Neo-5,22E-Cholestadienol Derivatives from Buthus martensii Karsch and Targeted Bactericidal Action Mechanisms

The discovery and search for new antimicrobial molecules from insects and animals that live in polluted environments is a very important step in the scientific search for solutions to the current problem of antibiotic resistance. Previously, we have reported that the secondary metabolite with the antibacterial action discovered in scorpion. The current study further isolated three new compounds from Buthus martensii karsch, while compounds 1 and 2 possessed 5,22E-cholestadienol derivatives whose structure demonstrated broad spectrum bactericide activities. To explore the antibacterial properties of these new compounds, the result shows that compound 2 inhibited bacterial growth of both S. aureus and P. aeruginosa in a bactericidal rather than a bacteriostatic manner (MBC/MIC ratio ≤ 2). Similarly, with compound 1, a ratio of MBC/MIC ≤ 2 indicates bactericidal activity inhibited bacterial growth of P. aeruginosa. Remarkably, this suggests that two compounds can be classified as bactericidal agents against broad spectrum bactericide activities for 5,22E-cholestadienol derivatives from Buthus martensii karsch. The structures of compounds 1–3 were established by comprehensive spectra analysis including two-dimensional nuclear magnetic resonance (2D-NMR) and high-resolution electrospray ionization-mass spectrometry (HRESI-MS) spectra. The antibacterial mechanism is the specific binding (various of bonding forces between molecules) using compound 1 or 2 as a ligand based on the different receptor proteins’—2XRL or 1Q23—active sites from bacterial ribosome unit A, and thus prevent the synthesis of bacterial proteins. This unique mechanism avoids the cross-resistance issues of other antibacterial drugs.


Introduction
The scorpion is an ancient arthropod and a source of bioactive functional ingredients, it has been traditionally long time used for medicinal benefits as analgesic and antitumor effects of scorpion toxin in traditional Chinese medicine (TCM). The secondary metabolites of insects and arthropods are an important resource for the study of natural products [1,2]. Insects, such as mosquito, flies, and bedbugs, adapted under the harshest living conditions on earth and developed bioactive secondary metabolites responding to environmental challenges [3]. However, due to the low content levels and difficulty of separation, the assessment of the diversity of active molecules and the identification of novel
Therefore, the planar structure of 3 was elucidated as a α-bisaboleneol acid and was reasonably assigned as 1β-methyl-6-hydroxyl-bicyclo[4,2,0]-hex-1-ene-4α-(2′-methyl)-isooctanoic acid. Analysis of the previously reported bisaboleneol acid revealed that almost all possessed hydroxyl group on ring, and the carboxyl group instead of the end of the carbon chain. However, it shows that compound 3 is a new compound isolated from the arthropods Buthus martensii kirsch, and has not been reported in the literature before.
The absolute configuration of 3 was established by comparison of the NOESY experimental with reference to the literature [14] and its CD spectra determined. The NOESY correlations of H-5eq /H-2′eq /H-4′, and H-5a/H-3eq (Figure 7a), as well as the coupling constants of H-5 and H-2′ value <10.0 Hz respectively in 1 H-NMR spectrum in CDCl3 (Table 3)   Therefore, the planar structure of 3 was elucidated as a α-bisaboleneol acid and was reasonably assigned as 1β-methyl-6-hydroxyl-bicyclo[4,2,0]-hex-1-ene-4α-(2 -methyl)-isooctanoic acid. Analysis of the previously reported bisaboleneol acid revealed that almost all possessed hydroxyl group on ring, and the carboxyl group instead of the end of the carbon chain. However, it shows that compound 3 is a new compound isolated from the arthropods Buthus martensii kirsch, and has not been reported in the literature before.

Assay of Antibacterial and Bactericidal Activities
The isolated compounds were tested for their antibacterial and germicide activities against four opportunistic pathogen strains-Bacillus subtilis ATCC 6051, Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853-as well as methicillin-resistant Staphylococcus aureus (MRSA) by the two fold serial dilution method [15]. The positive control, penicillin G (potassium salt, 1598 units per mg) gentamycin (sulfate 1000 units per mg), and vancomycin (50 units per mg) were purchased from Sigma-Aldrich Ltd., St. Louis, MO, USA.
The results indicated that compounds 1 and 2, both with 5,22E-cholestadienol structure, have a broad antibacterial spectrum against the opportunistic pathogen P. aeruginosa ATCC 27853 and S. aureus ATCC 6538. The minimal inhibitory concentrations (MICs) value of the sample was firstly measured by the dual-dilution method. In order to obtain the effective fungicide leading compound and further determine the MBC value, we selected the MIC value of less than 64 µg/mL for sustainable development and the testing sample. The results showed the significant antibacterial effect for the MIC values of 64 µg/mL of compound 1 against P. aeruginosa ATCC 27853 and 78 µg/mL against S. aureus ATCC 6538. Compound 2 displayed the stronger antibacterial effect with the MIC values of 16 µg/mL against both P. aeruginosa ATCC 27853 and S. aureus ATCC 6538. Then, the MBC values of 2 were at 32 µg/mL and less than 48 µg/mL, respectively. 5,22E-cholestadienol derivatives (compounds 1 and 2) are small molecules of natural products with the significant inhibitory effects against pathogenic bacteria S. aureus ATCC 6538 and P. aeruginosa ATCC 27853. And MBC/MIC ratios of two compounds were quantified by using a luciferase-based assay [16]. According to the ratio of MBC/MIC, it is possible to identify the antibacterial profile of compounds (bacteriostatic and/or bactericidal). The result shows that compound 2 inhibited bacterial growth of both S. aureus and P. aeruginosa in a bactericidal rather than a bacteriostatic manner (MBC/MIC ratio ≤ 2). Similarly, with compound 1, a ratio of MBC/MIC ≤ 2 indicates bactericidal activity inhibited bacterial growth of P. aeruginosa, whereas a ratio of MBC/MIC ≥ 4 defined a bacteriostatic effect [17]. Interestingly, this suggests that these two compounds with 5,22E-cholestadienol structure classified as bactericidal agents against broad spectrum bactericide activities from Buthus martensii karsch, which is a more interesting profile (Table 3). It was thus suggested that compound 2 had the most potent broad-spectrum bactericidal activity and need further investigation into the mechanistic insight. All in vitro experiments were performed in duplicate.      Table 2.
Supplementary Materials can include anything data of reported compounds and they have been designated open access or are freely available online.

General Experimental Procedure
Optical rotations were measured using a JASCO DIP-360 (Tokyo, Japan automatic digital polarimeter). IR and UV spectra were recorded using the JASCOFT-IR 620 spectrophotometer and UV-2600 instrument, respectively. 1D and 2D NMR spectra were recorded on Bruker DRX-400 spectrometer (400 MHz for 1 H-NMR, Karlsruhe, Germany) with TMS used as internal standard. The mass spectra were obtained on Agilent Series1100 SL mass spectrometer (Agilent Technologies Inc., Santa Clara, CA, USA), and Bruker Daltonics mass spectrometer (Bruker Daltonics Inc. Billerica, MA, USA) with an electrospray ionization source. Circular Dichroism (CD) was obtained using the Chirascan, Applied Photophysics Ltd. (Surrey, UK). HPLC was performed using a system comprised of a CCPM pump (Tosoh, Tokyo, Japan), a CCP PX-8010 controller (Tosoh), an RI-8010 detector (Tosoh) or a Shodex OR-2 detector (Showa-Denko, Tokyo, Japan), and a Rheodyne injection port. A Capcell Pak C18 UG120 column (10 mm i.d. × 250 mm, 5 µm, Shiseido, Tokyo, Japan) was employed for preparative HPLC. Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, SE, USA) was used for column chromatography (CC), and a silica gel GF 254 (10-40 mm, Haiyang Co., Qingdao, China) was used for preparative TLC as precoated plates. TLC spots were visualized under UV light through dipping into 5% H 2 SO 4 in alcohol. All chemicals used were analytical grade. In the antibacterial experiment, four selected pathogenic strains and one resistant strain were commonly used in clinic, including two Gram-positive bacteria (B. subtilis and S. aureus), two Gram-negative bacteria (E. coli and P. aeruginosa), and the multidrug-resistant strain (methicillin-resistant Staphylococcus aureus-MRSA). Four species of standard strains were acquired from Shanghai Biological Research Technology Co., Ltd. (Shanghai, China) and one species of MRSA strains was obtained from clinically isolated drug-resistant strain.

Determined Method of Antibacterial and the Bactericidal Activity
Antibacterial assays were performed using standard bacteria including B. subtilis ATCC 6051, S. aureus ATCC 6538, E. coli ATCC 25922, P. aeruginosa ATCC 27853, and methicillin-resistant Staphylococcus aureus (MRSA). The bacteria were isolated and cultivated using clinical and Laboratory Standards for antimicrobial susceptibility testing [15]. A single colony of bacteria were cultured from a master plate and put into a bottle of 10.0 mL LB. medium, cultured usually until the bacteria were growing well after shaking at 37 • C overnight or 28 • C. The UV absorbance was measured at 600 nm with 1.0 mL of the culture which the bacterial concentration controlled and determined to each well by ultraviolet spectrophotometer. The corresponding OD values of the different strains were respectively at Abs.600 nm as: B. subtilis ATCC 6051, A = 0.025; S. aureus ATCC 6538, A = 0.072; E. coli ATCC 25922, A = 0.055; P. aeruginosa ATCC 27853, A = 0.054; and MRSA (clinically isolated strain), A = 0.066.
The main purpose of the study was to determine the in vitro inhibitory and bactericidal activities and the level of tolerance to the three compounds observed by standardized MIC and MBC tests. MICs of each compound were determined by broth two fold serial dilution technique, in accordance with the guidelines of the Clinical and Laboratory Standards Institute [15]. Compounds 1-3 were tested at dilution ranges of 1000, 625, 312, 256, 128, 78, 64, 32, 16, 8, 3.12, to 1.56 µg/mL. Standard cation-adjusted penicillin G, gentamycin, and vancomycin were used as positive controls for MIC testing, with 70% methanol for the negative control of each petri dishes. Quantitative 3 µL of the tested sample was added to each well and cultured for 12 h at 37 • C or 28 • C. The plate was complemented with 1.00% tryptone, 0.55 g yeast extract, 15.00 g sodium chloride, adjusted to pH 7.5 and then add 1.50% agar as a semisolid medium for the bacterial detection. MBCs were determined in accordance with the guidelines of the Clinical and Laboratory Standards Institute [18]. The entire volume 100 µL of the MIC well and the wells with 4 dilutions above the MIC were spread across the center of a broth agar plate and allowed to dry for 20 min. Then, a sterile spreading rod was used to evenly disperse the inoculum over the entire surface of the plate, which was then incubated at 37 • C for 24 to 48 h. The MBCs were recorded as the lowest dilution to produce a 99.9% reduction in growth in comparison to the growth of the control. Furthermore, drug resistance can be determined when the tested drug MBC/MIC is at or over 32 times [19,20]. Vancomycin and ticlonin have been demonstrated. Vancomycin lacked bactericidal activity (defined as an MBC/MIC ratio of > or = 32) against two methicillin-resistant Staphylococcus aureus (MRSA) isolates from patients with bone and joint infection [21,22]. By contrast, MIC values of many strains were consistent with the tolerance in literature reported (MBC/MIC ratios ≥ 32) [23].

Molecular Docking Study
Compounds 1 and 2 belong to the 5,22E-cholestadienol derivatives have the broad-spectrum bactericide activity. This work aimed to evaluate the structure-activity relationships and to establish the mode of interaction by receptor-ligand interactions molecular modeling, thus revealing the action mechanism of target molecules.
Computational docking study was performed using PharmMapper method to detect and identify drug targets by Discovery Studio 3.0 (East China University of Science & Technology, and Shanghai institute of medicine, Chinese academy of sciences, Shanghai, China) [24]. Based on the possible targets screened, the target protein 2XRL of the doxycycline was found as the broad-spectrum antibiotics receptor [25] and 1Q23 protein of fusidic acid was the Gram-positive cocci receptor [26,27]. Both antibiotics structural was shown in Figure 8. According to the antibiotic drug doxycycline and the corresponding target protein (PDB ID: 2XRL) binding model, and fusidic acid with target protein (PDB ID: 1Q23) binding model, compounds 1 and 2 were further studied by molecular docking.
Molecules 2018, 23, x 10 of 17 when the tested drug MBC/MIC is at or over 32 times [19,20]. Vancomycin and ticlonin have been demonstrated. Vancomycin lacked bactericidal activity (defined as an MBC/MIC ratio of > or = 32) against two methicillin-resistant Staphylococcus aureus (MRSA) isolates from patients with bone and joint infection [21,22]. By contrast, MIC values of many strains were consistent with the tolerance in literature reported (MBC/MIC ratios ≥ 32) [23].

Molecular Docking Study
Compounds 1 and 2 belong to the 5,22E-cholestadienol derivatives have the broad-spectrum bactericide activity. This work aimed to evaluate the structure-activity relationships and to establish the mode of interaction by receptor-ligand interactions molecular modeling, thus revealing the action mechanism of target molecules.
Computational docking study was performed using PharmMapper method to detect and identify drug targets by Discovery Studio 3.0 (East China University of Science & Technology, and Shanghai institute of medicine, Chinese academy of sciences, Shanghai, China) [24]. Based on the possible targets screened, the target protein 2XRL of the doxycycline was found as the broad-spectrum antibiotics receptor [25] and 1Q23 protein of fusidic acid was the Gram-positive cocci receptor [26,27]. Both antibiotics structural was shown in Figure 8. According to the antibiotic drug doxycycline and the corresponding target protein (PDB ID: 2XRL) binding model, and fusidic acid with target protein (PDB ID: 1Q23) binding model, compounds 1 and 2 were further studied by molecular docking. Through some related data mining and receptor ligand interaction verification, drug targets of compounds 1 and 2 can be predicted involving the hydrogen bonds, electrostatic forces, van der Waals forces, etc., and the lead compound with 5,22E-cholesterol derivatives structure can be confirmed for the compounds 1 or 2. The active site of the target protein of two compounds had the broad-spectrum bactericide proteins 2XRL and anti-G+ bacteria 1Q23, which there were PDB site displayed in Figure 9. Regions of active site were functionally defined and confirmed independently through automatically parameters correction of the image processing computer. Through some related data mining and receptor ligand interaction verification, drug targets of compounds 1 and 2 can be predicted involving the hydrogen bonds, electrostatic forces, van der Waals forces, etc., and the lead compound with 5,22E-cholesterol derivatives structure can be confirmed for the compounds 1 or 2. The active site of the target protein of two compounds had the broad-spectrum bactericide proteins 2XRL and anti-G+ bacteria 1Q23, which there were PDB site displayed in Figure 9. Regions of active site were functionally defined and confirmed independently through automatically parameters correction of the image processing computer. The antibacterial activity of 5,22E-cholesteric compounds 1 and 2 was studied by reverse molecular docking. The doxycycline of an antibiotic clinically used in sensitive bacterial infection was used as reference. The 2XRL protein receptor of doxycycline is a homodimer and its catalytic site located in the active site BC4 of multiple areas (Figure 9a). A 3D graphic description of the docking structures for compounds 1 and 2 are shown in Figure 10. We can observe the scores ranking after molecular docking on the molecular absolute Energy, Conf. Number, Relative Energy, and LibDock in electrostatic 2D structure ( Table 4). The docking study showed that compound 2 presented a good score (98.2142), which is close to the score of the reference doxycycline control (99.0843). These results are in concordance with that obtained on in vitro assays (Table 3). Actual antimicrobial activity of compound 2 is stronger than compound 1. A 2D graphic description of the ligand and receptor protein interactions is shown in Figure 11. Although these new compounds are not as strong as the effective antimicrobial agents doxycycline, we made the hypothesis that compounds 1 and 2 could be the bactericide overcoming the issues of bacterial resistance through covalent binding interactions between their binding site The antibacterial activity of 5,22E-cholesteric compounds 1 and 2 was studied by reverse molecular docking. The doxycycline of an antibiotic clinically used in sensitive bacterial infection was used as reference. The 2XRL protein receptor of doxycycline is a homodimer and its catalytic site located in the active site BC4 of multiple areas (Figure 9a). A 3D graphic description of the docking structures for compounds 1 and 2 are shown in Figure 10. The antibacterial activity of 5,22E-cholesteric compounds 1 and 2 was studied by reverse molecular docking. The doxycycline of an antibiotic clinically used in sensitive bacterial infection was used as reference. The 2XRL protein receptor of doxycycline is a homodimer and its catalytic site located in the active site BC4 of multiple areas (Figure 9a). A 3D graphic description of the docking structures for compounds 1 and 2 are shown in Figure 10. We can observe the scores ranking after molecular docking on the molecular absolute Energy, Conf. Number, Relative Energy, and LibDock in electrostatic 2D structure ( Table 4). The docking study showed that compound 2 presented a good score (98.2142), which is close to the score of the reference doxycycline control (99.0843). These results are in concordance with that obtained on in vitro assays (Table 3). Actual antimicrobial activity of compound 2 is stronger than compound 1. A 2D graphic description of the ligand and receptor protein interactions is shown in Figure 11. Although these new compounds are not as strong as the effective antimicrobial agents doxycycline, we made the hypothesis that compounds 1 and 2 could be the bactericide overcoming the issues of bacterial resistance through covalent binding interactions between their binding site We can observe the scores ranking after molecular docking on the molecular absolute Energy, Conf. Number, Relative Energy, and LibDock in electrostatic 2D structure ( Table 4). The docking study showed that compound 2 presented a good score (98.2142), which is close to the score of the reference doxycycline control (99.0843). These results are in concordance with that obtained on in vitro assays (Table 3). Actual antimicrobial activity of compound 2 is stronger than compound 1. A 2D graphic description of the ligand and receptor protein interactions is shown in Figure 11. binding interaction with active pocket of amino acid residues as GlnA:109 and SerA:135 from doxycycline receptor proteins; compound 1 displayed the hydrogen binding interaction with GlnA:109 and van der Waals force; however, there were residues inthe SerA:135 hydrogen binding and π-π interaction of PheA:86 in compound 2, which would be a good explanation that 5,22E-cholestadienol compounds have the broad-spectrum of antibacterial activity and the different levels of inhibition are related to the interaction strength of those compounds.  Although these new compounds are not as strong as the effective antimicrobial agents doxycycline, we made the hypothesis that compounds 1 and 2 could be the bactericide overcoming the issues of bacterial resistance through covalent binding interactions between their binding site and the receptor protein. The interaction between each compound and the key amino acids residues of the active site of 2XRL can be clearly seen from 2D diagram of chemical bonding interaction ( Figure 11). Molecular docking of compounds showed important van der Waals and hydrogen binding interaction with active pocket of amino acid residues as GlnA:109 and SerA:135 from doxycycline receptor proteins; compound 1 displayed the hydrogen binding interaction with GlnA:109 and van der Waals force; however, there were residues inthe SerA:135 hydrogen binding and π-π interaction of PheA:86 in compound 2, which would be a good explanation that 5,22E-cholestadienol compounds have the broad-spectrum of antibacterial activity and the different levels of inhibition are related to the interaction strength of those compounds.
Secondly, based on the evaluation of the same skeleton antibiotic fusidic acid from PDB ID: 1Q23 (Figure 9b), the target molecular docking was used to demonstrate mechanisms of 5, 22E-cholestadienol compounds 1 and 2 against Gram-positive bacteria S. aureus ATCC 6538. The receptor protein 1Q23 of Fusidic acid bond site with amino acid residues for active pocket or catalytic site is located on the active site AC1 of multiple areas (Figure 9b). A 2D graphic description of the docking fusidic acid as a control for compounds 1 and 2 is shown in Figure 12. The docking site and binding of fusidic acid was selected as reference. Secondly, based on the evaluation of the same skeleton antibiotic fusidic acid from PDB ID: 1Q23 (Figure 9b), the target molecular docking was used to demonstrate mechanisms of 5, 22E-cholestadienol compounds 1 and 2 against Gram-positive bacteria S. aureus ATCC 6538. The receptor protein 1Q23 of Fusidic acid bond site with amino acid residues for active pocket or catalytic site is located on the active site AC1 of multiple areas (Figure 9b). A 2D graphic description of the docking fusidic acid as a control for compounds 1 and 2 is shown in Figure 12. The docking site and binding of fusidic acid was selected as reference.   (Table 5). These scores reveal that the chemical binding force of the extract substances is superior to the score of antibiotics  (Table 5). These scores reveal that the chemical binding force of the extract substances is superior to the score of antibiotics fluidic acid (122.250). These results are in concordance with those obtained on the in vitro assays (Table 3). A 2D graphic description of the ligand interactions are shown in Figure 12.  Table above indicates that compounds 1 and 2 are very strong antimicrobial agents even compared to a positive control. Based on the receptor-ligand interactions of compounds 1, 2 and the control with the active site of target protein 1Q23, the antibacterial activities and mechanism of action in the 2D diagram of chemical bonding interaction are evident ( Figure 13). Molecular docking of compounds showed important Van der Waals and hydrogen binding interaction with active pocket of amino acid residues as SerG:146 and ThruG:172 between fusidic acid and receptor proteins 1Q23; compound 1 displayed the stranger hydrogen binding interaction with HisH:193 Sigma-Pi interaction and Van der Waals force; compound 2 had similar Van der Waals force and Sigma-Pi interaction of TyrJ:33 with the amino acid residues. Therefore, compound 2 is another strong Gram-positive bactericide according to the discovery of molecular docking mechanism, which explains the strong bactericidal activity against S. aureus ATCC 6538 of compound 2, which was observed in the previous antibacterial experiments with MICs of 16 µg/mL and MBCs of 32 µg/mL.  (Table 3). A 2D graphic description of the ligand interactions are shown in Figure 12.  Table above indicates that compounds 1 and 2 are very strong antimicrobial agents even compared to a positive control. Based on the receptor-ligand interactions of compounds 1, 2 and the control with the active site of target protein 1Q23, the antibacterial activities and mechanism of action in the 2D diagram of chemical bonding interaction are evident ( Figure 13). Molecular docking of compounds showed important Van der Waals and hydrogen binding interaction with active pocket of amino acid residues as SerG:146 and ThruG:172 between fusidic acid and receptor proteins 1Q23; compound 1 displayed the stranger hydrogen binding interaction with HisH:193 Sigma-Pi interaction and Van der Waals force; compound 2 had similar Van der Waals force and Sigma-Pi interaction of TyrJ:33 with the amino acid residues. Therefore, compound 2 is another strong Gram-positive bactericide according to the discovery of molecular docking mechanism, which explains the strong bactericidal activity against S. aureus ATCC 6538 of compound 2, which was observed in the previous antibacterial experiments with MICs of 16 μg/mL and MBCs of 32 μg/mL.

Conclusions
Currently, there is great interest in the study of insects and related arthropods to develop new natural products for various medicinal purposes. In this work, we found three new compounds from the crude extract of scorpion Buthus martensii karsch. Among them, compounds 1 and 2 of the 5,22E-cholestadienol derivatives were identified as broad-spectrum bactericides, and their activity showed significant concentration-dependent effects against Staphylococcus aureus ATCC 6538 and Pseudomonas aeruginosa ATCC 27853.
Compound 2 was found to fit well in the active site of binding pocket both target proteins (PDB ID:2XRL and PDB ID:1Q23) from the chemical bonds formed intermolecular forces. This molecular docking study demonstrated that compound 2 is an effective lead compound for natural antibiotics, whether it is as a broad-spectrum antibiotic potence. The antibacterial mechanism is the specific binding (various of bonding forces between molecules) using compound 2 as a ligand based on the different receptor proteins 2XRL or 1Q23 active sites from bacterial ribosome unit A, and thus prevent the synthesis of bacterial proteins. This unique mechanism avoids the cross-resistance issues of other antibacterial drugs.
In the present stereochemical studies, we established molecular configuration of new compounds 1 and 3. The absolute configurations of compound 1 were confirmed by comparing the known chiral analogs of C-24R orientation (24α for the ethyl group) with its J value analysis of chemical correlation between H-17ax and H-20eq in the 1 H-NMR spectrum of compound 1. Similarly, the absolute configuration of compound 3 proposed the α-bisaboleneol structure as a reference on the Raharivelomanana's group, and we report the stereochemical determination of compound 3. Furthermore, The CD spectra data of compounds 1-3 also fully support the stereochemical assignments.

Conclusions
Currently, there is great interest in the study of insects and related arthropods to develop new natural products for various medicinal purposes. In this work, we found three new compounds from the crude extract of scorpion Buthus martensii karsch. Among them, compounds 1 and 2 of the 5,22E-cholestadienol derivatives were identified as broad-spectrum bactericides, and their activity showed significant concentration-dependent effects against Staphylococcus aureus ATCC 6538 and Pseudomonas aeruginosa ATCC 27853.
Compound 2 was found to fit well in the active site of binding pocket both target proteins (PDB ID:2XRL and PDB ID:1Q23) from the chemical bonds formed intermolecular forces. This molecular docking study demonstrated that compound 2 is an effective lead compound for natural antibiotics, whether it is as a broad-spectrum antibiotic potence. The antibacterial mechanism is the specific binding (various of bonding forces between molecules) using compound 2 as a ligand based on the different receptor proteins 2XRL or 1Q23 active sites from bacterial ribosome unit A, and thus prevent the synthesis of bacterial proteins. This unique mechanism avoids the cross-resistance issues of other antibacterial drugs.
In the present stereochemical studies, we established molecular configuration of new compounds 1 and 3. The absolute configurations of compound 1 were confirmed by comparing the known chiral analogs of C-24R orientation (24α for the ethyl group) with its J value analysis of chemical correlation between H-17ax and H-20eq in the 1 H-NMR spectrum of compound 1. Similarly, the absolute configuration of compound 3 proposed the α-bisaboleneol structure as a reference on the Raharivelomanana's group, and we report the stereochemical determination of compound 3. Furthermore, The CD spectra data of compounds 1-3 also fully support the stereochemical assignments.