4-Iminooxazolidin-2-one as a Bioisostere of the Cyanohydrin Moiety: Inhibitors of Enterovirus 71 3C Protease

Article abstract of DOI:10.1021/acs.jmedchem.8b01335

A recently reported potent inhibitor of enterovirus 71 3C protease, (R)-1, was found to have stability and potential toxicity issues due to the presence of a cyanohydrin moiety. Modifying the labile cyanohydrin moiety, by serendipity, led to the discovery of 4-iminooxazolidin-2-one-based inhibitors 4e and 4g with potent inhibitory activity and significantly improved stability. In vivo pharmacokinetic studies of 4e also demonstrated high plasma exposure and moderate half-life. These compounds have shown potential of becoming anti-EV71 drug candidates.

Full text of DOI:10.1021/acs.jmedchem.8b01335

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Brief Article
4-Iminooxazolidin-2-one as a bioisostere of the cyanohydrin
moiety: inhibitors of enterovirus 71 3C protease
Yuying Ma, Chengyou Shang, Peng Yang, Linfeng Li,
Yangyang Zhai, Zheng Yin, Binghe Wang, and Luqing Shang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01335 • Publication Date (Web): 26 Oct 2018
Downloaded from http://pubs.acs.org on October 26, 2018
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Journal of Medicinal Chemistry
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4-Iminooxazolidin-2-one as a bioisostere of the cyanohydrin
moiety: inhibitors of enterovirus 71 3C protease
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†
†
†
‡
§
†
Yuying Ma, Chengyou Shang, Peng Yang, Linfeng Li, Yangyang Zhai, Zheng Yin, Binghe
*,||
*,†
Wang, and Luqing Shang
†College of Pharmacy, State Key Laboratory of Medicinal Chemical Biology and Tianjin Key Laboratory of Molecular
Drug Research, Nankai University, Tianjin, 300350, China
‡Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, United States
§Medical College, Henan Polytechnic University, Jiaozuo, 454000, China
||Department of Chemistry, Georgia State University, Atlanta, GA 30303, United States
Supporting Information Placeholder
ABSTRACT: A recently reported potent inhibitor of enterovirus 71 3C protease, (R)-1, was found to have stability and
potential toxicity issues due to the presence of a cyanohydrin moiety. Modifying the labile cyanohydrin moiety, by
serendipity, led to the discovery of 4-iminooxazolidin-2-one-based inhibitors 4e and 4g with potent inhibitory activity and
significantly improved stability. In vivo pharmacokinetic studies of 4e also demonstrated high plasma exposure and
moderate half-life. These compounds have shown potential of becoming anti-EV71 drug candidates.
INTRODUCTION
group has long been committed to the development of
pro
3
C
inhibitors with improved potency and drug-like
Hand, foot and mouth disease (HFMD), primarily caused
by coxsackievirus A16 (CVA16) and enterovirus 71 (EV71),
is a highly contagious infection that usually causes mild
symptoms such as fever and malaise. However, EV71-
infected cases tend to be more severe than the CVA16-
infected cases, and could have neurological or
9
properties. In 2015, (R)-1 was reported as a novel
inhibitor, consisting of a fine-tuned peptidyl scaffold and a
1
0
1
cyanohydrin
warhead.
Despite
its
significant
improvement in both potency and selectivity for cysteine
pro
pro
protease (EV71 3C , SARS-CoV M ) over serine protease
(human neutrophil elastase, human trypsin etc.), the
presence of a cyanohydrin group gives rise to stability and
2
cardiopulmonary complications. Although two inactivated
EV71 vaccines have been approved in China for HFMD
prevention, the development of antiviral drugs is still of
great urgency since no specific clinical treatment is
11
potential toxicity issues.
In this work, we undertook an effort to optimize (R)-1
for improved stability and pharmacokinetic properties.
Very excitingly, after several rounds of modification, we
identified a 4-iminooxazolidin-2-one moiety that serves as
an excellent replacement of the labile cyanohydrin group,
allowing for maintaining comparable potency as (R)-1.
Thus, 4-iminooxazolidin-2-one does appear to serve as a
nonclassical bioisostere of protected cyanohydrin. Such a
discovery solved one critical problem with the original
structural scaffold of this class of otherwise very
promising anti-EV71 compounds. These findings also have
opened doors for further studies to examine whether the
3
available.
The EV71 genome encodes a large polyprotein precursor
that is subsequently processed into four structural
proteins (VP1-VP4) and seven non-structural proteins
4
pro
(
2A-2C and 3A-3D). Viral 3C protein (3C ) is a cysteine
protease with chymotrypsin-like specificity, and is critical
for most of the cleavage events in polyprotein processing.
pro
In addition to acting on virus itself, 3C can hamper host
cell function in different ways such as interfering with host
gene expression or disturbing the innate immune system.
Therefore, the importance of 3C in the viral life cycle
5
pro
4
-iminooxazolidin-2-one moiety could serve as a generally
makes it an attractive target for anti-EV71 drug discovery.
useful alternative to acylated cyanohydrin, which offers
diverse structural features in a small space, but is
problematic in drug design due to stability issues. Such a
concept is similar to using hydroxyisooxazole as
nonclassical bioisosteres of a carboxyl group and may find
The approach of structure-based design has generated
quite a few promising inhibitors of EV71 3C . Rupintrivir,
pro
originally developed as an inhibitor of human rhinovirus
6
(
HRV) protease, can effectively inhibit EV71 replication
and is deemed an archetype in the design of
peptidomimetic inhibitor of EV71 3C
crystal structure of EV71 3C -rupintrivir complex, our
12
additional applications.
pro 7
. Based on the
pro
8
RESULTS AND DISCUSSION
1
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Page 2 of 12
a
R

CN
R1
As previously reported, (R)-1 proved to be one of the
H
O
N
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
most potent EV71 inhibitors currently known (Fig. 1A).
Unfortunately, in vitro stability studies demonstrated that
(R)-1 was rapidly degraded in plasma and liver microsome
as shown in Fig. 1B. The half-life (t1/2) of (R)-1 in human
and mouse plasma was merely 11 min and 7 min,
respectively. Its metabolic rate in human and mouse liver
microsome was even faster (t1/2 = 8 min and 6 min,
respectively).
O
O
O
H
O
R =
N
2
a-c
N
N
H
O
R
 CN
R

CN
b
F
H
O
N
R2
OH
2a (R) R1=Me
(R) R1=i-Pr
3h (S) R2=n-Bu
3i (R) R2=i-Pr
1
O
2b
2c
3a
3a-m
(R) R1=c-Pr
(R) R2=Me
(S) R2=i-Pr
3j
3k
3l
(R) R2=c-Pr
3b (S) R2=Me
(S) R2=c-Pr
(R) R2=Et
3m
R
CN
3c
(R)
R
2=Ph

c
R3
3d (S) R2=Et
3n (R)
3o (R) R3=Me R4=Et
3p
R
3=R4=Me
O
N
(R) R2=n-Pr
R4
3e
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3f (S) R2=n-Pr
(R) R3/R4=4-Morph
O
(R) R2=n-Bu
3g
3
n-p
a
Reagents and conditions: (a) Alkyl chloride, TEA, DCM, 75%; (b) CDI,
amine, DCM, 60%; (c) Carbamoyl chloride, DMAP, DCM, 70%.
Table 1. Enzyme inhibitory activity, antiviral activity
and cytotoxicity of 2a-c, 3a-p as inhibitors of EV71
pro
3
C
Figure 1. A) Structure of (R)-1; B) The stability of (R)-1 in
plasma and microsome. Vertical bars represent the standard
deviation of each data point (n=3).
H
O
N
O
H
O
N
R =
N
Such instability can be attributed to either chemical or
metabolic reasons. Physiological pH was shown to enable
the reverse reaction of cyanohydrin formation, resulting in
the slow release of aldehyde and cyanide (Figure S1), both
of which are poisonous and not well-tolerated in drug
N
H
O
F
NO.
Ki
(µM)
EC50
CC50
a
a
a
(µM)
(µM)
>100
13
design. This process was further facilitated in plasma or
R
CN
OH
0.004 ± 0.001
0.088 ± 0.006
microsome (Figure S2, S3) probably owing to specific
(R)-1
enzymes. In the meantime, a hydroxyl group is susceptible
R
CN
to a series of hepatic metabolic reactions.14 On the other
0.064 ± 0.005
0.067 ± 0.008
2.61 ± 0.12
4.93 ± 0.23
0.62 ± 0.05
1.00 ± 0.09
0.75 ± 0.34
3.58 ± 0.43
0.16 ± 0.06
2.21 ± 0.05
>100
>100
>100
>100
>100
>100
>100
>100
>100
2
a
O
side, our previous research suggests that the hydroxyl
group could tolerate chemical modification to some extent
whereas the cyano group should be kept intact to retain
O
R
CN
0
.24 ± 0.047
2b
O
O
O
O
1
0
O
the binding affinity. Hence, the hydroxyl group was first
derivatized into an ester or a carbamate group in hope of
improving chemical or metabolic stability as well as
lowering the risk of toxic species (Table 1).
R
CN
0
.56 ± 0.15
2
c
O
R
R
CN
0.071 ± 0.017
H
3a
3b
3c
N
The preparation of (R)-1 was according to the
1
0
O
literature. The synthetic route to different derivatives of
R)-1 is outlined in Scheme 1. The esterified derivatives
a-c were generally obtained by reacting (R)-1 with acyl
CN
0
0
.046 ± 0.008
.051 ± 0.011
(
2
H
N
O
chloride under basic conditions. The conditions for
preparing carbamate derivatives varied with substitutions
on the nitrogen. To synthesize N-monosubstituted
R
R
CN
H
O
N
O
CN
carbamates 3a-m,
1
was treated with N, N’-
0.19 ± 0.037
H
3
d
O
N
carbonyldiimidazole (CDI) to form an active intermediate,
which then was reacted with different amines. N-
Disubstituted carbamates 3n-p were synthesized by
reacting (R)-1 with corresponding carbamoyl chlorides in
presence of 4-dimethylaminopyridine (DMAP).
O
R
CN
0
.085 ± 0.017
H
3
e
O
O
N
O
R
CN
0.183 ± 0.015
H
Scheme 1. Synthetic Scheme of 2a-c, 3a-pa
3f
N
O
R
CN
0
4
.45 ± 0.007
.78 ± 0.036
0.69 ± 0.08
8.46 ± 1.52
0.65 ± 0.07
>100
>100
>100
H
3
g
O
N
O
R
CN
H
3
h
O
R
N
O
O
CN
0
.036 ± 0.004
H
3
i
N
O
2
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Journal of Medicinal Chemistry
R
R
R
CN
0
.093 ± 0.011
1.73 ± 0.21
>100
>100
>100
>100
disubstituted carbamate). As shown in Figure 2A, 3i (t1/2,
H
3
j
O
O
O
N
1
2
3
4
5
6
7
8
9
mouse = 31 min) was more stable than 3e (t1/2, mouse = 12 min)
and the same trend was observed in human plasma
(Figure S5). Interestingly, N-disubstituted carbamate
inhibitors 3o and 3p showed extremely high plasma
stability. Plasma metabolite characterization was
performed for 3i using a tandem MS method to gain a
further understanding of the substituent effect. The peak
shift in LC indicated 3i (RT = 6.41 min) was partially
converted to another structure (RT = 5.41 min) as shown
in Figure 2B. To our surprise, these two structures had an
identical m/z value of 556 that was assigned to their
molecular ions according to the analysis of primary and
secondary mass spectra (Figure S7). The same conversion
was observed in PBS buffer (pH = 7.4) for 3e and 3i, but
the conversion of 3e was remarkably more rapid than 3i,
which coincided with the difference in their plasma half-
life. In addition, 3o and 3p did not undergo such
conversion in PBS buffer (pH = 7.4) (Figure S6). These
findings suggest that the conversion of carbamate was
plasma-independent and likely an intramolecular reaction
involving the carbamate nitrogen. After an extensive
O
CN
1
6
.28 ± 0.14
5.11 ± 0.62
12.83 ± 0.72
13.18 ± 2.35
H
3k
N
O
CN
.21 ± 0.42
H
3l
N
O
R
CN
N.D.b
H
3m
O
N
O
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
R
CN
N.D.b
70.40 ± 8.35
>100
>100
>100
>100
3n
N
O
R
CN
N.D.b
N.D.b
3o
O
O
N
O
R
CN
O
>100
3p
N
O
a
Each data presents the average results from three independent
experiments, and error bars represent SEMs (n=3). Not Detected.
b
Considering the relatively confined space at the S1’
pocket of 3C (Figure 3A), esterification was preliminarily
limited to several small groups, i.e., methyl, isopropyl and
cyclopropyl group. Compared with cyanohydrin (R)-1, 2a
1
5
pro
literature survey,
a
prototropic tautomerization
mechanism was proposed. Specifically, the nitrogen of N-
monosubstituted carbamate could nucleophilically attack
the well-oriented nitrile under basic condition thus
undergoing a cyclization. To validate this assumption, a
cyclized carbamate derivative 4e was chemically
synthesized, and it was confirmed by secondary mass
spectrometry that the synthesized 4e and the product
auto-converted from 3e were exactly the same (Figure S8).
This mechanism not only explains the identical m/z found
in original peak and product peak, but also sheds light on
the outstanding stability of N-disubstituted carbamate
derivatives in that at least one hydrogen was required for
the proton transfer.
i
(K = 0.064 ± 0.005 µM, EC50 = 0.067 ± 0.008 µM) showed
pro
slightly weaker inhibition against 3C . However, both of
them exhibited similar antiviral activity, probably because
the acetylation of hydroxyl group improved cell
permeability of 2a. Additionally, bulkier groups (2b and 2c)
led to reduced inhibition effect, which was in agreement
with the narrow S1’ pocket. The stability of 2a in plasma
and microsome was examined thereafter (Figure S4).
Compared with cyanohydrin (R)-1, the plasma stability of
2a was improved by over 5-fold in plasma different
sources (t1/2, human = 50 min and t1/2, mouse = 42 min).
The metabolic half-life of 2a in human liver microsome
was also extended to 64 min, but the improvement of its
metabolic stability in mouse liver microsome was
negligible. The insufficient stability in mouse microsome
has limited study on this class of compounds in the mouse
model. Further, the ester group is prone to hydrolysis and
could contribute to stability issues in vivo.
With the hope of further improving hydrolytic stability, a
series of N-monosubstituted and N-disubstituted
carbamate derivatives of 1 were synthesized and tested.
From the perspective of steric effect, long-chain alkyl
groups (3h) or bulky aromatic groups (3m) were not
easily accommodated by the S1’ pocket, similar with the
ester derivatives. The impact of α-carbon stereochemistry
on binding was trivial unless the substituent was oversized
(e.g., 3g vs 3h). In addition, there emerged almost total loss
of activity when the carbamate nitrogen was disubstituted
(e.g., 3n vs 3a). At first glance, the poor activity of 3n
seems aberrant since its structural analogue 2b (K
0.047 µM) still possesses fair potency. This is possibly
because, compared with 2b, the planar carbamate scaffold
of 3n restricts the adaptation of two methyl groups thus
leading to substantial steric clash with enzyme pocket.
Figure 2. A) The stability of represent compounds in mouse
plasma. B) LC/MS profile showing the disappearance of 3i
i
= 0.24
±
(
Retention time = 6.41min) and the appearance of product
peak (Retention time = 5.41min) after 60 min of incubation
with mouse plasma.
The plasma stability of this class was studied by
examining representatives 3e (best cellular activity), 3i
best enzyme inhibitory activity), 3o, and 3p (N-
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Scheme 2. Synthetic Scheme of 4a-j
(
3

Journal of Medicinal Chemistry
Page 4 of 12
O
NH
CN
O
NH
NH
4
b
0.21 ± 0.04
0.22 ± 0.05
3.53 ± 0.24
0.083 ± 0.01
2.60 ± 0.81
0.033± 0.008
0.061 ± 0.06
1.71 ± 0.11
2.54 ± 0.32
1.03 ± 0.21
4.37 ± 0.60
17.16 ± 0.80
0.21 ± 0.05
12.67 ± 1.71
0.10 ± 0.02
0.51 ± 0.08
8.81 ± 1.42
12.73 ± 2.83
>100
>100
>100
>100
>100
>100
>100
>100
>100
R
R
R
R
R
R
R
R
R
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
N
O
O
O
NH
O
O
O
O
O
O
O
O
O
O
MeOH
O
NH
NH
N
NH
N
NH
N
R'
PBS buffer(pH=7.4)
O
O
F
NH
O
O
F
NH
R'
4c
O
O
N
3
a-l
4a-j
O
NH
4d
4e
4f
The intriguing auto-conversion of carbamate derivatives
N
encouraged us to study their completely cyclized forms. A
series of cyclic carbamate compounds 4 containing the
same substituents corresponded to 3 were chemically
synthesized and tested (Scheme 2 and Table 2). The SAR of
4 shares a similar trend with that of 3, i.e. small or
branched groups are favored at the S1’ pocket. The
isopropyl substituted 4g exhibited the most potent
inhibitory activity (K
.02 µM) among three classes of compounds discussed in
this work. A flexible-receptor docking simulation yielded
the best binding pose of 4g that bears some resemblance
of a cyanohydrin inhibitor complexed with 3Cpro (Figure
O
NH
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
NH
N
O
i
= 0.033 ± 0.008 µM, EC50 = 0.10 ±
NH
0
4g
4h
4i
N
O
NH
3
A). In particular, the atoms of imine and bridging oxygen
in 4-iminooxazolidin-2-one moiety has a very similar
spatial arrangement with corresponding atoms in
N
O
NH
cyanohydrin moiety.
The carbonyl oxygen of 4-
N
iminooxazolidin-2-one moiety forms a strong hydrogen
bond (2.2 Å) with His40, which is also an important
interaction found in the crystal structure. Based on these
features, 4-iminooxazolidin-2-one can be regarded as a
nonclassical bioisostere of the cyanohydrin structure.
Furthermore, the side chains of Phe25 and Ala144 provide
the isopropyl group with a favorable hydrophobic
environment (Fig. 3B). Compared with 3, a noteworthy
detail was that the superiority of (R)-4 over (S)-4 became
more obvious. For example, 4c (K
e (K = 0.083 ± 0.01 µM) were substantially more potent
than their counterparts 4d (K = 3.53 ± 0.24 µM) and 4f (K
2.60 ± 0.81 µM). In contrast, the differences between
diastereomers of the corresponding uncyclized carbamate
compounds (3c vs 3d and 3e vs 3f) were relatively minor.
Apparently, the noncyclic structure of 3 allowed for
adequate adjustment of substituents during induced-fit
process, whereas the inflexibility of cyclized structure of 4
likely prevents such adjustment. On account of the fact that
preceding SAR discussion was premised on noncovalent
O
NH
4j
N
O
a
Each data presents the average results from three independent
experiments, and error bars represent SEMs (n=3).
i
= 0.22 ± 0.05 µM) and
4
i
i
i
=
pro
interaction, this issue was investigated by incubating 3C
with (R)-1 or 4e prior to trypsin digestion (Figure S11,
S12). The LC-MS/MS results of digested peptide fragments
indicated that neither was bonded to the catalytic cysteine
compared to AG7088 (a covalent inhibitor of EV71 3C ),
ruling out the possibility of covalent inhibitors.
pro
16
Table 2. Enzyme inhibitory activity, antiviral activity
and cytotoxicity of 4a-j as EV71 3C inhibitors
pro
H
O
N
O
H
O
N
R =
N
N
H
O
F
NO.
K
i
(µM)a
0.28 ± 0.07
EC50 (µM)a
3.97 ± 0.50
CC50 (µM)a
>100
NH
N
4a
R
Figure 3. Docking result. A) The docked 4g (yellow) were
superimposed with a co-crystal structure of a cyanohydrin
O
O
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Journal of Medicinal Chemistry
inhibitor (magenta) complexed with 3Cpro (PDB code: 5BPE);
B) Key interactions between 4g and surrounding residues.
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No spontaneous ring-opening was observed for 4e and
g in human and mouse plasma within at least 2 hours
the duration of the study, Figure 4A). The excellent
4
(
stability of both 4 and N-disubstituted 3 implies that a
tertiary amine structure could drastically prevent the
hydrolysis in plasma. In liver microsomal stability assays,
both compounds showed substantially improved
metabolic stability compared with (R)-1. The clearance in
microsomal incubation (Clint) of 4e and 4g was determined
to be 9-20 µL/min/mg (Figure 4B). As an estimation of the
risk of in vivo first pass metabolism, the data suggested
these compounds were subject to low risk of first pass
0
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metabolism according to a general classification of Clint
Moreover, unlike 2a, the data consistency across species of
e and 4g manifested the robustness of this class of
.
4
compounds.
Figure 5. A) Plasma concentration time profile following
Intravenous Administration of 20mg/kg 4e; B) Plasma
concentration time profile following Oral Administration of 20
mg/kg 4e. Each point represents mean ± SD (n =3).
CONCLUSION
An important class of EV71 3Cpro inhibitors such as (R)-1
is very promising for further development, but suffer from
inherent stability and potential toxicity problems because
of the use of a cyanohydrin warhead. The known stability
and toxicity of cyanohydrins, which releases cyanide upon
hydrolysis, led to the work to either stabilize this group or
find a replacement. In doing so, we first took advantage of
the hydroxyl group in (R)-1 for further derivatization for
improved stability. In the course of modification, we find
nonclassical bioisosteric replacements of cyanohydrin
moiety with 4-iminooxazolidin-2-one, which resulted in
identification of novel compound series 4. Among them, 4e
and 4g showed the most potent inhibitory activity with
greatly improved stability. Preliminary in vivo
pharmacokinetic studies of 4e also manifested moderate
PK properties. Overall, 4-iminooxazolidin-2-one moiety
does appear to serve as a nonclassical bioisostere to the
labile cyanohydrin moiety for circumventing potential
disadvantages. Further, the successful replacement of acyl
cyanohydrin with 4-iminooxazolidin-2-one also opens
doors to its further development as a generally applicable
bioisostere of the cyanohydrin moiety.
Figure 4. A) The stability of 4e, 4g in human and mouse
plasma; B) The stability of 4e, 4g in human and mouse
microsome (1.0 mg/mL). Vertical bars represent the standard
deviation of each data point (n=3).
In view of the potent activity as well as improved
microsomal and plasma stability of 4-iminooxazolidin-2-
one inhibitors, 4e was first selected for in vivo
pharmacokinetic studies in mice. 4e was administered
orally (PO) and intravenous (IV) to groups of male mice
with a single dose of 20 mg/kg (Figure 5). The results
showed that, after IV administration of 20.00 mg/kg 4e,
the peak plasma concentration (Cmax) in mice was 8.2
mg/mL at 5 min. Presenting high drug exposure level
EXPERIMENTAL SECTION
General. All reagents were purchased from commercial suppliers and
used as received. NMR spectra were recorded on a Bruker AVANCE-
1
13
4
00 (400 MHz for H and 101 MHz for C) (Bruker, Karlsruhe,
Germany) NMR spectrometer. Molecular mass was determined by ESI
mass spectrometry using a Shimadzu LCMS-2020 (Shimadzu, Kyoto,
Japan). Optical rotations were measured with an Insmark IP-120
Automatic Polarimeter (Insmark, Shanghai, China). Measurements
-1
(
AUC0-t = 140.1 mg·mL ·min), and the terminal elimination
half-life (t1/2) was 51 min. Moreover, the compound was
quickly absorbed after oral dosing at 20 mg/kg 4e and
reached a peak plasma concentration of 0.15 mg/mL at 15
min. At the same time, 4e possessed a moderate half-life of
D
were collected at 20 °C in MeOH at 589 nm. [α] values are given in
units of (° × mL)/(g × dm). HRMS were recorded on a high-resolution
ESI-FTICR mass spectrometer (Varian 7.0 T, Varian, USA). All tested
compounds exhibited purities of > 95% as analyzed by HPLC (Dionex
UltiMate 3000, Germany).
-
6
·
0 min and a reasonable plasma exposure of 7.9 mg·mL
1
min, yet its absolute bioavailability was relatively low
General Procedure for the Synthesis of ester derivatives 2a-c.
and that needs to be improved in the followed studies.
Preparation of 2a. A solution of (R)-1 (200 mg, 0.42 mmol) in
anhydrous CH Cl (50 mL), acetyl chloride (0.06 mL, 0.84 mmol) and
2 2
TEA (0.18 mL, 1.27 mmol) were added dropwise at 0 °C. Then the
5
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Journal of Medicinal Chemistry
Page 6 of 12
2
0
reaction mixture was stirred at room temperature for 2 h, followed by
washing with H O (100 mL×2), saturated citric acid solution (100
mL×2), saturated NaHCO solution (100 mL×2) and brine (100 mL×2).
The organic phase was dried over Na SO and concentrated, and the
to afford the pure product as a white solid 4a (128 mg, 64%). [훼]퐷 = -
15.83 (c = 0.063, MeOH). H NMR (CDCl -d) δ 8.49 (d, J = 8.2 Hz, 1H),
1
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
3
3
7.70 (d, J = 8.7 Hz, 1H), 7.60 (s, 1H), 7.14 (dd, J = 8.4, 5.3 Hz, 2H), 6.91
(t, J = 8.3 Hz, 2H), 6.58 (s, 1H), 6.31 (s, 1H), 4.95 (d, J = 2.7 Hz, 1H),
4.88 (dt, J = 8.5, 4.4 Hz, 1H), 4.55 (s, 1H), 3.27 (dd, J = 9.5, 4.7 Hz, 2H),
3.14 – 3.01 (m, 2H), 2.94 (s, 3H), 2.42 (d, J = 3.4 Hz, 4H), 2.17 – 2.11
(m, 1H), 2.03 (ddt, J = 12.6, 5.9, 2.9 Hz, 1H), 1.89 – 1.80 (m, 1H), 1.67
(q, J = 11.2, 9.9 Hz, 1H), 1.58 (ddd, J = 14.2, 8.4, 3.1 Hz, 1H), 1.50 (dd, J
2
4
residue was purified by silica gel column chromatography (MeOH:
DCM = 1: 70 v/v) to afford the pure product as a white solid 2a (174
2
0
[
훼]퐷
= -43.95 (c = 0.012, MeOH). H NMR (CDCl
1
3
-d) δ 8.33
mg, 80%).
(
d, J = 7.2 Hz, 1H), 7.57 (d, J = 8.3 Hz, 1H), 7.24 – 7.14 (m, 2H), 7.02 –
1
3
6
1
.90 (m, 2H), 6.79 (s, 1H), 6.32 (d, J = 1.0 Hz, 1H), 5.32 (d, J = 5.0 Hz,
H), 4.93 (q, J = 7.2 Hz, 1H), 4.23 (s, 1H), 3.28 (p, J = 5.7, 4.5 Hz, 2H),
3
= 12.1, 9.1 Hz, 1H). C NMR (CDCl -d) δ 174.8, 171.7, 171.2, 161.9 (d, J
= 245.3 Hz), 161.5, 159.0, 158.2, 156.1, 131.9 (d, J = 3.1 Hz), 130.8,
3.11 (qd, J = 13.9, 6.7 Hz, 2H), 2.43 (s, 3H), 2.33 (s, 1H), 2.22 (ddt, J =
130.7, 115.6, 115.3, 101.6, 79.5, 54.7, 47.7, 42.3, 37.8, 37.5, 31.6, 26.8,
+
1
1
1
1
3.7, 10.8, 4.5 Hz, 1H), 2.12 (s, 3H), 2.00 (d, J = 8.7 Hz, 1H), 1.85 (dt, J =
2.9, 4.8 Hz, 1H), 1.71 (ddd, J = 13.8, 7.7, 3.2 Hz, 2H), 1.49 (dtd, J =
26.3, 21.2, 12.3. HRMS (ESMS): C25
H
29FN
6
NaO
6
(M + Na) , calcd.
551.2025, found 551.2030.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
3
3.5, 10.9, 3.1 Hz, 1H). C NMR (CDCl -d) δ 175.0, 171.5, 171.2, 168.7,
62.0 (d, J = 245.3 Hz), 158.9, 158.2, 131.8 (d, J = 3.1 Hz), 130.9, 130.9,
ASSOCIATED CONTENT
Supporting Information.
115.7, 115.4, 115.1, 101.4, 62.7, 54.4, 48.5, 42.2, 37.8, 37.5, 31.0, 27.0,
+
2
5
1.3, 20.3, 12.3. HRMS (ESMS): C25
36.1916, found 536.1920.
5 6
H28FN NaO (M + Na) , calcd.
Detailed synthetic procedures, the stability study of (R)-1, the
stability study of 2a and 3, mass spectra of original and
product peak of 3i, mass spectra of 3e and 4e, bioassays,
plasma and microsome assay, pharmacokinetic studies, MS
General Procedure for the Synthesis of carbamate derivatives 3a-
p.
Preparation of 3a. To a solution of (R)-1 (200 mg, 0.42 mmol) in
anhydrous CH
mmol) in dry CH
under nitrogen atmosphere at
temperature for 30 min, methylamine (2M solution in THF) (0.64 mL,
.27 mmol) was added and the reaction mixture was stirred at room
temperature for 2 h, followed by washing with H O (100 mL×2),
saturated citric acid solution (100 mL×2), saturated NaHCO solution
100 mL×2) and brine (100 mL×2). The organic phase was dried over
Na SO and concentrated, and the residue was purified by column
chromatography (MeOH: DCM = 1: 60 v/v) to afford the pure product
2
Cl
2
(50 mL), N,N'-carbonyldiimidazole (103.2 mg, 0.64
Cl was added dropwise over a period of 30 min
°C. After stirring at room
1
spectrum of modified peptide from trypsin digestion, and H
2
2
13
NMR, C NMR data for tested compounds (PDF).
0
Molecular formula strings and some data (CSV).
This material is available free of charge via the Internet at
http://pubs.acs.org.
1
2
3
(
AUTHOR INFORMATION
Corresponding Author
2
4
2
0
as a white solid 3a (143 mg, 64%). [훼]퐷 = -48.71 (c = 0.058, MeOH).
1
3
H NMR (CDCl -d) δ 8.13 (d, J = 8.0 Hz, 1H), 7.64 (d, J = 8.6 Hz, 1H),
*
For L.S.: shanglq@nankai.edu.cn; for B.W.: wang@gsu.edu.
7
5
4
.24 – 7.11 (m, 2H), 6.94 (t, J = 8.6 Hz, 2H), 6.62 (s, 1H), 6.30 (s, 1H),
.33 – 5.26 (m, 1H), 5.23 (q, J = 4.7 Hz, 1H), 5.03 (q, J = 7.4 Hz, 1H),
.34 (dp, J = 12.8, 3.7 Hz, 1H), 3.25 (d, J = 6.9 Hz, 2H), 3.16 (dd, J = 13.9,
Notes
The authors declare no competing financial interests.
6.3 Hz, 1H), 3.06 (dd, J = 13.8, 7.4 Hz, 1H), 2.80 (s, 3H), 2.42 (s, 3H),
.35 (td, J = 13.0, 3.9 Hz, 1H), 2.22 (tt, J = 10.0, 6.5 Hz, 1H), 2.07 – 1.93
2
1
3
(
(
m, 1H), 1.90 – 1.76 (m, 1H), 1.71 – 1.57 (m, 2H), 1.44 (s, 1H). C NMR
CDCl -d) δ 174.5, 171.4, 171.2, 161.9 (d, J = 245.1 Hz), 158.9, 158.2,
ACKNOWLEDGMENTS
3
154.4, 132.0 (d, J = 3.1 Hz), 131.0, 130.9, 115.9, 115.5, 115.3, 101.3,
4.0, 54.3, 48.2, 42.2, 38.1, 37.4, 31.3, 27.7, 26.6, 21.4, 12.3. HRMS
This work was supported by the National Key Research and
Development Program of China (Grant No. 2018YFA0507204),
the National Natural Science Foundation of China (Grant No.
21672115) and the State Key Laboratory of Medicinal
Chemical Biology.
6
+
(
ESMS): C25
Preparation of 3n. To a solution of (R)-1 (200 mg, 0.42 mmol) in
anhydrous CH Cl (50 mL), dimethylcarbamoyl chloride (0.08 mL,
.85 mmol) in dry CH Cl was added dropwise at 0 °C, followed by
addition of DMAP (0.21 mg, 1.7 mmol). Then the reaction mixture was
stirred at room temperature for 4 h, followed by washing with H
6 6
H29FN NaO (M + Na) , calcd. 551.2025, found 551.2029.
2
2
0
2
2
ABBREVIATIONS USED
2
O
(
100 mL×2), saturated citric acid solution (100 mL×2), saturated
EV71, enterovirus 71; 3Cpro, 3C protease; SARS CoV Mpro
,
NaHCO
3
solution (100 mL×2) and brine (100 mL×2). The organic
SO and concentrated, and the residue was
purified by column chromatography (MeOH: DCM = 1: 60 v/v) to
severe acute respiratory syndrome coronaviruses main
protease; Clint, intrinsic clearance; CDI, N, N’-carbonyl-
diimidazole; AUC0-t, area under the curve between 0-t.
phase was dried over Na
2
4
2
0
afford the pure product as a white solid 3n (140 mg, 61%). [훼]퐷 = -
1
4
7
2
5.29 (c = 0.057, MeOH). H NMR (CDCl
3
-d) δ 8.20 (d, J = 8.0 Hz, 1H),
.62 (d, J = 8.5 Hz, 1H), 7.17 (dd, J = 8.2, 5.3 Hz, 2H), 6.93 (t, J = 8.5 Hz,
H), 6.57 (s, 1H), 6.29 (s, 1H), 5.38 – 5.22 (m, 1H), 4.97 (q, J = 7.4 Hz,
REFERENCES
1H), 4.35 (tt, J = 11.7, 5.1 Hz, 1H), 3.31 – 3.20 (m, 2H), 3.15 (dd, J =
(
1) Zhu, Y.; Jiang, Z.; Xiao, G.; Cheng, S.; Wen, Y.; Wan, C.
1
6
2
3.9, 6.1 Hz, 1H), 3.04 (dd, J = 13.9, 7.6 Hz, 1H), 2.89 (d, J = 15.7 Hz,
H), 2.41 (s, 3H), 2.35 (dd, J = 13.6, 3.9 Hz, 1H), 2.28 – 2.13 (m, 1H),
.00 (h, J = 5.0 Hz, 1H), 1.82 (dt, J = 14.5, 4.4 Hz, 1H), 1.75 – 1.58 (m,
Circadian rhythm disruption was observed in hand, foot, and
mouth disease patients. Medicine (Baltimore) 2015, 94, e601.
(
2) (a) Shang, L.; Xu, M.; Yin, Z. Antiviral drug discovery for the
treatment of enterovirus 71 infections. Antiviral Res. 2013, 97,
83-194; (b) Chang, L. Y.; Huang, L. M.; Gau, S. S.; Wu, Y. Y.; Hsia, S.
1
3
2H), 1.56 – 1.39 (m, 1H). C NMR (CDCl
3
-d) δ 174.4, 171.4, 171.1,
61.9 (d, J = 245.1 Hz), 158.8, 158.2, 153.7, 132.0 (d, J = 3.1 Hz), 130.9,
30.8, 116.0, 115.5, 115.3, 101.4, 64.4, 54.4, 48.5, 42.2, 37.9, 37.5, 36.8,
1
1
3
1
+
H.; Fan, T. Y.; Lin, K. L.; Huang, Y. C.; Lu, C. Y.; Lin, T. Y.
Neurodevelopment and cognition in children after enterovirus 71
infection. N. Engl. J. Med. 2007, 356, 1226-1234.
6 6
6.0, 31.4, 26.7, 21.3, 12.2. HRMS (ESMS): C26H31FN NaO (M + Na) ,
calcd. 565.2181, found 565.2184.
General Procedure for the Synthesis cyclic carbamate (4-
iminooxazolidin-2-one) derivatives of 4a-h.
(
3) (a) Liu, L.; Zhang, Y.; Wang, J.; Zhao, H.; Jiang, L.; Che, Y.; Shi,
H.; Li, R.; Mo, Z.; Huang, T.; Liang, Z.; Mao, Q.; Wang, L.; Dong, C.;
Liao, Y.; Guo, L.; Yang, E.; Pu, J.; Yue, L.; Zhou, Z.; Li, Q. Study of the
integrated immune response induced by an inactivated EV71
vaccine. PLoS One 2013, 8, e54451; (b) Li, J. X.; Mao, Q. Y.; Liang, Z.
L.; Ji, H.; Zhu, F. C. Development of enterovirus 71 vaccines: from
Preparation of 4a. A solution of compound 3a (200.0 mg, 0.38 mmol)
in MeOH: PBS buffer (pH = 7.4) = 1:2 v/v, the reaction mixture was
stirred at room temperature for 4 h. The solvent was removed under
vacuum. The aqueous layer was extracted with DCM (100 mL×3). The
combined organic layer was dried over anhydrous sodium sulfate and
concentrated, and the residue was purified by ether recrystallization
6
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the lab bench to Phase III clinical trials. Expert. Rev. Vaccines
2014, 13, 609-618.
4) Brown, B. A., Pallansch, M.A. Complete nucleotide sequence
of enterovirus 71 is distinct from poliovirus. Virus Res. 1995, 195-
05.
5) Li, M. L.; Hsu, T. A.; Chen, T. C.; Chang, S. C.; Lee, J. C.; Chen, C.
(15) (a) Kurz, T.; Widyan, K. O-Protected 3-hydroxy-oxazolidin-
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2,4-diones: novel precursors in the synthesis of alpha-
hydroxyhydroxamic acids. Org. Biomol. Chem. 2004, 2, 2023-
2027; (b) Kurz, T.; Widyan, K. Microwave-assisted conversion of
N-substituted oxazolidin-2,4-diones into α-hydroxyamides.
Tetrahedron 2005, 61, 7247-7251.
(16) Lu, G.; Qi, J.; Chen, Z.; Xu, X.; Gao, F.; Lin, D.; Qian, W.; Liu,
H.; Jiang, H.; Yan, J.; Gao, G. F. Enterovirus 71 and coxsackievirus
A16 3C proteases: binding to rupintrivir and their substrates and
anti-hand, foot, and mouth disease virus drug design. J. Virol.
2011, 85, 10319-10331.
(17) Gising, J.; Belfrage, A. K.; Alogheli, H.; Ehrenberg, A.;
Akerblom, E.; Svensson, R.; Artursson, P.; Karlen, A.; Danielson, U.
H.; Larhed, M.; Sandstrom, A. Achiral pyrazinone-based inhibitors
of the hepatitis C virus NS3 protease and drug-resistant variants
with elongated substituents directed toward the S2 pocket. J. Med.
Chem. 2014, 57, 1790-1801.
(
2
(
C.; Stollar, V.; Shih, S. R. The 3C protease activity of enterovirus 71
induces human neural cell apoptosis. Virology 2002, 293, 386-
3
95.
6) (a) Dragovich, P. S.; Prins, T. J.; Zhou, R.; Fuhrman, S. A.;
(
Patick, A. K.; Matthews, D. A.; Ford, C. E.; Meador, J. W., 3rd; Ferre,
R. A.; Worland, S. T. Structure-based design, synthesis, and
biological evaluation of irreversible human rhinovirus 3C
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
protease
ketomethylene-containing peptidomimetics. J. Med. Chem. 1999,
2, 1203-1212; (b) Dragovich, P. S.; Webber, S. E.; Babine, R. E.;
inhibitors.
3.
Structure-activity
studies
of
4
Fuhrman, S. A.; Patick, A. K.; Matthews, D. A.; Reich, S. H.;
Marakovits, J. T.; Prins, T. J.; Zhou, R.; Tikhe, J.; Littlefield, E. S.;
Bleckman, T. M.; Wallace, M. B.; Little, T. L.; Ford, C. E.; Meador, J.
W., 3rd; Ferre, R. A.; Brown, E. L.; Binford, S. L.; DeLisle, D. M.;
Worland, S. T. Structure-based design, synthesis, and biological
evaluation of irreversible human rhinovirus 3C protease
inhibitors. 2. Peptide structure-activity studies. J. Med. Chem.
TOC
1
998, 41, 2819-2834; (c) Dragovich, P. S.; Prins, T. J.; Zhou, R.;
Fuhrman, S. A.; Patick, A. K.; Matthews, D. A.; Ford, C. E.; Meador, J.
W., 3rd; Ferre, R. A.; Worland, S. T. Structure-based design,
synthesis, and biological evaluation of irreversible human
rhinovirus 3C protease inhibitors. 1. Structure-activity studies of
ketomethylene-containing peptidomimetics. J. Med. Chem. 1999,
4
2, 1203-1212.
(7) Kuo, C. J.; Shie, J. J.; Fang, J. M.; Yen, G. R.; Hsu, J. T.; Liu, H. G.;
Tseng, S. N.; Chang, S. C.; Lee, C. Y.; Shih, S. R.; Liang, P. H. Design,
synthesis, and evaluation of 3C protease inhibitors as anti-
enterovirus 71 agents. Bioorg. Med. Chem. 2008, 16, 7388-7398.
(
8) (a) Wu, C.; Cai, Q.; Chen, C.; Li, N.; Peng, X.; Cai, Y.; Yin, K.;
Chen, X.; Wang, X.; Zhang, R.; Liu, L.; Chen, S.; Li, J.; Lin, T.
Structures of enterovirus 71 3C proteinase (strain E2004104-TW-
CDC) and its complex with rupintrivir. Acta Crystallogr. D Biol.
Crystallogr. 2013, 69, 866-871; (b) Wang, J.; Fan, T.; Yao, X.; Wu,
Z.; Guo, L.; Lei, X.; Wang, J.; Wang, M.; Jin, Q.; Cui, S. Crystal
structures of enterovirus 71 3C protease complexed with
rupintrivir reveal the roles of catalytically important residues. J.
Virol. 2011, 85, 10021-10030.
(
9) (a) Zhai, Y.; Ma, Y.; Ma, F.; Nie, Q.; Ren, X.; Wang, Y.; Shang,
L.; Yin, Z. Structure-activity relationship study of peptidomimetic
aldehydes as enterovirus 71 3C protease inhibitors. Eur. J. Med.
Chem. 2016, 124, 559-573; (b) Zeng, D.; Ma, Y.; Zhang, R.; Nie, Q.;
Cui, Z.; Wang, Y.; Shang, L.; Yin, Z. Synthesis and structure-activity
relationship of alpha-keto amides as enterovirus 71 3C protease
inhibitors. Bioorg. Med. Chem. Lett. 2016, 26, 1762-1766.
(
10) Zhai, Y.; Zhao, X.; Cui, Z.; Wang, M.; Wang, Y.; Li, L.; Sun, Q.;
Yang, X.; Zeng, D.; Liu, Y.; Sun, Y.; Lou, Z.; Shang, L.; Yin, Z.
Cyanohydrin as an anchoring group for potent and selective
inhibitors of enterovirus 71 3C protease. J. Med. Chem. 2015, 58,
9414-9420.
(
11) Fomunyam, R. T.; Adegbola, A. A.; Oke, O. L. The stability of
cyanohydrins. Food Chem. 1985, 17, 221-225.
12) (a) Krehan, D.; Frølund, B.; Ebert, B.; Nielsen, B.;
(
Krogsgaard-Larsen, P.; Johnston, G. A. R.; Chebib, M. Aza-THIP and
related analogues of THIP as GABA C antagonists. Biorg. Med.
Chem. 2003, 11, 4891-4896; (b) Lima, L. M.; Barreiro, E. J.
Bioisosterism: a useful strategy for molecular modification and
drug design. Curr. Med. Chem. 2005, 12, 23-49.
(
13) De Nicola, G. R.; Leoni, O.; Malaguti, L.; Bernardi, R.;
Lazzeri, L. A simple analytical method for dhurrin content
evaluation in cyanogenic plants for their utilization in fodder and
biofumigation. J. Agric. Food Chem. 2011, 59, 8065-8069.
(
14) Kerns, E. H.; Di, L., Chapter 11 - Metabolic Stability. In
Drug-like Properties: Concepts, Structure Design and Methods,
Kerns, E. H.; Di, L., Eds. Academic Press: San Diego, 2008; pp 137-
II.
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A) Structure of (R)-1; B) The stability of (R)-1 in plasma and microsome. Vertical bars represent the
standard deviation of each data point (n=3).
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A) The stability of represent compounds in mouse plasma. B) LC/MS profile showing the disappearance of 3i
(Retention time = 6.41min) and the appearance of product peak (Retention time = 5.41min) after 60 min of
incubation with mouse plasma.
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Docking result. A) The docked 4g (yellow) were superimposed with a co-crystal structure of a cyanohydrin
inhibitor (magenta) complexed with 3Cpro (PDB code: 5BPE); B) Key interactions between 4g and
surrounding residues.
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A) The stability of 4e, 4g in human and mouse plasma; B) The stability of 4e, 4g in human and mouse
microsome (1.0 mg/mL). Vertical bars represent the standard deviation of each data point (n=3).
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A) Plasma concentration time profile following Intravenous Administration of 20mg/kg 4e; B) Plasma
concentration time profile following Oral Administration of 20 mg/kg 4e. Each point represents mean ± SD
(n =3).
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