Synthesis and antibacterial activities of pleuromutilin derivatives with modified 7-aminocephalosporin acid and thioether moiety

Article abstract of DOI:10.14233/ajchem.2013.13120

In order to study the effect of heterocyclic carboxamide group on pleuromutilin's antibacterial activity, seven novel pleuromutilin derivatives with modified 7-aminocephalosporin acid and thioether moiety were designed and synthesized. The antibacterial a

Full text of DOI:10.14233/ajchem.2013.13120

Asian Journal of Chemistry; Vol. 25, No. 1 (2013), 397-400
http://dx.doi.org/10.14233/ajchem.2013.13120
Synthesis and Antibacterial Activities of Pleuromutilin Derivatives with
Modified 7-Aminocephalosporin Acid and Thioether Moiety
1
2
1
1,*
1
2
A-RONG GUO , HUA-HONG ZHOU , KUI-KUI DONG , KAI-QUN WU , YU-LIANG WANG and TIAN CHEN
1Faculty of Chemistry, Sichuan University, Chengdu 610065, P.R. China
2Department of Pathogenic Biology, School of Biomedical Science, Chengdu Medical College, Chengdu 610083, P.R. China
*Corresponding author: Tel: +86 13708058490; E-mail: wukaiqun@tom.com; gyr9.221@163.com
(Received: 26 November 2011;
Accepted: 28 July 2012)
AJC-11887
In order to study the effect of heterocyclic carboxamide group on pleuromutilin's antibacterial activity, seven novel pleuromutilin derivatives
with modified 7-aminocephalosporin acid and thioether moiety were designed and synthesized. The antibacterial activities of the new
compounds were tested via agar-well diffusion method in vitro under different concentrations. The results showed that three target
compounds (5a, 5d, 5f) still had antibacterial activity against Staphylococcus aureus SC and Staphylococcus aureus ATCC26112 at the
concentration 2.0 µg/mL.
Key Words: Pleuromutilin derivatives, 7-Aminocephalosporin acid, Synthesis, Antibacterial activity, Agar-well diffusion.
In our previous works, some pleuromutilin derivatives
containing substituent pyrazole carboxamide12 and substituent
thiazole carboxamide13 (Fig. 1) were reported to exist excellent
antibacterial activity. Herein, for the propose of further
exploring the effect of heterocyclic carboxamide groups on
the antibacterial activity of pleuromutilin, some modified
7-aminocephalosporin acids (7-ACA) was introduced into
pleuromutilin by amido bond. The synthetic route was showed
in Scheme-I, the antibacterial activities of the target compounds
were tested via agar-well diffusion method.
INTRODUCTION
The antibiotic pleuromutilin (Fig. 1) was first isolated in
1951 by Kavanagh and co-workers1. Early research showed
that this kind of antibiotic has modest activity in vitro against
Gram-positive pathogens and mycoplasmas2,3. Pleuromutilin
selectively inhibits bacterial protein synthesis through inter-
action with prokaryotic ribosome4.A Sandoz group5 indicated
that the modification at C14 side chain could be the most
promising approach to obtain optimum activity and the combi-
nations of both the thioether group and the basic group in the
side chain would give excellent bioactivity. Based on these
structure and activity relationships, tiamulin6, valunemulin7,8
and retapamulin9,10 were successfully developed as therapeutic
pharmaceuticals. In 2008, pleuromutilin analogs with purine
ring possessing excellent antibacterial activity had been
reported by Hirokawa and coworkers11.
EXPERIMENTAL
Compounds 5a-g were prepared as shown in Scheme-I.
Intermediates 2a-g were prepared according to the literature
with little modification14. Intermediates 2 was suspended in
dried dichloromethane and Et3N at 0 ºC where 1-(3-dimethyl-
aminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and
O
R
3
Me
O
O
Me
OH
S
(5): R =
S
S
N
R
Valunemulin(3): R =
N
H
H
R
O
1
Me
N
N
H N
2
Me
Me
R
2
O
O
R
S
Me
Pleuromutilin(1): R = OH
N
S
(6): R =
Ratapamulin(4): R =
N
H
Me
Me
N
S
Tiamutilin(2): R =
N
Fig. 1. Pleuromutilin and pleuromutilin derivaties

398 Guo et al.
Asian J. Chem.
H
N
O
R
H2N
S
O
a
S
O
R
N
O
+
Cl
O
N
O
O
O
COOH
O
COOH
O
2a-g
1
7-ACA
O
O
O
Me
S
Me
O
Me
H2N
O
OH
OH
O
OH
Ts
O
OH
Me
Me
Me
Me
Me
Me
Me
Me
Me
O
O
4
OAc
O
Pleuromutilin
3
O
O
S
Me
S
N
H
N
O
OH
b
HN
O
Me
Me
O
O
Me
R
O
5a-g
2a,5a: R=CH3; 2b,5b: R=CH3CH2; 2c,5c: R=(CH3)2CH; 2d,5d: R=(CH3)2CH2CH; 2e,5e: R=(CH3)3C; 2f,5f: R=C6H5CH2; 2g,5g: R=CCl3CH2
Scheme-I: Synthetic route of compound 5a-g
1-hydroxybenzotriazole (HOBT) were rapidly added and the
reaction mixture was maintained at room temperature for 2 h.
Then compound 4 (which was synthesized according to the
literature12) was added into the solution at 0 ºC and the resulting
solution was allowed to be stirred at room temperature for
another 24 h. The solution was then washed with H2O and
saturated sodium chloride solution. The organic layer was
dried, filtered and evaporated under reduced pressure. The
crude products were purified on silica-gel column (petroleum
ether/ethyl acetate as the eluent).
2.09 (m, 3H), 2.12-2.33 (m, 3H), 3.06-3.18 (m, 3H), 3.20-
3.29 (m, 3H), 3.31-3.34 (m, 2H), 3.65 (m, 2H), 4.61 (d, 1H,
J = 12.8 Hz), 4.84 (d, 1H, J = 12.8 Hz), 4.90 (s, 1H), 5.14 (dd,
1H, J1 = 17.2 Hz, J2 = 9.2 Hz), 5.23 (dd, 1H, J1 = 10.4 Hz, J2
= 8.4 Hz), 5.40 (m, 1H), 5.48-5.51 (m, 1H), 6.56 (s, 1H), 7.60
(s, 1H); HR-MS (-ESI): Calcd. for C39H56N3O10S2 [M-H]¯:
790.3406, Found: 790.3412.
Mutilin-14-O-[1-(7-isopropoxycarbamidocephalo-
sporanic carboxamide)-2-methylpropane-2-yl] thioacetate
(5c):Yellow solid; yield: 61.28 %; m.p.: 98-102 ºC; IR (KBr,
Mutilin-14-O-[1-(7-methoxycarbamidocephalos-
poranic carboxamide)-2-methylpropane-2-yl] thioacetate
(5a): Orange solid; yield: 76.34 %; m.p.: 106-108 ºC; IR (KBr,
ν
max, cm-1): 3441, 2961, 2927, 2857, 1778, 1726, 1532, 1459,
1
1379, 1260, 1111, 1024, 914, 803, 616; H NMR spectrum
(400 MHz; d1-CDCl3; TMS): δ (ppm) 0.73 (d, 3H, J = 6.8
Hz), 0.90 (d, 3H, J = 7.2 Hz), 1.11-1.14 (m, 1H), 1.17 (s, 3H),
1.22-1.28 (m, 6H), 1.30 (s, 3H), 1.32-1.42 (m, 3H), 1.46 (s,
3H), 1.56 (s, 3H), 1.62-1.70 (m, 3H), 1.80-1.83 (m, 1H), 2.05
(s, 3H), 2.10-2.16 (m, 3H), 2.17-2.44 (m, 3H), 3.14-3.30 (m,
6H), 3.34-3.56 (m, 2H), 4.69 (d, 1H, J = 12.8 Hz), 4.90-5.03
(m, 2H), 5.20 (dd, 1H, J1 = 17.6 Hz, J2 = 9.6 Hz), 5.31 (d, 1H,
J = 7.6 Hz), 5.36-5.49 (m, 1H), 5.80 (d, 1H, J = 8.8 Hz), 6.44
(dd, 1H, J1 = 17.2 Hz, J2 = 11.2 Hz), 7.52 (s, 1H), 7.56 (s, 1H);
HR-MS (-ESI): Calcd. for C40H58N3O10S2 [M-H]¯: 804.3563,
Found: 804.3567.
ν
max, cm-1): 3402, 3084, 2931, 2870, 1729, 1664, 1537, 1459,
1
1375, 1280, 1117, 1022, 915, 587; H NMR spectrum (400
MHz; d1-CDCl3; TMS): δ (ppm) 0.73 (d, 3H, J = 6.8 Hz),
0.90 (d, 3H, J = 6.8 Hz), 1.10-1.15 (m, 1H), 1.18 (s, 3H),
1.26-1.28 (m, 3H), 1.31 (s, 3H), 1.33-1.45 (m, 3H), 1.47 (s,
3H), 1.60 (s, 3H), 1.65-1.71 (m, 3H), 1.77-1.81 (m, 1H), 2.06
(s, 3H), 2.07-2.14 (m, 3H), 2.18-2.35 (m, 3H), 3.12-3.30 (m,
6H), 3.32-3.38 (m, 2H), 3.73 (s, 3H), 5.19-5.23 (m, 2H), 5.32
(dd, 1H, J1 = 10.8 Hz, J2 = 1.2 Hz), 5.74-5.78 (m, 2H), 6.48
(dd, 1H, J1 = 17.6 Hz, J2 = 11.2 Hz); HR-MS (-ESI): Calcd.
for C38H54N3O10S2 [M-H]¯: 776.3251, Found: 776.3265.
Mutilin-14-O-[1-(7-ethoxycarbamidocephalosporanic
carboxamide)-2-methylpropane-2-yl] thioacetate (5b):
Mutilin-14-O-[1-(7-isobutoxycarbamidocephalos-
poranic carboxamide)-2-methylpropane-2-yl] thioacetate
(5d): Yellow solid; yield: 63.57 %; m.p.: 71-73 ºC; IR (KBr,
ν
max, cm-1): 3403, 3079, 2921, 2928, 2865, 1729, 1663, 1540,
1461, 1375, 1280, 1117, 1023, 915; 1H NMR spectrum (400
MHz; d1-CDCl3; TMS): δ (ppm) 0.73 (d, 3H, J = 7.2 Hz),
0.90 (d, 3H, J = 6.8 Hz), 0.99 (d, 6H, J = 6.8 Hz), 1.10-1.15
(m, 1H), 1.18 (s, 3H), 1.21-1.28 (m, 3H), 1.31 (s, 3H), 1.35-
1.43 (m, 3H), 1.46 (s, 3H), 1.48-1.62 (m, 3H), 1.65-1.70 (m,
3H), 1.76-1.80 (m, 1H), 1.99 (s, 3H), 2.05-2.18 (m, 3H), 2.20-
Yellow solid; yield: 75.83 %; m.p.: 94-96 ºC; IR (KBr, νmax
,
cm-1) : 3393, 3075, 2960, 2929, 2857, 1775, 1726, 1677, 1537,
1
1457, 1377, 1262, 1115, 1022, 914, 802, 731; H NMR
spectrum (400 MHz; d1-CDCl3; TMS): δ (ppm) 0.67 (d, 3H, J
= 6.8 Hz), 0.85 (d, 3H, J = 6.8 Hz), 1.02-1.08 (m, 1H), 1.12
(m, 3H), 1.18 (s, 3H), 1.19 (s, 3H), 1.26-1.37 (m, 3H), 1.40 (s,
3H), 1.43-1.57 (m, 3H), 1.65-1.77 (m, 1H), 1.99 (s, 3H, 2.02-

Vol. 25, No. 1 (2013)
Synthesis and Antibacterial Activities of Pleuromutilin Derivatives 399
2.35 (m, 4H), 3.11-3.31 (m, 6H), 3.36-3.40 (m, 2H), 4.09 (d,
2H, J = 6.8 Hz), 5.27 (dd, 1H, J1 = 17.6 Hz, J2 = 1.6 Hz), 5.32
(dd, 1H, J1 = 11.2 Hz, J2 = 1.6 Hz), 5.74 (d, 1H, J = 8.4 Hz),
6.43 (dd, 1H, J1 = 17.2 Hz, J2 = 10.8 Hz), 7.52 (m, 1H); HR-
MS (-ESI): Calcd. for C41H60N3O10S2 [M-H]¯: 818.3719,
Found: 818.3725.
covered with bacteria suspension in advance and the plates
were incubated at 37 ºC for 24 h. The results of average
diameters of the bacteriostatic circle were listed in Tables 1
and 2.
TABLE-1
In vitro ANTIBACTERIAL ACTIVITY OF 5a-g AT
THE CONCENTRATION 20 µg/mL
Mutilin-14-O-[1-(7-t-butoxycarbamidocephalo-
sporanic carboxamide)-2-methylpropane-2-yl] thioacetate
(5e): Yellow solid; yield: 56.78 %; m.p.: 86-88 ºC; IR (KBr,
Diameter of inhibition zone (mm)
Compound
max, cm-1): 3419, 2959, 2924, 2855, 1726, 1526, 1459, 1372,
Staphylococcus
aureus SC
Staphylococcus aureus
ATCC26112
ν
1261, 1110, 1023, 914, 803, 618, 588; 1H NMR spectrum (400
MHz; d1-CDCl3; TMS): δ (ppm) 0.73 (d, 3H, J = 6.8 Hz),
0.88 (d, 3H, J = 6.8 Hz), 1.10-1.14 (m, 1H), 1.17 (s, 3H),
1.21-1.24 (m, 3H), 1.25 (s, 9H), 1.28 (s, 3H), 1.36-1.39 (m,
3H), 1.45 (s, 3H), 1.48-1.69 (m, 3H), 2.05 (s, 3H), 2.10 (s,
3H), 2.17-2.33 (m, 3H), 3.12-3.31 (m, 6H), 3.33-3.80 (m, 2H),
4.67-4.79 (m, 2H), 4.96 (d, 1H, J = 11.6 Hz), 5.19 (dd, 1H, J1
= 17.6 Hz, J2 = 8.0 Hz), 5.47 (d, 1H, J = 8.4 Hz), 5.55 (m,
1H), 5.76 (d, 1H, J = 8.4 Hz), 6.45 (dd, 1H, J1 = 16.4 Hz, J2 =
6.0 Hz); HR-MS (-ESI): Calcd. for C41H60N3O10S2 [M-H]¯:
818.3719, Found: 818.3728.
21
20
14
20
15
24
15
22
-
17
15
12
16
12
19
13
13
-
5a
5b
5c
5d
5e
5f
5g
Pleuromutilina
Ethanolb
aConcentration of pleuromutilin: 20.0 µg/mL; as the positive control;
bNegative control: ethanol; Diameter of the well in each plate: 6 mm
Mutilin-14-O-[1-(7-benzoxycarbamidocephalos-
poranic carboxamide)-2-methylpropane-2-yl] thioacetate
(5f): Yellow solid; yield: 64.66 %; m.p.: 58-60 ºC; IR (KBr,
TABLE-2
In vitro ANTIBACTERIAL ACTIVITY OF 5a, 5d and 5f
AT THE CONCENTRATION 2 µg/mL
ν
max, cm-1): 3388, 2962, 2924, 2855, 1729, 1661, 1524, 1458,
Diameter of inhibition zone (mm)
1377, 1261, 1097, 1022, 912, 800, 698, 586; 1H NMR spec-
trum (400 MHz; d1-CDCl3; TMS): δ (ppm) 0.73 (d, 3H, J =
6.8 Hz), 0.90 (d, 3H, J = 6.8 Hz), 1.10-1.15 (m, 1H), 1.18 (s,
3H), 1.26-1.28 (m, 3H), 1.31 (s, 3H), 1.36-1.43 (m, 3H), 1.46
(s, 3H), 1.49-1.62 (m, 3H), 1.65-1.70 (m, 3H), 1.76-1.80 (m,
1H), 2.02 (s, 3H), 2.05-2.13 (m, 3H), 2.20-2.35 (m, 3H), 3.11-
3.27 (m, 6H), 3.29-3.37 (m, 2H), 5.21 (dd, 2H, J1 = 17.2 Hz,
J2 = 1.2 Hz), 5.33 (d, 2H, J = 12.0 Hz), 5.75 (d, 1H, J = 8.8
Hz), 6.48 (dd, 1H, J1 = 17.6 Hz, J2 = 11.2 Hz), 7.34 (m, 5H);
HR-MS (-ESI): Calcd. for C44H58N3O10S2 [M-H]¯: 852.3563,
Found: 852.3581.
Compound
Staphylococcus
aureus SC
Staphylococcus
aureus ATCC26112
10
11
14
7
7
7
5a
5d
11
-
5f
Pleuromutilina
Ethanolb
-
-
aConcentration of pleuromutilin: 2.0 µg/mL; as the positive control;
bNegative control: ethanol; Diameter of the well in each plate: 6 mm
RESULTS AND DISCUSSION
Mutilin-14-O-[1-(7-N-carbamate-2,2,2-trichloroethyl)
-cephalosporanic carboxamide)-2-methylpropane-2-yl]
thioacetate (5g): Yellow solid; yield: 57.89 %; m.p.: 68-70
ºC; IR (KBr, νmax, cm-1): 3347, 2962, 2926, 2858, 1733, 1679,
1533, 1457, 1380, 1261, 1101, 1020, 866, 799, 702, 570; 1H
NMR spectrum (400 MHz; d1-CDCl3; TMS): δ (ppm) 0.74
(d, 3H, J = 6.8 Hz), 0.88 (d, 3H, J = 6.8 Hz), 1.10-1.15 (m,
1H), 1.17 (s, 3H), 1.31 (s, 3H), 1.33 (s, 3H), 1.37-1.43 (m,
3H), 1.47 (s, 3H), 1.49-1.58 (m, 3H), 1.61-1.70 (m, 3H), 1.76-
1.80 (m, 1H), 2.02 (s, 3H), 2.05-2.20 (m, 3H), 2.22-2.35 (m,
3H), 3.16-3.30 (m, 6H), 3.35 (d, 1H, J = 6.4 Hz), 3.48 (dd, 1H,
J1 = 14.1 Hz, J2 = 6.8 Hz), 5.17 (d, 1H, J = 17.5 Hz), 5.26 (d,
1H, J = 11.12 Hz), 5.77 (d, 1H, J = 8.5 Hz), 6.47 (dd, 1H, J1 =
17.6 Hz, J2 = 11.2 Hz), 7.67 (s, 1H), 7.89 (d, 1H, J = 8.0 Hz);
HR-MS (-ESI): Calcd. for C39H53N3O10S2Cl3 [M-H]¯: 892.2237,
894.2208. Found: 892.2247, 894.2225 [isotope peaks].
The antibacterial activities of the target compounds were
tested via agar-well diffusion method in vitro. Target compound
(1000 µg) was dissolved with ethanol (1 mL) and diluted to
20 µg/mL with ethanol, later, 5a, 5d and 5f were further diluted
to 2 µg/mL. A 50 µL solution of each compound (target com-
pounds or pleuromutilin) was injected into the corresponding
well in the Luria Bertani (LB) culture medium, which was
The results of antibacterial activities showed that all the
target compounds exhibited antibacterial activity against two
bacterial strains at the concentration 20 µg/mL. Three comp-
ounds (5a, 5d, 5f) were chosen to further test the antibacterial
activity at the concentration 2 µg/mL, they still displayed
antibacterial activity against Staphylococcus aureus SC and
Staphylococcus aureus ATCC26112.
Conclusion
In conclusion, seven novel pleuromutilin derivatives with
modified 7-aminocephalosporin acid and thioether moiety in
the C14 side chain were successfully synthesized. The results
of antibacterial activities indicated that three target compounds
(5a, 5d, 5f) still exist antibacterial activity against Staphylo-
coccus aureus SC and Staphylococcus aureus ATCC26112 at
the concentration 2.0 µg/mL. Our exploration has further
enriched the content of SAR of pleuromutilin and it is helpful
for us to design more pleuromutilin derivatives.
ACKNOWLEDGEMENTS
The authors appreciated the financial support from the
National Science Foundation of China (No. 21072135), the
antibacterial activity test of Chengdu Medical College (cx

400 Guo et al.
Asian J. Chem.
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