Design, synthesis and antibacterial activity evaluation of pleuromutilin derivatives according to twin drug theory

Article abstract of DOI:10.1080/10286020.2021.1920581

A series of novel pleuromutilin derivatives were designed and synthesized based on the twin drugs theory. Piperazinyl and thioether were used as the linkage to connect the pleuromutilin nuclear and sulfonamide to improve the biological activity and water

Full text of DOI:10.1080/10286020.2021.1920581

Journal of Asian Natural Products Research
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ganp20
Design, synthesis and antibacterial activity
evaluation of pleuromutilin derivatives according
to twin drug theory
Hui-Xian Liu, Ge Cui, Dong-Lai Ma, Yue Zhang & Fei-Qun Xue
To cite this article: Hui-Xian Liu, Ge Cui, Dong-Lai Ma, Yue Zhang & Fei-Qun Xue (2021): Design,
synthesis and antibacterial activity evaluation of pleuromutilin derivatives according to twin drug
theory, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2021.1920581
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JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
https://doi.org/10.1080/10286020.2021.1920581
Design, synthesis and antibacterial activity evaluation of
pleuromutilin derivatives according to twin drug theory
a
b
a
b
c
Hui-Xian Liu , Ge Cui , Dong-Lai Ma , Yue Zhang and Fei-Qun Xue
a
b
College of Pharmacy, Hebei University of Chinese Medicine, Shijiazhuang 050200, China; College of
Chemical and Pharmaceutical Engineering, Hebei University of Science and Technology, Shijiazhuang
c
0
50018, China; Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences,
Shanghai 200241, China
ABSTRACT
ARTICLE HISTORY
A series of novel pleuromutilin derivatives were designed and
synthesized based on the twin drugs theory. Piperazinyl and thio-
ether were used as the linkage to connect the pleuromutilin
nuclear and sulfonamide to improve the biological activity and
water solubility of derivatives. The in vitro antibacterial activities
against drug-sensitive and drug-resistance bacteria were eval-
uated by agar dilution method. Most of the 25 compounds dis-
played excellent antibacterial activities against drug-sensitive
bacteria, particularly, five compounds (9, 10, 11, 14a and 14b)
exerted the excellent antibacterial activities against drug-resist-
ance bacteria.
Received 21 March 2020
Accepted 18 April 2021
KEYWORDS
Pleuromutilin derivatives;
antibacterial activity; design;
synthesis; twin drugs theory
1. Introduction
More and more drug-resistant pathogenic bacteria yielded in the past years because
of the abuse of antibiotics, which resulted in many available drugs ineffective on
those bacteria. More than two million people die from that each year and human
CONTACT Yue Zhang
yuezhang@hebust.edu.cn; Fei-Qun Xue
fqxue@outlook.com
Supplemental data for this article is available online at https://doi.org/10.1080/10286020.2021.1920581.
ß 2021 Informa UK Limited, trading as Taylor & Francis Group

2
H.-X. LIU ET AL.
Figure 1. The structures of pleuromutilin and related drugs.
health was under dangerous severely [1]. Pleuromutilin and its derivatives showed
great activities against the drug-resistant Gram-positive bacteria in vitro and had no
target-specific cross-resistance to other antibiotics [2, 3].
Pleuromutilin was first isolated from Basidiomycetes Pleurotusmutilus in 1951 [4].
The unusual tricyclic diterpenoid structure of pleuromutilin can disturb the protein
synthesis via interacting with the 50 s bacterial ribosome to exert its antibacterial
activity against Gram-positive bacteria and mycoplasmas [5], therefore, it has become
a lead compound of antibiotics. But pleuromutilin has many disadvantages, such as
poor water solubility and pharmacokinetic properties, low oral bioavailability and
mild toxicity, etc., so it is difficult to be developed directly as an antibacterial drug.
In recent years, some new pleuromutilin derivatives were developed with higher anti-
bacterial activities. According to the studies, the best design idea was to effectively
modify the side chain at C-14 and the breakthroughs in antibacterial activity were
made when both of the thioether and basic group were arranged in the same side
chain. Further modifications within these groups led to the development of four
drugs tiamulin, valnemulin, retapamulin and lefamulin (Figure 1). Tiamulin and val-
nemulin were approved as therapeutic agents for veterinary in 1974 and 1999,
respectively. They display good antibacterial activities against mycoplasma and some
gram-positive bacteria [6, 7]. In April 2007, retapamulin ointment (Altabax) was
developed by Glaxo Smith Kline and approved by the Food and Drug Administration
(FDA) for the treatment of skin and skin-structure infections (SSSIs) caused by
Staphylococcus aureus and Streptococcus pyogenes infection. It became the first pleuro-
mutilin approved for human use [8]. Lefamulin was approved by FDA on 19 August
2019 for the treatment of community-acquired bacterial pneumonia (CABP) [9]. The
successful marketing of the above drugs further demonstrated the modification of
C14 site was a great idea to develop drugs.
Piperazine was widely used in many antimicrobial drugs because of its good water
solubility and electron-rich property, such as temafloxacin, piperaquine, delavirdine,
norfloxacin and ketoconazole, etc. (Figure 2). We were inspired by those structures
and eager to improve water solubility of pleuromutilin, and piperazine was also intro-
duced to pleuromutilin as the linkage of C14 side chain in our previous work and
some modified compounds showed good biological activity [10, 11].

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
Figure 2. Antimicrobial drugs with piperazine rings.
In the 1970s, a number of pleuromutilin derivatives were synthesized and the
structure-activity relationships (SAR) studies implied that introduction of thioether
and alkaline group in the C14 side chain would improve antibacterial activity [6].
Beyond that, lots of studies have also confirmed this opinion. Such as Cheng-Hong
Li’s team reported the novel semisynthetic pleuromutilin analogues embracing 7H-
pyrrolo[2,3-d]pyrimidine backbone in the C14 side chain [12]. A series of novel
acetamide phenyl pleuromutilin derivatives incorporating 2-aminothiophenol moieties
into the C14 side chain were synthesized by You-zhi Tang’s team and one of them
has potential drug-like properties for the treatment of MRSA infection [13].
The (SAR) studies showed that the ester group at C14, the carbonyl group at C3
and the hydroxyl group at C11 are all required for antibacterial activity, and the only
valid modification site is C22 side chain on C14 group [14–16]. This can be con-
firmed by the four marketed drugs (tiamulin, valnemulin, retapamulin, lefamulin).
So, in our study, the modification of pleuromutilin derivatives was focused on the
hydroxy group of C22 that links in C14 side chain. A new class of pleuromutilin
derivatives was designed and synthesized based on the previous SAR studies and twin
drug theory. We spared no effort to link the benzene-sulfonamide group to pipera-
zine and thioether group, and then the combined two groups were linked together
with C14 side chain of pleuromutilin, which aims to improve water solubility and
antibacterial activity of pleuromutilin. The design, synthesis as well as detailed anti-
bacterial activity of these novel pleuromutilin derivatives will be reported and dis-
cussed in this paper.

4
H.-X. LIU ET AL.
Figure 3. The synthetic rout of derivatives containing piperazinyl and benzenesulfonyl: (a) TsCl,
triethylamine, DCM, r.t., (b) acetonitrile, r.f., (c) K CO , acetonitrile, r.f., (d) Fe powder, NH Cl,
2
3
4
ꢀ
CH COOH, ethyl alcohol, 80 C, (e) 2-chloroacetyl chloride, DCM, triethylamine, r.t., (f) alkylamine or
3
ꢀ
arylamine, triethylamine, DMF, 100 C.
2. Results and discussion
2.1. Chemistry
As shown in Figure 3, pleuromutilin (1) reacts with p-toluene sulfochloride in the
presence of triethylamine in CH Cl to provide 22-O-(p-toluenesulfonyl) pleuromuti-
2
2
lin (2) in 52% yield. Compound 2 reacts with piperazine in acetonitrile to obtain
compound 3. Compound 4 was prepared by electrophilic addition of p-nitrobenzene
sulfonyl chloride with 3 under a basic condition in 90% yield. Compound 4 was
reduced to 5 by iron powder. Compound 5 reacts with chloroacetyl chloride to afford
6
, and then the novel derivatives were obtained after electrophilic substitution reac-
tions of 6.
The SAR studies indicated that thioether group has great value in improving the
activity of pleuromutilin, so in the following compounds we retained the thioether
group and it was built by p-aminothiophenol. p-Nitrobenzene sulfonyl chloride
was connected to the end of the side chain as twin drug component. As shown in
Figure 4, compound 2 was prepared in the same method as Figure 3. Compound 8
was obtained in the present of base by substitution reaction. The obtained crude
product was treated with trifluoroacetic acid at room temperature for 8 hours to give
compound 9. Next, K CO was employed to promote the condensation of p-nitroben-
2
3
zene sulfonyl chloride with compound 9 in acetonitrile to yield 10, and the nitryl of

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
Figure 4. The synthetic rout of derivatives containing thioether and benzenesulfonyl: (a) TsCl, trie-
ꢀ
thylamine, DCM, r.t., (b) (Boc) O, triethylamine, acetonitrile, 0 C-r.t., (c) anhydrous K CO , aceto-
2
2
3
nitrile, r.f., (d) CF COOH, DCM, r.t., (e) anhydrous K CO , acetonitrile, r.t., (f) Fe powder, NH Cl,
3
2
3
4
ꢀ
ꢀ
CH COOH, ethyl alcohol, 80 C, (g) halohydrocarbon, anhydrous K CO , DMF, 100 C, (h) 2-chloroa-
cetyl chloride, DCM, triethylamine, r.t., (i) alkylamine, anhydrous K CO , DMF, 100 C.
3
2
3
ꢀ
2
3
1
1
0 was catalyzed by iron powder to obtain amino of compound 11. Then compound
1 was reacted with some different halohydrocarbon in dimethylformamide (DMF)
to give compounds 12a–f. Compound 11 was also reacted with chloroacetyl chloride
in the presence of triethylamine and CH Cl , and the obtained crude product was
2
2
treated with K CO and some different alkylamine in DMF to give com-
2
3
pounds 14a–f.
2
.2. In vitro antibacterial activity
The antibacterial properties of 25 novel pleuromutilin derivatives 4, 5, 6, 7a–f, 9–11,
2a–f, 13 and 14a–f were assessed in vitro by the broth microdilution method and
1

6
H.-X. LIU ET AL.
Table 1. The MIC value (lg/ml) of novel pleuromutilin derivatives.
S. aureus
ATCC 25923
MRSA
NY3
S. epidermidis
ATCC 12228
S. suis
ATCC 43765
E. coli
CVCC 231
Cpd. No.
4
5
6
0.5
>64
>64
>64
32
0.06
0.06
0.06
0.06
0.25
1
0.06
0.25
0.25
0.03
0.06
0.06
0.25
1
0.5
4
0.25
1
0.25
0.03
0.03
0.06
0.25
0.25
0.06
0.125
0.125
>64
>64
>64
8
>64
>64
>64
32
0.06
0.06
0.06
0.25
1
0.06
0.06
0.06
0.03
0.125
0.06
0.25
0.5
0.5
4
0.25
2
0.25
0.03
0.03
0.25
0.25
0.25
0.03
0.125
0.125
7
7
7
7
7
7
9
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
a
b
c
d
e
f
32
32
64
>64
>64
>64
>64
4
2
4
>64
>64
>64
>64
>64
>64
32
4
2
64
64
>64
>64
16
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
64
64
64
64
64
64
64
32
0.5
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
64
64
8
64
64
64
64
16
16
0
1
2a
2b
2c
2d
2e
2f
3
4a
4b
4c
4d
4e
4f
Tiamulin fumaric
Erythromycin thiocyanate
8
determined in comparison with tiamulin fumaric and erythromycin thiocyanate. The
MIC values are provided in Table 1. The data show that most of the tested com-
pounds display good to excellent in vitro antibacterial activities against Gram-positive
bacteria except S. suis to those of the positive controls. The MICs of 25 new pleuro-
mutilin derivatives in vitro against MSSA and S. epidermidis are from ꢁ 8 lg/ml to
0.03 lg/ml. Compound 5 displays better MIC value against MSSA compared with the
control drugs, while compound 4 shows worse antibacterial activity against MSSA in
comparison to that of compound 5. This data demonstrate that derivatives containing
amino group exhibit better activity compared to those with nitro group. The alkyl
amino groups were connected on the benzene ring of compound 6 and resulted in
compounds 7a–f, and most of these derivatives possess no significant increase in
activity against drug-resistant bacteria compared with both tiamulin and compound
8
. The MIC value of the most active compounds in this series is 0.06 lg/ml.
The activity of compound 9 was evaluated (bioactivities: 9 > 5), and this observa-
tion suggests that the replacement of the piperazine on the starting position of the
C14 side chain with diphenyl sulfide can increase the electron densities of C14 side
chain of pleuromutilin derivatives, which resulted in compound 9 possessing higher
antibacterial activities compared with compound 5. The activity decreases when the
amino group of diphenyl sulfide was replaced by p-toluensulfonyl chloride. The nitro
group on benzene of compound 10 was reduced to amino group to obtain compound
1
1, leading to increased activity, and it is proved that the alkaline center is the key
factor to improve antibacterial activity. Subsequent derivatives 12a–f exhibiting poor
activity may be due to the steric hindrance. Unexpectedly, compounds 14a and 14b

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
show excellent antibacterial activity, their MIC against MSSA and MSSE are
.03 lg/ml, compound 14f (0.03 lg/ml) displayed 4-fold enhanced antibacterial
0
activity against MSSA in comparison with that of tiamulin fumaric and erythro-
mycin thiocyanate. It is worth mentioning that compounds 9, 10, 11, 14a and 14b
show the excellent activity (2 lg/ml to 4 lg/ml) to drug-resistance bacteria, 2–8
fold enhance than the control drugs. This result indicates the increase in the elec-
tron cloud density affects the binding strength of receptor pocket with pleuromuti-
lin derivatives. We can see from 14a–f that the antibacterial activity decreases with
the increase of steric hindrance and increases with the increase of electron
cloud density.
2
.3. Molecular docking study
Crystal structure studies have proposed that pleuromutilin derivatives block peptide-
bond formation directly by interfering with the substrate binding at the ribosome’s
peptidyl-transferase center (PTC) domain. Since their excellent anti-bacterial activity,
we conducted the molecular docking research of 9, 10, 11, 14a and 14b, and tiamulin
was selected as the control. As shown in Figure 4, the molecular docking of 9 bearing
p-amino thiophenol displays four interaction forces (Figure 5A), three of them are
hydrogen bonds and formed between pleuromutilin nucleus and residues G2484
(1.65 Å) and G2044 (3.1 Å). The other one is H-p interaction, and it is formed
between C14 side chain and residue A2045.
There are seven interaction forces between compound 11 (Figure 5C) and the
active pocket, including four hydrogen bonds and three H-p interactions. It was
found that OH on C11 of pleuromutilin had a strong hydrogen bond (1.91 Å) with
residue C2565. It should be notable that two hydrogen bonds are formed between
C14 side chain and active pocket, NH with U2485 (2.37 Å) and S ¼ O with G2426
(2.22 Å), respectively. The calculation showed that the binding energy of 11 with ribo-
some is ꢂ9.78 kcal/mol, which is equivalent to 9 (ꢂ9.65 kcal/mol). In terms of 14a
(Figure 5D), there are three hydrogen bonds between compound and active pocket.
One of the three hydrogen bond is formed between NH of acylamino and residues
C2565 (2.24 Å), another one is formed between C22-S- and G2484 (3.83 Å) and the
last one is formed between C22-H- and G2484 (2.35 Å). It is worth mentioning that
thioether plays a role in the formation of hydrogen bonding. The CH between thio-
ether and ester plays a crucial role, and it forms a hydrogen bond with G2484 at a
distance of 2.35 Å. There are also three H-p interactions that are attached by the
nuclear to the residue. Furthermore, the total score of the optimal conformation of
1
4a is calculated to be ꢂ11.82 kcal/mol, which indicates that the affinity activity of
1
4a with 23S rRNA is significantly higher than those of tiamulin (ꢂ8.62 kcal/mol)
and compound 11 (ꢂ9.78 kcal/mol). Though compound 14a owns fewer bonds, the
lower binding energy may explain why it shows better in vitro antibacterial activity
than others. It can be explained that why 14b (Figure 5E) owns the equal antibacter-
ial activity.
The docking research of the positive control compound tiamulin was also made to
compare its binding mode with that of above docking compounds. The result showed

8
H.-X. LIU ET AL.
Figure 5. The molecular docking results of compounds 9 (A), 10 (B), 11 (C), 14a (D), 14b (E), and
tiamulin (F).
that tiamulin formed three hydrogen bonds with the 23S rRNA and rendered a simi-
lar docking mode to that of 9, 10, 11, 14a and 14b in general. The calculated binding
free energy of tiamulin was ꢂ8.62 kcal/mol, being higher than that of 9, 10, 11, 14a
and 14b. It is consistent with the in vitro assay that the antibacterial activity of these
five compounds were better than that of tiamulin. It can be observed that the con-
formation of all the above docking compounds are consistent with that of tiamulin in
the pocket, and formed some hydrogen bonds with G2044, G2484, U2483, C2565,
A2430, U2485, G2426 and A2045, which are rendered a similar docking mode to that
of tiamulin (Figure 5F) in general. The docking result may explain why 9, 10, 11, 14a
and 14b exhibit better in vitro antibacterial activity and suggest that the introduction
of sulfonyl and NH to the C14 side chain would be effective tactics for the design of
2
pleuromutilin derivatives.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
9
2.4. Conclusion
In summary, a series of piperazine type and thioether type new pleuromutilin deriva-
tives containing sulfanilamide substituents were designed and synthesized. In compar-
able with tiamulin and erythromycin, 10 compounds (5, 6, 7a, 7d, 9, 10, 11, 14a, 14b
and 14f) showed good to excellent anti-bacterial activities to most of the tested
Gram-positive pathogens and of them, 5 compounds (9, 10, 11, 14a and 14b) exhib-
ited good antibacterial activities against the drug-resistant Gram-positive bacteria.
Therefore, the result of anti-bacterial test revealed that thioether group in pleuromuti-
lin played more important role in antibacterial activity than piperazinyl in
pleuromutilin.
3. Experimental
3.1. General experimental procedures
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). p-toluene sulfochloride, chloroacetyl chloride, p-aminothiophenol
and alkylamine were obtained from Aladdin Reagent Co. (Shanghai, China). p-
Nitrobenzene sulfonyl chloride and piperazine were purchased from Adamas Reagent
Co. (Shanghai, China).
Routine monitoring of reaction was performed by TLC using pre-coated GF254
TLC plate, which were purchased from Qingdao Ocean Chemical Co., Ltd. (Qingdao,
China). NMR spectra were recorded on a Bruker AVANCE 600 spectrometer at
6
00 MHz instrument (Bruker Daltonik, Bremen, Germany) in CDCl using tetrame-
3
thylsilane (TMS) as the internal reference. MS was performed with electron spray ion-
ization (ESI) mode and recorded on a high performance liquid chromatography
tandem mass spectrometer by Waters (Waters, Milford, Massachusetts, America).
Melting points of all compounds were defined using Kerui X-5 apparatus (Gongyi
Kerui, Gongyi, Henan, China) without correction. Elemental analysis was performed
on an Elementar vario EL cube elemental analyzer (Elementar, Germany). IR was per-
formed by Nicolet IS10 Fourier Infrared transform spectrometer (Thermo Fisher
Scientific, Waltham, America).
3
.2. Synthesis
Compounds 2 and 3 were synthesized from pleuromutilin according to the pathway
in our previous work [10], and the yield and melting point were consistent with
the reference.
3
.2.1. Synthesis of 22-(4-(p-nitrobenzene sulfonyl) piperazinyl)-22-deoxypleuromutilin (4)
Compound 3 (6 g, 13.4 mmol) and anhydrous K CO (1.86 g, 13.4 mmol) were added
2
3
into 30 ml acetonitrile, followed by stirring and heating under reflux. p-
Nitrobenzenesulfonyl chloride (2.98 g, 13.4 mmol) was dissolved in acetonitrile and
dropwise added into the above reaction mixture. Most acetonitrile was evaporated in

1
0
H.-X. LIU ET AL.
vacuum after TLC test showed the reaction was completed. The slurry was cooled to
room temperature and treated with water (100 ml) and extracted with ethyl acetate
(
50 ml ꢃ 3). Yellow solid (7.54 g) was obtained after concentrating the organic phase.
The yield of the crude product was 90%. The next step of reaction could be carried
1
out directly without purification. H NMR (600 MHz, CDCl ) d 8.37 (d, J ¼ 8.4 Hz,
3
2
1
H, phenyl H-3, 5), 7.93 (d, J ¼ 8.4 Hz, 2H, phenyl H-2, 6), 6.43 (dd, J ¼ 17.4,
0.8 Hz, 1H, H-19), 5.74 (d, J ¼ 8.4 Hz, 1H, H-14), 5.29 (d, J ¼ 10.8 Hz, 1H, 1H from
H-20), 5.16 (d, J ¼ 17.4 Hz, 1H, 1H from H-20), 3.33 (dd, J ¼ 10.8, 6.6 Hz, 1H, H-11),
3
.14 (d, J ¼ 16.8 Hz, 1H, 1H from H-22), 3.11–3.13 (m, 4H, piperazinyl H-3, 5), 3.04
(
d, J ¼ 16.8 Hz, 1H, 1H from H-22), 2.58 ꢂ 2.70 (m, 4H, piperazinyl H-2, 6),
2
3
1
.27–2.32 (m, 1H), 2.14 ꢂ 2.25 (m, 2H), 2.02 ꢂ 2.06 (m, 2H), 1.75 (dd, J ¼ 14.4,
.0 Hz, 1H), 1.64 ꢂ 1.66 (m, 1H), 1.60 ꢂ 1.62 (m, 1H), 1.48 ꢂ 1.53 (m, 1H),
.42 ꢂ 1.47 (m, 2H), 1.40 (s, 3H), 1.32 ꢂ 1.35 (m, 1H), 1.23 (d, J ¼ 15.6 Hz, 1H), 1.14
13
(
s, 3H), 1.08 ꢂ 1.11 (m, 1H), 0.86 (d, J ¼ 7.2 Hz, 3H), 0.66 (d, J ¼ 7.2 Hz, 3H). C
NMR (151 MHz, CDCl ) d 217.1, 168.8, 150.4, 141.7, 139.1, 129.1 (phenyl C-3, 5),
3
124.4 (phenyl C-2, 6), 117.4, 74.7, 68.7, 59.3, 58.2, 51.9 (piperazinyl C-2, 6), 45.9
(
1
7
piperazinyl C-3, 5), 45.5, 45.1, 44.0, 41.8, 36.7, 36.1, 34.5, 30.5, 26.9, 26.5, 24.9, 16.8,
þ
4.9, 11.6. ESIMS: m/z 654.7 [M þ Na] . Elemental analysis: Found: C, 60.78%, H,
.24%, N, 6.71%, S, 5.16%; calcd for C H N O S, C, 60.84%, H, 7.18%, N, 6.65%,
3
2
45 3 8
S, 5.07%.
3
.2.2. Synthesis of 22-(4-(p-aminobenzene sulfonyl) piperazinyl)-22-deoxypleuro-
mutilin (5)
To a solution of ammonium chloride (0.95 g, 17.4 mmol) and reduced iron powder
1.46 g, 26.1 mmol) in acetic acid (10 ml), compound 4 (5.5 g, 8.7 mmol) was dissolved
(
in absolute ethyl alcohol (20 ml) and then added slowly to the above mixed solution.
ꢀ
The mixture was stirred and heated to 80 C. After TLC test showed the reaction was
completed, saturated sodium bicarbonate was employed to adjust the pH of the mix-
ture to 7. The ethyl acetate extract was washed with water (30 ml ꢃ 3), and the
organic phase was concentrated under reduced pressure, equivalent petroleum ether
was added, followed by recrystallizing to give the pure product (3.96 g) in 75.6% yield.
1
H NMR (600 MHz, CDCl ) d 7.48 (d, J ¼ 8.4 Hz, 2H, phenyl H-2, 6), 6.62
3
(
(
(
d, J ¼ 8.4 Hz, 2H, phenyl H-3, 5), 6.43 (dd, J ¼ 17.4, 10.8 Hz, 1H, H-19), 5.74
d, J ¼ 8.4 Hz, 1H, H-14), 5.29 (dd, J ¼ 10.8, 1.4 Hz, 1H, 1H from H-20), 5.17
d, J ¼ 17.4 Hz, 1H, 1H from H-20), 4.18 (s, 2H, phenyl-NH ), 3.33 (dd, J ¼ 10.8,
2
6
.6 Hz, 1H, CHOH), 3.12 (d, J ¼ 17.4 Hz, 1H, 1H from H-22), 3.02 ꢂ 3.04 (m, 4H,
piperazinyl H-3, 5), 3.01 (d, J ¼ 17.4 Hz, 1H, 1H from H-22), 2.56 ꢂ 2.61 (m, 4H,
piperazinyl H-2, 6), 2.28 ꢂ 2.31 (m, 1H), 2.16 ꢂ 2.23 (m, 2H), 2.02 ꢂ 2.08 (m, 2H),
1
1
.73 ꢂ 1.75 (m, 2H), 1.59 ꢂ 1.65 (m, 1H), 1.47 ꢂ 1.51 (m, 1H), 1.42 ꢂ 1.45 (m, 2H),
.39 (s, 3H), 1.32 ꢂ 1.34 (m, 1H), 1.23 (d, J ¼ 16.2 Hz, 1H), 1.13 (s, 3H), 1.08 ꢂ 1.11
13
(
m, 1H), 0.85 (d, J ¼ 6.6 Hz, 3H), 0.66 (d, J ¼ 6.6 Hz, 3H). C NMR (151 MHz,
CDCl ) d 217.2, 169.0, 150.9, 139.1, 130.1 (phenyl C-3, 5), 123.2 (phenyl C-2, 6),
3
1
4
17.4, 114.0, 74.7, 68.6, 59.5, 58.3, 52.2 (piperazinyl C-2, 6), 45.5 (piperazinyl C-3, 5),
5.9, 45.1, 44.0, 41.8, 36.8, 36.1, 34.6, 30.5, 26.7, 26.5, 24.9, 16.8, 14.9, 11.6. ESIMS:

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
11
þ
m/z 624.5 [M þ Na] . Elemental analysis: Found: C, 63.96%, H, 7.75%, N, 6.95%, S,
5
.29%; calcd for C H N O S, C, 63.87%, H, 7.87%, N, 6.98%, S, 5.33%.
32 47 3 6
3
.2.3. Synthesis of 22-((4-(4-chloroacetyl amino) benzenesulfonyl) piperazinyl)-22-
deoxypleuromutilin (6)
To a stirred solution of compound 5 (7.1 g, 11.8 mmol) in CH Cl (100 ml) were
2
2
added chloroacetyl chloride (1.33 g, 11.8 mmol) and triethylamine (0.6 g, 5.9 mmol) at
room temperature. After the materials were consumed completely, water (100 ml) was
added to the reaction system. The resulting solution was extracted with CH Cl2
2
(
50 ml ꢃ 3). The combined organic layers were dried over anhydrous Na SO and
2
4
evaporated in vacuum, to obtain the crude yellowish-brown solid (6.98 g) in 87.2%
yield, which was directly used in the next step without purification. H NMR
1
(
600 MHz, CDCl ) d 8.46 (s, 1H, NHCO), 7.71 ꢂ 7.75 (m, 4H, phenyl), 6.44 (dd,
3
J ¼ 17.4, 10.8 Hz, 1H, H-19), 5.74 (d, J ¼ 8.4 Hz, 1H, H-14), 5.28 (d, J ¼ 10.8 Hz, 1H,
1
3
3
2
2
H from H-20), 5.17 (d, J ¼ 17.4 Hz, 1H, 1H from H-20), 4.22 (s, 2H, NHCOCH Cl),
2
.33 (dd, J ¼ 10.8, 6.6 Hz, 1H, CHOH), 3.13 (d, J ¼ 17.4 Hz, 1H, 1H from H-22),
.04 ꢂ 3.06 (m, 4H, piperazinyl H-3, 5), 3.03 (d, J ¼ 17.4 Hz, 1H, 1H from H-22),
.57 ꢂ 2.66 (m, 4H, piperazinyl H-2, 6), 2.29 ꢂ 2.31 (m, 1H), 2.16 ꢂ 2.23 (m, 2H),
.02 ꢂ 2.07 (m, 2H), 1.75 (dd, J ¼ 14.4, 3.0 Hz, 1H), 1.60 ꢂ 1.66 (m, 2H), 1.50 (dd,
J ¼ 14.4, 3.0 Hz, 1H), 1.41 ꢂ 1.47 (m, 2H), 1.39 (s, 3H), 1.33 (dd, J ¼ 14.4, 3.0 Hz, 1H),
1
0
1
5
3
.24 (d, J ¼ 15.6 Hz, 1H), 1.14 (s, 3H), 1.08 ꢂ 1.12 (m, 1H), 0.85 (d, J ¼ 7.2 Hz, 3H),
1
3
.67 (d, J ¼ 7.2 Hz, 3H). C NMR (151 MHz, CDCl ) d 217.2, 168.9, 164.3, 141.0,
3
39.1, 131.3, 129.3 (phenyl C-3, 5), 119.7 (phenyl C-2, 6), 117.4, 74.7, 68.7, 59.5, 58.3,
2.1 (piperazinyl C-2, 6), 45.9 (piperazinyl C-3, 5), 45.5, 45.0, 44.0, 42.9, 41.8, 36.8,
6.1, 34.6, 30.5, 26.9, 26.5, 24.9, 16.8, 14.9, 11.6. MS (ESI, m/z): 700.8 [M þ Na] .
þ
Elemental analysis: Found: C, 60.23%, H, 7.18%, N, 6.28%, S, 4.71%; calcd for
C H ClN O S, C, 60.21%, H, 7.13%, N, 6.20%, S, 4.73%.
3
4
48
3 7
3
.2.4. General procedure for the synthesis of 7a–g
A three-neck round bottom flask equipped with a magnetic stirrer was added com-
pound 6 (1.2 g, 1.8 mmol) and 20 ml of DMF. The stirred solution was then added
anhydrous K CO (0.19 g, 1.4 mmol) and equal molar quantities of alkylamine/aryl-
2
3
ꢀ
amine at 100 C for 5 h. After completion, about 15 ml DMF was evaporated in vac-
uum. The residue was treated with 20 ml H O and stirred, the solid was filtered,
2
ꢀ
rinsed with water and dried at 80 C. The residue was purified by silica gel chroma-
tography with ethyl acetate/petroleum ether (V/V 20/1 to 5/1) as eluent to give 7a–g
as a white solid.
3
.2.4.1. 22-(4-(4-(2-Ethylamino acetamino) benzenesulfonyl) piperazinyl)-22-deoxy-
ꢀ
pleuromutilin (7a). White powdery solid, yield 73.5%. m.p.: 180 ꢂ 182 C. IR (KBr,
ꢂ1
cm ): 3372, 2958, 2935, 2861, 1733, 1694, 1593, 1520, 1456, 1403, 1348, 1306, 1204,
1
1
163, 1118, 1016, 940, 738. H NMR (600 MHz, CDCl ) d 9.79 (s, 1H, NHCO), 7.76
3
(
d, J ¼ 8.4 Hz, 2H, phenyl H-3, 5), 7.68 (d, J ¼ 8.4 Hz, 2H, phenyl H-2, 6), 6.44 (dd,
J ¼ 17.4, 10.8 Hz, 1H, H-19), 5.73 (d, J ¼ 8.4 Hz, 1H, H-14), 5.28 (d, J ¼ 10.8 Hz, 1H,
H-20), 5.16 (d, J ¼ 17.4 Hz, 1H, H-20), 3.47 (s, 2H, H-22), 3.33 (d, J ¼ 6.0 Hz, 1H, H-

1
2
H.-X. LIU ET AL.
1
1), 3.13 (d, J ¼ 17.4 Hz, 1H, H-22), 3.04 ꢂ 3.06 (m, 4H, piperazinyl H-3, 5), 3.02 (d,
J ¼ 17.4 Hz, 1H, H-22), 2.78 (q, J ¼ 7.2 Hz, 2H, NH-CH -CH ), 2.57 ꢂ 2.64 (m, 4H,
2
3
piperazinyl H-2, 6), 2.28 ꢂ 2.31 (m, 1H), 2.14 ꢂ 2.24 (m, 3H), 2.02 ꢂ 2.07 (m, 2H),
1
1
3
.74 (d, J ¼ 12.6 Hz, 1H), 1.60 ꢂ 1.66 (m, 2H), 1.49 ꢂ 1.53 (m, 1H), 1.43 ꢂ 1.45 (m,
H), 1.39 (s, 3H), 1.32 ꢂ 1.34 (m, 1H), 1.23 (d, J ¼ 15.6 Hz, 1H), 1.19 (t, J ¼ 7.2 Hz,
H, NH-CH -CH ), 1.13 (s, 3H), 1.08 ꢂ 1.11 (m, 1H), 0.85 (d, J ¼ 7.2 Hz, 3H), 0.66
2
3
1
3
(
(
d, J ¼ 7.2 Hz, 3H). C NMR (151 MHz, CDCl ) d 217.2 (C-3), 170.1 (C-21), 168.9
3
CONH), 141.9 (C-19), 139.1 (phenyl C-1), 129.9 (phenyl C-4), 129.3 (phenyl C-3, 5),
1
6
2
19.1 (phenyl C-2, 6), 117.4 (C-20), 74.7, 68.6, 59.5, 58.3, 52.6, 52.1 (piperazinyl C-2,
), 45.9 (piperazinyl C-3, 5), 45.5, 45.1, 44.7, 44.0, 41.8, 36.7, 36.1, 34.6, 30.5, 26.9,
þ
6.4, 24.9, 16.8, 15.1, 14.9, 11.6. ESIMS: m/z 687.3 [M þ H] . Elemental analysis:
Found: C, 62.82%, H, 7.96%, N, 8.10%, S, 4.61%; calcd for C H N O S, C, 62.95%,
36
54 4 7
H, 7.92%, N, 8.16%, S, 4.67%.
The spectral data of 7b–7f are listed in the supplementary information.
3
.2.5. Synthesis of 22-(4-(N-Boc) thiophenyl)-22-deoxypleuromutilin (8)
To a stirred solution of K CO (2.01 g, 14.6 mmol) and compound 2 (9.7 g,
2
3
18.2 mmol) in acetonitrile (30 ml), 4-N-Boc thiophenol (4.1 g, 18.2 mmol) was dis-
solved in acetonitrile (5 ml) and added at reflux temperature. After the materials were
consumed completely, about 15 ml acetonitrile was evaporated in vacuum, and then
water (150 ml) was added to the residue. The resulting solution was extracted with
ethyl acetate (50 ml ꢃ 3). The combined organic layers were evaporated in vacuum to
obtain the crude yellowish-brown solid (9.94 g) in 93.2% yield.
3
.2.6. Synthesis of 22-(4-amino thiophenyl)-22-deoxypleuromutilin (9)
Compound 8 (13.3 g, 22.7 mmol) was dissolved into CH Cl (50 ml) and trifluoroace-
2
2
tic acid (10 ml) was dropwise added into the mixture at room temperature. After the
materials were consumed completely, the formed slurry was treated with water
(150 ml) and saturated sodium bicarbonate was employed to adjust the pH of the
mixture to 7. The resulting solution was extracted with CH Cl (50 ml ꢃ 3). The
2
2
combined organic layers were dried over anhydrous Na SO and evaporated in vac-
2
4
1
uum, to obtain the crude yellowish-brown solid (9.52 g) in 86.3% yield. H NMR
600 MHz, CDCl ) d 7.18 (d, J ¼ 8.4 Hz, 2H, phenyl H-2, 6), 6.50 (d, J ¼ 8.4 Hz, 2H,
(
3
phenyl H-3, 5), 6.39 (dd, J ¼ 17.4, 10.8 Hz, 1H, H-19), 5.64 (d, J ¼ 8.4 Hz, 1H, H-14),
5
3
1
.27 (d, J ¼ 10.8 Hz, 1H, 1H from H-20), 5.12 (d, J ¼ 17.4 Hz, 1H, 1H from H-20),
.67 (s, 2H, phenyl NH ), 3.32 (dd, J ¼ 15.0, 10.8 Hz, 2H), 3.26 (dd, J ¼ 10.2, 6.6 Hz,
2
H, CHOH), 2.23 ꢂ 2.27 (m, 1H), 2.08 ꢂ 2.20 (m, 2H), 2.00 (s, 1H), 1.91 (dd,
J ¼ 15.6, 8.4 Hz, 1H), 1.68 (dd, J ¼ 14.4, 2.4 Hz, 1H), 1.53 ꢂ 1.59 (m, 2H), 1.42 ꢂ 1.49
(
m, 1H), 1.35 ꢂ 1.40 (m, 2H), 1.32 (s, 3H), 1.26 (dd, J ¼ 14.4, 3.0 Hz, 1H), 1.11 (d,
J ¼ 16.2 Hz, 1H), 1.07 (s, 3H), 1.01 ꢂ 1.05 (m, 1H), 0.79 (d, J ¼ 7.2 Hz, 3H), 0.59 (d,
1
3
J ¼ 7.2 Hz, 3H). C NMR (151 MHz, CDCl ) d 217.3, 168.8, 146.8, 139.2, 134.7 (phe-
3
nyl C-3, 5), 121.8, 117.2 (phenyl C-2, 6), 115.6, 74.7, 69.2, 58.3, 45.5, 44.7, 43.9, 41.8,
3
9.7, 36.9, 36.1, 34.6, 30.5, 26.9, 26.4, 24.9, 16.8, 15.0, 11.6. ESIMS: m/z 508.5
þ
[
M þ Na] . Elemental analysis: Found: C, 69.32%, H, 8.02%, N, 2.91%, S, 6.67%; calcd
for C H NO S, C, 69.24%, H, 8.09%, N, 2.88%, S, 6.60%.
2
8
39
4

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
13
3.2.7. Synthesis of 22-(4-(p-nitrobenzene sulfamine) thiophenyl)-22-deoxypleuro-
mutilin (10)
Compound 9 (7.05 g, 14.5 mmol) and anhydrous K CO (2.0 g, 14.5 mmol) were
2
3
added in acetonitrile (30 ml) and stirred at room temperature. p-Nitrobenzene sul-
fonyl chloride (3.22 g, 14.5 mmol) was dissolved in acetonitrile (10 ml) and dropwise
added into the mixture. After the materials were consumed completely, about 25 ml
acetonitrile was evaporated in vacuum, and then the formed slurry was treated with
water (150 ml) and extracted by ethyl acetate (50 ml ꢃ 3). The combined organic
layers were evaporated in vacuum to obtain the crude yellowish-brown solid (8.74 g)
1
0
in 89.7% yield. H NMR (600 MHz, CDCl ) d 8.11 (d, J ¼ 6.6 Hz, 2H, phenyl H-3 ,
3
0
0
0
5
6
), 7.90 (d, J ¼ 7.8 Hz, 2H, phenyl H-2 , 6 ), 7.19 (d, J ¼ 7.8 Hz, 2H, phenyl H-2, 6),
.93 (d, J ¼ 7.8 Hz, 2H, phenyl H-3, 5), 6.35 (dd, J ¼ 16.8, 11.4 Hz, 1H, H-19), 5.65 (d,
J ¼ 7.2 Hz, 1H, H-14), 5.21 (d, J ¼ 11.4 Hz, 1H, 1H from H-20), 5.10 (d, J ¼ 16.8 Hz,
H, 1H from H-20), 3.47 (t, J ¼ 16.8 Hz, 2H), 3.32 (s, 1H), 2.15 ꢂ 2.25 (m, 3H), 2.03
s, 1H), 1.94 (q, J ¼ 7.2 Hz, 1H), 1.73 (d, J ¼ 13.2 Hz, 1H), 1.59 ꢂ 1.63 (m, 2H),
.42 ꢂ 1.53 (m, 3H), 1.28 (s, 5H), 1.15 (d, J ¼ 15.6 Hz, 1H), 1.09 (s, 4H), 0.85 (d,
1
(
1
1
3
J ¼ 5.4 Hz, 3H), 0.56 (d, J ¼ 5.4 Hz, 3H). C NMR (151 MHz, CDCl ) d 217.8, 168.4,
3
0 0
1
49.9, 145.9, 139.1, 131.6 (phenyl C-2, 6), 128.5 (phenyl C-2 , 6 ), 124.3, 122.6 (phenyl
0
0
C-3 , 5 ), 117.2 (phenyl C-3, 5), 74.7, 69.8, 60.6, 58.3, 53.8, 45.6, 44.9, 44.0, 41.8, 37.6,
þ
3
6.9, 36.1, 34.7, 30.5, 26.9, 26.6, 24.9, 16.8, 14.9, 11.6. ESIMS: m/z 693.8 [M þ Na] .
Elemental analysis: Found: C, 60.93%, H, 6.39%, N, 4.24%, S, 9.49%; calcd for
C H N O S , C, 60.88%, H, 6.31%, N, 4.18%, S, 9.56%.
3
4
42 2 8 2
3.2.8. Synthesis of 22-(4-(p-aminobenzene sulfamine) thiophenyl)-22-deoxypleuro-
mutilin (11)
Reduced iron powder (2.4 g, 42.9 mmol) and ammonium chloride (1.54 g, 28.6 mmol)
were added into acetic acid (30 ml) and water (20 ml). Acetonitrile solution of com-
pound 10 (9.6 g, 14.3 mmol) was dropwise added into the above mixture and stirred
ꢀ
at 80 C for 3 h. After completion, saturated sodium bicarbonate was employed to
adjust the pH of the mixture to 7 and the mixture was extracted with ethyl acetate
(
30 ml ꢃ 3). The combined organic layers were washed with brine, dried over anhyd-
rous Na SO and concentrated in vacuum. The residue was purified by recrystalliza-
2
4
tion with ethyl acetate/petroleum ether (V/V: 2/3) to give 11 as a white solid (6.32 g,
1
6
7
(
8.9% yield). H NMR (600 MHz, CDCl ) d 7.53 (d, J ¼ 8.4 Hz, 2H, phenyl H-2, 6),
3
0
0
.21 (d, J ¼ 8.4 Hz, 2H, phenyl H-2 , 6 ), 6.97 (d, J ¼ 8.4 Hz, 2H, phenyl C-3, 5), 6.55
0
0
d, J ¼ 8.4 Hz, 2H, phenyl C-3 , 5 ), 6.36 (dd, J ¼ 17.4, 10.8 Hz, 1H, H-19), 5.65 (d,
J ¼ 8.4 Hz, 1H, H-14), 5.23 (d, J ¼ 10.8 Hz, 2H, 1H from H-20), 5.11 (d, J ¼ 17.4 Hz,
1
2
1
(
(
1
7
1
H, 1H from H-20), 4.23 (s, 2H, phenyl NH ), 3.42 ꢂ 3.48 (m, 2H), 3.32 (s, 1H),
2
.13 ꢂ 2.28 (m, 3H), 2.03 (s, 1H), 1.89 (dd, J ¼ 15.6, 8.4 Hz, 1H), 1.72 (d, J ¼ 13.8 Hz,
H), 1.55 ꢂ 1.65 (m, 3H), 1.40 ꢂ 1.50 (m, 2H), 1.29 (s, 3H), 1.24 ꢂ 1.28 (m, 1H), 1.08
13
s, 4H), 1.05 (s, 1H), 0.84 (d, J ¼ 6.6 Hz, 3H), 0.55 (d, J ¼ 6.6 Hz, 3H). C NMR
151 MHz, CDCl ) d 217.7, 168.5, 151.1, 138.9, 137.0, 131.8 (phenyl C-2, 6), 130.0,
3
0
0
0
0
29.5 (phenyl C-2 , 6 ), 126.9, 121.2 (phenyl C-3, 5), 117.4, 114.1 (phenyl C-3 , 5 ),
4.6, 69.6, 58.2, 45.5, 44.7, 43.9, 41.8, 37.8, 36.8, 36.0, 34.6, 30.5, 26.9, 26.5, 24.9, 16.7,
4.9, 11.6. ESIMS: m/z 663.8 [M þ Na] . Elemental analysis: Found: C, 63.79%, H,
þ

1
4
H.-X. LIU ET AL.
6
.86%, N, 4.32%, S, 10.06%; calcd for C H N O S , C, 63.72%, H, 6.92%, N, 4.37%,
3
4
44 2 6 2
S, 10.01%.
3
.2.9. General procedure for the synthesis of 12a–f
Compound 11 (1.0 g, 1.56 mmol), anhydrase K CO (0.17 g, 1.25 mmol) and equal
2
3
molar quantities of halohydrocarbon were added into 15 ml DMF and stirred at
ꢀ
1
00 C for 5 h. After completion, about 10 ml DMF was evaporated in vacuum. The
residue was treated with 30 ml H O and stirred, the solid was filtered, rinsed with
2
ꢀ
water and dried at 80 C. The residue was purified by silica gel chromatography with
ethyl acetate/petroleum ether (V/V 20/1 to 3/1) as eluent to give 12a–f as a
white solid.
3
.2.9.1. 22-(4-(p-ethylamine benzene sulfamine) thiophenyl)-22-deoxypleuromutilin
ꢀ
ꢂ1
(
3
1
12a). White powdery solid, yield 67.2%. m.p.: 198 ꢂ 199.5 C. IR (KBr, cm ): 3470,
376, 2982, 2934, 2880, 1725, 1629, 1596, 1491, 1457, 1340, 1321, 1281, 1147, 1116,
1
087, 1015, 938, 739, 679, 548. H NMR (600 MHz, CDCl ) d 7.33 (d, J ¼ 8.4 Hz, 2H,
3
0
0
phenyl H-2 , 6 ), 7.27 (d, J ¼ 8.4 Hz, 2H, phenyl H-2, 6), 6.97 (d, J ¼ 8.4 Hz, 2H, phe-
0
0
nyl H-3 , 5 ), 6.61 (d, J ¼ 8.4 Hz, 2H, phenyl H-3, 5), 6.42 (dd, J ¼ 17.4, 10.8 Hz, 1H,
H-19), 5.73 (d, J ¼ 8.4 Hz, 1H, H-14), 5.29 (d, J ¼ 10.8 Hz, 1H, H-20), 5.15 (d,
J ¼ 17.4 Hz, 1H, H-20), 4.16 (s, 2H), 3.56 (s, 2H, H-22), 3.52 (q, J ¼ 7.2 Hz, 2H,
NHCH CH ), 3.33 (dd, J ¼ 10.2, 6.6 Hz, 1H, H-11), 2.28 ꢂ 2.33 (m, 1H), 2.21 ꢂ 2.27
2
3
(
1
m, 1H), 2.14 ꢂ 2.20 (m, 1H), 2.00 ꢂ 2.07 (m, 2H), 1.75 (dd, J ¼ 14.4, 2.4 Hz, 1H),
.60 ꢂ 1.66 (m, 3H), 1.47 (m, 1H), 1.41 ꢂ 1.44 (m, 1H), 1.39 (s, 3H), 1.34 (dd,
J ¼ 14.4, 2.4 Hz, 1H), 1.23 (d, J ¼ 15.6 Hz, 1H), 1.13 (s, 3H), 1.08 ꢂ 1.11 (m, 1H), 1.02
13
(
t, J ¼ 7.2 Hz, 3H, NHCH CH ), 0.86 (d, J ¼ 6.6 Hz, 3H), 0.65 (d, J ¼ 6.6 Hz, 3H).
C
2
3
0
NMR (151 MHz, CDCl ) d 217.2 (C-3), 168.2 (C-21), 150.7 (phenyl C-4 ), 139.0 (C-
3
0 0
19), 137.9 (phenyl C-4), 134.8, 129.8 (phenyl C-2, 6), 129.6 (phenyl C-2 , 6 ), 129.5
0 0
(phenyl C-3, 5), 126.4, 117.3, 113.9 (phenyl C-3 , 5 ), 74.7, 69.9, 58.3, 45.5, 45.2, 44.9,
4
4.0, 41.9, 37.0, 36.8, 36.1, 34.6, 30.5, 26.9, 26.6, 24.9, 16.9, 14.9, 14.0, 11.6. ESIMS:
þ
m/z 691.8 [M þ Na] . Elemental analysis: Found: C, 64.69%, H, 7.16%, N, 4.17%, S,
9.66%; calcd for C H N O S , C, 64.64%, H, 7.23%, N, 4.19%, S, 9.59%.
36 48 2 6 2
The spectral data of 12b–12f are listed in the supplementary information.
3
.2.10. Synthesis of 22-(4-(4-(2-chloroacetamide) benzene sulfonamido) thio-
phenyl)-22-deoxypleuromutilin (13)
Compound 11 (5.2 g, 8.1 mmol) and triethylamine (1.23 g, 12.2 mmol) were dissolved
into 50 ml CH Cl . Chloroacetyl chloride (1.38 g, 12.2 mmol) was then dropwise
2
2
added into the mixture and stirred for about 5 h. After completion, the formed slurry
was treated with water (50 ml) and extracted with CH Cl (50 ml ꢃ 3). The combined
2
2
organic layers were dried over anhydrous Na SO and concentrated in vacuum to
2
4
obtain the crude yellowish-brown solid (4.73 g) in 81.3% yield. The next step of reac-
tion could be carried out directly without purification.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
15
3.2.11. General procedure for the synthesis of 14a–f
Compound 13 (1.0 g, 1.4 mmol), anhydrous K CO (0.15 g, 1.1 mmol) and equimolar
2
3
ꢀ
alkylamine/arylamine were added in 20 ml DMF and stirred at 100 C for 5 h. After
the materials were consumed completely, about 15 ml DMF was evaporated in vac-
uum. The residue was treated with 30 ml H O and stirred, the solid was filtered,
2
ꢀ
rinsed with water and dried at 80 C. The residue was purified by silica gel column
chromatography with ethyl acetate/petroleum ether (V/V 20/1 to 5/1) as eluent to
give 14a–f as a white solid.
3
2
.2.11.1. 22-(4-(4-(2-Dimethylamino acetamino) benzene sulfonamido) thiophenyl)-
ꢀ
2-deoxypleuromutilin (14a). White powdery solid, yield 62.1%. m.p.: 208 ꢂ 209.5 C.
ꢂ1
IR (KBr, cm ): ꢀ
3262, 2982, 2934, 2860, 1717, 1593, 1517, 1493, 1457, 1402,
max
1
1
9
336, 1278, 1159, 1116, 1093, 1015, 917, 828, 638, 574. H NMR (600 MHz, CDCl ) d
3
.37 (s, 1H, NHCO), 7.71 (d, J ¼ 8.4 Hz, 2H, phenyl H-2 , 6 ), 7.60 (d, J ¼ 8.4 Hz, 2H,
0
0
0
0
phenyl H-3 , 5 ), 7.19 (d, J ¼ 7.8 Hz, 2H, phenyl H-2, 6), 6.97 (d, J ¼ 7.8 Hz, 2H, phe-
nyl H-3, 5), 6.35 (dd, J ¼ 17.4, 10.8 Hz, 1H, H-19), 5.66 (d, J ¼ 7.8 Hz, 1H, H-14), 5.22
(
d, J ¼ 10.8 Hz, 1H, H-20), 5.09 (d, J ¼ 17.4 Hz, 1H, H-20), 3.44 ꢂ 3.49 (m, 2H), 3.32
(s, 1H), 3.07 (s, 2H, CH CONH), 2.35 (s, 6H, N(CH ) ), 2.22 ꢂ 2.27 (m, 2H),
2
3 2
2
2
2
3
.15 ꢂ 2.19 (m, 1H), 2.05 (s, 1H), 1.93 (dd, J ¼ 15.6, 8.4 Hz, 1H), 1.73 (dd, J ¼ 14.4,
.4 Hz, 1H), 1.57 ꢂ 1.65 (m, 3H), 1.41 ꢂ 1.49 (m, 2H), 1.31 (s, 3H), 1.25 ꢂ 1.29 (m,
H), 1.09 ꢂ 1.12 (m, 2H), 1.08 (s, 3H), 0.85 (d, J ¼ 6.6 Hz, 3H), 0.59 (d, J ¼ 6.6 Hz,
1
3
H). C NMR (151 MHz, CDCl ) d 217.5 (C-3), 169.9 (NHCO), 168.4 (C-21), 141.6
3
0
0
0
(
1
phenyl C-4 ), 139.1 (C-19), 134.7, 131.8 (phenyl C-2, 6), 128.6 (phenyl C-2 , 6 ),
0 0
21.9 (phenyl C-1), 119.3 (phenyl C-3 , 5 ), 117.3 (phenyl C-3, 5), 74.7, 69.7, 63.6
(
3
COCH N(CH ) ), 58.3, 53.6, 46.2 (CH N(CH ) ), 45.6, 44.8, 43.9, 41.8, 37.9, 36.9,
2 3 2 2 3 2
6.1, 34.6, 30.5, 29.81, 26.9, 26.6, 24.9, 16.8, 14.9, 11.6. ESIMS: m/z 748.8 [M þ Na] .
þ
Elemental analysis: Found: C, 62.81%, H, 7.02%, N, 5.73%, S, 8.78%; calcd for
C H N O S , C, 62.87%, H, 7.08%, N, 5.79%, S, 8.83%.
3
8
51 3 7 2
The spectral data of 14b–14f are listed in the supplementary information.
3
.3. Minimum inhibitory concentrations (MIC) testing
The evaluation of the in vitro anti-bacterial activity of all prepared derivatives was
performed against a panel of well characterized clinical susceptible (S.aureus,
S.epidermidis and S.suis) and resistant Gram-positive (MRSA-NY3) bacteria isolated
from clinic as well as against one Gram-negative (E.coli) bacteria strain. The min-
imum inhibitory concentrations (MICs) of target compounds against above bacteria
were determined by broth dilution method, tiamulin fumaric and erythromycin thio-
cyanate were employed as the reference agents based on the Clinical and Laboratory
Standards Institute (CLSI) [17]. The MIC values are showed in Table 1.
3
.4. Molecular modeling
Molecular docking study was performed by Molecular Operating Environment
MOE) 2018.0101 release of Chemical Computing Group Inc., Montreal, Quebec,
(

1
6
H.-X. LIU ET AL.
ꢂ1
Table 2. Docking scores as “S” (kcal mol ) and bond interactions of 9, 10, 11, 14a, 14b and
tiamulin with active pocket of 23s rRNA.
No. of
bonds
Amino acids
involved
ꢂ1
Cpd. No.
S (kcal mol
)
Distance (Å)
2.34
Interacting groups
9
ꢂ9.6458
4
G2044
G2044
G2484
A2045
G2044
G2044
A2045
C2565
G2426
U2485
U2485
U2483
U2485
C2565
C2565
G2484
G2484
G2044
G2044
A2430
G2044
U2483
C2565
C2431
C2431
U2483
G2484
G2044
U2564
U2483
U2564
H-bond with C ¼ O of pleuromutilin C3
H-bond with C ¼ O of pleuromutilin C3
H-bond with OH of pleuromutilin C11
H-p interaction
2
1
.30
.65
1
0
1
ꢂ10.4873
ꢂ9.7800
3
7
2.40
H-bond with S ¼ O of pleuromutilin C14
H-p interaction
H-p interaction
1
1.91
H-bond with OH of pleuromutilin C11
H-bond with S ¼ O of pleuromutilin C14
H-bond with C ¼ O of pleuromutilin C3
2.22
2.34
2.37
H-bond with CH
H-p interaction
H-p interaction
H-p interaction
3
of pleuromutilin C14
1
4a
4b
ꢂ11.8241
ꢂ10.3751
ꢂ8.6283
6
6
5
2.24
H-bond with NH of pleuromutilin C14
H-bond with H of pleuromutilin C14
H-bond with S of pleuromutilin C14
H-p interaction
H-p interaction
H-p interaction
H-bond with C ¼ O of ester group
H-bond with OH of pleuromutilin C11
H-bond with NH of pleuromutilin C14
H-p interaction
H-p interaction
H-p interaction
2
3
.35
.83
1
2.02
2
2
.07
.07
Tiamulin
1.64
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
2
2
.04
.24
H-p interaction
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 [18]. 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 con-
struct the initial model [19]. Binding score, number and lengths of the hydrogen
bonds and amino acid interactions are determined and showed in Table 2. The dock-
ing method was validated by the redock of tiamulin, with the root mean square devi-
ations (RMSD) of tiamulin in crystal structure relative to tiamulin in the redock
model being 0.35 Å, which confirmed the approach was appropriate.
Disclosure statement
The authors declare that they have no conflict of interest.
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), Basic Research Program of Outstanding Young Teachers of Hebei University of

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
17
Chinese Medicine (No. YQ2018004) and Natural Science Foundation of Hebei Province
H2020423279).
(
References
[
[
[
1] P.J. Yeh, M.J. Hegreness, A.P. Aiden, and R. Kishony, Nat. Rev. Microbiol. 7, 460
2009).
2] S.M. Poulsen, M. Karlsson, L.B. Johansson, and B. Vester, Mol. Microbiol. 41, 1091
2001).
3] F. Schl u€ nzen, E. Pyetan, P. Fucini, A. Yonath, and J.M. Harms, Mol. Microbiol. 54,
287 (2004).
(
(
1
[
[
4] F. Kavanagh, A. Hervey, and W.J. Robbins, Proc. Natl. Acad. Sci. USA. 37, 570 (1951).
5] A.J. Birch, D.W. Cameron, C.W. Holzapfel, and R.W. Richards, Chem. Ind. 14, 374
(
1963).
[6] H. Egger and H. Reinshagen, J. Antibiot. 29, 923 (1976).
[7] L. Stipkovits, P.H. Ripley, J. Varga, and V. Palfi, Vet. Rec. 148, 399 (2001).
[8] R. Novak and D.M. Shlaes, Curr. Opin. Invest. Drugs 11, 182 (2010).
[9] R.L. Young and L.J. Katy, Drugs 79, 1867 (2019).
[
[
10] H.X. Liu, D.L. Ma, G. Cui, Y. Zhang, and F.Q. Xue, J. Asian Nat. Prod. Res. 23, 123
2021).
11] H.X. Liu, S. Xiao, D.P. Zhang, S.H. Mu, L.F. Zhang, X.Y. Wang, and F.Q. Xue, Biol.
Pharm. Bull. 38, 1041 (2015).
(
[
12] Y. Deng, X.Z. Wang, S.H. Huang, and C.H. Li, Eur. J. Med. Chem. 162, 194 (2019).
[
13] Z. Jin, L. Wang, H. Gao, Y.H. Zhou, Y.H. Liu, and Y.Z. Tang, Eur. J. Med. Chem. 181,
111594 (2019).
[
[
[
[
14] Y. Hirokawa, H. Kinoshita, T. Tanaka, T. Nakamura, K. Fujimoto, S. Kashimoto, T.
Kojima, and S. Kato, Bioorg. Med. Chem. Lett. 19, 175 (2009).
15] I. Dreier, S. Kumar, H. Sondergaard, M.L. Rasmussen, L.H. Hansen, N.H. List, J.
Kongsted, B. Vester, and P. Nielsen, J. Med. Chem. 55, 2067 (2012).
16] I. Dreier, L.H. Hansen, P. Nielsen, and B. Vester, Bioorg. Med. Chem. Lett. 24, 1043
(
2014).
17] Clinical and Laboratory Standard Institute. CLSI Document M100-S17. Performance
Standard for Antimicrobial Susceptibility Testing; Seventh Informational Supplement,
Vol. 27 (Clsi, Wayne, PA, January 2007, no. 1).
[
[
18] Chemical Computing Group. Available from https://www.chemcomp.com/.
19] Protein Data Bank. http://www.rcsb.org/pdb.