Thio-derived disulfides as potent inhibitors of botulinum neurotoxin type B: Implications for zinc interaction

Article abstract of DOI:10.1016/S0968-0896(03)00450-4

Botulinum neurotoxin type B causes the inhibition of acetylcholine release at the neuromuscular junction resulting in a flaccid paralysis designated botulism. This occurs through the cleavage of synaptobrevin, an intracellular critical component of neurotransmitter exocytosis, by the zinc-metallopeptidase activity of the smallest subunit of the toxin. Blocking the proteolytic activity may present an attractive approach to treat botulism as to date there is no efficient specific drug therapy avalaible. We have therefore recently described a series of β-amino-thiol derived pseudotripeptides able of inhibiting the toxin at low (10^-8 M) concentration. In this study, binding characteristics of the protein's active site are explored through various structural modifications of the thiol functionality which was supposed to be a key structural constituent for effective zinc-ion chelation. Surprisingly, sulfanyl-derivatives such as symmetric disulfides were shown to be better inhibitors than their thiol-counterparts, the most potent compound displaying a K_i value of 3.4 nM.

Full text of DOI:10.1016/S0968-0896(03)00450-4

Bioorganic & Medicinal Chemistry 11 (2003) 4655–4660
Thio-Derived Disulfides as Potent Inhibitors of Botulinum
Neurotoxin Type B: Implications for Zinc Interaction
Christine Anne, Armand Blommaert, Serge Turcaud, Anne-Sophie Martin,
Herve Meudal and Bernard P. Roques*
´ ´
Departement de Pharmacochimie Moleculaire et Structurale, INSERM U266, UFR des Sciences Pharmaceutiques et Biologiques,
4 avenue de l’Observatoire, 75270 Paris cedex 06, France
Received 1 April 2003; accepted 2 July 2003
Abstract—Botulinum neurotoxin type B causes the inhibition of acetylcholine release at the neuromuscular junction resulting in a
flaccid paralysis designated botulism. This occurs through the cleavage of synaptobrevin, an intracellular critical component of
neurotransmitter exocytosis, by the zinc-metallopeptidase activity of the smallest subunit of the toxin. Blocking the proteolytic
activity may present an attractive approach to treat botulism as to date there is no efficient specific drug therapy avalaible. We have
therefore recently described a series of b-amino-thiol derived pseudotripeptides able of inhibiting the toxin at low (10ꢀ8 M) con-
centration. In this study, binding characteristics of the protein’s active site are explored through various structural modifications of
the thiol functionality which was supposed to be a key structural constituent for effective zinc-ion chelation. Surprisingly, sulfanyl-
derivatives such as symmetric disulfides were shown to be better inhibitors than their thiol-counterparts, the most potent compound
displaying a Ki value of 3.4 nM.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
is an interesting target of therapeutical research. Botu-
linum neurotoxin type B (BoNT/B), which cleaves
synaptobrevin, an integral protein of synaptic vesicles,4
is the serotype responsible of the majority of cases of
human botulism in Europe.12
Botulinum neurotoxins are constituted of seven sero-
types (A to G) that are among the most poisonous sub-
stances for humans known to date.1 They cause
botulism, a disease characterized by a flaccid paralysis,
that can lead to death by respiratory arrest.2 These tox-
ins block the excitatory neurotransmission at the neu-
romuscular junction and at the autonomous nervous
system level by cleaving one of the proteins involved in
exocytosis.3ꢀ7 They are composed of two chains linked
by a disulfide bridge. The heavy chain (100 kDa) is
involved in binding and translocation while the light
chain (50 kDa) is endowed with the catalytic activity.8,9
These light chains were demonstrated to be zinc-metallo-
peptidases.10 To date, there is no specific drug therapy
against botulism, whereas the use of serum is effective
only in the first hour following the intoxication.2 More-
over these toxins are potential biological weapons.11
The design of compounds able of blocking their activity
Few selective and potent antagonists of this toxin have
been described. Indeed, classical inhibitors of metallo-
proteases are ineffective on BoNT/B and small peptides
derived from the substrate do not inhibit BoNT/B
either.4,13ꢀ17 Strong ligands of zinc are effective but have
major adverse effects.17ꢀ19 ICD 1578 (7-N-phenylcarba-
moyl-amino-4-chloro-propyloxyisocoumarine), an inhi-
bitor of the matrix metalloprotease elastase has only a
slight inhibitory potency on BoNT/B14,17 whereas inhi-
bitors of serine protease, such as BABIM or keto-
BABIM [respectively, bis(5-amidino-2-benzimidazolyl)-
methane and bis(5-amidino-2-benzimidazolyl)oxo-
methane] were proved to be more effective with
inhibitory activities of 1.6 and 0.8 mM, respectively.20
Buforin-1, a natural peptide, blocks the activity of
BoNT/B in the micromolar range21 and the thearubigin
fraction of black tea was only shown to be efficient
against the inhibiting activity of BoNT/B on the stimu-
lated contractions of the mouse hemidiaphragm.22
*Corresponding author. Tel.:+33-1-4325-5045; fax:+33-1-4326-6918;
e-mail: roques@pharmacie.univ-paris5.fr
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00450-4

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C. Anne et al. / Bioorg. Med. Chem. 11 (2003) 4655–4660
We have recently reported23 potent and selective inhibi-
tors of BoNT/B, the more potent being the pseudo-tri-
peptide compound 1 (Table 1). This compound displays
an inhibitory activity of 20 nM on BoNT/B. Thiol-
bearing inhibitors of zinc metalloproteases are generally
proposed to interact with the Zn2+ ion through chela-
tion with the thiolate whereas their disulfide derived
counterparts are inactive.24,25 In the case of matrix
metallopeptidases, it has however been shown that the
zinc present in the catalytic site must be chelated in a
different mode as inhibitors are devoid of non-oxidized
sulfur.26 Sulfanyl-derivatives such as symmetric and
dissymmetric disulfides and thio-ethers of some of the
pseudo-tripeptides described previously were therefore
synthesized as well as pseudo-tripeptide surrogates
lacking a sulfur atom. All compounds were evaluated27
for their ability to block the proteolytic activity of
BoNT/B light chain (LC), which revealed some inhibitory
potencies in the low nanomolar range.
Results and Discussion
The values of the BoNT/B LC binding affinities of the
newly synthesized compounds (Schemes 1 and 2) as well
as some previously prepared analogues (1, 8–10)23 are
listed in Table 1. Lead compound 1 showed high affinity
for BoNT/B LC. The thiol functionality of compound 1
was either eliminated (2) or replaced with a methyl
group (3) in order to demonstrate whether enzyme
recognition might imply classical zinc chelation. The
syntheses of compounds 2 and 3 are outlined in Scheme 1.
4-[(2S)-2-(1,1-Dimethylethyl)oxycarbonylamino-4-meth-
oxy-4-oxo]benzoic acid-(1,1-dimethylethyl)ester was
obtained as previously described23 and was either
directly hydrolyzed and subsequently coupled with the
dipeptide Bip-Bta-NHBn23 and deprotected to yield
compound 2 or submitted to intermediate alkylation
reaction conditions with methyliodide to yield com-
pound 3. Both compounds exhibit low inhibitory
potencies as compared to 1 which is not withstanding
the mode of metal complexation found for other metal-
lopeptidases.24,25 The thiol functionality of compound 1
was also derivatized into a symmetric disulfide, yielding
compound 4 (see Scheme 2). Interestingly compound 4
displays a gain of affinity over an order of magnitude
higher as compared to 1. This was confirmed by synth-
esis of other symmetric disulfides 5–7 which were pre-
pared from some previously described23 thiol-bearing
pseudo-tripeptides 8–10 (Scheme 2). As shown in Table 1
all disulfides are more potent inhibitors of BoNT/B LC
than their corresponding sulfanyl counterparts. It can
also be observed that the less potent the thiol is the
greater the gain of activity for the corresponding dis-
ulfide (factor, respectively of 6, 9, 23 and 58 for com-
pounds 4–7). Zinc chelation does therefore not seem as
essential for these large molecules which might perfectly
complement several supplementary subsites of the pro-
tein binding pocket as compared to shorter sequences.
Similarly, an acceptable inhibitory potency of BABIM
showing weak amidinium–zinc ion chelation was
explained through good enzyme subsite recognition.28 A
general sketch depicting the virtual pharmacophore is
represented in Figure 1.
Table 1. BoNT/B LC inhibitory potencies of compounds 1–12
a
Compd
R1
R2
KiꢁSEMb (nM)
1
2
3
4
5
6
7
8
–SH
–H
Benzo[b]thiophen-3-yl
Benzo[b]thiophen-3-yl
Benzo[b]thiophen-3-yl
Benzo[b]thiophen-3-yl
Phenyl
20ꢁ2
193ꢁ10
697ꢁ157
3.4ꢁ0.5
18ꢁ2
c
–CH3
–S)2
–S)2
–S)2
–S)2
–SH
–SH
–SH
But-2-yl
23ꢁ2
4-Hydroxyphenyl
Phenyl
But-2-yl
14ꢁ2
160ꢁ20
540ꢁ41
810ꢁ90
10.4ꢁ1.2
28ꢁ4
9
10
11
12
4-Hydroxyphenyl
Benzo[b]thiophen-3-yl
–S–(4-MeOBn) Benzo[b]thiophen-3-yl
–S–S–Bn
aSubstituents indicated with –S)2 represent the corresponding sym-
metric disulfide.
bKi values represent the meanꢁSEM of three separate experiments
each in triplicate.
cUndefined stereochemistry at the carbon atom connecting this
substituent.
Scheme 1. Synthetic pathway of compounds 2 and 3.

C. Anne et al. / Bioorg. Med. Chem. 11 (2003) 4655–4660
4657
A short benzyl-derived dissymmetric disulfide (11) was
prepared in order to reduce the number of putative
interactions with additional subsites. Benzyl thiol was
activated with S-chloro O-methyl thiocarbonate29 and
subsequently added to 1 as to enable the specific cou-
pling to the thiol functionality yielding compound 11
(Scheme 2). As shown in Table 1, this compound,
although very effective with a Ki of 10.4 nM, is a less
potent inhibitor of BoNT/B LC as compared to 4 but
slightly better in comparison to 1. One can conclude
that additional interactions of 4 with the binding pocket
subsites as compared to 1 are involved in the gain of
inhibitory potency but they do not seem to be the only
contributors to this gain. The presence of the disulfide
instead of the thiol might also be one essential factor. It
can be proposed that the zinc is chelated either by the
two pairs of electrons of the sulfur atoms or by only one
pair of one sulfur atom. This last type of chelation exists
for the copper ion, Cu2+ whereas bichelation of zinc by
separated sulfur atoms was shown in the case of cryp-
tants.30 In order to attempt to elucidate the mode of
chelation, a thio-ether (12) was prepared through
retaining para-methoxy benzyl substitution by submit-
ting the synthetic precursor23 of 1 to selective deprotec-
tion conditions using trifluoro-acetic acid instead of
hydrofluoric acid (Scheme 2). Compound 12 is nearly as
potent as the corresponding thiol and only 8 times less
potent as compared to the corresponding symmetric
disulfide. Moreover it is only 3 times less effective than
the dissymmetric disulfide 11 whose side chains are
quite similar except for the methoxy in the para position
of the benzyl group. The affinity of 12 suggests that the
free pairs of electrons of the sulfur atom belonging to
the thio-ether may be involved in zinc chelation as
Scheme 2. Synthesis of compounds 4–7, 11 and 12 from previously described precursors.23

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C. Anne et al. / Bioorg. Med. Chem. 11 (2003) 4655–4660
recorded on an Esquier-Brucker spectrometer. Flash
column chromatography was performed using 40–63
mm silica gel. Reaction progress was determined by
either TLC analysis or monitored using analytical
reverse-phase HPLC (Shimadzu, LC10 AD-vp with a
Class-VP5.03 software) using a Kromasil C18 column
(100 A, 5 mm, 250ꢂ4.6 mm, from Touzart-Matignon,
France) with a mobile phase consisting of water con-
taining 0.05% TFA (A), acetonitrile containing 0.05%
TFA (B) or acetonitrile/water (9/1) containing 0.038%
TFA (C). Preparative HPLC was performed on a Kro-
masil C18 column (100 A, 5 mm, 250ꢂ20 mm). Reagents
were obtained from commercial sources and are used
without further purification.
4-[(2S,3S)-2-Amino-3-sulfanyl-4-[(1S)-2-[(1S)-1-(benzo
[b]thiophen - 3 - ylmethyl) - 2 - (benzylamino) - 2 - oxoethyl]
amino]-1-(biphenyl-4-ylmethyl)-2-oxoethyl]amino]-4-oxo-
butyl]benzoic acid (2). 4-[(2S)-2-(1,1-Dimethylethyl)
oxycarbonylamino-4-methoxy-4-oxo]benzoic acid-(1,1-
dimethylethyl)ester was obtained as previously descri-
bed23 and treated overnight with NaOH (1.5 equiv) in a
mixture of MeOH/H2O at room temperature. The sol-
vents were evaporated and H2O (10 mL) was added.
The pH was adjusted to 3 with an aqueous 10% citric
acid solution and the product was extracted with EtOAc
(3ꢂ20 mL). The combined organic layers were washed
with water and brine, dried over Na2SO4, filtered and
evaporated to yield the corresponding acid as a white
solid. 1H NMR (DMSO-d6) d 1.25 (s, 9H), 1.50 (s, 9H),
2.30 (m, 2H), 2.70 (m, 2H), 3.90 (m, 1H), 6.75 (d, 1H),
7.20 (d, 2H), 7.74 (d, 2H). The acid was subsequently
coupled to dipeptide Bip-Bta-NHBn as previously
reported.23 The residual solid was purified by flash col-
umn chromatography with cHex/EtOAc (7/3) and trea-
ted for 4 h with trifluoroacetic acid. The solvent was
evaporated and the product was freeze dried to yield
compound 2 as a white solid. Yield 54%. 1H NMR
(DMSO-d6+TFA) d 2.3–3.3 (m, 8H), 3.48 (m, 1H), 4.2
(d, 2H), 4.55–4.75 (m, 2H), 7.0–7.45 (m, 19H), 7.7–7.95
(m, 6H), 8.4 (d, 1H), 8.5 (d, 1H), 8.55 (t, 1H). SM (ESI)
(M+H)+ m/z 739.3. HPLC (50% B): Rt=19.3 min.
Figure 1. General structure (R=various substituents; n=0, 1 or 2)
and proposed mode for the interaction of potent inhibitors with
0
0
0
BoNT/B LC. S1 S1 and S2 are schematic representations of subsites
present in the BoNT/B LC active site which are supposed to comple-
ment the sequence surrounding the scissible bond (Glu76-Phe77) of the
substrate synaptobrevin.
already described in the literature for copper Cu2+
ion30,31 and zinc Zn2+ion,32 giving rise to weaker chel-
ation, and thus affinity, in comparison with thiol 1. A
better complexation obviously enhances potency
partially in the case of disulfides through either bi-chel-
ation with two sulfurs or through the existence of an a-
effect, reinforcing the nucleophilicity of one sulfur by
the free pairs of the other one.
Conclusion
Although zinc chelation is strictly required for high
inhibitory activity, the thiol functionality which gen-
erally provides an effective metal ion chelating group
does not seem a prerequisite in our series of compounds
for optimum BoNT/B metallopeptidase subunit recog-
nition. Thus in disulfides the decrease of complexation
with the metal as found in free thiol inhibitors could be
compensated by multiplying interactions of the disulfide
side chains with enzyme binding pocket subsites. These
results suggest that BoNT/B recognition might be quite
different from that of the other members of the zinc-
metallopeptidase family as already assumed to explain
the weak inhibitory potencies of classical zinc-metallo-
peptidase inhibitors on this toxin.4,13ꢀ17 This is con-
sistent with the mode of interaction of compounds such
as BABIM studied by crystallography.28 The insights
provided by this study were taken into account for the
further development of inhibitors with optimal adapta-
tion to clefts and pockets of the enzyme active site and
will be published shortly.
4-[(2S,3S)-2-Amino-3-(methylsulfanyl)-4-[(1S)-2-[(1S)-1-
(benzo[b]thiophen-3-ylmethyl)-2-(benzylamino)-2-oxo-
ethyl]amino]-1-(biphenyl-4-ylmethyl)-2-oxoethyl]amino]-
4-oxobutyl]benzoic acid (3). 4-[(2S)-2-(1,1-dimethylethyl)-
oxycarbonylamino-4-methoxy-4-oxo]benzoic acid-(1,1-
dimethylethyl)ester23 was dissolved in anhydrous THF
and treated at ꢀ78 ꢃC with LDA which was freshly
prepared from nBuLi (2 equiv) and diisopropylamine (2
equiv). MeI (1.1 equiv) was added and the mixture was
allowed to warm to room temperature while stirring for
5 h. The solvent was then evaporated and EtOAc (10
mL) was added to the residue. The organic layer was
washed with a 10% aqueous citric acid solution (3ꢂ10
mL), water, brine and the organic solution was dried
over Na2SO4 and filtered. Evaporation of the solvent
yielded a pale yellow oil which was chromatographed
using cHex/EtOAc (9/1) as the eluting solvent. The
resulting oil (yield 27%) was characterized by NMR: 1H
NMR (CDCl3) d 1.12 (d, 3H), 1.30 (s, 9H), 1.52 (s, 9H),
2.50 (m, 1H), 2.60–2.90 (m, 2H), 3.54 (s, 3H), 3.85 (m,
Experimental
Chemistry
1H NMR spectra were measured on a Brucker AC
270MHz spectrometer using tetramethylsilane as inter-
nal standard. Electrospray mass spectra (MS-ES) were

C. Anne et al. / Bioorg. Med. Chem. 11 (2003) 4655–4660
4659
1H), 5.35 (d, 1H), 7.15 (d, 2H), 7.82 (d, 2H). The
obtained intermediate was hydrolyzed and subsequently
coupled with dipeptide Bip-Bta-NHBn followed by
deprotection as above in the synthesis of compound 2 to
4-[(2S,3S)-2-Amino-3-(benzyldisulfanyl)-4-[(1S)-2-[(1S)-1
-(benzo[b]thiophen-3-ylmethyl)-2-(benzylamino)-2-oxo-
ethyl]amino]-1-(biphenyl-4-ylmethyl)-2-oxoethyl]amino]-
4-oxobutyl]benzoic acid (11). To a solution of 1 (0.01
mmol) and triethylamine (0.012 mmol) in 2 mL of
chloroform was added benzylsulfanyl-O-methyl thio-
carbonate33 (0.011 mmol) at room temperature. The
reaction was stirred at room temperature for 3 h. After
evaporation in vacuo, the residue was purified by semi
preparative HPLC and freeze dried.
1
give compound 3 as white solid. Yield 54%. H NMR
(DMSO-d6+TFA) d 1.15–2.5 (m, 11H), 2.60–3.0 (m,
2H), 3.00–3.30 (m, 2H), 4.23 (d, 2H), 4.50 (m, 1H), 4.65
(m, 1H), 7.0-7.50 (m, 22H), 7.9 (m, 2H), 8.08 (bs, 1H),
8.45 (m, 2H). SM (ESI) (M+H)+ m/z 753.3. HPLC
(50% B): Rt=19.7 min.
Yield 44%. 1H NMR (DMSO-d6+TFA) d 2.70 (m,
1H), 2.80 (m, 2H), 3.10 (m, 3H), 3.30 (m, 2H), 3.60 (d,
1H), 3.70 (m, 1H), 3.80 (q, 2H), 4.25 (d, 2H), 4.75 (m,
1H), 4.85 (m, 1H), 7.10–7.50 (m, 24H), 7.80 (m, 2H), 8.0
(m, 3H), 8.70 (t, 1H), 8.80 (d, 1H), 8.90 (d, 1H). HPLC
(gradient 40–100% C in 20 min): Rt=16.0 min.
General procedure for the synthesis of compounds 4–7
Disulfides 4–7 were synthesized via dimerization of cor-
responding thiol monomers 1, 8–10 which have been
previously described.23 Thiol compounds were dissolved
in ethanol at a concentration of 1 mM. The mixture was
stirred at room temperature and a solution of 0.25 M
iodine in ethanol was added drop by drop until the
solution became slightly yellow. After 15 min, the sol-
vent was evaporated and the crude product was purified
by preparative HPLC and freeze dried.
4-[(2S,3S)-2-Amino-3-(4-methoxybenzylsulfanyl)-4-[(1S)-
2-[(1S)-1-(benzo[b]thiophen-3-ylmethyl)-2-(benzylamino)-
2-oxoethyl]amino]-1-(biphenyl-4-ylmethyl)-2-oxoethyl]-
amino]-4-oxobutyl]benzoic acid (12). The synthetic pre-
cursor23 of compound 1 (0.03 mmol) was stirred at 0 ꢃC
for 1 h with 1 mL of trifluoroacetic acid and 0.1 mL of
triisopropylsilane. After evaporation, the residue was
purified by semi-preparative HPLC and freeze-dried.
4,40-Bis[disulfan-diyl[(2S,3S)-2-amino-3-[(1S)-2-[(1S)-1-
(benzo[b]thiophen-3-ylmethyl)-2-(benzylamino)-2-oxo-
ethyl]amino]-1-(biphenyl-4-ylmethyl)-2-oxoethyl]carba-
1
moyl]propane-1,3-diyl]dibenzoic acid (4). Yield 71%. H
NMR (DMSO-d6+TFA) d 2.8 (m, 3H), 3.1 (m, 2H),
3.25 (m, 1H), 3.8 (d, 1H), 3.9 (m, 1H), 4.2 (d, 2H), 4.75
(m, 2H), 7.0-7.50 (m, 20H), 7.80 (2d, 3H), 8.00 (m, 3H),
8.80 (m, 3H). SM (ESI) (M+H)+ m/z 1541.0. HPLC
(gradient 40–100% C in 20 min): Rt=16.1 min.
Yield 52%. 1H NMR (DMSO-d6+TFA) d 2.65 (m,
1H), 2.85 (m, 2H), 3.10 (m, 2H), 3.30 (m, 2H), 3.40 (m,
2H), 3.60 (m, 4H), 4.25 (d, 2H), 4.75 (m, 2H), 6.60 (d,
2H), 6.80 (d, 2H), 7.10–7.45 (m, 19H), 7.80 (2d, 3H),
7.90 (m, 3H), 8.0 (d, 1H), 8.60 (d, 1H), 8.70 (m, 2H).
4,40-Bis[disulfan-diyl[(2S,3S)-2-amino-3-[(1S)-2-[(1S)-1-
(phenylmethyl)-2-(benzylamino)-2-oxoethyl]amino]-1-(bi-
phenyl-4-ylmethyl)-2-oxoethyl]carbamoyl]propane-1,3-
13C NMR (DMSO-d6) d31.4, 35.5, 37.0, 37.8, 42.0, 47.0,
53.0, 54.0, 55.0, 114.0, 122.0, 123.0, 123.8, 124.0, 126.4,
126.9, 127.1, 128.3, 128.6, 128.8, 129.6, 129.6, 129.9,
130.1, 131.8, 136.8, 138.0, 138.5, 139.0, 139.4, 139.8,
140.6, 158.0, 167.0, 169.0, 171.0. SM (ESI) (M+H)+
m/z 1005.2. HPLC (A/B: 15/85): Rt=6.5 min.
1
diyl]dibenzoic acid (5). Yield 58%. H NMR (DMSO-
d6+TFA) d 2.70 (m, 1H), 2.80 (m, 2H), 3.0 (m, 2H),
3.75 (d, 1H), 3.90 (m, 1H), 4.20 (d, 2H), 4.65 (m, 2H),
7.0-7.50 (m, 22H), 7.80 (d, 2H), 8.0 (m, 3H), 8.60 (t,
1H), 8.65 (d, 1H), 8.70 (d, 1H). SM (ESI) (M+H)+ m/z
1429.8. HPLC (A/C: 30/70): Rt=6.4 min.
Enzyme assay
The inhibitory potencies of all compounds were mea-
sured by using the light chain of BoNT/B as previously
described27 with some slight modifications. Briefly,
BoNT/B LC (0.35 ng) was preincubated for 30 min at
37 ꢃC in 90 mL of 20 mM Hepes, pH 7.4 with increasing
concentrations (10ꢀ10–10ꢀ5 M) of inhibitor. Com-
pounds containing a free thiol group were tested in the
presence of 0.1 mM DTT in order to avoid oxidation.
All other compounds were tested in the absence of
DTT. The fluorescent substrate Syb 60-94 [Pya74-Nop77]
(Km=47 mM) was then incubated for 30 min and the
reaction stopped by addition of 0.2 N HCl at 4 ꢃC. Ver-
ification through HPLC analysis showed no reduction
in situ of compounds containing disulfide bridges (data
not shown). The percentage of degradation was mea-
sured directly in a 96-well plate. The IC50 values were
determined from logarithmic dose–degradation curves
and the values of the inhibitory constant (Ki) of com-
pounds 1–12 were calculated according to the equation
of Cheng and Prusoff and are expressed as the mean-
ꢁSEM of three separate experiments each in triplicate.
4,40-Bis[disulfan-diyl[(2S,3S)-2-amino-3-[(1S)-2-[(1S)-1-
(but-2-ylmethyl)-2-(benzylamino)-2-oxoethyl]amino]-1-
(biphenyl-4-ylmethyl)-2-oxoethyl]carbamoyl]propane-1,3-
1
diyl]dibenzoic acid (6). Yield 66%. H NMR (DMSO-
d6+TFA) d 0.85 (m, 6H), 1.1 (m, 1H), 1.45 (m, 1H),
1.70 (m, 1H), 2.80–3.10 (m, 4H), 3.80 (d, 1H), 4.0 (m,
1H), 4.30 (m, 3H), 4.80 (m, 1H), 7.10–7.50 (m, 16H),
7.80 (d, 2H), 8.0 (m, 3H), 8.40 (d, 1H), 8.60 (t, 1H), 8.75
(d, 1H). SM (ESI) (M+H)+ m/z=1360.2. HPLC (gra-
dient 40–100% C in 20 min): Rt=13.6 min.
4,40-Bis[disulfan-diyl[(2S,3S)-2-amino-3-[(1S)-2-[(1S)-1-
(4-hydroxyphenylmethyl)-2-(benzylamino)-2-oxoethyl]a-
mino]-1-(biphenyl-4-ylmethyl)-2-oxoethyl]carbamoyl]pro-
1
pane-1,3-diyl]dibenzoic acid (7). Yield 60%. H NMR
(DMSO-d6+TFA) d 2.70 (m, 1H), 2.80 (m, 2H), 3.10 (m,
2H), 3.30 (m, 1H), 3.80 (d, 1H), 3.90 (m, 1H), 4.20 (d, 2H),
4.75 (m, 2H), 7.0–7.50 (m, 20H), 7.75 (2d, 3H), 8.0 (m, 3H),
8.60 (t, 1H), 8.70 (d, 2H). SM (ESI) (M+H)+ m/z 1461.1.
HPLC (gradient 40–100% C in 20 min): Rt=9.5 min.

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18. Adler, M.; Dinterman, R. E.; Wannemacher, R. W. Tox-
icon 1997, 35, 1089.
References and Notes
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