Design, synthesis and biological evaluation of pleuromutilin-Schiff base hybrids as potent anti-MRSA agents in vitro and in vivo

Article abstract of DOI:10.1016/j.ejmech.2021.113624

A series of pleuromutilin derivatives with 1,2,4-triazole-3-substituted Schiff base structure were designed and synthesized under mild conditions. The in vitro antibacterial activities of the synthesized derivatives against 4 strains of Staphylococcus aureus (MRSA ATCC 43300, S.aureus ATCC 29213, S.aureus 144 and S.aureus AD3) and 1 strain of E. coli (ATCC 25922) were evaluated by the broth dilution method. Among these derivatives, compound 60 exhibited superior in vitro antibacterial effect against MRSA (MIC = 0.25 μg/mL) than tiamulin (MIC = 0.5 μg/mL), and compound 60 (?2.28 log₁₀ CFU/mL) also displayed superior in vivo antibacterial efficacy than tiamulin (?1.40 log₁₀ CFU/mL) in reducing MRSA load in the mouse thigh infection model. The time-kill study and the post-antibiotic effect study indicated that compound 60 showed a faster bactericidal kinetic and longer PAE time (exposure to 2 × MIC and 4 × MIC for 2 h, the PAE was 4.06 and 4.27 h) against MRSA compared with tiamulin (exposure to 2 × MIC and 4 × MIC for 2 h, the PAE was 1.72 and 2.14 h). Meanwhile, most of these compounds had no significant inhibitory effect on RAW 264.7 cells and HepG2 cells at the concentration of 4 μg/mL. Additionally, the development of resistance study showed that MRSA did not easily develop resistance against compound 60 compared with tiamulin after induction for 8 passages.

Full text of DOI:10.1016/j.ejmech.2021.113624

European Journal of Medicinal Chemistry 223 (2021) 113624
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis and biological evaluation of pleuromutilin-Schiff
base hybrids as potent anti-MRSA agents in vitro and in vivo
Bo Li a, Zhe Zhang a, Jian-Feng Zhang a, Jie Liu a, Xiang-Yi Zuo a, Fang Chen a,
,
, b, *
Guang-Yu Zhang a, Han-Qing Fang a, Zhen Jin a b, You-Zhi Tang a
a Guangdong Provincial Key Laboratory of Veterinary Pharmaceutics Development and Safety Evaluation, College of Veterinary Medicine, South China
Agricultural University, Guangzhou, 510642, China
b Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, 510642, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A
series of pleuromutilin derivatives with 1,2,4-triazole-3-substituted Schiff base structure were
Received 5 February 2021
Received in revised form
30 May 2021
Accepted 4 June 2021
Available online 12 June 2021
designed and synthesized under mild conditions. The in vitro antibacterial activities of the synthesized
derivatives against 4 strains of Staphylococcus aureus (MRSA ATCC 43300, S.aureus ATCC 29213, S.aureus
144 and S.aureus AD3) and 1 strain of E. coli (ATCC 25922) were evaluated by the broth dilution method.
Among these derivatives, compound 60 exhibited superior in vitro antibacterial effect against MRSA
(MIC ¼ 0.25
m
g/mL) than tiamulin (MIC ¼ 0.5
m
g/mL), and compound 60 (ꢀ2.28 log10 CFU/mL) also
displayed superior in vivo antibacterial efficacy than tiamulin (ꢀ1.40 log10 CFU/mL) in reducing MRSA
load in the mouse thigh infection model. The time-kill study and the post-antibiotic effect study indi-
cated that compound 60 showed a faster bactericidal kinetic and longer PAE time (exposure to 2 ꢁ MIC
and 4 ꢁ MIC for 2 h, the PAE was 4.06 and 4.27 h) against MRSA compared with tiamulin (exposure to
2 ꢁ MIC and 4 ꢁ MIC for 2 h, the PAE was 1.72 and 2.14 h). Meanwhile, most of these compounds had no
Keywords:
Antibacterial activity
MRSA
Pleuromutilin
1,2,4-Triazole
Schiff base
significant inhibitory effect on RAW 264.7 cells and HepG2 cells at the concentration of 4 mg/mL.
Additionally, the development of resistance study showed that MRSA did not easily develop resistance
against compound 60 compared with tiamulin after induction for 8 passages.
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
was first isolated from two basidiomycetes species (Pleurotus
mutilus and Pleurotus passeckerianus) by Kavanagh et al., in 1951. It
Staphylococcus aureus is an important bacterium because it can
cause a wide range of diseases [1]. Penicillin and its derivatives,
including methicillin have been used to treat infections caused by
S. aureus. However, certain strains of S. aureus generated resistance
were called methicillin-resistant S. aureus (MRSA) [2]. MRSA in-
fections may develop more serious diseases, such as pneumonia,
endocarditis and osteomyelitis, etc. [3,4]. MRSA strains have been
reported resistant to many clinical antibiotics, such as linezolid,
vancomycin and daptomycin [5]. MRSA infections cause a serious
threat to public health security around the world [6]. Thus, it is
urgent to develop new antibiotics agents with novel modes of ac-
tion to treat infections caused by MRSA.
exhibited potent antibacterial activity against Gram-positive bac-
teria [7,8]. Pleuromutilin selectively interacts with the peptidyl
transferase center of the 50S bacterial ribosome subunit and in-
terferes with the translation process of bacterial protein to achieve
antibacterial activity [9]. This unique antibacterial mechanism has
caused pleuromutilin to receive extensive attention from
researchers.
Tiamulin (2, Fig. 1) and valnemulin (3, Fig. 1) were approved for
use as veterinary drugs in 1979 and 1999, respectively [10]. In 2007,
retapamulin (4, Fig. 1) was approved by the Food and Drug
Administration (FDA) as the first pleuromutilin derivative antibiotic
for the treatment of topical skin infection in humans [11]. Subse-
quently, in 2019, lefamulin (Fig. 5) was approved by the FDA for
intravenous and oral treatment of community-acquired bacterial
pneumonia (CABP), it was the first systemic pleuromutilin anti-
biotic in humans [12]. Unfortunately, azamulin (6, Fig. 1) has
excellent antibacterial potency, but development was terminated
Pleuromutilin (1, Fig. 1), a tricyclic diterpenoid natural product,
* Corresponding author. College of Veterinary Medicine, South China Agricultural
University, No. 483 Wushan Road, Tianhe District, Guangzhou, 510642, China.
E-mail address: youzhitang@scau.edu.cn (Y.-Z. Tang).
https://doi.org/10.1016/j.ejmech.2021.113624
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

B. Li, Z. Zhang, J.-F. Zhang et al.
European Journal of Medicinal Chemistry 223 (2021) 113624
Fig. 1. Structure of pleuromutilin (1), tiamulin (2), valnemulin (3), retapamulin (4), lefamulin (5) and azamulin (6).
after Phase I trials for the low oral bioavailability [13]. In our pre-
vious work, a variety of pleuromutilin derivatives were designed
and synthesized. These derivatives contain 2-aminophenylthiol,
piperazine ring and 1,2,3-triazole structures as linkers, respectively.
Some of these pleuromutilin derivatives showed potent antimi-
crobial activity [14e16].
Compounds containing 1,2,4-triazoles structures have broad-
spectrum activities such as antifungal, antibacterial, antitumor
and antiparasitic activities, and have potential applications in the
field of pharmaceutical chemistry [17]. In recent years, 1,2,4-
triazole Schiff bases were found with good pharmacological activ-
ity [18,19].
This background inspired us to develop novel pleuromutilin
derivatives containing 1,2,4-triazole Schiff bases. In this study, 25
pleuromutilin derivatives containing 1,2,4-triazole-3-substituted
Schiff base structure were designed and synthesized. We pre-
liminary evaluated their antibacterial activities in vitro and in vivo.
synthesis of compounds were consistent with the expected
structure.
2.2. In vitro antibacterial activity
The MIC and MBC values of those pleuromutilin derivatives
were tested to evaluate theirs in vitro antibacterial activity. The
results were shown in Table 1.
The MIC values of those new pleuromutilin derivatives in vitro
against the above Gram-positive bacteria ranged from 0.25 to 4
mL. The MICs of the 25 derivatives against E. coli (ATCC 25922) were
higher than 64 g/mL. Most of the derivatives exhibited potent
antibacterial activities against MRSA. Compounds 48 and 60
g/mL) showed the highest antibacterial activity
mg/
m
(MIC ¼ 0.25
m
against MRSA among these compounds.
25 pleuromutilin derivatives with 1,2,4-triazole-3-substituted
Schiff base structure, the substituent groups include electron-
donating groups (methyl, hydroxyl, etc.) and electron-
withdrawing groups (fluorine, chlorine, trifluoromethyl, nitro,
etc.). Compound 48 containing the hydroxyl group and compound
60 containing the pyridyl group were the most antibacterial activity
against MRSA compounds in this series. The results showed that
the activity of the compounds containing the electron-donating
group may be slightly higher than that containing the electron-
withdrawing group, which was consistent with previous research
[21].
The ratios of MBC to MIC of the target pleuromutilin derivatives
ranged from 1 to 4 indicated that these compounds have excellent
bactericidal ability [22]. The results indicated that 25 pleuromutilin
derivatives have good bactericidal ability against S. aureus. Among
these derivatives, compounds 48 and 60 were exhibited a greater
bactericidal effect than tiamulin. Therefore, we conducted an in-
depth study on the antibacterial activity of these two compounds.
The in vitro antibacterial activity of compounds 48 and 60 were
tested by the time-kill kinetic approach. The results were shown in
Fig. 2.
2. Results and discussion
2.1. Chemistry
The general synthetic methods for all target pleuromutilin de-
rivatives were illustrated in Scheme 1. Compound 8 was synthe-
sized by compound 7 and CS2 under heating conditions according
to reference [20]. Compound 8 was converted into compound 9
through a cyclization reaction under acidic conditions. Compounds
10e34 were obtained by nucleophilic substitution of the amino
group in compound 9 and the aldehyde group in a variety of
benzaldehyde derivatives. Compound 35 was synthesized from
compound 1 and p-toluenesulfonyl chloride under alkaline condi-
tions. The iodinated compound 35 and compound 10e34 were
transformed by nucleophilic substitution reaction to gain the target
pleuromutilin derivatives (compound 36e60).
To get the target pleuromutilin derivatives, all those crude
products were purified by silica column chromatography. The
structures of 25 compounds were characterized by 1H NMR, 13C
NMR, HR-MS (ESI) analysis, and the results confirmed that the
In a concentration of 1 ꢁ MIC, compounds 48 and 60 had a
significant inhibitory effect on MRSA. After exposure for 24 h,
2

B. Li, Z. Zhang, J.-F. Zhang et al.
European Journal of Medicinal Chemistry 223 (2021) 113624
Scheme 1. Reagent and conditions: (i) H2O, CS2, 90 °C, 1 h (ii) CH3COOH, 118 °C, 4 h; (iii) absolute ethyl alcohol, Benzaldehyde derivatives, H2SO4, reflux, 3 h; (iv)
acetonitrile, p-toluenesulfonyl chloride, NaOH, rt, 3 h; (v) a). acetonitrile, NaI, 70 °C, 1.5 h; b). acetonitrile, K2CO3, compound 10e34, 70 ꢂC, 2 h.
compounds 48 and 60 showed the bactericidal effect on MRSA and
killed 99.9% of MRSA at 4 ꢁ MIC and 2 ꢁ MIC concentrations,
respectively. Especially, compound 60 showed a faster bactericidal
kinetic against MRSA compared with tiamulin. However, after the
two compounds and tiamulin reach a certain concentration, the
bactericidal effect did not have significantly increased. The results
indicated that compounds 48, 60 and tiamulin were time-
dependent rather than the concentration-dependent antibacterial
agent. In clinical practice, multiple or continuous intravenous
administration of time-dependent antibacterial drugs may achieve
better therapeutic effects [23].
To evaluate the in vitro antibacterial pharmacodynamic activity
of compounds 48 and 60, we conducted the PAE of the above two
compounds. PAE refers to bacteria exposed to antibiotics some
time, after elimination of the drug, effects sustained by bacterial
growth inhibition [24]. The PAE results were shown in Table 2 and
the bacterial growth kinetics curve was exhibited in Fig. 3.
After compound 48 was treated with 2 ꢁ MIC and 4 ꢁ MIC
concentration for 2 h, the PAE was 2.55 and 3.22 h, respectively.
After exposure at the same concentrations for 2 h, the PAE of
compound 60 was 4.06 and 4.27 h, respectively. These results
indicated that compounds 48 and 60 exhibited a longer PAE than
tiamulin.
As the drug concentration drops below the MIC, it still has a
certain antibacterial effect. Therefore, in clinical medication, com-
bined PAE with MIC, MBC and other pharmacodynamic parameters
could comprehensively evaluate the efficacy of antibacterial drugs.
Besides, the PAE value can be used as a reference for the design of
clinical dosing regimen, and provide a theoretical basis for the
adjustment of medication interval, which is of great significance for
reducing the occurrence of adverse drug reactions [25].
The results of the above experiment showed that the adminis-
tration interval of compounds 48 and 60 may be longer than that of
tiamulin.
dose, compound 60 could reduce the bacterial load (ꢀ2.28
log10 CFU/mL) in thighs, compared with the no-drug control group
(P < 0.0001, n ¼ 6/group). Compound 60 exhibited a significant
treatment effect against MRSA in thighs compared to the tiamulin
group (P < 0.0001, n ¼ 6/group). The result showed that the anti-
bacterial activity of compound 60 against MRSA in vivo was higher
than that of tiamulin.
2.4. Cytotoxicity assay
The cytotoxicity of the 25 derivatives were evaluated using one
non-cancer cell line RAW 264.7 cells and one cancer cell line HepG2
cells by MTT assay. The results were shown in Figs. 5 and 6.
The results indicated that most of these compounds had no
significant inhibitory effect on RAW 264.7 cells and HepG2 cells at
the concentration of 4 mg/mL, which was an acceptable concen-
tration. The only exception was compound 39 that reduced RAW
264.7 cells viability by more than 30%.
2.5. Development of resistance
To evaluate if any resistance can be easily induced by those
compounds, the bacterial resistance against compound 60 and
tiamulin was studied by investigating the resistance development
rates of MRSA. The results were shown in Fig. 7.
After induction by sub-MIC concentrations for 8 passages,
compound 60 also showed good anti-MRSA activity, with the MIC
value reaches 2
(0.25 g/mL). However, the MRSA gradually developed resistance to
tiamulin (MIC ¼ 32 g/mL) which lose 64-fold potency compared to
the initial MIC value (0.5 g/mL) after 8 passages. This assay showed
mg/mL, which was 8 times the initial MIC value
m
m
m
that the MRSA did not develop significant resistance against com-
pound 60 as easily as it did against the tiamulin. Therefore, com-
pound 60 has the potential to overcome the resistance to tiamulin.
2.3. Neutropenic murine thigh infection model
3. Conclusions
Since compound 60 exhibited superior antibacterial activity
in vitro, the in vivo antibacterial activity of compound 60 was tested
by using the neutropenic murine thigh infection model experi-
ment. The results were shown in Fig. 4.
From Fig. 4, tiamulin at 20 mg/kg could reduce the MRSA load
(ꢀ1.40 log10 CFU/mL) obviously in mice thighs compared to the no-
drug control group (P < 0.0001, n ¼ 6/group). Whereas, at the same
The antibacterial activity of a series of novel pleuromutilin de-
rivatives against MRSA were studied in vitro and in vivo. The results
of the MIC and MBC revealed that compounds 48 and 60 exhibited
superior in vitro antibacterial activity against S. aureus and MRSA
than tiamulin. The time-kill study and PAE study indicated that
compound 60 showed a faster bactericidal kinetic and longer PAE
time for MRSA compared with tiamulin. Meanwhile, compound 60
3

B. Li, Z. Zhang, J.-F. Zhang et al.
European Journal of Medicinal Chemistry 223 (2021) 113624
Table 1
MIC and MBC (
(ATCC 25922).
mg/mL) values of compounds 36e60, pleuromutilin and tiamulin against MRSA (ATCC 43300), S. aureus (ATCC 29213), S. aureus (AD3), S. aureus (144) and E. coli
Compound No
R
MIC/MBC(mg/mL)
MRSA
ATCC
S.aureus ATCC 29213
S.aureus AD3
S.aureus
144
E. coli ATCC 25922
43300
36
37
0.5/1
1/2
1/2
1/2
1/4
1/4
2/4
2/4
>64/>64
>64/>64
38
39
40
1/2
1/2
1/2
1/2
1/4
1/4
1/4
2/4
2/4
2/4
>64/>64
>64/>64
>64/>64
0.5/1
0.5/1
41
42
0.5/1
1/2
1/4
1/4
1/4
1/4
2/4
2/4
>64/>64
>64/>64
43
1/2
1/2
1/4
2/4
>64/>64
44
45
1/2
1/4
1/2
2/4
1/4
2/4
2/4
>64/>64
>64/>64
0.5/2
46
0.5/1
1/2
1/4
2/4
>64/>64
47
48
0.5/1
0.5/2
0.5/2
1/4
2/4
1/2
>64/>64
>64/>64
0.25/1
0.5/2
49
0.5/1
1/4
2/8
2/8
>64/>64
4

B. Li, Z. Zhang, J.-F. Zhang et al.
European Journal of Medicinal Chemistry 223 (2021) 113624
Table 1 (continued)
Compound No
R
MIC/MBC(mg/mL)
MRSA
ATCC
43300
S.aureus ATCC 29213
S.aureus AD3
S.aureus
144
E. coli ATCC 25922
>64/>64
50
51
1/2
2/8
2/8
2/4
2/8
1/2
2/4
2/8
>64/>64
52
53
1/2
1/4
1/4
1/4
2/8
2/4
2/4
2/4
>64/>64
>64/>64
54
55
2/4
2/4
2/4
2/4
2/4
2/8
4/4
4/8
>64/>64
>64/>64
56
57
0.5/1
4/4
0.5/2
4/8
1/4
2/4
4/8
>64/>64
>64/>64
4/16
58
59
1/2
2/4
1/2
2/4
1/4
2/4
2/4
>64/>64
>64/>64
0.5/1
60
0.25/1
0.5/1
0.5/2
1/4
>64/>64
Pleuromulin
Tiamulin
2/4
0.5/1
2/4
1/1
2/4
1/2
2/4
1/2
>64/>64
>64/>64
was found to display a potent in vivo antibacterial effect in the
neutropenic murine thigh infection model study. In the cytotoxicity
assay study, most of these compounds had no significant inhibitory
effect on RAW 264.7 cells and HepG2 cells at the concentration of
4. Experimental
4.1. Materials
4
m
g/mL. The development of resistance study showed that MRSA
The raw material pleuromutilin (>90% pure) was purchased
from Great Enjoyhood Biochemical Co. Ltd., (Daying, China). The
benzaldehyde derivatives were purchased from Bide Technology
Co. Ltd., (Shanghai, China). The other general reagents were all of
the analytical grades and obtained from Guangzhou Chemical Re-
agent Factory (Guangzhou, China). Purification of the target
did not easily develop resistance against compound 60 compared
with tiamulin. According to the current experimental results,
compound 60 was worthy of further development as a potential
drug against MRSA infection.
5

B. Li, Z. Zhang, J.-F. Zhang et al.
European Journal of Medicinal Chemistry 223 (2021) 113624
Fig. 3. The bacterial growth kinetic curves for MRSA ATCC 43300 exposed to com-
pound 48 (a), compound 60 (b) and tiamulin (c) for 2 h.
Fig. 2. Time-kill curves for MRSA ATCC 43300 with different concentrations of com-
pounds 48 (a), 60 (b) and tiamulin (c).
Table 2
The PAEs values of compounds 48, 60 and tiamulin against MRSA ATCC 43300.
Compounds
Concentrations
PAE (h)
Exposure for 0 ha
Exposure for 2 h
Compound 48
Compound 60
Tiamulin
2 ꢁ MIC
4 ꢁ MIC
2 ꢁ MIC
4 ꢁ MIC
2 ꢁ MIC
4 ꢁ MIC
2.51
2.55
3.22
4.06
4.27
1.72
2.14
2.04
2.44
a
Exposure for 0 h is the time required for the bacteria in the control groups to
increase by 1 log10 CFU/mL.
Fig. 4. Efficacy of tiamulin and compound 60 against MRSA ATCC 43300 in murine
neutropenic thigh models: circular: growth control; inverted triangle: tiamulin
(20 mg/kg); regular triangle: compound 60 (20 mg/kg).
compounds by column chromatography was carried out using silica
gel (200e300 mesh, Branch of Qingdao Haiyang Chemical Co. Ltd.,
6

B. Li, Z. Zhang, J.-F. Zhang et al.
European Journal of Medicinal Chemistry 223 (2021) 113624
Fig. 7. Development of MRSA ATCC 43300 resistance to compound 60 and tiamulin,
each MIC value of every passage was an average of three parallels.
electrospray ionization (ESI) source.
4.2. Synthesis
A general synthetic route based on the compound 22-O-tosyl-
pleuromutilin (compound 35) and a variety of 4-amino-5-methyl-
1,2,4-triazole-3-substituted Schiff base derivatives (compound
10e34) were used to prepare a series of pleuromutilin derivatives.
4.2.1. Synthesis of 1,3-Diamino-2-Thiourea (8)
Fig. 5. The cytotoxicity assay of 25 pleuromutilin derivatives to RAW 264.7 cells at the
Compound 7 (20 mL, 400 mmol) was dissolved in water (60 mL).
Then CS2 (6 mL, 100 mmol) was added to the solution and stirred at
90 ꢂC for 1 h until the reaction was completed. The reaction mixture
was cooled to room temperature. The solid products were precip-
itated from the reaction solution. Then the above products were
collected by vacuum filtration. The crude products were recrystal-
lized with water to afford compound 8 (white solid, yield 83.7%).
concentration of 4 mg/mL.
4.2.2. Synthesis of 4-amino-5-methyl-1,2,4-triazole-3-thiol (9)
Compound 8 (3 g, 28.2 mmol) was dissolved in acetic acid
(10 mL, 17.5 mmol). The reaction was refluxed for 4 h. After com-
pound 8 was completely consumed, the reaction mixture was
cooled to room temperature. Then the unreacted acetic acid was
removed by vacuum distillation. The solids were precipitated from
the reaction solution and collected by vacuum filtration. The crude
products were purified by recrystallization with water to obtain
compound 9 (white solid, yield 72.5%).
4.2.3. General procedure for the synthesis of 4-amino-5-methyl-
1,2,4-triazole-3-substituted Schiff base derivatives (compounds
10e34)
Compound 9 (1 g, 7.7 mmol) was added to anhydrous ethanol
(30 mL). The reaction mixture was heated and stirred until
completely dissolved. Then Benzaldehyde derivatives (0.78 mL,
7.7 mmol) and concentrated sulfuric acid (1 mL) were slowly added
to the reaction solution. The reaction mixture was stirred for 4 h at
78 ꢂC until compound 9 completely disappeared. The reaction
mixture was cooled to room temperature. Then a large amount of
solid was precipitated from the reaction solution, the solid was
collected by vacuum filtration. The crude products were purified by
recrystallization with ethyl alcohol to afford compound 10e34 in
35%e78% yield.
Fig. 6. The cytotoxicity assay of 25 pleuromutilin derivatives to HepG2 cells at the
concentration of 4 g/mL.
m
Shandong, China). 1H NMR and 13C NMR spectra were measured
on Bruker AV-400 or Bruker AV-600 spectrometer in CDCl3. The
chemical shift values (d) are reported in ppm relative to tetrame-
thylsilane as an internal standard. High-resolution mass spectra
were recorded by Waters Acquity UPLC-LCT Premier XE with an
4.2.4. 22-O-tosylpleuromutilin (35)
Compound 35 (22-O-tosylpleuromutilin) was synthesized ac-
cording to a reported method [15]. Pleuromutilin 1 (5.0 g,
13.21 mmol) and p-toluenesulfonyl chloride (2.7 g, 14.53 mmol)
were dissolved in dichloromethane (15.0 mL) under an ice bath.
7

B. Li, Z. Zhang, J.-F. Zhang et al.
European Journal of Medicinal Chemistry 223 (2021) 113624
Then sodium hydroxide granules (1.5 g, 26.42 mmol) were dis-
solved in water (10 mL) and dropped slowly into the above mixture
solution. The mixture was stirred at room temperature until the
pleuromutilin was completely consumed. The solution was
extracted twice with CHCl3 (30 mL). Then the organic phases were
combined and washed three times with saturated brine. The
organic phases were collected and dried with dried over anhydrous
sodium sulfate and evaporation under reduced pressure. The crude
product was purified by recrystallization with isopropanol to obtain
a white solid powder in 85.7% yield.
(C12), 41.85 (C5), 36.67 (C6), 36.07, 36.02 (C10), 34.43 (C2), 30.39
(C8), 26.82 (C7), 26.43 (C18), 24.82 (C1), 19.85, 16.72 (C16), 14.76
(C15), 11.42 (C17), 11.18. HR-MS(ESIþ): Calcd for C33H44N4O4S
(MþHþ): 593.3156; Found: 593.3146.
4.2.8. 22-[(4-(3-methyl-benzylidene amino)-5-methyl-4H-1,2,4-
triazol-3-yl)thio] deoxy pleuromutilin (38)
White powder; yield: 62%; 1H NMR (600 MHz, Chloroform-d)
d
8.55 (1H, s), 7.73e7.62 (2 H, m), 7.48e7.35 (2 H, m), 6.42 (1 H,
dd, J ¼ 17.4, 11.0Hz, H19), 5.73 (1 H, d, J ¼ 8.5, H14), 5.30 (1 H, dd,
J ¼ 11.0, 1.5Hz, H20), 5.18 (1H, dd, J ¼ 17.5, 1.5Hz, H20), 4.11e3.96
(2H, m, H22), 3.35 (1H, dd, J ¼ 10.3, 6.5Hz, H11), 2.51 (3 H, s), 2.45
(3 H, d, J ¼ 0.9Hz), 2.33e2.15 (3 H, m, H2, H10), 2.09 (1H, d,
J ¼ 2.7Hz, 11-OH), 2.04 (1 H, dd, J ¼ 16.1, 8.6, H13), 1.79 (2 H, s, H7),
1.65 (2 H, dtd, J ¼ 11.1, 9.1, 5.3Hz, H1), 1.55e1.42 (3 H, m, H4, H6,
H13), 1.42 (3 H, s, H15), 1.38e1.32 (2 H, m, H8), 1.16 (3 H, s, H18),
0.87 (3 H, d, J ¼ 7.0Hz, H17), 0.71 (3 H, d, J ¼ 7.1Hz, H16). 13C NMR
4.2.5. General procedure for the synthesis of target pleuromutilin
derivatives (36e60)
Compound 6 (1 g, 1.87 mmol) was dissolved in acetonitrile
(10 mL). The sodium iodide (0.31 g, 2.07 mmol) was added into the
mixture solution and stirred at 70 ꢂC for 1 h. After the compound 6
reaction was completed. Then compounds 10e34 (0.38 g,
2.07 mmol) and potassium carbonate (0.52 g, 3.76 mmol) were
added to the above reaction. Stirring was continued for 2 h at 70 ꢂC.
After completion of the reaction, the ice water (50 mL) and CHCl3
(50 mL) were added to the solution. The organic phase was washed
with brine three times. Then the organic solvent was dried
(Na2SO4) and concentrated under reduced pressure to give the
crude product. The above crude product was purified by silica gel
column chromatography (dichloromethane: methanol ¼ 40: 1 v/v)
to obtain the pure compounds 36e60 in 61%e83% yield.
(151 MHz, CDCl3) d 216.81 (C3), 166.89 (C21), 163.03, 150.89, 144.72,
139.07 (C19), 138.86, 133.91, 131.66, 129.27, 129.03, 126.37, 117.19
(C20), 74.61 (C11), 70.50 (C14), 58.09 (C4), 45.42 (C9), 44.52 (C13),
44.02 (C12), 41.85 (C5), 36.68 (C6), 36.00 (C10), 34.43 (C2), 30.40
(C8), 26.83 (C7), 26.43 (C18), 24.82 (C1), 21.28, 16.73 (C16), 14.76
(C15), 11.43 (C17), 11.18. HR-MS(ESIþ): Calcd for C33H44N4O4S
(MþHþ): 593.3156; Found: 593.3145.
4.2.9. 22-[(4-(4-methyl-benzylidene amino)-5-methyl-4H-1,2,4-
triazol-3-yl)thio] deoxy pleuromutilin (39)
4.2.6. 22-[(4-(benzylidene amino)-5-methyl-4H-1,2,4-triazol-3-yl)
thio]deoxy pleuromutilin (36)
White powder; yield: 59%; 1H NMR (600 MHz, Chloroform-d)
d
8.54 (1H, s), 7.81e7.66 (2 H, m), 7.34e7.31 (2 H, m), 6.41 (1 H,
White powder; yield: 68%; 1H NMR (600 MHz, Chloroform-d)
dd, J ¼ 17.4, 11.0Hz, H19), 5.73 (1 H, d, J ¼ 8.5Hz, H14), 5.30 (1 H, dd,
J ¼ 11.0, 1.5Hz, H20), 5.18 (1H, dd, J ¼ 17.4, 1.5Hz, H20), 4.12e3.93
(2H, m, H22), 3.35 (1H, dd, J ¼ 10.4, 6.5Hz, H11), 2.50 (3H, s), 2.46
(3 H, s), 2.33e2.15 (3 H, m, H2, H10), 2.11e2.08 (1H, m, 11-OH), 2.03
(1 H, dd, J ¼ 16.1, 8.6Hz, H13),1.78 (2 H, s, H7),1.65 (2 H, dtd, J ¼ 11.0,
9.0, 5.3Hz, H1), 1.54e1.49 (2 H, m, H6, H13), 1.45 (1 H, ddd, J ¼ 13.4,
9.6, 2.7Hz, H4), 1.41 (3 H, s, H15), 1.38e1.31 (2 H, m, H8), 1.16 (3 H, s,
d
8.60 (1H, s), 7.90e7.86 (2 H, m), 7.62e7.58 (1 H, m), 7.55e7.50
(2 H, m), 6.42 (1 H, dd, J ¼ 17.4, 11.0Hz, H19), 5.73 (1 H, d, J ¼ 8.5Hz,
H14), 5.30 (1 H, dd, J ¼ 11.0, 1.5Hz, H20), 5.18 (1H, dd, J ¼ 17.5, 1.5Hz,
H20), 4.11e3.98 (2H, m, H22), 3.35 (1H, dd, J ¼ 10.0, 6.4Hz, H11),
2.51 (3H, s), 2.33e2.22 (2 H, m, H2), 2.21e2.16 (1H, m, H10), 2.09
(1 H, d, J ¼ 2.8Hz, 11-OH), 2.04 (1 H, dd, J ¼ 16.1, 8.6Hz, H13), 1.77
(2 H, s, H6, H13), 1.65 (2 H, dtd, J ¼ 11.1, 9.3, 5.3Hz, H1), 1.51 (2 H, dd,
J ¼ 12.9, 3.3Hz, H7), 1.48e1.43 (1 H, m, H4), 1.42 (3 H, s, H15),
1.37e1.32 (2 H, m, H8),1.16 (3 H, s, H18), 0.87 (3 H, d, J ¼ 7.0Hz, H17),
H18), 0.87 (3 H, d, J ¼ 7.0Hz, H17), 0.71 (3 H, d, J ¼ 7.1Hz, H16). 13
C
NMR (151 MHz, CDCl3)
d 216.82 (C3), 166.90 (C21), 162.95, 150.83,
144.73, 144.00, 138.86 (C19), 129.89, 129.05, 128.97, 117.19 (C20),
74.61 (C11), 70.48 (C14), 58.09 (C4), 45.42 (C9), 45.01, 44.52 (C13),
44.02 (C12), 41.85 (C5), 36.68, 36.54 (C6), 36.01 (C10), 35.95, 34.43
(C2), 30.40 (C8), 26.83 (C7), 26.42 (C18), 24.82 (C1), 21.77, 16.73
(C16), 14.76 (C15), 11.43 (C17), 11.16. HR-MS(ESIþ): Calcd for
0.71 (3 H, d, J ¼ 7.1Hz, H16). 13C NMR (151 MHz, CDCl3)
d 216.81
(C3), 166.88 (C21), 162.50, 150.97, 144.71, 138.85 (C19), 133.02,
131.76, 129.15, 128.94, 117.20 (C20), 99.99, 74.61 (C11), 70.52 (C14),
58.08 (C4), 52.53, 45.42 (C9), 44.52 (C13), 44.02 (C12), 41.85 (C5),
41.76, 36.67 (C6), 36.02 (C10), 34.43 (C2), 30.40 (C8), 26.83 (C7),
26.42 (C18), 24.82 (C1), 16.73 (C16), 14.76 (C15), 11.43 (C17), 11.20.
HR-MS(ESIþ): Calcd for C32H42N4O4S (MþHþ): 579.3000; Found:
579.2989.
C
33H44N4O4S (MþHþ): 593.3156; Found: 593.3146.
4.2.10. 22-[(4-(2-fluoro-benzylidene amino)-5-methyl-4H-1,2,4-
triazol-3-yl)thio] deoxy pleuromutilin (40)
White powder; yield: 67%; 1H NMR (600 MHz, Chloroform-d)
4.2.7. 22-[(4-(2-methyl-benzylidene amino)-5-methyl-4H-1,2,4-
triazol-3-yl)thio] deoxy pleuromutilin (37)
d
8.89 (1H, d, J 0.6), 8.21e7.98 (1 H, m), 7.58 (1 H, dddd, J ¼ 8.4,
7.3, 5.4, 1.8Hz), 7.30 (1 H, dddd, J ¼ 7.9, 7.4, 1.1, 0.7Hz), 7.21 (1 H, ddd,
J ¼ 10.3, 8.4, 1.0Hz), 6.41 (1 H, dd, J ¼ 17.4, 11.0Hz, H19), 5.73 (1 H, d,
J ¼ 8.5Hz, H14), 5.30 (1 H, dd, J ¼ 11.0, 1.5Hz, H20), 5.18 (1H, dd,
J ¼ 17.4, 1.5Hz, H20), 4.15e3.91 (2H, m, H22), 3.35 (1H, dd, J ¼ 10.2,
6.5Hz, H11), 2.53 (3H, s), 2.33e2.14 (3 H, m, H2, H10), 2.11e2.08
(1H, m, 11-OH), 2.04 (1 H, dd, J ¼ 16.1, 8.6Hz, H13), 1.78 (2 H, s, H7),
1.65 (2 H, tdd, J ¼ 11.1, 8.6, 5.3Hz, H1), 1.52 (2 H, dd, J ¼ 14.8, 10.5Hz,
H6, H13), 1.45 (1 H, ddd, J ¼ 13.3, 9.5, 2.6Hz, H4), 1.42 (3 H, s, H15),
1.37e1.33 (2 H, m, H8),1.16 (3 H, s, H18), 0.87 (3 H, d, J ¼ 7.0Hz, H17),
White powder; yield: 71%; 1H NMR (600 MHz, Chloroform-d)
d
8.90 (1H, d, J ¼ 0.5Hz), 8.00e7.98 (1 H, m), 7.46 (1 H, td, J ¼ 7.5,
1.4Hz), 7.36e7.29 (2 H, m), 6.41 (1 H, dd, J ¼ 17.5, 11.0Hz, H19), 5.73
(1 H, d, J ¼ 8.5Hz, H14), 5.31e5.27 (1 H, m, H20), 5.18 (1H, dd,
J ¼ 17.4, 1.5Hz, H20), 4.12e3.97 (2H, m, H22), 3.35 (1H, dd, J ¼ 10.1,
6.5Hz, H11), 2.58 (3H, s), 2.52 (3 H, s), 2.32e2.28 (1 H, m, H2),
2.27e2.22 (1H, m, H2), 2.18 (1H, dd, J ¼ 19.4, 9.4Hz, H10), 2.09 (1 H,
d, J ¼ 2.8Hz, 11-OH), 2.04 (1 H, dd, J ¼ 16.1, 8.6Hz, H13), 1.80 (2 H, s,
H7), 1.75 (1 H, dq, J ¼ 14.6, 3.2Hz, H6), 1.65 (2 H, dtd, J ¼ 11.1, 9.0,
5.3Hz, H1), 1.54e1.49 (1 H, m, H13), 1.48e1.42 (1 H, m, H4), 1.41
(3 H, s, H15), 1.37e1.31 (2 H, m, H8), 1.16 (3 H, s, H18), 0.87 (3 H, d,
J ¼ 7.0Hz, H17), 0.71 (3 H, d, J ¼ 7.1Hz, H16). 13C NMR (151 MHz,
0.71 (3 H, d, J ¼ 7.1Hz, H16). 13C NMR (151 MHz, CDCl3)
d 216.82
(C3), 166.76 (C21), 155.00, 154.96, 150.87, 145.08, 138.84 (C19),
134.79,127.49,124.90,117.19 (C20),116.37,116.24, 74.61 (C11), 70.52
(C14), 58.09 (C4), 45.42 (C9), 44.52 (C13), 44.01 (C12), 41.85 (C5),
36.69 (C6), 36.01 (C10), 35.91, 34.43 (C2), 30.40 (C8), 26.82 (C7),
26.42 (C18), 24.82 (C1), 16.71 (C16), 14.74 (C15), 11.42 (C17), 11.33.
HR-MS (ESIþ): Calcd for C32H41FN4O4S (MþHþ): 597.2906; Found:
CDCl3)
d 216.81 (C3), 166.80 (C21), 161.57, 151.07, 144.32, 139.41
(C19), 138.85, 132.64, 131.42, 129.97, 128.23, 126.62, 117.18 (C20),
74.61 (C11), 70.51 (C14), 58.09 (C4), 45.42 (C9), 44.53 (C13), 44.01
8

B. Li, Z. Zhang, J.-F. Zhang et al.
European Journal of Medicinal Chemistry 223 (2021) 113624
597.2896.
4.2.14. 22-[(4-(3-chloro-benzylidene amino)-5-methyl-4H-1,2,4-
triazol-3-yl)thio] deoxy pleuromutilin (44)
White powder; yield: 77%; 1H NMR (600 MHz, Chloroform-d)
4.2.11. 22-[(4-(3-fluoro-benzylidene amino)-5-methyl-4H-1,2,4-
triazol-3-yl)thio] deoxy pleuromutilin (41)
d
8.62 (1H, s), 7.90 (1 H, t, J ¼ 1.9Hz), 7.73 (1 H, dt, J ¼ 7.7, 1.4Hz),
7.56 (1 H, ddd, J ¼ 8.0, 2.1, 1.1Hz), 7.47 (1 H, t, J ¼ 7.9Hz), 6.41 (1 H,
dd, J ¼ 17.4, 11.0Hz, H19), 5.73 (1 H, d, J ¼ 8.5Hz, H14), 5.29 (1 H, dd,
J ¼ 11.0, 1.5Hz, H20), 5.18 (1H, dd, J ¼ 17.4, 1.5Hz, H20), 4.13e3.97
(2H, m, H22), 3.35 (1H, dd, J ¼ 10.4, 6.5Hz, H11), 2.52 (3H, s),
2.34e2.14 (3 H, m, H2, H10), 2.11e2.08 (1H, m,11-OH), 2.04 (1 H, dd,
J ¼ 16.1, 8.6Hz, H13), 1.77 (2 H, s, H7), 1.65 (2 H, dddd, J ¼ 13.1, 11.1,
7.9, 4.8Hz, H1), 1.54e1.43 (3 H, m, H4, H6, H13), 1.42 (3 H, s, H15),
1.38e1.31 (2 H, m, H8),1.16 (3 H, s, H18), 0.87 (3 H, d, J ¼ 7.1Hz, H17),
White powder; yield: 73%; 1H NMR (600 MHz, Chloroform-d)
d
8.64 (1H, d, J ¼ 1.0Hz), 7.64 (1 H, ddd, J ¼ 9.1, 2.6, 1.5Hz), 7.61
(1 H, dt, J ¼ 7.6, 1.2Hz), 7.51 (1 H, td, J ¼ 8.0, 5.4Hz), 7.31e7.28 (1 H,
m), 6.41 (1 H, dd, J ¼ 17.4, 11.0Hz, H19), 5.73 (1 H, d, J ¼ 8.5Hz, H14),
5.29 (1 H, dd, J ¼ 11.0, 1.4Hz, H20), 5.18 (1H, dd, J ¼ 17.5, 1.5Hz, H20),
4.14e3.97 (2H, m, H22), 3.35 (1H, dd, J ¼ 10.4, 6.5Hz, H11), 2.52 (3 H,
d, J ¼ 0.7Hz), 2.31e2.16 (3 H, m, H2, H10), 2.12e2.07 (1H, m, 11-OH),
2.04 (1 H, dd, J ¼ 16.1, 8.7Hz, H13), 1.76 (2 H, s, H7), 1.65 (2 H, tdd,
J ¼ 12.6, 7.1, 3.4Hz, H1), 1.54e1.43 (3 H, m, H4, H6, H13), 1.42 (3 H, s,
H15),1.37e1.32 (2 H, m, H8),1.16 (3 H, s, H18), 0.88 (3 H, d, J ¼ 7.0Hz,
H17), 0.71 (3 H, d, J ¼ 7.1Hz, H16). 13C NMR (151 MHz, CDCl3)
0.71 (3 H, d, J ¼ 7.1Hz, H16). 13C NMR (151 MHz, CDCl3)
d 216.77 (C3),
166.86 (C21), 160.16, 151.21, 144.62, 138.85 (C19), 135.42, 132.83,
130.41, 128.29, 127.24, 117.19 (C20), 74.60 (C11), 70.62 (C14), 58.07
(C4), 45.41 (C9), 44.53 (C13), 44.02 (C12), 41.84 (C5), 36.66 (C6),
36.24, 36.02 (C10), 34.42 (C2), 30.39 (C8), 26.83 (C7), 26.43 (C18),
24.81 (C1), 16.73 (C16),14.75 (C15), 11.43 (C17), 11.25. HR-MS (ESIþ):
Calcd for C32H41ClN4O4S (MþHþ): 613.2610; Found: 613.2595.
d
216.76 (C3), 166.85 (C21), 160.29, 160.27, 151.19, 144.65, 138.86
(C19), 130.86, 125.31, 120.06, 119.92, 117.18 (C20), 114.76, 74.60
(C11), 70.62 (C14), 58.07 (C4), 45.41 (C9), 44.53 (C13), 44.02 (C12),
41.84 (C5), 36.66 (C6), 36.19, 36.02 (C10), 34.42 (C2), 30.39 (C8),
26.83 (C7), 26.42 (C18), 24.81 (C1), 16.73 (C16), 14.75 (C15), 11.42
(C17), 11.23. HR-MS (ESIþ): Calcd for C32H41FN4O4S (MþHþ):
597.2906; Found: 597.2893.
4.2.15. 22-[(4-(4-chloro-benzylidene amino)-5-methyl-4H-1,2,4-
triazol-3-yl)thio] deoxy pleuromutilin (45)
White powder; yield: 64%; 1H NMR (600 MHz, Chloroform-d)
d
8.60 (1H, s), 7.88e7.74 (2 H, m), 7.62e7.40 (2 H, m), 6.41 (1 H,
dd, J ¼ 17.4, 11.0Hz, H19), 5.72 (1 H, d, J ¼ 8.5Hz, H14), 5.29 (1 H, dd,
J ¼ 11.0, 1.4Hz, H20), 5.18 (1H, dd, J ¼ 17.5, 1.5Hz, H20), 4.11e3.98
(2H, m, H22), 3.35 (1H, dd, J ¼ 10.0, 6.5Hz, H11), 2.51 (3 H, d,
J ¼ 0.6Hz), 2.31e2.15 (3 H, m, H2, H10), 2.09 (1H, d, J ¼ 2.8Hz, 11-
OH), 2.04 (1 H, dd, J ¼ 16.1, 8.6Hz, H13), 1.77 (2 H, s, H7),
1.69e1.60 (2 H, m, H1), 1.55e1.42 (3 H, m, H4, H6, H13), 1.42 (3 H, s,
H15),1.38e1.31 (2 H, m, H8),1.16 (3 H, s, H18), 0.88 (3 H, d, J ¼ 7.0Hz,
H17), 0.70 (3 H, d, J ¼ 7.1Hz, H16). 13C NMR (151 MHz, CDCl3)
4.2.12. 22-[(4-(4-fluoro-benzylidene amino)-5-methyl-4H-1,2,4-
triazol-3-yl)thio] deoxy pleuromutilin (42)
White powder; yield: 66%; 1H NMR (600 MHz, Chloroform-d)
d
8.59 (1H, s), 7.92e7.84 (2 H, m), 7.25e7.17 (2 H, m), 6.41 (1 H,
dd, J ¼ 17.4, 11.0Hz, H19), 5.72 (1 H, d, J ¼ 8.5Hz, H14), 5.29 (1 H, dd,
J ¼ 11.0, 1.5Hz, H20), 5.18 (1H, dd, J ¼ 17.5, 1.5Hz, H20), 4.11e3.96
(2H, m, H22), 3.35 (1H, dd, J ¼ 10.0, 6.4Hz, H11), 2.50 (3H, s),
2.34e1.96 (5 H, m, H2, H4, H10, 11-OH), 1.80 (1H, s, H7), 1.75 (1 H,
dq, J ¼ 14.5, 3.1Hz, H6), 1.65 (2 H, tdd, J ¼ 10.8, 8.2, 5.2Hz, H1),
1.54e1.48 (2 H, m, H7, H13), 1.45 (1 H, ddd, J ¼ 13.4, 9.7, 2.8Hz, H13),
1.41 (3 H, s, H15), 1.38e1.31 (2 H, m, H8), 1.16 (3 H, s, H18), 0.87 (3 H,
d, J ¼ 7.0Hz, H17), 0.70 (3 H, d, J ¼ 7.1Hz, H16). 13C NMR (151 MHz,
d
216.77 (C3), 166.85 (C21), 160.61, 151.06, 144.66, 139.29 (C19),
138.87, 130.27, 130.01, 129.55, 117.17 (C20), 74.60 (C11), 70.61 (C14),
58.07 (C4), 45.41 (C9), 44.53 (C13), 44.02 (C12), 41.85 (C5), 36.66
(C6), 36.11, 36.02 (C10), 34.42 (C2), 30.39 (C8), 29.67, 26.83 (C7),
26.44 (C18), 24.81 (C1), 16.73 (C16), 14.76 (C15), 11.42 (C17), 11.22.
HR-MS (ESIþ): Calcd for C32H41ClN4O4S (MþHþ): 613.2610; Found:
613.2600.
CDCl3)
d 216.78 (C3), 166.87 (C21), 164.79, 161.06, 150.96, 144.65,
138.88 (C19), 131.17, 131.11, 117.16 (C20), 116.62, 116.47, 74.60 (C11),
70.58 (C14), 58.07 (C4), 45.41 (C9), 44.53 (C13), 44.02 (C12), 41.85
(C5), 36.66 (C6), 36.07, 36.02 (C10), 34.43 (C2), 30.39 (C8), 26.83
(C7), 26.44 (C18), 24.81 (C1), 16.73 (C16), 14.75 (C15), 14.71, 11.42
(C17), 11.18. HR-MS (ESIþ): Calcd for C32H41FN4O4S (MþHþ):
597.2906; Found: 597.2894.
4.2.16. 22-[(4-(2-hydroxyl-benzylidene amino)-5-methyl-4H-1,2,4-
triazol-3-yl)thio] deoxy pleuromutilin (46)
White powder; yield: 58%; 1H NMR (600 MHz, Chloroform-d)
d
10.26 (1H, s), 8.76 (1 H, s), 7.52 (1 H, ddd, J ¼ 8.6, 7.3, 1.7Hz),
7.43 (1 H, dd, J ¼ 7.8, 1.7Hz), 7.12e7.08 (1 H, m), 7.05 (1 H, td, J ¼ 7.5,
1.1Hz), 6.41 (1 H, dd, J ¼ 17.4, 11.0Hz, H19), 5.72 (1 H, d, J ¼ 8.5Hz,
H14), 5.29 (1 H, dd, J ¼ 11.0, 1.5Hz, H20), 5.18 (1H, dd, J ¼ 17.5, 1.5Hz,
H20), 4.11e3.92 (2H, m, H22), 3.35 (1H, dd, J ¼ 10.4, 6.5Hz, H11),
2.51 (3H, s), 2.32e2.15 (3 H, m, H2, H10), 2.11e2.02 (2H, m, H13, 11-
OH), 1.78e1.73 (3 H, m, H7, H13), 1.65 (2 H, tdd, J ¼ 11.1, 7.5, 5.2Hz,
H1), 1.51e1.48 (1 H, m, H6), 1.45 (1 H, ddd, J ¼ 13.4, 9.6, 2.7Hz, H4),
1.42 (3 H, s, H15), 1.37e1.30 (2 H, m, H8), 1.16 (3 H, s, H18), 0.88 (3 H,
d, J ¼ 7.0Hz, H17), 0.70 (3 H, d, J ¼ 7.1Hz, H16). 13C NMR (151 MHz,
4.2.13. 22-[(4-(2-chloro-benzylidene amino)-5-methyl-4H-1,2,4-
triazol-3-yl)thio] deoxy pleuromutilin (43)
White powder; yield: 68%; 1H NMR (600 MHz, Chloroform-d)
d
9.08 (1H, d, J ¼ 0.5Hz), 8.17 (1 H, dt, J ¼ 7.7, 1.3Hz), 7.55e7.46
(2 H, m), 7.42 (1 H, dddd, J ¼ 7.8, 5.5, 2.9, 0.7Hz), 6.42 (1 H, dd,
J ¼ 17.4, 11.0Hz, H19), 5.74 (1 H, d, J ¼ 8.5Hz, H14), 5.30 (1 H, dd,
J ¼ 11.0, 1.5Hz, H20), 5.18 (1H, dd, J ¼ 17.4, 1.5Hz, H20), 4.14e3.99
(2H, m, H22), 3.35 (1H, dd, J ¼ 10.4, 6.5Hz, H11), 2.55 (3H, s),
2.33e2.14 (3 H, m, H2, H10), 2.10e2.08 (1H, m, 11-OH), 2.04 (1 H,
dd, J ¼ 16.1, 8.6Hz, H13), 1.76 (2 H, s, H7), 1.65 (2 H, tdd, J ¼ 10.8, 8.2,
5.3Hz, H1), 1.56e1.43 (3 H, m, H4, H6, H13), 1.42 (3 H, s, H15), 1.36
(2 H, dd, J ¼ 16.5, 3.1Hz, H8), 1.16 (3 H, s, H18), 0.87 (3 H, d, J ¼ 7.0Hz,
H17), 0.72 (3 H, d, J ¼ 7.1Hz, H16). 13C NMR (151 MHz, CDCl3)
CDCl3)
d 216.74 (C3), 167.48 (C21), 166.85, 159.91, 150.02, 145.00,
138.88 (C19), 135.33, 133.26, 120.26, 117.87, 117.17 (C20), 115.83,
74.59 (C11), 70.70 (C14), 58.05 (C4), 45.41 (C9), 44.54 (C13), 44.03
(C12), 41.83 (C5), 36.64 (C6), 36.18, 36.00 (C10), 34.42 (C2), 30.38
(C8), 26.82 (C7), 26.39 (C18), 24.81 (C1), 16.74 (C16), 14.75 (C15),
11.43 (C17), 11.07. HR-MS (ESIþ): Calcd for C32H42N4O5S (MþHþ):
595.2949; Found: 595.2935.
d
216.82 (C3), 166.68 (C21), 157.48, 150.96, 145.15, 138.83 (C19),
136.33, 133.67, 130.29, 129.59, 128.05, 127.43, 117.21 (C20), 74.62
(C11), 70.51 (C14), 58.10 (C4), 45.42 (C9), 44.53 (C13), 44.01 (C12),
41.87 (C5), 36.69 (C6), 36.02 (C10), 35.88, 34.44 (C2), 30.40 (C8),
29.67, 26.83 (C7), 26.43 (C18), 24.82 (C1), 16.73 (C16), 14.77 (C15),
11.42 (C17). HR-MS (ESIþ): Calcd for C32H41ClN4O4S (MþHþ):
613.2610; Found: 613.2595.
4.2.17. 22-[(4-(3-hydroxyl-benzylidene amino)-5-methyl-4H-1,2,4-
triazol-3-yl)thio] deoxy pleuromutilin (47)
White powder; yield: 56%; 1H NMR (600 MHz, Chloroform-d)
d
8.88 (1H, s), 8.54 (1 H, s), 7.48 (1 H, dd, J ¼ 2.5, 1.5Hz),
9

B. Li, Z. Zhang, J.-F. Zhang et al.
European Journal of Medicinal Chemistry 223 (2021) 113624
7.38e7.31 (2 H, m), 7.18 (1 H, ddd, J ¼ 7.9, 2.5, 1.3Hz), 6.39 (1 H, dd,
J ¼ 17.4, 11.0Hz, H19), 5.71 (1 H, d, J ¼ 8.5Hz, H14), 5.27 (1 H, dd,
J ¼ 11.0, 1.5Hz, H20), 5.16 (1H, dd, J ¼ 17.4, 1.5Hz, H20), 4.10e3.93
(2H, m, H22), 3.35 (1H, dd, J ¼ 9.9, 6.2Hz, H11), 2.52 (3H, s),
2.31e2.14 (3 H, m, H2, H10), 2.09e2.06 (1H, m, 11-OH), 2.01 (1 H,
dd, J ¼ 16.0, 8.6Hz, H13), 1.92 (2 H, s, H7), 1.74 (1 H, dt, J ¼ 14.5,
3.2Hz, H13), 1.65e1.58 (3 H, m, H1, H6), 1.44 (1 H, ddt, J ¼ 12.1, 9.5,
5.0Hz, H4), 1.39 (3 H, s, H15), 1.35e1.28 (2 H, m, H8), 1.14 (3 H, s,
2.33e2.14 (3 H, m, H2, H10), 2.11e2.08 (1H, m,11-OH), 2.05 (1 H, dd,
J ¼ 16.1, 8.6Hz, H13), 1.75 (2 H, d, J ¼ 4.5Hz, H7), 1.65 (2 H, dddd,
J ¼ 13.8, 10.9, 8.1, 3.6Hz, H1), 1.53e1.43 (3 H, m, H4, H6, H13), 1.42
(3 H, s, H15), 1.39e1.31 (2 H, m, H8), 1.16 (3 H, s, H18), 0.88 (3 H, d,
J ¼ 7.1Hz, H17), 0.71 (3 H, d, J ¼ 7.1Hz, H16). 13C NMR (151 MHz,
CDCl3)
d 216.73 (C3), 166.82 (C21), 158.08, 151.49, 148.80, 144.62,
138.87 (C19), 133.91, 133.67, 130.34, 126.95, 123.48, 117.15 (C20),
74.59 (C11), 70.76 (C14), 58.05 (C4), 45.41 (C9), 44.55 (C13), 44.03
(C12), 41.85 (C5), 36.63 (C6), 36.41, 36.02 (C10), 34.41 (C2), 30.37
(C8), 26.83 (C7), 26.44 (C18), 24.81 (C1), 16.73 (C16), 14.76 (C15),
11.42 (C17), 11.33. HR-MS (ESIþ): Calcd for C32H41N5O6S (MþHþ):
624.2851; Found: 624.2836.
H18), 0.87 (3 H, d, J ¼ 7.0Hz, H17), 0.68 (3 H, d, J ¼ 7.1Hz, H16). 13
C
NMR (151 MHz, CDCl3)
d 217.10 (C3), 166.79 (C21), 163.84, 157.64,
150.93, 145.18, 138.78 (C19), 132.69, 130.29, 121.34, 121.14, 117.26
(C20), 115.02, 74.63 (C11), 70.62 (C14), 58.09 (C4), 45.43 (C9), 44.49
(C13), 44.00 (C12), 41.84 (C5), 36.67 (C6), 36.00 (C10), 35.86, 34.45
(C2), 30.38 (C8), 26.80 (C7), 26.41 (C18), 24.81 (C1), 16.71 (C16),
14.76 (C15), 11.46 (C17), 11.08. HR-MS (ESIþ): Calcd for C32H42N4O5S
(MþHþ): 595.2949; Found: 595.2936.
4.2.21. 22-[(4-(4-nitro-benzylidene amino)-5-methyl-4H-1,2,4-
triazol-3-yl)thio] deoxy pleuromutilin (51)
White powder; yield: 58%; 1H NMR (600 MHz, Chloroform-d)
d
8.80 (1H, s), 8.40e8.35 (2 H, m), 8.09e8.03 (2 H, m), 6.40 (1 H,
4.2.18. 22-[(4-(4-hydroxyl-benzylidene amino)-5-methyl-4H-1,2,4-
triazol-3-yl)thio] deoxy pleuromutilin (48)
dd, J ¼ 17.4, 11.0Hz, H19), 5.73 (1 H, d, J ¼ 8.5Hz, H14), 5.28 (1 H, dd,
J ¼ 11.0, 1.5Hz, H20), 5.18 (1H, dd, J ¼ 17.5, 1.5Hz, H20), 4.17e4.03
(2H, m, H22), 3.36 (1H, dd, J ¼ 10.3, 6.5Hz, H11), 2.56 (3H, s),
2.31e2.15 (3 H, m, H2, H10), 2.10 (1H, d, J ¼ 2.7, 11-OH), 2.05 (1 H,
dd, J ¼ 16.1, 8.6Hz, H13), 1.75 (2 H, s, H7), 1.70e1.61 (2 H, m, H1),
1.54e1.48 (2 H, m, H6, H13), 1.48e1.43 (1 H, m, H4), 1.43 (3 H, s,
H15),1.38e1.32 (2 H, m, H8),1.17 (3 H, s, H18), 0.88 (3 H, d, J ¼ 7.1Hz,
H17), 0.71 (3 H, d, J ¼ 7.1Hz, H16). 13C NMR (151 MHz, CDCl3)
White powder; yield: 66%; 1H NMR (600 MHz, Chloroform-d)
d
9.97 (1H, s), 8.46 (1 H, s), 7.79e7.73 (2 H, m), 7.11e7.05 (2 H, m),
6.40 (1 H, dd, J ¼ 17.4,11.0Hz, H19), 5.72 (1 H, d, J ¼ 8.6Hz, H14), 5.29
(1 H, dd, J ¼ 11.0, 1.5Hz, H20), 5.17 (1H, dd, J ¼ 17.4, 1.5Hz, H20),
4.11e3.91 (2H, m, H22), 3.35 (1H, dd, J ¼ 10.3, 6.4Hz, H11), 2.51 (3H,
s), 2.32e2.14 (3 H, m, H2, H10), 2.11e2.07 (1H, m, 11-OH), 2.02 (1 H,
dd, J ¼ 16.1, 8.6Hz, H13), 1.85 (2 H, s, H7), 1.78e1.72 (1 H, m, H13),
1.68e1.60 (2 H, m, H1), 1.57 (1 H, d, J ¼ 10.4Hz, H8), 1.54e1.42 (2 H,
m, H4, H6), 1.41 (3 H, s, H15), 1.35e1.31 (1 H, m, H8), 1.14 (3 H, s,
d
216.74 (C3), 166.76 (C21), 157.40, 151.56, 150.13, 144.73, 138.89
(C19), 137.49, 129.49, 124.29, 117.15 (C20), 74.59 (C11), 70.79 (C14),
58.05 (C4), 53.40 (C22), 45.41 (C9), 44.53 (C13), 44.04 (C12), 41.85
(C5), 36.63 (C6), 36.31, 36.03 (C10), 34.41 (C2), 30.36 (C8), 26.83
(C7), 26.47 (C18), 24.81 (C1), 16.74 (C16), 14.76 (C15), 11.42 (C17),
11.36. HR-MS (ESIþ): Calcd for C32H41N5O6S (MþHþ): 624.2851;
Found: 624.2835.
H18), 0.87 (3 H, d, J ¼ 7.0Hz, H17), 0.70 (3 H, d, J ¼ 7.1Hz, H16). 13
C
NMR (151 MHz, CDCl3)
d 217.01 (C3), 166.87 (C21), 164.88, 162.44,
150.74, 145.25, 138.82 (C19), 131.41, 122.84, 117.24 (C20), 116.69,
74.63 (C11), 70.59 (C14), 58.10 (C4), 45.43 (C9), 45.03, 44.51 (C13),
44.01 (C12), 41.85 (C5), 41.46, 36.68 (C6), 36.01 (C10), 35.70, 34.45
(C2), 30.39 (C8), 26.82 (C7), 26.41 (C18), 24.81 (C1), 16.72 (C16),
14.77 (C15), 11.46 (C17), 10.93. HR-MS (ESIþ): Calcd for C32H42N4O5S
(MþHþ): 595.2949; Found: 595.2938.
4.2.22. 22-[(4-(3-nitrile-benzylidene amino)-5-methyl-4H-1,2,4-
triazol-3-yl)thio] deoxy pleuromutilin (52)
White powder; yield: 70%; 1H NMR (600 MHz, Chloroform-d)
d
8.73 (1H, s), 8.18 (1 H, t, J ¼ 1.7Hz), 8.10 (1 H, dt, J ¼ 7.9, 1.4Hz),
4.2.19. 22-[(4-(2-nitro-benzylidene amino)-5-methyl-4H-1,2,4-
triazol-3-yl)thio] deoxy pleuromutilin (49)
7.86 (1 H, dt, J ¼ 7.8, 1.3Hz), 7.67 (1 H, t, J ¼ 7.8Hz), 6.40 (1 H, dd,
J ¼ 17.4, 11.0Hz, H19), 5.73 (1 H, d, J ¼ 8.5Hz, H14), 5.28 (1 H, dd,
J ¼ 11.0, 1.4Hz, H20), 5.18 (1H, dd, J ¼ 17.4, 1.5Hz, H20), 4.21e3.90
(2H, m, H22), 3.36 (1H, dd, J ¼ 10.3, 6.5Hz, H11), 2.54 (3H, s),
2.32e2.15 (3 H, m, H2, H10), 2.10 (1H, d, J ¼ 2.7Hz,11-OH), 2.05 (1 H,
dd, J ¼ 16.1, 8.6Hz, H13), 1.78e1.73 (3 H, m, H7, H13), 1.68e1.61 (2 H,
m, H1), 1.51 (1 H, d, J ¼ 10.2Hz, H6), 1.45 (1 H, ddd, J ¼ 13.0, 9.5,
2.5Hz, H4), 1.42 (3 H, s, H15), 1.39e1.31 (2 H, m, H8), 1.16 (3 H, s,
White powder; yield: 57%; 1H NMR (600 MHz, Chloroform-d)
d
9.29 (1H, s), 8.21 (2 H, ddd, J ¼ 8.0, 6.9, 1.4Hz), 7.86e7.68 (2 H,
m), 6.42 (1 H, dd, J ¼ 17.4, 11.0Hz, H19), 5.74 (1 H, d, J ¼ 8.4Hz, H14),
5.30 (1 H, dd, J ¼ 11.0, 1.5Hz, H20), 5.18 (1H, dd, J ¼ 17.4, 1.5Hz, H20),
4.14e4.00 (2H, m, H22), 3.35 (1H, dd, J ¼ 10.3, 6.5Hz, H11), 2.59 (3H,
s), 2.33e2.14 (3 H, m, H2, H10), 2.10 (1H, d, J ¼ 2.8Hz, 11-OH), 2.05
(1 H, dd, J ¼ 16.1, 8.6Hz, H13), 1.77 (2 H, s, H7), 1.65 (2 H, dddd,
J ¼ 11.0, 9.0, 7.0, 3.5, H1), 1.53e1.49 (2 H, m, H6, H13), 1.48e1.43
(1 H, m, H4), 1.42 (3 H, s, H15), 1.39e1.33 (2 H, m, H8), 1.17 (3 H, s,
H18), 0.88 (3 H, d, J ¼ 7.0Hz, H17), 0.70 (3 H, d, J ¼ 7.1Hz, H16). 13
C
NMR (151 MHz, CDCl3)
d 216.73 (C3), 166.82 (C21), 158.23, 151.43,
144.60, 138.88 (C19), 135.56, 133.23, 132.56, 131.96, 130.11, 117.58,
117.15 (C20), 113.85, 74.59 (C11), 70.75 (C14), 58.05 (C4), 45.41 (C9),
44.54 (C13), 44.03 (C12), 41.84 (C5), 36.63 (C6), 36.37, 36.02 (C10),
34.41 (C2), 30.37 (C8), 26.84 (C7), 26.46 (C18), 24.81 (C1), 16.73
(C16), 14.76 (C15), 11.42 (C17), 11.30. HR-MS (ESIþ): Calcd for
H18), 0.87 (3 H, d, J ¼ 7.1Hz, H17), 0.72 (3 H, d, J ¼ 7.1Hz, H16). 13
C
NMR (151 MHz, CDCl3)
d 216.83 (C3), 166.74 (C21), 156.36, 150.71,
145.93, 138.85 (C19), 134.12, 132.71, 129.49, 127.28, 125.23, 117.19
(C20), 74.61 (C11), 70.59 (C14), 58.08 (C4), 45.42 (C9), 44.51 (C13),
44.02 (C12), 41.86 (C5), 36.68 (C6), 36.01 (C10), 35.77, 34.43 (C2),
30.40 (C8), 26.83 (C7), 26.42 (C18), 24.81 (C1), 16.73 (C16), 14.77
(C15), 11.66, 11.42 (C17). HR-MS (ESIþ): Calcd for C32H41N5O6S
(MþHþ): 624.2851; Found: 624.2838.
C
33H41N5O4S (MþHþ): 604.2952; Found: 604.2942.
4.2.23. 22-[(4-(4-nitrile-benzylidene amino)-5-methyl-4H-1,2,4-
triazol-3-yl)thio] deoxy pleuromutilin (53)
White powder; yield: 72%; 1H NMR (600 MHz, Chloroform-d)
4.2.20. 22-[(4-(3-nitro-benzylidene amino)-5-methyl-4H-1,2,4-
triazol-3-yl)thio] deoxy pleuromutilin (50)
d 8.73 (1H, s), 8.03e7.96 (2 H, m), 7.87e7.76 (2 H, m), 6.40 (1 H,
dd, J ¼ 17.5, 11.0Hz, H19), 5.73 (1 H, d, J ¼ 8.5Hz, H14), 5.27 (1 H, dd,
J ¼ 11.0, 1.5Hz, H20), 5.18 (1H, dd, J ¼ 17.5, 1.5Hz, H20), 4.16e4.00
(2H, m, H22), 3.36 (1H, dd, J ¼ 10.3, 6.4Hz, H11), 2.54 (3H, s),
2.31e2.14 (3 H, m, H2, H10), 2.09 (1H, d, J ¼ 2.8Hz,11-OH), 2.04 (1 H,
dd, J ¼ 16.1, 8.6Hz, H13), 1.76 (2 H, s, H7), 1.65 (2 H, dddd, J ¼ 13.7,
10.8, 8.1, 3.5Hz, H1), 1.54e1.43 (3 H, m, H4, H6, H13), 1.42 (3 H, s,
H15),1.39e1.29 (2 H, m, H8),1.16 (3 H, s, H18), 0.88 (3 H, d, J ¼ 7.0Hz,
White powder; yield: 69%; 1H NMR (600 MHz, Chloroform-d)
d
8.83 (1H, s), 8.72e8.71 (1 H, m), 8.43 (1 H, ddd, J ¼ 8.2, 2.3,
1.1Hz), 8.23 (1 H, dt, J ¼ 7.7, 1.4Hz), 7.75 (1 H, t, J ¼ 8.0Hz), 6.40 (1 H,
dd, J ¼ 17.4, 11.0Hz, H19), 5.73 (1 H, d, J ¼ 8.5Hz, H14), 5.28 (1 H, dd,
J ¼ 11.0, 1.5Hz, H20), 5.18 (1H, dd, J ¼ 17.5, 1.5Hz, H20), 4.15e4.01
(2H, m, H22), 3.36 (1H, dd, J ¼ 10.1, 6.4Hz, H11), 2.56 (3H, s),
10

B. Li, Z. Zhang, J.-F. Zhang et al.
European Journal of Medicinal Chemistry 223 (2021) 113624
H17), 0.70 (3 H, d, J ¼ 7.1Hz, H16). 13C NMR (151 MHz, CDCl3)
58.11 (C4), 45.42 (C9), 44.50 (C13), 44.01 (C12), 41.85 (C5), 40.07,
39.81, 36.71 (C6), 36.01 (C10), 35.63, 34.45 (C2), 30.42 (C8), 29.67,
26.84 (C7), 26.43 (C18), 24.82 (C1), 16.74 (C16), 14.78 (C15), 11.44
(C17), 11.02. HR-MS (ESIþ): Calcd for C34H47N5O4S (MþHþ):
622.3422; Found: 622.3410.
d
216.73 (C3), 166.76 (C21), 158.10, 151.45, 144.71, 138.89 (C19),
135.85, 132.81, 129.07, 117.81, 117.14 (C20), 115.99, 74.58 (C11), 70.76
(C14), 58.05 (C4), 45.41 (C9), 44.53 (C13), 44.03 (C12), 41.85 (C5),
40.81, 36.63 (C6), 36.27, 36.03 (C10), 34.42 (C2), 30.37 (C8), 26.83
(C7), 26.47 (C18), 25.48, 24.81 (C1), 16.73 (C16), 14.75 (C15), 11.42
(C17), 11.32. HR-MS (ESIþ): Calcd for C33H41N5O4S (MþHþ):
604.2952; Found: 604.2943.
4.2.27. 22-[(4-(4-diethylamino-benzylidene amino)-5-methyl-4H-
1,2,4-triazol-3-yl)thio]deoxy pleuromutilin (57)
White powder; yield: 65%; 1H NMR (600 MHz, Chloroform-d)
4.2.24. 22-[(4-(3-trifluoromethyl-benzylidene amino)-5-methyl-
4H-1,2,4-triazol-3-yl)thio]deoxy pleuromutilin (54)
d 8.28 (1H, s), 7.74e7.64 (2 H, m), 6.76e6.65 (2 H, m), 6.42 (1 H,
dd, J ¼ 17.5, 11.0Hz, H19), 5.72 (1 H, d, J ¼ 8.5Hz, H14), 5.31 (1 H, dd,
J ¼ 11.0, 1.5Hz, H20), 5.19 (1H, dd, J ¼ 17.4, 1.5Hz, H20), 4.09e3.89
(2H, m, H22), 3.46 (4H, q, J ¼ 7.1Hz), 3.35 (1 H, dd, J ¼ 10.4, 6.5Hz,
H11), 2.44 (3 H, s), 2.33e2.16 (3 H, m, H2, H10), 2.09 (1H, d,
J ¼ 2.7Hz, 11-OH), 2.03 (1 H, dd, J ¼ 16.1, 8.6Hz, H13), 1.82 (2 H, s,
H7), 1.75 (1 H, dq, J ¼ 14.3, 3.2Hz, H13), 1.68e1.61 (2 H, m, H1),
1.54e1.45 (2 H, m, H4, H6), 1.41 (3 H, s, H15), 1.35 (2 H, dd, J ¼ 15.6,
3.5Hz, H8), 1.24 (6 H, t, J ¼ 7.1Hz), 1.17 (3 H, s, H18), 0.87 (3 H, d,
J ¼ 7.0Hz, H17), 0.71 (3 H, d, J ¼ 7.1Hz, H16). 13C NMR (151 MHz,
White powder; yield: 55%; 1H NMR (600 MHz, Chloroform-d)
d
8.74 (1H, s), 8.13 (1 H, d, J ¼ 1.8Hz), 8.07 (1 H, dt, J ¼ 7.7, 1.5Hz),
7.85 (1 H, ddt, J ¼ 7.9,1.9,1.0Hz), 7.68 (1 H, t, J ¼ 7.8Hz), 6.41 (1 H, dd,
J ¼ 17.5, 11.0Hz, H19), 5.73 (1 H, d, J ¼ 8.5Hz, H14), 5.28 (1 H, dd,
J ¼ 11.0, 1.5Hz, H20), 5.18 (1H, dd, J ¼ 17.5, 1.5Hz, H20), 4.14e3.99
(2H, m, H22), 3.35 (1H, dd, J ¼ 10.4, 6.5Hz, H11), 2.54 (3H, s),
2.32e2.16 (3 H, m, H2, H10), 2.11e2.08 (1H, m,11-OH), 2.05 (1 H, dd,
J ¼ 16.1, 8.6Hz, H13), 1.75 (2 H, s, H7), 1.68e1.61 (2 H, m, H1),
1.54e1.43 (3 H, m, H4, H6, H13),1.42 (3 H, s, H15),1.39e1.30 (2 H, m,
H8), 1.16 (3 H, s, H18), 0.88 (3 H, d, J ¼ 7.1Hz, H17), 0.71 (3 H, d,
CDCl3)
d 216.88 (C3), 167.03 (C21), 165.00, 151.38, 150.40, 144.92,
138.88 (C19), 131.27, 117.99, 117.20 (C20), 111.13, 100.00, 74.63 (C11),
70.28 (C14), 58.12 (C4), 45.95, 45.42 (C9), 44.65 (C13), 44.50, 44.02
(C12), 41.85 (C5), 39.72, 36.71 (C6), 36.01 (C10), 35.57, 34.91, 34.45
(C2), 30.42 (C8), 26.84 (C7), 26.43 (C18), 24.82 (C1), 16.74 (C16),
14.79 (C15), 12.50, 11.43 (C17), 10.98. HR-MS (ESIþ): Calcd for
J ¼ 7.1Hz, H16). 13C NMR (151 MHz, CDCl3)
d 216.74 (C3), 166.85
(C21), 159.76, 151.30, 144.61, 138.85 (C19), 132.70, 131.80, 129.79,
129.25, 129.22, 125.49,125.46, 117.16 (C20), 74.60 (C11), 70.67 (C14),
58.06 (C4), 45.41 (C9), 44.54 (C13), 44.02 (C12), 41.84 (C5), 36.65
(C6), 36.31, 36.02 (C10), 34.42 (C2), 30.38 (C8), 26.82 (C7), 26.42
(C18), 24.81 (C1), 16.72 (C16), 14.75 (C15), 11.41 (C17), 11.26. HR-MS
(ESIþ): Calcd for C33H41F3N4O4S (MþHþ): 647.2874; Found:
647.2861.
C
36H51N5O4S (MþHþ): 650.3735; Found: 650.3723.
4.2.28. 22-[(4-(4-pyrrolidinyl-benzylidene amino)-5-methyl-4H-
1,2,4-triazol-3-yl)thio]deoxy pleuromutilin (58)
White powder; yield: 55%; 1H NMR (600 MHz, Chloroform-d)
4.2.25. 22-[(4-(4-trifluoromethyl-benzylidene amino)-5-methyl-
4H-1,2,4-triazol-3-yl)thio]deoxy pleuromutilin (55)
d 8.29 (1H, s), 7.79e7.61 (2 H, m), 6.66e6.52 (2 H, m), 6.42 (1 H,
dd, J ¼ 17.4, 11.0Hz, H19), 5.72 (1 H, d, J ¼ 8.5Hz, H14), 5.31 (1 H, dd,
J ¼ 11.0, 1.5Hz, H20), 5.19 (1H, dd, J ¼ 17.5, 1.5Hz, H20), 4.09e3.89
(2H, m, H22), 3.43e3.38 (4H, m), 3.37e3.32 (1H, m, H11), 2.44 (3 H,
s), 2.34e2.15 (3 H, m, H2, H10), 2.08 (5H, td, J ¼ 7.0, 3.5Hz, 11-OH),
2.03 (1 H, dd, J ¼ 16.1, 8.5Hz, H13), 1.83 (2 H, s, H7), 1.75 (1 H, dq,
J ¼ 14.5, 3.2Hz, H13), 1.68e1.60 (2 H, m, H1), 1.56e1.45 (2 H, m, H4,
H6), 1.41 (3 H, s, H15), 1.35 (2 H, dd, J ¼ 15.6, 3.2Hz, H8), 1.17 (3 H, s,
White powder; yield: 51%; 1H NMR (600 MHz, Chloroform-d)
d
8.73 (1H, s), 8.03e7.94 (2 H, m), 7.79 (2 H, d, J ¼ 8.1Hz), 6.41
(1 H, dd, J ¼ 17.4, 11.0Hz, H19), 5.73 (1 H, d, J ¼ 8.6Hz, H14), 5.29
(1 H, dd, J ¼ 11.0, 1.5Hz, H20), 5.18 (1H, dd, J ¼ 17.4, 1.5Hz, H20),
4.16e3.98 (2H, m, H22), 3.36 (1H, dd, J ¼ 10.3, 6.4Hz, H11), 2.54
(3 H, d, J ¼ 0.9Hz), 2.32e2.16 (3 H, m, H2, H10), 2.11e2.08 (1H, m,
11-OH), 2.05 (1 H, dd, J ¼ 16.1, 8.6Hz, H13), 1.74 (2 H, s, H7), 1.65
(2 H, dddd, J ¼ 13.8, 10.7, 8.1, 3.6Hz, H1), 1.53e1.43 (3 H, m, H4, H6,
H13),1.42 (3 H, s, H15),1.39e1.31 (2 H, m, H8),1.16 (3 H, s, H18), 0.88
(3 H, d, J ¼ 7.0Hz, H17), 0.71 (3 H, d, J ¼ 7.1Hz, H16). 13C NMR
H18), 0.87 (3 H, d, J ¼ 7.0Hz, H17), 0.71 (3 H, d, J ¼ 7.1Hz, H16). 13
C
NMR (151 MHz, CDCl3)
d 216.88 (C3), 167.04 (C21), 165.14, 151.19,
150.41, 144.93, 138.87 (C19), 131.10, 118.20, 117.20 (C20), 111.67,
74.63 (C11), 70.29 (C14), 58.12 (C4), 47.65, 45.53 (C9), 45.42, 44.50
(C13), 44.02 (C12), 42.21, 41.85 (C5), 36.93, 36.71 (C6), 36.01 (C10),
35.59, 34.45 (C2), 30.42 (C8), 27.20 (C7), 26.84, 26.43 (C18), 25.42,
24.82 (C1), 16.74 (C16), 14.79 (C15), 11.43 (C17), 11.01. HR-MS (ESIþ):
Calcd for C36H49N5O4S (MþHþ): 648.3578; Found: 648.3565.
(151 MHz, CDCl3) d 216.74 (C3), 166.80 (C21), 159.34, 151.32, 144.68,
138.87 (C19), 135.09, 129.03, 126.15, 126.13, 126.10, 126.08, 124.40,
117.16 (C20), 74.60 (C11), 70.69 (C14), 58.06 (C4), 45.41 (C9), 44.54
(C13), 44.03 (C12), 41.85 (C5), 36.65 (C6), 36.21, 36.02 (C10), 34.42
(C2), 30.38 (C8), 26.83 (C7), 26.44 (C18), 24.81 (C1), 16.73 (C16),
14.75 (C15), 11.41 (C17), 11.28. HR-MS (ESIþ): Calcd for
4.2.29. 22-[(4-(4-morpholinyl-benzylidene amino)-5-methyl-4H-
1,2,4-triazol-3-yl) thio]deoxy pleuromutilin (59)
C
33H41F3N4O4S (MþHþ): 647.2874; Found: 647.2862.
White powder; yield: 69%; 1H NMR (600 MHz, Chloroform-d)
4.2.26. 22-[(4-(4-dimethylamino-benzylidene amino)-5-methyl-
4H-1,2,4-triazol-3-yl)thio]deoxy pleuromutilin (56)
d 8.39 (1H, s), 7.80e7.71 (2 H, m), 6.97e6.92 (2 H, m), 6.42 (1 H,
dd, J ¼ 17.4, 11.0Hz, H19), 5.72 (1 H, d, J ¼ 8.5Hz, H14), 5.30 (1 H, dd,
J ¼ 11.0, 1.5Hz, H20), 5.18 (1H, dd, J ¼ 17.5, 1.5Hz, H20), 4.08e3.94
(2H, m, H22), 3.90e3.86 (4H, m), 3.37e3.32 (5H, m, H11), 2.46 (3 H,
s), 2.33e2.15 (3 H, m, H2, H10), 2.09 (1H, d, J ¼ 2.7Hz, 11-OH), 2.03
(1 H, dd, J ¼ 16.1, 8.6Hz, H13), 1.81 (2 H, s, H7), 1.75 (1 H, dq, J ¼ 14.5,
3.2Hz, H13), 1.68e1.61 (2 H, m, H1), 1.55e1.50 (1 H, m, H6),
1.50e1.42 (1 H, m, H4), 1.41 (3 H, s, H15), 1.34 (2 H, dd, J ¼ 15.1,
6.5Hz, H8), 1.16 (3 H, s, H18), 0.87 (3 H, d, J ¼ 7.1Hz, H17), 0.70 (3 H,
White powder; yield: 50%; 1H NMR (600 MHz, Chloroform-d)
d
8.32 (1H, s), 7.75e7.69 (2 H, m), 6.77e6.69 (2 H, m), 6.43 (1 H,
dd, J ¼ 17.4, 11.0Hz, H19), 5.73 (1 H, d, J ¼ 8.5Hz, H14), 5.32 (1 H, dd,
J ¼ 11.0, 1.5Hz, H20), 5.19 (1H, dd, J ¼ 17.4, 1.5Hz, H20), 4.07e3.94
(2H, m, H22), 3.35 (1H, s, H11), 3.11 (6H, s), 2.46 (3 H, s), 2.34e2.15
(3 H, m, H2, H10), 2.10 (1H, d, J ¼ 2.8Hz, 11-OH), 2.04 (1 H, dd,
J ¼ 16.1, 8.6Hz, H13), 1.77 (1 H, q, J ¼ 3.2Hz, H7), 1.75 (1 H, q,
J ¼ 3.2Hz, H7), 1.69e1.61 (2 H, m, H1), 1.56e1.43 (2 H, m, H6, H13),
1.42 (3 H, s, H15), 1.35 (2 H, dd, J ¼ 15.3, 3.9Hz, H4, H8), 1.17 (3 H, s,
H18), 1.12 (1 H, td, J ¼ 14.1, 4.4Hz, H8), 0.88 (3 H, d, J ¼ 7.0Hz, H17),
d, J ¼ 7.1Hz, H16). 13C NMR (151 MHz, CDCl3)
d 216.85 (C3), 166.96
(C21), 163.64, 154.37, 150.55, 144.81, 138.87 (C19), 130.76, 121.83,
117.19 (C20),114.11, 74.62 (C11), 70.40 (C14), 66.50, 58.10 (C4), 47.53,
45.42 (C9), 44.51 (C13), 44.02 (C12), 43.70, 41.85 (C5), 36.69 (C6),
36.01 (C10), 35.76, 34.79, 34.44 (C2), 30.41 (C8), 29.67, 26.84 (C7),
26.44 (C18), 24.82 (C1), 16.74 (C16), 14.77 (C15), 11.43 (C17), 11.08.
0.71 (3 H, d, J ¼ 7.1Hz, H16). 13C NMR (151 MHz, CDCl3)
d 216.88
(C3), 167.03 (C21), 164.81, 153.58, 150.44, 144.90, 138.87 (C19),
132.87, 130.94, 118.75, 117.20 (C20), 111.55, 74.63 (C11), 70.31 (C14),
11

B. Li, Z. Zhang, J.-F. Zhang et al.
European Journal of Medicinal Chemistry 223 (2021) 113624
HR-MS (ESIþ): Calcd for C36H49N5O5S (MþHþ): 664.3527; Found:
incubated at 37 ꢂC for 18e24 h. The total bacterial CFU/mL on the
plates were counted to calculate the bacterial colonies. The time-
kill curve was constructed by plotting log10 CFU/mL of bacteria
counts versus time.
664.3513.
4.2.30. 22-[(4-(4-(2-pyridine)-benzylidene amino)-5-methyl-4H-
1,2,4-triazol-3-yl) thio]deoxy pleuromutilin (60)
White powder; yield: 71%; 1H NMR (600 MHz, Chloroform-d)
d
8.75 (1H, dt, J ¼ 4.8, 1.4Hz), 8.66 (1 H, s), 8.19e8.16 (2 H, m),
4.3.3. Determination of the post-antibiotic effect (PAE)
8.00e7.96 (2 H, m), 7.84e7.81 (2 H, m), 7.34e7.30 (1 H, m), 6.42
(1 H, dd, J ¼ 17.4, 11.0Hz, H19), 5.73 (1 H, d, J ¼ 8.5Hz, H14), 5.30
(1 H, dd, J ¼ 11.0, 1.5Hz, H20), 5.18 (1H, dd, J ¼ 17.5, 1.5Hz, H20),
4.12e4.00 (2H, m, H22), 3.35 (1H, dd, J ¼ 10.4, 6.5Hz, H11), 2.54 (3H,
s), 2.33e2.14 (3 H, m, H2, H10), 2.11e2.08 (1H, m, 11-OH), 2.04 (1 H,
dd, J ¼ 16.1, 8.6Hz, H13), 1.82 (2 H, s, H7), 1.75 (1 H, dq, J ¼ 14.6,
3.2Hz, H13), 1.65 (2 H, dddd, J ¼ 13.6, 10.7, 8.1, 3.5Hz, H1), 1.52 (1 H,
d, J ¼ 10.4Hz, H6), 1.48e1.43 (1 H, m, H4), 1.42 (3 H, s, H15), 1.35
(2 H, dd, J ¼ 15.5, 4.0Hz, H8),1.16 (3 H, s, H18), 0.87 (3 H, d, J ¼ 7.0Hz,
H17), 0.72 (3 H, d, J ¼ 7.1Hz, H16). 13C NMR (151 MHz, CDCl3)
The post-antibiotic effect (PAE) of compounds 48 and 60 against
MRSA were determined using time-kill methods according to
previous methods [27]. In this experiment, MRSA ATCC 43300 in
the logarithmic phase was diluted to 106 CFU/mL with MH broth.
The above compounds at final concentrations of 2 ꢁ MIC and
4 ꢁ MIC were added to the tubes containing the inoculum. Then,
the samples were shaken at 37 ꢂC for 2 h. After the incubation was
completed. The drugs were removed from the sample by diluting
1000 times with the preheated MH broth. The tubes were placed in
37 ꢂC and the culture solution (100
mL) was extracted at times 0, 2,
d
216.80 (C3), 166.89 (C21), 161.72, 155.84, 151.03, 149.99, 144.77,
4, 6 and 8 h. After the culture solution was diluted 10-fold with
sterile saline and plated onto MH agar plates. The colonies were
counted after 20 h of incubation in 37 ꢂC. The PAE value was pre-
sented in the hour and calculated by the equation PAE ¼ TA - TC. (TA
and TC are the time required for the bacteria in the test and control
groups to increase by 1 log10 CFU/mL)
143.62, 138.86 (C19), 136.97, 132.06, 129.32, 127.53, 123.04, 120.91,
117.20 (C20), 74.61 (C11), 70.55 (C14), 58.08 (C4), 45.52, 45.42 (C9),
44.61 (C13), 44.53, 44.02 (C12), 41.85 (C5), 36.68 (C6), 36.07, 36.02
(C10), 34.43 (C2), 30.40 (C8), 26.83 (C7), 26.44 (C18), 24.81 (C1),
16.74 (C16), 14.77 (C15), 11.43 (C17), 11.26. HR-MS (ESIþ): Calcd for
C
37H45N5O4S (MþHþ): 656.3265; Found: 656.3253.
4.3. In vitro efficacy of pleuromutilin derivatives
4.4. Neutropenic murine thigh infection model
4.3.1. Minimal inhibitory concentration (MIC) and minimum
bactericidal concentration (MBC) testing
The neutropenic murine thigh infection model experiment was
performed as described in the literature [16]. Six-week-old SPF-ICR
female that weighing 23e27 g mice were used for this study. The
mice were injected with cyclophosphamide (Mead Johnson Phar-
maceuticals, Evansville, IN) at a dose of 150 mg/kg on 4 days and at
a dose of 100 mg/kg on 1 day before the experiment to reduce
The MIC and MBC values were determined according to the
Clinical and Laboratory Standards Institute (CLSI). The MIC values of
the target pleuromutilin derivatives against methicillin-resistant S.
aureus (ATCC 43300), S. aureus (ATCC 29213), S. aureus (AD3), S.
aureus (144) and E. coli (ATCC 25922) were determined using by
pleuromutilin and tiamulin as positive controls. The synthetic
compounds were dissolved in the aqueous solution of 2.5% DMSO
and 2.5% tween-80 to make a stock solution with a concentration of
neutrophils and achieve immunosuppression (<0.1
ꢁ
109/L).
0.1 mL MH broth (MRSA ATCC 43300 concentration was approxi-
mately 107 CFU/mL) was injected by intramuscular into both sides
of the posterior thigh of mice to establish an infection model,
respectively. After the thigh of mice were infected 2 h, these mice
were randomly divided into 3 groups (3 per group). The mice were
intravenously injected with 0.9% saline, compound 60 (20 mg/kg)
and tiamulin (20 mg/kg). 24 h after the completion of the drug, the
mice were euthanized and their thigh tissue were collected. Then
their thigh tissues were weighed and homogenized respectively in
3 mL of iced sterile saline. Tissue homogenate was serially diluted
10-fold and plated on MH agar. The resulting bacterial colonies
were determined after incubation for 24 h.
1280
mg/mL. The stock solution was diluted twice continuously
with sterile water and diluted 10 times with MH broth to give a
concentration range of 320.03 mg/mL. The amounts of bacteria were
added in each well was 5 ꢁ 105 CFU/mL. Three parallel experiments
were set for each compound concentration. The 96-well-plate was
incubated at 37 ꢂC for 18e24 h. The MIC values were recorded as
the lowest drug concentration that completely inhibits the visible
growth of test bacteria.
After recording the MIC value, the 96-well plate was placed at
37 ꢂC for 24 h, and the MBC values were determined by plating
The protocol for this study was reviewed and approved by the
Institutional Animal Care and Use Committee of the South China
Agricultural University.
100 mL aliquots from wells without visible growth onto the MH agar
plates. Then, the MH agar plate was further cultured at 37 ꢂC for
24 h, and the minimum concentration of the compound required to
reduce the bacterial count by more than 99.9% on the plate was
MBC.
4.5. Cytotoxicity assay
4.3.2. Constant concentration time-kill curves
The cytotoxicity of all target compounds was evaluated by the
traditional MTT assay as described in Ref. [28]. RAW 264.7 cells and
HepG2 cells were used in this experiment, respectively. The cells
were inoculated into 96-well plates at a density of 1.0 ꢁ 105 cells
per well. Then, the plates were incubated at 37 ꢂC for 24 h. The cells
Compounds 48 and 60 were screened according to MIC results.
The Timeekill curve experiments of the above two compounds
were performed in triplicate according to the previous report [26].
The logarithmic phase of MRSA ATCC 43300 was diluted to 106 CFU/
mL with MH broth. Compounds 48, 60 and tiamulin were added
into the bacteria solution with the final concentration of 1 ꢁ MIC,
2 ꢁ MIC, 4 ꢁ MIC, 8 ꢁ MIC, 16 ꢁ MIC, and 32 ꢁ MIC, respectively. All
were treated with these target pleuromutilin derivatives (4
and cultured for 16 h at 37 ꢂC. The cells were incubated with 100
well of MTT (0.5 mg/mL in PBS) and cultured for 4 h under 5% CO2
37 ꢂC. After the medium was removed, 150
L DMSO was added to
mg/mL)
mL/
samples were cultured at 37 ꢂC with shaking. Samples (100
m
L)
were taken from the mixtures at 0, 3, 6, 9 and 24 h and were
continuously diluted decuple with sterile saline. Then 25 L of the
diluted sample were plated on the sterile MH agar plate and
m
each well to dissolve the cells. After 30 min incubation, absorbance
at 490 nm was recorded using a microplate spectrophotometer
(BIO-TEK Instrument Inc., USA).
m
12

B. Li, Z. Zhang, J.-F. Zhang et al.
European Journal of Medicinal Chemistry 223 (2021) 113624
10.2174/138955712798868968.
[11] R. Shang, X. Pu, X. Xu, Z. Xin, C. Zhang, W. Guo, Y. Liu, J. Liang, Synthesis and
biological activities of novel pleuromutilin derivatives with a substituted
thiadiazole moiety as potent drug-resistant bacteria inhibitors, J. Med. Chem.
57 (2014) 5664e5678, https://doi.org/10.1021/jm500374c.
[12] Y.R. Lee, K.L. Jacobs, Leave it to lefamulin: a pleuromutilin treatment option in
community-acquired bacterial pneumonia, Drugs 79 (2019) 1867e1876,
https://doi.org/10.1007/s40265-019-01219-5.
[13] Y. Hirokawa, H. Kinoshita, T. Tanaka, T. Nakamura, K. Fujimoto, S. Kashimoto,
T. Kojima, S. Kato, Pleuromutilin derivatives having a purine ring. Part 3:
synthesis and antibacterial activity of novel compounds possessing a piper-
azine ring spacer, Bioorg. Med. Chem. Lett 19 (2009) 175e179, https://doi.org/
10.1016/j.bmcl.2008.10.127.
4.6. Development of resistance
The development of resistance test was performed as described
in the literature with some modifications [29]. The logarithmic
phase culutures of MRSA ATCC 43300 were diluted 1:1000 into
fresh MHB media with sub-MIC concentration (1/4 MIC) of com-
pound 60 or tiamulin. After stationary cultured at 37 ꢂC for 24 h, the
MIC of culture was determined by section 4.3 method. Meanwhile,
the freshly diluted MRSA in the broth medium was cultured in sub-
MIC concentration (1/4 MIC) of drugs for next passages. The process
was repeated for 8 passages.
[14] J. Luo, Q. Yang, Y. Yang, Y. Tang, Y. Liu, Design, synthesis, and structure-activity
relationship studies of novel pleuromutilin derivatives having a piperazine
ring, Chem. Biol. Drug Des. 88 (2016) 699e709, https://doi.org/10.1111/
cbdd.12799.
Declaration of competing interest
[15] Z. Zhang, Y. Huang, J. Luo, Z. Jin, Y. Liu, Y. Tang, Synthesis and antibacterial
activities of novel pleuromutilin derivatives bearing an aminothiophenol
moiety, Chem. Biol. Drug Des. 92 (2018) 1627e1637, https://doi.org/10.1111/
cbdd.13328.
[16] G. Zhang, Z. Zhang, K. Li, J. Liu, B. Li, Z. Jin, Y. Liu, Y. Tang, Design, synthesis and
biological evaluation of novel pleuromutilin derivatives containing piperazine
and 1,2,3-triazole linker, Bioorg. Chem. 105 (2020) 104398, https://doi.org/
10.1016/j.bioorg.2020.104398.
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgments
[17] X. Sun, Y. Tao, Y. Liu, B. Chen, Synthesis and biological activities of substituted
triazolethione Schiff base, Chin. J. Chem. 25 (2007) 1573e1576, https://
doi.org/10.1002/cjoc.200790290.
[18] B. Soni, M.S. Ranawat, R. Sharma, A. Bhandari, S. Sharma, Synthesis and
evaluation of some new benzothiazole derivatives as potential antimicrobial
agents, Eur. J. Med. Chem. 45 (2010) 2938e2942, https://doi.org/10.1016/
j.ejmech.2010.03.019.
This work was supported by the Guangdong Natural Science
Funds for Distinguished Young Scholar (No. 2019B151502002) and
the Guangdong Special Support Program innovation team (No.
2019BT02N054).
[19] Y. Ünver, K. Sancak, F. Çelik, E. Birinci, M. Küçük, S. Soylu, N.A. Burnaz, New
thiophene-1,2,4-triazole-5(3)-ones: highly bioactive thiosemicarbazides,
structures of Schiff bases and triazoleethiols, Eur. J. Med. Chem. 84 (2014)
639e650, https://doi.org/10.1016/j.ejmech.2014.01.014.
[20] A. Ameri, G. Khodarahmi, F. Hassanzadeh, H. Forootanfar, G.H. Hakimelahi,
Novel aldimine-type Schiff bases of 4-Amino-5-[(3,4,5-trimethoxyphenyl)
methyl]-1,2,4-triazole-3-thione/thiol: docking study, synthesis, biological
evaluation, and anti-tubulin activity, Arch. Pharm. (Weinheim) 349 (2016)
662e681, https://doi.org/10.1002/ardp.201600021.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113624.
References
[21] M. Gao, J. Zeng, X. Fang, J. Luo, Z. Jin, Y. Liu, Y. Tang, Design, synthesis and
antibacterial evaluation of novel pleuromutilin derivatives possessing piper-
azine linker, Eur. J. Med. Chem. 127 (2017) 286e295, https://doi.org/10.1016/
j.ejmech.2017.01.004.
[22] J. Cha, Y. Park, Y.S. Lee, G.T. Chung, In vitro biofilm formation and bactericidal
activities of methicillin-resistant Staphylococcus aureus clones prevalent in
Korea, Diagn. Microbiol. Infect. Dis. 70 (2011) 112e118, https://doi.org/
10.1016/j.diagmicrobio.2010.11.018.
[23] S.K. Kasiakou, K.R. Lawrence, N. Choulis, M.E. Falagas, Continuous versus
intermittent intravenous administration of antibacterials with time-
dependent action, Drugs 65 (2005) 2499e2511, https://doi.org/10.2165/
00003495-200565170-00006.
[24] F.J. Boswell, J.M. Andrews, R. Wise, Pharmacodynamic properties of far-
openem demonstrated by studies of time-kill kinetics and postantibiotic ef-
fect, J. Antimicrob. Chemother. 39 (1997) 415e418, https://doi.org/10.1093/
jac/39.3.415.
[25] A. Athamna, Vitro post-antibiotic effect of fluoroquinolones, macrolides,
-lactams, tetracyclines, vancomycin, clindamycin, linezolid, chloramphenicol,
quinupristin/dalfopristin and rifampicin on Bacillus anthracis, J. Antimicrob.
Chemother. 53 (2004) 609e615, https://doi.org/10.1093/jac/dkh130.
[26] Z. Jin, L. Wang, H. Gao, Y. Zhou, Y. Liu, Y. Tang, Design, synthesis and biological
evaluation of novel pleuromutilin derivatives possessing acetamine phenyl
linker, Eur. J. Med. Chem. 181 (2019) 111594, https://doi.org/10.1016/
j.ejmech.2019.111594.
[27] X. Zuo, X. Fang, Z. Zhang, Z. Jin, G. Xi, Y. Liu, Y. Tang, Antibacterial activity and
pharmacokinetic profile of a promising antibacterial agent: 22-(2-Amino-
phenylsulfanyl)-22-Deoxypleuromutilin, Molecules 25 (2020) 878, https://
doi.org/10.3390/molecules25040878.
[1] H.A. Grema, Methicillin resistant Staphylococcus aureus (MRSA): a review,
Adv. Anim. Vet. Sci. 3 (2015) 79e98, https://doi.org/10.14737/journal.aavs/
2015/3.2.79.98.
[2] S. Hamid, M.A. Bhat, I.A. Mir, A. Taku, G.A. Badroo, S. Nazki, A. Malik, Pheno-
typic and genotypic characterization of methicillin-resistant Staphylococcus
aureus from bovine mastitis, Vet. World 10 (2017) 363e367, https://doi.org/
10.14202/vetworld.2017.363-367.
[3] M. Morgan, Methicillin-resistant Staphylococcus aureus and animals: zoonosis
or humanosis? J. Antimicrob. Chemother. 62 (2008) 1181e1187, https://
doi.org/10.1093/jac/dkn405.
[4] S. Monecke, G. Coombs, A.C. Shore, D.C. Coleman, P. Akpaka, M. Borg, H. Chow,
M. Ip, L. Jatzwauk, D. Jonas, K. Kadlec, A. Kearns, F. Laurent, F.G. O'Brien,
J. Pearson, A. Ruppelt, S. Schwarz, E. Scicluna, P. Slickers, H.L. Tan, S. Weber,
R. Ehricht,
A field guide to pandemic, epidemic and sporadic clones of
methicillin-resistant Staphylococcus aureus, PloS One
https://doi.org/10.1371/journal.pone.0017936.
6 (2011), e17936,
[5] Y. Abe, K. Shigemura, H. Yoshida, M. Fujisawa, S. Arakawa, Risk factors for anti-
MRSA drug resistance, Int. J. Antimicrob. Agents 40 (2012) 423e426, https://
doi.org/10.1016/j.ijantimicag.2012.07.005.
[6] S. Stefani, D.R. Chung, J.A. Lindsay, A.W. Friedrich, A.M. Kearns, H. Westh,
F.M. MacKenzie, Meticillin-resistant Staphylococcus aureus (MRSA): global
epidemiology and harmonisation of typing methods, Int. J. Antimicrob. Agents
39 (2012) 273e282, https://doi.org/10.1016/j.ijantimicag.2011.09.030.
[7] F. Kavanagh, A. Hervey, W.J. Robbins, Antibiotic substances from basidiomy-
cetes: VIII. Pleurotus multilus (Fr.) sacc. And Pleurotus passeckerianus pilat,
Proc. Natl. Acad. Sci. Unit. States Am. 37 (1951) 570e574, https://doi.org/
10.1073/pnas.37.9.570.
[8] F. Kavanagh, A. Hervey, W.J. Robbins, Antibiotic substances from basidiomy-
cetes: IX. Drosophila subtarata. (Batsch ex Fr.) quel. Proc. Natl. Acad. Sci. Unit.
States Am. 38 (1952) 555e560, https://doi.org/10.1073/pnas.38.7.555.
[9] G. Hogenauer, The mode of action of pleuromutilin derivatives. Location and
properties of the pleuromutilin binding site on Escherichia coli ribosomes,
Eur. J. Biochem. 52 (1975) 93e98, https://doi.org/10.1111/j.1432-
1033.1975.tb03976.x.
[28] W. Wang, R.F. Yue, Z. Jin, L.M. He, R. Shen, D. Du, Y.Z. Tang, Efficiency com-
parison of apigenin-7-O-glucoside and trolox in antioxidative stress and anti-
inflammatory properties, J. Pharm. Pharmacol. 72 (2020) 1645e1656, https://
doi.org/10.1111/jphp.13347.
[29] S. Cui, D. Addla, C. Zhou, Novel 3-aminothiazolquinolones: design, synthesis,
bioactive evaluation, SARs, and preliminary antibacterial mechanism, J. Med.
Chem. 59 (2016) 4488e4510, https://doi.org/10.1021/acs.jmedchem.5b01678.
[10] Y. Tang, Y. Liu, J. Chen, Pleuromutilin and its derivatives-the lead compounds
for novel antibiotics, Mini Rev. Med. Chem. 12 (2012) 53, https://doi.org/
13