Synthesis and in vitro antibacterial activity of four novel pleuromutilin derivatives

Article abstract of DOI:10.4067/S0717-97072013000100008

Four novel pleuromutilin derivatives with butyl amine group in the C-14 side-chain were designed, synthesized and confirmed by FT-IR, 1H-NMR and HRMS. The results of antibacterial activity showed that the antibacterial activity of pleuromutilin derivative

Full text of DOI:10.4067/S0717-97072013000100008

J. Chil. Chem. Soc., 58, Nº 1 (2013)
SYNTHESIS AND IN VITRO ANTIBACTERIAL ACTIVITY OF FOUR NOVEL
PLEUROMUTILIN DERIVATIVES
YOUZHI TANG*#, JIAN LUO#, XIAOJIE CHEN, BO WANG, XIANGGUANG SHEN, JIANHUA LIU*
Guangdong Provincial Key Laboratory of Veterinary Pharmaceutics Development and Safety Evaluation,
College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China
(Received: May 4, 2012 - Accepted: October 4, 2012)
ABSTRACT
Four novel pleuromutilin derivatives with butyl amine group in the C-14 side-chain were designed, synthesized and confirmed by FT-IR, 1H-NMR and HR-
MS. The results of antibacterial activity showed that the antibacterial activity of pleuromutilin derivatives could be affected by the steric hindrance of their C-14
side-chain.
Key words: pleuromutilin derivatives, antibacterial, Staphylococcus aureus.
In order to develop new analogues with potent antibacterial activity, a
large number of pleuromutilin derivatives have been investigated. The most
successful group is semi-synthetic pleuromutilins with different substituent
groups on the C-14 ester side-chain of pleuromutilin.10 The C-14 sulfanyl-
acetate pleuromutilin derivatives of this group, including tiamulin, valnemulin,
azamulin and retapamulin, possess a broad-spectrum and excellent antibacterial
activity against various clinically relevant bacterial strains.11,12 However, these
C-14 sulfanyl-acetate pleuromutilin derivatives’ oral bioavailability is usually
limited because of their poor solubility in water. To overcome this problem,
Hirokawa and coworkers designed and synthesized a large number of C-14
sulfanyl-acetate pleuromutilin derivatives in 2008 and 2009. Several promising
compounds were got from C-14 sulfanyl-acetate pleuromutilin derivatives.13,14
But the C-14 acyl-carbamate derivatives, which include SB-571519 (6, Figure
2) and SB-222734 (7, Figure 2), possessed more metabolic stability than other
types of pleuromutilin derivatives. Such derivatives seemed more suitable for
oral administration.6,12
INTRODUCTION
Due to the dramatic increasing of antibiotic resistance among pathogenic
bacterial strains, research efforts are focusing on new antibiotics that have
unique modes of action and activity against resistant organisms. During the
past 25 years, the number of antibiotics which have been approved by the
1
Food and Drug Administration (FDA) decreased annually. Actually, after
the discovery of sulphonamide antibiotics in the 1930s and penicillin in
the 1940s, there were only three novel classes of antibacterials, the topical
antibiotic mupirocin, the oxazolidinones (linezolid) and the cyclic lipopeptides
(daptomycin) which had entered the market.2 This fact, as well as the demands
of new antibiotics discovery and development, have led to the reassessment of
previously discovered antibacterial agents which have not been used in humans
such as pleuromutilin (1, Figure 1).3
Pleuromutilin, a natural antibiotic with modest antibacterial activity,
was first isolated in 1951 from the basidiomycetes Pleurotus mutilis and P.
passeckerianus.4 Pleuromutilin derivatives selectively inhibit bacterial protein
synthesis through binding to the ribosomal peptidyl transferase center (PTC).5
This unique mode of action of pleuromutilin derivatives makes them to rarely
exhibit target-specific cross-resistance to other antibacterial agents. Two
pleuromutilin derivatives, timamulin (2, Figure 1) and valnemulin (3, Figure
1), have been used in veterinary medicine to treat serious infections in pigs and
poultry. Unfortunately, azamulin (4, Figure 1), the first human-used derivative
of pleuromutilin, failed in Phase I clinical trials for its rapid metabolism and
subsequent excretion in vivo.6 The semi-synthetic pleuromutilin derivative
retapamulin (5, Figure 1) was approved in 2007 by the FDA for the topical
treatment of uncomplicated skin and skin structure infections (SSSIs). Thus,
the pleuromutilin derivatives have regained interest as potential antibacterial
agents for human use. In addition to the structural modification of pleuromutilin,
more and more methods have also been reported to synthesize the molecular
framework of pleuromutilin.7-9
Figure 2. Chemical structures of compounds 6 and 7
Considering amines are usually as important functional groups to many
drugs, four novel pleuromutilin derivatives containing butyl amine in their
C-14 side-chain are described in this article, and the antibacterial activities of
these four derivatives against five sensitive strains were evaluated by a standard
procedure in vitro.
METHODS AND MATERIALS
Materials
Pleuromutilin (>90% pure) was obtained from Great Enjoyhood
Biochemical Co Ltd (Daying, China). The other reagents were all of analytical
grade and purchased from Guangzhou Chemical Reagent Factory (Guangzhou,
1
China). H-NMR spectra were measured on Bruker AV-300 or Bruker AV-
600 spectrometer in CDCl3. Chemical shift values (δ) were given in p.p.m.
and tetramethylsilane was used as internal standard. Fourier transform infrared
(FTIR) were obtained with a Nicolet 6700 Research FTIR spectrometer using
KBr pellets. Mass spectra were recorded with Agilent 6430 and LTQ-orbitrap
mass spectrometer (Thermo Fisher) using the electro spray ionization (ESI)
method.
Methods
Synthesis
The synthetic route was shown in Scheme 1.
Figure 1. Chemical structures of compounds 1~5
e-mail: youzhitang@scau.edu.cn
1537

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
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J. Chil. Chem. Soc., 58, Nº 1 (2013)
derivatives, with slightly modification.18 Treatment of butyl amine in acetonitrile followed by nucleophilic substitution reaction with compound 1a at 80ºC for 4
hours led to the desired compounds 8-11. Finally, the pure products could be obtained in good yield (53.1% for n-butyl amine, 62.0% for iso-butyl amine, 53.1
% for sec-butyl amine and 44.3% for tert-butyl amine) by silica gel column chromatography using petroleum ether/ethyl acetate/ethanol (5:1:0.2) as eluent. The
chemical structures of compounds 8-11 were confirmed by FT-IR, 1H-NMR, and HR-MS.
MIC testing
The in vitro antibacterial activity of compounds 8-11, pleuromutilin, tiamulin and valnemulin were tested. Their MIC values against five tested bacteria were
showed in Table 1.
Table 1. The MIC values of compounds 8~11, pleuromutilin, tiamulin and valnemulin against five tested bacteria (µg mL-1)
MIC values/(µg mL-1)
Compound
S. agalactiae CVCC
S. pyogenes
E. faecalis ATCC
E. coli ATCC
S. aureus ATCC
586
CVCC 593
29212
25922
29213
8
9
>64
>64
32
>64
>64
32
>64
>64
32
>64
>64
32
4
2
10
11
1
4
>64
32
>64
32
>64
32
>64
32
32
0.5
2
64
>64
64
64
32
0.125
>0.125
3
32
16
16
DISCUSSION
All four target compounds showed some antibacterial activity against all
five tested bacteria. The MIC values of compounds 8-11 against Staphylococcus
aureus ATCC 29213 were 4 µg mL-1, 2 µg mL-1, 4 µg mL-1 and 32 µg mL-
1, respectively. The pleuromutilin derivatives selectively inhibit bacterial
protein synthesis through their interaction with prokaryotic ribosome. The
C-14 side-chain extension of pleuromutilin derivatives is likely to enhance
their antimicrobial activity through strengthening the interaction between
pleuromutilin derivatives and ribosomal peptidyl transferase in bacteria.19
Considering our current results, it is proposed that the steric hindrance of tert-
butyl impedes the interaction between compound 11 and the ribosomal peptidyl
transferase of Staphylococcus aureus ATCC 29213, and then decreases the
antibacterial activity of compound 11.
The antibacterial activity of compounds 8-10 against Staphylococcus
aureus ATCC 29213 were much higher than the antibacterial activity of
them against the other four tested bacteria. It is supposed that the different
antibacterial activity of the same compound is caused by the difference of the
ribosome of the five tested bacterial. For example, the interaction between
compound 8 and the ribosomal peptidyl transferase of Staphylococcus
aureus ATCC 29213 is mode A (Figure 3), while the interaction between this
compound and the ribosomal peptidyl transferase of Escherichia coli ATCC
25922 is mode B (Figure 3). Such different mode of interaction might be the
reason of differential activity of compound 8 against these two bacteria.12,20
CONCLUSION
Four pleuromutilin derivatives were designed, synthesized and identified
in this paper. The MIC values of compound 8, 10 against Staphylococcus
aureus ATCC 29213 were 4 µg mL-1. The MIC value of compound 9 was 2
µg mL-1 for Staphylococcus aureus ATCC 29213, while that of compound 11
reached 32 µg mL-1. Antibacterial activity of pleuromutilin derivatives could
be affected by the steric hindrance of their C-14 side-chain. In addition, the
MIC values of compound 10 against other four tested bacterial reached 32 µg
mL-1, while that of compounds 8, 9 and 11 were greater than 64 µg mL-1. Thus,
pleuromutilin derivatives with short C-14 side-chain might not be good choice
for developing new antibacterial drugs against these four tested bacterial in this
paper. Our results could be used to enrich the content of SAR of pleuromutilin
and promote rational design of novel potent pleuromutilin-based antibiotic.
Figure 3. Supposed interaction between compound 8 and the ribosome
of bacterial. A: Interaction between compound 8 and the ribosome of
Staphylococcus aureus ATCC 29213; B: Interaction between compound 8 and
the ribosome of Escherichia coli ATCC 25922
ACKNOWLEDGMENTS
This work was supported by Program Foundation for Distinguished Young
Talents in Higher Education of Guangdong, China (No. LYM09032), National
Natural Science Foundation of China (No. 31001085), Foundation of President
of South China Agricultural University (No. 5500-K11046), The undergraduate
innovative experiment project of Guangdong Province (No. SCAU109).
REFERENCES
1. R. Novak, Ann. N. Y. Acad. Sci. 1241, 71, (2011)
2. M. S. Butler, A. D. Buss, Biochem. Pharmacol. 71, 919, (2006)
3. B. Boyd, J. Castaner, Drugs Future 31, 107, (2006)
1539

J. Chil. Chem. Soc., 58, Nº 1 (2013)
4. F. Kavanagh, A. Hervey, W. J. Robbins, Proc. Natl. Acad. Sci. U. S. A. 37,
570, (1951)
5. L. Lolk, J. Pohlsgaard, A. S. Jepsen, L. H. Hansen, H. Nielsen, S. I.
Steffansen, L. Sparving, A. B. Nielsen, B. Vester, P. Nielsen, J. Med.
Chem. 51, 4957, (2008)
6. G. Brooks, W. Burgess, D. Colthurst, J. D. Hinks, E. Hunt, M. J. Pearson,
B. Shea, A. K. Takle, J. M. Wilson, G. Woodnutt, Bioorg. Med. Chem. 9,
1221, (2001)
7. J. Liu, S. D. Lotesta, E. J. Sorensen, Chem. Commun. 47, 1500, (2011)
8. S. D. Lotesta, J. Liu, E. V. Yates, I. Krieger, J. C. Sacchettini, J. S.
Freundlich, E. Sorensen, J. Chem. Sci. 2, 1258, (2011)
9. T. J. K. Findley, D. Sucunza, L. C. Miller, M. D. Helm, M. Helliwell, D.
T. Davies, D. J. Procter, Org. Biomol. Chem. 9, 2433, (2011)
10. O. A. Phillips, L. H. Sharaf, Expert Opin. Ther. Pat. 17, 429, (2007)
11. E. Hunt, Drugs Future 25, 1163, (2000)
12. C. Davidovich, A. Bashan, T. Auerbach-Nevo, R. D. Yaggie, R. R.
Gontarek, A. Yonath, Proc. Natl. Acad. Sci. U. S. A. 104, 4291, (2007)
13. Y. Hirokawa, H. Kinoshita, T. Tanaka, T. Nakamura, K. Fujimoto, S.
Kashimoto, T. Kojima, S. Kato, Bioorg. Med. Chem. Lett. 18, 3556, (2008)
14. Y. Hirokawa, H. Kinoshita, T. Tanaka, T. Nakamura, K. Fujimoto, S.
Kashimoto, T. Kojima, S. Kato, Bioorg. Med. Chem. Lett. 19, 170, (2009)
15. Institute, C. A. L. S. (2008)
16. N. Rodger, S. D. M, Curr. Opin. Invest. Drugs 11, 182, (2010)
17. Y. Z. Tang, Y. H. Liu, J. X. Chen, Mini-Rev. Med. Chem. 12, 53, (2012)
18. P. Xu, Y. Zhang, Y. Sun, J. Liu, B. Yang, Y. Wang, Y. Wang, Chem. Biol.
Drug Des. 73, 655, (2009)
19. K. S. Long, L. H. Hansen, L. Jakobsen, B. Vester, Antimicrob. Agents
Chemother. 50, 1458, (2006)
20. G. Hogenauer, C. Ruf, Antimicrob. Agents Chemother. 19, 260, (1981)
1540