Synthesis and antibacterial evaluation of novel pleuromutilin derivatives

Article abstract of DOI:10.1016/j.ejmech.2013.01.048

A series of novel pleuromutilin derivatives possessing thioether moiety has been synthesized via acylation reaction under mild conditions. Their in vitro antibacterial activity against methicillin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis, Escherichia coli, and Streptococcus agalactiae were tested by agar dilution method and Oxford cup assay. Among the 17 compounds screened, 14-O-[(4-methoxybenzamide-2- methylpropane-2-yl) thioacetate] mutilin 4i, 14-O-[(2-aminobenzamide-2- methylpropane-2-yl) thioacetate] mutilin 5a and 14-O-[(4-aminobenzamide-2- methylpropane-2-yl) thioacetate] mutilin 5c were resulted as most active antibacterial agents.

Full text of DOI:10.1016/j.ejmech.2013.01.048

European Journal of Medicinal Chemistry 63 (2013) 231e238
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antibacterial evaluation of novel pleuromutilin
derivatives
,
Ruofeng Shang a 1, Yu Liu a, Zhijun Xin b, Wenzhu Guo a, Zhiting Guo a, Baocheng Hao a,
,
Liang Jianping a
*
a Key Laboratory of New Animal Drug Project, Gansu Province, Key Laboratory of Veterinary Pharmaceutics Discovery, Ministry of Agriculture,
Lanzhou Institute of Animal Science and Veterinary Pharmaceutics Science, Chinese Academy of Agricultural Sciences, Jiangouyan, Qilihe District,
Lanzhou 730050, PR China
b Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel pleuromutilin derivatives possessing thioether moiety has been synthesized via acyl-
ation reaction under mild conditions. Their in vitro antibacterial activity against methicillin-resistant
Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis, Escherichia coli, and Strepto-
coccus agalactiae were tested by agar dilution method and Oxford cup assay. Among the 17 compounds
screened, 14-O-[(4-methoxybenzamide-2- methylpropane-2-yl) thioacetate] mutilin 4i, 14-O-[(2-
aminobenzamide-2-methylpropane-2-yl) thioacetate] mutilin 5a and 14-O-[(4-aminobenzamide-2-
methylpropane-2-yl) thioacetate] mutilin 5c were resulted as most active antibacterial agents.
Crown Copyright Ó 2013 Published by Elsevier Masson SAS. All rights reserved.
Received 16 August 2012
Received in revised form
7 January 2013
Accepted 22 January 2013
Available online 14 February 2013
Keywords:
Pleuromutilin derivative
Antibacterial activity
Synthesis
Antibiotics
Acylation reaction
1. Introduction
reportedthatmodifications of thismoietywitha thioethergroupand
a basic group together offered the best bioactivities [8]. Further al-
The abuse of antibiotics has made more pathogenic bacteria
resistance to drugs, which leads to many available drugs reducing
or losing curative effect. Drug-resistance bacteria, especially the
Staphylococcus aureus, Staphylococcus pneumoniae, Mycobacterium
tuberculosis, etc. kill more than two million people each year and
endanger human health seriously [1]. Pleuromutilin and its de-
rivatives (Fig.1) display high activities against drug-resistant Gram-
positive bacteria and mycoplasmas in vitro and in vivo [2], phar-
macodynamic properties [3] and no target-specific cross-resistance
to other antibiotics [4].
Pleuromutilinwas first isolated in a crystalline form from cultures
of two species of basidiomycetes, Pleurotus mutilus and
P. passeckerianus in 1951 [5]. Pleuromutilin is a diterpene, constituted
of a rather rigid 5e6e8 tricyclic carbon skeleton with eight stereo-
genic centers [6,7]. The molecular modifications of pleuromutilin
were focused essentially on the C-14 glycolic acid chain. Research
terations within this group led to the development of three drugs:
tiamulin, valnemulin, and retapamulin (Fig. 1). The first two drugs
were used as veterinary medicines. And the retapamulin became the
first pleuromutilin approved for human use in 2007. Besides reta-
pamulin, BC-3781, BC-3205 and BC-7013 (Fig. 1) are developing for
human use [9,10]. BC-3781 is especially currently under a clinical
phase II for complicated skin and skin structure infections and severe
respiratory infections by the biopharmaceutical company Nabriva
Therapeutics [9,11]. Another primary modification of pleuromutilin
was the synthesis of tricyclic carbon core or its analogs in order to
improve pharmacokinetic properties [12]. According to structure
characteristics of the core, 5e6 fused bicyclic framework was con-
structed first. Then a condensation reaction was employed to form
the pleuromutilin core or its analogs [12e14].
Further studies have shown that tiamulin, valnemulin and
others pleuromutilin derivatives interfered with bacterial protein
synthesis via a specific interaction with the 23S rRNA of the 50S
bacterial ribosome subunit [15e17]. The domain V of 23S rRNA at
the peptidyl transferase center (PTC) is mutilins derivatives binding
site, in which the tricyclic core of the pleuromutilin is positioned in
a pocket close to the A-tRNA binding site, whereas the C-14
extension points toward the P-tRNA binding site [11]. Thus these
* Corresponding author. Tel.: þ86 931 2115287; fax: þ86 931 2115951.
E-mail addresses: shangrf1974@163.com (R. Shang), Liangjp100@sina.com
(L. Jianping).
1
Lanzhou Institute of Animal Husbandry & Veterinary Pharmaceutics Science,
Chinese Academy of Agricultural Sciences, No. 335, Qilihe District, Lanzhou 730050,
PR China. Tel.: þ86 931 2117871; fax: þ86 931 2115951.
0223-5234/$ e see front matter Crown Copyright Ó 2013 Published by Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.01.048

232
R. Shang et al. / European Journal of Medicinal Chemistry 63 (2013) 231e238
CH2
CH3
OH
O
12
11
10
13
14
HO
O
CH3
22
H
5
9
H3C
H3C
4
1
7
8
6
3
2
O
Pleuromutilin
CH2
H
CH2
CH2
H
CH3
OH
CH3
OH
CH3
OH
O
O
O
O
N
H3C
S
H2N
S
S
N
O
O
O
N
H
CH3
CH3
CH3
H
H3C
H3C
H3C
H3C
H3C
H3C
O
O
O
Retapamulin
Valunemulin
Tiamulin
CH2
H
CH2
H
CH2
H
CH3
OH
CH3
CH3
OH
OH
O
O
O
OH
S
S
S
O
O
O
CH3
CH3
CH3
H3C
H3C
H3C
H2N
H3C
N
H3C
H3C
NH2
HO
O
O
O
O
BC-3781
BC-3205
Fig. 1. Structures of pleuromutilin and its derivative drugs.
BC-7013
compounds prevent the correct positioning of the tRNAs for pep-
tide transfer, and inhibit the peptidyl transferase [2,18].
amino-group was protected by tert-butoxycarbonyl (BOC). Conden-
sation reaction between the amino-group of compound 3 and
carboxyl group of amino acid derivative was carried out
in the presence of N,N0-dicyclohexylcarbodiimide (DCC) and
1-Hydroxybenzotriazole (HOBt) which is used to suppress racemiza-
tion and improve the efficiency of amide synthesis, followed by the
treatment of trifluoroacetic acid (TFA) for 30 min. Then the compound
5aef wasobtained. Thereactionofcompound 3 withchloroformatein
the presence of triethylamine resulted in the compound 6aeb.
All the formed amides were treated by distilled water and
saturated NaHCO3 or/and NH4Cl washing, followed by purification
with column chromatography. All derivatives were fully charac-
terized by means of IR, 1H NMR and 13C NMR spectral analysis.
Compound 3 is the most important intermediate. This compound
was synthesized in strong alkaline environment but was not stable in
such conditions for the carbonyl group at C21 position might be
attacked by the terminal amine, giving six-membered ring amide [22].
So the controls of reaction time and temperature were very necessary.
The compound 3 was purified by column chromatography according
to some reports [22,23]. But we obtained this compound with good
yield and convenience by recrystallization with ethyl acetate.
Structure activity relationship (SAR) studies show that the
presence of thioether group at C-22 position of pleuromutilin en-
hances antibacterial activity. The thioether group moiety is key to
their pharmacological properties, especially with side chain [11,19].
For example, antibacterial activity of valnemulin containing
dimethyl propane moiety is more effective than that of tiamulin
in vitro as well in vivo [20]. However related analogs of valnemulin
are still scarce. A serial of new pleuromutilin derivatives (4aei, 5ae
f, 6aeb) were synthesized based on the structure of valnemulin.
After bacteriostatic test in vitro, we demonstrated an attempt to
identify novel lead structure for antibiotics against some Gram-
positive bacteria, such as methicillin-resistant S. aureus (MRSA),
methicillin-resistant Staphylococcus epidermidis (MRSE), Escherichia
coli (E. coli), and Streptococcus agalactiae (S. agalactiae).
2. Results and discussion
2.1. Chemistry
Compounds 4aei also can be obtained by the condensation
method with N,N0-dicyclohexylcarbodiimide (DCC) as condensa-
tion agent. But the by-product 1,3-dicyclohexylurea (DCU) which
chemical polarity is close to the target compounds is very difficult
to remove by purification with column chromatography.
All pleuromutilin derivatives were synthesized by acylation re-
action starting from 14-O-[(1-Amino-2-methylpropan-2-yl) thio-
acetyl] mutilin 3, which was synthesized with 22-O-tosylpleuro-
mutilin 2 and dimethylcysteamine-hydrochloride [21] according to
the pathway reported in Scheme 1.
Compound 4aei was obtained in two steps: First, the correlative
carboxylic acids were converted into acyl chlorides with SOCl2 in the
refluxing condition and excess SOCl2 was evaporated. Then the acyl
chloride was directly reacted with compound 3 to form amide 4aei.
Compound 5aef was obtained from amino acid derivative which
2.2. Biological evaluation
All the synthesized pleuromutilin derivatives 4aei, 5aef, and
6aeb were screened for their in vitro antibacterial activity against

R. Shang et al. / European Journal of Medicinal Chemistry 63 (2013) 231e238
233
CH2
CH2
CH3
OH
CH3
OH
O
O
O
S
H3C
O
HO
ii
i
O
CH3
O
CH3
H
O
H
H3C
H3C
H3C
H3C
O
2
O
1
CH2
H
CH3
OH
O
O
S
N
H
O
CH3
R
H3C
H3C
4a-i
O
iii
CH2
CH2
H
CH3
OH
O
O
CH3
OH
O
S
iv
S
O
R
N
H
CH3
H
H2N
O
CH3
H3C
H3C
H3C
H3C
v
O
5a-f
1
O
3
CH2
CH3
OH
O
O
S
R
O
N
H
O
CH3
H
H3C
H3C
O
6a-b
NH2
5e: R=
5f: R=
6a: R=
4a-c: R= -Cl
5a-c: R=
NH2
4d-f: R= -CH3
6b: R=
5d: R=
4g-i: R= -OCH3
N
H
NH2
Scheme 1. Reagent and condition: (i) TsCl, NaOH, H2O, t-butyl methyl ether, reflux, 1 h; (ii) HSC(CH3)2CH2NH2$HCl, EtONa, EtOH, 0 ꢀC, 2.5 h; (iii) aromatic acyl chloride, N(CH2CH3)3,
CH2Cl2, 0 ꢀC, 3 h; (iv) amino acid derivative, DCC, HOBt, CH2Cl2, rt, 15h, then TFA, CH2Cl2, rt, 30 min; (v): Chloroformate, N(CH2CH3)3, CH2Cl2, 0 ꢀC or rt, 4.5 h.
MRSA, MRSE, E. coli, and S. agalactia by agar dilution method ac-
cording to the National Committee for Clinical Laboratory Stan-
dards (NCCLS). Minimum inhibitory concentration (MIC) is defined
as the minimum concentration of the compound required to
completely inhibit the bacterial growth. The determination of MIC
values was performed in triplicate at pH 7.40. The MICs of the
synthesized compounds 4aei, 5aef, and 6aeb along with tiamulin
and valnemulin which were used as reference drugs are depicted in
Table 1. The MICs of 17 new pleuromutilin derivatives in vitro
against MRSA, MRSE, E. coli, and S. agalactia ranged from 8 to
the others showed moderate antibacterial activity as indicated
by MIC values and the zones of inhibition. Compounds 4i, 5a
and 5c inhibited the growth of pathogen particularly MRSA and
MRSE, even better than the reference drug tiamulin at the
same concentration. However, all the synthesized compounds
showed lower antibacterial activity than valnemulin. From
MIC values and the zones of inhibition it was observed that
the substituents, especially chlorine substituent, in the ben-
zene ring of ortho para showed stronger antibacterial activity
than that in the benzene ring of meta. Also it was observed
that the stronger conjugation effect of substituents, the more
excellent antibacterial activity. For example, compounds 4gei
and 5aec showed stronger antibacterial activity than com-
pound 4aec.
The compounds 4a, 5a, 5c, 6a and 6b have been reported by
Zhang and coworkers [22e24]. Here we synthesized these
compounds again for the supplements to compounds 4b, 4c and
5b and compared their antibacterial activities. Furthermore we
supplemented their 13C NMR spectrum data and MIC except 6b
which was reported its MIC for different bacterial strains.
0.25
mg/mL, 32 to 0.5 mg/mL, 32 to 1 mg/mL, and 32 to 0.5 mg/mL
respectively.
Antibacterial activity for all the synthesized compounds was
evaluated against the above mentioned four bacterial strains. Ox-
ford cup assay was carried out and the zones of inhibition for
different concentrations of the synthetic compounds were
measured and the results are given in Table 2. Also tiamulin and
valnemulin were used as reference drugs.
Among all the pleuromutilin derivatives examined, three
compounds 4i, 5a, 5c showed good antibacterial activity, while

234
R. Shang et al. / European Journal of Medicinal Chemistry 63 (2013) 231e238
Table 1 (continued)
Table 1
Antibacterial activity of the novel pleuromutilin derivatives.
Compound no.
R
MIC (
m
g/mL)
CH2
MRSA
MRSE E. coli S. agalactia
CH3
OH
O
O
5f
2
16
8
16
8
S
NH
R
N
H
O
CH3
H
H3C
O
6a
6b
2
16
32
4
H3C
4
32
2
16
Compound no.
R
MIC (
m
g/mL)
O
MRSA
MRSE E. coli S. agalactia
Tiamulin
Valnemulin
0.5
0.125 0.5
2
0.5
2
0.5
Cl
4a
0.5
1
4
8
8
3. Conclusions
Cl
Cl
4b
8
16
32
We have synthesized a series of novel pleuromutilin derivatives
containing thioether moiety by three methods of acylation reaction
under very mild conditions. All these new derivatives have been
investigated for their antibacterial activity in vitro against some
Gram-positive bacteria, the investigation of MIC and antibacterial
activity revealed that all the synthesized compounds showed
moderate to good inhibitory characteristics. Among all the 17
compounds screened, compound 4i, 5a and 5c with stronger
conjugation effect of methoxy or amino in the in the benzene ring
of ortho para were resulted as most active antibacterial agents
against MRSA, MRSE, E. coli and S. agalactia.
4c
1
0.5
16
2
2
4
CH3
4d
0.25
0.5
H3C
H3C
4e
4f
1
16
16
8
8
4
4
2
4. Experimental section
0.25
0.25
0.5
2
4.1. General
OCH3
Melting points were determined on a Tianda Tianfa YRT-3
apparatus (China) with open capillary tubes and are uncorrected.
IR spectra were obtained on a Thermo Nicolet NEXUS-670 spec-
trometer and recorded as KBr thin film and absorptions are re-
ported in cmꢁ1. NMR spectra were recorded on a Bruker-400 MHz
4g
H3CO
H3CO
4h
4i
2
32
2
8
1
2
4
spectrometers in appropriate solvents. Chemical shifts (d) were
expressed in parts per million (ppm) relative to the tetrame-
thylsilane. Multiplicities of NMR signals are designated as s
(singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br
(broad), etc. 13C NMR spectra were recorded on 100 MHz spec-
trometers. All reactions were monitored by TLC on 0.2 mm thick
silica gel GF254 pre-coated plates. After elution, plate was visual-
ized under UV illumination at 254 nm for UV active materials.
Further visualized was achieved by staining with 0.05% KMnO4
aqueous solution or 0.5% phosphomolybdic acid. Column chroma-
tography was carried out on silica gel (200e300 mesh). The prod-
ucts were eluted in appropriate solvent mixture under air pressure.
Concentration and evaporation of the solvent after reaction or
extraction was carried out on a rotary evaporator. All reagents were
purchased from Aladdin (China) and used without further
purification.
0.25
1
0.5
0.5
NH2
5a
0.5
H2N
H2N
5b
5c
2
1
8
2
0.5
0.5
0.25
0.5
5d
5e
2
16
4
8
4
16
8
4.2. 14-O-(p-toluene sulfonyloxyacetyl) mutilin (2)
NH2
40% sodium hydroxide solution (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 t-butyl methyl ether
(20 mL) and water (5 mL). The mixture was stirred vigorously under
reflux for 1 h, then diluted with water (50 mL) and stirred under an
0.25
NH2

R. Shang et al. / European Journal of Medicinal Chemistry 63 (2013) 231e238
235
Table 2
The zone of inhibition for MRSA, MRSE, E. coli, and S. agalactia in mm.
Compound
MRSA (
320
m
g/mL)
160
MRSE (
m
g/mL)
160
E. coli (
m
g/mL)
160
S. agalactia (mg/mL)
80
320
80
320
80
320
160
80
4a
4b
4c
4d
4e
4f
4g
4h
4i
5a
5b
5c
5d
5e
5f
6a
14.16
13.50
14.62
12.27
13.09
12.61
12.38
12.81
19.72
19.56
10.22
22.25
10.25
10.28
8.52
12.19
13.11
13.79
11.85
13.21
11.49
12.06
12.98
18.35
18.92
10.77
21.60
9.98
10.11
8.77
9.56
11.53
17.64
22.89
14.11
11.87
11.75
10.28
11.20
10.41
9.50
11.80
15.29
16.55
9.56
20.65
9.41
9.82
9.05
9.64
13.36
11.93
12.45
13.77
12.41
12.05
13.54
12.50
18.33
17.36
11.21
17.89
10.85
11.21
9.00
12.20
10.62
12.67
12.88
10.75
10.35
12.50
11.69
15.25
16.02
9.72
18.13
10.02
10.63
8.84
10.96
10.16
15.59
25.95
11.40
9.45
8.72
10.70
9.90
14.48
15.68
12.57
15.00
15.52
16.59
17.42
14.30
18.50
13.75
10.68
18.83
10.84
10.66
10.80
10.32
14.66
18.31
24.50
11.91
10.65
12.88
13.65
13.74
15.07
15.50
13.34
17.07
13.68
12.46
17.76
11.31
9.89
11.31
11.95
11.77
12.08
11.30
9.80
12.40
12.18
15.69
13.07
9.94
10.80
12.17
14.00
12.40
13.62
12.26
13.71
13.42
16.58
11.88
9.66
11.45
11.03
11.19
9.25
10.20
11.48
12.32
15.79
11.23
13.91
12.05
10.87
12.97
10.95
12.98
12.40
15.92
11.75
9.80
10.55
10.44
10.77
10.00
13.01
9.82
11.79
12.63
16.01
12.02
8.86
10.91
10.77
11.38
14.41
15.32
9.63
17.31
10.90
10.29
10.35
10.60
11.00
14.50
22.55
18.09
9.20
10.04
8.87
11.62
10.61
10.43
9.62
10.95
10.11
10.68
9.31
9.52
9.55
15.31
16.52
23.71
10.88
12.69
18.26
25.76
11.10
10.98
16.41
27.46
9.91
9.01
9.85
6b
10.40
16.52
20.43
11.69
15.21
19.54
10.55
11.43
14.51
10.70
11.07
12.75
Tiamulin
Valnemulin
ice bath for 15 min, followed by washing with water (50 mL) and
cold t-butyl methyl ether (20 mL). Filtration afforded the title
compound as white solid (9.8, 93%). It was used in the next step
without further purification. mp: 147e148 ꢀC; IR (KBr): 3446, 2924,
2863, 1732, 1633, 1597, 1456, 1371, 1297, 1233, 1117, 1035, 832, 664,
J2 ¼ 10.8 Hz); 13C NMR (100 MHz, CDCl3)
d 216.9, 169.4, 139.0, 117.1,
74.6, 69.3, 58.2, 51.7, 48.5, 45.4, 44.7, 43.9, 41.8, 36.7, 35.9, 34.4, 31.2,
30.4, 26.8, 26.3, 26.2, 24.8, 16.8, 14.9, 11.4.
4.4. General procedure for the synthesis of compounds 4aei
560 cmꢁ1; 1H NMR (400 MHz, CDCl3)
d
0.63 (d, 3H, J ¼ 6.8 Hz), 0.87
(d, 3H, J ¼ 6.8 Hz), 1.11e1.15 (m, 1H), 1.22e1.26 (s, 5H), 1.33e1.36 (m,
1H), 1.41e1.44 (m, 1H), 1.46e1.50 (m, 5H), 1.63e1.65 (dd, 2H,
J1 ¼10 Hz, J2 ¼ 7.2 Hz), 2.01e2.08(m, 3H), 2.21e2.29 (m, 3H), 2.45 (s,
3H), 3.34 (d, 1H, J ¼ 6.4 Hz), 4.48 (s, 2H), 5.17e5.21 (d, 1H,
J ¼ 8.8 Hz), 5.31e5.34 (d, 1H, J ¼ 6.4 Hz), 5.75e5.78 (d, 1H,
J ¼ 4.2 Hz), 6.43 (q, 1H, J ¼ 17.2 Hz, 10.8 Hz); 7.35e7.37 (d, 2H,
J ¼ 4.0 Hz), 7.80e7.82 (d, 2H, J ¼ 4.0 Hz); 13C NMR (100 MHz, CDCl3)
A mixture of modified benzoic acid (5.0 mmol) and SOCl2
(10 mL) was heated under reflux for 5 h. The excess SOCl2 was
evaporated in vacuum and the resulting oil was cooled to room
temperature. To the cooled oil dichloromethane (60 mL), triethyl-
amine (2.5 g, 24.7 mmol) and compound 3 (1.6 g, 3.5 mmol) were
added and stirred at 0 ꢀC for 3 h. The mixture was quenched with
saturated aqueous NH4Cl and washed with water, followed by
separation of organic layer. The aqueous layer was extracted with
dichloromethane and the combined organic layer extracts were
washed with water again. The obtained organic solvent was dried
with anhydrous Na2SO4 overnight. After evaporation in vacuum of
the organic solvent, the residue was purified by column chroma-
tography (silica gel, petroleum ether:ethyl acetate 1:1 v/v) to afford
the desired compounds.
d
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.
4.3. 14-O-[(1-amino-2-methylpropane-2-yl) thioacetyl] mutilin (3)
Metallic sodium (0.69 g, 30 mmol) was cut into small pieces and
added portion wise to absolute ethanol (150 mL). After metallic
sodium had been dissolved completely at room temperature, the
solution was filtered and 1-amino-2-methyl-2-propanethiol hy-
drochloride (2.12 g, 15 mmol) was added. The mixture was stirred
for 0.5 h at room temperature, followed by freezing to 0 ꢀC and
compound 2 (7.99 g, 15 mmol) was added. The mixture was stirred
in an ice bath for 2.5 h, evaporated under reduced pressure to
50 mL, extracted with ethyl acetate (100 mL) and washed with
water (200 mL). The organic layer was separated and dried with
anhydrous Na2SO4 overnight. The organic solvent was evaporated
in vacuum and the residue was purified by recrystallization with
ethyl acetate or column chromatography (silica gel, ethyl aceta-
te:ethanol 10:1 v/v). the product was obtained (5.03 g, 72%). mp:
154e155 ꢀC; IR (free base, KBr): 3351, 2956, 2864, 1734, 1721, 1634,
1456, 1373, 1274, 1209, 1112, 1033, 982, 955, 941, 916 cmꢁ1; 1H NMR
4.4.1. 14-O-[(2-chlorobenzamide-2-methylpropane-2-yl) thioacetate]
Mutilin (4a)
White solid; yield: 66%; mp: 66e68 ꢀC; IR (KBr): 3417, 2928,
2889, 1728, 1651, 1594, 1537, 1469, 1385, 1282, 1202, 1184, 1117, 1017,
980, 939, 917, 751 cmꢁ1 1H NMR (400 MHz, CDCl3)
; d 0.70 (d, 3H,
J ¼ 7.2 Hz), 0.87 (d, 3H, J ¼ 7.2 Hz), 1.12e1.14 (m, 4H), 1.24e1.29 (t,
2H), 1.35 (s, 7H), 1.42e1.44 (d, 5H), 1.64e1.76 (m, 4H), 2.03e2.07 (d,
2H), 2.21e2.28 (d, 3H), 3.14e3.24 (m, 2H), 3.33 (s, 1H), 3.43e3.50 (t,
2H, J ¼ 6.8 Hz), 5.08e5.18 (m, 2H), 5.69(d, 1H), 6.34e6.41 (q, 1H,
J1 ¼ 10.4 Hz, J2 ¼ 10. Hz); 7.06 (s, 1H), 7.34e7.42 (m, 2H, J ¼ 7.2 Hz),
7.65 (s, 1H); 13C NMR (100 MHz, CDCl3)
d 216.9, 169.8, 166.7, 138.8,
135.4, 131.2, 130.9, 130.3, 129.9, 126.9, 117.2, 74.5, 69.9, 58.1, 48.1,
47.1, 45.4, 44.8, 43.9, 41.7, 36.7, 35.9, 34.4, 31.5, 30.4, 26.8, 26.5, 26.4,
24.8, 16.8, 14.8, 11.5.
(400 MHz, CDCl3)
d
0.73 (d, 3H, J ¼ 7.2 Hz), 0.87 (d, 3H, J ¼ 7.2 Hz),
1.09e1.16 (m, 1H), 1.23 (s, 6H), 1.30e1.38 (m, 2H), 1.45 (s, 1H), 1.52e
1.53 (m, 7H), 1.55e1.60 (m, 1H), 1.63e1.69 (m, 2H), 1.74e1.78(q, 1H,
J ¼ 0.8 Hz), 2.04e2.10 (q, 2H), 2.18e2.25(m, 2H), 2.32e2.59 (q, 1H,
J ¼ 6.8 Hz), 3.09 (s, 2H), 3.13e3.17 (t, 2H, J ¼ 1.6 Hz), 3.35 (d, 1H,
J ¼ 6.4 Hz), 5.17e5.22 (q, 1H, J ¼ 1.6 Hz), 5.31e5.34 (q, 1H,
J ¼ 1.2 Hz), 5.74(d, 1H, J ¼ 8.4), 6.44e6.51 (q, 1H, J1 ¼ 11.2 Hz,
4.4.2. 14-O-[(3-chlorobenzamide-2-methylpropane-2-yl)thioacetate]
Mutilin (4b)
White solid; yield: 74%; mp: 92e94 ꢀC; IR (KBr): 3323, 3084,
2924, 2863, 1718, 1649, 1567, 1538, 1454, 1369, 1292, 1238, 1144,
1117, 1015, 979, 940, 917, 883, 753, 681, 610 cmꢁ1 1H NMR
;
(400 MHz, CDCl3)
d
0.71 (d, 3H, J ¼ 7.2 Hz), 0.87 (d, 3H, J ¼ 7.2 Hz),

236
R. Shang et al. / European Journal of Medicinal Chemistry 63 (2013) 231e238
1.14 (s, 4H), 1.29e1.32 (d,7H), 1.45 (s, 4H), 1.60e1.63 (d, 1H), 1.65e
1.68 (t, 3H), 1.75e1.78 (d, 1H), 2.06e2.18 (m, 2H), 2.20e2.25(m,
2H), 2.29e2.34 (q, 1H, J ¼ 6.0 Hz), 3.16e3.24 (d, 2H), 3.26e3.31
(m, 1H), 3.35 (d, 1H, J ¼ 6.4), 3.45e3.50 (q, 1H), 5.09 (s, 1H), 5.13
(d, 1H, J ¼ 4.0), 5.76(d, 1H, J ¼ 8.4), 6.38e6.45 (q, 1H, J1 ¼ 10.4 Hz,
J2 ¼ 10.8 Hz), 7.38e7.42 (t, 2H), 7.48e7.50 (m, 1H), 7.64e7.67 (t, 1H,
J ¼ 5.6 Hz), 7.79e7.93 (m, 1H), 7.94 (s, 1H); 13C NMR (100 MHz,
1.25e1.33 (m, 8H), 1.38e1.53 (q, 5H), 1.61e1.79 (m, 4H), 2.05e2.33
(m, 5H), 2.42 (d, 3H), 3.15e3.35 (m, 4H), 3.47-3.52 (q, 1H, J ¼ 6.4),
5.11e5.16 (t, 2H), 5.77(d, 1H, J ¼ 8.4), 6.39e6.46 (q, 1H, J ¼ 11.2 Hz),
7.34 (d, 2H), 7.41 (s, 1H), 7.68e7.74(q, 2H, J1 ¼ 8.0 Hz, J1 ¼ 6.0 Hz);
13C NMR (100 MHz, CDCl3)
d 216.8, 170.4, 167.4, 138.7, 138.3, 134.5,
132.1, 128.4, 127.9, 124.1, 117.2, 74.5, 70.1, 58.1, 47.7, 47.5, 45.4, 44.9,
43.9, 41.8, 36.6, 35.9, 34.4, 31.6, 30.4, 26.8, 26.4, 26.3, 24.8, 21.4, 16.9,
14.8, 11.5.
CDCl3)
d 216.8, 170.8, 165.8, 138.7, 136.3, 134.6, 131.4, 129.8, 127.5,
125.3, 117.2, 74.5, 70.3, 58.0, 47.7, 45.4, 44.9, 43.9, 41.7, 36.6, 35.9,
34.4, 31.5, 30.3, 26.8, 26.4, 26.2, 24.8, 16.9, 14.8, 11.5.
4.4.7. 14-O-[(2-methoxybenzamide-2-methylpropane-2-yl) thioace-
tate] Mutilin (4g)
4.4.3. 14-O-[(4-chlorobenzamide-2-methylpropane-2-yl) thioacetate]
Mutilin (4c)
White solid; yield: 81%; mp: 60e62 ꢀC; IR (KBr): 3389, 2931,
2878, 1732, 1650, 1600, 1537, 1483, 1463, 1373, 1285, 1241, 1183,
White solid; yield: 67%; mp: 68e70 ꢀC; IR (KBr): 3395, 3081,
2929, 2864, 1727, 1651, 1596, 1539, 1486, 1455, 1384, 1284, 1190,
1116, 1021, 980, 917, 758, 656 cmꢁ1 1H NMR (400 MHz, CDCl3)
;
d
0.70 (d, 3H, J ¼ 5.6 Hz), 0.85 (d, 3H, J ¼ 6.0 Hz), 1.09e1.13 (t, 3H),
1147, 1116, 1014, 980, 938, 916, 846, 757, 678, 607 cmꢁ1
;
1H NMR
1.21e1.39 (m, 9H), 1.43e1.44 (t, 4H), 1.49e1.76 (m, 5H), 2.01e2.07
(m, 3H), 2.15e2.30 (m, 3H), 3.17e3.25 (t, 2H), 3.33 (t, 1H, J ¼ 8.0),
3.40e3.52 (m, 2H), 3.98 (q, 3H), 5.08e5.21 (q, 2H), 5.73(d, 1H,
J ¼ 7.2), 6.38e6.46 (m, 1H), 7.40e7.44 (q, 1H), 8.14e8.16 (q, 1H,
J1 ¼ 2.0 Hz, J1 ¼ 2.4 Hz), 8.48(s, 1H); 13C NMR(100 MHz, CDCl3)
(400 MHz, CDCl3)
d
0.69 (d, 3H, J ¼ 7.2 Hz), 0.89 (m, 6H, J ¼ 7.2 Hz),
1.13e1.18 (m, 4H), 1.25 (s, 4H), 1.29 (s, 3H), 1.30e1.51 (m, 4H), 1.61e
1.69 (m, 2H), 1.75e1.79 (q, 1H, J ¼ 2.8 Hz), 2.06e2.12 (q, 2H), 2.16-
2.31(m, 3H), 3.09 (s, 2H), 3.16e3.29 (m, 3H), 3.33e3.37 (t, 1H),
3.46e3.51 (q, 1H, J ¼ 6.4), 5.08e5.15 (t, 2H), 5.73(d, 1H, J ¼ 8.8),
6.36e6.44 (q, 1H, J1 ¼ 11.2 Hz, J2 ¼ 10.8 Hz), 7.43e7.45 (d, 2H,
J ¼ 8.4 Hz), 7.52e7.55 (t,1H, J ¼ 6.0 Hz), 7.86e7.88 (d,1H, J ¼ 8.4 Hz);
d
216.9,169.1,165.3, 157.6,138.8,132.6,132.1,121.5, 127.1,117.0,111.2,
74.4, 69.4, 58.1, 55.9, 47.9, 46.8, 45.3, 44.6, 43.8, 41.7, 36.6, 35.9, 34.3,
31.5, 30.3, 26.7, 26.5, 26.2, 24.7, 16.7, 14.8, 11.4.
13C NMR (100 MHz, CDCl3)
d 216.7, 170.6, 165.9, 138.7, 137.6, 133.9,
128.7, 128.6, 117.2, 74.5, 70.3, 58.0, 47.7, 47.6, 44.9, 43.9, 41.7, 36.6,
35.9, 34.4, 31.6, 30.3, 26.8, 26.4, 26.2, 24.8, 16.9, 14.8, 11.5.
4.4.8. 14-O-[(3-methoxybenzamide-2-methylpropane-2-yl) thioace-
tate] Mutilin (4h)
White solid; yield: 78%; mp: 59e61 ꢀC; IR (KBr): 3400, 2931,
2857, 1729, 1651, 1583, 1537, 1485, 1463, 1372, 1287, 1244, 1189, 1117,
4.4.4. 14-O-[(2-methylbenzamide-2-methylpropane-2-yl) thioacetate]
Mutilin (4d)
1037, 980, 917, 752, 689 cmꢁ1; 1H NMR (400 MHz, CDCl3)
d 0.71 (d,
White solid; yield: 79%; mp: 133e134 ꢀC; IR (KBr): 3459, 3330,
2953, 2883, 1730, 1650, 1601, 1539, 1485, 1455, 1406, 1371, 1301,
3H, J ¼ 6.8 Hz), 0.87 (d, 3H, J ¼ 6.4 Hz), 1.08e1.13 (d, 4H), 1.23e1.27
(m, 2H), 1.30e1.31 (d, 7H), 1.45 (d, 4H), 1.48e1.54 (t, 2H), 1.60e1.65
(t, 2H), 1.74e1.78 (d, 1H), 2.03e2.11 (m, 3H), 2.18e2.31 (m, 3H),
3.16e3.34 (m, 4H), 3.46e3.51 (q, 1H), 3.86 (t, 3H), 5.09e5.14 (t, 2H),
5.74 (d, 1H, J ¼ 8.0), 6.37e6.44 (m, 1H, J1 ¼ 11.2 Hz, J2 ¼ 11.6 Hz),
7.04e7.06 (d, 1H), 7.34e7.38 (q, 1H), 7.45e7.49 (t, 3H); 13C NMR
1284, 1185, 1147, 1115, 1037, 985, 940, 915, 771, 744, 723, 667 cmꢁ1
;
1H NMR (400 MHz, CDCl3)
d
0.69 (d, 3H, J ¼ 7.2 Hz), 0.86 (d, 3H,
J ¼ 7.2 Hz), 1.07e1.18 (m, 4H), 1.21e1.27 (m, 2H), 1.33 (d, 7H), 1.41 (d,
3H), 1.43e1.53 (m, 2H), 1.59e1.67 (m, 2H), 1.73e1.77(q, 1H,
J ¼ 2.4 Hz), 2.06e2.08 (m, 2H), 2.12e2.32(m, 3H), 2.49 (s, 3H), 3.13e
3.24 (q, 2H, J ¼ 16.0 Hz), 3.31e3.36 (q, 2H), 3.44e3.49 (q, 1H,
J ¼ 6.4), 5.09e5.16 (t, 2H), 5.69(d, 1H, J ¼ 8.8), 6.34e6.41 (q, 1H,
J1 ¼11.2 Hz, J2 ¼ 10.8 Hz), 6.79e6.82 (t, 1H), 7.20e7.24 (t, 2H), 7.30e
7.34 (t, 1H), 7.48 (d, 1H, J ¼ 7.6 Hz); 13C NMR (100 MHz, CDCl3)
(100 MHz, CDCl3)
d 216.8, 170.4, 166.9, 159.8, 138.6, 136.0, 129.5,
118.9, 117.7, 117.3, 112.3, 74.5, 70.1, 58.0, 55.3, 47.7, 47.5, 45.3, 44.9,
43.9, 41.7, 36.6, 35.9, 34.4, 31.5, 30.3, 26.8, 26.4, 26.2, 24.8, 16.8, 14.8,
11.5.
d
216.8, 169.9, 169.8, 138.7, 136.3, 136.2, 131.0, 129.8, 126.9, 125.7,
4.4.9. 14-O-[(4-methoxybenzamide-2-methylpropane-2-yl) thioace-
tate] Mutilin (4i)
117.1, 74.5, 69.9, 58.0, 47.5, 47.4, 45.3, 44.8, 43.9, 41.7, 36.6, 35.9, 34.4,
31.5, 30.3, 26.8, 26.4, 26.3, 24.8, 20.1, 16.8, 14.8, 11.4.
White solid; yield: 83%; mp: 60e63 ꢀC; IR (KBr): 3406, 2931,
2885, 1732, 1646, 1607, 1540, 1505, 1456, 1417, 1373, 1286, 1255,
4.4.5. 14-O-[(3-methylbenzamide-2-methylpropane-2-yl) thioacetate]
Mutilin (4e)
1185, 1179, 1116, 1028, 979, 938, 916, 845, 767, 610 cmꢁ1 1H NMR
;
(400 MHz, CDCl3)
d
0.71 (t, 3H, J ¼ 6.0 Hz), 0.87 (t, 3H, J ¼ 5.2 Hz),
White solid; yield: 74%; mp: 67e69 ꢀC; IR (KBr): 3396, 3328,
2928, 2861, 1728, 1650, 1628, 1613, 1573, 1539, 1505, 1456, 1417,
1385, 1285, 1189, 1117, 1018, 980, 916, 751 cmꢁ1; 1H NMR (400 MHz,
1.13e1.14 (d, 4H), 1.24e1.38 (m, 9H), 1.44e1.46 (t, 4H), 1.52e1.78 (m,
4H), 2.03e2.06 (m, 3H), 2.19e2.31 (m, 3H), 3.15e3.36 (m, 4H),
3.46e3.49 (q, 1H), 3.86 (t, 3H), 5.11e5.15 (m, 2H), 5.73(s, 1H), 6.37e
6.46 (m, 1H), 6.93e6.97 (q, 2H), 7.34 (s, 1H), 7.87e7.89 (m, 2H); 13C
CDCl3)
d
0.72 (d, 3H, J ¼ 7.2 Hz), 0.87 (d, 3H, J ¼ 6.8 Hz), 1.09e1.13 (d,
4H),1.23e1.31 (m, 7H),1.38e1.45 (t, 3H, J ¼ 14.4),1.49e1.78 (m, 6H),
2.03e2.32 (m, 6H), 2.40 (d, 3H), 3.15e3.30 (m, 2H), 3.34 (d, 2H,
J ¼ 6.0), 3.45e3.50 (q, 1H, J ¼ 6.4), 5.10e5.14 (t, 2H), 5.73(d, 1H,
J ¼ 8.4), 6.37e6.45 (q, 1H, J1 ¼ 11.6 Hz, J2 ¼ 11.2 Hz), 7.24e7.27 (t,
NMR (100 MHz, CDCl3) d 216.8, 170.4, 166.7, 162.2, 138.7, 128.9,
126.8, 117.3, 113.7, 74.5, 70.2, 60.3, 58.1, 55.4, 47.8, 47.5, 45.4, 44.9,
43.9, 41.8, 36.6, 35.9, 34.4, 31.5, 30.4, 26.9, 26.4, 26.2, 24.8, 16.9, 14.8,
14.2, 11.5.
1
2H), 7.38 (t, 1H), 7.80 (d, 1H, J ¼ 8.0 Hz); 3C
)
NMR (100 MHz, CDCl
3
d
216.8, 170.3, 167.1, 141.7, 138.7, 131.6, 129.1, 127.1, 117.2, 74.5, 70.1,
4.5. General procedure for the synthesis of compounds 5aef
58.0, 47.7, 47.4, 45.3, 44.9, 43.9, 41.7, 36.6, 35.9, 34.4, 33.8, 31.5, 30.3,
26.8, 26.3, 26.2, 25.6, 24.9, 21.4, 16.8, 14.8, 11.5.
A suspension of amino acid derivative (5 mmol) was dissolved in
a mixture of tetrahydrofuran (50 mL) and water (20 mL). 1 N NaOH
(6 mL) was added followed by adding 1.1 g tertebutoxycarbonyl
(5 mmol) dropwise at room temperature. After stirring for 4 h,
the tetrahydrofuran was evaporated in vacuum from the reaction
mixture. The residue was added ethyl acetate (50 mL) and 5% citric
acid (30 mL). The organic layer was separated, washed with water
(20 mL), dried with anhydrous Na2SO4 and rotary evaporated to
4.4.6. 14-O-[(4-methylbenzamide-2-methylpropane-2-yl) thioacetate]
Mutilin (4f)
White solid; yield: 77%; mp: 63e66 ꢀC; IR (KBr): 3403, 2928,
2854,1732,1651,1615,1539,1506,1456,1417,1386,1286,1189,1149,
1117, 1019, 980, 917, 836, 752, 667 cmꢁ1 1H NMR (400 MHz,
;
CDCl3)
d
0.73 (d, 3H, J ¼ 6.8 Hz), 0.88 (d, 3H, J ¼ 6.8 Hz), 1.15 (s, 4H),

R. Shang et al. / European Journal of Medicinal Chemistry 63 (2013) 231e238
237
dryness. Crude residue was used into next reaction without
purification.
917, 699 cmꢁ1; 1H NMR (400 MHz, CDCl3)
d
0.72 (d, 3H, J ¼ 7.2 Hz),
0.90 (d, 3H, J ¼ 6.8 Hz), 1.11e1.18 (q, 10H), 1.24e1.39 (m, 5H), 1.44 (d,
4H), 1.48e1.56 (t, 2H), 1.61e1.69 (q, 2H), 1.78 (d, 1H), 2.04 (s, 3H),
2.08 (d, 2H), 2.14e2.26 (m, 2H), 2.31e2.34 (t, 1H), 3.04e3.30(m,
9H), 3.36 (d, 1H), 4.59 (s, 1H), 5.16e5.26 (q, 2H), 5.72(d, 1H),
6.39e6.46 (q, 1H, J1 ¼10.8 Hz, J2 ¼ 11.2 Hz), 7.29-7.36 (m, 3H), 7.43e
A mixture of the above N-Boc protected amino acids (3.7 mmol),
1.63 g compound 3 (3.5 mmol), 0.72 g N,N0-dicyclohexylcarbodii-
mide (3.5 mmol), 0.47 g 1-Hydroxybenzotriazole (3.5 mmol) and
60 mL dichloromethane was stirred at room temperature for 15 h.
The mixture was filtered to remove dicyclohexylurea which was
produced by the reaction and the filtrate was washed with satu-
rated aqueous NaHCO3 and water. The solvent was evaporated in
vacuum and the residue was treated with a mixture of 10 mL tri-
fluoroacetic acid and 10 mL dichloromethane at room temperature
for 30 min. The reaction mixture was quenched with 25% aqueous
NaHCO3 (50 mL) and washed with water, dried with anhydrous
Na2SO4 overnight and rotary evaporated to dryness. Crude residue
thus obtained was purified over silica gel column chromatography
(petroleum ether:ethyl acetate 1:1 v/v) afford the desired
compounds.
7.45 (d, 2H); 13C NMR (100 MHz, CDCl3)
d 216.9, 169.8, 138.9, 128.8,
128.0, 126.9, 117.2, 74.6, 69.7, 58.1, 47.5, 47.1, 45.4, 44.8, 43.9, 41.8,
36.7, 35.9, 34.4, 31.4, 30.4, 26.8, 26.3, 26.2, 24.8, 16.8, 14.9, 11.5.
4.5.5. 14-O-[L(ꢁ)-phenylglycinamide-2-methylpropane-2-yl) thioa-
cetate] Mutilin (5e)
White solid; yield: 79%; mp: 62e64 ꢀC; IR (KBr): 3376, 22930,
1731, 1669, 1522, 1456, 1417, 1387, 1282, 1190, 1117, 1019, 981, 954,
917, 699 cmꢁ1; 1H NMR (400 MHz, CDCl3)
d
0.70 (d, 3H, J ¼ 6.4 Hz),
0.89 (d, 3H, J ¼ 6.4 Hz), 1.15e1.23 (t, 14H), 1.44e1.54 (t, 5H), 1.63e
1.79 (q, 4H), 1.99e2.09 (t, 3H), 2.23e2.32 (t, 7H), 3.06e3.25(m,
4H), 3.35 (d, 1H), 4.56 (s, 1H), 5.14e5.26 (q, 2H), 5.72(d, 1H),
6.39e6.46 (q, 1H, J1 ¼10.8 Hz, J2 ¼ 11.2 Hz), 7.27e7.33 (q, 3H), 7.43e
4.5.1. 14-O-[(2-aminobenzamide-2-methylpropane-2-yl) thioacetate]
Mutilin (5a)
7.44 (d, 2H), 7.63 (s, 1H); 13C NMR (100 MHz, CDCl3)
d 216.9, 173.0,
White solid; yield: 81%; mp: 70e72 ꢀC; IR (KBr): 3342, 2929,
2871, 1717, 1633, 1584, 1532, 1455, 1372, 1292, 1189, 1159, 1117, 1019,
169.6, 138.9, 128.7, 127.9, 126.8, 117.1, 74.5, 69.6, 59.9, 58.1, 47.4, 47.0,
45.4, 44.7, 43.8, 41.7, 36.6, 35.9, 34.4, 31.4, 30.3, 29.6, 26.8, 26.3, 26.2,
24.8, 16.8, 14.8, 11.5.
980, 936, 916, 746 cmꢁ1 1H NMR (400 MHz, CDCl3)
; d 0.72 (t, 3H,
J ¼ 7.2 Hz), 0.86 (t, 3H, J ¼ 6.8 Hz), 1.11e1.15 (q, 4H), 1.12e1.25 (q,
4H), 1.29e1.38 (q, 7H), 1.44e1.52 (q, 4H), 1.62e1.78 (m, 6H), 2.04e
2.30 (m, 6H), 3.15e3.34 (m, 4H), 3.39e3.44 (q, 1H, J ¼ 6.4 Hz),
3.68e3.72 (q, 2H, J ¼ 6.8 Hz), 5.11e5.19 (m, 2H), 5.72(d, 1H), 6.40e
6.45 (q, 1H, J1 ¼11.2 Hz, J2 ¼ 10.8 Hz), 6.66e6.99 (m, 2H), 7.19e7.27
4.5.6. 14-O-[(2-Indoleamide-2-methylpropane-2-yl) thioacetate]
Mutilin (5f)
White solid; yield: 71%; mp: 116e119 ꢀC; IR (KBr): 3327, 2927,
2851, 1731, 1627, 1573, 1549, 1456, 1419, 1374, 1308, 1282, 1189,
(m, 2H), 7.52e7.54 (q,1H); 13C NMR (100 MHz, CDCl3)
d
216.9,170.2,
1116, 1017, 979, 938, 946, 919, 705, 756, 747, 681, 617 cmꢁ1 1H
;
169.2, 148.8, 138.7, 132.2, 127.4, 117.3, 117.2, 116.6, 116.0, 74.5, 70.1,
58.2, 58.0, 47.6, 47.1, 45.4, 44.8, 43.9, 41.7, 36.6, 35.9, 34.4, 31.5, 30.3,
26.8, 26.4, 26.3, 24.7, 18.3, 16.8, 14.8, 11.4.
NMR (400 MHz, CDCl3)
d
0.77 (d, 3H, J ¼ 7.2 Hz), 0.88 (d, 3H,
J ¼ 7.2 Hz), 1.7 (s, 3H), 1.33e1.35 (d, 7H), 1.49 (s, 4H), 1.53e1.79 (m,
6H), 2.05e2.26 (m, 4H), 2.31e2.36 (t, 1H), 3.20e3.27 (t, 2H), 3.32e
3.38 (m, 2H), 3.49e3.54 (t, 1H), 5.15e5.24 (q, 2H), 5.80(d, 1H),
6.46e6.53 (q, 1H, J1 ¼ 11.2 Hz, J2 ¼ 10.8 Hz), 7.10 (d, 1H), 7.13e7.17
(t. 1H), 7.31 (d, 1H), 7.46 (d, 1H), 7.65e7.69 (t, 2H); 13C NMR
4.5.2. 14-O-[(3-aminobenzamide-2-methylpropane-2-yl) thioacetate]
Mutilin (5b)
White solid; yield: 55%; mp: 77e79 ꢀC; IR (KBr): 3380, 2931,
2873,1726,1666,1586,1537,1487,1454,1372,1285,1201,1138,1017,
(100 MHz, CDCl3) d 216.7, 170.8, 138.8, 130.9, 127.8, 124.4, 121.9,
120.6, 117.4, 111.9, 102.5, 74.6, 70.4, 58.1, 47.7, 47.6, 45.4, 44.9, 44.0,
41.8, 36.7, 36.0, 34.4, 31.6, 30.4, 26.9, 26.4, 26.3, 24.8, 16.9, 14.9,
14.2, 11.5.
980, 938, 917, 748, 722 cmꢁ1 1H NMR (400 MHz, CDCl3)
; d 0.71 (d,
3H, J ¼ 6.8 Hz), 0.86 (d, 3H, J ¼ 6.4 Hz), 1.13 (s, 4H), 1.24e1.30 (m,
9H), 1.37e1.45 (t, 5H), 1.52e1.77 (m, 4H), 2.04e2.09 (t, 2H), 2.18e
2.31 (m, 3H), 3.15e3.28(q, 3H), 3.35 (s, 1H), 3.45 (br, 1H), 5.09e
5.13 (t, 2H), 5.72(d, 1H), 6.36e6.43 (q, 1H, J1 ¼ 11.6 Hz,
J2 ¼ 11.2 Hz), 7.03 (br, 1H), 7.28 (d, 1H), 7.37e7.53 (m, 3H); 13C NMR
4.6. General procedure for the synthesis of compounds 6aeb
Chloroformate (3.7 mmol) was added dropwise to a solution of
compound 3 (1.6 g, 3.5 mmol) and triethylamine (0.81 g, 8 mmol) in
dichloromethane (60 mL) and was stirred at 0 ꢀC or room tem-
perature for 4.5 h. Then the reaction mixture was washed with
saturated aqueous NH4Cl and water and dried with anhydrous
Na2SO4 overnight. The solvent was evaporated in vacuum and the
residue was chromatographed on silica gel (petroleum ether:ethyl
acetate 2:1 v/v) to afford a pure product.
(100 MHz, CDCl3) d 216.9, 170.4, 138.7, 130.9, 129.6, 117.2, 74.6, 70.2,
60.4, 58.0, 47.7, 47.6, 45.4, 44.8, 43.9, 41.7, 36.6, 35.9, 34.8, 31.6, 30.5,
30.3, 26.8, 26.3, 26.2, 24.8, 19.1, 16.9, 14.8, 11.5.
4.5.3. 14-O-[(4-aminobenzamide-2-methylpropane-2-yl) thioacetate]
Mutilin (5c)
White solid; yield: 65%; mp: 86e85 ꢀC; IR (KBr): 3389, 2929,
2872, 1725, 1633, 1606, 1538, 1506, 1456, 1385, 1287, 1187, 1117, 1018,
980, 917, 842, 768 cmꢁ1
;
1H NMR (400 MHz, CDCl3)
d
0.72 (d, 3H,
4.6.1. 14-O-[1-N-carbamate-isobutyl-2-methylpropane-2-yl] thioa-
cetate] Mutilin (6a)
J ¼ 6.8 Hz), 0.87 (d, 3H, J ¼ 7.2 Hz), 1.14 (s, 4H), 1.25e1.31 (q, 9H), 1.45
(s, 4H), 1.50e1.79 (m, 5H), 2.05e2.24 (m, 5H), 2.29e2.33 (t, 1H),
3.15e3.28(m, 3H), 3.35 (s,1H), 3.44e3.49 (q,1H, J ¼ 6.4 Hz), 4.01 (br,
1H), 5.11e5.18 (t, 2H), 5.73 (d, 1H), 6.39e6.46 (q, 1H, J1 ¼ 10.8 Hz,
J2 ¼ 11.2 Hz), 6.67e6.69 (d, 2H), 7.16e7.19 (t, 1H), 7.71e7.74 (d, 2H);
White solid; yield: 89%; mp: 75e77 ꢀC; IR (KBr): 3481, 3440,
2959,1719,1698,1519,1456,1416,1385,1367,1286,1232,1189, 1119,
1038, 982, 942, 917 cmꢁ1; 1H NMR (400 MHz, CDCl3)
d 0.72 (t, 3H,
J ¼ 6.0 Hz), 0.88 (d, 3H, J ¼ 6.4 Hz), 0.91e0.94 (q, 6H), 1.09e1.12 (d,
1H), 1.63 (s, 3H), 1.25 (s, 6H), 1.31e1.38 (d, 2H), 1.44 (d, 4H), 1.47e
1.55 (q, 2H), 1.61e1.66 (t, 2H), 1.75e1.78 (d, 1H), 1.89e1.93 (t, 1H),
2.06e2.09 (d, 2H), 2.16e2.24 (m, 2H), 2.34 (s, 1H), 3.09e3.15(q, 4H),
3.33e3.35 (d 1H), 3.84 (d, 2H), 5.16e5.21 (d, 1H), 5.31e5.39 (t, 2H),
5.74(d, 1H), 6.42e6.49 (m, 1H, J1 ¼ 11.2 Hz, J2 ¼ 11.2 Hz), 13C
13C NMR (100 MHz, CDCl3)
d 216.8, 170.2, 166.9, 149.6, 138.7, 128.8,
124.1,117.3,114.1, 74.5, 70.1, 58.1, 53.8, 47.8, 47.4, 45.4, 44.9, 43.9, 41.8,
36.6, 35.9, 34.4, 31.5, 30.4, 29.2, 26.8, 26.4, 26.3, 24.8, 16.9, 14.8, 11.5.
4.5.4. 14-O-[D(ꢁ)-phenylglycinamide-2-methylpropane-2-yl) thioa-
cetate] Mutilin (5d)
NMR(100 MHz, CDCl3)
d 216.8, 169.6, 157.0, 138.9, 117.2, 74.6, 71.0,
White solid; yield: 79%; mp: 59e61 ꢀC; IR (KBr): 3373, 2891,
1731, 1667, 1519, 1455, 1415, 1385, 1282, 1190, 1117, 1018, 981, 954,
69.7, 58.1, 49.4, 47.2, 45.4, 44.8, 43.9, 41.7, 36.7, 35.9, 34.4, 31.3, 30.4,
28.0, 26.8, 26.3, 26.2, 26.1, 24.8, 19.0, 16.8, 14.8, 11.4.

238
R. Shang et al. / European Journal of Medicinal Chemistry 63 (2013) 231e238
4.6.2. 14-O-[1-N-carbamate-benzyl-2-methylpropane-2-yl]
thioacetate] Mutilin (6b)
Acknowledgments
White solid; yield: 78%; mp: 60e63 ꢀC; IR (KBr): 3418, 2931,
1727, 1519, 1455, 1417, 1385, 1282, 1251, 1140, 1117, 1016, 981,
This work was supported by Basic Scientific Research Funds in
Central Agricultral Scientific Research Institutions (no. 161032201
3004) and “Five-Year” plan of national science and technology
projects in rural areas (no. 2011AA10A214). We appreciate the 1H
NMR and 13C NMR analysis by Lanzhou University Analytical &
Testing Center, China.
917,698, 668 cmꢁ1 1H NMR (400 MHz, CDCl3)
; d 0.71 (d, 3H,
J ¼ 6.8 Hz), 0.87 (d, 3H, J ¼ 6.8 Hz), 1.12e1.17 (t, 5H), 1.26e1.33 (t,
7H), 1.38e1.42 (t, 2H), 1.45e1.50 (t, 5H), 1.55 (s, 2H), 1.61e1.77 (m,
4H), 2.04e2.10 (d, 2H), 2.14e2.25 (m, 2H), 2.31e2.34 (t, 1H), 3.09e
3.24(m, 4H), 3.32e3.36 (q 1H), 5.11e5.13 (d, 2H), 5.17 (s, 1H),
5.26e5.29 (d, 1H), 5.54 (s, 1H), 5.72e5.74 (d, 1H), 6.40e6.48 (q,
1H, J1 ¼ 11.2 Hz, J2 ¼ 10.8 Hz), 7.31e7.37 (m, 5H). 13C NMR
Appendix A. Supplementary data
(100 MHz, CDCl3)
d 216.8, 169.6, 156.6, 138.8, 136.6, 128.4128.1,
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2013.01.048.
117.2, 74.6, 69.7, 66.7, 58.1, 49.5, 47.1, 45.8, 45.4, 44.8, 43.8, 41.7,
36.7, 35.9, 34.4, 31.3, 30.4, 26.8, 26.3, 26.2, 26.1, 24.8, 16.8, 14.8,
11.5, 8.6.
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4.7.1. MIC of the compounds by agar dilution method
The minimum inhibitory concentration (MIC) values were
determined using agar dilution method according to NCCLS.
12800
mg synthesized compounds were weighed accurately and
dissolved in about 5 mL ethanol. Then distilled water was added to
the solution to 10 mL. Tiamulin fumarate and valnemulin hydro-
chloride as contrast drugs were dissolved in 10 mL distilled water.
Then all the solutions were diluted with distilled water by two fold.
2 mL of the 2-fold serial dilutions of each test compound/drug were
incorporated into 18 mL hot Mueller-Hintion agar medium. Inoc-
ulum of MRSA, MRSE, E. coli, and S. agalactiae which were all
separated from the clinic were prepared from blood slants and
adjusted to approximate 105e106 cfu/mL with sterile saline (0.90%
NaCl). A 10
mL amount of bacterial suspension was spotted into
Mueller-Hintion agar plates containing 2-fold serial dilutions of
compounds/drugs. The plates were incubated at 36.5 ꢀC for 48 h.
The MIC is defined as the minimum concentration of compound to
give complete inhibition of bacterial growth. The same procedure
was repeated in triplicate.
4.7.2. Oxford cup assay
Oxford cup assay was performed to evaluate the rate of inhibi-
tion in the growth of bacteria. Inoculum was prepared in 0.9% saline
using McFarland standard and spread uniformly on nutrient agar
plates. All the compounds were prepared as the same as MIC test by
agar dilution method and the resulting solution with varying con-
centration was added to the Oxford cups which were placed at
equidistance on the above agar surface. The zone of inhibition for
each concentration was measured after 24 h incubation at 37 ꢀC.