, 27.53, 29.81, 33.49,
34.08, 72.26, 73.46, 85.51, and 102.99.
Per-6-(6-aminohexylthio)-␤-cyclodextrin (cyclodextrin 5e). A suspension of
1.0 g (0.29 mmol) of cyclodextrin 4e in 15 ml of 1:1 H2O-EtOH was treated with
15 ml of hydrazine monohydrate and heated at 70°C for 20 h. The mixture was
cooled to room temperature, and the solvent was concentrated under diminished
pressure. The residue was triturated with 40 ml of 1 N HCl, and the mixture was
stirred at 23°C for 18 h. The insoluble material was removed by centrifugation,
and the supernatant was treated with acetone (200 ml, or until the product
precipitated). The product was collected, washed with three 25-ml portions of
acetone, and dried under vacuum. Cyclodextrin 5e was obtained as a colorless
solid: yield 523 mg (82%); mp 216 to 218°C (decomp.); 13C NMR (DMSO-d6) ␦
25.67, 26.86, 27.81, 29.13, 32.56, 33.09, 71.46, 72.22, 72.49, 84.47, and 101.97;
mass spectrum (ESI), m/z found 1,942.1 [M]ϩ (theoretical 1,941.7).
Per-6-(7-aminoheptylthio)-␤-cyclodextrin (cyclodextrin 5f). A suspension of
850 mg (0.24 mmol) of cyclodextrin 4f in 12 ml of 1:1 H2O-EtOH was treated
with 12 ml of hydrazine monohydrate and heated at 70°C for 18 h. The cooled
reaction mixture was concentrated under diminished pressure. The residue was
triturated with 40 ml of 1 N HCl, and the mixture was stirred at 23°C for 4 h. The
insoluble material was removed by centrifugation, and the supernatant was
treated with acetone until the product precipitated. The product was collected,
washed with three 20-ml portions of acetone, and dried under vacuum. Cyclo-
dextrin 5f was obtained as a yellow solid: yield 340 mg (61%); mp 186 to 188°C
(decomp.); 13C NMR (DMSO-d6) ␦ 26.65, 27.62, 28.96, 29.80, 33.48, 73.10, 85.21,
and 102.98.
Per-6-(8-aminooctylthio)-␤-cyclodextrin (cyclodextrin 5g). A suspension of
0.8 g (0.22 mmol) of cyclodextrin 4g in 12 ml of 1:1 H2O-EtOH was treated with
12 ml of hydrazine monohydrate and heated at 80°C for 14 h. The cooled
reaction mixture was concentrated under diminished pressure. The residue was
triturated with 25 ml of 1 N HCl, and the reaction mixture was stirred at 23°C for
2 h. The insoluble material was removed by centrifugation, and the supernatant
was treated with acetone (150 ml) or until the product precipitated. The product
was collected, washed with three 25-ml portions of acetone, and dried under
diminished pressure. Cyclodextrin 5g was obtained as a colorless solid: yield 433
mg (82%); mp 168 to 172°C (decomp.); 13C NMR (DMSO-d6) ␦ 26.95, 27.85,

VOL. 50, 2006
CYCLODEXTRIN-BASED INHIBITORS FOR ANTHRAX
3743
29.23, 29.60, 30.33, 33.73, 73.47, 85.53, and 102.98; mass spectrum (ESI), m/z
2,138.1 [M]ϩ (theoretical 2,138.0).
was added 0.8 ml of concentrated aqueous ammonia with stirring at 0°C. After
1 h, 400 mg (0.22 mmol) of compound 5a (15) was added and the reaction
mixture was stirred at 85°C for 72 h. The solvent was concentrated under
diminished pressure, and 2 to 3 ml of 1 N HCl was added with stirring for 1 h.
Acetone (40 ml) was then added, and the precipitated material was collected by
filtration. The product was washed with three 20-ml portions of acetone and
dried under vacuum. Cyclodextrin 9a was obtained as a tan powder: yield 240 mg
(53%); mp 182 to 184°C (decomp.); 13C NMR (DMSO-d6) ␦ 30.5, 31.2, 32.9,
33.9, 72.1, 73.4, 85.5, 103.0, and 158.1.
Per-6-(3-aminopropylthioguanidino)-␤-cyclodextrin (cyclodextrin 9b). To a
solution of 600 mg (4.31 mmol) of S-methylisothiourea hemisulfate in 15 ml of
H2O was added 0.6 ml of concentrated aqueous ammonia with stirring at 0°C.
After 1 h, 300 mg (0.158 mmol) of compound 5b (15) was added. The reaction
mixture was stirred at 85°C for 72 h. The solvent was concentrated under
diminished pressure, and 2 to 3 ml of 1 N HCl was added with stirring for 1 h.
Acetone (40 ml) was then added, and the precipitated material was collected by
filtration. The product was washed with three 20-ml portions of acetone and
dried under vacuum. Cyclodextrin 9b was obtained as a yellow foam: yield 150
mg (44%); 13C NMR (DMSO-d6) ␦ 27.7, 30.5, 31.5, 33.9, 38.6, 72.3, 73.0, 85.4,
102.9, and 155.5.
Per-6-(9-aminononylthio)-␤-cyclodextrin (cyclodextrin 5h). A suspension of
790 mg (0.21 mmol) of cyclodextrin 4h in 12 ml of 1:1 H2O-EtOH was treated
with 12 ml of hydrazine monohydrate and heated at 90°C for 15 h. The reaction
mixture was cooled to room temperature and then concentrated under dimin-
ished pressure. The residue was triturated with 25 ml of 1 N HCl, and the mixture
was stirred at 23°C for 2 h. The insoluble material was removed by centrifugation,
and the supernatant was treated with acetone (150 ml, or until the product
precipitated). The product was collected, washed with three 25-ml portions of
acetone, and dried under vacuum. Cyclodextrin 5h was obtained as a colorless
solid: yield 356 mg (67%); mp 182 to 186°C (decomp.); 13C NMR (DMSO-d6) ␦
26.29, 27.70, 29.16, 29.48, 30.24, 33.55, 73.05, 85.46, and 102.74; mass spectrum
(ESI), m/z 2,237.1 [MϩH]ϩ (theoretical 2,236.2).
Per-6-(10-aminodecylthio)-␤-cyclodextrin (cyclodextrin 5i). A suspension of
760 mg (0.19 mmol) of cyclodextrin 4i in 11 ml of 1:1 H2O-EtOH was treated
with 11 ml of hydrazine monohydrate and heated at 80°C for 15 h. The cooled
reaction mixture was concentrated under diminished pressure. The residue was
triturated with 15 ml of 1 N HCl, and the mixture was stirred at 23°C for 1 h. The
insoluble material was removed by centrifugation, and the supernatant was
treated with acetone (150 ml, or until the product precipitated). The product was
collected, washed with three 20-ml portions of acetone, and dried under vacuum.
Cyclodextrin 5i was obtained as a yellow solid: yield 174 mg (34%); mp 148 to
150°C (decomp.); 26.73, 27.68, 29.74, 33.57, 73.40, 85.43, and 102.86.
Per-6-(3-aminopropylthio)-␣-cyclodextrin (cyclodextrin 8b). To a mixture of
300 mg (0.141 mmol) of peracetylated 6-iodo-␣-CD 7 (29) and 800 mg (2.2
mmol) of the thiuronium salt 2b in 10 ml of anhydrous DMF was added 1.18 g
(3.62 mmol) of Cs2CO3. The reaction mixture was stirred at 23°C under argon for
48 h. The insoluble material was removed by filtration through Celite, and the
solvent was concentrated under diminished pressure. The residue was parti-
tioned between EtOAc (75 ml) and water (75 ml). The organic layer was con-
centrated under diminished pressure. The crude product was dissolved in 5 ml of
pyridine and 5 ml of Ac2O. A catalytic amount of dimethylaminopyridine was
added, and the reaction mixture was stirred at 23°C under argon for 3 days. The
reaction was quenched by slow addition of 3 ml of MeOH, and the solvent was
concentrated under diminished pressure. The residue was partitioned between
water (75 ml) and EtOAc (65 ml). The organic layer was dried (MgSO4) and
concentrated under diminished pressure. The crude product was purified on a
silica gel column (17 by 3 cm), eluting with EtOAc. A suspension of the purified
material in 6 ml of 1:1 H2O-EtOH was treated with 5 ml of hydrazine monohy-
drate and heated at 80°C for 16 h. The cooled reaction mixture was concentrated
under diminished pressure. The residue was triturated with 3 ml of 1 N HCl, and
the mixture was stirred at 23°C for 1 h. The insoluble material was removed by
centrifugation, and the supernatant was treated with acetone (150 ml, or until the
product precipitated). The product was collected, washed with three 20-ml por-
tions of acetone, and dried under vacuum. Cyclodextrin 8b was obtained as a
yellow oil: yield 60 mg (48%).
Per-6-(6-aminohexylthio)-␣-cyclodextrin (cyclodextrin 8e). To a mixture of
210 mg (0.098 mmol) of peracetylated 6-iodo-␣-CD 7 (29) and 610 mg (1.48
mmol) of the thiuronium salt 2e in 10 ml of anhydrous DMF was added 802 mg
(2.46 mmol) of Cs2CO3. The reaction mixture was stirred at 23°C under argon for
48 h. The insoluble material was removed by filtration through Celite, and the
solvent was concentrated under diminished pressure. The residue was parti-
tioned between EtOAc (75 ml) and water (75 ml). The organic layer was sepa-
rated, dried (MgSO4), and concentrated under diminished pressure. The crude
product was dissolved in 5 ml of pyridine and 5 ml of Ac2O. A catalytic amount
of dimethylaminopyridine was added, and the reaction mixture was stirred at
23°C under argon for 3 days. The reaction was quenched by slow addition of 2 ml
of MeOH, and the solvent was concentrated under diminished pressure. The
residue was partitioned between water (75 ml) and EtOAc (65 ml). The organic
layer was dried (MgSO4) and concentrated under diminished pressure. The
crude material was purified on a silica gel column (17 by 3 cm), eluting with
EtOAc. A suspension of the purified cyclodextrin in 6 ml of H2O-EtOH (1:1,
vol/vol) was treated with 5 ml of hydrazine monohydrate and heated at 80°C for
16 h. The cooled reaction mixture was concentrated under diminished pressure.
The residue was triturated with 2 ml of 1 N HCl, and the reaction mixture was
stirred at 23°C for 1 h. The insoluble material was removed by centrifugation, and
the supernatant was treated with acetone (150 ml, or until the product precipi-
tated). The product was collected, washed with three 20-ml portions of acetone,
and dried under vacuum. Cyclodextrin 8e was obtained as a yellow oil: yield 68
mg (36%).
Per-6-(4-aminobutylthioguanidino)-␤-cyclodextrin (cyclodextrin 9c). To a so-
lution of 390 mg (2.80 mmol) of S-methylisothiourea hemisulfate in 10 ml of H2O
was added 0.4 ml of concentrated aqueous ammonia with stirring at 0°C. After
1 h, 200 mg (0.10 mmol) of compound 5c (15) was added. The reaction mixture
was stirred at 85°C for 72 h. The solvent was concentrated under diminished
pressure, and 1 to 2 ml of 1 N HCl was added with stirring for 1 h. Acetone (40
ml) was then added, and the precipitated material was collected by filtration. The
product was washed with three 20-ml portions of acetone and dried under
vacuum. Cyclodextrin 9c was obtained as a yellow foam: yield 99 mg (43%); 13
C
NMR (DMSO-d6) ␦ 18.5, 26.1, 27.7, 30.7, 32.5, 56.0, 72.6, 73.1, 85.9, 102.3, and
158.1.
Per-6-(6-aminohexylthioguanidino)-␤-cyclodextrin (cyclodextrin 9e). To a so-
lution of 640 mg (4.60 mmol) of S-methylisothiourea hemisulfate in 20 ml of H2O
was added 0.6 ml of concentrated aqueous ammonia with stirring at 0°C. After
1 h, 360 mg (0.164 mmol) of compound 5e was added. The reaction mixture was
stirred at 85°C for 72 h. The solvent was concentrated under diminished pressure,
and 2 to 3 ml of 1 N HCl was added with stirring for 1 h. Acetone (40 ml) was
then added, and the precipitated material was collected by filtration. The product
was washed with three 20-ml portions of acetone and dried under vacuum.
Cyclodextrin 9e was obtained as a yellow foam: yield 196 mg (48%); 13C NMR
(DMSO-d6) ␦ 25.8, 27.9, 28.5, 29.4, 30.7, 32.8, 33.3, 71.6, 72.3, 72.6, 84.5, 102.1,
and 156.9.
2-{[2-(Bromomethyl)phenyl]methyl}-1H-isoindole-1,3(2H)-dione (phthal-
imide 11a). To a suspension of 10 g (37.8 mmol) of ␣,␣-dibromo-o-xylene
(compound 10a) in 40 ml of DMF was added 7.0 g (37.8 mmol) of potassium
phthalimide followed by 627 mg (3.78 mmol) of potassium iodide. The reaction
mixture was stirred at 100°C for 17 h. The cooled reaction mixture was diluted
with 200 ml of EtOAc and washed with four 200-ml portions of water. The
formed emulsion was removed by filtration through Celite. The organic layer was
separated, washed with 150 ml of brine, dried (MgSO4), and concentrated under
diminished pressure. The crude product was purified on a silica gel column (19
by 4.5 cm), eluting with EtOAc in hexane (10 to 50%). The product was isolated
in an impure form (contaminated with the unreacted starting material). It was
purified by trituration with cold MeOH. Phthalimide 11a was obtained as a
colorless solid: yield 2.44 g (20%); mp 153 to 155°C; 1H NMR (DMSO-d6) ␦ 4.94
(s, 2H), 4.97 (s, 2H), 7.26 (dd, 1H), 7.33 (dd, 2H, J ϭ 5.7, 3.6 Hz), 7.50 (dd, 1H,
J ϭ 5.7, 3.6 Hz), and 7.93 (m, 4H).
2-{[3-(Bromomethyl)phenyl]methyl}-1H-isoindole-1,3(2H)-dione (phthal-
imide 11b). To a suspension of 10 g (37.8 mmol) of ␣,␣-dibromo-m-xylene
(compound 10b) in 40 ml of DMF was added 7.0 g (37.8 mmol) of potassium
phthalimide followed by 627 mg (3.78 mmol) of potassium iodide. The reaction
mixture was stirred at 100°C for 16 h. The cooled reaction mixture was diluted
with 200 ml of EtOAc and washed with four 200-ml portions of water. The
formed emulsion was removed by filtration through Celite. The organic layer was
separated, washed with 150 ml of brine, dried (MgSO4), and concentrated under
diminished pressure. The crude product was purified on a silica gel column (19
by 4 cm), eluting with EtOAc in hexane (10 to 40%). The product was isolated
in an impure form (contaminated with unreacted starting material). It was
purified by trituration with cold MeOH. Phthalimide 11b was obtained as a
colorless solid: yield 3.6 g (28%); mp 146 to 148°C; 1H NMR (DMSO-d6) ␦ 4.69
(s, 2H), 4.78 (s, 2H), 7.25 to 7.39 (m, 4H), and 7.83 to 7.95 (m, 4H).
Per-6-(2-aminoethylthioguanidino)-␤-cyclodextrin (cyclodextrin 9a). To a so-
lution of 864 mg (6.20 mmol) of S-methylisothiourea hemisulfate in 20 ml of H2O

3744
KARGINOV ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
2-{[4-(Bromomethyl)phenyl]methyl}-1H-isoindole-1,3(2H)-dione (phthal-
imide 11c). To a suspension of 10 g (37.8 mmol) of ␣,␣-dibromo-p-xylene
(compound 10c) in 80 ml of DMF was added 7.0 g (37.8 mmol) of potassium
phthalimide followed by 627 mg (3.78 mmol) of potassium iodide. The reaction
mixture was stirred at 100°C for 18 h. The cooled reaction mixture was diluted
with 200 ml of EtOAc and washed with four 200-ml portions of water. The
formed emulsion was removed by filtration through Celite. The organic layer was
separated, washed with 150 ml of brine, dried (MgSO4), and concentrated under
diminished pressure. The crude product was purified on a silica gel column (20
by 4.5 cm), eluting with EtOAc in hexane (10 to 30%). Phthalimide 11c was
obtained as a colorless solid: yield 1.82 g (15%); mp 233 to 235°C; 1H NMR
(DMSO-d6) ␦ 4.68 (s, 2H), 4.78 (s, 2H), 7.36 (m, 4H), and 7.87 (m, 4H).
1-(Phthalimidomethyl)-2-[(isothiouronyl)methyl]benzene hydrobromide (thi-
uronium salt 12a). A suspension of 2.84 g (8.63 mmol) of phthalimide 11a and
624 mg (8.19 mmol) of thiourea was heated at reflux in 18 ml of EtOH for 18 h.
The solvent was concentrated under diminished pressure, and the residue was
triturated with 22 ml of acetone. The product was filtered, washed with two 10-ml
portions of acetone, and dried under vacuum. Thiuronium salt 12a was obtained
as a colorless solid: yield 2.15 g (61%); mp 214 to 216°C; 1H NMR (DMSO-d6)
␦ 4.71 (s, 2H), 4.95 (s, 2H), 7.28 (m, 1H), 7.35 (m, 2H), 7.48 (m, 1H), 7.93 (m,
4H), and 9.17 (br s, 4H).
1-(Phthalimidomethyl)-3-[(isothiouronyl)methyl]benzene hydrobromide (thi-
uronium salt 12b). A mixture of 2.59 g (7.84 mmol) of phthalimide 11b and 568
mg (7.47 mmol) of thiourea in 16 ml of EtOH was stirred at reflux for 18 h. The
solvent was concentrated, and the residue was triturated with 25 ml of Et2O and
filtered. Thiuronium salt 12b was obtained as a colorless solid: yield 0.97 g (30%);
mp 169 to 172°C; 1H NMR (DMSO-d6) ␦ 4.47 (s, 2H), 4.78 (s, 2H), 7.28 to 7.39
(m, 4H), 7.86 to 7.94 (m, 4H), 8.98 (br s, 2H), and 9.16 (br s, 2H).
1-(Phthalimidomethyl)-4-[(isothiouronyl)-methyl]benzene hydrobromide (thi-
uronium salt 12c). A mixture of 2.7 g (8.17 mmol) of phthalimide 11c and 593 mg
(7.78 mmol) of thiourea in 16 ml of EtOH was stirred at reflux for 18 h, at which
time the product was precipitated. The cooled reaction mixture was filtered. The
product was washed with three 25-ml portions of acetone and dried under
vacuum. Thiuronium salt 12c was obtained as a colorless solid: yield 2.8 g (84%);
mp 230 to 232°C; 1H NMR (DMSO-d6) ␦ 4.49 (s, 2H), 4.80 (s, 2H), 7.38 (m, 4H),
7.91 (m, 4H), and 9.09 (br s, 4H).
with EtOAc. Cyclodextrin 13b was obtained as a yellow foam: yield 835 mg
(51%); 13C NMR (acetone-d6) ␦ 20.40, 20.49, 33.32, 37.51, 41.37, 71.03, 72.35,
79.27, 97.15, 123.30, 126.81, 128.56, 128.62, 128.98, 129.23, 132.43, 134.38, 134.50,
137.25, 139.69, 167.90, 169.51, and 170.37; mass spectrum (MALDI), m/z 3,608.2
[Mϩ4HϩNa]ϩ (theoretical 3,607.8).
Heptakis{2,3-di-O-acetyl-6-deoxy-6-[4-(phthalimidomethyl)-benzyl]-thio}-
cyclomaltoheptaose (cyclodextrin 13c). To a mixture of 332 mg (0.13 mmol) of
peracetylated 6-iodo-␤-CD and 975 mg (2.39 mmol) of the thiuronium salt 12c in
13 ml of anhydrous DMF was added 1.08 g (3.32 mmol) of Cs2CO3. The reaction
mixture was stirred at 23°C under argon for 48 h. The insoluble material was
filtered, and the solvent was concentrated under diminished pressure. The res-
idue was partitioned between EtOAc (50 ml) and water (50 ml). The organic
layer was separated, dried (MgSO4), and concentrated under diminished pres-
sure. The crude product was dissolved in 8 ml of pyridine and 12 ml of Ac2O, and
then 10 mg of 4-dimethylaminopyridine was added and the reaction mixture was
stirred at 40°C under argon for 20 h. The cooled reaction mixture was quenched
by slow addition of 25 ml of MeOH, and the solvent was concentrated under
diminished pressure. The residue was partitioned between water (45 ml) and
EtOAc (30 ml). The organic layer was dried (MgSO4) and concentrated under
diminished pressure. The crude product was purified on a silica gel column (16
by 3 cm), eluting with EtOAc. Cyclodextrin 13c was obtained as a colorless foam:
yield 178 mg (37%); 13C NMR (acetone-d6) ␦ 20.34, 20.44, 33.51, 37.34, 41.12,
70.98, 72.07, 78.88, 96.96, 123.24, 128.43, 129.51, 132.42, 134.32, 135.70, 138.61,
167.88, 169.44, and 170.31; mass spectrum (MALDI), m/z 3,604.5 [MϩNa]ϩ
(theoretical 3,603.8).
Per-6-S-[(2-aminomethyl)benzyl]-␤-cyclodextrin hydrochloride (cyclodextrin
14a). A suspension of 600 mg (0.17 mmol) of cyclodextrin 13a in 10 ml of 1:1
H2O-EtOH was treated with 10 ml of hydrazine monohydrate and heated at 70°C
for 18 h. The cooled reaction mixture was concentrated under diminished pres-
sure. The residue was triturated with 30 ml of 1 N HCl, and the mixture was
stirred at 23°C for 3 h. The insoluble material was removed by centrifugation, and
the supernatant was treated with acetone until the product precipitated. The
product was collected, washed with three 20-ml portions of acetone, and dried
under vacuum. Cyclodextrin 14a was obtained as a colorless solid: 306 mg (78%);
mp 185 to 187°C (decomp.); 13C NMR (DMSO-d6) ␦ 33.14, 35.02, 72.82, 73.05,
73.23, 85.18, 102.92, 128.13, 128.94, 130.10, 131.27, 133.14, and 137.46; mass
spectrum (MALDI), m/z 2,082.0 [Mϩ] (theoretical 2,081.6).
Heptakis{2,3-di-O-acetyl-6-deoxy-6-[2-(phthalimidomethyl)-benzyl]-thio}-
cyclomaltoheptaose (compound 13a). To a mixture of 0.80 g (0.32 mmol) of
peracetylated 6-iodo-␤-CD 3 and 2.78 g (6.72 mmol) of the thiuronium salt 12a
in 30 ml of anhydrous DMF was added 2.6 g (8.0 mmol) of Cs2CO3. The mixture
was stirred at 23°C under argon for 64 h. The insoluble material was filtered, and
the solvent was concentrated under diminished pressure. The residue was par-
titioned between EtOAc (50 ml) and water (50 ml). The organic layer was dried
(MgSO4) and concentrated under diminished pressure. The crude product was
dissolved in 24 ml of pyridine and 36 ml of Ac2O, and then 20 mg of 4-dimethyl-
aminopyridine was added and the reaction mixture was stirred at 23°C under
argon for 48 h. The reaction was quenched by slow addition of 35 ml of MeOH,
and the solvent was concentrated under diminished pressure. The residue was
partitioned between water (75 ml) and EtOAc (65 ml). The organic layer was
dried (MgSO4) and concentrated under diminished pressure. The crude product
was purified on a silica gel column (20 by 4 cm), eluting with 1:4 hexane-EtOAc
and then EtOAc. Compound 13a was obtained as a colorless foam: yield 0.85 g
(73%); 1H NMR (CDCl3) ␦ 1.97 (s, 3H), 2.05 (s, 3H), 2.06 (m, 2H), 4.00 (m, 2H),
4.18 (m, 2H), 4.79 (m, 3H), 5.06 (d, 1H, J ϭ 3.9 Hz), 5.26 (t, 1H, J ϭ 8.8 Hz), 6.90
(m, 2H), 7.12 (m, 2H), and 7.52 (m, 4H); 13C NMR (acetone-d6) ␦ 21.03, 21.10,
34.44, 36.27, 39.24, 71.63, 73.00, 79.51, 97.91, 123.84, 128.26, 128.43, 129.58,
131.62, 133.00, 134.92, 136.16, 137.15, 168.65, 170.18, and 170.96; mass spectrum
(MALDI), m/z 3,604.8 [MϩHϩNa]ϩ (theoretical 3,604.8).
Heptakis{2,3-di-O-acetyl-6-deoxy-6-[3-(phthalimidomethyl)-benzyl]-thio}-
cyclomaltoheptaose (cyclodextrin 13b). To a mixture of 1.14 g (0.45 mmol) of
peracetylated 6-iodo-␤-CD 3 and 3.9 g (9.60 mmol) of the thiuronium salt 12b in
45 ml of anhydrous DMF was added 3.65 g (11.2 mmol) of Cs2CO3. The reaction
mixture was stirred at 23°C under argon for 48 h. The insoluble material was
filtered, and the solvent was concentrated under diminished pressure. The res-
idue was partitioned between EtOAc (50 ml) and water (50 ml). The organic
layer was dried (MgSO4) and concentrated under diminished pressure. The
crude product was dissolved in 26 ml of pyridine and 40 ml of Ac2O, and then 20
mg of 4-dimethylaminopyridine was added and the reaction mixture was stirred
at 40°C under argon for 48 h. The cooled reaction mixture was quenched by slow
addition of 50 ml of MeOH, and the solvent was concentrated under diminished
pressure. The residue was partitioned between water (50 ml) and EtOAc (50 ml).
The organic layer was dried (MgSO4) and concentrated under diminished pres-
sure. The crude product was purified on a silica gel column (20 by 4 cm), eluting
Per-6-S-[(3-aminomethyl)-benzyl]-␤-cyclodextrin hydrochloride (cyclodextrin
14b). A suspension of 757 mg (0.21 mmol) of cyclodextrin 13b in 12 ml of 1:1
H2O-EtOH was treated with 12 ml of hydrazine monohydrate and heated at 70°C
for 18 h. The cooled reaction mixture was concentrated under diminished pres-
sure. The residue was triturated with 40 ml of 1 N HCl, and the mixture was
stirred at 23°C for 21 h. The insoluble material was removed by centrifugation,
and the supernatant was treated with acetone until the product precipitated. The
product was collected, washed with three 20-ml portions of acetone, and dried
under vacuum. Cyclodextrin 14b was obtained as a colorless solid: yield 316 mg
(64%); mp 190 to 192°C (decomp.); 13C NMR (DMSO-d6) ␦ 33.82, 37.61, 42.90,
72.90, 73.22, 73.37, 85.40, 103.06, 128.15, 129.46, 129.79, 130.25, 134.98, and
139.90; mass spectrum (MALDI), m/z 2,082.0 [Mϩ] (theoretical 2,081.6).
Per-6-S-[(4-aminomethyl)-benzyl]-␤-cyclodextrin hydrochloride (cyclodextrin
14c). A suspension of 144 mg (0.04 mmol) of cyclodextrin 13c in 3 ml of 1:1
H2O-EtOH was treated with 3 ml of hydrazine monohydrate and heated at 65°C
for 19 h. The cooled reaction mixture was concentrated under diminished pres-
sure. The residue was triturated with 8 ml of 1 N HCl, and the mixture was stirred
at 23°C for 22 h. The insoluble material was removed by centrifugation, and the
supernatant was treated with acetone until the product precipitated. The product
was collected, washed with three 10-ml portions of acetone, and dried under
vacuum. Cyclodextrin 14c was obtained as a colorless solid: 66 mg (70%); mp 190
to 192°C (decomp.); 13C NMR (DMSO-d6) ␦ 33.77, 37.46, 42.60, 72.79, 73.07,
73.20, 85.37, 102.94, 129.74, 133.19, and 139.68; mass spectrum (MALDI), m/z
2,082.4 [Mϩ] (theoretical 2,081.6).
Hexakis [6-deoxy-6-amino]-␣-cyclodextrin (compound 16). To a mixture of
202 mg (0.124 mmol) of hexakis [2,3-di-O-acetyl-6-deoxy-6-amino]-␣-cyclodex-
trin (29) in 7 ml of dry DMF was added 525 mg (2.0 mmol) of PPh3 with stirring
at 23°C for 1.5 h. Concentrated aqueous ammonia was added dropwise until the
solution became a white suspension (7 ml), and the resulting mixture was stirred
for 18 h. The mixture was concentrated, and ethanol was added to form a white
precipitate. The solid was filtered, washed twice with ethanol, and dried to give
a white solid: yield 75 mg (55%); mass spectrum m/z 989.3 [MϩNa]ϩ (theoretical
989.4).
Cells and cell culture. The murine RAW 264.7 monocyte/macrophage cell line
(ATCC TIB-71) and the Chinese hamster ovary (CHO-K1) cell line (ATCC
CCL-61) were obtained from the American Type Culture Collection (Manassas,

VOL. 50, 2006
CYCLODEXTRIN-BASED INHIBITORS FOR ANTHRAX
TABLE 1. Activities of the compounds in this study
3745
Inhibition of cytotoxicity
Inhibition of transmembrane
conductance (IC50 [nM])a
Group and compound no.
R
(IC50 [␮M])b
Hepta-6-aminoalkyl ␤-cyclodextrin derivatives
15
5a
5b
5c
5d
5e
5f
5g
5h
5i
NH2
12.1 Ϯ 3.5
7.8 Ϯ 2.4
2.9 Ϯ 1.0
5.1 Ϯ 2.4
7.5 Ϯ 2.4
0.6 Ϯ 0.3
1.9 Ϯ 1.1
0.3 Ϯ 0.1
0.8 Ϯ 0.1
2.6 Ϯ 0.7
32 Ϯ 15
3.5 Ϯ 0.9
0.6 Ϯ 0.4
1.1 Ϯ 0.5
3.8 Ϯ 1.0
1.0 Ϯ 0.4
4.6 Ϯ 3.2
2.4 Ϯ 1.0
14.7 Ϯ 9.7
27.0 Ϯ 17.0
S(CH2)2NH2
S(CH2)3NH2
S(CH2)4NH2
S(CH2)5NH2
S(CH2)6NH2
S(CH2)7NH2
S(CH2)8NH2
S(CH2)9NH2
S(CH2)10NH2
Hepta-6-aminoalkyl ␣-cyclodextrin derivatives
16
8b
8e
NH2
ϪS(CH2)3NH2
ϪS(CH2)6NH2
Ͼ400
Ͼ400
160 Ϯ 60
1,300 Ϯ 500
10.9 Ϯ 2.5
Hepta-6-guanidinealkyl ␤-cyclodextrin derivatives
9a
8.9 Ϯ 6.0
5.3 Ϯ 3.2
9b
9c
9e
12.2 Ϯ 2.9
3.8 Ϯ 2.3
2.3 Ϯ 0.4
12.6 Ϯ 9.0
Hepta-6-arylamine ␤-cyclodextrin derivatives
17
Ͼ200
18
2.3 Ϯ 1.2
Hepta-6-alkylarylamine ␤-cyclodextrin derivatives
14a
1.7 Ϯ 0.4
14b
14c
0.5 Ϯ 0.2
0.7 Ϯ 0.5
0.07 Ϯ 0.05
a All data were calculated as averages from three to seven experiments; the errors are root mean square deviations.

3746
KARGINOV ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
VA). Raw 264.7 cells were cultured in phenol red-free Dulbecco’s modified
Eagle’s medium (DMEM) (Mediatech, Inc., Herndon, VA) supplemented with
10% heat-inactivated fetal bovine serum, 100 U/ml:100 ␮g/ml penicillin-strepto-
mycin, 0.1 mM nonessential amino acids, and 0.5 mM 2-mercaptoethanol at 37°C
in a 5% CO2 atmosphere. The cells were harvested by gentle scraping with a cell
scraper and were then washed once with media. RAW 264.7 cells were plated in
96-well, flat-bottomed tissue culture plates from Becton Dickinson (San Jose,
CA) at a concentration of 105 cells/well in the DMEM mentioned above and
incubated overnight at 37°C in 5% CO2. The CHO-K1 cell line was maintained
in Kaighn’s modification of Ham’s F-12 medium (F-12K) (ATCC 30-2004) sup-
plemented with 10% heat-inactivated fetal bovine serum and 100 U/ml:100
␮g/ml penicillin-streptomycin in 5% CO2 at 37°C. The CHO-K1 cells were
harvested by a brief wash with phosphate-buffered saline (Ca2ϩ and Mg2ϩ free)
and then addition of 0.05% trypsin-EDTA (Mediatech). F-12K medium was then
FIG. 2. Treatment of the N-bromoalkylphthalimides (1a to 1i) with
thiourea in EtOH at reflux to afford the corresponding thiuronium
salts (2a to 2i) in excellent yields. Compound designations are shown
in bold type.
FIG. 3. Synthesis of cyclodextrins 4d to 4i and 5d to 5i.

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CYCLODEXTRIN-BASED INHIBITORS FOR ANTHRAX
3747
FIG. 4. Synthesis of two ␣-cyclodextrins, done in order to explore the importance of sevenfold versus sixfold symmetry.
added, and the cells were washed once. The cells were plated in 24-well plates at
a concentration of 106 cells/well and incubated overnight at 37°C in 5% CO2.
Cytotoxicity neutralization assay. RAW 264.7 cells were preincubated with
different concentrations of tested compounds in DMEM for 1 h at 37°C in a 5%
CO2 atmosphere. Then, DMEM or LeTx (LF, 32 ng/ml; PA, 500 ng/ml) in the
medium was added, and the plate was incubated under the same conditions for
4 h. Cell viability was monitored using CellTiter 96 AQueous non-radioactive cell
proliferation assay (MTS) kit from Promega (Madison, WI). A ␮ Quant spectro-
photometer from Bio-Tek Instruments, Inc. (Winooski, VT), was used for readings
of optical density at 570 nm.
volume. Different aspects of cyclodextrin chemistry, such as a recently reported
modification of the polymer surface by the incorporation of cyclodextrins (9) and
an undesirable reaction of aqueous cyclodextrin solutions with polypropylene
(24), suggest the possible loss of cyclodextrins in low-cyclodextrin-containing
solutions due to adsorption or other kinds of interaction with the tubes or tips
surfaces. To minimize this problem, low-binding-polymer technology products
were used for cyclodextrin storage, dilution, and sampling (Sorenson BioScience,
Inc., Salt Lake City, UT).
RESULTS AND DISCUSSION
cAMP immunoassay. The CHO-K1 cells were preincubated with different
concentrations of tested compounds in F-12K medium for 30 min at 37°C in 5%
CO2. Medium or EdTx (EF, 500 ng/ml; PA, 500 ng/ml) was added, and the plate
was incubated for an additional 4 h. After incubation, the medium/EdTx was
removed and 0.1 M HCl plus 0.5% Triton X-100 was added to each well to lyse
the cells. The plate was centrifuged at 600 ϫ g for 10 min. The supernatants were
collected, and cyclic AMP (cAMP) production was evaluated using a cAMP
immunoassay kit purchased from R&D Systems, Inc. (Minneapolis, MN).
Channel reconstitution assay. Channel reconstitution experiments were per-
formed as described previously (22). To form “solvent-free” planar lipid bilayers
with the lipid monolayer opposition technique (21), we used a 5% solution of
diphytanoyl phosphatidylcholine in pentane. Membranes were formed on a 60-
␮m-diameter (for single-channel measurements) or a 150-␮m-diameter (for mul-
tichannel measurements) aperture in the 15-␮m-thick Teflon film that separated
two compartments. PA63 was prepared from PA83 by limited trypsin digestion
(23) or purchased directly from List Biological Laboratories, Inc. (Campbell,
CA), in the purified form. For multichannel experiments, about 1 to 2 ␮l of
0.2-mg/ml stock PA63 was applied to the cis side of the membrane. Single
channels were formed by adding 0.5 to 1 ␮l of 20-␮g/ml stock solution of PA63
to 1.5 ml aqueous phase in the cis half of the chamber. For all experiments, the
symmetrical solutions of 0.1 M KCl (multichannel) or 1.0 M KCl (single channel)
at pH 6.6 were employed.
Under this protocol, PA channel insertion was always directional. The elec-
trical potential difference across the bilayer lipid membrane was applied with a
pair of Ag-AgCl electrodes in 2 M KCl, 1.5% agarose bridges. All reported
multichannel experiments were performed at 20-mV applied voltage, positive
from the side of PA63 addition (cis side).
Conductance measurements were done using an Axopatch 200B amplifier
(Axon Instruments, Inc., Foster City, CA) in the voltage clamp mode. Signals
were filtered by a low-pass, eight-pole Butterworth filter (model 9002; Frequency
Devices, Inc., Haverhill, MA) at 15 Hz and 15 kHz for multichannel and single-
channel measurements, correspondingly, and directly saved into the computer
memory with sampling frequencies of 50 Hz and 50 kHz for multichannel and
single-channel experiments, respectively.
Chemistry. ␤-Cyclodextrin is a naturally occurring cyclooli-
gosaccharide containing seven ␣-(1,4)-D-glucopyranose sub-
units linked through ␣-(1,4) glucosidic bonds (28). The primary
(C-6) and secondary (C-2 and C-3) hydroxyl groups may be
used as points of functionalization (Fig. 1). The hydroxyl
groups at positions 2 and 3 form hydrogen bonds and are
required to keep the molecule rigid, making the 6-OH group a
favorable site for modifications. Here, we synthesized a series
of hepta-6-thioaminoalkyl and hepta-6-thioguanidinoalkyl de-
rivatives of ␤-cyclodextrin with alkyl spacers of various lengths
and tested them for the ability to inhibit the cytotoxicity of
LeTx as well as to block ion conductance through PA channels
reconstituted in planar bilayer lipid membranes. The com-
pounds synthesized and tested in this study are presented in
Table 1.
Our synthetic approach involved the preparation of thio-
lated alkylphthalimide building blocks in order to perform
attachment to per-6-iodo-␤-CD by nucleophilic substitution.
Thus, the N-bromoalkylphthalimides (1a to 1i) were treated
with thiourea in EtOH at reflux to afford the corresponding
thiuronium salts (2a to 2i) in excellent yields (Fig. 2). The use
of thiuronium salts provides an advantage in that these salts
are stable solids. The synthesis of the fully protected cyclodex-
trins (4d to 4i), in which the C-6 position of the sugar is linked
to alkylphthalimide through a sulfur atom, was carried out as
shown in Fig. 3. Linkage of the alkylphthalimide moiety was
achieved by reaction of the corresponding pseudothioureas (2d
to 2i) with peracetylated 6-iodo-␤-CD 3. Cyclodextrins 4d to 4i
were obtained in good yields (62 to 75%) when the reactions
were performed in DMF in the presence of Cs2CO3 at room
At low (nM) cyclodextrin concentrations, a certain nonadditivity of cyclodex-
trin additions was observed. For example, in some cases the inhibitory effect on
channel conductance was more significant with 1 ␮l of 1 ␮M cyclodextrin stock
solution addition than with 10 ␮l of 100 nM solution addition to the same

3748
KARGINOV ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 5. Cyclodextrins 5a to 5c and 5e, which were successfully guanidinylated with S-methylisothiourea hemisulfate and treated with 1 N HCl
to afford ␤-cyclodextrin derivatives 9a to 9c and 9e as the hydrochloride salts.
temperature. Simultaneous hydrazinolysis and deacetylation of
cyclodextrins 4d to 4i were carried out by heating with hydra-
zine hydrate in aqueous EtOH overnight. The fully depro-
tected cyclodextrins 5d to 5i were treated with 1 N HCl and
collected by precipitation with acetone. Cyclodextrins 5d to 5i
were obtained in 35 to 80% yields as the hydrochloride salts.
The synthesis of several ␣-cyclodextrins was also completed to
explore the importance of sevenfold versus sixfold symmetry
(Fig. 4). Compounds 8b and 8e were obtained as the hydro-
chloride salts by use of the same methods outlined for the
synthesis of ␤-cyclodextrins, starting from the known hexakis
(2,3-di-O-acetyl-6-deoxy-6-iodo)-␣-cyclodextrin 7 (29).
The aminoethyl (compound 5a), aminopropyl (compound
5b), aminobutyl (compound 5c), and aminohexyl (compound
5e) derivatives of ␤-cyclodextrin (compound 8) were success-
fully guanidinylated with S-methylisothiourea hemisulfate and
treated with 1 N HCl to afford ␤-cyclodextrin derivatives 9a to
9c and 9e as the hydrochloride salts (Fig. 5). The structures of
the final compounds were characterized only by 13C NMR and
MALDI mass spectrometry since the 1H NMR spectra showed
broad and unresolved signals in DMSO-d6 at room tempera-
ture.
were introduced to the ortho, meta, and para positions of the
phenyl ring.
␣,␣-Dibromoxylenes 10a to 10c were treated with potassium
phthalimide in DMF to give compounds 11a to 11c in 20 to
30% yields (Fig. 6). Treatment of phthalimides 11a to 11c with
thiourea in EtOH at reflux afforded isothioureas 12a to 12c in
61, 30, and 84% yields, respectively. Compounds 12a to 12c
reacted smoothly in DMF in the presence of Cs2CO3 with
peracetylated 6-iodo-␤-CD 3 to give the desired products (13a,
13b, and 13c) (Fig. 7). Finally, simultaneous removal of the
phthalimide and acetate groups by use of a large excess of
hydrazine monohydrate in aqueous EtOH gave the desired
products (14a to 14c), isolated as the hydrochlorides. The
structures and symmetrical substitution have been confirmed
by mass spectrometry and 13C NMR spectroscopy.
Activities of hepta-6-aminoalkyl ␤-cyclodextrin derivatives.
Earlier, we synthesized and tested a group of hepta-6-amino-
alkyl derivatives of ␤-cyclodextrin that successfully blocked the
action of anthrax lethal toxin (13, 15). That study showed that
the inhibitory activity of the derivatives depended on hydro-
carbon chain length. To further investigate this phenomenon,
we compared aminoalkyl ␤-cyclodextrin derivatives with hy-
drocarbon chains having up to 10 carbons. Table 1 summarizes
the experimental data on the inhibitory activity of cationic
cyclodextrins against lethal toxin-mediated cytotoxicity (Table
In order to define structure-activity relationships, another
set of compounds were also prepared, incorporating a rigid
spacer in lieu of the alkyl chain. Primary aminomethyl groups
FIG. 6. Treatment of ␣,␣-dibromoxylenes 10a to 10c with potassium phthalimide in DMF to give compounds 11a to 11c and treatment of
phthalimides 11a to 11c with thiourea in EtOH to afford isothioureas 12a to 12c.

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CYCLODEXTRIN-BASED INHIBITORS FOR ANTHRAX
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FIG. 7. Treatment of compounds 12a to 12c with peracetylated 6-iodo-␤-CD 3 in DMF in the presence of Cs2CO3 to give the products 13a to
13c. Subsequent simultaneous removal of the phthalimide and acetate groups by use of a large excess of hydrazine monohydrate in aqueous EtOH
gave the products 14a to 14c.
1, column 3) and the blocking activity studied by channel re-
constitution (Table 1, column 4). The cytotoxicity inhibition
data showed a general trend toward the increasing of the
inhibitory activity against lethal toxin with increasing alkyl
chain length. A possible interpretation is that the increase in
the overall radius of the aminoalkyl ␤-cyclodextrins provides a
better geometric fit to the binding site in the channel lumen.
These experiments also showed that compounds 15 and 5a
to 5f were not toxic to RAW 264.7 cells up to a 50 to 100 ␮M
concentration, while their 50% inhibitory concentration (IC50)
values were as low as 0.6 to 12 ␮M. The higher hydrocarbon
homologues 5g to 5i exhibited a very potent activity against
lethal toxin (0.3 to 2.6 ␮M) but also demonstrated an increased
toxicity against RAW 264.7 cells (with 50% cytotoxic concen-
tration values as low as 12 to 25 ␮M).
These compounds were also tested for the ability to block
ion conductance through the PA63 channels reconstituted in
planar bilayer lipid membrane (Table 1, column 4) in 0.1 M
KCl solutions at pH 6.6. Figure 8 (top) gives a typical titration
curve in a multichannel experiment with compound 5b as a

3750
KARGINOV ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 9. Typical multichannel titration curves calculated from data
similar to those shown in Fig. 8. The applied voltage was ϩ20 mV;
membrane-bathing solution was 0.1 M KCl.
FIG. 8. Conductance of the membranes containing multiple PA
channels as functions of compound 5b and 5i concentrations in the cis
side of the chamber. The current record was filtered by averaging over
100-ms time intervals. It is shown that compound 5i causes membrane
instability, inducing current increase and rupture already at submicro-
molar concentrations. Similar destabilizing effects were detected for
the other two compounds with long aminoalkyl chains, 5g and 5h. The
applied voltage was ϩ20 mV; membrane-bathing solution was 0.1
M KCl.
able to change the actual concentration of the compound at
the channel entrance quite significantly.
Activities of hepta-6-aminoalkyl ␣-cyclodextrin derivatives.
To address the importance of the size and sevenfold symmetry
of ␤-cyclodextrin derivatives for strength of interaction with
heptameric PA63 pores, we synthesized aminoalkyl derivatives
of ␣-cyclodextrin, a molecule similar to ␤-cyclodextrin but hav-
ing sixfold symmetry (see compounds 16, 8b, and 8e in Table
1). When tested with RAW 264.7 cells (Table 1, column 3),
cationic ␣-cyclodextrins displayed no (compounds 16 and 8b)
or weak (compound 8e) inhibitory activity against anthrax le-
thal toxin. Furthermore, the ion conductance studies (Table 1,
column 4) show that compounds 16 and 8e block PA-induced
ion conductance 40 and 11 times less effectively than com-
pounds 15 and 5e, respectively (compare, e.g., curves 16 and 15
in Fig. 9).
blocker. Titration curves obtained for other compounds looked
very similar except for the higher hydrocarbon homologues
(compounds 5g to 5i), where we observed membrane instability
(current increase) with the subsequent rupture (in 9 out of 10
experiments) at cyclodextrin concentrations ranging from 2
nM to 0.8 ␮M. Figure 8 (bottom) also shows an example of
such a titration curve for compound 5i. This finding suggests
that the higher hydrocarbon homologues interact with the lipid
bilayer by adsorbing to its surface and destabilizing its struc-
ture. It is quite possible that the destabilizing effect found in
the channel reconstitution assay is related to the increased
toxicity of these compounds revealed by experiments with
RAW 264.7 cells.
Activities of hepta-6-guanidinealkyl ␤-cyclodextrin deriva-
tives. In order to check the effects of the type of positive charge
on the PA63 pore blockage, we synthesized and tested a group
In the aggregate, the data obtained in the channel reconsti-
tution assay support the conclusion that cyclodextrin inhibitory
activity is related to the interaction of the compounds with the
PA63 channel. Indeed, the interaction increases with the
lengthening of the hydrocarbon chain: compounds 5b (IC50,
0.6 Ϯ 0.4 nM), 5c (IC50, 1.1 Ϯ 0.5 nM), and 5e (IC50, 1.0 Ϯ 0.4
nM) are about 30 to 50 times more effective than the smallest
compound, compound 15 (IC50, 32 Ϯ 15 nM). In Fig. 9, we
illustrate this observation by comparing the inhibition of PA-
induced conductance by compound 5b with that by compound
15. The increase in IC50 values for the long-chain derivatives
(compounds 5g to 5i) may be related to the depletion of the
free compound concentration at the membrane surface as a
consequence of adsorption to the membrane. These kinds of
factors are difficult to explain quantitatively, but it is clear that
at low nominal concentrations (pM and nM) the adsorption is
FIG. 10. Protection of RAW 264.7 cells from LeTx-induced cell
death by compound 14b. RAW 264.7 cells were incubated with differ-
ent concentrations of the ␤-CD derivative with or without LeTx. Each
experimental condition was performed in triplicate. Cell viability was
determined by an MTS colorimetric assay. Error bars represent stan-
dard deviations. OD, optical density.

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3751
FIG. 12. Inhibition of EdTx activity by compound 5b. CHO-K1
cells were incubated with different concentrations of the compound
with or without EdTx. cAMP production was determined using a
cAMP immunoassay kit. Each experimental condition was performed
in duplicate. Error bars represent standard deviations.
Activities of hepta-6-arylamine ␤-cyclodextrin derivatives.
To test the importance of the positive charge, we designed
compound 17, in which the arylamine group is less basic due to
the delocalization of the nonbonding electrons from the nitro-
gen atoms to the aromatic ring. Remarkably, this compound
did not display any significant inhibitory activity in the cell-
based assay (Table 1, column 3). At the same time, compound
18, with a similar structure but an additional CH2 group sep-
arating the nitrogen atom from the aromatic ring, displayed
potent inhibitory activity (IC50, 2.3 Ϯ 1.2 ␮M) against anthrax
lethal toxin. The last finding is even more interesting taking
into account that a related compound, compound 15, with
unsubstituted amino groups, was more than five times less
active than compound 18, which suggests a potential role for
aromatic groups in binding to the PA pore lumen. Recently,
Krantz and colleagues (17) found a “major conductance-block-
ing site” for hydrophobic cations. This binding site is formed by
seven phenylalanine-427 residues (so-called ␸ clamp), gener-
ating a radially symmetric heptad of solvent-exposed aromatic
rings. The favorable interaction of substances with aromatic
moieties with the ␸ clamp has also been shown. Our data show
the benefits of introducing the aromatic ring to the alkyl chains
of cyclodextrin derivatives.
Activities of hepta-6-alkylarylamine ␤-cyclodextrin deriva-
tives. To study the possible involvement of the aromatic groups
in cyclodextrin binding to the PA channel lumen, we designed
and tested a group of compounds with the phenyl rings in-
serted into the alkyl chains (compounds 14a to 14c). All three
compounds demonstrated potent inhibitory activities against
anthrax lethal toxin, having IC50 values as low as 0.5 to 1.7 ␮M.
At the same time, they displayed no signs of toxicity toward
RAW 264.7 cells up to a 100 ␮M concentration (Fig. 10).
One of the compounds from this group (compound 14b) has
been tested for the ability to block the current through the PA
channel on both the multichannel and the single-channel level.
Interestingly, in multichannel experiments this compound dis-
played extremely potent blocking activity, with an IC50 as low
as 70 Ϯ 50 pM, which is almost 1 order of magnitude more
effective than compound 5b, which had an IC50 of 0.6 Ϯ 0.4
FIG. 11. (A) Current recordings of a single PA channel at different
concentrations of compounds 14b and 5b in 1 M KCl solution. In the
absence of cyclodextrins, the ion movement is determined mainly by
the geometry and the surface properties of the pore (topmost tracks).
The conductance and noise difference between the two PA channels
presented here are within regular divergence between different exper-
iments (18). Fast flickering between open and closed states inherent to
PA channels (so-called voltage gating [10]) was mainly removed by
averaging over a time interval of 100 ms. In the presence of cationic
cyclodextrins in the cis side of the chamber, the channel is spontane-
ously blocked, with the frequency of blockages depending on com-
pound concentration. Transmembrane voltage of 50 mV was positive
from the side of protein addition. Compound 14b was used at a 120 nM
concentration and compound 5b at 100 nM. (B) Typical statistical
analyses of ␤-CD-induced blockages (see reference 27 for details). The
fits represent “variable metric” as a search method and “maximum
likelihood” as a minimization method. Note the difference in the
residence times for compounds 14b and 5b.
of hepta-6-guanidine ␤-cyclodextrin derivatives in which posi-
tive charges are distributed between the nitrogens of the gua-
nidine moiety (see compounds 9a to 9e in Table 1). The ac-
tivities of these compounds were quite comparable to those of
the aminoalkyl ␤-cyclodextrins with one possible exception,
namely, compound 9b, which was less active than the closely
related amino derivative compound 5b. This was shown both
with RAW 264.7 cells (Table 1, column 3) and in the channel
reconstitution assay (Table 1, column 4).

3752
KARGINOV ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
Inhibition of EdTx activity. Compound 5b was tested for its
ability to block EdTx activity. In this experiment, CHO-K1
cells were incubated with different concentrations of the com-
pound with or without EdTx and cAMP production was deter-
mined using a cAMP immunoassay kit. Cyclodextrin 5b inhib-
ited EdTx at the same concentration range as LeTx (IC50,
2.2 Ϯ 0.2 ␮M) (Fig. 12). These data demonstrate that ␤-cyclo-
dextrin derivatives are not specific inhibitors of LF, providing
additional support for the originally proposed mechanism of
action, which involves the blocking of PA, a common subunit
for both LeTx and EdTx.
Cytotoxicity inhibition versus channel blocking experi-
ments. Figure 13 compares the data on cytotoxicity inhibition
with the data obtained from multichannel lipid membrane
experiments for the corresponding cationic derivatives. A rea-
sonably good correlation between the IC50 values can be ob-
served if one takes into account the tremendous difference in
these two methods. This finding supports the concept that
cationic cyclodextrins inhibit anthrax toxins by means of block-
ing the PA channel. The aminoalkyl ␤-CD derivatives 5g to 5i,
with longer alkyl chains, fall outside the shaded region; the
nominal solution IC50 concentrations obtained in the channel
reconstitution assay are too high. This deviation may be ex-
plained by the concentration loss of the compounds in the
vicinity of the PA channel. Indeed, lipid membranes are de-
stabilized by these derivatives (Fig. 8, bottom), which suggests
their strong membrane adsorption. Strong adsorption at nM
concentrations in the bulk may lead to a reduced compound
concentration at the channel entrance because of a longer
equilibration between the bulk and the membrane surface
and/or compound-compound electrostatic repulsion.
FIG. 13. Correlation between the inhibition of cytotoxicity in RAW
264.7 cells and blockage of PA channels in the multichannel reconsti-
tution assay (in 0.1 M KCl membrane-bathing solution) by cationic
␤-cyclodextrins. Compound 14b was the most effective cationic deriv-
ative tested in the channel reconstitution experiments, with an IC50 as
low as 0.07 Ϯ 0.05 nM. The cytotoxicity inhibition experiments gave an
IC50 of 0.5 Ϯ 0.2 ␮M. The vertical arrow for compound 16 shows the
absence of protective activity in the cell-based assay up to a 400 ␮M
concentration. The shaded space represents the area of good correla-
tion between the two techniques. It includes all data points except for
the derivatives with longer aminoalkyl chains (compounds 5g, 5h, and
5i), which showed a pronounced interaction with the lipid membrane
(see text and legend for Fig. 8). With these three points excluded,
linear correlation analysis [lg(IC50) obtained in cell inhibition versus
lg(IC50) obtained in channel reconstitution] of all data points in the
shaded area gives a correlation coefficient of 0.84 with a slope of 0.67.
ACKNOWLEDGMENTS
This research was supported by grant 2R44AI052894-02 from the
National Institute of Allergy and Infectious Diseases, National Insti-
tutes of Health, and by the Intramural Research Program of the NIH,
National Institute of Child Health and Human Development.
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