Novel pleuromutilin derivatives with substituted 6-methylpyrimidine: Design, synthesis and antibacterial evaluation

Article abstract of DOI:10.1016/j.ejmech.2020.112735

A series of novel pleuromutilin derivatives with substituted 6-methylpyrimidine moieties was designed, synthesized, and evaluated for their antibacterial activities. Most of the tested compounds exhibited potent antibacterial activities against Staphyloco

Full text of DOI:10.1016/j.ejmech.2020.112735

Journal Pre-proof
Novel pleuromutilin derivatives with substituted 6-methylpyrimidine: Design, synthesis
and antibacterial evaluation
Yuan Fan, Yu Liu, Hao Wang, Tao Shi, Feng Cheng, Baocheng Hao, Yunpeng Yi,
Ruofeng Shang
PII:
S0223-5234(20)30707-8
DOI:
https://doi.org/10.1016/j.ejmech.2020.112735
EJMECH 112735
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 June 2020
Revised Date: 15 July 2020
Accepted Date: 5 August 2020
Please cite this article as: Y. Fan, Y. Liu, H. Wang, T. Shi, F. Cheng, B. Hao, Y. Yi, R. Shang, Novel
pleuromutilin derivatives with substituted 6-methylpyrimidine: Design, synthesis and antibacterial
evaluation, European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112735.
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Novel pleuromutilin derivatives with substituted
6-methylpyrimidine: Design, synthesis and antibacterial
evaluation
*
*
Yuan Fana, Yu Liua, Hao Wanga, Tao Shib, Feng Chenga, Baocheng Haoa, Yunpeng Yia , Ruofeng Shanga
aKey Laboratory of New Animal Drug Project, Gansu Province; Key Laboratory of Veterinary Pharmaceutical Development, Ministry
of Agriculture and Rural Affairs, China; Lanzhou Institute of Husbandry and Pharmaceutical Sciences of CAAS, Lanzhou, 730050,
China
bSchool of Pharmacy, Lanzhou University, Lanzhou, 730000, China
Corresponding author: Tel.: +86 931 2115253; fax: +86 931 2115951. E-mail address: shangrf1974@163.com
(Ruofeng Shang); yiyp@foxmail.com (Yunpeng Yi).
Author: Tel. +86 931 2115253; fax: +86 931 2115951. E-mail address: fanyuan666@163.com (Yuan Fan).
Address: Lanzhou Institute of Husbandry and Pharmaceutical Sciences of CAAS, No. 335, Qilihe District,
Lanzhou, 730050, P.R. China.
Abstract: A series of novel pleuromutilin derivatives with substituted 6-methylpyrimidine moieties was
designed, synthesized, and evaluated for their antibacterial activities. Most of the tested compounds exhibited
potent antibacterial activities against Staphylococcus aureus ATCC 25923 (S. aureus-25923), methicillin-resistant
Staphylococcus epidermidis ATCC 51625 (MRSE-51625), methicillin-resistant Staphylococcus aureus BNCC
337371 (MRSA-337371), Streptococcus dysgalactiae (S. dysgalactiae) and Streptococcus agalactiae (S.
agalactiae). Compounds 5c and 5g were the most active and displayed bacteriostatic activities against MRSA. In
vivo mouse systemic infection experiment showed that 5c significantly improved the survival rate of mice (ED50 =
18.02 mg/kg), reduced the bacterial load and alleviated the pathological changes in the lungs of the affected mice.
Keywords: Pleuromutilin derivatives, Synthesis, Antibacterial activity, In vivo efficacy.

1. Introduction
A major challenge to global health and wealth has been caused by the increasing bacterial resistance to currently
used antibiotics [1,2]. Multidrug resistant (MDR) bacteria, especially methicillin-resistant Staphylococcus aureus
(MRSA), are a growing concern internationally [3]. Moreover, most antibiotics, including vancomycin which is
indicated for the treatment of life-threatening infections by Gram-positive bacteria, are losing their efficiency
because of the wide-spreading bacterial resistance [4]. Therefore, there is an urgent need for novel antimicrobial
agents against the resistant bacteria without cross resistance to currently used antibiotics.
Pleuromutilin (1) (Fig. 1), a natural compound isolated from Pleurotus mutilus and P. passeckerianus in 1951 [5],
is characterized as the glycolic ester of the diterpene (+)-mutilin [6]. Pleuromutilin derivatives selectively inhibited
bacterial protein synthesis through interaction with prokaryotic ribosomes and preventing the peptidyl transferase
reaction [7,8]. The crystallography data, utilizing a structure of 50S ribosomal subunit from Deinococcus
radiodurans in complex with tiamulin, further confirmed this interaction mode in which the tricyclic core of the
tiamulin are mediated through hydrophobic interactions and hydrogen bond formed mainly by the nucleotides of
domain V [9,10]. Preliminary studies showed that the modifications of pleuromutilin side chain improved its
biological activity [11,12]. Furthermore, the special action mode of pleuromutilin encourages researchers to modify
it for new antibiotics to reduce cross-resistance in clinical use [13]. Such modifications successfully prompted the
approvals of tiamulin, valnemulin, retapamulin and lefamulin (Fig. 1) [14-17].
It is well known that heterocyclic ring bearing polar groups at the C-14 side chain of pleuromutilin derivatives
raise their antibacterial activities [18]. A series of novel pleuromutilin derivatives with pyrimidine moieties was
reported in our lab and further confirmed that these derivatives presented improved activities against resistant
Gram-positive bacterial strains [18,19]. In view of the above findings, we now report the design, synthesis, and
antibacterial activities of novel pleuromutilin derivatives with substituted 6-methylpyrimidine moieties.

Fig. 1. Structural formulas of pleuromutilin, tiamulin, valnemulin, retapamulin and lefamulin.
2. Results and Discussion
2.1. Chemistry
The synthetic approaches for the preparation of target pleuromutilin derivatives 5a-m and their intermediates
were illustrated in Scheme 1. The pleuromutilin 1 was converted into 22-O-tosylpleuromutilin 2, a key intermediate
used for synthesizing almost all pleuromutilin derivatives. Compound 14-O-[(4-hydroxy-6-methylpyrimidine-2-yl)
thioacetyl] mutilin (3) was prepared by nucleophilic substitution of 2 with
4-hydroxy-2-mercapto-6-methylpyrimidine under basic conditions. However, we used the one pot method to
synthesize 3 from 1 in 76% yield. The target pleuromutilin derivatives (5a-m) were directly prepared with one pot
in 41-66% yield using compound 3, the commercially available 2,4,6-trimethylbenzenesulphonyl chloride and
various secondary amines in the presence of triethylamine and 1-methylpyrrolidine.

Scheme 1. General synthetic scheme for the pleuromutilin derivatives 5a-m.
O-Alkylation of compound 3 with alcohols using Mitsunobu reaction directly afforded the target pleuromutilin
derivatives 6a-d (Scheme 2). However, 6e was obtained in three steps, including the amino protection of
2-aminoethanol with benzyl carbonochloridate (CbzCl), O-alkylation and deprotection employing catalytic
hydrogenolysis (Scheme 3).
Scheme 2. Synthesis of pleuromutilin derivatives 6a-d.

Scheme 3. Preparation of pleuromutilin derivative 6e.
All the structures of the synthesized compounds were characterized by IR, 1H NMR, 13C NMR (Supplementary
data) and HRMS spectra. Furthermore, a crystal of compound 3 was obtained as a colorless, block-like from a
solution of ethanol and acetone by slow evaporation method at room temperature (Fig. 2).
Fig. 2. ORTEP diagram for compound 3 with ellipsoids set at 75% probability.
2.2. Antibacterial testing of pleuromutilin derivatives
The minimum inhibitory concentrations (MICs) of the synthesized pleuromutilin derivatives, as well as tiamulin
fumarate and retapamulin used as reference drugs, were assessed against a panel of predominantly Gram-positive
bacteria, including Staphylococcus aureus ATCC 25923 (S. aureus-25923), methicillin-resistant Staphylococcus
epidermidis ATCC 51625 (MRSE-51625), methicillin-resistant Staphylococcus aureus BNCC 337371
(MRSA-337371), Streptococcus dysgalactiae (S. dysgalactiae), Streptococcus agalactiae (S. agalactiae) and
Enterococcus faecalis (E. faecalis), as well as Pasteurella multocida CVCC 1659 (P. multocida-1659) and
Escherichia coli CICC 10389 (E. coli-10389) used as representative Gram-negative strains. The results of MICs for
all screened compounds were listed in Table 1. In general, all the compounds showed higher antibacterial activities
against S. aureus-25923, MRSE-51625, MRSA-337371, S. dysgalactiae and S. agalactiae than that against E.
faecalis, P. multocida-1659 and E. coli-10389. Compounds with N-alkylation of 6-methylpyrimidine (5a-m) and 3

exhibited excellent antibacterial efficiency against S. aureus-25923, MRSE-51625, MRSA-337371, S. dysgalactiae
and S. agalactiae with MIC values ranging from 0.05 to 0.84 μM. However, Compounds with O-alkylation of
6-methylpyrimidine (6a-e) showed comparatively lower antibacterial efficiency against these strains with MIC
values ranging from 0.12 to 3.58 μM. For S. aureus-25923, MRSE-51625, MRSA-337371, S. dysgalactiae and S.
agalactiae, compounds 5c and 5g showed the highest antibacterial activities than the other synthesized compounds
and tiamulin fumarate, but comparable antibacterial activities to that of retapamulin. The MIC results indicated that
the introduction of amine groups into the C-14 glycolic acid side chain of pleuromutilin could enhance antibacterial
activities, which was consistent with previous reported [18,20].
TABLE 1. Antibacterial activities of the synthesized pleuromutilin derivatives.
MIC (µM)
Compounds
S. aureus MRSE
MRSA
0.25
0.24
0.45
0.06
0.22
0.11
0.43
0.11
0.21
0.21
0.21
0.84
0.20
0.41
0.24
0.12
0.23
3.58
0.92
0.82
0.12
S. dysgalactiae
0.12
0.12
0.22
0.06
0.22
0.05
0.43
0.05
0.11
S. agalactiae
0.12
0.12
0.11
0.06
0.11
0.11
0.11
0.05
0.05
0.11
0.05
0.84
0.05
0.10
0.24
0.24
0.23
0.90
0.46
0.20
0.06
E. faecalis
15.96
15.11
28.71
14.41
28.00
14.03
54.76
14.00
27.34
13.67
26.74
53.48
26.09
26.04
30.99
15.09
58.79
57.32
58.68
52.48
15.45
P. multocida
7.95
E. coli
3
0.25
0.12
0.22
0.06
0.11
0.11
0.43
0.11
0.21
0.21
0.10
0.21
0.10
0.10
0.24
0.12
0.46
1.79
0.92
0.82
0.24
0.25
0.24
0.45
0.11
0.11
0.22
0.43
0.11
0.21
0.21
0.21
0.42
0.20
0.20
0.24
0.24
0.46
1.79
0.92
0.82
0.12
> 63.84
> 60.46
> 57.42
> 57.63
> 56.01
> 56.11
> 54.76
> 56.01
> 54.67
> 54.67
> 53.48
> 53.48
> 52.17
> 52.09
> 61.98
> 60.35
> 58.79
> 57.32
> 58.68
52.48
5a
30.23
57.42
14.41
14.00
28.05
54.76
14.00
27.34
27.34
26.74
53.48
52.17
26.04
61.98
> 60.35
> 58.79
> 57.32
> 58.68
3.28
5b
5c
5d
5e
5f
5g
5h
5i
0.11
5j
0.05
0.42
0.05
0.10
0.12
0.12
0.92
0.45
0.23
0.20
0.06
5k
5l
5m
6a
6b
6c
6d
6e
Tiamulin
Retapamulin
0.97
30.90

Compounds 5c and 5g displayed promising antibacterial activities and therefore were further evaluated for their
in vitro time-kill assay. The time-kill curves of different concentrations of 5c and 5g, as well as tiamulin fumarate
used as reference drug, against MRSA-337371 displayed concentration-dependent bacteriostatic effects (Fig. 3).
Although 0.5 × and 1 × MIC of compound 5c, 5g and tiamulin fumarate slowed bacterial propagation when
compared to control, their higher concentrations (2 × and 4 × MIC) showed relatively bacteriostatic kinetics against
MRSA. Treated with 5c and 5g, living cell counts of MRSA were reduced by at least 2 log10 at 2× and 4 × MIC
nearly within 8 h (Fig. 3A and B). However, the bacteriostatic effect of tiamulin reduced living cell count only 1
log10 under the same conditions (Fig. 3C).
Fig. 3. Time-kill analysis of compounds 5c (A), 5g (B) and tiamulin fumarate (C) against MRSA-337371.
2.3. In vivo efficacy in mouse model
Bearing the excellent in vitro antibacterial activities, compound 5c was assessed for its in vivo efficacy by
measuring the survival of mice after a single lethal challenge of MRSA-337371 (3 × 108 CFU in 0.1 mL saline),
and tiamulin fumarate used as the reference drug. After being infected with MRSA, mice were intraperitoneally
treated with different doses of 5c dissolved in vehicle. Mice injected with vehicle alone showed 100% mortality in
this model. Treatment with 5c and tiamulin fumarate displayed dose-dependent protection and led to the survival of
the mice (Fig. 4), with ED50 of 18.02 and 25.85 mg/kg body weight, respectively.

Fig. 4. Efficacy of compound 5c (A) and tiamulin fumarate (B) in mouse systemic infection model.
For further evaluating the in vivo efficacy of 5c, the lung, kidney and liver of infection model mice were
examined for their MRSA load after treatment for 2 d. Treatment with 5c at 40 and 20 mg/kg doses extremely
significantly reduced MRSA load in lung compared to that in the control group (~ 4.8 log10 CFU/mL, p < 0.01) and
the tiamulin groups (~3.5 log10 CFU/mL, p < 0.01). Tiamulin at 40 and 20 mg/kg showed significantly treatment
effects (1.1-1.6 log10 CFU/mL) against MRSA in lung compared to that in the control group (p < 0.05) (Fig. 5A). In
the kidney, MRSA load in the two 5c groups and tiamulin group (40 mg/kg) were extremely significantly lower
than that in the control group (Fig. 5B). However, no significant difference of MRSA load in liver was observed
among the five groups (Fig. 5C).

Figure 5. The bacterial load of compound 5c and tiamulin in lung (A), kidney (B) and liver (C) after challenge of
MRSA.
To assess whether compound 5c attenuates lung tissue damage caused by MRSA, histopathological changes
from the non-treatment and treatment groups were assessed by light microscopy. Compared to the normal control
group (Fig. 6A), the predominant changes in the lungs of mice in the control group were congestion, edema, diffuse
infiltration of neutrophils cells within the alveolar septa and alveolar lumen, fibrous tissue hyperplasia and alveolar
wall thickening (Fig. 6B). It is worth noting that the lungs of mice at the group treated with 40 mg/kg of 5c were
histologically normal with no significant inflammatory cell infiltration in pulmonary interstitium (Fig. 6C).
Infiltration of a few inflammatory cells, slightly thickening of alveolar septum and wall were found in the lungs of
mice treated with 20 mg/kg of 5c (Fig. 6D). However, lungs of mice at the tiamulin groups (40 and 20 mg/kg)
showed mild to moderate infiltration of neutrophils cells and local congestion (Fig. 6E and F).

Fig. 6. Representative lung histology of mice in normal control group (A), control group (B), 5c treated group
with 40 mg/kg (C), 5c treated group with 20 mg/kg (D), tiamulin treated group with 40 mg/kg (E), and tiamulin
treated group with 20 mg/kg (F) treatment.
3. Conclusion
In summary, we have synthesized a series of novel pleuromutilin derivatives possessing 6-methylpyrimidine
moieties. These derivatives were initially evaluated for their in vitro antibacterial activities against S. aureus-25923,
MRSE-51625, MRSA-337371, S. dysgalactiae, S. agalactiae, E. faecalis, P. multocida-1659 and E. coli-1659. The
MIC values demonstrated that all the synthesized derivatives possessed potent antibacterial activity properties.
Compounds 5c and 5g were the most active antibacterial agents. In time-to-kill assays, 5c and 5g showed relatively
bacteriostatic kinetics against MRSA. In further in vivo antibacterial study, compound 5c displayed potent efficacy
to improve the survival rate of mice, reduce the bacterial load and alleviate the pathological damage in the lungs of
the infected mice. The antibacterial effect of 5c found in this study suggested that it merited further drug
development as a potential agent against MRSA infections.
4. Materials and methods
4.1. General
All reactions were performed using standard laboratory equipment and glassware. Reagents and solvents were
obtained commercially and used without further purification. The progress of all reactions and purity of the
compounds were monitored by thin layer chromatography (TLC) on silica gel plates (GF254; Qingdao Haiyang
Chemical Co., Ltd, Shandong, China), followed by visualization after spraying with a 0.05% KMnO4 aqueous
solution or under UV illumination directly. Infrared (IR) spectra were obtained on a Thermo Nicolet NEXUS-670
1
spectrometer using KBr thin films, and the absorptions are reported in cm-1. All H NMR and 13C NMR spectra
were recorded using Bruker 400 MHz and 100 MHz spectrometers in CDCl3, respectively. The chemical shifts (δ)

were reported in parts per million (ppm) relative to tetramethylsilane. High-resolution mass spectra (HRMS) were
obtained with a Bruker Daltonics APEX II 47e mass spectrometer equipped with an electrospray ion source.
4.2. Chemistry
4.2.1. 14-O-[(4-ol-6-methylpyrimidine-2-yl) thioacetyl] mutilin (3)
A 10 mL of NaOH aqueous solution (10 M) was added dropwise to a mixture of pleuromutilin (18.93 g, 50
mmol) and p-toluenesulfonyl chloride (10.49 g, 55 mmol) in methyl isobutyl ketone (150 mL). The mixture was
vigorously stirred for 45 min at 65 ◦C, followed by washing with 60 mL water for 10 min and separation. The
organic layer was added to a solution of sodium salt of 2-mercapto-6-methylpyrimidin-4-ol which was obtained by
2-mercapto-6-methylpyrimidin-4-ol (7.11, 50 mmol) and 5.5 mL NaOH aqueous solution (10 M) in 50 mL
methanol. The mixture was stirred for 6 h at 65 oC and evaporated under reduced pressure to dryness. The crude
product was extracted with 100 mL ethyl acetate and then cooled on an ice bath for 2 h. The target compound 3 was
then precipitated without further purification to yield 19.12 g (76%). IR (KBr): 3489, 2985, 2932, 2864, 1718, 1657,
1582, 1541, 1460, 1363, 1284, 1180, 1118, 1019, 914, 869 cm-1; 1H NMR (400 MHz, Chloroform-d) δ 13.09 (s,
1H), 6.47 (dd, J = 17.4, 11.0 Hz, 1H), 6.07 (d, J = 1.1 Hz, 1H), 5.76 (dd, J = 8.6, 3.2 Hz, 1H), 5.34 (dd, J = 11.0,
1.6 Hz, 1H), 5.23 – 5.13 (m, 1H), 3.95 – 3.81 (m, 2H), 3.37 (d, J = 6.3 Hz, 1H), 2.38 – 2.23 (m, 2H), 2.21 (d, J =
2.8 Hz, 4H), 2.07 (dq, J = 16.2, 2.9 Hz, 2H), 1.77 (dt, J = 14.4, 3.1 Hz, 1H), 1.69 – 1.61 (m, 2H), 1.56 – 1.38 (m,
6H), 1.36 – 1.23 (m, 2H), 1.16 (d, J = 2.9 Hz, 4H), 0.87 (dd, J = 7.2, 2.5 Hz, 3H), 0.75 (dd, J = 7.1, 2.8 Hz, 3H);
13C NMR (101 MHz, Chloroform-d) δ 216.9, 166.8, 165., 165.1, 158.8, 138.9, 117.3, 108.6, 74.5, 70.1, 58.1, 45.4,
44.5, 43.9, 41.9, 36.7, 36.0, 34.4, 33.2, 30.4, 26.8, 26.3, 24.8, 24.1, 16.8, 14.8, 11.5; HRMS (ES) calcd [M + H]+
for C27H38N2O5S 503.2576, found 503. 2569.
4.2.2. General procedure for the synthesis of compounds 5a-m
To a solution of compound 3 (0.75 g, 1.5 mmol) in 20 mL dry DCM, triethylamine (0.73 g, 7.2 mmol),
4-dimethylaminopyridine (0.01 g, 0.1 mmol) and mesitylene-2-sulfonyl chloride (0.50 g, 2.3 mmol) were added
and stirred at room temperature for 2-3 h. Then, amine (7.5 mmol) and 1-methylpyrrolidine (1.27 g, 15 mmol) were
added in one portion and the reaction was stirred at 0 oC for 1-2 h. The mixture was washed with 5% citric acid,
followed by drying with Na2SO4 overnight and rotary evaporation to dryness. The crude residue obtained was
purified by silica gel column chromatography (petroleum ether: ethyl acetate 1:1-1:10 v/v) to afford the desired
compounds.
4.2.2.1. 14-O-[(4-(Dimethylamino)-6-methylpyrimidine-2-yl) thioacetyl] mutilin (5a)
White solid, 0.32 g (yield 41%); IR (KBr): 3448, 2930, 2864, 1733, 1671, 1591, 1500, 1459, 1407, 1371, 1292,

1153, 1117, 1031, 916, 807 cm-1; 1H NMR (400 MHz, Chloroform-d) δ 6.50 (ddd, J = 17.0, 11.0, 5.7 Hz, 1H), 5.82
(s, 1H), 5.71 (d, J = 8.4 Hz, 1H), 5.30 (dt, J = 11.0, 1.4 Hz, 1H), 5.16 (dd, J = 17.4, 1.7 Hz, 1H), 3.93 – 3.75 (m,
2H), 3.58 (t, J = 6.1 Hz, 1H), 3.33 (dd, J = 10.7, 6.5 Hz, 1H), 3.05 (s, 2H), 2.29 (dd, J = 11.0, 5.4 Hz, 4H), 2.24 –
2.12 (m, 2H), 2.07 (s, 1H), 1.97 (t, J = 8.1 Hz, 2H), 1.76 – 1.70 (m, 1H), 1.66 – 1.58 (m, 2H), 1.55 – 1.35 (m, 7H),
1.25 (td, J = 8.2, 7.7, 2.8 Hz, 3H), 1.11 (d, J = 2.4 Hz, 4H), 0.85 (d, J = 7.0 Hz, 3H), 0.71 (dd, J = 6.8, 1.4 Hz, 3H);
13C NMR (101 MHz, Chloroform-d) δ 216.0, 167.4, 158.6, 138.2, 115.9, 97.1, 73.6, 68.3, 57.1, 57.14, 45.4, 44.4,
43.4, 42.9, 40.9, 36.2, 35.8, 34.9, 33.5, 33.0, 29.4, 25.9, 25.2, 23.8, 15.8, 13.9, 13.2, 10.4; HRMS (ES) calcd [M +
H]+ for C29H43N3O4S 530.3044, found 530.3047.
4.2.2.2. 14-O-[(4-(Diethylamino)-6-methylpyrimidine-2-yl) thioacetyl] mutilin (5b)
White solid, 0.49 g (yield 59%); IR (KBr): 3448, 3082, 2930, 2864, 1732, 1 1582, 1499, 1459, 1416, 1371, 1292,
1153, 1117, 1017, 915, 807, 547 cm-1; 1H NMR (400 MHz, Chloroform-d) δ 6.44 (dd, J = 17.3, 11.1 Hz, 1H), 5.75
(s, 1H), 5.64 (d, J = 8.5 Hz, 1H), 5.23 (dd, J = 10.9, 1.6 Hz, 1H), 5.09 (dd, J = 17.5, 1.7 Hz, 1H), 3.88 – 3.68 (m,
2H), 3.50 (d, J = 6.3 Hz, 1H), 3.26 (dd, J = 10.8, 6.6 Hz, 2H), 2.27 – 2.16 (m, 4H), 2.16 – 2.07 (m, 2H), 1.99 (d, J =
12.2 Hz, 3H), 1.91 (dd, J = 14.9, 7.6 Hz, 4H), 1.70 – 1.65 (m, 1H), 1.56 (ddt, J = 11.2, 8.1, 4.1 Hz, 2H), 1.48 – 1.27
(m, 7H), 1.22 – 1.14 (m, 3H), 1.04 (d, J = 2.9 Hz, 4H), 0.78 (d, J = 7.0 Hz, 3H), 0.65 (dd, J = 6.9, 4.1 Hz, 3H).; 13C
NMR (101 MHz, Chloroform-d) δ 216.0, 167.5, 158.8, 138.2, 115.9, 97.0, 73.5, 68.2, 59.4, 57.1, 45.3, 44.4, 43.4,
42.9, 40.9, 35.8, 34.9, 33.5, 33.1, 33.0, 29.4, 25.9, 25.3, 23.8, 22.8, 15.8, 13.9, 13.2, 10.4; HRMS (ES) calcd [M +
H]+ for C31H47N3O4S 558.3319, found 558.3326.
4.2.2.3. 14-O-[(4-(Pyrrolidine-1-yl)-6-methylpyrimidine-2-yl) thioacetyl] mutilin (5c)
White solid, 0.45 g (yield 54%); IR (KBr): 3448, 2928, 2864, 1734, 1592, 1499, 1459, 1416, 1370, 1293, 1153,
1117, 1030, 981, 915 cm-1; 1H NMR (400 MHz, Chloroform-d) δ 6.51 (dd, J = 17.4, 11.0 Hz, 1H), 5.82 (s, 1H),
5.71 (d, J = 8.5 Hz, 1H), 5.30 (dd, J = 10.9, 1.7 Hz, 1H), 5.16 (dd, J = 17.4, 1.7 Hz, 1H), 3.94 – 3.75 (m, 2H), 3.54
(s, 2H), 3.33 (dd, J = 10.7, 6.4 Hz, 2H), 2.33 – 2.24 (m, 4H), 2.22 – 2.11 (m, 2H), 2.06 (d, J = 13.1 Hz, 2H), 1.99 –
1.92 (m, 3H), 1.75 (dd, J = 14.6, 3.2 Hz, 1H), 1.62 (td, J = 11.0, 9.1, 4.6 Hz, 2H), 1.55 – 1.37 (m, 7H), 1.32 – 1.23
(m, 3H), 1.11 (s, 4H), 0.85 (d, J = 7.0 Hz, 3H), 0.72 (d, J = 6.8 Hz, 3H); 13C NMR (101 MHz, Chloroform-d) δ
216.0, 167.5, 158.9, 138.3, 115.9, 97.1, 73.7, 68.4, 59.4, 57.2, 45.3, 44.5, 43.5, 43.0, 41.0, 35.9, 35.0, 33.5, 33.1,
29.5, 26.0, 25.4, 23.9, 22.8, 20.1, 15. 9, 14.0, 13.3, 10.5; HRMS (ES) calcd [M + H]+ for C31H45N3O4S 556.3218,
found 556.3223.
4.2.2.4. 14-O-[(4-(2-(3S)-3-Hydroxypyrrolidine-1-yl)-6-methylpyrimidine-2-yl) thioacetyl] mutilin (5d)

White solid, 0.49 g (yield 57%); IR (KBr): 3386, 2928, 2863, 1718, 1592, 1498, 1456, 1417, 1370, 1294, 1220,
1153, 1117, 1019, 969, 915, 806 cm-1;1H NMR (400 MHz, Chloroform-d) δ 6.55 – 6.42 (m, 1H), 5.85 (s, 1H), 5.74
– 5.65 (m, 1H), 5.36 – 5.27 (m, 1H), 5.16 (dd, J = 17.4, 1.7 Hz, 1H), 3.94 – 3.80 (m, 2H), 3.73 (t, J = 7.0 Hz, 2H),
3.58 (s, 2H), 3.34 (s, 1H), 2.28 (s, 4H), 2.20 (dt, J = 9.3, 4.5 Hz, 2H), 2.10 – 2.05 (m, 2H), 1.75 (d, J = 12.7 Hz, 1H),
1.66 – 1.60 (m, 2H), 1.53 – 1.36 (m, 7H), 1.24 (t, J = 7.0 Hz, 5H), 1.11 (d, J = 2.6 Hz, 4H), 0.86 (d, J = 7.0 Hz, 3H),
0.72 (d, J = 6.9 Hz, 3H)；13C NMR (101 MHz, Chloroform-d) δ 216.1, 167.4, 159.0, 138.4, 116.0, 97.2, 73.6, 69.4,
68.5, 57.2, 57.2, 53.8, 44.5, 43.6, 43.0, 43.0, 35.8, 35.1, 33.5, 33.1, 32.8, 32.8, 29.5, 26.0, 25.4, 23.9, 22.5, 17.5,
15.9, 14.0, 10.5; HRMS (ES) calcd [M + H]+ for C31H45N3O5S 572.3131, found 572.3136.
4.2.2.5. 14-O-[(4-(Piperazine-1-yl)-6-methylpyrimidine-2-yl) thioacetyl] mutilin (5e)
White solid, 0.38 g (yield 45%); IR (KBr): 3448, 2926, 2863, 1734, 1591, 1499, 1459, 1416, 1372, 1291, 1226,
1153, 1117, 1018, 981, 915, 809 cm-1; 1H NMR (400 MHz, Chloroform-d) δ 6.44 (dd, J = 17.4, 11.0 Hz, 1H), 5.75
(s, 1H), 5.64 (d, J = 8.5 Hz, 1H), 5.29 – 5.22 (m, 1H), 5.09 (dd, J = 17.4, 1.7 Hz, 1H), 3.85 – 3.69 (m, 2H), 3.49 (s,
2H), 3.26 (dd, J = 10.6, 6.6 Hz, 2H), 2.26 – 2.17 (m, 4H), 2.13 (t, J = 7.5 Hz, 2H), 1.99 (d, J = 10.4 Hz, 2H), 1.91
(dd, J = 15.2, 7.9 Hz, 4H), 1.68 (dd, J = 14.7, 3.2 Hz, 2H), 1.61 – 1.54 (m, 2H), 1.46 – 1.28 (m, 7H), 1.19 (dd, J =
9.2, 6.9 Hz, 3H), 1.04 (s, 4H), 0.78 (d, J = 7.0 Hz, 3H), 0.65 (d, J = 6.9 Hz, 3H); 13C NMR (101 MHz,
Chloroform-d) δ 216.0, 167.5, 158.7, 138.2, 115.9, 97.1, 73.6, 68.3, 60.9, 57.1, 45.3, 44.4, 43.4, 42.9, 40.9, 37.0,
35.8, 34.9, 33.5, 33.0, 29.4, 28.7, 25.9, 25.2, 23.8, 22.7, 15.8, 13.9, 10.4; HRMS (ES) calcd [M + H]+ for
C31H46N4O4S 571.3208, found 571.302.
4.2.2.6. 14-O-[(4-(4-Methylpiperazine-1-yl)-6-methylpyrimidine-2-yl) thioacetyl] mutilin (5f)
White solid, 0.37 g (yield 42%); IR (KBr): 3547, 2937, 2865, 1735, 1671, 1591, 1499, 1459, 1416, 1373, 1291,
1222, 1148, 1117, 1018, 980, 914, 814, 700 cm-1; 1H NMR (400 MHz, Chloroform-d) δ 6.45 (dd, J = 17.4, 11.0 Hz,
1H), 5.75 (s, 1H), 5.64 (d, J = 8.5 Hz, 1H), 5.27 – 5.19 (m, 2H), 5.09 (dd, J = 17.5, 1.7 Hz, 1H), 3.87 – 3.69 (m,
2H), 3.50 (s, 2H), 3.26 (dd, J = 10.9, 6.5 Hz, 3H), 2.29 – 2.10 (m, 6H), 1.99 (d, J = 9.6 Hz, 2H), 1.93 – 1.80 (m,
4H), 1.68 (dd, J = 14.6, 3.2 Hz, 1H), 1.56 (td, J = 10.5, 9.8, 3.5 Hz, 2H), 1.37 (d, J = 7.4 Hz, 7H), 1.23 – 1.12 (m,
3H), 1.04 (s, 4H), 0.78 (d, J = 7.0 Hz, 3H), 0.65 (d, J = 6.9 Hz, 3H); 13C NMR (101 MHz, Chloroform-d) δ 216.1,
167.5, 158.7, 138.2, 115.9, 97.0, 73.5, 68.2, 57.1, 52.4, 47.5, 45.2, 44.4, 43.4, 42.9, 40.9, 35.8, 34.9, 33.5, 33.0,
29.4, 28.7, 25.9, 25.2, 24.3, 23.8, 22.8, 15.8, 13.9, 10.4; HRMS (ES) calcd [M + H]+ for C32H48N4O4S 585.3448,
found 585.3502.
4.2.2.7. 14-O-[(4- (Morpholine-4-yl)-6-methylpyrimidine-2-yl) thioacetyl] mutilin (5g)

White solid, 0.48 g (yield 56%); IR (KBr): 3547, 2927, 2861, 1732, 1584, 1490, 1448, 1417, 1372, 1307, 1274,
1151, 1117, 1018, 984, 917, 810, 552 cm-1; 1H NMR (400 MHz, Chloroform-d) δ 6.42 (dd, J = 17.4, 11.0 Hz, 1H),
5.97 (s, 1H), 5.64 (d, J = 8.5 Hz, 1H), 5.25 (dd, J = 11.0, 1.6 Hz, 1H), 5.11 (dd, J = 17.5, 1.6 Hz, 1H), 3.75 (d, J =
1.9 Hz, 2H), 3.68 (t, J = 4.9 Hz, 3H), 3.51 (q, J = 4.8 Hz, 3H), 3.27 (dd, J = 10.7, 6.5 Hz, 1H), 2.22 (d, J = 6.1 Hz,
4H), 2.18 – 2.07 (m, 2H), 1.99 (d, J = 14.3 Hz, 2H), 1.95 – 1.88 (m, 1H), 1.68 (dd, J = 14.4, 3.2 Hz, 1H), 1.56 (td, J
= 10.4, 10.0, 5.1 Hz, 2H), 1.38 (d, J = 16.0 Hz, 7H), 1.25 – 1.17 (m, 3H), 1.06 (s, 4H), 0.79 (d, J = 7.0 Hz, 3H),
0.64 (d, J = 6.9 Hz, 3H); 13C NMR (101 MHz, Chloroform-d) δ 216.0, 167.3, 160.9, 138.1, 116.1, 96.4, 73.5, 68.5,
65.5, 59.4, 57.1, 44.4, 43.5, 43.2, 42.9, 40.9, 35.7, 34.9, 33.5, 33.0, 29.4, 25.9, 25.3, 23.8, 20.0, 15.8, 13.9, 13.2,
10.4; HRMS (ES) calcd [M + H]+ for C31H45N3O5S 572.3169, found 572.3163.
4.2.2.8. 14-O-[(4-(3S)-3-Hydroxypiperidine-1-yl)-6-methylpyrimidine-2-yl) thioacetyl] mutilin (5h)
White solid, 0.54 g (yield 62%); IR (KBr): 3442, 2933, 2862, 1732, 1584, 1494, 1447, 1417, 1373, 1308, 1274,
1151, 1117, 1018, 980, 917, 807, 550 cm-1; 1H NMR (400 MHz, Chloroform-d) δ 6.52 – 6.38 (m, 1H), 6.08 (d, J =
1.8 Hz, 1H), 5.68 (d, J = 8.3 Hz, 1H), 5.30 (d, J = 6.5 Hz, 2H), 5.15 (ddd, J = 17.4, 5.9, 1.6 Hz, 1H), 3.87 – 3.75 (m,
3H), 3.67 (d, J = 10.4 Hz, 1H), 3.48 (d, J = 27.7 Hz, 1H), 3.40 – 3.27 (m, 2H), 2.25 (s, 4H), 2.19 (dd, J = 10.5, 6.8
Hz, 2H), 2.06 (d, J = 15.9 Hz, 2H), 2.02 – 1.90 (m, 2H), 1.88 – 1.79 (m, 1H), 1.75 (s, 1H), 1.67 – 1.61 (m, 2H),
1.54 – 1.39 (m, 7H), 1.30 – 1.20 (m, 3H), 1.12 (s, 4H), 0.86 (dt, J = 7.0, 3.3 Hz, 3H), 0.68 (t, J = 7.0 Hz, 3H); 13C
NMR (101 MHz, Chloroform-d) δ 216.7, 161.6, 138.9, 116.7, 97.2, 74.3, 71.9, 69.4, 65.9, 57.9, 53.1, 50.7, 45.2,
44.4, 43.7, 41.7, 36.5, 35.7, 34.2, 33.8, 32.3, 30.2, 26.6, 26.1, 24.6, 23.7, 21.9, 16.5, 14.7, 13.9, 11.2; HRMS (ES)
calcd [M + H]+ for C32H47N3O5S 586.3310, found 586.3316.
4.2.2.9. 14-O-[(4-(4-Hydroxypiperidine-1-yl)-6-methylpyrimidine-2-yl) thioacetyl] mutilin (5i)
White solid, 0.55 g (yield 63%); IR (KBr): 3438, 2930, 2863, 1719, 1585, 1497, 1453, 1417, 1370, 1301, 1206,
1153, 1117, 1088, 1026, 980, 915, 808, 556 cm-1; 1H NMR (400 MHz, Chloroform-d) δ 6.42 (dd, J = 17.4, 11.0 Hz,
1H), 6.01 (s, 1H), 5.64 (d, J = 8.5 Hz, 1H), 5.24 (d, J = 10.7 Hz, 1H), 5.10 (d, J = 17.4 Hz, 1H), 4.03 – 3.85 (m,
3H), 3.77 (s, 2H), 3.28 – 3.17 (m, 2H), 2.21 (d, J = 4.7 Hz, 4H), 2.14 (q, J = 9.1, 8.6 Hz, 2H), 1.99 (dd, J = 15.5,
2.9 Hz, 1H), 1.95 – 1.90 (m, 1H), 1.86 (s, 2H), 1.68 (d, J = 14.4 Hz, 1H), 1.57 (d, J = 11.7 Hz, 4H), 1.47 – 1.32 (m,
7H), 1.21 (d, J = 16.2 Hz, 3H), 1.06 (s, 4H), 0.79 (d, J = 7.0 Hz, 3H), 0.64 (d, J = 6.9 Hz, 3H); 13C NMR (101 MHz,
Chloroform-d) δ 216.1, 167.4, 158.3, 138.1, 116.0, 96.4, 73.6, 68.5, 66.3, 58.0, 57.1, 47.6, 44.4, 43.5, 42.9, 40.9,
40.4, 35.8, 34.9, 33.5, 33.0, 32.7, 31.5, 29.4, 25.9, 25.3, 23.8, 15.8, 13.9, 10.4; HRMS (ES) calcd [M + H]+ for
C32H47N3O5S 586.3310, found 586.3316.
4.2.2.10. 14-O-[(4-((3R)-3-Hydroxymethylpiperidine-1-yl)-6-methylpyrimidine-2-yl) thioacetyl] mutilin (5j)

White solid, 0.47 g (yield 53%); IR (KBr): 3448, 2931, 2862, 1732, 1585, 1496, 1445, 1418, 1308, 1207, 1179,
1117, 1019, 981, 917, 882, 549 cm-1; 1H NMR (400 MHz, Chloroform-d) δ 6.40 (dt, J = 10.9, 5.8 Hz, 1H), 6.00 (d,
J = 2.1 Hz, 1H), 5.62 (d, J = 8.5 Hz, 1H), 5.24 (td, J = 6.7, 3.3 Hz, 1H), 5.10 (ddd, J = 17.4, 5.8, 1.7 Hz, 1H), 3.79
– 3.67 (m, 2H), 3.52 – 3.44 (m, 1H), 3.45 – 3.36 (m, 1H), 3.26 (d, J = 8.3 Hz, 1H), 3.09 – 3.01 (m, 1H), 2.18 (s,
4H), 2.16 – 2.09 (m, 2H), 2.00 (d, J = 15.8 Hz, 3H), 1.93 (d, J = 7.4 Hz, 1H), 1.79 – 1.68 (m, 3H), 1.65 – 1.60 (m,
1H), 1.58 – 1.51 (m, 2H), 1.37 (s, 7H), 1.30 – 1.24 (m, 2H), 1.23 – 1.18 (m, 3H), 1.05 (s, 4H), 0.79 (d, J = 6.9 Hz,
3H), 0.66 – 0.58 (m, 3H); 13C NMR (101 MHz, Chloroform-d) δ 216.0, 167.7, 160.3, 138.2, 115.9, 96.4, 73.5, 68.6,
63.78, 59.4, 57.1, 46.2, 44.4, 43.5, 42.9, 42.9, 40.9, 37.3, 35.7, 34.9, 33.5, 29.4, 26.1, 25.9, 25.3, 23.8, 23.0, 22.9,
20.0, 15.8, 13.9, 13.2, 10.4; HRMS (ES) calcd [M + H]+ for C33H49N3O5S 600.3470, found 600.3464.
4.2.2.11. 14-O-[(4-((3R)-3-Hydroxymethylpiperidine-1-yl)-6-methylpyrimidine-2-yl) thioacetyl] mutilin (5k)
White solid, 0.40 g (yield 45%); IR (KBr): 3356, 2927, 2863, 1724, 1586, 1496, 1458, 1417, 1371, 1304, 1225,
1153, 1117, 1025, 981, 911, 806, 555 cm-1; 1H NMR (400 MHz, Chloroform-d) δ 6.42 (dd, J = 17.4, 11.0 Hz, 1H),
5.99 (s, 1H), 5.64 (d, J = 8.5 Hz, 1H), 5.24 (dd, J = 10.8, 1.7 Hz, 1H), 5.10 (dd, J = 17.5, 1.7 Hz, 1H), 4.32 (s, 1H),
3.77 (s, 2H), 3.45 (d, J = 5.8 Hz, 2H), 3.27 (dd, J = 10.6, 6.4 Hz, 1H), 2.84 – 2.71 (m, 2H), 2.26 – 2.17 (m, 4H),
2.17 – 2.09 (m, 2H), 1.97 (td, J = 16.1, 15.6, 5.0 Hz, 2H), 1.76 – 1.65 (m, 3H), 1.56 (ddd, J = 12.5, 7.3, 2.8 Hz, 3H),
1.48 – 1.32 (m, 7H), 1.26 (d, J = 19.5 Hz, 2H), 1.19 – 1.10 (m, 3H), 1.06 (s, 4H), 0.79 (d, J = 6.9 Hz, 3H), 0.65 (d,
J = 6.9 Hz, 3H); 13C NMR (101 MHz, Chloroform-d) δ 216.1, 167.4, 160.4, 138.1, 116.0, 96.5, 73.6, 68.4, 66.4,
57.2, 44.4, 43.5, 43.1, 43.1, 42.9, 40.9, 37.8, 35.8, 34.9, 33.5, 33.0, 29.4, 27.3, 25.9, 25.3, 23.8, 23.0, 15.8, 13.9,
13.2, 10.4; HRMS (ES) calcd [M + H]+ for C33H49N3O5S 600.3470, found 600.3464.
4.2.2.12. 14-O-[(4-(4-Hydroxyethylpiperidine-1-yl)-6-methylpyrimidine-2-yl) thioacetyl] mutilin (5l)
White solid, 0.61 g (yield 66%); IR (KBr): 3422, 2923, 2857, 1720, 1582, 1495, 1532, 1449, 1422, 1372, 1301,
1223, 1154, 1118, 1048, 1029, 971, 915, 819, 551 cm-1; 1H NMR (400 MHz, Chloroform-d) δ 6.42 (dd, J = 17.4,
11.0 Hz, 1H), 5.97 (s, 1H), 5.64 (d, J = 8.5 Hz, 1H), 5.24 (dd, J = 11.0, 1.6 Hz, 1H), 5.10 (dd, J = 17.4, 1.7 Hz, 1H),
4.28 (s, 1H), 3.77 (s, 2H), 3.66 (t, J = 6.5 Hz, 2H), 3.27 (dd, J = 10.4, 6.4 Hz, 1H), 2.76 (td, J = 12.8, 10.8, 4.1 Hz,
2H), 2.29 – 2.17 (m, 4H), 2.16 – 2.06 (m, 2H), 2.01 – 1.89 (m, 2H), 1.69 (dt, J = 11.5, 7.5 Hz, 4H), 1.63 – 1.49 (m,
3H), 1.48 – 1.33 (m, 8H), 1.31 – 1.23 (m, 2H), 1.21 – 1.10 (m, 3H), 1.06 (s, 4H), 0.79 (d, J = 7.0 Hz, 3H), 0.65 (d,
J = 6.9 Hz, 3H); 13C NMR (101 MHz, Chloroform-d) δ 216.1, 167.5, 160.4, 138.1, 116.0, 96.4, 73.6, 68.4 59.4,
59.2, 57.2, 44.4, 43.5, 42.9, 40.9, 38.1, 35.8, 34.9, 33.5 , 33.0, 31.7, 30.8, 29.4, 25.9, 25.3, 23.8, 23.0, 20.0, 15.8,
13.9, 13.2, 10.4; HRMS (ES) calcd [M + H]+ for C34H51N3O5S 614.3553, found 614.3559.
4.2.2.13. 14-O-[(4-(4-Hydroxyethylpiperazine-1-yl)-6-methylpyrimidine-2-yl) thioacetyl] mutilin (5m)

White solid, 0.46 g (yield 50%); IR (KBr): 3448, 2929, 2864, 1734, 1591, 1499, 1459, 1415, 1347, 1292, 1225,
1153, 1117, 1018, 981, 915, 807, 547 cm-1; 1H NMR (400 MHz, Chloroform-d) δ 6.44 (dd, J = 17.4, 11.0 Hz, 1H),
5.75 (s, 1H), 5.64 (d, J = 8.5 Hz, 1H), 5.23 (dd, J = 11.0, 1.7 Hz, 1H), 5.09 (dd, J = 17.5, 1.7 Hz, 1H), 3.88 – 3.69
(m, 2H), 3.50 (d, J = 6.4 Hz, 2H), 3.24 (td, J = 14.4, 12.7, 5.7 Hz, 3H), 2.22 (d, J = 14.9 Hz, 4H), 2.18 – 2.11 (m,
2H), 1.99 (d, J = 11.2 Hz, 2H), 1.94 – 1.82 (m, 4H), 1.70 – 1.64 (m, 1H), 1.60 – 1.50 (m, 3H), 1.48 – 1.29 (m, 7H),
1.26 – 1.15 (m, 4H), 1.04 (s, 5H), 0.78 (d, J = 7.0 Hz, 3H), 0.65 (d, J = 6.9 Hz, 3H); 13C NMR (101 MHz,
Chloroform-d) δ 216.0, 167.5, 158.7, 138.2, 115.9, 97.0, 73.6, 68.3, 59.4, 57.1, 45.3, 44.4, 43.4, 42.9, 42.0, 40.9,
35.8, 34.9, 33.5, 33.0, 29.4, 28.7, 25.9, 25.2, 24.2, 23.8, 20.0, 15.8, 13.9, 13.2, 10.4; HRMS (ES) calcd [M + H]+
for C33H50N4O5S 615.3568, found 615.3563.
4.2.3. General procedure for the synthesis of compounds 6a-e
To a solution of compound 3 (0.91 g, 1.8 mmol), alcohols (2 mmol) and triphenylphosphine (0.58 g, 2.2 mmol)
in 20 mL dry DCM, triethylamine (0.73 g, 7.2 mmol), diisopropyl azodiformate (0.45 g, 2.2 mmol) was added
dropwise and stirred at room temperature for 2-12 h. The mixture was washed with water, followed by drying with
Na2SO4 overnight and rotary evaporation to dryness. Before the synthesis of compounds 6e, the amino of
monoethanolamine was protected by benzyl carbonochloridate (CbzCl). After treatment with NaHCO3 and brine,
the residue was treated with Pd/C (0.1 g) and ammonium formate (1.14 g, 18 mmol) in 20 mL MeOH under reflux
for 2 h. The reaction mixture was filter and the filtrate was removed in vacuum. The obtained solid residue was
dissolved in DCM and washed with water, dried with anhydrous Na2SO4 overnight and rotary evaporated to
dryness. The crude residue obtained was purified by silica gel column chromatography (petroleum ether: ethyl
acetate 3:1-1:5 v/v) to afford the desired compounds.
4.2.3.1. 14-O-[(4-Methoxy-6-methylpyrimidine-2-yl) thioacetyl] mutilin (6a)
White solid, 0.32 g (yield 35%); IR (KBr): 3448, 2983, 2884, 1734, 1677, 1503, 1455, 1411, 1365, 1288, 1153,
1118, 1019, 980, 916, 754, 722, 542 cm-1; 1H NMR (400 MHz, Chloroform-d) δ 6.39 (dd, J = 17.4, 11.0 Hz, 1H),
5.97 (s, 1H), 5.69 (d, J = 8.5 Hz, 1H), 5.26 (dd, J = 11.0, 1.6 Hz, 1H), 5.12 (dd, J = 17.4, 1.6 Hz, 1H), 3.91 – 3.76
(m, 2H), 3.43 (s, 3H), 3.29 (dd, J = 10.3, 6.4 Hz, 1H), 2.27 – 2.12 (m, 3H), 2.09 – 1.93 (m, 6H), 1.70 (dd, J = 14.3,
3.3 Hz, 1H), 1.62 – 1.55 (m, 2H), 1.38 (s, 4H), 1.27 – 1.16 (m, 3H), 1.08 (s, 4H), 0.80 (d, J = 6.9 Hz, 3H), 0.67 (d,
J = 6.9 Hz, 3H); 13C NMR (101 MHz, Chloroform-d) δ 215.9, 165.7, 161.1, 158.8, 137.9, 116.2, 106.8 , 73.5, 69.1,
57.0, 44.4, 43.4, 42.9, 40.9, 35.6, 35.0, 33.8, 33.4, 29.3, 29.0, 25.8, 25.4, 23.8, 22.7, 15.9, 13.8, 13.2, 10.5; HRMS
(ES) calcd [M + H]+ for C29H42N2O5S 517.2747, found 517.2741.
4.2.3.2. 14-O-[(4-Ethoxy-6-methylpyrimidine-2-yl) thioacetyl] mutilin (6b)

White solid, 0.30 g (yield 31%); IR (KBr): 3468, 2935, 2860, 1730, 1664, 1493, 1456, 1402, 1372, 1310, 1290,
1201, 1168, 1110, 1015, 924, 846, 563 cm-1; 1H NMR (400 MHz, Chloroform-d) δ 6.47 (dd, J = 17.4, 11.0 Hz, 1H),
6.02 (s, 1H), 5.75 (d, J = 8.5 Hz, 1H), 5.34 (dd, J = 11.0, 1.5 Hz, 1H), 5.19 (dd, J = 17.4, 1.6 Hz, 1H), 4.16 – 4.02
(m, 2H), 3.99 – 3.79 (m, 2H), 3.35 (d, J = 6.5 Hz, 1H), 2.31 – 2.20 (m, 2H), 2.09 (d, J = 33.8 Hz, 4H), 1.76 (dd, J =
14.6, 3.1 Hz, 1H), 1.65 (d, J = 11.4 Hz, 2H), 1.45 (s, 4H), 1.34 (t, J = 7.1 Hz, 4H), 1.29 – 1.21 (m, 5H), 1.14 (s, 4H),
0.86 (d, J = 7.0 Hz, 3H), 0.74 (d, J = 7.0 Hz, 3H); 13C NMR (101 MHz, Chloroform-d) δ 215.8, 165.8, 160.9, 158.0,
137.9, 116.3, 107.3, 73.5, 69.1, 57.0, 44.4, 43.4, 42.9, 40.9, 38.6, 35.7, 35.0, 33.7, 33.4, 29.4, 28.7, 25.8, 25.3, 23.8,
22.6, 15.9, 13.8, 11.8, 10.4.; HRMS (ES) calcd [M + H]+ for C29H42N2O5S 531.2888, found 531.2884.
4.2.3.3. 14-O-[(4-Isopropoxy-6-methylpyrimidine-2-yl) thioacetyl] mutilin (6c)
White solid, 0.44 g (yield 45%); IR (KBr): 3448, 2925, 2864, 1733, 1676, 1500, 1452, 1413, 1376, 1362, 1297,
1263, 1176, 1116, 1058, 1019, 916, 731 cm-1; 1H NMR (400 MHz, Chloroform-d) δ 6.42 (dd, J = 17.4, 11.0 Hz,
1H), 5.87 (s, 1H), 5.67 (d, J = 8.5 Hz, 1H), 5.28 (dd, J = 11.0, 1.6 Hz, 1H), 5.13 (dd, J = 17.5, 1.6 Hz, 1H), 3.89 –
3.67 (m, 2H), 3.28 (d, J = 6.5 Hz, 1H), 2.24 – 2.15 (m, 2H), 2.02 (s, 3H), 1.97 – 1.89 (m, 1H), 1.69 (dd, J = 14.4,
3.1 Hz, 1H), 1.54 (dd, J = 6.7, 3.6 Hz, 6H), 1.38 (s, 7H), 1.18 (s, 4H), 1.08 (d, J = 7.0 Hz, 4H), 0.79 (dd, J = 6.9,
3.4 Hz, 3H), 0.66 (d, J = 7.0 Hz, 3H); 13C NMR (101 MHz, Chloroform-d) δ 215.9, 165.8, 159.9, 157.9, 137.9,
116.3, 73.5, 68.9, 57.0, 44.4, 43.3, 42.9, 40.9, 35.6, 34.9, 34.3, 33.4, 29.3, 28.7, 25.8, 25.3, 23.8, 22.2, 21.7, 18.2,
15.9, 13.8, 13.1, 10.4; HRMS (ES) calcd [M + H]+ for C30H44N2O5S 545.3009, found 545.3004.
4.2.3.4. 14-O-[(4-Butoxy-6-methylpyrimidine-2-yl) thioacetyl] mutilin (6d)
White solid, 0.24 g (yield 24%); IR (KBr): 3473, 2957, 2865, 1735, 1498, 1439, 1402, 1374, 1269, 1153, 1118,
1018, 981, 915, 839 cm-1; 1H NMR (400 MHz, Chloroform-d) δ 6.40 (dd, J = 17.4, 11.0 Hz, 1H), 5.94 (s, 1H), 5.68
(d, J = 8.5 Hz, 1H), 5.26 (dd, J = 11.0, 1.6 Hz, 1H), 5.12 (dd, J = 17.4, 1.6 Hz, 1H), 3.93 (td, J = 7.5, 5.6 Hz, 2H),
3.84 – 3.73 (m, 2H), 3.28 (d, J = 6.3 Hz, 1H), 2.24 – 2.15 (m, 2H), 2.06 (s, 4H), 1.98 (s, 2H), 1.62 (td, J = 14.6,
13.8, 9.5 Hz, 5H), 1.37 (d, J = 9.1 Hz, 8H), 1.21 – 1.14 (m, 3H), 1.07 (s, 3H), 0.90 (t, J = 7.4 Hz, 3H), 0.80 (d, J =
7.0 Hz, 3H), 0.67 (d, J = 7.0 Hz, 3H); 13C NMR (101 MHz, Chloroform-d) δ 215.9, 165.8, 160.9, 158.1, 137.9,
116.2, 107.2, 73.5, 69.0, 59.4, 57.0, 44.4, 43.4, 42.9, 40.8, 35.6, 35.0, 33.7, 33.4, 29.4, 28.6, 25.8, 25.4, 23.8, 22.6,
19.1, 15.8, 13.8, 13.2, 12.6, 10.4; HRMS (ES) calcd [M + H]+ for C31H46N2O5S 559.3122, found 559.3117.
4.2.3.5. 14-O-[(4-(2-aminoethoxy)-6-methylpyrimidine-2-yl) thioacetyl] mutilin (6e)
White solid, 036 g (yield 37%); IR (KBr): 3495, 3346, 2930, 2884, 1724, 1583, 1547, 1455, 1343, 1286, 1168,
1117, 1055, 1017, 980, 916, 736, 698 cm-1; 1H NMR (400 MHz, Chloroform-d) δ 6.48 (dd, J = 17.4, 11.0 Hz, 1H),
6.23 (s, 1H), 5.72 (d, J = 8.5 Hz, 1H), 5.30 (d, J = 2.9 Hz, 2H), 5.12 (d, J = 10.9 Hz, 3H), 4.39 (q, J = 5.9 Hz, 2H),

3.90 – 3.77 (m, 2H), 3.56 (q, J = 5.6 Hz, 2H), 3.33 (d, J = 6.3 Hz, 1H), 2.34 (s, 2H), 2.31 – 2.17 (m, 3H), 2.09 –
1.98 (m, 2H), 1.75 (dd, J = 14.3, 3.1 Hz, 1H), 1.68 – 1.58 (m, 2H), 1.49 – 1.39 (m, 5H), 1.26 (h, J = 5.1, 4.4 Hz,
3H), 1.12 (s, 4H), 0.85 (d, J = 7.0 Hz, 3H), 0.71 (d, J = 7.0 Hz, 3H); 13C NMR (101 MHz, Chloroform-d) δ 215.9,
166.9, 138.0, 127.5, 127.2, 116.1, 101.6, 73.5, 68.7, 65.9, 64.6, 57.1, 52.4, 44.4, 43.6, 42.9, 40.9, 35.7, 34.9, 33.4,
33.1, 29.4, 25.8, 25.3, 23.8, 22.6, 15.8, 13.7, 10.4; HRMS (ES) calcd [M + H]+ for C29H43N3O5S 546.2986, found
546.2991.
4.3. Biological evaluation
4.3.1. MIC testing
The MICs of the pleuromutilin derivatives were determined using a modified standard microtiter dilution method.
Briefly, compounds, as well as tiamulin fumarate and retapamulin used as reference drugs, were dissolved in 25%
DMSO to a solution with concentration of 64 μg/mL. All the solutions were then diluted two-fold with distilled
water to provide 11 dilutions (final concentration is 0.0613 μg/mL). Inoculums, including S. aureus-25923,
MRSE-51625, MRSA-337371, S. dysgalactiae, S. agalactiae, E. faecalis, P. multocida-1659 and E. coli-1659 were
incubated overnight at 37 °C and transferred to new nutrient broth (MHB) until the logarithmic phase of growth.
The bacteria were then diluted to 105-106 colony forming units (CFU)/mL in MHB. A 100 μL volume of bacteria in
MHB was mixed with 100 μL of serially diluted aliquots of the compounds in sterile 96-well plates, which resulted
in the final concentration of each dilutions decreasing two fold. The mixtures were then incubated at 37 °C for
18-24 h in an incubator. The MIC was determined by optical density (OD) measurements at a wavelength of 492
nm as the lowest concentration of the compounds that resulted in no bacterial growth. The results were expressed
as an average of the MICs obtained from three independent experiments.
4.3.2. Bactericidal time-kill kinetics
MRSA-337371 was cultured in MuellereHinton broth at 37 °C for 6 h with shaking and then diluted to
approximately 6 × 105 CFU/mL. Test compounds 5c, 5g and tiamulin fumarate with the final concentrations of 0.5
× MIC, 1 × MIC, 2 × MIC and 4 × MIC were inoculated with the aliquots of bacteria resuspended in fresh media.
After specified time intervals (0, 2, 4, 6, 8, 12 and 24 h), 0.5 mL aliquots were serially diluted to 10-1 to 10-8 by
10-fold in 0.9% saline. The resulted dilutions were plated on sterile MuellereHinton agar plates and incubated at
37 °C for 24 h. The viable colonies were counted and represented as log10 (CFU/mL). The same procedure was
repeated in triplicate.
4.4. Mouse systemic infection model
The in vivo efficacy of compound 5c was determined in 6-8 weeks old Kunming mice (specific pathogen-free,

Laboratory Animal Center of Lanzhou University, Lanzhou, China). All mice were rendered neutropenic upon
treatment with 150 mg/kg cyclophosphamide intraperitoneally for four days and with 100 mg/kg for one day prior
to inoculation, respectively. The neutropenic mice were anesthetized with isoflurane and then received a 0.5 mL
MRSA-337371 inoculum of 108 CFU/mL via intraperitoneal injection. Some animals were used for determining the
survival and ED50 which have been described in our previously work [18, 19]. Briefly, about 1 h after infection, the
mice were then intraperitoneal administered compound 5c dissolved in 0.5 mL vehicle
(DMSO:Tween-80:sterile:water = 0.5:0.5:9) at doses of 5, 8, 15, 25, and 40 mg/kg body weight (10 per group).
Tiamulin fumarate was used as a reference drug in the same manner at the same doses as 5c. All animals were
observed twice daily for symptoms and mortality for the animal procedures. The survival of the mice at 7 d after
infection was used as the end-point, and the ED50 was calculated by the Bliss method.
For the determination of MRSA in tissues and histological analyses, the neutropenic mice were euthanized and
sacrificed after treatment for 2 d. Then the kidney, liver and a part of lung were cut to two parts: one was weighted
and ground to homogenate with sterile saline, followed by 10-fold dilution. The resulted dilutions were plated on
sterile MuellereHinton agar plates and incubated at 37 °C for 24 h. The same procedure was repeated in triplicate.
The viable colonies were counted (represented as CFU/mL) and the bacterial load was calculated as follow: CFU/g
= (the mean CFU × 10 ×volume of homogenate × dilution factor)/weight of tissues. The other part of lung was
collected and fixed in 10% buffered formalin, embedded in paraffin, sectioned at 4 μm, and stained with
hematoxylin and eosin (H&E) and examined under light microscopy [21]. The protocol for this study was reviewed
and approved by the Laboratory Animal Ethical Commission of Lanzhou Institute of Husbandry and
Pharmaceutical Sciences of CAAS.
4.5. Statistical analysis
Statistical analysis was performed using IBM SPSS Statistics for Windows version 24.0 (SPSS Inc., Chicago,
USA). The data were analyzed by One-way analysis of variance (ANOVA), followed by Dunnett's post-hoc tests as
appropriate. Statistical significant difference was defined as a
defined as a < 0.01.
p < 0.05 and the extremely significant difference was
p
Acknowledgments
This work was financial supported by the National Natural Science Foundation of China (No. 31873027),
“Quancheng Scholar” Construction Project of Jinan (No. 00252019025) and Agricultural Science and Technology

Innovation Program (ASTIP, CAAS-LMY-03).
CONFLICT OF INTEREST
The authors declare no conflict of interest.
Appendix A. Supplementary data
Supplementary data related to this article can be found at:
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Figure Legends
Figure 1. Structural formulas of pleuromutilin, tiamulin, valnemulin, retapamulin and lefamulin.
Figure 2. ORTEP diagram for compound 3 with ellipsoids set at 75% probability.
Figure 3. Time-kill analysis of compounds 5c (A), 5g (B) and tiamulin fumarate (C) against
MRSA-337371.
Figure 4. Efficacy of compound 5c (A) and tiamulin fumarate (B) in mouse systemic infection model.
Figure 5. The bacterial load of compound 5c and tiamulin in lung (A), kidney (B) and liver (C) after challenge of
MRSA.
Figure 6. Representative lung histology of mice in normal control group (A), control group (B), 5c treated group
with 40 mg/kg (C), 5c treated group with 20 mg/kg (D), tiamulin treated group with 40 mg/kg (E), and tiamulin
treated group with 20 mg/kg (F) treatment.
Scheme 1. General synthetic scheme for the pleuromutilin derivatives 5a-m.
Scheme 2. Synthesis of pleuromutilin derivatives 6a-d.
Scheme 3. Preparation of pleuromutilin derivative 6e.

·A series of novel pleuromutilin derivatives with substituted 6-methylpyrimidine
moieties were synthesized.
·Synthesized compounds were evaluated for their antibacterial activity.
·Compounds 5c showed excellent antibacterial activity and potent therapeutic
efficacy in mouse systemic infection.

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: