Design, synthesis and antibacterial activities of pleuromutilin derivatives

Article abstract of DOI:10.1080/10286020.2020.1713764

We described the design, synthesis and antimicrobial activities of novel pleuromutilin derivatives with substituted piperazine substrate. Minimum inhibitory concentration (MIC) was used to evaluate the activity of the derivatives against six bacteria in?v

Full text of DOI:10.1080/10286020.2020.1713764

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: https://www.tandfonline.com/loi/ganp20
Design, synthesis and antibacterial activities of
pleuromutilin derivatives
Hui-Xian Liu, Dong-Lai Ma, Ge Cui, Yue Zhang & Fei-Qun Xue
To cite this article: Hui-Xian Liu, Dong-Lai Ma, Ge Cui, Yue Zhang & Fei-Qun Xue (2020): Design,
synthesis and antibacterial activities of pleuromutilin derivatives, Journal of Asian Natural Products
Research, DOI: 10.1080/10286020.2020.1713764
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JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
https://doi.org/10.1080/10286020.2020.1713764
Design, synthesis and antibacterial activities
of pleuromutilin derivatives
Hui-Xian Liua, Dong-Lai Maa, Ge Cuib, Yue Zhangb and Fei-Qun Xuec
b
aCollege of Pharmacy, Hebei University of Chinese Medicine, Shijiazhuang 050200, China; College of
Chemical and Pharmaceutical Engineering, Hebei University of Science and Technology, Shijiazhuang
c
050018, China; Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences,
Shanghai 200241, China
ABSTRACT
ARTICLE HISTORY
Received 8 September 2019
Accepted 6 January 2020
We described the design, synthesis and antimicrobial activities of
novel pleuromutilin derivatives with substituted piperazine sub-
strate. Minimum inhibitory concentration (MIC) was used to evalu-
ate the activity of the derivatives against six bacteria in vitro, and
KEYWORDS
Pleuromutilin derivatives;
antibacterial activity; design;
synthesis; Gram-
compound
8 was potent against Staphylococcus aureus and
Staphylococcus epidermidis with the MIC value of 0.0625lg/ml. 10a
and 10 b showed similar activity to positive control drugs (tiamulin,
erythromycin) against S. aureus with the MIC value of 0.125 lg/ml.
The binding mode of compound 8 and tiamulin to the ribosome
pocket showed the correlation between binding parameters and
the antibacterial activity, and more bonds and stronger combin-
ation could effectively enhance the activity of compounds.
positive bacteria
CONTACT Yue Zhang
University of Science and Technology, Shijiazhuang 050018, China; Fei-Qun Xue
yuezhang@hebust.edu.cn
College of Chemical and Pharmaceutical Engineering, Hebei
fqxue@outlook.com Shanghai
Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China
Supplemental data for this article is available online at https://doi.org/10.1080/10286020.2020.1713764.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
H.-X. LIU ET AL.
1. Introduction
The discovery of antibiotics is of great significance in human history, which not only
prolongs the life span of human beings, but also improves the quality of human lives.
However, the abuse of antibiotics makes many bacteria have increasingly serious drug
resistance, which poses a great threat to the safety of human life [1, 2]. It is estimated
that as a result of the increasing number of multidrug-resistant bacteria, the global
death toll from bacterial infections will climb to about 7 million to 10 million by
2050 [3]. Therefore, the Food and Drug Administration (FDA) launched an incentive
program in 2012 to promote antibiotic research and development (generating antibi-
otics now, GAIN), which aims to cope with the decrease of antibiotics and the
increasing number of drug-resistant bacteria [4]. According to related studies, the
vast majority of the 42 antibiotics currently in clinical trials are modified from previ-
ously marketed antibiotic drugs [5]. Researchers have concentrated their attention on
antibiotic analogues that are already on the market and compounds that have previ-
ously been ignored. One of them is pleuromutilin.
Pleuromutilin 1 (Figure 1) is a kind of natural product with antibacterial activity
discovered in the 1950s, which is a diterpene compound with parallel tricyclic skel-
eton produced by deep culture of Pleurotsmutilus and Pleurots passeckerianus from
basidiomycetes of higher fungi [6]. Studies have shown that pleuromutilin binds to
the 23 s rRNA of bacterial ribosomal 50 s subunit, locates in the peptidyl transferase
center of ribosomal 50 s subunit through its tricyclic mother nucleus, and forms a
tight pocket at site A. Meanwhile, the side chain partially covers the P site of tRNA
binding, which directly inhibits the formation of peptide bond, thus preventing the
synthesis of bacterial protein [7]. It is precisely because of this ingenious mode of
action that the compound and some of its derivatives have obvious antibacterial activ-
ity and excellent pharmacokinetic properties against drug-resistant Gram-positive bac-
teria and mycoplasma, and have less cross-drug resistance with other antibiotics [8].
In the 1970s, a number of pleuromutilin derivatives were synthesized and studied the
structure–activity relationship, implying that introduction of thioether and alkaline
group in the C14 side chain would improve antibacterial activity [9, 10].
Tiamulin 2 (Figure 1) is a derivative discovered by the Sandoz Institute in 1974,
which is 10 to 100 times higher than pleuromutilin in antibacterial activity [11]. It is
Figure 1. The structures of pleuromutilin and related drugs.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
Figure 2. Antimicrobial drugs with piperazine rings.
one of the commonly used veterinary antibiotics in the market and mainly used in
the form of feed additives to treat and prevent gastrointestinal and respiratory tract
infections in swine and poultry [11, 12]. In 1980, valnemulin 3 (Figure 1) was first syn-
thesized by Sandoz Institute, which is superior to tiamulin in antibacterial activity [13].
In addition, recent studies have shown that valnemulin is effective in the treatment of
antibiotic-resistant mycoplasma infections in immunocompromised patients [14].
Retapamulin 4 (Figure 1) is another pleuromutilin antibiotic developed by Glaxo Smith
Kline of the United Kingdom. It is the first topical antibiotic for human use and
was approved by US FDA in April 2007 [15, 16]. Lefamulin 5 (formally BC-3781)
(Figure 1) is a novel pleuromutilin antibiotic for community-acquired bacterial
pneumonia (CABP) as intravenous (IV) and oral (PO) formulations, and it is the
first to be used for systemic treatment of bacterial infections in humans [17].
Most of pleuromutilin derivatives usually contain sulfide linkage in C14 side chain,
either drugs that are on the market or have been reported, either for human or veterin-
ary use, however, it is reported that thioether group suffers from being rapidly and
extensively metabolized in vivo [18]. Piperazine substrate was widely used in many anti-
microbial drugs, such as piperaquine, norfloxacin, temafloxacin, delavirdine and keto-
conazole, etc. (Figure 2). We were inspired by those structures and tried to introduce
piperazine to pleuromutilin as the linkage of C14 side chain. It accorded with the SAR

4
H.-X. LIU ET AL.
Figure 3. Preparation of the target compounds. a: p-TsCl, Et3N, DCM; b: K2CO3, acetonitrile; c: Et3N,
DCM; d: Et3N, DMF; e: Fe, NH4Cl, CH3COOH, H2O; f: Et3N, DMF.
that alkaline group in C14 side chain is beneficial to activity improvement. It can be
found by observing pleuromutilin derivatives on the market that all of them owe amide
terminal group. Based on these points, we designed and synthesized a series of pleuro-
mutilin derivatives, and the antimicrobial activities were further measured.
2. Results and discussion
2.1. Chemistry
As depicted in Figure 2, pleuromutilin (1) was reacted with 1.5 equiv. of p-toluene
sulfonyl chloride in the presence of 1.5 equiv. of triethylamine to afford compound 2.
Compound 2 was reacted with piperazine and 2-methyl-4-nitroaniline to obtain com-
pound 8, and then it was reduced to afford compound 9. At last, we replaced com-
pound 9 with a series of halogenated hydrocarbons to obtain compounds 10a–f.
2.2. In vitro antibacterial activity
The evaluation of the in vitro antibacterial activities of the pleuromutilin derivatives
8, 9 and 10a–f was performed against five drug-susceptible Gram-positive pathogens

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
Table 1. The MIC values (lg/ml) of compounds 8, 9 and 10a–f.
Staphylococcus
aureus
ATCC 25923
Staphylococcus
epidermidis
ATCC 12228
Staphylococcus
suis
ATCC 43765
Enterococcus
faecalis
ATCC 19433
Escherichia
coli
CVCC 231
MRSA
NY3
Compounds
10a
10b
10c
10d
10e
10f
8
9
0.25
0.25
0.5
0.5
0.5
0.5
0.06
0.5
0.125
0.125
>64
>64
>64
>64
>64
>64
>64
>64
16
0.125
0.5
0.5
0.5
0.5
0.5
0.06
0.5
0.125
0.125
>64
>64
>64
>64
>64
>64
>64
>64
32
>64
>64
>64
>64
>64
>64
8
16
64
4
>64
>64
>64
>64
>64
>64
>64
>64
16
Tiamulin fumaric acid
Erythromycin sulfur
8
0.5
16
and one drug-resistant isolated from clinic as well as against one Gram-negative bac-
teria strain. Tiamulin fumaric acid and erythromycin sulfur were used as positive
control and the MIC values were provided in Table 1. Most of the derivatives showed
general in vitro antibacterial activity against the tested strains, and compound 8
showed excellent in vitro antibacterial activity against Staphylococcus aureus and
Staphylococcus epidermidis. However, these synthesized compounds and the reference
agents have minimal potency against Gram-negative bacterial, like Escherichia coli.
The results illustrate that compound 8 synthesized as a lead compound showed
almost two-fold increase in activity against S. aureus and S. epidermidis in compari-
son with that of reference drugs, and eight-fold increase in activity against
Enterococcus faecalis compared to that of tiamulin. 10a and 10 b showed the same
activity with two controlled drugs against S. aureus.
2.3. Molecular docking study
Conclusions can be drawn from Table 1 that compound 9 and 10c–f displayed moder-
ate antimicrobial activity and 10a–b owned better activity than 9 and 10c–f, while the
most active was compound 8. The results of molecular docking were consistent with the
results of activity determination. 10a and 10 b, 9 and 10c–f, displayed the similar inter-
action mode with the pocket, respectively. We compared the interaction of 10a, 10d, 8
and tiamulin with the active pocket. 10a was found to form one hydrogen bonds
(1.82 Å) with U2485 and two H-p interactions (Table 2). 10d was found to form two
H-p interactions (Table 2). 8 was found to form four hydrogen bonds (1.99 Å, 2.11 Å,
2.26 Å, 2.64 Å) with G2044, G2484, U2485, C2565 and two H-p interactions (Figures 4
and 5), its binding strength was the best of all the compounds. Tiamulin was found to
form three hydrogen bonds (1.64 Å, 2.04 Å, 2.24 Å) with G2484, G2044, U2564 and two
H-p interactions (Figures 6 and 7). The docking results can explain why 8 exhibited bet-
ter in vitro antibacterial activity, and more bonds and stronger combination could effect-
ively enhance the activity of compounds. At the same time, it is proved that the
existence of -NH- at C-14 is crucial. Almost all the designed molecules displayed inter-
actions with U2564, and among those compounds tiamulin and 8 displayed interactions
with both G2484 and G2044 (Figures 4–7). The compounds 8, 10a, 10d and tiamulin
were suggested a detailed analysis of their possible binding modes in the active pocket
of 23 s rRNA, and they showed almost comparable binding energy score (Table 2).

6
H.-X. LIU ET AL.
Table 2. Docking scores as “S” (kcal molꢀ1) and bond interactions of 8, 10a–f and tiamulin with
active pocket of 23 s rRNA.
Amino acids
involved
Cpd. No. S (Kcal mol ꢀ 1) No. of bonds
Distance (Aꢁ)
1.82
Interacting groups
10a
10b
10c
10d
˗ 9.7638
˗ 10.2427
˗ 10.5185
˗ 9.9347
3
3
2
2
U2485
H-bond with OH of
pleuromutilin C11 H-p
interaction H-p
U2564 A2430
interaction
2.46
1.79 2.39
–
G2484
G2044 A2430
H-bond with C ¼ O of
pleuromutilin C3 H-p
interaction H-p
interaction
G2044 G2044
H-bond with OH of
pleuromutilin C11 H-
bond with OH of
pleuromutilin C11
H-p interaction H-p
interaction
U2564 C2565
10e
10f
˗ 10.3751
˗ 10.9199
1
3
–
–
G2044
U2564 A2045
H-p interaction
H-p interaction H-p
interaction
8
˗ 9.9035
6
1.99 2.11 2.26 2.64
G2044 G2484
U2485 C2565
A2430 U2485
H-bond with C ¼ O of
ester group H-bond
with OH of
pleuromutilin C11 H-
bond with C ¼ O of
pleuromutilin C3 H-
bond with NH of
acylamino H-p
interaction H-p
interaction
tiamulin
˗8.6283
5
1.64 2.04 2.24
G2484 G2044
U2564
U2483 U2564
H-bond with OH of
pleuromutilin C11 H-
bond with C ¼ O of
ester group H-bond
with H of C14 side
chain H-p interaction
H-p interaction
3. Experimental
3.1. General experimental procedures
Routine monitoring of reaction was performed by TLC using pre-coated GF254 TLC
plate, which were purchased from Qingdao Ocean Chemical Co., Ltd. (Qingdao,
1
China). H-NMR spectrum was recorded on a Bruker AVANCE 600 spectrometer at
600 MHz instrument (Bruker Daltonics, Bremen, Germany) in CDCl3 using tetrame-
thylsilane (TMS) as the internal reference. MS were performed with electron spray
ionization (ESI) mode and recorded on a high performance liquid chromatography
tandem mass spectrometer by Waters (Waters, Milford, MA, USA).
All reagents were purchased from commercial sources and were used as received.
Pleuromutilin (>90% pure) was obtained from Beijing Ouhe Technology Co., Ltd.
(Beijing, China). 2-Methyl-4-nitroaniline, chloroacetyl chloride and alkylamine were
purchased from Aladdin Reagent Co. (Shanghai, China). Piperazine and p-methyl
benzene sulfonic chloride were from Sinopharm Chemical Reagent Co., Ltd.
(Shanghai, China).

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
Figure 4. 3 D diagram of compound 8 showing its interactions with the 23 s rRNA active site (dis-
tances in Å).
3.2. Synthesis
3.2.1. Synthesis of 14-O-(p-toluene-sulfonyloxyl) mulin (2)
As shown in Figure 1, pleuromutilin 1 (7.0 g, 18.5 mmol) was reacted with p-toluene
sulfonyl chloride (5.3 g, 28.3 mmol) in the presence of triethylamine (2.8 g, 27.5 mmol)
in dichloromethane (50 ml). The mixture was stirred overnight at room temperature,
then evaporated in vacuum and ethyl acetate (100 ml) was added to the residue. The
organic phase was washed with saturated salt solution, dried with anhydrous sodium
sulfate about 2 h, filtered, and the organic solvent was evaporated. The crude product
was recrystallized with mixed solvent of ethyl acetate and petroleum ether, and fil-
tered to afford the pure product (2, 9.0 g) as white solid in the yield of 91.2%. IR
(KBr, cmꢀ1): 3448, 2941, 2864, 1732, 1636, 1597, 1457, 1371, 1224, 1117, 1036, 832,
1
664, 552. H NMR (600 MHz; CDCl3): d (p.p.m.) 7.75 (d, J ¼ 7.2 Hz, 2 H, phenyl H-2,
6), 7.29 (d, J ¼ 8.4 Hz, 2 H, phenyl H-3, 5), 6.35 (dd, J ¼ 10.8, 6.6 Hz, 1 H, H-19), 5.71
(d, J ¼ 8.4 Hz, 1 H, C11-OH), 5.27 (d, J ¼ 10.8 Hz, 1 H, H-20), 5.14 (d, J ¼ 17.4 Hz,
1 H, H-20), 4.41 (s, 2 H, H-22), 3.27 (dd, J ¼ 6.6, 4.2 Hz, 1 H), 2.38 (s, 3 H), 2.09–2.22
(m, 3 H), 1.95–2.01 (m, 2 H), 1.70 (dd, J ¼ 1.8 Hz, 1 H), 1.55–1.60 (m, 2 H), 1.37–1.45
(m, 3 H), 1.34 (s, 3 H), 1.28 (dd, J ¼ 3.0 Hz, 1 H), 1.18 (d, J ¼ 16.2 Hz, 1 H), 1.09
(s, 3 H), 1.02–1.05 (m, 1 H), 0.81 (d, J ¼ 7.2 Hz, 3 H), 0.56 (d, J ¼ 7.2 Hz, 3 H). 13C
NMR (150 MHz; CDCl3): 216.8 (C-3, C ¼ O), 164.9 (C-21, C ¼ O), 145.3 (C-19),
138.7 (phenyl C-1), 132.6 (phenyl C-4), 129.9 (phenyl C-3, 5), 128.1 (phenyl C-2, 6),
117.4 (C-20), 74.5, 70.3, 65.0, 58.0, 45.4, 44.5, 43.9, 41.8, 36.5, 36.0, 34.4, 30.3, 26.8,
26.4, 24.8, 21.7, 16.5, 14.8, 11.5. MS (ESI, m/z): 555.3 [M þ Na]þ.
3.2.2. Synthesis of 14-O-(1-piperazinyl) mulin (3)
Anhydrous piperazine (3.0 g, 35 mmol) and K2CO3 (4.8 g, 35 mmol) were added into
acetonitrile (80 ml) in three-necked round bottom flask, stirred and heated to reflux.
Compound 2 (18.5 g, 35 mmol) was dissolved into acetonitrile (50 ml) and then added

8
H.-X. LIU ET AL.
Figure 5. 2 D diagram of compound 8 showing its interactions with the 23 s rRNA active site (dis-
tances in Å).
Figure 6. 3 D diagram of tiamulin showing its interaction with the 23 s rRNA active site.
dropwise to the above mixture. The mixture was reacted about 8 h, followed by cool-
ing to room temperature. Double volume of water was added and stirred for 1 h,
then filtered and the filter cake was washed to neutral with water. White solid
(12.7 g) in 82% yield was obtained after full drying at 80 ꢁC. The next step of reaction
could be carried out directly without purification. IR (KBr, cmꢀ1): 3422, 2930, 2860,
1
1734, 1458, 1287, 1194, 1153, 1117, 1016, 911, 852. H NMR (600 MHz; CDCl3): d
(p.p.m.) 6.44 (dd, J ¼ 10.8, 6.6 Hz, 1 H, H-19), 5.73 (d, J ¼ 8.4 Hz, 1 H, C11-OH), 5.27
(d, J ¼ 10.8 Hz, 1 H, H-20), 5.15 (d, J ¼ 17.4 Hz, 1 H, H-20), 3.58 (s, 1 H), 3.29
(d, J ¼ 6.6 Hz, 1 H), 3.10 (d, J ¼ 16.8 Hz, 1 H), 2.98 (d, J ¼ 16.8 Hz, 1 H), 2.94–2.95

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
9
Figure 7. 2 D diagram of tiamulin showing its interaction with the 23 s rRNA active site.
(m, 3 H), 2.49–2.59 (m, 4 H, piperazinyl H-2, 6), 2.24–2.30 (m, 2 H), 2.09–2.22
(m, 4 H, piperazinyl H-3, 5), 1.98–2.03 (m, 2 H), 1.72 (dd, J ¼ 3.0 Hz, 1 H), 1.56–1.64
(m, 2 H), 1.45–1.52 (m, 1 H,), 1.38–1.41 (m, 1 H), 1.37 (s, 3 H), 1.26–1.31 (m, 1 H),
1.24 (d, J ¼ 16.2 Hz, 1 H), 1.19–1.22 (m, 3 H), 0.81 (d, J ¼ 6.6 Hz, 3 H), 0.65
(d, J ¼ 6.6 Hz, 3 H). 13C NMR (150 MHz; CDCl3): 217.1 (C-3, C ¼ O), 169.0 (C-21,
C ¼ O), 139.0, 117.3, 74.6, 68.3, 60.2, 58.2 (C-22), 53.0 (piperazinyl C-2,6), 45.4
(piperazinyl C-3,5), 45.1, 45.0, 43.9, 41.7, 36.7, 36.0, 34.5, 30.4, 26.8, 26.3, 24.8, 16.7,
14.9, 11.5. MS (ESI, m/z): 447.3 [M þ H]þ.
3.2.3. Synthesis of 2-chloro-N-(2-methyl-4-nitrophenyl) acetamide (7)
2-Methyl-4-nitroaniline (9.8 g, 64.5 mmol) and triethylamine (7.2 g, 70.9 mmol) were
dissolved in CH2Cl2, and chloroacetyl chloride (7.28 g, 64.5 mmol) was added drop-
wise to the resultant mixture. The reaction was carried out at room temperature for
about 5 h, then most dichloromethane was evaporated in vacuum and ethyl acetate
(100 ml) was added to the residue. The extract was washed with saturated sodium
chloride to neutral and the organic phase was dried with anhydrous Na2SO4 for 2 h.
Vacuum concentration afforded the crude product (12.25 g) in 83.2% yield. The next
step of reaction could be carried out directly without purification.
3.2.4. Synthesis of 14-O-1-(4-(2-methyl-4-nitrophenyl) acetamide) piperazine
mulin (8)
Compound 7 (10 g, 43.8 mmol) and triethylamine (4.87 g, 48.1 mmol) were dissolved
in DMF (60 ml), and the mixture was stirred and heated to 80 ꢁC. Compound 3
(19.56 g, 43.8 mmol) was dissolved in DMF (40 ml), and then added dropwise to the
above mixture slowly. The majority of DMF was evaporated in vacuum after the end
of the reaction and water (100 ml) was added to the residue, then solid was precipi-
tated out by stirring. The crude target product was obtained by filtrating, and the fil-
ter cake was rinsed with water (30 ml ꢂ 3) and dried at 80 ꢁC. The crude product

10
H.-X. LIU ET AL.
was recrystallized using ethyl acetate and petroleum ether to afford pure product
1
(20.41 g), in 73.1% yield. H NMR (600 MHz, CDCl3): d 8.85 (d, J ¼ 13.2 Hz, 1 H,
phenyl H-5), 8.09–8.13 (m, 2 H, phenyl H-3, 6), 6.51 (dd, J ¼ 16.8, 9.6 Hz, 1 H, H-19),
5.78 (d, J ¼ 12.6 Hz, 1 H, H-19), 5.33 (d, J ¼ 16.2 Hz, 1 H, H-20), 5.19 (dd, J ¼ 12.6 Hz,
1 H, H-20), 3.36–3.38 (m, 1 H), 3.22–3.26 (m, 3 H), 3.09 (d, J ¼ 17.2 Hz, 1 H),
2.54–2.74 (m, 8 H, piperazinyl), 2.39 (s, 1 H), 2.31–2.35 (m, 1 H), 2.19–2.26 (m, 2 H),
2.07–2.10 (m, 2 H), 1.75–1.79 (m, 1 H), 1.62–1.69 (m, 3 H), 1.53–1.56 (m, 1 H),
1.47–1.49 (m, 1 H), 1.43–1.45 (m, 4 H), 1.34–1.38 (m, 1 H), 1.25–1.30 (m, 2 H), 1.16
(s, 3 H), 1.09–1.14 (m, 1 H), 0.88 (d, J ¼ 10.8 Hz, 3 H), 0.72 (d, J ¼ 10.8 Hz, 3 H). 13C
NMR (150 MHz, CDCl3) d 217.0 (C-3, C ¼ O), 168.8 (C-21, C ¼ O), 168.5 (C-24,
C ¼ O), 143.0 (C-19), 141.7 (phenyl C-4), 139.1 (phenyl C-1), 126.3 (phenyl C-2),
125.5 (phenyl C-3), 123.1 (phenyl C-5), 119.2 (C-20), 117.2 (phenyl C-6), 74.5, 68.4,
61.9 (C-23), 59.6, 58.1 (C-22), 53.2 (piperazinyl), 53.0, 45.4, 44.9, 43.9, 41.7, 36.6,
36.0, 34.4, 30.3, 26.8, 26.4, 24.8, 17.9, 16.7, 14.8, 11.5. MS (ESI, m/z):
661.6 [M þ Na]þ.
3.2.5. Synthesis of 14-O-1-(4-(2-methyl-4-aminophenyl)acetamide)piperazine
mulin (9)
Reduced iron powder (1.53 g, 27.3 mmol), ammonium chloride (2.19g, 40.9 mmol),
acetic acid (10 ml) and water (20 ml) were stirred together and heated to 90ꢁC.
Compound 8 (8.7 g, 13.6mmol) was dissolved in absolute ethyl alcohol (50ml) and
was added dropwise to the above mixed solution. Most ethyl alcohol was evaporated in
vacuum after complete reaction, and saturated sodium bicarbonate was added to the
residue to adjust pH to 7, followed by extracting with ethyl acetate and concentrating
the organic phase by reduced pressure distillation to give crude dark brown product. It
was chromatographed on silica gel and recrystallized with a mixture of ethyl acetate
1
and petroleum ether to give pure product (7.38g) in 89% yield. H NMR (600 MHz,
CDCl3) d 8.91 (s, 1 H, NHC ¼ O), 7.66 (d, J ¼ 12.0 Hz, 1 H, phenyl H-6), 6.56 (s, 1 H,
phenyl H-3), 6.48–6.53 (m, 1 H, phenyl H-5), 5.79 (d, J ¼ 12.6 Hz, 1 H, H-19), 5.34 (dd,
J ¼ 16.8, 1.8 Hz, 1 H, H-20), 5.20 (dd, J ¼ 17.4, 1.8 Hz, 1 H, H-20), 3.56 (s, 2 H, H-22),
3.35 (dd, J ¼ 9.6 Hz, 1 H), 3.22 (d, J ¼ 16.8Hz, 1 H), 3.15 (s, 2 H), 3.07 (d, J ¼ 16.8 Hz,
1 H), 2.70 (s, 6 H), 2.60 (s, 2 H), 2.31–2.38 (m, 1 H), 2.20–2.28 (m, 2 H), 2.18 (s, 3 H,
phenyl C-2, CH3), 2.04–2.10 (m, 2 H), 1.77 (dd, J ¼ 4.8 Hz, 1 H), 1.60–1.69 (m, 3 H),
1.51–1.57 (m, 1 H), 1.42–1.49 (m, 5 H), 1.34–1.39 (m, 1 H), 1.24–1.31 (m, 1 H), 1.16 (s,
3 H), 1.09–1.13 (m, 1 H), 0.87 (d, J ¼ 10.8 Hz, 3 H), 0.72 (d, J ¼ 10.8 Hz, 3 H). 13C NMR
(150MHz, CDCl3) d 217.3 (C-3), 169.1 (C-21), 168.0 (C-24, C ¼ O), 143.7, 139.2 (phe-
nyl C-4), 130.0 (phenyl C-2), 127.1 (phenyl C-1), 123.7 (phenyl C-6), 117.4 (phenyl C-
3), 117.0 (phenyl C-5), 113.5 (C-20), 74.7, 68.4, 62.0 (C-23), 59.8, 58.3 (C-22), 53.4
(piperazinyl), 53.2, 45.6, 45.1, 44.0, 41.9, 36.8, 36.1, 34.6, 30.5, 26.9, 26.5, 24.9, 18.1,
16.8, 15.0, 11.6. MS (ESI, m/z): 609.4 [Mþ H]þ.
3.2.6. Synthesis of 14-O-1-(4-(2-methyl-4-alkylamino phenyl) acetamide) pipera-
zine mulin (10a–f)
Compound 9 (2.7 g, 4.4 mmol) and equivalent halogenated alkane were dissolved in
DMF (15 ml) and triethylamine (0.49 g, 4.8 mmol) was added to the above mixture as

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
11
acid-binding agent. Water (20 ml) was added to the residue after complete reaction.
And solid was precipitated after stirring. The crude product was obtained by filtrat-
ing, rinsing the filter cake with water and drying at 80 ꢁC. It was purified by silica gel
chromatography that afforded the pure product.
3.2.6.1. 14-O-1-(4-(2-Methyl-4-methylamino phenyl) acetamide) piperazine mulin
1
(10a). White powdery solid, yield 55.2%. H NMR (600 MHz, CDCl3) d 8.87 (s, 1 H,
NHC ¼ O), 7.65 (d, J ¼ 8.4 Hz, 1 H, phenyl H-6), 6.49–6.53 (m, 1 H, phenyl H-3),
6.43–6.48 (m, 2 H, phenyl H-5 and -NH-), 5.79 (d, J ¼ 8.4 Hz, 1 H), 5.33 (dd, J ¼ 17.4,
1.2 Hz, 1 H, H-20), 5.19 (dd, J ¼ 17.4, 1.2 Hz, 1 H, H-20), 3.35 (s, 1 H), 3.20 (d,
J ¼ 16.8 Hz, 1 H), 3.15 (s, 2 H), 3.08 (d, J ¼ 16.8 Hz, 1 H), 2.78 (s, 3 H, NH-CH3),
2.60–2.71 (m, 8 H, piperazinyl), 2.34 (t, J ¼ 6.6 Hz, 1 H), 2.22–2.28 (m, 1 H), 2.17–2.20
(m, 3 H), 2.05–2.09 (m, 2 H), 1.77 (dd, J ¼ 14.4, 2.4 Hz, 1 H), 1.62–1.68 (m, 2 H),
1.54–1.57 (m, 1 H), 1.45–1.50 (m, 2 H), 1.44 (s, 3 H), 1.36 (dd, J ¼ 13.8, 3.0 Hz, 1 H),
1.28 (d, J ¼ 16.2 Hz, 1 H), 1.16 (s, 4 H), 0.87 (d, J ¼ 7.2 Hz, 3 H), 0.72 (d, J ¼ 7.2 Hz,
3 H). 13C NMR (150 MHz, CDCl3) d 217.3 (C-3 C ¼ O), 169.1 (C-21 C ¼ O), 168.0
(C-24 C ¼ O), 147.0, 139.2 (phenyl C-4), 130.2 (phenyl C-2), 126.0 (phenyl C-1),
124.0 (phenyl C-6), 117.4 (phenyl C-5), 114.2 (phenyl C-3), 110.8, 74.7, 68.5, 62.0,
59.9, 58.3, 53.4 (piperazinyl), 53.2, 45.6, 45.2, 44.1, 41.9, 36.9, 36.2, 34.6, 31.1, 30.6,
27.0, 26.5 (-NHCH3), 25.0, 18.3, 16.9, 15.0, 11.6. MS (ESI, m/z): 623.9 [M þ H]þ.
3.2.6.2. 14-O-1-(4-(2-Methyl-4-ethylaminophenyl) acetamide) piperazine mulin
1
(10 b). White powdery solid, yield 58.5%. H NMR (600 MHz, CDCl3) d 8.86 (s, 1 H,
NHC ¼ O), 7.64 (d, J ¼ 8.6 Hz, 1 H, phenyl H-6), 6.43–6.53 (m, 3 H, phenyl H-3, 5
and -NH-), 5.78 (d, J ¼ 8.5 Hz, 1 H), 5.32 (dd, J ¼ 17.4, 1.2 Hz, 1 H, H-20), 5.19 (dd,
J ¼ 17.4, 1.2 Hz, 1 H, H-20), 3.33–3.36 (m, 1 H), 3.21 (d, J ¼ 16.8 Hz, 1 H), 3.14 (s,
2 H), 3.10–3.14 (m, 2 H, -NHCH2CH3), 3.07 (d, J ¼ 16.8 Hz, 1 H) , 2.59–2.70 (m 8 H),
2.35 (t, J ¼ 6.6 Hz, 1 H), 2.20–2.28 (m, 2 H), 2.18 (s, 3 H), 2.04–2.09 (m, 2 H), 1.76
(dd, J ¼ 14.4, 2.4 Hz, 1 H), 1.62–1.67 (m, 2 H), 1.50–1.58 (m, 2 H), 1.45–1.50 (m, 2 H),
1.44 (s, 3 H), 1.36 (dd, J ¼ 13.8, 3.0 Hz, 1 H), 1.28 (d, J ¼ 16.2 Hz, 1 H), 1.23 (t,
J ¼ 7.2 Hz, 3 H, -NHCH2CH3), 1.14 (s, 4 H), 1.10–1.14 (m, 2 H), 0.87 (d, J ¼ 7.2 Hz,
3 H), 0.72 (d, J ¼ 7.2 Hz, 3 H). 13C NMR (150 MHz, CDCl3) d 217.3 (C-3 C ¼ O),
169.1 (C-21 C ¼ O), 168.0 (C-24 C ¼ O), 146.0, 139.2 (phenyl C-4), 130.2 (phenyl C-
2), 126.0 (phenyl C-1), 123.9 (phenyl C-6), 117.4 (phenyl C-5), 114.6 (phenyl C-3),
111.1, 74.7, 68.4, 62.0, 59.8, 58.3, 53.4, 53.2 (piperazinyl), 45.6, 45.1, 44.1, 41.9
(-NHCH2CH3), 38.8, 36.8, 36.2, 34.6, 30.5, 26.9, 26.5, 25.0, 18.3, 16.8 (-NHCH2CH3),
15.0, 11.6. MS (ESI, m/z): 637.8 [M þ H]þ.
3.2.6.3. 14-O-1-(4-(2-Methyl-4-propylamino phenyl) acetamide) piperazine mulin
1
(10c). White powdery solid, yield 62.3%. H NMR (600 MHz, CDCl3) d 8.85 (s, 1 H,
NHC ¼ O), 7.63 (d, J ¼ 8.6 Hz, 1 H, phenyl H-6), 6.43–6.53 (m, 3 H, phenyl H-3, 5
and -NH-), 5.79 (d, J ¼ 8.5 Hz, 1 H), 5.33 (dd, J ¼ 17.4, 1.2 Hz, 1 H, H-20), 5.19 (dd,
J ¼ 17.4, 1.2 Hz, 1 H, H-20), 3.35 (s, 1 H), 3.21 (d, J ¼ 16.8 Hz, 1 H), 3.12–3.16 (m,
2 H, -NHCH2CH2CH3), 3.03–3.09 (m, 3 H), 2.60–2.70 (m, 8 H, piperazinyl), 2.34 (t,
J ¼ 6.6 Hz, 1 H), 2.22–2.28 (m, 1 H), 2.20–2.26 (m, 2 H), 2.18 (s, 3 H), 2.04–2.09 (m,

12
H.-X. LIU ET AL.
2 H), 1.77 (dd, J ¼ 14.4, 2.4 Hz, 1 H), 1.64–1.67 (m, 2 H, -NHCH2CH2CH3), 1.60–1.62
(m, 2 H), 1.54–1.57 (m, 1 H), 1.49–1.51 (m, 1 H), 1.45–1.47 (m, 1 H), 1.44 (s, 3 H),
1.36 (dd, J ¼ 13.8, 3.0 Hz, 1 H), 1.28 (d, J ¼ 16.2 Hz, 1 H), 1.16 (s, 3 H), 1.10–1.15 (m,
1 H), 0.98 (t, J ¼ 7.2 Hz, 3 H, -NHCH2CH2CH3), 0.87 (d, J ¼ 7.2 Hz, 3 H), 0.72 (d,
J ¼ 7.2 Hz, 3 H). 13C NMR (150 MHz, CDCl3) d 217.3 (C-3 C ¼ O), 169.1 (C-21
C ¼ O), 168.0 (C-24 C ¼ O), 146.1, 139.2 (phenyl C-4), 130.2 (phenyl C-2), 125.8
(phenyl C-1), 124.0 (phenyl C-6), 117.4 (phenyl C-5), 114.6 (phenyl C-3), 111.0, 74.7,
68.4, 62.0, 59.9, 58.3, 53.4, 53.2 (piperazinyl), 46.2 (-NHCH2CH2CH3), 45.6, 45.1,
44.1, 41.9, 36.8, 36.2, 34.6, 30.6, 26.9, 26.5, 25.0 (-NHCH2CH2CH3), 22.8, 18.3, 16.9,
15.0, 11.7 (-NHCH2CH2CH3). MS (ESI, m/z): 673.7 [M þ Na]þ.
3.2.6.4. 14-O-1-(4-(2-Methyl-4-isopropylamino phenyl) acetamide) piperazine mulin
(10d). White powdery solid, yield 56.3%.1H NMR (600 MHz, CDCl3) d 8.86 (s, 1 H,
NHC ¼ O), 7.64 (d, J ¼ 8.6 Hz, 1 H, phenyl H-6), 6.52 (dd, J ¼ 11.4, 6.0 Hz, 1 H,
phenyl H-3), 6.42–6.46 (m, 2 H, phenyl H-5 and -NH-), 5.79 (d, J ¼ 8.5 Hz, 1 H),
5.34 (dd, J ¼ 17.4, 1.2 Hz, 1 H, H-20), 5.20 (dd, J ¼ 17.4, 1.2 Hz, 1 H, H-20),
3.57–3.61 (m, 1 H, -NHCH(CH3)2), 3.35 (dd, J ¼ 6.6, 4.2 Hz, 1 H), 3.21 (d,
J ¼ 16.8 Hz, 1 H), 3.15 (s, 2 H), 3.08 (d, J ¼ 16.8 Hz, 1 H), 2.61–2.67 (m, 8 H, pipera-
zinyl), 2.35 (t, J ¼ 7.2 Hz, 1 H), 2.21–2.29 (m, 2 H), 2.18–2.20 (m, 3 H), 2.05–2.10
(m, 2 H), 1.77 (dd, J ¼ 14.4, 2.4 Hz, 1 H), 1.63–1.68 (m, 2 H), 1.55–1.61 (m, 3 H),
1.45 (s, 3 H), 1.46–1.48 (m, 2 H), 1.35–1.38 (m, 1 H), 1.28 (d, J ¼ 16.2 Hz, 1 H), 1.19
(d, J ¼ 6.6 Hz, 6 H, -NHCH(CH3)2), 1.15 (s, 3 H), 0.88 (d, J ¼ 6.6 Hz, 3 H), 0.73 (d,
J ¼ 6.6 Hz, 3 H). 13C NMR (150 MHz, CDCl3) d 217.3 (C-3 C ¼ O), 169.2 (C-21
C ¼ O), 168.0 (C-24 C ¼ O), 145.1, 139.2 (phenyl C-4), 130.2 (phenyl C-2), 125.8
(phenyl C-1), 124.0 (phenyl C-6), 117.5 (phenyl C-5), 115.3 (phenyl C-3), 111.6,
74.7, 68.5, 62.1, 59.9, 58.3, 53.5, 53.3 (piperazinyl), 45.6 (-NHCH(CH3)2), 45.2,
44.6, 44.1, 41.9, 36.9, 36.2, 34.6, 30.6, 27.0, 26.5, 25.0 (-NHCH(CH3)2), 23.2, 18.3,
16.9, 15.1, 11.7. MS (ESI, m/z): 673.7 [M þ Na]þ.
3.2.6.5. 14-O-1-(4-(2-Methyl-4-allylamino phenyl) acetamide) piperazine mulin (10e).
White powdery solid, yield 79.3%. 1H NMR (600 MHz, CDCl3) d 8.87 (s, 1 H,
NHC ¼ O), 7.65 (d, J ¼ 8.4 Hz, 1 H, phenyl H-6), 6.49–6.56 (m, 3 H, phenyl H-3, 5 and
-NH-), 5.78–5.86 (m, 2 H, -NHCH2CH¼CH2 and H-19), 5.30–5.35 (m, 1 H,
-NHCH2CH ¼ CH2), 5.14–5.21 (m, 4 H, -NHCH2CH ¼ CH2 and C-11-OH and H-20),
3.88 (d, J¼ 4.8 Hz, 4 H), 3.34–3.37 (m, 1 H), 3.21 (d, J ¼ 16.8 Hz, 1 H), 3.15 (s, 2 H), 3.07
(d, J¼ 16.8 Hz, 1 H), 2.47–2.70 (m, 8 H, piperazinyl), 2.32–2.37 (m, 1 H), 2.22–2.25 (m,
1 H), 2.20 (s, 3 H), 2.05–2.09 (m, 2 H), 1.78 (dd, J¼ 14.4, 3.0 Hz, 1 H), 1.63–1.68 (m,
2 H), 1.52–1.59 (m, 1 H), 1.45–1.49 (m, 1 H), 1.44 (s, 3 H), 1.37 (dd, J¼ 13.8, 3.0 Hz,
1 H), 1.28 (d, J ¼ 16.2 Hz, 1 H), 1.10–1.18 (m, 4 H), 0.89 (d, J¼ 7.2 Hz, 3 H), 0.73 (d,
J ¼ 7.2 Hz, 3 H). 13C NMR (150 MHz, CDCl3) d 217.3 (C-3 C ¼ O), 169.1 (C-21 C ¼ O),
168.0 (C-24 C ¼ O), 146.4, 139.2 (phenyl C-4), 134.1 (-NHCH2CH ¼ CH2), 129.9 (phe-
nyl C-2), 125.2 (phenyl C-1), 123.8 (phenyl C-6), 117.4 (phenyl C-5), 116.1 (phenyl
C-3), 114.3 (-NHCH2CH¼CH2), 110.9, 74.7, 68.5, 62.0, 59.9, 58.3, 53.4, 53.2, 53.0
(piperazinyl), 45.6 (-NHCH2CH ¼ CH2), 45.2, 44.1, 41.9, 36.9, 36.2, 34.6, 30.6, 27.0, 26.5,
25.0, 18.6, 16.9, 15.0, 11.7. MS (ESI, m/z): 649.5 [M þ H]þ.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
13
3.2.6.6. 14-O-1-(4-(2-Methyl-4-benzylamino phenyl) acetamide) piperazine mulin
1
(10f). White powdery solid, yield 68.3%. H NMR (600 MHz, CDCl3) d 8.87 (s, 1 H,
NHC ¼ O), 7.65 (d, J ¼ 8.5 Hz, 1 H, phenyl H-6), 7.26–7.39 (m, 5 H, phenyl H-3, 5
and -NH- and phenyl H-3’, 5’) , 6.48–6.54 (m, 3 H, phenyl H-2’, 4’, 6’), 5.80 (d,
J ¼ 8.5 Hz, 1 H, H-19), 5.34 (dd, J ¼ 17.4, 1.2 Hz, 1 H), 5.18–5.21 (m, 1 H), 4.31 (s,
2 H), 3.36 (dd, J ¼ 6.6, 4.2, Hz, 1 H), 3.21 (d, J ¼ 16.8, 1 H), 3.15 (s, 2 H), 3.07 (d,
J ¼ 16.8, 1 H), 2.61–2.70 (m, 8 H, piperazinyl), 2.34–2.36 (m, 1 H), 2.18–2.24 (m, 4 H),
2.05–2.10 (m, 2 H), 1.78 (dd, J ¼ 14.4, 2.4 Hz, 1 H), 1.60–1.69 (m, 4 H), 1.53–1.58 (m,
1 H), 1.46–1.48 (m, 2 H), 1.45 (s, 3 H), 1.37 (dd, J ¼ 13.8, 2.4 Hz, 1 H), 1.29 (d,
J ¼ 16.2 Hz, 1 H), 1.11–1.17 (m, 4 H), 0.88 (d, J ¼ 7.2 Hz, 3 H), 0.73 (d, J ¼ 7.2 Hz,
3 H). 13C NMR (150 MHz, CDCl3) d 217.3 (C-3 C ¼ O), 169.1 (C-21 C ¼ O), 168.0
(C-24 C ¼ O), 145.7, 139.6 (phenyl C-1’), 139.2 (phenyl C-4), 130.2 (phenyl C-2),
128.8 (phenyl C-3’, 5’), 127.6 (phenyl C-2’, 6’), 127.5 (phenyl C-1), 124.0 (phenyl C-
6), 117.5 (phenyl C-4’), 114.7 (phenyl C-3), 111.2 (phenyl C-5), 74.7, 68.5, 62.0, 59.9,
58.3, 53.4, 53.2, 48.6, 45.6, 45.2, 44.1, 41.9, 36.9, 36.2, 34.6, 30.6, 27.0, 26.5, 25.0, 18.3,
16.9, 15.1, 11.7. MS (ESI, m/z): 699.7 [M þ H]þ.
3.3. Minimum inhibitory concentrations (MIC) testing
MICs of compounds 8, 9, 10a-f against Gram-positive bacteria (MSSA, MRSA, MSSE,
Staphylococcus suis and E. faecalis) and Gram-negative bacteria (E. coli) were investi-
gated using tiamulin fumaric acid and erythromycin sulfur as the reference agents
based on the Clinical and Laboratory Standards Institute (CLSI). The tested com-
pounds were dissolved in DMSO (dimethyl sulfoxide), and then diluted with sterile
water to make the concentration of stock solutions be 2560 lg/ml. Then, 1 ml of the
stock solution was added 9 ml sterile water and diluted to 256 lg/ml as the initial test
concentration. The above six bacteria were cultured in Mueller–Hinton Broth (MHB)
overnight and cell concentrations of those bacteria were diluted to 106–107 CFU/ml
as the test bacteria solution, respectively. Each well of the 96-well plates was added
100 ll of MHB and 100 ll initial solution was added to the first column to make the
concentration of the test compounds be 128 lg/ml. Then 100 ll of the solution in the
first column was pipetted into the second column and homogeneously mixed to
make the concentration of the test compounds in the second column be 64 lg/ml.
And the same two-fold dilution method was employed to adjust the concentrations of
the test compounds in columns 3–10 to 32, 16, 8, 4, 2, 1, 0.5 and 0.25 lg/ml, and
then each well of the columns 1–10 was added 100 ll of test bacteria solution.
Column 11 was added only 200 ll MHB and column 12 was added only 200 ll test
bacteria solution. The 96-well plates were cultured at 37 ꢁC for 24 h. The MIC values
were defined by the OD values of the lowest concentration which were compared to
that of MHB (blank control). All data were tested in parallel triplicate.
3.4. Molecular modeling
Molecular docking study was performed by Molecular Operating Environment
(MOE) 2018. 0101 release of Chemical Computing Group Inc., Montreal, Quebec,

14
H.-X. LIU ET AL.
Canada to fully understand the interaction between those new derivatives with recep-
tor targets and further study the structure–activity relationship of this series of com-
pounds [19]. The triangle matcher placement method and London dG scoring
function were used for evaluation of the binding pattern and binding affinity of the
ligands and refinement of the obtained results. The X-ray crystal structure of
Deinococcus radiodurans with tiamulin (PDB 1 D: 1XBP) was obtained from the
Protein Data Bank and was used to construct the initial model [20]. The co-crystal-
lized protein was prepared for docking study by removal of DNA chains, water mole-
cules and ligands, and then the enzyme was prepared using protonate 3 D protocol in
MOE with default options. Validation of the docking setup was done by redocking
the co-crystallized ligand (tiamulin) in the active site to study the scoring energy(s),
root mean standard deviation (RMSD) and amino acid interactions. The validated
process was used to predict the binding interactions and affinity of the designed com-
pounds at the active site. Binding score, number and lengths of the hydrogen bonds
and amino acid interactions were determined and shown in Table 2.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This research was funded by the Research Project of Hebei Administration of Chinese
Medicine [No. 2019080], Doctor Foundation of Hebei University of Chinese Medicine [No.
BSZ2018006] and Basic Research Program of Outstanding Young Teachers of Hebei
University of Chinese Medicine [No. YQ2018004].
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