Pleuromutilin Derivatives Research Articles

Semi-synthesis, antibacterial activity, and molecular docking study of novel pleuromutilin derivatives bearing cinnamic acids moieties.

To develop new antibiotics owning a special mechanism, we used the molecular assembly method to synthesize a series of novel pleuromutilin derivatives containing a cinnamic acid scaffold at the C-14 side chain. We evaluated their antibacterial activity and used in silico molecular docking to study their binding mode with the target. The structure-activity relationship (SAR) study suggested that compounds with NO2 (13e), OH (13u), and NH2 (13y) appeared more active (0.0625-2 µg/mL) in vitro against several penicillin-resistant Gram-positive bacteria and the position of the substituent on the benzene ring would affect the activity. The in vivo efficacy investigation of 13e, 13u, and 13y with once daily intragastric (i.g.) administration at 40 mg/kg for 3 consecutive days in a mouse systemic infection model showed that 13u had equal activity as valnemulin providing the mice with 60% survival, while 13e and 13y gave 30 and 40% survival, respectively. The molecular docking studies indicated that π-π stacking and hydrogen bond formation played important roles in improving the antibacterial activity.

Antibacterial activity evaluation of synthetic novel pleuromutilin derivatives in vitro and in experimental infection mice

A series of novel pleuromutilin derivatives embracing 7H-pyrrolo[2,3-d]pyrimidine moiety were synthesized and evaluated for their in vitro antibacterial activity against Gram-positive and Gram-negative pathogens as well as in vivo efficacy in lethal systemic infected mice. Most compounds displayed good in vitro potency against MSSA, MRSA, MSSE, MRSE and E. faecium (MIC = 0.0625–4 μg/mL), especially 15a, 15b and 15o showed excellent activity that even more active than the comparator valnemulin. The in vivo efficacy investigation exhibited compound 15a (ED50 = 16.0 mg/kg) had comparable activity to valnemulin (ED50 = 13.5 mg/kg). The results provided by the dose-response study demonstrated 15a can supply infected mice with 70% survival rate at dose of 40 mg/kg via intragastric (i.g.) administration.

An antimycobacterial pleuromutilin analogue effective against dormant bacilli

Pleuromutilin is a promising pharmacophore to design new antibacterial agents for Gram-positive bacteria. However, there are limited studies on the development of pleuromutilin analogues that inhibit growth of Mycobacterium tuberculosis (Mtb). In screening of our library of pleuromutilin derivatives, UT-800 (1) was identified to kill replicating- and non-replicating Mtb with the MIC values of 0.83 and 1.20 μg/mL, respectively. UT-800 also kills intracellular Mtb faster than rifampicin at 2× MIC concentrations. Pharmacokinetic studies indicate that 1 has an oral bioavailability with an average F-value of 27.6%. Pleuromutilin may have the potential to be developed into an orally administered anti-TB drug.

Synthesis and antibacterial activities of novel pleuromutilin derivatives.

Pleuromutilin derivatives 4a-h, 5a-g, and 6a-d were synthesized and characterized by IR, 1 H NMR, and 13 C NMR. All synthetic compounds were screened for their in vitro antibacterial activity against Staphylococcus aureus (ATCC 25923), methicillin-resistant S. aureus (MRSA, ATCC 43300), Pasteurella multocida (CVCC 408), Escherichia coli (ATCC 25922), and Salmonella typhimurium (ATCC 14028). Most compounds with quaternary amine showed higher antibacterial activities against both Gram-positive and Gram-negative bacteria strains. Among the screened compounds, compound 5a bearing an N,N,N-trimethyl group at the C-14 side chain of pleuromutilin was found to be the most active agent. Furthermore, preliminary molecular docking was performed to predict the binding interaction of the compounds in the binding pocket.

Synthesis and Antibacterial Activity Against MRSA of Pleuromutilin Derivatives Possessing a Mercaptoethylamine Linker.

Methicillin resistant Staphylococcus aureus (MRSA) usually invalidate powerful antibiotics in the clinic. Pleuromutilin derivatives have been reported to possess antibacterial activity against MRSA. The antibacterial activities against MRSA of a series of thirteen synthetic pleuromutilin derivatives were investigated through in vitro models. A series of novel thioehter pleuromutilin derivatives incorporating various aromatic substituents into the C14 side chain have been reported. The in vitro antibacterial activities of these derivatives against MRSA and Escherichia coli were tested by the broth dilution method. Twelve pleuromutilin derivatives were designed, synthesized and evaluated for in vitro antibacterial activities against four Staphylococcus aureus strains. From structure-activity relationship studies, compound 11c was identified as promising compounds with the most potent in vitro antibacterial activity among the series (MIC = 0.0625-0.125 µg/ml) against Staphylococcus aureus strains. The binding of compound 11c to the 50s ribosome was investigated by molecular modeling. It was found that there is a reasonable correlation between the binding free energy and the antibacterial activity.

Synthesis and antibacterial activities of novel pleuromutilin derivatives bearing an aminothiophenol moiety.

We synthesized a series of novel thioether pleuromutilin derivatives incorporating 2-aminothiophenol moieties into the C14 side chain via acylation reactions under mild conditions. We evaluated the in-vitro antibacterial activities of the derivatives against methicillin-resistant Staphylococcus aureus (MRSA, ATCC 43300), Staphylococcus aureus (ATCC 29213) and Escherichia coli (ATCC 25922). The majority of the synthesized derivatives possessed moderate antibacterial activities. Compound 8 was found to be the most active antibacterial derivative against MRSA. We conducted docking experiments to understand the possible mode of interactions between compounds 8, 9b, 11a and 50S ribosomal subunit. The docking results proved that there is a reasonable correlation between the binding free energy and the antibacterial activity. Compound 8 was evaluated for its in-vivo antibacterial activity and showed higher efficacy than tiamulin against MRSA in mouse infection model.

Determination of a New Pleuromutilin Derivative in Broiler Chicken Plasma by RP-HPLC-UV and Its Application to a Pharmacokinetic Study.

A simple, sensitive and reproducible high-performance liquid chromatography method was developed and validated for the determination of 14-O-[(2-amino-1,3,4-thiadiazol-5-yl) thioacetyl] mutilin (ATTM), a new synthesized pleuromutilin derivative with potent antibacterial activity, in broiler chicken plasma after a single intravenous (i.v.), intramuscular (i.m.) or oral (p.o.) administration. Satisfactory separation was achieved on a ZORBAX Ecliplus C18 column (250 × 4.6, 5 μm) with UV detection at 279 nm, using a mobile phase comprising acetonitrile and ultrapure water (50:50, v/v). The elution was isocratic at ambient temperature with a flow rate of 1.0 mL/min. The method exhibited good linearity (R2 > 0.999) over the assayed concentration range (0.12-120.00 μg/mL) and demonstrated good intra- and inter-day precision and accuracy. The method was validated and successfully applied to the pharmacokinetic study of ATTM in chicken plasma after i.v. and p.o. administration.

Unraveling the Metabolic Routes of Retapamulin: Insights into Drug Development of Pleuromutilins.

Retapamulin, a semisynthetic pleuromutilin derivative, is exclusively used for the topical short-term medication of impetigo and staphylococcal infections. In the present study, we report that retapamulin is adequately and rapidly metabolized in vitro via various metabolic pathways, such as hydroxylation, including mono-, di-, and trihydroxylation, and demethylation. Like tiamulin and valnemulin, the major metabolic routes of retapamulin were hydroxylation at the 2β and 8α positions of the mutilin moiety. Moreover, in vivo metabolism concurred with the results of the in vitro assays. Additionally, we observed significant interspecies differences in the metabolism of retapamulin. Until now, modifying the side chain was the mainstream method for new drug discovery of the pleuromutilins. This approach, however, could not resolve the low bioavailability and short efficacy of the drugs. Considering the rapid metabolism of the pleuromutilins mediated by cytochrome P450 enzymes, we propose that blocking the active metabolic site (C-2 and C-8 motif) or administering the drug in combination with cytochrome P450 enzyme inhibitors is a promising pathway in the development of novel pleuromutilin drugs with slow metabolism and long efficacy.

Heterologous expression reveals the biosynthesis of the antibiotic pleuromutilin and generates bioactive semi-synthetic derivatives

The rise in antibiotic resistance is a major threat for human health. Basidiomycete fungi represent an untapped source of underexploited antimicrobials, with pleuromutilin—a diterpene produced by Clitopilus passeckerianus—being the only antibiotic from these fungi leading to commercial derivatives. Here we report genetic characterisation of the steps involved in pleuromutilin biosynthesis, through rational heterologous expression in Aspergillus oryzae coupled with isolation and detailed structural elucidation of the pathway intermediates by spectroscopic methods and comparison with synthetic standards. A. oryzae was further established as a platform for bio-conversion of chemically modified analogues of pleuromutilin intermediates, and was employed to generate a semi-synthetic pleuromutilin derivative with enhanced antibiotic activity. These studies pave the way for future characterisation of biosynthetic pathways of other basidiomycete natural products in ascomycete heterologous hosts, and open up new possibilities of further chemical modification for the growing class of potent pleuromutilin antibiotics.

Antibacterial activity and pharmacokinetic profile of a promising antibacterial agent: 14-O-[(4-Amino-6-hydroxy-pyrimidine-2-yl)thioacetyl] mutilin

A new pleuromutilin derivative, 14-O-[(4-Amino-6-hydroxy-pyrimidine-2-yl)thioacetyl] mutilin (APTM), has been synthesized and proved most potent antibacterial agent in in vitro assays, suggesting that further development of this compound may lead to a promising antibacterial drug. In this study, we further evaluated the cytotoxicity, antibacterial efficacy and the pharmacokinetic profile of APTM. In BRL 3A cells, 50% of viability was obtained when 363μg/mL of APTM was used, while retapamulin and tiamulin fumarate needed 49 and 28μg/mL, respectively, to reach this viability. Compared to tiamulin fumarate, APTM showed higher inhibition efficacy and faster bactericidal activity against S. aureus and lower 50% effective dose (ED50) in mice after a lethal challenge with methicillin-resistant Staphylococcus aureus (MRSA). Docking experiment for APTM showed a similar binding pattern with tiamulin. Furthermore, a simple, accurate and sensitive HPLC method for the determination of APTM in rabbit plasma was developed and successfully applied to pharmacokinetic study, in which the half life (t1/2), clearance rate (Cl) and the area under the plasma concentration–time curve (AUC0→∞) were 3.37h, 0.35L/h/kg and 70.68μg·h/m, respectively.

Development of a Modular Synthetic Route to (+)-Pleuromutilin, (+)-12-epi-Mutilins, and Related Structures.

We describe the development of an enantioselective synthetic route to (+)-pleuromutilin (1), (+)-12-epi-mutilin, and related derivatives. A key hydrindanone was prepared in three steps and 48% overall yield from cyclohex-2-en-1-one. 1,4-Hydrocyanation provided a nitrile (53%, or 85% based on recovered starting material) that was converted to the eneimide 57 in 80% yield by the 1,2-addition of methyllithium to the nitrile function, cyclization, and in situ acylation with di-tert-butyldicarbonate. The eneimide 57 was employed in a 2-fold neopentylic coupling reaction with an organolithium reagent derived from the alkyl iodides (R)- or (S)-30, which contain the C11-C13 atoms of the target, to provide diastereomeric diketones in 60% or 48% yield (for coupling with (R)- or (S)-30, respectively). The diketone derived from (S)-30 contains the (S)-C12 stereochemistry found in pleuromutilin and was elaborated to an alkynylaldehyde. Nickel-catalyzed reductive cyclization of this alkynylaldehyde, to construct the eight-membered ring of the target, unexpectedly provided a cyclopentene (67%), which arises from participation of the C12-α-olefin in the transformation. The diketone derived from the enantiomeric C12-fragment (R)-30 underwent reductive cyclization to provide the desired product in 60% yield. This was elaborated to 12-epi-mutilin by a four-step sequence (39% overall). Installation of the glycolic acid residue followed by C12 epimerization (Berner et al. Monatsh. Chem. 1986, 117, 1073) generated (+)-pleuromutilin (1). (+)-12-epi-Pleuromutilin and (+)-11,12-di-epi-pleuromutilin were prepared by related sequences. This work establishes a convergent entry to the pleuromutilins and provides a foundation for the production of novel antibiotics to treat drug-resistant and Gram-negative infections.

3D-QSAR, Topomer CoMFA, Docking Analysis, and ADMET Predictio n of Thioether Pleuromutilin Derivatives as Antibacterial Agents

3D-QSAR, Topomer CoMFA, Docking Analysis, and ADMET Predictio n of Thioether Pleuromutilin Derivatives as Antibacterial Agents

Synthesis and Biological Activity Evaluation of Novel Heterocyclic Pleuromutilin Derivatives.

A series of pleuromutilin derivatives were synthesized by two synthetic procedures under mild reaction conditions and characterized by Nuclear Magnetic Resonance (NMR), Infrared Spectroscopy (IR), and High Resolution Mass Spectrometer (HRMS). Most of the derivatives with heterocyclic groups at the C-14 side of pleuromutilin exhibited excellent in vitro antibacterial activities against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE), and vancomycin-resistant Enterococcus (VRE) in vitro antibacterial activity. The synthesized derivatives which contained pyrimidine rings, 3a, 3b, and 3f, displayed modest antibacterial activities. Compound 3a, the most active antibacterial agent, displayed rapid bactericidal activity and affected bacterial growth in the same manner as that of tiamulin fumarate. Moreover, molecular docking studies of 3a and lefamulin provided similar information about the interactions between the compounds and 50S ribosomal subunit. The results of the study show that pyrimidine rings should be considered in the drug design of pleuromutilin derivatives.

A modular and enantioselective synthesis of the pleuromutilin antibiotics.

The tricyclic diterpene fungal metabolite (+)-pleuromutilin has served as a starting point for antibiotic development. Semisynthetic modification of its glycolic acid subunit at C14 provided the first analogs fit for human use, and derivatization at C12 led to 12-epi-pleuromutilins with extended-spectrum antibacterial activity, including activity against Gram-negative pathogens. Given the inherent limitations of semisynthesis, however, accessing derivatives of (+)-pleuromutilin with full control over their structure presents an opportunity to develop derivatives with improved antibacterial activities. Here we disclose a modular synthesis of pleuromutilins by the convergent union of an enimide with a bifunctional iodoether. We illustrate our approach through synthesis of (+)-12-epi-mutilin, (+)-11,12-di-epi-mutilin, (+)-12-epi-pleuromutilin, (+)-11,12-di-epi-pleuromutilin, and (+)-pleuromutilin itself in 17 to 20 steps.

A New Combination of a Pleuromutilin Derivative and Doxycycline for Treatment of Multidrug-Resistant Acinetobacter baumannii

Multidrug-resistant (MDR) Acinetobacter baumannii is one of the most difficult Gram-negative bacteria to treat and eradicate. In a cell-based screening of pleuromutilin derivatives against a drug sensitive A. baumannii strain, new molecules (2-4) exhibit bacteriostatic activity with 3.13 μg/mL concentration and 1 shows bactericidal activity with an MBC of 6.25 μg/mL. The pleuromutilin derivative 1 displays strong synergistic effects with doxycycline in a wide range of concentrations. A 35/1 ratio of 1 and doxycycline (1-Dox 35/1) kills drug susceptible A. baumannii with the MBC of 2.0 μg/mL and an MDR A. baumannii with the MBC of 3.13 μg/mL. In vitro anti-Acinetobacter activity of 1-Dox 35/1 is superior to that of clinical drugs such as tobramycin, tigecycline, and colistin. The efficacy of 1-Dox 35/1 is evaluated in a mouse septicemia model; treatment of the infected C57BL/6 mice with 1-Dox 35/1 protects from lethal infection of A. baumannii with an ED50 value of <2.0 mg/kg.

Design, synthesis and antibacterial evaluation of novel pleuromutilin derivatives possessing piperazine linker

A series of pleuromutilin derivatives bearing piperazine ring have been reported. The in vitro antibacterial activities of the synthetic derivatives against MRSA (ATCC 43300), Staphylococcus aureus (ATCC 29213), Enterococcus faecalis (ATCC 29212), Enterococcus faecium (ATCC35667) and Escherichia coli (ATCC25922) were evaluated by the broth dilution method. Most of the synthesized derivatives displayed potent activities. Compounds 11c, 12a and 12c were found to be the most active antibacterial derivatives against MRSA (minimum inhibitory concentration = 0.015 μg/mL). The binding of compounds 11c, 12a and 12c to the 50s ribosome were investigated by molecular modeling. Compound 11c possessed lower binding free energy compared with compounds 12a and 12c. Compound 11c was further evaluated in MRSA systemic infection model and displayed superior in vivo efficacy to that of tiamulin.

Synthesis and Biological Evaluation of Novel Thioether Pleuromutilin Derivatives.

To develop new pleuromutilin derivatives as veterinary antibiotic medicines, we designed and synthesized a series of new thioether pleuromutilin derivatives possessing acylthiazolyl moiety based on previously designed derivatives. The antibacterial properties of the prepared pleuromutilin derivatives were assessed in vitro by the broth dilution method against five kinds of bacteria and the mycoplasma Mycoplasma gallisepticum (MG). All of the tested compounds displayed moderate to good antibacterial activity to methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-sensitive Staphylococcus epidermidis (MSSE), methicillin-resistant S. aureus (MRSA), Streptococcus agalactiae (S. aga) and MG. However, the activity to Pyogeniccoccus (Pyogens) was generally poor. Compounds 13i and l showed potent antibacterial activity against MSSE and MRSA which are better than that of valnemulin. The structural modification for pleuromutilin affected the antibacterial activity. Amino substituents in the benzene ring can effectively improve activity. Compared with the analogue 13a that possesses unsubstitution benzoyl group, the nitro, methoxy, hydroxy and dichloro substituent contributed little to antibacterial activity. Increasing a methylene between benzene moiety and carbonyl group decreased the bioactivity of derivative. The analogues that obtained by the reaction of amino acids and intermediate 9 showed moderate activity.

Synthesis and antibacterial activities of novel pleuromutilin derivatives with a substituted pyrimidine moiety

The alarming growth of multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) has become a major global health hazard. Therefore, urgent demand for new antibiotics with a unique mechanism of action is very necessary. The present study reports the design, synthesis, and antibacterial studies of a series of novel pleuromutilin derivatives with substituted 6-amino pyrimidine moieties. Most of the tested compounds exhibited highly potent anti-MRSA or Staphylococcus aureus (S. aureus) activities. 14-O-[(4,6-Diamino -pyrimidine-2-yl) thioacetyl] mutilin (3) and 14-O-[(2-((3R)-3-Hydroxymethylpiperidine-1-yl)-acetamido-6-aminopyrimidine-2-yl) thioacetyl] mutilin (5h) were the most active compounds and showed higher antibacterial activities. Compound 3 displayed rapid bactericidal activity and affected bacterial growth with the same manner as tiamulin fumarate. Docking experiments for compounds 3 and 5h carried out on the peptidyl transferase center (PTC) of 23S rRNA provided the information about the binding model. In vivo mouse systemic infection experimental results confirmed the therapeutic efficacy of compound 3, with ED50 of 4.22 mg/kg body weight against MRSA.

Synthesis and Pharmacological Evaluation of Novel Pleuromutilin Derivatives with Substituted Benzimidazole Moieties.

A series of novel pleuromutilin derivatives with substituted benzimidazole moieties were designed and synthesized from pleuromutilin and 5-amino-2-mercaptobenzimidazole through sequential reactions. All the newly synthesized compounds were characterized by IR, NMR, and HRMS. Each of the derivatives was evaluated in vitro for their antibacterial activity against Escherichia coli (E. coli) and five Gram (+) inoculums. 14-O-((5-amino-benzimidazole-2-yl) thioacetyl) mutilin (3) was the most active compound and showed highest antibacterial activities. Furthermore, we evaluated the inhibition activities of compound 3 on short-term S. aureus and MRSA growth and cytochrome P450 (CYP). The bioassay results indicate that compound 3 could be considered potential antibacterial agents but with intermediate inhibition of CYP3A4.

Design, synthesis, and structure-activity relationship studies of novel pleuromutilin derivatives having a piperazine ring.

A series of novel pleuromutilin derivatives possessing piperazine moieties were synthesized under mild conditions. The in vitro antibacterial activities of these derivatives against Staphylococcus aureus and Escherichia coli were tested by the agar dilution method. Structure-activity relationship studies resulted in compounds 11b, 13b, and 14a with the most potent in vitro antibacterial activity among the series (minimal inhibitory concentration=0.0625-0.125μg/mL). The binding of compounds 11b, 13b, and 14a to the E.coli ribosome was investigated by molecular modeling, and it was found that there is a reasonable correlation between the binding free energy and the antibacterial activity.