Design, synthesis and biological activities of novel pleuromutilin derivatives with a substituted triazole moiety as potent antibacterial agents

Article abstract of DOI:10.1016/j.ejmech.2020.112604

A series of novel pleuromutilin derivatives possessing 1,2,3-triazole moieties were synthesized via click reactions under mild conditions. The in vitro antibacterial activities of these derivatives against 4 strains of S. aureus (MRSA ATCC 43300, ATCC 29213, AD 3, and 144) and 1 strain of E. coli (ATCC 25922) were tested by the broth dilution method. The majority of the synthesized derivatives displayed potent antibacterial activities against MRSA (MIC = 0.125–2 μg/mL). It was also found that most compounds had no significant inhibitory effect on the proliferation of RAW264.7 cells at the concentration of 8 μg/mL. Among these derivatives, compound 32 (～1.71 log₁₀ CFU/g) containing dimethylamine group side chain displayed more effective than tiamulin (～0.77 log₁₀ CFU/g) at the dose of 20 mg/kg in reducing MRSA load in thigh infected mice. Additionally, compound 32 (the survival rate was 50%) also displayed superior in vivo efficacy to that of tiamulin (the survival rate was 20%) in the mouse systemic model. Structure-activity relationship (SAR) studies resulted in compound 32 with the most potent in vitro and in vivo antibacterial activity among the series. Moreover, compound 32 was evaluated in CYP450 inhibition assay and showed moderate in vitro inhibition of CYP3A4 (IC₅₀ = 6.148 μM).

Full text of DOI:10.1016/j.ejmech.2020.112604

Journal Pre-proof
Design, synthesis and biological activities of novel pleuromutilin derivatives with a
substituted triazole moiety as potent antibacterial agents
Zhe Zhang, Kang Li, Guang-Yu Zhang, You-Zhi Tang, Zhen Jin
PII:
S0223-5234(20)30576-6
DOI:
https://doi.org/10.1016/j.ejmech.2020.112604
EJMECH 112604
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 March 2020
Revised Date: 16 June 2020
Accepted Date: 18 June 2020
Please cite this article as: Z. Zhang, K. Li, G.-Y. Zhang, Y.-Z. Tang, Z. Jin, Design, synthesis
and biological activities of novel pleuromutilin derivatives with a substituted triazole moiety as
potent antibacterial agents, European Journal of Medicinal Chemistry, https://doi.org/10.1016/
j.ejmech.2020.112604.
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Design, synthesis and biological activities of
novel pleuromutilin derivatives with a
substituted triazole moiety as potent
antibacterial agents
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Zhe Zhang , Kang Li , Guang-Yu Zhang , You-Zhi Tang *, Zhen Jin *
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Guangdong Provincial Key Laboratory of Veterinary Pharmaceutics Development
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and Safety Evaluation, College of Veterinary Medicine, South China Agricultural
University, Guangzhou 510642, China
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Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, 510642,
China
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Correspondence should be addressed:
College of Veterinary Medicine, South China Agricultural University, No. 483
Wushan Road, Tianhe District, Guangzhou 510642, China
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You-Zhi Tang,
Email: youzhitang@scau.edu.cn
or Zhen Jin,
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Email: jinzhenhami@scau.edu.cn
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Note: A series of novel pleuromutilin derivatives bearing both
aminophenylthiol and 1,2,3-triazole moiety were designed and
synthesized. Compound 32 from this series displayed superior in
vivo efficacy to the reference drug tiamulin against MRSA in
both the thigh infection model and the mouse systemic infection
model. Compound 32 possessed moderate in vitro inhibition of
CYP3A4.
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Design, synthesis and biological activities of
novel pleuromutilin derivatives with a
substituted triazole moiety as potent
antibacterial agents
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Zhe Zhang , Kang Li , Guang-Yu Zhang , You-Zhi Tang *, Zhen Jin *
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Guangdong Provincial Key Laboratory of Veterinary Pharmaceutics Development
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and Safety Evaluation, College of Veterinary Medicine, South China Agricultural
University, Guangzhou 510642, China
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Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, 510642, China
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Correspondence should be addressed:
College of Veterinary Medicine, South China Agricultural University, No. 483
Wushan Road, Tianhe District, Guangzhou 510642, China
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You-Zhi Tang,
Email: youzhitang@scau.edu.cn
or Zhen Jin,
Email: jinzhenhami@scau.edu.cn

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Abstract
A series of novel pleuromutilin derivatives possessing 1,2,3-triazole moieties were
synthesized via click reactions under mild conditions. The in vitro antibacterial
activities of these derivatives against 4 strains of S. aureus (MRSA ATCC 43300,
ATCC 29213, AD 3, and 144) and 1 strain of E. coli (ATCC 25922) were tested by the
broth dilution method. The majority of the synthesized derivatives displayed potent
antibacterial activities against MRSA (MIC = 0.125 ~ 2 μg/mL). It was also found
that most compounds had no significant inhibitory effect on the proliferation of
RAW264.7 cells at the concentration of 8 μg/mL. Among these derivatives,
compound 32 (~1.71 log10 CFU/g) containing dimethylamine group side chain
displayed more effective than tiamulin (~0.77 log CFU/g) at the dose of 20 mg/kg in
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reducing MRSA load in thigh infected mice. Additionally, compound 32 (the survival
rate was 50%) also displayed superior in vivo efficacy to that of tiamulin (the survival
rate was 20%) in the mouse systemic model. Structure-activity relationship (SAR)
studies resulted in compound 32 with the most potent in vitro and in vivo antibacterial
activity among the series. Moreover, compound 32 was evaluated in CYP450
inhibition assay and showed moderate in vitro inhibition of CYP3A4 (IC50 = 6.148
µM).
Keywords: Pleuromutilin; 1,2,3-triazole; MRSA; Synthesis; Antibiotics

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1. Introduction
As one of the prominent pathogen with high morbidity and mortality,
multidrug-resistant
Staphylococcus
aureus
(MRSA)
usually
cause
health-care-associated infections which posed a serious threat to human health around
the world [1]. Therefore, there is an urgent need to develop new antibiotics with new
mode of action that can be used to treat infections caused by multidrug-resistant S.
aureus (MRSA). Natural products have always played an important role in dealing
with human diseases, especially in the field of infectious diseases. Despite several
drawbacks, various natural products or their derivatives have been developed and
approved as antibacterial agents, such as penicillin G, tigecycline, vancomycin and
aztreonam [2]. The development of new antibacterial agents from natural products or
their semisynthetic derivatives is still the most effective way to deal with MRSA
infections.
Pleuromutilin (1, Figure 1) is a naturally tricyclic diterpenoid produced by the higher
fungi Basidiomycetes Pleurotus species Pleurotus mutilus and Pleurotus
Passeckerianus, first isolated in 1951 [3]. Pleuromutilin displays potent antibacterial
activity against gram-positive bacteria. It has been identified that pleuromutilin and its
derivatives could inhibit the bacterial protein synthesis through binding to the V
domain of the peptidyl transferase center (PTC) of the bacterial 50s ribosomal subunit
23s RNA [4-6]. Due to the unique mechanism of action, pleuromutilin and its
derivatives possess low propensity to select for cross-resistance to currently available
antibacterial agents. Thus, pleuromutilins have fascinated many researchers to explore

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and develop pleuromutilin derived effective antibacterial agents. The structural
modifications of the C14 side chain of pleuromutilin resulted in the discovery of
tiamulin (2, Figure 1) and valnemulin (3, Figure 1), which were approved and used as
veterinary antibiotics for poultry and pigs in 1979 and 1999, respectively [5]. In 2007,
another pleuromutilin derivative retapamulin (4, Figure 1) was approved for human
use. As the first pleuromutilin antibiotic for human use, retapamulin is used as a
topical antibiotic to treat skin infections [7]. Although pleuromutilin derivatives
usually exhibit potent antibacterial activity, most of them were discontinued develop
for human use due to their limited bioavailability and CYP450 inhibition [8]. More
recently, lefamulin (5, Figure 1), as the first systemic pleuromutilin antibiotic, was
approved by FDA for intravenous and oral treatment of community-acquired bacterial
pneumonia (CABP) on August 19, 2019 [6]. As many pleuromutilin derivatives have
poor bioavailability, lefamulin's success could be constituted as a milestone for the
pleuromutilin class of antibiotcs.
Figure 1 here
Previous work in our group has led to the synthesis and evaluation of various series of
novel pleuromutilin derivatives [9-11]. One of the derivatives containing the
2-aminophenylthiol in the C14 side chain (compound 7, Figure 1) displayed excellent
antibacterial activity in vitro and good efficacy in vivo against MRSA [11, 12].
Numerous pleuromutilin derivatives have been prepared and evaluated with excellent
antibacterial activity achieved by incorporating heteroaromatic substituents containing
a sulfide linkage into the C14 side chain [8, 13]. Additionally, 1,2,3-triazole moiety

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have been reported that might influence the antibacterial activity of the pleuromutilin
derivatives [14]. These research motivated us to develop the pleuromutilin derivatives
containing both aminophenylthiol and 1,2,3-triazole moiety.
We now report the synthesis and biological activities of novel pleuromutilin
derivatives with 1,2,3-triazole substituents incorporated into compound 7 and the
discovery of compound 32 which displayed good antibacterial activities against
MRSA both in vitro and in vivo.
2. Results and Discussion
2.1. Chemistry
A general synthesis strategy based on compound 22- (2-amino-phenylsulfanyl)
-22-deoxypleuromutilin (compound 7) and a variety of piperazine derivatives or
secondary amines were used (Scheme 1). Compound 7, based on which yielded a
series of pleuromutilin derivatives with potential antibacterial against MRSA in our
previous work [10], was used as a lead compound. Compound 8 was prepared by
condensation of acyl chloride group of chloroacetyl chloride with compound 7. Then,
compound 8 was converted into the azide compound 9 through a nucleophilic
substitution. This azide compound 9 was reacted with 27 different terminal alkynes
compounds 18-25, 53-71 that were all linked to various secondary amines or
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piperazine derivatives. A standard click reaction [15] based on the catalysis Cu ,
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which was produced in situ by Cu and sodium ascorbate, was applied for the
cycloaddition. Thus, pleuromutilin derivatives compounds 26-33 and 72-90 all
containing a 1,2,3-triazol linkage were prepared in good yields (Scheme 1). The

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structures of all those 27 pleuromutilin derivatives were confirmed by H NMR, C
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2.2. In vitro antibacterial activity
All the newly synthesized 1,2,3-triazole linked pleuromutilin derivatives were
evaluated for their antibacterial activity against methicillin-resistant S. aureus (ATCC
43300), S. aureus (ATCC 29213), E. coli (ATCC 25922) and two clinical strains of S.
aureus (AD3 and 144, isolated from Guangdong Province). The MICs and MBCs of
all synthesized pleuromutilin derivatives as well as the reference antibacterial drugs,
pleuromutilin and tiamulin were determined by the broth micro dilution methods
according to the Clinical and Laboratory Standards Institute (CLSI). S. aureus
(MRSA) ATCC 43300 was used as QC strain, and the quality control range of CLSI
quality control bacteria strain was taken as reference. The results of MIC and MBC
were shown in Table 1 and Table 2.
Table 1 here
Most of these new pleuromutilin derivatives showed potent antibacterial activities
against S. aureus (ATCC 43300), S. aureus (ATCC 29213), S. aureus (AD3) and S.
aureus (144) in vitro. The MIC value of all these 27 compounds against S. aureus
ranged from 2 to 0.125 μg/mL. The MIC value of all derivatives against E. coli
(ATCC 25922) are higher than 32 μg/mL. Among these compounds, compounds 26,
31, 32, 72, 86, 89 and 90 (MIC = 0.125 ~ 0.25 μg/mL) showed more potent
antibacterial activity against MRSA in comparison to that of tiamulin (MIC = 0.5
μg/mL). Compound 89 with 1-(3-nitrophenyl)-piperazine and compound 90 with

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1-(4-nitrophenyl)-piperazine exhibited the most potent activity with MIC value of
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Table 2 here
Firstly, compounds 26-33 were designed and prepared by using 1,2,3-triazole to link
various nitrogen heterocycles and secondary amine derivatives compound 7. These 8
compounds all showed potent activity against S. aureus (MIC = 0.25 ~ 1 μg/mL).
In order to explore SAR, different electron withdrawing and donating groups were
introduced. Then we borrowed from the previous work experience of the laboratory
and introduced a series of derivatives of piperazine [9] to obtain compounds 72-90.
Among them, compounds 77-82, 88-90, in which the electron-withdrawing group was
introduced into the phenylpiperazine of compound 73, resulting in a decrease in
antibacterial activity against MRSA. Nevertheless, compounds 89 and 90 showed
better antibacterial effect than compound 73. This may be explained by the strong
electron-withdrawing ability of the nitro group, which can generate local
electron-deficient sites in the molecule and interact with proteins and amino acids
present in the living system [16], other kinds of substituents or other substituent sites
may weaken these effects. However, in the face of the problem that drugs containing
nitro groups may cause serious adverse reactions in organisms, we still need to
explore in depth in the future development process of pleuromutilin derivatives.
Those ratios of MBC to MIC were all less than or equal to 4, indicating that all
synthesized pleuromutilin derivatives had good bactericidal ability [17]. MBC values
of all compounds for MRSA ranged 1×MIC from 4×MIC, which illustrated that these

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compounds have good bactericidal ability against MRSA. Among these derivatives,
compounds 26, 32, 89 and 90 exhibited more potent bactericidal effects than tiamulin.
Therefore, we carried out an in-depth study on the antibacterial activity of these four
compounds.
The time-kill kinetic approach was used to investigate the anti-MRSA activity of
compounds 26, 32, 89 and 90 in vitro. The experimental results were shown in
graphic form in Figure 2 and Figure SI 28. When the concentration of compounds 26
(Figure 2a) and 32 (Figure 2b) was 1×MIC, it had a definite inhibitory effect on
MRSA; when the concentration was 4×MIC or above, it had a good bactericidal effect
on MRSA and killed 99.9% of MRSA. However, compounds 89 (Figure SI 28a) and
90 (Figure SI 28b) can only play a role in killing MRSA at concentrations exceeding
8×MIC. However, from another aspect, after the compound reaches a certain
concentration, the bactericidal effect of these pleuromutilin derivatives and tiamulin
did not have a positive correlation with the increase of the compound concentration,
indicating that these derivatives and tiamulin were time-dependent antibacterial
agents. The key to the rational and scientific use of time-dependent antibacterial
agents was to optimize the time when bacteria were exposed to the effective
concentration of antibiotics, which can be achieved by multiple daily administration
in clinical practice. For pathogens with high MIC, even continuous intravenous
infusion was needed [18].
Figure 2 here
The postantibacterial effect (PAE) is an important indicator of antibiotic

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pharmacodynamics, and it has important guiding significance for the rational use of
antibiotics in clinical practice, evaluation of adverse reactions of antibiotics and
combined use [19]. Based on the test results that have been obtained, we performed
PAE tests on compounds 26 and 32. The bacterial growth kinetics curve was shown in
Figure 3 and the results of the PAEs were shown in Table 3. After espousing 4×MIC
for 2h, the PAE of compounds 26 and 32 were 1.84h and 2.74h, respectively. It is
worth mentioning that compound 32 showed better postantibacterial effect than
tiamulin at the concentrations of 2×MIC or 4×MIC.
Figure 3 here
Table 3 here
In the clinical use of antibacterial drugs, most of the pharmacodynamic indicators
referenced are the minimum antibacterial concentration (MIC) and the minimum
bactericidal concentration (MBC). However, this method of administration, which is
based on these two indicators, ignores the interaction between drugs and bacteria and
the potential of the drug to continue to inhibit the growth and reproduction of bacteria
[20]. To design the drug delivery regimen reasonably and provide a theoretical basis
for clinical adjustment of drug interval [21], we first performed time-kill kinetic tests
on four of this batch of compounds, and then PAE was tested on compounds 26 and
32 with stronger bactericidal effects. The above experimental results showed
compound 32 may be administered at longer intervals than tiamulin, suggesting that
compound 32 may have some potential value for clinical use in the treatment of
MRSA infections. Furthermore, a more scientific conclusion could be attained based

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on in vivo, dose-fractionation, PK/PD studies and target attainment studies as well as
PK studies.
2.3. In vivo antibacterial activity
2.3.1. Thigh infection model
In order to explore the in vivo efficacy of the drug, the mouse thigh infection model
with neutropenia was first established to evaluate the therapeutic effect of the drug on
the local infection in mice. Due to the longer PAE and the potent bacteriostatic ability
of compound 32 on MRSA in vitro, compounds 32 was tested in vivo at first in this
infection model.
First and foremost, a mice model of neutropenia was constructed by intraperitoneal
injection of cyclophosphamide into mice, and 0.9% saline was injected
intraperitoneally as a negative control group. The mice thigh muscle was injected with
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about 10 CFU/mL of the bacterial solution to establish a thigh infection model
caused by MRSA. The experimental results of this part were shown in Figure 4.
Figure 4 here
It can be concluded from Figure 4 that 20 mg/kg of compound 32 can significantly
reduce the bacterial load (~1.71 log CFU/g) in thighs, compared with the no drug
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control group the difference was statistically significant (P< 0.001, n = 6/group). The
bacterial clearance rate of tiamulin (~0.77 log CFU/g) in thigh muscle of mice was
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lower than that of compound 32, but compared with the control group without drugs,
tiamulin could still reduce the MRSA load in thighs (P< 0.01, n = 6/group). The
experimental results revealed that compound 32 displayed potency antibacterial

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activity than tiamulin in reducing MRSA load in the mouse thigh infection model.
Thus, compound 32 might be used in the treatment of MRSA infection.
2.3.2. MRSA infection model
After determining the potent antibacterial activity of compound 32 in the mice thigh
infection model with neutropenia, we further tested its therapeutic effect in mice in
the MRSA infection model with no drug treatment as a control group. As shown in
Figure 5, compound 32 (30 mg/kg) had a therapeutic effect on a mice model of
systemic infection with MRSA. After 7 days' administration, the survival rate of mice
in compound 32 group was 50%, which was significantly higher than that of tiamulin
group (20%), and the compound 32 did not directly cause animal death at this dose.
The result of in vivo efficacy demonstrated that compound 32 was a new antibiotic
candidate which was worth developing for clinical treatment of MRSA infections.
Figure 5 here
2.4. Cytotoxicity assay
Chemical substances act on the basic physiological processes of cells, which may lead
to reduced cell survival, inhibited proliferation and disturbance of physiological
functions, and trigger a series of adverse reactions [22]. Therefore, the antibiotics
approved for clinical use should have no inhibitory effect on cell proliferation within
a certain concentration range.
The cytotoxicity of these pleuromutilin derivatives to RAW 264.7 cells was evaluated
by MTT assay. The result of this part showed that most of these compounds including
compound 32 did not affect the viability of RAW 264.7 cells at the concentration of 8

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µg/mL (Figure SI 29), which was an acceptable starting point for further drug
discovery efforts.
2.5. Effect on liver microsomal CYP450 enzyme activity
During the process of pleuromutilin structural modification, azamulin (Figure 6a) was
screened out and entered the phase I clinical trial. Unfortunately, azamulin had been
eliminated during drug development due to its strong and irreversible inhibitory effect
on the CYP450 enzyme system (IC50 value for CYP3A is 0.03~0.24 µM) [23].
Additionally, pleuromutilin derivatives with thioether side chains had also been
reported to have strong inhibitory effects on CYP3A4 [8]. Therefore, we speculated
that these synthesized pleuromutilin derivatives might have inhibitory effect on
CYP3A4. Thus, compound 32 was selected to evaluate its inhibition potential against
CYP3A4. The CYP3A4 inhibition was analyzed by determining IC value in human
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liver microsomes through the use of testosterone as the probe substrate. The results of
this assay are shown in Figure 6b.
Figure 6 here
The results declared that compound 32 had an intermediate inhibitory effect on
CYP3A4 (IC50 = 6.148 µM). Compounds with 3 µM < IC50 <10 µM are generally
considered to be moderate CYP inhibitors, while compounds with IC > 10 µM are
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weak CYP inhibitors, compounds with IC < 3 µM are strong CYP inhibitors [24].
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[23], compound 32 could only inhibit the activity of CYP enzyme to some extent at a
certain concentration. Due to the complexity of living organisms, the results of in

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vitro experiments may be different from those of in vivo experiments, so further
clinical studies were still needed for comprehensive evaluation.
3. Conclusions
A series of novel pleuromutilin derivatives were designed and synthesized in few
steps. SAR studies have shown that the O atom at the C22 position was replaced by an
S atom, which contributes to the improvement of biological activity. The terminal was
connected to basic groups such as dimethylamine, diethylamine, tetrahydropyrrole
and a phenylpiperazine substituent bearing nitro groups have excellent in vitro
antibacterial activity against both sensitive and resistant S. aureus bacterial strains.
Among these prepared derivatives, compounds 26, 32, 89 and 90 exhibited the most
potent antibacterial activity and that were selected for the time-killing curve
determination. The results showed that compounds 26 and 32 manifested a more rapid
bactericidal kinetic effect on MRSA. Subsequently, the PAE (post-antibiotic effect) of
compounds 26 and 32 were determined. Compound 32 displayed a longer PAE time
than tiamulin against MRSA. The in vivo antibacterial activity of compound 32 was
further studied. The results indicated that compound 32 exhibited a potent in vivo
antibacterial effect than tiamulin in two MRSA infection mice models. CYP450
inhibition assay demonstrated that compound 32 had a moderate in vitro CYP3A4
inhibition. The current research results indicated that compound 32 might serve as a
possible lead compound for the development of novel pleuromutilin antibacterial
agent.
4. Experimental

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4.1. Materials
Pleuromutilin (>90% pure) was purchased from Great Enjoyhood Biochemical Co.
Ltd., (Sichuan, China). All analytical grade solvents were purchased from
Greagent-bate, and other reagents were purchased from Adamas. Purification of all
compounds by column chromatography was carried out using silica gel (200-300
1
mesh, Branch of Qingdao Haiyang Chemical Co. Ltd., Shandong, China). H-NMR
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and C-NMR spectra were recorded at Bruker AV-400 spectrometer. Among them,
the chemical shift value (δ) are reported in ppm, and the coupling constant (J) is in
Hertz. Tetramethylsilane was used as the internal standard in chloroform-d to analyze
the compounds. High-resolution mass spectra were conducted using Waters Acquity
UPLC-LCT Premier XE with an electro spray ionization (ESI) source.
4.2. Synthesis
The synthetic approaches for the preparation of the intermediates and the synthesis of
a series of novel pleuromutilin derivatives based on compound 1 were illustrated in
Scheme 1.
Scheme 1 here
Compound 1 (pleuromutilin) was purchased commercially. Compound 6
(22-O-tosylpleuromutilin)
and
compound
7
(22-(2-amino-phenylsulfanyl)-22-de-oxypleuromutilin) were synthesized according to
a reported method [11].
4.2.1. 22- (2-(2-chloroacetamido) phenyl) thioacety-l-yl-22-deoxypleuromutilin
(8)

3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Compound 22-(2-amino-phenylsulfanyl)-22-deoxypleuromutilin (compound 7) (1 g,
2.06 mmol) was dissolved in toluene (30 mL). Slowly added DIPEA (0.53 g, 4.12
mmol) and chloroacetyl chloride (0.47 g, 3.09 mmol) under ice bath, the remaining
mixture was reacted at room temperature for 0.5 h and then refluxed for 2 h. After the
reaction was completed, it was quenched with ice water, extracted three times with
chloroform, and the organic phases were combined. The organic phase was washed
successively with deionized water and saturated brine. The organic phase was dried
over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a
dark brown oil. The crude product was purified by silica gel column chromatography
using petroleum ether/ethyl acetate (2:1) as eluent to give a pure product, compound 8
(white solid, 85.1% yield).
4.2.2. 22- (2-(2- azido acetamido) phenyl) thioacety-l-yl-22-deoxypleuromutilin
(9)
Compound 8 (1 g, 1.78 mmol) was dissolved in acetone (20 mL), to which a solution
of sodium azide (0.58 g, 8.90 mmol) in deionized water (5 mL) was added. The
reaction was mixed in a round bottom flask and refluxed for 4 hours. After completion
of the reaction, the reaction solution was poured into a separating funnel, extracted
with chloroform, and washed twice with a saturated aqueous sodium chloride solution.
The organic phase was dried over anhydrous sodium sulfate and concentrated on a
rotary evaporator to give the crude product. The crude product was purified by silica
gel column chromatography using petroleum ether/ethyl acetate (4:1) as eluent to
obtain compound 9 (white solid, 73.5% yield).

3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
4.2.3. Synthesis of secondary amine analogs containing propynyl and piperazine
derivatives (18~25, 53~71)
Secondary amine derivatives (13.67 mmol) was dissolved in ethyl acetate (30 ml),
potassium carbonate (3.78 g, 27.34 mmol) was added, and then 3-bromopropyne (1.63
g, 13.67 mmol) was slowly added dropwise to the reaction system. The reaction was
performed at room temperature overnight. After the reaction was completed,
deionized water was added. Then the reaction solution was stirred for 0.5 h and
poured into a separating funnel, extracted twice with chloroform. The organic phase
was washed with saturated aqueous sodium chloride solution, then dried (Na SO )
2
4
and concentrated under reduced pressure to obtain a crude product. The crude product
was purified by column chromatography to obtain compound 18~25, 53~71,
respectively.
4.2.4. 22-(2-(2-(4-((diethylamino)methyl)-1H-1,2,3-triazol-1-yl)acetamido)phenyl)
thioacety-l-yl-22-deoxy pleuromutilin (26)
Compound 9 (1 g, 1.70 mmol) and compound 18 (0.19 g, 1.70 mmol) were added in a
mixture solution of t-butanol (10 mL) and of water (10 mL), and copper sulfate
pentahydrate (0.0033 g, 0.068 mmol) and sodium ascorbate ( 0.0013 g, 0.068 mmol),
and the reaction was stirred at room temperature for 3 h. After the reaction was
completed, the reaction solution was poured into a separating funnel, 40 ml of ethyl
acetate was added for extraction, and the organic phases were combined. The organic
phase was washed twice with a saturated aqueous sodium chloride solution, dried
over anhydrous sodium sulfate, and concentrated under reduced pressure to give the

3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
crude product. The crude product was purified by silica gel column chromatography
using petroleum dichloromethane/methanol (10: 1) as eluent to obtain the product
compound 26.
1
White powder; yield: 61%; H NMR (400 MHz, Chloroform-d) δ 9.54 (1 H, s),
8.37 (1 H, s), 8.25 (1 H, d, J = 8.3Hz), 7.56 (1 H, d, J = 7.8Hz), 7.36 (1 H, d, J =
7.8Hz), 7.07 (1 H, t, J = 7.6Hz), 6.44 (1 H, dd, J = 17.4, 10.9Hz, H19), 5.69 (1 H, d, J
=8.5Hz, H14), 5.43 (2 H, s, H20), 5.16 (1 H, d, J = 17.4Hz, H20), 4.23 (2 H, s), 3.53 –
3.31 (3 H, m, H11,H22), 2.98 (5 H, s, H2), 2.24 (3 H, ddt, J = 37.1, 19.5, 8.6Hz,
H4,H10,H13), 2.09 – 1.94 (2 H, m, H6,11-OH), 1.78 (1 H, d, J =14.7Hz, H8), 1.69 –
1.53 (3 H, m, H1,H7), 1.52 – 1.41 (2 H, m, H13), 1.38 (3 H, s, H15), 1.36 (9 H, s, H8),
13
1.12 (3 H, s, H18), 0.90 (3 H, d, J = 6.9Hz, H17), 0.53 (3 H, d, J = 7.0Hz, H16). C
NMR (101 MHz, Chloroform-d) δ 216.85 (C3), 169.70 (C21), 162.81, 139.20 (C19),
138.85, 136.45, 130.88, 130.19, 125.18, 122.49, 121.09, 117.25 (C20), 99.98, 74.55
(C11), 70.78 (C14), 66.04, 57.98 (C4), 53.43, 53.35 (C22), 46.18, 45.39 (C9), 44.58
(C13), 44.04 (C12), 41.75 (C15), 40.14, 36.53 (C6), 35.98 (C10), 34.41 (C2), 30.29
(C8), 28.27, 26.84 (C7), 26.39 (C18), 24.79 (C1), 16.52 (C16), 14.76 (C15), 11.60
+
(C17), 9.56. HR-MS (ESI): Calcd for C H N O S (M+H ): 680.3846; Found:
3
7
54
5
5
680.3839.
4.2.5. 22-(2-(2-(4-((morpholinomethy)methyl)-1H-1,2,3-triazol-1-yl)acetamido)
phenyl)thioacety-l-yl-22-deoxy pleuromutilin (27)
1
White powder; yield: 62%; H NMR (400 MHz, Chloroform-d) δ 9.42 (1 H, s),
8.28 (1 H, d, J = 8.3), 7.78 (1 H, s), 7.54 (1 H, d, J = 7.7Hz), 7.35 (1 H, t, J = 7.9Hz),

3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
7.06 (1 H, t, J = 7.6Hz), 6.42 (1 H, dd, J = 17.4, 11.0Hz, H19), 5.67 (1 H, d, J =
8.5Hz, H14), 5.35 (2 H, d, J = 3.6Hz, H20), 5.30 (1 H, t, J = 5.6Hz), 5.15 (1 H, d, J =
17.4Hz, H20), 3.72 (5 H, s), 3.40 (2 H, q, J = 16.1Hz, H22), 3.34 – 3.29 (1 H, m,
H11), 2.55 (4 H, t, J = 4.6Hz), 2.37 – 2.09 (4 H, m, H2,H4,H10,H13), 2.07 – 2.04 (1
H, m, 11-OH), 1.98 (1 H, dd, J = 16.1, 8.6Hz), 1.91 (1 H, s), 1.81 – 1.72 (1 H, m),
1.63 (2 H, d, J = 9.7Hz, H6,H8), 1.55 – 1.40 (4 H, m, H1,H7,H13), 1.37 (3 H, s, H15),
1.35 – 1.29 (1 H, m, H8), 1.11 (3 H, d, J = 4.2Hz, H18), 0.89 (3 H, d, J = 6.9Hz,
13
H17), 0.52 (3 H, d, J = 7.0Hz, H16). C NMR (101 MHz, Chloroform-d) δ 216.82
(C3), 169.34 (C3), 163.20, 144.78, 139.10 (C19), 138.86, 136.41, 130.82, 125.17,
124.46, 122.39, 120.96, 117.25 (C20), 77.36, 74.52 (C11), 70.59 (C14), 66.86, 57.97
(C4), 53.61, 53.41, 53.36 (C22), 45.38 (C9), 44.59 (C13), 43.99 (C12), 41.72 (C5),
39.80, 36.53 (C6), 35.96 (C10), 34.40 (C2), 30.29 (C8), 26.82 (C7), 26.51, 26.33
(C18), 24.78 (C1), 16.53 (C16), 14.76 (C15), 11.55 (C17). HR-MS (ESI): Calcd for
+
C H N O S (M+H ): 694.3638; Found: 694.3658.
37
52
5
6
4.2.6.
22-(2-(2-(4-((4-(2-hydroxyethyl)piperidin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)aceta
mido)phenyl)thioacety-l-yl-22-deoxy pleuromutilin (28)
1
White powder; yield: 57%; H NMR (400 MHz, Chloroform-d) δ 9.40 (1 H, s),
8.28 (1 H, d, J = 8.3Hz), 7.81 (1 H, s), 7.54 (1 H, s), 7.35 (1 H, t, J = 7.9Hz), 7.05 (1
H, t, J = 7.6Hz), 6.42 (1 H, dd, J = 17.4, 10.9Hz, H19), 5.67 (1 H, d, J = 8.5Hz, H14),
5.34 (1 H, d, J = 2.3Hz, H20), 5.28 (1 H, s), 5.15 (1 H, d, J = 17.3Hz, H20), 3.73 (2
H, s), 3.67 (2 H, t, J = 6.5Hz), 3.40 (2 H, t, J = 15.0Hz, H22), 3.36 – 3.29 (1 H, m,

3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
95
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
H11), 2.97 (2 H, d, J = 11.3Hz, H4,H10), 2.23 (4 H, ddd, J = 27.7, 13.1, 7.4Hz,
H2,H13), 2.11 (2 H, d, J = 11.5Hz), 2.05 (1 H, d, J = 3.7Hz, 11-OH), 1.98 (2 H, dd, J
= 16.1, 8.6Hz), 1.73 (4 H, dd, J = 25.6, 14.3Hz, H6,H8), 1.60 (3 H, dd, J = 20.2,
9.7Hz, H1,H7,H13), 1.51 (3 H, dd, J = 13.1, 6.7Hz H1,H7,H13), 1.46 – 1.41 (2 H, m),
1.37 (3 H, s, H15), 1.35 – 1.24 (4 H, m, H8,H18), 1.11 (3 H, s, H18), 0.89 (3 H, d, J =
1
3
6.9Hz, H17), 0.52 (3 H, d, J = 7.0Hz, H16). C NMR (101 MHz, Chloroform-d) δ
216.91 (C3), 169.26 (C21), 163.27, 145.12, 139.09 (C19), 138.85, 136.41, 130.80,
125.12, 124.61, 122.36, 120.93, 117.26 (C20), 99.98, 74.53 (C11), 70.51 (C14), 60.40,
57.99 (C4), 53.59, 53.50, 53.43 (C22), 45.38 (C9), 44.59 (C13), 43.98 (C12), 41.72
(C5), 39.68, 39.26, 36.55 (C6), 35.96 (C10), 34.41 (C2), 32.08, 32.03, 30.30 (C8),
26.81 (C7), 26.32 (C18), 24.78 (C1), 16.55 (C16), 14.78 (C15), 14.19, 11.56 (C17).
+
HR-MS (ESI): Calcd for C H N O S (M+H ): 736.4108; Found: 736.4126.
40
58
5
6
4.2.7.
22-(2-(2-(4-((3-(hydroxymethyl)piperidin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)aceta
mido)phenyl)thioacety-l-yl-22-deoxy pleuromutilin (29)
1
White powder; yield: 62%; H NMR (400 MHz, Chloroform-d) δ 9.38 (1 H, s),
8.26 (1 H, d, J = 8.3Hz), 7.80 (1 H, sHz), 7.52 (1 H, d, J = 7.8Hz), 7.33 (1 H, t, J =
7.9Hz), 7.04 (1 H, t, J = 7.6Hz), 6.41 (1 H, dd, J = 17.4, 11.0Hz, H19), 5.66 (1 H, d, J
= 8.5Hz, H14), 5.34 (2 H, s, H20), 5.28 (2 H, d, J = 11.6Hz), 5.13 (1 H, d, J =
17.3Hz, H20), 3.72 (2 H, s), 3.57 (1 H, d, J = 10.2Hz), 3.52 – 3.44 (2 H, m, H22,H11),
3.39 (2 H, d, J = 12.8Hz), 3.34 – 3.29 (1 H, m), 2.92 (1 H, d, J = 11.0Hz), 2.83 – 2.72
(1 H, m), 2.23 (5 H, ddd, J = 25.7, 15.5, 8.3Hz, H2,H4,H10,H13), 2.05 (2 H, s), 1.97

4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
(1 H, dd, J = 16.1, 8.6Hz, 11-OH), 1.75 (3 H, d, J = 14.7Hz, H6,H8), 1.66 – 1.57 (4
H, m, H1,H7,H13), 1.45 (3 H, d, J = 6.3Hz), 1.36 (3 H, s, H15), 1.27 (2 H, d, J =
13.0Hz, H8), 1.10 (3 H, s, H18), 0.88 (3 H, d, J = 6.9Hz, H17), 0.52 (3 H, d, J =
13
7.0Hz, H16). C NMR (101 MHz, Chloroform-d) δ 216.89 (C3), 169.24 (C21),
163.33, 145.05, 139.06 (C19), 138.91, 136.29, 130.74, 125.11, 124.62, 122.44, 120.97,
117.19 (C20), 74.54 (C11), 70.56 (C14), 66.51, 58.00 (C4), 56.89, 54.08, 53.70, 53.42
(C22), 45.39 (C9), 44.60 (C13), 43.99 (C12), 41.73 (C5), 39.64, 38.12, 36.55 (C6),
35.97 (C2), 34.41, 30.31 (C8), 27.11, 26.81 (C7), 26.37 (C18), 24.78 (C1), 24.54,
16.53 (C16), 14.77 (C15), 11.53 (C17). HR-MS (ESI): Calcd for C H N O S
39
56
5
6
+
(M+H ): 722.3951; Found: 722.3890.
4.2.8. 22-(2-(2-(4-((4-hydroxypiperidin-1-yl))methyl)-1H-1,2,3-triazol-1-yl)acet
amido)phenyl)thioacety-l-yl-22-deoxy pleuromutilin (30)
1
White powder; yield: 42%; H NMR (400 MHz, Chloroform-d) δ 9.41 (1 H, s),
8.26 (1 H, d, J = 8.4Hz), 7.80 (1 H, s), 7.52 (1 H, d, J = 7.8Hz), 7.34 (1 H, t, J =
7.9Hz), 7.05 (1 H, t, J = 7.6Hz), 6.40 (1 H, dd, J = 17.5, 10.9Hz, H19), 5.65 (1 H, d, J
= 8.5Hz, H14), 5.34 (2 H, s, H20), 5.28 (1 H, d, J = 10.5Hz), 5.13 (1 H, d, J =
17.3Hz, H20), 3.71 (3 H, d, J = 12.3Hz), 3.41 (2 H, t, J = 14.6Hz, H22), 3.30 (1 H, d,
J = 6.4Hz, H11), 2.84 (2 H, d, J = 12.1Hz), 2.31 – 2.22 (4 H, m, H2,H13), 2.21 –
2.15 (2 H, m, H4,H10), 2.04 (1 H, s, 11-OH), 1.97 (2 H, dd, J = 16.0, 8.5Hz), 1.91 –
1.85 (2 H, m), 1.75 (1 H, d, J = 14.5Hz), 1.64 – 1.55 (5 H, m, H1,H7,H13), 1.51 –
1.39 (3 H, m), 1.36 (3 H, s, H15), 1.33 – 1.24 (1 H, m, H8), 1.10 (3 H, s, H18), 0.88
1
3
(3 H, d, J = 6.9Hz, H17), 0.51 (3 H, d, J = 7.0Hz, H16). C NMR (101 MHz,

4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Chloroform-d) δ 216.94 (C3), 169.29 (C21), 163.29, 145.06, 139.05 (C19), 138.88,
137.71, 136.34, 130.76, 125.16, 124.62, 122.45, 120.99, 117.21 (C20), 111.33, 74.52
(C11), 70.56 (C14), 67.45, 57.99 (C4), 53.40, 53.06 (C22), 50.78, 45.38 (C9), 44.58
(C13), 43.98 (C12), 41.72 (C5), 39.70, 36.54 (C6), 35.96 (C10), 34.41 (C2), 34.21,
30.29 (C8), 26.81 (C7), 26.37 (C18), 24.78 (C1), 16.53 (C16), 14.77 (C15), 11.56
+
(C17). HR-MS (ESI): Calcd for C H N O S (M+H ): 708.3795; Found: 708.3699.
3
8
54
5
6
4.2.9.
22-(2-(2-(4-(pyrrolidin-1-yl-methyl)-1H-1,2,3-triazol-1-yl)acetamido)phenyl)
thioacety-l-yl-22-deoxy pleuromutilin (31)
1
White powder; yield: 40%; H NMR (400 MHz, Chloroform-d) δ 9.39 (1 H, s),
8.29 (1 H, dd, J = 8.3, 1.3Hz), 7.81 (1 H, s), 7.54 (1 H, dd, J = 7.8, 1.6Hz), 7.35 (1 H,
td, J = 7.9, 1.6Hz), 7.06 (1 H, td, J = 7.6, 1.4Hz), 6.43 (1 H, dd, J = 17.4,
11.0Hz,H19), 5.67 (1 H, d, J = 8.5 Hz, H14), 5.41 – 5.27 (3 H, m, H20), 5.15 (1 H, dd,
J = 17.5, 1.5Hz, H20), 3.86 (2 H, s), 3.46 – 3.28 (3 H, m, H11, H22), 2.63 (4 H, d, J
= 6.0Hz), 2.35 – 2.10 (3 H, m, H4,H10), 2.02 – 1.96 (4 H, m, 11-OH,H2,H13), 1.80
(4 H, h, J = 3.3Hz, H6,H8,H13), 1.70 – 1.57 (2 H, m, H1), 1.52 – 1.43 (2 H, m, H7),
1.38 (3 H, s, H15), 1.36 – 1.28 (1 H, m, H8), 1.12 (3 H, s, H18), 0.89 (3 H, d, J =
1
3
6.9Hz, H17), 0.53 (3 H, d, J = 7.0Hz, H16). C NMR (101 MHz, Chloroform-d) δ
216.89 (C3), 169.24 (C21), 163.27, 145.85, 139.08 (C19), 139.08, 138.85, 136.38,
130.77, 125.09, 124.33, 122.40, 122.40, 120.93, 117.21 (C20), 74.52 (C14), 70.49,
57.99, 53.94, 53.44 (C22), 50.49, 45.38 (C9), 44.59 (C13), 43.98 (C12), 41.72 (C5),
39.65, 36.55 (C6), 35.96 (C10), 34.41 (C2), 30.30 (C8), 26.81 (C7), 26.33 (C18),

4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
24.78 (C1), 23.49, 16.53 (C16), 14.77 (C15), 11.55 (C17). HR-MS (ESI): Calcd for
+
C H N O S (M+H ): 679.3689; Found: 679.3705.
37
52
5
5
4.2.10.
22-(2-(2-(4-((dimethylamino)methyl)-1H-1,2,3-triazol-1-yl)acetamido)phenyl)thio
acety-l-yl-22-deoxy pleuromutilin (32)
1
White powder; yield: 57%; H NMR (400 MHz, Chloroform-d) δ 9.36 (1 H, s),
8.26 (1 H, d, J =8.2 Hz), 7.76 (1 H, s), 7.51 (1 H, dd, J =7.7, 1.6 Hz), 7.32 (1 H, td, J=
7.9, 1.5 Hz), 7.06 – 7.00 (1 H, m), 6.39 (1 H, dd, J =17.4, 11.0 Hz, H19), 5.64 (1 H, d,
J =8.5 Hz, H14), 5.37 – 5.24 (4 H, m, H20), 5.12 (1 H, dd, J =17.4, 1.6 Hz, H20),
3.64 (2 H, s), 3.39 (1 H, d, J =16.2 Hz, H11), 3.35 – 3.29 (2 H, m, H22), 2.28 (6 H, s,
H2,H4,H10,H13), 2.27 – 2.10 (3 H, m), 2.06 – 2.02 (1 H, m, 11-OH), 1.95 (1 H, dd, J
=16.0, 8.6 Hz, H6), 1.74 (1 H, dq, J =14.6, 3.2 Hz, H8), 1.65 – 1.54 (2 H, m, H1),
1.50 – 1.38 (2 H, m, H7,H13), 1.35 (3 H, s, H15), 1.30 (1 H, dt, J =11.0, 4.0 Hz, H8),
1.09 (3 H, s, H18), 1.08 – 1.04 (1 H, m), 0.87 (3 H, d, J =6.9 Hz, H17), 0.50 (3 H, d, J
1
3
=7.1Hz, H16). C NMR (101 MHz, Chloroform-d) δ 216.78 (C3), 169.12 (C21),
163.28, 145.78, 139.07 (C21), 138.96, 136.31, 130.71, 125.07, 124.29, 122.43, 120.91,
117.11 (C20), 77.30, 77.09, 76.88, 74.49 (C11), 70.52 (C14), 57.99 (C4), 54.27, 53.44
(C22), 45.38 (C9), 45.13, 44.62 (C13), 44.00 (C12), 41.73 (C5), 39.59, 36.55 (C6),
35.99 (C10), 34.39 (C2), 30.30 (C8), 26.81 (C7), 26.42 (C18), 24.78 (C1), 16.50
+
(C16), 14.76 (C15), 11.51 (C17). HR-MS (ESI): Calcd for C H N O S (M+H ):
35
50
5
5
652.3533; Found: 652.3542.

4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
5
5
5
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
02
4.2.11.
22-(2-(2-(4-(piperidin-1-yl-methyl)-1H-1,2,3-triazol-1-yl)acetamido)phenyl)thioac
ety-l-yl-22-deoxy pleuromutilin (33)
1
White powder; yield: 47%; H NMR (400 MHz, Chloroform-d) δ 9.35 (1 H, s),
8.28 (1 H, d, J = 8.4Hz), 7.78 (1 H, s), 7.53 (1 H, d, J = 7.8Hz), 7.34 (1 H, t, J =
7.8Hz), 7.04 (1 H, t, J = 7.6Hz), 6.41 (1 H, dd, J = 17.4, 11.0Hz, H19), 5.66 (1 H, d, J
= 8.5Hz, H14), 5.33 (2 H, d, J = 2.4Hz, H20), 5.14 (1 H, d, J = 17.4Hz, H20), 3.69 (2
H, s), 3.45 – 3.27 (3 H, m, H22,H11), 2.52 – 2.44 (5 H, m, H4,H10), 2.24 (4 H, ddd, J
= 28.1, 10.6, 5.3Hz, H2,H13), 2.07 – 1.92 (2 H, m, 11-OH,H6), 1.80 – 1.71 (1 H, m),
1.61 – 1.56 (6 H, m, H1,H7,H13), 1.45 (4 H, ddd, J = 19.1, 8.6, 4.7Hz), 1.38 – 1.23 (4
H, m, ,H8,H15), 1.11 (4 H, s, H8,H18), 0.88 (3 H, d, J = 6.9Hz, H17), 0.52 (3 H, d, J
1
3
= 7.0Hz, H16). C NMR (101 MHz, Chloroform-d) δ 216.84 (C3), 169.15 (C21),
163.31, 145.43, 139.07 (C19), 138.88, 136.38, 130.76, 125.08, 124.47, 122.37, 120.90,
117.21 (C20), 74.52 (C11), 70.47 (C14), 57.99 (C4), 54.27, 53.93, 53.44, 51.63 (C22),
45.38 (C9), 44.60 (C13), 43.98 (C12), 41.72 (C5), 39.59, 36.55 (C6), 35.96 (C10),
34.40 (C2), 30.31 (C8), 26.81 (C7), 26.33 (C18), 25.89, 25.81, 24.78 (C1), 24.07,
16.53 (C16), 14.77 (C15), 11.53 (C17). HR-MS (ESI): Calcd for C H N O S
38
54
5
5
+
(M+H ): 692.3846; Found: 692.3719.
4.2.12.
22-(2-(2-(4-(4-methylpiperazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)acetamido)phe
nyl)thioacety-l-yl-22-deoxy pleuromutilin (72)

1
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
White powder; yield: 51%; H NMR (400 MHz, Chloroform-d) δ 9.39 (1 H, s),
8.27 (1 H, d, J = 8.3Hz), 7.77 (1 H, s), 7.53 (1 H, d, J = 7.7Hz), 7.34 (1 H, t, J =
7.9Hz), 7.05 (1 H, t, J = 7.6Hz), 6.41 (1 H, dd, J = 17.4, 11.0Hz, H19), 5.66 (1 H, d, J
= 8.5Hz, H14), 5.40 – 5.26 (3 H, m, H20), 5.15 (1 H, d, J = 17.4Hz, H20), 3.74 (2 H,
s), 3.38 (3 H, dt, J = 24.2, 12.1Hz, H11,H22), 2.60 (5 H, s), 2.48 (4 H, s), 2.29 (6 H, s,
H2,H4,H10,H13), 2.26 – 2.10 (2 H, m,H8), 2.07 – 1.92 (2 H, m, 11-OH,H6), 1.69 –
1.51 (3 H, m, H1,H7), 1.48 – 1.41 (1 H, m, H13), 1.37 (3 H, s, H15), 1.34 – 1.23 (1 H,
m,H8), 1.11 (3 H, s, H18), 0.89 (3 H, d, J = 7.0Hz, H17), 0.52 (3 H, d, J = 7.0Hz,
1
3
H16). C NMR (101 MHz, Chloroform-d) δ 216.79 (C3), 169.24 (C21), 163.24,
144.96, 139.09 (C19), 138.89, 136.35, 130.75, 125.13, 124.45, 122.42, 120.97, 117.22
(C20), 74.52 (C11), 70.55 (C14), 66.21, 57.98 (C4), 54.89, 53.41, 53.10, 52.69, 45.86,
45.38 (C9), 44.62 (C19), 43.99 (C12), 41.73, 39.71, 36.54 (C6), 35.97 (C10), 34.39
(C2), 30.30 (C8), 26.82 (C7), 26.35 (C18), 24.78 (C1), 16.52 (C16), 14.77 (C15),
+
14.74, 11.53 (C17). HR-MS (ESI): Calcd for C H N O S (M+H ): 707.3955; Found:
3
8
55
6
5
707.3823.
4.2.13.
22-(2-(2-(4-((4-phenylpiperazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)acetamido)phe
nyl)thioacety-l-yl-22-deoxy pleuromutilin (73)
1
White powder; yield: 53%; H NMR (400 MHz, Chloroform-d) δ 9.41 (1 H, s),
8.29 (1 H, d, J = 8.3Hz), 7.82 (1 H, s), 7.54 (1 H, d, J = 7.8Hz), 7.36 (1 H, t, J =
7.9Hz), 7.25 (1 H, d, J = 7.6Hz), 7.08 (1 H, q, J = 8.3, 7.6Hz), 6.92 (2 H, d, J =
8.2Hz), 6.85 (1 H, t, J = 7.3Hz), 6.43 (1 H, dd, J = 17.4, 11.0Hz, H19), 5.67 (1 H, d, J

5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
= 8.5Hz, H14), 5.37 – 5.32 (2 H, m, H20), 5.16 (1 H, d, J = 17.4Hz, H20), 3.82 (2 H,
s), 3.49 – 3.32 (3 H, m, H11,H22), 3.22 (4 H, q, J = 6.4, 4.8Hz), 2.73 (5 H, t, J =
4.9Hz, H2,H13), 2.24 (3 H, dq, J = 27.8, 9.3, 8.0Hz, H4,H10), 2.08 – 1.93 (2 H, m,
11-OH), 1.77 (1 H, dd, J = 14.5, 3.4Hz, H6), 1.63 (2 H, td, J = 12.3, 11.1, 7.1Hz, H8),
1.53 – 1.41 (4 H, m, H1,H7,H13), 1.38 (3 H, s, H15), 1.32 (1 H, dd, J = 14.1, 3.6Hz,
H8), 1.12 (3 H, s, H18), 0.90 (3 H, d, J = 7.0Hz, H17), 0.52 (3 H, d, J = 7.0Hz, H16).
1
3
C NMR (101 MHz, Chloroform-d) δ 216.75 (C3), 169.27 (C21), 163.23, 151.21,
144.89, 144.83, 139.12 (C19), 138.89, 136.37, 133.26, 130.79, 129.08, 126.03, 125.15,
124.54, 122.41, 120.97, 119.74, 117.23 (C20), 116.10, 74.54 (C11), 70.59 (C14),
57.98 (C4), 53.45, 53.23, 52.90 (C22), 49.06, 45.38 (C9), 44.62, 44.01 (C12), 41.74
(C5), 39.75, 36.54 (C6), 35.98 (C10), 34.40 (C2), 30.41, 30.31 (C8), 26.83 (C7),
26.35 (C18), 24.79 (C1), 16.53 (C16), 14.77 (C15), 11.54 (C17). HR-MS (ESI):
+
Calcd for C H N O S (M+H ): 769.4111; Found: 769.4102.
4
3
57
6
5
4.2.14.
22-(2-(2-(4-((4-(o-tolyl)piperazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)acetamido)ph
enyl)thioacety-l-yl-22-deoxy pleuromutilin (74)
1
White powder; yield: 57%; H NMR (400 MHz, Chloroform-d) δ 9.42 (1 H, s),
8.30 (1 H, d, J = 8.3Hz), 7.85 (1 H, s), 7.55 (1 H, d, J = 7.8Hz), 7.36 (1 H, t, J =
7.9Hz), 7.16 (2 H, t, J = 8.2Hz), 7.02 (3 H, ddt, J = 21.5, 14.8, 7.5Hz), 6.43 (1 H, dd,
J = 17.4, 10.9Hz, H19), 5.68 (1 H, d, J = 8.5Hz, H14), 5.42 – 5.31 (3 H, m, H20),
5.16 (1 H, d, J = 17.4Hz, H20), 3.84 (2 H, s), 3.51 – 3.29 (3 H, m, H11,H22), 2.97 (4
H, t, J = 4.8Hz), 2.75 (4 H, s), 2.30 (4 H, s, H2, H13), 2.28 – 2.13 (2 H, m, H4,H10),

5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
2.06 – 1.95 (2 H, m, 11-OH), 1.82 – 1.72 (1 H, m, H7), 1.68 – 1.56 (2 H, m, H1), 1.55
– 1.41 (2 H, m, H13), 1.38 (3 H, s, H15), 1.36 – 1.24 (1 H, m, H8), 1.12 (4 H, s, H18),
13
0.90 (3 H, d, J = 6.9Hz, H17), 0.53 (3 H, d, J = 7.0Hz, H16). C NMR (101 MHz,
Chloroform-d) δ 216.77 (C3), 169.28 (C21), 163.22, 151.35, 145.09, 139.13 (C19),
138.79, 136.40, 132.59, 131.03, 130.80, 126.53, 125.14, 124.58, 123.16, 122.41,
120.96, 120.90, 119.02, 117.13 (C20), 116.16, 110.50, 74.55 (C11), 70.58 (C14),
57.99 (C4), 53.53, 53.32 (C22), 51.52, 45.39 (C9), 44.61 (C13), 44.01 (C12), 41.74
(C5), 39.76, 36.55 (C6), 35.98 (C10), 34.40 (C2), 30.31 (C8), 26.83 (C7), 26.34 (C18),
24.80 (C1), 17.86, 16.53 (C16), 14.78 (C15), 11.54 (C17). HR-MS (ESI): Calcd for
+
C H N O S (M+H ): 783.4268; Found: 783.4269.
44
59
6
5
4.2.15.
22-(2-(2-(4-((4-(m-tolyl)piperazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)acetamido)p
henyl)thioacety-l-yl-22-deoxy pleuromutilin (75)
1
White powder; yield: 59%; H NMR (400 MHz, Chloroform-d) δ 9.41 (1 H, s),
8.30 (1 H, d, J = 8.3Hz), 7.83 (1 H, d, J = 2.4Hz), 7.55 (1 H, d, J = 7.8Hz), 7.36 (1 H,
t, J = 7.9Hz), 7.11 (2 H, dt, J = 31.9, 7.6Hz), 6.77 – 6.65 (3 H, m), 6.43 (1 H, dd, J =
17.5, 10.9Hz, H19), 5.67 (1 H, d, J = 8.5Hz, H14), 5.39 – 5.32 (2 H, m, H20), 5.16 (1
H, d, J = 17.4Hz, H20), 3.82 (2 H, d, J = 2.4Hz), 3.49 – 3.29 (3 H, m, H11,H22), 3.21
(4 H, t, J = 4.7Hz), 2.72 (4 H, t, J = 4.8Hz), 2.32 (4 H, s, H2,H13), 2.22 (2 H, dt, J =
16.8, 10.1Hz, H4,H10), 2.08 – 1.95 (2 H, m, 11-OH), 1.77 (1 H, d, J = 14.5Hz, H6),
1.62 (2 H, t, J = 11.0Hz, H8,H13), 1.54 – 1.40 (4 H, m, H1,H7), 1.38 (4 H, s,
H8,H15), 1.12 (4 H, d, J = 4.9Hz, H18), 0.90 (3 H, d, J = 6.9Hz, H17), 0.52 (3 H, d, J

1
3
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
= 7.0Hz, H16). C NMR (101 MHz, Chloroform-d) δ 216.78 (C3), 169.29 (C21),
163.22, 151.27, 144.89, 139.13 (C19), 138.87, 138.75, 136.59, 136.40, 130.80, 128.92,
125.15, 124.56, 122.39, 120.96, 120.68, 117.26 (C20), 116.96, 113.24, 74.54 (C11),
70.57 (C14), 57.98 (C4), 53.46, 53.23, 52.94 (C22), 49.14, 45.38 (C9), 44.61 (C13),
44.00 (C12), 41.73 (C5), 39.76, 36.54 (C6), 35.97 (C10), 34.41 (C2), 30.30 (C8),
26.83 (C7), 26.33 (C18), 24.79 (C1), 21.76, 16.54 (C16), 14.77 (C15), 13.28, 11.55
+
(C17). HR-MS (ESI): Calcd for C H N O S (M+H ): 783.4268; Found: 783.4270.
4
4
59
6
5
4.2.16.
22-(2-(2-(4-((4-(p-tolyl)piperazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)acetamido)ph
enyl)thioacety-l-yl-22-deoxy pleuromutilin (76)
1
White powder; yield: 59%; H NMR (400 MHz, Chloroform-d) δ 9.42 (1 H, s),
8.29 (1 H, d, J = 8.3Hz), 7.83 (1 H, s), 7.58 – 7.51 (1 H, m), 7.36 (1 H, t, J = 8.2Hz),
7.06 (3 H, dd, J = 7.9, 5.4Hz), 6.84 (2 H, d, J = 8.2Hz), 6.43 (1 H, dd, J = 17.4,
11.0Hz, H19), 5.67 (1 H, d, J = 8.5Hz, H14), 5.40 – 5.32 (2 H, m, H20), 5.16 (1 H, d,
J = 17.4Hz, H20), 3.82 (2 H, s), 3.43 (2 H, t, J = 15.6Hz, H11,H22), 3.33 (1 H, s),
3.17 (4 H, t, J = 5.0Hz), 2.73 (4 H, t, J = 5.0Hz), 2.27 (5 H, s, H2,H4,H13), 2.25 –
2.15 (1 H, m, H10), 2.08 – 1.93 (2 H, m, 11-OH), 1.93 – 1.83 (2 H, m, H8), 1.82 –
1.72 (1 H, m, H6), 1.63 (2 H, td, J = 12.1, 10.7, 6.2Hz, H1), 1.54 – 1.40 (3 H, m,
H7,H13), 1.38 (3 H, s, H15), 1.11 (4 H, d, J = 4.9Hz, H8,H18), 0.90 (3 H, d, J =
1
3
6.9Hz, H17), 0.52 (3 H, d, J = 7.0Hz, H16). C NMR (101 MHz, Chloroform-d) δ
216.82 (C3), 169.32 (C21), 163.22, 149.11, 144.85, 139.11 (C19), 138.81, 136.38,
130.80, 129.61, 129.31, 125.17, 124.59, 123.75, 122.41, 120.98, 117.21 (C20), 116.47,