Identification of Novel Tricyclic Benzo[1,3]oxazinyloxazolidinones as Potent Antibacterial Agents with Excellent Pharmacokinetic Profiles against Drug-Resistant Pathogens

Article abstract of DOI:10.1021/acs.jmedchem.0c02153

A series of conformationally constrained novel benzo[1,3]oxazinyloxazolidinones were designed, synthesized, and evaluated on their activities against Mycobacterium tuberculosis, Gram-positive bacteria, and Gram-negative bacteria. The studies identified a new compound 20aa that displayed good to excellent antibacterial and antitubercular profiles against drug-resistant TB strains (MIC = 0.48-0.82 μg/mL), MRSA (MIC = 0.25-0.5 μg/mL), MRSE (MIC = 1 μg/mL), VISA (MIC = 0.25 μg/mL), and VRE (MIC = 0.25 μg/mL) and some linezolid-resistant strains (MIC 1-2 μg/mL). Compound 20aa was demonstrated as a promising candidate through ADME/T evaluation including microsomal stability, cytotoxicity, and inhibition of hERG and monoamine oxidase. Notably, 20aa showed excellent mouse PK profile with high plasma exposure (AUC0-∞ = 78 669 h·ng/mL), high peak plasma concentration (Cmax = 10 253 ng/mL), appropriate half-life of 3.76 h, and superior oral bioavailability (128%). The present study not only successfully provides a novel benzo[1,3]oxazinyloxazolidinone scaffold with superior druggability but also lays a good foundation for new antibacterial drug development.

Full text of DOI:10.1021/acs.jmedchem.0c02153

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Article
Identification of Novel Tricyclic Benzo[1,3]oxazinyloxazolidinones as
Potent Antibacterial Agents with Excellent Pharmacokinetic Profiles
against Drug-Resistant Pathogens
Yongqi Wu, Bin Wang, Haijia Lu, Hongyi Zhao, Beibei Yang, Li Li, Yu Lu, Dongfeng Zhang,*
Ning Sun,* and Haihong Huang*
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ABSTRACT: A series of conformationally constrained novel
benzo[1,3]oxazinyloxazolidinones were designed, synthesized,
and evaluated on their activities against Mycobacterium tuberculosis,
Gram-positive bacteria, and Gram-negative bacteria. The studies
identified a new compound 20aa that displayed good to excellent
antibacterial and antitubercular profiles against drug-resistant TB
strains (MIC = 0.48−0.82 μg/mL), MRSA (MIC = 0.25−0.5 μg/
mL), MRSE (MIC = 1 μg/mL), VISA (MIC = 0.25 μg/mL), and VRE (MIC = 0.25 μg/mL) and some linezolid-resistant strains
(MIC 1−2 μg/mL). Compound 20aa was demonstrated as a promising candidate through ADME/T evaluation including
microsomal stability, cytotoxicity, and inhibition of hERG and monoamine oxidase. Notably, 20aa showed excellent mouse PK
profile with high plasma exposure (AUC0−∞ = 78 669 h·ng/mL), high peak plasma concentration (Cmax = 10 253 ng/mL),
appropriate half-life of 3.76 h, and superior oral bioavailability (128%). The present study not only successfully provides a novel
benzo[1,3]oxazinyloxazolidinone scaffold with superior druggability but also lays a good foundation for new antibacterial drug
development.
Figure 1, linezolid (1)5 was the first approved (2000)
oxazolidinone antibacterial drug for the treatment of Gram-
positive bacterial infections and has been off-label used in the
treatment of complicated MDR-TB and XDR-TB. As one
INTRODUCTION
■
Spanning decades, the use and misuse of antibiotics have
increased the occurrence of drug resistant bacterial strains.
This occurrence and prevalence of multidrug resistance strains
pose one of the greatest risks to human health1 and will
component in the Nix-TB regimen, consisting of bedaquiline,
pretomanid, and linezolid, for the treatment of XDR-TB or
treatment-intolerant/nonresponsive MDR-TB,6 linezolid plays
a key role in the efficacy of the treatment. As such, much effort
has been devoted to improving the activity and ADME/T
properties of linezolid. Extensive structural modifications along
with the structure−activity relationship (SAR) studies were
focused on the C-ring and C5 side chain (Figure 1) of the
parent scaffold.7−12 Tedizolid phosphate (2) was the second
oxazolidinone drug approved by FDA for the treatment of
MRSA skin infections in 2014.13 Contezolid (3), developed by
MicuRx Pharmaceuticals, phase III clinical trials were recently
finished and the New Drug Application was submitted to
regulators.14 Furthermore, three oxazolidinones have been
evaluated on their clinical effects with varying success such as
inevitably increase in the future. In 2017, the World Health
Organization released a list of 12 bacteria or bacterial families
that pose the greatest threat to human health and for which
new antibiotics are desperately needed. The list included a
range of bacteria. Among them a quarter of the bacteria were
Gram-positive bacteria, including methicillin-resistant Staph-
ylococcus aureus (MRSA), vancomycin-resistant Enterococci
(VRE) and penicillin-resistant Streptococcus pneumoniae
(PRSP). Mycobacterium tuberculosis (M. tuberculosis) was not
added to the list due to the fact that it is a well-established
priority for new drug discovery.2 In 2019 alone, there were
estimated 0.5 million new cases that displayed resistance to
rifampicin, the most effective first-line drug, of which 78% were
multidrug-resistant TB.3 There is therefore a clear and urgent
need to develop antibacterial agents bearing novel pharmaco-
phores or new modes of action to combat these multidrug
resistant bacteria.
Received: December 14, 2020
Published: March 11, 2021
Oxazolidinones were developed as a new class of synthetic
antibacterial agents and exhibited their antibacterial activity
through a unique mode of action. The compounds bind to 23S
rRNA of the bacterial 50S ribosomal subunit and inhibit the
bacterial protein synthesis at the initiation step.4 As shown in
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Figure 1. Representative structure modifications of linezolid focused on the C-ring and C5 side chain.
delpazolid (4) (for Gram-positive bacteria and TB),15,16
sutezolid (5) (for TB),17,18 and TBI-223 (6) (for TB).19
Oxazolidinones can therefore be described as the privileged
scaffold in antibacterial drug discovery, and further diversifi-
cation of this important pharmacophore shows the potential to
lead to the discovery of new antibacterial agents against both
Gram-positive bacteria and M. tuberculosis.
Recently, the conformationally unique benzoxazinyl-
oxazolidinone tricyclic scaffold has undergone intensive
investigation. Yang and co-workers reported a series of
benzo[1,4]oxazinyloxazolidinones20,21 as antibacterial agents
(Figure 2). The SAR showed that the compound with aryl
evaluation for antibacterial and antitubercular properties. We
aim to explore the effect of aromatic or nonaromatic groups
and F substitution on the B ring, as well as the key methylene
linkage between the B ring (phenyl ring) and A ring
(oxazolidinone ring) on the antibacterial activity, cytotoxicity,
and PK properties of this new type of molecule. Interestingly,
the newly designed benzo[1,3]oxazinyloxazolidinone scaffold
displayed differential SAR compared to that established by the
benzo[1,4]oxazinyloxazolidinones and the traditional phenyl-
oxazolidinones such as linezolid. In particular, this novel
scaffold exhibited good to excellent activity against both Gram-
positive bacteria and M. tuberculosis, particularly against drug-
resistant bacteria including MRSA, MRSE, VISA, VRE, and TB
strains. The present study demonstrated systematically that
compound 20aa exhibited not only highly potent antituber-
cular and antibacterial activities including moderate activity
against linezolid-resistant isolated clinical strains but also
superior PK profiles with oral bioavailability up to 128% in
mice.
CHEMISTRY
■
Figure 2. Design of novel conformational constraint oxazolidinones.
The synthetic route of the critical tricyclic fused benzo[1,3]-
oxazinyloxazolidinone intermediates ( )-18a and ( )-18b is
depicted in Scheme 1. Compound 8a was prepared from 4-
fluoro-2-hydroxybenzoic acid (7a) according to the liter-
ature.24 After esterification, the amination of 8a gave the
desired amide 9a in 83% yield. The amide group of 9a was
subsequently reduced with BH3 and followed by the protection
with benzyl chloroformate (CbzCl) to afford compound 10a at
room temperature. The key oxazolidinone fragment 5-
(hydroxymethyl)-3-(4-methoxybenzyl)oxazolidin-2-one (13)
was synthesized from 4-methoxybenzylamine (11) and 2-
(chloromethyl)oxirane (12) in the presence of K2CO3 as
base.25 The key intermediate 14 was produced from 13 via a
Swern oxidation.26 This was followed by a tandem
condensation/cyclization strategy to construct the 1,3-
oxazinane ring from 10a and 14.27 As compound 14 has a
chiral center, the cyclization will produce four distinct
stereoisomers: two racemic diastereomers, ( )-15a (trans)
and ( )-15b (cis), which were isolated by silica gel column
chromatography directly. After cleavage of the 4-methoxyben-
zyl (PMB) group, the nitrogen atom in the intermediates
( )-16a and ( )-16b was Boc-protected to give ( )-17a and
( )-17b, respectively.
group as the C ring was more active against MRSA and MRSE
than that with the corresponding alkyl group, while the
derivatives with fluorine substitution on the B ring generated
reduced activity. Interestingly, our previous work identified
that the promising candidates exhibiting highly potent
antitubercular activity contained an aliphatic heterocyclic
group as the C ring of this interesting motif. The SAR
expressed that F substitution on the B ring was beneficial for
improving the antitubercular activity and significantly reducing
overall cytotoxicity.22,23 Although there are some differences in
the SAR described between Yang’s work and our reported
work, this conformationally constrained tricyclic scaffold
exhibited good to excellent antibacterial activities, with notably
superior PK properties. These prominent characteristics
displayed by this aforementioned benzo[1,4]oxazinyl-
oxazolidinones led us to conclude that the exploration of
chemical space between the phenyl (B ring) and the
oxazolidinone (A ring) motif would pay dividends in relation
to the discovery of potentially novel antibacterial agents
bearing novel scaffolds.
Herein, we disclose our design and synthesis of novel
oxazolidinones containing unique benzo[e]oxazolo[4,3-b]-
[1,3]oxazin-1-one scaffold (Figure 2), and their subsequent
The benzo[e]oxazolo[4,3-b][1,3]oxazin-1-one scaffold was
constructed by an ingenious tandem reaction. The oxazolidi-
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a
Scheme 1. Synthesis of Benzo[1,3]oxazinyloxazolidinone Intermediates ( )-18a and ( )-18b
a
Reagents and conditions: (a) NBS, conc H2SO4, MeCN, rt, 1 h, 84%; (b) (i) conc H2SO4, MeOH, reflux, 32 h; (ii) NH4OH, MeOH, 55 °C, 6 h,
83% of two steps; (c) (i) BH3, THF, reflux, 5 h; (ii) CbzCl, NaHCO3, THF/H2O = 5:1, rt, 3 h, 72% of two steps; (d) K2CO3, TEA, MeOH, reflux,
12 h, 60%; (e) (COCl)2, DMSO, TEA, THF, −78 °C to rt, 90%; (f) TsOH·H2O, toluene, reflux, 6 h, 49% for ( )-15a, 25% for ( )-15b; (g) ceric
ammonium nitrate (CAN), MeCN/H2O = 9:1, rt, 2 h, 78% for ( )-16a, 76% for ( )-16b; (h) (Boc)2O, TEA, DMAP, DCM, rt, 2 h, 78% for
( )-17a, 82% for ( )-17b; (i) Cs2CO3, MeOH, rt, 3h, 89% for ( )-18a, 91% for ( )-18b.
a
Scheme 2. Synthesis of Compounds ( )-20a and ( )-20b
a
Reagents and conditions: (a) 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)picolinonitrile, Pd(PPh3)4, Na2CO3, DMSO/H2O = 6:1, 100 °C, 5 h,
84% for ( )-19a, 81% for ( )-19b; (b) (i) TFA, DCM, rt, 2 h; (ii) acetyl chloride, TEA, DCM, 0 °C, 2 h, 87% for ( )-20a, 80% for ( )-20b.
a
Scheme 3. Separation of Compound ( )-20a
a
Reagents and conditions: (a) CHIRALPAK IA column, MeOH/EtOAc = 80:20, 1.0 mL/min, λ = 254 nm, 25 °C.
none ring of ( )-17a and ( )-17b was opened in the
presence of Cs2CO3 to afford the required oxazolidinone ring
immediately and gave the key intermediates ( )-18a and
( )-18b with the novel tricyclic scaffold, respectively.
The coupling of ( )-18a or ( )-18b with boronic acid
ester afforded compounds ( )-19a or ( )-19b. Finally,
( )-20a and ( )-20b were obtained via cleavage of the
protective carbamate group, followed by acetylation with acetyl
chloride (Scheme 2). Compound ( )-20a was separated as
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the pure enantiomers 20aa and 20ab with chiral HPLC
(Scheme 3). The absolute configurations of the above
prepared compounds 20aa and 20ab were determined by the
comparison of the experimental ECD spectrum and the
corresponding calculated ECD spectra of compounds 20aa and
20ab (Figure 3). The ECD spectrum recorded for 20aa
acetylation of the NH2 group with acetyl chloride. The
absolute configuration of the synthetic target compound was
determined by chiral HPLC compared with the optically pure
20aa obtained by chiral chromatography separation (Figure
S4).
Our next goal was to produce compounds ( )-45−48, with
the fluorine on the phenyl ring at the para position relative to
the oxygen of benzo[e]oxazolo[4,3-b][1,3]oxazin-1-one scaf-
fold. As illustrated in Scheme 6, 4-bromo-5-fluoro-2-hydrox-
ybenzamide (9c) was used as the starting material and the
target compounds ( )-45−48 were prepared in a similar
procedure as shown in Scheme 1. Compound 9c was
synthesized from 4-bromo-5-fluoro-2-hydroxybenzoic acid via
esterification, amination nucleophilic substitution, and depro-
tection sequentially (Supporting Information, Scheme S1).
RESULTS AND DISCUSSION
■
As mentioned, the benzo[1,3]oxazinyloxazolidinone scaffold
was first designed to elucidate its antibacterial activity, with the
aim to characterize the intrinsic ability of this novel tricyclic
fused oxazolidinone as an antibacterial agent. Due to the N,O-
acetal fragment within the benzo[1,3]oxazinyloxazolidinone
scaffold, we initially explored its microsome stability, as this
would determine the subsequent success of this scaffold. We
subjected compound 49 (Supporting Information, Scheme S2)
with the key pharmacophore in the scaffold to two stability
tests. As shown in Table 1, compound 49 exhibited excellent
stability not only in mouse liver microsomes but also in human
liver microsomes compared to the known compound benzo-
[1,4]oxazinyloxazolidinone 50.
Figure 3. ECD spectra of compounds 20aa and 20ab.
matches the calculated ECD curve of 20aa but is opposite to
that of 20ab. Therefore, compounds 20aa and 20ab were
deduced to have the (3S,3aS) and (3R,3aR) absolute
configuration, respectively.
Supported by the positive results of the above microsomal
stability assay, the target compounds with the benzo[e]-
oxazolo[4,3-b][1,3]oxazin-1-one scaffold were synthesized and
screened for their in vitro activities against Mtb H37Rv to
confirm our molecular design strategy (Table 2). Compound
trans ( )-20a displayed good MIC (1.58 μg/mL) against Mtb
H37Rv, while compound cis ( )-20b showed low activity.
These results clearly indicated that the trans configuration was
optimal for anti-TB activity. Subsequently, compound trans
( )-20a was separated into optical isomers 20aa and 20ab
through chiral resolution. To our delight, compound 20aa
(3S,3aS) showed more potent in vitro anti-TB activity than
linezolid. By contrast, compound 20ab (3R,3aR) was found to
be virtually inactive (MIC > 32 μg/mL). The results disclosed
The synthetic route of the fluoride-free compound ( )-24 is
outlined in Scheme 4. Compound ( )-24 was prepared from
5-bromo-2-hydroxybenzamide (9b) in a similar procedure as
shown in Scheme 1. The synthesis of the chiral pure target
compounds 20aa, 30−38 are outlined in Scheme 5. First, the
pure enantiomers 18a and 24 were obtained by chiral HPLC
separation and then reacted with arylboronate ester or
morpholine to afford intermediates 25−29 followed by
subsequent deprotection of the N-Boc group to deliver
amine compounds 30−34. The desired products, 20aa and
35−38, were obtained from compounds 30−34 via the
a
Scheme 4. Synthesis of Compound ( )-24
a
Reagents and conditions: (a) (i) BH3, THF, reflux, 5 h; (ii) CbzCl, NaHCO3, THF/H2O = 5:1, rt, 3 h, 72% of two steps; (b) TsOH·H2O,
toluene, reflux, 6 h, 50%; (c) CAN, MeCN/H2O = 9:1, rt, 2 h, 76%; (d) (Boc)2O, TEA, DMAP, DCM, rt, 2 h, 90%; (e) Cs2CO3, MeOH, rt, 3 h,
84%.
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a
Scheme 5. Synthesis of Target Compounds 20aa and 30−38
a
Reagents and conditions: (a) CHIRALPAK AD-H column, MeOH = 100%, 1.0 mL/min, λ = 214 nm, 35 °C, for 18a; CHIRALPAK IA column,
hexane/EtOH = 50:50, 1.0 mL/min, λ = 254 nm, 25 °C, for 24; (b) (i) arylboronic acid ester, Pd(PPh3)4, Na2CO3, DMSO/H2O = 6:1, 100 °C, 5
h, 57−89%, for 25, 26, and 28; (ii) morpholine, X-PHOS, Pd(OAc)2, Cs2CO3, toluene, reflux, 100 °C, 6 h, 44−66%, for 27 and 29; (c) TFA,
DCM, rt, 3 h, 63−94%; (d) acetyl chloride, TEA, DCM, 0 °C, 2 h, 78−99%.
a
Scheme 6. Synthesis of Target Compounds ( )-45−48
a
Reagents and conditions: (a) (i) BH3, THF, reflux, 5 h; (ii) CbzCl, NaHCO3, THF/H2O = 5:1, rt, 3 h, 71% of two steps; (b) TsOH·H2O,
toluene, reflux, 6 h, 51%; (c) CAN, MeCN/H2O = 9:1, rt, 2 h, 67%; (d) (Boc)2O, TEA, DMAP, DCM, rt, 2 h, 74%; (e) Cs2CO3, MeOH, rt, 3h,
82%; (f) (i) 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)picolinonitrile, Pd(PPh3)4, Na2CO3, DMSO/H2O = 6:1, 100 °C, 5 h, 84%, for ( )-43;
(ii) morpholine, X-PHOS, Pd(OAc)2, Cs2CO3, toluene, reflux, 100 °C, 6 h, 38%, for ( )-44; (g) TFA, DCM, rt, 3 h, 78−86%; (h) acetyl chloride,
TEA, DCM, 0 °C, 2 h, 76−86%.
that the absolute configuration (3S,3aS) for this novel tricyclic
fused oxazolidinone was essential for potency in accordance
with that previously observed for the benzo[1,4]oxazinyl-
oxazolidinone scaffold.23 Compounds 20aa and 20ab were also
evaluated for their antibacterial activities against Gram-positive
bacteria S. aureus ATCC 29213. As anticipated, compound
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Table 1. Microsome Stability of Compounds 49 and 50
Table 3. Antibacterial Activities of Target Compounds
a
b
c
Mouse liver microsome. Human liver microsome. Substrate
concentrations were determined in incubations with NADPH after
d
30 min and normalized to concentrations at time zero. Stability was
e
determined without the NADPH cofactor. Compound 50 was
prepared according to the literature.23,28
Table 2. Antibacterial Activities of Different Isomers of 20a
configuration
chiral
purity
MIC (μg/mL), MIC (μg/mL),
compd
(3,3a)
Mtb H37Rv
S. aureus 29213
(
(
)-20a
)-20b
trans
cis
(3S,3aS)
(3R,3aR)
racemic
racemic
99.97%
99.94%
1.58
15.51
0.40
>32
20aa
20ab
0.125
>64
linezolid
0.88
0.125
20aa displayed potent antibacterial activity (MIC = 0.125 μg/
mL), while its enantiomer, 20ab, showed no activity (MIC >
64 μg/mL). This result further demonstrated that the 3S,3aS-
configuration is essential for maintaining potency.
After confirming 20aa, the 3S, 3aS-isomer, as the most
potent compound, we conducted extensive SAR studies on the
phenyl ring and C5 side chain. The target compounds were
evaluated for their activities against a series of bacteria
including Mtb, Gram-positive and Gram-negative strains
(Table 3). Keeping fluoro substitution at R2, compounds
with R1 as aryl group showed more potent in vitro anti-TB
activity (20aa, 30, 31, and 35), whether R3 was H or an acetyl
group. In contrast, when R1 was morpholinyl, compounds 32
and 36 had no activity. For Gram-positive bacteria, compounds
20aa and 35 containing an aryl group at R1 displayed equal or
more potent activity (MIC = 0.125 μg/mL) compared to the
reference linezolid (MIC = 0.125−0.25 μg/mL). Compound
36 bearing a morpholine motif showed moderate antibacterial
activity but lower than compound 20aa. Fluorine on the
phenyl ring has been illustrated to have a beneficial effect on
the antibacterial activity for linezolid analogues;5 thus we
assessed the activities of the fluoride-free compounds (33, 37
and 34, 38). As illustrated in Table 3, compounds 33 and 37
containing a pyridine group displayed significantly reduced
anti-TB activity, while compounds 34 and 38 bearing a
morpholine group totally lost anti-TB activity. Although
compound 37 showed good activity against Gram-positive
pathogens (S. aureus and B. subtilis), its activity was lower
a
b
c
Staphylococcus aureus 29213. Bacillus subtilis 168. Escherichia coli
25922.
compared to 20aa. These results implied that fluorine on the
phenyl ring of this novel tricyclic fused oxazolidinone was not
only essential for the anti-TB activity but also critical for its
antibacterial activity. The effect of fluorination on anti-TB
activity was consistent with benzo[1,4]oxazinyl-
oxazolidinones;22,23 however, for antibacterial activity, the
effect was contrary to that previously reported.20 Subsequently,
we investigated the effect on the activity by switching the F and
pyridine (or morpholine) positions. Obviously, compounds
( )-45−48 with F at the R1 position resulted in a complete
loss of antitubercular activity (MIC > 32 μg/mL); ( )-45 and
( )-47 even with a pyridine group at R2 displayed no
antibacterial activity (MIC > 64 μg/mL), indicating that the
positions of the substituents on the B ring also have an
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Table 4. Activities against Drug-Resistant Bacteria of Compounds 20aa and 35
MIC (μg/mL)
a
b
c
d
e
f
compd
20aa
35
INH
RIF
Mtb 13946
Mtb 14862
MRSA
MRSE
VISA
VRE
0.48
0.39
2.38
>10
0.82
0.85
>10
>10
0.25−0.5
0.5−4
1
1
0.25
0.5
0.25
0.25
ampicillin
vancomycin
>32
0.5−1
>32
<2
>32
>32
a
Resistance to isoniazid (INH), streptomycin (SM), rifampicin (RFP), ethambutol (EMB), rifabutin (RBT), p-aminosalicylate (PAS), and
b
c
ofloxacin (OLFX). Resistance to INH, SM, RFP, EMB, PAS, prothionamide (1321), and capreomycin (CPM). Methicillin resistant
Staphylococcus aureus, four strains. Methicillin resistant Staphylococcus epidermidis. Vancomycin intermediate Staphylococcus aureus. Vancomycin
resistant Enterococcus.
d
e
f
important effect on activity. Unsurprisingly, all these
compounds were inactive against Gram-negative bacteria E.
coli similar to the reference linezolid.
20aa (MIC = 3.63 μg/mL) and 35 (MIC = 2.32 μg/mL)
exhibited some activity against linezolid-resistant TB strain, for
drug-sensitive strain Mtb H37Rv, these two compounds
displayed reduced activity as about 3- to 7-fold increase in
MIC values (20aa 0.48 μg/mL and 35 0.54 μg/mL). In
addition, we evaluated the activities of 20aa and 35 against
linezolid-resistant Enterococcus and Staphylococcus strains. The
results showed that 20aa and 35 exhibited superior activities
against linezolid-resistant Enterococcus (MIC = 1−2 μg/mL)
and linezolid-resistant Staphylococcus (MIC = 2−4 μg/mL).
However, the antibacterial activity of 20aa and 35 against
linezolid-resistant Gram-positive bacteria was substantially
inferior compared to that against the standard strains. The
results were similar to antituberculosis activities of compounds
20aa and 35 against linezolid-resistant TB strain, indicating
that this novel benzo[1,3]oxazinyloxazolidinone scaffold might
have a similar mode of action as linezolid.
As we desired to assess the druggability of the most potent
compounds, their safety profile should be conducted as the first
pass assessment. Compound 20aa was selected for evaluation
on in vitro toxicity, due to its highly potent antibacterial
activity, particularly against MRSA. As shown in Table 6,
compound 20aa showed no cytotoxicity against Vero cell lines
(IC50 > 64 μg/mL). Its low hERG K+ channel inhibition
precluded its low QT prolongation risk. Compound 20aa
showed comparable mitochondrial protein synthesis (MPS)
inhibition compared to linezolid (IC50 = 9.28 μM).29 This
suggests that compound 20aa has a similar safety profile as
linezolid in terms of myelosuppression risk. Monoamine
oxidase (MAO) inhibition is one of the adverse effects of
oxazolidinone during long-term dosing; therefore, the MAO-A
and MAO-B inhibition profiles of compound 20aa were
evaluated to assess the potential risk of toxicity. Compound
20aa showed no activity against MAO-A and moderate activity
against MAO-B, which implies that compound 20aa could
have an improved safety property. Bearing good safety profile,
compound 20aa was subsequently tested for its metabolic
Prompted by two promising compounds 20aa and 35 which
displayed significant potency against Mtb H37Rv and Gram-
positive bacteria, we subsequently concentrated on evaluating
these two compounds for their activities against drug-resistant
TB strains and drug-resistant Gram-positive strains (Table 4).
We were pleased to find that compounds 20aa and 35
displayed equivalent potency against two clinically isolated
strains 13946 and 14862 (extensively drug-resistant TB)
compared with the drug-sensitive strain. Meanwhile, as
displayed in Table 4, 20aa and 35 exhibited excellent
antibacterial activity against all tested Gram-positive drug-
resistant bacteria, with MIC values of 0.25−0.5 μg/mL (20aa)
and 0.5−4 μg/mL (35) against MRSA, 1 μg/mL (20aa and
35) against MRSE, 0.25 μg/mL (20aa) and 0.5 μg/mL (35)
against VISA, and 0.25 μg/mL (20aa and 35) against VRE.
Furthermore, we investigated the activities of compounds 20aa
and 35 against linezolid-resistant bacteria (Table 5). Although
Table 5. Activities Against Linezolid-Resistant Bacteria of
Compounds 20aa and 35
MIC (μg/mL)
linezolid
20aa
35
Mtb
14.54
2
4−8
2
8
3.63
0.5
1−2
1
2
2−4
2.32
0.5
1−2
1
2
2−4
a
E. f
E. f
E. f
E. f
b
c
d
e
S. c
16 to >16
a
b
E. faecalis ATCC 29212. Linezolid-resistant E. faecalis, six strains.
E. faecium ATCC 49624. Linezolid-resistant E. faecium 13549.
Linezolid-resistant S. capitis, five strains.
c
d
e
Table 6. Representative Properties of Compound 20aa
a
b
MLM
HLM
IC50 (μM)
substrate remaining
substrate remaining
Vero cytotoxicity,
hERG K+
inhibition
c
d
c
d
e
f
f
compd
(%)
stability
108
(%)
stability
106
IC50 (μg/mL)
MPS inhibition MAO-A MAO-B
1.63 >50 6.07
20aa
95.5
118
>64
17.82
a
b
c
Mouse liver microsome. Human liver microsome. Substrate concentrations were determined in incubations with NADPH after 30 min and
d
e
f
normalized to concentrations at time zero. Stability was determined without the NADPH cofactor. Mitochondrial protein synthesis. Positive
control, leflunomide (IC50): 9.40 μM for MAO-A, 5.58 μM for MAO-B.
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stability. It was noted that this compound showed excellent
stability against mouse and human liver microsomes. This
further demonstrated that this novel tricyclic fused benzo-
[1,3]oxazinyloxazolidinone scaffold may have an acceptable PK
profile for further development.
The aforementioned results encouraged us to further
investigate the pharmacokinetic properties of compound
20aa in BALB/c mice by following a single oral administration
(dose, 10 mg/kg) and an intravenous injection (dose, 1 mg/
kg). As illustrated in Table 7, compound 20aa exhibited
tuberculosis. It was found that a (3S,3aS) absolute configuration
for the novel tricyclic fused oxazolidinone is required for
potency. The systematic SAR evaluation exhibited that these
new oxazolidinones differ from the previously described
benzo[1,4]oxazinyloxazolidinones. Fluorine on the phenyl
ring was not only essential for anti-TB activity but also
significant for antibacterial activity. Compounds with hetero-
aromatic substituents at the para-position relative to the
oxygen of benzo[1,3]oxazinyl framework exhibited relatively
high potency against M. tuberculosis and Gram-positive strains;
however, the compounds bearing a morpholine group lost
antituberculosis activity and displayed moderate activity
against S. aureus and B. subtilis. Two compounds, 20aa and
35, displayed good to excellent in vitro antibacterial activity
including against drug-resistant TB strains, MRSA, MRSE,
VISA, and VRE, as well as certain activities to some linezolid-
resistant strains. The most superior compound 20aa exhibited
good druggability exemplified by potent antibacterial activity,
excellent microsomal stability, no cytotoxicity against Vero
cells, low hERG K+ channel inhibition, comparable MPS
inhibition, and acceptable inhibitory activity of MAO.
Moreover, compound 20aa exhibited an excellent mouse PK
profile with high plasma exposure, high maximal plasma
concentration, appropriate half-life, and excellent oral bioavail-
ability after oral administration. Thus, we believe that 20aa is a
promising lead compound for further investigation and
subsequent development. This novel conformationally con-
strained benzo[1,3]oxazinyloxazolidinone scaffold could pro-
vide new insight into the identification of new generation
oxazolidinone for the treatment of serious infection caused by
drug-resistant bacteria, which is still the unmet medical needs
worldwide.
Table 7. Mouse PK Properties of Compound 20aa
parameter
unit
iv (1 mg/kg)
4.08
po (10 mg/kg)
a
t1/2
h
h
3.76
1.33
Tmax
C0/Cmax
AUC(0−t)
AUC(0−∞)
ng/mL
h·ng/mL
h·ng/mL
h
L/kg
(mL/min)/kg
%
1791
6080
6162
4.71
0.762
2.71
10253
77649
78669
5.86
b
c
MRT(0−∞)
d
V
e
CL
F
f
128
a
b
c
Plasma elimination half-life. Plasma exposure. Mean residence
d
e
f
time. Apparent volume of distribution. Clearance rate. Oral
bioavailability.
excellent PK profiles with high plasma exposure (AUC0−∞
78 669 h·ng/mL), high maximal plasma concentration (Cmax
=
=
10 253 ng/mL), appropriate half-life (t1/2 = 3.76 h), and
excellent oral bioavailability (128%). Furthermore, compound
20aa was rapidly absorbed after oral administration, with 8290
ng/mL plasma concentration after 15 min, and remained at
1730 ng/mL after 12 h (Figure 4, Supporting Information
EXPERIMENTAL SECTION
■
Chemistry. General. All solvents and reagents were obtained
commercially and used without further purification unless otherwise
stated. 1H NMR or 13C NMR spectra were recorded on Varian 400 or
500 MHz spectrometer using CDCl3, DMSO-d6, or acetone-d6 as
solvent and tetramethylsilane (TMS) as an internal standard.
Chemical shifts (δ) were referenced to the residual solvent peak
and reported in ppm, and all coupling constantd (J) were given in Hz.
The following multiplicity abbreviations are used: (s) singlet, (d)
doublet, (t) triplet, (q) quartet, (m) multiplet, and (brs) broad. High-
resolution mass spectra were recorded using a Thermo Exactive
Orbitrap plus mass spectrometer (ESI). TLC was performed on silica
gel plates (GF254) with visualization of components by UV light (254
nm). Silica gel 300−400 mesh was used for all flash column
chromatography experiments. Melting points were determined on
Yanaco MP-J3 microscope melting point apparatus. The purity of all
final compounds (≥95%) was established by high-performance liquid
chromatography (HPLC), which was carried out on a Thermo Fisher
Accela HPLC system with an Agilent Zorbax SB-C18 column (5 μm,
2.1 mm × 50 mm), a column temperature of 40 °C, detection
wavelength at 254 nm, flow rate = 0.3 mL/min, and a gradient of 5−
95% MeCN in water (both containing 0.1 vol% of HCOOH) in 10
min.
Figure 4. Mean blood concentration−time profiles of compound
20aa in mouse after oral administration (10 mg/kg) and intravenous
injection (1 mg/kg) of compound 20aa (n = 3). The data are
presented as the means SD (Table S1).
Table S1). The high plasma concentration and quick
absorption indicated that compound 20aa could be an
excellent antibacterial agent in a clinical setting.
Synthetic Procedure for Compounds ( )-20a and ( )-20b.
Benzyl 6-Bromo-7-fluoro-2-(3-(4-methoxybenzyl)-2-oxooxazolidin-
5-yl)-2H-benzo[e][1,3]oxazine-3(4H)-carboxylate (( )-15a) and
(( )-15b). A mixture of compound 14 (9.2 g, 39.1 mmol), 10a (5.5
g, 15.5 mmol), and p-toluenesulfonic acid monohydrate (0.3 g, 1.6
mmol) in toluene (80 mL) was refluxed under continuous removal of
water using Dean−Stark apparatus for 6 h. After cooling down to
room temperature, the mixture was washed with water and dried over
sodium sulfate. The products of ( )-15a and ( )-15b were obtained
by chromatography eluting with petroleum/ethyl acetate. ( )-15a
CONCLUSIONS
■
In summary, we have developed a series of novel
oxazolidinones containing the benzo[e]oxazolo[4,3-b][1,3]-
oxazin-1-one scaffold and assessed them as highly potent
antibacterial agents against both Gram-positive strains and M.
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(4.4 g, yield: 49%), white solid. Mp: 150−152 °C. LC−MS (ESI) m/z
[M + H]+: 571.0879 and 573.0859. 1H NMR (400 MHz, acetone-d6)
δ: 7.52 (d, J = 7.6 Hz, 1 H), 7.45−7.34 (m, 5 H), 7.21 (d, J = 8.0 Hz,
2 H), 6.89 (d, J = 8.4 Hz, 2 H), 6.78 (d, J = 9.6 Hz, 1 H), 6.05 (d, J =
8.4 Hz, 1 H), 5.23 (d, J = 12.4 Hz, 1 H), 5.19 (d, J = 12.4 Hz, 1 H),
5.07 (d, J = 17.6 Hz, 1 H), 4.86 (td, J = 5.2, 8.4 Hz, 1 H), 4.46 (d, J =
17.2 Hz, 1 H), 4.38 (d, J = 14.4 Hz, 1 H), 4.32 (d, J = 14.8 Hz, 1 H),
3.78 (s, 3 H), 3.62 (t, J = 9.2 Hz, 1 H), 3.49 (dd, J = 5.2, 9.2 Hz, 1 H).
( )-15b (2.2 g, yield: 24.7%), colorless oil. LC−MS (ESI) m/z [M +
precipitate was filtered and washed with water and dried to afford
( )-18a as a white solid (1.4 g, 89%). Mp: 182−184 °C. LC−MS
(ESI) m/z [M + Na]+: 439.0265 and 441.0246. 1H NMR (400 MHz,
CDCl3) δ: 7.26 (d, J = 7.2 Hz, 1 H), 6.70 (d, J = 8.8 Hz, 1 H), 5.45 (s,
1 H), 4.89 (brs, 1 H), 4.77 (d, J = 16.8 Hz, 1 H), 4.63 (t, J = 4.8 Hz, 1
H), 4.37 (d, J = 16.8 Hz, 1 H), 3.62−3.48 (m, 2 H), 1.44 (s, 9 H). 13C
NMR (100 MHz, CDCl3) δ: 158.3 (d, JC−F = 247.0 Hz), 156.3, 156.2,
152.4 (d, JC−F = 11.0 Hz), 130.8 (d, JC−F = 1.0 Hz), 116.2 (d, JC−F
=
4.0 Hz), 106.6 (d, JC−F = 26.0 Hz), 102.1 (d, JC−F = 21.0 Hz), 83.6,
80.5, 78.9, 41.4, 39.8, 28.3.
1
H]+: 571.0879 and 573.0859. H NMR (400 MHz, acetone-d6) δ:
7.47 (d, J = 7.6 Hz, 1 H), 7.41−7.34 (m, 5 H), 7.23 (d, J = 8.4 Hz, 2
H), 6.93 (d, J = 8.4 Hz, 2 H), 6.80 (d, J = 9.6 Hz, 1 H), 6.08 (d, J =
6.8 Hz, 1 H), 5.22 (d, J = 12.4 Hz, 1 H), 5.17 (d, J = 12.4 Hz, 1 H),
5.05 (d, J = 17.2 Hz, 1 H), 4.95−4.89 (m, 1 H), 4.45 (d, J = 17.2 Hz,
1 H), 4.33 (d, J = 14.8 Hz, 1 H), 4.28 (d, J = 15.2 Hz, 1 H), 3.79 (s, 3
H), 3.66 (t, J = 8.8 Hz, 1 H), 3.36 (dd, J = 6.0, 9.2 Hz, 1 H).
Benzyl 6-Bromo-7-fluoro-2-(-2-oxooxazolidin-5-yl)-2H-benzo[e]-
[1,3]oxazine-3(4H)-carboxylate (( )-16a). To a mixed solvent
(MeCN/H2O = 85.0 mL:9.5 mL) were added ( )-15a (3.4 g, 5.9
mmol) and CAN (12.9 g, 23.6 mmol). The mixture was stirred for 2 h
at room temperature and then poured into water (200 mL). The
resulting solution was extracted with DCM (100 mL × 3). The
combined organic layer was washed with water, brine and dried over
sodium sulfate. The crude product was purified by chromatography
(DCM/MeOH = 100:1) to afford ( )-16a as a white solid (2.1 g,
78%). Mp: 85−87 °C. LC−MS (ESI) m/z [M + H]+: 451.0318 and
453.0296. 1H NMR (400 MHz, acetone-d6) δ: 7.55 (d, J = 7.6 Hz, 1
H), 7.45−7.42 (m, 2 H), 7.39−7.33 (m, 3 H), 6.83 (d, J = 10.0 Hz, 1
H), 6.69 (s, 1 H), 6.18 (d, J = 8.4 Hz, 1H), 5.25 (d, J = 12.4 Hz, 1 H),
5.21 (d, J = 12.8 Hz, 1 H), 5.10 (d, J = 17.2 Hz, 1 H), 4.95 (td, J =
5.6, 8.8 Hz, 1 H), 4.49 (d, J = 17.2 Hz, 1 H), 3.77 (td, J = 0.8, 9.2 Hz,
1 H), 3.70−3.66 (m, 1 H).
Benzyl 6-Bromo-7-fluoro-2-(-2-oxooxazolidin-5-yl)-2H-benzo[e]-
[1,3]oxazine-3(4H)-carboxylate (( )-16b). Compound ( )-16b (1.6
g, 76%) was prepared from ( )-15b (2.5 g, 4.3 mmol) according to
the procedure described for ( )-16a. Mp: 80−82 °C. LC−MS (ESI)
m/z [M + H]+: 451.0280 and 453.0260. 1H NMR (400 MHz,
acetone-d6) δ: 7.49 (d, J = 7.6 Hz, 1 H), 7.45−7.34 (m, 5H), 6.88 (d,
J = 10.0 Hz, 1 H), 6.61 (s, 1 H), 6.12 (d, J = 7.2 Hz, 1 H), 5.24 (s, 2
H), 5.09 (d, J = 17.2 Hz, 1 H), 5.04−4.98 (m, 1 H), 4.48 (d, J = 17.2
Hz, 1 H), 3.80 (t, J = 9.2 Hz, 1 H), 3.47 (dd, J = 6.4, 9.2 Hz,1 H).
Benzyl 6-Bromo-2-(-3-(tert-butoxycarbonyl)-2-oxooxazolidin-5-
yl)-7-fluoro-2H-benzo[e][1,3]oxazine-3(4H)-carboxylate (( )-17a).
A mixture of ( )-16a (2.2 g, 4.8 mmol), TEA (1.0 mL, 7.3 mmol),
(Boc)2O (1.2 g, 5.7 mmol), DMAP (49.0 mg, 0.1 mmol) in DCM
(20.0 mL) was stirred at room temperature for 2 h. The solution was
washed with water and brine, dried over sodium sulfate, filtered, and
concentrated. The residue was purified by chromatography (PE/
DCM = 6:4) to give ( )-17a as a white solid (2.1 g, 78%). LC−MS
(ESI) m/z [M + Na]+: 573.0637 and 575.0617. Mp: 161−163 °C. 1H
NMR (400 MHz, acetone-d6) δ: 7.56 (d, J = 7.6 Hz, 1 H), 7.44−7.41
(m, 2 H), 7.40−7.32 (m, 3 H), 6.88 (d, J = 9.6 Hz, 1 H), 6.28 (d, J =
8.8 Hz, 1 H), 5.25 (d, J = 12.4 Hz, 1 H), 5.21 (d, J = 12.8 Hz, 1 H),
5.11 (d, J = 17.2 Hz, 1 H), 4.98−4.92 (m, 1 H), 4.50 (d, J = 16.8 Hz,
1 H), 4.16−4.08 (m, 2 H), 1.50 (s, 9 H).
tert-Butyl ((7-Bromo-6-fluoro-1-oxo-3,3a-dihydro-1H,9H-benzo-
[e]oxazolo[4,3-b][1,3]oxazin-3-yl)methyl)carbamate (( )-18b).
Compound ( )-18b (1.1 g, 91%) was prepared from ( )-17b (1.6
g, 2.9 mmol) according to the procedure described for ( )-18a. Mp:
88−90 °C. LC−MS (ESI) m/z [M + Na]+: 439.0258 and 441.0236.
1H NMR (400 MHz, CDCl3) δ: 7.28 (d, J = 7.2 Hz, 1 H), 6.72 (d, J =
9.2 Hz, 1 H), 5.43 (d, J = 5.2 Hz,1 H), 4.91 (brs, 1 H), 4.83−4.77 (m,
2 H), 4.41 (d, J = 16.4 Hz, 1 H), 3.85−3.78 (m, 1 H), 3.62−3.55 (m,
1 H),1.46 (s, 9 H). 13C NMR (100 MHz, CDCl3) δ: 158.3 (d, JC−F
247.0 Hz), 156.3, 155.8, 152.1 (d, JC−F = 11.0 Hz), 130.9 (d, JC−F
=
=
2.0 Hz), 116.2 (d, JC−F = 4.0 Hz), 106.7 (d, JC−F = 25.0 Hz), 102.4 (d,
JC−F = 22.0 Hz), 82.0, 80.3, 75.8, 40.0, 39.0, 28.4.
tert-Butyl ((7-(6-Cyanopyridin-3-yl)-6-fluoro-1-oxo-3,3a-dihydro-
1H,9H-benzo[e]oxazolo[4,3-b][1,3]oxazin-3-yl)methyl)carbamate
(( )-19a). To a three-necked bottle containing ( )-18a (270 mg,
0.65 mmol), 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
picolinonitrile (194 mg, 0.84 mmol), Pd(PPh3)4(74.8 mg, 0.06
mmol), Na2CO3(137.3 mg, 1.30 mmol) was added solvent (DMSO/
H2O = 9.0 mL: 1.5 mL) under an atmosphere of argon. The reaction
was stirred for 5 h at 100 °C and then cooled to room temperature.
The resulting mixture was poured into water (30 mL) and extracted
with DCM (15 mL × 3). The combined organic layer was washed
with water and brine, dried over sodium sulfate, filtered, and
concentrated. The residue was purified by chromatography (DCM/
MeOH = 100:1) to afford ( )-19a as an off-white solid (240 mg,
yield: 84%). Mp: 167−169 °C. LC−MS (ESI) m/z [M + H]+:
1
441.1558. H NMR (400 MHz, CDCl3) δ: 8.83 (s, 1 H), 7.97−7.94
(m, 1 H), 7.77 (dd, J = 0.8, 8.0 Hz, 1 H), 7.17 (d, J = 8.0 Hz, 1 H),
6.79 (d, J = 11.2 Hz, 1 H), 5.56 (s, 1 H), 4.90 (brs, 1 H), 4.86 (d, J =
16.8 Hz, 1 H), 4.67 (t, J = 4.8 Hz, 1 H), 4.47 (d, J = 16.4 Hz, 1 H),
3.65−3.50 (m, 2 H), 1.45 (s, 9 H).
tert-Butyl ((7-(6-Cyanopyridin-3-yl)-6-fluoro-1-oxo-3,3a-dihydro-
1H,9H-benzo[e]oxazolo[4,3-b][1,3]oxazin-3-yl)methyl)carbamate
(( )-19b). Compound ( )-19b (155 mg, 81%) was prepared from
( )-18b (200 mg, 0.48 mmol) in the same manner as described for
( )-19a. Mp: 98−100 °C. LC−MS (ESI) m/z [M + H]+: 441.1562.
1H NMR (400 MHz, CDCl3) δ: 8.83 (s, 1 H), 7.97−7.94 (m, 1 H),
7.77 (dd, J = 0.8, 8.0 Hz, 1 H), 7.19 (d, J = 8.4 Hz, 1 H), 6.81 (d, J =
11.2 Hz, 1 H), 5.52 (d, J = 4.8 Hz, 1 H), 4.94−4.88 (m, 2 H), 4.84−
4.80 (m, 1 H), 4.51 (d, J = 16.8 Hz, 1 H), 3.90−3.82 (m, 1 H), 3.65−
3.59 (m, 1 H), 1.47 (s, 9 H).
N-((7-(6-Cyanopyridin-3-yl)-6-fluoro-1-oxo-3,3a-dihydro-1H,9H-
benzo[e]oxazolo[4,3-b][1,3]oxazin-3-yl)methyl)acetamide
(( )-20a). Trifluoroacetic acid (7.0 mL) was added to a solution of
compound ( )-19a (404.4 mg, 0.91 mmol) in DCM (20 mL). The
reaction mixture was stirred for 2 h at room temperature and then
neutralized with saturated sodium bicarbonate aqueous solution. The
resulting mixture was extracted with DCM (10 mL × 3). The
combined organic layer was wash with water, brine, dried over sodium
sulfate, and filtered. To this filtered solution were added TEA (190.0
μL, 1.4 mmol) and acetyl chloride (84.0 μL, 1.2 mmol) sequentially.
The mixture was stirred for 2 h at 0 °C. Then the solution was washed
with water and brine, dried over sodium sulfate, filtered, and
concentrated. The residue was purified by chromatography (DCM/
MeOH = 100:1) to give ( )-20a as an off-white solid (305 mg, 87%).
Mp: 210−212 °C. HRMS (ESI) m/z [M + H]+: calcd for
C19H16FN4O4 383.1156, found 383.1144. 1H NMR (400 MHz,
CDCl3) δ: 8.82 (s, 1 H), 7.95 (dt, J = 1.6, 8.0 Hz, 1 H), 7.76 (dd, J =
0.4, 8.0 Hz, 1 H), 7.16 (d, J = 8.0 Hz, 1 H), 6.79 (d, J = 11.2 Hz, 1
H), 6.05 (t, J = 6.4 Hz,1 H), 5.51 (d, J = 1.2 Hz, 1 H), 4.84 (d, J =
Benzyl 6-Bromo-2-(-3-(tert-butoxycarbonyl)-2-oxooxazolidin-5-
yl)-7-fluoro-2H-benzo[e][1,3]oxazine-3(4H)-carboxylate (( )-17b).
In a similar manner to ( )-17a, compound ( )-17b (1.4 g, 82%)
was prepared from ( )-16b (1.4 g, 3.1 mmol). Mp: 92−94 °C. LC−
1
MS (ESI) m/z [M + Na]+: 573.0620 and 575.0604. H NMR (400
MHz, acetone-d6) δ: 7.51 (d, J = 7.6 Hz, 1 H), 7.45−7.43 (m, 2 H),
7.40−7.34 (m, 3 H), 6.88(d, J = 10.0 Hz, 1 H), 6.24 (d, J = 7.2 Hz, 1
H), 5.25 (s, 2 H), 5.10 (d, J = 16.8 Hz, 1 H), 5.04−4.98 (m, 1 H),
4.52 (d, J = 17.2 Hz, 1 H), 4.19 (dd, J = 8.8, 10.4 Hz, 1 H), 3.86 (dd, J
= 6.4, 10.4 Hz, 1 H), 1.49 (s, 9 H).
tert-Butyl ((7-Bromo-6-fluoro-1-oxo-3,3a-dihydro-1H,9H-benzo-
[e]oxazolo[4,3-b][1,3]oxazin-3-yl)methyl)carbamate (( )-18a). To
a solution of MeOH (20 mL) containing ( )-17a (2.1 g, 3.8 mmol)
was added Cs2CO3(4.5 g, 7.6 mmol). The mixture was stirred for 3 h
at room temperature, then poured into water (100 mL). The
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16.4 Hz, 1 H), 4.70 (t, J = 4.8 Hz, 1 H), 4.49 (d, J = 16.4 Hz, 1 H),
3.71 (t, J = 5.6 Hz, 2 H), 2.04 (s, 3 H). 13C NMR (100 MHz, CDCl3)
δ: 171.1, 159.1 (d, JC−F = 250.0 Hz), 156.2, 154.2 (d, JC−F = 12.0 Hz),
150.6 (d, JC−F = 3.0 Hz),136.7 (d, JC−F = 4.0 Hz), 134.2 (d, JC−F = 2.0
Hz), 132.5, 128.2, 128.1 (d, JC−F = 4.0 Hz), 118.7 (d, JC−F = 15.0 Hz),
117.2, 115.8 (d, JC−F = 4.0 Hz), 106.5 (d, JC−F = 25.0 Hz), 83.8, 78.9,
40.2, 40.1, 23.1.
N-((7-(6-Cyanopyridin-3-yl)-6-fluoro-1-oxo-3,3a-dihydro-1H,9H-
benzo[e]oxazolo[4,3-b][1,3]oxazin-3-yl)methyl)acetamide
(( )-20b). Compound ( )-20b (154 mg, 80%) was prepared from
( )-19b (222 mg, 0.50 mmol) in the same manner as described for
( )-20a. Mp: 204−206 °C. HRMS (ESI) m/z [M + H]+: calcd for
C19H16FN4O4 383.1156, found 383.1141. 1H NMR (400 MHz,
CDCl3) δ: 8.83 (s, 1 H), 7.97−7.94 (m, 1 H), 7.77 (dd, J = 0.8, 8.0
Hz, 1H), 7.20 (d, J = 8.4 Hz, 1 H), 6.81 (d, J = 10.8 Hz, 1 H), 5.94 (t,
J = 6.8 Hz, 1 H), 5.53 (d, J = 4.8 Hz, 1 H), 4.90 (d, J = 16.4 Hz, 1 H),
4.86−4.81 (m, 1 H), 4.52 (d, J = 16.8 Hz, 1 H), 4.20−4.14 (m, 1 H),
3.57−3.50 (m, 1 H), 2.06 (s, 3 H). 13C NMR (100 MHz, CDCl3) δ:
170.7, 159.2 (d, JC−F = 250.0 Hz), 156.2, 153.9 (d, JC−F = 12.0 Hz),
150.6 (d, JC−F = 3.0 Hz), 136.7 (d, JC−F = 4.0 Hz), 134.1, 132.6,
128.2, 128.1, 119.0 (d, JC−F = 14.0 Hz), 117.1, 115.8 (d, JC−F = 4.0
Hz), 106.6 (d, JC−F = 26.0 Hz), 82.1, 75.8, 40.2, 38.1, 23.2.
MHz, CDCl3) δ: 156.4, 156.1, 151.3, 131.5, 129.4, 120.8, 119.8,
114.9, 83.4, 80.4, 79.0, 41.5, 40.2, 28.3.
Synthetic Procedure for Compounds 20aa and 35−38. tert-
Butyl (((3S,3aS)-7-bromo-6-fluoro-1-oxo-3,3a-dihydro-1H,9H-
benzo[e] oxazolo[4,3-b][1,3]oxazin-3-yl)methyl)carbamate (18a).
Chiral resolution of ( )-18a afforded 18a. HPLC condition:
CHIRALPAK AD-H column, MeOH = 100%, 1.0 mL/min, λ =
29
214 nm, 35 °C. Rt: 5.5 min. Mp: 130−132 °C. [α]D = −204.9 (c
0.287, CHCl3).
tert-Butyl (((3S,3aS)-7-Bromo-1-oxo-3,3a-dihydro-1H,9H-benzo-
[e]oxazolo[4,3-b][1,3]oxazin-3-yl)methyl)carbamate (24). Chiral
resolution of ( )-24 afforded 24. HPLC condition: CHIRALPAK
IA column, hexane/EtOH = 50:50, 1.0 mL/min, λ = 254 nm, 25 °C.
29
Rt: 9.3 min. Mp: 114−115 °C. [α]D = −200.7 (c 0.300, CHCl3).
tert-Butyl (((3S,3aS)-7-(6-Cyanopyridin-3-yl)-6-fluoro-1-oxo-
3,3a-dihydro-1H,9H-benzo[e]oxazolo[4,3-b][1,3]oxazin-3-yl)-
methyl)carbamate (25). Compound 25 (185 mg, 88%) was prepared
from 18a (200 mg, 0.48 mmol) according to the procedure described
for ( )-19a. Mp: 148−150 °C. LC−MS (ESI) m/z [M + H]+:
441.1573.
tert-Butyl (((3S,3aS)-6-Fluoro-7-(6-(2-methyl-2H-tetrazol-5-yl)-
pyridin-3-yl)-1-oxo-3,3a-dihydro-1H,9H-benzo[e]oxazolo[4,3-b]-
[1,3]oxazin-3-yl)methyl)carbamate (26). Compound 26 (150 mg,
57%) was prepared from 18a (220 mg, 0.53 mmol) and 2-(2-methyl-
2H-tetrazol-5-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
pyridine (181.6 mg, 0.63 mmol) according to the procedure described
for ( )-19a. Mp: 95−97 °C. LC−MS (ESI) m/z [M + H]+:
498.1878. 1H NMR (400 MHz, CDCl3) δ: 8.87 (s, 1 H), 8.30 (d, J =
8.0 Hz,1 H), 7.98 (dt, J = 8.0, 1.6 Hz, 1 H), 7.20 (d, J = 8.0 Hz, 1 H),
6.79 (d, J = 11.2 Hz, 1 H), 5.54 (s, 1 H), 4.92−4.85 (m, 2 H), 4.67
(td, J = 1.2, 4.8 Hz, 1 H), 4.48 (d, J = 16.4 Hz, 1 H), 4.47 (s, 3 H),
3.64−3.51 (m, 2 H), 1.45 (s, 9 H).
N-(((3S,3aS)-7-(6-Cyanopyridin-3-yl)-6-fluoro-1-oxo-3,3a-dihy-
dro-1H,9H-benzo[e]oxazolo[4,3-b][1,3]oxazin-3-yl)methyl)-
acetamide (20aa) and N-(((3R,3aR)-7-(6-Cyanopyridin-3-yl)-6-
fluoro-1-oxo-3,3a-dihydro-1H,9H-benzo[e]oxazolo[4,3-b][1,3]-
oxazin-3-yl)methyl)acetamide (20ab). Chiral resolution of racemic
( )-20a afforded 20aa and 20ab. HPLC separation condition:
CHIRALPAK IA column, MeOH/EtOAc = 80:20, 1 mL/min, λ =
29
254 nm, 25 °C. Rt (20aa) = 2.95 min, Rt (20ab) = 3.52 min. [α]D
(20aa) = −298.8 (c 0.332, CHCl3), [α]D29 (20ab) = +268.9 (c 0.238,
CHCl3).
tert-Butyl (((3S,3aS)-6-Fluoro-7-morpholino-1-oxo-3,3a-dihydro-
1H,9H-benzo[e]oxazolo[4,3-b][1,3]oxazin-3-yl)methyl)carbamate
(27). To a three necked-bottle containing 18a (300 mg, 0.72 mmol),
morpholine (125.0 mg, 1.44 mmol), X-PHOS (103.0 mg, 0.22
mmol), Pd(OAc)2 (24.2 g, 0.11 mmol), and Cs2CO3(351.6 mg, 1.08
mmol) was added toluene (15.0 mL) under an atmosphere of argon.
The mixture was stirred at 100 °C for 6 h and then filtered. The
filtered solution was concentrated and purified by chromatography
(PE/EtOAc = 3:1) to give 27 as an off-white solid (134 mg, 44%).
Synthetic Procedure for Compound ( )-24. Benzyl 6-Bromo-
2-(3-(4-methoxybenzyl)-2-oxooxazolidin-5-yl)-2H-benzo[e][1,3]-
oxazine-3(4H)-carboxylate (( )-21). Compound ( )-21 (3.7 g,
50%) was prepared from 10b and 14 in the same manner as described
for ( )-15a. Mp: 134−136 °C. LC−MS (ESI) m/z [M + Na]+:
1
575.0792 and 577.0781. H NMR (400 MHz, acetone-d6) δ: 7.42−
7.25 (m, 7 H), 7.19 (d, J = 8.0 Hz, 2 H), 6.85 (d, J = 8.0 Hz, 2 H),
6.74 (d, J = 8.8 Hz, 1 H), 5.98 (d, J = 8.8 Hz, 1 H), 5.21 (d, J = 12.4
Hz, 1 H), 5.16 (d, J = 12.4 Hz, 1 H), 5.03 (d, J = 17.2 Hz, 1 H),
4.83−4.77 (m, 1 H), 4.46 (d, J = 16.8 Hz, 1 H), 4.35 (d, J = 14.8 Hz,
1 H), 4.28 (d, J = 15.2 Hz, 1 H), 3.75 (s, 3 H), 3.57 (t, J = 9.2 Hz, 1
H), 3.49−3.43 (m, 1 H).
1
Mp: 89−91 °C. LC−MS (ESI) m/z [M + H]+: 424.1880. H NMR
(400 MHz, CDCl3) δ: 6.67−6.62 (m, 2 H), 5.38 (s, 1 H), 4.87 (s, 1
H), 4.73 (d, J = 16.4 Hz, 1 H), 4.61 (t, J = 4.8 Hz, 1 H), 4.37 (d, J =
16.4 Hz, 1 H), 3.88−3.86 (m, 4 H), 3.56−3.49 (m, 2 H), 3.02−3.0
(m, 4 H), 1.44 (s, 9 H).
tert-Butyl (((3S,3aS)-7-(6-Cyanopyridin-3-yl)-1-oxo-3,3a-dihy-
dro-1H,9H-benzo[e]oxazolo[4,3-b][1,3]oxazin-3-yl)methyl)-
carbamate (28). Compound 28 (187 mg, 89%) was prepared from
24 (200 mg, 0.5 mmol) according to the procedure described for 25.
Mp: 151−153 °C. LC−MS (ESI) m/z [M + H]+: 423.1658. 1H NMR
(400 MHz, CDCl3) δ: 8.88 (dd, J = 0.8, 2.4 Hz, 1 H), 7.94 (dd, J =
2.4, 8.0 Hz, 1 H), 7.75 (dd, J = 0.8, 8.4 Hz, 1 H), 7.43 (dd, J = 2.0, 8.4
Hz, 1 H), 7.32 (d, J = 2.4 Hz,1 H), 7.05 (d, J = 8.4 Hz, 1 H), 5.52 (s,
1 H), 4.91−4.87 (m, 2 H), 4.68 (td, J = 4.8, 1.2 Hz, 1 H), 4.52 (d, J =
17.2 Hz, 1 H), 3.65−3.51 (m, 2 H), 1.45 (s, 9 H).
tert-Butyl (((3S,3aS)-7-Morpholino-1-oxo-3,3a-dihydro-1H,9H-
benzo[e]oxazolo[4,3-b][1,3]oxazin-3-yl)methyl)carbamate (29).
Compound 29 (283 mg, 66%) was prepared from 24 (400 mg, 1
mmol) according to the procedure described for 27. Mp: 85−87 °C.
LC−MS (ESI) m/z [M + H]+: 406.1983. 1H NMR (400 MHz,
CDCl3) δ: 6.84 (d, J = 8.8 Hz, 1 H), 6.79 (dd, J = 2.8, 8.8 Hz, 1 H),
6.59 (d, J = 2.8 Hz, 1 H), 5.33 (s, 1 H), 4.87 (brs, 1 H), 4.76 (d, J =
16.4 Hz, 1 H), 4.61 (td, J = 1.2, 4.8 Hz, 1 H), 4.40 (d, J = 16.4 Hz, 1
H), 3.86−3.84 (m, 4 H), 3.55−3.52 (m, 2 H), 3.07−3.05 (m, 4 H),
1.44 (s, 9 H).
5-((3S,3aS)-3-(Aminomethyl)-6-fluoro-1-oxo-3,3a-dihydro-
1H,9H-benzo[e]oxazolo[4,3-b][1,3]oxazin-7-yl)picolinonitrile (30).
Trifluoroacetic acid (4.0 mL) was added to a solution of compound
25 (240 mg, 0.54 mmol) in DCM (15 mL). The reaction mixture was
stirred for 2 h at room temperature and then neutralized with
Benzyl 6-Bromo-2-(2-oxooxazolidin-5-yl)-2H-benzo[e][1,3]-
oxazine-3(4H)-carboxylate (( )-22). Compound ( )-22 (1.9 g,
76%) was prepared from ( )-21 (3.2 g, 5.7 mmol) in the same
manner as described for ( )-16a. Mp: 72−74 °C. LC−MS (ESI) m/
1
z [M + H]+: 433.0388 and 435.0368. H NMR (400 MHz, acetone-
d6) δ: 7.45−7.30 (m, 6 H), 6.83 (d, J = 8.8 Hz, 1 H), 6.67 (s, 1 H),
6.14 (d, J = 8.4 Hz, 1 H), 5.25 (d, J = 12.4 Hz, 1 H), 5.21 (d, J = 12.4
Hz, 1 H), 5.08 (d, J = 17.2 Hz, 1H), 4.95−4.89 (m, 1 H), 4.52 (d, J =
17.6 Hz, 1 H), 3.77−3.65 (m, 2 H).
Benzyl 6-Bromo-2-(3-(tert-butoxycarbonyl)-2-oxooxazolidin-5-
yl)-2H-benzo[e][1,3]oxazine-3(4H)-carboxylate (( )-23). Com-
pound ( )-23 (2.0 g, 90%) was prepared from ( )-22 (1.8 g, 4.1
mmol) in the same manner as described for ( )-17a. Mp: 135−137
°C. LC−MS (ESI) m/z [M + H]+: 555.0742 and 557.0724. 1H NMR
(400 MHz, acetone-d6) δ: 7.44−7.33 (m, 7 H), 6.87 (d, J = 8.4 Hz, 1
H), 6.23 (d, J = 8.8 Hz, 1 H), 5.25 (d, J = 12.4 Hz, 1 H), 5.22 (d, J =
12.4 Hz, 1 H), 5.10 (d, J = 17.2 Hz, 1 H), 5.00−4.85 (m, 1 H), 4.53
(d, J = 17.2 Hz, 1 H), 4.10 (d, J = 7.2 Hz, 2 H), 1.50 (s, 9 H).
tert-Butyl ((7-Bromo-1-oxo-3,3a-dihydro-1H,9H-benzo[e]-
oxazolo[4,3-b][1,3]oxazin-3-yl)methyl)carbamate (( )-24). Com-
pound ( )-24 (1.2 g, 84%) was prepared from ( )-23 (1.9 g, 3.5
mmol) in the same manner as described for ( )-18a. Mp: 165−167
°C. LC−MS (ESI) m/z [M + Na]+: 421.0364 and 423.0343. 1H
NMR (400 MHz, CDCl3) δ: 7.28 (dd, J = 2.4, 8.8 Hz, 1 H), 7.21 (d, J
= 2.0 Hz, 1 H), 6.78 (d, J = 8.8 Hz, 1 H), 5.40 (s, 1 H), 4.91 (brs, 1
H), 4.77 (d, J = 16.8 Hz, 1 H), 4.63 (t, J = 4.8 Hz, 1 H), 4.39 (d, J =
16.8 Hz, 1 H), 3.61−3.49 (m, 2 H), 1.44 (s, 9 H). 13C NMR (100
3243
https://dx.doi.org/10.1021/acs.jmedchem.0c02153
J. Med. Chem. 2021, 64, 3234−3248

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
saturated sodium bicarbonate aqueous solution. The resulting mixture
was extracted with DCM (15 mL × 3). The combined organic layer
was washed with water, brine, dried over sodium sulfate, filtered, and
concentrated. The residue was purified by chromatography (DCM/
MeOH/NH4OH = 100:2:0.5) to afford 30 as a white solid (175 mg,
94%). Mp: 157−159 °C. HRMS (ESI) m/z [M + H]+: calcd for
C17H14FN4O3 341.1050, found 341.1039. 1H NMR (400 MHz,
CDCl3) δ: 8.84 (s, 1 H), 7.96 (dt, J = 2.0, 8.0 Hz, 1 H), 7.76 (dd, J =
0.8, 8.4 Hz, 1 H), 7.19 (d, J = 8.0 Hz, 1 H), 6.80 (d, J = 11.2 Hz, 1
H), 5.41 (d, J = 1.2 Hz,1 H), 4.86 (d, J = 16.4 Hz, 1 H), 4.61 (td, J =
1.2, 5.2 Hz, 1 H), 4.52 (d, J = 16.4 Hz, 1 H), 3.18−3.06 (m, 2 H),
1.29 (brs, 2 H). 13C NMR (125 MHz, CDCl3) δ: 159.1 (d, JC−F = 250
Hz), 156.2, 154.4 (d, JC−F = 12.5 Hz), 150.6, 136.7, 134.3, 132.5,
128.2, 118.6 (d, JC−F = 13.8 Hz), 117.2, 116.0, 106.4 (d, JC−F = 25
Hz), 84.0, 81.0, 42.7, 39.9.
(3S,3aS)-3-(Aminomethyl)-6-fluoro-7-(6-(2-methyl-2H-tetrazol-
5-yl)pyridin-3-yl)-3,3a-dihydro-1H,9H-benzo[e]oxazolo[4,3-b][1,3]-
oxazin-1-one (31). Compound 31 (85 mg, 63%) was prepared from
26 (170 mg, 0.34 mmol) according to the procedure described for 30.
Mp: 125−127 °C. HRMS (ESI) m/z [M + H]+: calcd for
C18H17FN7O3 398.1377, found 398.1353. 1H NMR (400 MHz,
CDCl3) δ: 8.88 (s, 1 H), 8.30 (dd, J = 0.8, 8.0 Hz, 1 H), 7.99 (dt, J =
8.0, 1.6 Hz, 1 H), 7.22 (d, J = 8.0 Hz, 1 H), 6.79 (d, J = 10.8 Hz, 1
H), 5.40 (d, J = 1.6 Hz, 1 H), 4.87 (d, J = 16.4 Hz, 1 H), 4.61 (td, J =
1.2, 5.6 Hz, 1 H), 4.53 (d, J = 16.4 Hz, 1 H), 4.47 (s, 3 H), 3.17−3.05
(m, 2 H), 1.34 (brs, 2 H). 13C NMR (100 MHz, CDCl3) δ: 164.8,
159.2 (d, JC−F = 248.0 Hz), 156.4, 153.6 (d, JC−F = 12.0 Hz), 149.9
(d, JC−F = 4.0 Hz), 145.7, 137.1, 132.1, 128.2 (d, JC−F = 4.0 Hz),
122.1, 119.9 (d, JC−F = 15.0 Hz), 115.7, 106.3 (d, JC−F = 25.0 Hz),
84.0, 81.1, 42.9, 40.1, 39.8.
sodium sulfate, filtered, and concentrated. The residue was purified by
chromatography (DCM/MeOH = 100:1) to give 20aa as an off-white
solid (66.0 mg, 78%). Mp: 159−161 °C. [α]D = −294.8 (c 0.384,
19
CHCl3). HRMS (ESI) m/z [M + H]+: calcd for C19H16FN4O4
383.1156, found 383.1146. 1H NMR (400 MHz, CDCl3) δ: 8.83
(s, 1 H), 7.95 (dt, J = 1.6, 8.0 Hz, 1 H), 7.76 (d, J = 8.0 Hz, 1 H), 7.16
(d, J = 8.0 Hz, 1 H), 6.79 (d, J = 11.2 Hz, 1 H), 5.91 (t, J = 5.6 Hz, 1
H), 5.51 (s, 1 H), 4.85 (d, J = 16.4 Hz, 1 H), 4.70 (t, J = 4.8 Hz,1 H),
4.49 (d, J = 16.8 Hz, 1 H), 3.72 (t, J = 5.2 Hz, 2 H), 2.05 (s, 3 H).
N-(((3S,3aS)-6-Fluoro-7-(6-(2-methyl-2H-tetrazol-5-yl)pyridin-3-
yl)-1-oxo-3,3a-dihydro-1H,9H-benzo[e]oxazolo[4,3-b][1,3]oxazin-
3-yl)methyl)acetamide (35). Compound 35 (44 mg, 89%) was
prepared from 31 (45 mg, 0.11 mmol) according to the procedure
described for 20aa. Mp: 108−110 °C. HRMS (ESI) m/z [M + H]+:
1
calcd for C20H19FN7O4 440.1483, found 440.1455. H NMR (400
MHz, CDCl3) δ: 8.87 (s, 1 H), 8.30 (d, J = 8.4 Hz, 1 H), 7.98 (dt, J =
1.6, 8.0 Hz, 1 H), 7.20 (d, J = 8.0 Hz, 1 H), 6.77 (d, J = 10.8 Hz, 1
H), 6.05 (t, J = 6.4 Hz, 1 H), 5.48 (d, J = 1.2 Hz, 1 H), 4.84 (d, J =
16.4 Hz, 1 H), 4.70 (t, J = 4.4 Hz, 1 H), 4.50 (d, J = 16.4 Hz, 1 H),
4.47 (s, 3 H), 3.73−3.70 (m, 2 H), 2.05 (s, 3 H). 13C NMR (100
MHz, CDCl3) δ: 171.1, 164.7, 159.2 (d, JC−F = 249.0 Hz), 156.3,
153.4 (d, JC−F = 12.0 Hz), 149.9, 145.7, 137.1 (d, JC−F = 3.0 Hz),
132.0, 128.1 (d, JC−F = 5.0 Hz), 122.0, 120.0 (d, JC−F = 15.0 Hz),
115.5 (d, JC−F = 4.0 Hz), 106.2 (d, JC−F = 25.0 Hz), 83.7, 78.9, 40.3,
40.2, 39.8, 23.1.
N-(((3S,3aS)-6-Fluoro-7-morpholino-1-oxo-3,3a-dihydro-1H,9H-
benzo[e]oxazolo[4,3-b][1,3]oxazin-3-yl)methyl)acetamide (36).
Compound 36 (58.4 mg, 86%) was prepared from 32 (60 mg, 0.14
mmol) according to the procedure described for 20aa. Mp: 118−120
°C. HRMS (ESI) m/z [M + H]+: calcd for C17H21FN3O5 366.1465,
1
found 366.1453. H NMR (400 MHz, CDCl3) δ: 6.65−6.59 (m, 2
(3S,3aS)-3-(Aminomethyl)-6-fluoro-7-morpholino-3,3a-dihydro-
1H,9H-benzo[e]oxazolo[4,3-b][1,3]oxazin-1-one (32). Compound
32 (75 mg, 70%) was prepared from 27 (140 mg, 0.33 mmol)
according to the procedure described for 30. Mp: 152−157 °C.
HRMS (ESI) m/z [M + H]+: calcd for C15H19FN3O4 324.1360,
H), 6.32 (brs, 1 H), 5.32 (d, J = 1.2 Hz, 1 H), 4.70 (d, J = 16.4 Hz, 1
H), 4.63 (t, J = 5.2 Hz, 1 H), 4.38 (d, J = 16.4 Hz, 1 H), 3.86−3.84
(m, 4 H), 3.67 (t, J = 5.6 Hz, 2 H), 2.99−2.97 (m, 4 H), 2.02 (s, 3
H). 13C NMR (100 MHz, CDCl3) δ: 171.2, 156.6, 155.1 (d, JC−F
=
1
found 324.1347. H NMR (400 MHz, CDCl3) δ: 6.66−6.62 (m, 2
246.0 Hz), 147.4 (d, JC−F = 11.0 Hz), 135.9 (d, JC−F = 9.0 Hz), 116.3
(d, JC−F = 4.0 Hz), 114.3 (d, JC−F = 3.0 Hz), 106.5 (d, JC−F = 24.0
Hz), 83.5, 79.0, 67.0, 51.4, 51.3, 40.4, 40.3, 23.1.
H), 5.25 (d, J = 1.6 Hz, 1 H), 4.74 (d, J = 16.4 Hz, 1 H), 4.55 (td, J =
1.2, 4.8 Hz, 1 H), 4.41 (d, J = 16.4 Hz,1 H), 3.87−3.85 (m, 4 H),
3.14−2.98 (m, 6 H), 1.37 (brs, 2 H). 13C NMR (100 MHz, CDCl3)
δ: 156.5, 155.0 (d, JC−F = 247.0 Hz), 147.5 (d, JC−F = 12.0 Hz), 135.7
(d, JC−F = 10.0 Hz), 116.3 (d, JC−F = 4.0 Hz), 114.4 (d, JC−F = 4.0
Hz), 106.4 (d, JC−F = 24.0 Hz), 83.6, 81.0, 67.0, 51.4, 51.3, 42.9, 40.2.
5-((3S,3aS)-3-(Aminomethyl)-1-oxo-3,3a-dihydro-1H,9H-benzo-
[e]oxazolo[4,3-b][1,3]oxazin-7-yl)picolinonitrile (33). Compound
33 (98 mg, 69%) was prepared from 28 (187 mg, 0.44 mmol)
according to the procedure described for 30. Mp: 154−156 °C.
HRMS (ESI) m/z [M + H]+: calcd for C17H15N4O3 323.1144, found
323.1130. 1H NMR (400 MHz, CDCl3) δ: 8.89 (s, 1 H), 7.94 (d, J =
8.0 Hz, 1 H), 7.75 (d, J = 8.0 Hz, 1 H), 7.44 (d, J = 7.6 Hz, 1 H), 7.34
(s, 1 H), 7.06 (d, J = 8.8 Hz, 1 H), 5.39 (s, 1 H), 4.90 (d, J = 16.8 Hz,
1 H), 4.64−4.55 (m, 2 H), 3.18−3.06 (m, 2 H), 1.32 (brs, 2 H). 13C
NMR (400 MHz, CDCl3) δ: 156.4, 153.6, 149.3, 138.8, 134.5, 132.1,
130.2, 128.5, 127.4, 125.8, 120.1, 119.3, 117.4, 83.9, 81.1, 42.9, 40.4.
(3S,3aS)-3-(Aminomethyl)-7-morpholino-3,3a-dihydro-1H,9H-
benzo[e]oxazolo[4,3-b][1,3]oxazin-1-one (34). Compound 34 (154
mg, 77%) was prepared from 29 (267 mg, 0.66 mmol) according to
the procedure described for 30. Mp: 173−175 °C. LC−MS (ESI) m/
N-(((3S,3aS)-7-(6-Cyanopyridin-3-yl)-1-oxo-3,3a-dihydro-1H,9H-
benzo[e]oxazolo[4,3-b][1,3]oxazin-3-yl)methyl)acetamide (37).
Compound 37 (81 mg, 93%) was prepared from 33 (77 mg, 0.24
mmol) according to the procedure described for 20aa. Mp: 229−231
°C. HRMS (ESI) m/z [M + H]+: calcd for C19H17N4O4 365.1250,
found 365.1240. 1H NMR (400 MHz, CDCl3) δ: 8.88 (d, J = 1.6 Hz,
1 H), 7.94 (dd, J = 2.4, 8.0 Hz, 1 H), 7.75 (d, J = 8.0 Hz, 1 H), 7.43
(dd, J = 2.4, 8.8 Hz, 1 H), 7.32 (d, J = 2.4 Hz, 1 H), 7.05 (d, J = 8.8
Hz, 1 H), 5.93 (t, J = 6.4 Hz, 1 H), 5.46 (d, J = 1.2 Hz, 1 H), 4.88 (d,
J = 16.8 Hz, 1 H), 4.71 (t, J = 4.4 Hz, 1 H), 4.54 (d, J = 16.8 Hz, 1
H), 3.75−3.67 (m, 2 H), 2.05 (s, 3 H). 13C NMR (400 MHz, CDCl3)
δ: 171.2, 156.3, 153.4, 149.3, 138. 8, 134.5, 132.2, 130.4, 128.5, 127.5,
125.7, 119.9, 119.3, 117.3, 83.6, 78.94, 40.6, 40.3, 23.2.
N-(((3S,3aS)-7-Morpholino-1-oxo-3,3a-dihydro-1H,9H-benzo[e]-
oxazolo[4,3-b][1,3]oxazin-3-yl)methyl)acetamide (38). Compound
38 (82 mg, 99%) was prepared from 34 (74 mg, 0.24 mmol)
according to the procedure described for 20aa. Mp: 160−162 °C.
HRMS (ESI) m/z [M + H]+: calcd for C17H22N3O5 348.1560, found
348.1554. 1H NMR (400 MHz, CDCl3) δ: 6.85−6.79 (m, 2 H), 6.59
(s, 1 H), 5.94 (t, J = 6.0 Hz, 1 H), 5.27 (d, J = 1.6 Hz, 1 H), 4.75 (d, J
= 16.4 Hz, 1 H), 4.65−4.62 (m, 1 H), 4.42 (d, J = 16.8 Hz, 1 H),
3.87−3.85 (m, 4 H), 3.76−3.70 (m, 1 H), 3.65−3.58 (m, 1 H), 3.07−
3.05 (m, 4 H), 2.03 (s, 3 H). 13C NMR (100 MHz, CDCl3) δ: 170.8,
156.5, 119.3, 118.7, 117.2, 113.70, 83.4, 78.9, 66.9, 50.3, 40.9, 40.4,
23.2.
1
z [M + H]+: 306.1148. H NMR (400 MHz, CDCl3) δ: 6.84 (d, J =
8.8 Hz, 1 H), 6.79 (dd, J = 2.8, 8.8 Hz, 1 H), 6.60 (d, J = 2.8 Hz, 1
H), 5.23 (d, J = 1.2 Hz, 1 H), 4.75 (d, J = 16.4 Hz, 1 H), 4.59−4.56
(td, J = 1.6, 4.8 Hz, 1 H), 4.44 (d, J = 16.8 Hz, 1 H), 3.86−3.84 (m, 4
H), 3.15−3.02 (m, 6 H), 1.57 (brs, 2 H). 13C NMR (400 MHz,
CDCl3) δ: 156.6, 147.0, 146.1, 119.5, 118.6, 117.0, 113.7, 83.6, 81.1,
66.9, 50.3, 43.0, 40.8.
N-(((3S,3aS)-7-(6-Cyanopyridin-3-yl)-6-fluoro-1-oxo-3,3a-dihy-
dro-1H,9H-benzo[e]oxazolo[4,3-b][1,3]oxazin-3-yl)methyl)-
acetamide (20aa). To a solution of 30 (75 mg, 0.22 mmol) in DCM
(10.0 mL) were added TEA (45.9 μL, 0.33 mmol) and acetyl chloride
(20.4 μL, 0.29 mmol) sequentially. The mixture was stirred for 2 h at
0 °C. Then the solution was washed with water and brine, dried over
Synthetic Procedure for Compounds ( )-47 and ( )-48.
Benzyl 7-Bromo-6-fluoro-2-(3-(4-methoxybenzyl)-2-oxooxazolidin-
5-yl)-2H-benzo[e][1,3]oxazine-3(4H)-carboxylate (( )-39). Com-
pound ( )-39 (3.2 g, 51%) was prepared from 10c (3.9 g, 11.0
mmol) according to the procedure described for ( )-15a. Mp: 110−
1
112 °C. LC−MS (ESI) m/z [M + H]+: 571.0892 and 573.0837. H
NMR (400 MHz, acetone-d6) δ: 7.45−7.32 (m, 5 H), 7.22−7.19 (m,
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3 H), 7.10 (d, J = 6.0 Hz, 1 H), 6.88 (d, J = 8.0 Hz, 2 H), 6.02 (d, J =
8.8 Hz, 1 H), 5.22 (d, J = 12.0 Hz, 1 H), 5.19 (d, J = 12.4 Hz, 1 H),
5.05 (d, J = 17.6 Hz, 1 H), 4.86−4.80 (m, 1 H), 4.45 (d, J = 17.6 Hz,
1 H), 4.37 (d, J = 14.8 Hz, 1 H), 4.31 (d, J = 14.8 Hz, 1 H), 3.78 (s, 3
H), 3.61 (t, J = 9.2 Hz, 1 H), 3.50−3.45 (m, 1 H).
Benzyl 7-Bromo-6-fluoro-2-(2-oxooxazolidin-5-yl)-2H-benzo[e]-
[1,3]oxazine-3(4H)-carboxylate (( )-40). Compound ( )-40 (1.9
g, 67%) was prepared from ( )-39 (3.6 g, 6.3 mmol) according to the
procedure described for ( )-16a. Mp: 150−152 °C. LC−MS (ESI)
m/z [M + H]+: 451.0299 and 453.0280. 1H NMR (400 MHz,
acetone-d6) δ: 7.45−7.42 (m, 2 H), 7.39−7.33 (m, 3 H), 7.22 (d, J =
8.8 Hz, 1 H), 7.16 (d, J = 6.0 Hz, 1 H), 6.67 (s, 1 H), 6.15 (d, J = 8.8
Hz, 1 H), 5.25 (d, J = 12.4 Hz, 1 H), 5.21 (d, J = 12.4 Hz, 1 H), 5.08
(d, J = 17.6 Hz, 1 H), 4.95−4.90 (m, 1 H), 4.48 (d, J = 17.2 Hz, 1 H),
3.76 (t, J = 8.8 Hz, 1 H), 3.69−3.65 (m, 1 H).
( )-46 (72 mg, 86%) was prepared from ( )-44 (110 mg, 0.26
mmol) according to the procedure described for 30. Mp: 64−66 °C.
HRMS (ESI) m/z [M + H]+: calcd for C15H19FN3O4 324.1360,
found 324.1349. 1H NMR (400 MHz, CDCl3) δ: 6.77 (d, J = 12.4 Hz,
1 H), 6.45 (d, J = 7.6 Hz, 1 H), 5.24 (d, J = 1.6 Hz, 1 H), 4.71 (d, J =
16.4 Hz, 1 H), 4.53 (td, J = 1.2, 5.6 Hz, 1 H), 4.38 (d, J = 16.4 Hz, 1
H), 3.85 (t, J = 4.8 Hz, 4 H), 3.12−2.99 (m, 6 H), 1.25 (brs, 2 H).
13C NMR (100 MHz, CDCl3) δ: 156.5, 151.1 (d, JC−F = 241.0 Hz),
148.5 (d, JC−F = 2.0 Hz), 140.1 (d, JC−F = 10.0 Hz), 113.6 (d, JC−F
=
23.0 Hz), 111.9 (d, JC−F = 7.0 Hz), 108.0 (d, JC−F = 3.0 Hz), 83.7,
81.1, 66.9, 50.8, 50.7, 43.0, 40.0.
N-((6-(6-Cyanopyridin-3-yl)-7-fluoro-1-oxo-3,3a-dihydro-1H,9H-
benzo[e]oxazolo[4,3-b][1,3]oxazin-3-yl)methyl)acetamide (( )-47).
Compound ( )-47 (68 mg, 76%) was prepared from ( )-45 (80 mg,
0.24 mmol) according to the procedure described for 20aa. Mp: 115−
117 °C. HRMS (ESI) m/z [M + H]+: calcd for C19H16FN4O4
383.1156, found 383.1141. 1H NMR (400 MHz, CDCl3) δ: 8.85
(s, 1 H), 7.98 (d, J = 8.0 Hz, 1 H), 7.78 (d, J = 8.0 Hz, 1 H), 7.00−
6.97 (m, 2 H), 6.22 (brs, 1 H), 5.45 (s, 1 H), 4.84 (d, J = 17.2 Hz, 1
H), 4.69 (t, J = 4.8 Hz, 1 H), 4.49 (d, J = 17.2 Hz, 1 H), 3.72−3.69
(m, 2 H), 2.04 (s, 9 H). 13C NMR (100 MHz, CDCl3) δ: 171.2,
156.4, 154.8 (d, JC−F = 245.0 Hz), 150.7 (d, JC−F = 4.0 Hz), 148.8 (d,
JC−F = 2.0 Hz), 137.0 (d, JC−F = 4.0 Hz), 134.1, 132.9, 128.2, 124.0
(d, JC−F = 15.0 Hz), 121.6 (d, JC−F = 8.0 Hz), 119.2 (d, JC−F = 3.0
Hz), 117.1, 114.4 (d, JC−F = 25.0 Hz), 83.7, 79.1, 40.5, 40.2, 23.1.
N-((7-Fluoro-6-morpholino-1-oxo-3,3a-dihydro-1H,9H-benzo[e]-
oxazolo[4,3-b][1,3]oxazin-3-yl)methyl)acetamide (( )-48). Com-
pound ( )-48 (44 mg, 86%) was prepared from ( )-46 (45 mg,
0.14 mmol) according to the procedure described for 20aa. Mp: 202−
205 °C. HRMS (ESI) m/z [M + H]+: calcd for C17H21FN3O5
366.1465, found 366.1443. 1H NMR (400 MHz, CDCl3) δ: 6.76
(d, J = 12.0 Hz, 1 H), 6.48 (d, J = 7.2 Hz, 1 H), 5.93 (brs, 1 H), 5.30
(d, J = 1.2 Hz, 1 H), 4.70 (d, J = 16.4 Hz, 1 H), 4.63 (t, J = 5.2 Hz, 1
H), 4.36 (d, J = 16.4 Hz, 1 H), 3.87 (t, J = 4.4 Hz, 4 H), 3.75−3.60
(m, 2 H), 3.07−3.04 (m, 4 H), 2.03 (s, 3 H). 13C NMR (100 MHz,
CDCl3) δ: 170.9, 156.4, 151.1 (d, JC−F = 241.0 Hz), 150.8, 148.3 (d,
JC−F = 2.0 Hz), 113.5 (d, JC−F = 24.0 Hz), 111.9 (d, JC−F = 7.0 Hz),
108.2 (d, JC−F = 3.0 Hz), 83.5, 78.9, 66.8, 50.8, 50.7, 40.3, 40.2, 23.2.
Minimum Inhibitory Concentration Testing (Antitubercular
Activity). MICs of compounds against M. tuberculosis were
determined by the microplate alamar blue assay (MABA). INH and
RFP were included as positive controls. Compound stock solutions
and the range of final testing concentrations were 32−0.5 μg/mL. For
the active compounds, the stock concentration and final testing
concentration range were lowered to 2−0.016 μg/mL. M. tuberculosis
H37Rv or clinical isolates were grown to late log phase (70 to 100
Klett units) in Difco Middlebrook 7H9 broth supplemented with
0.2% (v/v) glycerol, 0.05% Tween 80, and 10% (v/v) albumin-
dextrose-catalase (7H9-ADC-TG). Two-fold dilution of compounds
was prepared in 7H9-ADC-TG in a volume of 100 μL in 96-well,
black, clear-bottom microplates. M. tuberculosis (100 μL containing 2
× 105 CFU) was added, yielding a final testing volume of 200 μL. The
plates were incubated at 37 °C, and on day 7 of incubation, 12.5 μL of
20% Tween 80 and 20 μL of alamar blue were added to all wells. After
incubation at 37 °C for 16 h, the fluorescence was read at an
excitation of 530 nm and an emission of 590 nm. The MIC was
defined as the lowest concentration effecting a reduction in
fluorescence of ≥90% relative to controls.
Benzyl 7-Bromo-2-(3-(tert-butoxycarbonyl)-2-oxooxazolidin-5-
yl)-6-fluoro-2H-benzo[e][1,3]oxazine-3(4H)-carboxylate (( )-41).
Compound ( )-41 (3.2 g, 74%) was prepared from ( )-40 (3.4 g,
7.6 mmol) according to the procedure described for ( )-17a. Mp:
170−172 °C. LC−MS (ESI) m/z [M + Na]+: 573.0613 and
1
575.0594. H NMR (400 MHz, acetone-d6) δ: 7.44−7.32 (m, 5 H),
7.24−7.21 (m, 2 H), 6.24 (d, J = 8.8 Hz, 1 H), 5.25 (d, J = 12.4 Hz, 1
H), 5.21 (d, J = 12.4 Hz, 1 H), 5.08 (d, J = 17.6 Hz, 1 H), 4.96−4.90
(m, 1 H), 4.50 (d, J = 17.6 Hz, 1 H), 4.16−4.08 (m, 2 H), 1.50 (s, 9
H).
tert-Butyl ((6-Bromo-7-fluoro-1-oxo-3,3a-dihydro-1H,9H-benzo-
[e]oxazolo[4,3-b][1,3]oxazin-3-yl)methyl)carbamate (( )-42).
Compound ( )-42 (2.2 g, 82%) was prepared from ( )-41 (3.7 g,
6.7 mmol) according to the procedure described for ( )-18a. Mp:
107−109 °C. LC−MS (ESI) m/z [M + Na]+: 439.0256 and
1
441.0236. H NMR (400 MHz, CDCl3) δ: 7.11 (d, J = 6.0 Hz,1 H),
6.86 (d, J = 8.0 Hz,1 H), 5.40 (s,1 H), 4.88 (brs, 1 H), 4.75 (d, J =
16.8 Hz, 1 H), 4.62 (td, J = 1.2, 5.2 Hz,1 H), 4.35 (d, J = 17.2 Hz, 1
H), 3.60−3.48 (m, 2 H), 1.44 (s, 9 H). 13C NMR (100 MHz, CDCl3)
δ: 156.3, 156.1, 154.5 (d, JC−F = 242.0 Hz), 148.6 (d, JC−F = 2.0 Hz),
122.7, 119.2 (d, JC−F = 7.0 Hz), 113.6 (d, JC−F = 24.0 Hz), 108.3 (d,
JC−F = 23.0 Hz), 83.6, 80.5, 79.0, 41.4, 40.4, 40.3, 28.3.
tert-Butyl ((6-(6-Cyanopyridin-3-yl)-7-fluoro-1-oxo-3,3a-dihydro-
1H,9H-benzo[e]oxazolo[4,3-b][1,3]oxazin-3-yl)methyl)carbamate
(( )-43). Compound ( )-43 (160 mg, 84%) was prepared from
( )-42 (180 mg, 0.43 mmol) according to the procedure described
for ( )-19a. Mp: 60−62 °C. LC−MS (ESI) m/z [M + H]+:
1
441.1574. H NMR (400 MHz, CDCl3) δ: 8.86 (s,1 H), 8.00−7.97
(m, 1 H), 7.78 (dd, J = 0.8, 8.0 Hz, 1 H), 7.01−6.90 (m, 2 H), 5.49
(s, 1 H), 4.90 (s, 1 H), 4.86 (d, J = 17.2 Hz, 1 H), 4.66 (t, J = 4.8 Hz,
1 H), 4.47 (d, J = 17.6 Hz, 1 H), 3.64−3.52 (m, 2 H), 1.45 (s, 9 H).
tert-Butyl ((7-Fluoro-6-morpholino-1-oxo-3,3a-dihydro-1H,9H-
benzo[e]oxazolo[4,3-b][1,3]oxazin-3-yl)methyl)carbamate
(( )-44). Compound ( )-44 (35 mg, 38%) was prepared from
( )-42 (90 mg, 0.22 mmol) according to the procedure described for
1
27. Mp: 135−137 °C. LC−MS (ESI) m/z [M + H]+: 424.1871. H
NMR (400 MHz, CDCl3) δ: 6.75 (d, J = 12.0 Hz, 1 H), 6.44 (d, J =
7.2 Hz, 1 H), 5.35 (s, 1 H), 4.92 (brs, 1 H), 4.70 (d, J = 16.4 Hz, 1
H), 4.60 (t, J = 4.8 Hz, 1 H), 4.3 (d, J = 16.4 Hz, 1 H), 3.85 (t, J = 4.4
Hz, 4 H), 3.55−3.51 (m, 2 H), 3.05−3.02 (m, 4 H), 1.43 (s, 9 H).
5-(3-(Aminomethyl)-7-fluoro-1-oxo-3,3a-dihydro-1H,9H-benzo-
[e]oxazolo[4,3-b][1,3]oxazin-6-yl)picolinonitrile (( )-45). Com-
pound ( )-45 (91 mg, 78%) was prepared from ( )-43 (150 mg,
0.43 mmol) according to the procedure described for 30. Mp: 84−86
°C. HRMS (ESI) m/z [M + H]+: calcd for C17H14FN4O3 341.1050,
found 341.1042. 1H NMR (400 MHz, CDCl3) δ: 8.86 (s, 1 H), 8.01−
7.98 (m, 1 H), 7.78 (dd, J = 0.8, 8.0 Hz, 1 H), 7.02−7.00 (m, 2 H),
5.35 (d, J = 1.2 Hz, 1 H), 4.87 (d, J = 17.6 Hz, 1 H), 4.60 (td, J = 1.6,
5.6 Hz, 1 H), 4.53 (d, J = 17; 2 Hz, 1 H), 3.17−3.05 (m, 2 H), 1.49
Minimum Inhibitory Concentration Testing (Antibacterial
Activity). Antimicrobial susceptibility tests were conducted in 96-well
microplates using the broth microdilution procedure. Cation-adjusted
Mueller−Hinton broth for all the S. aureus strains or brain heart
infusion broth for VRE or Mueller−Hinton broth for the other strains
was used in the assays. After incubation at 37 °C for 18 h, the
absorbance at 600 nm was recorded using a microplate reader (Bio-
Rad Laboratory Ltd., U.K.) and then the percentage of bacterial cell
inhibition versus vehicles (1% DMSO) was calculated. The MIC was
defined as the lowest compound concentration at which the growth of
bacteria was inhibited by ≥90%. According to Methods for Dilution
Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically
(brs, 2 H). 13C NMR (100 MHz, CDCl3) δ: 156.3, 154.7 (d, JC−F
=
244.0 Hz), 150.7 (d, JC−F = 4.0 Hz), 148.9 (d, JC−F = 2.0 Hz), 136.9
(d, JC−F = 4.0 Hz), 134.1, 133.0, 128.2, 123.9, 121.8 (d, JC−F = 8.0
Hz), 119.1 (d, JC−F = 2.0 Hz), 117.1, 114..4 (d, JC−F = 26.0 Hz), 83.9,
81.1, 42.8, 40.3.
3-(Aminomethyl)-7-fluoro-6-morpholino-3,3a-dihydro-1H,9H-
benzo[e]oxazolo[4,3-b][1,3]oxazin-1-one (( )-46). Compound
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(29th edition, M100), for Enterococcus, linezolid resistant MIC ≥ 8
μg/mL, linezolid intermediate MIC = 4 μg/mL, linezolid susceptible
MIC ≤ 2 μg/mL; for Staphylococcus, linezolid resistant MIC ≥ 8 μg/
mL, linezolid susceptible MIC ≤ 4 μg/mL.
1 μL of the test compound/control working solution or DMSO
(vehicle control), mix well, and preincubate at 37 °C for 5 min. Add
100 μL of preincubated MAO-A or MAO-B working solution to
incubation tubes to start the reaction, mix well, and incubate at 37 °C
for 15 min. Following the incubation, immediately add 200 μL of
quenching solution and vortex to mix. Centrifuge all samples at 4 °C
for 15 min, then transfer 100 μL of supernatant with 100 μL of water
for LC−MS/MS analysis. Corresponding metabolite 4-hydroxyqui-
nolinol is detected. The inhibition potentials (% inhibition) are
calculated using formation with test compound or control compound
compared to that with the vehicle control. IC50 values are calculated
using GraphPad Prism.
Pharmacokinetic Studies in Mouse. The animal protocol was
approved by Institute Animal Care and Welfare Committee.
Compound 20aa was subjected to pharmacokinetic studies in
BALB/c mouse (female) weighing 16−21 g with three mice in oral
administration group and three mice in intravenous injection group.
Compound 20aa was formulated at a concentration of 5 mg/mL for a
dose of 10 mg/kg given orally (po) and at 1 mg/mL for a dose of 1
mg/kg given intravenously (iv). Compound 20aa was formulated with
0.5% carboxymethyl cellulose for po administration and with 10%
DMSO/50% PEG400/40% water for iv administration. Blood
samples were collected at 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h,
and 24 h after oral dosing and 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8
h, 12 h, and 24 h after iv administration. Analyte quantification was
performed by a LC/MS/MS Quantum Access mass spectrometer (AB
Sciex 5500). Compound detection on the mass spectrometer was
performed in electrospray positive ionization mode. The pharmaco-
kinetic parameters were calculated using WinNonlin software version
6.3 based on noncompartmental analysis (Pharsight Corporation,
Mountain View, USA). The oral bioavailability was calculated as the
ratio between the area under the curve (AUC) following intravenous
administration corrected for dose, F = [(AUCpo × doseiv)/(AUCiv ×
dosepo)] × 100%.
Cytotoxicity Assay. Vero cells were cultured in RPMI1640
medium supplemented with 10% fetal bovine serum (FBS). The cells
were incubated in a humidified atmosphere of 5% CO2 at 37 °C.
Stocks of cells were cultured in 25 cm2 tissue culture flasks and
subcultured two to three times per week. Cytotoxicity testing was
performed in a transparent 96-well microplate. Outer perimeter wells
were filled with sterile water to prevent dehydration in experimental
wells. The cells were incubated at 37 °C under 5% CO2 until
confluent and then diluted with culture medium to 4 × 105 cells/mL.
Three-fold serial dilutions of the stock solutions resulted in final
concentrations of 64−0.26 μg/mL in a final volume of 100 μL. After
incubation at 37 °C for 48 h, the medium was removed, and the
monolayers were washed twice with 100 μL of warm Hanks’ balanced
salt solution (HBSS). Warm medium (100 μL) and 10 μL of freshly
made methylthiazolyldiphenyltetrazolium bromide (MTT) were
added to each well, and then the plates were incubated for 4 h.
The absorbance was determined at 492 nm.
Liver Microsome Stability Assay. The assay was performed with
liver microsomes from male CD-1 mouse and pooled human. The
incubation was performed as follows: microsomes in 0.05 M
phosphate buffer, pH 7.4 (1 mg/mL microsomal protein), tested
compounds (final concentration 0.1 μM, cosolvent (DMSO)), and
then NADPH (1 mM) at 37 °C for 30 min. The reaction can be
started by the addition of NADPH or the same volume of buffer.
Aliquots were sampled at 0 and 30 min incubation, and enzymatic
reaction was quenched by addition of acetonitrile. After centrifuga-
tion, samples were then analyzed by LC−MS. The assay evaluated the
metabolic stability of compounds by measuring the substrate
remaining with or without NADPH cofactor.
Inhibition Evaluation on hERG K+ Channel. HEK 293 cells
̀
were stably transfected with human ether-a-go-go-related gene
(hERG) channel. The voltage-gated hERG potassium channel current
was recorded at room temperature (25 °C) from randomly selected
transfected cells under whole-cell manual patch clamp systems
equipped with EPC10 USB (HEKA) or Multiclamp 700B amplifier
(Molecular Devices), while electrical data were digitalized by
Digidata1440A with sampling frequency at 10 kHz using Patchmaster
or pClamp10. hERG current inhibition in the presence of five
concentrations, including 30, 10, 3.0, 1.0, and 0.3 μM, was tested for
IC50 determination. Dofetilide was also included as a positive control
to ensure the accuracy and sensitivity of the test system.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02153.
■
sı
Synthesis of compounds 10a−c, 14, and 49; NMR
spectra of all compounds; chiral HPLC of compounds
( )-18a, 18a, ( )-20a, 20aa, 20ab, ( )-24, and 24;
plasma exposure of compound 20aa in mouse (PDF)
Molecular formula strings (CSV)
Mitochondrial Protein Synthesis Inhibition. Mitochondrial
protein synthesis was measured on Cardiac (H9C2) cell line that was
obtained from National Platform of Experimental Cell Resources for
Sci-Tech. The H9C2 cells were kept in DMEM (Gibico) with 10%
FBS (Hyclone) and 1× glutamine and NEAA at 37 °C, 5% CO2. The
cell monolayer was washed with prewarmed PBS and detached using
trypsin. After centrifugation (1000 rpm for 5 min at rt), the cells were
resuspended with cell culture medium and seeded at 1500 cells/well
into a 384-well plate. After 18 h, compounds 20aa and 35, with final
concentrations of 200 μM, 66.67 μM, 22.22 μM, 7.41 μM, 2.47 μM,
0.82 μM, 0.27 μM, 0.09 μM, 0.03 μM, were added to the appropriate
well and then incubated at 37 °C 5% CO2 for 5 days. A negative
control with 0.5% DMSO was performed for every compound. MPS
inhibition was evaluated by MitoBiogenesis In-Cell ELISA. IC50
calculation was performed using GraphPad Prism 5 software.
MAO Inhibition. Test compound working solutions were made
(4-fold dilution, six nonzero concentrations) from 10 mM test
compound DMSO stock with DMSO. Control compound working
solution was made (4-fold dilution, six nonzero concentrations) from
40 mM leflunomide DMSO stock with DMSO. 40 μM substrate
working solution was made from 20 mM kynuramine DMSO stock
with phosphate buffer. Thaw MAO-A and MAO-B recombinase in 37
°C water bath. 0.01 mg/mL MAO-A and 0.04 mg/mL MAO-B
working solutions were made with phosphate buffer. Add 99 μL of
substrate working solution into 1.1 mL incubation tubes and then add
AUTHOR INFORMATION
Corresponding Authors
■
Dongfeng Zhang − Beijing Key Laboratory of Active
Substance Discovery and Druggability Evaluation and
Chinese Academy of Medical Sciences Key Laboratory of
Anti-DR TB Innovative Drug Research, Institute of Materia
Medica, Peking Union Medical College and Chinese Academy
of Medical Sciences, Beijing 100050, P. R. China;
orcid.org/0000-0003-0870-3782; Phone: +86-10-
63165254; Email: zdf@imm.ac.cn
Ning Sun − The State Key Laboratory of Chemical Biology
and Drug Discovery, Department of Applied Biology and
Chemical Technology, The Hong Kong Polytechnic
University, Kowloon, Hong Kong, P. R. China; The Fifth
Affiliated Hospital of Guangzhou Medical University,
Guangzhou 440112, P. R. China; Phone: +86-
13312853311; Email: ning.sun@connect.polyu.hk
Haihong Huang − Beijing Key Laboratory of Active Substance
Discovery and Druggability Evaluation and Chinese Academy
of Medical Sciences Key Laboratory of Anti-DR TB
3246
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Article
Innovative Drug Research, Institute of Materia Medica,
Peking Union Medical College and Chinese Academy of
Medical Sciences, Beijing 100050, P. R. China; Phone: +86-
10-63165244; Email: joyce@imm.ac.cn
CbzCl, benzyl chloroformate; PMB, 4-methoxybenzyl; Boc,
tert-butoxycarbonyl; DMAP, 4-dimethylaminopyridine; DCM,
dichloromethane; EtOAc, ethyl actate; THF, tetrahydrofuran;
CAN, ceric ammonium nitrate; ECD, electrostatic circular
dichroism; TFA, trifluoroacetic acid; MIC, minimum inhib-
itory concentration; MPS, mitochondrial protein synthesis;
MAO, monoamine oxidase; PK, pharmacokinetic; NADPH,
nicotinamide adenine dinucleotide phosphate
Authors
Yongqi Wu − Beijing Key Laboratory of Active Substance
Discovery and Druggability Evaluation and Chinese Academy
of Medical Sciences Key Laboratory of Anti-DR TB
Innovative Drug Research, Institute of Materia Medica,
Peking Union Medical College and Chinese Academy of
Medical Sciences, Beijing 100050, P. R. China
Bin Wang − Beijing Key Laboratory of Drug Resistance
Tuberculosis Research, Department of Pharmacology, Beijing
Tuberculosis and Thoracic Tumor Research Institute, Beijing
Chest Hospital, Capital Medical University, Beijing 101149,
P. R. China
Haijia Lu − Beijing Key Laboratory of Active Substance
Discovery and Druggability Evaluation and Chinese Academy
of Medical Sciences Key Laboratory of Anti-DR TB
Innovative Drug Research, Institute of Materia Medica,
Peking Union Medical College and Chinese Academy of
Medical Sciences, Beijing 100050, P. R. China
Hongyi Zhao − Beijing Key Laboratory of Active Substance
Discovery and Druggability Evaluation and Chinese Academy
of Medical Sciences Key Laboratory of Anti-DR TB
Innovative Drug Research, Institute of Materia Medica,
Peking Union Medical College and Chinese Academy of
Medical Sciences, Beijing 100050, P. R. China
Beibei Yang − Beijing Key Laboratory of Active Substance
Discovery and Druggability Evaluation, Institute of Materia
Medica, Peking Union Medical College and Chinese Academy
of Medical Sciences, Beijing 100050, P. R. China
Li Li − Beijing Key Laboratory of Active Substance Discovery
and Druggability Evaluation, Institute of Materia Medica,
Peking Union Medical College and Chinese Academy of
Medical Sciences, Beijing 100050, P. R. China; orcid.org/
0000-0002-9496-2280
Yu Lu − Beijing Key Laboratory of Drug Resistance
Tuberculosis Research, Department of Pharmacology, Beijing
Tuberculosis and Thoracic Tumor Research Institute, Beijing
Chest Hospital, Capital Medical University, Beijing 101149,
P. R. China
REFERENCES
■
(1) Brown, E. D.; Wright, G. D. Antibacterial drug discovery in the
resistance era. Nature 2016, 529, 336−343.
(2) World Health Organization. WHO Publishes List of Bacteria for
Which New Antibiotics Are Urgently Needed; WHO, Geneva,
Switzerland, 2017.
(3) Global Tuberculosis Report 2020; World Health Organization:
Geneva, Switzerland, 2020.
(4) Chellat, M. F.; Raguz, L.; Riedl, R. Targeting antibiotic
resistance. Angew. Chem., Int. Ed. 2016, 55, 6600−6626.
(5) Barbachyn, M. R.; Ford, C. W. Oxazolidinone structure-activity
relationships leading to linezolid. Angew. Chem., Int. Ed. 2003, 42,
2010−2023.
(6) Keam, S. J. Pretomanid: First Approval. Drugs 2019, 79, 1797−
1803.
(7) Phillips, O. A.; Sharaf, L. H. Oxazolidinone antimicrobials: a
patent review (2012−2015). Expert Opin. Ther. Pat. 2016, 26, 591−
605.
(8) Barbachyn, M. R. The oxazolidinones. Top. Med. Chem. 2017,
26, 97−122.
(9) Yang, T.; Chen, G.; Sang, Z.; Liu, Y.; Yang, X.; Chang, Y.; Long,
H.; Ang, W.; Tang, J.; Wang, Z.; Li, G.; Yang, S.; Zhang, J.; Wei, Y.;
Luo, Y. Discovery of a teraryl oxazolidinone compound (S)-N-((3-(3-
fluoro-4-(4-(pyridin-2-yl)-1H-pyrazol-1-yl)phenyl)-2-oxooxazolidin-
5-yl)methyl)acetamide phosphate as a novel antimicrobial agent with
enhanced safety profile and efficacies. J. Med. Chem. 2015, 58, 6389−
6409.
(10) Deshmukh, M. S.; Jain, N. Design, synthesis, and antibacterial
evaluation of oxazolidinones with fused heterocyclic C-ring
substructure. ACS Med. Chem. Lett. 2017, 8, 1153−1158.
(11) Renslo, A. R.; Jaishankar, P.; Venkatachalam, R.; Hackbarth, C.;
Lopez, S.; Patel, D. V.; Gordeev, M. F. Conformational constraint in
oxazolidinone antibacterials. Synthesis and structure-activity studies of
(azabicyclo[3.1.0]hexylphenyl)oxazolidinones. J. Med. Chem. 2005,
48, 5009−5024.
(12) Suzuki, H.; Utsunomiya, I.; Shudo, K.; Fujimura, T.; Tsuji, M.;
Kato, I.; Aoki, T.; Ino, A.; Iwaki, T. Potent oxazolidinone
antibacterials with heteroaromatic C-ring substructure. ACS Med.
Chem. Lett. 2013, 4, 1074−1078.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c02153
(13) Im, W. B.; Choi, S. H.; Park, J.-Y.; Choi, S. H.; Finn, J.; Yoon,
S.-H. Discovery of torezolid as a novel 5-hydroxymethyl-oxazolidi-
none antibacterial agent. Eur. J. Med. Chem. 2011, 46, 1027−1039.
(14) Gordeev, M. F.; Yuan, Z. Y. New potent antibacterial
oxazolidinone (MRX-I) with an improved class safety profile. J.
Med. Chem. 2014, 57, 4487−4497.
(15) Jeong, J. W.; Jung, S. J.; Lee, H. H.; Kim, Y. Z.; Park, T. K.;
Cho, Y. L.; Chae, S. E.; Baek, S. Y.; Woo, S. H.; Lee, H. S.; Kwak, J. H.
In vitro and in vivo activities of LCB01-0371, a new oxazolidinone.
Antimicrob. Agents Chemother. 2010, 54, 5359−5362.
(16) Zong, Z.; Jing, W.; Shi, J.; Wen, S.; Zhang, T.; Huo, F.; Shang,
Y.; Liang, Q.; Huang, H.; Pang, Y. Comparison of in vitro activity and
MIC distributions between the novel oxazolidinone delpazolid and
linezolid against multidrug-resistant and extensively drug-resistant
Mycobacterium tuberculosis in China. Antimicrob. Agents Chemother.
2018, 62, No. e00165-18.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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The research is supported by the National Natural Science
Foundation of China (Grant 81502917) and the National
Natural Science Foundation of China (Grant 81703333). The
linezolid resistant strains are gifts from Prof. Min Li of Renji
Hospital of Shanghai Jiaotong University and Dr. Wenchang
Yuan of Guangzhou Medical University.
ABBREVIATIONS USED
■
MRSA, methicillin-resistant Staphylococcus aureus; VISA,
vancomycin intermediate Staphylococcus aureus; VRE, vanco-
mycin resistant Enterococcus; MRSE, methicillin-resistant
Staphylococcus epidermidis; TB, tuberculosis; MDR-TB, multi-
drug-resistant TB; XDR-TB, extensively drug resistant TB;
(17) Barbachyn, M. R.; Hutchinson, D. K.; Brickner, S. J.; Cynamon,
M. H.; Kilburn, J. O.; Klemens, S. P.; Glickman, S. E.; Grega, K. C.;
Hendges, S. K.; Toops, D. S.; Ford, C. W.; Zurenko, G. E.
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J. Med. Chem. 2021, 64, 3234−3248

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Identification of a novel oxazolidinone (U-100480) with potent
antimycobacterial activity. J. Med. Chem. 1996, 39, 680−685.
(18) Wallis, R. S.; Jakubiec, W. M.; Kumar, V.; Silvia, A. M.; Paige,
D.; Dimitrova, D.; Li, X.; Ladutko, L.; Campbell, S.; Friedland, G.;
Mitton-Fry, M.; Miller, P. F. Pharmacokinetics and whole-blood
bactericidal activity against Mycobacterium tuberculosis of single doses
of PNU-100480 in healthy volunteers. J. Infect. Dis. 2010, 202, 745−
751.
(19) U.S. National Library of Medicine. ClinicalTrials.gov. https://
clinicaltrials.gov/ct2/show/NCT03758612 (accessed Nov 29, 2018).
(20) Xin, Q.; Fan, H.; Guo, B.; He, H.; Gao, S.; Wang, H.; Huang,
Y.; Yang, Y. Design, synthesis, and structure-activity relationship
studies of highly potent novel benzoxazinyl-oxazolidinone antibacte-
rial agents. J. Med. Chem. 2011, 54, 7493−7502.
(21) Guo, B.; Fan, H.; Xin, Q.; Chu, W.; Wang, H.; Huang, Y.;
Chen, X.; Yang, Y. Solubility-driven optimization of (pyridin-3-yl)
benzoxazinyl-oxazolidinones leading to a promising antibacterial
agent. J. Med. Chem. 2013, 56, 2642−2650.
(22) Zhao, H.; Lu, Y.; Sheng, L.; Yuan, Z.; Wang, B.; Wang, W.; Li,
Y.; Ma, C.; Wang, X.; Zhang, D.; Huang, H. Discovery of fluorine-
containing benzoxazinyl-oxazolidinones for the treatment of multi-
drug resistant tuberculosis. ACS Med. Chem. Lett. 2017, 8, 533−537.
(23) Zhao, H.; Wang, B.; Fu, L.; Li, G.; Lu, H.; Liu, Y.; Sheng, L.; Li,
Y.; Zhang, B.; Lu, Y.; Ma, C.; Huang, H.; Zhang, D.; Lu, Y. Discovery
of a conformationally constrained oxazolidinone with improved safety
and efficacy profiles for the treatment of multidrug-resistant
tuberculosis. J. Med. Chem. 2020, 63, 9316−9339.
(24) Wu, Y.-Q.; Lu, H.-J.; Zhao, W.-T.; Zhao, H.-Y.; Lin, Z.-Y.;
Zhang, D.-F.; Huang, H.-H. A convenient and efficient H2SO4-
promoted regioselective monobromination of phenol derivatives using
N-bromosuccinimide. Synth. Commun. 2020, 50, 813−822.
(25) Osa, Y.; Hikima, Y.; Sato, Y.; Takino, K.; Ida, Y.; Hirono, S.;
Nagase, H. Convenient synthesis of oxazolidinones by the use of
halomethyloxirane, primary amine, and carbonate salt. J. Org. Chem.
2005, 70, 5737−5740.
(26) Junquera, F.; Merchán, F. L.; Merino, P.; Tejero, T. A
straightforward synthesis of L-isoserinal. Tetrahedron 1996, 52, 7045−
7052.
(27) Zubkov, F. I.; Nikitina, E. V.; Galeev, T. R.; Zaytsev, V. P.;
Khrustalev, V. N.; Novikov, R. A.; Orlova, D. N.; Varlamov, A. V.
General synthetic approach towards annelated 3a,6-epoxyisoindoles
by tandem acylation/IMDAF reaction of furylazaheterocycles. Scope
and limitations. Tetrahedron 2014, 70, 1659−1690.
(28) Zhao, H.; Zhao, W.; Cheng, S.; Lu, H.; Zhang, D.; Huang, H.
Efficient and stereoselective one-pot synthesis of benzo[b]oxazolo-
[3,4-d][1,4]oxazin-1-ones. RSC Adv. 2020, 10, 24037−24044.
(29) McKee, E. E.; Ferguson, M.; Bentley, A. T.; Marks, T. A.
Inhibition of mammalian mitochondrial protein synthesis by
oxazolidinones. Antimicrob. Agents Chemother. 2006, 50, 2042−2049.
3248
https://dx.doi.org/10.1021/acs.jmedchem.0c02153
J. Med. Chem. 2021, 64, 3234−3248