Synthesis/biological evaluation of hydroxamic acids and their prodrugs as inhibitors for Botulinum neurotoxin A light chain

Article abstract of DOI:10.1016/j.bmc.2013.11.053

Botulinum neurotoxin A (BoNT/A) is the most potent toxin known Unfortunately, it is also a potential bioweapon in terrorism, which is without an approved therapeutic treatment once cellular intoxication takes place Previously, we reported how hydroxamic acid prodrug carbamates increased cellular uptake, which translated to successful inhibition of this neurotoxin Building upon this research, we detail BoNT/A protease molecular modeling studies accompanied by the construction of small library of hydroxamic acids based on 2,4-dichlorocinnamic hydroxamic acid scaffold and their carbamate prodrug derivatization along with the evaluation of these molecules in both enzymatic and cellular models

Full text of DOI:10.1016/j.bmc.2013.11.053

Bioorganic & Medicinal Chemistry 22 (2014) 1208–1217
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis/biological evaluation of hydroxamic acids and their
prodrugs as inhibitors for Botulinum neurotoxin A light chain
Hajime Seki a, Sabine Pellett b, Peter Šilhár a, G. Neil Stowe a, Beatriz Blanco c, Matthew A. Lardy a,
Eric A. Johnson b, Kim D. Janda a,d,
⇑
a Departments of Chemistry and Immunology, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037,
United States
b Department of Bacteriology, University of Wisconsin, 1550 Linden Drive, Madison, WI 53706, United States
c Centro Singular de Investigación en Química Biológica y Materiales Moleculares (CIQUS), Universidad de Santiago de Compostela, calle Jenaro de la Fuente s/n,
15782 Santiago de Compostela, Spain
d Worm Institute for Research and Medicine (WIRM), The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Botulinum neurotoxin A (BoNT/A) is the most potent toxin known. Unfortunately, it is also a potential
bioweapon in terrorism, which is without an approved therapeutic treatment once cellular intoxication
takes place. Previously, we reported how hydroxamic acid prodrug carbamates increased cellular uptake,
which translated to successful inhibition of this neurotoxin. Building upon this research, we detail BoNT/
A protease molecular modeling studies accompanied by the construction of small library of hydroxamic
acids based on 2,4-dichlorocinnamic hydroxamic acid scaffold and their carbamate prodrug derivatiza-
tion along with the evaluation of these molecules in both enzymatic and cellular models.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 11 October 2013
Accepted 28 November 2013
Available online 8 December 2013
Keywords:
Botulinum neurotoxin
SNARE
SNAP-25
Protease inhibitor
Zinc-dependent metalloprotease
Hydroxamic acid
Carbamate prodrug
1. Introduction
which can lead to fatal flaccid paralysis. The serotypes A, B, and E
are known to cause Botulism in humans. Among the SNARE com-
Botulinum neurotoxin, a protein produced by Clostridium botu-
linum, is the most potent toxin known to date.1 Although the toxin
has been historically recognized in food poisoning, its application
to medical maladies such as chronic pain and migraines; as well as
cosmetic improvementsincluding facial wrinkles, provides a clinical
significance that has led to its commercial production.2,3 This, how-
ever, also implicatesthe increased potential use of this toxin as a bio-
weapon in terrorism.4 Indeed, Botulinum neurotoxin has been
classified as a category A agent, the highest class for potential misuse
as a biothreat by the Center for Disease Control and Prevention.
Botulinum neurotoxin consists of 100 kDa heavy chain and
50 kDa light chain, which are appended to each other by a disulfide
linkage.5 In brief, the etiology of this neurotoxin consists of the
heavy chain acting to deliver the light chain into the cytosol by
translocation through target receptors on nerve terminals, and
depending on the serotype, the light chain cleaves one of the
proteins necessary for SNARE complex formation.6 As a result,
the release of acetylcholine into neuromuscular junction ceases,
ponents, type A and E target SNAP-25 whereas type B cleaves
VAMP.7,8 In addition to the aforementioned human intoxication,
its potency (LD50 value of 0.001 l
g per kg)9 and the length of dura-
tion (up to several months) sets Botulinum neurotoxin A apart
from the other serotypes and renders it to be a focal point of many
laboratories including our own.10
While there are known treatments such as vaccination and anti-
toxin treatment for serotype A, no therapeutic is currently available
once BoNT/A cellular intoxication takes place. Because the BoNT
protease is responsible for the pathology seen with all neurotoxins,
it is incumbent to develop small molecules to counteract its mech-
anism of action. To date, several potent light chain protease inhib-
itors have been reported based on in vitro enzyme assays, including
peptidic and non-peptidic molecules.11,12 However, the develop-
ment of efficacious inhibitors that can function intracellularly still
remains a major hurdle to overcome in the scientific community.
Hydroxamic acids developed in our laboratory as BoNT/A light
chain protease inhibitors have suffered from a lack of cellular pro-
tection, presumably due to their poor cellular uptake and toxic-
ity.11 However, we recently discovered that these issues could be
resolved and ultimately circumvented through protection of the
⇑
Corresponding author. Tel.: +1 858 784 2516; fax: +1 858 784 2595.
E-mail address: kdjanda@scripps.edu (K.D. Janda).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.11.053

H. Seki et al. / Bioorg. Med. Chem. 22 (2014) 1208–1217
1209
O
O
b-(2,4-dichlorophenyl)-hydroxamic acid O-carbamates along with
their inhibitory activity as determined through enzymatic as well
as cellular assays.
O
NHBn
prodrug
approach
NHOH
N
H
O
2. Results and discussion
2.1. Computational studies
A
A'
Ki = 460 nM
EC50 = 20 µM
cell permeable
The program Autocorrelator was utilized to construct computa-
tional models predicting and ranking potency of derived structures
against BoNT LC/A using 2 as a starting point for synthetically
accessible targets, amides 3 and ethers 4.18 In brief, a model was
developed through Autocorrelator using the 2IMB co-crystal struc-
ture of BoNT LC/A with a computational pipeline consisting of
Omega v2.4.6 and Fred v2.2.5 (R2 = 0.757).16,19,20 Autocorrelator
was also used to derive a new scoring function, produced by the
Scheme 1. O-Carbamation of the hydroxamic acid moiety as a prodrug approach.
hydroxamic acid moiety as a carbamate (Scheme 1).13 Thus, the
N-benzyl carbamate of hydroxamic acid A became cell-permeable
and now demonstrated inhibition of BoNT/A with the EC50 value
of 20 l
M in human induced pluripotent stem cells (hiPSCs).14
With this prodrug approach in mind, we next turned our focus
to additional druggable scaffolds (Scheme 2). Previously our labo-
ratory disclosed cinnamic hydroxamic acid 1 as a potent hit from
hydroxamic acid library screening,15,16 and a congener of this mol-
ecule, hydroxyethyl hydroxamate 2 as an inhibitor with an addi-
tional element of chemical functionality.17 To further diversify
this line of research, we devised two other scaffolds: amides (3)
and ethers (4) as a means to further increase hydrophobic interac-
tions within the active site of enzyme. Herein, we communicate
the synthesis/molecular modeling of amides (3), ethers (4), and
LARS methodology in R, shown in Eq. 121,22
:
pIC50 ¼ ꢀ0:43 þ Chemgauss3 ꢁ ꢀ 0:0714
þ Chemgauss3:Desolvation ꢁ ꢀ 0:142
þ Chemgauss3:Metal ꢁ ꢀ 0:0388
ð1Þ
Thus, computational modeling provided us a series of structures
as potential inhibitors with predicted IC50 values (Fig. 1). From this
computational exercise, aryl groups or planar structures seemed to
O
O
O
O
O
NHBn
NHBn
O
NHBn
NHBn
Cl
Cl
NHOH
NHOH
Cl
Cl
NHOH
OH
Cl
Cl
N
H
Cl
Cl
N
H
O
O
O
O
Cl
Cl
1
Cl
Cl
2
Cl
Cl
Cl
Cl
OH
prodrug
1'
2'
derivatization
O
O
O
O
O
O
N
H
NHOH
OR'
N
H
R
N
R
O
O
N
H
OR'
H
3
4
3'
4'
"amides"
"ethers"
in vitro evaluation
Scheme 2. Application of a prodrug approach to b-(2,4-dichlorophenyl)-hydroxamic acids.
Br
O
O
N
OMe
N
H
Cl
NHOH
O
N
3c
O
F
R
Cl
O
N
H
N
N
O
OMe
3a
3f
3b
4a
F
O
N
S
N
Cl
NHOH
OR
N
SO2
O
N
O
O
O
O
Cl
O
N
H
HO CF3
N
N
NH
OH
N
S
Predicted IC50
:
< 10 nM
< 100 nM
Figure 1. Predicted BoNT/A protease inhibitors and their IC50 values.
< 1µM

1210
H. Seki et al. / Bioorg. Med. Chem. 22 (2014) 1208–1217
Figure 2. The docking solutions of 3a and 3c within the 2IMB co-crystal using the automated pipeline derived by Autocorrelator.
Amides:
F
O
Br
MeO
Me
Cl
NHOH
NHR
3a
3d
3b
3e
3c
3f
F
OMe
Cl
O
Ethers:
Br
O
F
Cl
NHOH
OR'
Bn
allyl
4c
4a
4b
4d
Cl
Figure 3. Selected synthetic targets as possible protease inhibitors.
be important for engaging active site interactions. Two docking
solutions are shown in Figure 2, and based on these modeling
parameters, compounds such as 3a and 3c displayed optimized
steric and hydrogen-bonding interactions within the active site.
The size of the substituents also affected both the orientation
and hydrogen-bonding partners of our docked poses. Thus, larger
substituents forced molecules to lose interactions with Thr11,
Arg56, and Thr6, although this should have only a modest impact
on potency, as predicted molecules were able to still maintain
the key hydroxamate-zinc interaction. Hence, among the com-
pounds shown in Figure 1, we selected 3a–c, 3f, and 4a due to
the synthetic accessibility. Lastly, as many of the structures shown
in Figure 1 were synthetically challenging, close congeners (3d, 3e,
4b–d, Fig. 3) were prepared for examination.
converted to various amides 9 under the conventional coupling
conditions. Finally, each amide was reacted with hydroxylamine
to provide the desired hydroxamic acid 3.23
In terms of a similar set of ethers, we initially envisioned the
alkylation of alcohol B or C as a convergent approach (Scheme 4).
However, either route was found to be untenable, likely due to
the instability of starting materials under basic conditions. Thus,
although slightly more cumbersome, an alternative strategy was
undertaken. Hence, diacid 7 was reduced to diol 10, which was
then mono-alkylated with several electrophiles to provide alcohols
11. Each alcohol was subjected to three transformations in one-
pot: (1) oxidation of the alcohol to an acid by Jones’ reagent; (2)
formation of methyl ester using diazomethane; (3) conversion of
the ester to hydroxamic acid 4. We note that using this sequence
all attempts to isolate the methyl ester were not successful.
2.2. Synthesis of hydroxamic acids
2.3. Screening of selected hydroxamic acids against the BoNT/A
light chain 1–425
Synthesis of the target amide molecules was conducted using a
similar strategy to our previous report (Scheme 3).17 Thus, dichlo-
robenzaldehyde was condensed with dimethyl malonate in the
With desired hydroxamic acids in hand, their inhibitory activ-
ity was evaluated in vitro using an established FRET-based assay
(Table 1).24 The compounds were tested against truncated Bot-
lulinum neurotoxin A light chain (1–425 residues) in the pres-
ence of SNAPtide, a 13mer SNAP-25 pseudosubstrate containing
a FITC fluorophore and a DABCYL quencher. Based on the results
of the SNAPtide assay, the aryl moiety seems to be important.
presence of piperidine to provide the a,b-unsaturated diester 5. Di-
methyl malonate underwent a Michael addition with 5 under basic
conditions, affording tetraester 6, which was then subjected to
decarboxylation to furnish diacid 7. This diacid was mono-esteri-
fied via the initial formation of an anhydride with TFAA and subse-
quent nucleophilic opening with MeOH. The resulting acid 8 was

H. Seki et al. / Bioorg. Med. Chem. 22 (2014) 1208–1217
1211
Cl
Cl
O
CO2H
CO2H
MeO2C
NaOMe
CO2Me
CO2Me
CO2Me
CO2Me
Cl
O
O
piperidine
benzene
97%
conc. HCl
88%
dimethyl malonate
CO2Me
+
H
MeO
OMe
Cl
MeOH
98%
Cl
Cl
Cl
Cl
dimethyl
malonate
5
6
7
O
O
CO2Me
CO2H
i) (COCl)2, DMF, CHCl3
Cl
i) TFAA, CHCl3
ii) amine, DMAP, TEA, CHCl3
Cl
OMe
NHR
KCN, aq. NH2OH
THF, MeOH
Cl
NHOH
NHR
ii) TEA, MeOH
quant.
or
EDC, HOBt, amine
NMM, DCM
Cl
Cl
O
Cl
O
8
9
3
Scheme 3. Synthesis of amides 3.
Scheme 4. Synthesis of ethers 4.
Table 1
In vitro evaluation of amides 3 and ethers 4
This was true with either the amide or ether appendage,
whereas simple alkyl chains did not contribute to the inhibition
seen. Based on these findings, the most potent amide and ether
homologue of 2 were further evaluated in terms of Ki using our
66mer assay, where a cleaved product from 66 residues found
within SNAP-25 (141–206 residues) was quantified by LCMS
analysis.25 As anticipated, inhibitors 3a and 4a showed
competitive inhibition with Ki values of 1.0 and 2.1 lM respec-
tively (Table 1 and Fig. 4).
2.4. Synthesis of BoNT/A protease prodrugs
With in vitro assessment accomplished, several of the hydroxa-
mic acids (1, 2, 3a–3d, 4a–b, 4d) were converted to the

1212
H. Seki et al. / Bioorg. Med. Chem. 22 (2014) 1208–1217
further determined for these compounds with 3c0 being the most
potent, 198 l
M (Table 2). Disappointingly, prodrug ethers (4a0,
4b0 and 4d0) did not improve their cellular activity. This could par-
tially be due to a lack of solubility, which may also contribute to
cytotoxicity seen.
3. Conclusions
In summary, we have applied molecular modeling and a prodrug
strategy to our 2,4-dichlorocinnamic hydroxamic acid scaffold, thus
preparing a series of amide and ether O-carbamate hydroxamic
acids. The computational modeling studies granted insights into
the BoNT/A protease’s active site, which translated to a more effec-
tive approach, generating molecules for our screening efforts. In
general, these molecules as hydroxamic acids were toxic to the
cells, whereas their prodrug O-carbamates showed modest inhibi-
tory activity without major cellular toxicity. While we were pleased
to see our prodrug approach was translatable to alternate scaffolds,
in vitro cellular potency was marginal. We surmise this could be
due to the inefficient release of the hydroxamic acid warhead from
the corresponding prodrug, presumably enzyme-assisted within
the cell. Future research will entail exploration of the enzyme
responsible for carbamate hydrolysis as well as an alternative Zn
chelator to avoid the inherent toxicity of hydroxamic acids.
Figure 4. Kinetic analysis of inhibitor 3a.
O
O
CDI, NH2Bn
O
N
NHBn
NHOH
ACN
H
O
O
1, 2, 3a-d
4a-b, 4d
1', 2', 3a'-d'
4a'-b', 4d'
O
O
N
Scheme 5. Generic scheme showing synthetic access to the carbamate prodrugs.
4. Experimental section
4.1. Chemistry
Table 2
Prodrug screening using hiPSC-derived neurons
Prodrug
Inhibition
10
Cytotoxic at 150 l
Ma
Partial inhibition at 300 lM
4.1.1. Tetramethyl 2-(2,4-dichlorophenyl)propane-1,1,3,3-
tetracarboxylate (6)
20
3a0
3b0
3c0
3d0
4a0
4b0
4d0
EC50 > 300
EC50 = 209
EC50 = 198
EC50 > 300
lM
lM
lM
lM
To a stirred solution of dimethylmalonate (26.2 mmol, 3.46 g,
1.20 equiv) in NaOMe/MeOH solution (0.5 M, 26.2 mmol, 52 mL),
dimethyl 2-(2,4-dichlorobenzylidene)malonate (21.9 mmol, 6.32 g)
was added dropwise at ambient temperature. After 1 h, the reac-
tion mixture was cooled to 0 °C and quenched by the addition of
AcOH (87.4 mmol, 5.24 g, 4.0 equiv). Upon evaporation of volatiles,
reaction mixture was re-dissolved in dichloromethane and H2O.
The partitioned organic layer was dried over MgSO4, and concen-
trated in vacuo. The crude mixture was subjected to column chro-
matography (hexanes/EtOAc) to afford the titled compound as
clear oil (9.02 g, 98%).
Cytotoxic at 1 mMa
Cytotoxic at 1 mMa
EC50 >1 mM
a
No inhibition was observed up to the cytotoxic concentration examined.
corresponding benzylcarbamates (10, 20, 3a0-3d0, 4a0-b0, 4d0) as pro-
drugs via the formation of a carbonate intermediate and subse-
quent nucleophilic addition of benzylamine (Scheme 5).26 This
protocol achieves a selective O-carbamation (vs N-carbamation)
as shown previously.14
1H NMR (600 MHz, CDCl3) d 7.38 (s, 2H), 7.19 (d, J = 8.5 Hz, 1H),
4.79 (s, 1H), 4.37ꢀ4.12 (m, 2H), 3.70 (s, 6H), 3.57 (s, 6H); 13C NMR
(151 MHz, CDCl3) d 168.1, 167.8, 135.9, 134.5, 134.2, 130.0, 129.9,
127.4, 54.0, 53.0, 52.8; HRMS (ESI-TOF) m/e calcd for [M+H]+ C17-
H19Cl2O8: 421.0451, found 421.0450.
2.5. Testing of selected protease inhibitors in a BoNT/A cell
based assay
4.1.2. 3-(2,4-Dichlorophenyl)pentanedioic acid (7)
Tetraester 6 (10.4 mmol, 4.40 g) was dissolved in aq. HCl (37%,
30 mL) and heated to reflux overnight. The white solid was col-
lected by filtration to provide the titled compound as white solid
(2.56 g, 88%).
Selected hydroxamic acids and corresponding prodrugs were
tested in a hiPSC-derived neuron assay. The importance of this cell
line and assay is that it expresses the necessary receptors and sub-
strates for BoNT/A intoxication. The intoxicated cells were incu-
bated with each compound for 8 h, and upon cell lysis, the SNAP-
25 cleavage was analyzed by Western blot. The initial screening
was conducted at one or two concentrations (typically 0.1 and
1.0 mM). Unfortunately this assay revealed that most of the
hydroxamic acids were not effective due to cytotoxicity. Interest-
ingly, 2, was an exception, as it did not show cytotoxicity, although
1H NMR (600 MHz, MeOD-d4) d 7.42 (s, 1H), 7.36 (d, J = 8.0 Hz,
1H), 7.27 (d, J = 8.4 Hz, 1H), 4.06 (p, J = 6.9 Hz, 1H), 2.70 (m, 4H);
13C NMR (151 MHz, MeOD-d4) d 174.9, 140.6, 135.8, 133.9, 130.4,
130.3, 128.4, 39.4, 35.4; HRMS (ESI-TOF) m/e calc’d for [M+H]+ C11-
H11Cl2O4: 277.0029, found 277.0033.
no inhibition was observed at 200
l
M. Next, the carbamate pro-
4.1.3. 3-(2,4-Dichlorophenyl)-5-methoxy-5-oxopentanoic acid
(8)
To a solution of diacid 7 (3.00 mmol, 831 mg, 1.0 equiv) in chlo-
roform (20 mL) at 0 °C, TFAA (6.00 mmol, 1.26 g, 2.0 equiv) was
added dropwise. The reaction was stirred at ambient temperature
for 2 h and concentrated in vacuo. The resulting acid anhydride
drugs were examined in this cellular assay (Table 2). Carbamate
prodrugs 10 and 20 were found to be ineffective inhibitors due to
cytotoxicity and potency respectively, whereas amide prodrugs
3a0–3d0 showed reasonable protection of SNAP-25 from the
neurotoxin without apparent cytotoxicity. The EC50 values were

H. Seki et al. / Bioorg. Med. Chem. 22 (2014) 1208–1217
1213
was dissolved in MeOH (40 mL) at 0 °C. TEA (12.0 mmol, 1.21 g,
4.0 equiv) was added to this solution, and the reaction mixture
was stirred at ambient temperature overnight. Upon evaporation
of solvent, the crude mixture was re-dissolved in EtOAc, washed
with aq. HCl (1 M), brine, dried over MgSO4, and concentrated to
afford the titled product (900 mg, quant.) as yellow oil, which
was used to next reaction without further purification.
1H NMR (600 MHz, CDCl3)
d
7.23–7.13 (m, 2H), 7.04 (t,
J = 8.2 Hz, 2H), 4.67 (t, J = 6.8 Hz, 2H), 3.11 (t, J = 6.8 Hz, 2H); 13C
NMR (151 MHz, CDCl3) d 162.3 (JC-F = 246 Hz), 130.67, 130.67 (JC-F
= 7.55 Hz), 118.7 (q, JC-F = 320 Hz), 115.8 (JC-F = 21.1 Hz), 77.1,
35.1; HRMS (ESI-TOF) m/e calc’d for [M+H]+ C9H9F4O3S:
273.0209, found none.
1H NMR (600 MHz, CDCl3) d 7.39 (s, 1H), 7.24ꢀ7.15 (m, 2H),
4.07 (m, 1H), 3.61 (s, 3H), 2.76 (m, 4H); 13C NMR (151 MHz, CDCl3)
d 177.1, 171.7, 138.1, 134.6, 133.4, 130.0, 128.9, 127.5, 52.0, 38.4,
38.2, 34.0; HRMS (ESI-TOF) m/e calcd for [M+H]+ C12H13Cl2O4:
290.0185, found 290.0189.
4.1.8. 5-(Benzyloxy)-3-(2,4-dichlorophenyl)pentan-1-ol (11b)
47.4 mg, 47% 1H NMR (600 MHz, CDCl3) d 7.39–7.29 (m, 3H),
7.27 (s, 2H), 7.24–7.13 (m, 2H), 4.40 (t, J = 8.2 Hz, 2H), 3.50 (m,
4H), 3.37 (m, 1H), 3.30 (m, 1H), 1.93 (m, 5H); 13C NMR
(151 MHz, CDCl3) d 140.7, 138.4, 135.1, 132.4, 129.5, 128.5,
128.4, 127.8, 127.7, 127.6, 73.2, 68.2, 60.8, 39.1, 35.9, 34.5; HRMS
(ESI-TOF) m/e calcd for [M+H]+ C18H21Cl2O2: 339.0913, found
339.0908.
4.1.4. 3-(2,4-Dichlorophenyl)pentane-1,5-diol (10)
To a solution of diacid 7 (4.00 mmol, 1.11 g, 1.0 equiv) in THF
(40 mL), BH3 (THF solution of dimethyl sulfide complex, 2 M,
12.0 mmol, 6.00 mL, 3.0 equiv) was added at ꢀ78 °C. The reaction
mixture was warmed to ambient temperature and stirred for 3 h,
which was quenched by the addition of aq NaHCO3 at ꢀ78 °C. This
crude mixture was extracted with EtOAc, washed with brine, dried
over MgSO4, and concentrated in vacuo. The resulting crude prod-
uct was purified by column chromatography (MeOH/dichloro-
methane) to afford the titled compound (918 mg, 92%) as clear oil.
1H NMR (600 MHz, CDCl3) d 7.37 (s, 1H), 7.23 (d, J = 8.4 Hz, 1H),
7.18 (d, J = 8.4 Hz, 1H), 3.61–3.41 (m, 5H), 1.97 (m, 2H), 1.90–1.70
(m, 4H); 13C NMR (151 MHz, CDCl3) d 140.7, 135.0, 132.5, 129.5,
129.1, 127.8, 60.6, 38.8, 33.9; HRMS (ESI-TOF) m/e calcd for
[M+H]+ C11H15Cl2O2: 249.0444, found 249.0448.
4.1.9. 5-(Allyloxy)-3-(2,4-dichlorophenyl)pentan-1-ol (11c)
44.2 mg, 51% 1H NMR (600 MHz, CDCl3) d 7.37 (s, 1H), 7.23 (d,
J = 8.4 Hz, 1H), 7.18 (d, J = 8.3 Hz, 1H), 5.85 (ddt, J = 15.8, 10.3,
5.6 Hz, 1H), 5.21 (d, J = 17.2 Hz, 1H), 5.13 (d, J = 10.4 Hz, 1H), 3.87
(d, J = 4.5 Hz, 2H), 3.57–3.43 (m, 3H), 3.31 (q, J = 7.1 Hz, 1H), 3.24
(q, J = 8.9, 7.9 Hz, 1H), 1.99 (m, 2H), 1.85 (m, 2H); 13C NMR
(151 MHz, CDCl3) d 140.7, 135.1, 134.8, 132.4, 129.5, 129.1,
127.6, 117.1, 72.1, 68.1, 60.8, 39.1, 36.0, 34.4; HRMS (ESI-TOF) m/
e calcd for [M+H]+ C14H19Cl2O2: 289.0757, found 289.0754.
4.1.10. ((1r,3r)-3-Bromoadamantan-1-yl)methyl
trifluoromethanesulfonate
To a solution of alcohol (1.10 mmol, 270 mg, 1.0 equiv) in
dichloromethane (10 mL) were added Tf2O (1.32 mmol, 372 mg,
1.2 equiv) and 2,6-lutidine (1.43 mmol, 153 mg, 1.3 equiv) at 0 °C.
After stirring for 3 h, the reaction was concentrated in vacuo, re-
dissolved in Et2O, washed with aq. citric acid (10%), aq. NaHCO3,
brine, dried over MgSO4, and again concentrated. The obtained
crude product (377 mg, 91%, pinkish oil) was used for the synthesis
of 11d without further purification. The alcohol, starting material,
was prepared from the corresponding acid according to the litera-
ture procedure.27
4.1.5. General procedure for mono-alkylation of diol 10
To a solution of diol 10 (1.0 equiv) in THF (0.1 M) was added
NaH (2.0 equiv) at 0 °C. After stirring for 0.5 h, electrophile
(1.0 equiv) was added, and the solution was stirred at ambient
temperature overnight. Upon evaporation of solvent, crude mix-
ture was directly subjected to column chromatography (Hexanes/
EtOAc) to afford mono-alkylated product as clear oil.
ꢂ For the synthesis of 11c, TBAI (1.0 equiv) was added together
with electrophile.
1H NMR (500 MHz, CDCl3) d 4.11 (s, 2H), 2.35 (d, J = 11.7 Hz,
2H), 2.30–2.21 (m, 4H), 2.18 (s, 2H), 1.78–1.71 (m, 1H), 1.69–
1.62 (m, 1H), 1.59 (m, 4H); 13C NMR (126 MHz, CDCl3) d 118.8 (q,
JC-F = 320 Hz), 84.2, 62.7, 49.6, 48.2, 38.5, 36.7, 34.7, 31.5; HRMS
(ESI-TOF) m/e calcd for [M+H]+ C12H17BrF3O3S: 375.9956, found
none.
ꢂ For the synthesis of 11d, 18-Crown-6 (1.0 equiv) was added
together with electrophile.
4.1.6. 3-(2,4-Dichlorophenyl)-5-(4-fluorophenethoxy)pentan-1-
ol (11a)
43.8 mg, 49% 1H NMR (600 MHz, CDCl3) d 7.36 (s, 1H), 7.21 (d,
J = 8.4 Hz, 1H), 7.13 (d, J = 7.4 Hz, 3H), 6.96 (t, J = 8.2 Hz, 2H),
3.60–3.38 (m, 6H), 3.31 (q, J = 6.5, 5.9 Hz, 1H), 3.23 (q, J = 8.0,
7.5 Hz, 1H), 2.79 (t, J = 6.9 Hz, 2H), 1.96 (tq, J = 13.9, 6.7 Hz, 2H),
1.82 (m, 2H); 13C NMR (151 MHz, CDCl3) d 161.6 (JC–F = 243 Hz),
140.7, 135.1, 134.8 (JC–F = 3.02 Hz), 132.4, 130.4 (JC–F = 7.55 Hz),
129.4, 129.1, 127.6, 115.2 (JC–F = 21.1 Hz), 72.0, 68.7, 60.8, 39.0,
35.9, 35.6, 34.4; HRMS (ESI-TOF) m/e calc’d for [M+H]+ C19H22Cl2-
FO2: 371.0975, found 371.0973.
4.1.11. 5-(((1r,3r)-3-Bromoadamantan-1-yl)methoxy)-3-(2,4-
dichlorophenyl)pentan-1-ol (11d)
98.4 mg, 51% 1H NMR (600 MHz, CDCl3) d 7.37 (s, 1H), 7.23 (d,
J = 8.3 Hz, 1H), 7.18 (d, J = 8.3 Hz, 1H), 3.51 (m, 3H), 3.24 (m, 2H),
2.91 (s, 2H), 2.36–2.21 (m, 4H), 2.12 (m, 4H), 2.03–1.91 (m, 2H),
1.91–1.75 (m, 2H), 1.67 (m, 1H), 1.60 (m, 2H), 1.47 (m, 4H); 13C
NMR (151 MHz, CDCl3) d 140.9, 135.0, 132.3, 129.5, 129.1, 127.6,
80.6, 69.4, 66.4, 60.8, 51.3, 48.9, 39.5, 39.3, 37.9, 35.9, 35.3, 34.6,
32.3; HRMS (ESI-TOF) m/e calcd for [M+H]+ C22H30BrCl2O2:
475.0801, found 475.0807.
4.1.7. 4-Fluorophenethyl trifluoromethanesulfonate
To a solution of 4-fluorophenethyl alcohol (1.00 mmol, 140 mg,
1.0 equiv) in chloroform (5 mL) were added Tf2O (1.50 mmol,
423 mg, 1.5 equiv) and 2,6-lutidine (1.70 mmol, 182 mg, 1.7 equiv)
at 0 °C. After stirring for 1 h, the reaction was concentrated in va-
cuo, re-dissolved in hexanes/EtOAc (2:1), washed with aq. citric
acid (10%), aq NaHCO3, brine, dried over MgSO4, and again concen-
trated. Due to the instability of titled compound on SiO2, the ob-
tained crude product (244 mg, 90%, reddish oil) was used for the
synthesis of 11a without further purification.
4.1.12. General procedure for amide coupling
4.1.12.1. EDC coupling.
To a mixture of acid (1.0 equiv),
amine (1.2 equiv), HOBt (2.0 equiv), NMM (2.0 equiv) in DCM
(0.1 M) was added EDC (2.0 equiv) at 0 °C. The reaction was
warmed to ambient temperature and stirred overnight. Upon evap-
oration, crude mixture was re-dissolved in EtOAc, washed with aq
HCl (1 M), aq NaHCO3, brine, dried over MgSO4, and concentrated
in vacuo. The crude product was purified by column chromatogra-
phy (Hexanes/EtOAc) to afford the amide product as white solid.

1214
H. Seki et al. / Bioorg. Med. Chem. 22 (2014) 1208–1217
4.1.12.2. Coupling via acid chloride formation.
To a solution
(s, 3H), 3.59 (s, 3H), 3.49–3.36 (m, 2H), 2.81 (dd, J = 15.9, 6.6 Hz,
of acid 8 (1.0 equiv), DMF (1 drop) in chloroform (0.1 M) at 0 °C
was added (COCl)2 (4.0 equiv). The resulting solution was warmed
to ambient temperature and stirred for 1 h. Upon evaporation, this
acid chloride was dissolved in chloroform (0.1 M). The solution was
cooled to 0 °C, followed by the addition of TEA (1.2 equiv), DMAP (1
crystal) and aniline (1.0 equiv), and stirred at ambient temperature
overnight. Upon evaporation, the crude mixture was dissolved in
EtOAc/H2O. The partitioned organic layer was washed with brine,
dried over MgSO4, and concentrated. The crude product was puri-
fied by column chromatography (Hexanes/EtOAc) to provide amide
product as white solid.
1H), 2.75–2.62 (m, 3H), 2.52 (s, 2H); 13C NMR (151 MHz, CDCl3) d
172.1, 170.3, 158.4, 138.5, 134.5, 133.2, 130.7, 129.9, 129.7,
129.1, 127.5, 114.2, 55.4, 51.9, 40.8, 40.8, 38.1, 35.1, 34.7; HRMS
(ESI-TOF) m/e calcd for [M+H]+ C21H24Cl2NO4: 424.1077, found
424.1080.
4.1.18. Methyl 5-((2-cyclopropylethyl)amino)-3-(2,4-
dichlorophenyl)-5-oxopentanoate (9f)
60.6 mg, 85% 1H NMR (600 MHz, CDCl3) d 7.38 (s, 1H), 7.19 (s,
2H), 5.56 (s, 1H), 4.07 (pd, J = 7.2, 1.9 Hz, 1H), 3.60 (s, 3H), 3.25
(q, J = 6.5 Hz, 2H), 2.86 (dd, J = 15.9, 6.7 Hz, 1H), 2.75 (dd, J = 15.9,
7.5 Hz, 1H), 2.57 (h, J = 7.4 Hz, 2H), 1.30 (q, J = 6.9 Hz, 2H), 0.58–
0.49 (m, 1H), 0.42 (d, J = 7.8 Hz, 2H), 0.01 (d, J = 4.9 Hz, 2H); 13C
NMR (151 MHz, CDCl3) d 172.1, 170.2, 138.5, 134.5, 133.3, 129.9,
129.2, 127.5, 51.9, 40.9, 39.9, 38.1, 35.2, 34.4, 8.6, 4.3, 4.3; HRMS
(ESI-TOF) m/e calcd for [M+H]+ C17H22Cl2NO3: 358.0971, found
358.0976.
4.1.13. Methyl 3-(2,4-dichlorophenyl)-5-((4-fluoro-2-
methoxyphenyl)amino)-5-oxopentanoate (9a)
The aniline was prepared from the corresponding nitro com-
pound according to the literature procedure.28
55.0 mg, 66% 1H NMR (600 MHz, CDCl3) d 8.20 (t, J = 6.8 Hz, 1H),
7.56 (s, 1H), 7.39 (s, 1H), 7.21 (q, J = 8.2 Hz, 2H), 6.60 (m, 2H), 4.18
(q, J = 6.5 Hz, 1H), 3.83 (s, 3H), 3.60 (s, 3H), 2.90 (m, 1H), 2.84–2.75
(m, 3H); 13C NMR (151 MHz, CDCl3) d 172.0, 168.4, 159.3 (JC–F
= 243 Hz), 149.1 (JC–F = 9.06 Hz), 138.4, 134.6, 133.3, 130.0, 129.1,
127.5, 123.5 (JC–F = 3.02 Hz), 120.9 (JC–F = 9.06 Hz), 106.8 (JC–
F = 22.7 Hz), 98.8 (JC–F = 27.2 Hz), 56.1, 51.9, 41.8, 38.1, 35.0; HRMS
(ESI-TOF) m/e calcd for [M+H]+ C19H19Cl2FNO4: 414.0670, found
414.0678.
4.1.19. General procedure for the synthesis of hydroxamic
acid23
To a solution of methyl ester 9 (1.0 equiv) and KCN (3.0 equiv)
in THF/MeOH (1 mL/1 mL) was added aq NH2OH (50%, 0.5 mL).
This solution was stirred overnight at ambient temperature. Upon
evaporation of solvent, crude mixture was purified by preparative
TLC (MeOH/dichloromethane) to afford hydroxamic acid 3.
4.1.14. Methyl 3-(2,4-dichlorophenyl)-5-oxo-5-(o-
tolylamino)pentanoate (9b)
4.1.20. 3-(2,4-Dichlorophenyl)-N1-(4-fluoro-2-methoxyphenyl)-
N5-hydroxypentanediamide (3a)
114 mg, 84% 1H NMR (600 MHz, CDCl3) d 7.71 (d, J = 8.0 Hz, 1H),
7.41 (s, 1H), 7.23 (m, 2H), 7.17 (m, 2H), 7.07 (m, 2H), 4.21–4.11 (m,
1H), 3.62 (s, 3H), 2.86 (m, 4H), 2.14 (s, 3H); 13C NMR (151 MHz,
CDCl3) d 172.2, 168.8, 138.3, 135.5, 134.5, 133.5, 130.6, 130.0,
129.2, 129.1, 127.6, 126.9, 125.5, 123.2, 52.0, 41.5, 38.1, 35.4,
17.7; HRMS (ESI-TOF) m/e calcd for [M+H]+ C19H20Cl2NO3:
380.0815, found 380.0811.
39.4 mg, 45% 1H NMR (600 MHz, DMSO-d6) d 10.39 (s, 1H), 9.12
(s, 1H), 8.71 (s, 1H), 7.68 (t, J = 7.7 Hz, 1H), 7.53 (s, 1H), 7.41 (q,
J = 8.9 Hz, 2H), 6.93 (d, J = 10.8 Hz, 1H), 6.68 (t, J = 9.0 Hz, 1H),
4.03 (p, J = 7.9 Hz, 1H), 3.79 (d, J = 2.0 Hz, 3H), 2.72 (d, J = 7.5 Hz,
2H), 2.40 (dd, J = 14.9, 7.7 Hz, 1H), 2.32 (dd, J = 14.8, 6.8 Hz, 1H);
13C NMR (151 MHz, DMSO-d6) d 169.0, 166.8, 159.1 (JC–F = 242 Hz),
151.5, 139.9, 139.2, 134.1, 131.4, 128.7, 128.0, 127.2, 124.9, 105.8
(JC–F = 21.1 Hz), 99.6 (JC–F = 27.2 Hz), 56.1, 40.4, 36.7, 34.4; HRMS
(ESI-TOF) m/e calcd for [M+H]+ C18H18Cl2N2O4: 415.0622, found
415.0616.
4.1.15. Methyl 5-((4-bromophenyl)amino)-3-(2,4-
dichlorophenyl)-5-oxopentanoate (9c)
159 mg, 71% 1H NMR (600 MHz, CDCl3) d 7.51–7.30 (m, 6H),
7.24–7.16 (m, 2H), 4.19–4.09 (m, 1H), 3.62 (s, 3H), 2.92–2.69 (m,
4H); 13C NMR (151 MHz, CDCl3) d 172.4, 168.7, 138.2, 136.8,
134.4, 133.5, 132.1, 130.0, 129.1, 127.6, 121.5, 117.1, 52.1, 41.6,
38.1, 35.0; HRMS (ESI-TOF) m/e calcd for [M+H]+ C18H17BrCl2NO3:
443.9763, found 443.9758.
4.1.21. 3-(2,4-Dichlorophenyl)-N1-hydroxy-N5-(o-tolyl)
pentanediamide (3b)
The titled compound was purified by preparative HPLC (aceto-
nitrile/water) due to its limited solubility in organic media.
80.4 mg, 70% 1H NMR (600 MHz, DMSO-d6) d 10.31 (s, 1H), 9.16
(s, 1H), 8.62 (s, 1H), 7.40 (s, 1H), 7.32–7.24 (m, 2H), 7.07 (d,
J = 7.6 Hz, 1H), 7.01 (d, J = 7.2 Hz, 1H), 6.97 (t, J = 7.4 Hz, 1H), 6.90
(t, J = 7.2 Hz, 1H), 3.97–3.85 (m, 1H), 2.56 (m, 2H), 2.32–2.17 (m,
2H), 1.86 (s, 3H); 13C NMR (151 MHz, DMSO-d6) d 168.9, 167.0,
139.8, 136.2, 134.2, 131.9, 131.6, 130.3, 129.9, 128.9, 127.3,
126.0, 125.4, 125.3, 40.3, 37.1, 34.6, 17.7; HRMS (ESI-TOF) m/e
calcd for [M+H]+ C18H19Cl2N2O4: 381.0767, found 381.0763.
4.1.16. Methyl 3-(2,4-dichlorophenyl)-5-((4-
fluorophenethyl)amino)-5-oxopentanoate (9d)
56.7 mg, 80% 1H NMR (600 MHz, CDCl3) d 7.37 (s, 1H), 7.19 (d,
J = 8.4 Hz, 1H), 7.15 (d, J = 8.4 Hz, 1H), 7.06 (t, J = 6.2 Hz, 2H), 6.97
(t, J = 8.1 Hz, 2H), 5.46 (s, 1H), 4.03 (p, J = 6.9 Hz, 1H), 3.59 (s,
3H), 3.43 (dh, J = 14.1, 6.4 Hz, 2H), 2.80 (dd, J = 16.1, 6.3 Hz, 1H),
2.70 (q, J = 7.3 Hz, 3H), 2.53 (h, J = 7.6 Hz, 2H); 13C NMR
(151 MHz, CDCl3) d 172.1, 170.3, 161.8 (JC–F = 246 Hz), 138.5,
134.5, 134.4 (JC–F = 3.02 Hz), 133.3, 130.2 (JC–F = 7.55 Hz), 129.9,
129.1, 127.5, 115.6 (JC–F = 21.1 Hz), 51.9, 40.8, 40.7, 38.1, 35.1,
34.9; HRMS (ESI-TOF) m/e calcd for [M+H]+ C20H21Cl2FNO3:
412.0877, found 412.0875.
4.1.22. N1-(4-Bromophenyl)-3-(2,4-dichlorophenyl)-N5-
hydroxypentanediamide (3c)
The titled compound was purified by recrystallization from
MeOH.
36 mg, 80% 1H NMR (600 MHz, DMSO-d6) d 10.39 (s, 1H), 10.06
(s, 1H), 8.71 (s, 1H), 7.53 (s, 1H), 7.50–7.41 (m, 4H), 7.38 (s, 2H),
4.13–3.96 (m, 1H), 2.70 (d, J = 7.1 Hz, 2H), 2.40 (dd, J = 14.4,
8.1 Hz, 1H), 2.34 (dd, J = 14.5, 6.9 Hz, 1H); 13C NMR (151 MHz,
DMSO-d6) d 169.1, 166.7, 139.7, 138.3, 134.0, 131.5, 129.6, 128.8,
127.2, 124.9, 121.0, 114.6, 40.7, 36.6, 34.1; HRMS (ESI-TOF) m/e
calcd for [M+H]+ C17H16BrCl2N2O3: 444.9716, found 444.9717.
4.1.17. Methyl 3-(2,4-dichlorophenyl)-5-((4-
methoxyphenethyl)amino)-5-oxopentanoate (9e)
58.8 mg, 81% 1H NMR (600 MHz, CDCl3) d 7.37 (s, 1H), 7.19 (d,
J = 8.4 Hz, 1H), 7.15 (d, J = 8.4 Hz, 1H), 7.01 (d, J = 1.5 Hz, 2H),
6.83 (d, J = 7.8 Hz, 2H), 5.45 (s, 1H), 4.03 (p, J = 7.4 Hz, 1H), 3.79

H. Seki et al. / Bioorg. Med. Chem. 22 (2014) 1208–1217
1215
4.1.23. 3-(2,4-Dichlorophenyl)-N1-(4-fluorophenethyl)-N5-
hydroxypentanediamide (3d)
J = 7.3 Hz, 2H), 7.23 (m, 3H), 4.38–4.29 (m, 2H), 3.72 (p,
J = 7.7 Hz, 1H), 3.28 (m, 1H), 3.23 (m, 1H), 2.29 (hept, J = 7.6 Hz,
2H), 1.96–1.86 (m, 1H), 1.86–1.77 (m, 1H); 13C NMR (151 MHz,
DMSO) d 167.0, 140.4, 138.4, 134.2, 131.3, 129.8, 128.7, 128.1,
127.42, 127.35, 127.3, 71.8, 67.3, 40.4, 37.7, 34.4; HRMS (ESI-
TOF) m/e calcd for [M+H]+ C18H20Cl2NO3: 368.0815, found
368.0813.
28.1 mg, 60% 1H NMR (600 MHz, DMSO-d6) d 10.36 (s, 1H), 8.69
(s, 1H), 7.88 (s, 1H), 7.52 (s, 1H), 7.39–7.26 (m, 2H), 7.12 (t,
J = 6.1 Hz, 2H), 7.06 (t, J = 8.2 Hz, 2H), 3.93 (p, J = 7.7 Hz, 1H), 3.16
(d, J = 5.9 Hz, 2H), 2.57 (t, J = 6.9 Hz, 2H), 2.37 (m, 3H), 2.24 (dd,
J = 14.5, 6.6 Hz, 1H); 13C NMR (151 MHz, DMSO-d6) d 169.7,
166.8, 160.8 (JC–F = 240 Hz), 139.9, 135.6 (JC–F = 3.02 Hz), 134.0,
131.3, 130.3 (JC–F = 7.55 Hz), 129.6, 128.7, 127.1, 114.9 (JC–F = 21.1 -
Hz), 40.1, 40.0, 36.6, 34.2, 34.1; HRMS (ESI-TOF) m/e calcd for
[M+H]+ C19H20Cl2FN2O3: 413.0829, found 413.0823.
4.1.29. 5-(Allyloxy)-3-(2,4-dichlorophenyl)-N-
hydroxypentanamide (4c)
11.2 mg, 27% 1H NMR (600 MHz, DMSO-d6) d 10.36 (s, 1H), 8.69
(s, 1H), 7.53 (s, 1H), 7.37 (d, J = 13.4 Hz, 2H), 5.80 (td, J = 10.9, 5.3 Hz,
1H), 5.16 (d, J = 17.2 Hz, 1H), 5.07 (d, J = 10.3 Hz, 1H), 3.81 (s, 2H),
3.75–3.61 (m, 1H), 3.20 (dt, J = 28.2, 8.2 Hz, 2H), 2.38–2.20 (m,
2H), 1.87 (dd, J = 13.7, 6.8 Hz, 1H), 1.78 (p, J = 7.1 Hz, 1H); 13C
NMR (151 MHz, DMSO) d 167.0, 140.5, 135.2, 134.1, 131.3, 129.7,
128.7, 127.4, 116.1, 70.8, 67.1, 37.7, 34.5, 34.2; HRMS (ESI-TOF)
m/e calcd for [M+H]+ C14H18Cl2NO3: 318.0658, found 318.0658.
4.1.24. 3-(2,4-Dichlorophenyl)-N1-hydroxy-N5-(4-
methoxyphenethyl)pentanediamide (3e)
22.7 mg, 46% 1H NMR (600 MHz, DMSO-d6) d 10.36 (s, 1H), 8.69
(s, 1H), 7.87 (s, 1H), 7.53 (s, 1H), 7.39–7.28 (m, 2H), 7.01 (d,
J = 7.4 Hz, 2H), 6.81 (d, J = 7.3 Hz, 2H), 4.00–3.90 (m, 1H), 3.71 (s,
3H), 3.13 (d, J = 6.0 Hz, 2H), 2.55–2.47 (m, 2H), 2.45–2.30 (m,
3H), 2.25 (dd, J = 14.3, 5.6 Hz, 1H); 13C NMR (151 MHz, DMSO-d6)
d 169.6, 166.8, 157.6, 139.9, 134.0, 131.3, 131.3, 129.6, 129.5,
128.7, 127.1, 113.7, 55.0, 40.3, 40.01, 36.6, 34.19, 34.17; HRMS
(ESI-TOF) m/e calcd for [M+H]+ C20H23Cl2N2O4: 425.1029, found
425.1023.
4.1.30. 5-((3-Bromoadamantan-1-yl)methoxy)-3-(2,4-
dichlorophenyl)-N-hydroxypentanamide (4d)
53.5 mg, 55%1H NMR (600 MHz, DMSO-d6) d 10.35 (s, 1H), 8.69
(s, 1H), 7.50 (s, 1H), 7.35 (q, J = 8.6 Hz, 2H), 3.68 (s, 1H), 3.17 (m,
2H), 2.92–2.77 (m, 2H), 2.23 (d, J = 12.2 Hz, 4H), 2.16 (d,
J = 11.2 Hz, 2H), 2.02 (m, 4H), 1.84 (s, 1H), 1.77 (s, 1H), 1.62 (d,
J = 12.4 Hz, 1H), 1.52 (d, J = 12.0 Hz, 1H), 1.40 (d, J = 11.9 Hz, 2H),
1.34 (s, 2H); 13C NMR (151 MHz, DMSO-d6) d 167.0, 140.6, 134.1,
131.2, 129.8, 128.7, 127.3, 79.4, 68.6, 67.9, 50.8, 48.4, 39.0, 38.0,
37.0, 34.5, 34.3, 31.7, 29.1; HRMS (ESI-TOF) m/e calcd for [M+H]+
C22H29BrCl2NO3: 504.0702, found 504.0697.
4.1.25. N1-(2-Cyclopropylethyl)-3-(2,4-dichlorophenyl)-N5-
hydroxypentanediamide (3f)
14.3 mg, 40% 1H NMR (600 MHz, DMSO-d6) d 10.37 (s, 1H), 8.68
(s, 1H), 7.79 (s, 1H), 7.51 (s, 1H), 7.39–7.29 (m, 2H), 4.03–3.87 (m,
1H), 2.99 (s, 2H), 2.37 (m, 3H), 2.28 (dd, J = 14.8, 6.8 Hz, 1H), 1.16
(d, J = 6.8 Hz, 2H), 0.51 (s, 1H), 0.32 (d, J = 7.3 Hz, 2H), ꢀ0.04 (s,
2H); 13C NMR (151 MHz, DMSO-d6) d 169.5, 166.9, 139.9, 134.0,
131.3, 129.6, 128.7, 127.1, 40.1, 38.6, 36.6, 34.3, 34.0, 8.4, 4.1;
HRMS (ESI-TOF) m/e calcd for [M+H]+ C16H21Cl2N2O3: 359.0924,
found 359.0926.
4.1.31. General procedure to synthesize carbamate prodrug23,26
To a solution of hydroxamic acid (1.0 equiv) in acetonitrile
(0.05 M) was added CDI (1.0 equiv) at 0 °C. After 0.5 h, BnNH2
was added to the solution, which was stirred overnight at ambient
temperature. Upon evaporation, crude mixture was subjected to
column chromatography (Hexanes/EtOAc) to provide carbamate
prodrug.
4.1.26. General procedure to synthesize hydroxamic acid from
alcohol 11
To a solution of alcohol 11 (1.0 equiv) in acetone (0.1 M) was
added Jones’ reagent (2.0 M solution, 2.0 equiv) at 0 °C.29 The reac-
tion was stirred at ambient temperature for 1 h, and quenched by
the addition of IPA (a few drops). The organic layer was washed
with water, brine, dried over MgSO4, and concentrated in vacuo.
The resulting acid was dissolved in THF (0.1 M), and diazomethane
(excess) in Et2O was added at 0 °C until the yellow color stays in
the solution. After stirring overnight, upon evaporation, crude
methyl ester was subjected to the conditions above to form
hydroxamic acid as reddish oil.
Hydroxamic acid 1 and 2 were synthesized based on the litera-
ture procedure.17,30
4.1.32. (E)-N-((Benzylcarbamoyl)oxy)-3-(2,4-
dichlorophenyl)acrylamide (10)
200 mg, 71% 1H NMR (500 MHz, D2O) d 11.89 (s, 1H), 8.39 (s,
1H), 7.86–7.71 (m, 3H), 7.51 (dd, J = 8.5, 2.0 Hz, 1H), 7.40–7.22
(m, 5H), 6.63 (d, J = 15.8 Hz, 1H), 4.26 (d, J = 6.1 Hz, 2H); 13C NMR
(151 MHz, D2O) d 162.7, 155.2, 139.0, 135.0, 134.6, 134.3, 131.3,
129.5, 129.2, 128.4, 128.1, 127.1, 127.0, 121.5, 44.2; HRMS (ESI-
TOF) m/e calcd for [M+H]+ C17H15Cl2N2O3: 365.0454, found
365.0450.
4.1.27. 3-(2,4-dichlorophenyl)-5-(4-fluorophenethoxy)-N-
hydroxypentanamide (4a)
20.0 mg, 42% 1H NMR (600 MHz, DMSO-d6) d 10.35 (s, 1H), 8.70
(s, 1H), 7.53 (d, J = 2.3 Hz, 1H), 7.38 (dd, J = 8.4, 2.2 Hz, 1H), 7.32 (d,
J = 8.5 Hz, 1H), 7.21 (dd, J = 8.5, 5.7 Hz, 2H), 7.07 (t, J = 8.9 Hz, 2H),
3.66 (p, J = 7.6 Hz, 1H), 3.49–3.39 (m, 2H), 3.23 (dt, J = 12.5, 6.3 Hz,
1H), 3.21–3.13 (m, 1H), 2.72 (t, J = 6.8 Hz, 2H), 2.29 (qd, J = 14.5,
7.6 Hz, 2H), 1.84 (dq, J = 13.0, 6.6 Hz, 1H), 1.75 (dt, J = 14.8,
7.3 Hz, 1H); 13C NMR (151 MHz, DMSO) d 167.0, 160.8 (JC–F = 242 -
Hz), 140.5, 135.2 (JC–F = 3.02 Hz), 134.1, 131.3, 130.5 (JC–F = 9.06 -
Hz), 129.8, 128.7, 127.4, 114.8 (JC–F = 21.1 Hz), 70.9, 67.7, 40.4,
37.6, 34.6, 34.5; HRMS (ESI-TOF) m/e calcd for [M+H]+ C19H21Cl2-
FNO3: 400.0877, found 400.0895.
4.1.33. N-((Benzylcarbamoyl)oxy)-3-(2,4-dichlorophenyl)-5-
hydroxypentanamide (20)
33 mg, 34% 1H NMR (400 MHz, DMSO-d6) d 11.42 (s, 1H), 8.19 (t,
J = 6.1 Hz, 1H), 7.53 (d, J = 1.5 Hz, 1H), 7.37 (s, 2H), 7.36–7.29 (m,
2H), 7.25 (d, J = 8.0 Hz, 3H), 4.42 (t, J = 5.0 Hz, 1H), 4.21 (d,
J = 6.1 Hz, 2H), 3.65 (s, 1H), 3.25 (s, 2H), 2.43 (d, J = 7.8 Hz, 2H),
1.78 (d, J = 33.4 Hz, 2H); 13C NMR (151 MHz, D2O) d 168.1, 155.2,
140.5, 139.0, 134.0, 131.2, 129.8, 128.7, 128.3, 127.4, 127.03,
126.98, 58.4, 44.1, 37.4, 37.2, 33.9; HRMS (ESI-TOF) m/e calcd for
[M+H]+ C19H21Cl2N2O4: 411.0873, found 411.0865.
4.1.28. 5-(Benzyloxy)-3-(2,4-dichlorophenyl)-N-
hydroxypentanamide (4b)
4.1.34. N1-((Benzylcarbamoyl)oxy)-3-(2,4-dichlorophenyl)-N5-
(4-fluoro-2-methoxyphenyl)pentanediamide (3a0)
17 mg, 36% 1H NMR (600 MHz, DMSO-d6) d 10.36 (s, 1H), 8.69
(s, 1H), 7.52 (d, J = 1.8 Hz, 1H), 7.38–7.32 (m, 2H), 7.30 (t,
7.1 mg, 43% 1H NMR (600 MHz, CDCl3) d 10.20 (s, 1H), 8.07 (dd,
J = 8.9, 6.3 Hz, 1H), 7.61 (s, 1H), 7.41 (s, 1H), 7.34 (t, J = 7.5 Hz, 2H),

1216
H. Seki et al. / Bioorg. Med. Chem. 22 (2014) 1208–1217
7.29 (dt, J = 6.6, 3.3 Hz, 3H), 7.15 (s, 2H), 6.64 (td, J = 8.4, 2.4 Hz,
1H), 6.58 (dd, J = 10.1, 2.6 Hz, 1H), 5.65 (s, 1H), 4.41 (d, J = 5.9 Hz,
2H), 4.12 (m, 1H), 3.76 (s, 3H), 3.05 (m, 1H), 2.96–2.82 (m, 2H),
2.67–2.48 (m, 1H); 13C NMR (151 MHz, CDCl3) d 171.3, 169.9,
159.9 (JC–F = 246 Hz), 155.4, 149.8, 138.0, 137.3, 134.1, 133.5,
J = 14.3, 8.0 Hz, 1H), 2.52 (m, 1H), 2.07 (m, 2H); 13C NMR
(151 MHz, CDCl3) d 169.8, 155.2, 139.0, 138.0, 137.4, 134.7,
132.9, 129.7, 129.4, 128.9, 128.6, 128.0, 127.90, 127.88, 127.6,
127.4, 73.3, 68.0, 45.5, 37.9, 35.6, 33.3; HRMS (ESI-TOF) m/e calcd
for [M+H]+ C26H27Cl2N2O4: 501.1342, found 501.1339.
129.8, 129.4, 128.9, 128.0, 127.7, 127.5, 122.7, 122.3, 107.0 (JC–
-
F = 21.1 Hz), 99.0 (JC–F = 27.2 Hz), 56.1, 45.7, 39.9, 35.8, 35.6; HRMS
(ESI-TOF) m/e calcd for [M+H]+ C26H25Cl2FN3O5: 548.1150, found
548.1138.
4.1.40. N-((Benzylcarbamoyl)oxy)-5-(((1r,3r)-3-bromoadamantan-
1-yl)methoxy)-3-(2,4-dichlorophenyl)pentanamide (4d0)
5.0 mg, 50% 1H NMR (600 MHz, CDCl3) d 9.05 (s, 1H), 7.35 (dd,
J = 16.2, 9.0 Hz, 3H), 7.29 (dd, J = 13.0, 7.2 Hz, 3H), 7.20 (q,
J = 8.2 Hz, 2H), 5.46 (s, 1H), 4.40 (d, J = 4.8 Hz, 2H), 3.87–3.77 (m,
1H), 3.40–3.23 (m, 2H), 2.95 (q, J = 9.0 Hz, 2H), 2.68–2.52 (m,
2H), 2.30 (d, J = 11.8 Hz, 2H), 2.25 (d, J = 11.8 Hz, 2H), 2.12 (m,
4H), 1.68 (d, J = 12.7 Hz, 1H), 1.65–1.42 (m, 7H); 13C NMR
(151 MHz, CDCl3) d 172.0, 155.7, 139.8, 137.9, 135.3, 133.6,
130.5, 130.2, 129.7, 128.7, 128.4, 128.2, 81.5, 70.2, 61.3, 52.0,
49.5, 46.4, 40.1, 39.2, 38.6, 35.9, 34.1, 33.0, 30.6; HRMS (ESI-TOF)
m/e calcd for [M+H]+ C30H36BrCl2N2O4: 637.1230, found 637.1232.
4.1.35. N1-((benzylcarbamoyl)oxy)-3-(2,4-dichlorophenyl)-N5-
(o-tolyl)pentanediamide (3b0)
16.4 mg, 83% 1H NMR (600 MHz, DMSO-d6) d 11.46 (s, 1H), 9.29
(s, 1H), 8.23 (s, 1H), 7.55 (s, 1H), 7.46 (d, J = 7.7 Hz, 1H), 7.41 (d,
J = 8.3 Hz, 1H), 7.33 (m, 2H), 7.23 (m, 4H), 7.14 (d, J = 7.1 Hz, 1H),
7.09 (t, J = 7.3 Hz, 1H), 7.03 (t, J = 6.7 Hz, 1H), 4.22 (s, 2H), 4.06 (s,
1H), 2.78 (d, J = 8.3 Hz, 2H), 2.50 (m, 2H), 1.99 (s, 3H); 13C NMR
(151 MHz, DMSO-d6) d 168.7, 167.7, 155.2, 139.5, 139.0, 136.1,
134.0, 131.8, 131.6, 130.2, 129.7, 128.8, 128.5, 128.3, 127.0,
127.0, 125.8, 125.2, 125.2, 44.1, 40.1, 36.7, 34.3, 17.6; HRMS (ESI-
TOF) m/e calcd for [M+H]+ C26H26Cl2N3O4: 514.1295, found
514.1294.
4.2. Biology
4.2.1. SNAPtide assay and 66mer assay
Recombinant Botulinum neurotoxin light chain A (1-425) was
prepared as previously described.31 SNAPtide assay was conducted
according to the literature procedure with the enzyme concentra-
tion of 37 nM.31 66mer assay was conducted according to the liter-
ature procedure with the enzyme concentration of 0.5 nM. Prism 5
was used for Ki determination.25
4.1.36. N1-((Benzylcarbamoyl)oxy)-N5-(4-bromophenyl)-3-(2,4-
dichlorophenyl)pentanediamide (3c0)
18.6 mg, 87% 1H NMR (600 MHz, DMSO-d6) d 11.47 (s, 1H),
10.07 (s, 1H), 8.22 (s, 1H), 7.54 (s, 1H), 7.48–7.35 (m, 6H), 7.32 (t,
J = 7.2 Hz, 2H), 7.24 (d, J = 6.5 Hz, 3H), 4.21 (s, 2H), 4.04 (ddt,
J = 15.1, 9.0, 4.5 Hz, 1H), 2.77 (s, 2H), 2.50 (s, 2H); 13C NMR
(151 MHz, DMSO-d6) d 169.0, 167.7, 155.2, 139.5, 139.0, 138.3,
134.0, 131.5, 131.5, 129.6, 128.8, 128.3, 127.3, 127.0, 127.0,
121.0, 114.7, 44.1, 40.3, 36.5, 33.9; HRMS (ESI-TOF) m/e calcd for
[M+H]+ C25H23BrCl2N3O4: 578.0243, found 578.0240.
4.2.2. Cell assay
Pure Botulinum neurotoxin (BoNT)
A was prepared from
C. botulinum strains Hall A hyper as previously described.32 The
toxin was dissolved in phosphate buffered saline, pH 7.4 and 40%
glycerol, and stored at ꢀ20 °C until use. Activity of the BoNT/A
preparation was determined by the mouse bioassay,33,34 and
specific toxicity was about 1.25 ꢃ 108 mouse LD50 Units/mg. The
inhibitors were dissolved in 100% DMSO to 100 mM and stored
at 4 °C.
4.1.37. N1-((Benzylcarbamoyl)oxy)-3-(2,4-dichlorophenyl)-N5-
(4-fluorophenethyl)pentanediamide (3d0)
9.4 mg, 77% 1H NMR (600 MHz, CDCl3) d 10.22 (s, 1H), 7.40–7.27
(m, 5H), 7.16 (d, J = 6.6 Hz, 2H), 7.08–7.00 (m, 2H), 6.97 (d,
J = 8.8 Hz, 2H), 5.68 (d, J = 10.7 Hz, 2H), 4.40 (d, J = 6.1 Hz, 2H),
3.94 (t, J = 7.3 Hz, 1H), 3.41 (dt, J = 17.7, 6.8 Hz, 2H), 2.91–2.59
(m, 5H), 2.59–2.41 (m, 1H); 13C NMR (151 MHz, CDCl3) d 171.8,
169.9, 161.8 (JC–F = 245 Hz), 155.3, 138.2, 137.3, 134.1, 134.1,
133.5, 130.2 (JC–F = 7.55 Hz), 129.8, 129.3, 128.9, 128.0, 127.6,
127.4, 115.6 (JC–F = 21.1 Hz), 45.7, 40.7, 38.9, 35.9, 35.5, 34.7; HRMS
(ESI-TOF) m/e calcd for [M+H]+ C27H27Cl2FN3O4: 546.1357, found
546.1361.
The hiPSC derived neurons and culture medium were purchased
from Cellular Dynamics International (Madison, WI), and cultured
in 96-well plates as described for 5 days prior to the assay.35 For
the inhibition assay, 200 LD50 units of BoNT/A1 was added to the
cells in 50 ll stimulation medium (modified neurobasal containing
2.2 mM CaCl2 and 56 mM KCl (Invitrogen) and supplemented with
B27 and glutamax), and the cells were incubated at 37 °C in a
humidified 5% CO2 atmosphere for 7.5 min. The toxin was removed
and cells were washed 3 times in 200 ll of culture medium (pro-
4.1.38. N-((Benzylcarbamoyl)oxy)-3-(2,4-dichlorophenyl)-5-(4-
fluorophenethoxy)pentanamide(4a0)
vided by Cellular Dynamics), and the inhibitors were added in cul-
ture medium, 1% DMSO at the indicated concentrations. Cells were
incubated for 8 h at 37 °C, 5% CO2 to allow for SNAP-25 cleavage,
9.4 mg, 77% 1H NMR (600 MHz, CDCl3) d 9.20 (s, 1H), 7.39–7.26
(m, 6H), 7.16 (m, 3H), 7.03 (d, J = 8.4 Hz, 1H), 6.97 (t, J = 8.6 Hz, 2H),
5.55 (s, 1H), 4.39 (d, J = 5.6 Hz, 2H), 3.76 (m, 1H), 3.65–3.51 (m,
2H), 3.34 (s, 2H), 2.83 (t, J = 6.4 Hz, 2H), 2.61 (dd, J = 14.1, 8.1 Hz,
1H), 2.49 (dd, J = 13.2, 4.7 Hz, 1H), 2.06 (m, 2H); 13C NMR
(151 MHz, CDCl3) d 170.0, 161.7 (JC–F = 245 Hz), 155.0, 139.0,
137.3, 134.7, 134.6, 132.9, 130.3 (JC–F = 7.55 Hz), 129.7, 129.4,
128.9, 128.0, 127.7, 127.3, 115.3 (JC–F = 21.1 Hz), 72.0, 68.9, 45.6,
37.9, 35.8, 35.5, 32.8; HRMS (ESI-TOF) m/e calcd for [M+H]+ C27H28-
Cl2FN2O4: 533.1405, found 533.1399.
and the inhibitor mixtures were aspirated and cells lysed in 50 ll
of 1x LDS lysis buffer (Invitrogen). The samples were analyzed by
Western blot using a monoclonal anti-SNAP-25 antibody (Synaptic
Systems, Germany) as described previously,36,37 except that Phos-
phaglo chemiluminescent reagent (KPL) was used and the bands
visualized on a Fotoanalyst FX (Fotodyne) equipped with a CCD
camera and GraphQuant software (Fotodyne) for densitometry.
GrahPad Prism 6 software was used for graph creation and data
analysis.
4.1.39. N-((Benzylcarbamoyl)oxy)-5-(benzyloxy)-3-(2,4-
dichlorophenyl)pentanamide (4b0)
Acknowledgements
9.5 mg, 46% 1H NMR (600 MHz, CDCl3) d 9.36 (s, 1H), 7.36–7.30
(m, 5H), 7.28 (d, J = 8.9 Hz, 6H), 7.14 (dd, J = 8.4, 2.2 Hz, 1H), 7.08
(d, J = 8.4 Hz, 1H), 5.68 (s, 1H), 4.49–4.39 (m, 2H), 4.36 (d,
J = 5.9 Hz, 2H), 3.84 (p, J = 6.3 Hz, 1H), 3.40 (m, 2H), 2.64 (dd,
The authors gratefully acknowledge support of this project by
the National Institute of Allergy and Infectious Diseases, National
Institute of Health and the Department of Health and Human Ser-
vices under contract number AI080671. The authors thank Regina

H. Seki et al. / Bioorg. Med. Chem. 22 (2014) 1208–1217
1217
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