Serotype-selective, small-molecule inhibitors of the zinc endopeptidase of botulinum neurotoxin serotype A

Article abstract of DOI:10.1016/j.bmc.2005.08.018

Botulinum neurotoxin serotype A (BoNTA) is one of the most toxic substances known. Currently, there is no antidote to BoNTA. Small molecules identified from high-throughput screening reportedly inhibit the endopeptidase-the zinc-bound, catalytic domain of

Full text of DOI:10.1016/j.bmc.2005.08.018

Bioorganic & Medicinal Chemistry 14 (2006) 395–408
Serotype-selective, small-molecule inhibitors of the
zinc endopeptidase of botulinum neurotoxin serotype A
Jewn Giew Park,a Peter C. Sill,a Edward F. Makiyi,a Alfonso T. Garcia-Sosa,a
Charles B. Millard,b James J. Schmidtc,* and Yuan-Ping Panga,*
aComputer-Aided Molecular Design Laboratory, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905, USA
bWalter Reed Army Institute of Research, Division of Biochemistry, 503 Robert Grant Avenue, Silver Spring, MD 20910, USA
cDepartment of Cell Biology and Biochemistry, Toxinology and Aerobiology Division, United States Army Medical Research
Institute of Infectious Diseases, 1425 Porter Street, Frederick, MD 21702, USA
Received 30 June 2005; revised 8 August 2005; accepted 9 August 2005
Available online 3 October 2005
Abstract—Botulinum neurotoxin serotype A (BoNTA) is one of the most toxic substances known. Currently, there is no antidote to
BoNTA. Small molecules identified from high-throughput screening reportedly inhibit the endopeptidase—the zinc-bound, catalytic
domain of BoNTA—at a drug concentration of 20 lM. However, optimization of these inhibitors is hampered by challenges includ-
ing the computational evaluation of the ability of a zinc ligand to compete for coordination with nearby residues in the active site of
BoNTA. No improved inhibitor of the endopeptidase has been reported. This article reports the development of a serotype-selective,
small-molecule inhibitor of BoNTA with a Ki of 12 lM. This inhibitor was designed to coordinate the zinc ion embedded in the
active site of the enzyme for affinity and to interact with a species-specific residue in the active site for selectivity. It is the most potent
small-molecule inhibitor of BoNTA reported to date. The results suggest that multiple molecular dynamics simulations using the
cationic dummy atom approach are useful to structure-based design of zinc protease inhibitors.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
Development of such antidotes has long been challeng-
ing because BoNTA is not a ꢀdrug-friendlyꢁ protein.5
The crystal structure of the holo BoNTA comprises
two chains that are linked by a disulfide bond.6 The light
chain (50 kDa) is a zinc endopeptidase (hereafter the
endopeptidase) that specifically cleaves neuronal pro-
teins responsible for acetylcholine release.7 The heavy
chain (100 kDa) mediates selective binding to neuronal
cells via specific gangliosides and translocates the light
chain into the cytosol after receptor-mediated endocyto-
sis of the entire molecule.8 Although both chains can be
used as targets for developing small-molecule antidotes
to BoNTA,9–11 it is technically less challenging to use
the light chain as a target for four reasons.
Botulinum neurotoxin serotype A (BoNTA) is a highly
toxic by-product of a naturally occurring, spore-forming
anaerobic bacterium, Clostridium botulinum. BoNTA
inhibits the release of acetylcholine from presynaptic
nerve terminals at neuromuscular junctions thus causing
flaccid paralysis and leading to death by respiratory ar-
rest.1 BoNTA appears to be effective in treating muscle
dysfunctions2 and has been widely used as a cosmetic
known as Botox to temporarily diminish facial lines.3
However, BoNTA is fatal when misused.4 Currently,
there is no chemical antidote to BoNTA. Small-mole-
cule inhibitors of BoNTA as chemical antidotes are
highly desirable.
First, a theoretical 3D model of the endopeptidase com-
plexed with a P4–P30 substrate fragment has been devel-
oped and made available at the Protein Data Bank
(PDB code: 1UEE, manuscript submitted). This model
offers insights into how the substrate interacts with the
active-site residues of the endopeptidase and which re-
gion of the active site is worth targeting to improve affin-
ity and selectivity of a small-molecule inhibitor of the
Keywords: Countermeasures; Antidotes; Protease; Zinc protein simu-
lations; Structure-based drug design.
*
Corresponding authors. Tel.: +1 30 16 19 42 40; fax: +1 30 16 19 23
48 (J.J.S.); tel.: +1 50 72 84 78 68; fax: +1 50 72 84 91 11 (Y.P.P.)
e-mail addresses: james.schmidt@det.amedd.army.mil; pang@mayo.
edu
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.08.018

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J. G. Park et al. / Bioorg. Med. Chem. 14 (2006) 395–408
endopeptidase. The model suggests that the deep active
site of the endopeptidase is filled with a rod-shaped hep-
tapeptide. This structural feature requires that a mole-
cule be at least 10 carbons long to be an effective
inhibitor of the endopeptidase. This is consistent with
the fact that small-molecule inhibitors reported to date
are more than 10 carbons long.11 The model also sug-
gests that the interaction of Phe193 of the endopeptidase
with a substrate or an inhibitor partly establishes speci-
ficity of a substrate or selectivity of inhibitors.
hampered by some challenges including the computation-
al evaluation of the ability of a zinc coordinate to compete
for zinc coordination with other zinc coordinates in the
active site of the endopeptidase. To date, no improved
small-molecule inhibitor for BoNTA has been reported.
This article reports the design, synthesis, and testing of
serotype-specific small-molecule inhibitors of the endo-
peptidase of BoNTA. The results provides insights for
the development of better antidotes to BoNTA and to
the development of other metalloenzyme inhibitors.
Second, there are examples12,13 to support the notion that
the affinities of the small molecules binding at the active
site of the endopeptidase could be greatly improved by
these moleculesꢁ coordination to the zinc ion at the active
site. The presence of a zinc ion in the active site of the
endopeptidase permits the design of small molecules with
affinities that are high enough for these molecules to com-
pete with protein substrates for binding to the endopepti-
dase. The affinities of the small molecules binding at the
heavy-chain domain are, however, difficult to improve
to the extent that these molecules can compete with neu-
ronal cells for binding to the heavy-chain domain. This
is because the heavy-chain domain lacks a deep pocket
at the interface of its complex.5,9
2. Results
2.1. Design
A computational screen of a chemical database was first
carried out to identify molecules capable of coordina-
tion with the zinc ion in the active site of the endopepti-
dase. This screen was performed using an in-house
database of 2.5 million chemical structures and the crys-
tal structure of the zinc-bound endopeptidase6 accord-
ing to a published protocol.31 It had been reported
that the interaction energies between zinc and its coordi-
nates were markedly underestimated by molecular
mechanics calculations if the zinc ion was modeled tra-
ditionally with a one-atom-zinc representation.32 To
minimize the underestimation that would hamper the
search of zinc coordinates, the CaDA approach14–18
was used in the screen to estimate the zinc affinities of
potential endopeptidase inhibitors.
Third, the four-ligand coordination of the zinc ion
embedded in the active site of the endopeptidase
can be computationally simulated with the cationic
dummy atom (CaDA) approach14–18 without resorting
to the use of a covalent bond between zinc and its
coordinate19–21 or to semiempirical calculations.22 This
approach uses four identical dummy atoms tetrahedri-
cally attached to the zinc ion and transfers all the atomic
charge of the zinc divalent cation evenly to the four
dummy atoms. The four peripheral atoms are ꢀdummyꢁ
in that they interact with other atoms electrostatically
but not sterically, thus mimicking zincꢁs 4s4p3 vacant
orbitals that accommodate the lone-pair electrons of
zinc coordinates.14–18,23 The CaDA approach enables
(i) refinement of the endopeptidase, (ii) identification
of small molecules that are able to coordinate the zinc
ion upon binding to the active site of the endopeptidase
via docking and molecular dynamics simulations, and
(iii) optimization of the zinc-coordinating molecules
via free energy perturbation study.24
The top eight compounds identified in the screen were
purchased or synthesized and tested as potential inhibi-
tors of the endopeptidase. Of the eight compounds,
compound 1 (Fig. 1) showed 15% inhibition of the endo-
peptidase at a drug concentration of 100 lM and the
rest showed 5–12% inhibition. The reason why only
weak inhibitors were identified was in part due to the
competition for zinc coordination by a water molecule
or neighboring Glu223 in the active site of the endopep-
tidase, which was supported by subsequent multiple
molecular dynamics simulations (MMDSs) described
below. The docking-based screen predicts whether or
not a functional group such as a carboxylate can coordi-
nate zinc. It does not predict whether the functional
group can compete for zinc coordination with a water
molecule or Glu223. Therefore, an inhibitor identified
from the screen would fail to inhibit the endopeptidase
via zinc coordination if its affinity for zinc were lower
than that of water or Glu223.
Fourth, the X-ray structure of an inactive variant of the
endopeptidase in complex with a substrate has been
reported.25 In addition to the use of the active site of
the endopeptidase as a target described above, the X-
ray structure suggests that one can develop selective
inhibitors targeting the substrate-interacting surface re-
gions remote to the active site.25
To address this issue 20 different molecular dynamics
simulations were carried out for each of the eight com-
puter-identified compounds in complex with the endo-
peptidase. Each of these simulations was carried out
for 2.0 ns with a 1.0-fs time step and different initial
velocities using the CaDA approach according to a pub-
lished protocol.18,33 Interestingly, only 1 was able to
coordinate zinc throughout all 20 simulations; the others
failed to coordinate zinc due to the replacement of
the zinc coordinate by either a water molecule or the
Indeed, small peptides and their analogs are able to
inhibit the endopeptidase of BoNTA and a closely relat-
ed zinc endopeptidase of botulinum neurotoxin serotype
B (BoNTB) at nanomolar concentrations.26–30 Recently,
a high-throughput screen targeting the endopeptidase of
BoNTA has identified small molecules that inhibit the
endopeptidase at a drug concentration of 20 lM.11
However, optimization of such small molecules is

J. G. Park et al. / Bioorg. Med. Chem. 14 (2006) 395–408
397
the zinc coordination using better zinc coordinates such
as hydroxamates and its selectivity could be improved
by increasing the p–p interaction with Phe193 using
additional aromatic rings.
X
Y
Z
S
O
Analog 2 (Fig. 1) with a hydroxamate group as a better
zinc ligand34 was made. A high-pressure-liquid-chroma-
tography-based (HPLC-based) assay showed that 2 is
indeed more active in inhibiting the endopeptidase than
1. Analogs 3–7 (Fig. 1) were made by minor variations
of the synthesis of 2. No MMDS studies were performed
on these analogs. In vitro testing revealed that only 4
was more active in inhibiting the endopeptidase than
2. Without the 3D models of the endopeptidase in com-
plex with 3–7, respectively, the structure–activity rela-
tionship of these analogs offered little guidance in
optimization of 2.
1. X = CO2H, Y = Cl, Z = H, 15±0(1)% inhibition
2. X = CONHOH, Y = Cl, Z = H, 30±1(2)% inhibition
3. X = CONHOH, Y = Br, Z = H, 11±2(2)% inhibition
4. X = CONHOH, Y = I, Z = NH2, 40±3(3)% inhibition
5. X = CONHOH, Y = I, Z = NO2, 3±1(2)% inhibition
6. X = CONHNH2, Y = Br, Z = H, 5±1(2)% inhibition
7. X = CONHNH2, Y = I, Z = NH2, 4±1(2)% inhibition
X
N
Y
S
O
Visual inspection of the MMDS-generated 3D model
of the endopeptidase in complex with 1 identified a
cavity adjacent to the chlorine-substituted phenyl
group of 1. Given the synthetic feasibility, the chlo-
rine-substituted phenyl ring of 1 was replaced by 2-
phenylindole to make a rod-shaped analog 8 (Fig. 1)
that satisfies the structural requirement of the endo-
peptidase inhibitors suggested by the 3D model of a
peptide–substrate-bound endopeptidase as described
above. MMDSs on the endopeptidase in complex with
8 and its hydroxamic analog 9 (Fig. 1) were per-
formed. The results of these simulations suggested that
8 and 9 were able to fit the active site of the enzyme
and coordinate zinc. These compounds were subse-
quently synthesized and were unexpectedly found to
have poor water solubilities. Testing the partially dis-
solved 8 and 9 showed that both were less potent in
inhibiting the endopeptidase than 1 and 2 (Fig. 1).
8. X = CO2H, Y = H, 10±0(2)% inhibition
9. X = CONHOH, Y = H, 4±1(2)% inhibition
10. X = CO2H, Y = (CH2)4NH2, 30±1(2)% inhibition
11. X = CONHNH2, Y = (CH2)4NH2, 89±0(2)% inhibition
12. X = CONHOH, Y = (CH2)4NH2, 96±6(2)% inhibition
13. X = CONHOH, Y = (CH2)5NH2, 96±4(2)% inhibition
14. X = CONHOH, Y = (CH ) NH2, 97±2(2)% inhibition
Figure 1. Chemical structures of inhibitors 1–14 and their inhibition
on the zinc endopeptidase of botulinum neurotoxin serotype A at a
drug concentration of 100 lM.
carboxylate of Glu223. The correlation between the
ability to coordinate zinc in computer simulations and
the ability to inhibit the endopeptidase in experiments
suggested that MMDSs using the CaDA approach are
useful in triaging the docking-identified compounds that
have lower affinities for zinc than water and carboxylate.
To increase water solubility and possibly affinity, an
aminobutyl group was attached to the indole nitrogen
of 9, which led to the design of 12. As depicted in Figure
2, MMDSs of 12 showed that: (i) the hydroxamate
group was able to coordinate the active-site zinc, (ii)
The MMDSs showed that 1 has a carboxylate coordi-
nating the active-site zinc and a phenyl group interacting
with Phe193 of the endopeptidase. This suggested that
the affinity of 1 could be improved by strengthening
Figure 2. A close-up view of inhibitor 12 (ball-and-stick model) binding at the active site of the endopeptidase of BoNTA (stick model). The 3D
structure model was generated by averaging 5000 instantaneous structures obtained at 1.0-ps intervals during the last 500-ps period of 10 molecular
dynamics simulations each of which lasted for 2.0 ns with a 1.0-fs time step.

398
J. G. Park et al. / Bioorg. Med. Chem. 14 (2006) 395–408
the phenyl group substituted at the thiophene ring had a
p–p interaction with Phe193 and a cation–p interaction
with Arg362, (iii) the indole ring was engaged in a cat-
ion–p interaction with Lys165, (iv) the phenyl group at-
tached to the indole ring has a van der Waals interaction
with the side chain of Leu527 and a cation–p interaction
with Lys165, and (v) the ammonium group interacted
with the carboxylates of Glu54 and Glu55. The energet-
ically favorable 3D model of the 12-bound endopepti-
dase prompted for the synthesis and testing of analogs
10–14 (Fig. 1), where 10 is a synthetic precursor of 12,
and 11 is an analog of 12 made by a minor variation
of the synthesis of 12 using hydrazine as a known zinc
coordinate.35 Analogs 13 and 14 were also made to
probe the amino chain length.
2.2. Synthesis
The retro synthetic analysis for 1–14 is shown in
Scheme 1. Compounds 1–7 and compounds 8–14 were
prepared in good yields according to Schemes 2 and 3,
respectively.
Intermediate 16 was prepared according to a literature
procedure.36–38 To improve the reported yield for 15,39
cyclization of 16 to 15 was carried out by the treatment
of mercaptoacetic acid and triethylamine in refluxing
THF for 4 h followed by removing THF via distillation
and heating at 130 ꢁC in DMF for 18 h. The cyclization
reaction inevitably produced a UV-inactive, sulfur-con-
taining impurity. This impurity interfered with the
O
O
O
Cl
MeO2C
Y
Ph
O
X
X
OEt
Ph
Ph
Ph
Ph
Ph
N
Y
S
S
CHO O
Z
S
OEt
O
O
8: X=OH, Y=H
9: X=NHOH, Y=H
10: X=OH, Y=(CH2)4NH2
11: X=NHNH2, Y=(CH2)4NH2
12: X=NHOH, Y=(CH2)4NH2
13: X=NHOH, Y=(CH2)5NH2
14: X=NHOH, Y=(CH2)6NH2
1: X=OH, Y=Cl, Z=H
15
16
17
2: X=NHOH,Y=Cl, Z=H
3: X=NHOH, Y=Br, Z=H
4: X=NHOH, Y=I, Z=NH2
5: X=NHOH, Y=I, Z=NO2
6: X=NHNH2, Y=Br, Z=H
7: X=NHNH2, Y=I, Z=NH2
Scheme 1.
O
O
MeO2C
Cl
Y
Z
Ph
O
f,c (for 1 or 18)
g,c (for 19)
X
a
b,c,d,e
OEt
Ph
Ph
Ph
S
CHO O
S
OEt
O
17
15
16
1: X=OH, Y=Cl, Z=H
h
2: X=NHOH,Y=Cl, Z=H
18: X=OH, Y=Br, Z=H
19: X=OH, Y=I, Z=NO2
20: X=OH, Y=I, Z=NHBoc
i,j
HO
N
O
O
l (18 to 3)
l (19 to 5)
m (19 to 6)
NO2
KR
18,19, or 20
Y
Z
Y
X
O
Ph
S
Ph
k
n,l (20 to 4)
n,m (20 to 7)
S
Z
Kaiser Resin (KR)
O
O
3: X=NHOH, Y=Br, Z=H
4: X=NHOH,Y=I, Z=NH2
5: X=NHOH, Y=I, Z=NO2
6: X=NHNH2, Y=Br, Z=H
7: X=NHNH2, Y=I, Z=NH2
Scheme 2. Reagents and conditions: (a) DMF/POCl3; (b) HSCH2CO2H/NEt3/THF and DMF; (c) NaOH/MeOH; (d) purification; (e) HCl/MeOH;
(f) 4-Cl/Br-benzoyl chloride/AlCl3/CH2Cl2, rt; (g) 4-iodo-3-nitrobenzoyl chloride/AlCl3/CH2Cl2, rt; (h) NH2OHÆHCl/EDCI/HOBT/NMM/CH2Cl2;
(i) SnCl2Æ2H2O/EtOAc, reflux; (j) (Boc)2O/DIEA/DMAP/THF; (k) EDCI/HOBt/NMM/CH2Cl2; (l) NH2OH/CHCl3; (m) NH2NH2/EtOH/THF; (n)
TFA/CH2Cl2.

J. G. Park et al. / Bioorg. Med. Chem. 14 (2006) 395–408
399
Scheme 3. Reagents and conditions: (a) 4-iodo-3-nitrobenzoyl chloride/AlCl3/CH2Cl2, rt; (b) SnCl2 2H2O/EtOAc, reflux; (c) PhCCH/Pd(Ph3)4/CuI/
Et2NH; (d) PdCl2(PhCN)2/DMF, 80 ꢁC; (e) NaOH/MeOH; (f) Trt-ONH2/HATU[or HBTU]/HOBt/DIEA/CH2Cl2; (g) AcOH/TFE (1:2); (h) N-Boc-
4-bromobutyl-amine/CsF–Celite/CH3CN, reflux; (i) HCl/MeOH; (j) N2H4/EtOH/THF; (k) TMSI/CHCl3; (l) N-Boc-5-bromopentyl-amine/CsF–
Celite/CH3CN, reflux; (m) concd HCl/EtOAc; (n) N-Boc-6-bromohexyl-amine/CsF–Celite/CH3CN, reflux.
subsequent Friedel–Crafts reaction and was inseparable
from 15 by common purification procedures such as
vacuum distillation or column chromatography. This
problem was eventually solved by saponification of the
ethyl ester group of 15. The resulting acid was separated
by filtration from the sulfur-containing impurity and
then converted to a methyl ester.
nitrogen and the competition for alkylation at the meth-
ylene group under basic conditions. Weak bases such as
K2CO3/DMF, phase-transfer catalyst (PTC), or K2CO3/
18-crown-6 failed to yield the desired product. Ultimate-
ly, CsF–Celite/CH3CN46 was found to be effective in N-
alkylation of 23. Precursor 10 was lastly converted to 11
and 12 using hydrazine and O-tritylhydroxylamine,
respectively. Analogs 13 and 14 were then made accord-
ing to the procedure for 12.
Friedel–Crafts acylation of 1540,41 yielded 1 and inter-
mediates (8 and 19) for making 3–7. In preparing 19,
the Friedel–Crafts reaction gave two regioisomers of
the thiophene ring. The unwanted minor isomer had a
0.01-ppm-downfield shift for the thiophene proton and
was readily removed by chromatography. Conversion
of 19 to 20 was carried out in 95% yield by reduction
with stannous chloride42 as sulfur was feared to poison
the Pd catalyst, followed by protection of the amine
function with (Boc)2O.
2.3. Testing
HPLC-based assays47 were used to measure the inhibi-
tion of botulinum neurotoxins (BoNT) by 1–14. This
is because these compounds contain an indole and/or
phenyl ring (Fig. 1) that would interfere with fluores-
cence-based assays of BoNT inhibition.48
According to the HPLC-based assays, compounds 1–14
demonstrate inhibition of the endopeptidase of BoNTA
at the drug concentration of 100 lM, and hydroxamates
are more potent than the corresponding carboxylates or
hydrazines (Fig. 1). Furthermore, compounds 2, 4, and
12 inhibit the endopeptidase of BoNTA but have no ef-
fect on BoNTB (Table 1).
Reaction of 1 with hydroxylamine hydrochloride readily
gave rise to 2. However, a solid-phase synthesis of
hydroxamates using the Kaiser resin43 turned out to
be necessary in converting 18–20 to 3–5, respectively.
The Kaiser resin also was used to convert 19 and 20
to 6 and 7, respectively.
The indole moiety of 23 was constructed in 61% yield by
a two-step literature procedure using (Pd(Ph3)4/CuI/
Et2NH and PdCl2(PhCN)2/DMF).44 A recently reported
one-step procedure45 had a comparable yield (63%), but
its operation was more tedious than that of the two-step
synthesis. Precursor 23 was converted to 9 in a high yield
by basic hydrolysis followed by coupling with O-trit-
ylhydroxylamine. Deprotection of the trityl group was
carried out effectively by using a 1:2 mixture of AcOH/
2,2,2-trifluoroethanol.
Compounds 12–14 are equally active and they are the
most potent of the tested series. Kinetics studies show
that the Ki value for the inhibition of the endopeptidase
of BoNTA by 12 is 12 2.6 lM (average standard
Table 1. Inhibition of the endopeptidase (LC) in botulinum neuro-
toxin serotypes A and B (BoNTA and BoNTB) by hydroxamate-
containing inhibitors
Inhibitor
% Inhibition at 100 lM
BoNTA LC BoNTB LC
2
4
30 1 (2)
40 3 (3)
96 6 (2)
0
0
0
Introduction of an aminobutyl group to the indole
nitrogen of 23 to make 10–14 was initially problematic,
presumably due to the poor nucleophilicity of the indole
12

400
J. G. Park et al. / Bioorg. Med. Chem. 14 (2006) 395–408
deviation). Adding more zinc to the assays, up to
0.1 mM, had no effect on the inhibition by 12 (data
not shown), confirming that the inhibition was not due
to non-specific metal chelation. This is further supported
by the finding that 12 did not inhibit the endopeptidase
of BoNTB, even when no additional zinc was present.
According to the current literature, 12 has been the most
potent and serotype-specific small-molecule inhibitor of
the endopeptidase of BoNTA.
The free energy perturbation method24 is perhaps most
effective in assessing the relative free energy of binding
to zinc for structure-based design of zinc enzyme inhib-
itors; however, its application to even a truncated BoN-
TA endopeptidase that comprises 496 amino acid
residues is discouragingly expensive. Alternatively, this
work suggests that the competitiveness for zinc coordi-
nation can be evaluated by MMDSs using the CaDA
approach. It is computationally less expensive than the
free energy perturbation method, and yet accounts for
the entropy and solvent effects in assessing ligandsꢁ abil-
ity to compete for zinc coordination. Given the success
of 12 as the most serotype-specific and potent small-
molecule inhibitor of BoNTA to date, the CaDA-based
MMDS strategy appears to be useful to structure-based
design of zinc enzyme inhibitors.
3. Discussion
3.1. Toxicity
Hydroxamates are known as promiscuous zinc coordi-
nates34 as exemplified by matrix metalloprotease inhibi-
tors.49–52 Therefore, one concern of hydroxamates as
inhibitors of the endopeptidase is potential toxicity.
4. Conclusions
The endopeptidases of BoNTA and BoNTB are closely
related.6,53 Only two residues are different in the active
site between the two enzymes: Phe162 and Phe193 in
BoNTA correspond to Asn169 and Ser200 in BoNTB.
One would expect that the hydroxamate-containing
inhibitors of BoNTA would inhibit BoNTB indiscrimi-
nately. Interestingly, as a good inhibitor of BoNTA,
12 showed no inhibition on BoNTB (Table 1).
A novel small-molecule inhibitor of the endopeptidase
of BoNTA has been developed according to the zinc-
containing active site of the enzyme. This inhibitor selec-
tively inhibits the endopeptidase of BoNTA with a Ki of
12 2.6 uM. It was designed to coordinate the zinc ion
embedded in the active site of the enzyme for affinity
and to interact with a species-specific residue in the ac-
tive site for selectivity. It is the most potent and sero-
type-specific small-molecule inhibitor of BoNTA
reported to date. This work suggests that MMDSs using
the CaDA approach are useful to structure-based design
of zinc protease inhibitors.
This result confirms that hydroxamates can be selective
if they are well crafted according to the active sites of
their targets.54,55 While further experimental studies on
the interaction of 12 with other metalloenzymes are nec-
essary, according to Table 1 it is unlikely that 12 inter-
acts with other metalloenzymes. The toxicity concern
regarding hydroxamate-containing BoNTA inhibitors
is remote, especially when more functional groups are
introduced to 12 to further improve its affinity and
selectivity.
5. Methods and materials
Preparation of the zinc- and inhibitor-bound endopepti-
dase. Limited computing resources required that a trun-
cated endopeptidase (residues 1–429, 453–471, and 497–
544) be used in this study. The initial 3D structure of the
truncated zinc-containing endopeptidase was taken
from an available crystal structure of BoNTA (PDB
code: 3BTA)6 and modified according to a published
procedure.15 The zinc divalent cation in the crystal
structure was replaced by the tetrahedron-shaped zinc
divalent cation that has four cationic dummy atoms sur-
rounding the central zinc ion.14 The first-shell zinc coor-
dinates (His222 and His226; and Glu261) were
deprotonated as histidinate and glutamate, respective-
ly,14,15,56–58 Glu260 and Glu350, which form a hydrogen
bond with His222 and His226, respectively, were pro-
tonated as glutamic acid.14,15,56–58 All other His residues
were protonated as histidinium. Inhibitor 12 was manu-
ally docked into the active site according to the 1-bound
endopeptidase complex generated by the EUDOC pro-
gram.59 Four sodium ions were added to the surface
of the protein to neutralize the protein. The published
force field parameters of the tetrahedron-shaped zinc
divalent cation16 were used in energy minimizations
and in the following described molecular dynamics sim-
ulations, with the exception that the force constants for
DZ–DZ and ZN–DZ bonds were changed from 540
to 640 kcal/mol. The RESP charges and force field
3.2. New strategy to zinc enzyme inhibitors
Structure-based design of zinc enzyme inhibitors has
long been a challenging pursuit for two main reasons.
One is that the interaction energies between zinc and
its coordinates are markedly underestimated by molecu-
lar mechanics calculations using the one-atom-zinc rep-
resentation.32 The other is that the four-ligand
coordination of zinc identified in X-ray structures of
zinc enzymes inevitably changes to a six-ligand coordi-
nation during energy minimization or molecular dynam-
ics simulations.14–17 However, these problems can be
solved by using the CaDA approach.14–18
The present work demonstrates that the ability of a mol-
ecule to coordinate the active-site zinc ion is governed by
(i) the fitness of the molecule to the zinc-containing ac-
tive site, (ii) the intrinsic affinity of the molecule for zinc,
and (iii) the competitiveness for zinc coordination rela-
tive to nearby zinc ligands available in the active site.
The last factor is difficult to address structurally and
represents a new challenge of structure-based design of
zinc enzyme inhibitors.

J. G. Park et al. / Bioorg. Med. Chem. 14 (2006) 395–408
401
parameters of inhibitor 12 were generated by the AN-
TECHAMBER module of AMBER 7 program60 using
the structure of 12 optimized at the HF/6-31G* level
by the Gaussian 98 Program.61 A 10,000-step energy
minimization was first performed on inhibitor 12 with
a positional constraint applied to the rest of the com-
plex. A 50-step energy minimization was then performed
on the tetrahedron-shaped zinc divalent cation and 12
with a positional constraint applied to the endopepti-
dase only. A 100-step energy minimization was lastly
performed on the entire complex without any restraint
or constraint.
spectrometers. 13C NMR spectra were recorded on Bru-
ker Avance 500 (125 MHz), Bruker Avance 600
(150 MHz), or Varian Mercury 400 (100 MHz) spec-
trometers. Chemical shifts are reported in ppm from
the solvent resonance as the internal standard. Data
are reported as follows: chemical shift, multiplicity (s,
single; d, doublet; t, triplet; q, quartet; br, broad; and
m, multiplet), coupling constants (Hz), integration,
and assignment. Mass spectra were obtained on an
HP5973 mass-selective detector with an SIS DIP-MS.
Anhydrous THF and CH2Cl2 were obtained through
activated alumina columns by the Solv-Tech Inc. Anhy-
drous DMF and CH3CN were obtained by distillation
from CaH2 under N2. POCl3 was distilled under N2
immediately before use. All other commercially ob-
tained reagents were used as received. Flash chromatog-
raphy was performed on silica gel 60 (EM Science, 230–
400 mesh).
5.1. Virtual screening
Computational screening of an in-house database of 2.5
million chemical structures for inhibitor leads of BoN-
TA was carried out according to a published protocol.31
In this study, the EUDOC program59 was used on a
dedicated terascale computer with 420 Intel Xeon pro-
5.4. (2-Phenylthiophene-3-yl)acetic acid methyl ester (15)
3
˚
cessors (2.2 GHz). A docking box (3.5 · 3.5 · 8.0 A )
was defined in the active site of the zinc-bound endopep-
tidase that was modified from the crystal structure of
BoNTA (PDB code: 3BTA)6 using the procedure de-
scribed above. The active-site residues that surrounded
the docking box were Ile160, Phe162, Phe193, Thr214,
Thr219, His222, Glu223, Arg362, Tyr365, Asp369, and
Zn(II). The translational and rotational increments for
To a solution of 15.5 mL (3 equiv) of freshly distilled
DMF in a flame-dried flask at 5 ꢁC was added
15.5 mL (2.5 equiv) of freshly distilled POCl3 under
N2. The POCl3 mixture was warmed to room tempera-
ture followed by adding 13.75 g (66.66 mmol) of ethyl
3-benzoylpropionate (17). The resulting mixture was
stirred at 80 ꢁC for 16 h, cooled to room temperature,
poured onto ice, and basified to pH 6 with solid NaOAc.
The organic phase was extracted with EtOAc, washed
with a saturated NaHCO3 solution, dried over MgSO4,
filtered, and concentrated in vacuo. Kugelrohr distilla-
tion (195–200 ꢁC/0.1 mmHg) of the concentrated residue
gave 14.84 g (88%) of 16 as a brown oil. The product
which consisted of E and Z isomers at a ratio of 2:1
was subjected to next reaction without separation.
˚
the screening were set at 1.0 A and 10ꢁ of arc,
respectively.
5.2. Multiple molecular dynamics simulations
The multiple molecular dynamics simulations (MMDSs)
were performed according to a published protocol16,33
using the parallel PMEMD module of the AMBER 8
program60 with the Cornell et al. force field (parm96.-
dat)62 The zinc- and 12-containing endopeptidase com-
plex was solvated with 14,972 TIP3P water molecules63
and neutralized with four sodium ions, resulting in a sys-
tem of 52,993 atoms. The topology and coordinate files
of the zinc- and 12-containing endopeptidase were gen-
erated by the LINK, EDIT, and PARM modules of
the AMBER5 program.60 All simulations used (1) a
dielectric constant of 1.0, (2) the Berendsen coupling
algorithm,64 (3) a periodic boundary condition at a con-
stant temperature of 300 K and a constant pressure of
1 atm with isotropic molecule-based scaling, (4) the par-
ticle mesh Ewald method to calculate long-range electro-
static interactions,65 (5) iwrap = 1 to generate the
trajectories for interaction energy calculations using
the EUDOC program,59 (6) a time step of 1.0 fs, (7)
the SHAKE-bond-length constraints applied to all
bonds involving the hydrogen atom,66 and (8) default
values of all other inputs of the PMEMD module.
To a solution of 50 mL THF was added 5.00 g of 16
(19.79 mmol) followed by adding 2.06 mL (29.68 mmol)
of mercaptoacetic acid and 8.27 mL NEt3 (59.36 mmol).
The resulting solution was refluxed for 4 h. After the
removal of THF by distillation, the residue was dis-
solved in 50 mL DMF and the resulting solution was
heated at 130 ꢁC for 18 h. After DMF was removed in
vacuo, the residue was diluted with 50 mL H2O and
extracted with EtOAc. The organic layer was dried over
MgSO4, filtered, and concentrated in vacuo. The residue
was purified by flash chromatography (EtOAc/Hex 1:4)
to give 2.71 g (56%) of (2-phenylthiophene-3-yl)acetic
1
acid ethyl ester as a brown oil: H NMR (500 MHz,
DCDl3) d 7.47 (d, J = 7.5 Hz, 2H), 7.42 (d, J = 7.4 Hz,
2H), 7.34 (t, J = 7.4 Hz, 1H), 7.25 (d, J = 5.2 Hz, 1H),
7.08 (d, J = 5.2 Hz, 1H), 4.15 (q, J = 7.1 Hz, 2H), 3.64
(s, 2H), 1.24 (t, J = 7.1 Hz, 3H); 13C NMR (125 MHz,
CDCl3) d 171.2, 140.6, 133.8, 129.7, 129.4, 128.5,
127.7, 124.0, 60.8, 34.5, 14.1.
5.3. Synthesis
To a solution of 4.34 g (17.63 mmol) of the above ethyl
ester in 50 mL of MeOH was added 1.48 g (26.44 mmol)
KOH. The resulting mixture was refluxed for 3 h. After-
ward brown precipitates from the mixture were removed
by filtration and the filtrate was concentrated in vacuo.
The residue was dissolved in 20 mL of H2O, washed
Melting points are uncorrected. Infrared spectra were
record on a Thermo Nicolet Avatar 370 FT-IR. 1H
NMR spectra were recorded on Bruker Avance 500
(500 MHz), Bruker Avance 600 (600 MHz), or Varian
Mercury 400 (400 MHz), or Varian AM60 (60 MHz)

402
J. G. Park et al. / Bioorg. Med. Chem. 14 (2006) 395–408
1
for making 1. Methyl ester of 19: H NMR (60 MHz,
with 20 mL of EtOAc, and acidified to pH ꢀ 2–3 with
1 N HCl. The resulting mixture was extracted with
30 mL EtOAc and the organic phase was dried over
MgSO4, filtered, and concentrated in vacuo. The residue
was dissolved in 50 mL MeOH followed by adding
1.5 mL of concentrated HCl. The resulting solution
was refluxed for 5 h. The solvent was removed in vacuo,
and the residue was diluted with 20 mL H2O, neutral-
ized with a saturated NaHCO3 solution, and extracted
with 30 mL CH2Cl2. The organic layer was dried over
MgSO4, filtered, and concentrated in vacuo. The residue
was purified by flash chromatography (EtOAc/Hex 1:4)
to give 3.30 g (80%) of 15 as a white solid: mp 29–31 ꢁC;
1H NMR (400 MHz, CDCl3) d 7.48–7.33 (m, 5H), 7.27
(d, J = 5.2 Hz, 1H), 7.07 (d, J = 5.2 Hz, 1H), 3.70 (s,
3H), 3.66 (s, 2H); 13C NMR (100 MHz, CDCl3) d
171.8, 140.7, 133.7, 129.7, 129.4, 129.2, 128.6, 127.8,
124.2, 52.1, 34.2; IR (KBr) cmꢁ1 3113, 2941, 1728.
CDCl3) d 7.71 (m, 4H), 7.61 (s, 1H), 7.47 (m, 5H),
3.71 (s, 3H), 3.67 (s, 2H); LREIMS C20H15BrO3S re-
quires 413.99. Found: 412 ([Mꢁ1+], 100%), 413 (80%).
19 as a pale yellow solid: mp 147–150 ꢁC; 1H NMR
(400 MHz, CDCl3) d 7.77 (d, J = 8.2 Hz, 2H), 7.66 (d,
J = 8.2 Hz, 2H), 7.62 (s, 1H), 7.48 (m, 5H), 3.72 (s,
2H); 13C NMR (100 MHz, CDCl3) d 186.8, 175.5,
150.6, 140.9, 137.3, 136.6, 132.5, 131.8, 130.7, 129.8,
129.3, 129.2, 129.1, 127.4, 33.9; IR (KBr) cmꢁ1 2932,
1714, 1625; LREIMS C19H13BrO3S requires 399.98.
Found: 400 ([M+], 100%).
5.7. 2-[5-(4-Chlorobenzoyl)-2-phenylthiophene-3-yl]-N-
hydroxyacetamide (2)
To a stirred mixture of 30 mg (0.08 mmol) of 1, 11 mg
(0.08 mmol) of N-hydroxybenzotriazole (HOBt), 21 mg
(0.11 mmol) of 1-ethyl-3-(30-dimethylaminopropyl)car-
bodiimide hydrochloride (EDCIÆHCl), and 28 mL
(0.25 mmol) of 4-methylmorpholine in 5 mL dry CH2Cl2
was added 6 mg (0.08 mmol) of HONH2 Æ HCl at room
temperature. The resulting mixture was stirred overnight
and washed with 5 mL of H2O. The organic layer was
dried over MgSO4, filtered, and concentrated in vacuo.
The residue was purified by flash chromatography
(EtOAc) to give 6 mg (20%) of 2 as a white viscous res-
idue that solidified slowly at room temperature: 1H
NMR (400 MHz, DMSO-d6) d 10.69 (s, 1H), 8.90 (br
s, 1H), 7.86 (d, J = 8.6 Hz, 2H), 7.72 (s, 1H), 7.68–7.50
(m, 7H), 3.39 (s, 2H); 13C NMR (100 MHz, DMSO-
d6) d 185.9, 166.3, 148.8, 139.7, 138.6, 137.4, 136.0,
133.0, 132.3, 130.6, 129.2, 129.2, 129.1, 128.9; IR
(KBr) cmꢁ1 3305, 3223, 3060, 2921, 1746, 1631, 1436;
LREIMS C19H14ClNO3S requires 371.04. Found: 371
([M+], 9%), 355 (100%).
5.5. [5-(4-Chlorobenzoyl)-2-phenylthiophene-3-yl]acetic
acid (1)
To a stirred solution of 0.05 g (0.22 mmol) of 15 and
27 lL (0.22 mmol) of 4-chlorobenzoyl chloride in 5 mL
of dry CH2Cl2 was added 15 mg (0.11 mmol) of AlCl3
in three portions at room temperature. The resulting
mixture was stirred overnight. The reaction mixture
was slowly poured onto 5 g of ice and allowed to warm
to room temperature. The organic layer was dried over
MgSO4, filtered, and concentrated in vacuo. The residue
was purified by flash chromatography (EtOAc/Hex 1:9)
to give 0.04 g (50%) of [5-(4-chlorobenzoyl)-2-phenylthi-
1
ophene-3-yl]acetic acid methyl ester as a white solid: H
NMR (600 MHz, CDCl3) d 7.84 (d, J = 8.2 Hz, 2H),
7.61 (s, 1H), 7.50–7.43 (m, 3H), 3.71 (s, 3H), 3.67 (s,
2H); 13C NMR (150 MHz, CDCl3) d 186.5, 171.1,
150.1, 140.8, 138.6, 137.3, 136.2, 132.6, 130.5, 129.2,
128.9, 128.8, 128.7, 52.2, 34.1; IR (KBr) cmꢁ1 2949,
1737, 1623; LREIMS C20H15ClO3S requires 370.04.
Found: 370 ([M+], 100%), 310 (40%), 139 (59%).
5.8. [5-(4-Iodo-3-nitrobenzoyl)-2-phenylthiophene-3-yl]ace-
tic acid (19)
To 3.00 g (10.24 mmol) of 4-iodo-3-nitrobenzoic acid
was added 20 mL SOCl2. The resulting solution was re-
fluxed for 2 h. The excess thionyl chloride was removed
by distillation. A residual amount of thionyl chloride
was removed by N2 stream and thereafter by high vacu-
um manifold (0.2 mmHg). The resulting acid chloride
was dissolved in 30 mL of dry CH2Cl2, followed by add-
ing 2.38 g (10.24 mmol) of 15. To the resulting mixture
was added 5.46 g (40.95 mmol, 4.0 equiv) of AlCl3 in five
portions at room temperature. The reaction was moni-
tored by TLC. The completed reaction was quenched
by pouring the reaction mixture onto 50 g of ice. The
aqueous phase was extracted with 50 mL CH2Cl2. The
organic layer was dried over MgSO4, filtered, and con-
centrated in vacuo. The residue was purified by flash
chromatography (EtOAc/Hex 1:4) to give 3.86 g (74%)
To a stirred solution of 0.15 g (0.40 mmol) of the above
ester in a mixture of 2 mL THF and 1 mL H2O was add-
ed 0.60 mL (0.60 mmol) of 1 N NaOH at room temper-
ature. The resulting mixture was refluxed for 3 h. The
solvent was evaporated in vacuo. The residue was dis-
solved in 5 mL H2O and acidified with 0.11 mL of 1 N
HCl to give 0.14 g (100%) of 1 as a pale yellow solid:
mp 142–143 ꢁC; 1H NMR (400 MHz, CDCl3) d 9.75
(br s, 1H), 7.84 (d, J = 8.6 Hz, 2H), 7.62 (s, 1H), 7.462
(d, J = 8.6 Hz, 2H), 7.46 (m, 5H), 3.71 (s, 2H); 13C
NMR (100 MHz, CDCl3) d 186.6, 176.5, 150.6, 140.9,
138.8, 137.2, 136.1, 132.4, 130.6, 129.7, 129.3, 129.2,
129.1, 128.9, 34.0; IR (KBr) cmꢁ1 3058, 2921, 1715,
1628; LREIMS C19H13ClO3S requires 356.03. Found:
355 ([M+], 100%), 310 (25%), 244 (25%), 139 (42%).
1
of 21 as a yellow solid: H NMR (500 MHz, CDCl3) d
5.6. [5-(4-Bromobenzoyl)-2-phenylthiophene-3-yl]acetic
acid (18)
8.34 (d, J = 1.9 Hz, 1H), 8.23 (d, J = 8.1 Hz, 1H), 7.76
(dd, J = 1.9, 8.1 Hz, 1H), 7.64 (s, 1H), 7.48 (m, 5H),
3.73 (s, 3H), 3.69 (s, 2H); 13C NMR (125 MHz, CDCl3)
d 184.3, 170.9, 152.9, 151.4, 142.5, 139.7, 138.8, 137.8,
132.9, 132.3, 131.0, 129.3, 129.2, 129.0, 125.6, 91.1,
52.3, 34.1.
Compound 18 was obtained in 74% yield using 4-bro-
mobenzoyl chloride (from 4-bromobenzoic acid and
SOCl2) according to the same procedure as the one used

J. G. Park et al. / Bioorg. Med. Chem. 14 (2006) 395–408
403
Saponification of 21 using a stoichiometric amount of
1 N NaOH in a 1:1 mixture of MeOH and THF gave
19 as a yellow solid: mp 100–103 ꢁC; 1H NMR
(400 MHz, DMSO-d6) d 12.52 (br s, 1H), 7.75 (d,
J = 8.2 Hz, 1H), 7.72 (s, 1H), 7.52 (m, 5H), 7.21 (d,
J = 1.6 Hz, 1 H), 6.78 (dd, J = 1.6, 8.2 Hz, 1H), 3.66
(s, 2H); 13C NMR (100 MHz, DMSO-d6) d 186.7,
171.9, 148.8, 148.4, 139.9, 138.9, 138.6, 138.1, 132.4,
132.3, 129.2, 129.1, 128.7, 117.9, 113.7, 88.5, 34.0; IR
(KBr) cmꢁ1 3445, 3357, 2920, 1718, 1694; LREIMS
C19H12INO5S requires 492.95. Found: 492 ([M+],
100%).
g) packed in a Macro Kan (from IRORI Inc., San Die-
go, CA) was loaded with 18–20 according to a literature
procedure43 followed by cleavage with hydroxylamine to
make 3, 4, and 5 or with hydrazine to make 6 and 7.
5.11. 2-[5-(4-Bromobenzoyl)-2-phenylthiophene-3-yl]-N-
hydroxyacetamide (3)
1H NMR (400 MHz, DMSO-d6) d 10.68 (s, 1H), 8.89 (s,
1H), 7.81 (d, J = 8.6 Hz, 2H), 7.78 (d, J = 8.6 Hz, 2H),
7.72 (s, 1H), 7.67 (dd, J = 1.4, 8.1 Hz, 2H), 7.54–7.47
(m, 3H), 3.69 (s, 2H); 13C NMR (100 MHz, DMSO-
d6) d 186.1, 166.3, 148.9, 139.6, 138.6, 136.4, 133.0,
132.3, 131.8, 130.7, 129.21, 129.15, 129.06, 126.4, 32.5;
IR (KBr) cmꢁ1 3264, 2914, 1635, 1435; LREIMS
C19H14BrNO5S requires 414.99. Found: 417, 415
([M+], 5%), 402, 400 ([MꢁNH+], 100%).
5.9. [5-(3-tert-Butoxycarbonylamino-4-iodobenzoyl)-2-
phenylthiophene-3-yl]acetic acid (20)
To a solution of 5.54 g (10.92 mmol) of 21 in 100 mL
EtOAc was added 12.32 g (54.60 mmol, 5.0 equiv) of
stannous chloride dihydrate. The resulting mixture was
refluxed for 30 min under N2, afterward quenched by
pouring onto 100 g of ice, and basified to pH 8 with a
saturated NaHCO3 solution. The white milky mixture
was filtered through a Celite pad to remove the tin oxi-
des. The organic layer was dried over MgSO4, filtered,
and concentrated in vacuo to give 5.02 g (96%) of 22
5.12. 2-[5-(3-Amino-4-iodobenzoyl)-2-phenylthiophene-3-
yl]-N-hydroxyacetamide (4)
Mp >170 ꢁC (decomp); 1H NMR (400 MHz, DMSO-d6)
d 10.69 (s, 1H), 8.87 (s, 1H), 7.71 (d, J = 8.2 Hz, 1H),
7.70 (s, 1H), 7.67 (d, J = 7.5 Hz, 2H), 7.51 (m, 3H),
7.17 (d, J = 2.0 Hz, 1H), 6.75 (dd, J = 2.0, 8.0 Hz, 1H),
3.38 (s, 2H); 13C NMR (100 MHz, DMSO-d6) d 186.8,
166.3, 149.0, 148.3, 139.9, 138.8, 138.2, 138.1, 132.8,
132.3, 129.1, 129.0, 117.7, 113.5, 88.2, 32.5; IR (KBr)
cmꢁ1 3446, 3358, 3218, 2920, 1654, 1608, 1432.
1
as a yellow solid: H NMR (400 MHz, CDCl3) d 7.79
(d, J = 8.2 Hz, 1H), 7.64 (s, 1H), 7.45 (m, 5H), 7.22 (d,
J = 1.5 Hz, 1H), 6.97 (dd, J = 1.5, 8.2 Hz, 1H), 4.33
(br s, 2H), 3.72 (s, 3H), 3.68 (s, 2H); 13C NMR
(100 MHz, CDCl3) d 187.5, 171.5, 150.3, 147.4, 141.2,
139.3, 137.7, 132.9, 130.7, 129.5, 129.3, 129.2, 120.4,
114.6, 89.2, 52.6, 34.4; IR (KBr) cmꢁ1 3457, 3358,
1734, 1626; LREIMS C20H16INO3S requires 476.99.
Found: 477 ([M+], 100%).
5.13. 2-[5-(3-Nitro-4-iodobenzoyl)-2-phenylthiophene-3-
yl]-N-hydroxyacetamide (5)
Mp 147–151 ꢁC; 1H NMR (400 MHz, DMSO-d6) d
10.66 (s, 1H), 8.88 (s, 1H), 8.34 (d, J = 7.8 Hz, 1H),
8.30 (s, 1H), 7.79 (m, 2H), 7.68–7.50 (m, 5H), 3.39 (s,
2H); 13C NMR (100 MHz, DMSO-d6) d 184.4, 166.2,
153.4, 149.7, 141.8, 139.3, 138.9, 138.0, 133.2, 132.9,
132.1, 129.3, 129.2, 129.0, 124.6, 93.4, 32.4; IR (KBr)
cmꢁ1 3245, 2922, 1629, 1533, 1433; LREIMS C19H13I-
N2O5S requires 507.96. Found: 508 ([M+], 5%), 493
([MꢁNH+], 100%).
To a solution of 0.25 g (0.53 mmol) of 22 in 5 mL of dry
THF were added sequentially 0.07 g (0.05 mmol)
DMAP, 0.26 g (1.19 mmol) (Boc)2O, and 0.37 mL
(2.21 mmol) DIEA. The resulting solution was stirred
at room temperature for 12 h and afterward concentrat-
ed in vacuo. The residue was dissolved in a mixture of
2 mL of MeOH and 2 mL THF. To the mixture was
added 0.1 mL (1.0 mmol) of 1 N NaOH solution. The
resulting solution was stirred at room temperature for
3 h. Afterward the color changed from orange to deep
red. After the solvent was removed in vacuo, the residue
was dissolved in 10 mL EtOAc and washed with 10 mL
of a saturated NH4Cl solution. The organic layer was
dried over MgSO4, filtered, and concentrated in vacuo.
The residue was purified by flash chromatography
5.14. [5-(4-Bromobenzoyl)-2-phenylthiophene-3-yl]acetic
acid hydrazide (6)
Mp 157–161 ꢁC; 1H NMR (400 MHz, DMSO-d6) d 9.23
(s, 1H), 7.80 (d, J = 8.4 Hz, 2H), 7.77 (d, J = 8.4 Hz,
2H), 7.72 (s, 1H), 7.67 (d, J = 7.6 Hz, 2H), 7.49 (m,
3H), 4.25 (br s, 2H), 3.43 (s, 2H); 13C NMR
(100 MHz, CDCl3) d 186.6, 170.6, 150.3, 140.7, 137.2,
136.3, 132.3, 131.7, 131.6, 130.8, 130.6, 129.1, 129.0,
128.9, 128.8, 127.3, 34.2; IR (KBr) cmꢁ1 3294, 3048,
1635, 1435.
1
(EtOAc/Hex 1:1) to give 0.26 g (86%) of 20: H NMR
(400 MHz, CDCl3) d 8.57 (d, J = 2.2 Hz, 1H), 7.90 (d,
J = 8.2 Hz, 1H), 7.71 (s, 1H), 7.49 (m, 5H), 7.26 (dd,
J = 2.2, 8.2 Hz, 1H), 6.95 (s, 1H), 3.69 (s, 2H), 1.52 (s,
9H); 13C NMR (100 MHz, CDCl3) d 186.7, 176.1,
152.4, 150.6, 140.9, 139.1, 139.0, 138.6, 137.8, 132.6,
129.9, 129.2, 129.0, 128.9, 124.6, 120.5, 81.6, 34.4,
28.2; IR (KBr) cmꢁ1 3387, 2978, 1723.
5.15. [5-(3-Amino-4-iodobenzoyl)-2-phenylthiophene-3-
yl]acetic acid hydrazide (7)
Mp 159–165 ꢁC; 1H NMR (400 MHz, DMSO-d6) d 9.13
(s, 1H), 7.62 (d, J = 8.0 Hz, 1H), 7.58 (s, 1H), 7.55 (d,
J = 7.6 Hz, 2H), 7.37 (m, 3H), 7.05 (s, 1H), 6.62 (d,
J = 7.6 Hz, 1H), 4.12 (br s, 2H), 3.30 (s, 2H); 13C
5.10. Solid-phase synthesis of 3–7
Kaiser Oxime PS resin (purchased from Senn Chemi-
cals, Switzerland, 1% DVB, 100–200 mesh, 1.6 mmol/

404
J. G. Park et al. / Bioorg. Med. Chem. 14 (2006) 395–408
1
NMR (100 MHz, DMSO-d6) d 186.9, 168.8, 148.9,
148.3, 139.8, 138.9, 138.2, 133.1, 132.4, 129.1, 128.9,
117.8, 113.5, 88.2, 33.5; IR (KBr) cmꢁ1 3432, 1628,
1431; LREIMS C19H16IN3O2S requires 477.00. Found:
477 ([M+], 100%), 446 ([MꢁN2H3+], 45%), 246
([C6H3(NH2)I+], 100%).
to afford 0.36 g (87%) of 8: mp 238–240 ꢁC; H NMR
(400 MHz, DMSO-d6) d 12.5 (br s, 1H), 12.04 (s, 1H),
8.00 (s, 1H), 7.93 (d, J = 7.4 Hz, 2H), 7.80 (s, 1H),
7.70 (d, J = 8.2 Hz, 1H), 7.59–7.48 (m, 9H), 7.38 (t,
J = 7.1 Hz, 1H), 7.07 (d, J = 1.2 Hz, 1H), 3.69 (s, 2H);
13C NMR (100 MHz, CDCl3) d 186.6, 172.0, 147.4,
141.6, 141.0, 136.3, 132.6, 132.2, 131.9, 131.4, 130.4,
129.2, 129.1, 128.8, 128.3, 125.6, 125.4, 120.5, 99.3,
34.1; IR (KBr) cmꢁ1 3417, 3054, 1713, 1596.
5.16. [2-Phenyl-5-(2-phenyl-1H-indole-6-carbonyl)thio-
phene-3-yl]acetic acid methyl ester (23)
To 0.48 g (1.0 mmol) 22 placed in a flask was added
30 mL Et2NH under stream of N2. The resulting solu-
tion was degassed for 20 min with a stream of N2. To
this solution was added 164.7 lL (1.5 mmol) of phenyl-
acetylene. The resulting solution was then degassed for
5 min followed by adding 20.9 mg (0.11 mmol) of CuI
and 0.12 g (0.1 mmol) of Pd(PPh3)4 at room tempera-
ture. The resulting mixture was degassed for 10 min
and stirred at room temperature for 4 h. The solvent
was evaporated in vacuo, and the residue was purified
by flash chromatography (EtOAc/Hex 1:4) to give
0.44 g (98%) of [5-(3-amino-4-phenylethynylbenzoyl)-2-
phenyl-thiophene-3-yl]-acetic acid methyl ester as a yel-
low solid: mp 146–148 ꢁC; 1H NMR (400 MHz, CDCl3)
d 7.66 (s, 1H), 7.55 (m, 2H), 7.47 (m, 5H), 7.37 (m, 4H),
7.23 (m, 2H), 4.48 (s, 2H), 3.71 (s, 3H), 3.68 (s, 2H); 13C
NMR (100 MHz, CDCl3) d 187.45, 171.3, 149.9, 147.8,
141.2, 138.7, 137.4, 132.8, 132.1, 131.6, 130.4, 129.3,
128.98, 128.94, 128.7, 128.5, 122.8, 118.8, 114.4, 111.8,
97.2, 85.2, 52.3, 34.2.
5.18. N-Hydroxy-2-[2-phenyl-5-(2-phenyl-1H-indole-6-
carbonyl)thiophene-3-yl]acetamide (9)
To a stirred solution of 40.1 mg (0.09 mmol) of 8 in
5 mL of dry CH2Cl2 was added sequentially 140 mg
(0.37 mmol) of O-(7-azabenzotriazole-1-yl)-N,N,N0,N0-
tetramethyluronium hexafluorophosphate (HATU),
49.5 mg (0.37 mmol) of HOBt, and 91 lL (0.55 mmol)
DIEA. The resulting solution was stirred at room tem-
perature for 10 min followed by adding 50.5 mg
(0.18 mmol) of O-tritylhydroxylamine. After stirring at
the same temperature for 30 min, the solvent was re-
moved in vacuo and the residue was purified by flash
chromatography (EtOAc/Hex 1:4) to give O-trityl
hydroxyamic acid of 8 in a quantitative yield. The result-
ing oily product was treated with 5 mL of 1:2 mixture of
AcOH/2,2,2-trifluoroethanol at room temperature for
24 h. After evaporation of the solvent in vacuo, the res-
idue was dissolved in CH2Cl2 and washed with 10 mL of
a saturated NaHCO3 solution. The organic layer was
dried over MgSO4, filtered, and concentrated in vacuo.
The residue was purified by flash chromatography
(MeOH/EtOAc 1:9) to give 12.7 mg (54% over two
steps) of 9 as a bright yellow powder: mp ꢀ150 ꢁC (de-
A solution of 0.81 g (1.79 mmol) of the above com-
pound in 20 mL of dry DMF was degassed with N2
for 15 min followed by adding 0.14 g (0.36 mmol) of
PdCl2(PhCN)2. The resulting mixture was degassed for
10 min, heated at 80 ꢁC for 30 min, cooled to room tem-
perature, diluted with 50 mL of H2O, extracted with 3·
30 mL of EtOAc, and concentrated in vacuo. The resi-
due was purified by flash chromatography (EtOAc/
Hex 1:4) to give 0.49 g (61%) of 23 as a bright yellow sol-
1
comp); H NMR (400 MHz, DMSO-d6) d 12.1 (s, 1H),
11.68 (br s, 1H), 8.95 (br s, 1H), 7.99 (s, 1H), 7.93 (d,
J = 7.8 Hz, 2H), 7.81 (s, 1H), 7.70 (m, 3H), 7.66–7.48
(m, 6H), 7.38 (t, J = 7.0 Hz, 1H), 7.08 (s, 1H), 3.41 (s,
2H); 13C NMR (100 MHz, CDCl3) d 187.4, 167.1,
147.9, 142.3, 141.6, 138.1, 136.9, 133.3, 133.2, 132.8,
132.1, 131.4, 131.0, 129.8, 129.7, 129.0, 127.7, 126.2,
121.1, 120.7, 113.9, 99.9, 33.2; IR (KBr) cmꢁ1 3422–
3241, 2902, 1596, 1433, 1322.
1
id: mp 183–185 ꢁC; H NMR (400 MHz, CDCl3) d 9.50
(s, 1H), 8.19 (s, 1H), 7.79 (d, J = 8.3 Hz, 2H), 7.71 (m,
3H), 7.47 (m, 6H), 7.35 (t, J = 7.1 Hz, 1H), 6.89 (s,
1H), 3.71 (s, 3H), 3.68 (s, 2H); 13C NMR (100 MHz,
CDCl3) d 188.2, 171.5, 149.0, 142.1, 141.9, 137.2,
136.4, 132.95, 132.93, 131.7, 131.5, 130.2, 129.3, 129.1,
128.9, 128.8, 128.4, 125.7, 121.9, 120.0, 113.4, 99.9,
52.3, 34.3; IR (KBr) cmꢁ1 3350, 3054, 2947, 1737,
1596, 1436, 1314; LREIMS C28H21INO3S requires
451.12. Found: 451 ([M+], 100%).
5.19. {5-[1-(4-Aminobutyl)-2-phenyl-1H-indole-6-carbon-
yl]-2-phenylthiophene-3-yl}acetic acid hydroiodide (10)
To a stirred solution of 0.20 g (0.45 mmol) of 23 in
10 mL of anhydrous CH3CN was added 1.36 g of
CsF–Celite under N2 followed by adding 0.11 g
(0.45 mmol) of N-Boc-1-bromo-4-butylamine. The
resulting mixture was refluxed under N2 for 48 h. The
solvent was evaporated under reduced pressure, and
the residue was purified by flash chromatography
(EtOAc/Hex 1:4) to give 0.07 g (33% based on the recov-
ered starting material) of {5-[1-(4-N-Boc-aminobutyl)-2-
phenyl-1H-indole-6-carbonyl]-2-phenylthiophene-3-yl}ace-
tic acid methyl ester as a yellow foam: 1H NMR
(400 MHz, CDCl3) d 8.04 (s, 1H), 7.73 (m, 3H), 7.50
(m, 8H), 6.60 (s, 1H), 4.51 (br s, 1H), 4.28 (t,
J = 6.7 Hz, 2H), 3.72 (s, 3H), 3.70 (s, 2H), 2.97 (m,
2H), 1.73 (q, J = 7.5 Hz, 2H), 1.38 (s, 9H), 1.31 (q,
J = 6.3 Hz, 2H); 13C NMR (100 MHz, CDCl3) d 188.1,
5.17. [2-Phenyl-5-(2-phenyl-1H-indole-6-carbonyl)thio-
phene-3-yl]acetic acid (8)
To a stirred solution of 0.44 g (0.97 mmol) of 23 in 7 mL
MeOH and 7 mL THF was added 1.5 mL (1.5 mmol) of
1 N NaOH solution. The resulting solution was stirred
at room temperature for 4 h. The solvent was removed
in vacuo, and the residue was diluted with 10 mL
H2O, acidified to pH ꢀ 3 with 1 N HCl, and extracted
with 30 mL EtOAc. The organic layer was dried over
MgSO4, filtered, and concentrated in vacuo. The residue
was purified by flash chromatography (EtOAc/Hex 1:1)

J. G. Park et al. / Bioorg. Med. Chem. 14 (2006) 395–408
405
171.5, 155.8, 148.8, 144.9, 142.2, 136.9, 136.6, 132.9,
132.4, 131.7, 131.1, 130.1, 129.3, 128.9, 128.8, 128.7,
128.6, 121.4, 120.2, 112.3, 102.7, 79.1, 52.3, 43.7, 39.9,
34.3, 28.3, 27.4, 27.2; IR (KBr) cmꢁ1 3410–3380, 2930,
1738, 1709, 1625, 1600, 1437.
d 11.35 (s, 1H), 10.3 (br s, 3H), 8.13 (s, 1H), 7.89–7.49
(m, 14H), 6.70 (s, 1H), 4.36 (t, J = 7.4 Hz, 2H), 3.74
(s, 2H), 2.62 (m, 2H), 1.68 (m, 2H), 1.35 (m, 2H); 13C
NMR (100 MHz, DMSO-d6) d 186.9, 168.9, 147.9,
144.6, 141.1, 137.9, 136.2, 132.3, 131.8, 131.3, 131.1,
130.4, 129.2, 129.18, 129.0, 128.9, 128.8, 120.6, 120.3,
112.6, 102.5, 43.2, 38.3, 32.9, 26.8, 24.3; IR (KBr)
cmꢁ1 3421, 2936, 1700, 1594, 1446.
To a stirred solution of 56.9 mg (9.1 · 10ꢁ5 mol) of the
above ester in 2 mL MeOH was added 91.4 lL of 1 N
NaOH. The resulting mixture was refluxed for 2.5 h.
The solvent was removed in vacuo. The residue was
diluted with 2.5 mL H2O, neutralized with 91.4 lL of
1 N HCl, and extracted with 20 mL EtOAc. The organic
layer was dried over MgSO4, filtered, and concentrated
in vacuo to give 41.3 mg (75%) of {5-[1-(4-tert-butoxy-
carbonylamino-butyl)-2-phenyl-1H-indole-6-carbonyl]-
2-phenylthiophene-3-yl}acetic acid as a yellow solid: mp
167–169 ꢁC; 1H NMR (400 MHz, CDCl3) d 8.00 (s, 1H),
7.73 (m, 2H), 7.49 (m, 11H), 6.60 (s, 1H), 4.14 (t,
J = 7.0 Hz, 2H), 3.68 (s, 2H), 3.14 (m, 2H), 1.96 (m,
2H), 1.45 (s+m, 9+2H); IR (KBr) cmꢁ1 3433, 3303,
2977, 1714, 1601.
5.21. 2-{5-[1-(4-Aminobutyl)-2-phenyl-1H-indole-6-carbon-
yl]-2-phenylthiophene-3-yl}-N-hydroxyacetamide hydrochlo-
ride (12)
To a stirred solution of 43.0 mg (7.1 · 10ꢁ5 mol) of {5-
[1-(4-tert-butoxycarbonylamino-butyl)-2-phenyl-1H-in-
dole-6-carbonyl]-2-phenylthiophene-3-yl}acetic acid (N-
Boc-protected 10) in 5 mL of dry CH2Cl2 was added
sequentially 107.4 mg (2.83 · 10ꢁ4 mol) HATU,
38.2 mg
(2.83 · 10ꢁ4 mol)
HOBt,
and
70 lL
(4.24 · 10ꢁ4 mol) DIEA at room temperature. The
resulting mixture was stirred at room temperature for
10 min followed by adding 38.9 mg (1.41 · 10ꢁ4 mol)
of O-tritylhydoxylamine. After stirring at room temper-
ature for 30 min, the solvent was removed in vacuo, and
the residue was purified by flash chromatography
(EtOAc/Hex 1:4) to give 32.0 mg (57%) of O-tritylhydr-
oxamate as a yellow foam. The resulting material was
treated with 1.5 mL of a mixture of AcOH/TFE (1:2)
at room temperature for 48 h. The resulting mixture
was concentrated under reduced pressure, and the resi-
due was purified by flash chromatography (MeOH/
AcOEt 1:9) to afford 23.2 mg (92%) of the hydroxamate:
To a stirred solution of 30.0 mg of the above acid
(4.93 · 10ꢁ5 mol) in 1.0 mL CHCl3 was added 14 lL
(9.86 · 10ꢁ5 mol) TMSI at room temperature. After stir-
ring at room temperature for 6 h, precipitates were col-
lected by filtration, washed with CHCl3, and dried under
high vacuum to give 14.9 mg (47%) of 10 in its HI salt
1
form as a brown powder: mp >150 ꢁC (decomp); H
NMR (400 MHz, DMSO-d6) d 12.9 (br s, 1H), 8.12 (s,
1H), 7.81 (s, 1H), 7.75 (d, J = 8.2 Hz, 1H), 7.62–7.49
(m, 11H), 6.71 (s, 1H), 4.36 (t, J = 7.0 Hz, 2H), 3.70
(s, 2H), 3.44 (br s, 3H), 2.62 (h, J = 5.1 Hz, 2H), 1.65
(m, 2H), 1.29 (m, 2H); 13C NMR (100 MHz, DMSO-
d6) d 186.9, 172.1, 147.5, 144.5, 140.9, 138.5, 136.2,
132.5, 132.1, 131.8, 131.1, 130.5, 129.2, 129.16, 129.06,
128.9, 128.8, 120.6, 120.2, 112.5, 102.6, 43.1, 38.4,
34.1, 26.7, 24.3; IR (KBr) cmꢁ1 3438, 3057, 1718,
1590; LREIMS C31H28N2O3S (neutral form) requires
508.18. Found: 490 ([M+ꢁH2O], 60%).
1
mp ꢀ95 ꢁC (decomp); H NMR (400 MHz, DMSO-d6)
d 10.72 (s, 3H), 8.90 (s, 1H), 8.11 (s, 1H), 7.82 (s, 1H),
7.45–7.48 (m, 12H), 6.68 (s, 1H), 1.67 (s, 1H), 4.31 (t,
J = 7.0 Hz, 2H), 3.42 (s, 2H), 2.74 (q, J = 6.2 Hz, 2H),
1.58 (p, J = 7.4 Hz, 2H), 1.29 (s, 9H), 1.16 (m, 2H);
13C NMR (100 MHz, DMSO-d6) d 186.9, 166.4, 155.5,
147.4, 144.6, 140.9, 137.9, 136.2, 132.6, 132.5, 131.9,
130.9, 130.5, 129.1, 129.06, 128.9, 128.8, 128.6, 120.5,
120.2, 112.5, 102.4, 77.4, 43.3, 32.6, 28.2, 27.1, 26.7;
IR (KBr) cmꢁ1 3370–3260, 2929, 1677, 1448.
5.20. {5-[1-(4-Aminobutyl)-2-phenyl-1H-indole-6-carbonyl]-
2-phenylthiophene-3-yl}acetic acid hydrazide hydrochloride
(11)
To a stirred solution of 75 mg (0.12 mmol) of 2-{5-[1-(4-
N-Boc-aminobutyl)-2-phenyl-1H-indole-6-carbonyl]-2-
phenylthiophene-3-yl}-N-hydroxyacetamide in ethyl
acetate (3 mL) was added 100 lL (1.2 mmol) of 12 N
HCl. The reaction mixture was stirred at room temper-
ature for 30 min. The solvent was removed in vacuo and
the residue was then dissolved in water. Some insolubles
were observed and were removed by filtration through a
pad of Celite. The filtrate was freeze-dried under high
vacuum to give 61 mg (91% yield) of the desired product
as a fine brown powder; mp >155 ꢁC (decomp); 1H
NMR (400 MHz, DMSO-d6) d 10.89 (s, 1H), 9.02 (s,
1H), 8.12 (s, 1H), 7.84 (s, 1H), 7.76–7.49 (m, 12H),
6.70 (s, 1H), 4.34 (t, J = 6.9 Hz, 2H), 3.45 (s, 2H), 2.64
(h, J = 6.4 Hz, 2H), 1.69 (m, 2H), 1.36 (m, 2H); 13C
NMR (100 MHz, DMSO-d6) d 187.7, 167.4, 148.0,
145.2, 141.8, 138.4, 136.8, 133.3, 133.2, 132.5, 131.7,
131.1, 120.8, 129.7, 129.6, 129.4, 121.2, 121.1, 113.3,
103.3, 43.9, 39.1, 33.2, 27.5, 25.0; IR (KBr) cmꢁ1 3404,
3252, 3053, 2925, 1617, 1586, 1448.
To a stirred solution of 53.2 mg (8.54 · 10ꢁ5 mol) of {5-
[1-(4-N-Boc-aminobutyl)-2-phenyl-1H-indole-6-carbon-
yl]-2-phenylthiophene-3-yl} acetic acid methyl ester in
1.5 mL MeOH and 1.5 mL THF was added 415 lL of
hydrazine hydrate at room temperature. After stirring
at room temperature for 4.5 h, the solvent was removed
in vacuo. The residue was purified by flash chromatog-
raphy (EtOAc) to afford 49.7 mg (93%) of acid hydra-
zide as a yellow foam. To a solution of 60.0 mg of the
acid hydrazide (9.6 · 10ꢁ5 mol) in 1 mL MeOH was
added 16 lL (19.3 · 10ꢁ5 mol) of 12 N HCl at room
temperature. After stirring at room temperature for
17 h, the solvent was removed in vacuo. The deep red
residue was dissolved in 10 mL EtOAc followed by
extraction with 5 mL H2O. The aqueous layer was fil-
tered through a Celite pad. The filtrate was freeze-dried
to afford 11.5 mg (20%) of 11 as a dark red-brown pow-
1
der (dihydrochloride): H NMR (400 MHz, DMSO-d6)

406
J. G. Park et al. / Bioorg. Med. Chem. 14 (2006) 395–408
5.22. 2-{5-[1-(5-Aminopentyl)-2-phenyl-1H-indole-6-carbon-
yl]-2-phenylthiophene-3-yl}-N-hydroxy-acetamide hydro-
chloride (13)
aqueous phase was washed with DCM (20 mL). The
organic phases were combined and concentrated in vac-
uo. The residue was purified by flash chromatography
(SiO2, 2:1 Hex/EtOAc) to give 0.21 g (69% yield) of 2-
{5-[1-(5-N-Boc-aminopentyl)-2-phenyl-1H-indole-6-car-
bonyl]-2-phenylthiophene-3-yl}-O-tritylhydroxy-acetam-
ide as a yellow solid. Rf = 0.39 (2:1 Hex/EtOAc); mp
A suspension of 0.37 g (0.81 mmol) of 23, 0.23 g
(0.88 mmol, 1.1 equiv) of N-Boc-1-bromo-5-pentyl-
amine, and 2.00 g (8.0 mmol, 10 equiv) of CsF–Celite
in acetonitrile (15 mL) was refluxed under N2 for
24 h. The suspension was then allowed to cool to
room temperature and filtered through a pad of Cel-
ite. The filtrate was concentrated in vacuo and the res-
idue was purified by flash chromatography (SiO2, 4:1
Hex/EtOAc) to give 0.40 g (77% yield) of 2-{5-[1-(5-
N-Boc-aminopentyl)-2-phenyl-1H-indole-6-carbonyl]-2-
phenylthiophene-3-yl}acetic acid methyl ester as a yel-
low foam. Rf = 0.10 (4:1 Hex/EtOAc); 1H NMR
(400 MHz, CDCl3) d 7.96 (s, 1H), 7.70–7.62 (m,
3H), 7.47–7.34 (m, 10H), 6.52 (s, 1H), 4.37 (br s,
1H), 4.17 (t, J = 7.0 Hz, 2H), 3.64 (s, 3H), 3.62 (s,
2H), 2.90 (dt, J = 4.7, 5.6, 2H), 1.69–1.62 (m, 2H),
1.34 (s, 9H), 1.28–1.21 (m, 2H), 1.12–1.04 (m, 2H);
13C NMR (100 MHz, CDCl3) d 188.2, 171.7, 156.2,
149.0, 145.1, 142.2, 137.2, 137.0, 133.2, 133.0, 132.4,
131.9, 131.3, 130.4, 129.6, 129.5, 129.2, 129.1, 129.0,
128.8, 121.7, 120.3, 112.5, 102.8, 52.5, 44.1, 34.5,
30.0, 29.7, 28.6, 24.1; IR (KBr) cmꢁ1 3376, 2929,
2858, 1739, 1708, 1624, 1561, 1513.
1
ꢀ110 ꢁC; H NMR (400 MHz, CDCl3) d 7.97 (s, 1H),
7.68–7.18 (m, 23H), 6.56 (s, 1H), 4.37 (br s, 1H), 4.16
(t, J = 7.0 Hz, 2H), 3.60 (s, 2H), 2.90 (dt, J = 4.7, 5.6,
2H), 1.68–1.59 (m, 2H), 1.33 (s, 9H), 1.29–1.19 (m,
2H), 1.12–1.02 (m, 2H); 13C NMR (100 MHz, CDCl3)
d 188.4, 169.7, 156.2, 147.1, 145.1, 142.2, 137.1, 136.9,
136.8, 136.5, 133.5, 133.2, 132.7, 131.9, 131.3, 130.4,
129.6, 129.4, 129.2, 129.1, 129.0, 128.9, 128.7, 128.4,
128.3, 128.2, 128.1, 127.8, 127.7, 127.4, 121.7, 120.3,
112.6, 103.0, 102.9, 64.2, 49.1, 48.9, 46.1, 44.1, 40.5,
36.5; IR (KBr) cmꢁ1 3349, 2972, 2931, 2863, 1708,
1624, 1479, 1390.
A solution of 0.20 g (0.22 mmol) of the above product
in 2:1 mixture of 2,2,2-triflouroethanol/acetic acetic
(40 mL) was stirred at room temperature overnight.
Then the solvent was removed in vacuo and the resi-
due was dried under high vacuum for 1.5 h to get rid
of most of the acetic acid. The crude dry residue was
then purified by flash chromatography (SiO2, 1:1 Hex/
EtOAc) to give 87 mg (61% yield) of 2-{5-[1-(5-N-Boc-
aminopentyl)-2-phenyl-1H-indole-6-carbonyl]-2-phenyl-
thiophene-3-yl}-N-hydroxy-acetamide as a yellow sol-
id. Rf = 0.15 (1:1 Hex/EtOAc); mp ꢀ223 ꢁC; 1H
NMR (400 MHz, CDCl3) d 9.62 (br s, 1H), 8.51 (br
s, 1H), 7.99 (s, 1H), 7.78–7.36 (m, 13H), 6.57 (s,
1H), 4.58 (br s, 1H), 4.21 (t, J = 7.0 Hz, 2H), 3.52
(s, 2H), 2.96 (t, J = 5.7, 2H), 1.79–1.64 (m, 2H),
1.36 (s, 9H), 1.31–1.23 (m, 2H), 1.14–1.08 (m, 2H);
13C NMR (100 MHz, CDCl3) d 188.0, 168.2, 156.4,
149.1, 145.0, 143.1, 136.5, 133.0, 132.7, 132.0, 131.1,
130.2, 129.6, 129.2, 129.0, 128.7, 121.4, 120.3, 113.1,
103.0, 79.6, 64.2, 60.8, 44.2, 40.5, 29.9, 28.6, 24.0;
IR (KBr) cmꢁ1 3245, 2927, 2856, 1674, 1623, 1533,
1478, 1390.
To a stirred solution of 0.26 g (0.40 mmol) of the
above methyl ester in methanol (20 mL) was added
1.5 mL (1.5 mmol, 3.7 equiv) of 1.0 N NaOH. The
resulting solution was stirred under reflux for 4 h
and then allowed to cool to room temperature. The
solvent was removed in vacuo and the residue was dis-
solved in 40 mL EtOAc plus 30 mL water. To this
biphasic mixture, 1.5 mL (1.5 mmol) of 1.0 N HCl
was added. The phases were separated and the aque-
ous phase was washed with 40 mL EtOAc. The organ-
ic phases were combined and concentrated in vacuo.
The residue was purified by flash chromatography
(SiO2, 1:1 Hex/EtOAc) to give 0.25 g (quantitative
yield) of 2-{5-[1-(5-N-Boc-aminopentyl)-2-phenyl-1H-
indole-6-carbonyl]-2-phenylthiophene-3-yl}acetic acid
as a yellow foam. Rf = 0.29 (1:1 Hex/EtOAc); 1H
NMR (400 MHz, CDCl3) d 12.58 (br s, 1H) 8.03 (s,
1H), 7.78–7.31 (m, 13H), 6.53 (s, 1H), 4.78 (br s,
1H), 4.11 (t, J = 8.0 Hz, 2H), 3.58 (s, 2H), 3.02 (m,
2H), 1.81–1.71 (m, 2H), 1.42 (s, 9H), 1.40–1.34 (m,
2H), 1.18–1.09 (m, 2H); 13C NMR (100 MHz, CDCl3)
d 188.2, 175.5, 159.2, 148.8, 144.8, 143.1, 136.8, 136.4,
133.3, 132.8, 131.8, 130.9, 130.7, 129.7, 129.4, 129.2,
129.0, 128.9, 121.3, 120.3, 113.4, 103.0, 81.4, 64.6,
45.1, 41.3, 34.5, 30.5, 29.9, 28.6, 24.5; IR (KBr)
cmꢁ1 3371, 2928, 2856, 1711, 1624, 1533, 1479.
To a stirred solution of 69 mg (0.11 mmol) of the
above product in EtOAc (3 mL) was added 90 lL
(1.1 mmol, 10 equiv) of 12 N HCl. The reaction mix-
ture was stirred at room temperature for 30 min. The
solvent was removed in vacuo and the residue was then
dissolved in water. Some insolubles were observed and
were removed by filtration through a pad of Celite.
The filtrate was freeze-dried under high vacuum to give
54 mg (87% yield) of the desired final product 13 as a
1
fine brown powder; mp ꢀ139 ꢁC (decomp); H NMR
(400 MHz, DMSO-d6) d 10.87 (s, 1H), 8.97 (br s,
1H), 8.08 (s, 1H), 7.82–7.47 (m, 13H), 6.68 (s, 1H),
4.29 (t, J = 7.0 Hz, 2H), 3.44 (s, 2H), 3.35 (s, 2H),
2.62 (h, J = 6.0, 2H), 1.64–1.58 (m, 2H), 1.45–1.36
(m, 2H), 1.21–1.09 (m, 2H); 13C NMR (100 MHz,
DMSO-d6) d 187.6, 167.3, 148.1, 145.2, 141.6, 138.6,
136.8, 133.4, 133.2, 132.6, 131.6, 131.0, 129.8, 129.7,
129.6, 129.5, 129.4, 121.2, 121.0, 113.1, 103.1, 39.6,
39.1, 33.2, 29.8, 27.1, 23.7; IR (KBr) cmꢁ1 3395,
2936, 1662, 1616, 1596, 1477, 1380.
To a stirred solution of 0.22 g (0.35 mmol) of the above
acid in DCM (20 mL) were added sequentially 0.15 g
(0.39 mmol, 1.1 equiv) of HBTU, 0.11 g (0.39 mmol,
1.1 equiv) of O-tritylhydroxylamine, and 0.12 mL
(0.68 mmol, 1.9 equiv) of diisopropylethylamine
(DIEA). The reaction mixture was stirred at room tem-
perature for 1.5 h, and then water (15 mL) and brine
(5 mL) were added. The phases were separated and the

J. G. Park et al. / Bioorg. Med. Chem. 14 (2006) 395–408
407
5.23. 2-{5-[1-(6-Aminohexyl)-2-phenyl-1H-indole-6-carbon-
yl]-2-phenylthiophene-3-yl}-N-hydroxyacetamide hydrochlo-
ride (14)
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This work was supported by the Defense Advanced Re-
search Projects Agency (DAAD19-01-1-0322), the U.S.
Army Medical Research Acquisition Activity
(W81XWH-04-2-0001), the National Institutes of
Health/National Institute of Allergy and Infectious Dis-
eases (2R01AI054574-02), the Aeronautical Systems
Center of the High Performance Computing Moderniza-
tion Program of the U.S. Department of Defense, and
the University of Minnesota Supercomputing Institute.

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