Synthesis, biological activities and docking studies of pleuromutilin derivatives with piperazinyl urea linkage

Article abstract of DOI:10.1080/14756366.2021.1900163

Antibiotics resistance is becoming increasingly common, involving almost all antibiotics on the market. Diseases caused by drug resistant bacteria, such as MRSA, have high mortality and negatively affect public health. The development of new drugs would be an effective means of solving this problem. Modifications based on bioactive natural products could greatly shorten drug development time and improve success rate. Pleuromutilin, a natural product from the basidiomycete bacterial species, is a promising antibiotic candidate. In this study, a series of novel pleuromutilin derivatives possessing piperazinyl urea linkage were efficiently synthesised, and their antibacterial activities and bactericidal properties were evaluated via MIC, MBC and Time-kill kinetics assays. The results showed that all compounds exhibited potent activities against tested strains, especially MRSA strains with MIC values as low as 0.125 μg/mL; 8 times lower than that of marketed antibiotic Tiamulin. Docking studies indicate substituted piperazinyl urea derivatives could provide hydrogen bonds and initiate π-π stacking between molecules and surrounding residues.

Full text of DOI:10.1080/14756366.2021.1900163

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Synthesis, biological activities and docking studies
of pleuromutilin derivatives with piperazinyl urea
linkage
Yuanyuan Zhang, Chuan Xie, Yang Liu, Feng Shang, Rushiya Shao, Jing Yu,
Chunxia Wu, Xinghui Yao, Dongfang Liu & Zhouyu Wang
To cite this article: Yuanyuan Zhang, Chuan Xie, Yang Liu, Feng Shang, Rushiya Shao, Jing Yu,
Chunxia Wu, Xinghui Yao, Dongfang Liu & Zhouyu Wang (2021) Synthesis, biological activities
and docking studies of pleuromutilin derivatives with piperazinyl urea linkage, Journal of Enzyme
Inhibition and Medicinal Chemistry, 36:1, 764-775, DOI: 10.1080/14756366.2021.1900163
To link to this article: https://doi.org/10.1080/14756366.2021.1900163
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2021, VOL. 36, NO. 1, 764–775
https://doi.org/10.1080/14756366.2021.1900163
RESEARCH PAPER
Synthesis, biological activities and docking studies of pleuromutilin derivatives
with piperazinyl urea linkage
Yuanyuan Zhanga,b, Chuan Xiea, Yang Liua, Feng Shanga, Rushiya Shaoa, Jing Yuc, Chunxia Wua, Xinghui Yaoa,
Dongfang Liua and Zhouyu Wanga,b
b
aDepartment of Chemistry, School of Science, Xihua University, Chengdu, China; Yibin Key Laboratory of Research and Application of Small
c
Organic Chiral Molecules, Yibin Research Institute of Xihua University, Yibin, China; Kampo Medicine Pharmacology Research Laboratory,
Graduate School of Pharmaceutical Sciences, Yokohama University of Pharmacy, Yokohama-shi, Japan
ABSTRACT
ARTICLE HISTORY
Received 8 January 2021
Revised 25 February 2021
Accepted 3 March 2021
Antibiotics resistance is becoming increasingly common, involving almost all antibiotics on the market.
Diseases caused by drug resistant bacteria, such as MRSA, have high mortality and negatively affect public
health. The development of new drugs would be an effective means of solving this problem. Modifications
based on bioactive natural products could greatly shorten drug development time and improve success
rate. Pleuromutilin, a natural product from the basidiomycete bacterial species, is a promising antibiotic
candidate. In this study, a series of novel pleuromutilin derivatives possessing piperazinyl urea linkage were
efficiently synthesised, and their antibacterial activities and bactericidal properties were evaluated via MIC,
MBC and Time-kill kinetics assays. The results showed that all compounds exhibited potent activities
against tested strains, especially MRSA strains with MIC values as low as 0.125lg/mL; 8 times lower than
that of marketed antibiotic Tiamulin. Docking studies indicate substituted piperazinyl urea derivatives could
provide hydrogen bonds and initiate p-p stacking between molecules and surrounding residues.
KEYWORDS
Pleuromutilin; piperazinyl
urea; synthesis; biological
activities; MRSA
Introduction
disease in poultry10,11. Retapamulin (Figure 1, 4) is used for the
treatment of skin impetigo12,13
Lefamulin (Figure 1, 5) is
.
Current available antibiotics are losing their effectiveness due to
increasing bacterial resistance. Diseases caused by multi-drug
resistant bacteria are particularly difficult to prevent and treat, and
across the world, approximately 700,000 people die annually as a
result of them1. Among drug-resistant strains, methicillin-resistant
Staphylococcus aureus (MRSA) is one of the most problematic,
with pathogens that spread rapidly and cause severe
diseases such as septicaemia, pneumonia, osteomyelitis and endo-
carditis2,3. Average treatment time and MRSA patient mortality
are, respectively, five and three times higher than those of
patients infected by common pathogens. Antibiotics are generally
used to control MRSA infections, however the majority of common
antibiotics used in clinical treatment were ineffective against
MRSA. Therefore, it is crucial to develop and discover novel anti-
bacterial agents4.
approved for treatment of community-acquired bacterial pneumo-
nia (CABP)14. These derivatives can inhibit bacterial protein synthe-
sis via specific interaction with 23S rRNA of the 50S bacterial
ribosome unit15, which are unaffected by resistance to major anti-
biotic classes, such as beta-lactam antibiotics, tetracyclines, macro-
lides, fluoroquinolones, and others. Thus, pleuromutilin is
a
promising candidate for treating drug-resistant bacteria infec-
tions16, and many compounds derived from four marketed drugs
using the thioether as the linkage at the C14 side chain were
developed in recent years17–20. To increase side chain diversity
and improve modification success rate, the bioactive moiety piper-
azinyl urea, displaying multiple biological activities such as anti-
fungal21, analgesic22, antibacterial23 and antitumour24 effects
was introduced. In this study, a series of pleuromutilin derivatives
6aꢀz with piperazinyl urea (Scheme 1) were efficiently
synthesised. Their activities were evaluated against MRSA and
Gram-negative strains, and interactions were examined by molecu-
lar docking.
Natural
products
such
as
Penicillin,
Erythromycin,
Cephalosporin C and Kanamycin are often used as main com-
pounds in drug discoveries due to their potent bioactivity.
Corresponding
semi-synthesised
derivatives
Amoxicillin,
Azithromycin, Cefpirome and Amikacin all exhibit higher potency
than natural products5–7. Pleuromutilin (Figure 1, 1), a diterpenoid
natural product from the basidiomycete species, shows moderate
activities against Gram-positive strains and mycoplasmas8,9. Four
semi-synthesised marketed drugs based on its structure have
already been developed. Tiamulin and valnemulin (Figure 1, 2 and
3) can effectively prevent and control swine dysentery, mycoplas-
Results and discussion
Chemistry
The piperazinyl urea linkage can be constructed with substituted
amines via three strategies (Scheme 1). Pleuromutilin 1 was
mal diseases such as enzootic pneumonia and chronic respiratory reacted with tosyl chloride and triethylamine in DCM to form
CONTACT Dongfang Liu
No. 999, Jinzhou Road, Chengdu, 610039, China
ß 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
ldf@mail.xhu.edu.cn; Zhouyu Wang
zhouyuwang@mail.xhu.edu.cn
Department of Chemistry, School of Science, Xihua University,
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
765
pleuromutilin-22-O-tosylate 7 in 78.3% yield, which could convert 12fꢀu with trichloroacetyl chloride in DCM with 54.7 ꢀ 97.2%
to 22-(piperazine-1-yl)-22-deoxypleuromutilin 8 by treatment with yield. Moreover, in regard to the secondary amine, the expected
NaI, piperazine and potassium carbonate in dry THF with a 75.2% product was rarely obtained using the above two strategies. N-
yield. Compounds 6a ~ e were synthesised by treating 7 with N- methylaniline 12v was treated with 4-nitrophenyl carbonochlori-
substituted-1-carboxamide 9a ~ e in the presence of a catalytic date in the presence of N-methylmorpholine (NMM) in DCM to
amount of NaI and K2CO3 in MeCN with 78.3 ꢀ 92.3% yield. Here, give 11, which was condensed without further purification and
the primary amines 12aꢀe reacted with bis(trichloromethyl)car- reacted with 22-(piperazine-1-yl)-22-deoxypleuromutilin 8 to form
bonate (BTC) or N, N’-carbonyldiimidazole (CDI) to form the corre- N-methyl derivative 6v in 40.5% yield. Additionally, 6wꢀz were
sponding isocyanate, which was used to form piperazinyl urea synthesised from corresponding Boc-protected amine and nitro
moiety by reacting with N-Boc-piperazine in the presence of trie- molecules (Scheme 2). 6w was gained from deprotecting 6g in
thylamine with 56.2 ꢀ 82.3% yield. Afterwards, 9a ~ e were the presence of TFA in 95.6% yield, and two other amine deriva-
obtained via removal of the above Boc protected group using tri- tives 6x and 6y were attained by stannous chloride reduction of
fluoroacetic acid (TFA) in 93.7 ꢀ 97.2% yield. Compounds 6f ~ u 6d and 6p in 92.5% and 75.5% yields, respectively. 6y was used
were synthesised in 45.6 ꢀ 82.7% yield by treating 8 in the pres- to react with chloracetyl chloride to get 14 in the presence of trie-
ence of potassium carbonate with substituted 2,2,2-trichloro-aceta- thylamine with 70.1% yield, which was treated with morpholine to
mide 10fꢀu, which were acquired by reacting primary amines produce 6z in 50.3% yield.
19
1
: R=
HO
4
S
: R=
OH
O
S
12
N
N
R
14
5
2: R=
OH
O
22
H
9
NH2
S
H
N
1
5: R=
H2N
3
: R=
S
O
O
Figure 1. Structures of pleuromutilin derivatives 2 ꢀ 5 developed from natural Pleuromutilin 1.
OH
OH
OH
O
HN
O
O
i
ii
HO
TsO
N
O
H
O
O
H
H
O
O
O
1
8
7
R
N
1
R
1
Cl
NO
2
NH
Cl
Cl
O
N
N
R
2
R
2
N
O
O
O
9a~e
v
Boc
iii
iv
R1
N
R1
N
11
10f~u
N
NH
vii
vi
N
N
R2
R2
O
13a~e
O
9a~e
O
R1
OH
N
N
O
R1
Cl
N
Cl
Cl
R2
N
H
N
viii
ix
O
H
R2
R1
R2
O
10f~u
12a~v
NO2
O
O
N
O
6a~v
11
H
H
N
N
F
Boc
6e
6a
6d: R1=
: R1=
R2=H
6g
: R1=
6b: R1=
R2=H
6c: R1=
6f: R1=
: R1=
R2=H
6h: R1=
N
O
NO2
Br
R2=H
OMe
R2=H
R2=H
R2=H
R2=H
OMe
CF3
6p
NO2
Me
CF3
NH2
N
N
: R1=
R2=H
6o: R1=
R2=H
6l: R1=
R2=H
6n
6j: R1=
6m: R1=
: R1=
R2=H
6i
: R1=
6k: R1=
N
R2=H
R2=H
R2=H
R2=H
OMe
N
N
6q: R1=
6r
: R1=
6v: R1=
R2=CH3
6t: R1=
R2=H
6u: R1=
6s: R1=
O
R2=H
R2=H
R2=H
R2=H
CF3
Scheme 1. Synthesis route of target compounds 6a ~ v. Reagents and conditions: (i) TsCl, triethylamine, DCM, 25 ꢁC; (ii) Piperazine, K2CO3, NaI, THF, reflux; (iii) 9a ~ e,
NaI, K2CO3, MeCN, 70 ꢁC; (iv) 10f~ u, K2CO3, DMF, 80 ꢁC; (v) 11, DMAP, MeCN, 70 ꢁC; (vi) CDI, triethylamine, N-Boc piperazine, MeCN and DMF, 25 ꢁC for 13a and 13b,
or BTC, triethylamine, N-Boc piperazine, THF and DCM, 25ꢁC for 13c ~ e, (vii) TFA, DCM, 25 ꢁC; (viii) Trichloroacetyl chloride, triethylamine, DCM, 25ꢁC; (ix) 4-
Nitrophenyl carbonochloridate, NMM, DCM, 25ꢁC.

766
Y. ZHANG ET AL.
O
x
OH
H2N
N
H
N
O
O
N
O
H
R1
OH
N
N
O
6w
R2
N
O
H
O
O
OH
O
N
N
n
O
6g 6d 6p
, or
xi
H
N
H2N
O
H
6x
: n=0
6y
: n=1
O
O
O
OH
OH
O
N
N
O
O
N
N
O
O
xii
H
xiii
H
6y
Cl
N
N
N
N
N
O
O
H
H
H
H
14
6z
O
O
Scheme 2. Synthesis route of target compounds 6w ~ z. Reagents and conditions: (x) TFA, DCM, 25ꢁC from 6g; (xi) SnCl2, EtOH, reflux from 6d or 6p; (xii)
Chloroacetyl chloride, triethylamine, THF, 25ꢁC; (xiii) Morpholine, K2CO3, THF, reflux.
Biological activities
Table 1. MIC and MBC values of pleuromutilin derivatives possessing piperazinyl
urea linkage.
MIC and MBC values of the synthesised compounds against three
Gram-positive bacteria (i.e.: Staphylococcus aureus (S. aureus) ATCC
25923, MRSA ATCC 33591 and ATCC 43300) and one Gram-nega-
tive bacterium (i.e.: Escherichia coli (E. coli) ATCC 25922) were
determined using the broth microdilution method with the mar-
keted antibiotic Tiamulin as reference. The lipophilicities (ClogP)
were predicted by ACD/Labs (https://www.acdlabs.com/resources/
ilab/).
As Table 1 indicates, almost all the synthesised compounds
exhibited broad-spectrum antibacterial activities and showed high
potency against both Gram-positive and Gram-negative bacteria
with MIC values of 0.5 ꢀ 4 lg/mL. Moreover, the majority of syn-
thesised compounds displayed bactericidal activities against MRSA
ATCC 33591, as MBC/MIC values were less than 4. Among them,
the antibacterial activities of 6d, 6o, 6p and 6q were at least 2
times stronger than those of Tiamulin. In particular, compound 6p
had a 0.125 lg/mL MIC value (8 times lower than that of Tiamulin)
and displayed the best performance against MRSA ATCC 33591.
6p could also be considered as a bactericide, since MBC/MIC val-
ues against four tested strains were less than 2. Furthermore, its
activity against E. coli ATCC 25922 was 16 times stronger than
those of Tiamulin.
MRSA
MRSA
S. aureus
E.coli
ATCC25922
ATCC33591 ATCC43300 ATCC25923
Cpd
MIC MBC MIC MBC MIC MBC
MIC
MBC CLogP
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
1
1
0.5
0.5
1
2
1
1
1
4
1
1
2
0.5
0.5
2
2
1
1
2
4
4
2
4
8
2
2
4
2
2
1
>4
2
2
2
>4
4
4
1
>4
16
2
>4
4
1
1
1
2
2
4
1
1
0.5
0.5
1
4
1
1
4
32
1
1
1
0.5
0.5
>4
>4
2
4.94
4.78
5.28
5.47
4.56
4.35
6.26
5.33
6.32
4.42
3.68
5.22
5.74
5.19
5.85
0.5
0.5
0.5
1
2
1
0.5
1
4
0.5
1
1
0.5
1
2
2
2
4
2
1
1
1
4
1
1
4
2
1
2
16
4
4
>16
>4
>4
>16
32
>4
4
0.5
0.5
0.5
2
2
0.5
>4
2
1
2
0.5 >2
2
0.125 0.125 0.25
0.25 0.125 0.25 0.125
0.125 5.01
0.5
1
1
2
4
2
2
1
1
2
4
1
2
8
8
4
4
1
1
4
0.25
2
0.25 0.125 0.5
4
2
1
2
1
2
4
4
2
1
1
1
2
>4
5.85
6.02
5.93
5.24
3.59
4.94
3.85
3.25
4.00
4.30
5.93
1
1
2
4
4
2
1
1
1
1
4
4
4
4
16
8
>4
>4
4
8
4
4
8
6s
6t
6u
6v
6w
6x
6y
1
2
4
4
1
1
1
1
8
4
16
2
4
2
4
>4
16
>8
4
The effects of terminal ring types (e.g. phenyl, pyridinyl, mor-
pholinyl and piperidinyl), ring substituents and distance between
rings and urea groups on changes in activities were investigated. 6z
4
4
1
Tiamulin (2) 1
1
>4
>8
MIC value of phenyl piperazinyl urea pleuromutilin 6a was 1 lg/
mL against four strains and when substituents were introduced to
the terminal benzene, activities varied within a small range.
Compound 6d with electron-withdrawing 4-nitro groups was the
most potent among all phenyl derivatives and exhibited an MIC
value of 0.5 lg/mL. However, the introduction of N-methyl groups
to the urea led to decreased activities, since actions of compound
6v was 1/4 or 1/2 of compound 6a. To render the side chain
more flexible, a CH2 group was introduced to increase the dis-
noticeably improve activity level. In addition, similar as the effect
of substituents on phenyl piperazinyl urea pleuromutilin activity,
compound 6p with the 4-nitro benzyl group exhibited the best
performance among all the benzyl derivatives. However, continu-
ously extending the space between the benzene and piperazinyl
tance between terminal rings and urea groups. As result, the ben- urea exerts a slight influence on activities. Activity declined when
zyl derivatives with an additional methylene group could the phenyl group was replaced by 4-pyridnyl group, but phenyl

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
767
group replacement by substituting 2-pyridinyl, 3-pyridinyl or 5- assay25,26 (Figure 3). The CCK-8 assay is a colorimetric assay based
quinolinyl groups had nearly zero impact on activities. Activity on the reduction of dye WST-8 [2–(2-methoxy-4-nitrophenyl)-3–(4-
Figure 2. Time-kill kinetics of compound 6p against MRSA ATCC 33591(a) and E. coli ATCC 25922 (b).
level visibly decreased when aliphatic rings such as piperidine and
morpholine were added. The majority of target molecules with an
aromatic terminal ring were fairly lipophilic with a CLogP value of
greater than 5. To enhance hydrophilic properties, compounds
6wꢀy with amino groups were produced from corresponding
Boc-protected amines and nitro compounds. Their ClogP values
were approximately equal to or less than 4. Furthermore, a water-
soluble morpholine ring was attached to the terminal of 6y
through N-acetyl group. Although hydrophilic properties were
clearly optimised via the above modification, activity levels were
only slightly changed.
Since compound 6p showed the most potent antibacterial
activities against MRSA ATCC 33591 and E. coli ATCC 25922, its
bactericidal properties were examined by in vitro time-kill assay.
Bactericidal properties of 6p against two strains were evaluated at
concentrations of 1 ꢂ MIC and 6 ꢂ MIC, and two concentrations of
Tiamulin were set as controls (Figure 2). The MRSA ATCC 33591
propagation-inhibition effect of 6p was visibly higher at the
1 ꢂ MIC concentration than that of the Grown control, which
shows similarity to inhibition effects of Tiamulin at 1 ꢂ MIC.
Meanwhile, at 6 ꢂ MIC, compound 6p achieved complete bacteri-
cidal effects in 12 h, a much faster rate than that under 1 ꢂ MIC
(24 h). In addition, compound 6p displayed better bactericidal
effects against E. coli ATCC 25922 compared with Tiamulin at
1 ꢂ MIC, and was capable of eliminating total bacteria colony in
24 h, while Tiamulin could only limit bacterial propagation speed.
Outcomes were nearly identical at the 6 ꢂ MIC concentration.
Figure 3. Effects of compound 6p on cell viability by CCK-8 assay.
nitrophenyl)-5–(2,4disulfophenyl)-2H-tetrazolium,
monosodium
salt] to a water-soluble orange-coloured formazan via dehydro-
genase in viable cells. The amount of formazan dye produced by
cellular dehydrogenase is correlated with the number of living
cells. The result showed that 6p hardly affected cell viability at
concentrations ranging from 0.125 lg/mL to 4 lg/mL in either of
the two cell lines. More than 90% of cells remained alive at the
4 lg/mL concentration, indicating 6p exhibited almost no cytotox-
icity even at the concentration of 32 ꢂ MIC.
Cytotoxicity evaluation
Docking studies
The cytotoxicity of the most potent compound 6p was investi-
gated in normal human hepatic cell line LO2 and human embryo
Docking investigation was conducted to examine the binding
kidney cell line HEK293T using a cell counting kit-8 (CCK-8) mode between synthesised compounds and the peptidyl

768
Y. ZHANG ET AL.
transferase centre (PTC) of the 50S ribosomal subunit. The method nearly edge-to-face stacking. Moreover, the terminal nitro group
was confirmed by re-docking, revealing that the docking pose of was able to form one additional hydrogen bond by interacting
with C2565 or A2045. 6z (Figure 5(c)) was able to interact with
residues G2484, U2564 and C2565 by three hydrogen bonds, how-
ever the p-p stacking was missing. This might explain why the
Grid_vdw_energy of 6z (-98.69 kcal/mol) was worse than 6d
(-103.75 kcal/mol) and 6p (-111.64 kcal/mol). The hydrophobic
environment of the most potent compound 6p is shown in Figure
5(d). Residues G2484, U2483, C2431, U2485, A2430, U2564, C2565,
A2045, A2418, C2420, C2046, G2044 and A2482 were involved in
the hydrophobic interactions, which provided strong van der
Waals forces in conjunction with the above mentioned p–p stack-
ing of 6p.
the original ligand Tiamulin nearly coincided with the X-ray struc-
ture conformation of RMSD 0.9 Å. All of the title compounds were
used for the docking evaluation (Figs. S36–39). Among them, phe-
nyl derivative 6d and benzyl derivative 6p with the most potent
antibacterial activity, and aliphatic rings derivative 6z with moder-
ate activity were chosen for the binding mode study. The favour-
ably docked molecules were ranked according to their Grid_Score,
which consisted of two parameters: Grid_vdw_energy (mainly
referring to hydrophobic interactions and p–p stacking) and
Grid_es_energy (often containing hydrogen bonds and salt
bridges). The results presented in Table 2 showed that the order
of Grid_Scores went in decreasing order (6p > 6d > 6z>Tiamulin),
which matched those found in the biological assay.
As shown by the superimposed poses in Figures 4(a,b), three
synthesised ligands 6d (green), 6p (yellow) and 6z (magenta)
were located at the same pocket of PTC as Tiamulin (blue). The
tricyclic skeleton of the synthesised compounds was in good
agreement with that of Tiamulin, while the side chain at C14 var-
ied noticeably. Many active interactions occurred continuously
between the above-mentioned ligands and residues C2565,
U2564, G2484, G2044 and A2405.
Experimental
Synthesis
Analytical grade reagents were used and purchased from Energy
Chemical (Shanghai, China) and Kelong Chemical (Chengdu,
China). Melting points were determined on an Shenguang WRS-1B
apparatus (Shanghai, China). 1H NMR and 13 C NMR spectra were
measured on a Bruker AV400 spectrometer in CDCl3 or DMSO-d6.
Mass spectra were recorded with a X500R mass spectrometer (AB
SCIEX) using the electro spray ionisation (ESI) method.
The important intermolecular PTC-ligand interactions are
depicted in Figure 5. In addition to hydrogen bonds between the
hydroxyl group on C11 and residue G2484, and between the car-
bonyl group on C21 and residue G2044, 6d (Figure 5(a)) and 6p
(Figure 5(b)) were able to interact with A2045 by p-p stacking; the
former adopting face-to-face stacking, and the latter displaying
Synthesis of compound 7 and 8
Tosyl chloride (1.91 g, 10.0 mmol) in DCM (10 ml) was added to a
solution of pleuromutilin (3.56 g, 9.40 mmol) in DCM (10 ml) con-
taining triethylamine (4.54 g, 12.0 mmol) at 0 ꢁC, then the reaction
was stirred at 25 ꢁC for 20 h and the resultant was washed with
water (50 ml). The organic phase was collected, dried over Na2SO4
and evaporated under vacuum to produce yellow oil, which could
be purified by recrystallisation in ethanol to obtain white solid 7
(Yield: 78.3%). Spectral data of 7 were identical to those from
reports in the literature27.
Table 2. Molecular docking analysis of PTC with selected ligands (kcal/mol).
Cpd
Grid_Score
Grid_vdw_energy
Grid_es_energy
6d
6p
6z
Tiamulin
ꢃ107.27
ꢃ112.91
ꢃ105.50
ꢃ95.34
ꢃ103.75
ꢃ111.64
ꢃ98.69
ꢃ91.14
ꢃ3.52
ꢃ1.27
ꢃ6.81
ꢃ4.20
Figure 4. Superimposed poses of selected ligands in the PTC of the 50 s ribosomal subunit. (a) The interaction groove; (b) Residues involved in the interactions
between ligands and PTC.

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7 (533 mg, 1.0 mmol) and NaI (30.0 mg, 0.2 mmol) were stirred
10g: white powder; yield: 97.2%; mp: 124.9–126.3 ꢁC; 1H NMR
in dry THF (5 ml) at 25 ꢁC for 0.5 h, then K2CO3 (276 mg, 2.0 mmol) (400 MHz, CDCl3): d (ppm) 8.33 (s, 1H), 7.78 (t, J ¼ 2.0 Hz, 1H),
and piperazine (172 mg, 2.0 mmol) were added to the aforemen- 7.42 ꢃ 7.35 (m, 1H), 7.29 (t, J ¼ 8.0 Hz, 1H), 7.08 ꢃ 7.00 (m, 1H), 6.62
tioned solution. The mixture was heated to reflux and stirred for (s, 1H), 1.52 (s, 9H); 13 C NMR (100 MHz, CDCl3): d (ppm) 159.2,
6 h. After cooling to room temperature, the resulting mixture was 152.7, 139.3, 136.7, 129.8, 115.7, 114.7, 110.2, 92.8, 81.0, 28.3;
washed with water and condensed until dry. Compound 8 was HRMS: calculated for C13H15Cl3N2NaO3 ([M þ Na]þ): 375.0040,
collected after silica gel column chromatography (Yield: 75.2%). found 375.0046.
10k: white powder; yield: 54.1%; mp: 148.1–149.3 ꢁC; 1H NMR
Spectral data results of 8 were identical to those from reports in
the literature28.
(400 MHz, CDCl3): d (ppm) 8.57 (s, 1H), 7.56 ꢃ 7.45 (m, 2H), 6.35 (d,
J ¼ 7.6 Hz, 1H), 4.41 (s, 2H); 13 C NMR (100 MHz, CDCl3): d (ppm)
159.2, 157.5, 148.3, 140.5, 106.1, 103.3, 92.8; HRMS: calculated for
C7H6Cl3N3O ([M þ H]þ): 253.9649, found 253.9652.
Synthesis of compounds 9a ꢀ e
10n: white powder; yield: 88.1%; mp: 84.9–85.6 ꢁC; 1H NMR
(400 MHz, CDCl3): d (ppm) 7.26 ꢃ 7.22 (m, 2H), 6.92 ꢃ 6.88 (m, 2H),
4.48 (d, J ¼ 5.6 Hz, 2H); 13 C NMR (100 MHz, CDCl3): d (ppm) 161.8,
159.6, 129.3, 128.3, 114.4, 92.6, 55.4, 45.0; HRMS: calculated for
C10H10Cl3NNaO2 ([M þ Na]þ): 303.9669, found 303.9685.
Compounds 9a ~ d were synthesised according to previous litera-
ture29,30, and 9e was synthesised as follows:
BTC (386 mg, 1.3 mmol) in dry THF (4 ml) was added dropwise
to a mixture of triethylamine (526 mg, 5.2 mmol) and 12e (173 mg,
1.2 mmol) in dry THF (6 ml) solution under an ice-water bath. The
mixture was then stirred for 0.5 h. After condensation, N-Boc
piperazine (503 mg, 2.7 mmol), triethylamine (546 mg, 5.4 mmol)
and DCM (10 ml) were added and stirred for 2 h at 25 ꢁC. The solv-
ent was then evaporated before crude product purification via sil-
ica gel column chromatography to produce compound 13e.
Compound 13e (356 mg, 1.0 mmol) was added to the mixture
of 6 ml DCM and TFA (v/v ¼ 10/1) and stirred at 25 ꢁC until 13e
completely converted. The resulting solution was washed with
saturated aqueous NaHCO3 to neutralise TFA, then f the organic
phase was collected and dried over anhydrous Na2SO4. After filter-
ing, the filtrate was concentrated to acquire the crude product,
which was purified via silica gel chromatography to produce com-
pound 9e.
10p: light yellow powder; yield: 85.2%; mp: 87.2–88.4 ꢁC; 1H
NMR (400 MHz, CDCl3): d (ppm) 8.24 (d, J ¼ 8.8 Hz, 2H), 7.49 (d,
J ¼ 8.8 Hz, 2H), 4.67 (d, J ¼ 6.0 Hz, 2H); 13 C NMR (100 MHz, CDCl3):
d (ppm) 162.4, 147.7, 143.7, 128.3, 124.2, 92.2, 44.5; HRMS: calcu-
lated for C9H7Cl3N2O3 ([M þ H]þ): 296.9595, found 296.9601.
10q: white powder; yield: 87.5%; mp: 77.3–78.7 ꢁC; 1H NMR
(400 MHz, CDCl3): d (ppm) 7.70 (d, J ¼ 7.6 Hz, 1H), 7.59 ꢃ 7.57 (m,
2H), 7.48 ꢃ 7.44 (m, 1H), 4.73 (dd, J ¼ 6.0, 0.8 Hz, 2H), 1.25 (s, 1H);
13 C NMR (100 MHz, CDCl3): d (ppm) 162.0, 134.5, 132.7, 130.8,
128.4, 128.3 (q, J ¼ 31 Hz), 126.4 (q, J ¼ 6 Hz), 124.3 (q, J ¼ 272 Hz),
92.4, 42.1; HRMS: calculated for C10H7Cl3F3NO ([M þ H]þ):
319.9618, found 319.9624.
10s: white powder; yield: 86.8%; mp: 48.1–49.5 ꢁC; 1H NMR
(400 MHz, CDCl3): d (ppm) 7.26 ꢃ 7.25 (m, 1H), 6.84 ꢃ 6.83 (m, 2H),
6.78 (t, J ¼ 2.0 Hz, 1H), 3.83 (s, 3H), 3.65 (q, J ¼ 6.8 Hz, 2H), 2.92 (d,
J ¼ 6.8 Hz, 2H); 13 C NMR (100 MHz, CDCl3): d (ppm) 161.8, 160.0,
139.4, 129.9, 121.1, 114.4, 112.4, 92.6, 55.2, 42.5, 35.1; HRMS: calcu-
lated for C11H12Cl3NO2 ([M þ H]þ): 296.0006, found 296.0022.
13e: white powder; yield: 56.2%; mp: 222.9–223.8 ꢁC; 1H NMR
(400 MHz, CDCl3): d (ppm) 8.86 (dd, J ¼ 4.0, 1.4 Hz, 1H), 8.18 ꢃ 8.05
(m, 1H), 7.92 (d, J ¼ 8.4 Hz, 1H), 7.65 ꢃ 7.52 (m, 1H), 7.43 (d,
J ¼ 7.2 Hz, 1H), 7.33 (dd, J ¼ 8.4, 4.0 Hz, 1H), 6.89 (s, 1H), 3.45 (s,
8H), 1.48 (s, 9H); 13 C NMR (100 MHz, CDCl3): d (ppm) 156.2, 154.7,
150.4, 148.8, 134.2, 131.0, 129.2, 127.3, 124.4, 122.4, 121.0, 80.5,
28.5; HRMS: calculated for C19H24N4O3 ([M þ H]þ): 357.1921,
found 357.1929.
Synthesis of compound 1133
9e: yellow liquid; yield: 96.3%; 1H NMR (400 MHz, DMSO-d6): d 4-Nitrophenyl chloroformate (97 mg, 0.48 mmol) and NMM
(ppm) 9.08 (s, 1H), 8.87 (d, J ¼ 4.0 Hz, 1H), 8.35 (d, J ¼ 8.4 Hz, 1H), (143 mg, 1.41 mmol) were added to a DCM (5 ml) solution contain-
7.84 (d, J ¼ 8.4 Hz, 1H), 7.70 (t, J ¼ 8.0 Hz, 1H), 7.54 ꢃ 7.44 (m, 2H), ing N-methylaniline 12v (51 mg, 0.47 mmol) at 0 ꢁC. The subse-
3.80 ꢃ 3.72 (m, 4H), 3.24 ꢃ 3.17 (m, 4H); 13 C NMR (100 MHz, quent mixture was stirred for 2 h before pouring into water (5 ml).
DMSO-d6): d (ppm) 156.3, 150.7, 148.6, 136.1, 132.9, 129.4, 126.3, The organic layer was separated and dried over anhydrous
124.8, 123.1, 121.2, 43.1, 41.5; HRMS: calculated for C14H16N4O Na2SO4 After solvent evaporation, the residue was directly used
([M þ H]þ): 257.1391, found 257.1402.
for the next step (Yield: 97.5%).
Synthesis of compounds 10f ꢀ u
Synthesis of compounds 6a ꢀ e
Compounds 10f, 10h, 10i, 10j, 10l, 10m, 10o, 10r, 10t and 10u Pleuromutilin 22-tosylate 7 (213 mg, 0.4 mmol) and NaI (14.9 mg,
were synthesised as in literature31,32. The remaining compounds 0.1 mmol) in MeCN (5 ml) were stirred at 25 ꢁC for 0.5 h, then com-
were synthesised as follows:
pounds 9aꢀe (0.5 mmol) and K2CO3 (111 mg, 0.8 mmol) were
added. The mixture was stirred under 70 ꢁC for 5 h. After solvent
evaporation, the crude product was purified via silica gel column
chromatography to obtain pure products 6aꢀe.
A mixture of trichloroacetyl chloride (218 mg, 1.2 mmol) in an
appropriate amount of DCM was added to a DCM (3 ml) solution
containing compounds 12g (208 mg, 1.0 mmol) and triethylamine
(121 mg, 1.2 mmol) under an ice-water bath. Next, the mixture
6a: white powder; yield: 78.3%; mp: 98.6–100.4 ꢁC; 1H NMR
was stirred at 25 ꢁC until 12g was completely dispersed. The reac- (400 MHz, CDCl3): d (ppm) 7.39 ꢃ 7.11 (m, 4H), 6.95 (t, J ¼ 6.8 Hz,
tion mixture was evaporated by vacuum and residue was dis- 1H), 6.61 (s, 1H), 6.43 (dd, J ¼ 17.2, 11.2 Hz, 1H), 5.73 (d, J ¼ 8.0 Hz,
solved in DCM. After washing using water and saturated aqueous 1H), 5.27 (d, J ¼ 10.8 Hz, 1H), 5.13 (d, J ¼ 17.2 Hz, 1H), 3.47 (s, 4H),
ammonium chloride, the organic layer was separated and dried 3.29 (s, 1H), 3.11 (ABq, J ¼ 17.2 Hz, 2H), 2.70 ꢃ 2.38 (m, 4H),
over anhydrous Na2SO4. The crude compound was attained after 2.36 ꢃ 1.92 (m, 5H), 1.81 ꢃ 1.26 (m, 11H), 1.10 (s, 3H), 1.07 ꢃ 0.98
evaporating the solvent, which was recrystallised by ethanol/water (m, 1H), 0.82 (d, J ¼ 6.8 Hz, 3H), 0.66 (d, J ¼ 6.8 Hz, 3H); 13 C NMR
to give 10g. Compounds 10k, 10n, 10p, 10q and 10s were syn- (100 MHz, CDCl3): d (ppm) 217.3, 169.0, 155.1, 139.2, 139.1, 128.9,
thesised in the same manner.
123.2, 120.2, 117.3, 74.6, 68.6, 59.8, 58.3, 52.6, 45.5, 45.1, 44.0, 43.9,

770
Y. ZHANG ET AL.
Figure 5. Interactions between ligands and residues of the 50s ribosomal subunit. (a) 6d and site; (b) 6p and site; (c) 6z and site; (d) Hydrogen bonds and hydropho-
bic interactions.
41.8, 36.8, 36.1, 34.5, 30.5, 26.9, 26.5, 24.9, 16.8, 15.0, 11.6; HRMS: 11.6; HRMS: calculated for C34H49N3O6 ([M þ H]þ): 596.3694,
calculated for C33H47N3O5 ([M þ H]þ): 566.3588, found 566.3588.
6b: white powder; yield: 87.6%; mp: 121.6–122.4 ꢁC; 1H NMR
found 596.3693.
6c: white powder; yield: 86.4%; mp: 107.9–109.1 ꢁC; 1H NMR
(400 MHz, DMSO-d6): d (ppm) 8.33 (s, 1H, NH), 7.33 (d, J ¼ 8.8 Hz, (400 MHz, CDCl3): d (ppm) 7.42 ꢃ 7.13 (m, 5H), 6.46 (dd, J ¼ 17.2,
2H), 6.81 (d, J ¼ 8.8 Hz, 2H), 6.18 (dd, J ¼ 17.2, 11.2 Hz, 1H), 5.60 (d, 11.2 Hz, 1H), 5.74 (d, J ¼ 8.4 Hz, 1H), 5.29 (d, J ¼ 11.2 Hz, 1H), 5.15
J ¼ 8.0 Hz, 1H), 5.09 ꢃ 5.05 (m, 2H), 4.54 (d, J ¼ 6.0 Hz, 1H), 3.70 (s, (d, J ¼ 17.2 Hz, 1H), 4.75 (d, J ¼ 5.2 Hz, 1H), 4.37 (d, J ¼ 5.2 Hz, 2H),
3H), 3.53 ꢃ 3.34 (m, 5H), 3.30 ꢃ 3.01 (m, 2H), 2.45 ꢃ 2.35 (m, 4H), 3.39 (t, J ¼ 4.8 Hz, 4H), 3.31 (s, 1H), 3.11 (ABq, J ¼ 17.2 Hz, 2H),
2.28 ꢃ 2.01 (m, 5H), 1.75 ꢃ 1.21 (m, 11H), 1.07 (s, 3H), 1.03 ꢃ 0.95 2.71 ꢃ 2.40 (m, 4H), 2.40 ꢃ 1.96 (m, 5H), 1.83 ꢃ 1.29 (m, 11H), 1.12
(m, 1H), 0.83 (d, J ¼ 6.8 Hz, 3H), 0.64 (d, J ¼ 6.8 Hz, 3H); 13 C NMR (s, 3H), 1.10 ꢃ 1.02 (m, 1H), 0.84 (d, J ¼ 6.8 Hz, 3H), 0.67 (d,
(100 MHz, DMSO-d6): d (ppm) 217.3, 155.2, 154.4, 141.0, 133.5, J ¼ 6.8 Hz, 3H); 13 C NMR (100 MHz, CDCl3): d (ppm) 217.2, 169.0,
121.5, 115.2, 113.5, 72.6, 68.3, 59.8, 58.9, 57.3, 55.1, 51.8, 45.0, 44.1, 157.6, 139.4, 139.2, 128.7, 127.9, 127.4, 117.4, 74.7, 68.5, 59.9, 58.3,
43.7, 41.5, 36.5, 36.4, 34.0, 30.2, 28.6, 26.6, 24.5, 20.8, 16.0, 14.6, 52.6, 45.6, 45.1, 44.1, 43.8, 41.9, 36.8, 36.2, 34.6, 30.5, 26.9, 26.5,

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24.9, 16.8, 15.0, 11.6; HRMS: calculated for C34H49N3O5 ([M þ H]þ): 34.6, 30.6, 28.5, 27.0, 26.5, 25.0, 16.9, 15.0, 11.6; HRMS: calculated
for C38H56N4O7 ([M þ H]þ): 681.4222, found 681.4258.
580.3745, found 580.3741.
6h: white powder; yield: 82.3%; mp: 103.2–104.4 ꢁC; 1H NMR
(400 MHz, CDCl3): d (ppm) 7.30 ꢃ 7.27 (m, 1H),7.23 ꢃ 7.15 (m, 1H),
6.99 (dd, J ¼ 8.0, 1.2 Hz, 1H), 6.71 (td, J ¼ 8.4, 2.0 Hz, 1H),
6.56 ꢃ 6.43 (m, 2H), 5.79 (d, J ¼ 8.4 Hz, 1H), 5.34 (dd, J ¼ 11.2,
1.2 Hz, 1H), 5.20 (dd, J ¼ 17.2, 1.6 Hz, 1H), 3.53 (t, J ¼ 5.2 Hz, 4H),
3.35 (d, J ¼ 5.6 Hz, 1H), 3.15 (ABq, J ¼ 17.2 Hz, 2H), 2.71 ꢃ 2.50 (m,
4H), 2.39 ꢃ 2.04 (m, 5H), 1.85 ꢃ 1.33 (m, 11H), 1.16 (s, 3H),
1.14 ꢃ 1.07 (m, 1H), 0.88 (d, J ¼ 6.8 Hz, 3H), 0.72 (d, J ¼ 6.8 Hz, 3H);
6d: yellow powder; yield: 88.5%; mp: 87.6–88.9 ꢁC; 1H NMR
(400 MHz, CDCl3): d (ppm) 8.19 ꢃ 8.09 (m, 2H), 7.55 ꢃ 7.46 (m, 2H),
6.94 (s, 1H), 6.49 (dd, J ¼ 17.2, 11.2 Hz, 1H), 5.79 (d, J ¼ 8.4 Hz, 1H),
5.33 (dd, J ¼ 11.2, 1.2 Hz, 1H), 5.19 (dd, J ¼ 17.2, 1.2 Hz, 1H), 3.57 (t,
J ¼ 4.8 Hz, 4H), 3.41 ꢃ 3.30 (m, 1H), 3.15 (ABq, J ¼ 17.2 Hz, 2H),
2.71 ꢃ 2.53 (m, 4H), 2.39 ꢃ 2.02 (m, 5H), 1.93 ꢃ 1.32 (m, 11H), 1.16
(s, 3H), 1.14 ꢃ 1.07 (m, 1H), 0.87 (d, J ¼ 6.8 Hz, 3H), 0.71 (d,
J ¼ 6.8 Hz, 3H); 13 C NMR (100 MHz, CDCl3): d (ppm) 217.3, 169.0,
153.8, 145.5, 142.6, 139.2, 125.2, 118.6, 117.4, 74.7, 68.7, 59.7, 58.3,
52.5, 45.6, 45.1, 44.2, 44.1, 41.9, 36.8, 36.2, 34.6, 30.5, 27.0, 26.5,
25.0, 16.9, 15.0, 11.6; HRMS: calculated for C33H46N4O7 ([M þ H]þ):
611.3439, found 611.3440.
13 C NMR (100 MHz, CDCl3):
d (ppm) 217.2, 169.0, 163.2
(J ¼ 244 Hz), 154.6, 140.8, 140.7, 139.2, 130.0 (J ¼ 9.4 Hz), 117.4,
115.0 (J ¼ 3.2 Hz), 114.9, 109.8 (J ¼ 21 Hz), 107.4, 107.1, 74.7, 68.7,
60.5, 59.8, 58.3, 52.6, 45.6, 44.1, 41.9, 36.8, 36.2, 34.6, 30.6, 27.0,
26.5, 25.0, 16.9, 15.0, 11.6; HRMS: calculated for C33H46FN3O5
([M þ H]þ): 584.3494, found 584.3488.
6e: white powder; yield: 92.3%; mp: 138.1–139.3 ꢁC; 1H NMR
(400 MHz, CDCl3): d (ppm) 8.98 ꢃ 8.85 (m, 1H), 8.22 ꢃ 812 (m, 1H),
7.96 ꢃ 7.89 (m, 1H), 7.69 ꢃ 7.60 (m, 1H), 7.57 ꢃ 7.48 (m, 1H),
7.43 ꢃ 7.34 (m, 1H), 6.70 (d, J ¼ 16.0 Hz, 1H), 6.59 ꢃ 6.43 (m, 1H),
5.80 (d, J ¼ 8.0 Hz, 1H), 5.35 (d, J ¼ 11.2 Hz, 1H), 5.21 (d, J ¼ 17.2 Hz,
1H), 3.59 (d, J ¼ 4.8 Hz, 4H), 3.42 ꢃ 3.30 (m, 1H), 3.15 (ABq,
J ¼ 17.2 Hz, 2H), 2.77 ꢃ 2.51 (m, 4H), 2.45 ꢃ 2.00 (m, 5H),
1.87 ꢃ 1.33 (m, 11H), 1.17 (s, 3H), 1.14 ꢃ 1.07 (m, 1H), 0.88 (d,
J ¼ 5.6 Hz, 3H), 0.73 (d, J ¼ 5.6 Hz, 3H); 13 C NMR (100 MHz, CDCl3):
d (ppm) 217.2, 169.1, 155.9, 150.4, 148.9, 139.2, 134.2, 130.9, 129.3,
127.2, 122.1, 121.0, 117.4, 74.7, 68.7, 59.9, 58.3, 52.6, 45.6, 45.2,
44.3, 44.1, 41.9, 36.8, 36.2, 34.6, 30.6, 27.0, 26.6, 25.0, 16.9, 15.0,
11.6; HRMS: calculated for C36H48N4O5 ([M þ H]þ): 617.3697,
found 617.3695.
6i: white powder; yield: 81.5%; mp: 116.3–117.7 ꢁC; 1H NMR
(400 MHz, CDCl3): d (ppm) 7.62 (s, 1H), 7.55 (d, J ¼ 8.0 Hz, 1H), 7.37
(t, J ¼ 8.0 Hz, 1H), 7.30 ꢃ 7.26 (m, 1H), 6.61 (s, 1H), 6.50 (dd,
J ¼ 17.2, 11.2 Hz, 1H), 5.79 (d, J ¼ 8.4 Hz, 1H), 5.34 (dd, J ¼ 11.2,
1.2 Hz, 1H), 5.20 (dd, J ¼ 17.2, 1.6 Hz, 1H), 3.55 (t, J ¼ 5.2 Hz, 4H),
3.35 (s, 1H), 3.27 (ABq, J ¼ 17.2 Hz, 2H), 2.73 ꢃ 2.51 (m, 4H),
2.39 ꢃ 2.04 (m, 5H), 1.83 ꢃ 1.33 (m, 11H), 1.16 (s, 3H), 1.14 ꢃ 1.08
(m, 1H), 0.88 (d, J ¼ 6.8 Hz, 3H), 0.72 (d, J ¼ 6.8 Hz, 3H); 13 C NMR
(100 MHz, CDCl3): d (ppm) 217.2, 169.0, 154.6, 139.7, 139.2, 131.5,
131.2, 129.5, 123.0, 119.8, 117.4, 116.6, 74.7, 68.7, 60.5, 59.8, 58.3,
52.6, 45.6, 45.2, 44.1, 41.9, 36.9, 36.2, 34.6, 30.6, 27.0, 26.5, 25.0,
16.9, 15.0, 11.6; HRMS: calculated for C34H46F3N3O5 ([M þ H]þ):
634.3462, found 634.3468.
6j: white powder; yield: 73.2%; mp: 127.9–129.5 ꢁC; 1H NMR
(400 MHz, CDCl3):
d
(ppm) 8.33 (d, J ¼ 6.6 Hz, 2H), 7.66 (d,
Synthesis of compounds 6f ꢀ u
J ¼ 6.2 Hz, 2H), 6.50 (dd, J ¼ 17.2, 11.2 Hz, 1H), 5.80 (d, J ¼ 8.4 Hz,
1H), 5.34 (dd, J ¼ 11.2, 1.6 Hz, 1H), 5.20 (dd, J ¼ 17.2, 1.6 Hz, 1H),
3.64 (t, J ¼ 4.8 Hz, 4H), 3.36 (d, J ¼ 6.6 Hz, 1H), 3.16 (ABq,
J ¼ 17.2 Hz, 2H), 2.72 ꢃ 2.54 (m, 4H), 2.41 ꢃ 2.05 (m, 5H),
1.83 ꢃ 1.33 (m, 11H), 1.17 (s, 3H), 1.15 ꢃ 1.08 (m, 1H), 0.88 (d,
J ¼ 6.8 Hz, 3H), 0.72 (d, J ¼ 6.8 Hz, 3H); 13 C NMR (100 MHz, CDCl3):
d (ppm) 217.2, 169.0, 153.8, 150.4, 146.7, 139.2, 117.4, 113.4, 74.7,
68.7, 59.8, 58.3, 52.5, 45.6, 45.2, 44.2, 44.1, 41.9, 36.8, 36.2, 34.6,
30.6, 27.0, 26.6, 25.0, 16.9, 15.0, 11.6; HRMS: calculated for
C32H46N4O5 ([M þ H]þ): 567.3541, found 567.3546.
Compounds 10f ~ u (1.0 mmol) and K2CO3 (276 mg, 2.0 mmol)
were added to a stirred solution of compound 8 (402 mg,
0.9 mmol) in anhydrous DMF (10 ml) at 25 ꢁC. The mixture was
then heated to 80 ꢁC until 8 was fully converted. Afterwards, the
mixture was diluted by water and extracted via DCM (20 ml ꢂ 3).
Subsequently, the combined organic layer was washed with brine
and dried over anhydrous MgSO4. Solvent evaporation was com-
pleted before the crude product was purified by silica gel column
chromatography to produce compounds 6f ~ u.
6f: white powder; yield: 81.5%; mp: 110.9–113.2 ꢁC; 1H NMR
(400 MHz, CDCl3): d (ppm) 7.65 (s, 1H), 7.59 (s, 1H), 7.20 ꢃ 7.03 (m,
3H), 6.65 (s, 1H), 6.50 (dd, J ¼ 17.2, 11.2 Hz, 1H), 5.79 (d, J ¼ 8.4 Hz,
1H), 5.33 (dd, J ¼ 11.2, 1.2 Hz, 1H), 5.20 (dd, J ¼ 17.2, 1.6 Hz, 1H),
3.55–3.50 (m, 4H), 3.36 (s, 1H), 3.15 (ABq, J ¼ 17.2 Hz, 2H),
2.68 ꢃ 2.46 (m, 4H), 2.40 ꢃ 2.04 (m, 8H), 1.81 ꢃ 1.32 (m, 11H), 1.16
(s, 3H), 1.14 ꢃ 1.08 (m, 1H), 0.87 (d, J ¼ 6.8 Hz, 3H), 0.72 (d,
J ¼ 6.8 Hz, 3H); 13 C NMR (100 MHz, CDCl3): d (ppm) 217.2, 169.1,
168.7, 155.1, 139.7, 139.2, 138.6, 129.4, 117.4, 116.1, 114.7, 111.9,
74.7, 68.7, 59.9, 58.3, 52.7, 45.6, 45.2, 44.1, 44.0, 41.9, 36.9, 36.2,
34.6, 30.6, 27.0, 26.6, 25.0, 24.6, 16.9, 15.0, 11.6; HRMS: calculated
for C35H50N4O6 ([M þ H]þ): 623.3803, found 623.3804.
6k: white powder; yield: 67.8%; mp: 129.6–130.9 ꢁC; 1H NMR
(400 MHz, CDCl3): d (ppm) 7.43 ꢃ 7.35 (m, 1H), 7.31 (d, J ¼ 8.0 Hz,
1H), 6.96 (s, 1H), 6.49 (dd, J ¼ 17.2, 11.2 Hz, 1H), 6.14 (d, J ¼ 7.6 Hz,
1H), 5.78 (d, J ¼ 8.4 Hz, 1H), 5.33 (d, J ¼ 11.2 Hz, 1H), 5.19 (dd,
J ¼ 17.2, 1.2 Hz, 1H), 4.25 (s, 2H), 3.54 (t, J ¼ 4.8 Hz, 4H), 3.42 ꢃ 3.29
(m, 1H), 3.12 (ABq, J ¼ 17.2 Hz, 2H), 2.72 ꢃ 2.39 (m, 4H), 2.39 ꢃ 2.01
(m, 5H), 1.82 ꢃ 1.31 (m, 11H), 1.15 (s, 3H), 1.13 ꢃ 1.06 (m, 1H), 0.86
(d, J ¼ 6.8 Hz, 3H), 0.70 (d, J ¼ 6.8 Hz, 3H); 13 C NMR (100 MHz,
CDCl3): d (ppm) 217.3, 169.0, 157.0, 153.9, 151.2, 140.2, 139.1,
117.5, 102.9, 102.5, 74.7, 68.6, 59.8, 58.3, 52.6, 45.6, 45.1, 44.1, 43.9,
41.9, 36.8, 36.2, 34.6, 30.5, 26.9, 26.5, 25.0, 16.9, 15.0, 11.6; HRMS:
calculated for C32H47N5O5 ([M þ H]þ): 582.3650, found 582.3658.
6l: white powder; yield: 77.2%; mp: 149.9–151.8 ꢁC; 1H NMR
(400 MHz, CDCl3): d (ppm) 8.20 (d, J ¼ 2.4 Hz, 1H), 7.88 (dd, J ¼ 8.8,
2.8 Hz, 1H), 7.38 (d, J ¼ 8.8 Hz, 1H), 6.91 (s, 1H), 6.50 (dd, J ¼ 17.2,
11.2 Hz, 1H), 5.80 (d, J ¼ 8.4 Hz, 1H), 5.34 (dd, J ¼ 11.2, 1.2 Hz, 1H),
5.21 (dd, J ¼ 17.2, 1.2 Hz, 1H), 3.56 (t, J ¼ 4.8 Hz, 4H), 3.45 ꢃ 3.31
(m, 1H), 3.15 (ABq, J ¼ 17.2 Hz, 2H), 2.69 ꢃ 2.51 (m, 4H), 2.40 ꢃ 2.06
(m, 5H), 1.84 ꢃ 1.34 (m, 11H), 1.17 (s, 3H), 1.15 ꢃ 1.07 (m, 1H), 0.89
(d, J ¼ 6.8 Hz, 3H), 0.72 (d, J ¼ 6.8 Hz, 3H); 13 C NMR (100 MHz,
CDCl3): d (ppm) 217.3, 169.0, 154.4, 141.1, 139.2, 135.9, 134.6,
6g: white powder; yield: 77.4%; mp: 109.7–111.2 ꢁC; 1H NMR
(400 MHz, CDCl3): d (ppm) 7.51 (s, 1H), 7.20 ꢃ 7.10 (m, 2H), 6.88
(dd, J ¼ 7.6, 1.6 Hz, 1H), 6.63 ꢃ 6.36 (m, 3H), 5.79 (d, J ¼ 8.4 Hz, 1H),
5.34 (dd, J ¼ 11.2, 1.6 Hz, 1H), 5.20 (dd, J ¼ 17.2, 1.6 Hz, 1H), 3.51 (t,
J ¼ 5.0 Hz, 4H), 3.35 (s, 1H), 3.13 (ABq, J ¼ 17.2 Hz, 2H), 2.68 ꢃ 2.47
(m, 4H), 2.42 ꢃ 2.04 (m, 5H), 1.90 ꢃ 1.33 (m, 20H), 1.16 (s, 3H),
1.14 ꢃ 1.08 (m, 1H), 0.87 (d, J ¼ 6.8 Hz, 3H), 0.72 (d, J ¼ 6.8 Hz, 3H);
13 C NMR (100 MHz, CDCl3): d (ppm) 217.2, 169.1, 154.8, 152.9,
139.8, 139.2, 139.0, 129.5, 117.5, 114.5, 113.1, 109.8, 80.7, 74.7,
68.6, 60.5, 59.9, 58.3, 52.7, 45.6, 45.2, 44.1, 44.0, 41.9, 36.9, 36.2, 130.3, 127.9, 117.4, 74.7, 68.7, 59.8, 58.3, 52.5, 45.6, 45.1, 44.1, 41.9,

772
Y. ZHANG ET AL.
36.8, 36.2, 34.6, 30.5, 26.9, 26.5, 24.9, 16.9, 15.0, 11.6; HRMS: calcu- (100 MHz, CDCl3): d (ppm) 217.1, 168.9, 157.2, 139.1, 137.9, 132.3,
lated
645.2648, 647.2638.
for
C32H46BrN4O5
([M þ H]þ):
645.2646,
found 130.6, 127.4, 125.9, 125.9, 117.3, 74.6, 68.5, 59.8, 58.2, 52.5, 45.5,
45.0, 44.0, 43.6, 41.8, 41.4, 36.7, 36.1, 34.5, 30.4, 26.8, 26.4, 24.8,
6m: white powder; yield: 56.2%; mp: 102.3–103.7 ꢁC; 1H NMR
(400 MHz, CDCl3):
(ppm) 7.19 (d, J ¼ 8.0 Hz, 2H), 7.13 (d,
16.7, 14.9, 11.5; HRMS: calculated for C35H48F3N3O5 ([M þ H]þ):
648.3619, found 648.3620.
d
6r: white powder; yield: 65.1%; mp: 136.5–137.9 ꢁC; 1H NMR
(400 MHz, CDCl3): d (ppm) 7.32 ꢃ 7.27 (m, 2H), 7.24 ꢃ 7.15 (m, 3H),
6.49 (dd, J ¼ 17.2, 11.2 Hz), 5.78 (d, J ¼ 8.4 Hz), 5.33 (dd, J ¼ 11.2,
1.2 Hz), 5.19 (dd, J ¼ 17.2, 1.2 Hz), 4.44 (t, 1H, J ¼ 5.6 Hz), 3.47 (dd,
J ¼ 12.4, 6.8 Hz, 2H), 3.35 (t, J ¼ 4.8 Hz, 4H), 3.10 (ABq, J ¼ 17.2 Hz,
2H), 2.81 (t, J ¼ 6.8 Hz, 2H), 2.58 ꢃ 2.42 (m, 4H), 2.37 ꢃ 2.02 (m, 5H),
1.79 ꢃ 1.24 (m, 11H), 1.15 (s, 3H), 1.13 ꢃ 1.07 (m, 1H), 0.87 (d,
J ¼ 6.4 Hz, 3H), 0.70 (d, J ¼ 6.8 Hz, 3H); 13 C NMR (100 MHz, CDCl3):
d (ppm) 217.3, 169.0, 157.6, 139.1, 139.5, 129.0, 128.7, 126.5, 117.5,
74.7, 68.5, 59.9, 58.3, 52.6, 45.6, 45.1, 44.1, 43.6, 42.1, 41.9, 36.8,
36.4, 36.2, 34.6, 30.5, 26.9, 26.5, 25.0, 16.9, 15.0, 11.6; HRMS: calcu-
J ¼ 8.0 Hz, 2H), 6.50 (dd, J ¼ 17.2, 11.2 Hz, 1H), 5.79 (d, J ¼ 8.4 Hz,
1H), 5.33 (dd, J ¼ 11.2, 1.2 Hz, 1H), 5.19 (dd, J ¼ 17.2, 1.2 Hz, 1H),
4.63 (t, J ¼ 5.2 Hz, 1H), 4.37 (d, J ¼ 5.2 Hz, 2H), 3.47 ꢃ 3.31 (m, 5H),
3.13 (ABq, J ¼ 17.2 Hz, 2H), 2.57–2.50 (m, 4H), 2.33 (s, 3H),
2.25 ꢃ 2.04 (m, 5H), 1.80 ꢃ 1.74 (m, 1H), 1.70 ꢃ 1.42 (m, 10H), 1.16
(s, 3H), 1.13 ꢃ 1.08 (m, 1H), 0.87 (d, J ¼ 6.8 Hz, 3H), 0.71 (d,
J ¼ 6.8 Hz, 3H); 13 C NMR (100 MHz, CDCl3): d (ppm) 217.2, 169.0,
157.4, 139.0, 137.1, 136.3, 129.3, 127.9, 117.4, 74.6, 68.4, 59.9, 58.2,
52.6, 45.5, 45.0, 44.8, 44.0, 43.6, 41.8, 36.7, 36.1, 34.5, 30.4, 26.8,
26.4, 24.9, 21.1, 16.8, 14.9, 11.6; HRMS: calculated for C35H51N3O5
([M þ H]þ): 594.3901, found 594.3904.
6n: white powder; yield: 60.1%; mp: 104.2–105.8 ꢁC; 1H NMR
lated
found 594.3905.
for
C35H51N3O5
([M þ H]þ):
594.3901[M þ H]þ,
(400 MHz, CDCl3):
d
(ppm) 7.22 (d, J ¼ 8.4 Hz, 2H), 6.85 (d,
6s: white powder; yield: 61.7%; mp: 105.8–107.1 ꢁC; 1H NMR
(400 MHz, CDCl3): d (ppm) 7.22 (t, J ¼ 7.6 Hz, 1H), 6.78 ꢃ 6.73 (m,
3H), 6.50 (dd, J ¼ 17.2, 11.2 Hz, 1H), 5.78 (d, J ¼ 8.4 Hz, 1H), 5.33 (d,
J ¼ 11.2 Hz, 1H), 5.20 (d, J ¼ 17.2 Hz, 1H), 4.42 (t, J ¼ 5.6 Hz, 1H),
3.79 (s, 3H), 3.47 (dd, J ¼ 12.4, 6.8 Hz, 2H), 3.36 (t, J ¼ 4.8 Hz, 4H),
3.12 (ABq, J ¼ 17.2 Hz, 2H), 2.79 (t, J ¼ 6.8 Hz, 2H), 2.54 ꢃ 2.49 (m,
4H), 2.37 ꢃ 2.03 (m, 5H), 1.80 ꢃ 1.42 (m, 11H), 1.16 (s, 3H),
1.13 ꢃ 1.09 (m, 1H), 0.87 (d, J ¼ 6.8 Hz, 3H), 0.71 (d, J ¼ 6.8 Hz, 3H);
13 C NMR (100 MHz, CDCl3): d (ppm) 217.3, 169.1, 159.9, 157.6,
139.1, 141.1, 129.7, 121.3, 114.6, 111.9, 117.5, 74.7, 68.5, 59.9, 58.3,
55.3, 52.6, 45.6, 45.1, 44.1, 43.6, 42.0, 41.9, 36.8, 36.5, 36.2, 34.6,
30.5, 26.9, 26.5, 25.0, 16.9, 15.0, 11.6; HRMS: calculated for
C36H53N3O6 ([M þ H]þ): 624.4007, found 624.3995.
J ¼ 8.4 Hz, 2H), 6.49 (dd, J ¼ 17.2, 11.2 Hz, 1H), 5.78 (d, J ¼ 8.4 Hz,
1H), 5.33 (d, J ¼ 11.2 Hz, 1H), 5.19 (d, J ¼ 17.2 Hz, 1H), 4.61 (t,
J ¼ 5.6 Hz, 1H), 4.34 (d, J ¼ 5.2 Hz, 2H), 3.79 (s, 3H), 3.45 ꢃ 3.41 (m,
4H), 3.35 (dd, J ¼ 10.4, 6.4 Hz, 1H), 3.15 (ABq, J ¼ 17.2 Hz, 2H),
2.62 ꢃ 2.46 (m, 4H), 2.36 ꢃ 2.03 (m, 5H), 1.81 ꢃ 1.40 (m, 11H), 1.16
(s, 3H), 1.13 ꢃ 1.08 (m, 1H), 0.87 (d, J ¼ 6.8 Hz, 3H), 0.71 (d,
J ¼ 6.8 Hz, 3H); 13 C NMR (100 MHz, CDCl3): d (ppm) 217.2, 169.0,
157.4, 139.0, 131.4, 129.2, 117.3, 114.0, 74.6, 68.4, 59.9, 58.2, 55.3,
45.5, 45.0, 44.5, 44.0, 43.6, 41.8, 36.7, 36.1, 34.5, 30.4, 26.8, 26.4,
24.9, 16.8, 14.9, 11.5; HRMS: calculated for C35H51N3O6 ([M þ H]þ):
610.3851, found 610.3831.
6o: white powder; yield: 53.1%; mp: 109.6–110.7 ꢁC; 1H NMR
(400 MHz, CDCl3):
d
(ppm) 7.58 (d, J ¼ 8.4 Hz, 2H), 7.41 (d,
6t: white powder; yield: 82.7%; mp: 119.5–122.2 ꢁC; 1H NMR
(400 MHz, CDCl3): d (ppm) 6.49 (dd, J ¼ 17.2, 11.2 Hz, 1H), 5.77 (d,
J ¼ 8.4 Hz, 1H), 5.35 (s, 1H), 5.32 (dd, J ¼ 11.2, 1.6 Hz, 1H), 5.18 (dd,
J ¼ 17.2, 1.6 Hz, 1H), 3.40 (t, J ¼ 4.8 Hz, 4H), 3.36 ꢃ 3.24 (m, 3H),
3.12 (ABq, J ¼ 17.2 Hz, 2H), 2.63 ꢃ 1.99 (m, 16H), 1.83 ꢃ 1.31 (m,
17H), 1.15 (s, 3H), 1.13 ꢃ 1.03 (m, 1H), 0.86 (d, J ¼ 6.8 Hz, 3H), 0.70
(d, J ¼ 6.8 Hz, 3H); 13 C NMR (100 MHz, CDCl3): d (ppm) 217.3,
169.1, 162.6, 157.9, 139.2, 117.4, 77.5, 77.2, 76.8, 74.7, 68.5, 60.0,
58.3, 57.6, 54.3, 52.7, 45.6, 45.1, 44.0, 43.6, 41.9, 37.3, 36.8, 36.6,
36.2, 34.6, 31.5, 30.5, 26.9, 26.5, 26.0, 25.0, 24.4, 16.8, 15.0, 11.6;
HRMS: calculated for C34H56N4O5 ([M þ H]þ): 601.4323,
found 601.4326.
J ¼ 8.4 Hz, 2H), 6.50 (dd, J ¼ 17.2, 11.2 Hz, 1H), 5.79 (d, J ¼ 8.4 Hz,
1H), 5.34 (dd, J ¼ 11.2, 1.2 Hz, 1H), 5.20 (d, J ¼ 17.2 Hz, 1H), 4.83 (t,
J ¼ 5.6 Hz, 1H), 4.48 (d, J ¼ 5.6 Hz, 2H), 3.48(t, J ¼ 4.4 Hz, 4H), 3.35
(d, J ¼ 6.2 Hz, 1H), 3.25 (ABq, J ¼ 17.2 Hz, 2H), 2.67 ꢃ 2.45 (m, 4H),
2.38 ꢃ 2.03 (m, 5H), 1.81 ꢃ 1.37 (m, 11H), 1.16 (s, 3H), 1.14 ꢃ 1.09
(m, 1H), 0.88 (d, J ¼ 6.4 Hz, 3H), 0.72 (d, J ¼ 6.8 Hz, 3H); 13 C NMR
(100 MHz, CDCl3): d (ppm) 217.1, 168.3, 157.4, 143.6, 139.1, 129.7
(q, J ¼ 33 Hz), 127.9, 125.6 (q, J ¼ 4 Hz), 117.5, 74.7, 69.0, 59.3, 58.3,
52.7, 45.6, 45.1, 44.6, 44.1, 43.5, 41.9, 36.8, 36.2, 34.6, 30.5, 26.9,
26.5, 25.0, 16.9, 15.0, 11.6; HRMS: calculated for C35H48F3N3O5
([M þ H]þ): 648.3619, found 648.3597.
6p: light yellow powder; yield: 55.1%; mp: 115.2–116.5 ꢁC; 1H
NMR (400 MHz, CDCl3): d (ppm) 8.14 (d, J ¼ 8.4 Hz, 2H), 7.44 (d,
J ¼ 8.4 Hz, 2H), 6.49 (dd, J ¼ 17.2, 11.2 Hz, 1H), 5.79 (d, J ¼ 8.4 Hz,
1H), 5.33 (d, J ¼ 11.2 Hz, 1H), 5.21–5.15 (m, 2H), 4.50 (d, J ¼ 5.6 Hz,
2H), 3.46 (t, J ¼ 4.4 Hz, 4H), 3.36 (s, 1H), 3.15 (ABq, J ¼ 17.2 Hz, 2H),
2.62–2.50 (m, 4H), 2.38 ꢃ 2.05 (m, 5H), 1.82 ꢃ 1.41 (m, 11H), 1.16 (s,
3H), 1.11 (d, J ¼ 13.6 Hz, 1H), 0.88 (d, J ¼ 6.8 Hz, 3H), 0.71 (d,
J ¼ 6.8 Hz, 3H); 13 C NMR (100 MHz, CDCl3): d (ppm) 217.1, 168.9,
157.3, 147.4, 147.1, 139.1, 128.0, 123.8, 117.2, 74.6, 68.5, 60.41,
59.7, 58.2, 52.4, 45.5, 45.0, 44.2, 44.0, 43.7, 41.8, 36.7, 36.1, 34.5,
30.4, 26.8, 26.5, 24.8, 16.7, 14.9, 11.5; HRMS: calculated for
C34H48N4O7 ([M þ H]þ): 625.3596, found 625.3596.
6u: white powder; yield: 79.2%; mp: 92.1–93.8 ꢁC; 1H NMR
(400 MHz, CDCl3): d (ppm) 7.98 (s, 1H), 6.47 (dd, J ¼ 17.2, 11.2 Hz,
1H), 5.76 (d, J ¼ 8.4 Hz, 1H), 5.30 (d, J ¼ 11.2 Hz, 1H), 5.22 ꢃ 5.07 (m,
2H), 3.78 ꢃ 3.62 (m, 4H), 3.49 ꢃ 3.24 (m, 6H), 3.10 (ABq, J ¼ 17.2 Hz,
2H), 2.63 ꢃ 2.37 (m, 10H), 2.36 ꢃ 1.96 (m, 5H), 1.81 ꢃ 1.29 (m, 11H),
1.13 (s, 3H), 1.11 ꢃ 1.04 (m, 1H), 0.85 (d, J ¼ 6.8 Hz, 3H), 0.69 (d,
J ¼ 6.8 Hz, 3H); 13 C NMR (100 MHz, CDCl3): d (ppm) 217.2, 169.0,
157.8, 139.2, 117.3, 74.6, 68.5, 67.0, 59.9, 58.3, 57.6, 53.4, 52.6, 45.5,
45.1, 44.0, 43.6, 41.9, 37.0, 36.1, 34.5, 30.5, 26.9, 26.5, 24.9, 16.8,
15.0, 11.6; HRMS: calculated for C33H54N4O6 ([M þ H]þ): 603.4116,
found 603.4144.
6q: white powder; yield: 45.6%; mp: 107.6–108.7 ꢁC; 1H NMR
(400 MHz, CDCl3):
d
(ppm) 7.61 (d, J ¼ 7.6 Hz, 1H), 7.57 (d,
Synthesis of compound 6v
The solution of 11 (128 mg, 0.47 mmol) in acetonitrile (10 ml) was
J ¼ 7.6 Hz, 1H), 7.49 (t, J ¼ 7.6 Hz, 1H), 7.34 (t, J ¼ 7.6 Hz, 1H), 6.47
(dd, J ¼ 17.2, 11.2 Hz, 1H), 5.76 (d, J ¼ 8.4 Hz, 1H), 5.31 (d,
J ¼ 11.2 Hz, 1H), 5.17 (d, J ¼ 17.2 Hz, 1H), 4.93 (d, J ¼ 5.6 Hz, 1H), treated with 22-(piperazine-1-yl)-22-deoxypleuromutilin 8 (210 mg,
4.57 (d, J ¼ 5.6 Hz, 2H), 3.40 (t, J ¼ 4.8 Hz, 4H), 3.33 (dd, J ¼ 10.4, 0.47 mmol) and DMAP (57 mg, 0.47 mmol), which was heated to
6.8 Hz, 1H), 3.11 (ABq, J ¼ 17.2 Hz, 2H), 2.58 ꢃ 2.44 (m, 4H), reflux for 24 h. After solvent removal, the residue was dissolved in
2.35 ꢃ 2.04 (m, 5H), 1.80 ꢃ 1.39 (m, 11H), 1.14 (s, 3H), 1.12 ꢃ 1.06 EtOAc (10 ml). The organic phase was washed with 1 N NaOH and
(m, 1H), 0.86 (d, J ¼ 6.8 Hz, 3H), 0.69 (d, J ¼ 6.8 Hz, 3H); 13 C NMR brine, followed by drying over anhydrous Na2SO4. The filtrate

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
773
obtained via filtration was concentrated until dry, and then purifi- (m, 6H), 1.74– 1.27 (m, 10H), 1.15 (s, 3H), 1.12 ꢃ 1.06 (m, 1H), 0.86
(d, J ¼ 6.8 Hz, 3H), 0.70 (d, J ¼ 6.8 Hz, 3H); 13 C NMR (100 MHz,
cation by silica gel chromatography was conducted to obtain
1
compound 6v. white powder; yield: 40.5%; mp: 105.1–106.3 ꢁC; H
CDCl3) d (ppm) 217.1, 168.9, 157.4, 145.8, 139.1, 129.2, 129.1,
NMR (400 MHz, CDCl3): d (ppm) 7.33 ꢃ 7.31 (m, 2H), 7.11 ꢃ 7.06 (m,
117.3, 115.2, 74.6, 68.4, 59.9, 58.2, 52.6, 45.5, 45.0, 44.7, 44.0, 43.6,
3H), 6.47 (dd, J ¼ 17.2, 11.2 Hz, 1H), 5.74 (d, J ¼ 8.4 Hz, 1H), 5.30
41.8, 36.7, 36.1, 34.5, 30.4, 26.8, 26.4, 24.9, 16.7, 14.9, 11.5; HRMS:
calculated for C34H50N4O5 ([M þ H]þ): 595.3854, found 595.3860.
(dd, J ¼ 11.2, 1.2 Hz, 1H), 5.17(dd, J ¼ 17.2, 1.2 Hz, 1H), 3.33 (dd,
J ¼ 10.4, 6.8 Hz, 1H), 3.25 (t, J ¼ 4.9 Hz, 4H), 3.21 (s, 3H), 3.02 (ABq,
J ¼ 17.2 Hz, 2H), 2.40 ꢃ 2.23 (m, 7H), 2.26 ꢃ 1.99 (m, 2H),
1.80 ꢃ 1.38 (m, 11H), 1.14 (s, 3H), 1.12 ꢃ 1.05 (m, 1H), 0.85 (d,
J ¼ 7.2 Hz, 3H), 0.66 (d, J ¼ 7.2 Hz, 3H); 13 C NMR (100 MHz, CDCl3):
d (ppm) 217.2, 168.9, 160.9, 146.7, 139.0, 129.5, 124.6, 123.8, 117.3,
74.6, 68.3, 59.8, 58.2, 52.5, 45.5, 45.4, 44.9, 43.9, 41.8, 39.6, 36.7,
36.0, 34.5, 30.4, 26.8, 26.4, 24.9, 16.7, 14.9, 11.5; HRMS: calculated
for C34H49N3O5 ([M þ H]þ): 580.3745, found 580.3744.
Synthesis of compound 6z
6y (190 mg, 0.32 mmol) and triethylamine (49 mg, 0.48 mmol) were
dissolved in dry THF, and chloroacetyl chloride (54 mg, 0.48 mmol)
was added under an ice bath condition to the above solution. The
mixture was stirred at 25 ꢁC for about 4 h before water was added.
The resulting mixture was extracted via DCM, washed with brine,
and dried over anhydrous Na2SO4. Then, the organic phase was
concentrated to obtain the crude intermediate, which was purified
by silica gel column chromatography to produce 14.
Synthesis of compound 6w
Compound 6g (136 mg, 0.2 mmol) was added to a mixture of 6 ml
DCM and TFA (v/v ¼ 10/1) and stirred at 25 ꢁC until completely
converted. After neutralisation by saturated aqueous NaHCO3, the
organic phase was collected and dried over anhydrous Na2SO4.
Then, the filtrate obtained via filtration was concentrated to gen-
erate the crude product, which was purified by silica gel chroma-
tography to produce compound 6w. white powder; yield: 95.6%;
mp: 146.2–147.5 ꢁC; 1H NMR (400 MHz, CDCl3): d (ppm) 7.02 (t,
J ¼ 8.0 Hz, 1H), 6.95 (t, J ¼ 2.0 Hz, 1H), 6.56 ꢃ 6.44 (m, 2H),
6.38 ꢃ 6.31 (m, 2H), 5.79 (d, J ¼ 8.4 Hz, 1H), 5.34 (dd, J ¼ 11.2,
1.6 Hz, 1H), 5.20 (dd, J ¼ 17.2, 1.2 Hz, 1H), 3.52 (t, J ¼ 4.8 Hz, 4H),
3.35 (d, J ¼ 6.0 Hz, 1H), 3.12 (ABq, J ¼ 17.2 Hz, 2H), 2.68 ꢃ 2.49 (m,
4H), 2.41 ꢃ 2.02 (m, 7H), 1.86 ꢃ 1.21 (m, 13H), 1.16 (s, 3H),
1.14 ꢃ 1.07 (m, 1H), 0.87 (d, J ¼ 6.8 Hz, 4H), 0.72 (d, J ¼ 6.8 Hz, 3H);
13 C NMR (100 MHz, CDCl3): d (ppm) 217.3, 169.1, 155.0, 147.3,
140.0, 139.2, 129.7, 117.5, 110.2, 109.9, 106.8, 74.7, 68.6, 59.9, 58.3,
52.7, 45.6, 45.2, 44.1, 44.0, 41.9, 36.8, 36.2, 34.6, 30.6, 27.0, 26.5,
25.0, 16.9, 15.0, 11.6; HRMS: calculated for C33H48N4O5 ([M þ H]þ):
581.3697, found 581.3704.
Compound 14 (201 mg, 0.30 mmol), morpholine (52 mg,
0.60 mmol), and K2CO3 (82 mg, 0.60 mmol) were dissolved in dry
THF (5 ml) and maintained at reflux for about 6 h. After the solu-
tion was concentrated, the residue was purified by silica gel col-
umn chromatography to generate 6z.
14: white powder; yield: 70.1%; mp: 118.1–119.2 ꢁC; 1H NMR
(400 MHz, CDCl3): d (ppm) 8.25 (s, 1H), 7.49 (d, J ¼ 8.4 Hz, 2H), 7.29
(d, J ¼ 8.4 Hz, 2H), 6.49 (dd, J ¼ 17.2, 11.2 Hz, 1H), 5.79 (d,
J ¼ 8.4 Hz, 1H), 5.34 (d, J ¼ 11.2 Hz, 1H), 5.20 (d, J ¼ 17.2 Hz, 1H),
4.77 (s, 1H), 4.39 (d, J ¼ 5.6 Hz, 2H), 4.18 (s, 2H), 3.48 (s, 4H),
3.39 ꢃ 3.32 (m, 1H), 3.30 ꢃ 3.07 (m, 2H), 2.64 (br, 4H), 2.38 ꢃ 2.15
(m, 3H), 2.08 (dd, J ¼ 16.4, 8.0 Hz, 2H), 1.80 ꢃ 1.47 (m, 11H), 1.16 (s,
3H), 1.13 ꢃ 1.09 (m, 1H), 0.88 (d, J ¼ 6.8 Hz, 3H), 0.71 (d, J ¼ 6.8 Hz,
3H); 13 C NMR (100 MHz, CDCl3): d (ppm) 216.7, 188.9, 164.0, 157.4,
139.0, 135.8, 128.6, 120.6, 117.4, 74.6, 58.2, 52.3, 45.5, 45.0, 44.5,
44.0, 42.9, 41.8, 36.7, 36.1, 34.5, 30.4, 26.8, 26.4, 24.9, 16.8, 14.9,
11.5; HRMS: calculated for C35H51ClN4O6 ([M þ H]þ): 671.3586,
found 671.3570.
6z: white powder; yield: 50.3%; mp: 120.1–121.5 ꢁC; 1H NMR
(400 MHz, CDCl3): d (ppm) 9.02 (s, 1H), 7.49 (d, J ¼ 8.4 Hz, 2H), 7.26
(d, J ¼ 8.4 Hz, 2H), 6.48 (dd, J ¼ 17.2, 11.2 Hz, 1H), 5.77 (d,
J ¼ 8.4 Hz, 1H), 5.31 (dd, J ¼ 11.2, 1.2 Hz, 1H), 5.18 (dd, J ¼ 17.2,
1.2 Hz, 1H), 4.79 (t, J ¼ 5.6 Hz, 1H), 4.35 (d, J ¼ 5.6 Hz, 2H),
3.78 ꢃ 3.74 (m, 4H), 3.41 (t, J ¼ 5.0 Hz, 4H), 3.34 (dd, J ¼ 9.6, 6.8 Hz,
1H), 3.21 ꢃ 3.02 (m, 4H), 2.63 ꢃ 2.58 (m, 4H), 2.58 ꢃ 2.45 (m, 4H),
2.36 ꢃ 2.01 (m, 6H), 1.89 (s, 1H), 1.76 (dd, J ¼ 14.2, 2.4 Hz, 1H),
1.69 ꢃ 1.51 (m, 5H), 1.49 ꢃ 1.43 (m, 1H), 1.38 ꢃ 1.31 (m, 1H), 1.15
(s, 3H), 1.12 ꢃ 1.06 (m, 1H), 0.86 (d, J ¼ 6.8 Hz, 3H), 0.70 (d,
J ¼ 6.8 Hz, 3H); 13 C NMR (100 MHz, CDCl3): d (ppm) 217.1, 168.9,
167.9, 157.4, 139.1, 136.6, 135.5, 128.5, 119.8, 117.3, 74.6, 68.5,
67.0, 59.8, 58.2, 53.8, 52.6, 45.5, 45.0, 44.5, 44.0, 43.7, 41.8, 36.7,
36.1, 34.5, 30.4, 29.7, 26.8, 26.5, 24.9, 16.7, 14.9, 11.5; HRMS: calcu-
lated for C40H59N5O7 ([M þ H]þ): 722.4487, found 722.4497.
Synthesis of compound 6x and 6y
A mixture of 6d (122 mg, 0.2 mmol), SnCl2 (474 mg, 2.5 mmol) and
ethanol (5 ml) was stirred under reflux for 5 h. After cooling to
25 ꢁC, 1 N NaOH was added. The resultant mixture was extracted
with DCM (10 ml ꢂ 3) and separated. After the organic phase was
dried over Na2SO4 and concentrated, the residue was purified via
silica gel column chromatography to acquire 6x. 6y was synthes-
ised by a similar method used for 6p.
6x: yellow powder; yield: 92.5%; mp: 137.4–138.7 ꢁC; 1H NMR
(400 MHz, CDCl3):
d
(ppm) 7.08 (d, J ¼ 8.4 Hz, 2H), 6.62 (d,
J ¼ 8.4 Hz, 2H), 6.50 (dd, J ¼ 17.2, 11.2 Hz, 1H), 6.18 (s, 1H), 5.79 (d,
J ¼ 8.4 Hz, 1H), 5.34 (d, J ¼ 11.2 Hz, 1H), 5.20 (d, J ¼ 17.2 Hz, 1H),
3.50 (t, J ¼ 4.8 Hz, 4H), 3.35 (d, J ¼ 5.2 Hz, 1H), 3.14 (ABq,
J ¼ 17.2 Hz, 2H), 2.67 ꢃ 2.49 (m, 4H), 2.41 ꢃ 2.01 (m, 5H),
1.83 ꢃ 1.32 (m, 13H), 1.16 (s, 3H), 1.14 ꢃ 1.07 (m, 1H), 0.87 (d,
J ¼ 6.8 Hz, 3H), 0.72 (d, J ¼ 6.8 Hz, 3H); 13 C NMR (100 MHz, CDCl3):
d (ppm) 217.3, 169.1, 155.8, 142.9, 139.2, 130.2, 123.1, 117.5, 115.7,
74.7, 68.6, 59.9, 58.3, 52.7, 45.6, 45.2, 44.1, 44.0, 41.9, 36.8, 36.2,
34.6, 30.6, 29.8, 27.0, 26.5, 25.0, 16.9, 15.0, 11.6; HRMS: calculated
for C33H48N4O5 ([M þ H]þ): 581.3697, found 581.3705.
Biological assay
Bacterial solution preparation
Upon overall recovery of S. aureus ATCC 25923, MRSA ATCC
33591, MRSA ATCC 43300 and E.coli ATCC 25922, the single
selected colony was inoculated in sterile Luria-Bertani (LB) broth
and incubated at 37 ꢁC for 1 8 ꢀ 20 h. The bacterial solution was
adjusted to 0.5 McFarland standard with saline and subsequently
diluted with LB broth to the approximate concentration of
6y: light yellow powder; yield: 75.5%; mp: 118.5–119.3 ꢁC; 1H
NMR (400 MHz, CDCl3): d (ppm) 7.08 (d, J ¼ 8.4 Hz, 2H), 6.65 ꢃ 6.60
(m, 2H), 6.48 (dd, J ¼ 17.2, 11.2 Hz, 1H), 5.77 (d, J ¼ 8.4 Hz, 1H), 5.32
(dd, J ¼ 11.2, 1.2 Hz, 1H), 5.18 (dd, J ¼ 17.2, 1.2 Hz, 1H), 4.59 (t,
J ¼ 5.6 Hz, 1H), 4.27 (d, J ¼ 5.2 Hz, 2H), 3.40 (t, J ¼ 5.2 Hz, 4H), 3.35
(s, 1H), 3.11 (ABq, J ¼ 17.2 Hz, 2H), 2.56 ꢃ 2.46 (m, 4H), 2.36 ꢃ 2.02 106ꢀ107 CFUꢄmLꢃ1
.

774
Y. ZHANG ET AL.
MIC And MBC assays
Conclusions
MIC values were established in vitro using the agar dilution
method34,35. Moreover, tiamulin fumarate was selected as the ref-
erence drug. All compounds were dissolved in a small amount of
ethanol and diluted in distilled water to a concentration of
1280 mgꢄmLꢃ1. Next, two-fold serially diluted pleuromutilin com-
pound solutions containing distilled water were produced until
the final concentration reached 0.625 mgꢄmLꢃ1. After ten-fold dilu-
tion with LB broth, the two-fold serially diluted solutions were
successively added to plate wells containing 10 ml of the above
four bacterial solutions; then, solutions were incubated at 37 ꢁC
for 1 6 ꢀ 18 h and data was recorded. Each procedure was
repeated 3 times. MBC was considered when the compounds
killed over 99% of the tested bacterial culture. MIC-corresponding
wells and three previous wells were homogenised, serially diluted,
and plated on Mueller-Hinton (MH) agar to determine MBC. Data
was recorded after plates underwent incubation at 37 ꢁC for
18–24 h. Each procedure was repeated 3 times.
In summary, a series of pleuromutilin derivatives possessing the
piperazinyl urea linkage were designed and synthesised, and their
antibacterial activities against Gram-positive and Gram-negative
strains were evaluated. Nearly all synthesised compounds exhib-
ited broad-spectrum antibacterial activities, and particularly, more
potent activities against MRSA strains. Activities of 6p were most
effective with a MIC concentration of 0.1 2 5 ꢀ 0.25 lg/mL, which
was 8 ꢀ 16 times that of Tiamulin. Time-kill kinetics indicated that
at 1 ꢂ MIC, compound 6p and Tiamulin had similar bactericidal
effects against MRSA ATCC 33591; however compound 6p was
more effective than Tiamulin against E.coli ATCC 25922, since it
eliminated total bacteria colony in 24 h while Tiamulin could not.
Molecular docking demonstrated that aside from hydrophobic
interactions, benzyl urea linkage and terminal nitro groups from
the side chain could produce several hydrogen bonds and p-p
stacking with surrounding residues.
Author contributions
Time-kill kinetics assay
Y.Z., C.X., F.S. and R.S. designed and synthesised the compounds.
Y.L. and D.L. performed the biological evaluation. C. W. imple-
mented the cytotoxicity test. X. Y. performed the molecular dock-
ing. Y.Z., J.Y. and Z.W. interpreted the data and wrote the paper.
Compounds 6p and tiamulin fumarate were dissolved in MH broth
to generate different solutions of 1 ꢂ MIC and 6 ꢂ MIC, followed
by inoculation with the above bacteria suspension at 37 ꢁC; After
specified time intervals (0, 1, 2, 4, 6, 12 and 24 h), 10 mL of the All authors have read and agreed to the published version of
the manuscript.
above mixture was serially diluted 10-fold with saline and incu-
bated at 37 ꢁC for 24 h on MH agar plates. Following this, viable
colonies were counted and expressed as log 10 CFUꢄmLꢃ1. Each
procedure was done a total of 3 times.
Disclosure statement
There are no conflicts to declare.
CCK-8 assay
Funding
All cell lines were cultured in Dulbecco’s modified Eagle’s medium
(DMEM), with 10% foetal bovine serum (FBS) and supplemented
with 1% penicillin-streptomycin (P/S). The cell counting kit-8 (CCK-
8) was purchased from Dojindo Laboratories (Kumamoto, Japan).
Briefly, a 100 lL suspension of 10000 LO2 cells and 20000
HEK293T cells were seeded in 96-well plates, respectively. They
were treated with six different concentrations of 6p (0.125 lg/mL,
0.25 lg/mL, 0.5 lg/mL, 1 lg/mL, 2 lg/mL and 4 lg/mL) and incu-
bated at 37 ꢁC in a CO2 incubator for 48 h. Afterwards, the CCK-8
solution (10 lL) was added to each well, and plates were incu-
bated for an additional 1 h at 37 ꢁC. The absorbance at 450 nm
was then recorded using a SpectraMax-190 microplate reader
(Molecular Devices, USA). Each procedure was done a total of
3 times.
This research was funded by Chunhui Project of Ministry of
Education of China [Z2016164], Scientific Research Foundation of
the Education Department of Sichuan Province [12ZB128 and
18TD0023], National Undergraduate Training Program for
Innovation and Entrepreneurship [202010650016], National Natural
Science Foundation of China [81703355] and Scientific Research
Project from Science and Technology Department of Yibin
[2019GY007 and 2018ZGY013].
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