Synergistic activity of nitroimidazoleoxazolidinone conjugates against anaerobic bacteria

Article abstract of DOI:10.3390/molecules25102431

The introductions of the bicyclic 4nitroimidazole and the oxazolidinone classes of antimicrobial agents represented the most significant advancements in the infectious disease area during the past two decades. Pretomanid, a bicyclic 4nitroimidazole, and linezolid, an oxazolidinone, are also part of a combination regimen approved recently by the US Food and Drug Administration for the treatment of pulmonary, extensively drug resistant (XDR), treatmentintolerant or nonresponsive multidrugresistant (MDR) Mycobacterium tuberculosis (TB). To identify new antimicrobial agents with reduced propensity for the development of resistance, a series of dualacting nitroimidazoleoxazolidinone conjugates were designed, synthesized and evaluated for their antimicrobial activity. Compounds in this conjugate series have shown synergistic activity against a panel of anaerobic bacteria, including those responsible for serious bacterial infections.

Full text of DOI:10.3390/molecules25102431

Article
Synergistic Activity of Nitroimidazole‐Oxazolidinone
Conjugates against Anaerobic Bacteria
1
1
1
1
1
1
1
Zhijun Zhuang , Dawei Wan , Jun Ding , Shijie He , Qian Zhang , Xiaomei Wang , Ying Yuan ,
2
3
4
5
5
Yu Lu , Charles Z. Ding , Anthony Simon Lynch , Anna M. Upton , Christopher B. Cooper ,
William A. Denny and Zhenkun Ma *
6
1,
1
TenNor Therapeutics Limited, 218 Xinghu Street, Building B2, Suite 711, Suzhou Industrial Park,
Suzhou 215123, China; zhijun.zhuang@tennorx.com (Z.Z.); dawei.wan@tennorx.com (D.W.);
jun.ding@tennorx.com (J.D.); shijie.he@tennorx.com (S.H.); qian.zhang@tennorx.com (Q.Z.);
xiaomei.wang@tennorx.com (X.W.); ying.yuan@tennorx.com (Y.Y.)
2
Department of Pharmacology, Beijing Tuberculosis and Thoracic Tumor Research Institute,
Beijing Chest Hospital, Capital Medical University, 97 Ma Chang Street, Beijing 101149, China;
luyu4876@hotmail.com
WuXi AppTec. Co. Ltd., 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131, China;
3
charles_ding@wuxiapptec.com
Janssen Research & Development LLC., 1400 McKean Road, Spring House, PA 18940, USA;
4
alynch2@its.jnj.com
5
Global Alliance for TB Drug Development, 40 Wall Street, New York, NY 10005, USA;
anna.upton@tballiance.org (A.M.U.); christopher.cooper@tballiance.org (C.B.C.)
Auckland Cancer Society Research Centre, School of Medical Sciences, University of Auckland,
6
Private Bag 92019, Auckland 1142, New Zealand; b.denny@auckland.ac.nz
* Correspondence: zhenkun.ma@tennorx.com; Tel.: +86‐512‐8686‐1980
Academic Editors: Larry Wakelin and Sandra Gemma
Received: 8 May 2020; Accepted: 19 May 2020; Published: 22 May 2020
Abstract: The introductions of the bicyclic 4‐nitroimidazole and the oxazolidinone classes of
antimicrobial agents represented the most significant advancements in the infectious disease area
during the past two decades. Pretomanid, a bicyclic 4‐nitroimidazole, and linezolid, an
oxazolidinone, are also part of a combination regimen approved recently by the US Food and Drug
Administration for the treatment of pulmonary, extensively drug resistant (XDR),
treatment‐intolerant or nonresponsive multidrug‐resistant (MDR) Mycobacterium tuberculosis (TB).
To identify new antimicrobial agents with reduced propensity for the development of resistance, a
series of dual‐acting nitroimidazole‐oxazolidinone conjugates were designed, synthesized and
evaluated for their antimicrobial activity. Compounds in this conjugate series have shown
synergistic activity against a panel of anaerobic bacteria, including those responsible for serious
bacterial infections.
Keywords: drug conjugates; anaerobic bacterium; synergy; nitroimidazole and oxazolidinone
1. Introduction
Oxazolidinones, as represented by linezolid (1) and tedizolid (2), and bicyclic 4‐nitroimidazoles,
as represented by pretomanid (3) and delamanid (4), are two relatively new classes of antimicrobial
agents (Figure 1). Linezolid, the first oxazolidinone approved for clinical use, was introduced in 2000
for the treatment of Gram‐positive bacterial infections, including those that are resistant to other
classes of antibiotics [1]. This drug class inhibits bacterial protein synthesis by binding to rRNA on
both the 30S and 50S ribosomal subunits and preventing the formation of a translation initiation
Molecules 2020, 25, 2431; doi:10.3390/molecules25102431
www.mdpi.com/journal/molecules

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complex. The first bicyclic 4‐nitroimidazole, delamanid, was introduced in 2014 for the treatment of
drug‐resistant tuberculosis (TB). This drug class utilizes a deazaflavin‐dependent nitroreductase
(Ddn) to catalyze the bioreduction of the 4‐nitroimidazole core, leading to the intracellular
generation of reactive chemical species which are toxic to bacterial cells [2]. However, the primary
mechanism of action of the bicyclic 4‐nitroimidazole class against M. tuberculosis appeared to be
different under aerobic and anaerobic conditions. Inhibition of mycolic acid synthesis appeared to be
the main mechanism under aerobic conditions, while the generation of reactive nitrogen species and
inhibition of energy metabolism appeared to be the main mechanism under anaerobic conditions [3].
Figure 1. Structures of Linezolid, Tedizolid, Pretomanid, Delamanid and Conjugate Molecule 5.
Resistance to oxazolidinones and bicyclic 4‐nitroimidazoles is relatively uncommon. Both drug
classes have been used for the treatment of multidrug‐resistant (MDR) and extensively drug
resistant (XDR) TB. Recently, a three‐drug combination containing both pretomanid and linezolid
was approved by the US Food and Drug Administration for the treatment of pulmonary XDR,
treatment‐intolerant or nonresponsive MDR‐TB [4].
Previously, a drug conjugation strategy was utilized to identify the dual‐acting molecule
TNP‐2092 for the treatment of bacterial biofilm infections [5]. This strategy provides several
advantages compared to drug combination therapy, including matched pharmacokinetics, tissue
distribution and potential synergistic activity. Considering the importance of the oxazolidinone and
the bicyclic 4‐nitroimidazole classes, a series of oxazolidinone‐nitroimidazole conjugate molecules
was designed, synthesized and evaluated.
2. Results and Discussion
2.1. Chemistry
The safety and efficacy of the oxazolidinone and bicyclic 4‐nitroimidazole classes have been
validated clinically. The structure–activity relationships (SARs) of both drug classes have been
extensively studied in the past, which serve as the foundation for the design of the conjugation
molecules.
As a protein synthesis inhibitor, linezolid utilizes hydrogen bonding and hydrophobic
interactions to bind to a binding domain located within the ribosomal peptidyltransferase center. A
high resolution analysis of the of crystal structure of linezolid bound to the 50S ribosomal subunit
indicated that the oxazolidinone ring and the acetamide group on the right side of the molecule are
essential for the target interactions. The fluorophenyl moiety in the middle is also important.
However, the morpholino ring on the left side does not appear to have significant interactions with
the binding site, which is consistent with known SARs whose various structures can be used to

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substitute the morpholine group without significant loss of activity [6]. This position was therefore
identified as the linking point for conjugation to a bicyclic 4‐nitroimidazole core.
On the bicyclic 4‐nitroimidazole side, the nitroimidazole group and its fused oxazine or oxazole
ring are essential and directly responsible for the formation of intracellular reactive species via a
bioreduction process. The stereochemistry of the substituents connected to the oxazine or oxazole
ring also plays an important role for the antimicrobial activity. However, the structure of the
substituents on the right‐hand side are highly variable, and can tolerate many functional groups.
This site was hence identified as the linking point for the bicyclic 4‐nitroimidazole class.
A series of drug conjugate was therefore designed and synthesized by connecting the
right‐hand side of the bicyclic 4‐nitroimidazole and the left‐hand side of the oxazolidinone core
through various linkers. The syntheses of these conjugate molecules are illustrated in Scheme 1–3.
Compound 5 (Figure 1) is a previously known oxazolidinone‐metronidazole conjugate; it has not
been evaluated for its activity against M. tuberculosis or anaerobic organisms [7]. Compound 5 was
also prepared in the current study and its activity against M. tuberculosis isogenic mutant panel and
anaerobic bacterial panel was evaluated.
Scheme 1. Synthesis of Conjugate Molecules 8a–b and 11a–b.
The syntheses of the conjugate molecules formed by the oxazine fused nitroimidazole and the
oxazolidinone core 8a–b and 11a–b are summarized in Scheme 1. The known
(S)‐2‐nitro‐6,7‐dihydro‐5H‐imidazo[2,1‐b][1,3]oxazin‐6‐ol core (6) was prepared according to
literature procedures [8]. Intermediates 7a–b and 10a–b were prepared by aromatic or nucleophilic
substitution reaction. Suzuki coupling with the oxazolidinone piece 9a or 9b provided conjugate
molecules 8a–b and 11a–b.

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Scheme 2. Synthesis of Conjugate Molecules 14a–d and 16.
The syntheses of the conjugate molecules formed by the oxazole‐fused nitroimidazole and the
oxazolidinone core 14a–d and 16 are summarized in Scheme 2. The known racemate
2‐bromo‐1‐((2‐methyloxiran‐2‐yl)methyl)‐4‐nitro‐1H‐imidazole (12) was prepared according to a
method described in the literature [9]. Intermediate 13a–d were prepared by epoxide opening
followed by intramolecular cyclization. Suzuki or copper‐catalyzed coupling provided
diastereomeric conjugate molecules 14a–d and 16. The ratio of the two diastereomers was not
determined due to the fact that the two chiral centers in the molecules were distant from each other,
and the two diastereomers could not be distinguished from each other spectroscopically.
Scheme 3. Synthesis of Conjugate Molecules 19a–c.
Scheme 3 summarizes the syntheses of a third series of conjugate molecules 19a–c. This series
contains the same oxazine fused nitroimidazole core as shown in Scheme 1, but connects to the
oxazolidinones via
(2‐nitro‐6,7‐dihydro‐5H‐imidazo[2,1‐b][1,3]oxazin‐7‐yl)methanol
a
different linking point. The known racemate intermediates
(17a) and
(7‐methyl‐2‐nitro‐6,7‐dihydro‐5H‐imidazo[2,1‐b][1,3]oxazin‐7‐yl)methanol (17b) were prepared
according to a procedure described in the literature [10]. Aromatic substitution provided
intermediates 18a–c. Suzuki coupling with the oxazolidinone precursor 9a provided diastereomeric
conjugate molecules 19a–c.
2.2. Mechanism of Action
The mechanisms of action of the precursor antibiotics of the conjugate molecules are well
established. In order to understand whether the conjugate molecules synthesized in the current
study maintained antibacterial activity and were enhanced by both precursor antibiotic classes, an
isogenic panel of resistant mutant strains was prepared from wild‐type strain M. tuberculosis H37Rv

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by stepwise resistance induction with linezolid or pretomanid (Table 1). Resistant mutant strains L1
and L3 were induced by linezolid with mutations on rplC gene encoding the 50S ribosomal protein
L3. The minimum inhibitory concentrations (MICs) of linezolid against L1 and L3 were 4.80 and 8.18
μg/mL, respectively, as compared to 0.15 μg/mL against the wild‐type H37Rv strain. The resistant
mutant strains P1 and P3 were induced by pretomanid with mutations in the ddn gene encoding the
deazoflavin‐dependent nitroreductase. The MICs of pretomanid against P1 and P3 strains were >20
and >20 μg/mL respectively, as compared to 0.07 μg/mL for the H37Rv strain.
All four conjugate compounds listed in Table 1 (8a, 8b, 14c and 14d) were highly active against
the H37Rv strain with MICs similar to those of linezolid and pretomanid. More importantly, all
compounds were significantly more potent than linezolid against the L1 and L3, strains and
significantly more potent than pretomanid against the P1 and P3 mutant strains, indicating that the
antimicrobial activity of the conjugate molecules benefits from contributions from both parental
antibiotic pharmacophores.
Compounds 8a and 8b were formed by oxazine‐fused 4‐nitroimidazole and the oxazolidinone
core. These compounds, particularly compound 8a, appeared to be more potent against the linezolid
resistant strains L1 and L3 than the pretomanid resistant strains P1 and P3, suggesting that the
4‐nitroimidazole function contributes relatively more significantly to the antibacterial activity than
the oxazolidonone function in these conjugate molecules. However, compounds 14c and 14d,
formed by the oxazole fused 4‐nitroimidazole and the oxazolidinone core, demonstrated balanced
activity against the linezolid resistant strains L1 and L3 and the pretomanid resistant strains P1 and
P3, suggesting that these conjugate molecules exhibit a balanced contribution from the two parental
antibiotic pharmacophores.
Table 1. Minimum inhibitory concentrations of selected conjugate molecules against isogenic
resistant mutant strains of M. tuberculosis.
M. tuberculosis Isogenic Mutant Panel MIC (μg/mL)
Compounds
H37Rv
L1
L3
P1
P3
Linezolid
Pretomanid
0.15
0.07
0.06
0.20
0.08
0.33
4.80
0.15
1.95
0.91
1.84
1.88
8.18
0.24
1.78
1.87
0.81
1.46
0.29
>20
6.48
1.04
1.77
1.93
0.31
>20
5.96
3.30
1.80
2.94
8a
8b
14c
14d
2.3. Spectrum of Activity
Selected compounds from the current study were tested against a panel of representative
pathogens (Table 2). All eight conjugate compounds tested showed a similar spectrum of activity as
linezolid against the ESKAPE pathogens: Enterococcus faecium (Ef), Staphylococcus aureus (Sa),
Klebsiella pneumoniae (Kp), Acinetobacter baumannii (Ab), Pseudomonas aeruginosa (Pa), and Escherichia
coli (Ec). Specifically, these compounds were active against Ef and Sa, but inactive against Kp, Ac, Pa
and Ec strains. Several compounds (8a, 8b, 11a and 14c) were substantially more active than
linezolid against Ef and Sa. All conjugate compounds were substantially more active than linezolid
against the obligate anaerobic pathogen Clostridium difficile (Cd). The activities of these compounds
were similar to those of metronidazole. Compound 11a, with an acetamido group at the
oxazolidinone ring, appeared to be more active than its hydroxyl counterpart 11b against aerobic
bacteria Ef and Sa. However, 11a was similar or slightly less active than 11b against anaerobic
bacterium Cd. The different SARs between these two compounds against aerobic and anaerobic
bacteria could be a result of the different mechanisms of action. In an aerobic bacterium, the activity
is driven by the oxazolidinone pharmacophore, while in anaerobic bacteria, the activity is mainly
driven by the nitroimidazole pharmacophore. The conjugate compounds were not cytotoxic, with
IC50s > 64 μg/mL for all compounds tested against the Vero cell‐line.

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Table 2. Minimum inhibitory concentrations (MIC) of selected conjugate molecules against
representative strains from seven major pathogen classes.
Representative of Major Pathogens MIC (μg/mL)
Vero
IC50
μg/mL
Ef
ATCC
Sa
ATCC
29213
8
0.25
64
1
Kp
ATCC
43816
>64
0.063
>64
Ab
ATCC
19606
>64
0.5
>64
Pa
ATCC
27853
>64
1
>64
>64
>64
>64
>64
>64
>64
>64
>64
Ec
ATCC
25922
>64
<0.016
>64
Cd
ATCC
43255
4
Compounds
708221
Linezolid
Levofloxacin
Metronidazole
4
>16
>64
0.5
2
>64
‐
‐
4
0.25
0.125
0.25
0.25
0.125
0.25
0.5
8a
8b
>64
>64
>64
>64
>64
‐
>64
>64
>64
>64
>64
2
>64
>64
>64
11a
11b
14a
14b
14c
14d
1
4
4
8
1
4
2
>64
>64
>64
8
4
>64
>64
>64
>64
>64
>64
16
2
>64
>64
>64
>64
>64
>64
0.125
0.5
4
>64
>64
>64
2.4. Anaerobic Activity
The promising activity of the 4‐nitromidazole‐oxazolidinone conjugate series against the
anaerobic pathogen C. difficile encouraged us to explore a broader panel of clinically important
anaerobic bacteria (Table 3). The test panel included 13 strains of anaerobic bacteria and Helicobacter
pylori (Hp), a Gram‐negative microaerophilic bacterium. Seven of the 13 anaerobic bacterial strains
were Gram‐positive and the rest were Gram‐negative. The full names of the anaerobic bacterial
panel strains are listed in Table 4.
Table 3. Minimum inhibitory concentrations (MIC) of 12 conjugate molecules against clinically
important anaerobic and microaerophilic bacterial strains.
Anaerobic and Microaerophilic Bacterial Panel, MIC (ug/mL)
Micro‐
Aerophilic
Gram‐Positive
Gram‐Negative
Compounds
Cd
ATCC
700057
La
ATCC
4356
El
ATCC
43055
Gv
ATCC
14018
Pm
ATCC
23195
Pas
ATCC
3553
Pac
ATCC
11829
Bf
ATCC
25285
Bl
ATCC
15707
Fn
ATCC
10953
Mm
ATCC
35243
Pv
ATCC
29303
Vp
ATCC
17745
Hp
ATCC
43504
Metronidazole
Pretomanid (P)
Linezolid (L)
L + P (1:1)
5
≤0.25
2
0.5
>256
>32
2
0.5
8
1
2
>32
0.25
0.5
>256
>32
1
0.5
2
0.5
>256
>32
0.06
0.25
1
≤0.03
0.06
0.06
0.12
0.12
0.25
0.06
0.25
>32
≤0.25
16
2
4
0.25
1
4
2
4
1
4
0.5
8
>32
0.12
1
4
>32
0.5
2
0.5
0.25
0.12
0.25
0.25
0.25
0.5
0.25
0.5
>32
0.06
0.06
0.12
≤0.25
4
0.25
1
0.06
≤0.03 ≤0.03
≤0.03
0.06
≤0.03
0.12
1
≤0.03
0.12
>32
8
2
32
2
4
0.5
4
8
4
8
16
>32
8
16
>32
2
>256
>32
0.5
1
64
16
8
16
‐
0.5
1
2
2
2
8
1
>32
0.12
0.25
0.25
1
4
2
2
1
0.06
≤0.03
≤0.03
0.12
0.06
0.25
0.5
0.06
0.25
4
NA
0.5
0.5
0.5
2
1
2
0.5
2
>32
0.25
0.5
0.25
0.06
≤0.03
≤0.03
0.06
0.06
0.12
0.12
≤0.03
0.12
16
0.12
0.06
0.06
0.06
0.12
0.12
0.25
0.12
0.12
>32
0.25
0.12
0.25
0.5
1
0.5
1
0.5
1
>32
0.12
0.12
0.06
0.06
0.06
≤0.03
0.06
≤0.03
0.12
0.25
≤0.03
0.12
8
1
8a
8b
0.25
0.25
0.25
1
0.5
1
0.25
0.5
>32
0.25
0.25
0.25
0.06
0.06
0.25
0.25
0.5
0.12
0.25
>32
11a
11b
14a
14b
14c
14d
16
19a
19b
19c
1
16
0.25
0.25
0.5
≤0.03
≤0.03
≤0.03
≤0.06
0.06
≤0.03 ≤0.03
0.06
0.06
≤0.03 ≤0.03
≤0.03 0.06
≤0.03 ≤0.03
≤0.03
≤0.03
≤0.03
0.06
0.06
0.06
4
2
0.5
Metronidazole, a 5‐nitroimidazole, is one of the most important drugs for the treatment of
anaerobic bacterial infections. The activity of metronidazole against this panel was inconsistent, with
virtually no activity against Lactobacillus acidophilus (La), Peptostreptococcus micros (Pm),
Propionibacterium acnes (Pas) and Veillonella parvula (Vp). Pretomanid, a 4‐nitroimidazole, exhibited a
similar spectrum of activity as metronidazole against this panel, but with higher MICs. Linezolid

Molecules 2020, 25, 2431
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was active against all the strains, with MICs ranging from 0.06 to 2 μg/mL against the 13 anaerobic
bacteria. Interestingly, the 1:1 combination of linezolid and pretomanid was about one dilution less
active than linezolid, virtually reflecting the activity of linezolid in the mixture. Pretomanid did not
appear to contribute to the activity of the combination.
Compound 5, a conjugate molecule between linezolid and metronidazole, a 5‐nitroimidazole,
appeared to be more potent than either of its precursor antibiotics against all strains tested, with
MICs ranging from 0.06 to
1
μg/mL. The apparent synergistic effect of the
4‐nitroimidazole‐oxazolidione conjugate was more profound. The majority of the 12 compounds
were significantly more potent than the combination of linezolid and pretomanid, with the
exception of compound 16. Several compounds (8a, 8b, 11a, 14c, 19a, 19b and 19c) were 10–100‐fold
more potent against the majority of the tested strains than the combination.
One of the limitations of the current study is that compounds 14a–d, 16 and 19a–c are
diastereomers which may have an impact to the interpretation of the SARs, as one diastereomer may
be more active than another.
The underlying mechanism for the apparent synergistic effect of this conjugate series is still
unclear. A number of hypotheses are currently under consideration. The first is that conjugation of a
nitroimidazole to the oxazolidinone simply makes the oxazolidinone more potent. The
nitroimidazole group plays the role of a substituent that makes the oxazolidinone bind to the
ribosomal RNA better. This hypothesis appears to be less plausible, as the synergistic effect was only
observed with anaerobic bacteria. We did not observe the same level of improvement of potency
against aerobic bacteria Enterococcus faecium and Staphylococcus aureus (Table 2). The second
hypothesis is that the conjugate molecules bring additional nitroimidazole inside the cells by better
penetration or avoidance of efflux. A third hypothesis is that the nitroimidazole moiety acts
synergistically when conjugated to an oxazolidinone pharmacophore. The high binding affinity of
the oxazolidinone moiety to ribosomes or ribosomal components could bring the reactive species
generated from the nitroimidazole moiety into close affinity with the transcription/translation
machinery and make them work more efficiently. This includes the possibility for irreversible
covalent linking to the ribosome or other enzymes associated with intrinsic oxazolidinone resistance.
The last two hypotheses are supported by the mechanism‐of‐action study which indicated that both
the nitroimidazole and the oxazolidinone functions contribute the antibacterial activity inside a M.
tuberculosis cell (Table 1).
3. Materials and Methods
3.1. Chemistry
General: Reference compounds linezolid, levofloxacin and metronidazole were purchased from
ChemPacific (Baltimore, MD, USA) or Sigma‐Aldrich (St. Louis, MO, USA). Pretomanid (PA‐824)
and compound 5 were prepared according to published procedures [7]. All other compounds were
synthesized by TenNor Therapeutics (Suzhou, China).
All starting materials were either purchased from commercial sources or prepared according to
published procedures. Operations involving moisture‐ and/or oxygen‐sensitive materials were
conducted under an atmosphere of nitrogen. Flash chromatography was performed using silica gel
60 as normal phase adsorbent or C18 silica gel as reverse phase adsorbent. Nuclear magnetic
resonance (NMR) spectra were recorded on a Varian (Palo Alto, CA, USA) 400 MHz magnetic
1
resonance spectrometer. H‐NMR chemical shift is given in parts per million (δ) downfield from
1
TMS. H‐NMR information was tabulated in the following format: number of protons, multiplicity
(s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; td, triplet of doublet; dt, doublet of triplet),
coupling constant(s) (J) in hertz. The prefix app is occasionally applied in cases where the true signal
multiplicity is unresolved, and prefix br indicates a broad signal. A high performance liquid
chromatography (HPLC) analysis for the final compound was performed on an Agilent 1100
instrument using a Waters (Milford, MA, USA) Xterra RP18 column (5 μm, 4.6 mm × 250 mm) and

Molecules 2020, 25, 2431
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gradient elution (solvent A, 20 mM NaH2PO4/acetonitrile, 60:40 v/v; solvent B, acetonitrile). HPLC
purities for the final compound were ≥95%.
(R)‐3‐(3‐fluoro‐4‐(6‐(((S)‐2‐nitro‐6,7‐dihydro‐5H‐imidazo[2,1‐b][1,3]oxazin‐6‐yl)oxy)pyridin‐3‐yl)phe
nyl)‐5‐(hydroxymethyl)oxazolidin‐2‐one
(S)‐6‐((5‐bromopyridin‐2‐yl)oxy)‐2‐nitro‐6,7‐dihydro‐5H‐imidazo[2,1‐b][1,3]oxazine (1.01 g, 2.96
mmol) and
(8a)
[11].
To
a
solution
of
(R)‐3‐(3‐fluoro‐4‐(4,4,5,5‐tetramethyl‐1,3,2‐dioxaborolan‐2‐yl)phenyl)‐5‐(hydroxymethyl)‐oxazolidin
‐2‐onein DMF (30 mL) was added a solution of K2CO3 (0.90 g, 6.51 mmol) in water (1 mL) and the
mixture was purged with nitrogen. Pd(PPh3)4 (0.17 g, 0.15 mmol) was then added and the mixture
was warmed with stirring under nitrogen at 80 °C for 3 h. The solvents were completely removed
under reduced pressure and the residue was partitioned between EtOAc and water. The extract was
worked up and chromatographed on silica. Elution with EtOAc gave four fractions, then elution
with (1:20) MeOH/DCM gave 8a as a yellow powder (460 mg, 33%). This general procedure of
1
Suzuki coupling reaction was also applied for the synthesis of 8b, 11b, 14a–d, and 19a–c. H‐NMR
(400 MHz, DMSO) δ 8.38 (s, 1H), 8.06(s, 1H), 7.94–7.91 (m, 1H), 7.63 (dt, J = 13.6, 2.4 Hz, 1H), 7.57 (d,
J = 8.8 Hz, 1H), 7.46–7.43 (m, 1H), 6.97 (d, J = 9.2 Hz, 1H), 5.76 (s, 1H), 5.24 (t, J = 5.6 Hz, 1H),
4.74–4.67 (m, 2 H), 4.48–4.38 (m, 2 H), 4.13–4.09 (m, 1H), 3.86 (dd, J = 8.8, 6.0 Hz, 1H), 3.70–3.64 (m,
+
1H), 3.56–3.52 (m, 1H), 3.14 (d, J = 5.2 Hz, 1H). LC‐MS (ESI): m/z = 472 (M + H) .
(R)‐3‐(3‐fluoro‐4‐(2‐(((S)‐2‐nitro‐6,7‐dihydro‐5H‐imidazo[2,1‐b][1,3]oxazin‐6‐yl)oxy)pyrimidin‐5‐yl)p
henyl)‐5‐(hydroxymethyl)oxazolidin‐2‐one (8b) [11]. The title compound was prepared by following the
same
procedure
as
that
described
for
the
preparation
of
8a,
except
(S)‐6‐((5‐bromopyrimidin‐2‐yl)oxy)‐2‐nitro‐6,7‐dihydro‐5H‐imidazo[2,1‐b][1,3]oxazine was used
instead of (S)‐6‐((5‐bromopyridin‐2‐yl)oxy)‐2‐nitro‐6,7‐dihydro‐5H‐imidazo[2,1‐b][1,3]oxazine. (580
1
mg, 41%). H‐NMR (400 MHz, DMSO) δ 8.86 (s, 2 H), 8.06 (s, 1H), 7.68 (d, J = 8.8 Hz, 1H), 7.66 (t, J =
2.8 Hz, 1H), 7.47 (dd, J = 8.8, 2.4 Hz, 1H), 5.74 (s, 1H), 5.23 (t, J = 5.6 Hz,1H), 4.77–4.70 (m, 2 H), 4.47 (s,
1H), 4.1 1 (t, J = 9.0 Hz, 1H), 3.86 (dd, J = 8.8, 6.0 Hz, 1H), 3.71–3.65 (m, 1H), 3.57–3.53 (m, 1H),
+
3.38–3.35 (m, 1H). LC‐MS (ESI): m/z = 473 (M + H) .
N‐(((S)‐3‐(2‐fluoro‐4′‐((((S)‐2‐nitro‐6,7‐dihydro‐5H‐imidazo[2,1‐b][1,3]oxazin‐6‐yl)oxy)methyl)‐[1,1′‐
biphenyl]‐4‐yl)‐2‐oxooxazolidin‐5‐yl)methyl)acetamide
(11a)
[11].
To
a
solution
of
(S)‐6‐((4‐bromobenzyl)oxy)‐2‐nitro‐6,7‐dihydro‐5H‐imidazo[2,1‐b][1,3]oxazine (70.4 mg, 0.21 mmol)
and (S)‐N‐((3‐(3‐fluoro‐4‐(4,4,5,5‐tetramethyl‐1,3,2‐dioxaborolan‐2‐yl)phenyl)‐2‐oxooxazolidin‐5‐yl)
methyl)acetamide (86.0 mg, 0.227 mmol) in DMF (8 mL) was added a solution of Na2CO3 (57.0 mg,
0.538 mmol) in water (1 mL) and the mixture was purged with nitrogen. Pd(PPh3)4 (24.0 mg, 0.021
mmol) was then added and the mixture was warmed with stirring under nitrogen at 85 °C for 90
min. The solvents were completely removed under reduced pressure and the residue was
partitioned between EtOAc and water. The extract was worked up and chromatographed on silica.
Elution with EtOAc gave four fractions, then elution with (1:9) MeOH/EtOAc gave 11a as an
1
off‐white powder. (59.4 mg, 44%). H‐NMR (DMSO‐D6, 400 MHz) δ 8.22 (t, J = 5.8 Hz, 1H), 8.07 (s,
1H), 7.60–7.50 (m, 4H), 7.39 (dd, J = 8.6, 2.2 Hz, 1H), 7.15 (d, J = 8.8 Hz, 2H), 5.29 (br s, 1H), 4.79–4.70
(m, 2H), 4.70–4.64 (m, 2H), 4.42 (dd, J = 13.8, 3.2 Hz, 1H), 4.35 (br d, J = 13.8 Hz, 1H), 4.16 (t, J = 9.0
Hz, 1H), 40.03 (M, 1H), 3.78 (dd, J = 9.2, 6.5 Hz, 1H), 3.43 (t, J = 5.5 Hz, 2H), 1.84 (s, 3H). LC‐MS (ESI):
+
m/z = 526 (M + H) .
(R)‐3‐(3‐fluoro‐4‐(6‐((((S)‐2‐nitro‐6,7‐dihydro‐5H‐imidazo[2,1‐b][1,3]oxazin‐6‐yl)oxy)methyl)pyridin‐
3‐yl)phenyl)‐5‐(hydroxymethyl)oxazolidin‐2‐one (11b) [11]. The title compound was prepared by
following the same procedure as that described for the preparation of 8a except
(S)‐6‐((5‐bromopyridin‐2‐yl)methoxy)‐2‐nitro‐6,7‐dihydro‐5H‐imidazo[2,1‐b][1,3]oxazine was used
instead of (S)‐6‐((5‐bromopyridin‐2‐yl)oxy)‐2‐nitro‐6,7‐dihydro‐5H‐imidazo[2,1‐b][1,3]oxazine. (360
1
mg, 38%). H‐NMR (400 MHz, DMSO) δ 8.68 (s, 1H), 8.04 (s, 1H), 7.96 (dt, J = 8.8, 1.6 Hz, 1H), 7.65
(dd, J = 13.6, 2.4 Hz, 1H), 7.60 (d, J = 8.8 Hz, IH), 7.46 (dd, J = 8.4, 2.8 Hz, 2 H), 5.74 (s, 1H), 5.24 (t, J =
5.6 Hz, 1H), 4.77–4.69 (m, 3 H), 4.48 (d, J = 12.0 Hz, 1H), 4.35–4.22 (m, 3 H), 4.12 (t, J = 9.2 Hz, 1H), 3.86
(dd, J = 8.8, 5.6 Hz, 1H), 3.70–3.65 (m, 1H), 3.58–3.52 (m, 1H), 3.16–3.12 (m, 1H). LC‐MS (ESI): m/z =
+
486 (M + H) .

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(5R)‐3‐(2‐fluoro‐4′‐((2‐methyl‐6‐nitro‐2,3‐dihydroimidazo[2,1‐b]oxazol‐2‐yl)methoxy)‐[1,1′‐biphenyl]‐
4‐yl)‐5‐(hydroxymethyl)oxazolidin‐2‐one (14a). The title compound was prepared by following the
same
(S)‐6‐((5‐bromopyridin‐2‐yl)methoxy)‐2‐nitro‐6,7‐dihydro‐5H‐imidazo[2,1‐b][1,3]oxazine was used
instead of (S)‐6‐((5‐bromopyridin‐2‐yl)oxy)‐2‐nitro‐6,7‐dihydro‐5H‐imidazo[2,1‐b][1,3]oxazine
procedure
as
that
described
for
the
preparation
of
8a,
except
1
(2.87g, 56.3%). H‐NMR (400 MHz, DMSO) δ 8.14 (s, 1H), 7.67–7.26 (m, 5H), 6.96 (d, J = 8.8 Hz, 2H),
5.20 (t, J = 5.6 Hz, 1H), 4.69 (td, J = 9.3, 3.6 Hz, 1H), 4.39–4.28 (m, 3H), 4.17 (d, J = 11.0 Hz, 1H), 4.07 (t, J
= 9.0 Hz, 1H), 3.82 (dd, J = 8.8, 6.3 Hz, 1H), 3.65 (ddd, J = 12.2, 5.4, 3.2 Hz, 1H), 3.58–3.48 (m, 1H), 1.66
+
(s, 3H). LC‐MS (ESI): m/z = 485 (M + H) .
(5R)‐3‐(2′‐chloro‐2‐fluoro‐4′‐((2‐methyl‐6‐nitro‐2,3‐dihydroimidazo[2,1‐b]oxazol‐2‐yl)methoxy)‐[1,1′‐b
iphenyl]‐4‐yl)‐5‐(hydroxymethyl)oxazolidin‐2‐one (14b). The title compound was prepared by following
the same procedure as that described for the preparation of 8a, except
2‐((4‐bromo‐3‐chlorophenoxy)methyl)‐2‐methyl‐6‐nitro‐2,3‐dihydroimidazo[2,1‐b]oxazole was used
instead of (S)‐6‐((5‐bromopyridin‐2‐yl)oxy)‐2‐nitro‐6,7‐dihydro‐5H‐imidazo[2,1‐b][1,3]oxazine (346
1
mg, 66.7%). H‐NMR (400 MHz, DMSO) δ 8.13 (s, 1H), 7.55 (dd, J = 12.5, 2.0 Hz, 1H), 7.37 (dd, J = 8.6,
2.0 Hz, 1H), 7.28 (dd, J = 15.5, 8.5 Hz, 2H), 7.13 (d, J = 2.5 Hz, 1H), 6.93 (dd, J = 8.6, 2.5 Hz, 1H), 5.20 (t,
J = 5.6 Hz, 1H), 4.69 (dd, J = 9.1, 5.6 Hz, 1H), 4.41–4.29 (m, 3H), 4.16 (d, J = 11.0 Hz, 1H), 4.08 (t, J = 9.0
Hz, 1H), 3.82 (dd, J = 8.9, 6.1Hz, 1H), 3.69–3.60 (m, 1H), 3.58–3.49 (m, 1H), 1.65 (s, 3H). LC‐MS (ESI):
+
m/z = 567 (M + H) .
(5R)‐3‐(3‐fluoro‐4‐(6‐((2‐methyl‐6‐nitro‐2,3‐dihydroimidazo[2,1‐b]oxazol‐2‐yl)methoxy)pyridin‐3‐yl)p
henyl)‐5‐(hydroxymethyl)oxazolidin‐2‐one (14c). The title compound was prepared by following the
same
procedure
as
that
described
for
the
preparation
of
8a,
except
2‐(((5‐bromopyridin‐2‐yl)oxy)methyl)‐2‐methyl‐6‐nitro‐2,3‐dihydroimidazo[2,1‐b]oxazole was used
instead of (S)‐6‐((5‐bromopyridin‐2‐yl)oxy)‐2‐nitro‐6,7‐dihydro‐5H‐imidazo[2,1‐b][1,3]oxazine (3.5 g,
1
51.5%). H‐NMR (400 MHz, DMSO ) δ 8.34 (s, 1H), 8.15 (s, 1H), 7.94–7.87 (m, 1H), 7.68–7.55 (m, 2H),
7.45 (dd, J = 8.6, 2.1 Hz, 1H), 6.86 (d, J = 8.6 Hz, 1H), 5.22 (t, J = 5.6 Hz, 1H), 4.74 (td, J = 5.8, 2.7 Hz, 1H),
4.63 (s, 2H), 4.41 (d, J = 11.0 Hz, 1H), 4.25–4.09 (m, 2H), 3.88 (dd, J = 8.9, 6.2 Hz, 1H), 3.70 (ddd, J =
+
12.3, 5.5, 3.4 Hz, 1H), 3.58 (ddd, J = 12.3, 5.7, 4.1 Hz, 1H), 1.71 (s, 3H). LC‐MS (ESI): m/z = 486 (M + H) .
(5R)‐3‐(3‐fluoro‐4‐(5‐((2‐methyl‐6‐nitro‐2,3‐dihydroimidazo[2,1‐b]oxazol‐2‐yl)methoxy)pyridin‐2‐yl)p
henyl)‐5‐(hydroxymethyl)oxazolidin‐2‐one (14d). The title compound was prepared by following the
same
procedure
as
that
described
for
the
preparation
of
8a,
except
2‐(((6‐bromopyridin‐3‐yl)oxy)methyl)‐2‐methyl‐6‐nitro‐2,3‐dihydroimidazo[2,1‐b]oxazole was used
instead of (S)‐6‐((5‐bromopyridin‐2‐yl)oxy)‐2‐nitro‐6,7‐dihydro‐5H‐imidazo[2,1‐b][1,3]oxazine (280
1
mg, 57.7%). H‐NMR (400 MHz, DMSO) δ 8.32 (d, J = 2.9 Hz, 1H), 8.16 (d, J = 17.1 Hz, 1H), 7.90 (t, J =
9.0 Hz, 1H), 7.69 (d, J = 7.6 Hz, 1H), 7.58 (dd, J = 14.2, 1.9 Hz, 1H), 7.48–7.38 (m, 2H), 5.21 (t, J = 5.6 Hz,
1H), 4.70 (dd, J = 9.0, 5.7 Hz, 1H), 4.40 (dt, J = 11.0, 9.4 Hz, 3H), 4.17 (d, J = 11.0 Hz, 1H), 4.08 (t, J = 9.0
Hz, 1H), 3.83 (dd, J = 8.7, 6.3 Hz, 1H), 3.65 (ddd, J = 12.2, 5.2, 3.4 Hz, 1H), 3.59–3.49 (m, 1H), 1.67 (s,
+
3H). LC‐MS (ESI): m/z = 486 (M + H) .
(5R)‐3‐(3‐fluoro‐4‐((5‐((2‐methyl‐6‐nitro‐2,3‐dihydroimidazo[2,1‐b]oxazol‐2‐yl)methoxy)pyridin‐2‐yl)o
xy)phenyl)‐5‐(hydroxymethyl)oxazolidin‐2‐one (16).
2‐(((5‐bromopyridin‐2‐yl)oxy)methyl)‐2‐methyl‐6‐nitro‐2,3‐dihydroimidazo[2,1‐b]oxazole (532 mg, 1.5
mmol), (R)‐3‐(3‐fluoro‐4‐hydroxyphenyl)‐5‐(hydroxymethyl)oxazolidin‐2‐one (376 mg, 1.65 mmol ),
Potassium carbonate (622 mg, 4.5mmol), Cuprous iodide (286 mg, 1.5 mmol) and
N,N,N′,N′‐Tetramethylethylenediamine (TMEDA) (174 mg, 1.5 mmol) were added in
N,N‐dimethylformamide (5 mL). The mixture was stirred at 90 °C for 12 h. The mixture was cooled
to room temperature, then poured into water (50 mL), the solvent was filtered and the filtered solid
was pump dried. The crude product was further purified by silica chromatography column
(DCM:MeOH
=
100:2
)
to
give
(5R)‐3‐(3‐fluoro‐4‐((5‐((2‐methyl‐6‐nitro‐2,3‐dihydroimidazo‐[2,1‐b]oxazol‐2‐yl)methoxy)pyridin‐2‐y
1
l)oxy)phenyl)‐5‐(hydroxymethyl)oxazolidin‐2‐one(200 mg, 26.6%). H NMR (500 MHz, DMSO) δ
8.29 (s, 1H), 8.05 (s, 1H), 7.88 (d, J = 8.4 Hz, 1H), 7.64–7.50 (m, 2H), 7.41 (d, J = 8.5 Hz, 1H), 6.92 (d, J =
8.5 Hz, 1H), 4.70 (s, 1H), 4.48 (s, 2H), 4.23–4.01 (m, 3H), 3.83 (t, J = 7.3 Hz, 1H), 3.65 (d, J = 12.4 Hz,

Molecules 2020, 25, 2431
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1H), 3.53 (d, J = 12.2 Hz, 1H), 2.40–2.29 (m, 1H), 2.22–2.09 (m, 1H), 1.46 (s, 3H). LC‐MS (ESI): m/z = 502
+
(M + H) .
(5R)‐3‐(3‐fluoro‐4‐(6‐((2‐nitro‐6,7‐dihydro‐5H‐imidazo[2,1‐b][1,3]oxazin‐7‐yl)methoxy)pyridin‐3‐yl)p
henyl)‐5‐(hydroxymethyl)oxazolidin‐2‐one (19a). The title compound was prepared by following the
same
procedure
as
that
described
for
the
preparation
of
8a,
except
7‐(((5‐bromopyridin‐2‐yl)oxy)methyl)‐2‐nitro‐6,7‐dihydro‐5H‐imidazo[2,1‐b][1,3]oxazine was used
instead of (S)‐6‐((5‐bromopyridin‐2‐yl)oxy)‐2‐nitro‐6,7‐dihydro‐5H‐imidazo[2,1‐b][1,3]oxazine (162
1
mg, 56.3%). H‐NMR (400 MHz, DMSO) δ 8.32 (s, 1H), 8.06 (s, 1H), 7.91 (d, J = 8.2 Hz, 1H), 7.58 (dd, J =
22.0, 11.8 Hz, 2H), 7.42 (d, J = 8.0 Hz, 1H), 6.97 (d, J = 8.5 Hz, 1H), 5.21 (t, J = 5.4 Hz, 1H), 4.91 (s, 1H),
4.71 (d, J = 2.9 Hz, 1H), 4.57 (dt, J = 12.1, 7.7 Hz, 2H), 4.21–4.01 (m, 3H), 3.89–3.80 (m, 1H), 3.66 (d, J =
11.5 Hz, 1H), 3.60–3.49 (m, 1H), 2.29 (d, J = 13.4 Hz, 1H), 2.18 (dd, J = 15.1, 9.6 Hz, 1H). LC‐MS (ESI):
+
m/z = 486 (M + H) .
(5R)‐3‐(3‐fluoro‐4‐(2‐((2‐nitro‐6,7‐dihydro‐5H‐imidazo[2,1‐b][1,3]oxazin‐7‐yl)methoxy)pyrimidin‐5‐yl
)phenyl)‐5‐(hydroxymethyl)oxazolidin‐2‐one (19b). The title compound was prepared by following the
same
procedure
as
that
described
for
the
preparation
of
8a,
except
7‐(((5‐bromopyrimidin‐2‐yl)oxy)methyl)‐2‐nitro‐6,7‐dihydro‐5H‐imidazo[2,1‐b][1,3]oxazine
was
used instead of (S)‐6‐((5‐bromopyridin‐2‐yl)oxy)‐2‐nitro‐6,7‐dihydro‐5H‐imidazo[2,1‐b][1,3]oxazine
1
(674 mg, 50.3%). H‐NMR (400 MHz, DMSO) δ 8.81 (d, J = 0.9 Hz, 2H), 8.07 (s, 1H), 7.70–7.62 (m, 2H),
7.48–7.41 (m, 1H), 5.22 (t, J = 5.6 Hz, 1H), 5.01–4.90 (m, 1H), 4.76–4.61 (m, 3H), 4.20–4.06 (m, 3H), 3.85
(dd, J = 8.8, 6.3 Hz, 1H), 3.66 (ddd, J = 12.3, 5.2, 3.4 Hz, 1H), 3.54 (ddd, J = 12.4, 5.6, 4.1 Hz, 1H), 2.30
+
(dd, J = 11.9, 2.4 Hz, 1H), 2.24–2.10 (m, 1H). LC‐MS (ESI): m/z = 487 (M + H) .
(5R)‐3‐(3‐fluoro‐4‐(6‐((7‐methyl‐2‐nitro‐6,7‐dihydro‐5H‐imidazo[2,1‐b][1,3]oxazin‐7‐yl)methoxy)pyrid
in‐3‐yl)phenyl)‐5‐(hydroxymethyl)oxazolidin‐2‐one (19c). The title compound was prepared by
following the same procedure as that described for the preparation of 8a except
7‐(((5‐bromopyridin‐2‐yl)oxy)methyl)‐7‐methyl‐2‐nitro‐6,7‐dihydro‐5H‐imidazo[2,1‐b][1,3]oxazine
was
used
instead
of
(S)‐6‐((5‐bromopyridin‐2‐yl)oxy)‐2‐nitro‐6,7‐dihydro‐5H‐imidazo[2,1‐b][1,3]oxazine (260 mg, 68.6%).
1
H‐NMR (500 MHz, DMSO) δ 8.29 (s, 1H), 8.05 (s, 1H), 7.88 (d, J = 8.4 Hz, 1H), 7.64–7.50 (m, 2H), 7.41
(d, J = 8.5 Hz, 1H), 6.92 (d, J = 8.5 Hz, 1H), 4.70 (s, 1H), 4.48 (s, 2H), 4.11 (dt, J = 17.0, 7.2 Hz, 3H), 3.83
(t, J = 7.3 Hz, 1H), 3.65 (d, J = 12.4 Hz, 1H), 3.53 (d, J = 12.2 Hz, 1H), 2.40–2.29 (m, 1H), 2.22–2.09 (m,
+
1H), 1.46 (s, 3H). LC‐MS (ESI): m/z = 500 (M + H) .
3.2. Biology
3.2.1. Bacterial Strains
The bacterial strains used in the current study are listed in Table 4. Strains in the Mycobacterium
tuberculosis isogenic resistant mutant panel used for the mechanism of action study were generated
and tested at Beijing Tuberculosis and Thoracic Tumor Research Institute (BTTTRI, Bejing, China).
Strains of the spectrum panel were all ATCC stains, and testing was conducted at HD Biosciences
(Shanghai, China). Strains of the anaerobic bacterial panel were ATCC strains, and the test was
conducted at Micomyx Inc. (Kalamazoe, MI, USA).

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Table 4. List of bacterial strains used in the current study.
Panel
Organism
Enterococcus faecalis (Ef)
Staphylococcus aureus (Sa)
Klebciella pneumonia (Kp)
Acinetobacter baumannii (Ab)
Pseudomonas aeruginosa (Pa)
Escherichia coli (Ec)
ATCC No.
700221
292213
43816
19606
27853
25922
43255
27294
Isogenic
Isogenic
Isogenic
Isogenic
700057
4356
Testing Facility
Spectrum Panel
HD Biosciences
BTTTRI
Clostrium difficile (Cd)
M. tuberculosis H37Rv (Wild‐Type)
M. tuberculosis P1 (Pretomanid‐R)
M. tuberculosis P3 (Pretomanis‐R)
M. tuberculosis L1 (Linezolid‐R)
M. tuberculosis L3 (Linezolid‐R)
Clostridium difficile (Cd)
M. tuberculosis
Isogenic Resistant
Mutant Panel
Lactobacillus acidophilus (Lc)
Eggerthella lenta (El)
43055
Gardnerella vaginalis (Gv)
Peptostreptococcus micros (Pm)
Porphyromonas asaccharolytica (Pas)
Propionibacterium acnes (Pac)
Bacteroides fragilis (Bf)
Bifidobacterium longum (Bl)
Fusobacterium nucleatum (Fn)
Mobiluncus mulieris (Mm)
Prevotella bivia (Pb)
14018
23195
M3553
11892
25285
15707
10953
35243
Anaerobic Bacterial
Panel
Micromyx
29303
Veillonella parvula (Vp)
17745
Helicobacter pylori (Hp)
43504
3.2.2. Generation of Isogenic Mutant Panel
The methods to induce and characterize the Mtb isogenic mutant strains resistant to
4
5
pretomanid or linezolid have been described previously [12,13]. Briefly, 200 μL of 10 –10 CFU/mL
of M. tuberculosis H37Rv (ATCC27294) in 7H9 liquid culture medium at logarithmic growth phase
were evenly spread on a 7H11 solid medium containing 1 MIC (0.07 μg/mL) of pretomanid and
blank control medium. After sealing, the plates were incubated at 37 °C under 5% CO2 for 3 to 4
weeks. Single colonies with good growth were collected from the drug‐containing solid medium,
4
5
milled and diluted to a concentration of 10 –10 CFU/mL, and then inoculated onto 7H11 solid
medium with pretomanid at 2 MIC (0.15 μg/mL) and blank control medium. The plates were then
incubated under the same conditions for another 3 to 4 weeks to observe the growth of single
colonies. A single colony with robust growth was collected from the drug‐containing medium and
further subcultured on the drug‐containing solid medium with a 2‐fold increase of concentration of
pretomanid until the selection of a single colonies that grew well on solid medium with pretomanid
concentration of 16 MIC. The phenotypic confirmation of the pretomanid resistant mutant strains
was performed by the Alamar Blue double dilution method [14]. The genotypic change of the
resistant mutant strains were confirmed to be the T265C point mutation (TAC→CAC, Y89H) in the
ddn gene. A similar sequential drug selection method was applied to obtain linezolid resistant
mutant trains. The genotypic change of the linezolid resistant mutant strains was found to be the
point mutation of the rplC (T460C).
3.2.3. Media
Media were prepared according to guidelines from CLSI. The test medium used for the
anaerobic bacterial was supplemented Brucella broth (BD, Lot No. 6278735) containing 5 μg/mL

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hemin (Sigma (St. Louis, MO. USA), Lot No. SLBC4685V), 1 mg/mL Vitamin K1 (Sigma, Lot No.
MKBN5958V), and 5% (v/v) laked horse blood (LHB, Cleveland Scientific (Bath, OH, USA), Lot No.
385663). Brucella broth (BD) supplemented with 10% fetal bovine serum (FBS, Gibco, Lot No.
1709261) was used for testing of H. pylori. Cation‐adjusted Mueller Hinton Broth (CAMHB) media
was used for the spectrum determination against the spectrum panel. Difco Middlebrook
(Waltham, MA, USA) 7H9 Broth (Catalog No. 271310) supplemented with 0.2% (v/v) glycerol, 0.05%
Tween 80, and 10% (v/v) albumin‐dextrosecatalase (BBL Middlebrook (Waltham, MA, USA) ADC
Enrichment, Catalog No. 212352) (7H9‐ADC‐T) was used for the MIC assay against the M.
tuberculosis isogenic resistant mutant panel.
3.2.4. Minimum Inhibitory Concentration Testing
The MIC test against the spectrum panel was conducted at HB Biosciences, following the broth
microdilution method per CLSI guidance. The six ESKAPE ATCC strains were recovered in
Trypticase Soy Agar (TSA) plates and tested with drug in CAMHB. Anaerobic bacterial C. difficile
strains were recovered on TSA agar, grown and tested with compounds in Blucella broth with 5
μg/mL chlorhematin and 10 μg/mL vitamin K1 in an anaerobic chamber.
The MIC assay against the Mtb isogenic resistant mutant strains was performed at BTTTRI by
the microplate Alamar blue assay [14]. Pretomanid, linezolid and metronidazole were used as
comparators. The H37Rv strain and its derived isogenic resistant mutant strains were grown for 1–2
7
weeks at 37 °C and adjusted to a turbidity of McFarland 1 at 10 CFU/mL and diluted 1:20. Twofold
dilutions of testing compounds and comparators were prepared in 7H9‐ADC‐TG in volumes of 100
μL in 96‐well, black, clear‐bottom microplates (BD Biosciences, Franklin Lakes, NJ, USA). Bacterial
5
cells (100 μL containing 2 × 10 CFU) was added, yielding a final testing volume of 200 μL. The plates
were incubated at 37 °C, and on day 7, 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 to 24 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 the mean of replicate bacterium‐only controls.
The MIC assay against anaerobic bacterial strains was performed at Micromyx by following the
procedure described by CLSI [15–17]. A standardized inoculum of each organism was prepared as
per CLSI methods. Colonies were picked from the primary plate and a suspension was prepared to
equal to a 0.5 McFarland turbidity standard. Anaerobic suspensions were diluted 1:10 in Brucella
broth with 5% laked horse blood, and each well was inoculated with 10 μL using a multichannel
5
pipette in the Bactron anaerobe chamber, resulting in a final cell density of approximately 5  10
4
CFU/mL (5  10 CFU/mL for Clostridium spp.). For the H. pylori strain, colonies were picked from the
primary plate and a suspension was prepared to equal a 2.0 McFarland turbidity standard.
Suspensions were diluted 1:15 in Brucella broth with 10% FBS, and then transferred to
compartments of sterile reservoirs divided by length (Beckman Coulter). The Biomek 2000 (Brea,
CA, United States) was used to inoculate the plates. Daughter plates were placed on the Biomek 2000
work surface in reverse orientation so that plates were inoculated from low to high drug
concentration. The Biomek 2000 delivered 10 μL of standardized inoculum into each well of the
appropriate daughter plate for an additional 1:20 dilution. Anaerobe plates were placed in an
anaerobic box with GasPak sachets (BD), and were incubated anaerobically for 46–48 h at 35–37 °C.
H. pylori was incubated for 72 h in a microaerophilic atmosphere using boxes with GasPak EZ
Campy sachets (BD) prior to reading. After incubation, plates were viewed from the bottom using a
plate viewer. An uninoculated solubility control plate was observed for evidence of drug
precipitation. MIC values were read and recorded as the lowest concentration of drug that inhibited
the visible growth of the organism.
3.2.5. Cytotoxicity Assay
The cytotoxicity assay followed the previously published protocol [14]. Briefly, Vero cells in
RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) were incubated in a
5
humidified atmosphere of 5% CO2 at 37 °C to reach confluent, and then diluted to 4 × 10 cells/mL.

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Threefold serial dilutions of the stock solutions resulted in final concentrations of 64 to 0.26 μg/mL in
a final volume of 100 μL. Cytotoxicity testing was performed in a transparent 96‐well microplate.
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 and freshly made
methyl‐thiazolyldiphenyl‐tetrazolium bromide (MTT) were added to each well, and then the plates
were incubated for 4 h, after which the absorbance was determined at 492 nm.
4. Conclusions
A series of 4‐nitroimidazole and oxazolidione conjugate molecules was designed, synthesized
and evaluated based on previous structure‐activity relationship information. The dual mechanism of
action of this series was demonstrated against Mycobacterium tuberculosis by utilizing an isogenic
mutant panel resistant to either pretomanid or linezolid. Compounds in the series are highly active
against a panel of clinically important anaerobic bacteria. A strong synergistic effect was observed
compared to the combination of linezolid and pretomanid. It can be concluded that the
nitroimidazole‐oxazolidinone conjugate molecules hold potential for the treatment of anaerobic
bacterial infections.
Author Contributions: Z.M., C.Z.D., A.S.L., A.M.U., C.B.C. and W.A.D. conceived the compound series and
were responsible for the initial design, synthesis and evaluation of the conjugate series. Z.Z., D.W., J.D. and Q.Z.
conducted the synthesis and characterization of the conjugate molecules of the current study. S.H., X.W. and
Y.Y. coordinated and oversaw compound testing. Y.L. generated and tested against the Mtb isogenic resistant
mutant panel. All authors have read and agreed to the published version of the manuscript.
Funding: We thank the National Science and Technology Project of China (No.2015ZX09102007‐007) for partial
financial support of this project.
Conflicts of Interest: The authors declare no conflicts of interest.
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Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).