Novel Oxazolidinone Antibacterial Analogues with a Substituted Ligustrazine C-ring Unit

Article abstract of DOI:10.1111/cbdd.12537

A series of novel oxazolidinone compounds with a substituted ligustrazine C-ring unit and different substituted groups at the C-5 side chain were designed and synthesized using linezolid as a lead and based on a scaffold hopping strategy. Their antibacterial and anti-inflammatory activities were evaluated. The results of in vitro antibacterial assays showed that all fourteen target compounds displayed potent activity against Gram-positive pathogens, particularly 8b, 13b, 14a, 14b, 15a, and 15b. Moreover, 14a and 14b exhibited significant inhibitory activities on the production of inflammatory mediators, including nitric oxide, interleukin-6, and tumor necrosis factor-alpha. Thus, these derivatives could serve as valuable candidates to develop anti-infective agents for the treatment of chronic wounds.

Full text of DOI:10.1111/cbdd.12537

Received Date : 21-Oct-2014
Revised Date : 13-Jan-2015
Accepted Date : 16-Jan-2015
Article type
: Research Article
Novel oxazolidinone antibacterial analogues with a substituted
ligustrazine C-ring unit
Yan Chen+, Zhi-Xiong Ruan+, Fang Wang, De-Sheng Huangfu, Ping-Hua Sun, Jing Lin,
Wei-Min Chen*
College of Pharmacy, Jinan University, Guangzhou 510632, P. R. China
+These authors contributed equally.
*Corresponding author. Tel.: +86 20 8522 4497; Fax: +86 20 8522 4766.
E-mail address: twmchen@jnu.edu.cn
Abstract: A series of novel oxazolidinone compounds with a substituted ligustrazine C-ring
unit and different substituted groups at the C-5 side chain were designed and synthesized by
using linezolid as a lead and based on a scaffold hopping strategy. Their antibacterial and
anti-inflammatory activities were evaluated. The results of in vitro antibacterial assays
showed that all fourteen target compounds displayed potent activity against gram-positive
pathogens, particularly 8b, 13b, 14a, 14b, 15a and 15b. Moreover, 14a and 14b exhibited
significant inhibitory activities on the production of inflammatory mediators, including nitric
oxide (NO), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α). Thus, these
derivatives could serve as valuable candidates to develop anti-infective agents for the
treatment of chronic wounds.
Keywords: Antibacterial; Anti-inflammatory; Linezolid; Ligustrazine; Oxazolidinone;
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
an 'Accepted Article', doi: 10.1111/cbdd.12537
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Introduction
The alarming increasing rate of bacterial pathogens resistant to existing antimicrobials has
become a serious problem in current medicine. In particular, the emergence of drug-resistant
bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and epidermis
Staphylococcus (MRSE) severely reduce the efficacy of existing drugs, which leads to the
significantly extension of treatment time and the increase of mortality. Therefore, it is urgent
to discover novel antibacterial agents that are effective against the resistance strains to
overcome the growing problem of antibiotics resistance. A possible approach of solving this
problem is to develop antibacterial drugs with novel structure and unique mechanism (1, 2).
Linezolid, which is the first oxazolidinone antibacterial agent (Figure 1), has shown
promising effect for the treatment of multiple drug-resistant infections caused by
gram-positive bacteria. Oxazolidinones are a new class of antibacterial agents, and their
action sites are located inside the ribosomal 50S subunit near the peptidyl transferase center
(PTC) and peptidyl site (P), overlapping with peptide acyl transferase inhibitor (such as
clindamycin and chloramphenicol) loci (3). However, with excessive use of linezolid, it has
raised serious concerns that the emergence of linezolid-resistant strains such as
Staphylococcus aureus (4, 5) and Enterococcus (6) in the clinic. In the last few years, a
variety of structural modifications of linezolid has been carried out towards broadening the
antibacterial spectrum, re-establishing sensitivity to linezolid-resistant strains (7-10).
The infections caused by drug-resistant bacteria, whatever gram-positive or gram-negative
bacterium, often lead to chronic inflammation, even aggravate diseases or induce permanent
organ damage (11), and bacterial profile often observed in many chronic wounds, as an
example, Staphylococcus aureus (in 93.5% of the investigated ulcers), Enterococcus faecalis
(71.7%), Pseudomonas aeruginosa (52.2%), coagulase-negative staphylococci (45.7%),
Proteusspecies (41.3%), and anaerobic bacteria (39.1%) were found in venous leg ulcers (12).
Currently, to cure these chronic infection phlogosis such as venous leg ulcers, pressure ulcers,
and diabetic foot ulcers is still with great difficulties (13, 14). Thus, there is an urgent need to
develop drugs with both antibacterial and anti-inflammation effects to treat the chronic
wounds.
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Inflammation is very common and important basic pathological process and a fundamental
defensive response of the immune system to pathogens or harmful irritants (15). The classical
phenomena of inflammation are redness, swelling, heat, and pain (16). For example,
excessive production of inflammatory mediators, such as nitric oxide (NO), interleukin-6
(IL-6) and tumor necrosis factor-alpha (TNF-α) (17-19) may be observed after infection or
injury, which have been implicated in many inflammatory diseases (20, 21).
Ligustrazine (tetramethylpyrazine, TMP; Figure 1) is one of the major efficient component
from traditional Chinese medicine herb Chuanxiong (Ligusticum wallichii Franchat) (22). It
has been found that ligustrazine shows a certain degree of anti-inflammatory effects both in
vivo and in vitro (23-28). This encouraged us to design new compounds using ligustrazine
moiety to hybridize with oxazolidinone for exploring their anti-inflammatory activity.
Therefore, a series of novel oxazolidinone compounds with a substituted ligustrazine C-ring
unit and different substituted groups at the C-5 side chain were designed and synthesized
based on the template compound linezolid so as to obtain novel oxazolidinone anti-infective
drugs with both antibacterial and anti-inflammatory activities.
Materials and Methods
General synthetic procedure
All reagents and solvents used were purchased from common commercial of analytical grade.
1H NMR and 13C NMR were recorded on a 300 MHz spectrometer with TMS as the internal
standard. Proton and carbon chemical shifts are expressed in parts per million (ppm) and
coupling constants in Hz. Mass spectra (ESI-MS) and high resolution mass spectrometry
(HRMS) were performed.
Detailed synthetic procedures and characterization data for the synthesized compounds are
available in the Supporting Information (Appendix S1) of this manuscript.
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Antibacterial assay
In this study, thirteen strains including nine different MRSA strains, S.aureus ATCC 25923,
S.epidermidis ATCC 12228, Enterococcus faecalis ATCC 29212 and Escherichia coli ATCC
25922, were tested and stored at -80 oC. Tryptic soy broth (TSB) was prepared according to
the manufacturer’s instructions. After all bacterial were recovered, all strains were inoculated
into 5 mL sterilized TSB, cultured at 37 oC for 16-18 h.
Minimum Inhibitory Concentration (MIC) was determined by the micro-broth dilution
methodology, according to the guideline of the Clinical and Laboratory Standards Institute
(CLSI) (29). Each tested compound was dissolved in a certain amount of dimethyl sulphoxide.
Linezolid was chosen as a positive control drug. The bacteria solution was corrected to 0.5
McFarland standard (amount of bacteria about 108 colony-forming units/mL) using TSB.
Then, it was diluted 1:100 with TSB broth and inoculated immediately. First, the
concentration of each compound including linezolid was severally allocated to 256 μg/mL,
128 μg/mL, 64 µg/mL, 32 µg/mL, 16 µg/mL, 8 µg/mL, 4 µg/mL, 2 µg/mL and 1μg/mL. 100
µL different concentrations of each compound including linezolid was added into the holes of
1-12 rows of a 96-hole plate, then, 100 µL diluted bacterium solution was also added into
each hole and tapped to mix sufficiently. The seventh line of the 96 well plate was used for
the blank control group and the eighth line was used for the negative control (TSB broth 100
µL, diluted bacterium solution 100 µL). Finally, the plate was kept in incubator at 37 oC for
18-24 h. Meanwhile, each bacterium was repeated for three times.
Anti-inflammation assay
RAW264.7 murine macrophages were cultured in DMEM containing 10% new-born calf
o
serum, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37 C in a 5% CO2
humidified atmosphere.
Assay for NO production
RAW264.7 cells were inoculated at 10×104 cells per well in 96-well plate and cultured for 18
h. The concentration of each compound including the positive drugs (linezolid, TMP and
indomethacin) was designed to 100 µM, 50 µM, 20 µM, 10 µM, 5 µM, 2 µM and 1 µM.
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Then, the cells were pre-handled with different concentration compounds for 2 h and
stimulated with LPS (100 ng/mL) for 24 h. Then, the Griess reagent was used to determine
the NO produced in the culture medium, which was made up of 1% sulfanilamide and 0.1%
N-(1-naphthyl)ethylenediamine dihydrochloride in 5% phosphoric acid), that is to say, added
100 µL Griess reagent to 100 µL cultural supernatants and mixed sufficiently at room
temperature. Then the plate was measured absorbance of the samples at 540 nm (OD540) in a
microplate reader (Bio-Rad Laboratories, CA, USA). Meanwhile, the results were evaluated
from three independent experiments.
Cell Cytotoxicity
Cell cytotoxicity was evaluated by methyl thiazolyl tetrazolium (MTT) assay. RAW264.7
cells were inoculated at 8×103 cells per well in 96-well plate. The cells were treated with
different compounds which were diluted in DEME for 48 h until the cells were cultured
overnight. Then, 20 µL of 0.5 mg/mL MTT reagent was added into the each well and
incubated for 4 h, followed by removing the cell supernatant and adding 150 µL DMSO to
dissolve the formazan. The optical density was measured at 570 nm (OD570). Cell viability
was assessed from three independent experiments. The density of formazan formed in control
group was set as 100% of viability.
Cell viability (%) = [compound (OD570)-blank (OD570)] / [control (OD570)-blank (OD570)] ×
100%
Blank: fresh medium only
Control: the cells cultured with fresh medium only
Compound: treated with compounds
Measurement of IL-6 and TNF-α
RAW264.7 cells were inoculated at 5×105 cells per well in 24-well plate and pretreated with
different concentrations (10 µM, 5 µM, 2.5 µM, 1.25 µM) of compounds, except TMP (200
µM to 20 µM) for 2 h, and then added LPS. The production of IL-6 and TNF-α was
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stimulated by the addition of 100 ng/mL LPS and incubated for 24 h. The content of IL-6 and
TNF-α in the supernatant were determined using the mouse ELISA kit (TNF-α; IL-6) which
were operated according to the manufacturer’s instructions.
Results and discussion
Design of compounds
In order to develop novel anti-infective agents that are effective against the resistance strains,
drug designs were carried out based on linezolid (Figure 2). Firstly based on a scaffold
hopping strategy, the C-ring unit of linezolid was replaced with ligustrazine fragment
(3,5,6-trimethyl-pyrazin-2-ylmethoxy group) which has been reported to show
anti-inflammatory activity. Meanwhile, further modifications of the new skeleton compound
were carried out at C-5 side chain, since the reported structural modifications of
oxazolidinone compounds were mainly concentrated in the C-5 side chain and the C-ring unit
(30-34) to optimize their biological activities. It has been reported that the introducing of
acetamide group (3, 35, 36) or triazole group (37-40) at the C-5 position in oxazolidinone
compounds shows good antibacterial activities, thus acetamide group or triazole group were
chosen for the derivatization of C-5 side chain. Additionally, a growing body of evidence
suggests that the introduction of halogen group in the anti-bacterial agents could improve
their biological activities (41), thus, F atom and Cl atom were introduced to some designed
compound.
Synthesis
The synthetic route of the novel oxazolidinone compounds is depicted in Scheme 1.
Ligustrazine (TMP) was used as a starting material. It was firstly brominated to produce
TMP-Br, which was then reacted with p-nitrophenol or 2-fluoro-4-nitrophenol to give the
intermediate A1 and B1. After a few reaction steps including reduction of the nitro group,
carbamate protection of amino group with benzyl chloroformate and a ring closure using
(R)-glycidyl butyrate and LiHMDS, the 5-hydroxymethyl oxazolidinone derivatives 1a and
1b were obtained.
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Fluoromethyl oxazolidinones 2a and 2b were prepared from the reaction of 1a and 1b with
diethylaminosulphurtrifluoride (DAST) as fluorinating agent. After the hydroxyl groups of 1a
and 1b were converted into the corresponding mesylate group using mesyl chloride, the
resulting compounds 3a and 3b were reacted with a variety of nucleophiles to yield the
5-substituted oxazolidinone derivatives. Replacement of methanesulfonate group in
compounds 3a and 3b with a methoxy group was easily accomplished to get methoxy methyl
oxazolidinones 4a and 4b using sodium methoxide in methanol at room temperature.
Dimethylamino-substituted methyl oxazolidinones 5a and 5b was obtained from the reaction
of 3a and 3b with dimethylamine hydrochloride under K2CO3 in DMF. To prepare
compounds containing 1,2,4-triazole and 1,2,3-triazole moieties, unsubstituted triazole
compounds 6a, 6b, 7a, 7b, 8a and 8b were prepared according to the literature (42). The
substituted triazole compounds 10a, 10b, 11a and 11b were easily synthesized using
1,3-dipolar cycloaddition condition (40) on azides 9a and 9b, which were obtained from
reacting 3a and 3b with sodium azide in DMF. Finally, several 5-substituted amide
compounds (13a-17a and 13b-17b) were synthesized by acylation of compounds 12a and
12b, which were prepared from reduction of 9a and 9b in Pd/C under hydrogen at room
temperature in THF.
Antibacterial activity
The in vitro antibacterial activity of compounds 1a-17a and 1b-17b were evaluated and the
results were summarized in Table 1. Compounds 1b, 8a, 8b, 10b, 13a-17a and 13b-17b
showed potential antibacterial activities against gram-positive bacteria, while they were
inactive against gram-negative bacteria. Meanwhile, compounds 1a-7a, 10a, 11a, 2b-7b and
11b showed no antimicrobial activity which were not listed in Table 1. Among these active
compounds, 8b, 13b, 14a, 14b, 15a and 15b which were listed and bolded in Table 1,
exhibited potent antibacterial activities against gram-positive bacteria. 13b, 14b and 15b
showed the most prominent activities against S. epidermidis. 15b showed the best activity
against S. aureus which was comparable to linezolid. 14b and 15b also showed potent
activities against Enterococcus faecalis. Moreover, 13b, 14b and 15b exhibited almost the
same activities against nine different MRSA strains. It is obvious that compounds 14b and
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15b provided significant activities against resistant strains, which were equivalent to that of
linezolid.
Inhibition of NO production in (LPS)-stimulated RAW264.7 cells
Nitric oxide (NO) is an important indicator of inflammatory determination, which is one of
cytokines produced by active cells in the inflammation process. Excessive production of NO
was needed to be connected with the pathogenesis of inflammatory diseases, and it is
reported that NO inhibitors may provide the possibility of new therapeutic method for the
inflammatory diseases (43). Meanwhile, it has been reported that TMP shows
anti-inflammatory effects (44, 45). The NO levels were decreased after treatment with 10 μM
or 100 μM TMP in UVA treated keratinocyte monoculture cells or melanoma/keratinocyte
co-culture cells, respectively (45). Meanwhile, TMP could also decrease the UV-induced
increase of cytokines such as TNF-α (45).
So all synthesized compounds were tested for their inhibitory activities against
lipopolysaccharide (LPS)-induced NO release in RAW264.7 cells (LPS treatment caused
significant changes in cell morphologies, which indicated that inflammation could be induced
by LPS). Thus, linezolid, ligustrazine and indomethacin (Figure 1) were chosen as positive
controls. The results show that most of the novel oxazolidinone compounds did not show the
NO inhibitory activity. While, as shown in Table 2, compounds 14a and 14b (Figure 1)
displayed improved NO inhibitory activity compared to linezolid, TMP and indomethacin.
Cytotoxicity in RAW264.7 cells
To confirm whether the NO inhibitory activities of 14a and 14b at the concentration of 10
μM were related to their effects on the cell viability, their cytotoxicities in RAW264.7 cells
were examined by methyl thiazolyl tetrazolium (MTT) assay. As shown in Table 3 and Figure
3, all the agents (linezolid, TMP, indomethacin, 14a and 14b) at the concentration we used
here had no obviously cytotoxicity in RAW264.7 cell, the relative cell viabilities of the
treated cells were all more than 90%. Besides, it was shown that TMP did not induce
significant cell death and with no obvious cytotoxicity to cells even up to 200 μM. These
results indicated that the NO inhibitory effects of 14a and 14b were likely to be attributed to
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the interaction of the substituent group of these two compounds with their specific target.
These non-toxic concentrations were further used in subsequent experiment processes.
Inhibition of IL-6 and TNF-α production in RAW264.7 cells
IL-6 and TNF-α are two other critical pro-inflammatory cytokines in inflammatory diseases,
and it is widely accepted that the inhibition of their generation would be an effective method
for treatment of inflammation (46). Therefore, the inhibitory effects of 14a and 14b on IL-6
and TNF-α production were investigated. Linezolid, TMP and indomethacin were also used
as positive reference drugs. As shown in Table 4, both 14a and 14b showed better inhibitory
effects on the LPS-induced production of IL-6 and TNF-α than the positive control drugs
(linezolid and indomethacin) at both concentrations of 10 µM and 5 µM. Meanwhile, TMP
could decrease the LPS-induced TNF-α and IL-6 production but in a weak degree in a
concentration-dependent manner (200 μM to 20 μM). It is worth noting that compound 14b
exhibited significant improvement on inhibition of IL-6 production compared to control
drugs (Figure 4).
Taken together, these results suggest that there is a feeble anti-inflammatory activity of TMP
and indomethacin in the LPS-induced RAW264.7. However, compounds 14a and 14b with
TMP unit exhibit significant anti-inflammatory activities while linezolid does not show.
Besides, 14a and 14b possess potent antibacterial activities which are equivalent to linezolid.
These results suggest that the combination of ligustrazine moiety not only make the novel
oxazolidinone derivatives (especially 14a and 14b) keep the antibacterial activities as
linezolid, but also significantly enhance their anti-inflammatory activities.
Additionally, some preliminary structure-activity relationship (SAR) of the novel
oxazolidinone could be summarized: (a) introducing fluorine into molecule would increase its
antibacterial and anti-inflammatory activities, such as compounds 14a and 14b; (b) the
volume of the chlorine substituent of acetyl group exerted influences on the antibacterial and
anti-inflammatory activities. Apparently, monochloro and dichloroacetamide derivatives
(compounds 14a, 14b ,15a and 15b) showed better antibacterial activities, and monochloro
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derivatives with TMP(compounds 14a and 14b) also exhibited better anti-inflammatory
activities than linezolid; (c) different N-substituted triazole exerted different antibacterial
activities (compounds 8a and 8b compared to 6a-7a and 6b-7b); (d) the introduction of a
furan ring could exhibited potent antibacterial activities, such as compounds 17a and 17b.
Conclusions
In this study, a series of novel oxazolidinone derivatives with a substituted ligustrazine C-ring
unit were synthesized by using linezolid as a lead and based on a scaffold hopping strategy.
Meanwhile their antibacterial and anti-inflammatory activities were evaluated. Most of these
compounds showed significant antibacterial activities against gram-positive pathogens. It's
worth noting that two promising compounds 14a and 14b with ligustrazine fragment replaced
C-ring and chloroacetamide group at the C-5 side chain, not only showed potent antibacterial
activity, but also exhibited significant anti-inflammatory activity. And these results revealed
that combination of the structural features with anti-inflammatory fragment into the potential
anti-bacterial agents is likely to be a valuable strategy to obtain drug with both anti-bacterial
and anti-inflammatory effects for the treatment of chronic wounds.
Acknowledgment
This work was supported by the National Natural Science Foundation of China
(No.81072554).
Conflict of interest
The authors have declared no conflict of interest.
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Supporting Information
Additional Supporting Information may be found in the online version of this article:
Appendix S1. Experimental details and characterization for the synthesized compounds.
Figure Captions
Figure 1. Chemical structures of linezolid, ligustrazine, indomethacin, 14a and 14b.
Figure 2. Design strategy for oxazolidinone derivatives
Figure 3. Effects of compounds 14a, 14b, linezolid and indomethacin on the viability of
RAW264.7 cells at the concentration of 10 µM, except TMP of 200 µM. Data were presented
as means SD (n=3). CK was indicated that the cells were cultured with fresh medium only.
Figure 4. The effects of compounds 14a, 14b, linezolid, TMP and indomethacin on the
LPS-induced production of IL-6 in RAW264.7 cells. RAW264.7 cells were treated with 14a,
14b, linezolid, indomethacin (at the concentrations of 10 µM, 5 µM, 2.5 µM, 1.25 µM), TMP
(at the concentrations of 200 µM, 100µM, 50 µM and 20µM) and LPS (100 ng/mL) for 24 h.
Data were presented as means SD (n=3). *P < 0.05, **P < 0.01 versus the LPS (treated with
LPS only) group.
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Table 1. Antibacterial activity in vitro of the synthesized compounds
MIC (μg/mL)
Compd
X
R
MRSA
e
SAj SEk EFm ECn
a
b
c
d
f
g
h
i
1b
-F
-OH
64 64 64 64 64 64 64 64 64 64 128 64 >16
8a
8b
-H
32 32 32 32 64 64 32 32 32 64
64 128 >16
-F
8
4
8
4
8
8
8
8
8
8
8
32 >16
>16
10b -F
13a -H -NHCOCH3
128
16
Y
Y
Y
8
Y
Y
Y
Y
Y
Y
Y
Y
8
16
16 16 16 16 16 16 16 32 >16
13b -F -NHCOCH3
14a -H -NHCOCH2Cl
14b -F -NHCOCH2Cl
15a -H -NHCOCHCl2
15b -F -NHCOCHCl2
16a
4
8
4
4
4
4
4
8
2
8
2
4
4
8
4
8
4
Y
4
8
2
4
4
4
8
4
8
4
4
8
4
8
4
4
8
2
4
2
8
8
8
8
4
4
8
16 >16
16 >16
4
2
4
2
8
2
4
2
4
8
>16
8
16 >16
4
8
>16
>16
-H -NHCOCH2OCH3 64 64 128 128
128 128 128 128
Y
128
Y
16b -F -NHCOCH2OCH3 32 32 64 32 64 64 32 32 64 128 32 128 >16
17a
-H
64 64 64 128 128 128 64 64 64 128 64
Y >16
17b -F
32 32 32 32 32 32 32 32 32 32 16 128 >16
>16
Linezolid
1
1
1
1
1
1
1
1
1
4
2
2
Y: >128 μg/mL.
MRSA: Methicillin-resistant Staphylococcus aureus (a: ATCC 43300; b: ATCC 60202; c: ATCC 51345; d:
ATCC 510019; e: ATCC 52056; f: ATCC 52351-2; g: ATCC 51599-2; h: ATCC 51033; i: ATCC 405055).
gSA: Staphylococcus aureus (ATCC 25923). kSE: Staphylococcus epidermidis (ATCC 12228).
mEF: Enterococcus faecalis (ATCC 29212). nEC: E. coli (ATCC 25922).
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Table 2. The inhibitory effects of the synthesized compounds on NO production in
LPS-stimulated RAW264.7 cells.
Compounds
IC50 (µM)a
Linezolid
>100
TMP
>100
Indomethacin
>100
14a
14b
7.21 0.97
3.45 0.37
aResults were showed as means SD (n=4) of at least three independent experiments.
Table 3. Effects of compounds on the viability of RAW264.7 cells
Compounds
Cell viability (%)a
Concentrations (μM)
CKb
Linezolid
Indomethacin
TMP
100 3.2
107.3 2.9
108.9 5.1
97.5 1.1
95.5 0.8
94.1 1.3
10
10
200
10
14a
10
14b
aResults were expressed as means SD (n=3) of three independent experiments.
bCK: Control group (the cells cultured with fresh medium only).
Table 4. The inhibitory effects of linezolid, TMP, indomethacin, 14a and 14b on
LPS-induced IL-6 and TNF-α production in RAW264.7 cells.
TNF-α (pg/mL)a
Concentrations (µM)
Compounds
10
5
2.5
1.25
Blank
LPS
58.31 7.2
8756.7 167.4
8827.9 11.9 8888.7 16.2
LPS+Linezolid
8702.7 88.1
8974.4 27.4
LPS+Indomethacin 8588.6 134.5 8636.8 154.6 8670.7 130.5 8778.5 127.7
LPS+14a
LPS+14b
8487.6 18.8
8180.2 33.7** 8559.2 116.2 8672.2 18.1
200 100 50
8578.1 67.5 8667.4 155.7 8777.6 188.8
8756.2 52.2
20
LPS+TMP
7968.6 405.4* 8782.2 257.6 9208.7 216.8 9814.6 338.8
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IL-6 (pg/mL)a
Concentrations (µM)
2.5
Compounds
10
5
1.25
Blank
LPS
45.2 5.9
5750.9 75.4
LPS+Linezolid
5694..7 261.5 5829.5 419.6 5985.5 540.3 6398.3 378.2
LPS+Indomethacin 5564.5 468.0 5987.4 682.3 6116.5 680.1 6307.9 554.6
LPS+14a
LPS+14b
4907.8 152.4** 5688.7 364.6 5715.7 310.5 5907.7 448.5
3823.2 273.3** 5103.7 79.7** 5911.7 477.5 6203.7 594.1
200
100
50
20
LPS+TMP
5062 229.2** 5508.5 363.3 5657.3 173.4 5952.6 146.5
*P < 0.05, **P < 0.01 versus the LPS (treated with LPS only) group.
aResults were showed as means SD (n=3) of three independent experiments.
Figure 1. Chemical structures of linezolid, ligustrazine, indomethacin, 14a and 14b.
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Figure 2. Design strategy for oxazolidinone derivatives
Figure 3. Effects of compounds 14a, 14b, linezolid and indomethacin on the viability of
RAW264.7 cells at the concentration of 10 µM, except TMP of 200 µM. Data were presented
as means SD (n=3). CK was indicated that the cells were cultured with fresh medium only.
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Figure 4. The effects of compounds 14a, 14b, linezolid, indomethacin and TMP on the
LPS-induced production of IL-6 in RAW264.7 cells. RAW264.7 cells were treated with 14a,
14b, linezolid, indomethacin (at the concentrations of 10 µM, 5 µM, 2.5 µM and 1.25 µM),
TMP (at the concentrations of 200 µM, 100µM, 50 µM and 20µM) and LPS (100ng/mL) for
24 h. Data were presented as means SD (n=3). *P < 0.05, **P < 0.01 versus the LPS (treated
with LPS only) group.
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Scheme 1. Reagent and condition: (a) NBS, (PhCO)2O2, 24 h, reflux; (b) p-nitrophenol or
2-fluoro-4-nitrophenol, K2CO3, DMF, 85 oC; (c) H2, 10% Pd/C, THF; (d) Cbz-Cl, Na2CO3,
acetone-H2O (10:1); (e) LiN(Si(CH3)3)2, (R)-glycidyl butyrate, THF, -78 oC to r.t.；(f) DAST,
Et3N, CH2Cl2, r.t.; (g) MeSO2Cl, Et3N, CH2Cl2, 0 oC to r.t.; (h) NaOMe, MeOH, r.t., 24 h; (i)
o
K2CO3, dimethylamine hydrochloride, DMF, 60 C; (j) NaH, triazole, DMF, r.t.; (k) NaN3,
DMF, 90 oC, 3 h; (l) 1-hexyne, CuSO4·5H2O, sodium ascorbate, β-CD, H2O; (m)
2-cyanoacetamide, K2CO3, DMSO, r.t.; (n) H2, 10% Pd/C, THF, 24 h; (o) R3COCl, Et3N,
CH2Cl2, 0 oC to r.t.
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