Protostane and Fusidane Triterpenes: A Mini-Review

Protostane triterpenes belong to a group of tetracyclic triterpene that exhibit unique structural characteristics. Their natural distribution is primarily limited to the genus Alisma of the Alismataceae family, but they have also been occasionally found in other plant genera such as Lobelia, Garcinia, and Leucas. To date, there are 59 known protostane structures. Many of them have been reported to possess biological properties such as improving lipotropism, hepatoprotection, anti-viral activity against hepatitis B and HIV-I virus, anti-cancer activity, as well as reversal of multidrug resistance in cancer cells. On the other hand, fusidanes are fungal products characterized by 29-nor protostane structures. They possess antibiotic properties against staphylococci, including the methicillin-resistant Staphylococcus aureus (MRSA). Fusidic acid is a representative member which has found clinical applications. This review covers plant sources of the protostanes, their structure elucidation, characteristic structural and spectral properties, as well as biological activities. The fungal sources, structural features, biological activities of fusidanes are also covered in this review. Additionally, the biogenesis of these two types of triterpenes is discussed and a refined pathway is proposed.

Fusidane triterpene (FT) belongs to another small group of tetracyclic nor-triterpenes, which can be structurally considered as 29-nor protostane triterpenes ( Figure 1). To date, 18 naturally occurring FTs have been reported. Among them, fusidic acid has been used in the clinic as an antibiotic for decades; and it remains a unique and promising agent due to the significant potencies against staphylococci, especially the methicillin-resistant Staphylococcus aureus (MRSA). Fusidic acid has relatively low toxicity; it is non-allergic and has little cross-resistance with other clinically used antibiotics.
We herein present the first comprehensive review on these two groups of triterpenes. This paper deals with their natural occurrence, isolation and structure elucidation, structural and spectral characteristics, biological activities, as well as a proposed biogenetic pathway.
The typical UV spectrum of a PT may display absorption maxima at 243-246 nm and around 285 nm. These bands are indicative of enone functionality and dienone group, respectively.
General speaking, the molecular ion of PT can readily be observed by EI-MS. When soft ionization techniques such as ESI-MS and FAB-MS are used, the mass spectra will display the corresponding quasi-molecular ions, [M+H] + , [M+Na] + , or [M−H] − . In Q-TOF-MS, the protonated molecular ion [M+H] + is readily detectable, and collision-induced dissociation tandem mass spectrometry (CID-MS-MS) can produce characteristic fragments resulting from the dissociation of the bond between C-23 and C-24, which is useful for differentiation of isomers containing an acetyl unit on the C-23 or C-24 positions [40].

Nuclear Magnetic Resonance (NMR) Spectra
NMR has proved to be the most powerful tool for structural elucidation of organic compounds. As a matter of fact, most PT structures were elucidated primarily through interpretation of their NMR spectroscopic data. NMR analysis also provides an additional tool for stereochemical determination.
In the 1 H-NMR spectra (typically acquired in CDCl 3 ), distinctive signals can be observed for the methyl groups in the neighborhood of δ H 0.8-1.5. In addition, the presence of two broad singlets around δ H 4.93 and 4.97 are indicative of an olefinic CH 2 group [the terminal double bond present at the 25(26) position].
Alizexol A (alisol F 24-acetate, 12) was isolated in 1995 [22], but it was not until 2001 that the C 24 -R absolute configuration was determined using a chemical correlation method [42]. A unique structure bearing a C 23 -keto group, alisol H (13) [23], as well as alismaketone B 23-acetate (14) [11], possessing a seven-membered 16,24-epoxy ring system, were also isolated from the rhizome of A. orientale. The epoxy-ring connection between C-16 and C-24 was confirmed by HMBC correlations observed between 16-H and C-24. The stereochemistry of 14 was determined to be the same as that of alisol A (1) by chemical correlation, i.e., reduction of 14 with Li in ethylenediamine resulted in dihydroalisol A [11].
25-Anhydroalisol A 11-acetate (15) [24] is the only example of a PT structure that possesses an acetate group at the C-11 position; all other PTs bear acetates on C-23 or C-24, if present. 25-Anhydroalisol A 24-acetate (16) was identified as a new naturally occurring product [24], but it had been previously reported as an anodic oxidation product of alisol A (1) [43].

Alisol B Series
In contrast to the alisol A series, alisol B PTs ( Figure 3) possess a 24,25-epoxy group in their structure. Alisol B (24) and alisol B 23-acetate (25) were isolated from the rhizome of A. orientale in 1968, but their structures were later revised [9,17]. An X-ray crystallographic analysis of 25 was published in 2003 [44]; however, the stereochemistry was erroneously assigned as 5β-H, 8β-CH 3 , 9α-H, 13β-H, and 14α-CH 3 , which is opposite to the configurations of other PTs. 11-Deoxyalisol C (26) and alisol D (13β,17β-epoxyalisol B 23-acetate) (27) were isolated from A. planta-aquatica [34]. The former compound possesses a 13(17)-double bond and a 16-keto group whereas the latter contains a 13,17-epoxy group. When alisol B 23-acetate (25) was treated with m-chloroperbenzoic acid, alisol D (27) was obtained, presumably due to acid attack at the 13 (17) double bond from the hindered β-side. The presence of a 13β,17β-epoxy group in the structure of 27 was further confirmed by X-ray crystallographic analysis [45]. The structure elucidation of 16β-hydroxyalisol B 23-acetate (28) and 16β-methoxyalisol B 23-acetate (29) from A. planta-aquatica were primarily based on NMR spectroscopic data. The 11-OH was assigned to the β-configuration due to a large coupling constant for 11-H (J = 11.1 and 10.0 Hz). The β-configuration of the methoxy or hydroxy group at the C-16 position was determined based on NOE enhancement of the 16-H and 11-H signals upon irradiation of the 28-CH 3 [35].
11-Deoxyalisol B (30) and its 23-acetate derivative (31) were isolated from the fresh rhizome of A. orientale in 1993 [36]. This was the first investigation on fresh Alisma plant materials.
Alismaketone A 23-acetate (38) possesses a keto group and a hydroxy group at positions C-2 and C-3, respectively. This is in contrast with most PTs previously isolated from Alisma plants, all of which bear a keto group only at C-3. The 2-keto-3-ol structure of 38 was elucidated based on detailed examination of its COSY and HMBC spectra. The absolute configuration at C-3 was determined to be S based on the result of modified Mosher's method [37].

Alisol E Series
Protostanes of the alisol E series (Figure 4) possess an absolute configuration of 24S. Alisol E (epi-alisol A, 46) was first isolated from the rhizome of A. orientale in 1968 and later identified as a C 24 -S epimer of alisol A (1) [9,37]. It was the first example of a C 24 -S PT isolated from natural source. Detailed NMR spectroscopic data were not reported until 1993 in the study of alisol E 23-acetate (47) as the second C 24 -S protostane [19]. The stereochemistry at C-24 was confirmed by applying the modified Mosher's method. In addition to chemical methods, NMR data provided further evidence for the differentiation between the R and S configuration at C-24 in these molecules [8,39]. The isolation of alisol E 24-acetate (48) was reported in 2002 [25].

Biogenesis of Protostanes
In higher plants, 2,3-(S)-oxidosqualene is generally considered to be the biosynthetic precursor of triterpenes and phytosterols through a cascade of cyclizations and rearrangements. Squalene epoxidase is responsible for the conversion of 2,3-(S)-oxidosqualene to 2,3-(S)-22,23-(S)-bis-oxidosqualene prior to the cyclization steps [47]. At the start of triterpene synthesis, 2,3-(S)-oxidosqualene (Scheme 1; structure A) adopts a pre-organized chair-boat-chair conformation, followed by the protonation of the epoxy ring, which triggers a cascade of ring-forming reactions resulting in a 6.6.6.5-fused tetracyclic protosteryl C-20 cation with a 17β-side chain (Scheme 1; structure B). Under the control of the specific "protosterol synthase", this cation is directly deprotonated, either without rearrangement or with a 17α-hydride shift, to yield protosterol (Scheme 1; structure C), which is presumed to be the initial intermediate in the biosynthesis of protostane skeleton. The formation of the protosteryl C-20 cation is a key step in the synthesis pathway and is also an important intermediate in the biosyntheses of phytosterols and steroidal triterpenes. The cyclization of oxidosqualene (Scheme 2; structure A) to yield the protosteryl C-20 cation (Scheme 2; structure E) was initially considered to be a concerted reaction, i.e., a non-stop process without passing through stabilized intermediates. Due to a growing body of experimental and theoretical evidence, it is now largely accepted that the cyclization first yields a tricyclic Markovnikov tertiary cation possessing a five-membered C-ring (Scheme 2; structure B). However, how the six-membered C-ring and five- The isolation of alismaketone C 23-acetate (52) from A. orientale seemed to have provided a key piece of evidence to support the Corey hypothesis. Thus, the biosynthesis was presumed to originate from a 24,25-epoxy anti-Markovnikov cation (Scheme 3, structure C) by direct elimination of a proton, followed by oxidative structure modifications (Scheme 3). In the fresh rhizomes of Alisma plants, protostanes belonging to the alisol B series have been demonstrated to be the major components [53,54]. It was speculated that these 24,25-epoxides turn into compounds of the alisol A series during the drying process of the rhizomes, thus leading to the increase in the amounts of the latter in dried rhizome samples.
It is now proposed that the biogenesis of Alisma PTs starts with the 2,3-(S)-22,23-(S)-bisoxidosqualene pathway (Scheme 1; structure D). This is in accordance with the reported biosynthesis of 24,25-epoxycholesterol (which is processed in a shunt of the mevalonate pathway, as a parallel pathway to cholesterol synthesis) [55,56]. Squalene monoxoygenase has been reported to catalyze the downstream reactions leading to 2,3-(S)-22,23-(S)-bis-oxidosqualene, which subsequently undergoes a number of transformation steps which are catalyzed by a yet unidentified enzyme complex, but via the known 24,25-epoxy protosterol.

Lipotropic and Liver-Protective Activity
Compounds belonging to the alisol A group 1, and 2 and the alisol B derivatives 25 and 33 were found to display marked anticholesterolemic effects. Inclusion of 0.1% of either of these compounds in the diet for hypercholesterolemic rats would reduce the cholesterol levels by more than 50%, compound 2 being most potent resulting in 61% reduction [3]. In addition, 2, 25 and 33 were able to protect mice against CCl 4 -induced liver damage, as indicated by a modulation of serum glutaminepyruvic transaminase and triglyceride levels, with 33 being the most effective protectant [33]. Moreover, alisol A derivatives 5 and 9 were found to inhibit 100% and 60% of D-galactosamineinduced liver damage in vitro, respectively [4].
A number of synthetic alisol A derivatives have been prepared and evaluated for their in vitro anti-HBV activity and cytotoxicity in a structure-activity relationship study [57][58][59]. The results suggested that acylation of the hydroxy groups at positions 11, 23, and 24 decreased the cytotoxicity. It was concluded that the carbonyl function at C-3 might is of importance for the activity. On the contrary, the 25(26) double bond of alisol A analogues might be crucial for the anti-HBV activity [57][58][59].

Anti-HIV-I Activity
Two PT derivatives isolated from plants outside the Alisma genus, 55 and 57, were reported to display inhibitory activity against HIV-I reverse transcriptase with an IC 50 of 15.5 and 12.2 μg/mL [7].

Anti-Tumor Activity
The alisol B derivative 24 exhibited cytotoxic activity against several cancer cell lines SK-OV3 (a human ovary adenocarcinoma cell line), B16-F10 (a murine melanoma cell line), and HT1080 (a human fibrosarcoma cell line), showing ED 50 value of 7.5, 7.5, and 4.9 μg/mL, respectively. The alisol A analogue 2, as well as the alisol B analogues 25 and 33, showed only weak activities in the same cell lines with ED 50 values of 10-20 μg/mL [15].
In addition, compound 25 was found to induce apoptotic cell death in human hormone-resistant prostate cancer PC-3 cells in a time-and concentration-dependent manner. The mechanism was described to be mitochondria-mediated, causing the activation of caspases-3, -8, and -9. Compound 25 was found not only to induce Bax (a member of the Bcl-2 gene family of apoptosis regulatory proteins) expression, but also to cause the translocation of Bax from the cytosol to the nucleus [32].

Multi-Drug Resistance Reversal Activity in Cancer Therapy
The alisol B analogue 25 was suggested to have effects on reversing the multidrug resistance (MDR) of certain cancer cell lines towards standard chemo-therapy. Thus it was found to restore the sensitivity of two MDR cell lines, HepG 2 -DR and K562-DR, towards anti-tumor agents which are substrates of P-glycoprotein (P-gp) but have different modes of action. For example, 25 restored the activity of vinblastine in causing G 2 /M arrest in MDR cells. 25 increased doxorubicin accumulation in a dose dependent manner, and slowed down the efflux of rhodamin-123 from MDR cells. In addition, 25 inhibited the photoaffinity labeling of P-gp by [ 125I ]iodoarylazidoprazosin and stimulated the ATPase activity of P-gp in a concentration-dependent manner. This suggested that it could be a transporter substrate for P-gp. 25 was also found to be a partial non-competitive inhibitor of P-gp when verapamil was used as a substrate [61].

Anti-Complement Activity
Alisol A analogues 1, and 2, as well as alisol B analogues 24, and 25, were reported to inhibit the complement-induced hemolysis through the classical pathway [12]. 2 and 24 exhibited anticomplement activity with IC 50 values of 130 μM and 150 μM, respectively.

Other Biological Activities
The alisol B derivatives 24 and 25 were reported to exhibit muscle relaxant effects on isolated rat ileum against contractions induced by 5-isoleucine-angiotensin I, bradykinin, and acetylcholine [30]. Alisol B analogue 38 and the seco-PT analogue 49 showed concentration-dependent (10 −5 -10 −4 M) inhibitory activities on the contractions induced by K + in isolated aortic strips of rats [37].
The alisol B derivatives 24, 25, 28, 30, and 33 were found to be effective in restoring choline acetyltransferase activity, and were suggested to have potential for the treatment of Alzheimer's disease, myasthenia gravis, and gastrointestinal disorders [31].
Compounds 2 and 24 produced a significant increase in Na + excretion in saline-loaded rats when administered orally at a dose of 30 mg/kg [16].
Alisol B derivative 24 was reported to inhibit cell proliferation and induce apoptosis in both rat aortic smooth muscle A7r5 cells and human CEM lymphocytes. The effect was suggested to be partly due to the induction of c-Myc expression as well as the collapse of Bax/Bcl-2-mediated mitochondrial membrane potentials. In addition to apoptotic effect, 24 showed hypolipidemic and anti-inflammatory effects, and it was proposed to be useful for the development of drugs to prevent pathological changes associated with atherosclerosis and post-angioplasty restenosis [62].
Compounds 1, 24, 25, and 33 were observed to regulate the 5-HT 3 A receptor expression in Xenopus oocytes. All were reported to regulate the 5-HT-induced inward peak current mediated by the human 5-HT 3 A receptor in a concentration-dependent and reversible, but non-competitive, manner with relatively low IC 50 values (1.7-3.5 μM) [14].
Compound 3 showed inhibitory activity to alpha-glucosidase in a dose-dependent manner (0.125-2.5 mM). Since the total aqueous ethanol extract of Alismatis Rhizoma (25 μg/mL) could inhibit alpha-glucosidase activity by 34.06%, comparing to acarbose (0.5 mM) by 47.08% [63], the PTs present in the plant are likely involved in the inhibitory process.

Fusidane Triterpenes
Fusidane triterpenes (FT) belong to a small group of 29-nor protostane triterpenes (Figure 8). They are antimicrobial agents produced by fungal species (Table 2)  . Though only a few structures of this class have been reported to date, FTs play important roles as antibiotic agents. The most important representative is fusidic acid (60).

Fusidic Acid
Fusidic acid (60) was first isolated from Fusidium coccineum by Godtfredsen [64] and also reported from several other fungal sources (Table 2) [65][66][67]. It has been clinically used as an antibiotic since 1962 in both systemic and topical therapies for staphylococcal infections [68]. It exhibits potent effects against staphylococci, including the methicillin-resistant Staphylococcus aureus (MRSA) and the coagulase-negative staphylococcal species.
Fusidic acid distributes well in various tissue, exhibits low toxicity and allergic reactions; and it has little cross-resistance with other clinically used antibiotics. Though never approved for use in the United States, fusidic acid is marketed in more than twenty countries with 21 million annual prescriptions [69]. The global problem of microbial resistance has now led to a renewed interest in its use. Since 2006, this "old" antibiotic has received attention in the United States mainly because no recommended oral antibiotics (such as oxacillin, cloxacillin, dicloxacillin, and cephalexin) have shown useful activity against MRSA. To date, phase 2 clinical trials has finished and the results supported proceeding to phase 3 studies [70].
Fusidic acid (60) acts as a protein synthesis inhibitor, binding to elongation factor G (EF-G). The binding site was identified to be a pocket between domains I, II, and III of EF-G (EF-G consists of 5 domains). This binding results in a conformational intermediate structure between the GDP-and GTP-bound forms [71]. Due to its unique action mechanism, 60 has shown no cross-resistance with any other class of antibiotic.
The structure-activity relationship of fusidic acid (60) and related compounds have been extensively studied. The tetracyclic fusidane skeleton, lipophilic side-chain, and the carboxylic acid group at C-20 seem to be essential for its biological activity. The orientation of the lipophilic side-chain, rather than the double bond, is crucial to the antibacterial activity [72].
Structural modifications of 60 have shown that, among 51 derivatives, none displayed antibacterial activity better than the parent compound, and only one derivative, 24,25-dihydrofusidic acid, turned out to be as active as 60 itself [73].

Other Fusidane Triterpenes
Helvolic acid (61) was isolated from Aspergillus fumigatus during World War II. It showed bacteriostatic activity against gram-positive organisms, but had no effect against gram-negatives [74,75]. Subsequently, it showed significant antimicrobial activity against a wide range of microorganisms including fungi [76][77][78][79]. Compound 61 also exhibited synergistic effects with erythromycin on all tested multi-drug resistant Staphylococcus aureus and with penicillin and tetracycline on some multidrug resistant S. aureus strains. Enhanced effect was also found in time-kill studies on multi-drug resistant S. aureus strains [80].
Cephalosporin P1 (62) was discovered from the culture fluid of Cephalosporium acremonium in 1951, which exhibited potent activity against methicillin-sensitive, methicillin-resistant, and vancomycin-intermediate Staphylococcus aureus [81][82][83]. The complete cross-resistance between 60 and 62 was reported, but the nature and location of fusA (the gene that encodes EF-G) mutations selected by these two agents in S. aureus appeared to be different. The interaction of them with EF-G may also differ based on the examination of their effects on translocation and peptide bond formation using cell-free assays [83].

Biosynthesis of Fusidane Triterpenes
FTs share a similar biosynthetic pathway with PTs leading to the formation of protosteryl C-20 cation. 3β-Hydroxy-protosta-17(20)Z, 24-diene is supposed to be the precursor of FTs. After the formation of 3β-hydroxy-protosta-17(20)Z, 24-diene, further demethylation process of C-29 is catalyzed to produce the FT skeleton. A novel oxidosqualene cyclase (OSC), namely oxidosqualene: protostadienol cyclase (OSPC), produced in Aspergillus fumigatus was reported to be involved in the biosynthesis of helvolic acid [88,89]. The stabilization of the C-20 protosteryl cation by the active site Phe701 of OSPC through cation-π interactions is important for the product outcome of protostadienol [90]. Three genes (AfuOSC3, AfuSDR1, and CYP5081A1) have been characterized in the early steps of helvolic acid biosynthesis. AfuOSC3 is responsible for the formation of the basic carbon skeleton 3β-hydroxy-protosta-17(20)Z, 24-diene, whereas both AfuSDR1 and CYP5081A1 presumably work together to catalyze the demethylation of C-29 [89].

Conclusions and Future Prospects
PTs represent a compound class with a unique triterpene structure and they have been found to exhibit diverse biological activities in a broad range of in vitro and in vivo studies. To date only 59 PTs have been reported, with the majority isolated from the genus Alisma, mainly A. orientale and A. plantago-aquatica. Further phytochemical investigations on other Alisma plant species are warranted.
Details of the PT biosynthetic pathway are lacking at this time. It is likely that the Alisma genus possesses unique and specific enzyme systems capable of catalyzing the complex biosynthetic pathway, making the PT biosynthetic pathway is worthy of further studies in the future.
Because most reported PTs have not been investigated conclusively for their biological activities, and because some derivatives have shown significant effects in the bioassay systems, further biological studies on these compounds are anticipated to reveal interesting results, especially with respect to lipotropism, liver protection, and anti-hepatitis B activity. A better understanding of their biological activities would shed light on the rational use of Alismatis Rhizoma.
Fusidic acid, after decades of clinical use, remains a promising antibiotic agent due to its potency against MRSA, low degree of toxicity and allergic reactions, and no cross-resistance with other clinically used antibiotics. A new dosing regimen of fusidic acid has been developed in the United States in order to minimize the fusidic acid resistance selection and obviate the negative effects of protein binding [91]. This new strategy warrants further development of this antimicrobial agent.