Design, Synthesis, and Antibacterial Evaluation of Novel Ocotillol Derivatives and Their Synergistic Effects with Conventional Antibiotics

The improper use of antibiotics has led to the development of bacterial resistance, resulting in fewer antibiotics for many bacterial infections. Especially, the drug resistance of hospital-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) is distinctly serious. This research designed and synthesized two series of 3-substituted ocotillol derivatives in order to improve their anti-HA-MRSA potency and synergistic antibacterial activity. Among the synthesized compounds, 20–31 showed minimum inhibitory concentration (MIC) values of 1–64 µg/mL in vitro against HA-MRSA 18–19, 18–20, and S. aureus ATCC29213. Compound 21 showed the best antibacterial activity, with an MIC of 1 μg/mL and had synergistic inhibitory effects. The fractional inhibitory concentration index (FICI) value was 0.375, when combined with chloramphenicol (CHL) or kanamycin (KAN). The structure–activity relationships (SARs) of ocotillol-type derivatives were also summarized. Compound 21 has the potential to be developed as a novel antibacterial agent or potentiator against HA-MRSA.


Introduction
Globally, most bacteria (e.g., Mycobacterium tuberculosis, Escherichia coli, and Staphylococcus aureus) can cause severe human diseases (e.g., tuberculosis, sepsis, and skin infections) [1,2]. Because of the improper use and abuse of antibiotics, many bacteria have become resistant or even cross-resistant to many antibacterial drugs on the market, such as methicillin, chloramphenicol (CHL), and kanamycin (KAN) [3,4]. Diseases and deaths caused by drug-resistant bacterial infections have resulted in great losses to human health and property [5]. The World Health Organization prioritized 20 drug-resistant bacteria on the basis of indicators, such as mortality, resistance rates, health care burden, 10-year resistance trends, and transmissibility, which showed that methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) ranked the highest [6].
Natural products are important in the development of new drugs. Over the past four decades, medicinal chemists have become increasingly interested in the development of new drugs derived from natural products [7,8]. One of the strategies to treat diseases caused by drug-resistant bacterial infections is to combine existing antibiotics with phytochemicals. Most phytochemicals possess antimicrobial activity and when these plant-derived compounds are used in combination with conventional antibiotics, they can achieve better antibacterial effects and prevent the development of bacterial resistance [9][10][11]. Betulinic acid isolated from the leaves of Vitex negundo reduces the MIC of methicillin against MRSA Natural active ingredients derived from terpenoids have good antibacterial activity against MRSA and VRE, such as oleanolic acid (OA) from Salvia officinalis, ursolic acid (UA) from Alstonia scholaris, and 16R-hydroxymollic from Acalypha communis [16,17] (Figure 2). Ginseng is a perennial herb, mainly distributed in China, Russia, and Korea [18]. Ginsenosides and their metabolites (such as G-Rb1, G-Rb2, G-Rd, G-Re, and G-Rg1) have good effects on the human nervous system, cardiovascular system, and endocrine system, among others [18][19][20]. Ocotillol-type ginsenosides found in Panax quinquefolius L., Vietnamese ginseng, and Panax japonicus have a wide range of excellent pharmacological activities (including anti-ischemic, anti-inflammatory, and antitumor activities) [21]. Our preliminary research found that ocotillol-type ginsenosides have good antibacterial activity, such as (20S, 24S)-ocotillol with an MIC value of 8 µ g/mL against MRSA USA300 [22]. After isonipecotic acid modification, compound 1a was synthesized with an MIC value of 8 µ g/mL against MRSA USA300. Compound 2a, composed of ocotillol connected with succinic anhydride, had an MIC value of 16 µ g/mL against RN4220. Additionally, when compound 1a was combined with KAN, a synergistic antibacterial effect was observed against MRSA USA300 with an FICI of 0.28 [23,24] (Figure 2).  Natural active ingredients derived from terpenoids have good antibacterial activity against MRSA and VRE, such as oleanolic acid (OA) from Salvia officinalis, ursolic acid (UA) from Alstonia scholaris, and 16R-hydroxymollic from Acalypha communis [16,17] (Figure 2). Ginseng is a perennial herb, mainly distributed in China, Russia, and Korea [18]. Ginsenosides and their metabolites (such as G-Rb1, G-Rb2, G-Rd, G-Re, and G-Rg1) have good effects on the human nervous system, cardiovascular system, and endocrine system, among others [18][19][20]. Ocotillol-type ginsenosides found in Panax quinquefolius L., Vietnamese ginseng, and Panax japonicus have a wide range of excellent pharmacological activities (including anti-ischemic, anti-inflammatory, and antitumor activities) [21]. Our preliminary research found that ocotillol-type ginsenosides have good antibacterial activity, such as (20S, 24S)-ocotillol with an MIC value of 8 µg/mL against MRSA USA300 [22]. After isonipecotic acid modification, compound 1a was synthesized with an MIC value of 8 µg/mL against MRSA USA300. Compound 2a, composed of ocotillol connected with succinic anhydride, had an MIC value of 16 µg/mL against RN4220. Additionally, when compound 1a was combined with KAN, a synergistic antibacterial effect was observed against MRSA USA300 with an FICI of 0.28 [23,24] (Figure 2). achieve better antibacterial effects and prevent the development of bacterial resistance [9][10][11]. Betulinic acid isolated from the leaves of Vitex negundo reduces the MIC of methicillin against MRSA to 1/64×MIC. When ferulic acid from Phenylpropanoids was combined with amikacin, a synergistic antibacterial effect was noticed against Staphylococcus aureus NCIM 5021 with a fractional inhibitory concentration index (FICI) of 0.16. Epipallocatechin gallate (EGCG), a principal ingredient of tea, drives the anti-MRSA MIC of imipenem from 128 μg/mL to 8 μg/mL. Therefore, it is necessary to develop alternative natural or combination drug therapies [12][13][14][15] (Figure 1).
Natural active ingredients derived from terpenoids have good antibacterial activity against MRSA and VRE, such as oleanolic acid (OA) from Salvia officinalis, ursolic acid (UA) from Alstonia scholaris, and 16R-hydroxymollic from Acalypha communis [16,17] (Figure 2). Ginseng is a perennial herb, mainly distributed in China, Russia, and Korea [18]. Ginsenosides and their metabolites (such as G-Rb1, G-Rb2, G-Rd, G-Re, and G-Rg1) have good effects on the human nervous system, cardiovascular system, and endocrine system, among others [18][19][20]. Ocotillol-type ginsenosides found in Panax quinquefolius L., Vietnamese ginseng, and Panax japonicus have a wide range of excellent pharmacological activities (including anti-ischemic, anti-inflammatory, and antitumor activities) [21]. Our preliminary research found that ocotillol-type ginsenosides have good antibacterial activity, such as (20S, 24S)-ocotillol with an MIC value of 8 µ g/mL against MRSA USA300 [22]. After isonipecotic acid modification, compound 1a was synthesized with an MIC value of 8 µ g/mL against MRSA USA300. Compound 2a, composed of ocotillol connected with succinic anhydride, had an MIC value of 16 µ g/mL against RN4220. Additionally, when compound 1a was combined with KAN, a synergistic antibacterial effect was observed against MRSA USA300 with an FICI of 0.28 [23,24] (Figure 2).  On the basis of the good anti-MRSA potency and synergistic antibacterial activity of the ocotillol-type ginsenosides [25], our group considered (20S, 24S)-ocotillol and its isomer (20S, 24R)-ocotillol as the lead compounds for structural modification. In our previous work, we synthesized different series of ocotillol-type derivatives, from which we identified certain SARs [22,23,26]. The antibacterial activity of ocotillol derivatives increases when the pharmacophore contains an amino group or is a hydrogen bond donor [27]. Additionally, studies have shown that nitrogen-containing heterocycles, such as triazoles, morpholines, succinimides, Fmoc-protected amino acids, and dansulfonyl chloride, have extensive and excellent pharmacological activities, especially in terms of their antibacterial properties [28][29][30][31]. It is well known that amino acids play important roles in various medical fields, particularly, in the antimicrobial [32]. Researchers have shown that Fmocconjugated amino acids (Fmoc-AA) have a broad range of antimicrobial effects, which linearly correlates with their surfactant property [33]. Therefore, in this research, to obtain compounds with good anti-drug-resistant bacterial potency or synergistic antibacterial activity and further explore the SARs, we synthesized two series of new 3-substituted ocotillol derivatives by introducing nitrogen-containing heterocycles and Fmoc-AA.
Because of the clinical misuse of antibiotics and the higher prevalence of bacteria in hospitals, hospital-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) spreads faster and is more resistant to marketed antibacterial drugs than common Staphylococcus aureus [34][35][36]. Therefore, we conducted antibacterial activity assays using S. aureus ATCC 29213, and HA-MRSA strains18-19 and 18-20 on the 18 compounds designed and synthesized in this work so that the experiment was representative of clinical conditions. Furthermore, compounds 20 and 21, which had good antibacterial activity, were selected for evaluation of their synergistic antibacterial activity with KAN and CHL.

Chemistry
Compounds 4 and 5 were synthesized by a straightforward three-step procedure. The synthesis of ocotillol derivatives 4 and 5 followed previously reported procedures as shown in Scheme 1. On the basis of the good anti-MRSA potency and synergistic antibacterial activity of the ocotillol-type ginsenosides [25], our group considered (20S, 24S)-ocotillol and its isomer (20S, 24R)-ocotillol as the lead compounds for structural modification. In our previous work, we synthesized different series of ocotillol-type derivatives, from which we identified certain SARs [22,23,26]. The antibacterial activity of ocotillol derivatives increases when the pharmacophore contains an amino group or is a hydrogen bond donor [27]. Additionally, studies have shown that nitrogen-containing heterocycles, such as triazoles, morpholines, succinimides, Fmoc-protected amino acids, and dansulfonyl chloride, have extensive and excellent pharmacological activities, especially in terms of their antibacterial properties [28][29][30][31]. It is well known that amino acids play important roles in various medical fields, particularly, in the antimicrobial [32]. Researchers have shown that Fmoc-conjugated amino acids (Fmoc-AA) have a broad range of antimicrobial effects, which linearly correlates with their surfactant property [33]. Therefore, in this research, to obtain compounds with good anti-drug-resistant bacterial potency or synergistic antibacterial activity and further explore the SARs, we synthesized two series of new 3-substituted ocotillol derivatives by introducing nitrogen-containing heterocycles and Fmoc-AA.
Because of the clinical misuse of antibiotics and the higher prevalence of bacteria in hospitals, hospital-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) spreads faster and is more resistant to marketed antibacterial drugs than common Staphylococcus aureus [34][35][36]. Therefore, we conducted antibacterial activity assays using S. aureus ATCC 29213, and HA-MRSA strains18-19 and 18-20 on the 18 compounds designed and synthesized in this work so that the experiment was representative of clinical conditions. Furthermore, compounds 20 and 21, which had good antibacterial activity, were selected for evaluation of their synergistic antibacterial activity with KAN and CHL.

Chemistry
Compounds 4 and 5 were synthesized by a straightforward three-step procedure. The synthesis of ocotillol derivatives 4 and 5 followed previously reported procedures as shown in Scheme 1.

Antibacterial Activity
The initial antibacterial activity results of the compounds against HA-MRSA 18-19, 18-20, and S. aureus ATCC 29213 are summarized in Table 1. Our previous research showed that ocotillol phthalic anhydride derivatives linked with N-containing heterocycles did not demonstrate antibacterial activity. Considering different functional groups

Antibacterial Activity
The initial antibacterial activity results of the compounds against HA-MRSA 18-19, 18-20, and S. aureus ATCC 29213 are summarized in Table 1. Our previous research showed that ocotillol phthalic anhydride derivatives linked with N-containing heterocycles did not demonstrate antibacterial activity. Considering different functional groups and our previous work [24], succinic anhydride was used as a linker. However, the MICs of compounds 8-15 were greater than 128 µg/mL, thus the linker had no influence on the antibacterial activity. Among the synthesized compounds, 20-31 inhibited the growth of HA-MRSAs 18-19, 18-20 and S. aureus ATCC 29213 in vitro, with MIC values of 1-64 µg/mL. Compound 21 using L-2,4-diaminobutyric acid hydrobromide as the linking chain to the Fmoc group showed better antibacterial activity, with an MIC value of 1 µg/mL, while compounds 22 and 23 had good antibacterial activities against HA-MRSA 18-20 with MICs of 2 µg/mL. Compared with compounds 20-23, compounds 28-31 showed weaker antibacterial activity. Analysis of the structures of compounds 20-23 and 28-31 indicated that compounds 20-23, which were linked to the Fmoc group, had better antibacterial activity than compounds 28-31, which were linked to the dansyl chloride. The stereochemistry at C-24 dramatically affects the antibacterial activity, with the S-configuration preferred when C-3 hydroxyl is not substituted. However, the dramatic difference in antibacterial activity between the S-configuration and R-configuration caused by the stereochemistry at C-24 seemed to be overpowered by substitutions at C-3 hydroxyl [22,37]. Compounds 20-23 and 28-31, which were substituted with Fmoc or dansyl chloride at the C-3 hydroxyl, showed no significant differences in activity among stereoisomers against the same drug-resistant strain.

Synergistic Antibacterial Activity
Antibiotics with different phytochemicals are often used in combination to achieve a synergistic antibacterial effect in the clinical treatment of infections caused by drug-resistant bacteria [38]. According to the results in Table 2 and Table 3, compounds 20 and 21 possessed synergistic activity when combined with KAN or CHL (FICI < 0.5). FICI was defined as FICA+ FICB = (MICA+B/MICA) + (MICA+B/MICB), and when the value of FICI was less than or equal to 0.5, compounds A and B were deemed to have a synergistic effect.
When compound 21 was used with CHL, the antibacterial activity of CHL against HA-MRSA 18-15 was enhanced from 8 to 1 μg/mL, and the FICI value was 0.375. When compounds 20 and 21 were used with KAN, the antibacterial activity of KAN against HA-MRSA 18-15 was significantly enhanced from 2 to 0.5 and 0.25 μg/mL, and the FICI value was 0.3125 and 0.375, respectively. This result showed that compounds 20 and 21 have good synergistic effects in the presence of KAN or CHL and compound 21 is expected to be developed as a synergistic antibacterial drug in the future, but the drug combination did not produce a bactericidal effect.

Synergistic Antibacterial Activity
Antibiotics with different phytochemicals are often used in combination to achieve a synergistic antibacterial effect in the clinical treatment of infections caused by drug-resistant bacteria [38]. According to the results in Tables 2 and 3, compounds 20 and 21 possessed synergistic activity when combined with KAN or CHL (FICI < 0.5). FICI was defined as FICA + FICB = (MIC A+B /MIC A ) + (MIC A+B /MIC B ), and when the value of FICI was less than or equal to 0.5, compounds A and B were deemed to have a synergistic effect.  When compound 21 was used with CHL, the antibacterial activity of CHL against HA-MRSA 18-15 was enhanced from 8 to 1 µg/mL, and the FICI value was 0.375. When compounds 20 and 21 were used with KAN, the antibacterial activity of KAN against HA-MRSA 18-15 was significantly enhanced from 2 to 0.5 and 0.25 µg/mL, and the FICI value was 0.3125 and 0.375, respectively. This result showed that compounds 20 and 21 have good synergistic effects in the presence of KAN or CHL and compound 21 is expected to be developed as a synergistic antibacterial drug in the future, but the drug combination did not produce a bactericidal effect.

Structure-Activity Relationships (SARs)
Based on previous data and our recent work, a more comprehensive structure-activity relationship for ocotillol-type derivatives was obtained as follows (Figure 4). (a) A hydrogen donor at C-3 and C-12 is preferred to maintain the antibacterial activity; (b) decreased antibacterial activity was observed when the functional groups at C-3 and C-12 were ketones; (c) ocotillol-type derivatives coupled with N-heterocycles containing a tertiary amine did not improve the antibacterial activity; (d) the stereochemistry at C-24 of ocotillol affected the antibacterial activity slightly when C-3 hydroxyl was substituted with Fmoc or dansyl chloride; (e) the antibacterial activity of ocotillol with a connecting Fmoc was better than those with a connecting dansyl chloride; (f) substitution at the C-3 hydroxyl with a primary amine enhanced the activity against Gram-positive bacteria; and(g) ocotillol-type derivatives with long-chain amino acid substituents at C-3 improved the anti-MRSA ability of KAN and CHL. FICI: Fractional inhibitory concentration index.

Structure-Activity Relationships (SARs)
Based on previous data and our recent work, a more comprehensive structure-activity relationship for ocotillol-type derivatives was obtained as follows (Figure 4). (a) A hydrogen donor at C-3 and C-12 is preferred to maintain the antibacterial activity; (b) decreased antibacterial activity was observed when the functional groups at C-3 and C-12 were ketones; (c) ocotillol-type derivatives coupled with N-heterocycles containing a tertiary amine did not improve the antibacterial activity; (d) the stereochemistry at C-24 of ocotillol affected the antibacterial activity slightly when C-3 hydroxyl was substituted with Fmoc or dansyl chloride; (e) the antibacterial activity of ocotillol with a connecting Fmoc was better than those with a connecting dansyl chloride; (f) substitution at the C-3 hydroxyl with a primary amine enhanced the activity against Gram-positive bacteria; and(g) ocotillol-type derivatives with long-chain amino acid substituents at C-3 improved the anti-MRSA ability of KAN and CHL.

Chemical Reagents and Instruments
Most of the chemicals and solvents were of analytical grade and the solvents were purified and dried using standard methods. All the structures were verified by nuclear magnetic resonance (NMR), such as 1 H-NMR, 13 C-NMR, and high-resolution mass spectrometry (HR-MS). Spectra for all the compounds were obtained at 400 MHz for the 1 H

Chemical Reagents and Instruments
Most of the chemicals and solvents were of analytical grade and the solvents were purified and dried using standard methods. All the structures were verified by nuclear magnetic resonance (NMR), such as 1 H-NMR, 13 C-NMR, and high-resolution mass spectrometry (HR-MS). Spectra for all the compounds were obtained at 400 MHz for the 1 H NMR spectra and at 100 MHz for the 13 C NMR using a JNM-ECZ400S (JEOL Ltd., Japan) in CDCl 3 . HRMS was performed using a Q Exactive TM Orbitrap MS system (Thermo Scientific, USA) in CH 3 OH.
White  Compound 4 or 5 (80 mg, 0.17 mmol) was added to anhydrous dichloromethane (4 mL), then a Boc-Fmoc-protected amino acid (0.25 mmol), DMAP (56 mg, 0.50 mmol), and EDCI (96 mg, 0.50 mmol) were added slowly. After stirring at room temperature for 3 h, the resulting mixture was washed with 10% HCl, water, and brine, and dried over anhydrous Na 2 SO 4 . The dichloromethane was evaporated in vacuo to obtain the intermediates 16-19, 32, and 33. Trifluoroacetic acid (1 mL, 13.43 mmol) was added slowly to a solution of each intermediate 16-19, 32, and 33 (0.17 mmol) in anhydrous dichloromethane (4 mL). After stirring at room temperature for 2 h, the resulting mixture was filtered and evaporated to obtain the target compounds 20-23, 34, and 35. For compounds 20-23, each compound (0.17 mmol), dansyl chloride (55 mg, 0.20 mmol), and N, N-diisopropylethylamine (106 µL, 0.65 mmol) were combined in anhydrous dichloromethane (4 mL). After stirring at room temperature for 2 h, the resulting mixture was washed with 5% HCl, water, and brine, dried over in anhydrous Na 2 SO 4 , filtered, and evaporated to provide intermediates 24-27 (Scheme 3). Compounds 28-31 were synthesized by adding intermediates 24-27 (0.17 mmol) and piperidine (1 mL, 10.88 mmol) to anhydrous dichloromethane (4 mL). After stirring at room temperature for 4 h, the resulting mixture was washed with water and brine, dried over anhydrous Na 2 SO 4 , filtered, and evaporated. The residue was purified over silica gel with chloroform: methanol (100:1-50:1) to provide compounds 28-31.    the mechanism of the antibacterial potency and synergistic effect of ocotillol derivatives is probably related to the bacterial cell membrane. Compound 21 is a good probe that can be used to investigate the antibacterial and synergistic antibacterial mechanisms of ocotillol antibacterial derivatives.