Design, synthesis and antibacterial evaluation of novel pleuromutilin derivatives possessing piperazine linker

Article abstract of DOI:10.1016/j.ejmech.2017.01.004

A series of pleuromutilin derivatives bearing piperazine ring have been reported. The in vitro antibacterial activities of the synthetic derivatives against MRSA (ATCC 43300), Staphylococcus aureus (ATCC 29213), Enterococcus faecalis (ATCC 29212), Enterococcus faecium (ATCC35667) and Escherichia coli (ATCC25922) were evaluated by the broth dilution method. Most of the synthesized derivatives displayed potent activities. Compounds 11c, 12a and 12c were found to be the most active antibacterial derivatives against MRSA (minimum inhibitory concentration = 0.015 μg/mL). The binding of compounds 11c, 12a and 12c to the 50s ribosome were investigated by molecular modeling. Compound 11c possessed lower binding free energy compared with compounds 12a and 12c. Compound 11c was further evaluated in MRSA systemic infection model and displayed superior in vivo efficacy to that of tiamulin.

Full text of DOI:10.1016/j.ejmech.2017.01.004

European Journal of Medicinal Chemistry 127 (2017) 286e295
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis and antibacterial evaluation of novel pleuromutilin
derivatives possessing piperazine linker
Mei-Ling Gao, Jie Zeng, Xi Fang, 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, 510642, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of pleuromutilin derivatives bearing piperazine ring have been reported. The in vitro antibac-
terial activities of the synthetic derivatives against MRSA (ATCC 43300), Staphylococcus aureus (ATCC
29213), Enterococcus faecalis (ATCC 29212), Enterococcus faecium (ATCC35667) and Escherichia coli
(ATCC25922) were evaluated by the broth dilution method. Most of the synthesized derivatives displayed
potent activities. Compounds 11c, 12a and 12c were found to be the most active antibacterial derivatives
Received 20 October 2016
Received in revised form
11 December 2016
Accepted 1 January 2017
Available online 3 January 2017
against MRSA (minimum inhibitory concentration ¼ 0.015
mg/mL). The binding of compounds 11c, 12a
and 12c to the 50s ribosome were investigated by molecular modeling. Compound 11c possessed lower
binding free energy compared with compounds 12a and 12c. Compound 11c was further evaluated in
MRSA systemic infection model and displayed superior in vivo efficacy to that of tiamulin.
© 2017 Elsevier Masson SAS. All rights reserved.
Keywords:
Antibiotics
MRSA
Pleuromutilin
Synthesis
Molecular docking
1. Introduction
Further studies demonstrated that the pleuromutilin class of anti-
biotics inhibits bacterial protein synthesis by interacting with the
Methicillin-resistant Staphylococcus aureus (MRSA) are those
strains of S. aureus that display intrinsic resistance to methicillin
and other beta-lactam antibiotics, such as oxacillin, nafcillin and
carbapenems. The emergence of MRSA infection has led to higher
costs, longer hospitalization courses, as well as higher morbidity
and mortality [1]. MRSA infection is now commonly acquired not
only in hospital but also in community setting and its prevalence
continues to increase in both hospital and community setting [2].
Although, there are several antibiotics have been proven effective
for management of MRSA infection, concerns regarding the emer-
gence of widespread resistance in MRSA to these antibiotics,
including vancomycin [3], linezolid [4] and daptomycin [5], have
also been raised. It is therefore urgent to discover and develop new
antibiotics agents with novel mode of action against MRSA.
23S rRNA of the 50S subunit of prokaryotic ribosomes [8]. Thus, the
pleuromutilin class possesses rarely resistance with other classes of
clinically used antibacterial drugs [9]. Additionally, this distinct
mechanism of action of pleuromutilin also made it an attractive
lead compound in the discovery and development of novel anti-
biotics to combat drug-resistant bacterial infections [10e12].
Thousands of semisynthetic pleuromutilin analogues have been
made and evaluated to improve the antibacterial activities and
in vivo efficacy after the clarification of the structure of pleuro-
mutilin [13e15]. This has led to the discovery of tiamulin [16,17] (2,
Fig. 1) and valnemulin [18] (3, Fig. 1) which were approved as
therapeutic agents for veterinary clinical use. Further chemical
modifications of pleuromutilin have been made for an attempt to
discover an agent for human use after the discovery of tiamulin and
valnemulin. These efforts resulted in the discovery of azamulin (4,
Fig. 1), which entered phase I clinical trials in volunteers in 1980s
[19,20]. Unfortunately, azamulin did not undergo further clinical
trials because of its limited bioavailability as well as its CYP450
inhibition [21]. In 2007, retapamulin (5, Fig. 1) was approved as a
topical antibiotic for human use to treat impetigo [22]. Although
pleuromutilin analogues containing the sulfide linkage display
potent in vitro antibacterial activity, their oral bioavailabilities were
usually limited for their strong hydrophobic nature [23]. To
Pleuromutilin (1, Fig. 1), a tricyclic diterpenoid natural product,
was first isolated from cultures of two basiomycetes species, Pleu-
rotus mutilis and Pleurotus passeckerianus, in 1951 [6]. Pleuromutilin
has good antibacterial activity in vitro against Gram-positive bac-
teria and mycoplasmas but insufficient potency in vivo [6,7].
* Corresponding author. College of Veterinary Medicine, South China Agricultural
University, No. 483 Wushan Road, Tianhe District, Guangzhou, 510642, China.
E-mail address: youzhitang@scau.edu.cn (Y.-Z. Tang).
http://dx.doi.org/10.1016/j.ejmech.2017.01.004
0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.

M.-L. Gao et al. / European Journal of Medicinal Chemistry 127 (2017) 286e295
287
O
O
O
O
HO
O
O
3
O
N
N
S
S
H
H2N
21
O
O
O
14
11
OH
OH
OH
1. Pleuro mutilin
2. Tiamulin
3. Valnemulin
O
O
H
N
O
O
N
H2N
S
S
N
N
O
O
OH
OH
4. Azamulin
5. Reta pamulin
Fig. 1. Structure of pleuromutilin (1), tiamulin (2), valnemulin (3), azamulin (4) and retapamulin (5).
overcome this problem, a series of pleuromutilin derivatives having
a piperazine ring linker had been prepared and evaluated by Hir-
okawa and coworkers [23,24]. Additionally, analogues having a
triazole linker had been made and evaluated by Dreier and co-
workers [25,26]. Considering those above pleuromutilin analogues
with C14 side chains, in which without sulfide linkage, also possess
good in vitro antibacterial activity, we have focused our efforts on
pleuromutilin derivatives including nitrogen in the C14 side chains
[27,28].
A general observation from our first series was that pleuro-
mutilin derivatives bearing butyl amine group in the C14 side chain
possessed moderate in vitro antibacterial activity [27]. In the second
series, we identified the structurally novel pleuromutilin de-
rivatives having a piperazine as linker and basic group. These
pleuromutilin analogues displayed potent in vitro antibacterial ac-
tivity against S. aureus (ATCC 29213) in our previous work [28]. As
an extension to that study, we directed our efforts to explore the
influence of the piperazine moiety as a linker in pleuromutilin
analogues on the in vitro antibacterial activities. In the present
study, we describe the design, synthesis, in vitro and in vivo anti-
bacterial activities of novel pleuromutilin analogues bearing an
additional piperazine ring.
and 15a-15c were tested against four Gram-positive bacteria,
including ATCC 43300, ATCC 29213, ATCC 29212 and ATCC 35667 by
the broth dilution methods. Pleuromutilin, tiamulin and valne-
mulin were used as positive controls. The results of these studies
are summarized in Table 1.
All target compounds showed potent activity against all four
Gram-positive bacteria except E. faecium (ATCC 35667). All com-
pounds possessed less potent against E. faecalis (ATCC 29212) than
S. aureus (ATCC 43300 and ATCC 29213). It could be obtained that
compounds 7 and 8 showed lower activities against S. aureus (ATCC
43300 and ATCC 29213) in comparison to that of tiamulin. Com-
pound 9 possessed the same MIC value against MRSA and ATCC
29213 compared with tiamulin. The replacement of the methyl (R
group in Table 1) of 9 with benzene ring resulted in compound 10,
which displayed nearly the same antibacterial activities compared
with 9. Considering that the substitution position in the aromatic
ring have influence on the antibacterial activity of pleuromutilin
analogues which bearing aromatic ring [10,28,30], further research
on the types and position of benzene ring of 10 was carried on.
Compounds 13a-13c, 14a-14c, and 15a-15c, in which the elec-
tron donating group was introduced on the benzene ring of 10,
were much less active against MRSA. All those mention compounds
except 15a possessed superior antibacterial activity against MRSA
in comparison to that of tiamulin and compound 10. To our sur-
prise, all of compounds 11a-11c and 12a-12c presented improved
activity against MRSA compared with the compound 10. The results
suggested that the aromatic substituents with electron with-
drawing groups could be introduced to design new pleuromutilin
derivatives with higher antibacterial activities in the future.
Compounds 11c, 12a and 12c possessed the most antibacterial
activities which were superior or the same antibacterial activities
against MRSA and ATCC 29213 to that of tiamulin. The MIC values
have demonstrated that 11c, 12a and 12c might act as potent
antibacterial agent against MRSA.
2. Results and discussion
2.1. Chemistry
The general synthetic route of all pleuromutilin analogues is
illustrated in Scheme 1. N-tert-butoxycarbonyl (Boc) -piperazine
was used to react with compound 6 at first, the Boc group of the
product was removed using TFA/DCM to yield compound 7. Com-
pound 8 was directly prepared by condensation reactions between
the amino group of compound 7 and the acyl chloride group of
chloracetyl chloride. Pleuromutilin derivatives 9, 10, 11a-11c, 12a-
12c, 13a-13c, 14a-14c and 15a-15c were prepared from the com-
pound 8 which was synthesized in turn from compound 7 ac-
cording to previous literature with modification [29].
2.3. Molecular docking study
For they displayed excellent antibacterial activities, 11c, 12a
and 12c were chosen for molecular docking investigations.
Compound 11c was found to exhibit a hydrogen bond with 50s
ribosome in the docking studies. As shown in Fig. 2, the oxygen
2.2. Antibacterial activity
The MIC values of 7, 8, 9, 10, 11a-11c, 12a-12c, 13a-13c, 14a-14c

288
M.-L. Gao et al. / European Journal of Medicinal Chemistry 127 (2017) 286e295
3
O
O
O
HN
O
O
O
(i)
(ii)
HO
TsO
N
14
21
O
11
O
O
OH
OH
OH
6
7
R
O
N
Cl
O
O
N
N
O
(iii)
(iv)
O
N
N
O
N
O
O
OH
OH
8
9, 10, 11a-11c, 12a-12c, 13a-13c,14a-14c,15a-15c
Scheme 1. Reagent and conditions: (i) p-toluenesulfonyl chloride, ethyl acetate, NaOH, 0 ꢁC, 3 h. (ii) a. N-Boc-piperazine, K2CO3, acetonitrile, 70 ꢁC; b. TFA/DCM, room temperature.
(iii) Chloroacetyl chloride, K2CO3, DCM. (iv) piperazine derivatives, K2CO3, acetonitrile, reflux.
atom of 11c (on the nitro group in the 4-position of the benzene
ring) formed one H-bound with the amino group of A2041 (O/
NH distance: 1.84 Å). The binding free energy of 11c with 50s
ribosome was calculated to be ꢀ8.67 kcal/mol. Compound 12a
3. Conclusions
A series of novel pleuromutilin derivatives bearing piperazine
ring have been reported. These new derivatives were investi-
gated for their antibacterial activity in vitro against MRSA,
S. aureus, E. faecalis, E. faecium and E. coli. The MIC values
demonstrated that all the synthesized derivatives possessed
potent antibacterial activity properties. Compounds 11c, 12a and
12c possessed the most antibacterial activity against MRSA.
Compounds 11c, 12a and 12c were chosen for a molecular
docking study. The results showed that the binding free en-
ergies were ꢀ8.67, ꢀ6.95 and ꢀ8.11 kcal/mol, respectively.
Compounds 11c was evaluated for in vivo efficacy using MRSA
infected mouse model. It is interesting to note that compound
11c exhibited superior in vivo efficacy to that of tiamulin against
MRSA at the dose of 40 mg/kg in the mouse systemic infection
model. The results indicated that the promising pleuromutilin
derivative 11c might serve as useful lead compound for further
optimization and discovery of new antibiotics.
exhibited
a hydrogen bond between its OH group (eight-
membered ring) and C2565 at a distance of 2.13 Å (Fig. 3).
Although, the antibacterial activity of 12a was the same as 11c,
the binding energy of 12a (ꢀ6.95 kcal/mol) was higher than that
of 11c. One possible reason was that the distance of the
hydrogen bond formed between the OH group of 12a and C2565
was longer than the distance of the hydrogen bond formed
between 11c's nitro group and A2041.
The molecular docking results of 12c were shown in Fig. 4.
Compound 12c and 12a displayed the similar docking mode.
One hydrogen bond was formed between the OH group (eight-
membered ring) of 12c with C2565 with distance of 2.09 Å.
Compound 12c (ꢀ8.11 kcal/mol) exhibited a lower binding free
energy than that of 12a (ꢀ6.95 kcal/mol) while a higher binding
free energy than that of 11c (ꢀ8.67 kcal/mol), possibly because
of the distance of the hydrogen bond formed by 12c (OH/O
distance: 2.09 Å) was longer than that of 11c (O/NH distance:
1.84 Å) and shorter than that of 12a (OH/O distance: 2.13 Å).
The docking results of compounds 11c suggested that the sub-
stituents with oxygen atom, which might form hydrogen bond
with residues, could be introduced to produce pleuromutilin
derivatives with excellent antibacterial activities as a potential
drug design strategy.
4. Experimental
4.1. Materials
Pleuromutilin (>90% pure) was purchased from Great
Enjoyhood Biochemical Co Ltd (Daying, China). Piperazine de-
rivatives and chloracetyl chloride were obtained from J&K Sci-
entific Ltd. The other reagents were all of analytical grade and
purchased from Guangzhou Chemical Reagent Factory
(Guangzhou, China). All reactions were monitored using thin-
layer chromatography(TLC) on silica gel pre-coated plates. Pu-
rification of all compounds by column chromatography was
carried out using silica gel (200e300 mesh, Branch of Qingdao
Haiyang Chemical Co., Ltd., Shandong, China). Melting points
2.4. In vivo efficacy of compound 11c
Considering compound 11c showed potent in vitro activity
against MRSA (MIC ¼ 0.015
mg/ml) and possessed lower binding
free energy compared with compounds 12a and 12c, the in vivo
efficacy of 11c and the reference agent tiamulin was examined in a
systemic infection model in which mice were inoculated with
MRSA. As shown in Fig. 5, intravenously administered with 40 mg/
kg of test compounds, 11c provided significant protection (50%)
while tiamulin provided weak protective effect with 10% survival
against MRSA in the mouse systemic model. Thus, the in vivo effi-
cacy of 11c was higher than that of tiamulin. The result of in vivo
efficacy demonstrated that 11c was able to protect MRSA infection
mice and might be a potent antibacterial drug candidate.
were determined with
a Shenguang X-4 apparatus (China)
without corrected. 1H-NMR and 13C-NMR spectra were
measured on a Bruker AV-600 spectrometer in CDCl3 with tet-
ramethylsilane as an internal standard. Chemical shifts (d) are
given in ppm and coupling constants (J) reported in hertz.
Fourier transform infrared (FT-IR) spectra were obtained on a
Nicolet 6700 Research FTIR spectrometer (Thermo Fisher) using
KBr pellets, and the absorptions are reported in cmꢀ1. High-
resolution mass spectra were obtained using a LTQ-Orbitrap

M.-L. Gao et al. / European Journal of Medicinal Chemistry 127 (2017) 286e295
289
Table 1
MIC ( g/ml) for MRSA (ATCC 43300), Staphylococcus aureus (S. aureus) ATCC 29213, HN15 and SA1 (S. aureus, Guangdong isolated).
m
Compound
R
MRSA
S.aureus
E.facalis
E.facium
ATCC 43300
ATCC 29213
ATCC 29212
ATCC 35667
7
8
9
1
2
>32
0.5
8
>32
2
>32
>32
>32
>32
0.125
10
0.125
0.25
4
>32
11a
0.0625
0.0625
2
>32
11b
11c
0.0625
0.015
0.25
2
1
>32
>32
0.125
12a
12b
0.015
0.03
0.5
4
2
>32
>32
0.25
12c
13a
13b
0.015
0.5
0.125
0.5
2
4
2
>32
>32
>32
0.25
0.25
13c
14a
14b
0.25
0.5
0.5
1
2
2
2
>32
>32
>32
0.25
0.5
14c
1
2
8
>32
(continued on next page)

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M.-L. Gao et al. / European Journal of Medicinal Chemistry 127 (2017) 286e295
Table 1 (continued )
Compound
R
MRSA
S.aureus
E.facalis
E.facium
ATCC 43300
ATCC 29213
ATCC 29212
ATCC 35667
15a
15b
0.0625
0.25
2
>32
0.25
0.5
4
>32
15c
0.125
0.25
2
>32
pleuromutilin
tiamulin
valnemulin
retapamulin
2
8
0.5
0.125
0.0625
16
2
1
>32
>32
>32
>32
0.125
0.03
0.0625
mass spectrometer (Thermo Fisher) with an electro spray ion-
ization(ESI) source. HPLC was carried out on an UltiMate 3000
separation system (Dionex, Germany).
dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to
give a residue. The residue was precipitated from isopropanol to
give a white solid (12.0 g, 85.71%).
4.2. Synthesis
4.2.2. 22-(Piperazine-1-yl)-22-deoxypleuromutilin (7)
Compound 6 (1 g, 1.87 mmol) was dissolved in acetonitrile
(10 mL) and sodium iodide (0.31 g, 2.07 mmol) was added and
stirred at 70 ꢁC for 1 h. N-Boc-piperazine (0.38 g, 2.07 mmol) and
potassium carbonate (0.52 g, 3.76 mmol) were added to the
mixture and stirred again for 2 h at 70 ꢁC. Chloroform (30 mL) was
added, and the mixture was then washed with water (30 mL). The
organic layer was dried over anhydrous Na2SO4, filtered and
concentrated under reduced pressure to give the crude oil. The
crude oil was dissolved in 15 mL dichloromethane and 5 mL of
trifluoroacetic acid was added and the mixture was stirred at room
temperature for 8 h. The mixture was concentrated in vacuo to
remove the trifluoroacetic acid and yield the crude product, which
was purified by column chromatography (dichloromethane:
methanol ¼ 5:1) using silica gel to give the desired compound.
A general synthesis strategy based on the usual 22-O-tosyl-
pheuromutilin and piperazine derivatives were used (Scheme 1).
4.2.1. 22-O-tosylpleuromutilin (6)
A solution of pleuromutilin 1 (10.0 g, 26.42 mmol) in ethyl ac-
etate (25.0 mL) was stirred at 0 ꢁC in a three-necked round bottom
flask, and p-toluenesulfonyl chloride (5.5 g, 29.06 mmol) was
added. Sodium hydroxide (2.1 g, 52.84 mmol) was dissolved in
10 mL of water and dropped into the aforementioned solution. The
mixture was then stirred at room temperature until complete
consumption of pleuromutilin was obtained. CHCl3(50 mL) and Ice-
cold water (50 mL) were added to the solution. The organic phase
was washed with brine and water. Then the organic phase was
Fig. 2. Result of the docking of 11c (green) into the PTC model binding site. Residue
numbers shown are according to E. coli 23s RNA numbering. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of
this article.)
Fig. 3. Result of the docking of 12a (orange) into the PTC model binding site. Residue
numbers shown are according to E. coli 23s RNA numbering. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of
this article.)

M.-L. Gao et al. / European Journal of Medicinal Chemistry 127 (2017) 286e295
291
4.2.3. 22-(4-(2-chloro-acetyl)-piperazine-1-yl)-22-
deoxypleuromutilin (8)
Compound 7 (1.0 g, 2.24 mmol) and potassium carbonate
(0.62 g, 4.48 mmol) were added into 10 mL of dichloromethane and
stirred at ꢀ15 ꢁC (ice-sodium chloride bath). Chloroacetyl chloride
(0.19 mL, 2.46 mmol) was added dropwise and the resulting
mixture were stirred for 2.5 h at room temperature until complete
consumption of compound 7 was obtained. The reaction mixture
was extracted with 30 mL of chloroform and washed with water.
The organic layer was dried using anhydrous Na2SO4, filtered and
evaporated in vacuum to yield the curde product, which was
chromatographed on silica gel (petroleum ether: ethyl
acetate ¼ 1:1) to obtain the pure product. White power; Yield:
60.7%; m.p.: 57.5e58.0 ꢁC; Purity of compound was 96.4% (deter-
mined by RP HPLC at 254 nm, tR ¼ 6.40 min); 1H NMR (600 MHz,
CDCl3):
d
(ppm) 6.51 (1H, dd, J ¼ 17.4, 11.0 Hz, H19), 5.78 (1H, d,
J ¼ 8.5 Hz, H14), 5.33 (1H, dd, J ¼ 11.0, 1.6 Hz, H20), 5.19 (1H, dd,
J ¼ 17.3, 1.7 Hz, H20), 3.35 (1H, dd, J ¼ 9.9, 6.3 Hz, H11), 3.16 (1H, d,
J ¼ 17.1 Hz, H22), 3.03 (1H, d, J ¼ 17.0 Hz, H22), 2.6 (4H, m), 2.34 (1H,
t, J ¼ 7.0 Hz, H2), 2.16 (4H, m, H2, H4, H10, H14), 1.77 (1H, dd,
J ¼ 14.6, 3.3 Hz, H6), 1.57 (4H, m, H1, H7), 1.44 (3H, s, H15), 1.31 (3H,
m, H8, H13, 11-OH), 1.16 (3H, s, H18), 1.12 (1H, dd, J ¼ 14.1, 4.2 Hz,
Fig. 4. Result of the docking of 12c (red) into the PTC model binding site. Residue
numbers shown are according to E. coli 23s RNA numbering. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of
this article.)
H8), 0.87 (3H, d, J ¼ 7.0 Hz, H17), 0.72 (3H, d, J ¼ 7.0 Hz, H16). 13
C
NMR (151 MHz, CDCl3):
d (ppm) 217.1 (C3), 169.1 (C21), 139.1 (C19),
117.2 (C20), 74.6 (C11), 68.3 (C14), 60.0, 58.2 (C4), 52.8 (C22), 45.5
(C9), 45.0 (C13), 44.0 (C12), 41.8 (C5), 36.8 (C6), 36.1 (C10), 34.5 (C2),
30.5 (C8), 26.8 (C7), 26.4 (C18), 24.9 (C1), 16.7 (C16), 14.9 (C15), 11.5
(C17). IR (KBr, cmꢀ1) 3353, 2930, 2863, 1736, 1457, 1210, 1172, 1118,
1017, 917, 838, 647. HR-MS (ESI): Calcd for C28H44ClN2O5 ([MþH]þ):
523.2933; Found: 523.2938.
4.2.4. General procedure for the synthesis of compounds 9, 10, 11a-
11c, 12a-12c, 13a-13c, 14a-14c and 15a-15c
Compound
8 (1 g, 1.91 mmol), piperazine derivatives
(2.00 mmol) and potassium carbonate (0.53 g, 3.82 mmol) were
added into acetonitrile (10 mL) and stirred at 70 ꢁC until complete
consumption of compound 8 was obtained. The reaction mixture
was extracted with chloroform (30 mL) and then washed with
water (30 mL) twice. The organic layer was dried with anhydrous
Na2SO4. After evaporation the organic solvent in vacuum, the res-
idue was purified by silica gel column chromatography to give a
pure product.
Fig. 5. Efficacy of compound 11c and tiamulin in mouse systemic infection model.
4.2.5. 22-(4-(2-(4-methyl-piperazin-1-yl)-acetyl)-piperazin-1-yl)-
22-deoxypleuromutilin (9)
White powder; Yield: 61.9%; m.p.: 56.7e57.2 ꢁC; Purity of com-
White powder; Yield: 40.8%; m.p.: 38.9e40.5 ꢁC; Purity of
compound was 96.4% (determined by RP HPLC at 219 nm,
pound was 96.8% (determined by RP HPLC at 210 nm,
tR ¼ 3.51 min); 1H NMR (600 MHz, CDCl3):
d (ppm) 7.75 (2H, m),
tR ¼ 7.20 min); 1H NMR (600 MHz, CDCl3):
d (ppm) 6.50 (1H, m,
7.22 (2H, d, J ¼ 7.9 Hz), 6.46 (1H, dd, J ¼ 17.4, 11.0 Hz, H19), 5.77 (1H,
d, J ¼ 8.4 Hz, H14), 5.33 (1H, dd, J ¼ 11.0, 1.6 Hz, H20), 5.19 (1H, dd,
J ¼ 17.3, 1.6 Hz, H20), 3.35 (1H, dd, J ¼ 10.0, 6.6 Hz, H11), 3.15 (1H, d,
J ¼ 17.2 Hz, H22), 3.05 (1H, d, J ¼ 17.1 Hz, H22), 2.38 (3H, s), 2.33 (1H,
t, J ¼ 7.0 Hz, H2), 2.16 (5H, m, H2, H4, H10, H14), 1.77 (1H, dd,
J ¼ 14.5, 3.2 Hz, H6), 1.56 (5H, m, H1, H7), 1.42 (3H, s, H15), 1.31 (3H,
m, H8, H13, 11-OH), 1.16 (3H, s, H18), 1.12 (1H, dd, J ¼ 14.1, 4.4 Hz,
H19), 5.79 (1H, t, J ¼ 8.9 Hz, H14), 5.34 (1H, m, H20), 5.20 (1H, m,
H20), 3.48 (4H, m), 3.34 (1H, m, H11), 3.19 (1H, d, J ¼ 17.1 Hz, H22),
3.05 (1H, d, J ¼ 17.1 Hz, H22), 2.54 (4H, m), 2.35 (1H, m, H4), 2.22
(2H, m, H2, H10), 2.08 (2H, m, H13), 1.77 (1H, dd, J ¼ 14.5, 2.9 Hz,
H6), 1.66 (2H, m, H1), 1.59 (7H, s, H7), 1.55 (2H, m), 1.45 (3H, s, H15),
1.37 (2H, m, H8, H13),1.27 (2H, dd, J ¼ 16.7, 9.7 Hz), 1.19 (1H, m), 1.16
(3H, d, J ¼ 6.3 Hz, H18), 1.12 (1H, dd, J ¼ 14.1, 4.3 Hz, H8), 0.89 (3H, d,
J ¼ 9.6 Hz, H17), 0.73 (3H, d, J ¼ 9.4 Hz, H16). 13C NMR (151 MHz,
H8), 0.88 (3H, d, J ¼ 7.0 Hz, H17), 0.68 (3H, d, J ¼ 7.1 Hz, H16). 13
C
NMR (151 MHz, CDCl3): d (ppm) 216.7 (C3),168.4 (C21),141.51,139.1
CDCl3):
d (ppm) 216.8 (C3), 169.0 (C21), 154.6, 139.2 (C19), 117.1
(C19), 129.0, 117.2 (C20), 74.6 (C11), 68.8 (C14), 59.3, 58.2 (C4), 49.5
(C22), 45.4 (C9), 45.1 (C13), 44.0 (C12), 43.7, 41.8 (C5), 36.7 (C6), 36.1
(C10), 34.4 (C2), 30.4 (C8), 26.8 (C7), 26.4 (C18), 24.9 (C1), 16.7
(C16), 14.8 (C15), 11.4 (C17). IR (KBr, cmꢀ1) 3438, 2985, 2862, 1731,
1460, 1285, 1177, 1122, 1010, 910, 817. HR-MS (ESI): Calcd for
(C20), 79.6, 74.6 (C11), 68.5 (C14), 65.8, 60.0, 58.2 (C4), 52.7 (C22),
45.5 (C9), 45.2 (C13), 44.0 (C12), 41.8 (C5), 36.8 (C6), 36.1 (C10), 34.4
(C2), 30.5 (C8), 28.4, 26.9 (C7), 26.4 (C18), 24.9 (C1), 16.7 (C16), 15.2,
14.9 (C15), 11.4 (C17). IR (KBr, cmꢀ1) 3479, 2930, 2865, 1732, 1673,
1456, 1434, 1366,1280, 1250, 1193, 1170, 982, 862, 763. HR-MS (ESI):
Calcd for C33H55N4O5 ([MþH]þ): 587.4167; Found: 587.4180.
C
26H43N2O4 ([MþH]þ): 447.3217; Found: 447.3201.

292
M.-L. Gao et al. / European Journal of Medicinal Chemistry 127 (2017) 286e295
4.2.6. 22-(4-(2-(4-phenyl-piperazin-1-yl)-acetyl)-piperazin-1-yl)-
22-deoxypleuromutilin (10)
26.8 (C7), 26.5 (C18), 25.3, 24.8 (C1), 16.7 (C16), 14.9 (C15), 11.5
(C17). IR (KBr, cmꢀ1) 3445, 2935, 2862, 1732, 1635, 1526, 1455, 1346,
1290, 1242, 1193, 1153, 1116, 1009, 914, 866, 761, 738, 669. HR-MS
(ESI): Calcd for C38H56N5O7 (M þ Hþ): 694.4174; Found: 694.4143.
White powder; Yield: 77.3%; m.p.: 73.8e74.9 ꢁC; Purity of
compound was 97.4% (determined by RP HPLC at 254 nm,
tR ¼ 3.82 min); 1H NMR (600 MHz, CDCl3):
d (ppm) 7.28 (1H, t,
J ¼ 6.0 Hz), 6.92 (2H, d, J ¼ 17.4 Hz), 6.49 (1H, m, H19), 5.79 (1H, dd,
J ¼ 8.7, 4.2 Hz, H14), 5.34 (1H, m, H20), 5.20 (1H, dd, J ¼ 17.4, 2.4 Hz,
H20), 3.64 (4H, m), 3.37 (4H, m), 3.23 (1H, m, H22), 3.10 (1H, m,
H22), 2.57 (4H, m), 2.34 (1H, t, J ¼ 6.9 Hz, H2), 2.16 (4H, m, H2, H4,
H10, H14), 1.77 (1H, dd, J ¼ 14.3, 3.2 Hz, H6), 1.58 (5H, m, H1, H7),
1.44 (3H, d, J ¼ 3.0 Hz, H15), 1.30 (4H, m, H8, H13, 11-OH), 1.17 (3H,
d, J ¼ 3.1 Hz, H18), 1.12 (1H, dd, J ¼ 14.1, 4.3 Hz, H8), 0.88 (3H, d,
J ¼ 6.9 Hz, H17), 0.72 (3H, d, J ¼ 7.0, H16). 13C NMR (151 MHz,
4.2.9. 22-(4-(2-(4-Nitrophenyl-piperazin-1-yl)-acetyl)-piperazin-
1-yl)-22-deoxypleuromutilin (11c)
Yellow powder; Yield: 49.3%; m.p.: 90.0e91.0 ꢁC; Purity of
compound was 97.7% (determined by RP HPLC at 215 nm,
tR ¼ 9.25 min); 1H NMR (600 MHz, CDCl3)
8.28 (1H, d, J ¼ 2.6 Hz),
d
8.15 (1H, m), 6.84 (1H, m), 6.48 (1H, m, H19), 5.80 (1H, m, H14), 5.34
(1H, m, H20), 5.21 (1H, dd, J ¼ 17.3, 1.6 Hz, H20), 4.15 (1H, s), 3.64
(1H, m), 3.45 (1H, m), 3.36 (1H, dd, J ¼ 10.4, 6.5 Hz, H11), 3.27 (1H,
m, H22), 3.17 (1H, dd, J ¼ 18.1,10.4 Hz, H22), 2.66 (3H, s), 2.44 (1H, d,
J ¼ 3.4 Hz), 2.34 (1H, t, J ¼ 6.7 Hz, H2), 2.16 (5H, m, H2, H4, H10,
H14), 1.78 (1H, m, H6), 1.57 (5H, m, H1, H7), 1.43 (3H, d, J ¼ 2.0 Hz,
H15),1.30 (3H, m, H8, H13,11-OH),1.17 (3H, s, H18),1.13 (1H, m, H8),
0.89 (3H, dd, J ¼ 7.0, 1.4 Hz, H17), 0.71 (3H, dd, J ¼ 7.0, 2.9 Hz, H16).
CDCl3):
d (ppm) 217.0 (C3), 168.9 (C21), 167.8, 151.2, 139.1 (C19),
129.1, 119.8, 117.3 (C20), 116.1, 74.6 (C11), 68.5 (C14), 65.8, 64.4, 61.3,
59.7, 58.2 (C4), 53.2, 52.7, 49.1 (C22), 46.1 (C9), 45.0 (C13), 44.0
(C12), 41.7 (C5), 41.7, 36.7 (C6), 36.1 (C10), 34.5 (C2), 31.6, 30.4 (C8),
26.8 (C7), 26.4 (C18), 24.9 (C1), 22.6, 16.7 (C16), 15.3, 14.9 (C15), 14.1,
11.5 (C17). IR (KBr, cmꢀ1) 3450, 2935, 2820, 1732, 1634, 1599, 1454,
1192, 1151, 1116, 1009, 917, 759, 693. HR-MS (ESI): Calcd for
13C NMR (151 MHz, CDCl3):
d (ppm) 217.0 (C3), 168.9 (C21), 167.4,
154.8, 139.1 (C19), 138.7, 125.9, 117.3 (C20), 112.8, 74.6 (C11), 68.5
(C14), 60.7, 59.7, 58.2 (C4), 53.0, 52.8, 52.5, 47.0 (C22), 46.1, 45.4
(C9), 45.0 (C13), 44.0 (C12), 41.7 (C5), 41.2, 36.7 (C6), 36.1 (C10), 34.5
(C2), 30.4 (C8), 29.7, 28.4, 26.8 (C7), 26.4 (C18), 25.4, 24.9 (C1), 21.3,
16.7 (C16), 14.9 (C15), 11.5 (C17). IR (KBr, cmꢀ1) 3445, 2929, 2862,
1732, 1636, 1597, 1455, 1373, 1325, 1243, 1194, 1154, 1115, 1006, 916,
829, 754, 693, 666. HR-MS (ESI): Calcd for C38H56N5O7 ([MþH]þ):
694.4174; Found: 694.4182.
C
38H57N4O5 ([MþH]þ): 649.4323; Found:649.4329.
4.2.7. 22-(4-(2-(2-Nitrophenyl-piperazin-1-yl)-acetyl)-piperazin-
1-yl)-22-deoxypleuromutilin (11a)
Yellow powder; Yield: 51.8%; m.p.: 79.3e80.5 ꢁC; Purity of
compound was 97.1% (determined by RP HPLC at 215 nm,
tR ¼ 7.53 min); 1H NMR (600 MHz, CDCl3):
d (ppm) 7.76 (1H, d,
J ¼ 8.0 Hz), 7.48 (1H, d, J ¼ 7.8 Hz), 7.13 (1H, d, J ¼ 8.3 Hz), 7.05 (1H, t,
J ¼ 7.7 Hz), 6.50 (1H, dd, J ¼ 17.4, 11.0 Hz, H19), 5.79 (1H, d,
J ¼ 8.5 Hz, H14), 5.33 (1H, J ¼ 11.0 Hz, H20), 5.20 (1H, d, J ¼ 17.3 Hz,
H20), 3.65 (4H, m), 3.38 (1H, m, H11), 3.25 (2H, s, H22), 3.09 (4H,
m), 2.60 (8H, m), 2.35 (1H, t, J ¼ 6.9 Hz, H2), 2.15 (5H, m, H2, H4,
H10, H14), 1.78 (1H, m, H6), 1.58 (5H, m, H1, H7), 1.44 (3H, s, H15),
1.31 (3H, m, H8, H13, 11-OH), 1.17 (3H, s, H18), 1.12 (1H, dd, J ¼ 14.1,
4.4 Hz, H8), 0.89 (3H, d, J ¼ 7.0 Hz, H17), 0.72 (3H, d, J ¼ 7.1 Hz, H16).
4.2.10. 22-(4-(2-(2-chlorophenyl-piperazin-1-yl)-acetyl)-
piperazin-1-yl)-22-deoxypleuromutilin (12a)
White powder; Yield: 55.8%; m.p.: 96.8e97.0 ꢁC; Purity of
compound was 96.0% (determined by RP HPLC at 254 nm,
tR ¼ 5.80 min); 1H NMR (600 MHz, CDCl3):
d
7.35 (1H, d, J ¼ 7.9,
1.5 Hz), 7.04 (1H, m), 6.97 (1H, t, J ¼ 7.6 Hz), 6.50 (1H, dd, J ¼ 17.4,
11.0 Hz, H19), 5.79 (1H, d, J ¼ 8.4 Hz, H14), 5.34 (1H, m, H20), 5.20
(1H, dd, J ¼ 17.4, 1.5 Hz, H20), 3.68 (4H, t, J ¼ 5.2 Hz), 3.36 (1H, d,
J ¼ 6.0 Hz, H11), 3.26 (2H, s, H22), 3.21 (1H, d, J ¼ 17.1 Hz), 3.09 (5H,
m), 2.71 (4H, s), 2.56 (4H, m), 2.34 (1H, t, J ¼ 7.0 Hz, H2), 2.16 (5H, m,
H2, H4, H10, H14), 1.77 (1H, dd, J ¼ 14.4, 3.1 Hz, H6), 1.59 (5H, m, H1,
H7), 1.44 (3H, s, H15), 1.29 (3H, m, H8, H13, 11-OH), 1.16 (3H, s, H18),
1.12 (1H, dd, J ¼ 14.1, 4.4 Hz, H8), 0.88 (3H, d, J ¼ 7.0 Hz, H17), 0.72
13C NMR (151 MHz, CDCl3):
d (ppm) 217.1 (C3), 168.9 (C21), 167.6,
145.8, 143.4, 139.1 (C19), 133.5, 125.9, 121.9, 121.1, 117.2 (C20), 74.6
(C11), 68.5 (C14), 64.3, 60.7, 59.7, 58.2 (C4), 53.1, 52.6, 51.5 (C22),
45.5 (C9), 45.4, 45.0 (C13), 44.03 (C12), 41.8 (C5), 41.6, 36.7 (C6),
36.1 (C10), 34.4 (C2), 30.4 (C8), 29.7, 26.8 (C7), 26.5 (C18), 25.3, 24.8
(C1), 16.7 (C16), 14.9 (C15), 11.5 (C17). IR (KBr, cmꢀ1) 3450, 2934,
2882,1732, 1642, 1604,1521,1455,1381, 1292,1192, 1154,1117,1009,
915, 668. HR-MS (ESI): Calcd for C38H56N5O7 ([MþH]þ): 694.4174;
Found: 694.4174.
(3H, d, J ¼ 7.1 Hz, H16). 13C NMR (151 MHz, CDCl3):
d (ppm) 217.0
(C3), 168.9 (C21), 167.9, 149.1, 139.1 (C19), 130.7, 128.8, 127.5, 123.7,
120.4, 117.2 (C20), 74.6 (C11), 68.5 (C14), 65.8, 61.1, 59.7, 58.2 (C4),
53.3, 53.2, 52.7, 51.1 (C22), 45.4 (C9), 45.0 (C13), 44.0 (C12), 41.7
(C5), 36.7 (C6), 36.1 (C10), 34.5 (C2), 30.4 (C8), 26.8 (C7), 26.5 (C18),
24.8 (C1), 16.7 (C16), 15.3, 14.9 (C15), 11.5 (C17). IR (KBr, cmꢀ1) 3450,
2935, 2819, 1734, 1639, 1453, 1230, 1192, 1153, 1121, 1010, 933, 751.
HR-MS (ESI): Calcd for C38H56ClN4O5 ([MþH]þ): 683.3934; Found:
683.3933.
4.2.8. 22-(4-(2-(3-Nitrophenyl-piperazin-1-yl)-acetyl)-piperazin-
1-yl)-22-deoxypleuromutilin (11b)
Yellow powder; Yield: 53.5%; m.p.: 71.6e72.8 ꢁC; Purity of
compound was 96.8% (determined by RP HPLC at 225 nm,
tR ¼ 4.57 min); 1H NMR (600 MHz, CDCl3):
d (ppm) 7.72 (1H, m),
7.65 (1H, dd, J ¼ 7.9, 2.0 Hz), 7.38 (1H, t, J ¼ 8.2 Hz), 7.21 (1H, m),
6.49 (1H, dd, J ¼ 17.4, 10.9 Hz, H19), 5.79 (1H, t, J ¼ 6.8 Hz, H14), 5.33
(1H, t, J ¼ 10.6 Hz, H20), 5.20 (1H, dd, J ¼ 17.4, 6.3 Hz, H20), 4.81 (1H,
s), 4.03 (1H, m), 3.67 (3H, dd, J ¼ 11.3, 6.2 Hz), 3.35 (1H, dd, J ¼ 10.0,
5.9 Hz, H11), 3.30 (3H, t, J ¼ 4.9 Hz), 3.26 (1H, d, J ¼ 2.4 Hz), 3.21 (1H,
m, H22), 3.09 (1H, m, H22), 2.83 (1H, d, J ¼ 5.1 Hz), 2.69 (3H, t,
J ¼ 4.9 Hz), 2.57 (3H, m), 2.35 (1H, t, J ¼ 6.6 Hz, H2), 2.17 (4H, m, H2,
H4, H10, H14), 1.91 (1H, s), 1.77 (1H, m, H6), 1.58 (5H, m, H1, H7),
1.43 (3H, d, J ¼ 3.6 Hz, H15) 1.32 (3H, m, H8, H13,11-OH),1.17 (3H, d,
J ¼ 5.4 Hz, H18), 1.12 (1H, d, J ¼ 4.4 Hz, H8), 0.88 (3H, d, J ¼ 7.0 Hz,
H17), 0.71 (3H, d, J ¼ 7.1 Hz, H16). 13C NMR (151 MHz, CDCl3):
4.2.11. 22-(4-(2-(3-chlorophenyl-piperazin-1-yl)-acetyl)-piperazin-
1-yl)-22-deoxypleuromutilin (12b)
White powder; Yield: 52.9%; m.p.: 56.1e57.5 ꢁC; Purity of
compound was 95.8% (determined by RP HPLC at 254 nm,
tR ¼ 4.77 min); 1H NMR (600 MHz, CDCl3):
7.27 (1H, d, J ¼ 1.4 Hz),
d
6.50 (1H, dd, J ¼ 17.3, 10.9 Hz, H19), 5.78 (1H, d, J ¼ 8.4 Hz, H14),
5.33 (1H, d, J ¼ 10.9 Hz, H20), 5.19 (1H, d, J ¼ 17.4 Hz, H20), 3.67 (4H,
m), 3.36 (1H, m, H11), 3.23 (1H, d, J ¼ 4.0 Hz, H22), 3.20 (6H, m),
3.07 (1H, m, H22), 2.65 (4H, t, J ¼ 4.9 Hz), 2.55 (3H, m), 2.34 (1H, t,
J ¼ 7.0 Hz, H2), 2.16 (5H, m, H2, H4, H10, H14), 1.78 (3H, m, H6), 1.58
(5H, m, H1, H7), 1.43 (3H, d, J ¼ 1.4 Hz, H15), 1.29 (3H, m, H8, H13,
11-OH), 1.16 (3H, s, H18), 1.12 (1H, dd, J ¼ 14.2, 4.4 Hz, H8), 0.88 (3H,
d, J ¼ 6.9 Hz, H17), 0.71 (3H, d, J ¼ 6.9 Hz, H16). 13C NMR (151 MHz,
d
(ppm) 217.1 (C3), 168.9 (C21), 167.6, 151.7, 149.2, 139.1 (C19), 129.7,
121.2,117.2 (C20),113.8,109.6, 74.6 (C11), 68.5 (C14), 64.3, 60.9, 59.7,
58.2 (C4), 53.1, 52.5, 48.5, 48.3 (C22), 45.4 (C9), 45.0 (C13), 44.0
(C12), 41.7 (C5), 39.7, 36.7 (C6), 36.1 (C10), 34.4 (C2), 30.4 (C8), 28.8,
CDCl3):
d (ppm) 217.0 (C3), 168.9 (21), 167.7, 152.2, 139.1 (C19),

M.-L. Gao et al. / European Journal of Medicinal Chemistry 127 (2017) 286e295
293
135.0, 130.0, 119.4, 117.3 (C20), 115.8, 114.0, 74.6 (C11), 68.5 (C14),
64.4, 61.2, 59.7, 58.2 (C4), 53.1, 52.9, 52.7, 48.7 (C22), 45.5 (C9), 45.1
(C13), 44.0 (C12), 41.7 (C5), 36.7 (C6), 36.1 (C10), 34.5 (C2), 30.4 (C8),
29.7, 26.8 (C7), 26.4 (C18), 25.4, 24.9 (C1), 16.7 (C16), 14.9 (C15), 11.5
(C17). IR (KBr, cmꢀ1) 3451, 2934, 2824, 1732, 1640, 1594, 1453, 1383,
1192, 1152, 1116, 914, 770. HR-MS (ESI): Calcd for C38H56ClN4O5
([MþH]þ): 683.3934; Found: 683.3940.
J ¼ 5.0 Hz), 2.54 (2H, m), 2.34 (1H, t, J ¼ 6.9 Hz, H2), 2.18 (5H, m, H2,
H4, H10, H14), 1.77 (1H, dd, J ¼ 14.5, 3.1 Hz, H6), 1.54 (5H, m, H1,
H7), 1.43 (3H, s, H15), 1.30 (3H, m, H8, H13, 11-OH), 1.16 (3H, s, H18),
1.12 (1H, dd, J ¼ 14.0, 4.4 Hz, H8), 0.88 (3H, d, J ¼ 7.0 Hz, H17), 0.71
(3H, d, J ¼ 7.0 Hz, H16). 13C NMR (151 MHz, CDCl3):
d (ppm) 217.0
(C3), 168.9 (C21), 167.8, 160.6, 152.6, 139.1 (C19), 129.8, 117.3 (C20),
108.9, 104.6, 102.6, 74.6 (C11), 68.5 (C14), 64.4, 61.3, 59.7, 58.2 (C4),
55.2, 53.1, 52.7, 49.0 (C22), 45.5 (C9), 45.0 (C13), 44.0 (C12), 41.7
(C5), 36.7 (C6), 36.1 (C10), 34.5 (C2), 30.4 (C8), 28.4, 26.8 (C7), 26.4
4.2.12. 22-(4-(2-(4-chlorophenyl-piperazin-1-yl)-acetyl)-
piperazin-1-yl)-22-deoxypleuromutilin (12c)
(C18), 25.4, 24.9 (C1),16.7 (C16),14.9 (C15),11.5 (C17). IR (KBr, cmꢀ1
)
White powder; Yield: 63.7%; m.p.: 79.2e80.0 ꢁC; Purity of
compound was 96.4% (determined by RP HPLC at 254 nm,
3440, 2936, 2830, 1732, 1642, 1496, 1454, 1374, 1291, 1259, 1203,
1168, 970, 839, 757, 690.HR-MS (ESI): Calcd for C39H59N4O6
([MþH]þ): 679.4429; Found:679.4416.
tR ¼ 4.56 min); 1H NMR (600 MHz, CDCl3):
d (ppm) 7.29 (1H, s), 7.20
(1H, d, J ¼ 8.5 Hz), 6.83 (1H, d, J ¼ 8.6 Hz), 6.50 (1H, m, H19), 5.78
(1H, d, J ¼ 8.1 Hz, H14), 5.33 (1H, t, J ¼ 9.7 Hz, H20), 5.20 (1H, dd,
J ¼ 17.4, 5.8 Hz, H20), 4.79 (1H, m), 4.02 (1H, m)3.67 (3H, m), 3.36
(1H, s, H11), 3.24 (1H, d, J ¼ 3.9 Hz, H22), 3.18 (4H, m), 3.07 (1H, m,
H22), 2.79 (1H, t, J ¼ 4.9 Hz), 2.66 (3H, t, J ¼ 4.8 Hz), 2.55 (5H, m),
2.34 (1H, t, J ¼ 6.7 Hz, H2), 2.16 (5H, m, H2, H4, H10, H14), 1.78 (1H,
m, H6), 1.58 (5H, m, H1, H7), 1.43 (3H, s, H15), 1.32 (3H, m, H8, H13,
11-OH), 1.16 (3H, d, J ¼ 4.7 Hz, H18), 1.11 (1H, dd, J ¼ 14.0, 4.2 Hz,
4.2.15. 22-(4-(2-(4-methoxyphenyl-piperazin-1-yl)-acetyl)-
piperazin-1-yl)-22-deoxypleuromutilin (13c)
White powder; Yield: 55.7%; m.p.: 81.6e82.0 ꢁC; Purity of
compound was 96.2% (determined by RP HPLC at 215 nm,
tR ¼ 9.07 min); 1H NMR (600 MHz, CDCl3):
d (ppm) 7.02 (1H, d,
J ¼ 8.8 Hz), 6.95 (1H, d, J ¼ 2.9 Hz), 6.85 (2H, m), 6.78 (1H, dd, J ¼ 8.8,
2.9 Hz), 6.50 (1H, dd, J ¼ 17.4, 10.9 Hz, H19), 5.79 (1H, dd, J ¼ 8.6,
3.0 Hz, H14), 5.34 (1H, m, H20), 5.20 (1H, m, H20), 3.89 (1H, d,
J ¼ 4.8 Hz), 4.03 (1H, m), 3.77 (3H, d, J ¼ 4.3 Hz), 3.68 (2H, s), 3.60
(2H, s), 3.36 (1H, dd, J ¼ 10.6, 6.5 Hz, H11), 3.27 (2H, s), 3.21 (1H, d,
J ¼ 7.9 Hz, H22), 3.10 (1H, dd, J ¼ 17.2, 7.7 Hz, H22), 2.58 (3H, m),
2.34 (1H, t, J ¼ 7.0 Hz, H2), 2.17 (5H, m, H2, H4, H10, H14), 1.78 (1H,
dd, J ¼ 14.6, 3.1 Hz, H6), 1.57 (5H, m, H1, H7), 1.44 (3H, d, J ¼ 2.4 Hz,
H15), 1.30 (3H, m, H8, H13, 11-OH), 1.17 (3H, d, J ¼ 2.4 Hz, H18), 1.12
(1H, m, H8), 0.88 (3H, d, J ¼ 6.9 Hz, H17), 0.72 (3H, d, J ¼ 7.1 Hz,
H8), 0.88 (3H, d, J ¼ 7.0 Hz, H17), 0.71 (3H, d, J ¼ 6.2 Hz, H16). 13
C
NMR (151 MHz, CDCl3): d (ppm) 217.1 (C3), 168.9 (C21), 167.7, 149.8,
139.1 (C19), 128.9, 124.6, 117.2 (C20), 117.2, 74.6 (C11), 68.5 (C14),
68.3, 64.3, 61.1, 59.9, 59.7, 58.2 (C4), 53.1, 53.3, 52.8, 52.6, 49.1 (C22),
45.4 (C9), 45.0 (C13), 44.0 (C12), 41.7 (C5), 36.7 (C6), 36.1 (C10), 34.5
(C2), 30.4 (C8), 26.8 (C7), 26.4 (C18), 25.3, 24.8 (C1), 16.7 (C16), 14.9
(C15), 11.5 (C17). IR (KBr, cmꢀ1) 3451, 2935, 2882, 1732, 1596, 1496,
1454, 1383, 1304, 1237, 1192, 1151, 1116, 1009, 916, 819. HR-MS (ESI):
Calcd for C38H56ClN4O5 ([MþH]þ): 683.3934; Found: 683.3939.
H16). 13C NMR (151 MHz, CDCl3):
d (ppm) 217.0 (C3), 168.9 (C21),
167.8, 153.9, 145.6, 139.1 (C19), 118.2, 117.3 (C20), 114.5, 74.6 (C11),
68.5 (C14), 61.3, 59.8, 58.2 (C4), 55.6, 53.2, 52.7, 50.6 (C22), 45.5
(C9), 45.0 (C13), 44.0 (C12), 41.8 (C5), 36.7 (C6), 36.1 (C10), 34.5 (C2),
30.4 (C8), 26.8 (C7), 26.4 (C18), 25.4, 24.9 (C1),16.7 (C16),14.9 (C15),
11.5 (C17). IR (KBr, cmꢀ1) 3472, 2934, 2862, 1733, 1511, 1455, 1382,
1292, 1244, 1191, 1151, 1117, 1011, 915, 824, 668. HR-MS (ESI): Calcd
for C39H59N4O6 ([MþH]þ): 679.4429; Found: 679.4434.
4.2.13. 22-(4-(2-(2-methoxyphenyl-piperazin-1-yl)-acetyl)-
piperazin-1-yl)-22-deoxypleuromutilin (13a)
White powder; Yield: 43.8%; m.p.: 64.7e65.8 ꢁC; Purity of
compound was 95.7% (determined by RP HPLC at 254 nm,
tR ¼ 4.67 min); 1H NMR (600 MHz, CDCl3):
d (ppm) 7.01 (1H, m),
6.94 (2H, m), 6.87 (1H, dd, J ¼ 8.0, 1.2 Hz), 6.51 (1H, dd, J ¼ 17.4,
11.0 Hz, H19), 5.80 (1H, d, J ¼ 8.5 Hz, H14), 5.34 (1H, dd, J ¼ 11.0,
1.6 Hz, H20), 5.21 (1H, dd, J ¼ 17.4, 1.6 Hz, H20), 3.69 (4H, m), 3.37
(1H, dd, J ¼ 10.4, 6.5 Hz, H11), 3.29 (2H, m), 3.21 (1H, d, J ¼ 17.2 Hz,
H22), 3.12 (4H, s), 3.08 (1H, d, J ¼ 17.1 Hz, H22) 2.76 (4H, s), 2.57 (4H,
m), 2.35 (1H, t, J ¼ 6.9 Hz, H2), 2.16 (5H, m, H2, H4, H10, H14), 1.78
(1H, dd, J ¼ 14.4, 3.1 Hz, H6), 1.59 (5H, m, H1, H7), 1.45 (3H, s, H15),
1.30 (3H, m, H8, H13, 11-OH), 1.17 (3H, s, H18), 1.13 (1H, dd, J ¼ 14.0,
4.3 Hz, H8), 0.89 (3H, d, J ¼ 7.0 Hz, H17), 0.73 (3H, dd, J ¼ 108.5,
4.2.16. 22-(4-(2-(2-Hydroxyphenyl-piperazin-1-yl)-acetyl)-
piperazin-1-yl)-22-deoxypleuromutilin (14a)
White powder; Yield: 64.2%; m.p.: 104.0e106.1 ꢁC; Purity of
compound was 95.4% (determined by RP HPLC at 215 nm,
tR ¼ 8.67 min); 1H NMR (600 MHz, CDCl3):
d (ppm) 7.15 (1H, dd,
J ¼ 7.9, 1.5 Hz), 7.08 (1H, m), 6.94 (1H, dd, J ¼ 8.2, 1.5 Hz), 6.86 (1H,
m), 6.51 (1H, dd, J ¼ 17.4, 11.0 Hz, H19), 5.79 (1H, d, J ¼ 8.4 Hz, H14),
5.34 (1H, dd, J ¼ 10.9, 1.5 Hz, H20), 5.20 (1H, dd, J ¼ 17.3, 1.6 Hz,
H20), 4.03 (1H, m), 3.67 (4H, m), 3.36 (1H, dd, J ¼ 10.4, 6.5 Hz, H11),
3.27 (2H, s), 3.22 (1H, d, J ¼ 17.2 Hz, H22), 3.09 (1H, d, J ¼ 17.0 Hz,
H22), 2.91 (3H, t, J ¼ 4.8 Hz), 2.70 (3H, s), 2.58 (3H, m), 2.35 (1H, t,
J ¼ 7.0 Hz, H2), 2.17 (5H, m, H2, H4, H10, H14), 1.78 (1H, dd, J ¼ 14.8,
3.3 Hz, H6), 1.60 (5H, m, H1, H7), 1.45 (3H, s, H15), 1.35 (3H, m, H8,
H13, 11-OH), 1.17 (3H, s, H18), 1.13 (1H, dd, J ¼ 14.1, 4.4 Hz, H8), 0.88
(3H, d, J ¼ 7.1 Hz, H17), 0.73 (3H, d, J ¼ 7.0 Hz, H16). 13C NMR
7.0 Hz, H16). 13C NMR (151 MHz, CDCl3):
d (ppm) 217.0 (C3), 168.9
(C21), 167.8, 152.3, 141.1, 139.1 (C19), 123.0, 121.0, 118.2, 117.3 (C20),
111.3, 74.6 (C11), 68.5 (C14), 64.4, 61.2, 59.8, 58.2 (C4), 55.4, 53.3,
52.7, 50.5 (C22), 45.5 (C9), 45.0 (C13), 44.0 (C12), 41.7 (C5), 41.7, 36.7
(C6), 36.1 (C10), 34.5 (C2), 30.4 (C8), 29.7, 26.8 (C7), 26.4 (C18), 25.4,
24.9 (C1), 16.7 (C16), 14.9 (C15), 11.5 (C17). IR (KBr, cmꢀ1) 3452,
2936, 2817, 1728, 1500, 1453, 1302, 1242, 1191, 1152, 1117, 915, 749.
HR-MS (ESI): Calcd for C39H59N4O6 ([MþH]þ): 679.4429; Found:
679.4409.
(151 MHz, CDCl3): d (ppm) 217.0 (C3), 168.9 (C21), 151.4, 139.1 (C19),
138.7, 126.6, 121.4, 120.1, 117.3 (C20), 114.2, 74.6 (C11), 68.5 (C14),
60.7, 59.7, 58.2 (C4), 53.6, 53.1, 52.6, 52.3 (C22), 45.4 (C9), 45.4, 45.0
(C13), 44.0 (C12), 41.8 (C5), 41.6, 36.7 (C6), 36.1 (C10), 34.4 (C2), 30.4
(C8), 29.7, 26.8 (C7), 26.4 (C18), 24.8 (C1),16.7 (C16),14.9 (C15),14.2,
11.5 (C17). IR (KBr, cmꢀ1) 3450, 2934, 2882, 1732, 1604, 1521, 1455,
1381,1343,1292,1192,1154,1117,1009, 915, 668. HR-MS (ESI): Calcd
for C38H57N4O6 ([MþH]þ): 665.4273; Found: 665.4271.
4.2.14. 22-(4-(2-(3-methoxyphenyl-piperazin-1-yl)-acetyl)-
piperazin-1-yl)-22-deoxypleuromutilin (13b)
White powder; Yield: 46.1%; m.p.: 90.0e91.2 ꢁC; Purity of
compound was 97.1% (determined by RP HPLC at 254 nm,
tR ¼ 3.63 min); 1H NMR (600 MHz, CDCl3):
d (ppm) 7.17 (1H, t,
J ¼ 8.2 Hz), 6.49 (1H, m), 6.46 (1H, t, J ¼ 2.4 Hz, H19), 6.42 (1H, dd,
J ¼ 7.9, 2.3 Hz), 5.78 (1H, d, J ¼ 8.5 Hz, H14), 5.33 (1H, dd, J ¼ 10.9,
1.6 Hz, H20), 5.19 (1H, dd, J ¼ 17.4, 1.6 Hz, H20), 3.79 (3H, s), 3.66
(4H, m), 3.35 (1H, dd, J ¼ 10.5, 6.5 Hz, H11), 3.23 (1H, d, J ¼ 4.2 Hz,
H22), 3.19 (5H, m), 3.07 (1H, d, J ¼ 17.1 Hz, H22), 2.66 (4H, t,
4.2.17. 22-(4-(2-(3-Hydroxyphenyl-piperazin-1-yl)-acetyl)-
piperazin-1-yl)-22-deoxypleuromutilin (14b)
White powder; Yield: 68.3%; m.p.: 35.6e35.8 ꢁC; Purity of

294
M.-L. Gao et al. / European Journal of Medicinal Chemistry 127 (2017) 286e295
compound was 96.1% (determined by RP HPLC at 215 nm,
663.4485.
tR ¼ 6.32 min); 1H NMR (600 MHz, CDCl3):
d (ppm) 7.07 (1H, t,
J ¼ 8.0 Hz), 6.46 (3H, m), 6.36 (1H, d, J ¼ 7.9 Hz, H19), 5.78 (1H, d,
J ¼ 8.3 Hz, H14), 5.32 (1H, d, J ¼ 10.8 Hz, H20), 5.19 (1H, d,
J ¼ 17.4 Hz, H20), 4.79 (1H, s), 4.12 (1H, t, J ¼ 7.1 Hz), 3.66 (4H, d,
J ¼ 5.1 Hz), 3.49 (1H, t, J ¼ 7.1 Hz), 3.37 (1H, d, J ¼ 7.1 Hz, H11), 3.22
(2H, d, J ¼ 11.2 Hz, H22), 3.16 (4H, m), 3.09 (1H, d, J ¼ 17.2 Hz, H22),
2.64 (4H, t, J ¼ 4.7 Hz), 2.54 (2H, m), 2.34 (1H, t, J ¼ 7.0 Hz, H2), 2.15
(4H, m),1.77 (1H, d, J ¼ 14.5 Hz, H6),1.60 (4H, m, H1, H7),1.44 (3H, s,
H15), 1.30 (3H, m, H8, H13, 11-OH), 1.16 (3H, s, H18), 1.12 (1H, d,
J ¼ 4.5 Hz, H8), 0.88 (3H, d, J ¼ 6.9 Hz, H17), 0.71 (3H, d, J ¼ 7.0 Hz,
4.2.20. 22-(4-(2-(3-Methylphenyl-piperazin-1-yl)-acetyl)-
piperazin-1-yl)-22-deoxypleuromutilin (15b)
White powder; Yield: 60.6%; m.p.: 53.0e54.0 ꢁC; Purity of
compound was 95.9% (determined by RP HPLC at 254 nm,
tR ¼ 4.31 min); 1H NMR (600 MHz, CDCl3):
d (ppm) 7.15 (1H, t,
J ¼ 7.8 Hz), 6.73 (2H, dd, J ¼ 11.2, 2.7 Hz), 6.69 (1H, d, J ¼ 7.4 Hz), 6.50
(1H, dd, J ¼ 17.4, 11.0 Hz, H19), 5.78 (1H, d, J ¼ 8.4 Hz, H14), 5.33 (1H,
dd, J ¼ 10.9, 1.6 Hz, H20), 5.19 (1H, dd, J ¼ 17.4, 1.6 Hz, H20), 3.68
(4H, m), 3.35 (1H, dd, J ¼ 10.7, 6.5 Hz, H11), 3.23 (2H, d, J ¼ 4.0 Hz,
H22), 3.19 (5H, m), 3.06 (1H, d, J ¼ 17.1 Hz, H22), 2.66 (4H, t,
J ¼ 4.9 Hz), 2.53 (3H, m), 2.34 (1H, t, J ¼ 6.9 Hz, H2), 2.18 (7H, m, H2,
H4, H10, H14), 1.77 (1H, dd, J ¼ 14.5, 3.1 Hz, H6), 1.54 (14H, m, H1,
H7), 1.43 (3H, s, H15), 1.27 (4H, m, H8, H13, 11-OH), 1.16 (3H, s, H18),
1.12 (1H, dd, J ¼ 14.1, 4.4 Hz, H8), 0.88 (3H, d, J ¼ 7.0 Hz, H17), 0.71
H16). 13C NMR (151 MHz, CDCl3):
d (ppm) 217.4 (C3), 169.0 (C21),
168.0, 157.3, 152.6, 139.0 (C19), 129.9, 117.3 (C20), 108.1, 107.1, 103.4,
74.6 (C11), 68.6 (C14), 61.0, 59.7, 58.2 (C4), 53.0, 52.6 (C22), 49.0,
45.5 (C9), 45.1 (C13), 44.0 (C12), 41.8 (C5), 36.7 (C6), 36.1 (C10), 34.5
(C2), 30.4 (C8), 26.8 (C7), 26.4 (C18), 24.8 (C1), 16.8 (C16),15.0 (C15),
11.5 (C17). IR (KBr, cmꢀ1) 3437, 2936, 2823, 1731, 1629, 1453, 1194,
1153, 1117, 1009, 914, 866, 765, 690. HR-MS (ESI): Calcd for
(3H, d, J ¼ 7.0 Hz, H16). 13C NMR (151 MHz, CDCl3):
d (ppm) 217.0
(C3), 168.9 (C21), 167.8, 151.3, 139.1 (C19), 138.8, 129.0, 120.8, 117.3
(C20), 113.2, 74.6 (C11), 68.5 (C14), 64.4, 61.3, 59.8, 58.2 (C4), 52.9,
49.2 (C22), 45.3 (C13), 44.0 (C12), 41.8 (C5), 36.7 (C6), 36.1 (C10),
34.5 (C2), 30.4 (C8), 26.8 (C7), 26.4 (C18), 25.4, 24.9 (C1), 21.8, 16.7
(C16), 14.9 (C15), 11.5 (C17). IR (KBr, cmꢀ1) 3484, 2932, 2863, 1733,
1602, 1494, 1455, 1366, 1307, 1192, 1170, 1117, 1013, 941, 846, 772,
693. HR-MS (ESI): Calcd for C39H59N4O5 ([MþH]þ): 663.4480;
Found: 663.4470.
C
38H57N4O6 ([MþH]þ): 665.4273; Found: 665.4276.
4.2.18. 22-(4-(2-(4-Hydroxyphenyl-piperazin-1-yl)-acetyl)-
piperazin-1-yl)-22-deoxypleuromutilin (14c)
White powder; Yield: 66.8%; m.p.: 103.0e103.5 ꢁC; Purity of
compound was 96.8% (determined by RP HPLC at 215 nm,
tR ¼ 5.75 min); 1H NMR (600 MHz, CDCl3):
d (ppm) 6.79 (4H, m),
6.49 (1H, dd, J ¼ 17.4,11.0 Hz, H19), 5.78 (1H, d, J ¼ 8.5 Hz, H14), 5.32
(1H, d, J ¼ 10.9 Hz, H20), 5.19 (1H, d, J ¼ 17.4 Hz, H20), 3.66 (4H, m),
3.49 (1H, q, J ¼ 7.0 Hz), 3.37 (1H, d, J ¼ 7.4 Hz, H11), 3.24 (1H, s, H22),
3.19 (1H, d, J ¼ 17.2 Hz, H22), 3.06 (5H, m), 2.66 (4H, t, J ¼ 4.8 Hz),
2.55 (3H, m), 2.34 (1H, t, J ¼ 6.9 Hz, H4), 2.16 (5H, m, H2, H4, H10,
H14), 1.77 (1H, m, H6), 1.57 (5H, m, H1, H7), 1.43 (3H, s, H15), 1.27
(3H, m, H8, H13, 11-OH), 1.16 (3H, s, H18), 1.12 (1H, m, H8), 0.88 (3H,
d, J ¼ 7.0 Hz, H17), 0.71 (3H, d, J ¼ 7.0 Hz, H16). 13C NMR (151 MHz,
4.2.21. 22-(4-(2-(4-Methylphenyl-piperazin-1-yl)-acetyl)-
piperazin-1-yl)-22-deoxypleuromutilin (15c)
White powder; Yield: 48.9%; m.p.: 87.1e88.4 ꢁC; Purity of
compound was 96.3% (determined by RP HPLC at 254 nm,
tR ¼ 6.26 min); 1H NMR (600 MHz, CDCl3):
d (ppm) 7.16 (2H, m),
6.99 (2H, m), 6.50 (1H, dd, J ¼ 17.4, 11.0 Hz, H19), 5.79 (1H, d,
J ¼ 8.4 Hz, H14), 5.33 (1H, d, J ¼ 10.8 Hz, H20), 5.19 (1H, d,
J ¼ 17.3 Hz, H20), 3.69 (4H, m), 3.36 (1H, s, H11), 3.25 (2H, s), 3.21
(1H, d, J ¼ 17.2 Hz, H22), 3.08 (1H, d, J ¼ 17.1 Hz, H22), 2.93 (4H, d,
J ¼ 4.9 Hz), 2.64 (4H, d, J ¼ 26.4 Hz), 2.53 (4H, m), 2.34 (1H, t,
J ¼ 6.9 Hz, H2), 2.20 (7H, m, H2, H4, H10, H14), 1.77 (1H, d,
J ¼ 14.6 Hz, H6), 1.59 (4H, m, H1, H7), 1.44 (3H, s, H15), 1.27 (3H, m,
H8, H13, 11-OH), 1.16 (3H, s, H18), 1.12 (1H, m, H8), 0.88 (3H, d,
J ¼ 7.0 Hz, H17), 0.72 (3H, d, J ¼ 7.0 Hz, H16). 13C NMR (151 MHz,
CDCl3):
d (ppm) 217.3 (C3), 168.9 (C21), 168.1, 150.6, 145.0, 139.0
(C19), 118.5, 117.3 (C20), 116.0, 74.6 (C11), 68.6 (C14), 65.9, 61.1, 59.7,
58.2 (C4), 53.2, 52.6, 50.8 (C22), 45.3 (C9), 45.0 (C13), 44.9, 44.0
(C12), 41.7 (C5), 36.7 (C6), 36.0 (C10), 34.5 (C2), 30.4 (C8), 26.8 (C7),
26.4 (C18), 24.8 (C1), 16.7 (C16), 15.2, 14.9 (C15), 11.5 (C17). IR (KBr,
cmꢀ1) 3427, 2936, 2819, 1732, 1632, 1514, 1455, 1374, 1302, 1227,
1153, 1117, 1009, 916, 824, 723. HR-MS (ESI): Calcd for C38H57N4O6
([MþH]þ): 665.4273; Found: 665.4277.
CDCl3):
d (ppm) 217.1 (C3), 168.9 (C21), 168.0, 151.3, 139.1 (C19),
132.6, 131.1, 126.5, 123.2, 118.9, 117.2 (C20), 74.6 (C11), 68.5 (C14),
65.8, 61.4, 59.8, 58.2 (C4), 53.6, 53.2, 52.7, 51.7 (C22), 45.5 (C9), 45.1
(C13), 44.0 (C12), 41.9, 41.9 (C5), 41.7, 36.7 (C6), 36.1 (C10), 34.5 (C2),
30.4 (C8), 26.8 (C7), 26.5 (C18), 24.9 (C1), 17.9, 16.7 (C16), 15.3, 14.9
(C15), 11.5 (C17). IR (KBr, cmꢀ1) 3456, 2937, 2862, 1734, 1642, 1599,
1493, 1456, 1375, 1302, 1268, 1192, 1152, 1116,1011, 932, 822. HR-MS
(ESI): Calcd for C39H59N4O5 ([MþH]þ): 663.4480; Found: 663.4488.
4.2.19. 22-(4-(2-(2-Methylphenyl-piperazin-1-yl)-acetyl)-
piperazin-1-yl)-22-deoxypleuromutilin (15a)
White powder; Yield: 46.7%; m.p.: 68.2e70.3 ꢁC; Purity of
compound was 96.7% (determined by RP HPLC at 225 nm,
tR ¼ 6.15 min); 1H NMR (600 MHz, CDCl3):
d (ppm) 7.00 (1H, m),
6.92 (2H, dd, J ¼ 8.0, 2.0 Hz), 6.86 (1H, d, J ¼ 8.0 Hz), 6.50 (1H, dd,
J ¼ 17.4,11.0 Hz, H19), 5.79 (1H, dd, J ¼ 8.8, 5.2 Hz, H14), 5.34 (1H, m,
H20), 5.20 (1H, m, H20), 4.72 (1H, d, J ¼ 37.0 Hz), 4.01 (1H, m), 3.86
(2H, s), 3.68 (3H, m), 3.50 (1H, t, J ¼ 5.1 Hz), 3.36 (1H, dd, J ¼ 9.9,
6.2 Hz, H11), 3.24 (1H, d, J ¼ 2.0 Hz, H22), 3.20 (1H, m, H22), 3.08
(4H, m), 2.70 (3H, s), 2.55 (3H, m), 2.35 (1H, t, J ¼ 6.9 Hz, H2), 2.17
(5H, m, H2, H4, H10, H14), 1.91 (1H, s), 1.77 (1H, dd, J ¼ 14.5, 3.2 Hz,
H6), 1.57 (5H, m, H1, H7), 1.44 (3H, s, H15), 1.26 (3H, m, H8, H13, 11-
OH), 1.17 (3H, d, J ¼ 3.3 Hz, H18), 1.12 (1H, dd, J ¼ 13.9, 4.2 Hz, H8),
0.88 (3H, d, J ¼ 6.8 Hz, H17), 0.72 (3H, d, J ¼ 7.0 Hz, H16). 13C NMR
4.3. Minimum inhibitory concentration(MIC) testing
The MIC values of the target compounds against MRSA (ATCC
43300), Staphylococcus aureus (ATCC 29213), Enterococcus faecalis
(ATCC 29212), Enterococcus faecium (ATCC35667) and Escherichia
coli (ATCC25922) were determined using pleuromutilin, tiamulin
and valnemulin as positive controls. MIC values were determined
by broth dilution in accordance with the Clinical and Laboratory
Standards Institute (CLSI) [31].
(151 MHz, CDCl3): d (ppm) 217.1 (C3), 168.9 (C21), 168.0, 141.2, 139.1
(C19), 123.0, 121.0, 117.2 (C20), 111.3, 74.6 (C11), 68.5 (C14), 64.3,
61.3, 58.2 (C4), 55.4, 53.4, 53.2, 52.8, 52.5, 50.6 (C22), 46.1, 45.5 (C9),
45.0 (C13), 44.0 (C12), 41.8 (C5), 41.6, 41.2, 36.7 (C6), 36.1 (C10), 34.5
(C2), 30.4 (C8), 26.8 (C7), 26.4 (C18), 25.3, 24.8 (C1), 21.3, 16.7 (C16),
14.9 (C15), 11.5 (C17). IR (KBr, cmꢀ1) 3445, 2935, 2862, 1733, 1500,
1455, 1382, 1373, 1302, 1270, 1241, 1192, 1153, 1118, 1011, 915, 750.
HR-MS (ESI): Calcd for C39H59N4O5 ([MþH]þ): 663.4480; Found:
Stock solutions of these compounds were dissolved in methanol
to make a stock solution with a concentration of 1280
mg/mL. The
working solutions (320 g/mL) of these compounds were made by
m
diluting stock solutions in sterile Mueller-Hinton (MH) broth or
Brain Heart Infusion (BHI) broth (for ATCC 29212 and ATCC 35667).
The control test with the same medium as used in the MIC test was
carried out to eliminate the effect of methanol on the growth of

M.-L. Gao et al. / European Journal of Medicinal Chemistry 127 (2017) 286e295
295
bacteria.
The tested organisms were recovered and then incubated in
Development Program of China (No. 2016YFD0501300), the Pearl
River S&T Noval Program of Guangzhou (No. 2014J2200042) and
the National Natural Science Foundation of China (No. 31672602
and No. 21405051).
broth medium for 18e24 h at 37 ꢁC in 5% CO2 atmosphere. Inocula
was then prepared by transferring several colonies of the tested
organisms to saline. The bacterial suspensions were mixed and
corrected to 0.5 McF arland standard using saline. Further dilutions
in saline were made to get the required working suspensions
(106 CFU/mL).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2017.01.004.
The compounds (with a final concentration of 0.016e32 mg/mL)
and bacteria solutions were dispensed into 96-well plate with total
volume at 200
The minimum inhibitory concentration (MIC,
m
L, the mixture were then cultured for 24 h at 37 ꢁC.
References
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is the lowest concentration of the compound that inhibit the visible
growth of bacteria after overnight incubation. All dates were tested
in duplicate in each plate. Two columns served as drug-free con-
trols (no cultures were added in one column and drugs replaced by
blank solvent in the other column). Pleuromutilin, tiamulin and
valnemulin were used as positive controls against tested organ-
isms. The final concentration of methanol in the first well column
was 1.25%. Initially, preliminary analyses were conducted with
1.25% (v/v) methanol/broth and this did not affect neither the
growth of the tested bacteria nor the determination of MIC.
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4.5. MRSA infection model
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days prior to infection and with 100 mg/kg 1 day prior to infection.
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Acknowledgements
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This work was supported by the National Key Research and