Synthesis and biological activities of novel pleuromutilin derivatives with a substituted thiadiazole moiety as potent drug-resistant bacteria inhibitors

Article abstract of DOI:10.1021/jm500374c

A series of novel pleuromutilin derivatives possessing thiadiazole moieties were synthesized via acylation reactions under mild conditions. The in vitro antibacterial activities of the derivatives against methicillin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis, Escherichia coli, and Streptococcus agalactiae were tested by the agar dilution method and Oxford cup assay. The majority of the tested compounds displayed moderate antibacterial activities. Importantly, the three compounds with amino or tertiary amine groups in their side chains, 11, 13b, and 15c, were the most active antibacterial agents. Docking experiments carried out on the peptidyl transferase center (PTC) of 23S rRNA proved that there is a reasonable direct correlation between the binding free energy (ΔG_b, kcal/mol) and the antibacterial activity. Moreover, the pharmacokinetic profiles of 11 and 15c in rat were characterized by moderate clearance and oral bioavailability.

Full text of DOI:10.1021/jm500374c

Article
pubs.acs.org/jmc
Synthesis and Biological Activities of Novel Pleuromutilin Derivatives
with a Substituted Thiadiazole Moiety as Potent Drug-Resistant
Bacteria Inhibitors
Ruofeng Shang,† Xiuying Pu,‡ Ximing Xu,§ Zhijun Xin,∥ Chao Zhang,† Wenzhu Guo,† Yu Liu,†
and Jianping Liang*,†
†Key Laboratory of New Animal Drug Project of Gansu Province; Key Laboratory of Veterinary Pharmaceutical Development,
Ministry of Agriculture, Lanzhou Institute of Husbandry and Pharmaceutical Sciences of CAAS, 335 Jiangouyan, Lanzhou 730050,
China
‡College of Life Science and Engineering, Lanzhou University of Technology, 287 Langongping Road, Lanzhou 730050, China
§Department of Toxicology, School of Public Health, Guiyang Medical University, 9 Beijing Road, Guiyang 550004, China
∥Institute of Modern Physics, Chinese Academy of Sciences, 509 Nanchang Road, Lanzhou 730000, China
S
* Supporting Information
ABSTRACT: A series of novel pleuromutilin derivatives possessing thiadiazole moieties were synthesized via acylation reactions
under mild conditions. The in vitro antibacterial activities of the derivatives against methicillin-resistant Staphylococcus aureus,
methicillin-resistant Staphylococcus epidermidis, Escherichia coli, and Streptococcus agalactiae were tested by the agar dilution
method and Oxford cup assay. The majority of the tested compounds displayed moderate antibacterial activities. Importantly, the
three compounds with amino or tertiary amine groups in their side chains, 11, 13b, and 15c, were the most active antibacterial
agents. Docking experiments carried out on the peptidyl transferase center (PTC) of 23S rRNA proved that there is a reasonable
direct correlation between the binding free energy (ΔGb, kcal/mol) and the antibacterial activity. Moreover, the pharmacokinetic
profiles of 11 and 15c in rat were characterized by moderate clearance and oral bioavailability.
Pleuromutilin derivatives interact with the 23S rRNA of the
50S bacterial ribosome subunit and interfere with bacterial
protein synthesis.9,10 The pleuromutilin derivative binding site
is the domain V of 23S rRNA at the peptidyl transferase center
(PTC). Their tricyclic core is positioned in a pocket next to the
A-tRNA binding site, whereas the C-14 side chain extends to
the P-tRNA binding site to prevent the peptide transfer from
correct positioning of tRNAs and thus inhibit peptide bond
formation.11,12
The introduction of a thioether at the C-22 position of
pleuromutilin analogues and the presence of a basic group
enhance antibacterial activity.13−16 Thus, the structural
modifications have focused basically on their C-14 side chain.
INTRODUCTION
■
The abuse of antibiotics has resulted in the emergence and
spread of antibiotic resistance, leading to reductions or losses in
therapeutic effects. Drug-resistant bacteria, especially Staph-
ylococcus aureus, Streptococcus pneumoniae, and Mycobacterium
tuberculosis, kill more than two million people each year and
seriously endanger human health.1,2
Pleuromutilin (1) (Figure 1), a natural product composed of
a 5−6−8 tricyclic carbon skeleton,3,4 was first discovered and
isolated from Pleurotus mutilus or P. passeckerianus in 1951.5
The derivatives of pleuromutilin have received much
investigation due to their high activities against mycoplasmas
and drug-resistant Gram-positive bacteria,2,6 their pharmacody-
namic properties,7 and their lack of target-specific cross-
resistance to other antibiotics.8
Received: March 10, 2014
© XXXX American Chemical Society
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Figure 1. Structural formulas of pleuromutilin and derivatives thereof.
Further alterations on the C-14 side chain of pleuromutilin
have led to three drugs: tiamulin (2), valnemulin (3), and
retapamulin (4) (Figure 1). Tiamulin and valnemulin are used
in veterinary medicine as a drug for poultry and pigs.17−19
Retapamulin became the first pleuromutilin antibiotic to be
approved for human use in 2007 by the Food and Drug
Administration (FDA).20,21 In addition to retapamulin, 14-O-
{[(1R,2R,4R)-4-amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-
mutilin (BC-3781, 5),22 14-O-[(N-(3-methyl-2(S)-amino-bu-
turyl-piperidin-3(R)-yl)-sulfanyl)-acetyl]-mutilin (BC-3205,
6)23 and 14-O-[(3-hydroxymethyl-phenylsulfanyl)-acetyl]-mu-
tilin (BC-7013, 7)24 are being tested in clinical trial for human
use. Azamulin (8) (Figure 1) was the next pleuromutilin
designated for human use, and the drug was designed during
the early 1980s. It did not undergo further clinical trials because
of its poor solubility in water and its strong inhibition of human
cytochrome P450.25
Thiadiazole-based structural scaffolds form an essential
constituent of many synthetic drugs. Compounds containing
a 1,3,4-thiadiazole moiety exhibit anticancer, anti-inflammatory,
antibacterial, antifungal, antiviral, anticonvulsant, and antipar-
asitic activities (for some 1,3,4-thiadiazole-containing drugs
available on the market, see Figure S1 in the Supporting
Information).26 Furthermore, thiadiazoles are bioisosteres of
oxadiazole, oxazole, and benzene, which leads to analogues with
improved activities because the sulfur atom imparts improved
liposolubility.27,28
We have synthesized three types of pleuromutilin derivatives
bearing a thiadiazole moiety: compounds 12a−f, compounds
13a−c, and compounds 15a−f. All these compounds were
synthesized from compound 11, an important intermediate
synthesized from 14-O-(iodoacetyl) mutilin (10) and 2-amino-
5-mercapto-1,3,4-thiadiazole. In particular, compound 9 was
synthesized using an efficient method characterized by a higher
yield and shorter time compared with other published
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Scheme 1. Synthesis of Pleuromutilin Derivatives
Reagents and conditions: (i) TsCl, NaOH, t-butyl methyl ether/H2O, reflux, 1 h, 93%; (ii) KI, acetone, reflux, 1 h, 81%; (iii) 2-amino-5-mercapto-
1,3,4-thiadiazole, NaOH, THF, 0 °C, 2 h, 82%; (iv) benzoic acid, EDCI, HOBt, DCM, room temperature, 36−48 h, 62−75%; (v) N-Boc protected
amino acids, EDCI, HOBt, DCM, room temperature, 36−48 h, then TFA, DCM, room temperature, 0.5 h, 58−61%; (vi) 2-chloroacetyl chloride,
NMM, DCM, 0 °C, 2 h, 86%; (vii) secondary amines, TEA, THF, 45 °C, 2 h, 62−79%.
methods.14,29 In this study, the antibacterial properties of all the
above-mentioned synthesized compounds were evaluated using
the agar dilution method and Oxford cup assay against three
Gram-positive bacteria: methicillin-resistant Staphylococcus
aureus (MRSA), methicillin-resistant Staphylococcus epidermidis
(MRSE), and Streptococcus agalactiae (S. agalactiae) and one
Gram-negative bacteria: Escherichia coli (E. coli). Pleuromutilin
derivatives are known to bind to the 23S rRNA of the 50S
bacterial ribosome subunit. To explore the antibacterial
mechanisms of compounds displaying different antibacterial
activities but with similar substituents, compounds 12d−f,
13a−c, and 15a−d were chosen for molecular docking studies.
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Figure 2. ORTEP diagram for compound 12e with ellipsoids set at 50% probability (hydrogen atoms were omitted for clarity).
The antibacterial and docking results may provide new insights
into the design of novel pleuromutilin derivatives and lay the
basis for further studies of promising pleuromutilin antibiotics.
intermediate 14, which was directly treated with various amines
under alkaline conditions to give the target compounds 15a−f.
All derivatives were obtained by column chromatography,
1
followed by IR, H NMR, and 13C NMR spectral character-
ization as well as by high-resolution mass spectral (HRMS)
analysis (for the spectra of all the synthesized compounds, see
Supporting Information). A crystal of compound 12e was
formed as a colorless, block-like crystal in an acetone/ethanol
solution, and X-ray diffraction unambiguously confirmed the
structure (Figure 2; for discussion, see Supporting Informa-
tion).
RESULTS AND DISCUSSION
■
Chemistry. The general synthetic routes for all the
pleuromutilin derivatives are shown in Scheme 1. Almost all
the syntheses of pleuromutilin derivatives begin from 22-O-
tosylpleuromutilin (9), which was obtained by the reaction of
pleuromutilin and p-toluenesulfonyl chloride to activate the 22-
hydroxyl of pleuromutilin. In this paper, we describe a different
synthesis method using NaOH as a base, in which pleuro-
mutilin is rapidly converted to 9 in excellent yield. Intermediate
10 was prepared in good yield under reflux for 3 h in acetone,
as previously described.30,31 Compound 11 was then obtained
in good yield by the nucleophilic attack of 2-amino-5-mercapto-
1, 3, 4-thiadiazole on intermediate 10 under alkaline conditions
and without further separation by column chromatography. In
addition to the above synthetic method, compound 11 can be
synthesized directly from compound 9 in a strongly alkaline
environment. However, this method is undesirable because of
the many byproducts produced in the residue and its lower
yield.
Compounds 12a−f were directly obtained by condensation
reactions between the amino-group of compound 11 and the
carboxyl group of substituted aryl carboxylic acids with 1-ethyl-
3-(3-dimethyllaminopropyl) carbodiimide hydrochloride
(EDCI) and 1-hydroxybenzotriazole (HOBt). Compounds
12a−f could also be prepared by the reaction of compound
11 with correlative acyl chlorides, which were obtained from
the acyl chlorination of substituted aryl carboxylic acids by
SOCl2 in refluxing conditions. Compounds 13a−c were
obtained in three steps: first, the amino groups of the amino
acid derivative were protected by tert-butoxycarbonyl (BOC).
Then, an amide was formed via a condensation reaction
between compound 11 and the amino acid derivative using
EDCI and HOBt. Finally, the protected amino groups were
hydrolyzed with trifluoroacetic acid (TFA) and compounds
13a−c were obtained. Compound 11 was treated with 2-
chloroacetyl chloride using N-methylmorpholine to give the
Biological Assays. The synthesized pleuromutilin deriva-
tives (i.e., 12a−f, 13a−c, and 15a−f) were screened for their in
vitro antibacterial properties against MRSA, MRSE, S.
agalactiae, and E. coli by the agar dilution method. The
minimum inhibitory concentration (MIC) of the synthesized
compounds, 11, 12a−f, 13a−c, and 15a−f, along with tiamulin
and azamulin used as reference drugs, were determined in
triplicate at pH 7.40 (Table 1). The MICs of the 16 new
pleuromutilin derivatives in vitro against MRSA, MRSE,
S.agalactiae and E. coli ranged from 8 to 0.25 μg/mL, 8 to
0.5 μg/mL, 8 to 0.25 μg/mL, and 64 to 8 μg/mL, respectively.
The Oxford cup assays were carried out to evaluate the
antibacterial activities against the above-mentioned four
bacterial strains. The zones of inhibition for two concentrations
(320 and 160 μg/mL) of the synthetic compounds and
reference drugs were measured (Table 2). From Table 2, it can
be observed that most compounds exhibited relatively
moderate inhibitory activity against MRSA, MRSE, and S.
agalactiae and weak activity against E. coli. Good inhibitory
characteristics, exceeding those of tiamulin and azamulin, were
observed for compounds 13b and 15c
Among the examined pleuromutilin derivatives, three
compounds, 11, 13b, and 15c, showed excellent antibacterial
activity against all strains except E. coli. The others, especially
compounds with chlorbenzene and methoxybenzene, showed
moderate antibacterial activity. Compounds 11, 13b, and 15c
showed superior or comparable antibacterial activities to that of
tiamulin. Compounds 11 and 15c displayed the same
antibacterial activities as azamulin against MRSA but lower
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Table 1. In Vitro Antibacterial Activity (MIC) of the Novel Pleuromutilin Derivatives
antibacterial activities than azamulin against MRSE, E. coli, and
S. agalactiae. The antibacterial activities of compound 13b,
being the best antibacterial derivative, were superior or similar
to those of azamulin against MRSA and MRSE but lower than
those of azamulin against E. coli and S. agalactiae.
The compounds with chlorine or amino substituents in the
benzene ring at the position meta to the side chain showed
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Table 2. Zone of Inhibition for MRSA, MRSE, E. coli, and S. agalactiae in mm
MRSA (μg/mL)
MRSE (μg/mL)
E. coli (μg/mL)
S. agalactiae (μg/mL)
compound
320
160
320
160
320
160
320
160
11
15.64
14.98
14.66
13.70
14.77
13.09
15.54
15.50
19.23
13.22
17.62
12.35
18.82
13.25
15.44
11.97
17.23
17.98
13.01
12.25
13.36
11.87
12.16
11.42
12.18
12.88
17.48
11.39
15.44
10.98
13.75
13.07
13.39
11.02
16.94
16.52
15.15
12.61
15.02
14.10
14.25
13.78
14.44
15.16
17.82
14.76
16.02
12.85
17.37
12.77
10.83
13.12
15.58
17.02
12.75
11.89
12.94
11.34
11.92
12.26
9.52
14.33
10.26
12.60
10.02
11.54
10.89
11.18
11.27
13.71
12.21
11.97
10.23
13.31
11.14
11.90
11.25
15.85
17.20
12.36
0.00
17.30
15.49
13.54
13.41
13.37
12.75
14.32
13.75
18.38
14.02
14.51
12.72
17.69
13.82
13.25
12.60
16.08
18.86
14.98
12.74
12.69
11.42
11.29
11.66
11.53
12.42
17.42
12.30
11.54
10.44
13.54
12.75
10.91
11.19
14.60
17.16
12a
12b
12c
11.68
0.00
12d
12e
9.96
9.02
12f
10.72
10.09
12.65
10.65
10.10
9.13
13a
12.18
16.15
12.07
14.35
11.52
14.54
10.54
0.00
13b
13c
15a
15b
15c
11.70
9.86
15d
15e
0.00
15f
10.73
13.95
15.36
10.61
13.58
15.96
tiamulin
azamulin
Table 3. Binding Free Energy, Number of Noncovalent Molecular Interactions, and RMSD
noncovalent molecular interaction
compound
ΔGb (kcal/mol)
−10.88
hydro I interaction
atom of compound
residue
distance (Å)
RMSD (Å)
0.99
11
H-bonding
H-bonding
H-bonding
H-bonding
H-bonding
H-bonding
π−π interaction
H-bonding
H-bonding
π−π interaction
H-bonding
H-bonding
H-bonding
π−πinteraction
H-bonding
H-bonding
H-bonding
π−π interaction
H-bonding
H-bonding
π−π interaction
H-bonding
H-bonding
H-bonding
H-bonding
H-bonding
H-bonding
H-bonding
H-bonding
H-bonding
H-bonding
H-bonding
cation−π interaction
OH (eight-membered ring)
NH2 (triazole ring)
CO (ester)
G-2484
C-2565
G-2044
G-2044
G-2484
G-2044
C-2565
G-2484
C-2565
A-2045
G-2484
G-2044
C-2565
A-2045
G-2484
G-2484
C-2420
A-2045
G-2484
C-2420
A-2045
G-2484
G-2044
C-2565
G-2484
U-2564
G-2484
C-2431
U-2485
G-2484
G-2044
C-2565
G-2045
2.0
2.3
2.9
3.3
2.2
2.9
4.0
2.2
3.2
3.9
2.2
2.8
3.0
3.9
2.2
2.2
1.6
3.9
2.1
2.1
4.0
2.2
3.4
2.8
2.1
2.8
2.2
2.7
3.2
2.1
3.5
3.0
4.1
CO (ester)
12d
12e
12f
−9.46
−8.40
−9.52
OH (eight-membered ring)
CO (ester)
0.99
0.95
0.86
benzene ring
OH (eight-membered ring)
OCH3 (benzene ring)
benzene ring
OH (eight-membered ring)
CO (ester)
OCH3 (benzene ring)
benzene ring
13a
13b
−8.16
−11.46
OH (eight-membered ring)
OH (eight-membered ring)
NH2 (benzene ring)
benzene ring
0.92
0.81
13c
15a
−9.83
−9.76
OH (eight-membered ring)
NH2 (benzene ring)
benzene ring
0.75
0.87
OH (eight-membered ring)
CO (ester)
CO (amido bond)
OH (eight-membered ring)
CO (amido bond)
OH (eight-membered ring)
CO (five-membered ring)
CO (five-membered ring)
OH (eight-membered ring)
CO (ester)
15b
15c
−9.69
0.93
1.12
−11.13
15d
−9.85
0.93
CO (amido bond)
N (pyrrolidine)
stronger antibacterial activities than did those with ortho or para
substituents in the benzene ring. For example, compound 13b
showed stronger antibacterial activity than did compounds 13a
or 13c. In contrast, the compounds with a methoxyl group in
F
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Figure 3. (A) Docking mode of compound 11 (green) to 1XBP. (B) Docking modes of compounds 12d (green), 12e (yellow), and 12f (magenta)
to 1XBP. (C) Docking modes of compounds 13a (green), 13b (yellow), and 13c (magenta) to 1XBP. (D) Docking modes of compounds 15a
(green), 15b (cyan), 15c (magenta), and 15d (yellow) to 1XBP. Important residues are drawn as sticks with different colors. Hydrogen bonds are
shown as dashed red lines; π−π and cation−π interactions are shown as dashed green lines.
the benzene ring at the meta position showed lower
antibacterial activities than did those with ortho or para
substituents in the benzene ring. In addition, the compounds
with an amino group in the benzene ring preferentially
inhibited the growth of MRSE and E. coli. The antibacterial
activity presented obvious variations among the 2-(substituted
amino) acetyl amide analogues (15a−f) with similar sub-
stituents. The diethyl amino substitution (15b) and the
piperidine substitution (15c) noticeably increased the anti-
MRSA, MRSE and S. agalactiae activities compared with those
of the dimethyl amino (15a) and pyrrolidine (15d)
substitutions.
Compound 11 was structurally similar to azamulin but
showed lower antibacterial activities. One possible reason was
that 1,3,4-thiadiazole and 1,3,4-triazole were all mesoionic
compounds, which contain distinct regions of positive and
negative charges associated with a poly heteroatomic system,
enabling them to cross cellular membranes and interact with
biological targets with distinct affinities.32,33 The replacement of
the nitrogen in the triazole ring of azamulin with sulfur reduced
the degree of polarity, which resulted in compound 11
displaying lower affinities compared with azamulin to
biomolecules such as DNA and/or proteins.
compounds with living systems. In our experience, there is a
correlation between the two parameters and antibacterial
activity. The antibacterial activity improved as the π and σ
value of the substituted benzenes decreased below zero. For
example, compound 13b exhibited excellent antibacterial
activity, and the π and σ values of the amine substitution in
the benzene ring at the meta position were −1.23 and −0.16,
respectively. This research may help us to design more potent
antibacterial activities for pleuromutilin derivatives bearing
substituted benzenes.
Molecular Docking Study. Compounds with similar
substituents can exhibit large differences in antibacterial activity.
To understand this phenomenon, a series of docking studies
were conducted to compare the binding modes of 12d−f, 13a−
c, 15a−d, and 11. On applying Homdock software,34 the
redocking of tiamulin 2 into 1XBP37 placed the drug in the
same conformation as that in the X-ray structure. The results
for the 11 compounds present a similar binding mode, with a
RMSD range of 0.75 to 1.12 Å, as documented for tiamulin and
presented in Figure S2 in the Supporting Information, which
displys a superimposition of the 11 docked compounds.
The lowest binding free energies (ΔGb, kcal/mol) and the
lowest root-mean-square difference (RMSD) values in
comparison to the native cocrystallized ligand, were considered
as the best parameters to evaluate the dock binding affinities.36
Upon flexible docking into the 50S ribosomal subunit, the
above-mentioned compounds revealed binding free energies
(ΔGb) in the range of −8.40 to −11.46 kcal/mol. Moreover,
three types of noncovalent molecular interactions, namely,
To better understand the biological activities of the
compounds bearing a substituent in the benzene ring, 12a−f
and 13a−c, we examined their Hansch−Fujita hydrophobic
parameters (π) and Hammett parameters (σ), two physical
properties used to derive quantitative structure−activity
relationships (QSAR) for a host of interactions of organic
G
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hydrogen bonding and π−π and cation−π interactions, played
an important role in the binding of the compounds to 1XBP.
For all the docking models, at least one hydrogen bond was
formed between the hydroxyl group of the eight-membered
ring and the residue of G-2484 (Table 3 and Figure 3). In the
docking results for compounds 12d, 12e, 12f, 13b, and 13c, a
π−π interaction was formed between the benzene ring and the
purine of A-2045. Cation−π interactions between pyrrolidine
and the purine of A-2045 were only present in the docking
model for compound 15d.
Information). The results revealed a direct reasonable
correlation between the binding free energy and the zone of
inhibition, with correlation coefficients (R2) of 0.6626 and
0.7658 for compounds 11, 12d−f, 13a−c, and 15a−d, as
illustrated in Figure S4 in the Supporting Information.
Pharmacokinetics (PK) Studies. The intravenous and oral
pharmacokinetics of 11 and 15c were investigated in the rat.
The compounds were administered in either single intravenous
(iv) injections of 5 mg/kg or in an oral dose of 15 mg/kg. The
main pharmacokinetic profiles of 11 and 15c are displayed in
Table 4, and the kinetic profiles of distribution in plasma of 11
and 15c after intravenous (iv) and oral (po) administration are
shown in Figure S5 in the Supporting Information.
The molecular docking results revealed why compounds 11,
13b, and 15c were the most active agents. When compound 11
was docked, it exhibited four hydrogen bonds between its OH
(eight-membered ring), NH2 (triazole ring) and CO (ester)
groups with G-2484, G-2565, and G-2044, with an RMSD of
0.99 Å (see Figure 3A and Table 3). When compound 13b was
docked, it exhibited two hydrogen bonds between its OH
(eight-membered ring) and NH2 (benzene ring) groups with
the phosphates of G-2484 and C-2420 with distances of 2.2 and
1.6 Å, respectively. In addition, a π−π interaction was formed
between the benzene ring and the purine of A-2045 with a
distance of 3.9 Å. Although 13c showed the same hydrogen
bonds and π−π interactions as did 13b, it exhibited a higher
binding free energy than that of 13b, possibly because of the
greater distances of the hydrogen bond between the NH2
(benzene ring) and C-2420 and of the π−π interaction between
the benzene ring and the purine of A-2045 (see Figure 3C and
Table 3). Compounds 15a−d adopted very similar conforma-
tions, and their entire molecules were superimposed well.
Compound 15c formed three hydrogen bonds between its OH
(eight-membered ring) and CO (five-membered ring)
groups with G-2484, C-2431 and U-2485, with distances of
2.2, 2.7, and 3.2 Å, respectively. To our surprise, compound
15d showed a better docking model than did 15c, with three
hydrogen bonds and a cation-π interaction formed, but
displayed a lower binding affinity, perhaps because of the
longer distance of the hydrogen bond between its CO (ester
and amido bond) group and C-2420 and C-2565 (see Figure
3D and Table 3). Compounds 12d and 12f were not the most
active agents, but they showed higher antibacterial activities
than did their analogue, 12e. Molecular docking studies
revealed that 12f formed three hydrogen bonds (OH, CO,
and OCH3) and a π−π interaction. Compound 12e displayed a
docking model similar to that of 12f, except with the absence of
one hydrogen bond formed between CO (ester) and G-2044
(see Figure 3B and Table 3). The superior docking modes of
compounds 12d, 12f, 13b, and 13c suggested that the
substituents with hydrogen donators or with groups with
lower Hammett constants, which enhance π−π stacking
interactions,37 should be introduced to produce new analogues
with higher antibacterial activities as a potential design strategy.
Based on the docking results, the predicted binding affinities
(binding free energies) were in agreement with the biological
results, revealing that there is a direct and rational correlation
between the binding free energy and the antibacterial activity.
Because X-ray structures of the 50S ribosomal subunits are
available only for E. coli (PDB ID: 2AW4), we investigated the
linear relationships between the binding free energy (ΔGb,
kcal/mol) and the zone of inhibition (mm) for 320 and 160
μg/mL concentrations of E. coli on the basis of the high
similarity in domain V of 23S rRNA at the PTC between 1XBP
and 2AW4 (the superimposed results of the key residues of
1XBP and 2AW4 are shown in Figure S3 in the Supporting
Table 4. Pharmacokinetic Properties of 11 and 15c in Rats
11
15c
parameter
iv
po
iv
po
a
Cmax (μg/mL)
13.57
0.08
3.07
11.26
0.08
4.65
b
Tmax (h)
0.75
0.75
c
t1/2 (h)
3.40
6.07
3.89
5.98
d
Cl (mL/h·kg)
324.57
0.92
611.01
5.14
353.81
1.02
621.41
4.91
e
Vss (L/kg)
f
MRT (d)
2.84
8.42
2.88
7.91
g
AUC0→∞ (μg·h/mL)
15.41
24.55
35.81
14.13
24.14
39.73
h
F
(%)
a
b
c
d
Maximum concentration. Time to reach Cmax. Half-life. Clearance.
e
f
g
Volume of distribution at steady state. Mean resident time. Area
h
under the curve. Oral bioavailability.
After a single iv administration of 11 and 15c (5 mg/kg), the
mean values of t1/2 were 3.40 and 3.89 h, reflecting a high rate
of elimination. The similar basic nature and lipophilicity of 11
and 15c produced similar distribution volumes (0.92 and 1.02
L/kg) and AUC0→∞ values (15.41 and 14.13 μg·h/mL) after iv
administration. Following po administration, compounds 11
and 15c were absorbed rapidly, with Cmax values of 3.07 and
4.65 μg/mL, respectively, at 0.75 h (Tmax). Then, the
concentrations of the two compounds dropped rapidly, with
elimination half times (t1/2) of 6.07 and 5.89 h, respectively,
and moderate plasma clearance (CL) rates of 611.01 and
621.41 mL/h·kg, respectively.
Compounds 11 and 15c had higher Cmax values following
oral dosing than did valnemulin in ducks (0.12 μg/mL) and
chicken (0.66 μg/mL). Furthermore, the plasma t1/2 values for
11 and 15c after po dosing were longer than that of valnemulin
in ducks (4.83 h) but shorter than that of valnemulin in
chickens (11.10 h).38,39 Based on the MICs of compounds 11
and 15c against MRSA and S. agalactiae, the drug plasma
concentrations were all above the MIC for a minimum of 6 h
following iv and po administration. In clinical practice, a dose of
15 mg/kg body weight is suggested every 6 h following oral
administration. The systemic bioavailabilities of 11 and 15c
were 35.81% and 39.73% following oral administration in rats,
respectively. Our results are similar to those for valnemulin in
ducks (36.68%) but far lower than those for valnemulin in
chickens (74%) and in Sprague−Dawley (SD) rats (100%).38,39
One of the reasons was that the two compounds were
administered as the basic form, whereas valnemulin was
administered as the hydrochloride with a higher solubility in
water. These relatively low bioavailabilities indicate that it is
necessary to develop proper preparations of compounds 11and
H
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15c to increase their bioavailability if they are to serve as new
drugs.
1.46−1.50 (m, 5H), 1.63−1.65 (dd, 2H,J1 = 10 Hz,J2 = 7.2 Hz),
2.01−2.08(m, 3H), 2.21−2.29 (m, 3H), 2.45(s, 3H), 3.34 (d,
1H, J = 6.4 Hz), 4.48 (s, 2H), 5.17−5.21 (d, 1H, J = 8.8 Hz),
5.31−5.34 (d, 1H, J = 6.4 Hz), 5.75−5.78 (d, 1H, J = 4.2 Hz),
6.43(q, 1H, J = 17.2 Hz, 10.8 Hz); 7.35−7.37 (d, 2H, J = 4.0
Hz), 7.80−7.82 (d, 2H, J = 4.0 Hz). 13C NMR (100 MHz,
CDCl3) δ: 216.7, 164.8, 145.2, 138.6, 132.5, 129.9, 127.9, 117.2,
74.4, 70.2, 64.9, 57.9, 45.3, 44.4, 43.9, 41.7, 36.4, 35.9, 34.3,
30.2, 26.7, 26.3, 24.7, 21.6, 16.4, 14.6, 11.4. HRMS (ESI): [M +
Na]+ calcd for C29H40O7S, 533.2501; found, 533.2507.
CONCLUSION
■
New series of novel pleuromutilin derivatives containing
thiadiazole moieties were synthesized by acylation under very
mild conditions. These new derivatives were investigated for
their antibacterial activity in vitro against MRSA, MRSE, S.
agalactiae, and E. coli. The obtained MIC values and
antibacterial activities revealed that all the synthesized
compounds showed potent activity properties. Compounds
11, 13b, and 15c were the most active antibacterial agents
against MRSA, MRSE and S. agalactiae. Docking studies were
performed on some compounds (11, 12d−f, 13a−c, and 15a−
d) with similar substituents but differences in antibacterial
activity. The results showed that the binding free energies
(ΔGb) were in the range of −8.40 to −11.46 kcal/mol, with an
RMSD range of 0.75 to 1.12 Å. Furthermore, three interactions
were found to occur: hydrogen bonding, π−π interactions, and
cation−π interactions. These results reveal that there is a direct
and rational correlation between the binding free energy and
the antibacterial activity. Compounds 11 and 15c were further
profiled in a rat model for pharmacokinetic studies. The results
indicated that 11 and 15c may serve as possible lead
compounds for the development of antibacterial drugs.
14-O-(Iodoacetyl)-mutilin (10). Compound 10 was pre-
pared on the basis of a published method from a solution of
compound 9 and KI, which was refluxed for 1 h.30,31 The crude
production was purified by recrystallization with acetone to give
81% compound 10 as a pale yellow powder; mp: 117−119 °C.
IR (KBr): 3307, 2979, 2933, 1736, 1712, 1456, 1417, 1279,
1
1084, 1023, 985, 967 cm−1. H NMR (400 MHz, CDCl3) δ:
7.26 (s, 1H), 6.41 (dd, J = 17.4, 11.0 Hz, 1H), 5.70 (d, J = 8.5
Hz, 1H), 5.34 (d, J = 11.0 Hz, 1H), 5.20 (dd, J = 17.4, 1.1 Hz,
1H), 3.65 (d, J = 10.4 Hz, 1H), 3.57 (d, J = 10.5 Hz, 1H), 3.35
(s, 1H), 2.36−2.28 (m, 1H), 2.26−2.14 (m, 2H), 2.07 (dd, J =
16.0, 8.5 Hz, 2H), 1.79−1.60 (m, 4H), 1.57−1.47 (m, 2H),
1.47−1.42 (m, 4H), 1.35 (dd, J = 26.4, 9.7 Hz, 2H), 1.17 (s,
3H), 1.12 (d, J = 4.3 Hz, 1H), 0.87 (d, J = 7.0 Hz, 3H), 0.73 (d,
J = 6.9 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ: 216.76,
166.88, 138.57, 117.28, 74.71, 70.49, 58.20, 45.48, 44.20, 43.93,
41.98, 36.71, 35.86, 34.31, 30.10, 26.78, 26.42, 24.80, 16.75,
14.78, 11.46. HRMS (ESI): [M + NH4]+ calcd for C22H33IO4,
506.1762; found, 506.1758.
EXPERIMENTAL SECTION
■
Synthesis. The reagents and solvents were obtained
commerically and used without further purification. The
melting points were recorded on a Tianda Tianfa YRT-3
apparatus (China) with open capillary tubes and were
uncorrected. Infrared (IR) spectra were obtained on a Thermo
Nicolet NEXUS-670 spectrometer. NMR spectra were
recorded using Bruker 400 MHz spectrometers in CDCl3or
DMSO-d6. The chemical shifts (δ) were reported in parts per
million (ppm) relative to tetramethylsilane. 13C NMR spectra
were recorded on 100 MHz spectrometers. All reactions were
monitored by TLC (silica gel GF254) using elution (petroleum
ether-ethyl acetate) followed by visualization after spraying with
a 0.05% KMnO4 aqueous solution or under UV illumination
directly. The final compounds was determined to be ≥95%
pure by HPLC analysis using a Waters 2695 series instrument
equipped with a Waters 2998 DAD detector and a Hypersil
ODS column (4.6 mm × 250 mm, 5 μm) at a mobile phase
flow rate of 1 mL/min. Peaks were detected at 254 nm and the
system was operated at 25 °C. The mobile phase was a mixture
of acetonitrile (90%) and water (10%) containing 0.1%
disodium hydrogen phosphate.
14-O-[(2-Amino-1,3,4-thiadiazole-5-yl)-thioacetyl]-mutilin
(11). A solution of compound 10 (2.45 g, 5 mmol) in 60 mL of
tetrahydrofuran was added dropwise to a mixture of 2-amino-5-
mercapto-1,3,4-thiadiazole (0.73 g, 5.5 mmol) in 5 mL of 10%
NaOH. It was stirred at 0 °C for 2 h, then evaporated in
vacuum, and ethyl acetate (100 mL) was added to the residue.
The obtained mixture was quenched with saturated aqueous
NH4Cl and washed with water, followed by separation of the
organic layer and drying under anhydrous Na2SO4 overnight.
After evaporating most of the ethyl acetate under vacuum, the
residue was purified by recrystallization or silica gel column
chromatography (petroleum ether/ethyl acetate 1:2 v/v) to
afford compound 11 as a white solid in 82% yield (2.3 g); mp:
74−75 °C. IR (KBr): 3419, 3330, 2931, 1731, 1616, 1507,
1456, 1417, 1373, 1282, 1190, 1152, 1117, 1019, 980, 953,
1
938,916 cm−1. H NMR (400 MHz, DMSO) δ: 7.28 (s, 2H),
6.08 (dd, J = 17.8, 11.2 Hz, 1H), 5.52 (d, J = 8.1 Hz, 1H), 5.03
(dd, J = 21.0, 14.7 Hz, 2H), 4.51 (d, J = 5.9 Hz, 1H), 4.02 (q, J
= 7.1 Hz, 1H), 3.90 (q, J = 16.0 Hz, 2H), 3.45−3.32 (m, 2H),
2.39 (s, 1H), 2.19 (dd, J = 18.8, 10.8 Hz, 1H), 2.11−1.97 (m,
4H), 1.64 (dd, J = 18.4, 9.5 Hz, 2H), 1.54−1.42 (m, 1H), 1.36−
1.21 (m, 6H), 1.16 (d, J = 7.1 Hz, 1H), 1.08−0.94 (m, 4H),
0.82 (d, J = 6.7 Hz, 3H), 0.59 (d, J = 6.7 Hz, 3H). 13C NMR
(100 MHz, DMSO) δ: 217.08, 169.73, 166.70, 148.89, 140.68,
115.39, 72.63, 70.27, 59.75, 57.23, 44.95, 44.14, 41.51, 36.33,
34.00, 30.10, 28.70, 26.60, 24.47, 20.75, 16.09, 14.51, 14.08,
11.54. HRMS (ESI): [M + H]+ calcd for C24H35N3O4S2,
494.2142; found, 494.2139.
General Procedure for the Synthesis of Compounds 12a−
f. A mixture of benzoic acid (3.6 mmol), compound 11 (1.48 g,
3 mmol), EDCI (0.69 g, 3.6 mmol), HOBt (0.51 g, 3.6 mmol),
and 60 mL of dichloromethane was stirred at room temperature
for 36−48 h. The mixture was washed with saturated aqueous
NaHCO3 and water, dried with anhydrous Na2SO4 overnight
14-O-(p-Toluene-sulfonyloxyacetyl)-mutilin (9). A 10 M
NaOH (5 mL, 50 mmol) was added dropwise to a mixture of
pleuromutilin (7.57 g, 20 mmol) and p-toluenesulfonyl chloride
(4.2 g, 22 mmol) in water (5 mL) and t-butyl methyl ether (20
mL). The result mixture was stirred under reflux for 1 h
followed by dilution with water (50 mL) and stir under an ice
bath for 15 min. The result suspension was washed with water
(50 mL) and cold t-butyl methyl ether (20 mL) 3 times. The
crude production was filtered to give 93% (9.8 g) compound 9
as a white powder; mp: 147−148 °C. IR (KBr): 3446, 2924,
2863, 1732, 1633, 1597, 1456, 1371, 1297, 1233, 1117, 1035,
1
832, 664, 560 cm−1. H NMR (400 MHz, CDCl3) δ: 0.63 (d,
3H, J = 6.8 Hz), 0.87 (d, 3H, J = 6.8 Hz), 1.11−1.15 (m, 1H),
1.22−1.26 (s, 5H), 1.33−1.36 (m, 1H), 1.41−1.44 (m, 1H),
I
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and rotary evaporated to dryness. The residue was purified by
silica gel column chromatography (petroleum ether/ethyl
acetate 1:1 v/v) to afford the desired compounds 12a−12f.
14-O-[(2-Chlorobenzamide-1,3,4-thiadiazole-5-yl)-thioa-
cetyl]-mutilin (12a). Compound 12a was prepared according
to the general procedure from 14-O-[(2-amino-1,3,4-thiadia-
zole-5-yl)-thioacetyl]-mutilin (11) and o-chlorobenzoic acid.
The crude product was purified by silica gel column
chromatography in 75% yield (1.42 g) as a white solid; mp:
74−76 °C. IR (KBr): 3446, 2932, 1731, 1683, 1539, 1456,
1374, 1305, 1248, 1189, 1152, 1117, 1046, 1017, 980, 938, 916,
895, 746 cm−1. 1H NMR (400 MHz, CDCl3) δ: = 7.79 (s, 1H),
7.57−7.34 (m, 3H), 6.38 (dd, J = 17.4, 11.0, 1H), 5.73 (d, J =
8.4, 1H), 5.27 (d, J = 11.0, 1H), 5.13 (d, J = 17.4, 1H), 3.94−
3.77 (m, 2H), 3.32 (d, J = 6.1, 1H), 2.32−2.09 (m, 3H), 2.09−
1.96 (m, 3H), 1.78−1.68 (m, 1H), 1.65−1.47 (m, 3H), 1.45−
1.29 (m, 5H), 1.23 (dd, J = 12.2, 5.1, 2H), 1.10 (d, J = 8.5, 4H),
0.84 (d, J = 7.0, 3H), 0.67 (d, J = 6.9, 3H). 13C NMR (100
MHz, CDCl3) δ: 216.79, 166.40, 164.40, 160.13, 158.44,
138.71, 130.84, 130.47, 127.08, 117.20, 74.48, 70.36, 60.28,
57.98, 45.33, 44.62, 43.84, 41.77, 36.57, 35.85, 34.33, 30.28,
26.73, 26.35, 24.73, 16.64, 14.75, 14.10, 11.37. HRMS (ESI):
[M + Na]+ calcd for C31H38ClN3O5S2, 654.1834; found,
654.1828.
158.79, 139.91, 138.71, 130.18, 129.18, 128.85, 117.26, 74.53,
70.44, 58.03, 45.38, 44.59, 43.85, 41.80, 36.62, 35.87, 34.39,
30.33, 26.75, 26.28, 24.77, 16.69, 14.78, 11.44. HRMS (ESI):
[M + Na]+ calcd for C31H38ClN3O5S2, 654.1834; found,
654.1829.
14-O-[(2-Methoxybenzamide-1,3,4-thiadiazole-5-yl)-thio-
acetyl]-mutilin (12d). Compound 12d was prepared according
to the general procedure from 14-O-[(2-amino-1,3,4-thiadia-
zole-5-yl)-thioacetyl]-mutilin (11) and o-methoxybenzoic acid.
The crude product was purified by silica gel column
chromatography in 68% yield (1.28 g) as a white solid; mp:
89−92 °C. IR (KBr): 3304, 2934, 1732, 1662, 1601, 1527,
1487, 1466, 1398, 1373, 1288, 1242, 1184, 1163, 1118, 1044,
1017, 980, 938, 916, 895, 756, 666 cm−1. 1H NMR (400 MHz,
CDCl3) δ: = 11.18 (s, 1H), 8.20 (dd, J = 7.9, 1.7, 1H), 7.58−
7.53 (m, 1H), 7.11 (t, J = 7.6, 1H), 7.05 (d, J = 8.4, 1H), 6.38
(dd, J = 17.4, 11.0, 1H), 5.72 (d, J = 8.4, 1H), 5.29−5.23 (m,
1H), 5.14 (dd, J = 17.4, 1.2, 1H), 4.12−4.00 (m, 6H), 3.97 (t, J
= 10.2, 2H), 3.32 (d, J = 6.0, 1H), 2.29−2.24 (m, 1H), 2.16
(dd, J = 11.4, 6.3, 2H), 2.07−1.97 (m, 5H), 1.71 (dd, J = 14.4,
2.4, 2H), 1.64−1.53 (m, 4H), 1.52−1.45 (m, 1H), 1.43−1.27
(m, 9H), 1.21 (t, J = 7.1, 2H), 1.14−1.06 (m, 5H), 0.82 (s,
3H), 0.70 (d, J = 6.9, 3H). 13C NMR (100 MHz, CDCl3) δ:
216.91, 166.77, 162.45, 159.06, 158.51, 157.98, 138.83, 135.12,
132.74, 121.81, 118.16, 117.20, 111.76, 74.55, 70.38, 60.34,
58.08, 56.44, 45.40, 44.59, 43.96, 41.83, 36.69, 35.99, 34.42,
30.37, 26.82, 26.45, 24.80, 16.74, 14.79, 11.46. HRMS (ESI):
[M + Na]+ calcd for C32H41N3O6S2, 650.2329; found,
650.2322.
14-O-[(3-Chlorobenzamide-1,3,4-thiadiazole-5-yl)-thioa-
cetyl]-mutilin (12b). Compound 12b was prepared according
to the general procedure from 14-O-[(2-amino-1,3,4-thiadia-
zole-5-yl)-thioacetyl]-mutilin (11) and m-chlorobenzoic acid.
The crude product was purified by silica gel column
chromatography in 62% yield (1.18 g) as a white solid; mp:
115−118 °C. IR (KBr): 3427, 2928, 1731, 1673, 1538, 1454,
1411, 1309, 1250, 1188, 1152, 1116, 1019, 980, 914, 805, 733
14-O-[(3-Methoxybenzamide-1,3,4-thiadiazole-5-yl)-thio-
acetyl]-mutilin (12e). Compound 12e was prepared according
to the general procedure from 14-O-[(2-amino-1,3,4-thiadia-
zole-5-yl)-thioacetyl]-mutilin (11) and m-methoxybenzoic acid.
The crude product was purified by silica gel column
chromatography in 62% yield (1.17 g) as a white solid; mp:
142−145 °C. IR (KBr): 32894, 2934, 1728, 1678, 1585, 1557,
1489, 1456, 1407, 1310, 1273, 1226, 1186, 1153, 1114, 1042,
1009, 979, 922, 736, 675 cm−1. 1H NMR (400 MHz, CDCl3) δ:
7.81 (d, J = 8.1 Hz, 1H), 7.65−7.62 (m, 1H), 7.44 (t, J = 8.0
Hz, 1H), 7.15 (dd, J = 8.1, 2.2 Hz, 1H), 6.35 (dd, J = 17.4, 11.0
Hz, 1H), 5.72 (d, J = 8.4 Hz, 1H), 5.22 (dd, J = 11.0, 1.1 Hz,
1H), 5.08 (dd, J = 17.4, 1.2 Hz, 1H), 4.00 (dd, J = 36.4, 16.1
Hz, 2H), 3.84 (s, 3H), 3.35−3.27 (m, 1H), 2.29−2.10 (m, 3H),
2.03 (d, J = 5.7 Hz, 1H), 1.96 (dd, J = 16.0, 8.5 Hz, 1H), 1.72
(dd, J = 14.4, 2.2 Hz, 1H), 1.63−1.47 (m, 3H), 1.41−1.30 (m,
4H), 1.25−1.19 (m, 1H), 1.06 (s, 4H), 0.83 (d, J = 7.0 Hz,
3H), 0.65 (d, J = 7.0 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ:
216.39, 166.05, 164.73, 160.88, 159.22, 157.88, 138.12, 131.64,
129.29, 120.30, 119.08, 116.68, 112.92, 73.98, 69.78, 57.47,
54.89, 44.82, 43.99, 43.28, 41.22, 36.06, 35.40, 35.11, 33.84,
29.77, 26.19, 25.75, 24.22, 16.11, 14.20, 10.90. HRMS (ESI):
[M + K]+ calcd for C32H41N3O6S2, 666.2068; found, 666.2061.
14-O-[(4-Methoxybenzamide-1,3,4-thiadiazole-5-yl)-thio-
acetyl]-mutilin (12f). Compound 12f was prepared according
to the general procedure from 14-O-[(2-amino-1,3,4-thiadia-
zole-5-yl)-thioacetyl]-mutilin (11) and p-methoxybenzoic acid.
The crude product was purified by silica gel column
chromatography in 66% yield (1.24 g) as a white solid; mp:
131−133 °C. IR (KBr): 3426, 2933, 1731, 1666, 1606, 1537,
1513, 1456, 1416, 1374, 1299, 1256, 1176, 1117, 1027, 980,
1
cm−1. H NMR (400 MHz, CDCl3) δ: 8.13 (d, J = 6.5 Hz,
2H), 7.60 (d, J = 6.6 Hz, 1H), 7.51 (dd, J = 9.9, 5.3 Hz, 1H),
6.40−6.29 (m, 1H), 5.72 (d, J = 5.3 Hz, 1H), 5.27−5.19 (m,
1H), 5.09 (dd, J = 17.4, 1.8 Hz, 1H), 4.05−3.92 (m, 2H), 3.31
(s, 1H), 2.30−2.11 (m, 3H), 2.04 (s, 2H), 1.97 (dd, J = 14.6,
6.9 Hz, 1H), 1.73 (d, J = 14.3 Hz, 1H), 1.61 (d, J = 10.1 Hz,
2H), 1.46 (dd, J = 16.4, 13.0 Hz, 2H), 1.34 (t, J = 13.3 Hz, 4H),
1.28−1.16 (m, 3H), 1.09 (t, J = 10.4 Hz, 4H), 0.84 (d, J = 4.8
Hz, 3H), 0.65 (d, J = 3.2 Hz, 3H). 13C NMR (100 MHz,
CDCl3) δ: 166.20, 163.91, 161.20, 158.54, 138.37, 133.00,
132.35, 129.84, 128.48, 126.62, 116.93, 74.21, 70.06, 60.04,
57.71, 45.06, 44.25, 43.53, 41.47, 36.29, 35.58, 34.07, 30.00,
26.43, 25.99, 24.46, 20.71, 16.35, 14.44, 13.85, 11.12. HRMS
(ESI): [M + Na]+ calcd for C31H38ClN3O5S2, 654.1834; found,
654.1827.
14-O-[(4-Chlorobenzamide-1,3,4-thiadiazole-5-yl)-thioa-
cetyl]-mutilin (12c). Compound 12c was prepared according to
the general procedure from 14-O-[(2-amino-1,3,4-thiadiazole-
5-yl)-thioacetyl]-mutilin (11) and p-chlorobenzoic acid. The
crude product was purified by silica gel column chromatog-
raphy in 64% yield (1.21 g) as a white solid; mp: 101−103 °C.
IR (KBr): 3420, 2927, 1731, 1670, 1594, 1539, 1493, 1456,
1
1409, 1310, 1152, 1116, 1014, 980, 891, 846, 749 cm−1. H
NMR (400 MHz, CDCl3) δ: 8.19 (d, J = 8.6 Hz, 2H), 7.54 (d,
J = 8.3 Hz, 2H), 6.38 (dd, J = 17.4, 11.0 Hz, 1H), 5.74 (d, J =
8.4 Hz, 1H), 5.23 (d, J = 10.9 Hz, 1H), 5.10 (d, J = 17.3 Hz,
1H), 3.98 (s, 2H), 3.31 (d, J = 6.4 Hz, 1H), 2.29−2.15 (m,
3H), 2.05−1.94 (m, 2H), 1.73 (d, J = 14.3 Hz, 1H), 1.61 (d, J =
10.4 Hz, 2H), 1.50−1.37 (m, 5H), 1.33−1.20 (m, 3H), 1.08 (s,
4H), 0.84 (d, J = 6.9 Hz, 4H), 0.68 (d, J = 6.9 Hz, 3H). 13C
NMR (100 MHz, CDCl3) δ: 216.88, 166.48, 164.44, 161.65,
1
938, 892, 845, 759, 691, 649, 609 cm−1. H NMR (400 MHz,
CDCl3) δ: 8.20 (d, J = 8.8 Hz, 2H), 7.02 (d, J = 8.4 Hz, 2H),
6.35 (dd, J = 17.3, 11.0 Hz, 1H), 5.72 (d, J = 8.4 Hz, 1H), 5.21
J
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Journal of Medicinal Chemistry
Article
(d, J = 11.0 Hz, 1H), 5.08 (d, J = 17.4 Hz, 1H), 4.11 (q, J = 7.1
Hz, 1H), 3.98 (t, J = 9.3 Hz, 2H), 3.87 (d, J = 18.7 Hz, 3H),
3.30 (dd, J = 10.1, 6.6 Hz, 1H), 2.29−2.11 (m, 3H), 2.03 (s,
2H), 1.95 (dd, J = 16.0, 8.5 Hz, 1H), 1.72 (d, J = 14.2 Hz, 1H),
1.60 (dd, J = 21.1, 11.3 Hz, 2H), 1.54−1.40 (m, 3H), 1.37−
1.29 (m, 4H), 1.24 (t, J = 7.1 Hz, 2H), 1.15−1.02 (m, 4H),
0.84 (d, J = 6.9 Hz, 3H), 0.66 (d, J = 6.9 Hz, 3H). 13C NMR
(100 MHz, CDCl3) δ: 216.99, 166.64, 164.82, 163.89, 158.25,
138.77, 130.95, 123.07, 117.34, 114.19, 74.62, 70.47, 60.46,
58.12, 55.64, 45.47, 44.62, 43.93, 41.89, 36.72, 36.05, 34.49,
30.43, 26.85, 26.37, 24.87, 21.12, 16.75, 14.82, 14.27, 11.54.
HRMS (ESI): [M + K]+ calcd for C32H41N3O6S2, 666.2068;
found, 666.2059.
General Procedure for the Synthesis of Compounds 13a−
c. A 1 N NaOH solution (6 mL) was added to a suspension of
amino acid derivative (5 mmol) in tetrahydrofuran (50 mL)
and water (20 mL), followed by the addition of 1.1 g di-tert-
butyl dicarbonate (5 mmol) dropwise at room temperature.
The tetrahydrofuran was evaporated under reduced pressure
after stirring for 4 h, then ethyl acetate (50 mL) and 5% citric
acid (30 mL) were added to the result residue. The organic
layer was separated, washed with water (20 mL), dried over
Na2SO4, and rotary evaporated to dryness. The crude residue
was used in the next reaction without purification.
A mixture of the above N-Boc protected amino acids (3.6
mmol), 1.48 g compound 11 (3 mmol), 0.69 g EDCI (3.6
mmol), 0.51 g HOBt (3.6 mmol), and 60 mL of dichloro-
methane was stirred at room temperature for 36−48 h. The
mixture was washed with saturated aqueous NaHCO3 and
water and then evaporated under reduced pressure. The residue
was treated with a mixture of TFA and dichloromethane (1:1;
20 mL) at room temperature for 30 min. The reaction mixture
was quenched with 25% aqueous NaHCO3 (50 mL), washed
with water, dried with anhydrous Na2SO4 overnight, and rotary
evaporated to dryness. The residue was purified by silica gel
column chromatography (petroleum ether/ethyl acetate 1:2 v/
v) to give the desired compounds.
14-O-[(2-Aminobenzamide-1,3,4-thiadiazole-5-yl)-thioa-
cetyl]-mutilin (13a). Compound 13a was prepared according
to the general procedure from 14-O-[(2-amino-1,3,4-thiadia-
zole-5-yl)-thioacetyl]-mutilin (11) and o-aminobenzoic acid.
The crude product was purified by silica gel column
chromatography in 61% yield (1.12 g) as a white solid; mp:
146−149 °C. IR (KBr): 3473, 3367, 2931, 1731, 1651, 1617,
1557, 1525, 1455, 1385, 1297, 1241, 1161, 1117, 1017, 980,
937, 896, 751, 696, 670 cm−1. 1H NMR (400 MHz, CDCl3) δ:
7.91 (d, J = 8.2 Hz, 1H), 7.29 (dd, J = 15.0, 7.4 Hz, 1H), 6.72
(t, J = 6.9 Hz, 2H), 6.38 (dd, J = 17.4, 11.0 Hz, 1H), 5.74 (d, J
= 8.4 Hz, 1H), 5.26 (d, J = 11.0 Hz, 1H), 5.12 (d, J = 17.4 Hz,
1H), 3.99 (q, J = 16.1 Hz, 2H), 3.33 (d, J = 5.9 Hz, 1H), 2.35−
2.10 (m, 4H), 2.09−1.91 (m, 3H), 1.73 (d, J = 15.4 Hz, 1H),
1.57 (ddd, J = 27.7, 23.3, 12.8 Hz, 3H), 1.48−1.28 (m, 6H),
1.25 (t, J = 7.1 Hz, 1H), 1.19−0.99 (m, 4H), 0.95 (t, J = 7.4 Hz,
1H), 0.85 (d, J = 6.9 Hz, 3H), 0.69 (d, J = 6.9 Hz, 3H). 13C
NMR (100 MHz, CDCl3) δ: 217.14, 166.84, 166.65, 161.15,
158.06, 150.56, 138.82, 134.51, 129.17, 117.50, 116.83, 111.52,
74.71, 70.48, 58.21, 45.53, 44.69, 44.01, 41.95, 36.81, 36.09,
34.56, 30.49, 26.91, 26.45, 24.93, 16.84, 14.94, 11.60. HRMS
(ESI): [M + H]+ calcd for C31H40N4O5S2, 613.2513; found,
613.2509.
zole-5-yl)-thioacetyl]-mutilin (11) and m-aminobenzoic acid.
The crude product was purified by silica gel column
chromatography in 61% yield (1.12 g) as a white solid; mp:
135−138 °C. IR (KBr): 3432, 3370, 2927, 1729, 1667, 1626,
1587, 1531, 1456, 1410, 1374, 1305, 1189, 1152, 1117, 1049,
1
1017, 980, 939, 847, 743, 677 cm−1. H NMR (400 MHz,
CDCl3) δ: 7.59 (d, J = 1.4 Hz, 1H), 7.49 (d, J = 7.4 Hz, 1H),
7.31−7.25 (m, 1H), 6.89 (d, J = 7.9 Hz, 1H), 6.41−6.31 (m,
1H), 5.73 (d, J = 8.0 Hz, 1H), 5.25 (d, J = 10.9 Hz, 1H), 5.10
(d, J = 17.4 Hz, 1H), 4.11 (q, J = 7.1 Hz, 1H), 3.93 (dd, J =
20.6, 4.6 Hz, 2H), 3.31 (s, 1H), 2.32−2.10 (m, 3H), 2.01 (dt, J
= 26.7, 8.0 Hz, 3H), 1.72 (d, J = 14.4 Hz, 1H), 1.60 (dd, J =
20.2, 10.1 Hz, 2H), 1.52−1.29 (m, 6H), 1.28−1.18 (m, 3H),
1.10 (d, J = 14.2 Hz, 4H), 0.84 (d, J = 6.7 Hz, 3H), 0.66 (d, J =
6.6 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ: 216.90, 166.70,
165.57, 162.01, 158.11, 147.14, 138.68, 131.50, 129.71, 119.66,
118.47, 117.28, 114.54, 74.51, 70.41, 60.34, 58.01, 45.35, 44.58,
43.84, 41.76, 36.56, 35.94, 34.37, 30.31, 26.74 26.26, 24.75,
21.00, 16.68, 14.75, 14.15, 11.44. HRMS (ESI): [M + H]+ calcd
for C31H40N4O5S2, 613.2513; found, 613.2510.
14-O-[(4-Aminobenzamide-1,3,4-thiadiazole-5-yl)-thioa-
cetyl]-mutilin (13c). Compound 13c was prepared according to
the general procedure from 14-O-[(2-amino-1,3,4-thiadiazole-
5-yl)-thioacetyl]-mutilin (11) and p-aminobenzoic acid. The
crude product was purified by silica gel column chromatog-
raphy in 58% yield (1.07 g) as a white solid; mp: 151−153 °C.
IR (KBr): 3441, 3371, 2930, 1728, 1627, 1603, 1568, 1518,
1455, 1374, 1296, 1261, 1183, 1117, 1049, 980, 938, 890, 842,
760, 693, 609 cm−1. 1H NMR (400 MHz, CDCl3) δ: 8.06−7.96
(m, 2H), 6.69 (d, J = 8.6 Hz, 2H), 6.35 (dd, J = 17.4, 11.0 Hz,
1H), 5.71 (d, J = 8.1 Hz, 1H), 5.15 (dd, J = 49.9, 14.1 Hz, 2H),
4.35−4.23 (m, 2H), 4.01 (t, J = 9.9 Hz, 2H), 3.35−3.26 (m,
1H), 2.20 (ddd, J = 23.9, 11.0, 4.8 Hz, 3H), 2.07−1.81 (m,
3H), 1.72 (d, J = 14.2 Hz, 1H), 1.67−1.53 (m, 3H), 1.49−1.30
(m, 5H), 1.28−1.18 (m, 2H), 1.06 (s, 4H), 0.83 (d, J = 6.7 Hz,
3H), 0.67 (d, J = 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ:
216.92, 166.790, 164.770, 161.78, 157.81, 151.60, 138.73,
130.82, 119.69, 117.28, 114.06, 74.55, 70.48, 60.41, 58.04,
45.40, 44.52, 43.89, 41.83, 36.65, 35.91, 34.43, 30.34, 26.56,
24.79, 16.73, 14.78, 11.47. HRMS (ESI): [M + H]+ calcd for
C31H40N4O5S2, 613.2513; found, 613.2507.
14-O-[(2-Chloroacetamido-1,3,4-thiadiazole-5-yl)-thioa-
cetyl]-mutilin (14). To a solution of 1.48 g of compound 11 (3
mmol) and 0.61 g of N-methylmorpholine (6 mmol) in dry
dichloromethane (20 mL), 0.51 g of ClCH2COCl (4.5 mmol)
in dry dichloromethane (5 mL) was slowly dropped at 0 °C.
After stirring for 1 h, the solution was washed with water three
times, and then the organic layer was dried with MgSO4,
filtered, concentrated, and purified by column chromatography
(petroleum ether/ethyl acetate = 1:1) to yield 14 as a white
solid in 86% yield (1.47 g); mp: 82−84 °C. IR (KBr): 3446,
2936, 1731, 1652, 1558, 1540, 1456, 1405, 1286, 1189, 1153,
1117, 1056, 1017, 980, 668 cm−1. 1H NMR (400 MHz, CDCl3)
δ: 6.47−6.32 (m, 1H), 5.72 (s, 1H), 5.33−5.20 (m, 1H), 5.13
(d, J = 13.6 Hz, 1H), 4.38 (s, 2H), 4.14−4.03 (m, 1H), 3.98 (s,
2H), 3.33 (d, J = 5.6 Hz, 1H), 2.23 (d, J = 33.3 Hz, 3H), 2.03
(t, J = 11.2 Hz, 3H), 1.73 (d, J = 13.6 Hz, 1H), 1.62 (d, J = 7.5
Hz, 2H), 1.42 (t, J = 17.2 Hz, 6H), 1.10 (d, J = 4.0 Hz, 4H),
0.84 (d, J = 3.4 Hz, 3H), 0.69 (d, J = 6.8 Hz, 3H).13C NMR
(100 MHz, CDCl3) δ: 216.94, 171.07, 166.43, 164.90, 159.41,
138.80, 117.24, 74.47, 70.46, 60.43, 57.97, 45.46, 44.55, 43.87,
41.94, 41.79, 36.48, 35.89, 34.31, 30.28, 26.75, 26.34, 24.60,
14-O-[(3-Aminobenzamide-1,3,4-thiadiazole-5-yl)-thioa-
cetyl]-mutilin (13b). Compound 13b was prepared according
to the general procedure from 14-O-[(2-amino-1,3,4-thiadia-
K
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Journal of Medicinal Chemistry
Article
16.71, 14.74, 11.41. HRMS (ESI): [M + Na]+ calcd for
C26H36ClN3O5S2, 592.1678; found, 592.1669.
cm−1. 1H NMR (400 MHz, acetone) δ: 6.26 (dd, J = 17.7, 11.2
Hz, 1H), 5.72 (d, J = 8.4 Hz, 1H), 5.12 (dd, J = 32.7, 14.4 Hz,
2H), 4.16−4.03 (m, 2H), 3.60 (d, J = 5.9 Hz, 1H), 3.30 (s,
2H), 2.57 (s, 3H), 2.38 (s, 1H), 2.31−2.23 (m, 1H), 2.19−2.08
(m, 2H), 1.96 (s, 6H), 1.77 (d, J = 16.7 Hz, 1H), 1.69−1.51
(m, 6H), 1.50−1.35 (m, 7H), 1.31 (d, J = 15.8 Hz, 1H), 1.21−
1.09 (m, 4H), 0.94 (d, J = 7.0 Hz, 3H), 0.71 (d, J = 6.6 Hz,
3H). 13C NMR (100 MHz, acetone) δ: 217.24, 172.50, 170.26,
167.42, 159.22, 141.30, 116.40, 74.68, 71.60, 62.29, 58.48,
55.44, 46.31, 45.14, 42.85, 37.62, 36.81, 34.90, 28.67, 27.45,
27.83, 26.69, 25.61, 24.46, 20.62, 16.96, 15.25, 11.88. HRMS
(ESI): [M + H]+ calcd for C31H46N4O5S2, 619.2982; found,
619.2974.
General Procedure for the Synthesis of Compounds 15a−
f. The appropriate secondary amine (4.5 mmol) was added to a
solution of 1.76 g of compound 14 (3 mmol) and 0.61 g of
triethylamine (6 mmol) in tetrahydrofuran (60 mL) and stirred
at 45 °C for 2 h. Then, the solvent was removed under reduced
pressure, and ethyl acetate (50 mL) was added. The mixture
was quenched with saturated aqueous NH4Cl (30 mL). The
organic layer was separated, washed with water (20 mL) three
times, dried with anhydrous Na2SO4, and rotary evaporated to
dryness. The crude residue was purified by silica gel column
chromatography (ethyl acetate/ethanol 20:1 v/v) to afford the
desired compounds.
14-O-[(2-(Pyrrolidine-1-yl)-acetamido-1,3,4-thiadiazole-5-
yl)-thioacetyl]-mutilin (15d). Compound 15d was prepared
according to the general procedure from 14-O-[(2-chloroace-
tamido-1,3,4-thiadiazole-5-yl)-thioacetyl]-mutilin (14) and pi-
peridine. The crude product was purified by silica gel column
chromatography in 71% yield (1.29 g) as a white solid; mp:
82−84 °C. IR (KBr): 3418, 2937, 1732, 1519, 1456, 1402,
1374, 1289, 1167, 1149, 1117, 1013, 981, 954, 940, 918, 835,
671 cm−1. 1H NMR (400 MHz, acetone) δ: 6.27 (dd, J = 17.7,
11.2 Hz, 1H), 5.74 (d, J = 8.4 Hz, 1H), 5.22−5.05 (m, 2H),
4.11 (s, 1H), 4.06 (d, J = 7.1 Hz, 1H), 3.50 (d, J = 1.8 Hz, 2H),
2.74 (d, J = 6.5 Hz, 3H), 2.26 (dd, J = 12.7, 5.8 Hz, 1H), 2.11−
2.04 (m, 8H), 1.98 (s, 4H), 1.83 (d, J = 14.1 Hz, 4H), 1.74 (dd,
J = 19.5, 6.0 Hz, 1H), 1.63−1.53 (m, 1H), 1.44 (s, 4H), 1.32
(d, J = 14.0 Hz, 1H), 1.21 (t, J = 7.1 Hz, 3H), 1.14 (s, 3H), 0.95
(d, J = 7.1 Hz, 3H), 0.72 (d, J = 6.8 Hz, 3H). 13C NMR (100
MHz, CDCl3) δ: 216.89, 171.07, 169.17, 166.57, 158.51,
138.65, 117.12, 74.42, 70.26, 60.27, 58.00, 54.49, 45.28, 44.42,
43.80, 41.69, 36.54, 35.88, 34.31, 30.23, 26.67, 26.31, 24.66,
23.89, 20.94, 16.62, 14.65, 14.06, 11.37. HRMS (ESI): [M +
H]+ calcd for C30H44N4O5S2, 605.2826; found, 605.2818.
14-O-[(2-(Morpholine-4-yl)-acetamido-1,3,4-thiadiazole-
5-yl)-thioacetyl]-mutilin (15e). Compound 15e was prepared
according to the general procedure from 14-O-[(2-chloroace-
tamido-1,3,4-thiadiazole-5-yl)-thioacetyl]-mutilin (14) and
morpholine. The crude product was purified by silica gel
column chromatography in 73% yield (1.36 g) as a white solid;
mp: 69−71 °C. IR (KBr): 3438, 2927, 2892, 1731, 1519, 1454,
1403, 1295, 1190, 1152, 1116, 1015, 981, 954, 912, 897, 668
cm−1. 1H NMR (400 MHz, acetone) δ: 6.26 (dd, J = 17.7, 11.2
Hz, 1H), 5.72 (d, J = 8.4 Hz, 1H), 5.12 (ddd, J = 14.5, 12.5, 1.4
Hz, 2H), 4.12−4.03 (m, 2H), 3.74−3.67 (m, 4H), 3.59 (d, J =
6.0 Hz, 1H), 3.38 (s, 2H), 2.67−2.59 (m, 4H), 2.38 (s, 1H),
2.29−2.21 (m, 1H), 2.18−2.03 (m, 5H), 1.97 (s, 2H), 1.81−
1.64 (m, 2H), 1.56 (ddd, J = 16.2, 10.3, 4.1 Hz, 2H), 1.47−1.35
(m, 5H), 1.36−1.27 (m, 1H), 1.20 (t, J = 7.1 Hz, 1H), 1.17−
1.06 (m, 4H), 0.94 (d, J = 7.1 Hz, 3H), 0.71 (d, J = 6.7 Hz,
3H). 13C NMR (100 MHz, acetone) δ: 216.16, 168.81, 166.46,
158.32, 158.10, 140.33, 115.46, 73.65, 70.65, 66.36, 60.98,
57.52, 53.54, 45.34, 44.18, 41.89, 36.66, 35.84, 33.95, 30.22,
27.75, 26.86, 24.65, 19.97, 19.66, 16.01, 14.31, 10.93. HRMS
(ESI): [M + H]+ calcd for C30H44N4O6S2, 621.2775; found,
619.2770.
14-O-[(2-(bis(Methyl)-amino)-acetamido-1,3,4-thiadia-
zole-5-yl)-thioacetyl]-mutilin (15a). Compound 15a was
prepared according to the general procedure from 14-O-[(2-
chloroacetamido-1,3,4-thiadiazole-5-yl)-thioacetyl]-mutilin
(14) and dimethylamine. The crude product was purified by
silica gel column chromatography in 69% yield (1.20 g) as a
white solid; mp: 74−76 °C. IR (KBr): 3444, 2937, 1731, 1557,
1519, 1403, 1284, 1152, 1117, 1047, 1017, 980, 953, 916, 864,
665 cm−1. 1H NMR (400 MHz, acetone) δ: 6.36 (dd, J = 17.8,
11.2 Hz, 1H), 5.83 (d, J = 8.6 Hz, 1H), 5.22 (dd, J = 35.5, 14.5
Hz, 2H), 4.17 (dt, J = 14.2, 5.8 Hz, 4H), 3.70 (d, J = 5.4 Hz,
1H), 3.40 (s, 2H), 2.48 (s, 7H), 2.35 (dd, J = 18.0, 11.0 Hz,
2H), 2.25−2.10 (m, 14H), 2.06 (d, J = 1.4 Hz, 9H), 1.92−1.75
(m, 2H), 1.68 (dd, J = 34.3, 21.7 Hz, 2H), 1.56−1.34 (m, 6H),
1.30 (t, J = 7.1 Hz, 3H), 1.21 (d, J = 16.0 Hz, 4H), 1.04 (d, J =
7.0 Hz, 3H), 0.81 (d, J = 6.7 Hz, 3H). 13C NMR (100 MHz,
CDCl3) δ: 216.79, 175.22, 168.65, 166.36, 158.38, 138.44,
116.94, 74.23, 70.08, 61.50, 60.10, 57.74, 45.48, 45.09, 44.21,
43.60, 41.50, 36.34, 35.67, 34.12, 30.03, 26.47, 26.10, 24.46,
20.70, 16.43, 14.46, 13.87, 11.18. HRMS (ESI): [M + H]+ calcd
for C28H42N4O5S2, 579.2669; found, 579.2661.
14-O-[(2-(bis(Ethyl)-amino)-acetamido-1,3,4-thiadiazole-
5-yl)-thioacetyl]-mutilin (15b). Compound 15b was prepared
according to the general procedure from 14-O-[(2-chloroace-
tamido-1,3,4-thiadiazole-5-yl)-thioacetyl]-mutilin (14) and di-
ethylamine. The crude product was purified by silica gel column
chromatography in 64% yield (1.17 g) as a white solid; mp:
70−73 °C. IR (KBr): 3444, 2971, 2932, 1732, 1698, 1519,
1455, 1403, 1285, 1202, 1152, 1117, 1065, 1018, 980, 953, 938,
916, 724, 665 cm−1. 1H NMR (400 MHz, acetone) δ: 6.25 (t, J
= 14.0 Hz, 1H), 5.72 (s, 1H), 5.11 (dd, J = 32.6, 14.1 Hz, 2H),
4.18−4.02 (m, 2H), 3.59 (s, 1H), 3.38 (s, 2H), 3.11 (s, 1H),
2.69 (d, J = 5.3 Hz, 3H), 2.37 (s, 1H), 2.28−2.03 (m, 4H), 1.73
(dd, J = 27.5, 12.4 Hz, 2H), 1.63−1.49 (m, 2H), 1.39 (d, J =
20.0 Hz, 5H), 1.30 (d, J = 13.1 Hz, 1H), 1.11 (dd, J = 25.5, 14.0
Hz, 12H), 0.93 (s, 3H), 0.70 (s, 3H). 13C NMR (100 MHz,
acetone) δ: 217.09, 171.44, 167.44, 159.08, 141.33, 116.42,
74.70, 71.63, 58.52, 57.46, 49.35, 46.34, 45.18, 42.88, 37.65,
36.86, 34.94, 31.24, 30.56, 30.07, 28.74, 27.82, 27.38, 25.65,
16.88, 15.12, 12.51, 11.81. HRMS (ESI): [M + H]+ calcd for
C30H46N4O5S2, 607.2982; found, 607.2972.
14-O-[(2-(Piperidine-1-yl)-acetamido-1,3,4-thiadiazole-5-
yl)-thioacetyl]-mutilin (15c). Compound 15c was prepared
according to the general procedure from 14-O-[(2-chloroace-
tamido-1,3,4-thiadiazole-5-yl)-thioacetyl]-mutilin (14) and pi-
peridine. The crude product was purified by silica gel column
chromatography in 62% yield (1.15 g) as a white solid; mp:
49−51 °C. IR (KBr): 3434, 2932, 1731, 1557, 1519, 1455,
1403, 1286, 1188, 1152, 1117, 1019, 980, 954, 939, 916, 667
14-O-[(2-(4-Methylpiperazine-1-yl)-acetamido-1,3,4-thia-
diazole-5-yl)-thioacetyl]-mutilin (15f). Compound 15f was
prepared according to the general procedure from 14-O-[(2-
chloroacetamido-1,3,4-thiadiazole-5-yl)-thioacetyl]-mutilin
(14) and 4-methylpiperazine. The crude product was purified
by silica gel column chromatography in 79% yield (1.50 g) as a
white solid; mp: 70−71 °C. IR (KBr): IR (KBr): 3435, 2935,
L
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1731, 1556, 1519, 1455, 1403, 1284, 1189, 1153, 1117, 1018,
981, 955, 916, 863, 665 cm−1. 1H NMR (400 MHz, acetone) δ:
6.27 (dd, J = 17.7, 11.2 Hz, 1H), 5.74 (d, J = 8.4 Hz, 1H),
5.22−5.05 (m, 2H), 4.11 (d, J = 3.5 Hz, 1H), 4.06 (d, J = 7.1
Hz, 2H), 3.61 (s, 1H), 3.36 (s, 2H), 2.65 (s, 3H), 2.43 (d, J =
24.2 Hz, 4H), 2.30−2.16 (m, 5H), 2.14−2.05 (m, 6H), 1.98 (s,
4H), 1.83−1.66 (m, 2H), 1.56 (dt, J = 26.2, 10.6 Hz, 2H),
1.47−1.37 (m, 5H), 1.32 (d, J = 14.3 Hz, 1H), 1.21 (t, J = 7.1
Hz, 4H), 1.16−1.01 (m, 5H), 0.95 (d, J = 7.1 Hz, 3H), 0.72 (d,
J = 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ: 216.86,
168.43, 166.50, 158.58, 158.17, 138.66, 117.02, 74.33, 70.24,
60.48, 60.21, 57.89, 54.55, 53.30, 45.71, 45.23, 44.38, 43.77,
41.64, 36.49, 35.84, 34.27, 30.18, 26.63, 26.34, 24.63, 20.90,
16.59, 14.62, 14.03, 11.34. HRMS (ESI): [M + H]+ calcd for
C31H47N5O5S2, 634.3091; found, 634.3087.
Pharmacology. MIC Determination. The MIC values of
the synthesized compounds were determined using the agar
dilution method according to the National Committee for
Clinical Laboratory Standards (NCCLS). A 12 800 μg sample
of each synthesized compound was dissolved in approximately
5 mL of dimethyl sulfoxide (DMSO). Then, distilled water was
added to the solution to bring the volume to 10 mL. Tiamulin
fumarate and azamulin as reference drugs were dissolved in 10
mL of distilled water directly. All the obtained solutions were
then diluted 2-fold with distilled water. A 2 mL volume of the
2-fold serial dilution of each test compound/drug was
incorporated into 18 mL of hot Mueller−Hinton agar medium.
Inoculums of MRSA, MRSE, S. agalactiae, and E. coli, which
were all obtained from the clinic, were prepared from blood
slants and adjusted to approximately 105−106 cfu/mL with
saline solution (0.90% NaCl). A 10 μL amount of bacterial
suspension was spotted onto Mueller−Hinton agar plates
containing 2-fold serial dilutions of the compounds/drugs. The
plates were incubated at 36.5 °C for 48 h. The MIC was
defined as the minimum concentration of the compound
needed to completely inhibit bacterial growth. The same
procedure was repeated in triplicate.
The compounds were built with with Avogadro software,40 with
5000 steps Steepest Descent and 1000 steps Conjugate
Gradients optimization using MMFF94 force field.
Pharmacokinetic Studies. Intravenous and oral PK studies
(n = 3) were performed with male SD rats (Lanzhou Veterinary
Research Institute, CAAS, Lanzhou, China) weighing 220−250
g and approximately 6−8 weeks old. The rats were fasted
overnight before use and for 6 h after dosing. Compounds 8
and 15c were formulated as a solution of 5% Tween in water
for iv administration at a dose of 5 mg/kg and for po at 15 mg/
kg, respectively. Serial blood samples (0.5 mL) were collected
via the retrobulbar vein at various time points after the
compounds were administered and placed into EDTA−K2-
coated tubes. Then, the samples were centrifuged to yield
plasma samples, and the concentrations of the compounds in
plasma were determined by HPLC (for methods and
equipment, see Supporting Information). The pharmacokinetic
parameters were calculated from the mean plasma concen-
tration by noncompartmental analysis.
X-ray Diffraction. The crystals of compound 12e with
dimensions of 0.31 mm × 0.28 mm × 0.26 mm was mounted
on an Agilent SuperNova-CCD diffractometer equipped with a
mirror-monochromatic Mo Kα (λ = 0.7107 Å) radiation at
296.35(10) K. The total of reflections including 4476
independent scans (Rint = 0.0250) were collected in the range
of 3.07 ≤ θ ≤ 26.37° by using an ω scan mode, of which 3825
with I > 2σ(I) were observed and used in the succeeding
refinements. The structure was refined by full-matrix least-
squares techniques on F2 with the SHELXL program.41 The
final refinement gave R = 0.0399, wR = 0.0841 (w = 1/[σ2 (F0 )
2
2
+ (0.0405P)2 + 0.0182P], where P = (F0 + 2Fc2)/3), S = 1.033,
(Δρ)max = 0.179, and (Δρ)min = −0.225 e/Å3 (for more details
and for hydrogen bond information, see Tables S1 and S2 in
the Supporting Information). PLATON 1.1742 was used for the
molecular representations, and SHELXL41 was used for the
packing diagrams (for the molecular packing, see Figure S6 in
the Supporting Information).
Oxford Cup Assays. Inoculums were prepared in 0.9% saline
using McFarland standard and spread uniformly on nutrient
agar plates. All the compounds were prepared in the same
manner used for the MIC test, and the resulting solutions (320
and 160 μg/mL) were added individually to the Oxford cups,
which were placed at equal distances above the agar surfaces.
The zone of inhibition for each concentration was measured
after a 24 h incubation at 37 °C. The same procedure was
repeated in triplicate.
Molecular Modeling. Because all the compounds share a
common pleuromutilin scaffold, similarity-based molecular
docking was introduced to analyze the different activities of
compounds 11, 12d, 12e, 12f, 13a, 13b, 13c, 15a, 15b, 15c,
and 15d. The conformation of tiamulin in the X-ray crystal
structure (PDB ID: 1XBP)35 was used as a control to evaluate
the accuracy of the docking performance. Homdock software in
the Chil2 package34 was used for docking study. Briefly, the
starting conformations of all compounds were obtained from
alignment to tiamulin in the X-ray crystal stucture. Molecular
superposition was carried out by a graph-based molecular
alignment (GMA) method. Then the interaction between the
receptor and compounds was optimized by a 200 steps gradient
optimization to remove partial overlap. Optimization was
performed by Glamdock, and the binding affinity was evaluated
by Chil2 Score. The receptor used for docking was extracted
from the X-ray structure of a 50S ribosomal subunit (1XBP).
ASSOCIATED CONTENT
* Supporting Information
■
S
The biological activities of various compounds containing a
thiadiazole group, the correlations between the binding free
energy and the antibacterial activity, the kinetic profiles in rat
plasma, the data for hydrogen bonding and X-ray crystal
structure information, and the characterizations of the
synthesized compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: Liangjp100@sina.com. Fax: +86 931 2115951. Tel.:
+86 931 2115287.
■
Author Contributions
R.S. and Z.X. contributed to chemistry. J.L., X.P., C.Z., and E.G.
carried out biological evaluation and pharmacokinetics studies.
X.X. and Y.L. performed the molecular docking experiments.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by Basic Scientific Research Funds in
Central Agricultural Scientific Research Institutions (no.
■
M
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Journal of Medicinal Chemistry
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1610322014003), The Agricultural Science and Technology
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Center, China.
ABBREVIATIONS
■
BOC, tert-butoxycarbonyl; DMSO, dimethyl sulfoxide; EDCI,
1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochlor-
ide; FDA, Food and Drug Administration; GMA, graph-based
molecular alignment; HOBt, 1-hydroxybenzotriazole; HRMS,
high-resolution mass spectral; MC/SA, Monte Carlo/Simulated
Annealing; MRSA, Staphylococcus aureus; MRSE, methicillin-
resistant Staphylococcus epidermidis; MIC, minimum inhibitory
concentration; NCCLS, National Committee for Clinical
Laboratory Standards; QSAR, quantitative structure−activity
relationships; PTC, peptidyl transferase center; RMSD, root-
mean-square difference; SD, Sprague−Dawley; TFA, trifluoro-
acetic acid
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