Design, synthesis, and antibacterial activity of novel pleuromutilin derivatives bearing an amino thiazolyl ring

Article abstract of DOI:10.1002/ardp.201100430

A series of novel pleuromutilin derivatives containing the amino thiazolyl ring were designed, synthesized, and evaluated for their antibacterial activities in vitro against Gram-positive clinical bacteria. All the target compounds showed better aqueous s

Full text of DOI:10.1002/ardp.201100430

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–9
1
Full Paper
Design, Synthesis, and Antibacterial Activity of Novel
Pleuromutilin Derivatives Bearing an Amino Thiazolyl Ring
1
Yong Ling , Xinyang Wang , Hui Wang , Jianghe Yu , Junming Tang , Donggeng Wang ,
1
2
2
3
1
1
Guangtong Chen , Jinhua Huang , Yuqin Li , and Heng Zheng
1
1
4
1
Department of Pharmacy, School of Medical, Nantong University, Nantong, P. R. China
Department of Microbiology, College of Life Science and Technology, China Pharmaceutical University,
Nanjing, P. R. China
National Chemical Experimental Teaching Demonstration Center, College of Chemistry and Environmental
Science, Henan Normal University, Xinxiang, P. R. China
2
3
4
Department of Bioinformatics, College of Life Science and Technology, China Pharmaceutical University,
Nanjing, P. R. China
A series of novel pleuromutilin derivatives containing the amino thiazolyl ring were designed,
synthesized, and evaluated for their antibacterial activities in vitro against Gram-positive clinical
bacteria. All the target compounds showed better aqueous solubility compared with the lead
compound (10). Most compounds displayed strong antibacterial activities against both susceptible
and resistant bacteria, particularly for the compound (12f) which showed extraordinary antibacterial
properties superior to amoxicillin and tiamulin. Molecular docking studies revealed that the amino
thiazolyl ring, the side chains of the pleuromutilin derivatives, can be adopted in the binding pocket
of the 50S ribosomal subunit near the mutilin core. Therefore, our novel findings may provide new
insights into the design of novel pleuromutilin derivatives and lay the basis for further studies on
these promising antibiotics for human clinical use.
Keywords: 2-Aminothiazole / Antibacterial activity / Aqueous solubility / Gram-positive pathogens / Molecular docking /
Pleuromutilin derivatives
Received: November 27, 2011; Revised: February 26, 2012; Accepted: February 29, 2012
DOI 10.1002/ardp.201100430
Introduction
synthesis through interaction with the 50S subunits of pro-
karyotic ribosomes [3] and has no cross-resistance to marketed
antibacterial agents. Therefore, the design and development
of more potent derivatives of pleuromutilin will be crucial
for the clinical applications of pleuromutilin-based antibac-
terial agents. The first practical pleuromutilin derivative,
tiamulin 2, was developed by Sandoz in 1974. It is a prophy-
lactic and therapeutic agent for swine dysentery, and has
shown remarkable inhibiting potency (MIC <1 mg/mL)
against anaerobic bacteria, intestinal spirochetes, and
Mycoplasma spp. [4, 5]. But it can be rapidly metabolized
in vivo by cytochrome P450-mediated hydroxylation of the
tricyclic nucleus, and then eliminated [6]. The next potent
pleuromutilin derivative valnemulin 3 was used as an effec-
tive medicine in treatment of enzootic pneumonia of pigs
and originally approved in 1999 by the European Union [7, 8].
The antibiotic 3, also once used in some rare human cases,
was probably too toxic to be approved [9]. Afterwards,
azamulin 4 was designed to overcome the toxicity of
Antibacterial resistance of hospital-acquired Gram-positive bac-
terial pathogens, especially methicillin-resistant Staphylococcus
aureus (MRSA) and penicillin-resistant Streptococcus pneumonia
(PRSP), has become an increasingly serious medical issue
which has drawn a great concern in community settings
and intensive care in the hospitals [1, 2]. Therefore, develop-
ing novel classes of antibiotics against resistant bacteria has
grown to be a desperate mission for medicinal chemists.
The natural product pleuromutilin 1 (Fig. 1), isolated from
two basidiomycete species (e.g., Pleurotus mutilus and Pleurotus
passeckerianus), can inhibit the growth of a variety of Gram-
positive bacteria by selectively inhibiting bacterial protein
Correspondence: Xinyang Wang, Department of Pharmacy, School of
Medical, Nantong University, Nantong 226001, P. R. China.
E-mail: wangxy@ntu.edu.cn
Fax: þ86 513 85051892
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Y. Ling et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–9
notice that the new types of cephalosporin containing the
thiazolyl ring usually display higher bioactivities than the
former ones, and a lot of natural products of thiazolyl pep-
tide with thiazolyl ring have been isolated from marine
sources in recent years and show excellent bioactivities
against resistant bacteria strains [17, 18]. These results
indicate that modifying the side chain of pleuromutilin with
the aminothiazole ring moiety may improve their antibacte-
rial activities. With these ideas in mind, the new important
pleuromutilin derivative 10 was designed and synthesized as
our lead compound by coupling the C-22 position of pleuro-
mutilin with 2-amino thiazolyl group. Meanwhile, aimed to
obtain potent drug molecules with high aqueous solubility, a
total set of seven target compounds 12a–g (Fig. 2) were also
designed and synthesized with different alkanolamine sub-
stituted 2-amino acetyl amides as the polar and water soluble
group on the thiazolyl ring in the side chain. After unam-
biguous characterization, these target compounds were eval-
uated for their water solubility, and their antibacterial
activities were investigated with drug resistant pathogens
through standard procedure in vitro. Herein, we report the
synthesis and biological evaluation of these derivatives.
Figure 1. Chemical structures of pleuromutilin and derivatives
thereof.
valnemulin and it successfully entered Phase I clinical evalu-
ation. Unfortunately no more researches were conducted
due to its low solubility and a short half-life in vivo [10].
Retapamulin 5 was so far the first drug developed for human
use but only for topical treatment of skin infections [11]. Results and discussion
In recent years, some other derivatives like BC-3205 (6) and
BC-3781 (7) had already been under Phase I clinical studies,
especially which became the second pleuromutilin
Chemistry
7
The synthetic routes of lead compound 10 and target com-
pounds 12a–g are outlined in Scheme 1. The starting material
1 firstly reacted with p-toluene sulfonyl chloride (TsCl) in the
presence of triethylamine at room temperature for 17 h in
derivative that successfully entered Phase II clinical evalu-
ation [12, 13]. These results suggest that pleuromutilin
derivatives have the prospect of being developed as new
drugs for human use with improvement in pharmacology
and ability to counter the development of resistance.
However, given the fact that there has been no effective
pleuromutilin derivative for human oral use on the market
so far, the discovery of new pleuromutilin derivatives with
excellent antimicrobial activities against drug resistant
pathogens will be of great significance.
2 2
CH Cl to yield 14-O-(p-toluene sulfonyloxyacetyl) mutilin 8
followed by the displacement with potassium iodide to
give the 14-O-(2-iodoacetyl) mutilin 9 under reflux for 3 h
in acetone. The key intermediate 10 was obtained by the
nucleophilic attack of (2-aminothiazol-4-yl)methanethiol on
the latter 9 in the presence of 30% NaOH in an ice bath for 2 h
in tetrahydrofuran (THF). Furthermore, lead compound 10
was treated with 2-chloroacetyl chloride in the presence of
potassium carbonate to give the intermediate 11. Finally, the
resulting intermediate 11 was directly treated with different
substituted alkanolamines in the presence of KI and potass-
The previous structure–activity relationships (SARs) studies
of pleuromutilin analogues above revealed that the C-22
thioether side chain can tolerate a variety of modifications
to improve their antimicrobial activities [10, 14–16]. We also
Figure 2. The newly designed and synthe-
sized compounds.
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–9
Novel Pleuromutilin Derivatives as Antibiotic Agents
3
Scheme 1. Reaction conditions and
reagents: (a) TsCl, triethylamine, CH Cl , rt,
7 h, 50%; (b) KI, acetone, reflux, 3 h, 87%;
c) (2-aminothiazol-4-yl)methanethiol, 30%
NaOH, THF, 08C rt, 2 h, 72%; (d) 2-chloro-
acetyl chloride, K CO , CH Cl , 08C rt, 1 h,
CO , THF, 408C, 4–8 h,
2
2
1
(
2
3
2
2
1
2
9
5
2%; (e) R R NH, K
3–65%.
2
3
ium carbonate to generate the corresponding 12a–g in yields
of 53–65%, respectively. The final products were purified
by column chromatography and their structures were con-
aqueous solubility of the synthetic target compounds with a
solubility ranging from 1.08 to 1.32 mg/mL was significantly
higher than that of the lead compound with an equivalent of
27- to 33-fold solubility of the lead compound.
1
firmed on the basis of their spectral data of IR, H NMR, MS,
1
3
1
C NMR, and elementary analyses. In the H NMR spectrum,
three olefinic protons were observed, a double doublet in the
range of 6.50–6.53 ppm and two doublets in the range of
Biological evaluation
The target compounds were evaluated for their antibacterial
activities against a panel of susceptible and resistant Gram-
positive organisms using the minimum inhibitory concen-
tration (MIC). The results of these studies are summarized in
Tables 2 and 3. Most target compounds displayed excellent
in vitro antibacterial activities superior or similar to
amoxicillin and tiamulin. Generally, their antibacterial
activities against Staphylococcus were better than that against
Streptococcus. Moreover, the key intermediate compound 10
with (2-aminothiazol-4-yl)methyl thioether group at C-14 pos-
ition was 4–8-fold more potent than amoxicillin and tiamu-
lin against most of the selected Gram-positive pathogens.
Especially, compound 12f containing 4-OH-piperidinyl
5
.35–5.41 and 5.21–5.26 ppm, which indicated the (CH
2
–CH)
protons. Additionally, the C NMR spectra exhibited the
characteristic signals for CH
–CH in the range of 139.00–
39.10 and 117.05–117.23 ppm, and the characteristic
1
3
2
1
signals for C–O of cyclopentanone, ester or amide in the
range of 217.01–217.18 ppm, 169.14–170.32, or 168.43–
1
68.77 ppm, respectively, which confirmed the formation
of the compounds 12a–g.
Aqueous solubility
The aqueous solubility of the pleuromutilin derivatives was
measured using UV at 25 ꢀ 18C [19] (Table 1). As expected, the
Table 1. Solubility of the target compounds in water at 25 W 18C
Compd
10
12a
12b
12c
12d
12e
12f
12g
Solubility (mg/mL) <0.04
1.14
1.10
1.08
1.32
1.13
1.17
1.28
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Y. Ling et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–9
Table 2. Antibacterial activity (MIC, mg/mL) of pleuromutilin derivatives 12a–g in vitro
a)
MIC (mg/mL)
Compd
MSSA
MRSA
MSSE
b)
cpu0587
cpu0211
cpu1344
cpu0446
cpu0887
cpu1098
cpu3256
cpu0347
cpu2089
Amoxicillin
Tiamulin
2
8
2
4
2
0.5
0.25
0.125
0.25
0.125
0.0625
0.0625
0.125
1
2
4
2
0.25
1
0.25
1
1
0.125
0.125
0.125
0.125
0.5
0.0625
0.25
0.25
0.5
4
2
0.5
0.25
0.25
0.5
0.125
0.25
0.0625
0.25
8
2
0.5
0.25
0.25
0.5
0.125
0.125
0.0625
0.25
8
0.5
0.125
0.125
0.5
0.125
0.5
0.125
0.125
0.125
0.25
0.0625
0.25
0.125
0.125
1
1
1
1
1
1
1
1
0
0.125
0.25
0.25
0.25
0.125
0.125
0.0625
0.125
2a
2b
2c
2d
2e
2f
2g
0.125
0.125
0.25
0.125
0.0625
0.0625
0.125
0.5
0.5
0.125
0.0625
0.0625
0.125
0.25
0.125
0.25
a)
b)
Minimal inhibitory concentrations (MIC): lowest concentration of compound that inhibits visible growth of the organism.
MSSA, methicillin-susceptible Staphylococcus aureus; MRSA, methicillin-resistant S. aureus; MSSE, methicillin-susceptible Staphylococcus
epidermidis; MRSE, methicillin- resistant S. epidermidis; PSSP, penicillin-susceptible Streptococcus pneumonia; PRSP, penicillin-resistant
S. pneumonia. All strains were isolated from the clinical bacteria in the Nanjing Gulou hospital and reserved in the Department of
Microbiology, College of Life Science and Technology, China Pharmaceutical University. Clinical strains were identified by API
bacteria analysis system.
Table 3. Antibacterial activity (MIC, mg/mL) of pleuromutilin derivatives 12a–g in vitro
Compd
MIC (mg/mL)
MRSE
PSSP
PRSP
cpu2623
cpu3889
cpu3569
cpu2736
cpu1215
cpu1653
cpu2103
cpu2634
Amoxicillin
Tiamulin
8
1
8
1
8
0.125
0.0625
0.125
0.5
0.25
0.5
8
1
1
1
2
2
1
1
0.5
2
2
2
1
4
2
4
1
1
1
4
2
4
4
1
1
2
1
0.5
1
1
4
1
0.25
2
2
4
1
1
0.25
2
0.25
0.125
0.5
0.5
1
0.5
0.125
0.25
0.25
1
1
1
1
1
1
1
1
0
0.125
0.125
0.5
0.125
0.25
0.25
0.5
2a
2b
2c
2d
2e
2f
2g
2
0.125
0.0625
0.0625
0.125
0.5
0.5
0.25
0.125
0.25
0.25
0.0625
0.5
groups exhibited the strongest activity with MIC values of
.0625–0.25 mg/mL against MRSE, MRSE, MSSE, and MSSA, which
of pleuromutilin, compounds 12a, 12b, and 12d with a
terminal aminoethanol residue had higher antibacterial
activities against the selected Gram-positive pathogens than
those bearing a terminal aminopropanol residue linker, such
as 12c. Secondly, the bioactivities of the compounds 12b, 12d,
and 12e–g linked with tertiary amine substituent groups
were a little stronger than that of the derivatives 12a and
12c linked with secondary amine groups. In sharp contrast,
the tertiary amine derivatives of pleuromutilin such as 12f
showed the best activity among the compounds tested. The
higher antibacterial activity of 12f may also stem from
good lipid/water partition coefficient and the better binding
affinity between the molecular structure and the 50S
ribosomal subunit. We also endeavor to further determine
0
was almost twofold more potent than compound 10, 8–16-fold
more potent than amoxicillin (MIC ¼ 0.125–8 mg/mL), and
tiamulin (MIC ¼ 0.0625–2 mg/mL) against the same infections
pathogens, respectively.
Compared to other pleuromutilin analogues containing
different heterocycle from the literature, these novel amino
thiazole derivatives in the series exhibited excellent anti-
bacterial activity similar to purine derivatives [15], and better
than pyrazole derivatives [20]. Despite the excellent anti-
bacterial effect against both these susceptible and resistant
Gram-positive bacteria, they demonstrated few rules of SARs.
Among these different substituted alkanolamine derivatives
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–9
Novel Pleuromutilin Derivatives as Antibiotic Agents
5
the SARs of pleuromutilin derivatives, which will provide us a
new insight into the design of novel antibacterial drugs.
modes within the active pocket of the 50S ribosomal subunit.
All of the side chains were of the pharmacophores, and the
spatial orientations of the pharmacophores direct to the
active pocket. The thiazolyl heterocycle moiety at the C-22
side chain position of compound 12f bound with residue
A2045 via p–p stacking interaction. Besides, two hydrogen
bonds were also found. One formed between G2484 and the
oxygen of OH group in the mother ring of 12f (OꢁꢁꢁO distance:
Molecular docking
To further study the primary antibacterial activities mech-
anism and the interaction mode of the novel classes of the
pleuromutilin derivatives, molecular docking study was per-
formed using software package MOE 2008.10. The X-ray crys-
tal structure of the 50S ribosomal subunit from Deinococcus
radiodurans in complex with tiamulin (PDB code: 1XBP) was
obtained from the Protein Data Bank and applied to build the
starting model of the 50S ribosomal subunit.
˚
2.68 A). The other formed between G2044 and the oxygen of
C–O group in the side chain (NꢁꢁꢁO distance: 2.67 A). Thus,
compound 12f was found to be docked into the gorge of the
50S ribosomal subunit perfectly, which explained its high
antibacterial effect. All these results indicated that the side
chains of the compounds played an important role in adjust-
ing the physico-chemical properties of the whole molecule,
and furthermore, the amino thiazolyl ring in general can be
˚
The docking results are shown in Fig. 3, using compound
12f as a template which possesses the highest bioactivity to
susceptible and resistant bacteria, respectively. As shown in
Fig. 3A and B, compound 12f could adopt different binding
Figure 3. The docking conformation of com-
pound 12f in the active site of the 50S ribosomal
subunit from D. radiodurans (PDB code: 1XBP)
generated with MOE.
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6
Y. Ling et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–9
tion mixture was stirred in the ice bath environment for 2 h.
After the reaction, the solvent was removed under reduced
pressure. The crude residue was dissolved in ethyl acetate and
washed with water and saturated NaCl solution separately,
adopted near the mutilin core in the binding pocket and used
to further improve the binding affinity.
Conclusion
4
and then the organic layer was dried with MgSO , filtered,
concentrated, and purified by column chromatography (PE/
EtOAc ¼ 5:1–1:1) to yield 10 as a white solid 0.75 g, yield:
In summary, new classes of pleuromutilin derivatives have
been designed and synthesized with different 2-(substituted
amino) acetyl amide groups on the thiazolyl ring in the C-22
side chain, and their antibacterial activities have been eval-
uated in vitro against a number of Gram-positive clinical
pathogens from both sensitive and resistant strains. We
find that all the target compounds exhibited better aqueous
solubility than that of the lead compound and most of the
compounds displayed strong antibacterial activities against
these susceptible and resistant Gram-positive bacteria,
particularly for the compound 12f which showed excellent
antibacterial properties superior to the reference drug amox-
icillin and tiamulin. Molecular docking studies revealed that
the amino thiazolyl ring, the side chains of pleuromutilin
derivatives, can be adopted near the mutilin core in the
binding pocket of the 50S ribosomal subunit which may
clarify their high antibacterial effect. Therefore, our new
findings may provide potentially important information
for further development of pleuromutilin derivatives and
lay the basis for further studies on the promising antibiotics
for human clinical treatment.
ꢂ1
7
2%; m.p.: 67–708C. Analytical data for 10: IR (KBr, cm ):
3
H
436, 3353, 2984, 2856, 1716, 1617, 1524, 1280, and 685;
¼ 11.2 Hz,
1
NMR (CDCl3, 400 MHz):
¼ 17.6 Hz, H19), 6.33 (1H, s, H25), 5.77 (1H, d, J ¼ 8.0 Hz,
d
6.50 (1H, dd,
J
1
J
2
H14), 5.37 (1H, d, J ¼ 11.2 Hz, H20), 5.23 (1H, d, J ¼ 17.2 Hz,
H20), 3.67 (2H, s, H23), 3.08 (2H, s, H22), 3.38 (1H, m, H11),
2.38–2.05 (5H, m, H2, H4, H10, H13), 1.83–1.31 (11H, m, H1,
H6, H7, H8, H15, H13, 11-OH), 1.19 (3H, m, H, H18), 0.89 (3H,
13
d, J ¼ 6.8 Hz, H17), 0.74 (3H, d, J ¼ 6.8 Hz, H16); C NMR (CDCl3,
1
00 MHz): d 217.12 (C3), 168.82 (C21), 168.18 (C26), 147.13 (C24),
139.10 (C19), 117.27 (C20), 105.89 (C25), 74.82 (C11), 69.16 (C14),
8.20 (C4), 45.46 (C22), 44.76 (C9), 43.9 (C13), 41.73 (C12), 36.78
C5), 36.00 (C6), 34.48 (C10), 32.80 (C2), 31.77 (C23), 30.42 (C8),
5
(
2
6.84 (C7), 26.39 (C18), 24.85 (C1), 16.90 (C16), 14.99 (C15), 11.58
þ
(C17); ESI-MS (m/z): 507 [MþH] ; Anal. Calcd. for C26
38 2 4 2
H N O S : C,
6
1.63; H, 7.56; N, 5.53; Found: C, 61.41; H, 7.81; N, 5.46.
1
4-O-[2-((2-(2-Chloroacetamido)thiazol-4-yl)-
methylthio)acetyl]mutilin 11
To a solution of compound 10 (1.00 g, 1.98 mmol) and K
(0.41 g, 2.96mmol) in 20 mL dry CH Cl , ClCH COCl (0.33 g,
.96 mmol) in 5 mL dry CH Cl was slowly dropped at 08C.
2
CO
3
2
2
2
2
2
2
The reaction mixture was stirred for 1 h. After the reaction,
the solution was washed with water three times, and then the
4
organic layer was dried with NaSO , filtered, concentrated, and
Experimental
purified by column chromatography (PE/EtOAc ¼ 2:1) to yield 11
as a white waxy solid 1.06 g, yield: 92%. Analytical data for 11: IR
(KBr, cm ): 3482, 3204, 3078, 2933, 2864, 1728, 1552, 1455,
ꢂ1
Chemistry
Melting points were determined on a Mel-TEMP II melting point
1
411, 1281, 1115, 1016, 980; H NMR (CDCl
1
3
, 400 MHz): d 6.86
(
1H, s, H25), 6.51 (1H, dd, J
1
¼ 11.2 Hz, J ¼ 17.2 Hz, H19), 5.79
2
apparatus and uncorrected. Infrared (IR) spectra (KBr) were
1
(1H, d, J ¼ 8.4 Hz, H14), 5.39 (1H, d, J ¼ 11.2 Hz, H20), 5.24 (1H, d,
J ¼ 17.2 Hz, H20), 4.29 (2H, s, H28), 3.84 (2H, s, H23), 3.38 (1H, m,
H11), 3.05 (2H, s, H22), 2.38–2.05 (5H, m, H2, H4, H10, H13), 1.81–
recorded on a Nicolet Impact 410 instrument (KBr pellet).
1
H
3
NMR and C NMR spectra were recorded with a Bruker Avance
00 and 100 MHz spectrometer, respectively, at 300 K, using TMS
4
1
0
.32 (11H, m, H1, H6, H7, H8, H15, H13, 11-OH), 1.19 (3H, s, H18),
.89 (3H, d, J ¼ 6.8 Hz, H17), 0.74 (3H, d, J ¼ 6.8 Hz, H16);
as an internal standard. MS spectra were recorded on a Shimadzu
GC-MS 2010 (EI) or a Mariner Mass Spectrum (ESI). Elemental
analysis was performed on an Eager 300 instrument. All
compounds were routinely checked by TLC and preparative
TLC was performed on silica gel GF/UV 254, and the column
chromatography were conducted on silica gel (200–300 mesh)
and visualized under UV light at 254 and 365 nm. As determined
by elemental analysis, the purity of all target compounds is
1
3
3
C NMR (CDCl , 100 MHz): d 217.11 (C3), 168.55 (C21), 164.01
(C27), 157.31 (C26), 146.34 (C24), 139.11 (C19), 117.28 (C20),
1
4
11.94 (C25), 74.61 (C11), 69.29 (C14), 58.16 (C4), 45.44 (C22),
4.79 (C9), 43.89 (C13), 41.94 (C28), 41.72 (C12), 36.74 (C5), 35.99
(
2
C6), 34.46 (C10), 32.77 (C2), 31.35 (C23), 30.40 (C8), 26.83 (C7),
6.38 (C18), 24.84 (C1), 16.90 (C16), 14.93 (C15), 11.57 (C17);
ꢂ
ESI-MS (m/z): 581 [M–H] ; Anal. Calcd. for C28
5
H
39ClN
7.66; H, 6.74; N, 4.80; Found: C, 57.79; H, 6.94; N, 4.66.
2
O
5
S
2
: C,
>95%. All solvents were reagent grade and, when necessary,
were purified and dried by standard methods. Solutions after
reactions and extractions were concentrated using a rotary
evaporator operating at a reduced pressure of ca. 20 Torr.
Organic solutions were dried over anhydrous sodium sulfate.
Compound 1 was commercially available, and compounds 8 and
General procedure for the synthesis of the target
compounds 12a–g
A solution of compound 11 (1.72 mmol), R
and K CO (5.16 mmol) in 20 mL acetonitrile was stirred at 408C
1 2
R NH (8.60 mmol),
9
were prepared according to the literature [21].
2
3
for 4–8 h. The solvent was removed under reduced pressure. The
crude residue was dissolved in chloroform and washed with
saturated NaCl. The organic layer was dried with MgSO , filtered,
concentrated, and purified by column chromatography (EtOAc/
MeOH ¼ 20:1–10:1) to yield 12a–g.
1
4-O-[2-((2-Aminothiazol-4-yl)methylthio)acetyl]mutilin 10
To a solution of (2-aminothiazol-4-yl)methanethiol (0.33 g,
.24 mmol) in 2 mL 30% NaOH aqueous solution, compound 9
1.00 g, 2.04 mmol) in 25 mL THF was slowly dropped. The reac-
4
2
(
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–9
Novel Pleuromutilin Derivatives as Antibiotic Agents
7
1
58.38 (C26), 146.81 (C24), 139.10 (C19), 117.16 (C20), 111.36
C25), 74.53 (C11), 69.21 (C14), 59.87 (CH OH), 58.14 (C4), 57.98
C28), 45.41 (C22), 45.22 (NCH ), 44.74 (C9), 43.86 (C13), 41.65
C12), 36.75 (C5), 35.98 (C6), 34.43 (C10), 32.79 (C2), 31.70 (C23),
CH ),
14-O-[2-((2-(2-(2-Hydroxyethylamino)acetamido)-
thiazol-4-yl)methylthio)acetyl]mutilin 12a
(
(
(
2
2
Compound 12a was synthesized according to the general pro-
cedure. White foam solid; yield: 63%. Analytical data for 12a: IR
ꢂ1
30.46 (C8), 26.80 (C7), 26.52 (C18), 24.82 (C1), 21.15 (NCH
16.94 (C16), 14.96 (C15), 11.54 (C17). ESI-MS (m/z): 622 [MþH] ;
Anal. Calcd. for C31 : C, 59.87; H, 7.62; N, 6.76; Found: C,
9.61; H, 7.84; N, 6.57.
2
2
þ
(
1
KBr, cm ): 3316, 2931, 1728, 1685, 1447, 1413, 1373, 1324,
1
153, 1117, 937; H NMR (CDCl
3
, 400 MHz): d 6.78 (1H, s, H25),
47 3 6 2
H N O S
6
.51 (1H, dd, J ¼ 17.6 Hz, H19), 5.78 (1H, d,
1
¼ 11.2 Hz, J
2
5
J ¼ 8.4 Hz, H14), 5.37 (1H, d, J ¼ 10.8 Hz, H20), 5.23 (1H, d,
OH), 3.39
2H, m, OH, H11), 3.22 (2H, s, H22), 3.05 (2H, s, H28), 2.82 (2H, t,
J ¼ 17.2 Hz, H20), 3.79 (2H, s, H23), 3.44 (2H, m, CH
2
(
14-O-[2-((2-(2-(Bis(2-hydroxyethyl)amino)-
J ¼ 4.8 Hz, NCH
2
), 2.02–2.29 (5H, m, H2, H4, H10, H13), 1.23–1.81
acetamido)thiazol-4-yl)methylthio)acetyl]mutilin 12d
Compound 12d was synthesized according to the general pro-
cedure. White foam solid; yield: 58%. Analytical data for 12d: IR
(
(
(
11H, m, H1, H6, H7, H8, H15, H13, 11-OH), 1.19 (3H, s, H18), 0.89
1
3
3H, d, J ¼ 6.8 Hz, H17), 0.74 (3H, d, J ¼ 6.8 Hz, H16); C NMR
CDCl , 100 MHz): d 217.02 (C3), 169.14 (C21), 168.58 (C27),
58.15 (C26), 147.07 (C24), 139.00 (C19), 117.05 (C20), 111.11
C25), 74.32 (C11), 69.13 (C14), 59.21 (CH OH), 58.12 (C4), 57.97
C28), 51.37 (NCH ), 45.40 (C22), 44.72 (C9), 43.84 (C13), 41.63
C12), 36.73 (C5), 35.99 (C6), 34.42 (C10), 32.77 (C2), 31.71 (C23),
ꢂ1
3
(KBr, cm ): 3410, 2933, 2879, 2353, 1722, 1533, 1460, 1409,
, 400 MHz): d 6.71 (1H, s, H25), 6.50
1
280, 1114; H NMR (CDCl
1
1
3
(
(
(
2
(
1H, dd, J
1
¼ 11.2 Hz, J ¼ 17.6 Hz, H19), 5.77 (1H, d, J ¼ 8.4 Hz,
2
2
H14), 5.35 (1H, d, J ¼ 10.8 Hz, H20), 5.21 (1H, d, J ¼ 17.2 Hz, H20),
3.76 (2H, s, H23), 3.46 (4H, m, 2 ꢃ CH OH), 3.36 (3H, m, 2 ꢃ OH,
2
3
0.46 (C8), 26.80 (C7), 26.55 (C18), 24.80 (C1), 16.97 (C16), 14.95
þ
H11), 3.20 (2H, s, H22), 3.08 (2H, s, H28), 2.82 (4H, t, J ¼ 4.8 Hz,
(
C15), 11.50 (C17). ESI-MS (m/z): 608 [MþH] ; Anal. Calcd. for
2 ꢃ NCH ), 2.05–2.37 (5H, m, H2, H4, H10, H13), 1.80–1.25
2
C
30
H
45
N
3
O
6
S
H, 7.74; N, 6.76.
2
: C, 59.28; H, 7.46; N, 6.91; Found: C, 59.11;
(11H, m, H1, H6, H7, H8, H15, H13, 11-OH), 1.19 (3H, s, H18),
0.89 (3H, d, J ¼ 6.8 Hz, H17), 0.73 (3H, d, J ¼ 6.8 Hz, H16);
1
3
3
C NMR (CDCl , 100 MHz): d 217.18 (C3), 170.32 (C21), 168.77
(
1
(
(
C27), 158.55 (C26), 147.21 (C24), 139.13 (C19), 117.18 (C20),
11.02 (C25), 74.45 (C11), 69.36 (C14), 59.37 (2 ꢃ NCH ), 58.20
2 ꢃ CH OH), 58.15 (C4), 57.90 (C28), 45.44 (C22), 44.76 (C9), 43.88
C13), 41.72 (C12), 36.77 (C5), 35.97 (C6), 34.45 (C10), 32.79 (C2),
14-O-[2-((2-(2-((2-Hydroxyethyl)(methyl)amino)-
acetamido)thiazol-4-yl)methylthio)acetyl]mutilin 12b
Compound 12b was synthesized according to the general pro-
cedure. White foam solid; yield: 60%. Analytical data for 12b: IR
2
2
ꢂ1
31.79 (C23), 30.46 (C8), 26.81 (C7), 26.41 (C18), 24.82 (C1), 16.87
þ
(
1
(
KBr, cm ): 3427, 2934, 1732, 1628, 1451, 1408, 1280, 1165,
1
(C16), 14.93 (C15), 11.52 (C17). ESI-MS (m/z): 652 [MþH] ; Anal.
016, 980, 696; H NMR (CDCl
1H, dd, J ¼ 17.2 Hz, H19), 5.81 (1H, d, J ¼ 8.0 Hz,
¼ 11.2 Hz, J
H14), 5.41 (1H, d, J ¼ 10.8 Hz, H20), 5.26 (1H, d, J ¼ 17.2 Hz, H20),
.84 (2H, s, H23), 3.45 (2H, m, CH OH), 3.40 (2H, m, OH, H11), 3.28
2H, s, H22), 3.06 (2H, s, H28), 2.54 (2H, m, NCH ), 2.04–2.33
8H, m, H2, H4, H10, H13, NCH ), 1.17–1.81 (14H, m, H1, H6,
3
, 400 MHz): d 6.83 (1H, s, H25), 6.53
49 3 7 2
Calcd. for C32H N O S : C, 58.96; H, 7.58; N, 6.45; Found: C,
1
2
5
9.21; H, 7.33; N, 6.28.
3
(
(
2
14-O-[2-((2-(2-(4-Hydroxypiperidin-1-yl)acetamido)-
thiazol-4-yl)methylthio)acetyl]mutilin 12e
2
3
H7, H8, H15, H13, 11-OH, H18), 0.90 (3H, d, J ¼ 6.8 Hz, H17), 0.75
Compound 12e was synthesized according to the general pro-
cedure. White foam solid; yield: 64%. Analytical data for 12e: IR
1
3
(
3H, d, J ¼ 6.8 Hz, H16); C NMR (CDCl
70.08 (C21), 168.65 (C27), 158.07 (C26), 146.56 (C24), 139.06
C19), 117.23 (C20), 111.18 (C25), 74.56 (C11), 69.15 (C14),
0.71 (NCH ), 58.62 (CH OH), 58.43 (C4), 56.37 (C28), 45.66
NCH ), 45.48 (C22), 44.82 (C9), 43.86 (C13), 41.65 (C12),
3
, 100 MHz): d 217.10 (C3),
ꢂ1
1
(
(KBr, cm ): 3431, 2937, 1731, 1454, 1373, 1280, 1146, 1116,
1
1016; H NMR (CDCl , 400 MHz): d 6.80 (1H, s, H25), 6.51 (1H, dd,
3
6
2
2
J
1
¼ 11.2 Hz, J ¼ 17.2 Hz, H19), 5.79 (1H, d, J ¼ 8.4 Hz, H14),
2
(
3
5.38 (1H, d, J ¼ 10.8 Hz, H20), 5.24 (1H, d, J ¼ 17.2 Hz, H20), 4.14
(1H, m, CHOH), 3.83 (2H, m, H23, CH), 3.38 (1H, m, H11), 3.23
(2H, s, H22), 3.06 (2H, s, H28), 2.83 (4H, m, N (CH –C) –), 2.00–2.05
3
3
1
6.75 (C5), 35.99 (C6), 34.44 (C10), 32.76 (C2), 31.65 (C23),
0.44 (C8), 26.83 (C7), 26.51 (C18), 24.83 (C1), 16.97 (C16),
4.95 (C15), 11.52 (C17). ESI-MS (m/z): 622 [MþH] ; Anal. Calcd.
2
2
þ
(5H, m, H2, H4, H10, H13), 1.19–1.80 (18H, m, H1, H6, H7, H8,
47 3 6 2
for C31H N O S : C, 59.87; H, 7.62; N, 6.76; Found: C, 60.05;
H13, H15, H18, 11-OH, N (C–CH ) ), 0.90 (3H, d, J ¼ 6.8 Hz, H17),
2
2
1
3
H, 7.54; N, 6.52.
0.75 (3H, d, J ¼ 6.8 Hz, H16); C NMR (CDCl3, 100 MHz): d 217.17
C3), 168.95 (C21), 168.65 (C27), 157.76 (C26), 146.28 (C24),
39.12 (C19), 117.25 (C20), 111.33 (C25), 74.62 (C11), 69.27
C14), 66.61 (CHOH), 61.94 (C4), 58.21 (C28), 51.64 (N (CH ),
5.46 C22), 44.83 (C9), 43.92 (C13), 41.75 (C12), 36.77 (C5),
6.01 (C6), 34.47 (C10), 34.21 (N (C–CH ), 32.80 (C2), 31.63
(
1
14-O-[2-((2-(2-(2-Hydroxypropylamino)acetamido)-
thiazol-4-yl)methylthio)acetyl]mutilin 12c
(
2 2
)
4
3
Compound 12c was synthesized according to the general pro-
cedure. White foam solid; yield: 53%. Analytical data for 12c:
2 2
)
ꢂ1
(C23), 30.42 (C8), 26.85 (C7), 26.43 (C18), 24.84 (C1), 16.91
þ
IR (KBr, cm ): 3308, 2934, 1745, 1677, 1532, 1466, 1374, 1456,
1
(C16), 14.98 (C15), 11.54 (C17); ESI-MS (m/z): 648 [MþH] ; Anal.
1
066, 1017; H NMR (CDCl
3
, 400 MHz): d 6.75 (1H, s, H25), 6.50
¼ 17.2 Hz, H19), 5.78 (1H, d, J ¼ 8.4 Hz,
H14), 5.37 (1H, d, J ¼ 10.8 Hz, H20), 5.24 (1H, d, J ¼ 17.2 Hz, H20),
.80 (2H, s, H23), 3.45 (2H, m, CH OH), 3.37 (2H, m, OH, H11), 3.20
2H, s, H22), 3.05 (2H, s, H28), 2.76 (2H, t, J ¼ 4.8 Hz, NCH ), 2.04–
.31 (5H, m, H2, H4, H10, H13), 1.24–1.83 (13H, m, H1, H6, H7, H8,
H15, H13, 11-OH, NCH CH ), 1.18 (3H, s, H18), 0.88 (3H, d,
J ¼ 6.8 Hz, H17), 0.73 (3H, d, J ¼ 6.8 Hz, H16);
CDCl , 100 MHz): d 217.01 (C3), 169.03 (C21), 168.45 (C27),
49 3 6 2
Calcd. for C33H N O S : C, 61.18; H, 7.62; N, 6.49; Found: C,
(
1H, dd, J
1
¼ 11.2 Hz, J
2
5
9.92; H, 7.83; N, 6.26.
3
2
(
2
(S)-14-O-[2-((2-(2-(2-(Hydroxymethyl)pyrrolidin-1-yl)-
acetamido)thiazol-4-yl)methylthio)acetyl]mutilin 12f
2
2
2
Compound 12f was synthesized according to the general pro-
cedure. White foam solid; yield: 65%. Analytical data for 12f: IR
1
3
C NMR
ꢂ1
(
3
(
KBr, cm ): 3435, 2936, 2866, 1732, 1530, 1456, 1414, 1280,
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

8
Y. Ling et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–9
1
1
6
374, 1115, 1018; H NMR (CDCl3, 400 MHz): d 6.77 (1H, s, H25),
.50 (1H, dd, J
Aqueous solubility
1
¼ 10.8 Hz, J ¼ 17.2 Hz, H19), 5.78 (1H, d,
2
Each compound with the accurate weight of 200.0 mg was
placed in a 50 mL beaker. Then 10 mL water was added and
exposed to ultrasonic oscillation for 5 min and stirred for 24 h in
a water bath with a constant temperature of 258C. It was
centrifuged at 10 000 rpm for 10 min, and the supernatant
was filtered through a microporous membrane and injected into
the HPLC system for determination.
J ¼ 8.0 Hz, H14), 5.38 (1H, d, J ¼ 10.8 Hz, H20), 5.23 (1H, d,
J ¼ 17.2 Hz, H20), 3.81 (2H, d, H23), 3.76 (1H, m, NCH), 3.38
(
2
1H, m, H11), 3.34 (2H, m, CH OH), 3.19 (3H, m, H22, C–OH),
3
1
CH
.04 (2H, s, H28), 2.49–2.05 (7H, m, H2, H4, H10, H13, NCH
.80–1.14 (18H, m, H1, H6, H7, H8, H15, H13, 11-OH, H18,
CH
CH), 0.89 (3H, d, J ¼ 7.2 Hz, H17), 0.736 (3H, d,
J ¼ 6.8 Hz, H16); C NMR (CDCl
68.61 (C21), 168.43 (C27), 157.68 (C26), 146.28 (C24), 139.13
C19), 117.25 (C20), 111.45 (C25), 74.63 (C11), 69.29 (C14),
1.01 (NCH), 59.19 (C4), 58.19 (CH OH), 57.65 (C28), 52.61
NCH ), 45.46 (C22), 44.85 (C9), 43.93 (C13), 41.75 (C12), 36.78
C5), 36.02 (C6), 34.46 (C10), 32.82 (C2), 31.67 (C23), 30.42 (C8),
6.86 (C7), 26.42 (C18), 26.24 (CHCH ), 24.85 (C1), 21.20
NCH CH ), 16.92 (C16), 14.96 (C15), 11.54 (C17); ESI-MS (m/z):
48 [MþH] ; Anal. Calcd. for C33
2
),
2
2
1
3
3
, 100 MHz): d 217.05 (C3),
1
(
6
(
(
2
(
6
Molecular docking methodology
All calculations and analysis were conducted with MOE 2008.10
program (Chemical Computing Group, Montreal, Canada). The
crystal structure of the 50S ribosomal subunit from D. radiodurans
in complex with tiamulin (PDB code: 1XBP) used in the docking
study were obtained from the Protein Data Bank (www.rcsb.org).
Heteroatoms and water molecules in the PDB files were removed
at the beginning, and all hydrogen atoms were subsequently
added to the 50S ribosomal subunit. Amber99 forcefield was
assigned to the ribosomal subunit by MOE. Prior to the docking
calculations, an energy minimization using the MMFF94 force-
field was performed on the ribosomal subunit based on the
minimization protocol.
Compound 12f was drawn in MOE and all hydrogen atoms
were added subsequently. Then the compounds were protonated
using the protonate 3D protocol and energy minimized using
the MMFF94 forcefield in MOE. When the ribosomal subunit and
compound 12f were ready for the docking study, compound 12f
was modeled within the ribosomal subunit according to the MOE
docking protocol with the Alpha Triangle placement option and
the London dG scoring function.
2
2
2
2
2
þ
49 3 6 2
H N O S : C, 61.18; H, 7.62;
N, 6.49; Found: C, 59.92; H, 7.88; N, 6.22.
14-O-[2-((2-(2-(4-(2-Hydroxyethyl)piperazin-1-yl)-
acetamido)thiazol-4-yl)methylthio)acetyl]mutilin 12g
Compound 12g was synthesized according to the general pro-
cedure. White foam solid; yield: 53%. Analytical data for 12g: IR
ꢂ1
(
KBr, cm ): 3427, 2937, 2823, 1728, 1456, 1383, 1327, 1280,
1
1
156, 1116. H NMR (CDCl3, 400 MHz): d 6.81 (1H, s, H25), 6.51
1H, dd, J ¼ 17.2 Hz, H19), 5.79 (1H, d, J ¼ 8.0 Hz,
¼ 11.2 Hz, J
H14), 5.39 (1H, d, J ¼ 11.2 Hz, H20), 5.24 (1H, d, J ¼ 17.2 Hz, H20),
.83 (2H, s, H23), 3.39 (1H, m, H11), 3.27 (2H, s, H22), 3.05 (2H, s,
H28), 3.67 (2H, m, CH OH), 2.65–2.67 (10H, m, N(CH CH NCH ),
.05–2.38 (5H, m, H2, H4, H10, H13), 1.11–1.81 (14H, m, H1, H6,
H7, H8, H15, H13, 11-OH, H18), 0.89 (3H, d, J ¼ 6.8 Hz, H17), 0.75
(
1
2
3
2
2
2
)
2
2
2
1
3
(
1
(
3H, d, J ¼ 6.4 Hz, H16); C NMR (CDCl
70.15 (C21), 168.67 (C27),158.10 (C26), 146.08 (C24), 139.11
C19), 117.27 (C20), 111.21 (C25), 74.61 (C11), 69.19 (C14),
3
, 100 MHz): d 217.12 (C3),
We gratefully acknowledge the financial support by the Natural Science
Foundation of Jiangsu Province in China (Grant No. BK2011389), the Natural
Science Funds for Colleges and Universities in Jiangsu Province (No.
6
5
5.83 (HOCH ), 64.74 (NCH
8.19 (2 ꢃ NCH
2
2
), 60.41 (2 ꢃ NCH
2
), 58.84 (C4),
08KJD350003), and also thank for project funded by the Priority Academic
Programs Development of Jiangsu Higher Education Institutions (PAPD).
2
), 56.34 (C28), 45.46 (C22), 44.82 (C9), 43.92
(
(
C13), 41.75 (C12), 36.77 (C5), 36.01 (C6), 34.47 (C10), 32.78
C2), 31.67 (C23), 30.42 (C8), 26.85 (C7), 26.40 (C18), 24.85 (C1),
The authors have declared no conflict of interest.
þ
1
6.89 (C16), 14.96 (C15), 11.54 (C17); ESI-MS (m/z): 677 [MþH] ;
Anal. Calcd. for C34
52 4 6 2
H N O S : C, 60.33; H, 7.74; N, 8.28; Found: C,
60.41; H, 7.96; N, 8.05.
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