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

Abstract 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.


Introduction
Current available antibiotics are losing their effectiveness due to increasing bacterial resistance. Diseases caused by multi-drug resistant bacteria are particularly difficult to prevent and treat, and across the world, approximately 700,000 people die annually as a result of them 1 . Among drug-resistant strains, methicillin-resistant Staphylococcus aureus (MRSA) is one of the most problematic, with pathogens that spread rapidly and cause severe diseases such as septicaemia, pneumonia, osteomyelitis and endocarditis 2,3 . Average treatment time and MRSA patient mortality are, respectively, five and three times higher than those of patients infected by common pathogens. Antibiotics are generally used to control MRSA infections, however the majority of common antibiotics used in clinical treatment were ineffective against MRSA. Therefore, it is crucial to develop and discover novel antibacterial agents 4 . Natural products such as Penicillin, Erythromycin, Cephalosporin C and Kanamycin are often used as main compounds in drug discoveries due to their potent bioactivity. Corresponding semi-synthesised derivatives Amoxicillin, Azithromycin, Cefpirome and Amikacin all exhibit higher potency than natural products [5][6][7] . Pleuromutilin (Figure 1, 1), a diterpenoid natural product from the basidiomycete species, shows moderate activities against Gram-positive strains and mycoplasmas 8,9 . Four semi-synthesised marketed drugs based on its structure have already been developed. Tiamulin and valnemulin (Figure 1, 2 and 3) can effectively prevent and control swine dysentery, mycoplasmal diseases such as enzootic pneumonia and chronic respiratory disease in poultry 10,11 . Retapamulin (Figure 1, 4) is used for the treatment of skin impetigo 12,13 . Lefamulin (Figure 1, 5) is approved for treatment of community-acquired bacterial pneumonia (CABP) 14 . These derivatives can inhibit bacterial protein synthesis via specific interaction with 23S rRNA of the 50S bacterial ribosome unit 15 , which are unaffected by resistance to major antibiotic classes, such as beta-lactam antibiotics, tetracyclines, macrolides, fluoroquinolones, and others. Thus, pleuromutilin is a promising candidate for treating drug-resistant bacteria infections 16 , and many compounds derived from four marketed drugs using the thioether as the linkage at the C14 side chain were developed in recent years [17][18][19][20] . To increase side chain diversity and improve modification success rate, the bioactive moiety piperazinyl urea, displaying multiple biological activities such as antifungal 21 , analgesic 22 , antibacterial 23 and antitumour 24 effects was introduced. In this study, a series of pleuromutilin derivatives 6a$z with piperazinyl urea (Scheme 1) were efficiently synthesised. Their activities were evaluated against MRSA and Gram-negative strains, and interactions were examined by molecular docking.
pleuromutilin-22-O-tosylate 7 in 78.3% yield, which could convert to 22-(piperazine-1-yl)-22-deoxypleuromutilin 8 by treatment with NaI, piperazine and potassium carbonate in dry THF with a 75.2% yield. Compounds 6a~e were synthesised by treating 7 with Nsubstituted-1-carboxamide 9a~e in the presence of a catalytic amount of NaI and K 2 CO 3 in MeCN with 78.3 $ 92.3% yield. Here, the primary amines 12a$e reacted with bis(trichloromethyl)carbonate (BTC) or N, N'-carbonyldiimidazole (CDI) to form the corresponding isocyanate, which was used to form piperazinyl urea moiety by reacting with N-Boc-piperazine in the presence of triethylamine with 56.2 $ 82.3% yield. Afterwards, 9a~e were obtained via removal of the above Boc protected group using trifluoroacetic acid (TFA) in 93.7 $ 97.2% yield. Compounds 6f~u were synthesised in 45.6 $ 82.7% yield by treating 8 in the presence of potassium carbonate with substituted 2,2,2-trichloro-acetamide 10f$u, which were acquired by reacting primary amines 12f$u with trichloroacetyl chloride in DCM with 54.7 $ 97.2% yield. Moreover, in regard to the secondary amine, the expected product was rarely obtained using the above two strategies. Nmethylaniline 12v was treated with 4-nitrophenyl carbonochloridate in the presence of N-methylmorpholine (NMM) in DCM to give 11, which was condensed without further purification and reacted with 22-(piperazine-1-yl)-22-deoxypleuromutilin 8 to form N-methyl derivative 6v in 40.5% yield. Additionally, 6w$z were synthesised from corresponding Boc-protected amine and nitro molecules (Scheme 2). 6w was gained from deprotecting 6g in the presence of TFA in 95.6% yield, and two other amine derivatives 6x and 6y were attained by stannous chloride reduction of 6d and 6p in 92.5% and 75.5% yields, respectively. 6y was used to react with chloracetyl chloride to get 14 in the presence of triethylamine with 70.1% yield, which was treated with morpholine to produce 6z in 50.3% yield.  were predicted by ACD/Labs (https://www.acdlabs.com/resources/ ilab/). As Table 1 indicates, almost all the synthesised compounds exhibited broad-spectrum antibacterial activities and showed high potency against both Gram-positive and Gram-negative bacteria with MIC values of 0.5 $ 4 lg/mL. Moreover, the majority of synthesised compounds displayed bactericidal activities against MRSA ATCC 33591, as MBC/MIC values were less than 4. Among them, the antibacterial activities of 6d, 6o, 6p and 6q were at least 2 times stronger than those of Tiamulin. In particular, compound 6p had a 0.125 lg/mL MIC value (8 times lower than that of Tiamulin) and displayed the best performance against MRSA ATCC 33591. 6p could also be considered as a bactericide, since MBC/MIC values against four tested strains were less than 2. Furthermore, its activity against E. coli ATCC 25922 was 16 times stronger than those of Tiamulin.
The effects of terminal ring types (e.g. phenyl, pyridinyl, morpholinyl and piperidinyl), ring substituents and distance between rings and urea groups on changes in activities were investigated. MIC value of phenyl piperazinyl urea pleuromutilin 6a was 1 lg/ mL against four strains and when substituents were introduced to the terminal benzene, activities varied within a small range. Compound 6d with electron-withdrawing 4-nitro groups was the most potent among all phenyl derivatives and exhibited an MIC value of 0.5 lg/mL. However, the introduction of N-methyl groups to the urea led to decreased activities, since actions of compound 6v was 1/4 or 1/2 of compound 6a. To render the side chain more flexible, a CH 2 group was introduced to increase the distance between terminal rings and urea groups. As result, the benzyl derivatives with an additional methylene group could noticeably improve activity level. In addition, similar as the effect of substituents on phenyl piperazinyl urea pleuromutilin activity, compound 6p with the 4-nitro benzyl group exhibited the best performance among all the benzyl derivatives. However, continuously extending the space between the benzene and piperazinyl urea exerts a slight influence on activities. Activity declined when the phenyl group was replaced by 4-pyridnyl group, but phenyl  group replacement by substituting 2-pyridinyl, 3-pyridinyl or 5quinolinyl groups had nearly zero impact on activities. Activity level visibly decreased when aliphatic rings such as piperidine and morpholine were added. The majority of target molecules with an aromatic terminal ring were fairly lipophilic with a CLogP value of greater than 5. To enhance hydrophilic properties, compounds 6w$y with amino groups were produced from corresponding Boc-protected amines and nitro compounds. Their ClogP values were approximately equal to or less than 4. Furthermore, a watersoluble morpholine ring was attached to the terminal of 6y through N-acetyl group. Although hydrophilic properties were clearly optimised via the above modification, activity levels were only slightly changed.
Since compound 6p showed the most potent antibacterial activities against MRSA ATCC 33591 and E. coli ATCC 25922, its bactericidal properties were examined by in vitro time-kill assay. Bactericidal properties of 6p against two strains were evaluated at concentrations of 1 Â MIC and 6 Â MIC, and two concentrations of Tiamulin were set as controls ( Figure 2). The MRSA ATCC 33591 propagation-inhibition effect of 6p was visibly higher at the 1 Â MIC concentration than that of the Grown control, which shows similarity to inhibition effects of Tiamulin at 1 Â MIC. Meanwhile, at 6 Â MIC, compound 6p achieved complete bactericidal effects in 12 h, a much faster rate than that under 1 Â MIC (24 h). In addition, compound 6p displayed better bactericidal effects against E. coli ATCC 25922 compared with Tiamulin at 1 Â MIC, and was capable of eliminating total bacteria colony in 24 h, while Tiamulin could only limit bacterial propagation speed. Outcomes were nearly identical at the 6 Â MIC concentration.

Cytotoxicity evaluation
The cytotoxicity of the most potent compound 6p was investigated in normal human hepatic cell line LO2 and human embryo kidney cell line HEK293T using a cell counting kit-8 (CCK-8) assay 25,26 (Figure 3). The CCK-8 assay is a colorimetric assay based on the reduction of dye WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4nitrophenyl)-5-(2,4disulfophenyl)-2H-tetrazolium, monosodium salt] to a water-soluble orange-coloured formazan via dehydrogenase in viable cells. The amount of formazan dye produced by cellular dehydrogenase is correlated with the number of living cells. The result showed that 6p hardly affected cell viability at concentrations ranging from 0.125 lg/mL to 4 lg/mL in either of the two cell lines. More than 90% of cells remained alive at the 4 lg/mL concentration, indicating 6p exhibited almost no cytotoxicity even at the concentration of 32 Â MIC.

Docking studies
Docking investigation was conducted to examine the binding mode between synthesised compounds and the peptidyl  transferase centre (PTC) of the 50S ribosomal subunit. The method was confirmed by re-docking, revealing that the docking pose of the original ligand Tiamulin nearly coincided with the X-ray structure conformation of RMSD 0.9 Å. All of the title compounds were used for the docking evaluation (Figs. S36-39). Among them, phenyl derivative 6d and benzyl derivative 6p with the most potent antibacterial activity, and aliphatic rings derivative 6z with moderate activity were chosen for the binding mode study. The favourably docked molecules were ranked according to their Grid_Score, which consisted of two parameters: Grid_vdw_energy (mainly referring to hydrophobic interactions and p-p stacking) and Grid_es_energy (often containing hydrogen bonds and salt bridges). The results presented in Table 2 showed that the order of Grid_Scores went in decreasing order (6p > 6d > 6z>Tiamulin), which matched those found in the biological assay.
As shown by the superimposed poses in Figures 4(a,b), three synthesised ligands 6d (green), 6p (yellow) and 6z (magenta) were located at the same pocket of PTC as Tiamulin (blue). The tricyclic skeleton of the synthesised compounds was in good agreement with that of Tiamulin, while the side chain at C14 varied noticeably. Many active interactions occurred continuously between the above-mentioned ligands and residues C2565, U2564, G2484, G2044 and A2405.
The important intermolecular PTC-ligand interactions are depicted in Figure 5. In addition to hydrogen bonds between the hydroxyl group on C11 and residue G2484, and between the carbonyl group on C21 and residue G2044, 6d ( Figure 5(a)) and 6p ( Figure 5(b)) were able to interact with A2045 by p-p stacking; the former adopting face-to-face stacking, and the latter displaying nearly edge-to-face stacking. Moreover, the terminal nitro group was able to form one additional hydrogen bond by interacting with C2565 or A2045. 6z (Figure 5(c)) was able to interact with residues G2484, U2564 and C2565 by three hydrogen bonds, however the p-p stacking was missing. This might explain why the Grid_vdw_energy of 6z (-98.69 kcal/mol) was worse than 6d (-103.75 kcal/mol) and 6p (-111.64 kcal/mol). The hydrophobic environment of the most potent compound 6p is shown in Figure  5(d). Residues G2484, U2483, C2431, U2485, A2430, U2564, C2565, A2045, A2418, C2420, C2046, G2044 and A2482 were involved in the hydrophobic interactions, which provided strong van der Waals forces in conjunction with the above mentioned p-p stacking of 6p.

Synthesis
Analytical grade reagents were used and purchased from Energy Chemical (Shanghai, China) and Kelong Chemical (Chengdu, China). Melting points were determined on an Shenguang WRS-1B apparatus (Shanghai, China). 1 H NMR and 13 C NMR spectra were measured on a Bruker AV400 spectrometer in CDCl 3 or DMSO-d 6 . Mass spectra were recorded with a X500R mass spectrometer (AB SCIEX) using the electro spray ionisation (ESI) method.

Synthesis of compound 7 and 8
Tosyl chloride (1.91 g, 10.0 mmol) in DCM (10 ml) was added to a solution of pleuromutilin (3.56 g, 9.40 mmol) in DCM (10 ml) containing triethylamine (4.54 g, 12.0 mmol) at 0 C, then the reaction was stirred at 25 C for 20 h and the resultant was washed with water (50 ml). The organic phase was collected, dried over Na 2 SO 4 and evaporated under vacuum to produce yellow oil, which could be purified by recrystallisation in ethanol to obtain white solid 7 (Yield: 78.3%). Spectral data of 7 were identical to those from reports in the literature 27 .  7 (533 mg, 1.0 mmol) and NaI (30.0 mg, 0.2 mmol) were stirred in dry THF (5 ml) at 25 C for 0.5 h, then K 2 CO 3 (276 mg, 2.0 mmol) and piperazine (172 mg, 2.0 mmol) were added to the aforementioned solution. The mixture was heated to reflux and stirred for 6 h. After cooling to room temperature, the resulting mixture was washed with water and condensed until dry. Compound 8 was collected after silica gel column chromatography (Yield: 75.2%). Spectral data results of 8 were identical to those from reports in the literature 28 .
Synthesis of compounds 9a $ e Compounds 9a~d were synthesised according to previous literature 29,30 , and 9e was synthesised as follows: BTC (386 mg, 1.3 mmol) in dry THF (4 ml) was added dropwise to a mixture of triethylamine (526 mg, 5.2 mmol) and 12e (173 mg, 1.2 mmol) in dry THF (6 ml) solution under an ice-water bath. The mixture was then stirred for 0.5 h. After condensation, N-Boc piperazine (503 mg, 2.7 mmol), triethylamine (546 mg, 5.4 mmol) and DCM (10 ml) were added and stirred for 2 h at 25 C. The solvent was then evaporated before crude product purification via silica gel column chromatography to produce compound 13e.
Synthesis of compounds 6a $ e Pleuromutilin 22-tosylate 7 (213 mg, 0.4 mmol) and NaI (14.9 mg, 0.1 mmol) in MeCN (5 ml) were stirred at 25 C for 0.5 h, then compounds 9a$e (0.5 mmol) and K 2 CO 3 (111 mg, 0.8 mmol) were added. The mixture was stirred under 70 C for 5 h. After solvent evaporation, the crude product was purified via silica gel column chromatography to obtain pure products 6a$e.  MIC And MBC assays MIC values were established in vitro using the agar dilution method 34,35 . Moreover, tiamulin fumarate was selected as the reference drug. All compounds were dissolved in a small amount of ethanol and diluted in distilled water to a concentration of 1280 mgÁmL À1 . Next, two-fold serially diluted pleuromutilin compound solutions containing distilled water were produced until the final concentration reached 0.625 mgÁmL À1 . After ten-fold dilution with LB broth, the two-fold serially diluted solutions were successively added to plate wells containing 10 ml of the above four bacterial solutions; then, solutions were incubated at 37 C for 1 6 $ 18 h and data was recorded. Each procedure was repeated 3 times. MBC was considered when the compounds killed over 99% of the tested bacterial culture. MIC-corresponding wells and three previous wells were homogenised, serially diluted, and plated on Mueller-Hinton (MH) agar to determine MBC. Data was recorded after plates underwent incubation at 37 C for 18-24 h. Each procedure was repeated 3 times.

Time-kill kinetics assay
Compounds 6p and tiamulin fumarate were dissolved in MH broth to generate different solutions of 1 Â MIC and 6 Â MIC, followed by inoculation with the above bacteria suspension at 37 C; After specified time intervals (0, 1, 2, 4, 6, 12 and 24 h), 10 mL of the above mixture was serially diluted 10-fold with saline and incubated at 37 C for 24 h on MH agar plates. Following this, viable colonies were counted and expressed as log 10 CFUÁmL À1 . Each procedure was done a total of 3 times.
Briefly, a 100 lL suspension of 10000 LO2 cells and 20000 HEK293T cells were seeded in 96-well plates, respectively. They were treated with six different concentrations of 6p (0.125 lg/mL, 0.25 lg/mL, 0.5 lg/mL, 1 lg/mL, 2 lg/mL and 4 lg/mL) and incubated at 37 C in a CO 2 incubator for 48 h. Afterwards, the CCK-8 solution (10 lL) was added to each well, and plates were incubated for an additional 1 h at 37 C. The absorbance at 450 nm was then recorded using a SpectraMax-190 microplate reader (Molecular Devices, USA). Each procedure was done a total of 3 times.

Molecular docking
The binding of synthesised compound 6p was examined using the PTC ribosome complex and Tiamulin (PDB ID: 1XBP). In particular, the PTC ribosome model was constructed using residues within 30 Å from the binding site after extraction of the original ligand Tiamulin. In addition, both 6p and the above ribosome model were prepared via AutoDockTools by removing water molecules, adding polar hydrogens, and assigning Gasteiger charges. Finally, the box (52.791, 122.679, 114.289) was established using original ligand as the grid centre with box size (36.758, 44.448, 36.761). Docking was performed by DOCK 6.9, and the 2 D interaction diagrams was generated via LigPlot þ v1.4 36 .

Conclusions
In summary, a series of pleuromutilin derivatives possessing the piperazinyl urea linkage were designed and synthesised, and their antibacterial activities against Gram-positive and Gram-negative strains were evaluated. Nearly all synthesised compounds exhibited broad-spectrum antibacterial activities, and particularly, more potent activities against MRSA strains. Activities of 6p were most effective with a MIC concentration of 0.1 2 5 $ 0.25 lg/mL, which was 8 $ 16 times that of Tiamulin. Time-kill kinetics indicated that at 1 Â MIC, compound 6p and Tiamulin had similar bactericidal effects against MRSA ATCC 33591; however compound 6p was more effective than Tiamulin against E.coli ATCC 25922, since it eliminated total bacteria colony in 24 h while Tiamulin could not. Molecular docking demonstrated that aside from hydrophobic interactions, benzyl urea linkage and terminal nitro groups from the side chain could produce several hydrogen bonds and p-p stacking with surrounding residues.