Discovery, synthesis of novel fusidic acid derivatives possessed amino-terminal groups at the 3-hydroxyl position with anticancer activity

Article abstract of DOI:10.1016/j.ejmech.2018.10.059

A series of novel fusidic acid (FA) derivatives were synthesized and screened for their in vitro cytotoxicity against the Hela, U87, KBV and MKN45 cancer cell lines. Selected FA derivatives with anti-tumor activity were firstly identified including compound 4, which exhibited good anti-proliferative activity with IC₅₀ values in the range of 1.26–3.57 μM. Further research revealed that compound 4 induced Hela cells to undergo apoptosis by increasing the ratio of the cells in the Sub-G₀/G₁ phase via decreasing the neo-synthesized proteins in a dose-dependent manner from 1 to 10 μM. Compound 4 also showed good in vivo anti-tumor activity against the xenograft tumor of Hela cells and had no apparent toxicity. This study highlights the advantage of introducing the medium-length amino-terminal groups at the 3-OH position of FA to enhance its anti-tumor activity and suggests that compound 4 provides a starting point for designing more potent derivatives in the future.

Full text of DOI:10.1016/j.ejmech.2018.10.059

Accepted Manuscript
Discovery, synthesis of novel fusidic acid derivatives possessed amino-terminal
groups at the 3-hydroxyl position with anticancer activity
Jingxuan Ni, Mengqi Guo, Yucheng Cao, Lei Lei, Kangli Liu, Binghua Wang,
Fangfang Lu, Rong Zhai, Xiangwei Gao, Chunhong Yan, Hongbo Wang, Yi Bi
PII:
S0223-5234(18)30936-X
DOI:
https://doi.org/10.1016/j.ejmech.2018.10.059
EJMECH 10845
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 September 2018
Revised Date: 25 October 2018
Accepted Date: 27 October 2018
Please cite this article as: J. Ni, M. Guo, Y. Cao, L. Lei, K. Liu, B. Wang, F. Lu, R. Zhai, X. Gao, C. Yan,
H. Wang, Y. Bi, Discovery, synthesis of novel fusidic acid derivatives possessed amino-terminal groups
at the 3-hydroxyl position with anticancer activity, European Journal of Medicinal Chemistry (2018), doi:
https://doi.org/10.1016/j.ejmech.2018.10.059.
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ACCEPTED MANUSCRIPT
Discovery, synthesis of novel fusidic acid derivatives possessed amino-terminal
groups at the 3-hydroxyl position with anticancer activity
Jingxuan Ni1,#, Mengqi Guo1,#, Yucheng Cao1, Lei Lei1,2, Kangli Liu3, Binghua Wang1, Fangfang
Lu1, Rong Zhai1, Xiangwei Gao3, Chunhong Yan1,4, Hongbo Wang1,*, Yi Bi1,*
1School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University),
Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in
Universities of Shandong, Yantai University, Yantai, 264005, P. R. China
2State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica,
Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, P. R. China
3Institute of Environmental Medicine, Zhejiang University School of Medicine, Hangzhou, 310058, P. R. China
4Department of Biochemistry and Molecular Biology, Medical College of Georgia, Georgia Regents University,
Augusta, GA, 30912, USA
*Corresponding Authors: hongbowangyt@gmail.com (Hongbo Wang), beeyee_413@163.com (Yi Bi)
#Jingxuan Ni and Mengqi Guo have equally contributed to the work.
Graphical abstract
Mechanism of action of comopound 4 in Hela cells
Compound 4
Decreasing the neo-synthesized proteins
O
O
Increasing the ratio of the cells in the Sub-G0/G1 phase
Apoptosis
H
H
21
OH
O
HO
H
HO
H
O
O
O
H
H
O
O
HO
H2N
O
3
Compound 4
Fusidic acid (FA)
Hela
U87
KBV
IC50 = 1.26 ~ 3.57
M
IC50 > 100
M
MKN45
JHH-7
Compound 4 showed good anti-tumor activity
against the xenograft tumor of Hela cells in nude mice

ACCEPTED MANUSCRIPT
Discovery, synthesis of novel fusidic acid derivatives possessed amino-terminal
groups at the 3-hydroxyl position with anticancer activity
Jingxuan Ni1,#, Mengqi Guo1,#, Yucheng Cao1, Lei Lei1,2, Kangli Liu3, Binghua Wang1, Fangfang Lu1,
Rong Zhai1, Xiangwei Gao3, Chunhong Yan1,4, Hongbo Wang1,*, Yi Bi1,*
1School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry
of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of
Shandong, Yantai University, Yantai, 264005, P. R. China
2State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica,
Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, P. R. China
3Institute of Environmental Medicine, Zhejiang University School of Medicine, Hangzhou, 310058, P. R. China
4Department of Biochemistry and Molecular Biology, Medical College of Georgia, Georgia Regents University,
Augusta, GA, 30912, USA
*Corresponding Authors: hongbowangyt@gmail.com (Hongbo Wang), beeyee_413@163.com (Yi Bi)
#Jingxuan Ni and Mengqi Guo have equally contributed to the work.
ABSTRACT: A series of novel fusidic acid (FA) derivatives were synthesized and screened for their in vitro
cytotoxicity against the Hela, U87, KBV and MKN45 cancer cell lines. Selected FA derivatives with anti-tumor
activity were firstly identified including compound 4, which exhibited good anti-proliferative activity with IC50 values
in the range of 1.26-3.57 µM. Further research revealed that compound 4 induced Hela cells to undergo apoptosis by
increasing the ratio of the cells in the Sub-G0/G1 phase via decreasing the neo-synthesized proteins in a
dose-dependent manner from 1-10 µM. Compound 4 also showed good in vivo anti-tumor activity against the
xenograft tumor of Hela cells and had no apparent toxicity. This study highlights the advantage of introducing the
medium-length amino-terminal groups at the 3-OH position of FA to enhance its anti-tumor activity and suggests that
compound 4 provides a starting point for designing more potent derivatives in the future.
Key words: Fusidic acid, derivatives, synthesis, anti-tumor activity, apoptosis
INTRODUCTION
Cancer is a particularly complex, widespread and fatal disease. There were 14.1 million new cancer cases, 8.2
million cancer deaths and 32.6 million cancer patients (within 5 years after diagnosis) worldwide in 2012, and cancer
deaths are expected to continue rising with an estimated 13.1 million deaths in 2030 [1,2]. Thus, cancer has become a

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major cause of mortality around the world. Several lines of evidence support the view that chemotherapy has become
one of the most important treatments for cancer [3-5]. Unfortunately, most anticancer drugs have serious side effects
making them less than ideal for the long term treatment of cancer [6]. Therefore, the identification of new agents that
are cytotoxic to cancer cells without inducing serious side effects is critical for the research and development of new
anti-tumor drugs.
Fusidic acid (FA) is a steroid-based, narrow spectrum bacteriostatic antibiotic with a tetracyclic ring system [7]. It
was first isolated from the fungus Fusidium coccineum in 1960 and has been in clinical use since 1962 for the
treatment of skin, bone and joint infections caused by Staphylococcus aureus as well as against several other
Gram-positive species [8-11]. Additionally, as FA is a marketed drug, it has been extensively studied and is known not
to be cytotoxic. Despite the extensive research on FA, no FA derivatives have appeared on the market, and there have
not been any reports in the literature that FA derivatives are cytotoxic.
In our group, we are committed to finding noncytotoxic FA derivatives with better antibacterial activity compared
with FA. Antibacterial screening for synthesized derivatives in the Community for Open Antimicrobial Drug Discovery
(http://www.co-add.org/), including antibacterial and cytotoxicity assay, was carried out to obtain derivatives with
better antibacterial activity and lower cytotoxicity compared with FA. However, the results of the antibacterial
screening showed that FA derivatives 1 and 10 did not possess the expected antibacterial activity but instead were
cytotoxic. Compared with FA that had inhibitory activity against S. aureus with a MIC ≤ 0.25 µg/mL and no
cytotoxicity against the human embryonic kidney cell line HEK293 with a CC50 > 32 µg/mL, compound 1 had no
inhibitory activity against S. aureus with a MIC > 32 µg/mL and cytotoxicity against HEK293 with a CC50 = 10.8
µg/mL. Similarly, compound 10 had weak inhibitory activity against S. aureus with a MIC = 32 µg/mL and
cytotoxicity against HEK293 with a CC50 = 20.6 µg/mL. According to the results of the antibacterial screening, we
speculated that introduction of a benzyl group at the 21-COOH site and amino-terminal groups or carboxyl-terminal
groups at the 3-OH site led to the cytotoxicities of compound 1 and 10. Therefore, FA derivatives with similar
structures to compound 1 or 10 were designed and synthesized to verify whether the introduction of similar structures
led to the cytotoxicity of the FA derivatives, and to further explore the mechanism of the cytotoxicity of the FA
derivatives.
In this work, a series of novel FA derivatives were synthesized, their cytotoxicities in vitro and anti-tumor activities

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in vivo were evaluated and the mechanism of their anti-tumor activity was explored. If novel FA derivatives with
anti-tumor activity and a defined mechanism of action can be obtained, it will further support the design and synthesis
of novel FA derivatives with better anti-tumor activity.
Figure 1. Structures of FA, 1, 10, glycyrrhetinic acid (GA) derivatives A, B and C.
RESULTS AND DISCUSSION
Synthetic chemistry
FA derivatives 1-18 were synthesized as shown in Scheme 1. First, commercially available FA was coupled with
benzyl bromide in acetone in the presence of an appropriate amount of K2CO3 to obtain the intermediate X1. Three
different types of derivatives can be synthesized using X1 as a raw material according to the following methods: (1)
Treatment of X1 with different t-butyloxy carbonyl (boc)-protected amino acids in anhydrous CH2Cl2 in the presence
of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and
a
catalytic amount of
dimethylaminopyridine (DMAP) produced intermediates. Next, intermediates were deprotected under acidic
conditions to produce compounds 1-3; (2) The amino-terminal acids X2-X6 with different chain lengths were reacted
with di-tert-butyl dicarbonate (boc anhydride) in tert-butyl alcohol (TBA) in the presence of sodium hydroxide
solution to obtain the amino-protected acids X7-X11, respectively. Then, treatment of X1 with X7-X11 in anhydrous
CH2Cl2 in the presence of EDCI and DMAP produced the corresponding intermediates. Next, the intermediates were
deprotected with trifluoroacetic acid (TFA) to produce compounds 4-8; (3) Treatment of X1 with different anhydrides
in anhydrous CH2Cl2 in the presence of DMAP produced compounds 9-11. Next, 9 or 10 was reacted with different
nitrogen-containing heterocyclic groups in anhydrous CH2Cl2 in the presence of EDCI produced compounds 12-18.

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Scheme 1. Synthesis of FA derivatives 1-18a
aReagent and conditions: (1) BrBn, K2CO3, acetone, 30 °C; (2) Boc-amino acids, DMAP, EDCI, CH2Cl2, r.t.; (3) Hydrochloric acid, ethyl
acetate, r.t.; (4) Boc anhydride, NaOH/H2O, TBA, 0 °C-r.t.; (5) DMAP, EDCI, CH2Cl2, r.t.; (6) TFA, CH2Cl2, 0 °C-r.t.; (7) Anhydrides, DMAP,
CH2Cl2, r.t.; (8) Nitrogen-containing heterocyclic groups, EDCI, CH2Cl2, r.t.
Cell growth inhibition
The anti-proliferative activity of all the synthesized compounds at 5 µM and 1.5 µM was determined by an MTT
assay in four cancer cell lines: Hela (cervical cancer), KBV (multidrug-resistant oral epidermoid carcinoma), U87
(glioma) and MKN45 (gastric cancer). The results are summarized in Table 1. FA showed no significant growth
inhibition effects against the Hela, KBV, U87 and MKN45 cell lines. Some of its derivatives displayed good growth
inhibition effects at 5 µM and even several derivatives showed good effects at 1.5 µM. In the assay of cell growth
inhibition at 5 µM, compounds 4-8 showed good anti-proliferative activity against four cancer cell lines that yielded
survival rates of not more than 23%. Compound 2 showed good growth inhibition effects against Hela, KBV, U87 cell
lines (cell viability ≤ 33% ) and it had no effect against MKN45 cell lines. Compound 1 showed good growth

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inhibition effect against KBV (cell viability was 28%) and moderate effect against U87 (cell viability was 41%).
Compound 3 showed good growth inhibition effect only against Hela (cell viability was 22%). Compound 13 showed
moderate effect against Hela (cell viability was 47%). Other compounds showed no significant growth inhibition
effects against Hela, KBV, U87 and MKN45 cell lines. In the assay of cell growth inhibition at 1.5 µM, compound 4
still exhibited good growth inhibition effects against Hela, KBV, U87 and MKN45 cell lines (cell viability were 32%,
14%, 10% and 23%, respectively). However, at a concentration of 1.5 µM, compound 5 exhibited good growth
inhibition effect only against U87 (cell viability was 10%), compound 6 exhibited good effect only against MKN45
(cell viability was 16%) and compound 7 exhibited moderate growth inhibition effects only against U87 (cell viability
was 49%). Other compounds showed no significant growth inhibition effects against the Hela, KBV, U87 and MKN45
cell lines. In previous antibacterial screening, compounds 1 and 10 had cytotoxicity against HEK293 with a CC50
=
10.8 µg/mL and 20.6 µg/mL, respectively. However, in the MTT assay at a concentration of 5 µM, compound 1
showed a growth inhibition effect against KBV and U87 cells but had no effect against Hela and MKN45 cells, while
compound 10 showed no significant growth inhibition effects against the Hela, KBV, U87 and MKN45 cell lines. Thus,
we infered that differences in the compound concentrations and cell lines led to the different cytotoxicity results.
The results of the MTT assay showed that derivatives with amino-terminal groups at the 3-OH site had better
anti-tumor activity compared with derivatives with carboxyl-terminal groups. And S. Schwarz et al reported that the
glycine substituted glycyrrhetinic acid (GA) derivative B had better anti-tumor activity and the hydrogen succinate C
had less anti-tumor activity compared with GA derivative A (Figure 1) [12,13]. The results of the above studies were
consistent, then a preliminary structure-activity relationship was developed that derivatives with amino-terminal
groups introduced at the 3-OH site had better anti-proliferative activity than derivatives with carboxyl-terminal groups
at the 3-OH site. In addition, carbon chains inserted between the amino and carboxyl groups that were too short
induced a decrease of anti-proliferative activity against the various cancer cell lines. 3-Glycine derivatives without a
branched-chain or with a short branched-chain showed better anti-proliferative activity than 3-glycine derivatives with
a long branched-chain. Derivatives with carboxyl-terminal groups or nitrogen-containing heterocyclic groups
introduced at the 3-OH site had no significant anti-proliferative activity, but introduction of fat chains and saturated
nitrogen-containing heterocyclic groups might favor anti-proliferative activity.
Compound 4 showed the best anti-proliferative activity against all four cancer cell lines among the eighteen FA

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derivatives. Therefore, we further extended our characterization of the anti-proliferative activity of 4 in five cancer cell
lines: Hela, U87, JHH-7 (liver cancer), KBV and MKN45. The IC50 (50% growth inhibition) values for 4 are shown in
Table 2. Compound 4 displayed good anti-proliferative activity in all the tested cell lines with IC50 values of 1.26
µM-3.57 µM. Thus compound 4 has an effective growth inhibitory effect on a variety of tumor cells, and we further
explored its possible mechanism of action and anti-tumor activity in vivo.
Table 1. In vitro cytotoxicities of FA and its derivatives against Hela, KBV, U87 and MKN45 at 5 µM and 1.5 µMa
Cell viability
Cpd
Hela
KBV
U87
MKN45
.
5 µM
1.5 µM
5 µM
1.5 µM
5 µM
1.5 µM
5 µM
1.5 µM
80%±3%
95% ± 1%
96% ± 2%
95% ± 1%
23% ± 2%
90% ± 1%
16% ± 2%
96% ± 1%
91% ± 1%.
74%±2%
78%±3%
95%±13%
82%±3%
81%±5%
88%±6%
104%±14%
104%±9%
101%±3%
FA
1
73%±2%
96% ± 5%
70%±2%
100% ± 1%
102% ± 5%
99% ± 1%
32% ± 14%
101% ± 6%
99% ± 2%
100% ± 4%
101% ± 8%
83%±18%
89%±18%
76%±7%
76%±4%
70%±2%
75%±0%
76%±7%
83%±5%
90%±2%
67%±2%
28% ± 9%
15% ± 2%
83% ± 2%
12% ± 8%
13% ± 9%
11% ± 6%
9% ± 3%
71%±4%
97% ± 1%
95% ± 3%
96% ± 1%
14% ± 6%
98% ± 4%
83% ± 8%
93% ± 5%
99% ± 7%
72%±5%
78%±9%
86%±5%
78%±6%
82%±5%
77%±1%
81%±2%
89%±9%
85%±3%
87%±2%
41% ± 7%
33% ± 8%
111% ± 3%
9% ± 1%
92%±2%
94% ± 3%
95% ± 2%
106% ± 4%
10% ± 0%
10% ± 0%
116% ± 4%
49% ± 9%
105% ± 1%
86%±2%
81%±2%
88%±3%
91%±5%
87%±6%
87%±3%
86%±3%
86%±5%
90%±0%
83%±2%
93% ± 1%
91% ± 1%
92% ± 2%
5% ± 0%
2
23% ± 10%
22% ± 1%
13% ± 9%
23% ± 16%
19% ± 4%
19% ± 17%
8% ± 1%
3
4
5
9% ± 1%
6% ± 1%
6
10% ± 0%
10% ± 0%
9% ± 1%
4% ± 0%
7
5% ± 0%
8
18% ± 16%
66%±5%
81%±11%
108%±26%
66%±8%
71%±12%
80%±6%
66%±12%
75%±3%
79%±2%
8% ± 2%
9
69%±2%
68%±2%
69%±1%
64%±6%
47%±2%
71%±2%
60%±5%
73%±2%
82%±3%
84%±2%
85%±13%
90%±8%
93%±11%
88%±7%
83%±2%
77%±2%
77%±3%
87%±3%
76%±4%
76%±1%
87%±4%
78%±3%
75%±6%
83%±12%
92%±9%
99%±13%
96%±4%
10
11
12
13
14
15
16
17

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18
63%±2%
84%±4%
69%±6%
81%±4%
77%±1%
85%±6%
92%±5%
103%±8%
aThe cell viabilities are presented as the mean ± SD of three independent experiments.
Table 2. The IC50 values of compound 4 against a panel of cancer cell lines
IC50 (µM)
Cancer cell lines
4
FA
Hela
U87
3.57 ± 1.27
2.40 ± 0.87
3.18 ± 1.16
1.26 ± 0.23
1.28 ± 0.25
> 100
> 100
> 100
> 100
> 100
JHH-7
KBV
MKN45
Compound 4 induced the Hela cells to undergo cell apoptosis
To explore the effect of compound 4 on the cell apoptosis, a TUNEL staining assay and flow cytometry were
conducted. As shown in Figure 2, compound 4 at 2 µM, but not the parental FA, could induced the Hela cells to
undergo apoptosis after 24 hours incubation as indicated by the green fluorescence cells. Similar results could be
observed in the flow cytometry assay, in which compound 4 at 5 µM, but not the parental FA, could significantly
increase the ratio of the cells in the Sub-G0/G1 phase after 48 h or 72 h incubation (Figure 3).
Figure 2. Effects of compound 4 on the early stage of cell apoptosis in Hela cells. Cells were seeded into 12-well plates with cover slips and
treated with compound 4 at 2 µM for 24 h. Then cells were subjected to the TUNEL staining assay to determine the early stage of cell
apoptosis. The red arrows indicate the TUNEL-positive cells.

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Figure 3. Effects of compound 4 on the late stage of cell apoptosis in Hela cells. Cells were seeded into 6-well plates and treated with
compound 4 at 5 µM for 48 h and 72 h. Cells were then subjected to flow cytometry to determine the distribution ratio of sub-G0/G1. *: p<0.05,
compared with the control group.
Compound 4 inhibited protein synthesis
As puromycin can be incorporated into neo-synthesized proteins, the anti-puromycin antibody was used to detect the
effect of compound 4 on the neo-synthesized proteins. As shown in Figure 4, compound 4 at 5 µM, but not the parental
FA, decreased the amount of puromycin-incorporated neo-synthesized proteins, which indicated that compound 4
could inhibit protein synthesis. Consistently, compound 4 decreased the neo-synthesized proteins in a dose-dependent
manner in Hela cells from 1-10 µM.
Figure 4. Effects of compound 4 on protein synthesis in Hela cells. Cells were seeded into plates and treated with compound 4 at the
concentration indicated for 2 h, and then the cells were incubated with puromycin for 10 min. After washing, cells were lysed, and the
puromycin-incorporated neo-synthesized proteins were detected.

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Compound 4 showed good anti-tumor activity against the xenograft tumor of Hela cells in vivo
A Hela cell xenograft model was established using female nude mice to explore the effect of compound 4 on the
anti-tumor activity in vivo. Considering that the half-life of FA is short, we adopted a twice daily dosing regimen. The
results showed that compound 4 had satisfactory anti-tumor activity in this xenograft tumor model (Figure 5).
Compared with the control, the anti-tumor activity of compound 4 was obvious and the compound had no apparent
toxicity. We determined anti-tumor activity of compound 4 was dose-dependent and its toxicity was not increased.
Figure 5. Effects of compound 4 on the anti-tumor activity of Hela xenograft tumors in nude mice. Mice were inoculated with Hela cells and
treated with at the indicated concentrations at a dose of 25 mg/kg and 50 mg/kg. At the end of treatment, the mice were euthanized and the
tumors were removed and weighed. All data are expressed as the mean ± SD (n = 5). *p < 0.05, compared with the control group.
Table 3. Inhibitory effects of 4 on the xenograft tumor growth of Hela in nude micea
Body weight (g)
Initial
Dosage
(mg/kg)
0
Number
Initial/end
5/5
Groups
Tumor weight (g)
IR (%)
End
Control
19.7 ± 0.8
19.6 ± 1.0
20.2 ± 0.8
21.2 ± 1.5
20.4 ± 0.7
20.5 ± 1.0
1.85 ± 0.36
1.19 ± 0.17*
1.01 ± 0.32*
4
4
25
5/5
35.4
45.5
50
5/5
aData are expressed as the mean ± SD (n = 5). *P < 0.05, compared with control.
CONCLUSION
A series of novel FA derivatives were synthesis. All the synthesized derivatives were screened for anti-proliferative
activity in Hela, KBV, U87 and MKN45 cell lines using the MTT assay. This study demonstrates that introducing
amino acids and amino-terminal groups at the 3-OH position of FA generated compounds that exhibited good
anti-proliferative activity, but introducing carboxyl-terminal groups or nitrogen-containing heterocyclic groups had no

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obvious effect on the anti-proliferative activity. We found that compound 4 had the best anti-proliferative activity
among those tested. Additionally, compound 4 induced the Hela cells to undergo apoptosis by increasing the ratio of
cells in the Sub-G0/G1 phase as shown by the flow cytometry and TUNEL assay experiments. Compound 4 was also
shown to decrease the neo-synthesized proteins in a dose-dependent manner in Hela cells from 1-10 µM. Moreover,
compound 4 shows good anti-tumor activity against a xenograft tumor of Hela cells in vivo. Based on these results,
work is ongoing to design and synthesize novel FA derivatives with the introduction of medium-length amino-terminal
groups to obtain derivatives with better anti-tumor activity.
EXPERIMENTAL SECTION
Chemistry
The chemicals and reagents were analytically pure or dried with standard methods when necessary. The progress of
all reactions were monitored by TLC on silica gel HSGF254 using fluorescence with a wavelength of 254 nm on a
ZF7-C three-purpose UV analyzer and 10% ethanol sulfate solution for detection of the spots. All the synthesized
compounds were purified by column chromatography on silica gel. All NMR spectra (1H and 13C NMR) were
routinely measured using Bruker av400 or JNM-ECZS 400 instruments using CDCl3 as solvent with TMS as the
internal standard. Chemical shifts are expressed in δ values (ppm) and the coupling constants (J) in Hertz. High
resolution mass spectra (HRMS) were recorded on Agilent QTOF 6520 or 6530 spectrometer. Melting points of
compounds were recorded on Micro melting point apparatus XT3A or SGW®X-4.
General procedure for the synthesis of 1-3
Synthetic method of X1
FA (10.0 g, 19.0 mmol) was dissolved in acetone (200.0 mL), potassium carbonate (5.4 g, 39.0 mmol) and benzyl
bromide (2.8 mL, 23.0 mmol) were added with stirring, and reacted at 30 °C for 6 hours. It was filtered, concentrated,
diluted with ethyl acetate (50.0 mL), washed successively with 10% hydrochloric acid, water and saturated brine, dried
over anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure. The residue was
purified by silica gel column chromatography to provide white solid X1.
Synthetic method of intermediates a-c
X1 (0.1 mmol) was dissolved in anhydrous dichloromethane (10.0 mL) and tert-butyloxy carbonyl protected amino
acids (0.2 mmol), DMAP (0.3 mmol) and EDCI (0.3 mmol) were added with stirring, reacted at room temperature for

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4 hours. It was washed successively with 10% hydrochloric acid, water and saturated brine, dried over anhydrous
sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel
column chromatography (Vchloroform: Vmethanol = 300:1-200:1) to give intermediates a-c, respectively.
3β-[(2-amino)acetoxy]-21-fusidic acid (benzyl) ester (1)
a (45.0 mg, 59.0 µmol) was dissolved in ethyl acetate (10.0 mL) and 37% hydrochloric acid was added dropwise,
reacted at room temperature for 5 hours. It was washed successively with saturated sodium bicarbonate solution, water
and saturated brine, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced
pressure. The residue was purified by silica gel column chromatography (Vchloroform: Vmethanol = 60:1) to give white solid
1 (31.0 mg, 80.3% yield). m.p. 237-239 °C. 1H-NMR (CDCl3, 400 MHz) δ: 7.31-7.36 (m, 5H, 5×Ar-H), 5.89 (d, J =
8.20 Hz, 1H, 16-H), 5.22 (dd, J = 5.17, 12.14 Hz, 1H, CHAr), 5.06 (t, J = 6.91 Hz, 1H, 24-H), 4.99 (d, J = 2.11 Hz, 1H,
11-OH), 4.92 (dd, J = 4.38, 12.18 Hz, 1H, CHAr), 4.65 (d, J = 23.65 Hz, 2H, -NH2), 4.31 (s, 1H, 11-H), 3.47 (s, 1H,
3-H), 3.02 (d, J = 11.43 Hz, 1H, 13-H), 2.38-2.48 (m, 2H, 2×22-H), 2.23-2.30 (m, 1H, 12-H), 2.01-2.20 (m, 5H, 1-H,
5-H, 15-H and 2×23-H), 1.93 (s, 3H, OCOCH3), 1.68-1.88 (m, 4H, 2×2-H, 7-H and 12-H), 1.63 (s, 3H, 27-CH3),
1.54-1.60 (m, 3H, 1-H, 6-H and 9-H), 1.52 (s, 3H, 26-CH3), 1.40-1.48 (m, 1H, 4-H), 1.35 (s, 3H, 30-CH3), 1.25-1.32
(m, 1H, 15-H), 1.04-1.18 (m, 2H, 6-H and 7-H), 0.98 (s, 3H, 19-CH3), 0.91 (s, 3H, 18-CH3), 0.82 (d, J = 6.68 Hz, 3H,
28-CH3). 13C-NMR (CDCl3, 100 MHz) δ: 175.20, 171.97, 171.39, 149.93, 137.19, 134.03, 131.88, 130.03, 129.97,
129.74, 124.49, 111.68, 76.60, 75.86, 69.55, 67.88, 50.48, 50.16, 45.39, 40.87, 40.47, 39.11, 38.81, 38.30, 37.34, 36.36,
33.98, 32.34, 31.17, 30.47, 29.82, 28.80, 27.20, 25.75, 24.13, 22.43, 22.07, 19.43, 19.20, 17.10. HR-MS (ESI) m/z:
calcd. for C40H57NO7 [M+H]+: 664.4208, found: 664.4221.
3β-[(2-amino)propionyloxy]-21-fusidic acid (benzyl) ester (2)
According to the synthesis method of 1, b was reacted with 37% hydrochloric acid to give white solid 2 (46.0 mg,
86.7% yield). m.p. 232-235 °C. 1H-NMR (CDCl3, 400 MHz) δ: 7.29-7.35 (m, 5H, 5×Ar-H), 5.88 (d, J = 8.27 Hz, 1H,
16-H), 5.20 (d, J = 12.18 Hz, 1H, CHAr), 5.05 (t, J = 7.16 Hz, 1H, 24-H), 4.91 (d, J = 2.46 Hz, 1H, 11-OH), 4.92 (d, J
= 12.14 Hz, 1H, CHAr), 4.64 (d, J = 24.20 Hz, 2H, -NH2), 4.31 (s, 1H, 11-H), 3.59 (q, J = 6.38 Hz, 1H, -CH-), 3.01 (d,
J = 11.13 Hz, 1H, 13-H), 2.38-2.49 (m, 2H, 2×22-H), 2.24-2.28 (m, 1H, 12-H), 2.05-2.19 (m, 5H, 1-H, 5-H, 15-H and
2×23-H), 1.92 (s, 3H, OCOCH3), 1.67-1.89 (m, 4H, 2×2-H, 7-H and 12-H), 1.62 (s, 3H, 27-CH3), 1.54-1.60 (m, 3H,
1-H, 6-H and 9-H), 1.51 (s, 3H, 26-CH3), 1.47 (s, 1H, 4-H), 1.36 (s, 3H, -CH3), 1.35 (s, 3H, 30-CH3), 1.24-1.31 (m, 1H,

ACCEPTED MANUSCRIPT
15-H), 1.09-1.17 (m, 2H, 6-H and 7-H), 0.97 (s, 3H, 19-CH3), 0.91 (s, 3H, 18-CH3), 0.83 (d, J = 6.69 Hz, 3H, 28-CH3).
13C-NMR (CDCl3, 100 MHz) δ: 177.50, 171.97, 171.38, 149.90, 137.20, 134.06, 132.07, 131.92, 130.04, 129.98,
129.75, 124.47, 111.69, 76.58, 75.85, 69.62, 67.90, 51.65, 50.44, 50.21, 45.39, 40.92, 40.48, 39.18, 38.33, 37.38, 36.29,
34.07, 32.31, 31.18, 30.47, 29.81, 28.67, 27.20, 25.74, 24.12, 22.44, 22.08, 19.42, 19.21, 17.19. HR-MS (ESI) m/z:
calcd. for C41H59NO7 [M+H]+: 678.4364, found: 678.4376.
3β-[(2-amino-4-methyl)pentanoyloxy]-21-fusidic acid (benzyl) ester (3)
According to the synthesis method of 1, c was reacted with 37% hydrochloric acid to give white solid 3 (28.0 mg,
79.4% yield). m.p. 248-250 °C. 1H-NMR (CDCl3, 400 MHz) δ: 7.30-7.36 (m, 5H, 5×Ar-H), 5.88 (d, J = 8.26 Hz, 1H,
16-H), 5.20 (d, J = 12.21 Hz, 1H, CHAr), 5.06 (t, J = 6.94 Hz, 1H, 24-H), 4.91-4.94 (m, 2H, CHAr and 11-OH), 4.64
(d, J = 23.61 Hz, 2H, -NH2), 4.31 (s, 1H, 11-H), 3.49 (t, J = 7.43 Hz, 1H, -CH-), 3.02 (d, J = 11.58 Hz, 1H, 13-H),
2.38-2.52 (m, 2H, 2×22-H), 2.26-2.29 (m, 1H, 12-H), 2.07-2.20 (m, 3H, 15-H and 2×23-H), 1.96-2.04 (m, 2H, 1-H and
5-H), 1.92 (s, 3H, OCOCH3), 1.68-1.88 (m, 4H, 2×2-H, 7-H and 12-H), 1.63 (s, 3H, 27-CH3), 1.54-1.61 (m, 3H, 1-H,
6-H and 9-H), 1.52 (s, 3H, 26-CH3), 1.38-1.46 (m, 1H, 4-H), 1.36 (s, 3H, 30-CH3), 1.25-1.33 (m, 1H, 15-H), 1.03-1.17
(m, 2H, 6-H and 7-H), 0.98 (s, 3H, 19-CH3), 0.94 (s, 3H, -CH3), 0.93 (d, J = 1.78 Hz, 3H, 18-CH3), 0.91 (s, 3H, -CH3),
0.83 (d, J = 6.66 Hz, 3H, 28-CH3). 13C-NMR (CDCl3, 100 MHz) δ: 177.58, 171.94, 171.36, 149.95, 137.21, 134.01,
131.88, 130.02, 129.94, 129.72, 124.49, 111.68, 76.60, 75.85, 69.55, 67.86, 54.45, 50.49, 50.19, 46.06, 45.39, 40.92,
40.50, 39.07, 38.30, 37.38, 36.30, 34.02, 32.29, 31.17, 30.46, 29.82, 28.61, 27.19, 26.47, 25.62, 24.44, 24.17, 23.54,
22.43, 22.06, 19.43, 19.20, 17.23. HR-MS (ESI) m/z: calcd. for C44H65NO7 [M+H]+: 720.4834, found: 720.4849.
General procedure for the synthesis of 4-8
Synthetic method of X7-X11
Aqueous sodium hydroxide (sodium hydroxide: 170.0 mg, 4.3 mmol; water: 2.0 mL) and di-tert-butyl dicarbonate
were dissolved in tert-butyl alcohol and X2-X6 (3.8 mmol) were added with stirring respectively, reacted at room
temperature for 18-24 hours. It was diluted with water and 1.0 mol/L hydrochloric acid, extracted with ethyl acetate
rapidly, washed successively with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and the
solvent was evaporated under reduced pressure. Then it gave intermediates X7-X11, respectively.
Synthetic method of intermediates d-h
X1 (0.1 mmol) was dissolved in anhydrous dichloromethane (8.0 mL) and compounds X10-X14 (0.2 mmol), DMAP

ACCEPTED MANUSCRIPT
(0.3 mmol) and EDCI (0.3 mmol) were added with stirring respectively, reacted at room temperature for 20-24 hours.
It was filtered, concentrated, diluted with ethyl acetate (10.0 mL), washed successively with water and saturated brine,
dried over anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure. The residue was
purified by silica gel column chromatography (Vpetroleum ether: Vethyl
respectively.
= 20:1-8:1) to give intermediates d-h,
acetate
3β-(4-aminopropionyloxy)-21-fusidic acid (benzyl) ester (4)
d (35.0 mg, 45.0 µmol) was dissolved in anhydrous dichloromethane (10.0 mL) and trifluoroacetic acid (0.9 ml)
was added at 0 °C, reacted at room temperature for 3 hours. The solvent was evaporated under reduced pressure. The
residue was purified by silica gel column chromatography (Vdichloromethane: Vmethanol = 100:1-50:1) to give white solid 4
1
(26.0 mg, 85.0% yield). m.p. 207-210 °C. H-NMR (CDCl3, 400 MHz) δ: 7.31-7.34 (m, 5H, 5×Ar-H), 5.89 (d, J =
8.24 Hz, 1H, 16-H), 5.20 (d, J = 12.11 Hz, 1H, CHAr), 4.94 (d, J = 12.08 Hz, 1H, CHAr), 4.33 (s, 1H, 11-H), 3.30 (s,
1H, 3-H), 3.01 (d, J = 11.51 Hz, 1H, 13-H), 4.47 (m, 2H, -CH2-), 4.33 (s, 1H, 11-H), 4.21-4.24 (m, 4H, 2×-CH2-), 2.80
(t, J = 7.12 Hz, 2H, -CH2-), 2.31-2.42 (m, 3H, 12-H and 2×22-H), 2.13-2.23 (m, 3H, 15-H and 2×23-H), 2.00-2.09 (m,
2H, 1-H and 5-H), 1.93 (s, 3H, OCOCH3), 1.66-1.87 (m, 3H, 2×2-H, 7-H and 12-H), 1.51-1.62 (m, 4H, 1-H, 4-H, 6-H
and 9-H), 1.47 (s, 3H, 27-CH3), 1.43 (s, 3H, 26-CH3), 1.32 (s, 3H, 30-CH3), 1.26-1.27 (m, 1H, 15-H), 1.07-1.16 (m,
2H, 6-H and 7-H), 0.97 (s, 3H, 19-CH3), 0.90 (s, 3H, 18-CH3), 0.80 (d, J = 6.39 Hz, 3H, 28-CH3). 13C-NMR (CDCl3,
100 MHz) δ: 170.88, 170.09, 169.27, 148.87, 135.13, 129.31, 128.09, 128.01, 127.93, 127.86, 88.59, 75.88, 73.82,
67.44, 65.90, 48.40, 48.21, 45.55, 43.43, 39.37, 38.90, 38.40, 36.90, 36.08, 35.35, 34.42, 31.51, 30.41, 30.04, 28.37,
26.43, 25.14, 24.68, 23.90, 23.34, 22.26, 20.44, 20.17, 17.26, 14.97, 7.95. HR-MS (ESI) m/z: calcd. for C41H59NO7
[M+H]+: 678.4364, found: 678.4348.
3β-(4-aminobutyryloxy)-21-fusidic acid (benzyl) ester (5)
According to the synthesis method of 4, e was reacted with trifluoroacetic acid to give white solid 5 (29.0 mg,
73.6% yield). m.p. 210-212 °C. 1H-NMR (CDCl3, 400 MHz) δ: 7.31-7.35 (m, 5H, 5×Ar-H), 5.88 (d, J = 8.32 Hz, 1H,
16-H), 5.20 (d, J = 12.13 Hz, 1H, CHAr), 4.95 (d, J = 12.21 Hz, 1H, CHAr), 4.93 (s, 1H, 11-OH), 4.36 (s, 1H, 11-H),
3.53 (t, J = 7.13 Hz, 2H, -CH2-), 3.13 (s, 1H, 3-H), 3.00 (d, J = 11.69 Hz, 1H, 13-H), 2.45-2.54 (m, 2H, 2×22-H),
2.31-2.44 (m, 1H, 12-H), 2.12-2.24 (m, 3H, 15-H and 2×23-H), 2.02-2.10 (m, 2H, 1-H and 5-H), 1.93 (s, 3H,
OCOCH3), 1.62-1.88 (m, 6H, 2×2-H, 6-H, 7-H, 9-H and 12-H), 1.50-1.58 (m, 2H, 1-H and 4-H), 1.47 (s, 3H, 27-CH3),

ACCEPTED MANUSCRIPT
1.43 (s, 3H, 26-CH3), 1.33 (s, 3H, 30-CH3), 1.27-1.31 (m, 1H, 15-H), 1.09-1.17 (m, 2H, 6-H and 7-H), 0.98 (s, 3H,
19-CH3), 0.90 (s, 3H, 18-CH3), 0.80 (d, J = 6.56 Hz, 3H, 28-CH3). 13C-NMR (CDCl3, 100 MHz) δ: 172.68, 170.70,
169.83, 149.25, 135.63, 129.86, 128.60, 128.38, 89.12, 75.63, 74.31, 68.07, 66.43, 49.03, 48.68, 46.14, 43.90, 43.42,
39.89, 39.62, 39.46, 38.90, 37.09, 36.49, 35.91, 35.25, 31.62, 30.43, 30.21, 28.84, 26.97, 25.61, 25.12, 24.40, 23.49,
23.18, 22.74, 20.94, 20.21, 17.61, 15.54, 8.45. HR-MS (ESI) m/z: calcd. for C42H61NO7 [M+H]+: 692.4521, found:
692.4505.
3β-(8-aminooctanoyloxy)-21-fusidic acid (benzyl) ester (6)
According to the synthesis method of 4, f was reacted with trifluoroacetic acid to give white solid 6 (25.0 mg, 81.1%
yield). m.p. 219-221 °C. 1H-NMR (CDCl3, 400 MHz) δ: 8.37 (s, 2H, -NH2), 7.32-7.34 (m, 5H, 5×Ar-H), 5.89 (d, J =
8.26 Hz, 1H, 16-H), 5.18 (d, J = 12.13 Hz, 1H, CHAr), 4.97 (d, J = 12.14 Hz, 1H, CHAr), 4.93 (d, J = 2.03 Hz, 1H,
11-OH), 4.36 (s, 1H, 11-H), 3.12 (q, J = 7.33 Hz, 2H, -CH2-), 2.99-3.01 (m, 3H, 13-H and -CH2-), 2.31-2.46 (m, 5H,
12-H, -CH2- and 2×22-H), 2.00-2.25 (m, 5H, 1-H, 5-H, 15-H and 2×23-H), 1.93 (s, 3H, OCOCH3), 1.72-1.87 (m, 4H,
2×2-H, 7-H and 12-H), 1.51-1.70 (m, 4H, 1-H, 4-H, 6-H and 9-H), 1.47 (s, 3H, 27-CH3), 1.44 (s, 3H, 26-CH3), 1.34 (s,
3H, 30-CH3), 1.26-1.33 (m, 1H, 15-H), 1.04-1.18 (m, 2H, 6-H and 7-H), 0.98 (s, 3H, 19-CH3), 0.91 (s, 3H, 18-CH3),
0.81 (d, J = 6.58 Hz, 3H, 28-CH3). 13C-NMR (CDCl3, 100 MHz) δ: 173.90, 170.73, 169.78, 161.59, 161.22, 156.39,
155.98, 149.67, 135.62, 129.85, 128.60, 128.49, 128.39, 123.00, 112.97, 89.12, 74.33, 68.03, 66.43, 48.84, 48.77,
46.08, 44.01, 40.08, 39.80, 39.38, 37.90, 36.91, 34.65, 34.62, 32.88, 28.60, 28.47, 27.26, 25.91, 25.68, 25.24, 24.82,
24.40, 24.37, 22.31, 20.94, 20.49, 18.01, 15.61, 8.46. HR-MS (ESI) m/z: calcd. for C46H69NO7 [M+H]+: 748.5147,
found: 748.5131.
3β-(11-aminoundecyloxy)-21-fusidic acid (benzyl) ester (7)
According to the synthesis method of 4, g was reacted with trifluoroacetic acid to give white solid 7 (27.0 mg,
1
78.8% yield). m.p. 223-225 °C. H-NMR (CDCl3, 400 MHz) δ: 7.32-7.34 (m, 5H, 5×Ar-H), 6.17 (s, 2H, -NH2), 5.88
(d, J = 8.25 Hz, 1H, 16-H), 5.18 (d, J = 12.15 Hz, 1H, CHAr), 4.95-4.98 (m, 2H, 11-OH and CHAr), 4.33 (s, 1H,
11-H), 2.97-3.05 (m, 3H, 13-H and -CH2-), 2.39-2.45 (m, 3H, 12-H and 2×22-H), 2.33 (t, J = 7.46 Hz, 2H, -CH2-),
2.04-2.24 (m, 5H, 1-H, 5-H, 15-H and 2×23-H), 1.92 (s, 3H, OCOCH3), 1.78-1.86 (m, 4H, 2×2-H, 7-H and 12-H),
1.52-1.75 (m, 4H, 1-H, 4-H, 6-H and 9-H), 1.48 (s, 3H, 27-CH3), 1.44 (s, 3H, 26-CH3), 1.36 (s, 3H, 30-CH3),
1.26-1.30 (m, 1H, 15-H), 1.03-1.18 (m, 2H, 6-H and 7-H), 0.98 (s, 3H, 19-CH3), 0.91 (s, 3H, 18-CH3), 0.82 (d, J =

ACCEPTED MANUSCRIPT
6.58 Hz, 3H, 28-CH3). 13C-NMR (CDCl3, 100 MHz) δ: 173.74, 171.05, 169.70, 156.39, 155.98, 149.76, 135.59,
128.89, 128.61, 128.48, 128.41, 115.81, 112.96, 89.04, 74.39, 74.13, 68.05, 66.46, 48.77, 44.05, 40.21, 39.78, 39.36,
38.92, 38.03, 37.01, 35.64, 35.00, 34.53, 33.24, 31.03, 29.40, 29.25, 29.16, 29.11, 28.79, 27.55, 27.40, 26.41, 25.69,
25.29, 25.12, 24.71, 24.34, 22.12, 20.91, 20.37, 18.08, 15.70. HR-MS (ESI) m/z: calcd. for C49H75NO7 [M+H]+:
790.5616, found: 790.5603.
3β-(L-lysinyloxy)-21-fusidic acid (benzyl) ester (8)
According to the synthesis method of 4, h was reacted with trifluoroacetic acid to give white solid 8 (28.0 mg,
80.5% yield). m.p. 221-222 °C. 1H-NMR (CDCl3, 400 MHz) δ: 8.53 (s, 2H, -NH2), 8.19 (s, 2H, -NH2), 7.29-7.33 (m,
5H, 5×Ar-H), 5.86 (d, J = 6.38 Hz, 1H, 16-H), 5.16 (d, J = 12.14 Hz, 1H, CHAr), 4.94-4.99 (m, 2H, 11-OH and CHAr),
4.31 (s, 1H, 11-H), 2.97-3.04 (m, 3H, 13-H and -CH2-), 2.39-2.41 (m, 2H, 2×22-H), 2.20-2.28 (m, 1H, 12-H),
2.03-2.12 (m, 5H, 1-H, 5-H, 15-H and 2×23-H), 1.90 (s, 3H, OCOCH3), 1.70-1.84 (m, 4H, 2×2-H, 7-H and 12-H),
1.54-1.62 (m, 4H, 1-H, 4-H, 6-H and 9-H), 1.45 (s, 3H, 27-CH3), 1.42 (s, 3H, 26-CH3), 1.31 (s, 3H, 30-CH3),
1.26-1.30 (m, 1H, 15-H), 1.05-1.13 (m, 2H, 6-H and 7-H), 0.97 (s, 3H, 19-CH3), 0.89 (s, 3H, 18-CH3), 0.80 (d, J =
4.30 Hz, 3H, 28-CH3). 13C-NMR (CDCl3, 100 MHz) δ: 170.80, 169.91, 169.13, 161.43, 156.37, 155.96, 149.66,
135.66, 129.81, 128.59, 128.39, 128.34, 117.48, 115.84, 114.59, 112.98, 89.24, 78.76, 74.28, 67.67, 67.09, 66.37,
52.69, 48.97, 48.66, 46.08, 43.86, 39.86, 39.34, 38.86, 36.42, 34.94, 31.64, 28.83, 28.23, 25.58, 25.02, 24.60, 23.64,
22.89, 20.90, 17.75, 15.27, 8.44. HR-MS (ESI) m/z: calcd. for C44H66N2O7 [M+H]+: 735.4943, found: 735.4931.
General procedure for the synthesis of 9-11
4-[21-fusidic acid (benzyl) ester-3β-oxy]-4-oxo-butyric acid (9)
X1 (420.0 mg, 0.7 mmol) was dissolved in anhydrous dichloromethane (20.0 mL), Succinic anhydride (346.0 mg,
3.5 mmol) and DMAP (254.0 mg, 2.1 mmol) were added with stirring respectively, reacted at room temperature for 10
hours. It was washed successively with 10% hydrochloric acid, water and saturated brine, dried over anhydrous
sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel
column chromatography (Vchloroform: Vmethanol = 150:1) to give white solid 9 (397.0 mg, 81.1% yield). m.p. 217-220 °C.
1H-NMR (CDCl3, 400 MHz) δ: 7.28-7.36 (m, 5H, 5×Ar-H), 5.88 (d, J = 8.36 Hz, 1H, 16-H), 5.21 (d, J = 12.20 Hz, 1H,
CHAr), 5.06 (t, J = 7.15 Hz, 1H, 24-H), 4.93 (d, J = 12.14 Hz, 1H, CHAr), 4.92 (d, J = 2.34 Hz, 1H, 11-OH), 4.38 (s,
1H, 11-H), 3.04 (d, J = 11.30 Hz, 1H, 13-H), 2.52-2.78 (m, 4H, 2×-CH2-), 2.38-2.48 (m, 2H, 2×22-H), 2.28-2.31 (m,

ACCEPTED MANUSCRIPT
1H, 12-H), 2.10-2.22 (m, 3H, 15-H and 2×23-H), 1.96-2.04 (m, 2H, 1-H and 5-H), 1.93 (s, 3H, OCOCH3), 1.74-1.77
(m, 2H, 2-H and 12-H), 1.66-1.72 (m, 2H, 2-H and 7-H), 1.63 (s, 3H, 27-CH3), 1.52 (s, 3H, 26-CH3), 1.39-1.48 (m, 4H,
1-H, 4-H, 6-H and 9-H), 1.37 (s, 3H, 30-CH3), 1.27-1.27 (m, 1H, 15-H), 1.08-1.16 (m, 2H, 6-H and 7-H), 0.98 (s, 3H,
19-CH3), 0.92 (s, 3H, 18-CH3), 0.82 (d, J = 6.66 Hz, 3H, 28-CH3). 13C-NMR (CDCl3, 100 MHz) δ: 175.53, 170.96,
170.04, 169.48, 147.73, 135.21, 132.09, 129.81, 128.06, 127.98, 127.76, 122.49, 74.50, 73.86, 68.14, 65.92, 48.63,
48.11, 43.43, 39.02, 38.42, 36.11, 35.84, 35.39, 35.09, 30.62, 29.84, 29.79, 29.58, 29.21, 28.64, 28.48, 27.91, 26.38,
25.23, 23.29, 22.44, 20.70, 20.45, 17.22, 16.93, 15.07. HR-MS (ESI) m/z: calcd. for C42H58O9 [M+Na]+: 729.39785,
found: 729.39569.
2-[21-fusidic acid (benzyl) ester-3β-oxy]-2-oxo-benzoic acid (10)
According to the synthesis method of 9, X1 was reacted with o-phthalic anhydride to give white solid 10 (410.0 mg,
1
73.2% yield). m.p. 227-228 °C. H-NMR (CDCl3, 400 MHz) δ: 7.97 (d, J = 7.40 Hz, 1H, Ar-H), 7.47-7.56 (m, 3H,
3×Ar-H), 7.30-7.36 (m, 5H, 5×Ar-H), 5.86 (d, J = 8.39 Hz, 1H, 16-H), 5.26 (d, J = 2.12 Hz, 1H, 11-OH), 5.20 (d, J =
12.20 Hz, 1H, CHAr), 5.04 (t, J = 7.10 Hz, 1H, 24-H), 4.93 (d, J = 12.19 Hz, 1H, CHAr), 4.53 (s, 1H, 11-H), 3.05 (d, J
= 10.89 Hz, 1H, 13-H), 2.41-2.44 (m, 1H, 22-H), 2.24-2.34 (m, 2H, 12-H and 22-H), 2.17-2.22 (m, 1H, 23-H),
2.04-2.13 (m, 2H, 5-H and 23-H), 1.96-2.02 (m, 2H, 1-H and 5-H), 1.92 (s, 3H, OCOCH3), 1.80-1.90 (m, 2H, 2-H and
12-H), 1.73-1.75 (m, 2H, 2-H and 7-H), 1.65-1.68 (m, 4H, 1-H, 4-H, 6-H and 9-H), 1.63 (s, 3H, 27-CH3), 1.51 (s, 3H,
26-CH3), 1.40 (s, 3H, 30-CH3), 1.26-1.29 (m, 1H, 15-H), 1.08-1.13 (m, 2H, 6-H and 7-H), 0.94 (s, 3H, 19-CH3), 0.93
(s, 3H, 18-CH3), 0.81 (d, J = 6.66 Hz, 3H, 28-CH3). 13C-NMR (CDCl3, 100 MHz) δ: 170.63, 169.98, 169.48, 165.76,
147.27, 135.17, 133.64, 132.17, 131.28, 130.14, 129.78, 129.43, 128.74, 128.08, 128.00, 127.79, 126.56, 122.39,
75.77, 73.74, 69.17, 65.95, 48.66, 48.09, 43.40, 39.24, 38.36, 38.20, 36.24, 35.73, 35.34, 34.85, 29.80, 29.26, 29.22,
28.44, 27.94, 26.40, 25.23, 24.47, 21.40, 21.12, 20.45, 17.23, 16.36, 14.88. HR-MS (ESI) m/z: calcd. for C46H58O9
[M+Na]+: 777.39785, found: 777.39504.
2-[21-fusidic acid (benzyl) ester-3β-oxy]-2-oxo-pyrazine acid (11)
According to the synthesis method of 9, X1 was reacted with 2,3-pyrazinedicarboxylic anhydride to give white solid
1
11 (250.0 mg, 83.7% yield). m.p. 232-234 °C. H-NMR (CDCl3, 400 MHz) δ: 8.69-8.72 (m, 2H, 2×Ar-H), 7.32-7.36
(m, 5H, 5×Ar-H), 5.84 (d, J = 7.76 Hz, 1H, 16-H), 5.71 (s, 1H, 11-OH), 5.23 (s, 1H, 11-H), 5.17 (d, J = 12.07 Hz, 1H,
CHAr), 5.02 (t, J = 7.11 Hz, 1H, 24-H), 4.94 (d, J = 12.12 Hz, 1H, CHAr), 3.18 (s, 1H, 3-H), 2.95 (d, J = 10.98 Hz, 1H,

ACCEPTED MANUSCRIPT
13-H), 2.63-2.66 (m, 3H, 12-H and 2×22-H), 2.36-2.46 (m, 5H, 1-H, 5-H, 15-H and 2×23-H), 2.05-2.11 (m, 4H,
2×2-H, 7-H and 12-H), 1.92 (s, 3H, OCOCH3), 1.79-1.83 (m, 4H, 1-H, 4-H, 6-H and 9-H), 1.54 (s, 3H, 27-CH3), 1.43
(s, 3H, 26-CH3), 1.26-1.31 (m, 1H, 15-H), 1.19 (s, 3H, 30-CH3), 1.11-1.14 (m, 2H, 6-H and 7-H), 1.05 (s, 3H, 19-CH3),
0.96 (s, 3H, 18-CH3), 0.87 (d, J = 4.40 Hz, 3H, 28-CH3). 13C-NMR (CDCl3, 100 MHz) δ: 170.55, 169.98, 166.73,
165.01, 164.53, 147.50, 146.13, 145.92, 144.82, 144.57, 135.57, 132.71, 131.01, 128.59, 128.35, 122.92, 77.75, 74.22,
73.94, 66.52, 39.35, 38.84, 37.84, 37.74, 36.72, 34.63, 32.81, 32.24, 32.13, 30.09, 30.02, 29.69, 28.99, 28.20, 26.35,
25.61, 24.16, 24.11, 22.36, 20.92, 20.29, 18.19, 17.58, 15.38. HR-MS (ESI) m/z: calcd. for C44H56N2O9 [M+H]+:
755.3913, found: 755.3941.
General procedure for the synthesis of 12-18
4-[21-fusidic acid (benzyl) ester-3β-oxy]-4-oxo-butyrylmorpholine (12)
9 (50.0 mg, 70.0 µmol) was dissolved in anhydrous dichloromethane, morpholine (12.0 µL, 140.0 µmol) and EDCI
(21.0 mg, 110.0 µmol) were added with stirring, reacted at room temperature for 6 hours. It was washed successively
with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under
reduced pressure. The residue was purified by silica gel column chromatography (Vchloroform: Vmethanol = 150:1) to give
white solid 12 (41.0 mg, 74.7%). m.p. 227-229 °C. 1H-NMR (CDCl3, 400 MHz) δ: 7.45-7.32 (m, 5H, 5×Ar-H), 5.92 (d,
J = 8.40 Hz, 1H, 16-H), 5.25 (d, J = 12.20 Hz, 1H, CHAr), 5.10 (t, J = 7.20 Hz, 1H, 24-H), 5.00-4.93 (m, 2H, CHAr
and 11-OH), 4.35 (s, 1H, 11-H), 3.75-3.47 (m, 8H, mor-H), 3.06 (d, J = 11.50 Hz, 1H, 13-H), 2.77-2.60 (m, 4H,
2×-CH2-), 2.56-2.42 (m, 2H, 2×22-H), 2.37-2.28 (m, 1H, 12-H), 2.26-2.00 (m, 5H,15-H, 2×23-H, 1-H and 5-H), 1.97
(s, 3H, OCOCH3), 1.93-1.76 (m, 4H, 2×2-H, 12-H and 7-H), 1.67 (s, 3H, 27-CH3), 1.65-1.57 (m, 4H, 1-H, 4-H, 6-H
and 9-H), 1.56 (s, 3H, 26-CH3),1.39 (s, 3H, 30-CH3), 1.36-1.27 (m, 3H,15-H, 6-H and 7-H), 1.01 (s, 3H, 19-CH3), 0.95
(s, 3H, 18-CH3), 0.86 (d, J = 6.70 Hz, 3H, 28-CH3). 13C-NMR (CDCl3, 100 MHz) δ: 172.64, 170.52, 170.02, 169.98,
148.50, 135.83, 132.59, 130.48, 128.62, 128.55, 128.31, 123.12, 74.59, 74.47, 68.18, 66.92, 66.59, 66.46, 49.19, 48.77,
45.78, 44.02, 42.15, 39.51, 39.10, 37.71, 36.90, 35.83, 34.99, 32.61, 30.99, 30.64, 29.76, 29.71, 29.08, 28.41, 28.03,
27.28, 25.79, 24.38, 22.78, 21.02, 20.68, 18.03, 17.79, 15.69. HR-MS (ESI) m/z: calcd. for C46H65NO9 [M+Na]+:
798.4552, found: 798.4558.
4-[21-fusidic acid (benzyl) ester-3β-oxy]-4-oxo-butyryl(4ʼ-methyl)piperazine (13)
According to the synthesis method of 12, 9 was reacted with N-methyl piperazine to give white solid 13 (45.0 mg,

ACCEPTED MANUSCRIPT
80.6% yield). m.p. 225-227 °C. 1H-NMR (CDCl3, 400 MHz) δ: 7.43-7.31 (m, 5H, 5×Ar-H), 5.90 (d, J = 8.40 Hz, 1H,
16-H), 5.23 (d, J = 12.20 Hz, 1H, CHAr), 5.08 (t, J = 7.10 Hz, 1H, 24-H), 4.98-4.91 (m, 2H, CHAr and 11-OH), 4.33
(s, 1H, 11-H), 3.73-3.48 (m, 4H, CON(CH2)2), 3.04 (d, J = 11.60 Hz, 1H, 13-H), 2.75-2.61 (m, 4H, 2×-CH2-),
2.51-2.35 (m, 7H, N(CH2)2, 2×22-H and 12-H), 2.32 (s, 3H, N-CH3), 2.25-1.98 (m, 6H, 15-H, 2×23-H, 1-H, 5-H and
12-H), 1.95 (s, 3H, OCOCH3), 1.90-1.67 (m, 6H, 2×2-H, 1-H, 4-H, 6-H and 7-H), 1.65 (s, 3H, 27-CH3), 1.63-1.58 (m,
1H, 9-H), 1.54 (s, 3H, 26-CH3), 1.37 (s, 3H, 30-CH3), 1.35-1.08 (m, 3H, 15-H, 6-H and 7-H), 0.98 (s, 3H, 19-CH3),
0.92 (s, 3H, 18-CH3), 0.83 (d, J = 6.70 Hz, 3H, 28-CH3). 13C-NMR (CDCl3, 100 MHz) δ: 174.01, 171.86, 171.32,
171.07, 149.87, 137.19, 133.93, 131.83, 129.96, 129.89, 129.65, 124.48, 75.82, 69.50, 67.79, 56.81, 56.39, 56.06,
55.64, 54.84, 50.56, 50.11, 47.42, 46.50, 45.37, 43.02, 40.86, 40.45, 39.05, 38.25, 37.17, 36.34, 33.94, 32.33, 31.17,
30.42, 29.75, 29.55, 28.61, 27.12, 25.71, 24.13, 22.36, 22.03, 19.37, 19.13, 17.02. HR-MS (ESI) m/z: calcd. for
C47H68N2O8 [M+H]+: 789.5049, found: 789.5049.
4-[21-fusidic acid (benzyl) ester-3β-oxy]-4-oxo-butyrylpyrazole (14)
According to the synthesis method of 12, 9 was reacted with pyrazole to give white solid 14 (40.0 mg, 74.7% yield).
m.p. 233-236 °C. 1H-NMR (CDCl3, 400 MHz) δ: 8.26 (d, J = 2.60 Hz, 1H, pyr-H), 7.73 (s, 1H, pyr-H), 7.41-7.30 (m,
5H, 5×Ar-H), 6.46 (dd, J = 2.8, 1.5 Hz, 1H, pyr-H), 5.90 (d, J = 8.30 Hz, 1H, 16-H), 5.23 (d, J = 12.20 Hz, 1H, CHAr),
5.08 (t, J = 7.20 Hz, 1H, 24-H), 4.99 (d, J = 2.40 Hz, 1H, 11-OH), 4.94 (d, J = 12.20 Hz, 1H, CHAr), 4.35 (s, 1H,
11-H), 3.54-3.47 (m, 2H, -CH2-), 3.02 (d, J = 11.10 Hz, 1H, 13-H), 2.88-2.81 (m, 2H,-CH2-), 2.56-2.40 (m, 2H,
2×22-H), 2.35-2.27 (m, 1H, 12-H), 2.23-2.00 (m, 5H, 15-H, 2×23-H, 1-H and 5-H), 1.95 (s, 3H, OCOCH3), 1.90-1.79
(m, 3H, 2×2-H and 12-H), 1.66 (s, 3H, 27-CH3), 1.63-1.56 (m, 1H, 7-H), 1.55 (s, 3H, 26-CH3), 1.28 (s, 3H, 30-CH3),
1.18-1.02 (m, 2H, 6-H and 7-H), 0.98 (s, 3H, 19-CH3), 0.92 (s, 3H, 18-CH3), 0.84 (d, J = 6.70 Hz, 3H, 28-CH3).
13C-NMR (CDCl3, 100 MHz) δ: 171.69, 171.06, 170.56, 170.00, 148.49, 144.24, 135.83, 132.61, 130.50, 128.64,
128.58, 128.44, 128.34, 123.12, 109.75, 74.90, 74.47, 68.24, 66.48, 49.16, 48.76, 44.02, 39.45, 39.08, 37.81, 36.92,
34.96, 32.60, 31.01, 30.65, 29.56, 29.09, 28.91, 28.41, 27.31, 25.79, 24.27, 22.75, 21.03, 20.67, 19.26, 18.00, 17.80,
15.64, 13.81. HR-MS (ESI) m/z: calcd. for C45H60N2O8 [M+Na]+: 779.4242, found: 779.4247.
2-[21-fusidic acid (benzyl) ester-3β-oxy]-2-oxo-benzoylmorpholine (15)
According to the synthesis method of 12, 10 was reacted with morpholine to give white solid 15 (44.0 mg, 80.6%
1
yield). m.p. 240-243 °C. H-NMR (CDCl3, 400 MHz) δ: 7.99 (d, J = 7.40 Hz, 1H, Ar-H), 7.60-7.52 (m, 1H, Ar-H),

ACCEPTED MANUSCRIPT
7.48-7.41 (m, 1H, Ar-H), 7.36-7.26 (m, 6H, Ar-H), 5.87 (d, J = 8.30 Hz, 1H, 16-H), 5.19 (m, 2H, 11-OH and CHAr),
5.03 (t, J = 7.20 Hz, 1H, 24-H), 4.92 (d, J = 12.20 Hz, 1H, CHAr), 4.32 (s, 1H, 11-H), 4.00-3.08 (m, 8H, mor-H), 3.04
(d, J = 11.20 Hz, 1H, 13-H), 2.53-2.35 (m, 2H, 2×22-H), 2.33-1.92 (m, 6H, 1-H, 2×5-H, 12-H and 2×23-H), 1.90 (s,
3H, OCOCH3), 1.89-1.82 (m, 4H, 2×2-H, 7-H and 12-H), 1.81-1.63 (m, 4H, 1-H, 4-H, 6-H and 9-H), 1.61 (s, 3H,
27-CH3), 1.50 (s, 3H, 26-CH3), 1.41 (s, 3H, 30-CH3), 1.19-1.03 (m, 2H, 6-H and 7-H), 1.00 (s, 3H, 19-CH3), 0.91 (s,
3H, 18-CH3), 0.85 (d, J = 6.70 Hz, 3H, 28-CH3). 13C-NMR (CDCl3, 100 MHz) δ: 170.55, 170.02, 169.97, 165.03,
148.54, 135.86, 132.92, 132.60, 130.99, 130.49, 130.17, 129.09, 128.63, 128.54, 128.32, 128.27, 127.16, 123.12,
75.65, 74.47, 68.13, 66.63, 66.45, 66.36, 49.32, 48.81, 47.24, 44.05, 42.22, 39.57, 39.11, 36.91, 35.87, 32.00, 31.13,
30.65, 29.77, 29.73, 29.43, 29.08, 28.46, 27.49, 25.78, 22.77, 21.02, 20.82, 17.97, 17.80, 15.86, 14.20. HR-MS (ESI)
m/z: calcd. for C50H65NO9 [M+H]+: 824.4732, found: 824.4730.
2-[21-fusidic acid (benzyl) ester-3β-oxy]-2-oxo-benzoyl(4ʼ-methyl)piperazine (16)
According to the synthesis method of 12, 10 was reacted with N-methyl piperazine to give white solid 16 (45.0 mg,
1
81.2% yield). m.p. 235-238 °C. H-NMR (CDCl3, 400 MHz) δ: 7.99 (d, J = 7.80 Hz, 1H, Ar-H), 7.59-7.53 (m, 1H,
Ar-H), 7.48-7.42 (m, 1H, Ar-H), 7.36-7.26 (m, 6H, Ar-H), 5.88 (d, J = 8.30 Hz, 1H, 16-H), 5.25-5.15 (m, 2H, 11-OH
and CHAr), 5.05 (t, J = 7.20 Hz, 1H, 24-H), 4.93 (d, J = 12.20 Hz, 1H, CHAr), 4.34 (s, 1H, 11-H), 4.20-3.15 (m, 4H,
CON(CH2)2), 3.06 (d, J = 11.50 Hz, 1H, 13-H), 2.55-2.40 (m, 4H, N(CH2)2), 2.35 (s, 3H, N-CH3), 2.32-1.96 (m, 8H,
2×22-H, 1-H, 2×5-H, 12-H and 2×23-H), 1.91 (s, 3H, OCOCH3), 1.90-1.72 (m, 8H, 2×2-H, 7-H, 12-H, 1-H, 4-H, 6-H
and 9-H), 1.62 (s, 3H, 27-CH3), 1.51 (s, 3H, 26-CH3), 1.41 (s, 3H, 30-CH3), 1.20-1.05 (m, 2H, 6-H and 7-H), 1.00 (s,
3H, 19-CH3), 0.91 (s, 3H, 18-CH3), 0.85 (d, J = 6.70 Hz, 3H, 28-CH3). 13C-NMR (CDCl3, 100 MHz) δ: 170.55, 170.00,
167.80, 165.20, 148.56, 135.87, 132.79, 132.60, 132.39, 130.99, 130.44, 128.99, 128.92, 128.63, 128.54, 128.31,
127.17, 123.14, 74.49, 68.09, 66.44, 65.65, 54.53, 54.40, 48.80, 46.49, 45.97, 44.06, 41.45, 39.58, 39.10, 36.85, 35.81,
32.00, 30.65, 29.77, 29.08, 28.47, 27.45, 25.79, 23.28, 22.74, 21.03, 20.92, 19.26, 17.91, 17.80, 16.64, 15.83, 14.20,
13.80. HR-MS (ESI) m/z: calcd. for C51H68N2O8 [M+H]+: 837.5049, found: 837.5047.
2-[21-fusidic acid (benzyl) ester-3β-oxy]-2-oxo-benzoylpyrazole (17)
According to the synthesis method of 12, 10 was reacted with pyrazole to give white solid 17 (39.0 mg, 73.2%
1
yield). m.p. 239-242 °C. H-NMR (CDCl3, 400 MHz) δ: 8.48 (d, J = 2.70 Hz, 1H, pyr-H), 8.08 (d, J = 8.40 Hz, 1H,
Ar-H), 7.72-7.62 (m, 3H, Ar-H), 7.58 (m, 1H, pyr-H), 7.42-7.32 (m, 5H, Ar-H), 6.50 (dd, J = 2.70, 1.40 Hz, 1H, pyr-H),

ACCEPTED MANUSCRIPT
5.92 (d, J = 8.20 Hz, 1H, 16-H), 5.24 (d, J = 12.20 Hz, 1H, CHAr), 5.13-5.05 (m, 2H, 24-H and 11-OH), 4.96 (d, J =
12.20 Hz, 1H, CHAr), 4.37 (s, 1H, 11-H), 3.10 (d, J = 12.20 Hz, 1H, 13-H), 2.58-2.43 (m, 2H, 22-H), 2.40-1.98 (m,
6H, 1-H, 2×5-H, 12-H and 2×23-H), 1.95 (s, 3H, OCOCH3), 1.92-1.69 (m, 4H, 2×2-H, 7-H and 12-H), 1.66 (s, 3H,
27-CH3), 1.65-1.60 (m, 4H, 1-H, 4-H, 6-H and 9-H), 1.55 (s, 3H, 26-CH3), 1.45 (s, 3H, 30-CH3), 1.22-1.05 (m, 2H,
6-H and 7-H), 0.99 (s, 3H, 19-CH3), 0.94 (s, 3H, 18-CH3), 0.80 (d, J = 6.70 Hz, 3H, 28-CH3). 13C-NMR (CDCl3, 100
MHz) δ: 170.54, 169.99, 168.41, 165.32, 148.47, 144.63, 135.86, 135.07, 132.63, 132.24, 130.99, 130.72, 130.53,
129.53, 129.25, 128.92, 128.64, 128.55, 128.32, 123.13, 109.92, 74.48, 68.26, 66.46, 65.65, 49.41, 48.79, 44.02, 39.51,
39.10, 37.68, 36.82, 35.54, 32.18, 31.26, 30.65, 29.10, 28.47, 27.14, 25.79, 24.05, 23.08, 21.03, 20.80, 19.26, 17.93,
17.81, 15.55, 13.81. HR-MS (ESI) m/z: calcd. for C49H60N2O8 [M+Na]+: 827.4242, found: 827.4240.
2-[21-fusidic acid (benzyl) ester-3β-oxy]-2-oxo-benzoylimidazole (18)
According to the synthesis method of 12, 10 was reacted with imidazole to give white solid 18 (40.0 mg, 75.0%
yield). m.p. 235-237 °C. 1H-NMR (CDCl3, 400 MHz) δ: 8.19-8.12 (m, 1H, IMZ-H), 7.84 (s, 1H, Ar-H), 7.80-7.70 (m,
2H, IMZ-H and Ar-H), 7.62-7.54 (m, 1H, Ar-H), 7.48 (s, 1H, Ar-H), 7.43-7.33 (m, 5H, Ar-H), 7.15 (s, 1H, IMZ-H),
5.94 (d, J = 8.40 Hz, 1H, 16-H), 5.25 (d, J = 12.20 Hz, 1H, CHAr), 5.16-5.05 (m, 2H, 24-H and 11-OH), 4.97 (d, J =
12.20 Hz, 1H, CHAr), 4.38 (s, 1H, 11-H), 3.10 (d, J = 11.40 Hz, 1H, 13-H), 2.58-2.43 (m, 2H, 22-H), 2.27-2.01 (m,
6H, 1-H, 2×5-H, 12-H and 2×23-H), 1.97 (s, 3H, OCOCH3), 1.94-1.69 (m, 6H, 2×2-H, 4-H, 6-H, 7-H and 12-H), 1.67
(s, 3H, 27-CH3), 1.64-1.59 (m, 2H, 1-H and 9-H), 1.56 (s, 3H, 26-CH3), 1.46 (s, 3H, 30-CH3), 1.25-1.05 (m, 2H, 6-H
and 7-H), 1.02 (s, 3H, 19-CH3), 0.96 (s, 3H,18-CH3), 0.84 (d, J = 6.70 Hz, 3H, 28-CH3). 13C-NMR (CDCl3, 100 MHz)
δ: 170.56, 169.97, 166.48, 164.72, 148.45, 135.85, 134.72, 132.88, 132.64, 131.44, 131.15, 130.54, 130.31, 129.84,
128.66, 128.64, 128.56, 128.33, 128.19, 123.91, 123.11, 74.46, 74.14, 68.14, 66.47, 49.30, 48.79, 44.02, 39.55, 39.11,
37.82, 36.81, 35.96, 35.47, 32.19, 32.00, 31.13, 29.77, 29.09, 28.45, 27.12, 25.78, 24.06, 23.06, 21.03, 20.83, 17.92,
17.81, 15.67. HR-MS (ESI) m/z: calcd. for C49H60N2O8 [M+H]+: 805.4423, found: 805.4423.
Biology
Cell culture and treatment
The cancer cell lines Hela, KBV, MKN45 and U87 were kindly provided by Dr. Xiaoguang Chen (Institute Materia
Medica, Chinese Academy of Medical Sciences) and cultured in our lab [14]. All the cells were maintained in DMEM
(Hyclone) with 10% heat-inactivated fetal calf serum (Life Technology), and 100 U/mL penicillin and l00 g/mL

ACCEPTED MANUSCRIPT
streptomycin in a humidified atmosphere of 5% CO2 at 37 °C. Cells in the logarithmic growth phase were used for
further experiments [14].
MTT assay
Cytotoxicity was detected using a MTT assay following our previous protocol [15,16]. Briefly, cells were seeded in
96-well plates and treated with the tested compounds at the desired concentration for 72 h. The MTT solution was then
added into the wells and incubated, and then DMSO was added, the optical density was determined at 570 nm, and the
cell survival rate was calculated. Data are expressed as average values from at least three independent experiments.
Protein synthesis assay
Hela cells at 70-80% confluence were treated with 10 µg/mL puromycin for 10 min. After washing twice with
ice-cold PBS, cells were lysed with SDS-PAGE sample buffer, and proteins were separated on SDS-PAGE and
transferred to Immobilon-P membranes. Membranes were blocked for 1 h in TBS containing 5% non-fat milk and
0.1% Tween-20, followed by incubation with puromycin antibodies (1:100 dilution) overnight at 4 °C. After
incubation with HRP-conjugated anti-mouse IgG (1:5000 dilution) for 1 h at room temperature, the membrane was
visualized using enhanced chemiluminescence.
Cell cycle analysis
A flow cytometry assay was used to analyze the cell apoptosis as previously reported [15]. Briefly, Hela cells (1×105
cells/well) were seeded in 6-well plates. After 24 h incubation, the cells were treated with compound 4 or FA (5 µM)
for 48 h and 72 h. The cells were fixed in 80% alcohol and overnight at 4 °C. After washing with PBS, cells were
treated with PI solution (20 mg/mL PI and 20 mg/mL RNaseA in PBS) for 1 h. Cells were detected in FACS Calibur
(Becton-Dickinson, C6, USA), and the cell cycle distribution was analyzed.
TUNEL staining
Cellular apoptosis was assessed by TUNEL staining using the In Situ Cell Death Detection Kit (Roche, Mannheim,
Germany) as previously described [17]. Briefly, Hela cells (2×104 cells/well) were seeded in 6-well plates. After 24 h
incubation, the cells were treated with compound 4 or FA (2 µM) for 24 h, and then were fixed and permeabilized. The
cells were incubated with 50 mL of reaction mixture containing the labeling enzyme and the TMR green labeled-dUTP
at 37 °C for 1 h. After extensive washing, the cells were counterstained with DAPI and observed under a fluorescence
microscope (Nikon, Japan).

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Xenograft model
Athymic nude mice (6-8 weeks old, BALB/c, female) were used to establish the xenograft tumors following our
published Protocol [18]. In brief, Hela cells (5 ×106) were inoculated subcutaneously in right frank regions. When the
relative tumor volume reached around 100-200 mm3 in size, the mice were randomly divided into three groups, control,
4 (25 mg/kg), and 4 (50 mg/kg) with five mice per group. The mice were intra-gastric administration twice a day with
compound 4, and the tumor appearance and size were measured every 3 days. Animals were sacrificed and the tumors
were stripped and weighed. The use of animals was approved by the Animal Experimentation Ethics Committee of
Yantai University (protocol number 20180607) in accordance with the guidelines for ethical conduct in the care and
use of animals.
ACKNOWLEDGMENTS
This work was partially supported by Taishan Scholar Project, NSFC (81773563, 81728020), Key Research Project of
Shandong Province (2017GSF18177), Natural Science Foundation of Shandong Province (ZR2018LH025). We thank
Renee Mosi, PhD, from Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), for editing the English text of a
draft of this manuscript.
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ACCEPTED MANUSCRIPT
[18] Y.T. Ma, Y.T. Yang, P. Cai, D.Y. Sun, P.A. Sánchez-Murcia, X.Y. Zhang, W.Q. Jia, L. Lei, M.Q. Guo, F. Gago, H.B.
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ACCEPTED MANUSCRIPT
Discovery, synthesis of novel fusidic acid derivatives possessed amino-terminal
groups at the 3-hydroxyl position with anticancer activity
Jingxuan Ni1,#, Mengqi Guo1,#, Yucheng Cao1, Lei Lei1,2, Kangli Liu3, Binghua Wang1, Fangfang
Lu1, Rong Zhai1, Xiangwei Gao3, Chunhong Yan1,4, Hongbo Wang1,*, Yi Bi1,*
1School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University),
Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in
Universities of Shandong, Yantai University, Yantai, 264005, P. R. China
2State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica,
Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, P. R. China
3Institute of Environmental Medicine, Zhejiang University School of Medicine, Hangzhou, 310058, P. R. China
4Department of Biochemistry and Molecular Biology, Medical College of Georgia, Georgia Regents University,
Augusta, GA, 30912, USA
*Corresponding Authors: hongbowangyt@gmail.com (Hongbo Wang), beeyee_413@163.com (Yi Bi)
#Jingxuan Ni and Mengqi Guo have equally contributed to the work.
Highlights：
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Fusidic acid (FA) derivatives with anti-tumor activity were first discovered.
A preliminary structure-activity relationship of the anti-tumor activity was described.
One novel derivative 4 exhibited good anti-tumor activity both in vitro and in vivo.
The mechanism that compound 4 induced Hela cells to undergo apoptosis was first proved.