J. Chil. Chem. Soc., 58, Nº 1 (2013)
1637.92, 1464.54, 1154.54, 1116.74, 1037.83; HR-MS (ESI): Calcd for
C26H44NO4 (M-H+):434.32648; Found:434.32648.
22-N-tert-butylpleuromutilin (11)
Prepared according to the above general preparation of pleutromutilin
derivatives 8-11. White powder; yield: 44.3%; m.p.: 174.7-176.4ºC; 1H-NMR
spectrum (600 MHz; d1-CDCl3; TMS): δ (ppm) 6.513 (1H, dd, J 10.8, 17.4,
H19), 5.776 (1H, d, J 8.4, H14), 5.341 (1H, d, J 11.4, H20), 5.195 (1H, dd, J
17.4, H20), 3.389-3.315 (2H, m, H22), 3.262 (1H, d, J 17.4, H11), 2.366-2.041
(5H, m, H2, H4, H10, H13), 1.785-1.756 (1H, m, H8), 1.684-1.440 (6H, m, H1,
H6, H7, 11-OH), 1.454 (3H, s, H15), 1.383-1.348 (1H, m, H8), 1.323 (1H, d, J
16.2, H13), 1.159 (3H, s, H18), 1.083 (9H, s, (CH3)3NH), 0.877 (3H, d, J 6.6,
H17), 0.717 (3H, d, J 7.2, H16); IR (KBr, cm-1) 3316.59, 2935.51, 2882.35,
1736.63, 1641.43, 1467.34, 1153.12, 1118.63, 1016.50; HR-MS (ESI): Calcd
for C26H44NO (M-H+):434.32648; Found:434.32626.
Scheme 1. The synthetic route of pleutromutilin derivatives 8~11
Minimal4inhibitory concentration (MIC) testing
22-O-tosylpleuromutilin (1a)
MIC values of 4 novel pleuromutilin derivatives, tiamulin, valnemulin
and pleuromutilin were determined by agar dilution in accordance with the
“Clinical and Laboratory Standards Institute” (CLSI, 2008).15 The following
reference strains were used for quality control: Streptococcus agalactiae
CVCC 586, Streptococcus pyogenes CVCC 593, Enterococcus faecalis ATCC
29212, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213.
Preparation of stock and working solutions of compounds 8-11
Stock solutions of compounds 8-11 were prepared in N,N-
Dimethylformamide (DMF) at the concentrations of 5120 µg/ml. The working
solutions (256 µg/ml) were obtained by diluting stock solutions in sterile
Mueller Hinton broth.
A solution of pleuromutilin 1 (4.8 g, 12.69 mmol) in pyridine (8.6 mL)
was stirred at 0ºC in a three-necked round bottom flask, and p-toluenesulfonyl
chloride (3.6 g, 18.87 mmol) was added. The solution was stirred at 0ºC for 3h.
CHCl3 (50 mL) and Ice-cold water (50 mL) were added to the solution. The
organic phase was washed with a 2 M aqueous solution of H2SO4, a saturated
aqueous solution of NaHCO3 and water, respectively. Then the organic phase
was dried with anhydrous Na2SO4 for 2 h and evaporated in vacuum. The
residue was precipitated from isopropanol to give a white solid (3.8 g, 56.3%),
MS (ESI) m/z 555.4 [M+Na]+.
The synthetic route of pleuromutilin derivatives 8-11
5
A solution of compound 1a (1.3 g, 2.4 mmol) in acetonitrile (10 mL)
was added a solution of a butylamine (n-butylamine, iso-butylamine, (±)-sec-
butylamine and tert-butylamine) (1.3 mL) in acetonitrile (20 ml) in three-
necked round bottom flask. The mixture was stirred at 80ºC for 4 h, followed
by cooling to below 60ºC. Water (50 mL) was added to the solution, which
was then extracted with CHCl3 (50 mL). The organic phase was washed with a
saturated aqueous solution of NaHCO , water, dried (Na SO4) and concentrated
under reduced pressure to give the 3crude product. T2he crude product was
purified by silica gel column chromatography using petroleum ether/ethyl
acetate/ethanol (5:1:0.2) as eluent to give a pure product.
Preparation of bacteria solution
After all five strains were all recovered, single colony selected was
inculated on Mueller Hinton agar respectively, follow by incubation for 18-
24 h at 37ºC. Then, inocula were prepared by transferring several colonies of
bacteria to saline. The suspensions were mixed for 15 s and then corrected to
0.5 McF arland standard using saline. Further dilutions in saline were made to
get the required working suspensions (105 CFU/ml).
The determination of MIC
The test was performed in 96-well plate. All dates were tested in duplicate
in each plate. 100 µl of Mueller Hinton broth was added into all the wells of the
96-well plate. 100 µl of the working solutions (256 µg/ml) of compounds 8-11
were added into the wells in rows A to H in column 1 and well mixed. Then
100 µl of the mixture in column 1 was inhaled to add into the wells in column
2, well mixed. The similar operation was repeated until the 10th row of wells
was filled. 100 µl of excess medium was discarded from the wells in column
10. 100 µl of the bacteria solution was added to the wells in rows A to H in
columns 1 to 10, well mixed.
Two columns served as drug-free controls (no cultures were added in one
column and drugs replaced by blank solvent in the other column). Tiamulin
and Valnemulin were used as positive controls against bacteria. The final
concentration of DMF in the first well column was 1.25%. Initially, preliminary
analyses were conducted with 1.25% (v/v) DMF/MHB and this did not affect
neither the growth of the tested bacteria nor the determination of MIC. The
concentration of drugs in each row of well were 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25,
0.125 µg/ml respectively. Each 96-well plate was covered and incubated for
18-24 h at 37ºC. The MIC value was defined as the lowest concentration of the
sample which inhibits the visible growth of test bacteria.
22-N-n-butylpleuromutilin (8)
Prepared according to the above general preparation of pleutromutilin
derivatives 8-11. Light pink powder; yield: 53.1%; m.p.: 55.0-57.4ºC; 1H-NMR
spectrum (300 MHz; d1-CDCl3; TMS): δ (ppm) 6.528 (1H, dd, J 11.1, 17.4,
H19), 5.797 (1H, d, J 8.4, H14), 5.357 (1H, dd, J 1.2, 10.8, H20), 5.205 (1H,
dd, J 1.5, 17.4, H20), 3.355 (2H, d, J 17.4, H22), 3.255 (1H, J 17.7, H11),
2.856-2.487 (2H, m, CH CH2CH CH NH), 2.386-2.039 (5H, m, H2,H4, H10,
H13), 1.807-1.471 (9H, 3m, H1, 2H6,2 H7, H8, 11-OH, CH3CH2CH2CH NH),
1.452 (3H, s, H15), 1.400-1.278 (4H, m, H8, H13, CH CH CH CH NH), 21.167
(3H, s, H18), 0.932-0.870 (6H, m, H17, CH3CH2CH32CH22NH2), 0.2724 (3H, d,
J 6.6, H16); IR (KBr, cm-1) 3448.44, 2955.14, 2864.59, 1728.97, 1637.45,
1458.33, 1153.60, 1117.08, 1018.74; HR-MS (ESI): Calcd for C26H44NO4 (M-
H+):434.32648; Found:434.32578.
22-N-iso-butylpleuromutilin (9)
Prepared according to the above general preparation of pleutromutilin
derivatives 8-11. White powder; yield: 62.0%; m.p.: 130.0-130.8ºC; 1H-NMR
spectrum (600 MHz; d1-CDCl3; TMS): δ (ppm) 6.523 (1H, dd, J 11.4, 17.4,
H19), 5.794 (1H, d, J 8.4, H14), 5.354 (1H, dd, J 1.2, 10.8, H20), 5.203 (1H,
dd, J 1.2, 17.4, H20), 3.371-3.324 (2H, m, H22), 3.245 (1H, d, J 17.4, H11),
2.437-2.054 (7H, m, H2, H4, H10, H13, (CH3)2CHCH2NH), 1.789-1.597
(6H, m, H1, H6, H7, 11-OH,), 1.579-1.529 (1H, m, H8), 1.476-1.456 (1H, m,
(CH3)2CHCH NH) 1.448 (3H, s, H15), 1.390-1.341 (1H, m, H8) 1.306 (1H, d,
J 16.2, H13), 21.165 (3H, s, H18), 0.909 (6H, dd, J 1.8, 2.4, (CH3)2CHCH2NH),
0.878 (3H, d, J 6.6, H17), 0.723 (3H, d, J 7.2, H16); IR (KBr, cm-1) 3448.52,
2954.54, 2870.12, 1727.50, 1637.61, 1459.23, 1150.84, 1115.58, 1017.46; HR-
MS (ESI): Calcd for C26H44NO4 (M-H+):434.32648; Found:434.32605.
22-N-sec-butylpleuromutilin (10)
Prepared according to the above general preparation of pleutromutilin
derivatives 8-11. White powder; yield: 53.1%; m.p.: 107.5-109.3ºC; 1H-NMR
spectrum (600 MHz; d1-CDCl3; TMS): δ (ppm) 6.532-6.478 (1H, m, H19),
5.806-5.759 (1H, m, H14), 5.354-5.318 (1H, m, H20), 5.216-5.170 (1H, m,
H20), 3.454-3.316 (3H, m, H11, H22), 2.628 (1H, br s, 11-OH), 2.377-2.034
(6H, m, H2, H4, H10, H13, CH3CH2CH(CH3)NH), 1.775 (1H, dd, J 3.0, 14.4,
H8), 1.686-1.464 (5H, m, H1, H6, H7), 1.451 (3H, s, H15), 1.438-1.303 (4H,
m, H8, H13, CH3CH2CH(CH3)NH ), 1.165 (3H, d, J 2.4, H18), 1.050 (3H, dd,
J 6.0, 6.6, CH3CH2CH(CH3)NH), 0.925-0.845 (6H, m, H17, CH3CH2CH(CH3)
NH), 0.706 (3H, dd, J 7.2, H16); IR (KBr, cm-1) 3452,18 2957.82, 1735.34,
RESULTS
Synthesis
The structural modification of pleuromutilin focused on variations of
the C-14 side-chain. Previous to this research, tiamulin and valnemulin were
successfully developed for veterinary use; retapamulin was successfully
approved for use in human skin infections.16,17 The success of these three
drugs showed that pleuromutilin derivatives with different C-14 side-chain
might yield other candidates suitable for developing new antibacterial drugs
in the future. The steric configuration of the C-14 side-chain of pleuromutilin
derivatives might influence their antibacterial activities. In order to clarify
this influence, four pleuromutilin derivatives which have n-butyl, iso-butyl,
sec-butyl and tert-butyl groups in their C-14 side-chain were designed and
synthesized (8-11). The results showed that the compound having a tert-butyl
amine side chain possessed less antibacterial activity against Staphylococcus
aureus than the other derivatives.
Reaction of pleuromutilin (1) with p-toluenesulfonyl chloride in pyridine
at 0ºC gave compound 1a. The compounds 8-11 were prepared via four
amines according to the method described in the literature for pleuromutilin
1538