Pleuromutilin Derivatives Research Articles

Antibacterial Activity of a Promising Antibacterial Agent: 22-(4-(2-(4-Nitrophenyl-piperazin-1-yl)-acetyl)-piperazin-1-yl)-22-deoxypleuromutilin.

A novel pleuromutilin derivative, 22-(4-(2-(4-nitrophenyl-piperazin-1-yl)-acetyl)-piperazin-1-yl)-22-deoxypleuromutilin (NPDM), was synthesized in our laboratory and proved excellent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). In this study, more methods were used to further study its preliminary pharmacological effect. The antibacterial efficacy and toxicity of NPDM were evaluated using tiamulin as the reference drug. The in vitro antibacterial activity study showed that NPDM is a potent bactericidal agent against MRSA that induced time-dependent growth inhibition and a concentration-dependent post-antibiotic effect (PAE). Toxicity determination showed that the cytotoxicity of NPDM was slightly higher than that of tiamulin, but the acute oral toxicity study proved that NPDM was a low-toxic compound. In an in vivo antibacterial effect study, NPDM exhibited a better therapeutic effect than tiamulin against MRSA in a mouse thigh infection model as well as a mouse systemic infection model with neutropenia. The 50% effective dose (ED50) of NPDM in a Galleria mellonella infection model was 50.53 mg/kg. The pharmacokinetic properties of NPDM were also measured, which showed that NPDM was a rapid elimination drug in mice.

In Vitro and In Vivo Activity of 14-O-[(4,6-Diamino-pyrimidine-2-yl) thioacetyl] Mutilin against Methicillin-Resistant Staphylococcus aureus.

Staphylococcus aureus (S. aureus) is a major human pathogen that requires new antibiotics with unique mechanism. A new pleuromutilin derivative, 14-O-[(4,6-Diamino-pyrimidine-2-yl) thioacetyl] mutilin (DPTM), has been synthesized and proved as a potent antibacterial agent using in vitro and in vivo assays. In the present study, DPTM was further in vitro evaluated against methicillin-resistant Staphylococcus aureus (MRSA) isolated from dairy farms and outperformed tiamulin fumarate, a pleuromutilin drug used for veterinary. Moreover, a murine skin wound model caused by MRSA infection was established, and the healing effect of DPTM was investigated. The results showed that DPTM could promote the healing of MRSA skin infection, reduce the bacterial burden of infected skin MRSA and decrease the secretion of IL-6 and TNF-α inflammatory cytokines in plasma. These results provided the basis for further in-depth drug targeted studies of DPTM as a novel antibacterial agent.

Design, synthesis and antibacterial activity evaluation of pleuromutilin derivatives according to twin drug theory

A series of novel pleuromutilin derivatives were designed and synthesized based on the twin drugs theory. Piperazinyl and thioether were used as the linkage to connect the pleuromutilin nuclear and sulfonamide to improve the biological activity and water solubility of derivatives. The in vitro antibacterial activities against drug-sensitive and drug-resistance bacteria were evaluated by agar dilution method. Most of the 25 compounds displayed excellent antibacterial activities against drug-sensitive bacteria, particularly, five compounds (9, 10, 11, 14a and 14b) exerted the excellent antibacterial activities against drug-resistance bacteria.

Design, synthesis, in vitro and in vivo evaluation against MRSA and molecular docking studies of novel pleuromutilin derivatives bearing 1, 3, 4-oxadiazole linker

A class of pleuromutilin derivatives containing 1, 3, 4-oxadiazole were designed and synthesized as potential antibacterial agents against Methicillin-resistant staphylococcus aureus (MRSA). The ultrasound-assisted reaction was proposed as a green chemistry method to synthesize 1, 3, 4-oxadiazole derivatives (intermediates 85–110). Among these pleuromutilin derivatives, compound 133 was found to be the strongest antibacterial derivative against MRSA (MIC = 0.125 μg/mL). Furthermore, the result of the time-kill curves displayed that compound 133 could inhibit the growth of MRSA in vitro quickly (- 4.36 log10 CFU/mL reduction). Then, compound 133 (- 1.82 log10 CFU/mL) displayed superior in vivo antibacterial efficacy than tiamulin (- 0.82 log10 CFU/mL) in reducing MRSA load in mice thigh model. Besides, compound 133 exhibited low cytotoxicity to RAW 264.7 cells. Molecular docking studies revealed that compound 133 was successfully localized in the binding pocket of 50S ribosomal subunit (ΔGb = -10.50 kcal/mol). The results indicated that these pleuromutilin derivatives containing 1, 3, 4-oxadiazole might be further developed into novel antibiotics against MRSA.

Evaluation of Ferroptocide (FTC) As a Potential Immunogenic Cell Death Inducing Agent in Prostate Cancer Cells

Prostate cancer (PCa) is one of the most common types of cancer in men, and its incidence continues to rise in many countries. PCa is the sixth most common cancer in the world. It is the third most common cancer in men and the second most leading cause of death in American males. The American cancer society's estimated 191,930 new cases and 33,330 death from prostate cancer. The existing treatments for PCa include surgery, radiation therapy, chemotherapy, and hormonal therapy. However, these treatments have limited efficacy and patients often experience side effects. Although these treatment options show effects in the beginning stages subsequently, the majority of the patients develop resistance. Therefore, effective treatments for PCa are urgently required. A novel synthetic small molecule compound ferroptocide (FTC), a derivative of diterpene pleuromutilin is one such potential therapeutic agent against PCa. It displays potent tumor-suppressive effects in a variety of cancer models. Immunogenic Cell Death (ICD) pathways result in a regulated activation of tumor immunity that could potentially eliminate PCa. Therefore, agents that promote ICD is highly desirable. We hypothesize that ferroptocide (FTC) would be an effective anticancer agent against prostate cancer cells. In this study, we have evaluated the ICD inducing potential of FTC using human-derived LNCaP and mouse-derived TRAMP-C1 PCa cellular models. Our results showed that FTC potently inhibits the cell viability of LNCaP and TRAMP-C1 cells. Interestingly, FTC elevated the levels of ROS by utilizing DCFH-2DA probe and analyzed by confocal and flow cytometry, intracellular ROS time point study by flow cytometry depicted that FTC robustly induces ROS within 1hr. Mitochondrial superoxide was generated using MitoSox probe suggesting FTC induces oxidative stress and leading to G2/M cell cycle arrest and activation of apoptosis in LNCaP cells. Notably, FTC treatment promoted the secretion of Adenosine Triphosphate (ATP), in both LNCaP and TRAMP-C1 cells in a dose-dependent manner. Furthermore, Western blot analysis of LNCaP cells treated with FTC showed increased expression of ICD markers such as HMGB1 and HSP70. Confocal immunofluorescence analysis confirmed the higher expression of these ICD markers in both the cellular models. Hence, this is the first study to demonstrate that FTC targeting mitochondria induce mitophagy as well as its ability to induce ICD in prostate cancer cells. Our data suggest that FTC is a promising anticancer agent with the potential to activate ICD to effectively combat PCa.

Design, synthesis and biological evaluation of novel pleuromutilin derivatives as potent anti-MRSA agents targeting the 50S ribosome

A series of novel pleuromutilin derivatives were designed and synthesized with 1,2,4-triazole as the linker connected to benzoyl chloride analogues under mild conditions. The in vitro antibacterial activities of the synthesized derivatives against four strains of Staphylococcus aureus (MRSA ATCC 43300, ATCC 29213, AD3 and 144) were tested by the broth dilution method. Most of the synthesized derivatives displayed potent activities, and 22-(3-amino-2-(4-methyl-benzoyl)-1,2,4-triazole-5-yl)-thioacetyl)-22-deoxypleuromutilin (compound 12) was found to be the most active antibacterial derivative against MRSA (MIC = 0.125 μg/mL). Furthermore, the time-kill curves showed compound 12 had a certain inhibitory effect against MRSA in vitro. The in vivo antibacterial activity of compound 12 was further evaluated using MRSA infected murine thigh model. Compound 12 exhibited superior antibacterial efficacy than tiamulin. It was also found that compound 12 had no significant inhibitory effect on the proliferation of RAW264.7 cells. Compound 12 was further evaluated in CYP450 inhibition assay and showed moderate inhibitory effect on CYP3A4 (IC50 = 3.95 μM). Moreover, seven candidate compounds showed different affinities with the 50S ribosome by SPR measurement. Subsequently, binding of compound 12 and 20 to the 50S ribosome was further investigated by molecular modeling. Three strong hydrogen bonds were formed through the interaction of compound 12 and 20 with 50S ribosome. The binding free energy of compound 12 and 20 with the ribosome was calculated to be −10.7 kcal/mol and −11.66 kcal/mol, respectively.

Determination of 8α-hydroxymutilin as a Marker Residue for Tiamulin in Swine Tissue by Liquid Chromatography-Tandem Mass Spectrometry

Tiamulin is a semi-synthetic derivative of the natural antibiotic pleuromutilin and is widely used as a veterinary drug for swine. Herein, we report the development of a sensitive and reliable method for determining 8α-hydroxymutilin as a marker residue for tiamulin in swine tissue using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method consists of sample extraction with acetone, defatting by acetonitrile/hexane partitioning, hydrolysis of the tiamulin metabolites to 8α-hydroxymutilin under alkaline conditions, liquid-liquid extraction with ethyl acetate, cleanup using a primary secondary amine cartridge, and LC-MS/MS analysis. The developed method was validated for 8α-hydroxymutilin in swine muscle, fat, and liver at two levels, namely 0.01 mg/kg and the maximum residue limits established in Japan (i.e., 0.1 mg/kg for swine muscle and fat, and 0.6 mg/kg for liver). The trueness ranged from 82 to 89%, and the relative standard deviations ranged from 1 to 3%. No chromatographic interference was observed near the retention time of 8α-hydroxymutilin, and matrix effects were negligible for all matrices, suggesting that the cleanup protocol was effective. The calibration curve was linear in the 0.005–0.5 μg/mL range, with a coefficient of determination greater than 0.997. The developed method enabled accurate quantification using solvent-based calibration without compensating for matrix effects and losses during sample preparation. The limit of detection of the method was 0.0005 mg/kg for each matrix. The developed method is suitable for regulatory-purpose analysis of 8α-hydroxymutilin as a marker residue for tiamulin as defined by the European Union and several other countries.

Synthesis, biological activities and docking studies of pleuromutilin derivatives with piperazinyl urea linkage

Antibiotics resistance is becoming increasingly common, involving almost all antibiotics on the market. Diseases caused by drug resistant bacteria, such as MRSA, have high mortality and negatively affect public health. The development of new drugs would be an effective means of solving this problem. Modifications based on bioactive natural products could greatly shorten drug development time and improve success rate. Pleuromutilin, a natural product from the basidiomycete bacterial species, is a promising antibiotic candidate. In this study, a series of novel pleuromutilin derivatives possessing piperazinyl urea linkage were efficiently synthesised, and their antibacterial activities and bactericidal properties were evaluated via MIC, MBC and Time-kill kinetics assays. The results showed that all compounds exhibited potent activities against tested strains, especially MRSA strains with MIC values as low as 0.125 μg/mL; 8 times lower than that of marketed antibiotic Tiamulin. Docking studies indicate substituted piperazinyl urea derivatives could provide hydrogen bonds and initiate π-π stacking between molecules and surrounding residues.

A click chemistry approach to pleuromutilin derivatives, evaluation of anti-MRSA activity and elucidation of binding mode by surface plasmon resonance and molecular docking

Novel series of pleuromutilin analogs containing substituted 1,2,3-triazole moieties were designed, synthesised and assessed for their in vitro antibacterial activity against Methicillin-resistant Staphylococcus aureus (MRSA). Initially, the in vitro antibacterial activities of these derivatives against 4 strains of S. aureus (MRSA ATCC 43300, ATCC 29213, AD3, and 144) were tested by the broth dilution method. Most of the synthesised pleuromutilin analogs displayed potent activities. Among them, compounds 50, 62, and 64 (MIC = 0.5∼1 µg/mL) showed the most effective antibacterial activity and their anti-MRSA activity were further studied by the time-killing kinetics approach. Binding mode investigations by surface plasmon resonance (SPR) with 50S ribosome revealed that the selected compounds all showed obvious affinity for 50S ribosome (KD = 2.32 × 10−8∼5.10 × 10−5 M). Subsequently, the binding of compounds 50 and 64 to the 50S ribosome was further investigated by molecular modelling. Compound 50 had a superior docking mode with 50S ribosome, and the binding free energy of compound 50 was calculated to be −12.0 kcal/mol.

Design, synthesis and biological evaluation of novel pleuromutilin derivatives containing piperazine and 1,2,3-triazole linker

A series of novel pleuromutilin derivatives containing piperazine ring, 1, 2, 3-triazoles and secondary amines on the side chain of C14 were synthesized under mild conditions via click reaction. The in vitro antibacterial activities of the synthesized derivatives against four strains of Staphylococcus aureus (MRSA ATCC 43300, ATCC 29213 ,144 and AD3) and one strain of Escherichia coli (ATCC 25922) were evaluated by the broth dilution method. Among these derivatives, 22–[2-(4-((4-nitrophenyl piperazine)methyl)-1,2,3-triazol-1-yl)-1-(piperazine-1-yl) ethyl-1-one] deoxy pleuromutilin (compound 59) showed the most prominent in vitro antibacterial effect against MRSA (MIC = 1 μg/mL). Furthermore, compound 59 displayed more rapid bactericidal kinetic than tiamulin time-kill studies and possessed a longer PAE than tiamulin against MRSA in vitro. In addition, in vivo antibacterial activities of compound 59 against MRSA were further evaluated employing thigh infection model. And compound 59 (-8.89 log10 CFU/mL) displayed superior activities than tiamulin. Compound 59 was further evaluated in CYP450 inhibition assay and the results showed that it exhibited low to moderate inhibitory effects on CYP1A2, CYP2E1, CYP2D6 and CYP3A4 enzymes. The PK properties of compound 59 were then measured. The half-life (t1/2), clearance rate (Cl) and the area under the plasma concentration time curve (AUC0→∞) of compound 59 were 0.74 h, 0.29 L/h/kg and 46.28 μg·h/mL, respectively.

Novel pleuromutilin derivatives with substituted 6-methylpyrimidine: Design, synthesis and antibacterial evaluation.

A series of novel pleuromutilin derivatives with substituted 6-methylpyrimidine moieties was designed, synthesized, and evaluated for their antibacterial activities. Most of the tested compounds exhibited potent antibacterial activities against Staphylococcus aureus ATCC 25923 (S.aureus-25923), methicillin-resistant Staphylococcus epidermidis ATCC 51625 (MRSE-51625), methicillin-resistant Staphylococcus aureus BNCC 337371 (MRSA-337371), Streptococcus dysgalactiae (S.dysgalactiae) and Streptococcus agalactiae (S.agalactiae). Compounds 5c and 5g were the most active and displayed bacteriostatic activities against MRSA. Invivo mouse systemic infection experiment showed that 5c significantly improved the survival rate of mice (ED50=18.02mg/kg), reduced the bacterial load and alleviated the pathological changes in the lungs of the affected mice.

Synergistic Effect of a Pleuromutilin Derivative with Tetracycline against Streptococcus suis In Vitro and in the Neutropenic Thigh Infection Model.

Tetracycline (TET) has been widely used in the treatment of Streptococcus suis (S. suis) infection. However, it was found that the efficacy of many antibiotics in S. suis decreased significantly, especially tetracycline. In this study, GML-12 (a novel pleuromutilin derivative) was used in combination with TET against 12 S. suis isolates. In the checkerboard assay, the TET/GML-12 combination exhibited synergistic and additive effects against S. suis isolates (n = 12). In vitro time-killing assays and in vivo therapeutic experiments were used to confirm the synergistic effect of the TET/GML-12 combination against S. suis strains screened based on an FICI ≤ 0.5. In time-killing assays, the TET/GML-12 combination showed a synergistic effect or an additive effect against three isolates with a bacterial reduction of over 2.4-log10 CFU/mL compared with the most active monotherapy. Additionally, the TET/GML-12 combination displayed potent antimicrobial activity against four isolates in a mouse thigh infection model. These results suggest that the TET/GML-12 combination may be a potential therapeutic strategy for S. suis infection.

Design, synthesis and biological activities of novel pleuromutilin derivatives with a substituted triazole moiety as potent antibacterial agents

A series of novel pleuromutilin derivatives possessing 1,2,3-triazole moieties were synthesized via click reactions under mild conditions. The in vitro antibacterial activities of these derivatives against 4 strains of S. aureus (MRSA ATCC 43300, ATCC 29213, AD 3, and 144) and 1 strain of E. coli (ATCC 25922) were tested by the broth dilution method. The majority of the synthesized derivatives displayed potent antibacterial activities against MRSA (MIC = 0.125–2 μg/mL). It was also found that most compounds had no significant inhibitory effect on the proliferation of RAW264.7 cells at the concentration of 8 μg/mL. Among these derivatives, compound 32 (∼1.71 log10 CFU/g) containing dimethylamine group side chain displayed more effective than tiamulin (∼0.77 log10 CFU/g) at the dose of 20 mg/kg in reducing MRSA load in thigh infected mice. Additionally, compound 32 (the survival rate was 50%) also displayed superior in vivo efficacy to that of tiamulin (the survival rate was 20%) in the mouse systemic model. Structure-activity relationship (SAR) studies resulted in compound 32 with the most potent in vitro and in vivo antibacterial activity among the series. Moreover, compound 32 was evaluated in CYP450 inhibition assay and showed moderate in vitro inhibition of CYP3A4 (IC50 = 6.148 μM).

Synthesis, biological activities, and docking study of novel chalcone-pleuromutilin derivatives.

The issue of antibiotic resistance is becoming progressively serious these days, and the feasible solution to address it is to develop and discover novel antibiotics. The diterpene natural pleuromutilin is a prominent candidate for its special mode of action by inhibiting protein synthesis. In this study, a series of novel pleuromutilin derivatives with chalcone moiety was designed and synthesized, and their antibacterial activities were assessed in vitro. As suggested from the results, most of compounds exhibited potent activities against two methicillin-resistant Staphylococcus aureus (MRSA) ATCC 33591 and 43300. The further modification of the chalcone structure, aza-cyclic derivatives were afforded and then assessed, and potent activities against the tested strains were reported. The preliminary docking studies were conducted to explore the interactions between target molecules and binding site.

Antibacterial Activity and Pharmacokinetic Profile of a Promising Antibacterial Agent: 22-(2-Amino-phenylsulfanyl)-22-Deoxypleuromutilin.

A new pleuromutilin derivative, 22-(2-amino-phenylsulfanyl)-22-deoxypleuromutilin (amphenmulin), has been synthesized and proved excellent in vitro and in vivo efficacy than that of tiamulin against methicillin-resistant Staphylococcus aureus (MRSA), suggesting this compound may lead to a promising antibacterial agent to treat MRSA infections. In this study, the effectiveness and safety of amphenmulin were further investigated. Amphenmulin showed excellent antibacterial activity against MRSA (minimal inhibitory concentration = 0.0156~8 µg/mL) and performed time-dependent growth inhibition and a concentration-dependent postantibiotic effect (PAE). Acute oral toxicity test in mice showed that amphenmulin was a practical non-toxic drug and possessed high security as a new drug with the 50% lethal dose (LD50) above 5000 mg/kg. The pharmacokinetic properties of amphenmulin were then measured. After intravenous administration, the elimination half-life (T1/2), total body clearance (Clβ), and area under curve to infinite time (AUC0→∞) were 1.92 ± 0.28 h, 0.82 ± 0.09 L/h/kg, and 12.23 ± 1.35 μg·h/mL, respectively. After intraperitoneal administration, the T1/2, Clβ/F and AUC0→∞ were 2.64 ± 0.72 h, 4.08 ± 1.14 L/h/kg, and 2.52 ± 0.81 μg·h/mL, respectively, while for the oral route were 2.91 ± 0.81 h, 6.31 ± 2.26 L/h/kg, 1.67 ± 0.66 μg·h/mL, respectively. Furthermore, we evaluated the antimicrobial activity of amphenmulin in an experimental model of MRSA wound infection. Amphenmulin enhanced wound closure and promoted the healing of wound, which inhibited MRSA bacterial counts in the wound and decreased serum levels of the pro-inflammatory cytokines TNF-α, IL-6, and MCP-1.

Design, synthesis and antibacterial activities of pleuromutilin derivatives

We described the design, synthesis and antimicrobial activities of novel pleuromutilin derivatives with substituted piperazine substrate. Minimum inhibitory concentration (MIC) was used to evaluate the activity of the derivatives against six bacteria in vitro, and compound 8 was potent against Staphylococcus aureus and Staphylococcus epidermidis with the MIC value of 0.0625 μg/ml. 10a and 10 b showed similar activity to positive control drugs (tiamulin, erythromycin) against S. aureus with the MIC value of 0.125 μg/ml. The binding mode of compound 8 and tiamulin to the ribosome pocket showed the correlation between binding parameters and the antibacterial activity, and more bonds and stronger combination could effectively enhance the activity of compounds.

Design, synthesis, and biological activity evaluation of a series of pleuromutilin derivatives with novel C14 side chains

In this work, according to the ‘me-too me-better’ design strategy, a peculiar side chain different from lefamulin at C14 position of pleuromutilin was introduced. A series of novel thioether pleuromutilin derivatives containing cyclohexane in the C14 chain was synthesized by ten-step synthesis reaction. All derivatives were characterized by Nuclear Magnetic Resonance (NMR) and High Resolution Mass Spectrometer (HRMS). Furthermore, majority of derivatives displayed moderate antibacterial activity in vitro. However, the compound 2C and 2J exhibited comparable or superior antibacterial activity to lefamulin. The summarized structure-activity relationship not only made the variety of pleuromutilin derivatives more diverse, but also provided new ideas for its design and development.

Efflux-Mediated Resistance to New Oxazolidinones and Pleuromutilin Derivatives in Escherichia coli with Class Specificities in the Resistance-Nodulation-Cell Division-Type Drug Transport Pathways.

A major contribution of the resistance-nodulation-cell division (RND)-transporter AcrB to resistance to oxazolidinones and pleuromutilin derivatives in Escherichia coli was confirmed. However, we discovered significant differences in efflux inhibitor activities, specificities of the homologous pump YhiV (MdtF), and the impact of AcrB pathway mutations. Particularly, entrance channel double-mutation I38F I671T and distal binding pocket mutation F615A revealed class-specific transport routes of oxazolidinones and pleuromutilin derivatives. The findings could contribute to the understanding of the RND-type multidrug transport pathways.

Design, synthesis and biological evaluation of novel pleuromutilin derivatives possessing acetamine phenyl linker

A series of novel acetamine phenyl pleuromutilin derivatives incorporating 2-aminothiophenol moieties into the C14 side chain were synthesized via acylation reactions under mild conditions. The in vitro antibacterial activities of the synthesized derivatives against three Staphylococcus aureus (MRSA ATCC 43300, ATCC 29213 and AD 3) and two Escherichia coli (ATCC 25922 and 9–1) were evaluated by the broth dilution method. Most of the synthesized derivatives displayed potent activities. Compound 27 was found to be the most active antibacterial derivative against MRSA (minimal inhibitory concentration = 0.015 μg/mL) which may lead to a promising antibacterial drug. Furthermore, compound 27 displayed more rapid bactericidal kinetic than tiamulin in in vitro time-kill studies and possessed a longer PAE than tiamulin against MRSA. The PK properties of compound 27 were then measured. The half life (t1/2), clearance rate (Cl) and the area under the plasma concentration–time curve (AUC0→∞) of compound 27 were 6.88 h, 21.64 L/h/kg and 0.48 μg h/mL, respectively. The in vivo antibacterial activities of compound 27 against MRSA were further evaluated using thigh infection model and systemic infection model. Compound 27 possessed superior antibacterial efficacy to tiamulin against MRSA infection in both model.

Antibacterial Activity and Structure-Activity Relationship of a Series of Newly Synthesized Pleuromutilin Derivatives.

A series of novel thioether or sulfoxide-type pleuromutilin derivatives containing heteroaromatic substituents at the end of C14 side chain were designed and synthesized. All of the derivatives were evaluated for their in vitro antibacterial activity. Some of them showed good to excellent antibacterial activity comparable to retapamulin and azamulin in most of the tested Gram-positive pathogens. In this work, a five-membered heterocyclic moiety, a pyrimidine-heterocyclic moiety, or a benzoheterocyclic moiety was introduced in the C14 side chain to increase the structural diversity of the pleuromutilin derivatives. The antibacterial results reveal that the thioether-containing pleuromutilin derivatives exert a more potency activity than the sulfoxide-type derivatives against Gram-positive pathogens. The structure-activity relationship summarized in this work may provide with some interesting clues as to which functionalities are beneficial for high antimicrobial activity of the pleuromutilin derivatives.