Design, synthesis and biological evaluation of novel pleuromutilin derivatives containing piperazine and 1,2,3-triazole linker

Article abstract of DOI:10.1016/j.bioorg.2020.104398

A series of novel pleuromutilin derivatives containing piperazine ring, 1, 2, 3-triazoles and secondary amines on the side chain of C14 were synthesized under mild conditions via click reaction. The in vitro antibacterial activities of the synthesized derivatives against four strains of Staphylococcus aureus (MRSA ATCC 43300, ATCC 29213, 144 and AD3) and one strain of Escherichia coli (ATCC 25922) were evaluated by the broth dilution method. Among these derivatives, 22–[2-(4-((4-nitrophenyl piperazine)methyl)-1,2,3-triazol-1-yl)-1-(piperazine-1-yl) ethyl-1-one] deoxy pleuromutilin (compound 59) showed the most prominent in vitro antibacterial effect against MRSA (MIC = 1 μg/mL). Furthermore, compound 59 displayed more rapid bactericidal kinetic than tiamulin time-kill studies and possessed a longer PAE than tiamulin against MRSA in vitro. In addition, in vivo antibacterial activities of compound 59 against MRSA were further evaluated employing thigh infection model. And compound 59 (-8.89 log₁₀ CFU/mL) displayed superior activities than tiamulin. Compound 59 was further evaluated in CYP450 inhibition assay and the results showed that it exhibited low to moderate inhibitory effects on CYP1A2, CYP2E1, CYP2D6 and CYP3A4 enzymes. The PK properties of compound 59 were then measured. The half-life (t_1/2), clearance rate (Cl) and the area under the plasma concentration time curve (AUC_0→∞) of compound 59 were 0.74 h, 0.29 L/h/kg and 46.28 μg·h/mL, respectively.

Full text of DOI:10.1016/j.bioorg.2020.104398

Bioorganic Chemistry 105 (2020) 104398
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis and biological evaluation of novel pleuromutilin
derivatives containing piperazine and 1,2,3-triazole linker
,
,b
Guang-Yu Zhanga, Zhe Zhanga, Kang Lia, Jie Liua, Bo Lia, Zhen Jina b, Ya-Hong Liua
,
,b,*
You-Zhi Tanga
a Guangdong Provincial Key Laboratory of Veterinary Pharmaceutics Development and Safety Evaluation, College of Veterinary Medicine, South China Agricultural
University, Guangzhou 510642, China
b Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou 510642, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of novel pleuromutilin derivatives containing piperazine ring, 1, 2, 3-triazoles and secondary amines on
the side chain of C14 were synthesized under mild conditions via click reaction. The in vitro antibacterial ac-
tivities of the synthesized derivatives against four strains of Staphylococcus aureus (MRSA ATCC 43300, ATCC
29213 ,144 and AD3) and one strain of Escherichia coli (ATCC 25922) were evaluated by the broth dilution
method. Among these derivatives, 22–[2-(4-((4-nitrophenyl piperazine)methyl)-1,2,3-triazol-1-yl)-1-(piperazine-
1-yl) ethyl-1-one] deoxy pleuromutilin (compound 59) showed the most prominent in vitro antibacterial effect
Antibacterial activity
MRSA
Pleuromutilin
1,2,3-Triazole
Click chemistry
against MRSA (MIC = 1 μg/mL). Furthermore, compound 59 displayed more rapid bactericidal kinetic than
tiamulin time-kill studies and possessed a longer PAE than tiamulin against MRSA in vitro. In addition, in vivo
antibacterial activities of compound 59 against MRSA were further evaluated employing thigh infection model.
And compound 59 (-8.89 log10 CFU/mL) displayed superior activities than tiamulin. Compound 59 was further
evaluated in CYP450 inhibition assay and the results showed that it exhibited low to moderate inhibitory effects
on CYP1A2, CYP2E1, CYP2D6 and CYP3A4 enzymes. The PK properties of compound 59 were then measured.
The half-life (t1/2), clearance rate (Cl) and the area under the plasma concentration time curve (AUC0→∞) of
compound 59 were 0.74 h, 0.29 L/h/kg and 46.28 μg⋅h/mL, respectively.
1. Introduction
of pleuromutilin has fascinated many researchers to explore and develop
pleuromutilin effective antibacterial agents derived from it for the
treatment of multidrug resistant bacterial infections. Further alterations
on the C-14 side chain of pleuromutilin have resulted in the discovery of
tiamulin (2, Fig. 1) and valnemulin (3, Fig. 1), which were approved as
therapeutic agents for pig and poultry in 1979 and 1999 [7]. In suc-
cession, retapamulin (4, Fig. 1) was successfully approved as the first
pleuromutilin derivative for treatment of human topical skin infection
[8]. More recently, lefamulin (5, Fig. 1) was approved by the Food and
Drug Administration (FDA) for treatment of community-acquired bac-
terial pneumonia (CABP) in humans [9]. Azamulin (6, Fig. 1) has
excellent potency against Gram-positive and some Gram-negative bac-
teria, but development was discontinued after Phase I trials for the
highly inhibition of human CYP3A enzyme [10]. The sulfur-containing
atom structure of azamulin may be the reason for its strong inhibitory
effect on CYP enzyme, therefore, we attempted to converted the C14
Among the multi-drug resistant Gram-positive bacteria, methicillin-
resistant S. aureus (MRSA) has become the most important issue of
concern [1]. MRSA possess intrinsic resistance to methicillin and other
β-lactam antibiotics [2]. MRSA infections are seriously endangering
public health safety all over the world [3]. Therefore, there is an urgent
need to discover and develop new antibacterial drugs with novel mode
of action against MRSA.
Pleuromutilin (1, Fig. 1) is a tricyclic diterpenoid natural product
and first isolated in 1951 from two basiomycetes species, Pleurotus
mutilus and Pleurotus passeckerianus, which displays modest in vitro ac-
tivity and weak in vivo activity against Gram-positive pathogens and
mycoplasmas [4,5]. Pleuromutilin interacts with the 23S rRNA of the
50S bacterial ribosome subunit and interferes with bacterial protein
synthesis to exert antibacterial activity [6]. The distinct mode of action
* Corresponding author at: College of Veterinary Medicine, South China Agricultural University, No. 483 Wushan Road, Tianhe District, Guangzhou 510642,
China.
E-mail address: youzhitang@scau.edu.cn (Y.-Z. Tang).
https://doi.org/10.1016/j.bioorg.2020.104398
Received 9 August 2020; Received in revised form 16 October 2020; Accepted 17 October 2020
Available online 21 October 2020
0045-2068/© 2020 Elsevier Inc. All rights reserved.

G.-Y. Zhang et al.
Bioorganic Chemistry 105 (2020) 104398
side chain of pleuromutilin into a nitrogen-containing heterocycle
without sulfur atom structure to reduce the inhibitory effect on CYP
enzyme.
2.2. In vitro antibacterial activity
The antibacterial activities of the synthesized pleuromutilin de-
rivatives 47–64, 81–88 were evaluated against MRSA ATCC 43300,
S. aureus ATCC 29213, E. coli ATCC 25,922 as well as S. aureus (AD3) and
S. aureus (144) which were isolated from Guangdong Province using
tiamulin and pleuromutilin as positive control. The MICs of those
pleuromutilin derivatives 47–64, 81–88, as well as tiamulin (2, Fig. 1)
and pleuromutilin (1, Fig. 1) were determined using the broth dilution
methods according to the Clinical and Laboratory Standards Institute
(CLSI). The MIC values of compounds 47–64, 81–88 are provided in
Table 1.
In our previous work, novel pleuromutilin derivatives having a
piperazine as linker were designed and synthesized. Some of these
pleuromutilin analogues displayed potent in vitro and in vivo antibacte-
rial activity against S. aureus (ATCC 29213) [11,12]. Since the above
pleuromutilin derivatives that without sulfur linkage on the C14 side
chains still keep well in vitro antibacterial activity, we exerted our effort
to transform the pleuromutilin C14 side chain with a nitrogen-
containing heterocycle to develop novel antibacterial pleuromutilin
derivatives with low degree of inhibition on CYP450 enzyme.
Compounds containing 1H-1,2,3-triazole functionality has been re-
ported to have antibacterial, β-lactamase inhibitory, anti-inflammatory,
antiviral, anticonvulsant and muscarinic biological activities [13].
Therefore, as an extension to that study, we determine to develop the
pleuromutilin analogue that has a piperazine ring linker, and secondary
amino which connected with 1H-1,2,3-triazole to improve antibacterial
activity and weaken the degree of inhibition on CYP450 enzyme. In the
present study, 26 pleuromutilin derivatives were designed, synthesized
and evaluated for their antibacterial activities against five strains
including MRSA in vitro and in vivo.
The MICs of the 26 novel pleuromutilin derivatives in vitro against
MRSA (ATCC 43300), S. aureus (ATCC 29213), S. aureus (AD3), S. aureus
(144), E. coli ATCC 25,922 ranged from ≥ 32 to 1 μg/mL. Compounds
47–64, 81, 87 and 88 exhibited relatively inhibitory activities against
S. aureus. The other compounds showed lower antibacterial activities. It
can be observed that compound 59 which bearing 4-nitrophenyl
piperazine group on the C-14 glycolic acid side chain possessed the
highest antibacterial activities against S. aureus (MIC = 1 μg/mL) in this
series, being comparable to tiamulin and pleuromutilin against MRSA.
Initially, compounds 47 and 48 were synthesized and evaluated for
their antibacterial activities. Compound 48 showed superior antibacte-
rial activity against MRSA in comparison to that of compound 47. The
results showed that derivatives containing benzene ring exhibited better
activity compared to those with methyl substituent group, which was
consistent with previous research [11,15].
2. Results and discussion
2.1. Chemistry
The general synthetic route used to prepare of all pleuromutilin de-
rivatives are illustrated in Scheme 1. Compound 8 and compound 9 were
synthesized from pleuromutilin according to the method in our previous
work by using N-tert-butoxycarbonyl -piperazine and chloroacetic
chloride [11,12]. Compound 9 was converted into the azide compound
10 through a nucleophilic substitution. Compounds 29–46, 73–80 were
synthesized by compounds 11–28, 65–72 and propargyl bromide under
alkaline conditions. The azide compounds 10 was reacted with com-
pounds 29–46, 73–80 to get the target compounds 47–64, 81–88 by a
standard click reaction, respectively [14].
The electron donating groups were introduced on the benzene ring of
compound 8 resulted in compounds 49–56. Compounds 57–59 with
nitro group on phenylpiperazine presented improved activity against all
the strains except E. coli compared with all other target compounds
bearing the methyl, fluorine, hydroxy, methoxy, or chlorine group on
phenylpiperazine. The results of in vitro antibacterial activities indicated
that the introduction of the aromatic substituents with electron with-
drawing groups could enhance antibacterial activity against MRSA,
which was consistent with previous reported [11].
Finally, compounds 81–88 were designed and prepared by replacing
the phenylpiperazine substitution with various nitrogen heterocyclic
rings, such as dimethylamine and dimethylamine substitutions. Com-
pounds 81–88 exhibited relatively moderate inhibitory activities against
MRSA, which was lower than that of tiamulin. Therefore, it is still
necessary to optimize the chemical composition of the linker and the
selection of different heterocyclic substitutes in the process of modifi-
cation of pleuromutilin derivatives in order to obtain compounds with
All those pleuromutilin derivatives were purified by silica column
chromatography to get the pure compounds. The structures of those
compounds were characterized by 1H NMR, 13C NMR, and high-
resolution mass spectral (HR-MS) analysis, and the analysis confirmed
that the synthesized compound was consistent with the expected
structure.
Fig. 1. Structure of pleuromutilin (1), tiamulin (2), valnemulin (3), retapamulin (4), lefamulin (5) and azamulin (6).
2

G.-Y. Zhang et al.
Bioorganic Chemistry 105 (2020) 104398
Scheme 1. Reagent and conditions: (i)
acetonitrile,
p-toluenesulfonyl
chloride,
NaOH, rt, 3 h; (ii) a). acetonitrile, NaI, 70 ℃,
1.5 h; b). acetonitrile, K2CO3, N-Boc-pipera-
zine, 70 ℃, 3 h; c). concentrated hydro-
chloric acid, rt, 8 h; (iii) ethyl acetate,
chloroacetyl chloride, K2CO3, rt, 3 h; (iv)
NaN3, acetone, H2O, 78 ℃,
4 h; (v)
dichloromethane, 3-Bromopropyne, K2CO3,
0 ℃-rt, 5 h; (vi) Tert-butyl alcohol/H2O =
1:1 (v/v), compound 10, copper(II) sulfate
pentahydrate, L (+)-ascorbic acid, rt, 3 h.
enhanced antibacterial activity in the future.
PAEs are shown in Table 2. Following exposure to 2 × MIC and 4 × MIC
for 2 h, the PAE of compound 59 was 3.20 and 3.24 h, respectively. The
PAE at the same concentrations of compound 61 against MRSA was 1.08
and 1.30 h. These results indicated that compound 59 possessed a longer
PAE than tiamulin [15] (exposure to 2 × MIC and 4 × MIC for 2 h, the
PAE was 1.65 and 2.04 h) and compound 61 against MRSA at the con-
centrations of 2 × MIC or 4 × MIC.
The MBC referred to the minimum drug concentration required to
kill 99.9% of the test microorganisms. The ratio of MBC to MIC values of
all synthesized pleuromutilin derivatives were 1–2, indicating that these
compounds have good bactericidal ability and the microorganism had
no drug resistance to the tested drugs [16].The results showed that all
synthetic pleuromutilin derivatives had a good killing effect on S. aureus.
Compounds 59 and 61 possessed the most prominent antibacterial ac-
tivity in this series, so we conducted a more in-depth study on the
antibacterial activity of these two compounds.
The traditional drug delivery regimen is only based on some phar-
macokinetic parameters such as plasma concentration, elimination rate
and tissue distribution. However, pharmacokinetic parameters could
only reflect the final result of the interaction between drugs and bac-
teria, but ignore the interaction process between them [17]. Therefore,
in clinical medication, we could refer to the PAE of drugs, and optimize
the drug administration scheme combined with pharmacokinetic and
pharmacokinetic parameters, so as to maximize the effect of drugs and
reduce side effects and medical expenses [18]. The results of the above
studies suggested that the administration interval of compound 59 may
be longer than that of tiamulin. The effective antibacterial activity of
compound 59 also prompted us to carry out further study in its potential
use in the clinical treatment of MRSA infection.
Time-kill kinetics assay was used to investigate the in vitro antibac-
terial activity of compound 59 and compound 61. The results of the
time-kill curves experiments are presented in Fig. 2. Compound 59 and
compound 61 at 2 × MIC induced marked MRSA killing (-1.40 log10
CFU/mL and ꢀ 1.73 log10 CFU/mL reduction), beginning at 3 h after
incubation, and complete bacterial growth inhibition was observed at
24 h (- 8.89 log10 CFU/mL and ꢀ 8.94 log10 CFU/mL reduction). In
contrast, a few colonies could be detected even after incubation for 24 h
in the tiamulin group at the concentration of 4 × MIC (the total bacterial
ꢀ 1.90 log10 CFU/mL [15]). Therefore, it can be seen that after 3 h of
incubation, at 2 × MIC, compounds 59 and 61 were able to display more
rapid bactericidal kinetic effects than tiamulin. To summarize, com-
pounds 59 and 61 induced dose- and time-dependent growth inhibition
against MRSA, with a bactericidal effect at the concentrations higher
than 2 × MIC.
2.3. In vivo antibacterial activity
Since compound 59 exhibited the best antibacterial activity against
MRSA in vitro among the 26 pleuromutilin derivatives synthesized in this
study, the in vivo antibacterial activity of compound 59 was further
evaluated by a neutropenic murine thigh infection model. Initially, the
mice model of neutropenia was established by intraperitoneal injection
of cyclophosphamide, and then the thigh infection model of mice
In order to clarify the potential of the drug for continuous inhibition
of bacteria and provide guidance for the rational formulation of dosing
regimens, we conducted PAE tests on compounds 59 and 61. The bac-
terial growth kinetics curve was shown in Fig. 3 and the results of the
3

G.-Y. Zhang et al.
Bioorganic Chemistry 105 (2020) 104398
Table 1
MIC and MBC (
μ
g/mL) values of compounds 47–64, 81–88, pleuromulin and tiamulin against S. aureus (ATCC 43300), S. aureus (ATCC 29213), S. aureus (AD3),
S. aureus (144) and E. coli (ATCC 25922).
Compound No
R
MIC/MBC(μg/mL)
MRSA
ATCC
S.aureus
S.aureus
S.aureus
E. coli
ATCC 29,213
AD3
144
ATCC 25,922
43,300
47
48
8/8
4/8
8/8
4/4
4/4
4/4
4/4
4/4
>32/>32
>32/>32
49
50
51
52
53
54
55
56
57
58
59
2/2
4/4
8/16
4/4
4/4
4/4
8/8
4/4
2/2
2/2
1/1
2/4
4/8
8/8
4/8
4/4
4/8
8/16
4/8
2/2
2/2
1/1
4/4
4/4
4/8
4/8
4/4
4/4
8/8
4/4
2/2
2/2
1/1
2/4
4/4
4/4
4/8
4/8
4/4
8/8
4/4
2/2
2/2
1/1
>32/>32
>32/>32
>32/>32
>32/>32
>32/>32
>32/>32
>32/>32
>32/>32
>32/>32
>32/>32
>32/>32
(continued on next page)
4

G.-Y. Zhang et al.
Bioorganic Chemistry 105 (2020) 104398
Table 1 (continued)
Compound No
R
MIC/MBC(μg/mL)
MRSA
ATCC
S.aureus
S.aureus
S.aureus
E. coli
ATCC 29,213
AD3
144
ATCC 25,922
43,300
60
61
62
63
64
8/8
1/2
8/8
4/4
4/8
8/16
1/2
8/8
4/4
4/8
8/8
1/1
8/16
4/4
4/4
8/8
2/2
8/16
4/4
4/4
>32/>32
>32/>32
>32/>32
>32/>32
>32/>32
81
82
83
84
85
4/4
4/4
4/4
2/2
>32/>32
>32/>32
>32/>32
>32/>32
>32/>32
4/4
4/4
4/8
4/8
8/8
8/8
8/16
16/32
16/32
8/8
16/16
16/16
16/16
16/32
16/16
16/16
86
87
88
16/16
4/4
16/16
4/4
16/16
4/8
16/16
4/4
>32/>32
>32/>32
>32/>32
4/8
4/4
4/4
4/4
Pleuromulin
Tiamulin
2/4
2/4
2/4
1/2
2/4
1/2
–
–
0.5/1
0.5/1
5

G.-Y. Zhang et al.
Bioorganic Chemistry 105 (2020) 104398
induced by MRSA was established by injecting bacteria solution about
107 CFU/mL into the thigh muscle of mice. Then mice were divided into
3 groups and intravenously (i.v.) injected saline, compound 59 (20 mg/
kg) and tiamulin (20 mg/kg), respectively. Saline and tiamulin treated
mice groups were established as a negative and positive control
respectively. The results are shown in Fig. 4.
Tiamulin at 20 mg/kg showed a treatment effect (-0.57 log10 CFU/
mL) against MRSA in thigh compared to no drug control group (P <
0.01, n = 6/group). However, the treatment with compound 59 at the
same dose could reduce MRSA load (-2.07 log10 CFU/mL) in thigh
compared to no drug control group (P < 0.001, n = 6/group). By force of
contrast, it can be concluded that compound 59 could treat MRSA
infection in vivo and was more effective than tiamulin in reducing the
MRSA load of thigh infected mice.
2.4. Effect on liver microsomal CYP450 enzyme activity
The cytochrome P450 (CYP450) proteins catalyze many reactions
likes xenobiotic metabolism and biosynthesis of cholesterol, steroids,
and other lipid components. Therefore, once the action of CYP450
enzyme is affected, it may cause a series of adverse reactions [19].
During the CYP450 enzyme inhibition assessment, it is commonly
Fig. 2. Time-kill curves for MRSA ATCC 43,300 with different concentrations
of tiamulin and compounds 59 (a) and 61 (b).
accepted that compounds with IC50 values higher than 10
CYP450 enzyme are weak CYP inhibitors; compounds with 3
< 10 M are moderate CYP inhibitors; while compounds with IC50 < 3
M are strong CYP inhibitors [20]. As azamulin was reported to have
μ
M against
μM < IC50
μ
μ
high CYP450 inhibition effect [10], the inhibition potential of com-
pounds 49, 59 and 61 on CYP450 enzyme were evaluated and tiamulin
was used as the control group. The inhibitory effect of compounds 49,
59, 61 and tiamulin on the four major CYP450 enzymes was analyzed by
determining IC50 values. The results are summarized in Table 3, as well
as the inhibition curve is shown in Figure SI 24.
As shown in Table 3, compounds 49, 59 and 61 showed low inhi-
bition on CYP450 enzymes CYP1A2, CYP2E1, CYP2D6, and CYP3A4.
Compounds 49, 59 and 61 displayed low to no significant inhibitory
effect on CYP3A4 enzyme (IC50 > 10
μ
M), whereas tiamulin showed a
M). Furthermore, the
inhibitory effect of tiamulin on CYP2E1 enzymes (IC50 < 3 M) was
high, while the inhibitory effect of compounds 49, 59 and 61 were low
(IC50 > 10 M). The results showed that the inhibitory rate of compound
strong inhibitory effect on CYP3A4 (IC50 = 1.60
μ
μ
μ
59 on CYP3A4 was much lower than that of tiamulin. The most likely
reason is that compound 59 does not contain sulfur atoms. The results
indicated that the safety and efficacy of compound 59 were better than
that of tiamulin when combined with drugs mainly metabolized by
CYP3A4 enzyme. However, due to the complexity of life organisms, it is
necessary to evaluate the inhibition rate of CYP enzyme in vivo in the
Fig. 3. The bacterial growth kinetic curves for MRSA ATCC 43,300 exposed to
compound 59 (a) and compound 61 (b) for 2 h.
Table 2
The PAEs values of compounds 59, 61 and tiamulin against MRSA ATCC 43300.
Compounds
Concentrations
PAE (h)
Exposure for 2 h
Compound 59
Compound 61
Tiamulin
2 × MIC
4 × MIC
2 × MIC
4 × MIC
2 × MIC
4 × MIC
3.20
3.24
1.08
1.30
1.65
2.04
Fig. 4. Efficacy of tiamulin and compound 59 against MRSA ATCC 43,300 in
murine neutropenic thigh models: circular: growth control; square: tiamulin
(20 mg/kg); triangle: compound 59 (20 mg/kg).
6

G.-Y. Zhang et al.
Bioorganic Chemistry 105 (2020) 104398
Table 3
CYP450 Inhibition by compounds 49, 59, 61 and tiamulin.
Compound
IC50
CYP3A4(T)b
(
μ
M)a
CYP2E1
CYP2D6
CYP1A2
49
23.60
20.49
17.00
1.60
> 50
> 50
> 50
> 50
> 50
27.22
8.67
59
10.95
24.31
0.045
61
22.29
> 50
tiamulin
a
IC50 refers to the concentration of compounds that causes 50% inhibition of
the enzyme activity.
b
Testosterone was used as substrate.
future to minimize the adverse reactions caused by the inhibition of CYP
enzyme.
2.5. Pharmacokinetic analysis of compound 59
Fig. 5. Plasma concentration–time curve (mean ± SE) following a single i.v.
administration of 10 mg/kg compound 59 to mice (n = 3/dose).
The PK profile of compound 59 was measured in mice following
intravenous (i.v.) administration at a dose of 10 mg/kg. The main
pharmacokinetic profiles of compound 59 were displayed in Table 4,
and the kinetic profiles of distribution in plasma of compound 59 after
intravenous (i.v.) administration were shown in Fig. 5.
Compound 59 showed a more rapid kinetics bactericidal against MRSA
and has a longer PAE than tiamulin. The in vivo antibacterial activity of
compound 59 was further studied in a neutropenic murine thigh infec-
tion model. The results indicated that compound 59 exhibited a potent
in vivo antibacterial effect than tiamulin. The pharmacokinetic studies
indicated that compound 59 possessed extremely high clearance (Cl¼
0.29 L/h/kg) and MRT of 0.74 h. Meanwhile, CYP450 inhibition ex-
periments showed that compound 59 inhibited CYP3A4 in vitro to the
same extent as compounds 49 and 61, and the degree of inhibition was
lower than that of tiamulin. The results indicated that the inhibition of
pleuromutilin derivatives containing piperazine side chain on CYP450
enzyme was lower than that of the pleuromutilin derivative containing
thioether side chain. Compound 59 might be served as a possible lead
compound for the development of antibacterial drugs and is worthy of
further clinical trials for the systematic treatment of infection in the
future.
After intravenous injection of compound 59, its plasma concentra-
tion decreased rapidly within the first 2 h, the maximum plasma con-
centration (Cmax) was 25.38 µg/mL at 0.083 h (Tmax). The half life (t1/2
)
of compound 59 was 0.74 h which reflected a high rate of elimination.
The clearance (Cl) value was 0.29 L/h/kg and higher than the mouse
hepatic blood flow (0.09 mL/min/kg). The area under curve until the
last measurable concentration (AUC0→t), and area under curve to
infinite time (AUC0→∞) were 34.27 µg⋅h/mL and 46.28 µg⋅h/mL,
respectively. The analysis of PK parameters is to deeply understand the
process of its absorption, distribution, metabolism and excretion by
analyzing the parameters, so as to clarify its value to safety and effec-
tiveness and how to obtain better clinical treatment effect. In one word,
these PK parameters can facilitate the evaluation of drug-like properties
of compound 59 as a potential MRSA infection treatment.
4. Experimental
3. Conclusions
4.1. Materials
New series of novel pleuromutilin derivatives containing piperazine
and 1,2,3-1H-triazole structure were synthesized by click reaction under
mild conditions. Initially, these derivatives were tested for the MIC and
MBC against methicillin-resistant S. aureus (ATCC 43300), S. aureus
(ATCC 29213), S. aureus (AD3), S. aureus (144) and E. coli (ATCC
25922). All the synthesized compounds showed moderate to good
antibacterial activity, and compound 59 and compound 61 displayed
better antibacterial activity than other compounds. Subsequently,
compound 59 and compound 61 were selected to evaluate the time-kill
curves and PAE for further investigation of antibacterial activity in vitro.
Pleuromutilin (>90% pure) was purchased from Great Enjoyhood
Biochemical Co. Ltd., (Daying, China). The piperazine derivatives or
secondary amines were purchased from TiTan Technology Co. Ltd.,
(Shanghai, China). The other analytical grade solvents were purchased
from Guangzhou General Reagent Factory (Guangzhou, China). The
target compounds were purified by silica gel column chromatography
(200–300 mesh, Branch of Qingdao Haiyang Chemical Co. Ltd., Shan-
dong, China). 1H NMR and 13C NMR spectra were processed with Bruker
AV-400 spectrometer in chloroform-d using trimethylsilane as an in-
ternal standard. Chemical shift values (δ) were reported in ppm. High-
resolution mass spectra were recorded by Waters Acquity UPLC-LCT
Premier XE with an electro spray ionization (ESI) source.
Table 4
Pharmacokinetic parameters of compound 59 in mice after i.
v. administration.
Parameters
i.v.
4.2. Synthesis
Cmax g⋅h/mL)
(
μ
25.38 ± 6.82
0.083
T
max (h)
A general synthetic route based on the compound 22–(piperazine-1-
yl)–22-deoxypleuromutilin (compound 8) and a variety of piperazine
derivatives or secondary amines were used to prepare pleuromutilin
derivatives (Scheme 1).
t1/2 (h)
0.74 ± 0.08
0.29 ± 0.06
34.27 ± 7.52
46.28 ± 8.94
Cl (L/h kg)
AUC0→t
(μ
g⋅h/mL)
AUC0→∞ (μg⋅h/mL)
Cmax, maximum concentration; Tmax, time to reach Cmax; t1/
2, half-life. Cl, clearance; AUC0→t, Area under the curve from
4.2.1. 22–(4-(2-chloro-acetyl)-piperazine-1-yl)–22-deoxypleuromutilin
(9)
zero to the last measurable plasma concentration; AUC0→∞
,
Compound 22–(piperazine-1-yl)–22-deoxypleuromutilin (compound
8) (1 g, 2.24 mmol) was dissolved in dichloromethane (20 mL). Then
Area under the curve from the last measurable plasma con-
centration to infifinity.
7

G.-Y. Zhang et al.
Bioorganic Chemistry 105 (2020) 104398
K2CO3 (0.62 g, 4.48 mmol) and chloroacetic chloride (0.28 g, 2.46
mmol) were added to the solution and stirred at room temperature for 3
h until reaction was completed. The reaction mixture was extracted with
dichloromethane (30 mL × 3) and washed with brine (50 mL × 3). The
organic phase was combined and dried with anhydrous Na2SO4, evap-
orated under reduced pressure to obtain crude product. The crude
product was purified by silica (200–300 mesh) gel column chromatog-
raphy using ethyl acetate / petroleum ether (3:1, v:v) to give a pure
product (yellow solid, yield 65.7%).
4.2.5. 22–[2-(4-((phenyl piperazine)methyl)-1,2,3-triazol-1-yl) ꢀ 1
-(piperazine-1-yl) ethyl-1-one] deoxy pleuromutilin (48)
White powder; yield: 67%; 1H NMR (400 MHz, Chloroform-d) δ 7.71
(1H, s), 7.25 (3H, d, J = 9.6 Hz), 6.98 – 6.78 (3H, m), 6.50 (1H, dd, J =
17.4, 10.9 Hz), 5.80 (1H, d, J = 8.4 Hz), 5.40 – 5.27 (2H, m), 5.21 (3H, d,
J = 14.0 Hz), 3.77 (2H, s), 3.65 (5H, d, J = 31.7 Hz), 3.37 (1H, t, J = 8.4
Hz), 3.27 – 3.05 (6H, m), 2.78 – 2.47 (9H, m), 2.35 (1H, t, J = 7.1 Hz),
2.22 (1H, dt, J = 19.4, 9.8 Hz), 2.08 (2H, d, J = 19.7 Hz), 1.83 (3H, dd, J
= 33.9, 9.7 Hz), 1.66 (1H, d, J = 10.7 Hz), 1.58 – 1.48 (1H, m), 1.44 (3H,
s), 1.28 (1H, d, J = 15.8 Hz), 1.18 (3H, s), 0.89 (3H, d, J = 6.9 Hz), 0.72
(3H, d, J = 7.0 Hz). 13C NMR (101 MHz, Chloroform-d) δ 217.01 (C3),
168.75 (C21), 163.30, 151.23, 144.45, 139.03 (C19), 129.06, 124.49,
119.69, 117.27 (C20), 116.07, 77.26, 74.56 (C11), 68.62 (C14), 59.43,
58.15 (C4), 53.43, 53.25, 53.18, 52.88 (C22), 52.49, 52.41, 52.12,
50.81, 49.04, 45.44 (C9), 45.22 (C13), 45.01, 43.96 (C12), 42.17, 41.76
(C5), 36.67 (C6), 36.06 (C10), 34.44 (C2), 30.40 (C8), 26.81 (C7), 26.43
(C18), 24.84 (C1), 16.72 (16), 14.86 (C15), 11.50 (C17). HR-MS(ESI+):
Calcd for C41H62N7O5(M + H+): 730.4656; Found: 730.4559.
4.2.2. 22–(4-(2-azidoacetyl))-piperazine-1-yl)–22-deoxypleuromutilin
(10)
Compound 9 (1 g, 1.91 mmol) was dissolved in acetone (10 mL), and
added with sodium azide (0.37 g, 5.73 mmol) in deionized water (10
mL). The reaction was refluxed for 4 h. After the reaction completed, the
mixture was extracted with ethyl acetate (30 mL × 3) and washed with
brine (50 mL × 3). The organic layer was combined and dried with
anhydrous Na2SO4. After the organic solvent was evaporated under
reduced pressure, the solid of crude compound 10 could be used in the
next step without any further purification.
4.2.6. 22–[2-(4-((2-methylphenyl piperazine)methyl)-1,2,3-triazol-1-yl)-
1-piperazi-ne-1-yl) ethyl-1-one] deoxy pleuromutilin (49)
4.2.3. General procedure for the synthesis of secondary amine analogs
containing propynyl and piperazine derivatives (compounds 29–46, 73–80)
Secondary amine derivatives or piperazine derivatives (13.67 mmol)
and potassium carbonate (27.34 mmol) were added to dichloromethane
(30 mL). Then propargyl bromide (1.63 g, 13.67 mmol) was dripped
slowly into the above solution under the condition of ice bath, and the
reaction mixture was stirred for 5 h at room temperature until the re-
action was completed. The reaction solution was washed with brine (50
mL × 3) and extracted with dichloromethane (30 mL × 3). The organic
phase was dried with anhydrous Na2SO4 and concentrated under
reduced pressure to obtain the crude product. Then it was purified by
silica gel column chromatography to obtain compound 29–46, 73–80,
respectively.
White powder; yield: 72%; 1H NMR (400 MHz, Chloroform-d) δ 7.72
(1H, s), 7.16 (2H, t, J = 7.8 Hz), 7.05 – 6.92 (2H, m), 6.50 (1H, dd, J =
17.4, 11.0 Hz, H19), 5.80 (1H, d, J = 8.4 Hz, H14), 5.36 (1H, dd, J =
10.9, 1.6 Hz), 5.27 – 5.15 (3H, m, H20), 3.78 (2H, s), 3.66 (4H, dt, J =
31.0, 5.2 Hz,), 3.37 (1H, dd, J = 10.7, 6.4 Hz, H11), 3.26 – 3.03 (2H, m,
H22), 2.95 (4H, t, J = 4.8 Hz), 2.70 (4H, s), 2.58 (4H, q, J = 9.3, 5.4 Hz),
2.36 (1H, q, J = 6.7 Hz), 2.29 (3H, s, H2,H4), 2.25 – 2.15 (1H, m, H10),
2.08 (2H, dd, J = 15.6, 5.6 Hz, H13), 1.82 (4H, s), 1.68 (2H, t, J = 11.2
Hz, H1), 1.58 – 1.47 (2H, m, H7), 1.44 (3H, s, H15), 1.41 – 1.35 (1H, m),
1.28 (1H, d, J = 15.9 Hz, H8), 1.18 (3H, s, H18), 0.89 (3H, d, J = 6.9 Hz,
H17), 0.72 (3H, d, J = 6.9 Hz, H16). 13C NMR (101 MHz, Chloroform-d)
δ 216.94 (C3), 168.75 (C21), 163.31, 151.43, 144.76, 139.03 (C19),
132.59, 131.01, 126.51, 124.35, 123.07, 118.97, 117.29 (C20), 77.22,
74.58 (C11), 68.62 (C14), 59.45, 58.15 (C4), 53.46, 53.38, 52.51 (C22),
52.13, 51.58, 50.86, 45.45 (C9), 45.28, 45.03, 44.66 (C13), 43.97 (C12),
42.18, 41.77 (C5), 36.67 (C6), 36.07 (C10), 34.44 (C2), 30.41 (C8),
26.82 (C7), 26.40 (C18), 24.85 (C1), 17.87, 16.72 (C16), 14.86 (C15),
11.49 (C17). HR-MS(ESI+): Calcd forC42H64N7O5(M + H+): 744.4812;
Found: 744.4714.
4.2.4. 22–[2-(4-((methyl piperazine)methyl)-1,2,3-triazol-1-yl) ꢀ 1
-(piperazine-1-yl) ethyl-1-one] deoxy pleuromutilin (47)
Compound 10 (1 g, 1.63 mmol) was dissolved in a mixture solution
of tert-butyl alcohol (10 mL) and water (10 mL), then acetylene amine
derivatives compounds 29 (0.18 g, 1.63 mmol), copper(II) sulfate pen-
tahydrate (0.008 g, 0.033 mmol) and sodium ascorbate (0.007 g, 0.033
mmol) were added into the solution. The reaction mixture was stirred
for 3 h at room temperature. After the reaction was completed, it was
extracted with ethyl acetate (30 mL × 3) and the organic phase was
washed twice with saturated sodium chloride solution. The organic
layers were dried with anhydrous Na2SO4 and concentrated under
reduced pressure. The residue was purified by silica (200–300 mesh) gel
column chromatography using petroleum dichloromethane/methanol
(20: 1, v:v) as eluent to give the product compound 47.
4.2.7. 22–[2-(4-((3-methylphenyl piperazine)methyl)-1,2,3-triazol-1-yl)-
1-(piperazine-1-yl) ethyl-1-one] deoxy pleuromutilin (50)
White powder; yield: 62%; 1H NMR (400 MHz, Chloroform-d) δ 7.71
(1H, s), 7.14 (1H, t, J = 7.7 Hz), 6.79 – 6.65 (3H, m), 6.50 (1H, dd, J =
17.4, 11.0 Hz, H19), 5.80 (1H, d, J = 8.5 Hz, H14), 5.36 (1H, dd, J =
10.9, 1.6 Hz), 5.25 – 5.16 (3H, m,H20), 3.77 (2H, s), 3.65 (4H, dt, J =
31.4, 5.1 Hz), 3.37 (1H, dd, J = 10.7, 6.4 Hz), 3.27 – 3.03 (7H, m, H22,
H11), 2.69 (4H, t, J = 5.0 Hz), 2.59 (2H, ddq, J = 16.9, 11.2, 5.7 Hz,),
2.42 – 2.15 (6H, m, H2,H4,H10,H13), 2.09 (2H, q, J = 8.4, 6.9 Hz), 1.87
– 1.74 (5H, m, H6,H8,11-OH), 1.72 – 1.45 (4H, m, H1,H7,H13), 1.44
(3H, s, H15), 1.41 – 1.25 (1H, m, H8), 1.18 (3H, s, H18), 0.89 (3H, d, J =
6.9 Hz, H17), 0.72 (3H, d, J = 6.9 Hz, H16). 13C NMR (101 MHz,
Chloroform-d) δ 216.97 (C3), 168.76 (C21), 163.29, 151.31, 144.54,
139.02 (C19), 138.74, 128.90, 124.42, 120.61, 117.30 (C20), 116.93,
113.21, 77.23, 74.57 (C11), 68.62 (C14), 59.44, 58.15 (C4), 53.28,
52.93, 52.50 (C22), 52.13, 50.82, 49.14, 45.44 (C9), 45.26, 45.01, 44.84
(C13), 43.96 (C12), 42.18, 41.76 (C5), 36.67 (C6), 36.06 (C10), 34.44
(C2), 30.41 (C8), 26.82 (C7), 26.40 (C18), 24.85 (C1), 21.75, 16.72
(C16), 14.86 (C15), 11.50 (C17). HR-MS(ESI+): Calcd for
White powder; yield: 47%; 1H NMR (400 MHz, Chloroform-d) δ 7.67
(1H, s), 6.49 (1H, dd, J = 17.4, 11.0 Hz, H19), 5.79 (1H, d, J = 8.4 Hz,
H14), 5.35 (1H, d, J = 11.0 Hz, H20), 5.19 (3H, q, J = 9.1 Hz, H20), 3.71
(2 H, s), 3.64 (5 H, dt, J = 30.7, 5.6 Hz, H22,H11), 3.49 – 3.33 (2 H, m),
3.27 – 3.03 (2 H, m), 2.72 – 2.42 (13H, m), 2.39 – 2.16 (5 H, m, H2,H4,
H10,H13), 2.07 (3 H, d, J = 19.8 Hz, H6,H8,11-OH), 1.84 – 1.46 (4 H, m,
H1,H7,H13), 1.43 (3 H, s, H15), 1.40 – 1.23 (2 H, m, H8), 1.17 (3 H, s,
H18), 0.88 (3 H, d, J = 6.9 Hz, H17), 0.71 (3 H, d, J = 6.9 Hz, H16). 13
C
NMR (101 MHz, Chloroform-d) δ 217.01 (C3), 168.75 (21), 163.29,
144.41, 139.01 (C19), 124.42, 117.29 (C20), 77.23, 74.56 (C11), 68.61
(C14), 59.43, 58.15 (C4), 54.84, 53.09, 52.61 (C22), 52.49, 52.12,
50.80, 45.84 (C9), 45.44, 45.23, 45.01 (C13), 43.95 (C12), 42.16, 41.76
(C5), 36.67 (C6), 36.05 (C10), 34.44 (C2), 30.40 (C8), 26.81 (C7), 26.40
(C18), 24.84 (C1), 16.72 (C16), 14.86 (C15), 11.50 (C17). HR-MS(ESI+):
Calcd for C36H60N7O5(M + H+): 668.4499; Found: 668.4506.
C
42H64N7O5(M + H+): 744.4812; Found: 744.4869.
4.2.8. 22–[2-(4-((4-methylphenyl piperazine)methyl)-1,2,3-triazol-1-yl)-
1-(piperazine-1-yl) ethyl-1-one] deoxy pleuromutilin (51)
White powder; yield: 58%; 1H NMR (400 MHz, Chloroform-d) δ 7.70
(1H, s), 7.06 (2H, d, J = 8.1 Hz), 6.83 (2H, d, J = 8.2 Hz), 6.50 (1H, dd, J
8

G.-Y. Zhang et al.
Bioorganic Chemistry 105 (2020) 104398
= 17.4, 11.0 Hz, H19), 5.80 (1H, d, J = 8.4 Hz, H14), 5.36 (1H, d, J =
11.0 Hz, H20), 5.25 – 5.17 (3H, m), 3.77 (2H, s), 3.65 (4H, dt, J = 31.3,
5.1 Hz, H22,H11), 3.37 (1H, dd, J = 10.6, 6.4 Hz), 3.26 – 3.03 (6H, m),
2.69 (4H, t, J = 5.0 Hz), 2.63 – 2.50 (3H, m), 2.35 (1H, p, J = 8.2, 7.6
Hz), 2.27 (3H, s, H,H2,H4), 2.25 – 2.14 (3H, m, H10,H13), 2.08 (2H, d, J
= 19.6 Hz,11-OH), 1.81 (4H, q, J = 12.3, 9.3 Hz,H1,H6,H8), 1.68 (2H,
dd, J = 12.7, 9.3 Hz, H7), 1.59 – 1.47 (1H, m), 1.44 (3H, s, H15), 1.41 –
1.24 (2H, m, H8), 1.18 (3H, s, H18), 0.89 (3H, d, J = 6.9 Hz, H17), 0.72
(3H, d, J = 6.9 Hz, H16). 13C NMR (101 MHz, Chloroform-d) δ 216.91
(C3), 168.74 (C21), 163.30, 149.19, 144.57, 139.04 (C19), 129.58,
129.19, 124.37, 117.27 (C20), 116.41, 77.22, 74.58 (C11), 68.63 (C14),
59.44, 58.15 (C4), 53.40, 53.28, 52.92, 52.50 (C22), 52.13, 50.82,
49.63, 45.45 (C9), 45.27, 45.04, 44.73 (C13), 43.97 (C12), 42.18, 41.77
(C5), 36.68 (C6), 36.08 (C10), 34.43 (C2), 30.42 (C8), 26.82 (C7), 26.41
(C18), 24.85 (C1), 20.39, 16.71 (C16), 14.86 (C15), 11.48 (C17). HR-MS
(ESI+): Calcd for C42H64N7O5(M + H+): 744.4812; Found: 744.4841.
Hz), 3.37 (1H, dd, J = 10.6, 6.4 Hz), 3.26 – 3.01 (6H, m), 2.64 (9H, dt, J
= 53.0, 9.6 Hz, H10), 2.35 (1H, p, J = 7.2 Hz, H13), 2.29 – 2.14 (2H, m,
H2), 2.12 – 2.03 (2H, m, H4), 1.92 – 1.69 (4H, m, H1, H6), 1.68 – 1.46
(3H, m, H13,H7), 1.44 (3H, s, H15), 1.40 – 1.24 (2H, m, H8), 1.17 (3H, s,
H18), 0.89 (3H, d, J = 6.9 Hz, H17), 0.72 (3H, d, J = 6.9 Hz, H16). 13C
NMR (101 MHz, Chloroform-d) δ 216.96 (C3), 168.75 (C21), 163.29,
153.77, 145.67, 144.54, 139.02 (C19), 124.39, 118.19, 117.30 (C20),
114.40, 77.23, 76.79, 74.57 (C11), 68.61 (C14), 59.44, 58.15 (C4),
55.55, 53.42, 53.25, 52.98, 52.87, 52.50 (C22), 52.13, 50.82, 50.53,
45.44 (C9), 45.26, 45.01 (C13), 43.96 (C12), 42.17, 41.76 (C5), 36.67
(C6), 36.06 (C10), 34.44 (C2), 30.41 (C8), 26.82 (C7), 26.41 (C18),
24.84 (C1), 16.72 (C16), 14.86 (C15), 11.50 (C17). HR-MS (ESI+): Calcd
for C38H64N6O6 (M + H+): 760.4762; Found: 760.4839.
4.2.12. 22–[2-(4-((2-hydroxyphenyl piperazine)methyl)-1,2,3-triazol-1-
yl)-1-(piperazine-1-yl) ethyl-1-one] deoxy pleuromutilin (55)
White powder; yield: 66%; 1H NMR (400 MHz, Chloroform-d) δ 7.72
(1H, s), 7.15 (1H, dd, J = 7.9, 1.6 Hz), 7.06 (1H, td, J = 7.8, 1.6 Hz), 6.93
(1H, dd, J = 8.0, 1.6 Hz), 6.85 (1H, td, J = 7.6, 1.6 Hz), 6.50 (1H, dd, J =
17.4, 11.0 Hz, H19), 5.79 (1H, d, J = 8.4 Hz, H14), 5.40 – 5.28 (2H, m,
H20), 5.23 (3H, s), 3.79 (2H, s), 3.65 (5H, dt, J = 30.6, 5.2 Hz), 3.37 (1H,
t, J = 8.4 Hz, H11), 3.25 – 3.07 (2H, m, H22), 2.91 (4H, t, J = 4.8 Hz),
2.79 – 2.48 (7H, m), 2.39 – 2.14 (3H, m, H2,H4), 2.08 (2H, d, J = 19.9
Hz, 11-OH,H7), 1.83 – 1.74 (1H, m, H13), 1.66 (2H, td, J = 15.1, 13.2,
7.0 Hz, H1), 1.55 (1H, d, J = 15.7 Hz), 1.51 – 1.46 (1H, m, H7), 1.44
(3H, s, H15), 1.41 – 1.30 (1H, m, H13), 1.28 (1H, d, J = 10.8 Hz, H8),
1.17 (4H, s, H18), 0.89 (3H, d, J = 6.9 Hz, H17), 0.72 (3H, d, J = 6.9 Hz,
H16). 13C NMR (101 MHz, Chloroform-d) δ 216.98 (C3), 168.75 (C21),
163.27, 151.47, 144.41, 139.02, 138.90 (C19), 126.42, 124.47, 121.46,
120.01, 117.29 (C20), 114.02, 77.24, 74.57 (C11), 68.62 (C14), 59.43,
58.14 (C4), 53.51, 53.43, 53.20, 52.48, 52.42, 52.11, 50.81 (C22),
45.44, 45.25 (C9), 45.01 (C13), 43.96 (C12), 42.19 (C5), 41.76 (C5),
36.67 (C6), 36.06 (C10), 34.44 (C2), 30.41 (C8), 26.81 (C7), 26.40
(C18), 24.84 (C1), 16.72 (16.53), 14.86 (C15), 11.49 (C17). HR-MS
(ESI+): Calcd for C41H62N7O6 (M + H+): 746.4605; Found:746.4660.
4.2.9. 22–[2-(4-((2-methoxyphenyl piperazine)methyl)-1,2,3-triazol-1-
yl)-1-(piperazine-1-yl) ethyl-1-one] deoxy pleuromutilin (52)
White powder; yield: 61%; 1H NMR (400 MHz, Chloroform-d) δ 7.71
(1H, s), 6.91 (5H, td, J = 26.4, 25.7, 7.2 Hz), 6.49 (1H, dd, J = 17.4,
11.0 Hz, H19), 5.79 (1H, d, J = 8.4 Hz, H14), 5.35 (1H, d, J = 11.0 Hz),
5.22 (3H, d, J = 4.5 Hz, H20), 5.19 (1H, s), 3.93 – 3.73 (5H, m), 3.68
(2H, d, J = 5.4 Hz), 3.62 (2H, d, J = 5.2 Hz, H22), 3.36 (1H, dd, J = 10.6,
6.5 Hz, H11), 3.25 – 2.98 (6H, m), 2.74 (4H, s), 2.58 (4H, ddq, J = 17.4,
11.5, 6.0, 5.6 Hz, H2,H4), 2.35 (1H, q, J = 7.0 Hz, H10), 2.21 (2H, hept,
J = 9.6, 9.2 Hz, H6,H1), 2.07 (2H, d, J = 20.5 Hz,H8), 2.01 – 1.87 (1H,
m, 11-OH), 1.78 (1H, d, J = 14.4 Hz, H1), 1.71 – 1.46 (3H, m, H13, H7),
1.43 (3H, s, H15), 1.41 – 1.23 (2H, m, H8), 1.17 (3H, s, H18), 0.89 (3H,
d, J = 6.9 Hz, H17), 0.71 (3H, d, J = 6.9 Hz, H16). 13C NMR (101 MHz,
Chloroform-d) δ 217.00 (C3), 168.76 (C21), 163.30, 152.22, 144.45,
141.23, 139.01 (C19), 124.46, 122.89, 120.95, 118.20, 117.30 (C20),
111.14, 77.24, 74.56 (C11), 68.61 (C14), 59.44, 58.15 (C4), 55.31,
53.43, 53.26, 53.04, 52.51 (C22), 52.14, 50.82, 50.49, 45.44 (C9),
45.26, 45.01 (C13), 43.96 (C12), 42.17, 41.76 (C5), 36.67 (C6), 36.06
(C10), 34.44 (C2), 30.41 (C8), 26.81 (C7), 26.40 (C18), 24.84 (C1),
16.72 (C16), 14.86 (C15), 11.50 (C17). HR-MS(ESI+): Calcd for
C38H64N6O6(M + H+): 760.4762; Found: 760.4810.
4.2.13. 22–[2-(4-((3-hydroxyphenyl piperazine)methyl)-1,2,3-triazol-1-
yl)-1-(piperazine-1-yl) ethyl-1-one] deoxy pleuromutilin (56)
White powder; yield: 52%; 1H NMR (400 MHz, Chloroform-d) δ 7.71
(1H, s), 7.06 (1H, td, J = 8.1, 2.4 Hz), 6.59 – 6.40 (2H, m, H19), 6.38 –
6.27 (2H, m), 5.80 (1H, d, J = 8.4 Hz, H14), 5.35 (1H, d, J = 11.1 Hz),
5.26 – 5.16 (3H, m, H20), 3.75 (4H, s), 3.64 (4H, dt, J = 35.5, 5.2 Hz,
H22,H11), 3.37 (1H, dd, J = 10.7, 6.5 Hz), 3.21 (1H, d, J = 17.2 Hz),
3.17 – 3.06 (6H, m,), 2.71 – 2.51 (9H, m, H2,H4,H10), 2.34 (1H, q, J =
7.0 Hz), 2.25 (1H, d, J = 8.0 Hz, 11-OH), 2.21 (1H, d, J = 9.4 Hz, H8),
2.14 – 2.03 (2H, m, H6), 1.79 (1H, d, J = 14.3 Hz, H1), 1.72 – 1.59 (2H,
m, H1), 1.59 – 1.47 (1H, m, H7), 1.44 (3H, s, H15), 1.41 – 1.33 (1H, m,
H13), 1.29 (1H, d, J = 16.0, H8), 1.18 (3H, s, H18), 0.89 (3H, d, J = 6.9,
H17), 0.72 (3H, d, J = 6.9 Hz, H16). 13C NMR (151 MHz, Chloroform-d)
δ 217.03 (C3), 168.80 (C21), 163.35, 156.99, 152.65, 144.27, 139.02
(C19), 129.91, 124.62, 117.32 (C20), 108.31, 106.78, 103.20, 74.61
(C11), 68.68 (C14), 59.46, 58.18 (C4), 53.40, 53.15, 52.68 (C22), 52.50,
52.13, 50.84, 48.77, 45.46 (C9), 45.27, 45.06 (C13), 44.79, 43.98 (C12),
42.24, 41.79 (C5), 36.69 (C6), 36.08 (C10), 34.45 (C2), 30.43 (C8),
26.83 (C7), 26.42 (C18), 24.86 (C1), 16.73 (C16), 14.88 (C15), 11.49
(C17). HR-MS (ESI+): Calcd for C41H62N7O6 (M + H+): 746.4605;
Found: 746.4666.
4.2.10. 22–[2-(4-((3-methoxyphenyl piperazine)methyl)-1,2,3-triazol-1-
yl)-1-(piperazine-1-yl) ethyl-1-one] deoxy pleuromutilin (53)
White powder; yield: 55%; 1H NMR (400 MHz, Chloroform-d) δ 7.70
(1H, s), 7.16 (1H, t, J = 8.2 Hz), 6.59 – 6.30 (4H, m,H19), 5.79 (1H, d, J
= 8.4, H14), 5.35 (1H, d, J = 11.0 Hz), 5.26 – 5.14 (3H, m), 3.77 (5H, d,
J = 8.7 Hz, H22,H11), 3.69 (2H, t, J = 5.1 Hz), 3.61 (2H, t, J = 5.1 Hz),
3.37 (1H, dd, J = 10.6, 6.4 Hz), 3.27 – 3.04 (6H, m), 2.73 – 2.49 (8H, m,
H4,H10), 2.35 (1H, p, J = 8.0, 7.4 Hz, H8), 2.28 – 2.14 (2H, m, H2), 2.08
(2H, d, J = 19.8 Hz, 11-OH), 1.88 (2H, d, J = 7.7 Hz, H6), 1.78 (1H, dd,
J = 14.5, 3.1 Hz, H13), 1.66 (2H, q, J = 11.4 Hz, H1), 1.58 – 1.45 (2H, m,
H7), 1.44 (3H, s, H15), 1.33 (1H, d, J = 22.4 Hz, H13), 1.26 (1H, s, H8),
1.17 (3H, s, H18), 0.89 (3H, d, J = 6.9 Hz, H17), 0.72 (3H, d, J = 7.0 Hz,
H16). 13C NMR (101 MHz, Chloroform-d) δ 216.98 (C3), 168.75 (C21),
163.28, 160.52, 152.64, 144.50, 139.02 (C19), 129.73, 124.43, 117.30
(C20), 108.86, 104.42, 102.47, 77.24, 74.56 (C11), 68.61 (C14), 59.44,
58.15 (C4), 55.16, 53.43, 53.26, 52.84, 52.49 (C22), 52.12, 50.81,
48.96, 45.44 (C9), 45.24, 45.01 (C13), 43.96 (C12), 42.17, 41.76 (C5),
36.67 (C6), 36.06 (C10), 34.44 (C2), 30.41 (C8), 26.82 (C7), 26.41
(C18), 24.84 (C1), 16.72 (C16), 14.86 (C15), 11.50 (C17). HR-MS
(ESI+): Calcd for C38H64N6O6 (M + H+): 760.4762; Found: 760.4810.
4.2.14. 22–[2-(4-((2-nitrophenyl piperazine)methyl)-1,2,3-triazol-1-yl)-
1-(piperazine-1-yl) ethyl-1-one] deoxy pleuromutilin (57)
4.2.11. 22–[2-(4-((4-methoxyphenyl piperazine)methyl)-1,2,3-triazol-1-
yl)-1-(piperazine-1-yl) ethyl-1-one] deoxy pleuromutilin (54)
White powder; yield: 75%; 1H NMR (400 MHz, Chloroform-d) δ 7.75
(1H, d, J = 8.1 Hz), 7.47 (1H, t, J = 7.8 Hz), 7.13 (1H, d, J = 8.3 Hz),
7.03 (1H, t, J = 7.7 Hz), 6.50 (1H, dd, J = 17.4, 10.9 Hz, H19), 5.80 (1H,
d, J = 8.4 Hz, H14), 5.35 (1H, d, J = 11.0 Hz), 5.21 (3H, d, J = 15.4 Hz,
H20), 3.77 (2H, s), 3.66 (4H, dt, J = 29.9, 5.2 Hz, H22,H11), 3.37 (1H,
dd, J = 10.7, 6.4 Hz), 3.22 (1H, d, J = 17.2 Hz), 3.09 (6H, dd, J = 7.7,
White powder; yield: 79%; 1H NMR (400 MHz, Chloroform-d) δ 7.70
(1H, s), 6.91 – 6.79 (4H, m), 6.50 (1H, dd, J = 17.4, 11.0 Hz, H19), 5.79
(1H, d, J = 8.4 Hz, H14), 5.35 (1H, dd, J = 10.9, 1.6 Hz), 5.26 – 5.15
(3H, m, H20), 3.76 (4H, s), 3.69 (2H, t, J = 5.1 Hz), 3.60 (2H, d, J = 5.2
9

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Bioorganic Chemistry 105 (2020) 104398
3.3 Hz), 2.72 – 2.51 (9H, m), 2.35 (1H, p, J = 7.2 Hz), 2.21 (2H, dq, J =
19.5, 9.3 Hz, H13,H10), 2.13 – 2.04 (2H, m, 11-OH,H8), 1.83 – 1.69 (4H,
m, H2,H4), 1.66 (1H, d, J = 11.1 Hz, H13), 1.58 – 1.47 (2H, m, H7), 1.44
(3H, s, H15), 1.42 – 1.26 (1H, m,H8), 1.18 (3H, s, H18), 0.89 (3H, d, J =
7.0 Hz, H17), 0.72 (3H, d, J = 6.9 Hz, H16). 13C NMR (101 MHz,
Chloroform-d) δ 216.97 (C3), 168.76 (C21), 163.29, 145.95, 144.39,
143.40, 139.01 (C19), 133.43, 125.83, 124.37, 121.72, 121.01, 117.31
(C20), 77.22, 76.74, 74.57 (C11), 68.60 (C14), 59.44, 58.15 (C4), 53.13,
52.74, 52.51 (C22), 52.13, 51.56, 50.83, 45.44 (C9), 45.27, 45.01 (C13),
43.96 (C12), 42.18, 41.76 (C5), 36.67 (C6), 36.06 (C10), 34.44 (C2),
30.41 (C8), 26.82 (C7), 26.39 (C18), 24.84 (C1), 16.72 (C16), 14.86
(C15), 11.50 (C17). HR-MS (ESI+): Calcd for C41H61N8O7 (M + H+):
775.4507; Found: 775.4559.
(2H, m, H6,11-OH), 1.91 (2H, s, H8,H13), 1.83 – 1.73 (1H, m, H13),
1.66 (2H, dd, J = 15.4, 7.4 Hz, H1), 1.58 – 1.45 (2H, m, H7), 1.43 (3H, s,
H15), 1.40 – 1.23 (2H, m, H8), 1.17 (3H, s, H18), 0.89 (3H, d, J = 7.0 Hz,
H17), 0.71 (3H, d, J = 6.9 Hz, H16). 13C NMR (101 MHz, Chloroform-d)
δ 216.96 (C3), 168.75 (C21), 163.31, 149.22, 144.58, 139.03 (C19),
130.60, 128.72, 127.54, 124.35, 123.61, 120.36, 117.28 (C20), 77.24,
74.56 (C11), 68.61 (C14), 59.44, 58.14 (C4), 53.42, 53.25, 53.04, 52.51
(C22), 52.13, 51.07, 50.86, 45.44 (C9), 45.27, 45.01 (C13), 43.96 (C12),
42.17, 41.76 (C5), 36.67 (C6), 36.06 (C10), 34.43 (C2), 30.41 (C8),
26.81 (C7), 26.41 (C18), 24.84 (C1), 16.71 (C16), 14.85 (C15), 11.49
(C17). HR-MS (ESI+): Calcd for C41H61ClN7O5 (M + H+): 764.4317;
Found: 764.4308.
4.2.18. 22–[2-(4-((4-chlorophenyl piperazine)methyl)-1,2,3-triazol-1-yl)-
1-(piperazine-1-yl) ethyl-1-one] deoxy pleuromutilin (61)
4.2.15. 22–[2-(4-((3-nitrophenyl piperazine)methyl)-1,2,3-triazol-1-yl)-
1-(piperazine-1-yl) ethyl-1-one] deoxy pleuromutilin (58)
White powder; yield: 68%; 1H NMR (400 MHz, Chloroform-d) δ 7.70
(1H, s), 7.19 (2H, d, J = 8.6 Hz), 6.82 (2H, d, J = 8.7 Hz), 6.49 (1H, dd, J
= 17.4, 11.0 Hz, H19), 5.79 (1H, d, J = 8.4 Hz, H14), 5.35 (1H, d, J =
11.0 Hz), 5.20 (3H, d, J = 13.3 Hz, H20), 3.76 (2H, s), 3.65 (4H, dt, J =
31.6, 5.2 Hz), 3.37 (1H, dd, J = 10.6, 6.5 Hz), 3.26 – 3.05 (6H, m), 2.63
(6H, dt, J = 38.2, 5.5 Hz, H2,H4), 2.34 (1H, q, J = 7.2 Hz, H13), 2.29 –
2.14 (2H, m, H10), 2.08 (2H, d, J = 19.7 Hz,11-OH), 1.87 – 1.78 (3H, m,
H6.H8), 1.77 – 1.67 (2H, m, H1), 1.66 – 1.56 (1H, m, H13), 1.55 – 1.46
(2H, m, H7), 1.44 (4H, s, H15), 1.41 – 1.24 (2H, m, H8), 1.17 (3H, s,
White powder; yield: 60%; 1H NMR (400 MHz, Chloroform-d) δ 7.76
– 7.67 (2H, m), 7.66 – 7.62 (1H, m), 7.36 (1H, t, J = 8.2 Hz), 7.17 (1H,
dd, J = 8.3, 2.5 Hz), 6.50 (1H, dd, J = 17.4, 10.9 Hz, H19), 5.80 (1H, d, J
= 8.4 Hz, H14), 5.35 (1H, d, J = 11.0 Hz), 5.21 (3H, d, J = 18.6 Hz), 3.78
(2H, s), 3.65 (4H, dt, J = 31.2, 5.1 Hz), 3.37 (1H, dd, J = 10.6, 6.5 Hz),
3.29 (4H, t, J = 5.0 Hz), 3.26 – 3.06 (3H, m), 2.76 – 2.52 (7H, m, H2),
2.34 (1H, q, J = 7.2 Hz, H10), 2.28 – 2.14 (2H, m, H4), 2.09 (2H, q, J =
8.5, 6.9 Hz, 11-OH,H8), 1.84 – 1.46 (8H, m, H1,H7,H13), 1.44 (3H, s,
H15), 1.41 – 1.24 (1H, m, H8), 1.17 (3H, s, H18), 0.89 (3H, d, J = 6.9 Hz,
H17), 0.72 (3H, d, J = 6.9, H16). 13C NMR (101 MHz, Chloroform-d) δ
216.96 (C3), 168.75 (C21), 163.24, 151.74, 149.24, 144.30, 139.02
(C19), 129.64, 124.50, 121.08, 117.29 (C20), 113.62, 109.59, 77.23,
74.57 (C11), 68.63 (C14), 59.43, 58.14 (C4), 53.42, 53.16, 52.47 (C22),
52.10, 50.79, 48.27, 45.44 (C9), 45.23, 45.01, 44.88 (C13), 43.96 (C12),
42.19, 41.76 (C5), 36.66 (C6), 36.06 (C10), 34.44 (C2), 30.40 (C8),
26.82 (C7), 26.40 (C18), 24.84 (C1), 16.72 (C16), 14.85 (C15), 11.49
(C17). HR-MS (ESI+): Calcd for C41H61N8O7 (M + H+): 775.4507;
Found: 775.4561.
H18), 0.89 (3H, d, J = 6.9 Hz, H17), 0.72 (3H, d, J = 6.9 Hz, H16). 13
C
NMR (101 MHz, Chloroform-d) δ 216.95 (C3), 168.75 (C21), 163.27,
149.87, 144.43, 139.03 (C19), 128.90, 124.45, 124.43, 117.29 (C20),
117.21, 77.23, 74.57 (C11), 68.62 (C14), 59.43, 58.14 (C4), 53.42,
53.21, 52.89, 52.70, 52.48 (C22), 52.18, 52.11, 50.80, 49.06, 45.44
(C9), 45.24, 45.01 (C13), 43.96 (C12), 42.18, 41.76 (C5), 36.66 (C6),
36.07 (C10), 34.44 (C2), 30.40 (C8), 26.82 (C7), 26.41 (C18), 24.84
(C1), 16.72 (C16), 14.86 (C15), 11.49 (C17). HR-MS (ESI+): Calcd for
C
41H61ClN7O5 (M + H+): 764.4317; Found: 764.4318.
4.2.19. 22–[2-(4-((2-fluorophenyl piperazine)methyl)-1,2,3-triazol-1-yl)-
1-(piperazine-1-yl) ethyl-1-one] deoxy pleuromutilin (62)
4.2.16. 22–[2-(4-((4-nitrophenyl piperazine)methyl)-1,2,3-triazol-1-yl)-
1-(piperazine-1-yl) ethyl-1-one] deoxy pleuromutilin (59)
White powder; yield: 49%; 1H NMR (400 MHz, Chloroform-d) δ 7.70
(1H, s), 7.09 – 6.97 (2H, m), 6.93 (2H, t, J = 8.1 Hz), 6.49 (1H, dd, J =
17.4, 10.9 Hz, H19), 5.79 (1H, d, J = 8.4,H14), 5.35 (1H, d, J = 11.0 Hz),
5.20 (3H, d, J = 13.8 Hz, H20), 3.78 (2H, s), 3.65 (4H, dt, J = 29.9, 5.1
Hz), 3.37 (1H, dd, J = 10.5, 6.4 Hz), 3.24 – 3.05 (6H, m, H22,H11), 2.72
(4H, t, J = 4.8 Hz), 2.58 (3H, ddp, J = 15.7, 11.1, 6.2 Hz), 2.36 (1H, d, J
= 7.0 Hz), 2.34 – 2.14 (2H, m, H2), 2.08 (2H, d, J = 20.9 Hz, H4), 1.91 –
1.69 (4H, m, H10,H13), 1.61 (2H, dd, J = 35.4, 7.1 Hz, H1), 1.55 – 1.48
(1H, m, H7), 1.43 (3H, s, H15), 1.40 – 1.33 (1H, m, H7), 1.28 (1H, d, J =
16.0 Hz, H8), 1.17 (4H, s, H18), 0.89 (3H, d, J = 7.0 Hz, H17), 0.71 (3H,
d, J = 6.9 Hz, H16). 13C NMR (101 MHz, Chloroform-d) δ 216.93 (C3),
168.75 (C21), 163.31, 154.47, 144.46, 139.04 (C19), 124.39, 124.36,
122.42, 118.95, 118.92, 117.27 (C20), 116.16 (d, JC-F = 21.21 Hz),
74.57 (C11), 68.62 (C14), 59.43, 58.15 (C4), 53.23, 52.89 (C22), 52.50,
52.13, 50.84, 50.42, 50.39, 45.44 (C9), 45.28, 45.03 (C13), 43.96 (C12),
42.18, 41.76 (C5), 36.67 (C6), 36.07 (C10), 34.43 (C2), 30.41 (C8),
26.82 (C7), 26.41 (C18), 24.84 (C1), 16.70 (C16), 14.85 (C15), 11.47
(C17). HR-MS (ESI+): Calcd for C41H61FN7O5 (M + H+): 748.4562;
Found: 748.4584.
White powder; yield: 53%; 1H NMR (400 MHz, Chloroform-d) δ 8.11
(2H, d, J = 9.0 Hz), 7.70 (1H, s), 6.80 (2H, d, J = 9.1 Hz), 6.49 (1H, dd, J
= 17.4, 10.9 Hz, H19), 5.79 (1H, d, J = 8.4, H14), 5.35 (1H, d, J = 11.1
Hz), 5.21 (3H, d, J = 19.3 Hz, H20), 3.77 (2H, s, H22), 3.68 (2H, d, J =
5.2 Hz,H11), 3.61 (2H, t, J = 5.2 Hz), 3.49 – 3.31 (5H, m), 3.28 – 3.06
(2H, m), 2.64 (9H, dt, J = 38.0, 9.1 Hz,H2), 2.34 (1H, p, J = 7.1 Hz),
2.28 – 2.15 (1H, m, H10), 2.08 (2H, d, J = 19.7 Hz, 11-OH,H13), 1.78
(4H, d, J = 17.1 Hz, H6,H8,H4), 1.65 (2H, dq, J = 19.4, 9.0, 8.0 Hz, H1),
1.53 (2H, dd, J = 16.2, 12.2 Hz, H7), 1.44 (3H, s, H15), 1.28 (1H, d, J =
16.2 Hz,H8), 1.17 (3H, s, H18), 0.89 (3H, d, J = 6.9 Hz, H17), 0.72 (3H,
d, J = 7.0 Hz, H16). 13C NMR (101 MHz, Chloroform-d) δ 216.97 (C3),
168.76 (C21), 163.24, 154.80, 144.08, 139.03 (C19), 138.40, 125.92,
124.56, 117.28 (C20), 112.64, 77.23, 74.56 (C11), 68.64 (C14), 59.42,
58.14 (C4), 53.43, 53.08, 52.56 (C22), 52.45, 52.26, 52.08, 52.01,
50.77, 46.94, 45.44 (C9), 45.21, 45.01, 43.96 (C13), 42.20 (C12), 41.76
(C5), 36.66 (C6), 36.07 (C10), 34.43 (C2), 30.40 (C8), 26.82 (C7), 26.42
(C18), 24.84 (C1), 16.72 (C16), 14.86 (C15), 11.49 (C17). HR-MS
(ESI+): Calcd for C41H61N8O7 (M + H+): 775.4507; Found: 775.4592.
4.2.17. 22–[2-(4-((2-chlorophenyl piperazine)methyl)-1,2,3-triazol-1-yl)-
1-(piperazine-1-yl) ethyl-1-one] deoxy pleuromutilin (60)
4.2.20. 22–[2-(4-((3-fluorophenyl piperazine)methyl)-1,2,3-triazol-1-yl)-
1-(piperazine-1-yl) ethyl-1-one] deoxy pleuromutilin (63)
White powder; yield: 45%; 1H NMR (400 MHz, Chloroform-d) δ 7.70
(1H, s), 7.34 (1H, d, J = 7.7 Hz), 7.21 (1H, t, J = 7.6 Hz), 7.03 (1H, d, J
= 7.9 Hz), 6.96 (1H, t, J = 7.6 Hz), 6.49 (1H, dd, J = 17.4, 11.0 Hz, H19),
5.79 (1H, d, J = 8.4 Hz), 5.35 (1H, d, J = 10.9 Hz), 5.20 (3H, d, J = 16.1
Hz,H20), 3.79 (2H, s), 3.65 (4H, dt, J = 29.7, 5.2 Hz, H22,H11), 3.36
(1H, dd, J = 10.7, 6.4 Hz), 3.21 (1H, d, J = 17.2 Hz), 3.10 (6H, d, J = 8.9
Hz), 2.78 – 2.68 (4H, m), 2.58 (3H, ddp, J = 16.0, 11.3, 6.2 Hz,H2), 2.34
(1H, dq, J = 10.2, 4.9, 3.0 Hz,H4), 2.29 – 2.13 (2H, m, H10), 2.12 – 2.03
White powder; yield: 62%; 1H NMR (400 MHz, Chloroform-d) δ 7.70
(1H, s), 7.17 (1H, q, J = 7.9), 6.65 (1H, dd, J = 8.4, 2.4 Hz,H19), 6.60 –
6.44 (3H, m), 5.80 (1H, d, J = 8.4 Hz, H14), 5.39 – 5.33 (1H, m), 5.26 –
5.17 (3H, m), 3.76 (2H, s), 3.65 (4H, dt, J = 31.6, 5.2 Hz, H22,H11),
3.37 (1H, dd, J = 10.6, 6.4 Hz), 3.21 (5H, dd, J = 11.3, 6.4 Hz), 3.10
(1H, d, J = 17.2 Hz), 2.74 – 2.51 (6H, m), 2.41 – 2.03 (5H, m, H2,H4,
H10,H13), 1.84 – 1.78 (4H, m, H6,H8,11-OH), 1.78 – 1.65 (3H, m, H1,
H13), 1.63 – 1.45 (2H, m, H7), 1.44 (3H, s, H15), 1.42 – 1.24 (2H, m,
10

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H8), 1.18 (3H, s, H18), 0.89 (3H, d, J = 6.9 Hz, H17), 0.72 (3H, d, J =
6.9 Hz, H16). 13C NMR (101 MHz, Chloroform-d) δ 216.94 (C3), 168.75
(C21), 165.02, 163.27, 152.93, 144.43, 139.03 (C19), 130.10, 124.43,
117.28 (C20), 111.09, 105.86, 105.65, 102.73 (d, JC-F = 24.24 Hz),
74.57 (C11), 68.63 (C14), 59.43, 58.15 (C4), 53.22, 52.66, 52.48 (C22)
52.11, 50.80, 48.53, 45.44 (C9), 45.25, 45.03 (C13), 43.96 (C12), 42.19,
41.77 (C5), 36.67 (C6), 36.07 (C10), 34.43 (C2), 30.41 (C8), 26.82 (C7),
26.41 (C18), 24.84 (C1), 16.72 (C16), 14.86 (C15), 11.48 (C17). HR-MS
(ESI+): Calcd for C41H61FN7O5 (M + H+): 748.4562; Found: 748.4573.
45.19, 45.00, 44.92 (C13), 43.95 (C12), 42.16, 41.75 (C5), 36.67 (C6),
36.05 (C10), 34.44 (C2), 30.40 (C8), 26.80 (C7), 26.40 (C18), 24.83
(C1), 16.71 (C16), 14.85 (C15), 11.56 (C17), 11.50. HR-MS (ESI+):
Calcd for C35H59N6O5 (M + H+): 641.4424; Found: 641.4440.
4.2.24. 22–[2-(4-((morpholine) methyl)-1,2,3-triazol-1-yl)-1-(piperazine-
1-yl) ethyl-1-one] deoxy pleuromutilin (83)
White powder; yield: 53%; 1H NMR (400 MHz, Chloroform-d) δ 7.68
(1H, s), 6.50 (1H, dd, J = 17.4, 11.0 Hz, H19), 5.80 (1H, d, J = 8.4 Hz,
H14), 5.35 (1H, dd, J = 10.9, 1.6 Hz), 5.25 – 5.16 (3H, m, H20), 3.77 –
3.64 (9H, m), 3.62 (2H, d, J = 5.2 Hz), 3.37 (1H, dd, J = 10.7, 6.5 Hz,
H22), 3.26 – 3.05 (2H, m, H11), 2.67 – 2.47 (9H, m), 2.35 (1H, p, J = 6.9
Hz, H2), 2.28 – 2.16 (1H, m, H4), 2.13 – 2.04 (2H, m, H10), 1.76 (4H, s,
H13,H6,H8,11-OH), 1.71 – 1.47 (3H, m, H1,H7,H13), 1.44 (3H, s, H15),
1.39 – 1.24 (1H, m, H8), 1.18 (3H, s, H18), 0.89 (3H, d, J = 7.0 Hz, H17),
0.72 (3H, d, J = 7.0 Hz, H16). 13C NMR (101 MHz, Chloroform-d) δ
216.99 (C3), 168.75 (C21), 163.26, 144.34, 139.01 (C19), 124.41,
117.30 (C20), 77.23, 74.56 (C11), 68.61 (C14), 66.86, 59.42, 58.14
(C4), 53.63, 53.34, 52.48 (C22), 52.10, 50.79, 45.44 (C9), 45.23, 45.00,
44.60 (C13), 43.95 (C12), 42.17, 41.76 (C5), 36.66 (C6), 36.06 (C10),
34.44 (C2), 30.40 (C8), 26.81 (7), 26.40 (C18), 24.84 (C1), 16.72 (C15),
14.85, 11.50 (C17). HR-MS (ESI+): Calcd for C35H57N6O6 (M + H+):
655.4183; Found: 655.4197.
4.2.21. 22–[2-(4-((4-fluorophenyl piperazine)methyl)-1,2,3-triazol-1-yl)-
1-(piperazine-1-yl) ethyl-1-one] deoxy pleuromutilin (64)
White powder; yield: 54%; 1H NMR (400 MHz, Chloroform-d) δ 7.71
(1H, s), 6.95 (2H, t, J = 8.7 Hz), 6.86 (2H, dd, J = 9.1, 4.4 Hz), 6.50 (1H,
dd, J = 17.4, 11.0 Hz, H19), 5.80 (1H, d, J = 8.4 Hz, H14), 5.39 – 5.32
(1H, m), 5.25 – 5.16 (3H, m), 3.77 (2H, s), 3.65 (4H, dt, J = 31.2, 5.2 Hz,
H22,H11), 3.37 (1H, dd, J = 10.5, 6.4 Hz), 3.26 – 3.05 (6H, m), 2.70
(4H, t, J = 4.9 Hz), 2.59 (2H, ddq, J = 17.2, 11.3, 5.7 Hz), 2.35 (1H, t, J
= 6.9 Hz), 2.32 – 2.14 (2H, m, H2), 2.08 (2H, d, J = 19.7 Hz, H4,11-OH),
1.90 – 1.59 (7H, m, H10,H13,H6,H8,H1), 1.59 – 1.51 (1H, m, H13), 1.48
(1H, d, J = 10.8 Hz, H7), 1.44 (3H, s, H15), 1.41 – 1.28 (1H, m, H7), 1.26
(1H, s, H8), 1.18 (3H, s, H18), 0.89 (3H, d, J = 6.9 Hz,H17), 0.72 (3H, d,
J = 6.9 Hz, H16). 13C NMR (101 MHz, Chloroform-d) δ 216.95 (C3),
168.75 (C21), 163.27, 158.33, 155.96, 147.90, 144.42, 139.02, 124.46,
117.87 (C20), 117.79, 117.29, 115.57 (d, JC-F = 22.22 Hz), 77.24, 74.57
(C11), 68.63 (C14), 59.43, 58.15 (C14),53.42, 53.19, 52.85, 52.49
(C22), 52.11, 50.81, 50.05, 45.44 (C9), 45.25, 45.02 (C13), 43.96 (C12),
42.18, 41.76 (C5), 36.67 (C6), 36.07 (C10), 34.43 (C2), 30.41 (C8),
26.82 (C7), 26.40 (C18), 24.84 (C1), 16.72 (C16), 14.86 (C15), 11.49
(C17). HR-MS (ESI+): Calcd for C41H61FN7O5 (M + H+): 748.4562;
Found: 748.4602.
4.2.25. 22–[2-(4-(4-(piperidine ethanol))methyl)-1,2,3-triazol-1-yl) ꢀ 1-
(piperazine-1-yl) ethyl-1-one] deoxy pleuromutilin (84)
White powder; yield: 57%; 1H NMR (400 MHz, Chloroform-d) δ 7.69
(1H, s), 6.47 (1H, dd, J = 17.4, 10.9 Hz, H19), 5.77 (1H, d, J = 8.4 Hz,
H14), 5.33 (1H, d, J = 11.0 Hz, H20), 5.26 – 5.14 (4H, m), 3.73 – 3.44
(9H, m, H22,H11), 3.36 (1H, dd, J = 10.4, 6.4 Hz), 3.23 – 3.04 (2H, m),
2.98 – 2.83 (2H, m), 2.56 (5H, ddt, J = 16.6, 11.6, 5.6 Hz), 2.40 – 2.13
(3H, m, H2,H4,H10), 2.12 – 2.01 (5H, m, H13), 1.82 – 1.73 (1H, m, H6),
1.62 (5H, dt, J = 27.1, 13.9 Hz, H8,11-OH), 1.49 (2H, q, J = 6.8 Hz, H1,
H7), 1.42 (3H, s, H13), 1.33 (3H, d, J = 39.1 Hz, H15), 1.25 (1H, d, J =
4.3 Hz, H8), 1.16 (4H, s, H18), 0.87 (3H, d, J = 6.9 Hz, H17), 0.70 (3H,
d, J = 6.9 Hz, H16). 13C NMR (101 MHz, Chloroform-d) δ 217.09 (C3),
168.75 (C21), 163.36, 144.51, 139.03 (C19), 124.59, 117.25 (C20),
77.25, 74.54 (C11), 68.63 (C14), 60.32, 59.43, 58.15 (C4), 53.50, 53.45,
52.48 (C22), 52.12, 50.83, 50.62, 45.44 (C9), 45.18, 45.00 (C13), 44.02
(C12), 43.95, 42.15, 41.75 (C5), 39.22, 36.66 (C6), 36.05 (C10), 34.44
(C2), 32.02 (C8), 30.40, 26.80 (C7), 26.43 (C18), 24.83 (C1), 16.70
(C16), 14.85 (C15), 11.49 (C17). HR-MS (ESI+): Calcd for C38H63N6O6
(M + H+): 697.4653; Found: 697.4692.
4.2.22. 22–[2-(4-((dimethylamino) methyl)-1,2,3-triazol-1-yl)-1-
(piperazine-1-yl) ethyl-1-one] deoxy pleuromutilin (81)
White powder; yield: 55%; 1H NMR (400 MHz, Chloroform-d) δ 7.66
(1H, s), 6.46 (1H, dd, J = 17.4, 11.0 Hz, H19), 5.76 (1H, d, J = 8.4 Hz,
H14), 5.31 (1H, dd, J = 10.9, 1.6 Hz), 5.21 – 5.15 (3H, m, H20), 3.67 –
3.63 (2H, m), 3.62 (2H, s), 3.57 (2H, t, J = 5.2 Hz), 3.35 (1H, d, J = 6.4
Hz, H21), 3.20 – 3.03 (2H, m, H22), 2.55 (3H, tp, J = 22.5, 5.9 Hz), 2.32
(1H, p, J = 6.9 Hz), 2.26 (7H, s,H2), 2.21 – 2.13 (1H, m), 2.10 – 2.02
(2H, m, 11-OH, H10), 1.77 (1H, q, J = 3.2 Hz, H13), 1.74 (1H, q, J = 3.1
Hz, H7), 1.63 (2H, tdd, J = 14.2, 6.6, 3.3 Hz, H1), 1.55 (1H, dd, J = 13.9,
3.6 Hz), 1.52 – 1.48 (1H, m), 1.46 – 1.42 (1H, m), 1.41 (3H, s), 1.38 –
1.31 (1H, m), 1.26 (1H, d, J = 16.0 Hz, H8), 1.14 (3H, s, H18), 1.10 (1H,
dd, J = 14.1, 4.3 Hz, H8), 0.86 (3H, d, J = 7.0 Hz, H17), 0.69 (3H, d, J =
7.1 Hz, H16). 13C NMR (101 MHz, Chloroform-d) δ 217.02 (C3), 168.75
(C21), 163.29, 144.86, 139.03 (C19), 124.40, 117.27 (C20), 74.54
(C11), 68.60 (C14), 59.44, 58.14 (C4), 54.14, 53.43, 52.50 (C22), 52.13,
50.90, 45.44 (C9), 45.20, 45.00 (C13), 44.93, 43.95 (C12), 42.15, 41.75
(C5), 36.67 (C6), 36.06 (C10), 34.44 (C2), 30.40 (C8), 26.81 (C7), 26.41
(C18), 24.84 (C1), 16.71 (C16), 14.85 (C15), 11.50 (C17). HR-MS
(ESI+): Calcd for C36H59N6O5 (M + H+): 653.4077; Found: 613.4072.
4.2.26. 22–[2-(4-(3-(piperidine methanol))methyl)-1,2,3-triazol-1-yl)-1-
(piperazine-1-yl) ethyl-1-one] deoxy pleuromutilin (85)
White powder; yield: 61%; 1H NMR (400 MHz, Chloroform-d) δ 7.69
(1H, s), 6.50 (1H, dd, J = 17.4, 11.0 Hz, H19), 5.79 (1H, d, J = 8.4 Hz,
H14), 5.35 (1H, d, J = 11.0 Hz, H20), 5.20 (3H, d, J = 11.6 Hz), 3.71
(2H, s), 3.69 (4H, s), 3.59 (2H, s), 3.59 – 3.50 (2H, m), 3.48 (2H, s, H22),
3.38 – 3.33 (1H, m, H11), 3.26 – 3.05 (2H, m), 2.94 – 2.48 (7H, m), 2.40
– 1.93 (8H, m, H2,H4,H10,H13), 1.85 – 1.46 (6H, m, H6,H8,11-OH,H8),
1.44 (3H, s, H1,H7,H13), 1.28 (1H, d, J = 16.1 Hz, H8), 1.17 (3H, s,
H18), 1.15 – 1.09 (2H, m), 0.89 (3H, d, J = 6.9 Hz, H17), 0.72 (3H, d, J
= 6.9 Hz, H16). 13C NMR (101 MHz, Chloroform-d) δ 217.00 (C3),
168.77 (C21), 163.34, 144.67, 139.01 (C19), 124.47, 117.31 (C20),
74.57 (C11), 68.61 (C14), 66.66, 59.44, 58.15 (C14), 56.71, 54.09,
53.74, 52.50 (C22), 52.13, 50.89, 45.45 (C9), 45.19 (C13), 45.01, 43.96
(C12), 42.17, 41.77 (C5), 37.98, 36.67 (C6), 36.06 (C10), 34.44 (C2),
30.41 (C8), 27.14 (C7), 26.81, 26.39 (C18), 24.84 (C1), 24.48,16.72
(C16),14.86 (C15), 11.50 (C17). HR-MS (ESI+): Calcd for C37H61N6O6
(M + H+): 683.4530; Found: 683.4556.
4.2.23. 22–[2-(4-((diethylamino) methyl)-1,2,3-triazol-1-yl)-1-
(piperazine-1-yl) ethyl-1-one] deoxy pleuromutilin (82)
White powder; yield: 47%; 1H NMR (400 MHz, Chloroform-d) δ 7.74
(1H, s), 6.49 (1H, dd, J = 17.4, 10.9 Hz, H19), 5.79 (1H, d, J = 8.4 Hz,
H14), 5.34 (1H, d, J = 11.0 Hz, H20), 5.22 (3H, s), 3.87 (2H, s), 3.64
(4H, dt, J = 32.2, 5.2 Hz), 3.36 (1H, dd, J = 10.4, 6.4 Hz), 3.26 – 2.97
(2H, m, H22,H21), 2.72 – 2.44 (8H, m), 2.39 – 1.99 (9H, m, H2,H4,H10,
H13), 1.83 – 1.48 (5H, m, H1,H6,H7,H8,11-OH,), 1.43 (4H, s, H15),
1.27 (1H, d, J = 16.1 Hz), 1.15 (4H, d, J = 11.4 Hz), 1.10 (3H, s,H18),
0.88 (3H, d, J = 7.0 Hz, H17), 0.71 (3H, d, J = 7.0 Hz, H16). 13C NMR
(101 MHz, Chloroform-d) δ 217.02 (C3), 168.75 (C21), 163.32, 144.33,
139.01 (C19), 124.72, 117.29 (C20), 77.24, 74.55 (C11), 68.60 (C14),
59.44, 58.14 (C4), 52.50 (C22), 52.13, 50.94, 47.36, 46.72, 45.44 (C9),
4.2.27. 22–[2-(4-(4-hydroxy-piperidine)methyl)-1,2,3-triazol-1-yl)-1-
(piperazine-1-yl) ethyl-1-one] deoxy pleuromutilin (86)
White powder; yield: 43%; 1H NMR (400 MHz, Chloroform-d) δ 7.70
11

G.-Y. Zhang et al.
Bioorganic Chemistry 105 (2020) 104398
(1H, s), 6.50 (1H, dd, J = 17.4, 10.9 Hz, H19), 5.80 (1H, d, J = 8.4 Hz,
H14), 5.36 (1H, d, J = 11.0 Hz, H20), 5.20 (4H, d, J = 10.1 Hz), 3.80 –
3.66 (6H, m), 3.60 (2H, d, J = 5.2 Hz, H22), 3.49 (1H, s, H11), 3.37 (1H,
dd, J = 10.6, 6.5 Hz), 3.26 – 3.04 (2H, m), 2.96 – 2.81 (3H, m),2.69 –
2.15 (10H, m, H2,H4,H10,H13), 2.08 (2H, d, J = 19.0 Hz), 1.97 – 1.73
(3H, m, H6,H8,11-OH), 1.64 (2H, dq, J = 19.2, 10.5, 9.6 Hz, H1), 1.54 –
1.48 (1H, m, H7), 1.44 (3H, s, H15), 1.38 – 1.22 (1H, m, H8), 1.18 (3H, s,
H18), 1.13 (2H, d, J = 15.3 Hz), 0.89 (3H, d, J = 6.9 Hz, H17), 0.72 (3H,
d, J = 7.0 Hz, H16). 13C NMR (101 MHz, Chloroform-d) δ 217.06 (C3),
168.76 (C21), 163.32, 144.44, 139.02 (C19), 124.60, 117.28 (C20),
68.62 (C11), 67.39 (C14), 59.43, 58.15 (C4), 53.03, 52.48 (C22), 52.11,
50.83, 50.68, 50.64, 45.44 (C9), 45.18, 45.00 (C13), 43.95 (C12), 42.16
(C5), 41.76, 36.67, 36.49 (C6), 36.05 (C10), 34.44 (C2), 34.13, 30.40
(C8), 26.80 (C7), 26.42 (C18), 24.83 (C1), 16.71 (C16), 14.86 (C15),
11.50 (C17). HR-MS (ESI+): Calcd for C36H59N6O6 (M + H+): 669.4340;
Found: 669.4382.
of 2 times were prepared in Mueller-Hinton broth medium (MHB). There
were three parallel experiments for each compound concentration. The
plate was incubated at 37 ℃ for 24 h, and the MIC value was recorded as
the lowest inhibitory concentration of the sample on the visible growth
of the tested bacteria.
After obtaining the MIC value, the 96-well plate was incubated at 37
ꢀ for 24 h, and the MBC was determined by inoculating the borehole
bacterial solution from no obvious bacterial growth on Mueller-Hinton
Agar plate (MHA) [21]. Next, the MH agar plate was further cultured
at 37 ℃ for 24 h, and the lowest concentration of the compound
required to reduce the bacterial count by>99.9% on the plate was MBC.
4.3.2. Constant concentration time-kill curves
The bactericidal activity of compound 59 and compound 61 was
determined by the constant concentration time-kill curve as reported in
our previous work [15]. MRSA (ATCC 43300) was diluted to about 1 ×
106 CFU/mL in MH broth and treated with compound 59, compound 61
and tiamulin of 1 × MIC, 2 × MIC, 4 × MIC, 8 × MIC, 16 × MIC, 32 ×
MIC. The sampling time was 0, 3, 6, 9, 12 and 24 h, respectively. After
4.2.28. 22–[2-(4-((pyrrolidine)methyl)-1,2,3-triazol-1-yl)-1-(piperazine-
1-yl) ethyl-1-one] deoxy pleuromutilin (87)
White powder; yield: 49%; 1H NMR (400 MHz, Chloroform-d) δ 7.80
(1H, s), 6.49 (1H, dd, J = 17.4, 10.9 Hz, H19), 5.79 (1H, d, J = 8.4 Hz,
H14), 5.35 (1H, d, J = 11.0 Hz, H20), 5.20 (3H, d, J = 13.8 Hz, H20),
4.47 (1H, s), 3.90 (2H, s), 3.63 (4H, dt, J = 32.8, 5.2 Hz, H22,H11), 3.36
(1H, s), 3.23 – 3.01 (2H, m), 2.70 (4H, d, J = 6.0 Hz), 2.64 – 2.42 (9H, m,
H2,H4,H10,H13), 2.34 (1H, p, J = 6.9 Hz), 2.27 – 2.16 (1H, m), 2.07
(2H, d, J = 20.7 Hz, 11-OH,H6), 1.91 – 1.74 (5H, m, H1,H7,H13), 1.72 –
1.61 (1H, m, H8), 1.59 – 1.47 (1H, m, H8), 1.43 (3H, s, H15), 1.36 – 1.25
(1H, m, H8), 1.17 (3H, s, H18), 0.88 (3H, d, J = 6.9 Hz, H17), 0.71 (3H,
d, J = 6.9 Hz, H16). 13C NMR (101 MHz, Chloroform-d) δ 217.02 (C3),
168.75 (C21), 163.25, 144.44, 139.01 (C19), 124.70, 117.30 (C20),
77.23, 74.55 (C11), 68.60 (C14), 59.45, 58.15 (C4), 53.75, 52.49 (C22),
52.13, 50.91, 50.32, 45.44 (C9), 45.17, 45.00, 44.96 (C13), 43.95 (C12),
42.16, 41.76 (C5), 36.67 (C6), 36.05 (C10), 34.44 (C2), 30.41 (C8),
26.81 (C7), 26.40 (C18), 24.84 (C1), 23.45, 16.72 (C16), 14.86 (C15),
11.50 (C17). HR-MS (ESI+): Calcd for C35H57N6O5 (M + H+): 639.4234;
Found: 639.4246.
specified time intervals, 100
in 0.9% saline and 25
μL of samples were serially diluted 10-fold
μ
L of them were plated on sterile MH agar plates.
After incubated 24 h at 37 ℃, the total bacterial CFU/mL on the plates
were counted to calculate the bacterial colonies. The time-kill curve was
constructed by plotting log10 CFU/mL of bacteria counts versus time.
4.3.3. Determination of the postantibiotic effect (PAE)
The PAE of compound 59, compound 61 and tiamulin against MRSA
were determined using time-kill methods according previous work [15].
At first MRSA in logarithmic phase was diluted to ca. 1 × 106 CFU/mL in
MH broth as the culture solution. Then concentrations of 2 × MIC and 4
× MIC of compound 59, compound 61 and tiamulin were added into test
tubes that contained the culture solution. The test tubes were incubated
at 37 ℃ for 2 h. After that, the test compounds and tiamulin were
removed by diluting 1000-fold into the pre-warmed MH broth. The
tubes were placed in 37 ℃ and the bacterial growth was estimated at
time 0, 1, 2, 4, 6 and 8 h. The sample solutions (100 μL) were taken and
diluted 10-fold in saline and then plated onto MH agar plates. After that
the MH agar plates were incubated at 37 ℃ for 24 h and the resulting
colonies on the plates were counted. The PAE value was calculated by
the equation PAE = TA - TC (TA was the time required for the increase of
1 log10 CFU/mL after the removal of drugs and TC was the time required
for the number of MRSA in the control tube to increase by 1 log10 CFU/
mL) and expressed in an hour.
4.2.29. 22–[2-(4-((piperidine)methyl)-1,2,3-triazol-1-yl)-1-(piperazine-1-
yl)ethyl-1-one] deoxy pleuromutilin (88)
White powder; yield: 68%; 1H NMR (400 MHz, Chloroform-d) δ 7.68
(1H, s), 6.49 (1H, dd, J = 17.4, 11.0 Hz, H19), 5.79 (1H, d, J = 8.4 Hz,
H14), 5.35 (1H, d, J = 11.0 Hz), 5.25 – 5.15 (3H, m, H20), 3.68 (4H, s),
3.60 (2H, t, J = 5.1 Hz), 3.36 (1H, dd, J = 10.3, 6.3 Hz, H21), 3.25 – 3.02
(2H, m, H22), 2.64 – 2.39 (9H, m), 2.34 (1H, p, J = 7.0 Hz,), 2.29 – 2.14
(2H, m, H2), 2.09 (2H, dd, J = 9.1, 5.3 Hz, H4), 2.03 (4H, d, J = 9.9 Hz,
H10,H13), 1.83 – 1.45 (8H, m, H6,H8,11-OH,H1,H7,H13), 1.43 (3H, s,
H15), 1.41 – 1.28 (1H, m), 1.26 (1H, s, H8), 1.17 (3H, s, H18), 0.89 (3H,
d, J = 6.9 Hz, H17), 0.71 (3H, d, J = 6.9 Hz, H16). 13C NMR (101 MHz,
Chloroform-d) δ 217.00 (C3), 168.74 (C21), 163.34, 144.81, 139.00
(C19), 124.36, 117.31 (C20), 77.23, 74.56 (C11), 68.59 (C14), 59.45,
58.15 (C4), 54.18, 53.92, 52.52 (C22), 52.14, 50.89, 45.44 (C9), 45.24,
45.00 (C13), 43.95 (C12), 42.15, 41.76 (C5), 36.67 (C6), 36.05 (C10),
34.44 (C2), 30.41 (C8), 26.81 (C7), 26.39 (C18), 25.77, 24.84 (C1),
24.06, 16.72 (C16), 14.85 (C15), 11.50 (C17). HR-MS (ESI+): Calcd for
4.4. In vivo efficacy of pleuromutilin derivatives
4.4.1. Neutropenic murine thigh infection model
Six-week-old, specific pathogen-free, female ICR/Swiss mice
weighing 22–26 g were fed, housed and rendered neutropenic (<0.1 ×
109/L) by injecting two doses of cyclophosphamide (Mead Johnson
Pharmaceuticals, Evansville, IN) ip 4 days (150 mg/kg) and 1 day (l00
mg/kg) before the experiment, as described previously [15]. Then each
mice thigh was injected with 0.1 mL MH broth (MASR concentration
approximately 107 CFU/mL) to establish the thigh infection model.
These mice were randomly divided into three groups (3 per group)
and intravenously injected (i.v.) 0.9% saline, compound 59 (20 mg/kg)
and tiamulin (20 mg/kg) as one control group and two experimental
groups respectively after 2 h of MRSA growth in the thigh. The mice
from each group were euthanized 24 h after drug injection. And their
thigh tissue of each group was removed and homogenized in 3 mL of
iced saline, respectively. Finally, Duplicate aliquots of five to six serial
10-fold dilutions were plated on agar for CFU determination.
The protocol for this study was reviewed and approved by the
Institutional Animal Care and Use Committee of the South China Agri-
cultural University.
C
36H59N6O5 (M + H+): 653.4390; Found: 653.4400.
4.3. In vitro efficacy of pleuromutilin derivatives
4.3.1. Minimal inhibitory concentration (MIC) testing and minimum
bactericidal concentration (MBC) testing
The minimal inhibitory concentrations MIC of the target compounds
against methicillin-resistant S. aureus (ATCC 43300), S. aureus (ATCC
29213), S. aureus (AD3), S. aureus (144) and E. coli (ATCC 25922) were
determined using pleuromulin and tiamulin as positive controls. MIC
values were determined following our previous work [11]. The amount
of inoculation in each well was 5 × 105 CFU/mL, and a series of diluents
12

G.-Y. Zhang et al.
Bioorganic Chemistry 105 (2020) 104398
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CYP450 was evaluated according previous work [22]. The experiments
were performed in 96-well plates with final incubation volume of 100 µL
per well. First, 20 µL of compound 59 or tiamulin was added to 96-well
plates, with final concentrations of 0.25, 0.5, 1, 5, 10, and 50 µM. And
then, 40 µL of human liver microsomes (final concentration of human
liver microsomes is 0.2 mg/mL) and 20 µL of probe substrates (final
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concentration of testosterone is 20 μmol/L) in 0.1 M Tris (pH = 7.4)
were added. After preincubation at 37 ℃ for 5 min, the reaction started
with the addition of 20 µL of NADPH (final concentration is 1 mmol/L).
Then the plate was incubated at 37 ℃ for 5 mins, and then 100 μL
acetonitrile (with loratadine used as an internal standard) was added to
terminate the reaction. After reactions were completed, the plates were
centrifuged and the supernatants were analyzed for the loratadine and
probe substrates testosterone by LC-MS/MS. The specific CYP substrates
used in the experiment included phenacetin (CYP1A2), chlorzoxazone
(CYP2E1), dextromethorphan (CYP2D6), and testosterone (CYP3A4
(T)).
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4.6. Pharmacokinetic studies of compound 59
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Appendix A. Supplementary data
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