Recent Advances in the Semisynthesis, Modifications and Biological Activities of Ocotillol-Type Triterpenoids

Ginseng is one of the most widely consumed herbs in the world and plays an important role in counteracting fatigue and alleviating stress. The main active substances of ginseng are its ginsenosides. Ocotillol-type triterpenoid is a remarkably effective ginsenoside from Vietnamese ginseng that has received attention because of its potential antibacterial, anticancer and anti-inflammatory properties, among others. The semisynthesis, modification and biological activities of ocotillol-type compounds have been extensively studied in recent years. The aim of this review is to summarize semisynthesis, modification and pharmacological activities of ocotillol-type compounds. The structure–activity relationship studies of these compounds were reported. This summary should prove useful information for drug exploration of ocotillol-type derivatives.


Introduction
From the 1940s to the middle of 2019, approximately 33.5% of approved drugs were either natural products or directly derived from them [1]. The development of new drug entities based on natural products as sources of novel structures is still an area of active research. Ginseng, including Asian ginseng (Panax vietnamensis HA et GRUSHV.) and American ginseng (Panax quinquefolium L.), is one of the most widely consumed herbs in the world and plays an important role in counteracting fatigue and alleviating stress [2,3]. Ginseng contains a variety of active ingredients, but its main active substances are attributed to its ginsenosides. The ginsenosides with the highest content in Vietnamese ginseng are protopanaxadiol, protopanaxatriol, oleanolic acid and 20,24-epoxydammarane (ocotillol) (Figure 1) [4,5].
Among the active components of ginseng, ocotillol-type compounds have received increasing attention because of their antibacterial, anticancer and anti-inflammatory properties [6,7]. Their different pharmacologic effects and potential molecular mechanisms have been gradually elucidated. Compared with the structure of dammarane ginsenosides (including the protopanaxadiol and protopanaxatriol types), ocotillol-type saponins are tetracyclic triterpenoid saponins containing a furan ring linked with aglycones.
Ocotillol-type saponins were first isolated from Fozrqwieria splendens Eliselm. in 1965 by Warnhoff et al. They were also found in Panax quinquefolium L, Panax vietnamensis HA et GRUSHV, and Panax japonicus var, to name a few [8][9][10][11][12][13][14][15][16][17]. However, because of the low content of ocotillol-type saponins in natural products, there were few studies on ocotillol-type derivatives in previous years [18,19]. Fueled by the growing use of semisynthetic methods for the preparation of ocotillol-type derivatives, increased research of ocotillol-type derivatives has been recently observed. In 2016, Liu et al. published a review that focused on the discovery, semisynthesis, biological activities and metabolism of ocotillol-type saponins [6]. However, the structure of most derivatives and its structure-activity relationship (SAR) were not mentioned in the article. Compared with the previous review, this review summarized the semisynthesis, modification and pharmacological activities of ocotillol-type derivatives. All the structures of ocotillol-type derivatives and their SARs in antibacterial, anti-inflammatory and tumor multidrug resistance reversal were summarized. This review provides useful information for the development of ocotillol-type derivatives and gives a direction for further inspiration to enrich its structures with good pharmacological activities.

Semisynthesis of Ocotillol-Type Compounds
Ocotillol-type sapogenins are less abundant in natural sources. Vietnamese ginseng contains higher amounts of ginseng saponins compared with other Panax genus species. The content of ocotillol-type saponins in Panax Vietnamese ginsengs is only 5.6%, while in Panax quinquefolius, it is less than 0.01% [20]. Additionally, 1 kg of fresh rhizome low-quality Vietnamese ginseng is about $1000 in 2019. These factors may have led to the slow development of ocotillol-type ginsenosides in previous years.
In 2005, 20(S)-protopanaxadiol (20(S)-PPD) was used as a raw material to obtain 4 and 5 by a semisynthetic method [21]. Yang et al. optimized and improved the synthetic process and achieved the industrial production of 4 and 5 [22].
Ocotillol-type sapogenins have been made using similar synthetic methods. 20(S)-PPD was used as the raw starting material and reacted with acetic anhydride, and then acetylated 20(S)-PPD was oxidized by m-CPBA. The molar ratio of the acetylated 20(S)-PPD to m-CPBA at −3°C is approximately 1:4, 3 h. The ocotillol-type epimers (4,5) were obtained by the hydrolysis of the oxidation products. The synthetic route is shown in Figure 2A [23].
After further research by Meng et al., the synthesis mechanism of ocotillol-type epimers was proposed as follows ( Figure 2B). 20(S)-PPD or 20(R)-PPD is oxidized by m-CPBA to generate the 24,25-epoxy intermediates, and then an intramolecular ring-opening loop reaction is carried out according to Baldwin's rule, and finally cyclization by a 5-exo-tet method forms a tetrahydrofuran ring [24][25][26].

Antibacterial Effects
Evidence has shown that ginseng has antibacterial properties, and its extract may be effective for treating bacterial infections in the future [33]. Compound 5 had strong antibacterial activities against Staphylococcus aureus (S. aureus) and Bacillus subtilis (B. subtilis) with minimum inhibitory concentration (MIC) values of 8 µg/mL [34]. Further research showed that 5 also had strong synergistic inhibition against community-associated methicillin-resistant S. aureus (MRSA; strain USA300), as 5 reduced the MIC of kanamycin (KAN) against MRSA USA300 from 1 µg/mL to 0.125 µg/mL giving a fractional inhibitory concentration index (FICI) of 0.14.
The furan ring, C-3 and C-12 are possible to explore in terms of chemical diversity as a modification of the furan ring, C-3, and C-12 significantly changed the antibacterial activity of ocotillol-type derivatives. Aromatic-substituted ocotillol-type derivatives 6-17 were synthesized by an esterification reaction, and their in vitro activity against Escherichia coli (E. coli), B. subtilis, S. aureus, Pseudomonas aeruginosa (P. aeruginosa) and Acinetobacter baumannii (A. baumannii) was determined ( Figure 4) [35]. Compounds 6 and 7 exhibited excellent antibacterial activities with MIC values of 1 µg/mL against S. aureus and B. subtilis, while compounds 9, 10, 12 and 16 exhibited moderate antibacterial activities against S. aureus. Further research showed that 6 and 7 displayed good antibacterial activities against MRSA USA300 with MIC values of 4 µg/mL. Additionally, 6 and 7 combined with KAN and chloramphenicol had strong synergistic inhibition against MRSA USA300 and reduced the MICs of KAN against MRSA USA300 from 1 µg/mL to 0.0156 and 0.0625 µg/mL (FICI = 0.078 and 0.020, respectively). with MIC values of 8 µg/mL. Most ocotillol-type derivatives with an amino group at C-3 displayed excellent antibacterial activities, while those with a carboxylic group at C-3 showed moderate activities. A synergistic effect was observed for compound 19 as it reduced the MIC of KAN against MRSA USA300 from 1 µg/mL to 0.25 µg/mL with a FICI of 0.28.
A series of ocotillol-type derivatives 34-55 with an amino group was also synthesized ( Figure 4) [39,40]. The antibacterial activity results showed that most of the ocotillol-type derivatives with an amino group had moderate to good inhibitory activities against Gram-positive bacteria but had no effect on Gram-negative bacteria. Compounds 38, 40 and 51 had good inhibitory activities against MRSA USA 300 with MICs ≤ 4 µg/mL, while 51 had the same antibacterial activity as KAN. A synergistic effect was observed for 39 when it was combined with KAN as shown by the significant enhancement of the MIC from 4 µg/mL to 0.25 µg/mL (FICI < 0.0088) against MRSA USA300.
A series of derivatives 57-61 were synthesized and screened. Among them, compound 58 had the best antibacterial activity against MRSA USA300 with a MIC of 8 µg/mL, and 60 had a moderate inhibitory effect against both Gram-positive and Gram-negative bacteria ( Figure 4). Additionally, 58 combined with KAN had strong synergistic inhibition against MRSA USA300 with a FICI of 0.008 [34].
The synthetic approaches to prepare compounds 6-56B are only slightly different. Compound 6 was synthesized by the treatment of compound 4, DMAP and phthalic anhydride in dry dichloromethane over 6 h to obtain 6 with 73% yield at room temperature. 1-ethyl-3(3-dimethylpropylamine) carbodiimide (EDCI) is an excellent dehydrating agent that can accelerate the esterification reaction. Compound 20 was synthesized by the treatment of 4, DMAP, N-Boc-isonipecotic acid and EDCI in dry dichloromethane over 3 h to obtain the intermediate with 80% yield at room temperature. The use of EDCI can increase the speed and yield of the esterification reaction. It is noteworthy that the hydroxyl group at the C-12 does not easily react with anhydride or acid because of steric hindrance and the formation of hydrogens bond. After the addition of 56A, DMAP and phthalic anhydride to anhydrous pyridine at 120 • C for 25 h, the yield of the intermediate is only 50%.
Ocotillol ketone derivatives 62-69 were synthesized by Zhou et al. ( Figure 5) [34,36]. Compound 4 (0.21 mmol) in dry dichloromethane (8 mL) was added to pyridinium chlorochromate (0.40 mmol), and the mixture was stirred at room temperature for 3 h to obtain compound 62 with 66% yield. While compound 64 was synthesized by combining 4 (0.33 mmol) and pyridinium chlorochromate (1.00 mmol) in dry dichloromethane (8 mL), the reaction takes about 8 h to obtain intermediate with 76% yield at room temperature. Compound 65 had excellent antibacterial activities against S. aureus with a MIC of 16 µg/mL, while compounds 67 and 69 had moderate inhibitory effects against S. aureus.   [42]. The results showed that 102 might exert its antibacterial effect by damaging bacterial cell membranes and disrupting the function of DNA. The precise mechanism of its DNA antibacterial action is currently under investigation. subtilis. An epifluorescent microscopy study showed that 109A was mainly distributed on the bacterial cell membrane rather than within the nucleoid (Figure 8) [4]. On this basis, Bi et al. synthesized the ocotillol-type probe 109B, which had a MIC of 1 µg/mL against MRSA 18-19 (Hospital-acquired methicillin-resistant Staphylococcus aureus, collected in Chengdu, China from 2018) [43]. The antibacterial mechanism of 109B against MRSA 18-19 is currently underway. The number of ocotillol-type probes is small, which limits the discovery of their antibacterial target. In 2017, 28-hydroxy protopanaxadiol was synthesized as a novel probe template [44]. The synthesis of new ocotillol-type probes employing 28-hydroxy protopanaxadiol may provide an effective means to enrich the structure of ocotillol-type probes. Additionally, functional probes that target the cell membrane are needed. Further research of ocotillol-type probes will promote the discovery of the target protein and provide a reference for the development of more effective drugs. Based on the present research of the ocotillol-type derivatives, a preliminary SAR of their antibacterial activities is summarized in Figure 9. The 24(S)-configuration is preferred, while substitution at the 3-OH changes the conformation to render the 24(R)-compound bioactive. A hydrogen donor at C-3 and C-12 are preferred to maintain the activity against Gram-positive bacteria. Decreased activity was observed when the functional groups at C-3 and C-12 were a ketone. When R 2 is an ester, mild activity against Gram-negative bacteria was observed.
Ocotillol-type derivatives with NO-inhibitory activity were further studied ( Figure 10 Based on the present research of ocotillol-type derivatives, a preliminary SAR of their anti-inflammatory effects is summarized, as shown in Figure 12. The 24(R)-configuration is preferred for the anti-inflammatory activity. An oxime at C-3 is preferred for good inhibitory activity of LPS-induced NO synthesis. Boc-amino groups seem to be preferred to inhibit the activity of LPS-induced NO synthesis than amino groups at C-3. A hydrogen donor at C-12 is preferred to inhibit LPS-induced NO synthesis. A fatty acid or amino acid group at C-3 has inhibitory effects on the expression of IL-6 and promotes the expression of IL-10 in the serum of a rat model of COPD induced by cigarettes.
Pharmacological results indicated that ocotillol-type derivatives had anticancer potential, and the configurations at C-20 or C-24 and the number of glycosyl units at C-3 could have an important influence on the cytotoxicity in vitro. There are only a small number of studies on ocotillol-type derivatives with anticancer activity, and thus, there is an opportunity to increase the number of ocotillol-type derivatives with anticancer activity.

Reversal of Multidrug Resistance in Cancer by Ocotillol-Type Derivatives
Ocotillol enhanced doxorubicin-induced cell death in p53 wild-type cancer cells [64]. Additionally, doxorubicin has a strong anticancer effect, but dose-dependent cardiotoxicity limits its clinical applications. Ocotillol-type ginseng reduced plasma creatine kinase and creatine kinase-MB isoenzyme levels and helped to reduce cardiotoxicity [65][66][67][68].
Wang et al. proved that ocotillol-type ginsenosides were substrates of P-glycoprotein (P-gp), and the pharmacological effects of ocotillol were the result of decreased efflux of digoxin across Caco-2 cell monolayers. In vivo experiments on mice showed that the inhibition of the 24(R)-epimer on P-gp was stronger than its counterpart [69]. This suggested that ocotillol-type ginseng may be a new type of drug resistance reversal agent.
Pharmacological experiments showed that 110 significantly reversed the resistance of ABCB1overexpressing SW620/Ad300 and HEK/ABCB1 cells to paclitaxel and vincristine ( Figure 13B). A further mechanistic study showed that 110 reversed ABCB1-mediated MDR by competitively inhibiting the drug efflux function of ABCB1 [70]. On this basis, Ren et al. synthesized a series of derivatives ( Figure 13B) .  Compounds 175-177, 185, 192, 194 and 203 have demonstrated a promising capability to reverse drug resistance, with compound 176 showing slight superiority [71,72]. Importantly, a xenograft model of KBV200 cells in nude mice showed that oral 176 significantly enhanced the inhibitory effect of paclitaxel on tumor growth. The inhibition of paclitaxel in vivo is 17.9%, while the inhibition of paclitaxel with 176 is 53.75%. In vitro, mechanistic studies suggested that 176 could inhibit P-gp-mediated rhodamine123 efflux function via stimulation of P-gp-ATPase activity ( Figure 14A). This indicated that ocotillol-type amide derivatives were substrates of P-gp, and it also showed that ocotillol-type amide derivatives were excellent drug resistance reversal agents.
Ocotillol ester derivatives with Boc-amino groups also have drug resistance reversal activity ( Figure 13B) [73]. Compared with the positive drug verapamil, compounds 206-212 showed good paclitaxel enhancing effect on KBv200 cells at a concentration of 10 µM. Generally speaking, compare with amide derivatives, ester derivatives are prone to hydrolysis in vivo; therefore, ester derivatives may not have drug resistance reversal activity in vivo. Compared with compounds 174-180 and 206-212, amide bond and ester bond have no effect on its activity in vitro, and almost all of these compounds with Boc-amino showed good drug resistance reversal activity, suggested that ocotillol-type derivatives containing Boc-amino group should be further enriched. Moreover, Bi et al. synthesized ring-A fused aminothiazole derivatives of ocotillol, compounds 215 and 216 possessed a remarkable multidrug resistance reversal activity higher than verapamil ( Figure 13B). SAR of ring-A fused aminothiazole derivatives needs further research [74].
Based on the present research of ocotillol-type derivatives, a preliminary SAR of their multidrug resistance reversal ability in cancer cells is summarized in Figure 14B. The 24(R)-configuration is preferred for the reversal of multidrug resistance in cancer. A linear alkyl amide containing a terminal Boc-protected amine at C-3 shows the best drug resistance reversal activity. Aromatic or heteroaromatic ring amide is better than linear alkyl amide or linear alkyl amide containing a terminal amine. Deprotection of Boc-protected amines obviously reduced the MDR reversal ability. A length of six carbon atoms in the alkyl chain of the linear alkyl amide is preferred, whether the N-terminus is Boc-protected or not. The ester derivatives and amide derivatives at the C-3 position may show similar activity trends in vitro.

Nervous System Effects of Ocotillol-Type Derivatives
Ginseng is a traditional herb and has been widely used for the treatment of neurological disorders [75]. In 2013, the protective effect of 109D on a rat model of Parkinson's disease was studied. This research showed that 109D had anti-Parkinson activity by inhibiting free radical formation and stimulating endogenous antioxidant release. Pretreatment based on oral administration of 109D significantly improved the motor balance, coordination and apomorphine-induced rotations in 6-OHDA-lesioned rats [76,77].
Compound 109D had inhibitory effects on the cognitive function of Tg-APPswe/PS1dE9 mice by inhibiting the expression of amyloid β-protein and amyloid-β-peptide  in the cortex and hippocampus, restoring the activities of superoxide dismutase and glutathione peroxidase, and decreasing the production of malondialdehyde in the cortex [78]. Compound 109D showed a protective effect against mild cognitive impairment (MCI-like pathological changes) by reducing the accumulation of advanced glycation end products and expression of the receptor of advanced glycation end-products [79]. Compound 109D attenuated memory disorders in the Morris water maze by promoting the transport of amyloid beta A4 and amyloid precursor protein from the cytoplasm to the plasma membrane and reducing the abnormally high expression of β-site APP cleaving enzyme 1 in the hippocampus and cortex of SAMP8 mice [80].
Compound 109D may also be a candidate for stroke treatment. Compound 109D inhibited the over-activation of µ-calpain and reduced the calcium calmodulin kinase II-α, reduced the degradation of sarcoplasmic/endoplasmic reticulum ATPase-2, and alleviated endoplasmic reticulum stress in transient middle cerebral artery occlusion rats [81]. Additionally, 109D also accelerated the oxygen-and glucose deprivation-induced promotion of microglial myelin debris phagocytosis and reinforced the RhoA-ROCK signaling pathway through the regulation of complement receptor 3 [81,82]. Neutrophils and macrophages are promising targets for the treatment of cerebral ischemia. Compound 109D inhibited the induction of neutrophils and macrophages to N1 and M1 phenotypes and promoted the polarization of neutrophils and macrophages to N2 and M2 phenotypes [83].
Quyen et al. evaluated the antidepressant-like activity of 109C and 218 by a tail suspension test and a forced swimming test in mice ( Figure 15B). The results showed that the stress model caused an increase of MDA and a decrease of glutathione levels in the mouse brain. This proved that 109C and 218 had antidepressant effects [84].

Effects of Ocotillol-Type Derivatives on the Cardiovascular System
Ocotillol-type derivatives protect from myocardial ischemic injury by reducing the area of the myocardial ischemia and the levels of necrosis and lactate dehydrogenase in the serum to enhance the anti-free-radical actions of heart tissues [85][86][87]. Bi et al. found that when 4 and its epimer 5 were tested in cultured myocardiocytes with anoxia/re-oxygen injury, only 4 had protective effects [88]. In 2017, Yang et al. synthesized an ocotillol-type small-molecule fluorescent probe 217B with anti-myocardial ischemia-reperfusion injury activity ( Figure 15A). This tool may help to understand the mechanism of how ocotillol-type derivatives protect against myocardial ischemia [89].
Oral administration of compound 4 ameliorated aconitine-induced arrhythmias [90]. Compound 4 reduced the incidence of arrhythmia in mice and shortened the duration time of ventricular tachycardia. Further research proved that oral administration of compound 4 prolonged action potential duration, reduced action potential amplitude in ventricular myocytes, reduced L-type calcium peak current in a dose-dependent manner, and inhibited delayed rectifier K + channels, but not inward rectifier K + channels.

Other Pharmacological Activities of Ocotillol-Type Derivatives
Ocotillol-type ginsenoside 219 ( Figure 15B), discovered from the stems and leaves of Panax quinquefolium L., increased the production of superoxide dismutase and glutathione, decreased malondialdehyde production, and increased the expression level of nuclear correlation factor 2 and heme oxygenase-1 in A549 cells. These results showed that compound 219 significantly inhibited hydrogen peroxide-induced oxidative stress and had a protective effect on the oxidative damage of lung epithelial cells [91].
Ocotillol-type ginsenosides have anti-melanogenic activity as 220 showed a good melanogenesis effect with an IC 50 value of 37 µM, but the mechanism of its anti-melanogenic effect is still not clear ( Figure 15B) [92]. Additionally, ocotillol may also have a protective effect against gastric ulcers. Ocotillol increased the expression of NO, superoxide dismutase, epidermal growth factor and the epidermal growth factor receptor, and decrease the expression of endotelin-1 and nitric oxide synthase, which is a similar effect as omeprazole [93]. In addition, ocotillol also has antiviral activity, and it could enhance the neuronal activity of mice [24,94,95].

Conclusions and Future Perspectives
In this review, the main chemical modifications of ocotillol-type derivatives and the SARs for their antibacterial, anti-inflammatory and reversal of multidrug resistance in cancer were summarized. In the past few years, ocotillol has attracted considerable interest in the medicinal chemistry society owing to its promising multiple pharmacological activities, especially antibacterial activity. Nevertheless, ocotillol-type derivatives exhibit limited water solubility, low systemic exposure, slow clearance and imprecise mechanism of action. Toxicity has greatly hindered its clinical applications [96][97][98][99][100][101][102]. To advance ocotillol-type derivatives into clinical therapies, there remain to be several issues and new directions for future research in the area.
(1) Rational design of new ocotillol-type derivatives with increased water solubility, good ADME.
For example, through polyethylene glycol modification or preparation techniques such as micronization, solid dispersion, self-microemulsion, inclusion techniques, etc., to improve water solubility. Formulation design of sustained-or controlled-release system should be used to maintain an effective blood concentration and decrease side effects. (2) Ocotillol, an active ingredient in ginseng, has already been proved to have multiple pharmacological activities; however, its precise molecular targets that responsible for the potent biological activity are currently not well understood. Therefore, it is important to further design and synthesize a new ocotillol-type probe to explore possible mechanisms and identify the molecular target.
(3) Currently, there is still much chemical space to be explored. The main chemical modifications performed to date have focused on the hydroxyl groups on ring A, while the skeleton structures and ring C modifications have been limited. (4) As many of the current studies are limited to in vitro studies, whether ocotillol is effective in vivo must be validated in the future. (5) Combination drugs have various significant advantages, including production additive or synergistic effects, reducing side effects, treatment failure rates and slow down the development of drug resistance [103]. The development of ocotillol-based combination drugs would be a useful strategy. For example, the combination of ocotillol with other antibacterial drugs to reduce treatment failure rates.

Conflicts of Interest:
Authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.