Synthesis, antibacterial activity, and biological evaluation of formyl hydroxyamino derivatives as novel potent peptide deformylase inhibitors against drug-resistant bacteria

Article abstract of DOI:10.1016/j.ejmech.2014.07.106

Peptide deformylase (PDF) has been identified as a promising target for novel antibacterial agents. In this study, a series of novel formyl hydroxyamino derivatives were designed and synthesized as PDF inhibitors and their antibacterial activities were evaluated. Among the potent PDF inhibitors (1o, 1q, 1o′, 1q′, and 1x), in vivo studies showed that compound 1q possesses mild toxicity, a good pharmacokinetic profile and protective effects. The good in vivo efficacy and low toxicity suggest that this class of compounds has potential for development and use in future antibacterial drugs.

Full text of DOI:10.1016/j.ejmech.2014.07.106

European Journal of Medicinal Chemistry 86 (2014) 133e152
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, antibacterial activity, and biological evaluation of formyl
hydroxyamino derivatives as novel potent peptide deformylase
inhibitors against drug-resistant bacteria
,
Shouning Yang a, Wei Shi a, Dong Xing a, Zheng Zhao b **, Fengping Lv a, Liping Yang a,
,
, c, *
Yushe Yang c **, Wenhao Hu a
a Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, Department of Chemistry, East China Normal University,
Shanghai 200062, China
b Key Laboratory of Brain Functional Genomics, Ministry of Education, Shanghai Key Laboratory of Brain Functional Genomics, East China Normal
University, Shanghai 200062, China
c State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 200031, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Peptide deformylase (PDF) has been identified as a promising target for novel antibacterial agents. In
this study, a series of novel formyl hydroxyamino derivatives were designed and synthesized as PDF
inhibitors and their antibacterial activities were evaluated. Among the potent PDF inhibitors (1o, 1q, 1o′,
1q′, and 1x), in vivo studies showed that compound 1q possesses mild toxicity, a good pharmacokinetic
profile and protective effects. The good in vivo efficacy and low toxicity suggest that this class of com-
pounds has potential for development and use in future antibacterial drugs.
Received 29 April 2014
Received in revised form
22 July 2014
Accepted 29 July 2014
Available online 12 August 2014
© 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
Drug resistant bacteria
Peptide deformylase inhibitor
Proline derivatives
1. Introduction
formyl moiety catalyzed by PDF is a crucial step in bacterial protein
biosynthesis and growth [10]. On the other hand, mammalian
The increased prevalence of multi drug-resistant (MDR) bacteria
from clinical isolates has made the search for new antibacterial
agents with novel modes of action even more important. One of the
new targets currently receiving widespread attention from both
academic and industrial research groups is peptide deformylase
(PDF) [1e4]. PDF is an iron-containing metalloenzyme involved in
the post-translational modification of nascent polypeptides in
bacterial cells [4e8]. The catalytic mechanism of PDF enzymes
containing zinc, iron, cobalt, and nickel dications was studied by
Nino Russo in 2006 [9]. Protein synthesis in bacterial cells is initi-
ated by ribosomal binding to a formyl methionine-charged transfer
RNA, but most mature proteins do not retain the N-formyl group or
the terminal methionine residue. Therefore, the removal of the N-
cytosolic protein synthesis does not produce N-formylated poly-
peptides and does not need the PDF enzyme [11]. The difference
between bacterial and mammalian protein synthesis makes PDF an
attractive and unique target for treating resistant bacteria [12,13].
Previous studies have shown that the potency of PDF inhibitors
is closely linked to the additive effects of several chemical groups
(Fig. 1): (a) metal-binding group: studies indicate that the best
metal-binding group for most peptide deformylase inhibitors are
hydroxamate or N-formyl hydroxylamine [14]; (b) P10 group: sub-
stituents in the P10 position that mimic the methionine residue in
the natural substrate closely, such as n-butyl and cyclo-
pentylmethyl, resulting in potent PDF inhibitors that display
promising antibacterial activity [15]; (c) P20 group: many active
substituents have been described at the P20 position and some in-
hibitors with proline at this position result in the desired combi-
nation of antibacterial activity and low toxicity [16e18], also the
preliminary evaluation of our synthesized PDF inhibitors with
proline derivatives at the P20 position showed good to excellent
antibacterial activity [19,20]; (d) P30 group: the P30 position is more
amenable to different substitutions, and appropriate modifications
* Corresponding author. Shanghai Engineering Research Center of Molecular
Therapeutics and New Drug Development, Department of Chemistry, East China
Normal University, Shanghai 200062, China.
** Corresponding authors.
E-mail addresses: zzhao@brain.ecnu.edu.cn (Z. Zhao), ysyang@mail.shcnc.ac.cn
(Y. Yang), whu@chem.ecnu.edu.cn (W. Hu).
http://dx.doi.org/10.1016/j.ejmech.2014.07.106
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

134
S. Yang et al. / European Journal of Medicinal Chemistry 86 (2014) 133e152
Fig. 1. Chemical structures of PDF inhibitors.
at the P30 position could significantly improve the antibacterial
activity of PDF inhibitors with only minor effects to the binding
affinity for the enzyme. The first PDF inhibitor, actinonin (Fig. 1),
was isolated from an actinomycete by Gordon in 1962 [21]. Acti-
nonin exhibited moderate antibacterial activity against several
Gram-positive and Gram-negative bacteria [22], but it did not show
good in vivo antibacterial activity due to poor pharmacokinetic
properties, which could be attributed to either poor absorption or
quick clearance [23]. In order to overcome these drawbacks, novel
PDF inhibitors were developed by many pharmaceutical companies
and academic institutions [24e27]. LBM415 (Fig. 1, discovered by
Vicuron Pharmaceuticals in collaboration with Novartis) [28,29]
and BB83698 (Fig. 1, discovered by British Biotech in collaboration
with Genesoft) [8,30] were the first two PDF inhibitors to undergo
human clinical trials, and they exhibited much better in vitro and
in vivo efficacies when compared to the original lead compound
actinonin, but drawbacks of LBM415 were still founded in the
further study [31]. GSK1322322 (Fig. 1, developed by Glax-
oSmithKline, Brentford, UK) completed a phase II trial for acute
bacterial skin and skin structure infections in April 2012 [32]. This
compound not only possesses potent activity against methicillin-
resistant Staphylococcus aureus (MRSA), but also exhibits activity
against the respiratory pathogens Haemophilus influenzae and
Streptococcus pneumoniae [33]. However, no PDF inhibitors are
currently marketed.
Among the PDF inhibitors that have undergone human clinical
trials, LBM415 gained widespread attention for its good activity
in vitro against a range of pathogens [34]. However, some concerns
remain about its in vivo stability (e.g., proteolysis of the peptide
bonds), solubility, and bioavailability, as these properties are closely
linked to medical efficacy. In continuation of our previous study
[19,20], we describe the design, synthesis, and biological evaluation
of a series of PDF inhibitors based on the modification of LBM415.
Five proline derivatives widely used in many biologically active
products that usually play positive roles in promoting pharmaco-
logical activities and other medicinal properties, specifically (2S)-
2,5-dihydro-1H-pyrrole-2-carboxylic acid, (2S)-4-methylene-pyr-
selection, and the in vivo antibacterial activities, acute toxicity,
solubility and stability, plasma protein binding rate, pharmacoki-
netic properties, and bioavailability of the selected representative
compounds were evaluated to identify new compounds with an
improved antibacterial profile and pharmacological properties.
2. Results and discussion
2.1. Chemistry
Scheme 1 outlines the retrosynthetic analysis of the PDF in-
hibitors. The illustrated bond disconnection resulted in three
fragments: A, B, and C. The target compound was assembled using
the intermolecular amide coupling reaction of A and B, followed by
hydrolysis of the ester group of the coupling product and subse-
quent coupling of the obtained carboxyl acids with amine C. The
last step was deprotection to obtain the desired target compounds.
Following the route elucidated by Joel Slade [38], the synthesis
of fragment A is illustrated in Scheme 2. Diethyl malonate was
chosen as the starting material to synthesize fragment A bearing n-
butyl (A1) or cyclopentylmethyl (A2) groups. Taking the synthesis of
fragment A1 as an example, the reaction of diethyl malonate with 1-
bromobutane in the presence of sodium ethoxide resulted in
dimethyl 2-butylmalonate
6 (61% yield). The dimethyl 2-
butylmalonate 6 was hydrolyzed with 25% aqueous sodium hy-
droxide to obtain 2-butylmalonic acid 7 (85% yield). Compound 7
was then converted to 2-methylenehexanoic acid 8 via treatment
with formaldehyde in the presence of diethylamine. The reaction of
rolidine-2-carboxylic
acid,
(2S,4S)-4-methylpyrrolidine-2-
carboxylic acid, (2S,4S)-4-fluoropyrrolidine-2-carboxylic acid, and
(2S,3aR,7aS)-octahydro-1H-indole-2-carboxylic acid, were carefully
selected to replace the proline at the P20 position of LBM415
[35e37]. Novel PDF inhibitors were prepared by introducing various
amines, such as aliphatic amines, aromatic amines, and heterocyclic
aromatic amines, into the P30 position. The in vitro antibacterial
activities of all these compounds were evaluated for a primary
Scheme 1. Retrosynthetic analysis of PDF inhibitors.

S. Yang et al. / European Journal of Medicinal Chemistry 86 (2014) 133e152
135
8 with pivaloyl chloride gave an anhydride intermediate, which
was treated with the anion of (S)-4-benzyl-2 -oxazolidinone at low
temperature (ꢀ78 ꢁC, THF) to yield conjugated amide 9 (63% yield).
The diastereoselective Michael addition reaction of 9 with O-(4-
methoxybenzyl)-hydroxyl amine resulted in 10 as a single diaste-
reomer with a yield of 59% after recrystallization in t-butyl methyl
ether. The chiral auxiliary was removed by LiOH and H2O2 to obtain
the corresponding carboxylic acid derivative 11 (80% yield). For-
mylation of 11 using ethyl formate at 60 ꢁC resulted in compound
A1 (75% yield). A2 was prepared similarly from diethyl malonate
and bromomethyl cyclopentane according to the same procedure
as illustrated in Scheme 2, and the total yield was 9.6%.
more efficacious PDF inhibitors through structural modification of
LBM415. N-Butyl and cyclopentylmethyl groups were selected as
part of the P10 group to mimic the methionine residue present in
the natural substrate. In order to improve stability and enhance
antibacterial activity, significant modification at the P20 position
was carried out by choosing five widely used L-proline derivatives
to replace the pyrrolidine functionality of LBM415. By introducing
various aliphatic amines, aromatic amines, and heterocyclic aro-
matic amines at the P30 position, 43 PDF inhibitor candidates were
designed and synthesized. The in vitro antibacterial activities were
evaluated and the results summarized in Tables 1e4.
Eighteen 2,5-dihydropyrrole formyl hydroxyamino derivatives
with n-butyl at the P10 position (1aer) were synthesized initially
for in vitro evaluation of antibacterial activity (Table 1). Among
these PDF inhibitor candidates, compounds 1a and 1b containing
aliphatic amines at the P30 position exhibited unsatisfactory anti-
bacterial activity against all tested bacterial strains. Moderate
antibacterial activity was observed when using aliphatic 1-
phenylethylamines containing an aromatic ring at the P30 posi-
tion (compounds 1c and 1d). Taking these results into account, we
decided to replace the aliphatic amines at the P30 position with
aromatic amines. Compounds 1eem, each bearing aromatic amide
moieties, were synthesized, and they exhibited moderate to good
antibacterial activity against the Gram-positive bacterial strains.
Finally, heterocyclic aromatic amines were introduced at the P30
position, resulting in compounds 1neq, and they exhibited com-
parable or better antibacterial activities than the positive controls.
Because compound 1f, which contains an electron-withdrawing
group, exhibited favorable antibacterial activity, we incorporated
the electron-withdrawing trifluoromethyl group in a heterocyclic
amine to synthesize compound 1r. Unfortunately, a dramatic
decrease in antibacterial activity was observed with 1r.
To further confirm the structureeactivity relationship (SAR) of
the P30 position, several derivatives of formyl hydroxyamino 3-
methylenepyrrolidine with n-butyl at the P10 position were
designed and synthesized (1sex) for evaluation of antibacterial
activity. Unlike compound 1a, which exhibited almost no antibac-
terial activity, compound 1s bearing an aliphatic amino morpholine
group at the P30 position exhibited moderate antibacterial activity.
Compounds bearing aromatic amines (1t, 1u) at the P30 position
typically resulted in moderate to good antibacterial activity. Good
to excellent antibacterial activities were observed when
Five proline derivatives (Scheme 3) were selected as fragment B
and prepared according to known methods [39e43]: B1 ((S)-methyl
2,5-dihydro-1H-pyrrole-2-carboxylate),
B2
((S)-methyl
4-
methylenepyrrolidine-2-carboxylate), B3 ((2S, 3aR, 7aS)-methyl
octahydro-1H-indole-2-carboxylate), B4 ((2S, 4S)-methyl 4-
fluoropyrrolidine-2-carboxylate), and B5 ((2S, 4S)-methyl 4-
methylpyrrolidine-2-carboxylate).
The synthesis of PDF inhibitors 1ae2l is outlined in Scheme 4.
Coupling of the proline derivative B (B1eB5) with fragment A
(A1eA2) using 1-hydroxy-benzotriazole monohydrate (HOBt) and 1-
(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride
(EDCI) in the presence of N-methylmorpholine (NMM) produced
compound 18 (65e78% yield). Hydrolysis of 18 with lithium hy-
droxide gave 19 in moderate to good yield. Compound 20 was made
from 19 and various amines (R3NH2) using the mixed anhydride
coupling method. Finally, removal of the PMB protecting group by
trifluoroacetic acid successfully provided the desired formyl
hydroxyamino derivatives. In order to obtain pyridine N-oxide
products, the PMB protected 5-fluoride pyridine derivatives were
oxidized by urea hydrogen peroxide in the presence of phthalic an-
hydride (85e95% yield). Removal of the PMB protecting group with
trifluoroacetic acid successfully provided the desired corresponding
N-oxide products (41e53% yield). All new compounds were unam-
biguously characterized by 1H, 13C NMR spectroscopy and HR-MS.
2.2. Pharmacology
2.2.1. Structureeactivity relationship study
As the binding potency of a PDF inhibitor depends on the ad-
ditive effects of several chemical groups, we decided to search for
Scheme 2. Synthesis of intermediate A.
Reagents and conditions: (a) R ¼ n-propyl: 1-bromobutane, EtOH, EtONa, reflux overnight, 61%; R ¼ cyclopentyl: bromomethyl cyclopentane, EtOH, EtONa, reflux overnight, 60%. (b)
NaOH, H2O, reflux, 4 h, 85%. (c) HCHO, Et2NH, EtOH, reflux overnight, 84%. (d) i, Pivaloyl chloride, Et3N, THF, ꢀ78 ꢁC, 2 h; ii, (S)-4-benzyl-2-oxazolidinone, BuLi, THF, ꢀ78 ꢁC,
overnight, 63%. (e) i, PMBONH2, 45 ꢁC, 24 h; ii, EtOAc, p-TsOH; iii, EtOAc, aqNa2CO3, 59.6%. (f) H2O2, LiOH, THF, H2O, 1 h, 80%. (g) Ethyl formate, 60 ꢁC, overnight, 75%.

136
S. Yang et al. / European Journal of Medicinal Chemistry 86 (2014) 133e152
Scheme 3. Chemical structures of intermediates B1eB5.
Scheme 4. Synthesis of PDF inhibitors 1ae2l.
Reagents and conditions: (a) HOBT, EDCI, NMM, CH2Cl2, rt, 12 h, 65e78%. (b) LiOH, THF, H2O, 2 h, rt, 85e95%. (c) i, Et3N, ClCO2Et, THF, 0e25 ꢁC, 0.5 h; ii, R3NH2, 12 h, rt, 55e75%. (d)
TFA, DCM, 0e25 ꢁC, 2 h, 90%. (e) CO(NH2)2eH2O2, phthalic anhydride, EA, rt, 85e95%. (f) TFA, DCM, 0e25 ꢁC, 2 h, 41e53%.
heterocyclic aromatic amines were introduced at the P30 position in
compounds 1vex. Among the screened compounds, compound 1x
bearing a 5-methylthiazol-2-amino group at the P30 position
exhibited the best antibacterial activity and was approximately
2e8-fold more potent than the positive control LBM415.
In summary, 3-methylenepyrrolidine formyl hydroxyamino
derivatives (1sex) and 2,5-dihydropyrrole formyl hydroxyamino
derivatives (1aer) exhibited the same tendency of SAR in the P30
position. In addition, aliphatic amine derivatives are poor antibac-
terial agents, whereas compounds bearing aromatic amines at the
P30 position typically exhibit moderate to good antibacterial ac-
tivity. Introducing heterocyclic aromatic amines into the P30 posi-
tion results in the best antibacterial activity.
In order to obtain more information regarding the SAR at the P20
position, three other proline derivatives, specifically (2S, 3aR, 7aS)-
octahydro-1H-indole-2-carboxylic
fluoropyrrolidine-2-carboxylic
acid,
acid, and
(2S,
(2S,
4S)-4-
4S)-4-
methylpyrrolidine-2-carboxylic acid, were selected at the P20 po-
sition to make new PDF inhibitors (2ael). The P30 position of these
new PDF inhibitors consisted mainly of heterocyclic aromatic
amines, as the SAR study of both 2,5-dihydropyrrole and 3-
methylenepyrrolidine formyl hydroxyamino derivatives showed
that heterocyclic aromatic amines, such as 5-methylthiazol-2-
amine and 5-fluoropyridin-2-amine, are the best candidates for
the P30 position. The new compounds (2ael) were screened against
a wide range of bacteria, including S. aureus, MSSA, PRSP, MRSA,
and MRSE, and the results are summarized in Table 2. Compounds
bearing octahydroindole at the P20 position (2a, 2b, 2f, 2g, 2h)
exhibited only moderate antibacterial activity, which may be
attributed to the large steric hindrance of the octahydroindole. The
3-fluoropyrrolidine formyl hydroxyamino derivatives (2c, 2d, 2i, 2j)
exhibited moderate to good antibacterial activity. Good to excellent
antibacterial activities were observed when 3-methylpyrrolidine
was introduced into the P20 position. Notably, compound 2e,
which contains 2-amino-5-fluoropyridine-N-oxide at the P30 po-
sition and an n-butyl group at the P10 position exhibited better
antibacterial activity than the positive control LBM415. The 3-
methylpyrrolidine formyl hydroxyamino derivatives with n-butyl
at the P10 position were more potent than the corresponding
cyclopentylmethyl derivatives (2e vs 2l), which is different from the
2,5-dihydropyrrole and other formyl hydroxyamino analogs.
To explore the SAR at the P10 position, several 2,5-
dihydropyrrole formyl hydroxyamino derivatives with a cyclo-
pentylmethyl group at the P10 position were synthesized (1a0eq0).
The cyclopentylmethyl compounds exhibited better in vitro anti-
bacterial activity (2e4 times) than the corresponding n-butyl an-
alogs (1m′ vs. 1m, 1o′ vs. 1o, 1p′ vs. 1p, and 1q′ vs. 1q). Preliminary
SAR at the P30 position of the cyclopentylmethyl compounds was
also studied, and the tendency of SAR in the P30 position was almost
identical to that of the n-butyl derivatives. The cyclopentylmethyl
compounds containing aliphatic amines, such as morpholine (1a0),
exhibited unsatisfactory antibacterial activity. Moderate antibac-
terial activities were observed when aromatic rings were intro-
duced at the P30 position. Notably, compounds containing
heterocyclic aromatic amines at the P30 position exhibited better
antibacterial activity than existing drugs, such as penicillin, cipro-
floxacin, linezolid, and vancomycin. In particular, compounds with
5-methylthiazol-2-amine at the P30 position (1o, 1o′) were
exceedingly potent PDF inhibitors and 4e8-fold more potent than
the positive control LBM415.
2.2.2. Broad-spectrum antimicrobial activity
After preliminary evaluation of the antimicrobial activities of
the 43 synthesized novel compounds, seven candidates (1q, 1e′, 1o

S. Yang et al. / European Journal of Medicinal Chemistry 86 (2014) 133e152
137
Table 1
In vitro minimum inhibitory concentration (MIC, m
g/ml)a values for 1aeq′ in various strains.
No.
1a
R1
X
R3
S. aureus
MSSAb
MRSAc
S. epidermidis
E. coli.1
>64
E. coli.2
>64
>64
64
4
>64
32
32
2
>64
4
1b
64
32
16
8
8
>64
>64
1c
4
4
16
8
2
1
4
2
>64
>64
>64
1d
64
1e
1f
0.5
0.5
32
8
2
2
4
1
0.5
1
0.5
2
>64
>64
>64
64
32
>64
>64
16
1g
1h
1
8
0.125
1
1i
4
1
0.5
2
>64
>64
1j
0.5
8
2
2
0.25
1
1
4
>64
>64
64
1k
>64
1l
2
2
1
1
4
>64
>64
32
64
32
16
1m
1n
0.125
0.0625
0.125
0.125
0.5
0.25
0.25
1o
(continued on next page)

138
S. Yang et al. / European Journal of Medicinal Chemistry 86 (2014) 133e152
Table 1 (continued )
No.
R1
X
R3
S. aureus
MSSAb
1
MRSAc
0.25
S. epidermidis
E. coli.1
>64
E. coli.2
0.5
0.5
64
1p
1q
0.5
32
1
0.5
8
1
>64
>64
64
16
16
>64
1r
1s
4
1
64
4
4
8
1
>64
>64
>64
0.5
64
1t
0.5
0.5
1
2
1
4
0.25
0.125
0.5
0.5
0.25
1
64
64
1u
1v
32
>64
>64
>64
1w
0.25
0.5
0.0312
0.0625
64
32
1x
64
4
64
16
32
4
64
8
>64
>64
>64
>64
1a′
1c′
4
4
1
0.5
1
32
32
32
64
1e′
0.25
0.5
0.25
1m′

S. Yang et al. / European Journal of Medicinal Chemistry 86 (2014) 133e152
139
Table 1 (continued )
No.
R1
X
R3
S. aureus
MSSAb
0.25
MRSAc
0.125
S. epidermidis
E. coli.1
E. coli.2
0.03125
0.0625
16
16
1o′
0.25
0.5
0.125
0.125
32
16
1p′
1q′
0.25
0.25
0.5
1
0.25
0.5
0.25
0.5
64
64
32
64
LBM415
Linezolid
Penicillin
2
2
4
0.5
2
>64
>64
>64
0.0625
0.5
1
64
1
>64
Ciprofloxacin
0.5
0.25
<0.0312
>64
<0.0312
>64
Vancomycin
1
1
1
2
a
MIC was determined using the broth microdilution technique. The MICs of 1aer in this table were reported previously by our group [18].
MSSA, methicillin-susceptible S. aureus.
MRSA, methicillin-resistant S. aureus.
b
c
Table 2
In vitro minimum inhibitory concentration (MIC, m
g/ml)a values for 2ael in various strains.
No.
2a
R1
X
R3
S. aureus
MSSAb
MSSEc
0.5
Enterococcus faecalis
MRSAd
MRSEe
16
Moraxella catarrhalis
PRSPf
0.03
0.03
0.06
8
8
8
8
8
8
>16
>16
>16
8
4
8
0.03
0.03
0.25
2b
0.5
16
4
8
2c
4
4
4
>16
4
16
0.25
0.03
0.06
0.03
2d
2e
0.25
0.25
0.0625
4
0.25
0.5
(continued on next page)

140
S. Yang et al. / European Journal of Medicinal Chemistry 86 (2014) 133e152
Table 2 (continued )
No.
R1
X
R3
S. aureus
MSSAb
4
MSSEc
0.5
Enterococcus faecalis
MRSAd
4
MRSEe
8
Moraxella catarrhalis
PRSPf
0.03
4
>16
0.5
2f
4
4
4
4
0.25
0.5
>16
>16
4
4
4
8
0.03
0.5
0.03
0.03
2g
2h
2i
1
2
0.5
0.5
0.125
0.25
>16
0.5
2
1
4
0.125
0.25
0.03
0.03
8
2j
1
0.5
0.5
0.25
4
2
1
2
1
0.5
0.5
0.03
0.03
2k
0.5
1
0.5
2l
0.5
0.5
0.25
4
0.5
2
0.25
0.03
LBM415
a
MIC was determined using the broth microdilution technique.
MSSA, methicillin-susceptible S. aureus.
MSSE, methicillin-sensitive Staphylococcus epidermidis.
MRSA, methicillin-resistant S. aureus.
MRSE, methicillin-resistant S. epidermidis.
PRSP, penicillin-resistant Streptococcus pneumoniae.
b
c
d
e
f
′, 1q′, 1m′, 1v, and 1x) were selected as representative compounds
based on their in vitro antibacterial activity. The seven represen-
tative compounds, along with the control compound amoxicillin,
were screened against a panel of 14 isolates representing a broad
spectrum of activity. The results are summarized in Table 3. Com-
pounds 1q, 1o′, 1q′, 1m′, and 1x exhibited good activity against all
isolates except Escherichia coli. However, compounds 1v and 1e′
exhibited unsatisfactory antibacterial activity against several tested
strains, such as Entc.faecalis I and Haem.influenzae H128.
Compound 1q was selected for further in vitro exploration of
antibacterial activity by screening against 31 clinical isolates of
S. aureus. The screening results are summarized in Table 4. Nineteen
clinical isolates exhibited resistance to amoxicillin, and nine strains
exhibited resistance to mupirocin. However, only one strain
exhibited moderate resistance to 1q. The Gmean values of 1q,
0.855 mg/mL, respectively. The results indicate that 1q has better
antibacterial potency and spectrum than amoxicillin and
mupirocin.
2.2.3. Pharmacokinetic study
The outstanding in vitro antibacterial activities of compounds
1o, 1q, 1o′, 1q′, and 1x encouraged us to evaluate their further
medicinal properties. Thus, in vivo pharmacokinetic study was
carried out with compounds 1o, 1q, 1o′, 1q′, 1x, and LBM415. Rats
were dosed with 5 mg/kg via intravenous (i.v.) routes and 25 mg/kg
via oral (p.o.) routes. Blood samples were collected from anes-
thetized rats via cardiac puncture at 0.083, 0.25, 0.5, 1, 2, 4, 6, and
24 h after dosing, and the concentrations of the compounds were
determined by HPLC analysis. The results are summarized in Table 5
and Fig. 2. The tested compounds were rapidly absorbed after oral
amoxicillin, and mupirocin were 0.625
m
g/mL, 5.72
mg/mL, and
administration
and
reached
maximum
concentrations

S. Yang et al. / European Journal of Medicinal Chemistry 86 (2014) 133e152
141
Table 3
In vitro minimum inhibitory concentration (MIC, m
g/mL)a values for seven typical compounds against 14 bacterial strains.a
Isolate
Compound MIC (
m
g/mL)
Genus_Species_Strain
Amoxicillin
1q
1o′
0.25
1e′
1q′
0.5
0.125
2
1
1
1m′
1v
1x
0.5
0.125
4
2
4
4
Staph. aureus OXFORD
Staph. aureus WCUH29
Entc.faecalis I
0.125
64
0.5
16
0.25
32
0.5
0.125
4
2
1
1
4
0.5
ꢂ0.06
0.125
>64
1
2
0.125
2
1
2
2
ꢂ0.06
ꢂ0.06
1
0.5
1
32
0.25
0.25
2
32
Entc.faecium X7501
0.5
2
2
4
Haem.influenzae Q1
Haem.influenzae H128
Haem.influenzae H128 Acr A-
Morax.catarrhalis 1502
Strep.pneumoniae 1629
Strep.pneumoniae N1387
Strep.pneumoniae ERY2
E. coli 3
1
16
16
0.5
0.25
16
2
ꢂ0.06
ꢂ0.06
1
0.5
1
64
2
2
2
2
64
ꢂ0.06
ꢂ0.06
0.5
0.25
0.5
32
0.25
0.5
ꢂ0.06
ꢂ0.06
2
0.5
1
64
0.25
0.5
ꢂ0.06
ꢂ0.06
0.5
0.125
0.25
64
ꢂ0.06
ꢂ0.06
ꢂ0.06
2
ꢂ0.06
2
1
2
4
8
ꢂ0.06
1
>64
4
64
2
2
Strep.pyogenes 1307006P
Strep.pyogenes 1308007P
ꢂ0.06
ꢂ0.06
0.25
0.5
4
a
MIC was determined using the broth microdilution technique.
22.5e41.3 min after dosing (Table 5a). In contrast, the Tmax for
LBM415 is 52 min after dosing, which is 11e30 min slower than the
other testing compounds. The oral bioavailability seems to be
closely related to the P10 group of the tested compounds. PDF in-
hibitors with n-butyl in the P10 position exhibited better oral
bioavailability (1o vs. 1o′, 1q vs. 1q′) than the corresponding
cyclopentylmethyl analogs (Table 5b). When the P30 group was
taken into consideration, we found that PDF inhibitors with 5-
methylthiazol-2-amine at the P30 position exhibited much higher
bioavailability than the corresponding 5-fluoropyridin-2-amino
analogs (1o vs. 1q, 1o′ vs. 1q′). The absolute oral bioavailability of
1o was calculated to be 88%, which was much higher than that of
the other PDF inhibitors and LBM415.
2.2.4. hERG Kþ inhibition study
Human ether-a-go-go-related gene1 (hERG) has received a
tremendous amount of attention since its discovery in 1994
because inherited mutations or drug-induced blockade of channels
increases the risk of lethal arrhythmia [44]. The outstanding in vitro
antibacterial activities and good pharmacokinetics of 1o, 1q, 1o′, 1q
′, and 1x prompted us to carry out the hERG Kþ inhibition study
(Table 6). Except for compound 1o′, which exhibited 15.86 1.27%
Table 4
In vitro minimum inhibitory concentration (MIC,
m
g/mL)a values for 1q against
Staphylococcus aureus isolates.a
Isolate
Compound MIC (
Amoxicillin
m
g/mL)
Mupirocin
Genus_Species_Strain
1q
ATCC 29213
A53
CARTER 37
CL 939
Ealing 23
Ealing 32
F89
1
0.125
0.25
2
0.5
1
0.25
1
inhibition at a concentration of 10 mM, the PDF inhibitors exhibited
0.125
0.5
0.5
0.125
0.5
0.25
1
0.5
0.5
0.5
0.5
0.25
0.125
1
only very mild or no hERG Kþ inhibition, making them unlikely to
cause serious lethal arrhythmia.
>16
0.25
>16
>16
>16
>16
0.125
>16
0.25
0.06
0.125
0.25
0.25
0.25
2
2.2.5. Solubility and stability
MILES HALL
OXFORD
RN4220 pmz1
RUSSELL
Smith ATCC 13709
SWEETING
CL 1033
CL 938
MN 1255
NEQAS 4026
OGA833
PAV 5
RN1024
RN1030
WCUH29
306
68/8684
929003
929035
934324
934334
934335
934387
NEQAS 4158
MIC 90
MIC 50
GMEAN
0.25
0.25
0.125
>16
0.125
0.125
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
5.720
0.125
>16
Solubility and stability of a medicine are closely linked to its
absorption and dosage. Thus, the solubility and stability of the three
most promising compounds (1o, 1p, and 1q) and the positive
control LBM415 were studied in artificial gastric juice and artificial
intestinal juice. As shown in Fig. 3, all tested compounds exhibited
better solubility than the positive control LBM415. Compound 1q
was 2-fold and 3-fold more soluble than LBM415 in artificial gastric
juice and artificial intestinal juice, respectively. Samples were taken
every 2 h for 12 h and evaluated by HPLC to investigate the stability
of these compounds (Fig. 3B and C). All compounds had similar or
better stability than LBM415.
1
0.25
1
0.5
1
>16
>16
0.125
0.25
0.25
0.125
0.5
0.5
0.125
2
>16
0.125
0.25
0.25
0.25
0.25
0.25
0.25
0.25
>16
2
1
1
1
0.25
0.5
1
2.2.6. In vivo efficacy study
2-Amino-5-fluoropyridine-N-oxide derivative 1q and 5-
methylthiazol-2-amino derivative 1x were selected as representa-
tive candidates to evaluate in vivo efficacy based on their good
pharmacokinetic properties and robust activities in broader anti-
bacterial profiling. The in vivo efficacy of 1q was evaluated in a
mouse MRSA08-12 infection model. Compound 1q and the refer-
ence inhibitor LBM415 were administered by i.v. to the infected
mice with single dose, and the results are summarized in Table 7a. It
is gratified to find that compound 1q had comparable protective
effects as LBM415. The 50% effective dose (ED50) of compound 1q
indicated good in vivo efficacy against MRSA (Table 7a).
>16
1
0.25
0.855
0.06
0.5
0.625
0.125
MIC Min
MIC Max
32
>16
a
MIC was determined using the broth microdilution technique.

142
S. Yang et al. / European Journal of Medicinal Chemistry 86 (2014) 133e152
Fig. 2. Mean plasma concentrationetime curve of (A) 1o, (B) 1q, (C) 1o′, (D) 1q′, (E) 1x, and (F) LBM415 in male Sprague Dawley mice. The mice received a 5 mg/kg injection or
25 mg/kg oral dose. Data are given as mean SD. n ¼ 4 mice in each group.
The in vivo efficacy of 1x was evaluated in a mouse MRSA (ATCC
33591) infection model with intragastric administration of single
doses (Table 7b). Although compound 1x was still efficacious
against MRSA, it had lower efficacies than the marketed drug
linezolid. The inconsistency between the in vitro potency and
in vivo efficacy of compound 1x was surprising. As the in vivo ef-
ficacy may be affected by the degree to which the drug binds to the
proteins within blood plasma, we decided to conduct a plasma
protein binding (PPB) rate test for compounds 1q and 1x.
2.2.7. Plasma protein binding rate study
PPB is an important biological property with implications on a
number of toxicological, pharmacological, and pharmacokinetic
parameters. Therefore, we evaluated the PPB rate of compounds 1q
and 1x with warfarin as a positive control agent, and the results are
summarized in Table 8. The high PPB rate of compound 1x (99% PPB
rate) may be the responsible factor for the lower efficacy in vivo
despite its good in vitro activity and pharmacokinetic properties.
Table 5b
Table 5a
The pharmacokinetics parameters following PDF inhibitor administration to male
Sprague Dawley mice (25 mg/kg oral).
The pharmacokinetics parameters following PDF inhibitor administration to male
Sprague Dawley mice (5 mg/kg intravenous).
Parameter (mean)
1o
1q
1o′
1q′
1x
LBM415
Parameter (mean)
1o
1q
1o′
1q′
1x
LBM415
Cmax (ng/mL)
tmax (h)
t1/2 (h)
12,365
0.625
2.56
21,834
21,862
2.45
1393
0.688
1.82
3315
3390
2.34
4565
0.625
3.31
12,243
12,295
3.39
956
0.375
2.58
1626
1808
2.01
15,700
0.688
3.02
58,108
58,312
3.41
1302
0.875
2.66
3689
3788
2.71
T1/2 (h)
MRT0-t (h)
4.08
1.17
2.68
1.24
4701
4704
1115
1373
0.807
0.503
1611
1615
3101
1601
3.21
2.03
19,752
19,795
290
0.784
0.562
2239
2244
2265
1305
0.952
4924
4931
1019
1007
0.564
1949
1960
2810
1715
AUC0-t
AUC0-∞
(
m
g.h/L)
g.h/L)
AUC0et g h/L)
(
m
(m
AUC0e∞ (mg h/L)
MRT0et (h)
F (%)
CLz (mL/h/kg)
Vss (mL/kg)
571
88.67
34.59
52.27
22.39
58.92
33.76

S. Yang et al. / European Journal of Medicinal Chemistry 86 (2014) 133e152
143
Table 6
The inhibition ratio of PDF inhibitors in various concentrations on hERG Kþ channels.
Concentration
Fractional block %
1o
1q
1o′
5.38 1.14
1q′
1x
LBM415
1
m
M
5.70 2.65
5.50 2.42
4.55 4.24
6.71 2.55
1.15 1.02
0.89 1.49
ꢀ0.35 2.16
ꢀ2.98 3.10
1.78 1.26
2.14 1.81
10
m
M
15.86 1.27
Fig. 3. The solubility and stability of 1o, 1p, 1q, and LBM415 in (A, B) artificial gastric juice and (A, C) artificial intestinal juice.
2.2.8. Acute toxicity study
1q at a dose of 250 mg/kg. On day 14, all animals were sacrificed
and gross necropsy performed for the skin, lungs, heart, liver,
kidneys, and spleen. No abnormalities were observed except dark
red plaques in the lungs of one of the three dead mice in the
250 mg/kg group. In general, the PDF inhibitors studied in this work
exhibited only mild toxicity toward the laboratory mice, with an
LD50 > 250 mg/kg.
Ideal antibacterial agents kill selectively and without being
harmful to the host. Therefore, we evaluated the acute toxic
behavior of our representative compounds (1q) toward KM mice in
SPF grade. During the 14 successive days of the acute toxicity study,
10 male and 9 female mice were injected with different concen-
trations of 1q and their behavior change, weight, coat color, anal
temperature, daily food intake, daily water intake, and daily weight
of urine and stool were checked to judge toxicity. The mice lived
without apparently abnormal behavior when 1q was given at a
dose of 100 mg/kg. When the dosage was increased to 250 mg/kg,
three male mice died after the injection, and three female mice
exhibited decreased activity but appeared normal 2 h later. No
adverse effects were observed for the remaining mice that received
3. Conclusion
In summary, 43 formyl hydroxyamino derivatives were ratio-
nally designed and synthesized, and an SAR study of the P10, P20,
and P30 positions carried out to provide crucial information for the
development of novel and potent PDF inhibitors. A preliminary SAR
Table 7a
In vivo efficacy of 1q and LMB415 in a rat MRSA infection model.
Strain (CFU/mL)
Drug
1q
Route
i.v.
Dose (mg/kg)
Rat amount
Death amount
Death rate (%)
ED50(mg/kg) (95% CI)
MRSA08-12 (5 ꢃ 107)
20
10
5
2.5
1.25
20
10
5
10
10
10
10
10
10
10
10
10
10
10
10
1
3
6
8
10
0
2
5
7
9
10
30
60
80
100
0
20
50
70
90
100
0
6.253 (4.307e9.077)
LBM415
i.v.
4.269 (2.934e6.211)
2.5
1.25
e
Control group
Blank control
(normal saline)
e
10
0
e
e
i.v.
e

144
S. Yang et al. / European Journal of Medicinal Chemistry 86 (2014) 133e152
Table 7b
line of the triplet at 77.23 ppm in CDCl3. Coupling constants (J
values) are reported in hertz.
In vivo efficacy of 1x and linezolid in a rat MRSA infection model.
Strain (CFU/ Drug
mL)
Route Dose
Rat
Death
Death
ED50(mg/kg)
(mg/kg) amount amount rate (%) (95% CI)
4.1.1. Diethyl 2-butylmalonate (6)
MRSA
(ATCC
33591)
1x
i.g.
20
10
5
6
6
6
6
6
3
4
5
1
4
50
66
83
16
66
19.58 (9.11
e42.09)
Diethyl malonate (8.3 g, 51.8 mmol) was added to a solution of
sodium ethoxide (3.52 g, 51.8 mmol) in EtOH (75 mL) and the re-
action mixture heated to reflux for 30 min. N-BuBr (8.64 g,
63.2 mmol) was added over an hour and the reaction mixture
refluxed overnight. The solvent was removed under reduced
pressure and then water (23 mL) added. The mixture was extracted
with ethyl acetate (43 mL). The organic layer was washed with
brine, dried over MgSO4, and concentrated under reduced pressure.
The crude product was purified by flash chromatography using the
solvent system hexane/ethyl acetate (5:1) to yield compound 6
(6.9 g, 61.2%) as a colorless oil. The NMR and MS data were the same
as previously reported [45].
Linezolid i.g.
20
10
5
11.90 (8.02
e17.66)
6
10
5
10
83
100
Control
group
Blank
e
e
e
e
i.g.
e
10
0
0
control
Table 8
Protein binding test for 1q, 1x, and warfarin in plasma using equilibrium dialysis.a
Drug
Species/matrix
%Unbound
SD %
% Recovery
SD
(n ¼ 3)
Bound (n ¼ 3)
4.1.2. 2-Methylenehexanoic acid (8)
1q
1x
Warfarin
CD-1 mouse
plasma
46.7
1.0
5.9
4.0 53.3
0.2 99.0
0.9 94.1
91.2
93.7
104.3
2.4
1.8
5.2
Compound 6 (8.6 g, 39.8 mmol) was added to a solution of NaOH
(8.6 g, 215 mmol) in water (25 mL) at room temperature. The re-
action mixture was heated to reflux for 4 h, and then cooled to
ambient temperature and the pH adjusted to 1e2 with concen-
trated hydrochloric acid. The mixture was extracted with ethyl
acetate (86 mL). The organic layer was washed with brine, dried
over MgSO4, and concentrated under reduced pressure. Ether
(35 mL) was added to the residue petroleum and stirred for 3 h. The
product was isolated by filtration and dried at 50 ꢁC to give 5.5 g
(85%) of 7.
Et2NH (0.6 mL, 5.88 mmol), HCHO (aq) (4.7 mL, 58.8 mmol), and
7 (4.7 g, 29.4 mmol) were mixed in EtOH (90 mL) and the reaction
mixture heated to reflux overnight. Volatiles were removed under
reduced pressure, the residue dissolved in water (23 mL), and the
pH adjusted to 3e4 with hydrochloric acid. The mixture was
extracted with ethyl acetate (40 mL). The organic layer was washed
with brine, dried over Na2SO4, and concentrated under reduced
pressure to yield 8 (3.2 g, 84%) as a colorless oil. The NMR data were
the same as reported [46].
a
The concentration was calculated using the peak area ratio of analyte and in-
ternal standard.
study of the P10 position found that the cyclopentylmethyl group
provided better in vitro antibacterial activity than the corre-
sponding n-butyl analogs. When the P20 group was taken into
consideration, we found that large steric hindrance and electron-
withdrawing groups in the P20 position decreases the antibacte-
rial activity of the PDF inhibitors, and ingenious modification of the
proline in P20 position can maximize antibacterial activity. The SAR
study of the P30 position indicated that compounds with hetero-
cyclic aromatic amines typically result in favorable antibacterial
activity, and the optimum groups in the P30 position were 5-
methylthiazol-2-amine and 2-amino-5-fluoropyridine-N-oxide.
The studies of solubility, stability, hERG Kþ channel-inhibiting
ability, in vivo pharmacokinetics, and acute toxicity of the repre-
sentative compounds showed that most of the representative
compounds possess very mild toxicity and good pharmacokinetic
profiles. We also noted that the 2-amino-5-fluoropyridine-N-oxide
compounds, such as compound 1q, exhibited comparable anti-
bacterial activities in vitro and in vivo with known compound
LBM415. The 5-methylthiazol-2-amino derivative, compound 1x,
which had a 99% PPB rate, still exhibited reasonable efficacy in vivo.
Studies on reducing the PPB rate of compound 1x by structural
modification are currently in progress. We believe that derivatives
of 1x with appropriate PPB rate will have robust activities against
drug-resistant bacteria both in vitro and in vivo.
4.1.3. (S)-3-(2-methylenehexanoyl)-4-benzyloxazolidin-2-one (9)
Et3N (5.5 mL, 38.0 mmol) was added to a solution of 8 (3.9 g,
30.5 mmol) in THF (90 mL) at ꢀ78 ꢁC under nitrogen atmosphere.
Pivaloyl chloride (3.8 mL, 31.7 mmol) was then added dropwise
below ꢀ60 ꢁC. The reaction mixture was stirred at ꢀ78 ꢁC for
30 min, warmed to room temperature and stirred for another 2 h,
then cooled to ꢀ78 ꢁC for further use (9a). In another flask, THF
(90 mL) and (S)-4-benzyloxazolidin-2-one (4.9 g, 27.6 mmol) were
combined and the mixture cooled to ꢀ78 ꢁC under nitrogen at-
mosphere. N-BuLi (13.2 mL, 32.8 mmol) was added dropwise
at ꢀ78 ꢁC and then warmed to room temperature and stirred for
another 30 min (9b).
4. Experimental
The two solutions (9a and 9b) were mixed together at ꢀ78 ꢁC
and then warmed to room temperature and stirred overnight.
Aqueous KHCO3 solution (40 mL, 40 mmol) was added to quench
the reaction and the solvent removed under reduced pressure.
Ethyl acetate (100 mL) and water (100 mL) were added to the
residue and stirred for 10 min. The water layer was extracted with
ethyl acetate (100 mL). The organic layer was combined and
washed with brine, dried over MgSO4, and concentrated under
reduced pressure to yield a yellow residue. Petroleum ether (20 mL)
was added to the residue and stirred for 2 h. The product was
isolated by filtration and dried to yield 9 (5.5 g, 63%) as a white
4.1. Chemistry
Unless otherwise described, all commercial reagents and sol-
vents were purchased from commercial suppliers and used without
further purification. Flash column chromatography was carried out
using silica gel 60 (200ꢀ400 mesh), and preparative thin layer
chromatography was carried out with glass-backed silica gel plates
(1 mm). Thin layer chromatography was performed to monitor
reactions. All 1H NMR spectra are reported in
d units ppm relative to
tetramethylsilane (TMS) in CDCl3. All chemical shift values are re-
ported with multiplicity, coupling constants, and proton count. All
power. 1H NMR (500 MHz, CDCl3)
2H), 4.44 (m, 1H), 4.22 (m, 2H), 3.37 (m, 1H), 2.82 (m, 1H), 2.39 (m,
2H), 1.43 (m, 4H), 0.93 (m, 3H); 13C NMR (125 MHz, CDCl3)
171.1,
d
7.29 (m, 5H), 5.40 (d, J ¼ 7 Hz,
13C NMR spectra are reported in
d
units ppm relative to the central
d

S. Yang et al. / European Journal of Medicinal Chemistry 86 (2014) 133e152
145
152.8, 144.3, 135.1, 129.4, 128.9, 127.4, 118.9, 66.4, 55.2, 37.6, 32.6,
29.9, 22.21, 13.8.
(m, 2H), 6.89 (d, J ¼ 8.5 Hz, 2H), 5.00e4.71 (m, 2H), 3.79 (s, 5H), 2.76
(m,1H),1.81e1.75 (m, 4H),1.60e1.50 (m, 4H),1.05e1.07 (m, 2H); 13
C
NMR (125 MHz, CDCl3) 180.0, 163.3, 160.2, 131.3, 128.9, 126.0, 114.1,
55.2, 45.3, 43.0, 37.7, 36.0, 32.7, 32.3, 25.1, 25.0; HR-MS(ESI) calc. for
C
4.1.4. (S)-3-((R)-2-((4-methoxybenzyloxyamino)methyl)hexanoyl)-
4-benzyloxazolidin-2-one (10)
18H25NNaO5 (MþNa)þ: 358.1625, found: 358.1617.
O-(4-Methoxybenzyl) hydroxylamine (30 g, 0.196 mol) and 9
(23 g, 0.08 mol) were mixed together under nitrogen atmosphere.
The mixture was warmed to 45 ꢁC and stirred for 24 h. The resulting
mixture was diluted with ethyl acetate (50 mL). p-Toluene sul-
phonic acid (68 g, 0.394 mol) in ethyl acetate (120 mL) was added to
this solution. The resulting mixture was stirred for 1.5 h, filtered,
and concentrated under reduced pressure. MTBE (550 mL) was
added to this residue and stirred overnight. The resulting mixture
was filtered and the filter cake added to a mixture solution of ethyl
acetate (150 mL) and aqueous sodium carbonate (0.36 mol/L,
150 mL). The reaction mixture was stirred for 30 min and the
aqueous layer separated. The organic layer was washed with brine,
dried over Na2SO4, filtered, and concentrated under reduced pres-
sure to yield 10 (21 g, 59.6%) as a colorless oil. 1H NMR (500 MHz,
4.1.8. General procedure for intermolecular amide coupling of A and
B
NMM (1.94 mL, 17.6 mmol) was added to a solution of A
(8.8 mmol), HOBt (1.1 g, 8.8 mmol), and EDCI (1.7 g, 8.8 mmol) in
DCM (20 mL) at room temperature, and then compound B (B1eB5,
8.8 mmol) in DCM (10 mL) was added and stirred for 12 h. The
mixture was washed with 10% citric acid aqueous solution (20 mL),
followed with saturated aqueous sodium carbonate (20 mL), the
organic layer was dried with MgSO4, and concentrated under
reduced pressure to give 18 in 65e78% yield.
4.1.9. General procedure for the hydrolysis of 18
Lithium hydroxide monohydrate (0.28 g, 6.8 mmol) aqueous
solution (15 mL) was added to a solution of 18 (6.8 mmol) in THF
(15 mL) and the reaction mixture stirred for 1 h at room temper-
ature and extracted with ethyl acetate (20 mL). The pH of the water
layer was adjusted to 4e5 and extracted with ethyl acetate (20 mL).
The organic layer was dried with MgSO4 and concentrated under
reduced pressure to obtain 19 (85e95% yield).
CDCl3):
d
7.32 (m, 5H), 7.19 (d, J ¼ 8.5, 2H), 6.82 (d, J ¼ 8.5, 2H),
5.75(br, 1H), 4.63 (m, 3H), 4.14 (m, 3H), 3.70(s, 3H), 3.36 (m, 1H),
3.20 (m, 2H), 2.43 (m, 1H), 1.75 (m, 1H), 1.55 (m, 1H), 1.31 (m, 4H),
0.89 (t, J ¼ 7 Hz, 3H); 13C NMR (125 MHz, CDCl3):
d 175.9, 159.3,
153.4, 135.7, 130.1, 129.8, 129.4, 128.8, 127.1, 113.7, 75.6, 65.9, 55.7,
55.1, 54.3, 42.0, 37.3, 30.6, 29.2, 22.7, 13.8.
4.1.5. (R)-2-((4-methoxybenzyloxyamino)methyl)hexanoic acid
(11)
4.1.10. General procedure for intermolecular amide coupling of C
(R3NH2) and 19
Hydrogen peroxide (4.9 mL, 43 mmol) was added to a solution of
10 (8.6 g, 19.5 mmol) in THF (50 mL) and water (12 mL) at 0 ꢁC, and
then lithium hydroxide monohydrate (0.9 g, 21.5 mmol) in water
(10 mL) was added. The mixture was stirred at 0 ꢁC for 1 h. Sodium
sulfite aqueous solution (0.42 mol/L, 70 mL) was added to this so-
lution and the resulting solution stirred for another hour. Volatiles
were distilled off and the resulting residue extracted with ethyl
acetate (30 mL). The pH of the water layer was adjusted to 4e5 with
hydrochloric acid and then extracted with ethyl acetate (3 ꢃ 30 mL).
The combined organic layers were washed with brine, dried over
MgSO4, filtered, and concentrated under reduced pressure to yield
Et3N (0.72 mL, 5.0 mmol) and ethyl chlorocarbonate (0.54 g,
5.0 mmol) were added to a solution of 19 (5.0 mmol) in THF (20 mL)
at 0 ꢁC and the reaction mixture stirred for 1 h at room tempera-
ture. R3NH2 (6 mmol) was added and the reaction mixture stirred
for another 12 h and concentrated under reduced pressure. Ethyl
acetate (20 mL) was added to the residue and stirred for 10 min. The
mixture was washed with 10% citric acid aqueous solution (20 mL),
followed with saturated sodium carbonate (20 mL) solution. The
organic layer was dried with MgSO4 and concentrated under
reduced pressure to obtain 20 (55e75% yield). The product was
used for the next step without further purification.
11 (4.4 g, 80%) as a yellow residue. 1H NMR (500 MHz, CDCl3):
d 7.28
(d, J ¼ 7 Hz, 2H), 6.88 (d, J ¼ 7 Hz, 2H), 4.68 (m, 2H), 3.80 (s, 3H), 3.14
4.1.11. General procedure for oxidation of 20
(m, 2H), 2.72 (m, 1H), 1.70 (m, 1H), 1.53 (m, 1H), 1.33 (m, 4H), 0.91 (t,
Urea-hydrogen peroxide (1.05 g, 12 mmol) was added to a so-
lution of 20 (4 mmol) in ethyl acetate (20 mL) at 0 ꢁC, followed by
the addition of phthalic anhydride, and the reaction mixture stirred
for 10 h at room temperature and diluted with sodium sulfite
(2.48 g, 20 mmol) aqueous solution. The water layer was separated,
the organic layer dried with MgSO4, and then concentrated under
reduced pressure to obtain oxynitride (85e95% yield).
J ¼ 7 Hz, 3H); 13C NMR (125 MHz, CDCl3):
d 180.3,159.4,130.1,129.4,
113.8, 75.8, 55.2 53.1, 43.0, 29.6, 29.2, 22.5,13.8; HR-MS(ESI) calc. for
C
15H24NO4 (MþH)þ: 282.1700, found: 282.1745.
4.1.6. (R)-2-[(4-methobenzyloxy-formyl-amino)-methyl]-hexanoic
acid (A1)
Compound 11 (4.5 g, 16 mmol) and ethyl formate (30 mL) were
mixed at room temperature, and then warmed to 60 ꢁC and stirred
overnight. The solvents were removed in vacuo and the crude
product purified by flash chromatography using the solvent system
hexane/ethyl acetate (1:1) to yield compound 12 (3.7 g, 75%) as a
4.1.12. General procedure for deprotection reaction of 20 or 21
TFA (5 mL, 67.5 mmol) was added to a solution of 20 or 21
(3.6 mmol) in DCM (10 mL) at 0 ꢁC and the reaction mixture stirred
for 2 h at room temperature. The mixture was diluted with DCM
(10 mL) and water (10 mL), stirred for 10 min, and the water layer
separated. The organic layer was dried with MgSO4 and concen-
trated under reduced pressure. The residue was subjected to
chromatography on silica gel, eluting with 10% methanol/DCM to
obtain 1ae2l (41e53% yield).
yellow oil. 1H NMR (500 MHz, CDCl3):
d 8.09 (br, 1H), 7.35 (d,
J ¼ 8.5 Hz, 2H), 6.90 (d, J ¼ 8.5 Hz, 2H), 4.89e4.69 (m, 2H),
3.81e3.64 (s, 5H), 2.72 (m, 1H), 1.62 (m, 1H), 1.48 (m, 1H), 1.24 (m,
4H), 0.88 (t, J ¼ 7 Hz, 3H); 13C NMR (125 MHz, CDCl3) 180.0, 163.4,
160.2, 131.2, 130.0, 114.1, 55.2, 45.1, 43.4, 29.6, 29.3, 28.9, 22.4, 13.7;
HR-MS(ESI) calc. for C16H23NNaO5 (MþNa)þ: 332.1468, found:
332.1542.
4.1.13. N-hydroxy-N-((R)-2-((S)-2-(morpholine-4-carbonyl)-2,5-
dihydro-1H-pyrrole-1-carbonyl)hexyl)formamide (1a)
4.1.7. (R)-3-cyclopentyl-2-((N-((4-methoxybenzyl)oxy)formamido)
methyl)propanoic acid(A2)
Prepared from A1, B1, and morpholine (R3NH2) according to the
general procedure and purified by flash column chromatography
on silica gel (MeOH/DCM ¼ 1:10) to obtain 1a (21% yield). 1H NMR
Prepared following the same procedure as A1, the total yield of
A2 was 9.6%. 1H NMR (500 MHz, CDCl3)
d
8.07 (bs, 1H), 7.34e7.23
(500 MHz, CDCl3) d 9.65 (s, 1H), 8.41 (s, 1H), 6.04 (m, 1H), 5.71 (m,

146
S. Yang et al. / European Journal of Medicinal Chemistry 86 (2014) 133e152
1H), 5.61 (m,1H), 4.39 (m, 2H), 4.15 (m,1H), 3.88 (m,1H), 3.75e3.65
(m, 5H), 3.61 (m, 2H), 3.39 (m, 1H), 3.02 (m, 1H), 1.48 (m, 1H),
1.31e1.24 (m, 4H), 0.88 (t, J ¼ 3.5 Hz, 3H); 13C NMR (125 MHz,
4.1.13.5. (S)-1-((R)-2-((N-hydroxyformamido)methyl)hexanoyl)-N-
(4-nitrophenyl)-2,5-dihydro-1H-pyrrole-2-carboxamide (1f).
Prepared from A1, B1, and 4-nitroaniline (R3NH2) according to the
general procedure and purified by flash column chromatography
on silica gel (MeOH/DCM ¼ 1:10) to obtain 1f (13% yield). 1H NMR
CDCl3) d 172.9, 168.6, 163.0, 129.54, 123.3, 66.6, 66.4, 64.1, 54.4, 52.3,
46.0, 43.1, 42.4, 29.6, 29.5, 22.7, 13.8; HR-MS(ESI) calc. for
C
17H27N3NaO5 (MþNa)þ: 376.1843, found: 376.1849.
(500 MHz, CDCl3) d 9.75 (s, 1H), 8.05 (m, 4H), 7.70 (s, 1H), 5.99 (m,
1H), 5.83 (m, 1H), 5.22 (m, 1H), 4.58 (m, 1H), 4.21 (m, 1H), 3.89 (m,
1H), 3.43 (m, 1H), 3.20 (m, 1H), 1.68 (m, 1H), 1.50e1.26 (m, 5H), 0.89
4.1.13.1. (S)-N-cyclopropyl-1-((R)-2-((N-hydroxyformamido)methyl)
hexanoyl)-2,5-dihydro-1H-pyrrole-2-carboxamide (1b).
(t, J ¼ 5 Hz, 3H); 13C NMR (125 MHz, CDCl3)
d 203.3, 169.3, 164.4,
140.1, 139.1, 125.0, 121.0, 120.7, 115.2, 114.9, 64.5, 50.9, 47.6, 37.0,
27.0, 26.3, 25.0, 18.8; HR-MS(ESI) calc. for C19H24N4NaO6 (MþNa)þ:
427.1588, found: 427.1620.
Prepared from A1, B1, and cyclopropanamine (R3NH2) according to
the general procedure and purified by flash column chromatog-
raphy on silica gel (MeOH/DCM ¼ 1:10) to obtain 1b (23% yield). 1H
NMR (500 MHz, CDCl3)
d 10.50e9.75 (bs, 1H), 7.59 (s, 1H), 7.06 (s,
4.1.13.6. (S)-1-((R)-2-((N-hydroxyformamido)methyl)hexanoyl)-N-
1H), 6.00e5.97 (m, 1H), 5.86e5.85 (m, 1H), 5.24 (m, 1H), 4.76 (m,
1H), 4.34 (m, 1H), 3.88 (m, 1H), 3.38 (m, 1H), 3.13 (m, 1H), 2.75 (m,
1H), 1.60 (m, 1H), 1.49 (m, 1H), 1.35e1.30 (m, 4H), 0.92 (t, J ¼ 6 Hz,
(4-methoxyphenyl)-2,5-dihydro-1H-pyrrole-2-carboxamide
(1g).
Prepared from A1, B1, and 4-methoxyaniline (R3NH2) according to
the general procedure and purified by flash column chromatog-
raphy on silica gel (MeOH/DCM ¼ 1:10) to obtain 1g (17% yield). 1H
3H), 0.75 (m, 2H), 0.52 (m, 2H); 13C NMR (125 MHz, CDCl3)
d 172.5,
171.4, 127.4, 126.2, 67.7, 53.9, 51.4, 40.5, 29.8, 28.9, 22.7, 22.5, 13.8,
6.5, 6.2; HR-MS(ESI) calc. for C16H25N3NaO4 (MþNa)þ: 346.1737,
found: 346.1728.
NMR (500 MHz, CDCl3) d 9.08 (s, 1H), 7.66 (s, 1H), 7.56 (m, 2H), 6.72
(s, 2H), 6.02e5.93 (m, 2H), 5.48 (s, 1H), 4.76 (s, 1H), 4.42 (m, 1H),
3.86e3.73 (m, 4H), 3.42 (m, 1H), 3.18 (m, 1H), 1.66e1.19 (m, 6H),
0.89 (m, 3H); 13C NMR (125 MHz, CDCl3)
d 172.6, 167.6, 155.8, 131.2,
4.1.13.2. (S)-1-((R)-2-((N-hydroxyformamido)methyl)hexanoyl)-N-
127.8, 125.9, 120.9, 113.6, 68.3, 55.2, 54.1, 51.4, 29.8, 29.5, 28.7, 22.6,
13.7; HR-MS(ESI) calc. for C20H27N3NaO5 (MþNa)þ: 412.1843,
found: 412.1865.
((S)-1-phenylethyl)-2,5-dihydro-1H-pyrrole-2-carboxamide
(1c).
Prepared from A1, B1, and (S)-1-phenylethanamine (R3NH2) ac-
cording to the general procedure and purified by flash column
chromatography on silica gel (MeOH/DCM ¼ 1:10) to obtain 1c (18%
4.1.13.7. (S)-1-((R)-2-((Formyl-hydroxy-amino)methyl)hexanoyl)-N-
yield). 1H NMR (500 MHz, CDCl3)
d 10.18 (bs, 1H), 7.60 (s, 1H), 7.25
(2-bromophenyl)-2,5-dihydro-1H-pyrrole-2-carboxamide
(1h).
(m, 5H), 5.94 (d, J ¼ 5 Hz, 1H), 5.81 (d, J ¼ 5 Hz, 1H), 5.29 (t, J ¼ 5 Hz,
1H), 5.02 (t, J ¼ 10 Hz, 1H), 4.75 (d, J ¼ 10 Hz, 1H), 4.28 (d, J ¼ 10 Hz,
1H), 3.84 (t, J ¼ 10 Hz, 1H), 3.35 (d, J ¼ 10 Hz, 1H), 3.13 (m, 1H), 1.55
(m, 1H), 1.44 (m, 5H), 1.30 (m, 4H), 0.86 (t, J ¼ 10 Hz, 3H); 13C NMR
Prepared from A1, B1, and 2-bromoaniline (R3NH2) according to the
general procedure and purified by flash column chromatography
on silica gel (MeOH/DCM ¼ 1:10) to obtain 1h (16% yield). 1H NMR
(500 MHz, CDCl3)
d
10.20 (bs, 1H), 8.77 (s, 1H), 8.23 (d, J ¼ 8 Hz, 1H),
(125 MHz, CDCl3)
d 172.5, 169.1, 158.1, 143.2, 128.3, 127.1, 126.9,
7.65 (s, 1H), 7.49 (d, J ¼ 8 Hz, 1H), 7.27 (d, J ¼ 7.5 Hz, 1H), 6.95 (t,
J ¼ 7.5 Hz, 1H), 6.06 (d, J ¼ 5 Hz, 1H), 6.01 (d, J ¼ 5 Hz, 1H), 5.60 (s,
1H), 4.83 (d, J ¼ 13.5 Hz, 1H), 4.43 (dd, J ¼ 10 Hz, 2.5 Hz, 1H), 3.92 (t,
J ¼ 10 Hz, 1H), 3.39 (dd, J ¼ 10 Hz, 2.5 Hz, 1H), 3.18 (m, 1H), 1.66 (m,
1H), 1.50 (m, 1H), 1.31 (m, 4H), 0.83 (t, J ¼ 7 Hz, 3H); 13C NMR
126.3, 125.9, 67.9, 53.8, 51.3, 48.9, 40.4, 29.8, 28.9, 22.5, 21.9, 13.6;
HR-MS(ESI) calc. for C21H29N3NaO4 (MþNa)þ: 410.2050, found:
410.2063.
4.1.13.3. (S)-1-((R)-2-((N-hydroxyformamido)methyl)hexanoyl)-N-
(125 MHz, CDCl3)
d 172.9, 168.4, 158.2, 135.5, 132.1, 128.0, 127.9,
((R)-1-phenylethyl)-2,5-dihydro-1H-pyrrole-2-carboxamide
(1d).
125.9, 125.4, 122.9, 114.0, 68.6, 53.8, 51.3, 40.5, 29.8, 28.9, 22.6, 13.7;
HR-MS (ESI) calc. for C19H24N3BrNaO4 (MþNa)þ: 460.0842, found:
460.0840.
Prepared from A1, B1, and (R)-1-phenylethanamine (R3NH2) ac-
cording to the general procedure and purified by flash column
chromatography on silica gel (MeOH/DCM ¼ 1:10) to obtain 1d
(17% yield). 1H NMR (500 MHz, CDCl3)
d 9.88 (bs, 1H), 7.58 (s, 1H),
4.1.13.8. (S)-1-((R)-2-((Formyl-hydroxy-amino)methyl)hexanoyl)-N-
7.21 (m, 5H), 5.89 (d, J ¼ 5 Hz, 1H), 5.81 (d, J ¼ 5 Hz, 1H), 5.32 (s, 1H),
4.99 (t, J ¼ 10 Hz, 1H), 4.67 (d, J ¼ 15 Hz, 1H), 4.24 (d, J ¼ 10 Hz, 1H),
3.83 (t, J ¼ 10 Hz, 1H), 3.32 (d, J ¼ 15 Hz, 1H), 3.05 (m, 1H), 1.49e1.19
(3-bromophenyl)-2,5-dihydro-1H-pyrrole-2-carboxamide
(1i).
Prepared from A1, B1, and 3-bromoaniline (R3NH2) according to the
general procedure and purified by flash column chromatography
on silica gel (MeOH/DCM ¼ 1:10) to obtain 1i (16% yield). 1H NMR
(m, 9H), 0.78 (t, J ¼ 10 Hz, 3H); 13C NMR (125 MHz, CDCl3)
d 169.0,
165.3, 154.2, 139.4, 124.8, 124.7, 124.5, 123.2, 122.6, 122.2, 64.0, 50.1,
47.5, 45.2, 36.8, 26.1, 25.1, 18.8, 18.1, 9.95; HR-MS(ESI) calc. for
(500 MHz, CDCl3)
d 9.25 (s, 1H), 7.71 (s, 1H), 7.59 (s, 1H), 7.28 (d,
J ¼ 9 Hz, 1H), 7.08 (d, J ¼ 8 Hz, 1H), 6.99 (t, J ¼ 8 Hz, 1H), 6.06 (d,
J ¼ 5 Hz, 1H), 5.90 (d, J ¼ 5 Hz, 1H), 5.44 (s, 1H), 4.79 (d, J ¼ 14 Hz,
1H), 4.45 (d, J ¼ 14 Hz,1H), 3.80 (t, J ¼ 13 Hz,1H), 3.45 (dd, J ¼ 10 Hz,
3 Hz, 1H), 3.24 (m, 1H), 1.70 (m, 1H), 1.68e1.25 (m, 5H), 0.90 (t,
C
21H29N3NaO4 (MþNa)þ: 410.2050, found: 410.2064.
4.1.13.4. (S)-1-((R)-2-((N-hydroxyformamido)methyl)hexanoyl)-N-
phenyl-2,5-dihydro-1H-pyrrole-2-carboxamide (1e). Prepared from
A1, B1, and aniline (R3NH2) according to the general procedure and
purified by flash column chromatography on silica gel (MeOH/
DCM ¼ 1:10) to obtain 1e (19% yield). 1H NMR (500 MHz, CDCl3)
J ¼ 7.5 Hz, 3H); 13C NMR (125 MHz, CDCl3)
d 169.1, 164.2, 153.6,
135.4, 126.0, 124.5, 123.0, 121.7, 118.5, 118.3, 113.9, 64.6, 50.6, 47.4,
36.8, 27.0, 25.0, 18.9, 10.0; HR-MS(ESI) calc. for C19H25N3BrO4
(MþH)þ: 438.1023, found: 438.1050.
d
10.02 (s, 1H), 9.54 (s, 1H), 8.50 (s, 1H), 7.40 (d, J ¼ 5 Hz, 2H), 7.03 (t,
J ¼ 5 Hz, 2H), 6.90 (t, J ¼ 5 Hz, 1H) 5.99 (d, J ¼ 5 Hz, 1H), 5.83 (d,
J ¼ 5 Hz, 1H), 5.48 (s, 1H), 4.49 (t, J ¼ 15 Hz, 1H), 4.37 (d, J ¼ 10 Hz,
1H), 4.17 (dd, J ¼ 10 Hz, 5 Hz,1H), 3.48 (t, J ¼ 10 Hz,1H), 3.10 (m,1H),
1.70 (m, 1H), 1.46 (m, 1H), 1.29 (m, 4H), 0.88 (t, J ¼ 5 Hz, 3H); 13C
4.1.13.9. (S)-1-((R)-2-((Formyl-hydroxy-amino)methyl)hexanoyl)-N-
(4-bromophenyl)-2,5-dihydro-1H-pyrrole-2-carboxamide
(1j).
Prepared from A1, B1, and 4-bromoaniline (R3NH2) according to the
general procedure and purified by flash column chromatography
on silica gel (MeOH/DCM ¼ 1:10) to obtain 1j (16% yield). 1H NMR
NMR (125 MHz, CDCl3)
d 173.7, 168.6, 163.4, 137.2, 128.4, 128.3,
125.4, 124.3, 119.7, 68.4, 54.8, 52.2, 42.4, 29.6, 29.3, 22.6, 13.7; HR-
(500 MHz, CDCl3) d 9.71 (bs, 1H), 9.35 (s, 1H), 7.57 (s, 1H), 7.26 (d,
MS(ESI) calc. for
382.1715.
C
19H25N3NaO4 (MþNa)þ: 382.1737, found:
J ¼ 5 Hz, 2H), 7.20 (d, J ¼ 5 Hz, 2H), 6.02 (s, 1H), 5.87 (s, 1H), 5.42 (s,
1H), 4.74 (d, J ¼ 12.5 Hz, 1H), 4.43 (d, J ¼ 13.5 Hz, 1H), 3.77 (t,

S. Yang et al. / European Journal of Medicinal Chemistry 86 (2014) 133e152
147
J ¼ 12 Hz, 1H), 3.45 (d, J ¼ 13 Hz, 1H), 3.21 (s, 1H), 1.67 (m, 1H),
1.49e1.42 (m, 5H), 0.91 (t, J ¼ 7 Hz, 3H); 13C NMR (125 MHz, CDCl3)
Prepared from A1, B1, and 5-methylthiazol-2-amine (R3NH2) ac-
cording to the general procedure and purified by flash column
chromatography on silica gel (MeOH/DCM ¼ 1:10) to obtain 1o (17%
d
172.8, 167.8, 157.5, 137.2, 131.5, 128.4, 125.4, 12.7, 116.3, 68.3, 54.4,
51.4, 40.6, 30.0, 28.8, 22.7, 13.8; HR-MS(ESI) calc. for
yield). 1H NMR (500 MHz, CDCl3)
d 10.48 (bs, 1H), 7.69 (s, 1H), 5.95
C
19H24N3BrNaO4 (MþNa)þ: 460.0842, found: 460.0855.
(s, 1H), 5.73 (s, 1H), 4.92 (s, 1H), 4.37 (d, J ¼ 14 Hz, 1H), 4.07 (m, 1H),
3.37 (d, J ¼ 13.5 Hz,1H), 3.17 (s, 1H), 2.37e2.28 (m, 4H), 1.69 (m, 1H),
1.54(m, 2H), 1.38 (m, 3H), 0.95 (s, 3H); 13C NMR (125 MHz, CDCl3)
4.1.13.10. (S)-1-((R)-2-((Formyl-hydroxy-amino)methyl)hexanoyl)-
N-(2-chlorophenyl)-2,5-dihydro-1H-pyrrole-2-carboxamide (1k).
d
173.8, 168.5, 158.7, 154.3, 128.6, 127.1, 125.0, 66.9, 53.2, 51.4, 40.2,
Prepared from A1, B1, and 2-chloroaniline (R3NH2) according to the
general procedure and purified by flash column chromatography
on silica gel (MeOH/DCM ¼ 1:10) to obtain 1k (18% yield). 1H NMR
29.6, 28.8, 22.7, 14.1; HR-MS (ESI) calc. for C17H24N4NaO4S
(MþNa)þ: 403.1410, found: 403.1409.
(500 MHz, CDCl3)
d 9.88 (bs, 1H), 9.29 (s, 1H), 7.61 (s, 1H), 7.38 (d,
4.1.13.15. (S)-1-((R)-2-(((formyl-hydroxyamino)methyl)hexanoyl)-N-
(5-fluoropyridin-2-yl)-2,5-dihydro-1H-pyrrole-2-carboxamide (1p).
Prepared from A1, B1, and 5-fluoropyridin-2-amine (R3NH2) ac-
cording to the general procedure and purified by flash column
chromatography on silica gel (MeOH/DCM ¼ 1:10) to obtain 1p
J ¼ 8 Hz, 2H), 7.20 (d, J ¼ 7.5 Hz, 2H), 6.06 (s,1H), 5.91 (s,1H), 5.46 (s,
1H), 4.77 (d, J ¼ 14 Hz, 1H), 4.44 (d, J ¼ 13.5 Hz, 1H), 3.79 (d,
J ¼ 12 Hz, 1H), 3.45 (d, J ¼ 12 Hz, 1H), 3.23 (m, 1H), 1.69 (m, 1H),
1.55e1.36 (m, 5H), 0.91 (t, J ¼ 7 Hz, 3H); 13C NMR (125 MHz, CDCl3)
(15% yield). 1H NMR (500 MHz, CDCl3)
d 10.50e9.75 (bs,1H), 9.66 (s,
d
172.8, 167.9, 157.6, 136.6, 128.7, 128.5, 128.3, 125.5, 120.4, 68.4,
54.4, 51.3, 40.5, 29.9, 28.8, 22.7, 13.8; HR-MS(ESI) calc. for
1H), 8.23 (m, 1H), 8.03 (s, 1H), 7.70 (s, 1H), 7.41 (m, 1H), 6.01 (m, 1H),
5.94 (m, 1H), 5.51 (m, 1H), 4.74 (m, 1H), 4.45 (m, 1H), 3.94 (m, 1H),
3.44 (m, 1H), 3.21 (m, 1H), 1.75 (m, 1H), 1.55 (m, 1H), 1.42e1.25 (m,
C
19H24N3ClNaO4 (MþNa)þ: 416.1384, found: 416.1374.
4H), 0.89 (t, J ¼ 7 Hz, 3H); 13C NMR (125 MHz, CDCl3)
d
172.9, 168.2,
4.1.13.11. (S)-1-((R)-2-((Formyl-hydroxy-amino)methyl)hexanoyl)-
N-(4-chlorophenyl)-2,5-dihydro-1H-pyrrole-2-carboxamide (1l).
Prepared from A1, B1, and 4-chloroaniline (R3NH2) according to the
general procedure and purified by flash column chromatography
on silica gel (MeOH/DCM ¼ 1:10) to obtain 1l (17% yield). 1H NMR
157.8, 155.0, 147.6, 135.2, 134.9, 128.1, 125.6, 115.1, 68.4, 54.0, 51.4,
40.7, 29.9, 28.9, 22.7, 13.7; HR-MS(ESI) calc. for C18H23FN4NaO4
(MþNa)þ: 401.1596, found: 401.1607.
(500 MHz, CDCl3)
d
8.91 (s, 1H), 8.27 (d, J ¼ 8 Hz, 1H), 7.66 (s, 1H),
4.1.13.16. (S)-1-((R)-2-(((Formyl-hydroxyamino)methyl)hexanoyl)-
N-5-fluoro-1-oxido-pyridin)-2-yl)-2,5-dihydro-1H-pyrrole-2-
carboxamide (1q). Prepared from A1, B1, and 5-fluoropyridin-2-
amine (R3NH2) according to the general procedure and purified
by flash column chromatography on silica gel (MeOH/DCM ¼ 1:10)
7.34 (m, 2H), 7.20 (m, 1H), 6.04 (s, 1H), 6.00 (s, 1H), 5.59 (s, 1H), 4.83
(d, J ¼ 14 Hz, 1H), 4.41 (d, J ¼ 13 Hz, 1H), 3.94 (q, J ¼ 12 Hz, 1H), 3.42
(d, J ¼ 13 Hz, 1H), 3.19 (m, 1H), 1.69 (m, 1H), 1.55 (m, 1H), 1.35 (m,
4H), 0.86 (t, J ¼ 7 Hz, 3H); 13C NMR (125 MHz, CDCl3)
d 173.1, 168.4,
158.2, 134.4, 129.0, 127.8, 127.4, 125.9, 124.8, 123.3, 122.2, 68.5, 53.9,
51.3, 40.5, 29.8, 28.9, 22.6, 13.7; HR-MS(ESI) calc. for
to obtain 1q (13% yield). 1H NMR (500 MHz, CDCl3)
d 10.58 (s, 1H),
8.47 (m, 1H), 8.17 (m, 1H), 7.65 (s, 1H), 7.15 (m, 1H), 6.08 (m, 1H),
6.00 (m, 1H), 5.67 (m, 1H), 4.85 (m, 1H), 4.44 (m, 1H), 3.94 (m, 1H),
3.40 (m, 1H), 3.18 (m, 1H), 1.75 (m, 1H), 1.53 (m, 1H), 1.42 (m, 4H),
C
19H24N3ClNaO4 (MþNa)þ: 416.1348, found: 416.1357.
4.1.13.12. (S)-N-(4-Fluorophenyl)-1-((R)-2-((N-hydroxyformamido)
methyl)hexanoyl)-2,5-dihydro-1H-pyrrole-2-carboxamide (1m).
0.90 (t, J ¼ 7 Hz, 3H); 13C NMR (125 MHz, CDCl3)
d 172.9, 168.5,
158.3, 156.0, 141.5, 128.7, 127.4, 127.0, 125.3, 114.9, 68.5, 53.8, 51.2,
40.4, 29.6, 28.9, 22.6, 13.8; HR-MS(ESI) calc. for C18H23FN4NaO5
(MþNa)þ: 417.1545, found: 417.1567.
Prepared from A1, B1, and 4-fluoroaniline (R3NH2) according to the
general procedure and purified by flash column chromatography
on silica gel (MeOH/DCM ¼ 1:10) to obtain 1m (14% yield). 1H NMR
(500 MHz, CDCl3)
d 9.89 (bs, 1H), 9.20 (s, 1H), 7.60 (s, 1H), 7.41 (t,
4.1.13.17. (S)-1-((R)-2-((N-hydroxyformamido)methyl)hexanoyl)-N-
(5-(trifluoromethyl)-1,3,4-thiadiazol-2-yl)-2,5-dihydro-1H-pyrrole-
2-carboxamide (1r). Prepared from A1, B1, and 5-(trifluoromethyl)-
1,3,4-thiadiazol-2-amine (R3NH2) according to the general proce-
dure and purified by flash column chromatography on silica gel
(MeOH/DCM ¼ 1:10) to obtain 1r (11% yield). 1H NMR (500 MHz,
J ¼ 5 Hz, 2H), 6.85 (t, J ¼ 8.5 Hz, 2H), 6.05 (s, 1H), 5.91 (s, 1H), 5.44 (s,
1H), 4.83 (d, J ¼ 14 Hz, 1H), 4.41 (d, J ¼ 13 Hz, 1H), 3.94 (q, J ¼ 12 Hz,
1H), 3.44 (d, J ¼ 13 Hz, 1H), 3.21 (m, 1H), 1.69 (m, 1H), 1.55 (m, 1H),
1.35 (m, 4H), 0.88 (t, J ¼ 7 Hz, 3H); 13C NMR (125 MHz, CDCl3)
d
172.8,167.8, 157.7,134.9,128.1, 125.7, 121.0,120.9,115.3,115.0, 68.4,
54.3, 51.3, 40.6, 29.9, 28.8, 22.6, 13.7; HR-MS(ESI) calc. for
19H24N3FNaO4 (MþNa)þ: 400.1643, found: 400.1653.
CDCl3) d 7.75 (bs, 1H), 6.09 (s, 1H), 5.89 (s, 1H), 5.56 (m, 1H), 4.72 (d,
C
J ¼ 12.8 Hz, 1H), 4.55 (d, J ¼ 12.8 Hz, 1H), 3.90 (m, 1H), 3.48 (m, 1H),
3.23 (s, 1H), 1.65e1.51(m, 2H), 1.33 (m, 4H), 0.90 (s, 3H); 13C NMR
4.1.13.13. (S)-1-((R)-2-((N-hydroxyformamido)methyl)hexanoyl)-N-
(pyridin-2-yl)-2,5-dihydro-1H-pyrrole-2-carboxamide (1n).
(125 MHz, CDCl3)
d 173.0, 168.8, 161.8, 158.1, 129.7, 123.8, 120.6,
118.4, 67.4, 54.1, 51.5, 40.5, 29.7, 28.8, 22.6, 13.8; HR-MS (ESI) calc.
Prepared from A1, B1, and pyridin-2-amine (R3NH2) according to the
general procedure and purified by flash column chromatography
on silica gel (MeOH/DCM ¼ 1:10) to obtain 1n (16% yield). 1H NMR
for C16H20F3N5NaO4S (MþNa)þ: 458.1080, found: 458.1086.
(500 MHz, CDCl3)
d
10.50e9.75 (bs, 1H), 9.48 (s, 1H), 8.19 (m, 2H),
4.1.13.18. N-hydroxy-N-((R)-2-((S)-4-methylene-2-(morpholine-4-
7.75 (s, 1H), 7.65 (t, J ¼ 8 Hz, 1H), 6.98 (m, 1H), 5.97 (s, 1H), 5.94 (s,
1H), 5.47 (s, 1H), 4.67 (d, J ¼ 14 Hz, 1H), 4.46 (d, J ¼ 14 Hz, 1H), 3.94
(t, J ¼ 13.7 Hz, 1H), 3.45 (d, J ¼ 11.7 Hz, 1H), 3.20 (m, 1H), 1.69 (m,
1H), 1.42 (m, 1H), 1.33 (m, 4H), 0.88(t, J ¼ 7 Hz, 3H); 13C NMR
carbonyl)pyrrolidine-1-carbonyl)hexyl)formamide
(1s).
Prepared from A1, B2, and morpholine (R3NH2) according to the
general procedure and purified by flash column chromatography
on silica gel (MeOH/DCM ¼ 1:10) to obtain 1s (19% yield). 1H NMR
(125 MHz, CDCl3)
d
172.8, 168.3, 157.6, 151.3, 147.4, 138.3, 127.9,
(500 MHz, CDCl3)
d
7.69 (s, 1H), 5.10e5.05 (m, 3H), 4.45 (t, J ¼ 14 Hz,
125.8, 119.6, 114.3, 68.4, 54.0, 51.4, 40.9, 29.9, 28.9, 22.7, 13.7; HR-
1H), 4.32 (t, J ¼ 14 Hz, 1H), 3.78 (m, 1H), 3.65 (m, 7H), 3.53 (m, 2H),
MS(ESI) calc. for
383.1692.
C
18H24N4NaO4 (MþNa)þ: 383.1690, found:
3.18 (m, 1H), 2.93 (m, 2H), 1.72 (m, 1H), 1.45e1.25 (m, 5H), 0.92 (t,
J ¼ 7 Hz, 3H); 13C NMR (125 MHz, CDCl3)
d 172.3, 169.5, 157.2, 142.5,
108.4, 66.7, 66.5, 56.2, 51.5, 51.2, 45.9, 42.3, 40.7, 35.1, 29.6, 28.9,
22.6, 13.8; HR-MS(ESI) calc. for C18H29N3NaO5 (MþNa)þ: 390.1999,
found: 390.2024.
4.1.13.14. (S)-1-((R)-2-((N-hydroxyformamido)methyl)hexanoyl)-N-
(5-methylthiazol-2-yl)-2,5-dihydro-1H-pyrrole-2-carboxamide (1o).

148
S. Yang et al. / European Journal of Medicinal Chemistry 86 (2014) 133e152
4.1.13.19. (S)-1-((R)-2-((N-hydroxyformamido)methyl)hexanoyl)-4-
methylene-N-phenylpyrrolidine-2-carboxamide (1t). Prepared from
A1, B2, and aniline (R3NH2) according to the general procedure and
purified by flash column chromatography on silica gel (MeOH/
DCM ¼ 1:10) to obtain 1t (22% yield). 1H NMR (500 MHz, CDCl3)
1H), 3.42 (d, J ¼ 12.5 Hz, 1H), 3.14 (m, 2H), 2.62 (d, J ¼ 15.0 Hz, 1H),
2.27 (s, 3H), 1.91 (m, 1H), 1.50e1.44 (m, 5H), 0.98 (s, 3H); 13C NMR
(125 MHz, CDCl3)
d 172.4, 168.2, 158.4, 157.2, 142.8, 126.9, 117.5,
108.5, 59.3, 51.4, 40.3, 29.0, 28.8, 22.6, 13.9, 11.4; HR-MS(ESI) calc.
for C18H26N4NaO4S (MþNa)þ: 417.1572, found: 417.1573.
d
9.19 (s, 1H), 7.76 (s, 1H), 7.48 (m, 2H), 7.24 (m, 2H), 7.04 (m, 1H),
5.12e5.08 (d, J ¼ 24 Hz, 2H), 4.92 (m, 1H), 4.51 (d, J ¼ 14 Hz, 1H),
4.22 (d, J ¼ 14 Hz, 1H), 3.91 (t, J ¼ 12 Hz, 1H), 3.46 (d, J ¼ 14 Hz, 1H),
3.18 (m, 1H), 2.97 (m, 1H), 2.84 (m, 1H), 1.66 (m, 1H), 1.52 (m, 1H),
4.1.13.24. N-((R)-2-(cyclopentylmethyl)-3-((S)-2-(morpholine-4-
carbonyl)-2,5-dihydro-1H-pyrrol-1-yl)-3-oxopropyl)-N-hydrox-
yformamide (1a0). Prepared from A2, B1, and morpholine (R3NH2)
according to the general procedure and purified by flash column
chromatography on silica gel (MeOH/DCM ¼ 1:10) to obtain 1a′
1.31 (m, 4H), 0.84 (s, 3H); 13C NMR (125 MHz, CDCl3)
d 173.6, 169.2,
157.7, 142.8, 138.0, 128.7, 123.9, 119.5, 108.5, 60.4, 51.6, 51.4, 40.8,
33.9, 30.0, 28.8, 22.7, 13.7; HR-MS (ESI) calc. for C20H27N3NaO4
(MþNa)þ: 396.1894, found: 396.1914.
(18% yield). 1H NMR (500 MHz, CDCl3)
d 9.70 (s, 1H), 8.43 (s, H), 6.06
(m, 1H), 5.73 (m, 1H), 5.62 (s, 1H), 4.42 (m, 2H), 4.13 (m, 1H), 3.86
(m, 1H), 3.75e3.65 (m, 5H), 3.62 (m, 2H), 3.38 (m, 1H), 3.15 (m, 1H),
1.78 (m, 4H), 1.61 (m, 5H), 1.26 (m, 2H); 13C NMR (125 MHz, CDCl3)
4.1.13.20. (S)-N-(4-fluorophenyl)-1-((R)-2-((N-hydroxyformamido)
methyl)hexanoyl)-4-methylenepyrrolidine-2-carboxamide
(1u).
d 172.8, 168.6, 163.0, 129.5, 123.3, 66.5, 66.3, 64.1, 54.3, 52.5, 45.9,
Prepared from A1, B2, and 4-fluoroaniline (R3NH2) according to the
general procedure and purified by flash column chromatography
on silica gel (MeOH/DCM ¼ 1:10) to obtain 1u (16% yield). 1H NMR
43.0, 41.5, 37.7, 35.9, 33.0, 32.5, 25.0; HR-MS(ESI) calc. for
C
19H29N3NaO5(MþNa)þ: 402.1999, found: 402.1999.
(500 MHz, CDCl3)
d
9.31 (s, 1H), 7.73 (s, 1H), 7.38 (m, 2H), 6.87 (t,
4.1.13.25. (S)-1-((R)-3-cyclopentyl-2-((N-hydroxyformamido)
methyl)propanoyl)-N-((S)-1-phenylethyl)-2,5-dihydro-1H-pyrrole-2-
carboxamide (1c0). Prepared from A2, B1, and (S)-1-
phenylethanamine (R3NH2) according to the general procedure
and purified by flash column chromatography on silica gel (MeOH/
DCM ¼ 1:10) to obtain 1c′ (16% yield). 1H NMR (500 MHz, CDCl3)
J ¼ 8 Hz, 2H), 5.10e5.06 (d, J ¼ 19 Hz, 2H), 4.88 (m, 1H), 4.50 (d,
J ¼ 14 Hz, 1H), 4.23 (d, J ¼ 14 Hz, 1H), 3.88 (t, J ¼ 12 Hz, 1H), 3.45 (d,
J ¼ 14 Hz, 1H), 3.17 (m, 1H), 2.93e2.81 (m, 2H), 1.66 (m, 1H), 1.51 (m,
1H), 1.30 (m, 4H), 0.88 (s, 3H); 13C NMR (125 MHz, CDCl3)
d 173.5,
169.2, 159.9, 157.9, 142.6, 134.1, 120.9, 115.2, 115.0, 108.5, 60.3, 51.7,
51.4, 40.8, 34.1, 29.9, 28.8, 22.6, 13.7; HR-MS (ESI) calc. for
d
10.06 (bs, 1H), 7.61 (s, 1H), 7.26 (m, 4H), 7.12 (d, J ¼ 5 Hz, 1H), 5.96
C
20H26N3NaO4 (MþNa)þ : 414.1800, found: 414.1799.
(d, J ¼ 5 Hz, 1H), 5.84 (d, J ¼ 5 Hz,1H), 5.29 (s, 1H), 5.03 (m, 1H), 4.75
(d, J ¼ 15 Hz, 1H), 4.33 (d, J ¼ 10 Hz, 1H), 3.83 (t, J ¼ 10 Hz, 1H), 3.42
(d, J ¼ 10 Hz, 1H), 3.15 (m, 1H), 1.80e1.76 (m, 4H), 1.65e1.50 (m, 5H),
4.1.13.21. (S)-N-(5-fluoropyridin-2-yl)-1-((R)-2-((N-hydrox-
yformamido)methyl)hexanoyl)-4-methylenepyrrolidine-2-
carboxamide (1v). Prepared from A1, B2, and 5-fluoropyridin-2-
amine (R3NH2) according to the general procedure and purified
by flash column chromatography on silica gel (MeOH/DCM ¼ 1:10)
1.26 (m, 3H), 1.13 (m, 2H); 13C NMR (125 MHz, CDCl3)
d 172.9, 169.1,
158.0, 143.2, 128.5, 127.1, 126.3, 126.0, 67.9, 53.9, 51.2, 48.9, 40.1,
37.4, 36.4, 33.0, 32.7, 24.9, 21.8; HR-MS (ESI) calc. for C23H31N3NaO4
(MþNa)þ: 436.2207, found: 436.2221.
to obtain 1v (14% yield). 1H NMR (500 MHz, CDCl3)
d 9.82 (s, 1H),
8.20 (m,1H), 8.07 (s,1H), 7.78 (s,1H), 7.41 (m,1H), 5.09 (m, 2H), 4.96
(m, 1H), 4.55 (d, J ¼ 13.8 Hz, 1H), 4.26 (d, J ¼ 13.8 Hz, 1H), 3.95 (t,
J ¼ 12 Hz,1H), 3.45 (d, J ¼ 12 Hz,1H), 3.20 (m, 1H), 2.88 (m, 2H), 1.67
(m, 1H), 1.51 (m, 1H), 1.32 (m, 4H), 0.88 (t, J ¼ 7 Hz, 3H); 13C NMR
4.1.13.26. (S)-1-((R)-3-cyclopentyl-2-((N-hydroxyformamido)
methyl)propanoyl)-N-phenyl-2,5-dihydro-1H-pyrrole-2-
carboxamide (1e0). Prepared from A2, B1, and aniline (R3NH2) ac-
cording to the general procedure and purified by flash column
chromatography on silica gel (MeOH/DCM ¼ 1:10) to obtain 1e′
(125 MHz, CDCl3)
d 173.4, 169.7, 157.2, 155.2, 147.6, 142.6, 135.0,
125.3, 115.0, 108.5, 60.2, 51.5, 40.8, 34.3, 29.8, 28.8, 22.6, 13.7; HR-
MS (ESI) calc. for C19H25FN4NaO4 (MþNa)þ: 415.1752, found:
415.1832.
(17% yield). 1H NMR (500 MHz, CDCl3)
d 10.00 (s, 1H), 9.52 (s, 1H),
8.50 (s, H), 7.32 (d, J ¼ 10 Hz, 2H), 7.04 (t, J ¼ 10 Hz, 2H), 6.91 (t,
J ¼ 10 Hz, 1H), 6.02 (s, 1H), 5.85 (s, 1H), 5.49 (s, 1H), 4.51 (m, 1H),
4.40 (d, J ¼ 15 Hz, 1H), 4.21 (dd, J ¼ 10 Hz, 5 Hz, 1H), 3.48 (t,
J ¼ 10 Hz, 1H), 3.15 (m, 1H), 1.78e1.76 (m, 4H), 1.61e1.45 (m, 5H),
4.1.13.22. 5-Fluoro-2-((S)-1-((R)-2-((N-hydroxyformamido)methyl)
hexanoyl)-4-methylenepyrrolidine-2-carboxamido)pyridine 1-oxide
(1w). Prepared from A1, B2, and 5-fluoropyridin-2-amine (R3NH2)
according to the general procedure and purified by flash column
chromatography on silica gel (MeOH/DCM ¼ 1:10) to obtain 1w
1.13 (m, 2H); 13C NMR (125 MHz, CDCl3)
d 173.8, 168.8, 163.4, 137.2,
128.5,128.3,125.4,124.4, 119.7, 68.5, 54.8, 52.4, 41.6, 38.1, 36.0, 32.8,
25.0; HR-MS(ESI) calc. for C21H27N3NaO4 (MþNa)þ: 408.1894,
found: 408.1897.
(12% yield). 1H NMR (500 MHz, CDCl3)
d 10.60 (s, 1H), 8.45 (m, 1H),
8.22 (s, 1H), 7.72 (s, 1H), 7.14 (m, 1H), 5.29 (m, 3H), 4.63 (d,
J ¼ 12.5 Hz, 1H), 4.23 (d, J ¼ 12.5 Hz, 1H), 3.95 (t, J ¼ 12.5 Hz, 1H),
3.42 (d, J ¼ 11.5 Hz, 1H), 3.16 (m, 1H), 2.99 (m, 1H), 2.86 (m, 1H), 1.68
(m, 1H), 1.50 (m, 1H), 1.32e1.30 (m, 4H), 0.84e0.88 (s, 3H); 13C NMR
4.1.13.27. (S)-1-((R)-3-cyclopentyl-2-((N-hydroxyformamido)
methyl)propanoyl)-N-(4-fluorophenyl)-2,5-dihydro-1H-pyrrole-2-
carboxamide (1m0). Prepared from A2, B1, and 4-fluoroaniline
(R3NH2) according to the general procedure and purified by flash
column chromatography on silica gel (MeOH/DCM ¼ 1:10) to
(125 MHz, CDCl3)
d 173.3, 170.2, 157.2, 155.2, 147.6, 142.6, 135.0,
127.6, 114.9, 108.5, 60.2, 51.4, 51.2, 40.6, 34.7, 29.7, 28.9, 22.6, 13.8;
HR-MS (ESI) calc. for C19H25FN4NaO5 (MþNa)þ: 431.1701, found:
431.1755.
obtain 1m′ (13% yield). 1H NMR (500 MHz, CDCl3)
d 9.87 (bs, 1H),
9.48 (s, 1H), 7.59 (s, 1H), 7.37 (m, 2H), 6.78 (m, 2H), 6.04 (m, 1H),
5.88 (m, 1H), 5.43 (s, 1H), 4.74 (d, J ¼ 14 Hz, 1H), 4.42 (d, J ¼ 15 Hz,
1H), 3.75 (t, J ¼ 12 Hz, 1H), 3.49 (dd, J ¼ 12 Hz, 2.5 Hz, 1H), 3.24 (m,
1H), 2.05e1.40 (m, 9H), 1.20 (m, 2H); 13C NMR (125 MHz, CDCl3)
4.1.13.23. (S)-1-((R)-2-((N-hydroxyformamido)methyl)hexanoyl)-4-
methylene-N-(5-methylthiazol-2-yl)pyrrolidine-2-carboxamide (1x).
Prepared from A1, B2, and 5-methylthiazol-2-amine (R3NH2) ac-
cording to the general procedure and purified by flash column
chromatography on silica gel (MeOH/DCM ¼ 1:10) to obtain 1x (19%
d
173.0, 167.8, 157.6, 134.0, 128.1, 125.6, 120.9, 115.2, 115.0, 68.4, 54.3,
51.3, 40.2, 37.3, 36.4, 33.1, 32.8, 25.0; HR-MS (ESI) calc. for
C
21H26FN3NaO4 (MþNa)þ: 426.1800, found: 426.1793.
yield). 1H NMR (500 MHz, CDCl3)
d
10.52 (s, 1H), 7.79 (m, 1H), 5.31
4.1.13.28. (S)-1-((R)-3-cyclopentyl-2-((N-hydroxyformamido)
methyl)propanoyl)-N-(5-methylthiazol-2-yl)-2,5-dihydro-1H-
(m, 2H), 4.96 (m, 1H), 4.77 (m, 1H), 4.26 (d, J ¼ 14.0 Hz, 1H), 4.11 (m,

S. Yang et al. / European Journal of Medicinal Chemistry 86 (2014) 133e152
149
pyrrole-2-carboxamide (1o0). Prepared from A2, B1, and 5-
methylthiazol-2-amine (R3NH2) according to the general proce-
dure and purified by flash column chromatography on silica gel
(MeOH/DCM ¼ 1:10) to obtain 1o′ (19% yield). 1H NMR (500 MHz,
d 173.0, 170.3, 156.6, 149.3, 147.0, 139.1, 128.8, 113.9, 60.6, 58.6, 52.4,
41.8, 37.1, 30.5, 29.6, 29.4, 28.7, 25.6, 23.9, 22.8, 19.8, 17.8, 13.7; HR-
MS (ESI) calc. for C23H34N4NaO4 (MþNa)þ: 453.2472, found:
453.2491.
CDCl3) d 10.50 (bs, 1H), 7.63 (s, 1H), 5.95 (m, 2H), 5.72 (m, 1H), 4.94
(m, 1H), 4.33 (m, 1H), 3.97 (m, 1H), 3.44 (m, 1H), 3.14 (m, 1H), 2.26
4.1.13.33. (2S,4S)-4-fluoro-1-((R)-2-((N-hydroxyformamido)methyl)
hexanoyl)-N-(5-methylthiazol-2-yl)pyrrolidine-2-carboxamide (2c).
Prepared from A1, B4, and 5-methylthiazol-2-amine (R3NH2) ac-
cording to the general procedure and purified by flash column
chromatography on silica gel (MeOH/DCM ¼ 1:10) to obtain 2c (17%
(s, 3H), 1.97 (m, 2H), 1.82 (m, 2H), 1.65e1.52 (m, 5H), 1.26 (m, 2H);
13C NMR (125 MHz, CDCl3)
d 171.5, 166.6, 159.0, 158.4, 133.8, 128.6,
127.0, 125.0, 66.6, 53.0, 51.1, 39.9, 37.6, 35.5, 33.3, 32.4, 29.5, 25.0,
11.2; HR-MS (ESI) calc. for C19H26N4NaO4 S (MþNa)þ: 429.1567,
found: 429.1582.
yield). 1H NMR (500 MHz, CDCl3)
d 10.58 (s,1H), 7.66 (s, 2H), 5.46 (d,
J ¼ 52.5 Hz,1H), 4.43 (m,1H), 4.05 (m,1H), 3.94 (dd, J ¼ 12.5 Hz,1H),
4.1.13.29. (S)-1-((R)-3-cyclopentyl-2-((N-hydroxyformamido)
methyl)propanoyl)-N-(5-fluoropyridin-2-yl)-2,5-dihydro-1H-pyr-
role-2-carboxamide (1p0). Prepared from A2, B1, and 5-
fluoropyridin-2-amine (R3NH2) according to the general proce-
dure and purified by flash column chromatography on silica gel
(MeOH/DCM ¼ 1:10) to obtain 1p′ (14% yield). 1H NMR (500 MHz,
3.48e3.40 (m, 2H), 3.11 (s, 1H), 2.46 (m, 2H), 2.25 (s, 3H), 1.59 (m,
2H), 1.43 (m, 4H), 0.98 (m, 3H); 13C NMR (125 MHz, CDCl3)
d 171.9,
167.5, 158.7, 133.5, 127.3, 93.3, 91.5, 58.2, 53.2, 51.4, 40.2, 29.3, 28.6,
22.6, 14.0, 11.4; HR-MS (ESI) calc. for C17H25FN4SNaO4 (MþNa)þ:
423.1473, found: 423.1457.
CDCl3)
d
10.22 (bs, 1H), 9.59 (s, 1H), 8.18 (s, H), 7.98 (s, 1H), 7.67 (s,
4.1.13.34. 5-Fluoro-2-((2S,4S)-4-fluoro-1-((R)-2-((N-hydrox-
yformamido)methyl)hexanoyl)pyrrolidine-2-carboxamido)pyridine
1-oxide (2d). Prepared from A1, B4, and 5-fluoropyridin-2-amine
(R3NH2) according to the general procedure and purified by flash
column chromatography on silica gel (MeOH/DCM ¼ 1:10) to
1H), 7.26 (s, 1H), 5.98 (s, 1H), 5.92 (s, 1H), 5.50 (s, 1H), 4.74 (d,
J ¼ 15 Hz,1H), 4.43 (d, J ¼ 15 Hz, 1H), 3.85 (m, 1H), 3.47 (m, 1H), 3.21
(m, 1H), 1.88e1.70 (m, 4H),1.65e1.50 (m, 5H),1.13 (m, 2H); 13C NMR
(125 MHz, CDCl3)
d 173.0, 168.0, 157.7, 154.9, 147.5, 134.8, 128.0,
125.5, 125.1, 124.9, 115.0, 68.2, 54.0, 51.3, 40.2, 37.4, 36.3, 33.0, 32.8,
29.5, 25.0; HR-MS(ESI) calc. for C20H25FN4NaO4 (MþNa)þ: 427.1752,
found: 427.1766.
obtain 2d (13% yield). 1H NMR (500 MHz, CDCl3)
d 10.53 (s, 1H), 8.47
(s, 1H), 8.18 (s, 1H), 7.71 (s, 1H), 7.12 (s, 1H), 5.38 (d, J ¼ 52.5 Hz, 1H),
4.99 (d, J ¼ 9.0 Hz, 1H), 4.19 (m, 1H), 3.95 (m, 2H), 3.42 (d, J ¼ 13 Hz,
1H), 3.14 (bs, 1H), 2.72 (m, 1H), 2.45 (m, 1H), 1.76 (bs, 1H), 1.56 (s,
4.1.13.30. 2-((S)-1-((R)-3-cyclopentyl-2-((N-hydroxyformamido)
methyl)propanoyl)-2,5-dihydro-1H-pyrrole-2-carboxamido)-5-
fluoropyridine 1-oxide (1q0). Prepared from A2, B1, and 5-
fluoropyridin-2-amine (R3NH2) according to the general proce-
dure and purified by flash column chromatography on silica gel
(MeOH/DCM ¼ 1:10) to obtain 1p′ (12% yield). 1H NMR (500 MHz,
1H), 1.35 (m, 4H), 0.90 (m, 3H); 13C NMR (125 MHz, CDCl3)
d 174.2,
169.4, 157.8, 156.0, 141.5, 127.3, 114.7, 93.3, 91.5, 60.0, 54.1, 51.3, 40.8,
34.9, 31.9, 29.6, 22.6, 13.8; HR-MS(ESI) calc. for C18H24F2N4NaO5
(MþNa)þ: 437.1607, found: 437.1635.
4.1.13.35. 5-Fluoro-2-((2S,4S)-1-((R)-2-((N-hydroxyformamido)
methyl)hexanoyl)-4-methylpyrrolidine-2-carboxamido)pyridine 1-
oxide (2e). Prepared from A1, B5, and 5-fluoropyridin-2-amine
(R3NH2) according to the general procedure and purified by flash
column chromatography on silica gel (MeOH/DCM ¼ 1:10) to
CDCl3) d 10.59 (s, 1H), 8.42 (m, 1H), 8.14 (s, 1H), 7.64 (s, 1H), 7.11 (m,
1H), 6.04 (s,1H), 5.95 (s,1H), 5.65 (s,1H), 4.82 (d, J ¼ 10 Hz,1H), 4.39
(d, J ¼ 10 Hz, 1H), 3.89 (m, 1H), 3.41 (d, J ¼ 10 Hz, 1H), 3.15 (m, 1H),
1.87 (m, 2H), 1.76 (m, 2H), 1.56 (m, 5H), 1.11 (m, 2H); 13C NMR
(125 MHz, CDCl3)
d
173.0, 168.4, 158.3, 155.8, 141.4, 128.5, 127.3,
obtain 2e (13% yield). 1H NMR (500 MHz, CDCl3)
d 10.46 (s, 1H), 8.50
127.0, 125.1, 114.9, 68.3, 53.7, 51.1, 39.9, 37.3, 35.9, 33.0, 32.5, 29.5,
24.9; HR-MS (ESI) calc. for C20H25FN4NaO5 (MþNa)þ: 443.1701,
found: 443.1700.
(m, 1H), 8.29 (s, 1H), 7.74 (s, 1H), 7.12 (m, 1H), 4.70 (t, J ¼ 8.0 Hz, 1H),
4.10 (t, J ¼ 8.0 Hz, 1H), 3.88 (m, J ¼ 13.0 Hz, 1H), 3.36 (d, J ¼ 13.1 Hz,
1H), 3.20 (m, 1H), 3.14 (m, 1H), 2.54 (m, 1H), 2.39 (m, 1H), 1.71 (m,
2H), 1.37 (m, 4H), 1.11 (d, J ¼ 13.8 Hz, 3H), 0.92 (t, J ¼ 6.6 Hz, 3H); 13
C
4.1.13.31. (2S,3aR,7aS)-1-((R)-2-((N-hydroxyformamido)methyl)hex-
anoyl)-N-(5-methylthiazol-2-yl)octahydro-1H-indole-2-carboxamide
(2a). Prepared from A1, B3, and 5-methylthiazol-2-amine (R3NH2)
according to the general procedure and purified by flash column
chromatography on silica gel (MeOH/DCM ¼ 1:10) to obtain 2a (21%
NMR (125 MHz, CDCl3) d 172.8, 171.0, 158.0, 141.6, 127.3, 127.0, 115.9,
115.0, 62.1, 54.7, 51.4, 40.5, 37.5, 33.8, 29.8, 28.7, 22.6, 16.6, 13.8; HR-
MS(ESI) calc. for
433.1863.
C
19H27FN4NaO5 (MþNa)þ: 433.1858, found:
yield). 1H NMR (400 MHz, CDCl3)
d
7.78 (bs, 1H), 6.92 (s, 1H), 4.59
4.1.13.36. (2S,3aR,7aS)-1-((R)-3-cyclopentyl-2-((N-hydrox-
yformamido)methyl)propanoyl)-N-(5-methylthiazol-2-yl)octahydro-
1H-indole-2-carboxamide (2f). Prepared from A2, B3, and 5-
methylthiazol-2-amine (R3NH2) according to the general proce-
dure and purified by flash column chromatography on silica gel
(MeOH/DCM ¼ 1:10) to obtain 2f (14% yield). 1H NMR (400 MHz,
(m, 1H), 3.97 (m, 1H), 3.73 (m, 1H), 3.33 (m, 1H), 3.06 (m, 1H), 2.34
(m, 1H), 2.28 (s, 3H), 2.08 (m, 2H), 1.62 (m, 5H), 1.25e1.18 (m, 9H),
0.82 (m, 3H); 13C NMR (125 MHz, CDCl3)
d 172.5, 170.3, 158.3, 157.8,
132.7, 127.0, 60.1, 58.1, 53.4, 41.7, 37.3, 30.6, 30.3, 29.6, 29.4, 29.3,
28.8, 25.6, 23.8, 22.8, 19.8, 14.1, 11.5; HR-MS (ESI) calc. for
C
21H32N4NaO4S (MþNa)þ: 459.2036, found: 459.2040.
CDCl3)
(m, 1H), 3.43 (m, 1H), 3.17 (m, 1H), 2.35 (s, 4H), 2.17 (m, 2H),
1.91e1.38 (m, 15H), 1.29 (m, 4H); 13C NMR (125 MHz, CDCl3)
172.8,
d 7.83 (bs, 1H), 6.99 (s, 1H), 4.63 (m, 1H), 4.06 (m, 1H), 3.75
4.1.13.32. (2S,3aR,7aS)-1-((R)-2-((N-hydroxyformamido)methyl)hex-
anoyl)-N-(5-methylpyridin-2-yl)octahydro-1H-indole-2-
carboxamide (2b). Prepared from A1, B3, and 5-methylpyridin-2-
amine (R3NH2) according to the general procedure and purified
by flash column chromatography on silica gel (MeOH/DCM ¼ 1:10)
d
170.1, 158.1, 157.8, 133.0, 127.0, 60.0, 58.2, 53.3, 41.0, 37.7, 37.3, 36.7,
33.2, 32.8, 30.2, 28.6, 25.6, 25.1, 24.9, 23.8, 19.8, 11.5; HR-MS (ESI)
calc. for C23H34N4NaO4S(MþNa)þ: 485.2193, found: 485.2171.
to obtain 2b (16% yield). 1H NMR (400 MHz, CDCl3)
d
9.82 (s, 1H),
4.1.13.37. (2S,3aR,7aS)-1-((R)-3-cyclopentyl-2-((N-hydrox-
yformamido)methyl)propanoyl)-N-(5-fluoropyridin-2-yl)octahydro-
1H-indole-2-carboxamide (2g). Prepared from A2, B3, and 5-
fluoropyridin-2-amine (R3NH2) according to the general proce-
dure and purified by flash column chromatography on silica gel
8.14 (d, J ¼ 8.0 Hz, 1H), 8.01 (s, 1H), 7.82 (s, 1H), 7.50 (d, J ¼ 8.0 Hz,
1H), 4.64 (m, 1H), 4.02 (m, 1H), 3.83 (m, 1H), 3.43 (m, 1H), 3.16 (m,
1H), 2.30 (m, 1H), 2.26 (s, 3H), 2.10 (m, 1H), 1.86 (m, 1H), 1.68 (m,
4H) 1.43e1.01 (m, 10H), 0.84 (m, 3H); 13C NMR (125 MHz, CDCl3)

150
S. Yang et al. / European Journal of Medicinal Chemistry 86 (2014) 133e152
(MeOH/DCM ¼ 1:10) to obtain 2g (13% yield). 1H NMR (400 MHz,
4.1.13.42. 2-((2S,4S)-1-((R)-3-cyclopentyl-2-((N-hydroxyformamido)
methyl)propanoyl)-4-methylpyrrolidine-2-carboxamido)-5-
fluoropyridine 1-oxide (2l). Prepared from A2, B5, and 5-
fluoropyridin-2-amine (R3NH2) according to the general proce-
dure and purified by flash column chromatography on silica gel
(MeOH/DCM ¼ 1:10) to obtain 2l (16% yield). 1H NMR (500 MHz,
CDCl3)
d 9.74 (s, 1H), 8.19e8.09 (m, 2H), 7.78 (s, 1H), 7.38 (m, 1H),
4.67 (m, 1H), 4.02 (bs, 1H), 3.79 (m, 1H), 3.51 (m, 1H), 3.21 (m, 1H),
2.44e2.28 (m, 2H), 2.03 (m, 1H), 1.74 (m, 8H), 1.47 (m, 7H), 1.07 (m,
2H); 13C NMR (125 MHz, CDCl3)
d 173.5, 169.6, 161.9, 147.7, 135.3,
135.1, 124.9, 114.8, 60.3, 59.0, 50.8, 41.1, 37.9, 37.0, 33.1, 32.7, 28.6,
28.5, 25.6, 25.1, 24.9, 23.9, 19.7; HR-MS (ESI) calc. for C24H33N4FO4
(MþNa)þ: 483.2378, found: 483.2344.
CDCl3) d 10.43 (s, 1H), 8.50 (m, 1H), 8.21 (s, 1H), 7.74 (s, 1H), 7.14 (m,
1H), 4.67 (t, J ¼ 8.0 Hz, 1H), 4.12 (m, J ¼ 8.0 Hz, 1H), 3.86 (m, 1H),
3.44 (m, J ¼ 13.7 Hz, 1H), 3.24 (m, 1H), 3.13 (m, 1H), 2.50 (m, 1H),
2.38 (bs, 1H), 1.90 (m, 3H), 1.70e1.54 (m, 7H), 1.10 (m, 5H); 13C NMR
4.1.13.38. (2S,3aR,7aS)-1-((R)-3-cyclopentyl-2-((N-hydrox-
yformamido)methyl)propanoyl)-N-(5-methylpyridin-2-yl)octahydro-
1H-indole-2-carboxamide (2h). Prepared from A2, B3, and 5-
methylpyridin-2-amine (R3NH2) according to the general proce-
dure and purified by flash column chromatography on silica gel
(MeOH/DCM ¼ 1:10) to obtain 2h (13% yield). 1H NMR (400 MHz,
(125 MHz, CDCl3) d 173.0, 170.9, 157.7, 155.9, 141.7, 127.4, 115.7, 114.9,
62.1, 54.7, 51.3, 40.2, 37.5, 37.4, 36.3, 33.8, 33.0, 32.8, 29.6, 25.1, 16.7;
HR-MS (ESI) calc. for C21H29FN4NaO5 (MþNa)þ: 459.2014, found:
459.2019.
4.2. Biology
CDCl3) d 9.65 (s, 1H), 8.08 (m, 2H), 7.80 (s, 1H), 7.49 (m, 1H), 4.66 (t,
J ¼ 8.4 Hz,1H), 4.03 (dd, J ¼ 6.0 Hz, 4.8 Hz,1H), 3.81 (m,1H), 3.51 (m,
1H), 3.21 (m, 1H), 2.48(m, 1H), 2.31 (m, 1H), 2.27 (s, 3H), 2.07 (m,
1H), 1.76 (m, 7H), 1.53 (m, 7H), 1.25 (m, 3H) 1.06 (m, 2H); 13C NMR
4.2.1. Antibiotic susceptibility tests
Whole-cell antimicrobial activity was determined by broth
microdilution using the procedure recommended by the National
Committee for Clinical Laboratory Standards (NCCLS; Document
M7-A4). All organisms were brought from The People's Hospital of
Jiangsu Province and consisted of ATCC strains and clinical isolates
of relevant pathogens, including methicillin-resistant S. aureus
(MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE),
enterococci, methicillin-susceptible S. aureus (MSSA), methicillin-
susceptible Staphylococcus haemolyticus (MSSE), penicillin-
resistant S. pneumonia (PRSP), Enterococcus faecalis, Moraxella
catarrhalis, and E. coli. The minimum inhibitory concentration
(MIC) was determined as the lowest concentration of compound
that inhibited visible growth after 24 h at 35 ꢁC.
(125 MHz, CDCl3)
d 172.7, 168.8, 156.5, 154.1, 146.3, 134.5, 124.2,
113.4, 63.3, 57.3, 52.7, 41.7, 36.7, 35.6, 32.1, 31.5, 30.9, 28.7, 28.3, 27.3,
24.5, 24.0, 23.5, 21.6, 18.7; HR-MS(ESI) calc. for C25H37N4O4S
(MþH)þ: 457.2809, found: 457.2807.
4.1.13.39. (2S,4S)-1-((R)-3-cyclopentyl-2-((N-hydroxyformamido)
methyl)propanoyl)-4-fluoro-N-(5-methylthiazol-2-yl)pyrrolidine-2-
carboxamide (2i). Prepared from A2, B4, and 5-methylthiazol-2-
amine (R3NH2) according to the general procedure and purified
by flash column chromatography on silica gel (MeOH/DCM ¼ 1:10)
to obtain 2i (19% yield). 1H NMR (500 MHz, CDCl3)
d 10.48 (s, 1H),
7.67 (s, 1H), 7.61 (s, 1H), 5.38e5.18 (m, 2H), 4.41 (m, 1H), 3.97 (m,
2H), 3.54 (m, 1H), 3.07 (m, 1H), 2.48 (m, 2H), 2.37 (m, 3H), 1.97 (m,
3H), 1.69 (m, 2H), 1.59 (m, 4H), 1.26 (m, 3H); 13C NMR (125 MHz,
4.2.2. Pharmacokinetics study
Briefly, outbred male SpragueeDawley rats (Shanghai Slaccas
Laboratory Animal Breeding Co., Ltd) were used for pharmacoki-
netic analysis of compounds 1o, 1q, 1o′, 1q′, 1x, and LBM415. The
compounds were formulated in PBS (pH 6.5, 20% DMSO) and filter
sterilized. Rats (weight, 274e375 g each) were administered 5 mg
of the chosen compound per kilogram of body weight in a volume
of 2 mL/kg by intravenous (i.v.) route, or 25 mg of the chosen
compound per kilogram of body weight in a volume of 10 mL/kg by
oral (p.o.) route. Blood samples were collected from anesthetized
rats via cardiac puncture 0.083, 0.25, 0.5, 1, 2, 4, 6, and 24 h after
dosing. Groups of four rats were used for each time point. The blood
was allowed to clot, and the serum samples collected and stored
immediately at ꢀ80 ꢁC. For analysis, standards were prepared by
spiking PDF inhibitor candidates in control serum and extracting
the samples with an equal volume of acetonitrile; the supernatant
was analyzed by UFLC (3.0 ꢃ 50 mm pro-C18 column from ACE on
HP1100 ChemStation from Agilent with UV detection at 240 nm).
The runs were performed with a linear gradient of A (10 mM
CDCl3)
d 172.3, 167.5, 158.9, 133.6, 127.2, 93.3, 91.5, 58.3, 53.3, 51.3,
40.2, 37.8, 35.5, 33.3, 32.4, 25.1, 25.0, 11.3; HR-MS(ESI) calc. for
C
19H27FN4NaO4S (MþNa)þ: 449.1629, found: 449.1622.
4.1.13.40. 2-((2S,4S)-1-((R)-3-cyclopentyl-2-((N-hydroxyformamido)
methyl)propanoyl)-4-fluoropyrrolidine-2-carboxamido)-5-
fluoropyridine 1-oxide (2j). Prepared from A2, B4, and 5-
fluoropyridin-2-amine (R3NH2) according to the general proce-
dure and purified by flash column chromatography on silica gel
(MeOH/DCM ¼ 1:10) to obtain 2j (15% yield). 1H NMR (500 MHz,
CDCl3)
d 10.57 (s, 1H), 8.50 (m, 1H), 8.21 (s, 1H), 7.74 (s, 1H), 7.14 (s,
1H), 5.40 (d, J ¼ 52.5 Hz, 1H), 5.00 (d, J ¼ 9.3 Hz, 1H), 4.21 (m, 1H),
3.93 (m, 2H), 3.50 (d, J ¼ 13.4 Hz, 1H), 3.19 (bs, 1H), 2.77 (t,
J ¼ 16.5 Hz 1H), 2.45 (m, 1H), 1.94e1.79 (m, 4H), 1.65 (m, 4H), 1.20
(m, 3H); 13C NMR (125 MHz, CDCl3)
d 174.3, 169.3, 158.0, 153.5,
141.6, 127.4, 114.8, 93.3, 91.5, 60.0, 54.1, 51.3, 40.4, 37.5, 36.4, 33.1,
32.7, 29.6, 25.0; HR-MS (ESI) calc. for C20H26F2N4NaO5 (MþNa)þ:
463.1763, found: 463.1759.
ammonium
formate
aqueous
solution)
and
B
(acetonitrile:methanol ¼ 7:3): t ¼ 0.1 min, 36% B; t ¼ 2 min, 95% B;
t ¼ 2.3 min, 95% B; t ¼ 2.4 min, 36% B; t ¼ 4 min, stop). The phar-
macokinetics parameters, including time to maximum concentra-
tion (Tmax), maximum concentration measured (Cmax), terminal
half-life (t1/2), and area under the curve (AUC) were calculated
using WinNorlin (Version 5.3, Pharsight). The oral bioavailability
was calculated as the ratio of the AUC for p.o. administration and
the AUC for i.v. administration.
4.1.13.41. (2S,4S)-1-((R)-3-cyclopentyl-2-((N-hydroxyformamido)
methyl)propanoyl)-4-methyl-N-(5-methylthiazol-2-yl)pyrrolidine-2-
carboxamide (2k). Prepared from A2, B5, and 5-methylthiazol-2-
amine (R3NH2) according to the general procedure and purified
by flash column chromatography on silica gel (MeOH/DCM ¼ 1:10)
to obtain 2k (22% yield). 1H NMR (500 MHz, CDCl3)
d 7.70 (m, 1H),
4.60 (m, 1H), 4.27 (m, 1H), 4.02 (m, 1H), 3.43 (m, 1H), 3.15 (m, 1H),
2.52 (m, 1H), 2.39 (m, 3H), 2.20 (m, 1H), 1.97 (m, 1H), 1.86 (m, 1H),
1.58 (m, 6H), 1.00 (m, 5H), 0.87 (m, 3H); 13C NMR (125 MHz, CDCl3)
4.2.3. hERG inhibition
d
173.3, 170.7, 157.6, 133.9, 125.3, 113.0, 59.3, 53.6, 52.8, 36.4, 31.8,
All compounds to be tested were dissolved in dimethyl sulph-
oxide at a concentration of 100 mM and then diluted to produce
stock concentrations. Fresh solutions were made each
30.9, 28.6, 24.1, 21.6, 15.6, 13.1, 10.5; HR-MS(ESI) calc. for
C
20H30N4NaO4S (MþNa)þ: 445.1880, found: 445.1873.

S. Yang et al. / European Journal of Medicinal Chemistry 86 (2014) 133e152
151
experimental day, and the final experimental concentrations for
testing were 1 M and 10 M. An Axopatch 200A (Axon In-
struments, CA) amplifier was used, controlled by a PC connected via
a Digidata 1322A A/D converter (Axon Instruments, CA), which was
also used for data acquisition and analysis. Fractional block of IhERG
was calculated using the following equation:
(containing 200 ng/mL tolbutamide plus 20 ng/mL buspirone) to
precipitate the plasma proteins. The sample collection plate was
shaken at 800 rpm for 5 min to mix the samples, and then centri-
m
m
fuged at 3220 RCF for 20 min. An aliquot of supernatant (100
was transferred from each well and mixed with 200 L ultra pure
water before being subjected to LC-MS/MS analysis (30 ꢃ 2.00 mm
Phenomenex Luna 5 m C18; UV detection at 240 nm). The runs
mL)
m
m
h
ꢀ
.
ꢁi
were performed with a linear gradient of A (0.1% FA in water) and B
(0.1% FA in acetonitrile): t ¼ 0.01 min, 10% B; t ¼ 0.8 min, 95% B;
t ¼ 1.0 min, 95% B; t ¼ 1.01 min, 10% B; t ¼ 1.2 min, stop). The
concentration was calculated using the peak area ratio of analyte
and internal standard. The percent of unbound, bound and recovery
were calculated using the following equations:
Fractional block % ¼ 1 ꢀ IhERGꢀDrug IhERGꢀControl
%
Where IhERG-control is the whole-cell peak tail current of hERG under
the conditions listed, and IhERG-drug is the peak tail current of that
cell under the same conditions after superfusion of the drug. Tail
current was used to quantify the current mediated by the mutant
channels as well as wild-type.
% Unbound ¼ 100*FC=TC;
4.2.4. Protection from infection in mouse septicemia model (MRSA)
A standard peritonitis model (Shanghai Slaccas Laboratory An-
imal Breeding Co., Ltd) of infection was used to evaluate the efficacy
of 1q. Briefly, outbred mice (KM, weight, 20e25 g each) were
% Bound ¼ 100 ꢀ % Unbound
% Recovery ¼ 100*ðFC þ TCÞ=T0
inoculated intraperitoneally with
a 90-to-100%-lethal dose
Where TC is the total compound concentration as determined by
the calculated concentration on the plasma side of the membrane,
FC is the free compound concentration as determined by the
calculated concentration on the buffer side of the membrane, And
T0 is the total compound concentration as determined before
dialysis.
(5 ꢃ 107 CFU/mL) of MRSA08-12 in 0.5 mL of brain heart infusion
broth containing 5% mucin. Compound 1q was dissolved in 20%
DMSO and administered at doses of 1.25, 2.5, 5, 10, and 20 mg/kg in
a dosing volume of 0.1 mL via i.v. injection. LBM415 was used as a
control antibiotic, and was dosed with the same formulation as the
drug treated group. Groups of 10 mice were used for each dosage.
Mice were monitored daily for 14 days and cumulative mortality
used to determine the 50% effective dose (ED50), which was
calculated using Bliss's method.
4.2.6. Acute toxicity study in mice
Nine male and ten female outbred KM mice in SPF grade
(weight, 20e25 g each, bought from Shanghai Slaccas Laboratory
Animal Breeding Co., Ltd) were used for this study. Compound 1q
was formulated in PBS (pH 6.5, 20% DMSO) and given in a single-
dose at 100 and 250 mg/kg, respectively, via intravenous injection
of the tail vein over approximately 60s. The animals were moni-
tored for 14 days and their behavior change, weight, coat color, anal
temperature, daily food intake, daily water intake, and daily weight
of urine and stool checked every day.
Similar procedures were employed to evaluate the in vivo effi-
cacy of 1x. Briefly, outbred mice were inoculated intraperitoneally
with a 90-to-100%-lethal dose (5 ꢃ 106 CFU/mL) of MRSA (ATCC
33591) in 0.5 mL of brain heart infusion broth containing 5% mucin
to establish the mouse septicemia model, and 1x was dissolved in
20% DMSO and administered at doses of 5, 10, and 20 mg/kg in a
dosing volume of 0.1 mL via the i.g. route after infection. Linezolid
was included as a control antibiotic, and was dosed with the same
formulation as the drug treated group. Groups of six mice were
used for each dosage. Mice were monitored daily for 14 days and
cumulative mortality used to determine the 50% effective dose
(ED50).
Acknowledgments
This work was supported by the NSF of China (21125209 and
21332003), the MOST of China (2011CB808600), STCSM
(12JC1403800) and State Key Laboratory of Drug Research
(SIMM1403KF-09). We thank GlaxoSmithKline for their kind sup-
port to test the antibacterial activity shown in Tables 3 and 4 of this
manuscript.
4.2.5. Plasma protein binding test of 1q, 1x, and warfarin
Pooled plasma (CD-1 Mouse Plasma purchased form Bio-
reclamation, frozen at <ꢀ70 ꢁC) was thawed in a water bath at 37 ꢁC
immediately prior to the experiment and centrifuged at 3220 RCF
for 5 min to remove the clots. A 200
by diluting the appropriate volume of stock solution with 50%
CH3CN/H2O. The 2 M final solution was made by diluting 8 L of
the working solution (200 M) with 792 L of blank plasma. The
dialysis instrument was assembled following the manufacturer's
instructions. Each cell of the dialysis plate was loaded with 150
of plasma solution (in triplicate) and dialyzed against an equal
volume of dialysis buffer (100 mM phosphate buffered saline, pH
7.4). Before dialysis, 50 mL aliquots of spiked plasma (in triplicate)
mM working solution was made
Appendix A. Supplementary data
m
m
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.07.106.
m
m
mL
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