Synthesis and antibacterial activities of novel pleuromutilin derivatives bearing an aminothiophenol moiety

Article abstract of DOI:10.1111/cbdd.13328

We synthesized a series of novel thioether pleuromutilin derivatives incorporating 2-aminothiophenol moieties into the C14 side chain via acylation reactions under mild conditions. We evaluated the in-vitro antibacterial activities of the derivatives against methicillin-resistant Staphylococcus aureus (MRSA, ATCC 43300), Staphylococcus aureus (ATCC 29213) and Escherichia coli (ATCC 25922). The majority of the synthesized derivatives possessed moderate antibacterial activities. Compound 8 was found to be the most active antibacterial derivative against MRSA. We conducted docking experiments to understand the possible mode of interactions between compounds 8, 9b, 11a and 50S ribosomal subunit. The docking results proved that there is a reasonable correlation between the binding free energy and the antibacterial activity. Compound 8 was evaluated for its in-vivo antibacterial activity and showed higher efficacy than tiamulin against MRSA in mouse infection model.

Full text of DOI:10.1111/cbdd.13328

PROFESSOR YOU-ZHI TANG (Orcid ID : 0000-0003-2293-0287)
Article type
: Research Article
Synthesis and antibacterial activities of novel
pleuromutilin derivatives bearing an
aminothiophenol moiety
Zhao-Sheng Zhang, Yun-Zhen Huang, Jian Luo, Zhen Jin, Ya-Hong Liu, You-Zhi Tang*
Guangdong Provincial Key Laboratory of Veterinary Pharmaceutics Development and Safety
Evaluation, College of Veterinary Medicine, South China Agricultural University, Guangzhou
5
10642, China
Correspondence should be addressed:
College of Veterinary Medicine, South China Agricultural University, No. 483 Wushan
Road, Tianhe District, Guangzhou 510642, China
You-Zhi Tang,
Fax: +86 20 85280665; Email: youzhitang@scau.edu.cn
Abstract
A series of novel thioether pleuromutilin derivatives incorporating 2-aminothiophenol
moieties into the C14 side chain were synthesized via acylation reactions under mild
conditions. The in vitro antibacterial activities of the derivatives against methicillin-resistant
Staphylococcus aureus (MRSA, ATCC43300), Staphylococcus aureus (ATCC 29213) and
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13328
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Escherichia coli (ATCC25922) were evaluated. The majority of the synthesized derivatives
possessed moderate antibacterial activities. Compound 8 was found to be the most active
antibacterial derivative against MRSA. Docking experiments were conducted to understand
the possible mode of interactions between compounds 8, 9b, 11a and 50S ribosomal subunit.
The docking results proved that there is a reasonable correlation between the binding free
energy and the antibacterial activity. Compound 8 was evaluated for its in vivo antibacterial
activity and showed higher efficacy than tiamulin against MRSA in mouse infection model.
Introduction
Multidrug-resistant bacteria represent a severe threat to human communities worldwide[1].
However, there have not been any developments for antibacterial against antibiotic-resistant
bacteria in recent decades [2]. Among those antibiotic-resistant bacteria, methicillin-resistant
Staphylococcus aureus (MRSA) strains have been reported resistance to clinical antibiotics,
including the class of β-lactam antibiotics[3], linezolid[4], vancomycin[5] and
daptomycin[6]. Infections with MRSA have been the key cause of higher costs, longer
hospitalization[7], immense morbidity and mortality, and so on [8]. Thus, there is an urgent
need for antibacterial agents with novel mechanisms of action to treat infections caused by
MRSA.
Pleuromutilin 1 (Figure 1) is a naturally tricyclic diterpene natural product, first isolated in
1
951 from Pleurotus mutiliz and Pleurotus passeckeranius, that displays potent antibacterial
activity against Gram-positive bacteria and mycoplasmas [9, 10]. The pleuromutilin class of
antibiotics selectively interferes bacterial protein synthesis through binding to the bacterial
5
0S ribosome subunit at the peptidyl transferase center (PTC) [11-13]. This distinct mode of
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action of pleuromutilin also made it an attractive lead compound in the discovery and
development of new antibacterial agents for the treatment of resistant bacterial infections [14,
1
5].
Numerous semisynthetic pleuromutilin derivatives have been prepared and evaluated [11,
5-18]. In order to improve the pharmacokinetic of pleuromutilin derivatives, much synthetic
1
effort has also been focused on total synthesis of pleuromutilin [19, 20]. The most successful
of pleuromutilin synthetic modications was the replacement of the hydroxyl group in C14
glycolic ester side chain of pleuromutilin with a substituent containing a sulfide linkage. This
has led to the discovery of three pleuromutilin drugs tiamulin (2, Figure 1) [21], valnemulin
(
3, Figure 1) [22] and retapamulin (4, Figure 1) [14]. Tiamulin and valnemulin are approved
in veterinary medicine for pigs and poultry. Retapamulin is the first pleuromutilin drug
approved for human use to treat topical skin infections both in USA and Europe [23].
Although it was not licensed for use in MRSA infections, retapamulin displayed clinical
successes in clinical trails for few cases (100%:8/8) [24]. Additionally, BC-3781 (5, Figure 1)
and BC-7013 (6, Figure 1) have been reported in clinical trail for human use [25, 26]. All
above pleuromutilin derivatives either licensed or in clinical trails vary only in the C14 side
chain of pleuromutilin, furthermore all of them have a thioether moiety.
Previous work in our group has led to several semisynthetic pleuromutilin derivatives with
powerful antibacterial activity against S. aureus including ATCC 29213 [27] and MRSA [28].
One of the synthesized pleuromutilin derivative in our group possessed superior in vivo
efficacy to that of tiamulin in MRSA systemic infection model [28]. This research
background motivated us to develop the pleuromutilin derivatives containing a thioether
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moiety, and thus, 13 pleuromutilin derivatives were designed, synthesized and evaluated for
their antibacterial activities against three strains including MRSA. The antibacterial and
docking results in this study may help us to design novel pleuromutilin derivatives.
Experimental
Materials
All reagents and solvents were purchased from commercial suppliers as reagent grade and
were used as supplied unless noted otherwise. Column chromatography was carried out using
silica gel (200-300 mesh). Melting points were measured on a Shenguang X-4 apparatus
1
13
(
China) and are uncorrected. H-NMR and C-NMR spectra were recorded at Bruker AV-400
or Bruker AV-600 spectrometer. The chemical shift values (δ) are reported in ppm relative to
tetramethylsilane as internal standard. Fourier transform infrared(FT-IR) were obtained on a
Nicolet 6700 Research FTIR spectrometer using KBr pellets, and the absorptions are reported
-1
in cm . High-resolution mass spectra were conducted using a LTQ-Orbitrap mass
spectrometer (Thermo Fisher) with an electro spray ionization (ESI) source.
Synthesis
A general synthesis strategy based on the usual 22-O-tosylpheuromutilin, 2-aminophenol and
a variety of phenylacetyl chlorides were used (Scheme 1).
Scheme 1 here
2
2-O-tosylpleuromutilin (7)
A solution of pleuromutilin 1(5.4g, 14.27 mmol) in pyridine (30.0 mL) was stirred at 0 ℃ in
a three-necked round bottom flask, and p-toluenesulfonyl chloride (8.6 g, 45.11 mmol) was
3
added. The solution was stirred at 0 ℃ for 3h. CHCl (50 mL) and Ice-cold water (50 mL)
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were added to the solution. The organic phase was washed with a 2 M aqueous solution of
SO , a saturated aqueous solution of NaHCO and water, respectively. Then the organic
phase was dried with anhydrous Na SO for 2 h and evaporated in vacuum. The residue was
precipitated from isopropanol to give a white solid (6.0 g, 79%) [29].
H
2
4
3
2
4
2
2-(2-Amino-phenylsulfanyl)-22-deoxypleuromutilin (8)
2
2-O-tosylpleuromutilin (1g, 1.87 mmol) was dissolved in ethyl acetate (35 mL) and sodium
iodide (0.31g, 2.07 mmol) was added. The mixture was stirred for 0.5 h at 70 ℃. After
2-O-tosylpleuromutilin had been dissolved completely, a solution of 2-aminothiophenol
0.35g, 2.79mmol) in 20% aqueous NaOH (10mL) was added dropwise, and the resulting
2
(
reaction mixture was stirred at 70 ℃ until complete consumption of 22-O-tosylpleuromutilin
was obtained. The reaction mixture was concentrated in vacuo. Chloroform was added, and
the mixture was washed with brine and water. The organic layer was dried over anhydrous
2 4
Na SO , filtered and concentrated under reduced pressure to give the crude product. The
crude product was purified by column chromatography (ethyl acetate: petroleum ether = 3:4)
using silica gel to afford the desired compounds (0.51g, 56%).
1
White powder; yield: 56%; m.p.: 184.5-186.8 ℃; H NMR (600 MHz, CDCl
3
) δ 7.38 (1H, dd,
J = 7.7, 1.6 Hz), 7.12 (1H, m), 6.68 (2H, m), 6.48 (1H, J = 17.4, 11.0 Hz, H19), 5.72 (1H, d, J
8.5 Hz, H14), 5.36 (1H, dd, J = 11.0, 1.5 Hz, H20), 5.21 (1H, dd, J = 17.4, 1.6 Hz, H20),
=
4
2
1
.40 (2H, s), 3.44 (1H, d, J = 14.8Hz, H22), 3.38 (1H, d, J = 14.9 Hz, H22), 3.35 (1H, s, H11),
.12 (5H, dd, J = 16.0, 8.6 Hz), 1.76 (1H, dd, J = 14.6, 3.3 Hz, H6), 1.56 (4H, m, H1, H7),
.39 (3H, s, H15), 1.28 (2H, m, H18, H13), 1.16 (3H, s, H18), 1.10 (1H, d, J = 13.9, 4.4 Hz,
1
3
H8), 0.88 (3H, d, J = 7.0 Hz, H17), 0.65 (3H, d, J = 7.0 Hz, H16) ; C NMR (151 MHz,
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CDCl
15.8, 74.6 (C11), 69.5 (C14), 58.1 (C4), 45.4 (C9), 44.6 (C13), 43.9, 41.7, 37.8, 36.7, 36.0
C10), 34.5 (C2), 30.4 (C8), 26.8 (C7), 26.3 (C18), 24.8 (C1), 16.6 (C16), 14.8 (C15), 11.5
3
) δ 217.0 (C3), 168.9 (C21), 147.4, 139.1 (C19), 136.4, 130.6, 119.4, 117.1 (C20),
1
(
(
-1
C17); IR (KBr, cm ) 3497, 3346, 2923, 1726, 1614, 1482, 1449, 1407, 1312, 1278, 1122,
+
1
020, 983, 964, 915, 751, 668; HR-MS (ESI): Calcd for C28
H40NO
4
S (M+H ): 486.2673;
Found: 486.2674.
General procedure for the synthesis of compounds 9a-9c, 10a-10c, 11a-11c and 12a-12c
Compound 8 (1g, 2.28mmol) was dissolved in ethyl acetate (35 mL), acyl chloride
derivatives (2.28mmol) was added dropwise, and the resulting reaction mixture was stirred at
7
0 ℃ for 1 h. The mixture was washed with water, followed by separation of organic layer.
The aqueous layer was extracted with chloroform and the combined organic layer extracts
were washed with brine, water and then dried with anhydrous Na SO . After evaporation the
2
4
organic solvent in vacuo, the residue was purified by silica gel column chromatography to
give a pure product.
2
2-(2-(2-Fluoro-benzoylamino)- phenylsulfanyl)-22-deoxypleuromutilin (9a)
1
White powder; yield: 62%; m.p.: 129.5-130.7 ℃; H NMR (600 MHz, CDCl
3
) δ 9.89 (1H, d,
J = 12.6 Hz), 8.59 (1H, d, J = 8.3 Hz), 8.19 (1H, m), 7.61 (1H, dd, J = 7.8, 1.6 Hz), 7.56 (1H,
m), 7.41 (1H, m), 7.34 (1H, m), 7.23 (1H, dd, J = 11.9, 8.2 Hz), 7.09 (1H, dd, J = 7.5, 1.4 Hz),
6
1
.36 (1H, dd, J = 17.4, 11.0 Hz, H19), 5.66 (1H, d, J = 8.5 Hz, H14), 5.24 (1H, dd, J = 10.9,
.6 Hz, H20), 5.12 (1H, dd, J = 17.3, 1.6 Hz, H20), 3.51 (1H, d, J = 15.3 Hz, H22), 3.46 (1H,
d, J = 15.4 Hz, H22), 3.31 (1H, d, J = 6.5 Hz, H11), 2.20 (5H, m), 1.95 (1H, dd, J = 16.0, 8.5
Hz), 1.75 (1H, m, H6), 1.53 (5H, m, H1, H7), 1.35 (3H, s, H15), 1.23 (2H, m, H18, H13),
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1
.10 (3H, s, H18), 1.06 (1H, d, J = 16.0 Hz, H8), 0.86 (3H, d, J = 7.0 Hz, H17), 0.58 (3H, d, J
13
=
3
7.1 Hz, H16) ; C NMR (151 MHz, CDCl ) δ 217.0 (C3), 168.1 (C21), 140.0, 138.8, 138.7
(
C19), 135.7, 133.7, 129.9, 124.6, 121.4, 117.2 (C20), 74.5 (C11), 69.8 (C14), 65.1 (C22),
8.0 (C4), 45.4 (C9), 44.5 (C13), 43.9 (C12), 41.7 (C5), 38.9, 36.6 (C6), 35.9 (C10), 34.4
C2), 30.4 (C8), 26.8 (C7), 26.4 (C18), 26.2, 24.8 (C1), 16.6 (C16), 14.7 (C15), 11.5 (C17);
5
(
-1
IR (KBr, cm ) 3529, 3334, 2941, 1728, 1674, 1581, 1529, 1454, 1435, 1375, 1311, 1192,
+
1
149, 1115, 1020, 918, 764, 663; HR-MS (ESI): Calcd for C35
H43FNO
5
S (M+H ): 608.2840;
Found: 608.2867.
2
2-(2-(3-Fluoro-benzoylamino)- phenylsulfanyl)-22-deoxypleuromutilin (9b)
1
Faint yellow powder; yield: 47%; m.p.: 130.9-132.1 ℃; H NMR (600 MHz, CDCl
3
) δ 9.90
(1H, s), 8.46 (1H, dd, J = 8.3, 1.4 Hz), 7.93 (1H, dd, J = 7.8, 1.2 Hz), 7.89 (1H, dd, J = 9.5,
2
.2 Hz), 7.61 (1H, dd, J = 7.8, 1.6 Hz), 7.53 (1H, m), 7.42 (1H, m), 7.32 (1H, m), 7.11 (1H,
m), 6.33 (1H, dd, J = 17.4, 11.0 Hz, H19), 5.64 (1H, d, J = 8.6 Hz, H14), 5.14 (1H, dd, J =
1.0, 1.4 Hz, H20), 5.10 (1H, dd, J = 17.5, 1.5 Hz, H20), 3.57 (1H, d, J = 16.3 Hz, H22), 3.50
1H, d, J = 16.0 Hz, H22), 3.30 (1H, d, J = 16.0 Hz, H11), 2.10 (5H, m), 1.74 (1H, dd, J =
1
(
1
1
4.7, 3.3 Hz, H6), 1.53 (4H, m, H1, H7), 1.36 (3H, s, H15), 1.22 (3H, m, H18, H13, 11-OH),
.09 (3H, s, H18), 1.01 (1H, d, J = 16.0 Hz,, H8), 0.86 (3H, d, J = 7.0 Hz, H17), 0.46 (3H, d,
1
3
J = 7.1 Hz, H16) ; C NMR (151 MHz, CDCl
3
) δ 216.7 (C3), 169.3 (C21),163.9, 140.3,
1
7
3
1
38.6 (C19), 137.3, 136.3, 130.7, 130.3, 124.9, 123.1, 122.5, 121.5, 118.8, 117.4 (C20), 114.8,
4.5 (C11), 70.3 (C14), 65.0 (C22), 58.0 (C4), 45.4(C9), 44.5 (C13), 43.9 (C12), 41.7, 40.5,
6.6 (C6), 36.0 (C10), 34.4 (C2), 30.4 (C8), 26.8 (C7), 26.2 (C18), 24.8 (C1), 16.5 (C16),
-1
4.8 (C15), 11.5 (C17) ; IR (KBr, cm ) 3567, 2937, 2864, 1732, 1675, 1598, 1579, 1510,
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1
455, 1372, 1291, 1190, 1176, 1117, 1037, 978, 775, 663; HR-MS (ESI) ): Calcd for
+
C
35
H43FNO
5
S (M+H ): 608.2840; Found: 608.2856.
2
2-(2-(4-Fluoro-benzoylamino)- phenylsulfanyl)-22-deoxypleuromutilin (9c)
1
Faint yellow powder; yield: 65%; m.p.: 129.5-130.7 ℃; H NMR (600 MHz, CDCl
3
) δ 9.82
(1H, s), 8.46 (1H, dd, J = 8.3, 1.3 Hz), 8.16 (2H, m), 7.60 (1H, dd, J = 7.7, 1.6 Hz), 7.41 (1H,
m), 7.23 (1H, s), 7.09 (1H, t, J = 7.6, 1.4 Hz), 6.31 (1H, dd, J = 17.4, 11.1 Hz, H19), 5.60 (1H,
d, J = 8.6 Hz, H14), 5.13 (1H, m, H20), 5.09 (1H, m, H20), 3.56 (1H, d, J = 16.2 Hz, H22),
3
1
0
.48 (1H, d, J = 16.2 Hz, H22), 3.29 (1H, d, J = 6.5 Hz, H11), 2.10 (5H, m), 1.73 (1H, m, H6),
.52 (4H, m, H1, H7), 1.35 (3H, s, H15), 1.20 (3H, m, H18, H13, 11-OH), 1.07 (3H, s, H18),
1
3
.96 (1H, m, H8), 0.85 (3H, d, J = 7.0 Hz, H17), 0.47 (3H, d, J = 7.1 Hz, H16) ; C NMR
(151 MHz, CDCl
3
) δ 216.8 (C3), 169.3 (C21), 165.9, 164.1, 138.6 (C19), 140.4, 136.2, 130.7,
1
4
2
1
29.9, 124.7, 122.3, 121.3, 117.2 (C20), 115.8, 77.4 (C11), 70.5 (C14), 57.9 (C4), 45.3 (C9),
4.5 (C13), 43.9 (C12), 41.6, 40.4, 36.5 (C6), 35.9 (C10), 34.4 (C2), 30.3 (C8), 26.8 (C7),
-
1
6.1 (C18), 24.8 (C1), 16.4 (C16), 14.8 (C15), 11.5 (C17) ; IR (KBr, cm ) 3508, 3331, 2939,
736, 1664, 1581, 1529, 1456, 1437, 1317, 1298, 1192, 1147, 1115, 1020, 918, 854, 766, 579;
+
HR-MS (ESI) ): Calcd for C35
H43FNO
5
S (M+H ): 608.2840; Found: 608.2836.
2
2-(2-(2-Chloro-benzoylamino)- phenylsulfanyl)-22-deoxypleuromutilin (10a)
1
Yellow powder; yield: 48%; m.p.: 157-160 ℃; H NMR (600 MHz, CDCl
3
) δ 9.91 (1H, s),
.44 (1H, dd, J = 8.3, 1.4 Hz), 8.19 (1H, m), 8.02 (1H, dd, J = 7.7, 1.4 Hz), 7.60 (2H, m),
.50 (1H, dd, J = 7.9, 1.3 Hz), 7.42 (1H, m), 7.10 (1H, t), 6.34 (1H, dd, J = 17.4, 11.0 Hz,
8
7
H19), 5.67 (1H, d, J = 8.5 Hz, H14), 5.32 (1H, s), 5.13 (1H, dd, J = 10.9, 1.5 Hz, H20), 5.09
1H, dd, J = 17.4, 1.6 Hz, H20), 3.56 (1H, d, J = 15.9 Hz, H22), 3.50 (1H, d, J = 15.9 Hz,
(
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H22), 3.30 (1H, d, J = 6.6 Hz, H11), 2.21 (5H, m,H2, H4, H10, H13), 1.74 (1H, dd, J = 14.4,
.2 Hz, H6), 1.53 (5H, m, H1, H7), 1.36 (3H, s, H15), 1.22 (3H, m, H18, H13), 1.09 (3H, s,
3
13
H18), 1.03 (1H, m, H8), 0.86 (3H, d, J = 7.0 Hz, H17), 0.48 (3H, d, J = 7.1 Hz, H16) ; C
NMR (151 MHz, CDCl ) δ 216.8 (C3), 169.4 (C21), 164.0, 140.3, 138.7 (C19), 136.8, 136.4,
34.9, 131.8, 130.7, 130.0, 127.9, 125.7, 124.9, 122.7, 121.6, 117.3 (C20), 74.5 (C11), 70.5
3
1
(
(
(
C22), 58.0 (C4), 45.4 (C9), 44.6 (C13), 43.9 (C12), 41.7, 40.6, 36.6 (C6), 36.0 (C10), 34.4
C2), 30.4 (C8), 26.8 (C7), 26.2 (C18), 24.8 (C1), 16.4 (C16), 14.8 (C15), 11.5 (C17) ; IR
-1
KBr, cm ) 3545, 3332, 2945, 1720, 1678, 1579, 1516, 1435, 1308, 1194, 1149, 1112, 1018,
+
9
6
2
17, 766, 747, 627; HR-MS (ESI): Calcd for C35
24.2575.
H
43ClNO
5
S (M+H ): 624.2545; Found:
2-(2-(3-Chloro-benzoylamino)- phenylsulfanyl)-22-deoxypleuromutilin (10b)
1
Faint yellow powder; yield: 35%; m.p.: 63.7-64.5 ℃; H NMR (600 MHz, CDCl
3
) δ 9.92
(
1H, s), 8.44 (1H, dd, J = 8.3, 1.3 Hz), 8.19 (1H, t, J = 1.9 Hz), 8.03 (1H, d, J = 7.8 Hz), 7.61
1H, dd, J = 7.8, 1.5 Hz), 7.58 (1H, m), 7.50 (1H, t, J = 7.9 Hz), 7.42 (1H, m), 7.11 (1H, d, J
7.6, 1.3 Hz), 6.34 (1H, dd, J = 17.4, 11.0 Hz, H19), 5.67 (1H, d, J = 8.5 Hz, H14), 5.13 (1H,
(
=
m, H20), 5.08 (1H, dd, J = 17.4, 1.5 Hz, H20), 3.56 (1H, d, J = 16.5 Hz, H22), 3.50 (1H, m,
H22), 3.30 (1H, d, J = 6.6 Hz, H11), 2.10 (6H, m), 1.74 (1H, d, J = 14.5, 3.3 Hz, H6), 1.53
(5H, m, H1, H7), 1.36 (3H, s, H15), 1.22 (2H, m, H18, H13), 1.08 (3H, s, H18), 1.02 (1H, d,
1
3
J = 16.0 Hz, H8), 0.86 (3H, d, J = 7.0 Hz, H17), 0.47 (3H, d, J = 7.1 Hz, H16) ; C NMR
151 MHz, CDCl ) δ 216.8 (C3), 169.4 (C21), 164.0, 140.3, 138.6 (C19), 136.3, 134.9, 130.7,
30.0, 127.9, 125.7, 124.9, 122.7, 121.6, 117.2 (C20), 74.5 (C11), 70.5 (C14), 58.0 (C4), 45.4
C9), 44.6 (C13), 43.9 (C12), 41.7 (C5), 40.6, 36.5 (C6), 36.0 (C10), 34.4 (C2), 30.3 (C8),
(
3
1
(
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-
1
2
3
7
2
6.8 (C7), 26.2 (C18), 24.8 (C1), 16.4 (C16), 14.8 (C15), 11.5 (C17) ; IR (KBr, cm ) 3458,
348, 2931, 1728, 1678, 1579, 1435, 1375, 1303, 1246, 1190, 1151, 1115, 1016, 979, 914,
+
52; HR-MS (ESI): Calcd for C35
H
43ClNO
5
S (M+H ): 624.2545; Found: 624.2562.
2-(2-(4-Chloro-benzoylamino)- phenylsulfanyl)-22-deoxypleuromutilin (10c)
1
Faint yellow powder; yield: 50%; m.p.: 155-157 ℃; H NMR (600 MHz, CDCl
3
) δ 9.85 (1H,
s), 8.47 (1H, d, J = 8.3 Hz), 8.10 (2H, m), 7.61 (1H, dd, J = 7.8, 1.6 Hz), 7.53 (2H, m), 7.41
1H, t, J = 8.0 Hz), 7.10 (1H, t, J = 7.6 Hz), 6.29 (1H, dd, J = 17.3, 11.1 Hz, H19), 5.59 (1H,
(
d, J = 8.6 Hz, H14), 5.12 (1H, d, J = 3.8 Hz, H20), 5.09 (1H, d, J = 10.8 Hz, H20), 3.56 (1H,
d, J = 16.2 Hz, H22), 3.48 (1H, d, J = 16.3 Hz, H22), 3.29 (1H, d, J = 6.5 Hz, H11), 2.09 (5H,
m), 1.73 (1H, m, H6), 1.52 (4H, m, H1, H7), 1.35 (3H, s, H15), 1.21 (3H, s, H15), 1.07 (3H, s,
H18), 0.96 (1H, d, J = 16.0 Hz, H8), 0.85 (3H, d, J = 7.0 Hz, H17), 0.46 (3H, d, J = 7.1 Hz,
1
3
H16) ; C NMR (151 MHz, CDCl
3
) δ 216.7 (C3), 169.3 (C21), 164.2, 140.3, 138.6 (C19),
38.2, 136.2, 133.4, 130.7, 129.0, 124.8, 122.3, 121.4, 117.1 (C20), 74.5 (C11), 70.5 (C14),
7.9 (C4), 45.3 (C9), 44.5 (C13), 43.9 (C12), 41.7 (C5), 40.4, 36.5 (C6), 35.9 (C10), 34.5
1
5
(C2), 30.3 (C8), 26.8 (C7), 26.2, 24.8 (C1), 16.5 (C16), 14.7 (C15), 11.5 (C17) ; IR (KBr,
-1
cm ) 3504, 3321, 2937, 1722, 1662, 1581, 1529, 1491, 1435, 1317, 1288, 1192, 1149, 1111,
+
1
6
2
014, 935, 914, 849, 766, 752, 642; HR-MS (ESI): Calcd for C35
24.2545; Found: 624.2540.
H
43ClNO
5
S (M+H ):
2-(2-(2-Methy-benzoylamino)- phenylsulfanyl)-22-deoxypleuromutilin (11a)
1
White powder; yield: 47%; m.p.: 76.6-77.4 ℃; H NMR (600 MHz, CDCl
3
) δ 7.19 (1H, dd, J
=
7.6, 1.5 Hz), 6.93 (1H, m), 6.63 (1H, d, J = 8.0 Hz), 6.51 (1H, t, J = 7.5 Hz), 6.24 (1H, dd,
J = 17.4, 11.0 Hz, H19), 5.48 (1H, d, J = 8.5 Hz, H14), 5.14 (1H, m, H20), 4.97 (1H, dd, J =
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1
7.4, 1.6 Hz, H20), 3.23 (1H, d, J = 15.8 Hz, H22), 3.17 (1H, d, J = 15.9 Hz, H22), 3.11 (1H,
d, J = 6.6 Hz, H11), 1.92 (5H, m), 1.53 (1H, m, H6), 1.33 (4H, m, H1, H7), 1.16 (3H, s, H15),
1
7
1
6
.04 (2H, m, H18, H13), 0.92 (3H, s, H18), 0.87 (1H, d, J = 16.0 Hz, H8), 0.65 (3H, d, J =
1
3
3
.0 Hz, H17), 0.38 (3H, d, J = 7.1 Hz, H16) ; C NMR (151 MHz, CDCl ) δ 217.0 (C3),
69.0 (C21), 139.1 (C19), 136.5, 130.6, 119.9, 117.2 (C20), 116.3, 74.6 (C11), 69.6 (C14),
5.0 (C22), 58.1 (C4), 45.4 (C9), 44.6 (C13), 43.9 (C12), 41.7 (C5), 38.0, 36.7 (C6), 36.0
(C10), 34.5 (C2), 30.4 (C8), 26.8 (C7), 26.3 (C18), 24.8 (C1), 16.6 (C16), 14.8 (C15), 11.5
-1
(
C17) ; IR (KBr, cm ) 3479, 3339, 2931, 1732, 1678, 1579, 1510, 1433, 1373, 1304, 1190,
+
1
6
2
176, 1117, 1016, 980, 914, 756, 661; HR-MS (ESI): Calcd for C36
04.3091; Found: 604.3117.
H
46NO
5
S (M+H ):
2-(2-(3-Methy-benzoylamino)- phenylsulfanyl)-22-deoxypleuromutilin (11b)
1
White powder; yield: 56%; m.p.: 65.8-66.7 ℃; H NMR (600 MHz, CDCl
3
) δ 7.41 (1H, dd, J
=
7.7, 1.5 Hz), 7.14 (1H, m), 6.80 (1H, d, J = 8.0 Hz), 6.71 (1H, t, J = 7.5 Hz), 6.47 (1H, dd,
J = 17.4, 11.0 Hz, H19), 5.71 (1H, d, J = 8.5 Hz, H14) , 5.37 (1H, dd, J = 11.1, 1.5 Hz, H20),
.20 (1H, dd, J = 17.5, 1.5 Hz, H20), 3.45 (1H, d, J = 16.0 Hz, H22), 3.39 (1H, d, J = 16.0 Hz,
H22), 3.34 (1H, d, J = 6.5 Hz, H11), 2.17 (5H, m), 1.76 (1H, m, H6), 1.55 (4H, m, H1, H7),
.39 (3H, s, H15), 1.24 (2H, m, H18, H13), 1.15 (3H, s, H18), 1.11 (1H, m, H8), 0.88 (3H, d,
5
1
1
3
J = 7.0 Hz, H17), 0.63 (3H, d, J = 7.1 Hz, H16) ; C NMR (151 MHz, CDCl
3
) δ 217.0 (C3),
69.0 (C21), 139.1 (C19), 136.4, 130.6, 119.5, 117.2 (C20), 115.9, 74.6 (C11), 69.7 (C14),
5.0 (C22), 58.1 (C4), 45.4 (C9), 44.6 (C13), 43.9 (C12), 41.8 (C5), 37.8 (C6), 36.7, 36.0
1
6
(C10), 34.5 (C2), 30.4 (C8), 26.8 (C7), 26.3 (C18), 24.8 (C1), 16.6 (C16), 14.8 (C15), 11.5
-1
(
C17) ; IR (KBr, cm ) 3487, 3349, 2929, 1732, 1674, 1579, 1518, 1435, 1373, 1304, 1190,
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+
1
176, 1117, 1018, 980, 914, 812, 756, 663; HR-MS (ESI): Calcd for C36
04.3091; Found: 604.3110.
5
H46NO S (M+H ):
6
2
2-(2-(4-Methy-benzoylamino)- phenylsulfanyl)-22-deoxypleuromutilin (11c)
1
White powder; yield: 53%; m.p.: 133.1-133.8 ℃; H NMR (600 MHz, CDCl
3
) δ 7.75 (1H, d,
J = 7.9 Hz), 7.15 (1H, dd, J = 7.7, 1.5 Hz), 7.03 (1H, m), 6.89 (1H, td, J = 7.7, 1.5 Hz), 6.47
1H, d, J = 8.0 Hz), 6.42 (1H, m), 6.25 (1H, dd, J = 17.4, 11.0 Hz, H19), 5.48 (1H, d, J = 8.6
Hz, H14), 5.13 (1H, dd, J = 11.1, 1.6 Hz, H20), 4.97 (1H, dd, J = 17.4, 1.6 Hz, H20), 3.20
(
(1H, d, J = 14.9 Hz, H22), 3.14 (1H, d, J = 14.9 Hz, H22), 3.12 (1H, d, J = 6.5 Hz, H11), 2.19
(1H, s), 1.93 (5H, m), 1.53 (1H, m, H6), 1.33 (4H, m, H1, H7), 1.16 (3H, s, H15), 1.04 (2H,
m, H18, H13), 0.92 (3H, s, H18), 0.87 (1H, dd, J = 13.9, 4.2 Hz, H8), 0.64 (3H, d, J = 7.0 Hz,
1
3
H17), 0.42 (3H, d, J = 7.1 Hz, H16); C NMR (151 MHz, CDCl
3
) δ 216.8 (C3), 168.9 (C21),
1
6
3
1
1
38.7 (C19), 136.0, 129.9, 129.4, 128.1, 127.5, 121.2, 117.3 (C20), 74.5 (C11), 70.3 (C14),
5.0 (C22), 58.0 (C4), 45.4 (C9), 44.5 (C13), 43.9 (C12), 41.9 (C5), 41.6, 40.1, 36.6 (C6),
5.9 (C10), 34.4 (C2), 30.3 (C8), 26.8 (C7), 26.4 (C18), 26.1, 24.8 (C1), 21.7, 16.6 (C16),
-1
4.8 (C15), 11.5 (C17) ; IR (KBr, cm ) 3491, 3332, 2939, 1728, 1660, 1579, 1529, 1506,
435, 1371, 1306, 1176, 1149, 1115, 1020, 914, 818, 764, 663; HR-MS (ESI): Calcd for
+
C
36
H46NO
5
S (M+H ): 604.3091; Found: 604.3110.
2
2- (2- (2-Methoxybenzamido) phenylsulfanyl) -22-deoxypleuromutilin(12a)
1
Yellow powder; yield: 42%; m.p.: 140.7-141.5 ℃; H NMR (600 MHz, CDCl
3
) δ 11.06 (1H,
s), 8.69 (1H, dd, J = 8.3, 1.3 Hz), 8.33 (1H, dd, J = 7.8, 1.8 Hz), 7.58 (1H, dd, J = 7.8, 1.6 Hz),
7
5
.53 (1H, m), 7.37 (1H, m), 7.16 (1H, d, J = 7.6, 1.0 Hz), 7.07 (2H, m), 6.40 (1H, m, H19),
.67 (1H, d, J = 8.5 Hz, H14), 5.26 (1H, dd, J = 11.0, 1.5 Hz, H20), 5.11 (1H, dd, J = 17.4,
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1
3
.6 Hz, H20), 4.12 (3H, s), 3.52 (1H, d, J = 15.0 Hz, H22), 3.47 (1H, d, J = 15.0 Hz, H22),
.30 (1H, d, J = 6.5 Hz, H11), 2.11 (5H, m), 1.75 (1H, dd, J = 14.7, 3.1 Hz, H6), 1.49 (5H, m,
H1, H7), 1.29 (3H, s, H15), 1.11 (2H, m, H18, H13), 1.05 (3H, d, J = 4.3 Hz, H18), 0.86 (3H,
1
3
d, J = 7.0 Hz, H17), 0.60 (3H, d, J = 7.1 Hz, H16); C NMR (151 MHz, CDCl
3
) δ 216.9 (C3),
68.0, 163.5 (C21), 157.5, 140.7, 138.9 (C19), 135.1, 133.4, 132.7, 130.2, 124.0, 121.4,
17.0 (C20), 111.4, 74.5 (C11), 69.7 (C14), 65.0 (C22), 58.0 (C4), 56.2, 45.4 (C9), 44.5
1
1
(C13), 43.9 (C12), 41.7, 38.5, 36.6 (C6), 35.9 (C10), 34.4 (C2), 30.4 (C8), 26.8 (C7), 26.1
-1
(
C18), 24.8 (C1), 16.6 (C16), 14.7 (C15), 11.5 (C17) ; IR (KBr, cm ) 3502, 3284, 2937, 1732,
1
666, 1579, 1529, 1485, 1439, 1373, 1308, 1176, 1117, 1018, 980, 914, 816, 756, 660;
+
HR-MS (ESI): Calcd for C36
H46NO
6
S (M+H ): 620.3040; Found: 620.3037.
2
2- (2- (3-Methoxybenzamido) phenylsulfanyl) -22-deoxypleuromutilin (12b)
1
White powder; yield: 45%; m.p.: 99.2-100.7 ℃; H NMR (600 MHz, CDCl
.49 (1H, d, J = 8.3 Hz), 7.69 (2H, m), 7.60 (1H, d, J = 7.6 Hz), 7.46 (1H, t, J = 8.1 Hz), 7.41
1H, t, J = 7.8 Hz), 7.14 (1H, dd, J = 8.1, 2.4 Hz), 7.09 (1H, t, J = 7.5 Hz), 6.34 (1H, dd, J =
7.4, 11.0 Hz, H19), 5.63 (1H, d, J = 8.5 Hz, H14), 5.32 (1H, s), 5.15 (1H, d, J = 11.0 Hz,
3
) δ 9.78 (1H, s),
8
(
1
H20), 5.09 (1H, d, J = 17.4 Hz, H20), 3.93 (3H, s), 3.55 (1H, d, J = 16.0 Hz, H22), 3.48 (1H,
d, J = 16.0 Hz, H22), 3.29 (1H, d, J = 6.5 Hz, H11), 2.09 (5H, m,H2, H4, H10, H13), 1.73
(1H, dd, J = 14.7, 3.3 Hz, H6), 1.52 (4H, m, H1, H7), 1.34 (3H, s, H15), 1.20 (3H, m, H18,
H13, 11-OH), 1.06 (3H, s, H18), 0.97 (1H, d, J = 16.0 Hz, H8), 0.85 (3H, d, J = 7.0 Hz, H17),
1
3
0
.49 (3H, d, J = 7.1 Hz, H16) ; C NMR (151 MHz, CDCl
65.0, 160.0, 140.3, 138.6 (C19), 136.4, 136.0, 130.6 , 129.7, 124.6, 122.2, 121.3, 119.3,
18.3, 117.2 (C20), 112.6, 74.5 (C11), 70.3 (C14) , 58.0 (C4) , 55.5 (C22) , 45.4 (C9), 44.6
3
) δ 216.8 (C3), 168.9 (C21),
1
1
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(C13), 43.9 (C12), 41.7 (C5), 40.2, 36.5 (C6), 35.9 (C10), 34.4 (C2), 30.3 (C8), 26.8 (C7),
-
1
2
1
6.1 (C18), 24.8 (C1), 16.4 (C16), 14.7 (C15), 11.5 (C17); IR (KBr, cm ) 3494, 3344, 2937,
720, 1655, 1579, 1522, 1489, 1435, 1385, 1306, 1192, 1151, 1115, 1036, 914, 796, 760, 683;
+
HR-MS (ESI): Calcd for C36
H46NO
6
S (M+H ):620.3040; Found: 620.3062.
2
2- (2- (4-Methoxybenzamido) phenylsulfanyl) -22-deoxypleuromutilin (12c)
1
Maple powder; yield: 37%; m.p.: 204.5-205.6 ℃; H NMR (600 MHz, CDCl
3
) δ 9.69 (1H, s),
8
7
5
1
.48 (1H, d, J = 8.3 Hz), 8.09 (2H, d, J = 8.5 Hz), 7.59 (1H, d, J = 7.8 Hz), 7.38 (1H, t, J =
.9 Hz), 7.05 (3H, m), 6.34 (1H, dd, J = 17.4, 11.0 Hz, H19), 5.62 (1H, d, J = 8.6 Hz, H14),
.15 (1H, d, J = 11.0 Hz, H20), 5.10 (1H, d, J = 17.4 Hz, H20), 3.91 (3H, s), 3.55 (1H, d, J =
5.9 Hz, H22), 3.48 (1H, d, J = 15.9 Hz, H22), 3.29 (1H, d, J = 6.5 Hz, H11), 2.09 (5H, m,H2,
H4, H10, H13), 1.73 (2H, m, H6), 1.51 (4H, m, H1, H7), 1.33 (3H, s, H15), 1.21 (3H, m, H18,
H13, 11-OH), 1.07 (3H, s, H18), 0.98 (1H, d, J = 16.0 Hz, H8), 0.84 (3H, d, J = 6.9 Hz, H17),
1
3
0
.49 (3H, d, J = 7.2 Hz, H16) ; C NMR (151 MHz, CDCl
C21), 162.6, 138.7 (C19), 136.0, 130.6, 129.3, 127.2, 124.3, 122.0, 121.2, 117.2 (C20),
13.9, 74.5 (C11), 70.3 (C14), 58.0 (C4), 55.5 (C22), 45.4 (C9), 44.6 (C13), 43.9 (C12), 41.7
C5), 40.1, 36.5 (C6), 35.9 (C10), 34.4 (C2), 30.3 (C8), 26.8 (C7), 26.1 (C18), 24.8 (C1),
3
) δ 216.8 (C3), 168.9, 164.8
(
1
(
-
1
1
1
6.5 (C16), 14.8 (C15), 11.5 (C17); IR (KBr, cm ) 3477, 3325, 2937, 1726, 1657, 1579,
531, 1458, 1435, 1387, 1319, 1174, 1149, 1115, 1020, 916, 816, 768, 663; HR-MS (ESI):
+
Calcd for C36
H46NO
6
S (M+H ): 620.3040; Found: 620.3067.
Minimal inhibitory concentration(MIC) testing
The MIC values of these novel pleuromutilin derivatives, and pleuromutilin were determined
on MRSA, E. coli and S. aureus by broth dilution in accordance with the “Clinical and
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Laboratory Standards Institute” (CLSI, 2008). Stock solutions of these compounds were
prepared in N,N-Dimethylformamide (DMF) at the concentrations of 5120 µg/mL. The
working solutions (256 µg/mL) were obtained by diluting stock solutions in sterile Mueller
Hinton broth.
The drug susceptibility testing was performed in 96-well plate. All dates were tested in
duplicate in each plate. Medium was inoculated with single colonies and incubated overnight.
1
00 µL of diluted culture was mixed with 100 µL of drug solutions in a series with 2-fold
concentration steps. The tested concentration ranges were 0.015-64 µg/mL. Valnemulin and
tiamulin were used as positive controls against bacteria. The final concentration of DMF in
the first well column was 1.25%. Initially, preliminary analyses were conducted with 1.25%
(v/v) DMF/MHB and this affect neither the growth of the tested bacteria nor the
determination of MIC. The MIC value was defined as the lowest concentration of the sample
which inhibits the visible growth of test bacteria.
Molecular Modeling
The docking experiments were performed by use of the AutoDockTools and Pymol. The
crystal structure of Deinococcus radiodurans in complex with tiamulin (PDB ID:1XBP) [13]
was used to build the peptidyl transferase center (PTC) mode. The PTC model was
constructed containing all residues within a spherical cut of 30 Å around the PTC binding site
of 1XBP. The compounds were built with the CORINA online service and optimized by
Avogadro 1.1.1 [30], with 5000 steps Steepest Descent and 1000 steps Conjugate Gradients
geometry optimization using MMFF94 force field.
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MRSA infection model
All experimental procedures and animal care were in accordance with South China
Agricultural University (SCAU) guidelines and were approved by the Animal Ethical and
Experimental Committee (AEEC) of SCAU. All mice were acclimatized to the new
environment for at least one week before treatment initiation. Institute of Cancer Research
mice (the Hunan SLAC Jingda Laboratory Animal Company, Changsha, China), weighing
2
5±1 g, were rendered neutropenic upon treatment with 150 mg/kg cyclophosphamide
intraperitoneally 4 days prior to infection and with 100 mg/kg 1 day prior to infection. The
neutropenic animals (10 per group) were inoculated intraperitoneally with 0.5 mL of an
7
inoculum containing ~10 CFU/mL MRSA. Intragastric administration of the test compounds
at dose of 50 mg/kg were given to the mice 1 h after infection. Tiamulin was used as a control
in the same manner at the same doses as 8. Formulation of the test compounds was dissolved
in vehicle (20% DMSO, 5% Tween-80, and 75% normal saline). The survival of the mice at
seventh day after infection was selected as the end-point.
Results
Chemistry
As shown in Scheme 1, compound 7 was prepared by the reaction of pleuromutilin 1 with
p-toluenesulfonyl chloride in pyridine. Compound 8 was then prepared by nucleophilic
substitution of 22-O-tosylpleuromutilin (compound 7) with 2-aminothiophenol under basic
conditions. Compounds 9a-9c, 10a-10c, 11a-11c and 12a-12c were prepared by acylation of
compound 8 with the corresponding acyl chloride derivatives.
All compounds were purified using silica gel column chromatography. The structure of all
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1
13
synthesized pleuromutilin derivatives were characterized by FT-IR, H-NMR, C-NMR and
HR-MS.
In vitro antibacterial activity
The minimum inhibitory concentrations (MIC) of these synthesized pleuromutilin derivatives
were tested against methicillin-resistant S. aureus ATCC 43300 (MRSA), S. aureus ATCC
2
9213 and E. coli ATCC 25922 by the broth dilution methods using tiamulin and valnemulin
as reference drugs. The results of these studies were summarized in Table 1. The MICs of
these synthesized pleuromutilin derivatives against MRSA and S. aureus ATCC 29213 ranged
from 0.0156~1 µg/mL. It could also be observed that all these derivatives possessed pretty
weak antibacterial activities against the gram-negative bacteria E. coli ATCC 25922.
Table 1 here
The antibacterial activities of compound 8, which was an intermediate of other pleuromutilin
derivatives, were evaluated at first. To our surprise, compound 8 displayed higher activity
against MRSA and ATCC 29213 than all the other pleuromutilin derivatives. The in vitro
antibacterial activities of compound 8 (MIC = 0.0156µg/mL) were even superior to that of
both tiamulin (MIC = 0.5µg/mL) and valnemulin (MIC = 0.0625µg/mL). Compounds 9b,
1
0b and 11a showed lower potent activity against both S. aureus when compared to that of
valnemulin while possessed higher activity when compared to that of tiamulin. All other
compounds displayed comparable or slightly less antibacterial activities against MRSA and
ATCC 29213 to that of tiamulin.
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Molecular Docking Study
Due to their superior in vitro antibacterial activity to that of other synthesized pleuromutilin
derivatives, compounds 8, 9b and 11a were selected for molecular docking studies.
Compounds8, 9b and 11apossessed multiple binding modes into 1XBP with the binding free
energies at -7.28, -6.93 and -6.94 kcal/mol, respectively. As shown in Figure 2, two strong
hydrogen bonds were formed through the interaction of 8 with G2044 (O/NH distance: 1.79Å)
and C2431 (OH/O distance: 1.99Å). It could also be observed that the amino group of 8
protruding into the area formed by resides of C2046 and U2564 with the distance at 2.55Å
and 2.43Å, respectively. This might be one possible reason that why compound 8 possessed
higher binding affinity as well as better in vitro antibacterial activities than that of 9b and
11a.
Figure 2, 3 and 4 here
In vivo efficacy of compound 8
Compound 8 (0.0156 μg/mL) possessed higher in vitro antibacterial activity against MRSA
3300 than tiamulin (0.5 μg/mL) in our experiment. The in vivo efficacy of compound 8 was
4
evaluated using a systemic MRSA infection mice model. Mice which were intragastric
administrated with vehicle alone showed 100% mortality in this MRSA infection model. As
shown in Figure 5, both tiamulin and compound 8 possessed in vivo antibacterial efficacy and
led to the survival of the MRSA infection mice. Treatment with tiamulin and compound 8
displayed protection with 30% and 50% survival of the MRSA infection mice in this model,
respectively. Thus, Compound 8 showed higher antibacterial activity than that of tiamulin
against MRSA-43300 in the mouse infection model. The result of in vivo efficacy
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demonstrated that compound 8 might act as a potent antibacterial drug candidate against
MRSA.
Figure 5 here
Discussion
As shown in Table 1, compounds 9b and 10b, bearing an electron withdrawing substituent in
the benzene ring at the meta- position to the side chain, presented improved activity against
MRSA and ATCC 29213 compared with the compounds 9a, 9c, 10a and 10c. The substitution
position on the benzene ring of the phenyl amide side chain appeared to have influence on the
antibacterial activity of these pleuromutilin derivatives. Electron donating substituents were
then introduced to investigate the optimal substituents of this series of pleuromutilin
derivatives. Compound 11a possessed superior antibacterial activity to that of tiamulin.
Compounds 11b, 11c and 12a-12c displayed lower or comparable antibacterial activities to
that of tiamulin.
Generally, all of the benzoylamino substituted pleuromutilin derivatives bearing
phenylsulfanyl linker in Table 1 showed less in vitro antibacterial activity against MRSA and
ATCC 29213 when compared to that of valnemulin. Some of them possessed slightly superior
activities to that of tiamulin. Compound 8 possessed the highest antibacterial activity against
MRSA and ATCC 29213 than all the other synthesized pleuromutilin derivatives. It displayed
even better in vitro antibacterial activity than both tiamulin and valnemulin against MRSA
and ATCC 29213. The results of MIC values from Table 1 indicated that the introduction of
both electron withdrawing and electron donating groups in benzene ring of the phenyl amide
could decrease antibacterial activity.
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Molecular docking studies revealed that compound 9b formed just one hydrogen bond
between its oxygen atom of OH (8-membered ring) with C2589 (O/NH distance: 1.91 Å).
The molecular docking results of compound 11a were shown in Figure 4. Two strong
hydrogen bonds were observed through the interaction of its amide with G2484 (NH/O
distance: 1.97 Å) and its oxygen atom of OH (8-membered ring) with C2565 (O/NH distance:
2
.12 Å). Although adopted one hydrogen bond more than 9b, compound 11a displayed
similar binding free energy as 9b (-6.94 kcal/mol vs -6.93 kcal/mol). The similar binding
affinity of 11a compared to 9b might be explained by the interaction between 9b and A2482
with the distance at 2.48 Å. Additionally, both 11a and 9a displayed lower antibacterial
activity when compared to 8, perhaps because of the longer distance of the hydrogen bonds
between both compounds and the residues around them (Figure 2, 3 and 4).
Conclusion
In conclusion, a series of novel pleuromutilin derivatives bearing 2-aminothiophenol moieties
have been reported. These synthesized derivatives were evaluated for their in vitro
antibacterial activities against MRSA, S. aureus and E. coli. The obtained MIC values
revealed that all the synthesized derivatives possessed moderate antibacterial activity.
Compound 8 was the most active pleuromutilin derivative, it possessed superior antibacterial
activity to that of both tiamulin and valnemulin against MRSA. Docking studies were
conducted on compounds 8, 9b and 11a. The docking results showed that the binding free
energies were -7.28, -6.93 and -6.94 kcal/mol, respectively. This demonstrates that there is a
rational correlation between the binding free energy and the antibacterial activity of
compounds 8, 9b and 11a. Compound 8 exhibited higher in vivo antibacterial activity than
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that of tiamulin against MRSA at th dose of 50 mg/kg in the mouse infection model. The
results indicated that compound 8 may serve as possible lead compound for the development
of new potent antibacterial drugs based on pleuromutilin. A more comprehensive study of
compound 8 will be reported in our future research.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (No.
3
1672602 and 21405051) and the National Key Research and Development Program of
China (No. 2016YFD0501300).
Conflict of interest
The authors declare that they have no conflict of interest.
Figure Captions
Scheme 1 Reagent and conditions: (i) p-toluenesulfonyl chloride, pyridine, 0℃, 3h; (ii)
2
-aminothiophenol, 20% aqueous NaOH (10ml), chloroforme, reflux; (iii) acyl chloride
derivatives, ethyl acetate, reflux.
Figure 1Structure of pleuromutilin (1), tiamulin (2), valnemulin (3), retapamulin (4)
BC-3004 (5) and BC-3080 (6).
Figure 2 Result of the docking of compound 8 into the PTC model binding site. Residue
numbers shown are according to E. coli 23s RNA numbering.
Figure 3 Result of the docking of 9b into the PTC model binding site. Residue numbers
shown are according to E. coli 23s RNA numbering.
Figure 4 Result of the docking of 11a into the PTC model binding site. Residue numbers
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shown are according to E. coli 23s RNA numbering.
Figure 5 Efficacy of compound 8 and tiamulin in mouse systemic infection model.
Table 1 MIC (μg/mL) for MRSA (ATCC 43300), Staphylococcus aureus (S. aureus) ATCC
2
9213 and E. coli ATCC 25922.
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2
012, 4.
MRSA
S.aureus
E. coli ATCC
Compounds
R
ATCC 29213
25922
ATCC 43300
9
9
a
0.5
0.5
>64
>64
F
b
0.125
0.125
F
9
1
c
1
1
>64
>64
F
0a
0.5
0.5
Cl
1
1
0b
0c
0.25
0.25
>64
Cl
0.5
0.5
>64
>64
Cl
11a
0.25
0.25
1
1b
0.5
0.5
1
>64
>64
11c
0.5
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1
1
2a
2b
2c
0.5
1
1
1
1
>64
>64
>64
O
O
O
1
8
1
0.0156
0.5
0.0156
1
>64
>64
>64
32
pleuromutilin
Tiamulin
0.5
0.5
Valnemulin
0.0625
0.0625
This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.