Design, synthesis, in vitro and in vivo evaluation against MRSA and molecular docking studies of novel pleuromutilin derivatives bearing 1, 3, 4-oxadiazole linker

Article abstract of DOI:10.1016/j.bioorg.2021.104956

A class of pleuromutilin derivatives containing 1, 3, 4-oxadiazole were designed and synthesized as potential antibacterial agents against Methicillin-resistant staphylococcus aureus (MRSA). The ultrasound-assisted reaction was proposed as a green chemistry method to synthesize 1, 3, 4-oxadiazole derivatives (intermediates 85–110). Among these pleuromutilin derivatives, compound 133 was found to be the strongest antibacterial derivative against MRSA (MIC = 0.125 μg/mL). Furthermore, the result of the time-kill curves displayed that compound 133 could inhibit the growth of MRSA in vitro quickly (- 4.36 log10 CFU/mL reduction). Then, compound 133 (- 1.82 log₁₀ CFU/mL) displayed superior in vivo antibacterial efficacy than tiamulin (- 0.82 log₁₀ CFU/mL) in reducing MRSA load in mice thigh model. Besides, compound 133 exhibited low cytotoxicity to RAW 264.7 cells. Molecular docking studies revealed that compound 133 was successfully localized in the binding pocket of 50S ribosomal subunit (ΔG_b = -10.50 kcal/mol). The results indicated that these pleuromutilin derivatives containing 1, 3, 4-oxadiazole might be further developed into novel antibiotics against MRSA.

Full text of DOI:10.1016/j.bioorg.2021.104956

Bioorganic Chemistry 112 (2021) 104956
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis, in vitro and in vivo evaluation against MRSA and
molecular docking studies of novel pleuromutilin derivatives bearing 1, 3,
4-oxadiazole linker
Jie Liua, Guang-Yu Zhanga, Zhe Zhanga, Bo Lia, Fei Chaia, Qi Wanga, Zi-Dan Zhoua,
,
b
,
,b,*
Ling-Ling Xua, Shou-Kai Wanga, Zhen Jina *, You-Zhi Tanga
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
Keywords:
A class of pleuromutilin derivatives containing 1, 3, 4-oxadiazole were designed and synthesized as potential
antibacterial agents against Methicillin-resistant staphylococcus aureus (MRSA). The ultrasound-assisted reaction
was proposed as a green chemistry method to synthesize 1, 3, 4-oxadiazole derivatives (intermediates 85–110).
Among these pleuromutilin derivatives, compound 133 was found to be the strongest antibacterial derivative
Pleuromutilin
1, 3, 4-Oxadiazole
Molecular docking
MRSA
against MRSA (MIC = 0.125 μg/mL). Furthermore, the result of the time-kill curves displayed that compound
Antibacterial activity
133 could inhibit the growth of MRSA in vitro quickly (- 4.36 log10 CFU/mL reduction). Then, compound 133 (-
1.82 log10 CFU/mL) displayed superior in vivo antibacterial efficacy than tiamulin (- 0.82 log10 CFU/mL) in
reducing MRSA load in mice thigh model. Besides, compound 133 exhibited low cytotoxicity to RAW 264.7 cells.
Molecular docking studies revealed that compound 133 was successfully localized in the binding pocket of 50S
ribosomal subunit (ΔGb = -10.50 kcal/mol). The results indicated that these pleuromutilin derivatives containing
1, 3, 4-oxadiazole might be further developed into novel antibiotics against MRSA.
1. Introduction
potent towards Gram-positive bacteria and Mycoplasma. Pleuromutilin
exhibited antibacterial activity through binding to the V domain of the
Methicillin-resistant staphylococcus aureus (MRSA) is one of the most
important community-acquired (CA) and hospital-acquired (HA) path-
ogens [1]. MRSA caused severe skin and soft-tissue infections but on
occasion causing life-threatening infections such as necrotizing pneu-
monia [2]. The annual cost of treating hospital acquired-MRSA was
estimated to be $ 9.7 billion in the United States [3]. Vancomycin is one
of the main drugs in the treatment for MRSA infection. However, it was
reported that MRSA might acquire the vanA gene cluster during anti-
biotic therapy and became resistant to vancomycin [2]. The prevalence
of MRSA has created a critical demand for the development of novel
antimicrobial agents [4].
peptidyl transferase center (PTC) of bacterial 50S ribosomal subunit
[7,8]. For the unique mechanism, pleuromutilin rarely exhibit cross-
resistance with other antibiotics. Thus, pleuromutilin has aroused
considerable research. Most modifications focus on the C14 side chain of
pleuromutilin [9]. These modifications have led to the approvement of
tiamulin (2, Fig. 1) and valnemulin (3, Fig. 1) which were authorized as
veterinary medicines in 1979 and 1999, respectively [10]. As the first
pleuromutilin drug used in human medicine, retapamulin (4, Fig. 1) has
been approved by the U.S. Food and Drug Administration (FDA) [11].
Lefamulin (5, Fig. 1) was approved by the FDA for treatment of
community-acquired bacterial pneumonia (CABP) in humans in 2019
[12]. Success of lefamulin attracted researchers to further focus the
development of pleuromutilin drugs.
Pleuromutilin, a natural product, was isolated from Pleurotus mutilus
and Pleurotus passeckerianus in 1951 [5]. It is composed of the tricyclic
core (five-, six- and eight-membered rings), a C14 extension and a C21
keto group [6]. The pleuromutilin exhibited especially antibacterial
Previous work in our laboratory has led to several semisynthetic
pleuromutilin derivatives with potent antimicrobial activity against
* Corresponding authors at: College of Veterinary Medicine, South China Agricultural University, No. 483 Wushan Road, Tianhe District, Guangzhou 510642,
China.
E-mail addresses: jinzhenhami@scau.edu.cn (Z. Jin), youzhitang@scau.edu.cn (Y.-Z. Tang).
https://doi.org/10.1016/j.bioorg.2021.104956
Received 17 February 2021; Received in revised form 22 April 2021; Accepted 27 April 2021
Available online 1 May 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

J. Liu et al.
Bioorganic Chemistry 112 (2021) 104956
MRSA [13]. One of these pleuromutilin derivatives, with five-membered
heterocyclic such as 1,2,3-triazole group in the C14 side chain [13]. The
series of compounds possessed superior in vivo efficacy to that of tia-
mulin in MRSA thigh infection model. 1, 3, 4-Oxadiazole is a hetero-
cyclic compound containing an oxygen atom and two nitrogen atoms in
(compounds 33–58, 59–84, 85–110) were disclosed in Table SI 1.
All pleuromutilin derivatives were purified by silica column chro-
matography. The structures of synthesized compounds were character-
1
ized by H NMR, 13C NMR and high-resolution mass spectral (HRMS)
analysis. All the spectra of synthesized pleuromutilin derivatives are
supplied in the Supporting Information.
the five-membered ring. Owing to the π-conjugated system, hydrogen-
bonding capacity, and rigidity, 1, 3, 4-oxadiazole possesses high affin-
ity and acts as important pharmacophores by interacting with the bio-
logical receptors [14,15]. The antibacterial activity of norfloxacin
against MRSA could be increased through the introduction of 1, 3, 4-oxa-
diazole and the target compound (6, Fig. 1) also displayed very low
cytotoxicity [16]. Thus, we speculated that the introduction of 1, 3, 4-
oxadiazole to C14 side chain of pleuromutilin might produce new de-
rivatives with enhanced antibacterial activity compared to the prototype
pleuromutilin.
2.2. In vitro antibacterial activity
The in vitro antibacterial activity of these pleuromutilin derivatives
containing 1, 3, 4-oxadiazole was evaluated against MRSA (ATCC
43300) and three S. aureus strains in accordance with the Clinical and
Laboratory Standards Institute (CLSI)[19]. The results were displayed as
the minimum inhibitory concentration (MIC) and the minimum bacte-
ricidal concentration (MBC) in Table 1. The pleuromutilin and tiamulin
were used as the positive control drugs.
In this study, a novel process for the synthesis of 5-substituted 1, 3, 4-
oxadiazol-2-thiol derivatives was proposed via ultrasound-assisted re-
action in the absence of basic or acidic. It is aligned with the principles of
green chemistry. Then, 26 pleuromutilin derivatives were designed,
synthesized and evaluated for their in vitro and in vivo antibacterial ac-
tivities against four strains including MRSA.
As illustrated in Table 1, most target compounds displayed stronger
antibacterial activity than pleuromutilin or tiamulin against MRSA and
S. aureus. The MIC values of 11 compounds are 0.125 μg/mL against
MRSA. The MBC/MIC ratios of most target compounds against MRSA
were ≤4. According to previous study, the antimicrobial drug could be
considered bacteriostatic when the MBC/MIC is ≥4, while being
bactericidal when the MBC/MIC is ≤4 [20]. Thus, the pleuromutilin
derivatives containing 1, 3, 4-oxadiazole displayed potent and stable
bactericidal effect.
2. Results and discussion
2.1. Chemistry
Meanwhile, the preliminary structure–activity relationships (SARs)
study was also explored. Among compounds 113–137, all the com-
pounds showed better antibacterial activity than compound 112.
Consistent with previous research [21], the results suggested that de-
rivatives containing aromatic rings showed stronger antibacterial ac-
tivity. The reason might be related to the exaltation of the binding ability
between the C14 side and the hydrophobic portion of ribosomal subunits
by aromatic rings. Then different electron-withdrawing and electron-
donating groups were introduced on the benzene ring. When the
hydrogen atom on the benzene ring was substituted by hydroxyl or nitro
group, the compounds 120–122 were obtained. Their MIC values
The synthesis methods of all 1, 3, 4-oxadiazole derivatives and
pleuromutilin derivatives were displayed in Scheme 1. All the syntheses
of pleuromutilin derivatives begun with 22-O-tosylpleuromutilin
(compound 111), which was obtained by the reaction of pleuromutilin
and p-toluenesulfonyl chloride [17].
As illustrated in Scheme 1, the intermediates 85–110 were prepared
in 3 steps starting from carboxylic acid derivatives (compounds 33–58).
First, different carboxylic acid reacted with EtOH to get the ethyl
carboxylate (compounds 33–58), respectively. These esters were
hydrazinolysised in EtOH to obtain carboxyhydrazide (compounds
59–84) under reflux. The compounds 85–110 were synthesized in the
absence of basic or acidic via ultrasound-assisted reaction. Compounds
85–110 were produced in good to excellent yields under easy workup
and purification conditions [18]. The target compounds 112–137 were
prepared through reaction of the compounds 85–110 with compound
111 by a nucleophilic substitution reaction, respectively. Structures of
starting materials (compounds 7–32) and corresponding intermediates
against MRSA were 0.125
μg/mL. Overall, the stronger electron-
withdrawing or electron-donating ability of introduced groups, the
better antibacterial activity of compounds. Increasing electron cloud
density might change the physical and chemical properties of the com-
pound molecules, which influenced pharmacological properties of target
compound [22]. When the benzene ring on the 1, 3, 4-oxadiazole was
substituted by thiophene group, the corresponding compound 133
Fig. 1. Structure of pleuromutilin (1), tiamulin (2), valnemulin (3), retapamulin (4), lefamulin (5) and norfloxacin (6).
2

J. Liu et al.
Bioorganic Chemistry 112 (2021) 104956
Scheme 1. Reagent and conditions: (i) ethanol, Concentrated sulfuric acid, 78 ℃, 2 h; (ii) ethanol, hydrazine,hydrate (1:1), 78 ℃, 6 h; (iii) DMF, CS2, ultrasound, 10
h; (iv) acetonitrile, p-toluenesulfonyl chloride, NaOH, rt, 3 h; (v) a). acetonitrile, NaI, 70 ℃, 1.5 h; b). acetonitrile, K2CO3, 70 ℃, 3 h.
showed the most potent anti-MRSA activity among compounds
112–133. The results might be related to the higher electron cloud
density of the thiophene ring than that of the benzene ring.
The time-kill kinetics assay was used to analyze the antibacterial
activity of compound 118 and compound 133 against MRSA. The results
are displayed in Fig. 2.
against MRSA in thigh muscle (P < 0.01, n = 6/group), and the exper-
imental group with compound 133 at 20 mg/kg performed a treatment
effect (ꢀ 1.82 log10 CFU/mL) in thigh (P < 0.01, n = 6/group).
Comparing the clearance of bacteria in thigh muscle, compound 133
possessed more efficient antibacterial activity than tiamulin in reduce
the MRSA load of thigh infected mice. It suggested compound 133 could
do therapy MRSA infection in vivo initially.
In a concentration of 2 × MIC, compounds 118 and 133 had a sig-
nificant inhibitory effect on MRSA. Compound 133 at 2 × MIC had
induced killing of MRSA (- 1.28 log10 CFU/mL reduction) after 3 h of
incubation, and conspicuous bacterial growth inhibition was observed
at 24 h (- 4.36 log10 CFU/mL reduction). 99.9% of MRSA was killed by 2
× MIC compound 133 at 24 h. In contrast, some colonies were also
detected after incubation for 24 h in the 4 × MIC tiamulin (the total
bacterial ꢀ 1.90 log10 CFU/mL) [22]. The further increases in the con-
centration of the compound 133 to 4 × MIC did not further increase the
antibacterial effect. According to time/concentration-killing bacteria
relationships, compounds 118 and 133 can be divided into time-
dependent groups, with a bactericidal effect at concentrations higher
than 2 × MIC.
2.4. Cytotoxicity assay
The presence of compounds can affect cellular basic physiological
processes, inhibited proliferation, even reduce cell survival, etc.
Macrophage generally exhibited microbicidal activity and was an
important line of defense against invading microorganism [23]. There-
fore, cytotoxicity was evaluated in order to explore the effect of com-
pounds 112–133 on the macrophage viability [24]. The cytotoxicity of
compounds 112–133 to RAW 264.7 cells was evaluated by MTT assay
[25,26]. Most compounds displayed slight inhibition of the viability of
RAW 264.7 cells at the concentration of 8.0 µg/mL and the result was
shown in Figure SI 27. The cytotoxicity of compounds 118, 133 and
tiamulin was measured by MTT assay within different concentrations
(Fig. 5). The cytotoxicity of compound was usually expressed as the IC50
value [27] (the concentration at which 50% of the cells are viable). The
PAE tests were conducted for compounds 118 and 133 to explore the
potential of the measured compounds for continuous inhibition of bac-
teria and provide a more rational dosing regimen. The bacterial growth
kinetics curve was displayed in Fig. 3 and the results of the PAEs are
tabulated in Table 2. Following exposure to 2 × MIC and 4 × MIC for 2 h,
the PAE of compound 118 was respectively 3.66 and 4.01 h, and the PAE
at the same concentrations of compound 133 was 3.42 and 3.74 h.
Correlatively, the PAE at the same concentrations of tiamulin was 1.65
and 2.04 h. The results indicated that the administration interval of
compounds 118 and 133 might be longer than that of tiamulin. The
results are displayed in Fig. 3.
IC50 values for compounds 118, 133 were calculated to be 16.41
μg/mL
and 17.88 g/mL, which were higher than that of tiamulin with 9.684
μ
μ
g/mL, respectively. The present study indicated that the compounds
118, 133 were less cytotoxic than tiamulin towards RAW 264.7 cells in
our system. The safety profiles of compounds 118, 133 were more
reliable than that of tiamulin.
2.5. Molecular docking study
2.3. In vivo antibacterial activity
Based on the above theoretical and experimental findings, molecular
docking experiments were carried out to predict the possible binding
mode of compound 133 to 50S ribosomes (PDB: 1XBP) [7]. Through
Autodock software, the redocking of tiamulin into 1XBP placed the
ligand in the same conformation as that in the X-ray structure (RMSD =
0.781). The results for compound 133 present a similar binding mode to
tiamulin and are displayed in Fig. 6a, which presented a superimposition
of 2 kinds of docked compounds.
Due to excellent in vitro antibacterial activity, the in vivo antibacterial
activity of compound 133 was further assessed by neutropenic murine
thigh infection model. The infection model was established by injecting
MRSA solution about 107 CFU/mL into the thigh muscle of neutropenic
mice. The mice were randomly divided into three groups and intrave-
nously injected sterile normal saline (as a negative control), compound
133 (20 mg/kg) and tiamulin (20 mg/kg), respectively. The results are
indicated in Fig. 4.
The binding free energy of compound 133 with the 50S ribosome
was calculated to be ꢀ 10.5 kcal/mol. As shown in Fig. 6b, four hydrogen
bonds played an important part in the binding of compound 133 to 50S
Compared to the negative control group, the positive control of tia-
mulin at 20 mg/kg could reduce MRSA load (- 0.85 log10 CFU/mL)
3

J. Liu et al.
Bioorganic Chemistry 112 (2021) 104956
Table 1
MIC and MBC (
(144).
μ
g/mL) values of compounds 112–137, pleuromulin and tiamulin against S. aureus (ATCC 43300), S. aureus (ATCC 29213), S. aureus (AD3) and S. aureus
Compound No
R
MIC/MBC(μg/mL)
MRSA ATCC 43300
S. aureus ATCC 29213
S. aureus AD3
S. aureus 144
112
113
-H
16/16
8/16
1/1
16/>16
16/>16
16/>16
0.25/1
2/8
114
1/1
16/16
16/16
16/>16
115
116
117
0.125/0.25
0.5/1
0.5/2
8/16
1/2
1/8
4/16
16/16
1/4
16/>16
8/16
0.25/0.25
118
119
0.125/0.125
0.125/0.5
0.25/1
0.5/2
0.125/0.5
0.5/8
0.25/2
2/4
120
121
0.125/0.125
0.125/0.5
0.5/1
0.5/2
0.5/4
0.5/4
8/16
8/16
122
123
124
0.125/0.125
0.25/0.5
0.25/1
0.5/2
0.5/2
0.125/1
0.5/4
0.5/2
8/16
2/4
0.125/0.25
0.5/8
(continued on next page)
4

J. Liu et al.
Bioorganic Chemistry 112 (2021) 104956
Table 1 (continued)
Compound No
R
MIC/MBC(μg/mL)
MRSA ATCC 43300
0.125/0.25
S. aureus ATCC 29213
S. aureus AD3
S. aureus 144
125
126
127
0.25/2
0.25/2
1/2
1/4
8/16
8/16
8/16
16/>16
16/16
16/>16
16/>16
128
129
130
131
0.25/1
16/16
16/16
>16/>16
8/16
16/16
16/>16
0.25/0.5
>16/>16
>16/>16
16/>16
8/16
>16/>16
16/>16
16/>16
4/8
132
133
134
0.125/0.25
0.125/0.125
0.25/0.5
0.25/1
0.5/2
0.5/1
0.25/2
0.5/1
0.5/4
0.25/0.5
1/2
1/4
135
136
137
2/4
8/16
0.5/2
0.5/2
8/16
0.5/2
1/1
16/>16
0.5/2
1/4
0.125/0.25
0.25/0.25
Pleuromutilin
Tiamulin
/
/
2/4
2/4
1/1
2/4
1/2
2/4
1/2
0.5/1
ribosome. A hydrogen bond (distance: 3.3 Å) was formed between the
ketone on the C3 atom of compound 133 (pleuromutilin core) and the
residue of U2485. A hydrogen bond (distance: 2.6 Å) was constituted by
the hydroxyl group on the C11 atom of compound 133 (pleuromutilin
core) and the residue of G2484. A hydrogen bond (distance: 3.4 Å) was
formed between the ketone on the C21 core and the residue of G2044.
The other hydrogen bond was constituted by 1, 3, 4-oxadiazole of
compound 133 and the residues of G2484 (distance: 3.4 Å). The results
suggested that substituted oxadiazole rings might be acted as hydrogen
bond acceptors forming hydrogen bonds with 50S ribosome residue to
improve antibacterial activity [28]. The interaction of compound 133
with 50S ribosome should be further studied in our future work.
3. Conclusions
In summary, a series of novel pleuromutilin derivatives containing 1,
3, 4-oxadiazole have been prepared and evaluated for their antimicro-
bial effects against MRSA and S. aureus. The results indicated that
5

J. Liu et al.
Bioorganic Chemistry 112 (2021) 104956
Fig. 2. Time-kill curves for MRSA ATCC 43300 with different concentrations of tiamulin and compounds 118 (a) and 133 (b).
Fig. 3. The bacterial growth kinetic curves for MRSA ATCC 43300 exposed to compound 118 (a) and 133 (b) for 2 h.
6

J. Liu et al.
Bioorganic Chemistry 112 (2021) 104956
Table 2
The PAEs values of compounds 118, 133 and tiamulin against MRSA ATCC
43300.
Compounds
Concentrations
PAE (h)
Exposure for 2 h
Compound 118
Compound 133
Tiamulin
2 × MIC
4 × MIC
2 × MIC
4 × MIC
2 × MIC
4 × MIC
3.66
4.01
3.42
3.74
1.65
2.04
Fig. 4. Efficacy of tiamulin and compound 133 against MRSA ATCC 43300 in
murine neutropenic thigh models: circular: growth control; square: tiamulin
(20 mg/kg); triangle: compound 133 (20 mg/kg).
Fig. 6. (a) Docking mode of tiamulin (purple) and compound 133 (yellow) to
1XBP. (b) 3D representation of docking poses for the compound 133 in the 50S
ribosome residues. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
4. Experimental
4.1. Materials
Pleuromutilin (>90% pure) was purchased from Great Enjoyhood
Biochemical Co. Ltd., (Daying, China). The carboxylic acids were pur-
chased from TiTan Technology Co. Ltd., (Shanghai, China). The other
analytical grade solvents were purchased from Guangzhou General Re-
agent Factory (Guangzhou, China). The target compounds were purified
by silica gel column chromatography (200–300 mesh, Branch of Qing-
dao Haiyang Chemical Co. Ltd., Shandong, China). 1H NMR and 13C
NMR spectra were processed with Bruker AV-600 spectrometer in
chloroform-d using trimethylsilane as an internal standard. Chemical
shift values (δ) were reported in ppm. High-resolution mass spectra were
recorded by Thermo Scientific Q Exactive Focus Orbitrap LC-MS/MS
with an electrospray ionization (ESI) source. Ultrasonic irradiation
was supplied by Backer vCLEAN1-L03 (35 kHz frequency and 50 W
output power).
Fig. 5. The cytotoxicity of compounds 118, 133 and tiamulin against RAW
264.7 cells was determined using different concentrations of compound113
and tiamulin.
compounds 118 and 133 displayed an excellent antimicrobial effect
against MRSA. The results of the time-killing curve indicated that
compounds 118 and 133 manifested a more rapid bactericidal kinetic
effect on MRSA than tiamulin. Compound 133 displayed a longer PAE
time than tiamulin against MRSA. Compound 133 also exhibited a
potent in vivo antibacterial effect than tiamulin in MRSA infection mice
thigh models. Four strong hydrogen bonds were formed through the
interaction of compound 133 with residues. In addition, compound 133
displayed lower cytotoxicity than tiamulin. Compound 133 could be
served as a possible lead compound for the development of antibacterial
drugs and is worthy of further clinical trials for the systematic treatment
of infection.
4.2. Synthesis
A general synthetic route based on the compound 22-O-tosylpleuro-
mutilin (compound 111) and a variety of 2-mercapto-1, 3, 4-oxadiazole
derivatives (compound 85–110) were used to prepare pleuromutilin
derivatives (Scheme 1).
The starting materials ethyl carboxylates (compound 33–58), car-
boxyhydrazides (compound 59–84), 22-O-tosylpleuromutilin (com-
pound 111) were prepared according to the literature.
7

J. Liu et al.
Bioorganic Chemistry 112 (2021) 104956
4.2.1. General procedure for the synthesis of 2-mercapto-1, 3, 4-oxadiazole
derivatives (compounds 85–110)
(3H, s, H22, H11), 2.32 (2H, td, J = 12.8, 12.0, 5.9 Hz, H2, H4), 2.25
(1H, d, J = 9.6 Hz, H10), 2.19 (1H, dd, J = 19.4, 9.4 Hz), 2.12 – 2.07
(2H, m, H13), 1.77 (2H, dd, J = 14.5, 3.0 Hz, H6, H8), 1.67 (1H, d, J =
3.2 Hz, H1), 1.65 – 1.64 (1H, m, H7), 1.54 (1H, s), 1.48 (1H, s), 1.45 (3H,
s, H13), 1.39 (1H, d, J = 7.0 Hz, H15), 1.17 (3H, s, H8), 1.14 – 1.11 (1H,
m, H18), 0.89 (3H, d, J = 7.0, H17), 0.76 (3H, d, J = 7.1, H16). 13C NMR
(151 MHz, Chloroform-d) δ 216.69(C3), 166.26(C21), 166.14, 162.32,
138.72(C19), 138.33, 131.73, 131.27, 128.75, 126.15, 122.54, 117.33
(C20), 74.60(C11), 70.89(C14), 58.07(C4), 45.42(C9), 44.63(C13),
44.00(C12), 41.90(C5), 36.67(C6), 36.03(C10), 35.02, 34.41(C2), 30.39
(C8), 26.84(C7), 26.39(C18), 24.82(C1), 22.01, 16.76(C16), 14.78
(C15), 11.44(C17); HR-MS (ESI): Calcd for C31H40N2O5S (M+H+):
553.2736; Found: 553.2721.
Carbon disulfide (50 mmol 4.7 mL) was added dropwise to the sus-
pension including carboxyhydrazides (compound 59–84, 50 mmol) in
DMF (5 mL) processed to an ultrasonic bath for 10 h. Hydrogen sulfide
emission was treated with a saturated solution of sodium bicarbonate.
The reaction mixture was added to 20 g crushed ice containing excess of
salt. The solid phase was filtered with Buchner funnel, washed with
water, and dried to obtain compound 85–110, respectively.
4.2.2. 22-[(1, 3, 4-oxadiazol-5-yl) thio] deoxy pleuromutilin (compound
112)
Compound 6 (1 g, 2.16 mmol) was dissolved in acetonitrile (10 mL)
and sodium iodide (0.35 g, 2.37 mmol) was added and stirred at 70 ℃
for 0.5 h. 2-mercapto-1, 3, 4-oxadiazole (0.24 g, 2.37 mmol) and po-
tassium carbonate (0.60 g, 4.32 mmol) were added to the mixture and
stirred again for 2 h at 70 ℃. Chloroform (30 mL) was added, and the
mixture was then washed with water (30 mL). The organic layer was
dried over anhydrous Na2SO4, filtered and concentrated under reduced
pressure to give the crude product. The crude product was concentrated
in vacuo to remove the trifluoroacetic acid and yield the product, which
was purified by column chromatography (dichloromethane: methanol
= 70: 1) using silica gel to give the desired compound.
4.2.5. 22-[(3-Methylphenyl-1, 3, 4-oxadiazol-5-yl) thio] deoxy
pleuromutilin (compound 115)
White powder; yield: 72%; 1H NMR (600 MHz, Chloroform-d) δ 7.83
– 7.78 (2H, m), 7.39 – 7.34 (2H, m), 6.43 (1H, dd, J = 17.4, 11.0 Hz,
H19), 5.79 (1H, d, J = 8.5 Hz, H14), 5.34 – 5.30 (1H, m, H20), 5.18 (1H,
dd, J = 17.4, 1.5 Hz, H20), 4.05 (2H, d, J = 2.5 Hz, H22), 3.38 – 3.34
(1H, m, H11), 2.43 (3H, s), 2.32 (1H, t, J = 6.9 Hz, H2), 2.26 – 2.23 (1H,
m, H4), 2.18 (1H, dd, J = 19.5, 9.4 Hz, H10), 2.09 (2H, d, J = 10.8 Hz,
H13, 11-OH), 1.79 – 1.76 (1H, m, H6), 1.73 (1H, s, H8), 1.71 – 1.66 (2H,
m), 1.66 – 1.63 (1H, m, H1), 1.54 (1H, dd, J = 13.4, 2.9 Hz, H7), 1.50
(1H, d, J = 10.5 Hz, H13), 1.45 (3H, s), 1.39 (1H, d, J = 3.1 Hz, H15),
1.37 (1H, d, J = 5.1 Hz, H18), 1.16 (3H, s, H18), 0.88 (3H, d, J = 7.1,
H17), 0.76 (3H, d, J = 7.1, H16). 13C NMR (151 MHz, Chloroform-d) δ
216.2(C3), 166.21(C21), 166.11, 162.57, 138.96(C19), 138.71, 132.60,
128.95, 127.20, 123.87, 123.32, 117.34(C20), 74.60(C14), 70.90(C14),
58.07(C4), 45.42(C9), 44.64(C13), 44.00(C12), 41.90(C5), 36.66(C6),
36.02(C10), 35.07, 34.41(C2), 30.39(C8), 26.83(C7), 26.41(C18), 24.82
(C1), 21.30, 16.75(C16), 14.77(C15), 11.44(C17); HR-MS (ESI): Calcd
for C31H40N2O5S (M+H+): 553.2736; Found: 553.2721.
White powder; yield: 58%; 1H NMR (600 MHz, Chloroform-d) δ 6.41
(1H, dd, J = 17.4, 11.0 Hz, H19), 5.77 (1H, d, J = 8.5 Hz), 5.31 – 5.30
(1H, m, H14), 5.20 – 5.17 (1H, m, H20), 4.08 – 3.96 (2H, m, H20, H22),
3.36 (1H, dd, J = 10.4, 6.6 Hz, H11), 2.32 (1H, q, J = 6.9 Hz, H2), 2.26
(1H, dd, J = 19.5, 11.0 Hz, H4), 2.20 (1H, q, J = 9.7 Hz), 2.10 (1H, s,
H10), 2.06 (1H, dd, J = 16.0, 8.6 Hz, H13), 1.77 (1H, dd, J = 14.4, 2.5
Hz, H6), 1.68 (1H, s, H8), 1.67 – 1.62 (2H, m, 11-OH), 1.58 – 1.52 (1H,
m), 1.49 (1H, q, J = 4.0 Hz), 1.46 (1H, d, J = 6.6 Hz, H1, H7), 1.44 (3H,
s, H13), 1.39 (1H, s), 1.36 (1H, s, H15), 1.18 (3H, s, H8), 1.13 (1H, td, J
= 14.1, 4.3 Hz, H18), 0.88 (3H, d, J = 7.0 Hz, H17), 0.73 (3H, d, J = 7.1
Hz, H16). 13C NMR (151 MHz, Chloroform-d) δ 216.76(C3), 166.38
(C21), 163.63, 138.809(C19), 117.24(C20), 74.62(C11), 70.65(C14),
58.08(C4), 53.41(C22), 45.42(C9), 44.59(C13), 43.98(C5), 41.86,
36.69, 36.26(C6), 36.02(C10), 34.42(C2), 30.39(C8), 26.84(C7), 26.44
(C18), 24.82(C1), 16.75(C16), 14.82(C15), 11.44(C17); HR-MS (ESI):
Calcd for C24H34N2O5S (M+K+): 501.1820; Found: 501.1842.
4.2.6. 22-[(4-Methylphenyl-1, 3, 4-oxadiazol-5-yl) thio] deoxy
pleuromutilin (compound 116)
White powder; yield: 71%; 1H NMR (600 MHz, Chloroform-d) δ 7.87
(1H, d, J = 1.6 Hz), 7.30 (1H, d, J = 0.6 Hz), 7.29 (1H, s), 6.42 (1H, dd, J
= 17.4, 11.0 Hz, H19), 5.78 (1H, d, J = 8.5 Hz, H14), 5.32 – 5.30 (1H, m,
H20), 5.18 (1H, dd, J = 17.4, 1.5 Hz, H20), 4.10 – 3.97 (2H, m), 3.37 –
3.34 (1H, m, H22), 2.42 (1H, s, H2), 2.34 – 2.28 (1H, m, H4), 2.24 (1H,
dd, J = 11.1, 1.4 Hz, H10), 2.18 (1H, dd, J = 19.5, 9.4, H13), 2.10 (1H,
s), 2.09 – 2.04 (1H, m,11-OH), 1.79 – 1.76 (1H, m, H6), 1.76 (1H, d, J =
2.6 Hz, H8), 1.69 – 1.61 (3H, m, H1, H7), 1.57 – 1.52 (1H, m), 1.51 (1H,
d, J = 10.5 Hz, H13), 1.46 (1H, dd, J = 9.9, 3.1 Hz), 1.44 (3H, s), 1.39
(1H, d, J = 3.5 Hz, H15), 1.36 (1H, d, J = 5.5 Hz, H8), 1.16 (3H, s), 1.15
– 1.09 (1H, m, H18), 0.88 (3H, d, J = 7.1 Hz, H17), 0.75 (3H, d, J = 7.1
Hz, H16). 13C NMR (151 MHz, Chloroform-d) δ 216.72(C3), 166.21
(C21), 166.13, 162.30, 142.36, 138.71(C19), 129.75, 126.67, 120.68,
117.33(C20), 74.59(C11), 70.88(C14), 58.07(C4), 45.42(C9), 44.64
(C13), 44.00(C12), 41.90(C5), 38.02, 36.66(C6), 36.02(C9), 35.09
(C10), 34.41(C2), 30.39(C8), 29.17, 26.83(C7), 26.41(C18), 24.81(C1),
21.61, 16.75(C16), 14.77(C15), 11.44(C17); HR-MS (ESI): Calcd for
4.2.3. 22-[(phenyl-1, 3, 4-oxadiazol-5-yl) thio] deoxy pleuromutilin
(compound 113)
White powder; yield: 72%; 1H NMR (600 MHz, Chloroform-d) δ 7.53
– 7.49 (3H, m), 6.43 (1H, dd, J = 17.4, 11.0 Hz, H19), 5.79 (1H, d, J =
8.5 Hz, H14), 5.33 – 5.30 (1H, m, H20), 5.19 (1H, dd, J = 17.4, 1.5 Hz,
H20), 4.05 (2H, d, J = 3.0 Hz), 3.36 (1H, d, J = 6.2 Hz, H22), 2.34 – 2.29
(1H, m, H2), 2.26 – 2.23 (1H, m, H4), 2.20 (1H, q, J = 9.7 Hz, H10), 2.10
(1H, s, H13), 1.79 – 1.76 (1H, m, H6), 1.68 (2H, ddd, J = 9.6, 7.1, 3.5,
H8), 1.66 – 1.62 (1H, m, 11-OH), 1.56 – 1.50 (2H, m, H1, H7), 1.49 –
1.46 (1H, m, H13), 1.45 (3H, s), 1.39 (1H, d, J = 3.1 Hz, H15), 1.37 (1H,
d, J = 5.2 Hz, H8), 1.16 (3H, s), 1.13 (1H, dd, J = 14.2, 9.8 Hz, H18),
0.88 (3H, d, J = 7.1 Hz, H17), 0.76 (3H, d, J = 7.1 Hz, H16). 13C NMR
(151 MHz, Chloroform-d) δ 216.69(C3), 166.09(C21), 166.05, 162.72,
138.70(C19), 131.77, 129.06, 126.71, 123.47, 117.35(C20), 109.11,
74.59(C11), 70.91(C14), 58.06(C4), 45.42(C9), 44.64(C13), 44.00
(C12), 41.90(C5), 36.66(C6), 36.03(C10), 35.08, 34.41(C2), 30.39(C8),
26.83(C7), 26.40(C18), 24.82(C1), 23.38, 16.76(C16), 14.77(C15),
11.44.(C17); HR-MS (ESI): Calcd for C30H38N2O5S (M+Na+): 561.2393;
Found: 561.2381.
C
31H40N2O5S (M+H+): 553.2736; Found: 553.2720.
4.2.7. 22-[(2-aminophenyl-1, 3, 4-oxadiazol-5-yl) thio] deoxy
pleuromutilin (compound 117)
White powder; yield: 74%; 1H NMR (600 MHz, Chloroform-d) δ 7.27
– 7.25 (1H, m), 6.81 – 6.73 (3H, m), 6.43 (1H, dd, J = 17.4, 11.0 Hz,
H19), 5.80 (1H, d, J = 8.5 Hz, H14), 5.34 – 5.31 (1H, m, H20), 5.20 –
5.17 (1H, m, H20), 4.08 – 3.99 (2H, m), 3.36 (1H, d, J = 6.3 Hz, H22),
2.33 (1H, q, J = 6.9 Hz, H2), 2.19 (1H, dd, J = 19.5, 9.4 Hz, H4), 2.09
(2H, d, J = 11.9 Hz, H10, H13), 1.79 – 1.76 (1H, m, H6), 1.69 – 1.67 (1H,
m, H8), 1.67 – 1.66 (1H, m), 1.65 – 1.61 (1H, m, H1), 1.57 (1H, dd, J =
14.1, 3.5 Hz), 1.54 – 1.51 (1H, m, H7), 1.47 (1H, d, J = 6.9 Hz), 1.45
(3H, s, H13), 1.39 (2H, d, J = 3.4 Hz, H15), 1.37 (1H, d, J = 5.5 Hz, H8),
4.2.4. 22-[(2-Methylphenyl-1, 3, 4-oxadiazol-5-yl) thio] deoxy
pleuromutilin (compound 114)
White powder; yield: 74%; 1H NMR (600 MHz, Chloroform-d) δ 7.42
(2H, td, J = 7.6, 1.2 Hz), 7.32 (2H, dd, J = 13.8, 6.4 Hz), 6.43 (1H, dd, J
= 17.4, 11.0 Hz, H19), 5.80 (1H, d, J = 8.5 Hz, H14), 5.32 – 5.30 (1H, m,
H20), 5.18 (1H, dd, J = 17.4, 1.3 Hz, H20), 4.11 – 4.01 (2H, m), 2.68
8

J. Liu et al.
Bioorganic Chemistry 112 (2021) 104956
1.16 (3H, s), 1.12 (1H, dd, J = 14.1, 4.4 Hz, H18), 0.88 (3H, d, J = 7.1
Hz, H17), 0.76 (3H, d, J = 7.1 Hz, H16) . 13C NMR (151 MHz, Chloro-
form-d) δ 216.70(C3), 166.12(C21), 166.01, 161.03, 146.73, 138.68
(C19), 132.57, 127.72, 117.36(C20), 116.85, 116.14, 105.30, 74.60
(C11), 70.88(C14), 58.07(C4), 45.42(C9), 44.64(C13), 43.99(C12),
41.90(C5), 36.67(C6), 36.03(C10), 35.03, 34.41(C2), 30.39(C8), 26.84
(C7), 26.39(C18), 24.82(C1), 16.75(C16), 14.78(C15), 11.45(C17); HR-
MS (ESI): Calcd for C30H39N3O5S (M+H+): 554.2688; Found: 554.2674.
(C17); HR-MS (ESI): Calcd for C30H37N3O7S (Mꢀ H+): 582.2274; Found:
582.2275.
4.2.11. 22-[(2-hydroxyphenyl-1, 3, 4-oxadiazol-5-yl) thio] deoxy
pleuromutilin (compound 121)
White powder; yield: 64%; 1H NMR (600 MHz, Chloroform-d) δ 6.42
(1H, dd, J = 17.4, 11.0 Hz, H19), 5.72 (1H, d, J = 8.5 Hz, H14), 5.36
(1H, dd, J = 11.0, 1.3 Hz, H20), 5.22 (1H, dd, J = 17.4, 1.4 Hz, H20),
3.67 (1H, d, J = 10.5 Hz), 3.59 (1H, d, J = 10.5 Hz), 3.38 (1H, dd, J =
10.4, 6.6 Hz, H11), 2.37 – 2.16 (3H, m, H2, H4, H10), 2.13 – 2.05 (2H,
m, H13, 11-OH), 1.78 (1H, dq, J = 14.4, 2.8 Hz, H6), 1.71 – 1.62 (3H, m,
H8, H1, H17), 1.56 (1H, qd, J = 13.9, 3.6 Hz, H13), 1.51 – 1.43 (5H, m),
1.47 (4H, s), 1.42 – 1.31 (2H, m, H15, H8), 1.21 – 1.10 (4H, m, H18),
0.90 (3H, d, J = 7.1, H17), 0.75 (3H, d, J = 7.1, H16). 13C NMR (151
MHz, Chloroform-d) δ 216.68(C3), 165.87, 165.47, 162.17, 157.24,
138.66(C19), 133.76, 126.43, 119.98, 117.57, 117.36(C20), 107.73,
74.58(C11), 71.06(C14), 58.05(C4), 45.42(C9), 44.63(C13), 43.98
(C12), 41.89(C5), 36.64(C6), 36.03(C10), 35.04(C2), 34.40, 30.38(C8),
26.83(C7), 26.40(C18), 24.81(C1), 16.74(C16), 14.77(C15), 11.44
(C17); HR-MS (ESI): Calcd for C30H38N2O6S (M+H+): 555.2529; Found:
555.2517.
4.2.8. 22-[(4-aminophenyl-1, 3, 4-oxadiazol-5-yl) thio] deoxy
pleuromutilin (compound 118)
White powder; yield: 74%; 1H NMR (600 MHz, Chloroform-d) δ 7.79
(2H, d, J = 8.7 Hz), 6.74 (2H, d, J = 8.3 Hz), 6.44 (1H, dd, J = 17.4, 11.0
Hz, H19), 5.79 (1H, d, J = 8.5 Hz, H14), 5.39 – 5.15 (3H, m, H20), 4.03
(2H, d, J = 2.5 Hz), 3.36 (1H, d, J = 6.5 Hz, H22), 2.34 – 2.31 (1H, m,
H11), 2.31 – 2.23 (1H, m, H2), 2.19 (1H, dd, J = 19.5, 9.3 Hz, H4), 2.12
– 2.07 (1H, m, H10), 1.78 (1H, dd, J = 14.4, 3.1 Hz, H6), 1.72 – 1.66
(2H, m, H8), 1.66 – 1.62 (1H, m, H1), 1.58 – 1.52 (1H, m, H7), 1.48 (1H,
dd, J = 9.6, 2.8 Hz, H13), 1.45 (3H, s), 1.44 (1H, d, J = 4.0 Hz), 1.41 –
1.38 (1H, m,H15), 1.38 – 1.36 (1H, m, H8), 1.27 (1H, s), 1.17 (3H, s),
1.13 (1H, dd, J = 14.0, 4.5 Hz, H18), 0.89 (3H, d, J = 7.1 Hz, H17), 0.76
(3H, d, J = 7.1 Hz, H16). 13C NMR (151 MHz, Chloroform-d) δ 216.79
(C3), 166.48, 166.25, 161.18, 149.83, 138.72(C19), 128.44, 117.35
(C20), 114.62, 113.04, 74.60(C11), 70.81(C14), 65.93, 58.08(C4),
53.39(C22), 45.42, 44.63(C13), 44.00(C12), 41.89(C5), 36.67(C6),
36.02(C10), 35.12, 34.42(C2), 30.39, 26.83(C7), 26.41(C8), 24.81(C1),
16.75(C16), 14.77(C15), 11.44(C17); HR-MS (ESI): Calcd for
4.2.12. 22-[(4-hydroxyphenyl ꢀ 1, 3, 4-oxadiazol-5-yl) thio] deoxy
pleuromutilin (compound 122)
White powder; yield: 67%; 1H NMR (600 MHz, Chloroform-d) δ 7.85
(3H, dd, J = 8.5, 6.6 Hz), 7.03 – 6.91 (2H, m), 6.42 (1H, dd, J = 17.4,
11.0 Hz, H19), 5.78 (1H, d, J = 8.5 Hz, H14), 5.39 – 5.07 (2H, m, H20),
4.10 – 3.97 (2H, m), 3.36 (1H, dd, J = 10.3, 6.5 Hz, H22), 2.34 – 2.27
(1H, m), 2.27 – 2.22 (1H, m), 2.19 (1H, dd, J = 19.5, 9.4 Hz, H2), 2.11 –
2.04 (2H, m, H4, H10), 1.93 (1H, s, H13), 1.77 (1H, dq, J = 14.6, 3.2 Hz,
H6), 1.68 – 1.63 (1H, m, H1), 1.61 (1H, d, J = 10.6, H7), 1.54 (1H, td, J
= 13.3, 12.6, 3.2 Hz, H13), 1.50 – 1.45 (1H, m), 1.43 (3H, s), 1.40 – 1.34
(2H, m, H15), 1.15 (3H, s, H8), 1.11 (1H, dd, J = 14.1, 4.4 Hz, H18),
0.88 (3H, d, J = 7.0 Hz, H17), 0.75 (3H, d, J = 7.1, H16). 13C NMR (151
MHz, Chloroform-d) δ 217.28(C3), 166.28, 162.00, 159.92, 138.59
(C19), 128.78, 117.49(C20), 116.29, 115.05, 74.66(C11), 70.99(C14),
58.13(C4), 45.45(C9), 44.62(C13), 43.98(C12), 41.91(C5), 36.68(C6),
36.00(C10), 35.74, 35.02, 35.02, 34.46(C2), 30.39(C8), 26.80(C7),
26.37(C18), 24.80(C1), 23.87, 16.75(C16), 14.77(C15), 11.48(C17);
HR-MS (ESI): Calcd for C30H38N2O6S (M+H+): 555.2529; Found:
555.2513.
C
30H39N3O5S (M+H+): 554.2688; Found: 554.2674.
4.2.9. 22-[((4-dimethylamino)phenyl-1, 3, 4-oxadiazol-5-yl) thio] deoxy
pleuromutilin (compound 119)
White powder; yield: 74%; 1H NMR (600 MHz, Chloroform-d) δ 7.84
– 7.82 (2H, m), 6.73 (1H, s), 6.71 (1H, d, J = 2.1 Hz), 6.43 (1H, dd, J =
17.4, 11.0 Hz, H19), 5.78 (1H, d, J = 8.5 Hz, H14), 5.32 (1H, dd, J =
11.0, 1.4 Hz, H20), 5.19 (1H, dd, J = 17.4, 1.5 Hz, H20), 4.01 (2H, d, J
= 2.9 Hz), 3.35 (1H, t, J = 7.6 Hz, H22), 3.06 (6H, s, H11), 2.32 (1H, q, J
= 6.9 Hz, H2), 2.20 (1H, q, J = 9.7 Hz, H4), 2.10 (1H, s, H10), 1.79 –
1.75 (1H, m, H6, H8), 1.74 (1H, s), 1.69 – 1.67 (1H, m, H1), 1.67 – 1.65
(1H, m, H7), 1.65 – 1.62 (1H, m), 1.54 – 1.52 (1H, m), 1.51 – 1.49 (1H,
m), 1.46 (1H, dd, J = 9.7, 2.8 Hz), 1.44 (3H, s, H13), 1.39 (1H, d, J = 3.8
Hz, H15), 1.36 (1H, d, J = 6.2 Hz, H8), 1.16 (3H, s), 1.15 – 1.10 (1H, m,
H18), 0.88 (3H, d, J = 7.1 Hz, H17), 0.75 (3H, d, J = 7.1 Hz, H16). 13
C
NMR (151 MHz, Chloroform-d) δ 216.77(C3), 166.79, 166.30, 160.81,
152.39, 138.71(C19), 128.12, 117.36(C20), 111.56, 110.46, 74.61
(C11), 70.75(C14), 58.08(C4), 45.42(C9), 44.63(C12), 44.40, 44.00,
42.66, 41.89(C5), 40.05, 36.68(C6), 36.02(C10), 35.16, 34.42(C2),
31.69, 30.40(C8), 26.83(C7), 26.40(C18), 24.82(C1), 16.75(C16), 14.78
(C15), 11.45(C17); HR-MS (ESI): Calcd for C32H43N3O5S (M+H+):
582.3001; Found: 582.2986.
4.2.13. 22-[(4-methoxyphenyl-1, 3, 4-oxadiazol-5-yl) thio] deoxy
pleuromutilin (compound 123)
White powder; yield: 64%; 1H NMR (600 MHz, Chloroform-d) δ 7.93
– 7.92 (2H, m), 7.00 – 6.98 (2H, m), 6.42 (1H, dd, J = 17.4, 11.0 Hz,
H19), 5.78 (1H, d, J = 8.5 Hz, H14), 5.18 (1H, dd, J = 17.4, 1.4 Hz,
H20), 4.03 (2H, d, J = 3.1 Hz), 3.88 (3H, s, H22), 3.36 (1H, s, H11), 2.32
(1H, q, J = 6.9 Hz, H2), 2.27 – 2.24 (1H, m, H4), 2.19 (1H, dt, J = 19.4,
9.4 Hz, H10), 2.10 (1H, s, H13), 1.77 (2H, dd, J = 14.5, 2.9 Hz, H6, H8),
1.67 (2H, dd, J = 8.1, 4.2 Hz, H1), 1.58 – 1.50 (3H, m, H7), 1.47 – 1.45
(1H, m, H13), 1.44 (3H, s), 1.39 (1H, d, J = 4.0, H15), 1.36 (1H, d, J =
6.1, H8), 1.16 (3H, s), 1.15 – 1.11 (1H, m, H18), 0.88 (3H, d, J = 7.1 Hz,
H17), 0.75 (3H, d, J = 7.1, H16). 13C NMR (151 MHz, Chloroform-d) δ
216.72(C3), 166.16, 166.01, 162.39, 161.92, 138.72(C19), 128.49,
117.33(C20), 115.97, 114.51, 74.60(C11), 70.86(C14), 58.07(C4),
55.46, 45.42(C9), 44.64(C13), 44.00(C2), 43.03, 41.90(C5), 36.66(C6),
36.33, 36.03(C10), 35.11, 34.41(C2), 30.39(C8), 26.83(C7), 26.41
(C18), 24.82(C1), 16.75(C16), 14.77(C15), 11.44(C17); HR-MS (ESI):
Calcd for C31H40N2O6S (M+H+): 569.2685; Found: 569.2671.
4.2.10. 22-[(3-nitrophenyl-1, 3, 4-oxadiazol-5-yl) thio] deoxy
pleuromutilin (compound 120)
White powder; yield: 74%; 1H NMR (600 MHz, Chloroform-d) δ 8.84
(1H, td, J = 1.9, 0.4 Hz), 8.52 – 8.19 (2H, m), 7.82 – 7.65 (1H, m), 6.44
(1H, dd, J = 17.4, 11.0 Hz, H19), 5.81 (1H, d, J = 8.5 Hz, H14), 5.32
(1H, dd, J = 11.0, 1.4 Hz, H20), 5.20 (1H, dd, J = 17.4, 1.5 Hz, H20),
4.17 – 3.91 (2H, m), 2.35 – 2.16 (3H, m, H2, H4, H10), 2.11 (3H, q, J =
8.6 Hz, H13, 11-OH), 1.83 – 1.75 (1H, m, H6), 1.69 (3H, dddd, J = 24.4,
13.2, 9.1, 6.5 Hz, H1), 1.56 (2H, td, J = 13.2, 12.5, 2.9 Hz, H7), 1.51 –
1.47 (1H, m), 1.46 (3H, s, H14), 1.43 – 1.37 (2H, m, H15), 1.19 (3H, s,
H8), 0.89 (3H, d, J = 7.1, H17), 0.78 (3H, d, J = 7.1 Hz) . 13C NMR (151
MHz, Chloroform-d) δ 216.68(C3), 165.88, 164.12, 148.68, 138.73
(C19), 132.12, 130.44, 126.15, 125.10, 121.58, 117.33(C20), 74.58
(C11), 71.11(C14), 58.04(C4), 55.04, 45.42(C9), 44.66(C13), 44.02
(C12), 41.91(C5), 36.64(C6), 36.03(C10), 35.08, 34.41(C2), 30.37(C8),
26.83(C7), 26.44(C18), 24.81(C1), 16.76(C16), 14.77(C15), 11.43
4.2.14. 22-[(2-fluorophenyl-1, 3, 4-oxadiazol-5-yl) thio] deoxy
pleuromutilin (compound 124)
White powder; yield: 81%; 1H NMR (600 MHz, Chloroform-d) δ 8.01
– 7.99 (1H, m), 7.55 – 7.52 (1H, m), 7.31 – 7.28 (1H, m), 7.26 – 7.23
(1H, m), 6.42 (1H, dd, J = 17.4, 11.0, H19), 5.79 (1H, d, J = 8.5 Hz,
9

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Bioorganic Chemistry 112 (2021) 104956
H14), 5.32 – 5.30 (1H, m, H20), 5.19 – 5.16 (1H, m, H20), 4.09 – 4.02
(2H, m), 3.36 (1H, d, J = 5.8 Hz, H22), 2.32 (1H, q, J = 7.0 Hz, H11),
2.26 – 2.23 (1H, m, H2), 2.22 – 2.17 (1H, m, H4), 2.09 (2H, d, J = 9.1 Hz,
H10, H13), 1.79 – 1.75 (1H, m, H6), 1.67 – 1.65 (1H, m, H1), 1.65 – 1.62
(1H, m, H7), 1.56 (1H, dd, J = 14.1, 3.7 Hz, H13), 1.54 – 1.51 (1H, m),
1.46 – 1.45 (1H, m), 1.44 (3H, s), 1.39 (1H, d, J = 4.1 Hz, H15), 1.37
(1H, d, J = 5.8H8), 1.16 (3H, s), 1.13 (1H, dd, J = 14.2, 9.8 Hz, H18),
0.88 (3H, d, J = 7.1 Hz, H17), 0.75 (3H, d, J = 7.1, H16). 13C NMR (151
MHz, Chloroform-d) δ 216.69(C3), 166.04, 163.32, 162.73, 160.68,
158.96, 138.71(C19), 133.58, 133.53, 129.53, 124.67, 117.31(C20),
112.03, 74.59(C11), 70.93(C14), 58.06(C4), 45.42(C9), 44.63(C13),
44.00(C12), 41.90(C5), 36.66(C6), 36.03(C10), 35.03, 34.41(C2), 30.39
(C8), 26.83,(C7) 24.82(C18), 16.73(C16), 14.75(C15), 11.44(C17); HR-
MS (ESI): Calcd for C30H37FN2O5S (M+Na+): 579.2299; Found:
579.2288.
1.46 (2H, m, H13), 1.45 (3H, s), 1.38 (2H, dd, J = 15.4, 4.2 Hz, H15,
H8), 1.16 (3H, s), 1.12 (1H, dd, J = 14.1, 4.4 Hz, H18), 0.88 (3H, d, J =
7.0 Hz, H17), 0.75 (3H, d, J = 7.1 Hz, H16).13C NMR (151 MHz,
Chloroform-d) δ 216.71(C3), 166.04, 164.34, 163.46, 138.70(C19),
133.08, 132.46, 131.24, 130.98, 127.07, 122.79, 117.35(C20), 74.60
(C11), 70.95(C14), 58.07(C4), 45.42(C9), 44.64(C13), 44.00(C12),
41.90(C5), 36.66(C6), 36.03(C10), 35.03, 34.41(C2), 30.39(C8), 26.83
(C7), 26.39(C18), 24.82(C1), 16.76(C16), 14.77(C15), 11.44(C17); HR-
MS (ESI): Calcd for C30H37ClN2O5S (M+Na +): 595.2004; Found:
595.1993.
4.2.18. 22-[(3-chlorophenyl-1, 3, 4-oxadiazol-5-yl) thio] deoxy
pleuromutilin (compound 128)
White powder; yield: 84%; 1H NMR (600 MHz, Chloroform-d) δ 7.99
(1H, td, J = 1.8, 0.5 Hz), 7.89 (1H, ddd, J = 7.8, 1.6, 1.1 Hz), 7.51 (1H,
ddd, J = 8.0, 2.1, 1.1 Hz), 7.48 – 7.39 (1H, m), 6.43 (1H, dd, J = 17.4,
11.0 Hz, H19), 5.79 (1H, d, J = 8.5, H14), 5.33 – 5.30 (1H, m, H20), 5.19
(1H, dd, J = 17.4, 1.5 Hz, H20), 4.12 – 4.00 (2H, m), 3.36 (1H, dd, J =
10.5, 6.5 Hz, H22), 2.35 – 2.29 (1H, m, H2), 2.28 – 2.22 (1H, m, H4),
2.19 (1H, dd, J = 19.5, 9.4 Hz, H10), 2.12 – 2.06 (2H, m, H13. 11-OH),
1.77 (1H, dq, J = 14.5, 3.1 Hz, H6, H8), 1.68 (1H, s, H1), 1.67 – 1.62
(1H, m, H7), 1.58 – 1.52 (1H, m), 1.51 – 1.46 (2H, m), 1.45 (3H, s, H13),
1.42 – 1.34 (2H, m, H15, H8), 1.17 (3H, s), 1.16 – 1.10 (1H, m, H18),
0.88 (3H, d, J = 7.1 Hz, H17), 0.76 (3H, d, J 7.1, H17).13C NMR (151
MHz, Chloroform-d) δ 216.69(C3), 165.99, 164.88, 163.32, 138.70
(C19), 135.25, 131.82, 130.44, 126.66, 125.05, 124.77, 117.36(C20),
74.59(C11), 71.00(C14), 58.06(C4), 45.42(C9), 44.65(C13), 44.01
(C12), 41.90(C5), 36.65(C6), 36.03(C10), 35.06, 34.41(C2), 30.38(C8),
26.83(C7), 26.41(C18), 24.82(C1), 16.76(C16), 14.77(C15), 11.44
(C17); HR-MS (ESI): Calcd for C30H37ClN2O5S (M+Na +): 595.2004;
Found: 595.1991.
4.2.15. 22-[(3-fluorophenyl-1, 3, 4-oxadiazol-5-yl) thio] deoxy
pleuromutilin (compound 125)
White powder; yield: 82%; 1H NMR (600 MHz, Chloroform-d) δ 7.80
(1H, ddd, J = 7.8, 1.6, 1.0 Hz), 7.70 (1H, ddd, J = 9.1, 2.6, 1.5 Hz), 7.49
(1H, td, J = 8.0, 5.5 Hz), 7.24 (1H, tdd, J = 8.4, 2.6, 1.0 Hz), 6.43 (1H,
dd, J = 17.4, 11.0 Hz, H19), 5.80 (1H, d, J = 8.5 Hz, H14), 5.35 – 5.10
(2H, m, H20), 4.14 – 3.99 (2H, m), 3.36 (1H, dd, J = 10.4, 6.5 Hz, H22),
2.33 – 2.23 (2H, m, H2, H4), 2.19 (1H, dd, J = 19.5, 9.4 Hz, H10), 2.12 –
2.05 (2H, m, H13, 11-OH), 1.77 (1H, dq, J = 14.5, 3.2 Hz, H6), 1.67 (2H,
s H8), 1.67 – 1.62 (1H, m, H1), 1.59 – 1.51 (1H, m, H7), 1.50 – 1.46 (1H,
m, H13), 1.45 (3H, s), 1.42 – 1.34 (2H, m, H15, H8), 1.17 (3H, s), 1.13
(1H, dd, J = 14.2, 4.4 Hz, H18), 0.88 (3H, d, J = 7.1 Hz, H17), 0.76 (3H,
d, J = 7.1 Hz, H16). 13C NMR (151 MHz, Chloroform-d) δ 216.69(C3),
166.00, 165.05, 163.26, 161.99, 138.70(C19), 130.96, 130.91, 125.32,
122.45, 118.93, 118.79, 117.35(C20), 113.84, 113.68, 74.59(C11),
70.99(C14), 58.06(C4), 45.42(C9), 44.01(C12), 41.90(C5), 36.65(C6),
36.03(C10), 34.41(C2), 30.38(C8), 26.83(C7), 24.82, 16.76(C16), 14.76
(C15), 11.43(C17); HR-MS (ESI): Calcd for C30H37FN2O5S (M+Na +):
579.2299; Found: 579.2288.
4.2.19. 22-[(4-chlorophenyl-1, 3, 4-oxadiazol-5-yl) thio] deoxy
pleuromutilin (compound 129)
White powder; yield: 82%; 1H NMR (600 MHz, Chloroform-d) δ 7.96
– 7.92 (2H, m), 7.51 – 7.47 (2H, m), 6.44 (1H, dd, J = 17.4, 11.0 Hz,
H19), 5.80 (1H, d, J = 8.5 Hz, H14), 5.34 – 5.31 (1H, m), 5.20 (1H, dd, J
= 17.4, 1.4 Hz, H20), 4.06 (2H, d, J = 3.0, H20, H22), 2.34 – 2.25 (2H,
m, H2, H4), 2.20 (1H, dd, J = 19.5, 9.4 Hz), 2.10 (2H, d, J = 8.7 Hz, H10,
H13), 1.78 (1H, dd, J = 14.5, 3.0 Hz, H6), 1.69 (2H, ddd, J = 12.9, 4.8,
2.8 Hz, H8), 1.60 – 1.54 (3H, m, 11-OH), 1.46 (3H, s, H1, H7), 1.41 –
1.35 (3H, m, H13, H15), 1.18 (3H, s, H8), 1.13 (1H, dd, J = 14.1, 4.4 Hz,
H18), 0.89 (3H, d, J = 7.1, H17), 0.77 (3H, d, J = 7.1, H16). 13C NMR
(151 MHz, Chloroform-d) δ 216.70(C3), 166.02, 165.24, 163.04, 138.71
(C19), 138.09, 129.48, 127.96, 121.91, 117.34(C20), 99.99, 74.59
(C11), 70.98(C14), 58.05(C4), 48.14, 45.42(C9), 44.64(C13), 44.01
(C12), 41.90(C5), 36.65(C6), 36.02(C10), 35.08, 34.41(C2), 30.38(C8),
26.83(C7), 26.42(C18), 24.81(C1), 16.76(C16), 14.77(C15), 11.44
(C17); HR-MS (ESI): Calcd for C30H37ClN2O5S (M+Na +): 595.2004;
Found: 595.1990.
4.2.16. 22-[(4-fluorophenyl-1, 3, 4-oxadiazol-5-yl) thio] deoxy
pleuromutilin (compound 126)
White powder; yield: 78%; 1H NMR (600 MHz, Chloroform-d) δ 8.00
(2H, td, J = 5.2, 2.8 Hz), 7.21 – 7.18 (2H, m), 6.43 (1H, dd, J = 17.4,
11.0 Hz, H19), 5.79 (1H, d, J = 8.5 Hz), 5.32 – 5.30 (1H, m, H20), 5.19
(1H, d, J = 17.4 Hz, H20), 4.05 (2H, d, J = 3.3 Hz), 3.36 (1H, dd, J =
10.4, 6.5 Hz, H22), 2.32 (1H, q, J = 6.9 Hz), 2.29 – 2.22 (1H, m, H2),
2.19 (1H, dd, J = 19.5, 9.4 Hz, H4), 2.09 (2H, d, J = 16.5 Hz, H10, H13),
1.77 (1H, dd, J = 14.5, 2.9 Hz, H6), 1.71 (1H, s, H8), 1.67 – 1.64 (1H, m,
H1), 1.57 – 1.49 (2H, m, H7, H13), 1.47 (1H, d, J = 11.6 Hz), 1.45 (3H,
s), 1.38 (1H, d, J = 5.2 Hz, H15), 1.38 – 1.35 (1H, m, H8), 1.17 (3H, s),
1.16 – 1.10 (1H, m, H18), 0.88 (3H, d, J = 7.0 Hz, H17), 0.75 (3H, d, J =
7.1 Hz, H16). 13C NMR (151 MHz, Chloroform-d) δ 216.71(C3), 166.05,
165.63, 165.23, 163.95, 162.79, 138.72(C19), 129.04, 128.98, 117.33
(C20), 116.51, 116.36, 74.59(C11), 70.95(C14), 58.06(C4), 45.42(C9),
44.64(C13), 44.00(C12), 41.90(C5), 36.65(C6), 36.02(C10), 35.08,
34.41(C2), 30.38(C8), 26.83(C7), 26.41(C18), 24.81(C1), 16.75(C16),
14.77(C15), 11.43(C17); HR-MS (ESI): Calcd for C30H37FN2O5S (M+Na
+): 579.2299; Found: 579.2289.
4.2.20. 22-[(3-bromophenyl-1, 3, 4-oxadiazol-5-yl) thio] deoxy
pleuromutilin (compound 130)
White powder; yield: 81%; 1H NMR (600 MHz, Chloroform-d) δ 8.15
– 8.14 (1H, m), 7.94 – 7.93 (1H, m), 7.67 – 7.66 (1H, m), 7.38 (1H, t, J =
7.9 Hz), 6.42 (1H, dd, J = 17.4, 11.0 Hz, H19), 5.79 (1H, d, J = 8.5 Hz),
5.31 (1H, dd, J = 9.6, 1.4 Hz, H14), 5.21 – 5.17 (1H, m, H20), 4.06 (2H,
d, J = 3.2, H20, H22), 3.36 (1H, d, J = 6.4, H11), 2.34 – 2.30 (1H, m,
H2), 2.26 – 2.23 (1H, m, H4), 2.20 (1H, q, J = 9.7, H10), 2.10 (2H, d, J =
8.6 Hz, H13), 1.79 – 1.75 (1H, m, H6), 1.67 (1H, dd, J = 5.2, 3.0 Hz, H8),
1.56 (1H, dd, J = 13.9, 3.7 Hz), 1.54 – 1.49 (2H, m), 1.48 – 1.46 (1H, m,
H1), 1.45 (3H, s, H7, H13), 1.39 (1H, d, J = 5.7, H15), 1.37 (1H, d, J =
7.9 Hz), 1.17 (3H, s), 1.12 (1H, dd, J = 14.1, 4.4 Hz, H8), 0.88 (3H, d, J
= 7.1, H17), 0.76 (3H, d, J = 7.1 Hz, H16). 13C NMR (151 MHz, Chlo-
roform-d) δ 216.66(C3), 165.97, 164.72, 163.32, 138.71(C19), 134.73,
130.63, 129.52, 125.27, 125.20, 123.11, 117.35(C20), 74.59(C11),
4.2.17. 22-[(2-chlorophenyl-1, 3, 4-oxadiazol-5-yl) thio] deoxy
pleuromutilin (compound 127)
White powder; yield: 80%; 1H NMR (600 MHz, Chloroform-d) δ 7.93
(1H, dd, J = 7.8, 1.7 Hz), 7.55 (1H, dd, J = 8.1, 1.3 Hz), 7.48 (1H, ddd, J
= 8.0, 7.4, 1.7 Hz), 7.41 (1H, td, J = 7.6, 7.6, 1.3 Hz), 6.43 (1H, dd, J =
17.4, 11.0 Hz, H19), 5.80 (1H, d, J = 8.5H14), 5.36 – 5.14 (2H, m, H20),
4.14 – 4.00 (2H, m), 3.36 (1H, dd, J = 10.6, 6.5 Hz), 2.32 (1H, p, J = 7.5,
7.5, 7.3, 7.3 Hz, H2), 2.27 – 2.22 (1H, m, H4), 2.19 (1H, dd, J = 19.5,
9.4 Hz, H10), 2.13 – 2.04 (2H, m, H13, 11-OH), 1.77 (1H, dq, J = 14.6,
3.2, 3.2, 3.2 Hz, H6), 1.67 (2H, s, H1, H7), 1.59 – 1.51 (1H, m), 1.50 –
10

J. Liu et al.
Bioorganic Chemistry 112 (2021) 104956
71.00(C14), 58.05(C4), 45.42(C9), 44.65(C13), 44.01(C12), 41.90(C5),
36.65(C6), 36.03(C10), 35.06, 34.41(C2), 30.38(C8), 26.83(C7), 26.43
(C18), 24.82(C1), 16.76(C16), 14.77(C15), 11.44(C17); HR-MS (ESI):
Calcd for C30H37BrN2O5S (M+H+): 619.1664; Found: 619.1647.
567.1946.
4.2.24. 22-[(2-pyridy-1, 3, 4-oxadiazol-5-yl) thio] deoxy pleuromutilin
(compound 134)
White powder; yield: 69%; 1H NMR (600 MHz, Chloroform-d) δ 8.78
– 8.77 (1H, m), 8.20 – 8.19 (1H, m), 7.89 (1H, td, J = 7.8, 1.7 Hz), 7.47
(1H, ddd, J = 7.6, 4.8, 1.1 Hz), 6.43 (1H, dd, J = 17.4, 11.0 Hz, H19),
5.80 (1H, d, J = 8.5 Hz, H14), 5.32 (1H, dd, J = 11.0, 1.4 Hz, H20), 5.20
(1H, dd, J = 17.4, 1.5 Hz, H20), 4.13 – 4.04 (2H, m), 3.36 (1H, d, J = 6.4
Hz, H22), 2.33 – 2.18 (3H, m, H2, H4, H10), 2.12 – 2.00 (3H, m, H13,
H6), 1.79 – 1.76 (1H, m, H8), 1.71 – 1.68 (1H, m, 11-OH), 1.67 – 1.65
(1H, m, H1), 1.65 – 1.62 (1H, m, H7), 1.54 (2H, qd, J = 14.0, 3.7, H13),
1.44 (3H, s), 1.42 – 1.38 (2H, m, H5, H8), 1.17 (3H, s, H18), 0.88 (3H, d,
J = 7.1 Hz, H17), 0.76 (3H, d, J = 7.1 Hz, H16). 13C NMR (151 MHz,
Chloroform-d) δ 216.72(C3), 166.00(C21), 165.14, 164.38, 150.26,
143.12, 138.71(C19), 137.24, 125.84, 122.88, 117.34(C20), 74.60,
70.94, 58.06, 45.42(C9), 44.61(C13), 44.00(C12), 41.90(C5), 36.66
(C6), 36.02(C10), 35.02, 34.41(C2), 30.38(C8), 26.82(C7), 26.42(C18),
24.81(C1), 16.75(C16), 14.75(C15), 11.43(C17); HR-MS (ESI): Calcd for
4.2.21. 22-[(4-bromophenyl-1, 3, 4-oxadiazol-5-yl) thio] deoxy
pleuromutilin (compound 131)
White powder; yield: 81%; 1H NMR (600 MHz, Chloroform-d) δ 7.96
– 7.92 (1H, m), 7.67 – 7.63 (1H, m), 7.38 (1H, t, J = 7.9 Hz), 6.43 (1H,
dd, J = 17.4, 11.0 Hz, H19), 5.80 (1H, d, J = 8.5 Hz), 5.33 – 5.30 (1H, m,
H14), 5.19 (1H, dd, J = 17.4, 1.4 Hz, H20), 4.09 – 4.03 (2H, m, H20,
H22), 3.36 (1H, s, H11), 2.32 (1H, q, J = 6.9 Hz, H2), 2.29 – 2.25 (1H, m,
H4), 2.21 (1H, q, J = 9.7 Hz, H10), 2.09 (2H, d, J = 16.0 Hz, H13), 1.77
(1H, dd, J = 14.5, 2.9 Hz, H6), 1.69 – 1.67 (1H, m, H8), 1.66 – 1.62 (1H,
m, 11-OH), 1.52 (1H, dd, J = 13.3, 2.9 Hz), 1.47 (2H, dd, J = 9.7, 2.8 Hz,
H1, H7), 1.45 (3H, s, H13), 1.40 (1H, d, J = 5.9 Hz, H15), 1.37 (1H, d, J
= 7.9 Hz), 1.17 (3H, s, H8), 1.13 (1H, dd, J = 14.1, 4.4 Hz, H18), 0.89
(3H, d, J = 7.1 Hz, H17), 0.76 (3H, d, J = 7.1, H16). 13C NMR (151 MHz,
Chloroform-d) δ 216.66(C3), 165.97, 164.72, 163.32, 138.71(C19),
134.73, 130.63, 129.52, 125.27, 125.20, 123.11, 117.35(C20), 74.59
(C11), 71.00(C14), 58.05(C4), 45.42(C9), 44.65(C13), 44.01(C12),
41.90(C5), 36.65(C6), 36.03(C10), 35.06, 34.41(C2), 30.38(C8), 26.83
(C7), 26.43(C18), 24.82(C1), 16.76(C16), 14.77(C15), 11.44(C17); HR-
MS (ESI): Calcd for C30H37BrN2O5S (M+Na+): 641.1478; Found:
641.1466.
C
29H37N3O5S (M+H+): 540.2532; Found: 540.2516.
4.2.25. 22-[(3-pyridy-1, 3, 4-oxadiazol-5-yl) thio] deoxy pleuromutilin
(compound 135)
White powder; yield: 68%; 1H NMR (600 MHz, Chloroform-d) δ 9.27
– 9.18 (1H, m), 8.78 (1H, dd, J = 4.9, 1.7 Hz), 8.30 (1H, dt, J = 8.0, 1.9
Hz), 7.47 (1H, ddd, J = 8.0, 4.9, 0.9 Hz), 6.43 (1H, dd, J = 17.4, 11.0 Hz,
H19), 5.80 (1H, d, J = 8.5 Hz, H14), 5.33 – 5.17 (2H, m, H20), 4.16 –
4.01 (2H, m), 3.37 (1H, s, H22), 2.35 – 2.24 (2H, m, H2), 2.20 (1H, dt, J
= 19.4, 9.3 Hz, H4), 2.13 – 2.06 (2H, m, H10, H13), 1.78 (1H, dq, J =
14.5, 3.1 Hz, H6), 1.71 – 1.64 (1H, m, H8), 1.57 – 1.48 (2H, m, H1, H7),
1.46 (3H, s, H13), 1.39 (2H, dd, J = 15.4, 3.7 Hz, H15), 1.31 – 1.25 (1H,
m, H8), 1.18 (4H, s, H18), 1.17 – 1.11 (1H, m), 0.89 (3H, d, J = 7.0 Hz,
H17), 0.77 (3H, d, J = 7.1, H16). 13C NMR (151 MHz, Chloroform-d) δ
216.67(C3), 165.94(C21), 163.94, 163.71, 152.47, 147.68, 138.70
(C19), 133.87, 123.77, 120.02, 117.36(C20), 74.60(C11), 71.05(C14),
58.06(C4), 45.42(C9), 44.66(C13), 44.01(C12), 41.91(C5), 36.65(C6),
36.03(C10), 35.09, 34.41(C2), 30.38(C8), 26.83(C7), 26.41(C18), 24.82
(C1), 16.76(C16), 14.77(C15), 11.43(C17); HR-MS (ESI): Calcd for
4.2.22. 22-[(2-furanyl-1, 3, 4-oxadiazol-5-yl) thio] deoxy pleuromutilin
(compound 132)
White powder; yield: 67%; 1H NMR (600 MHz, Chloroform-d) δ 7.64
(1H, dd, J = 1.8, 0.8 Hz), 7.13 (1H, dd, J = 3.6, 0.8 Hz), 6.60 (1H, dd, J
= 3.5, 1.8 Hz), 6.44 (1H, dd, J = 17.4, 11.0 Hz, H19), 5.80 (1H, d, J =
8.5 Hz), 5.33 (1H, dd, J = 11.0, 1.5 Hz, H14), 5.21 (1H, dd, J = 17.5, 1.5
Hz, H20), 4.05 (2H, d, J = 1.9 Hz, H20, H22), 3.36 (1H, s, H11), 2.33
(1H, p, J = 7.1 Hz, H2), 2.28 – 2.23 (1H, m, H4), 2.20 (1H, dd, J = 19.4,
9.4 Hz), 2.11 (1H, s, H10), 2.10 – 2.06 (1H, m, H13), 1.78 (1H, dd, J =
14.5, 3.2 Hz, H6), 1.70 – 1.64 (1H, m, H8), 1.55 (2H, d, J = 13.1 Hz, 11-
OH, H1), 1.52 – 1.46 (1H, m, H7), 1.45 (3H, s, H13), 1.44 – 1.39 (1H, m),
1.37 (1H, d, J = 5.5, H15), 1.28 (1H, d, J = 8.6 Hz), 1.18 (3H, s, H8),
1.17 – 1.11 (1H, m, H18), 0.89 (3H, d, J = 7.0 Hz, H17), 0.77 (3H, d, J =
7.1 Hz, H16). 13C NMR (151 MHz, Chloroform-d) δ 216.71(C3), 165.96
(C21), 162.27, 158.77, 145.75, 138.90(C19), 138.69, 117.35(C20),
114.10, 112.15, 74.60(C11), 70.97(C14), 58.06(C4), 45.42(C9), 44.64
(C13), 44.00(C12), 41.90(C5), 36.65(C6), 36.02(C10), 35.12, 34.41
(C2), 30.38(C8), 26.82(C7), 26.42(C18), 24.81(C1), 16.74(C16), 14.75
(C15), 11.44(C17); HR-MS (ESI): Calcd for C28H36N2O6S (M+Na+):
551.2186; Found: 551.2177.
C
29H37N3O5S (Mꢀ H+): 538.2376; Found: 538.2376.
4.2.26. 22-[(4-pyridy-1, 3, 4-oxadiazol-5-yl) thio] deoxy pleuromutilin
(compound 136)
White powder; yield: 73%; 1H NMR (600 MHz, Chloroform-d) δ 8.81
(2H, d, J = 5.5 Hz), 7.85 – 7.84 (2H, m), 6.42 (1H, dd, J = 17.4, 11.0 Hz,
H19), 5.80 (1H, d, J = 8.5 Hz, H14), 5.33 – 5.30 (1H, m, H20), 5.21 –
5.17 (1H, m, H20), 4.08 (2H, d, J = 3.7 Hz), 3.38 – 3.33 (2H, m, H22,
H11), 2.32 – 2.29 (1H, m,H2), 2.20 (1H, q, J = 9.7 Hz, H4), 2.11 (1H, s),
1.79 – 1.77 (1H, m), 1.76 (1H, d, J = 3.0 Hz,H10), 1.66 (1H, dd, J = 4.4,
2.8 Hz, H1), 1.56 (1H, dd, J = 14.0, 3.6, H7), 1.52 – 1.48 (2H, m, H13),
1.48 – 1.46 (1H, m), 1.45 (3H, s), 1.39 (1H, s), 1.37 (1H, d, J = 4.7 Hz,
H15), 1.17 (3H, s, H8), 1.12 (1H, dd, J = 14.1, 4.4 Hz, H18), 0.88 (3H, d,
J = 7.1 Hz, H17), 0.76 (3H, d, J = 7.1 Hz, H16). 13C NMR (151 MHz,
Chloroform-d) δ 216.63(C3), 165.84(C21), 164.44, 164.20, 150.89,
138.72(C19), 130.48, 120.02, 117.33(C20), 74.58(C11), 71.11(C14),
58.04(C4), 45.42(C9), 44.66(C13), 44.02(C12), 42.79, 41.90(C5), 36.63
(C6), 36.03, 35.99(C10), 35.06, 34.40(C2), 30.37(C8), 26.83(C7), 26.44
(C18), 24.81(C1), 16.75(C16), 14.76(C15), 11.43(C17); HR-MS (ESI):
Calcd for C29H37N3O5S (M+H+): 540.2532; Found: 540.2518.
4.2.23. 22-[(2-thienyl-1, 3, 4-oxadiazol-5-yl) thio] deoxy pleuromutilin
(compound 133)
White powder; yield: 67%; 1H NMR (600 MHz, Chloroform-d) δ 7.72
(1H, dd, J = 3.7, 1.2 Hz), 7.56 (1H, dd, J = 5.0, 1.2 Hz), 7.17 (1H, dd, J
= 5.0, 3.7 Hz), 6.44 (1H, dd, J = 17.4, 11.0 Hz, H19), 5.80 (1H, d, J =
8.5 Hz, H14), 5.33 (1H, dd, J = 11.0, 1.4 Hz, H20), 5.20 (1H, dd, J =
17.4, 1.4 Hz, H20), 4.04 (2H, d, J = 2.4 Hz), 3.36 (2H, s, H22, H11), 2.35
– 2.31 (1H, m, H2), 2.21 (1H, q, J = 9.8 Hz, H4), 2.10 (2H, d, J = 8.7 Hz,
H10, H13), 1.78 (1H, dd, J = 14.5, 3.1 Hz, H6), 1.68 – 1.63 (1H, m, H8),
1.57 (2H, d, J = 10.4, 11-OH), 1.54 – 1.49 (1H, m, H1), 1.46 (3H, s, H7,
H13), 1.40 (1H, d, J = 5.4 Hz), 1.38 – 1.35 (1H, m, H15), 1.28 (1H, d, J
= 7.9 Hz, H8), 1.18 (3H, s), 1.13 (1H, dd, J = 14.1, 4.4 Hz, H18), 0.89
(3H, d, J = 7.1 Hz, H17), 0.77 (3H, d, J = 7.1 Hz, H16). 13C NMR (151
MHz, Chloroform-d) δ 216.72(C3), 166.03(C21), 162.22, 162.12,
138.69(C19), 130.21, 129.78, 128.13, 124.60, 117.36(C20), 74.60
(C11), 70.94(C14), 58.06(C22), 45.42(C9), 44.64(C13), 44.01(C12),
41.90(C5), 36.66(C6), 36.02(C10), 35.11, 34.42(C2), 30.39(C8), 26.83
(C7), 26.40(C18), 24.82(C1), 16.76(C16), 14.77(C15), 11.44(C17); HR-
MS (ESI): Calcd for C28H36N2O5S2 (M+Na+): 567.1958; Found:
4.2.27. 22-[(pyrazinyl-1, 3, 4-oxadiazol-5-yl) thio] deoxy pleuromutilin
(compound 137)
White powder; yield: 73%; 1H NMR (600 MHz, Chloroform-d) δ 9.43
(1H, d, J = 1.5 Hz), 8.85 – 8.66 (2H, m), 6.44 (1H, dd, J = 17.4, 11.0 Hz,
H19), 5.81 (1H, d, J = 8.6 Hz, H14), 5.40 – 5.16 (2H, m, H20), 4.23 –
4.00 (2H, m), 3.37 (1H, t, J = 8.0 Hz, H22), 2.32 (1H, p, J = 7.0 Hz, H2),
2.28 – 2.23 (1H, m, H4), 2.20 (1H, dd, J = 19.4, 9.4, H10), 2.13 – 2.07
11

J. Liu et al.
Bioorganic Chemistry 112 (2021) 104956
(2H, m, H13), 1.78 (1H, dd, J = 14.5, 3.2 Hz, H6), 1.73 – 1.63 (2H, m,
H8, 11-OH), 1.55 (2H, d, J = 14.9 Hz, H1, H7), 1.53 – 1.46 (1H, m, H13),
1.46 (3H, s), 1.44 – 1.37 (2H, m, H15, H8), 1.19 (3H, s), 1.15 (1H, td, J
= 14.2, 4.5 Hz, H18), 0.89 (3H, d, J = 7.0 Hz, H17), 0.78 (3H, d, J = 7.1
Hz, H16). 13C NMR (151 MHz, Chloroform-d) δ 216.68(C3), 165.83
(C21), 165.25, 163.33, 146.49, 144.56, 143.92, 139.24(C19), 138.72,
117.33(C20), 74.59(C11), 71.09(C14), 58.04(C4), 45.41(C9), 44.63
(C13), 44.01(C12), 41.90(C5), 36.64(C6), 36.02(C10), 35.04, 34.40
(C2), 30.37(C8), 26.82(C7), 26.44(C18), 24.81(C1), 16.75(C16), 14.75
(C15), 11.43(C17); HR-MS (ESI): Calcd for C29H37N3O5S (M+Na+):
563.2298; Found: 563.2289.
The tubes were placed in 37 ℃ and the growth of bacteria colonies was
recorded from each tube at 0, 1, 2, 4, 6 and 8 h. The sample solutions
(100 μL) were diluted 10-fold by sterile normal saline and plated on MH
agar plates. After spreading plates, the MH agar plates were incubated at
37 ℃ for 20 h. Finally, the colonies on the plates were counted. The PAE
value was calculated by the equation PAE = TA - TC (TA is the time
required for the bacteria in the test and control groups to increase by 1
log10 CFU/mL) and expressed in an hour.
4.4. In vivo efficacy of pleuromutilin derivatives
4.4.1. Neutropenic murine thigh infection model
Five-week-old, specific-pathogen-free (SPF), female ICR/Swiss mice
weighing 24–26 g were used for the experiment. As described previ-
ously, Neutropenic was rendered (<0.1 × 109/L) by injecting intraper-
itoneally with 150 mg/kg and 100 mg/kg cyclophosphamide (Mead
Johnson Pharmaceuticals, Evansville, IN) 3–5 days and 1 day, before
4.3. In vitro efficacy of pleuromutilin derivatives
4.3.1. Minimal inhibitory concentration (MIC) testing and minimum
bactericidal concentration (MBC) testing
The minimal inhibitory concentrations MIC of the target compounds
against methicillin-resistant S. aureus (ATCC 43300), S. aureus (ATCC
29213), S. aureus (AD3) and S. aureus (1 4 4) were determined using
tiamulin as positive controls. MIC values were determined by broth
dilution in accordance with the Clinical and Laboratory Standards
Institute (CLSI). Stock solutions of these compounds were made a stock
solution with a concentration of 1280 mg/mL by ultrapure-water and
Tween 80. The working solutions (320 mg/mL) of these compounds
were made by diluting stock solutions in sterile Mueller-Hinton (MH)
broth. The amount of inoculation in each well was 5 × 105 CFU/mL, and
a series of diluents of 2 times were prepared in Mueller-Hinton broth
medium (MHB). There were three parallel experiments for each com-
pound concentration. The plate was incubated at 37 ℃ for 24 h, and the
MIC value was recorded as the lowest inhibitory concentration of the
sample on the visible growth of the tested bacteria.
MASR infection. Each mouse was injected with 100 μL MRSA solution
(approximately 107 CFU/mL) into the thigh to establish the thigh
infection model 2 h before dosing. Then these mice were randomly
divided into three groups (5 per group) and intravenously injected
sterile normal saline, compound 133 (20 mg/kg), and tiamulin (20 mg/
kg) as a control group and two experimental groups respectively. Mice
were euthanized 24 h after intravenous injection. Thigh tissue of each
mouse was removed, collected, weighed, and homogenized in 5 mL of
iced saline. In the end, aliquots of five to six serial dilutions (10-fold)
were plated in duplicate on MH agar.
This study protocol was reviewed and approved by the Institutional
Animal Care and Use Committee of the South China Agricultural
University.
4.5. Cytotoxicity assay
After obtaining the MIC value, the 96-well plate was incubated at 37
℃ in 5% CO2 atmosphere for 24 h, and the MBC was determined by
inoculating the borehole bacterial solution from no obvious bacterial
growth on Mueller-Hinton Agar plate (MHA). Next, the MH agar plate
was further cultured at 37 ℃ for 24 h according to previous reference,
and the lowest concentration of the compound required to reduce the
bacterial count by more than 99.9% on the plate was MBC.
As previously described, cell viability was assessed using the MTT
assay. The cell line used in the experiment was RAW 264.7 cells. The
cells were seeded into 96-well plates at a density of 105 cells per well and
incubated at 37 ℃ overnight. The cells were then treated with com-
pounds 112–137 (8 mg/mL) and cultured at 37 ℃. After incubation for
16 h, the cells were incubated with 100
μ
L per well of MTT (0.5 mg/mL
in PBS) for another 4 h at 37 ℃ under 5% CO2. The medium was
4.3.2. Constant concentration time-kill curves
removed, and the remaining cells were dissolved in 150
μL DMSO for 30
The bactericidal activities of compounds 118 and 133 were deter-
mined by the constant concentration time-kill curve as reported in our
previous work. MRSA (ATCC 43300) grown in Muller-Hinton (MH) was
diluted to about 1 × 106 CFU/mL and treated with compound 118,
compound 133 and tiamulin of 2 × MIC, 4 × MIC, 8 × MIC, 16 × MIC,
min at 37 ℃. Finally, the absorbance of the samples at 540 nm was
recorded by the microplate spectrophotometer (BIO-TEK Instrument
Inc., USA)[4].
The RAW 264.7 cells were incubated by the same method. The
compound 118, compound 133 and tiamulin was chosen as the com-
pound to be tested. The cells were then treated with the resulting so-
32 × MIC. Samples were taken 100
μL respectively from the sub-
culturing inoculums at 0, 3, 6, 9, 12 and 24 h and serially diluted 10-flod
lution with varying concentrations (2, 4, 6, 8, 16, 32 μg/mL) of these
with sterile normal saline. Then 25 μL of the dilutions were plated on
coumponds and cultured for 16 h incubation at 37 ℃. Then the cells
were incubated with 100 L per well of MTT (0.5 mg/mL in PBS) for
another 4 h at 37 ℃ under 5% CO2. The medium was removed, and the
sterile MH agar plates. The total bacterial CFU/mL on the plates were
counted to calculate the bacterial colonies after incubated at 37 ℃ for
20 h. The time-kill curve was constructed by plotting log10 CFU/mL of
bacteria counts versus time.
μ
remaining cells were dissolved in 150 μL DMSO for 30 min at 37 ℃.
Finally, the absorbance of the samples at 540 nm was recorded by the
microplate spectrophotometer (BIO-TEK Instrument Inc., USA).
4.3.3. Determination of the postantibiotic effect (PAE)
The PAE of compound 118, compound 133 and tiamulin against
MRSA (ATCC 43300) were determined using time-kill methods ac-
cording previous work.
4.6. Molecular modeling
At first, the MRSA suspension was diluted to 1 × 106 CFU/mL in MH
broth as the culture medium. Then each test compound at final con-
centrations of 2 × MIC and 4 × MIC was added into test tubes that
contained the culture solution. The test tubes were incubated at a 37 ℃
constant temperature vibration incubator for 2 h. After incubating, the
test compounds were removed by centrifuging the tubes for 10 min at
3600 revolutions per minute, decanting and suspending in pre-warmed
MH broth. The bacterial pellet was washed using sterile normal saline
and then suspended in 5 mL MH broth in new tubes, diluted 1000-fold.
In order to reveal the binding modes of synthesized pleuromutilin
analogs, docking was performed based on the crystal structure of
S. aureus 50S ribosomal in complex with tiamulin (PDB ID code: 1XBP).
The peptidyl transferase center (PTC) model was built that consists of all
residues within 40 Å around the tiamulin in 5HL7. The binding site of
tiamulin in 5HL7 was set to the docking position. All compounds were
prepared with Avogadro 1.1.1, with a 5000 steps Steepest Descent as
well as 1000 steps Conjugate Gradients geometry optimization using
MMFF94 force field. Docking experiments were performed using
12

J. Liu et al.
Bioorganic Chemistry 112 (2021) 104956
[13] G.-Y. Zhang, Z. Zhang, K. Li, J. Liu, B.o. Li, Z. Jin, Y.-H. Liu, Y.-Z. 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.
AutoDock[29] Vina and Pymol.
Declaration of Competing Interest
[14] S.H. Derawey, M.N. Mosa, E.Q. Jasim, R.M.O. Hraishawi, Synthesis,
characterization and antibacterial evaluation of 1,3,4-oxadiazole derivatives, Int.
J. Res. Pharmaceut. Sci. 10 (2019) 2342–2350, https://doi.org/10.26452/ijrps.
v10i3.1477.
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.
[15] B.B. Kashid, P.H. Salunkhe, B.B. Dongare, K.R. More, V.M. Khedkar, A.A. Ghanwat,
Synthesis of novel of 2, 5-disubstituted 1, 3, 4- oxadiazole derivatives and their in
vitro anti-inflammatory, anti-oxidant evaluation, and molecular docking study,
Bioorg. Med. Chem. Lett. 30 (12) (2020) 127136, https://doi.org/10.1016/j.
bmcl.2020.127136.
Acknowledgments
[16] Y. Guo, T. Xu, C. Bao, Z. Liu, J. Fan, R. Yang, S. Qin, Design and synthesis of new
norfloxacin-1,3,4-oxadiazole hybrids as antibacterial agents against methicillin-
resistant Staphylococcus aureus (MRSA), Eur. J. Pharm. Sci. 136 (2019) 104966,
https://doi.org/10.1016/j.ejps.2019.104966.
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.
[17] M. Gao, J. Zeng, X. Fang, J. Luo, Z. Jin, Y. Liu, Y. Tang, Design, synthesis and
antibacterial evaluation of novel pleuromutilin derivatives possessing piperazine
linker, Eur. J. Med. Chem. 127 (2017) 286–295, https://doi.org/10.1038/s41557-
019-0261-6.
Appendix A. Supplementary material
[18] E. Yarmohammadi, H. Beyzaei, R. Aryan, A. Moradi, Ultrasound-assisted, low-
solvent and acid/base-free synthesis of 5-substituted 1,3,4-oxadiazole-2-thiols as
potent antimicrobial and antioxidant agents, Mol. Divers (2020), https://doi.org/
10.1007/s11030-020-10125-y.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104956.
[19] K. Povilas, G. Birute, M. Vytautas, P. Ruta, G. Ramune, P. Rita, G. Philip, P. Alius,
P. Vidmantas, Synthesis, ADMET Properties, and In Vitro Antimicrobial and
Antibiofilm Activity of 5-Nitro-2-thiophenecarbaldehyde N-((E)-(5-Nitrothienyl)
methylidene)hydrazone (KTU-286) against Staphylococcus aureus with Defined
Resistance Mechanisms, Antibiotics 9 (2020), https://doi.org/10.3390/
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13