Mulinane- and Azorellane-Type Diterpenoids: A Systematic Review of Their Biosynthesis, Chemistry, and Pharmacology

Mulinane- and azorellane-type diterpenoids have unique tricyclic fused five-, six-, and seven-membered systems and a wide range of biological properties, including antimicrobial, antiprotozoal, spermicidal, gastroprotective, and anti-inflammatory, among others. These secondary metabolites are exclusive constituents of medicinal plants belonging to the Azorella, Laretia, and Mulinum genera. In the last 30 years, more than 95 mulinanes and azorellanes have been reported, 49 of them being natural products, 4 synthetics, and the rest semisynthetic and biotransformed derivatives. This systematic review highlights the biosynthetic origin, the chemistry, and the pharmacological activities of this remarkably interesting group of diterpenoids.


Introduction
For millennia, humankind has relied heavily on nature to provide for its basic needs, and to alleviate a wide spectrum of diseases. It is well documented that plants constitute the basis of traditional medicine systems; fossil records date the human use of plants as medicines to at least the Middle Paleolithic age, some 60,000 year ago [1,2]. In Mesopotamia, the uses of approximately 1000 plant-derived substances were documented around 2600 B.C. [3]. Currently, herbal remedies continue to be used for the treatment of different diseases by a large number of people; it has been reported that between 70% and 95% of the population, mainly in developing countries, still use traditional medicine as their primary health care when caring for their health-related needs and concerns [4].
Many plant species have been reported to possess pharmacological activities which are due to their content of natural products, broadly defined as small molecules derived from primary metabolites (e.g. carbohydrates, amino acids, etc.), used by the plant to mediate its interactions with the surrounding environment [5,6]. These natural products are genetically encoded and are produced by secondary metabolic pathways [6]. The four main families of secondary metabolites include polyketides, terpenoids, polyphenols derivatives, and alkaloids, and can be found in the leaves, stems, root, and bark of plants [7].

Mulinane and Azorellane Biosynthesis
While it has been suggested that mulinane biogenesis derives from the biogenetic transformation of a labdane derivative [17], a different proposal for the biosynthetic origin of mulinane and azorellane diterpenoids ( Figure 2) starts with the cyclization of the C-20 general precursor geranylgeranyl pyrophosphate (GGPP) to produce all trans-GGPP, which is then isomerized to S-geranyllinaloyl pyrophosphate (S-GLPP); an anti-Markovnikov cyclization generates the enantiomeric cation I that, by means of cyclization, yields the bicyclic system II a sigmatropic rearrangement, and leads to the stereoisomeric ion III having angular methyl groups considered the syn precursor of mulinanes and azorellanes. Cyclization of the side chain yields the tricyclic system IV, with an all-trans ring junctions' stereochemistry, that, following a series of 1,2-hydride and methyl shifts, leads to V. The intermediate V is the true precursor of mulinanes and azorellanes; a 1,2-hydride shift followed by deprotonation yields the mulinane skeleton, while the loss of the allylic proton produces the cyclopropane ring found in azorellanes [11].

Sources and Chemical Structures of Natural Mulinane and Azorellane Diterpenoids
Species of the genera Mulinum, Azorella, and Laretia are well-recognized sources of diterpenoids with mulinane and azorellane skeletons. While mulinanes are only present in Mulinum spp., the Azorella and Laretia genera are known to produce secondary metabolites with both mulinane and azorellane skeletons [10]. Azorella spp., Mulinum spp., and Laretia spp. include perennial shrubs, cushions, or mat-forming species that are adapted to cold and windy terrain and are often found at high-elevation habitats, particularly in the Andes mountain range of South America [18]. These species are distributed in southernmost South America and in the Subantarctic islands, as well as south of Australia and New Zealand. In South America, they extend from the Subantarctic region northward through the Patagonian steppes of Argentina and Chile and further north, they are restricted to the Andes Plateau highlands [19].

Synthetic, Semisynthetic, and Biotransformed Mulinane and Azorellane Diterpenoids
Recently, Liu et al. achieved the enantioselective total synthesis of natural mulinanes and analogous from cyclopentenone ( Table 2, and Figure 4) [53]. In addition, the presence of different functional groups, which include hydroxyl, carboxyl, acetoxy, and double bond, in the mulinane skeleton has allowed the preparation of a significant number of semisynthetic derivatives using dehydration, alkylation, hydrolysis, and oxidation reactions ( Table 3, and Figure 5). To date, only one semisynthetic azorellane derivative, 7β-deacetylazorellanol (16), has been reported; this can be explained by the fact that the cyclopropane ring in the azorellane skeleton can be easily open under weak acidic conditions, to produce semisynthetic derivatives having a mulinane skeleton [54]. However, the limited number of functionalized positions, and the type of functional groups, found in the chemical structures of natural azorellane and mulinane diterpenoids limit the number and type of semisynthetic derivatives that can be prepared through chemical modification [55]; because of this, in recent years, biotransformation using filamentous fungi such as Mucor plumbeus and M. circinelloides has been explored as a new strategy to obtain novel mulinane and azorellane derivatives [51,55,56] (Table 4, Figure 6). Table 2. Synthetic derivatives of mulinanes [53].

No
Synthetic Mulinanes

Pharmacological Activities of Mulinane and Azorellane Diterpenoids
Despite the development of drugs for treating diseases such as HIV/AIDS, malaria, tuberculosis, hypertension, diabetes, and cancer, these diseases continue to affect diverse populations worldwide with significant associated mortalities and the need to develop new and more effective pharmaceuticals is always present [61]. Currently, the importance of natural products and/or its derivatives in drug discovery and development is well recognized [8,62]. Since the structurally-unique mulinanes and azorellanes have displayed a wide variety of biological activities in both in vitro and in vivo pharmacological models, the preparation of mulinane and azorellane derivatives could yield new products with a stronger biological activity, a better solubility, or useful in determining structure-activity relationships or mode-of-action [63]. A summary of the pharmacological activities reported for mulinanes and azorellanes is listed in Table 5.

Antiprotozoal Activity
Parasitic diseases are a serious health problem that has had a deep impact on the global human population [65]. Among parasites, protozoal parasites such as Trypanosoma cruzi, Leishmania spp., Plasmodium falciparum, Giardia intestinalis, Trichomonas vaginalis, and Toxoplasma gondii, represent the major disease-causing organisms [65,66]. The infections caused by these parasites are responsible for 500 million deaths worldwide, especially in undeveloped countries, where a tropical or temperate climate and poor sanitary and hygiene conditions are common [67]. Globally, the burden of protozoal diseases is increasing and has been exacerbated by the limited number of pharmaceuticals available, the lack of effective medication due to drug resistance, the severity of side effects, the high costs, or their limited practicality for field use. These limitations have prompted many researchers to search for novel drugs against protozoal parasites [65,67]. Diverse studies provide support that mulinanes and azorellane represent a promising group of natural antiprotozoal agents [21,29,35,41]. Azorellanol (9) and mulin-11,13-dien-20-oic acid (6), both isolated from A. compacta, showed strong in vitro trypanocidal activity (IC 50 values of 20-87 µM) when tested against epimastigotes, trypomastigotes, and amastigotes of different strains (Tulahuen, SPA-14, and CL Brener) of T. cruzi. Both metabolites also showed activity against intracellular amastigotes of the CL Brener clone with an IC 50 of 32.3 µM and 29 µM, respectively [21]. Additionally, azorellanol (9) also had an effect on trophozoites of T. vaginalis Ant-1 strain (LD 50 = 40.5 mM) and T. gondii (ID 50 = 54 mM), but 7β-deacetylazorellanol (16) showed a stronger activity (ID 50 = 42 mM) against T. gondii [35,41]. Finally, 17-acetoxymulin-11,13-dien-20-oic acid (20) and 13α,14α-dihydroxymulin-11-en-20-oic acid (18), both from A. compacta, caused 60% and 42% growth inhibition of Plasmodium berghei NK 65 in infected mice, respectively, when tested at a dose of 10 mg/kg/day [29].   Azorellanol (

Spermicidal/Spermatostatic Activity
Mulinane and azorellane diterpenoids have been evaluated in terms of several parameters that characterize human sperm function, i.e., sperm motility and viability, sperm binding to the human zona pellucida, the progesterone-induced acrosome reaction, an increase in intracellular Ca 2+ concentration, and protease activity in the search for a contraceptive method to inhibit, in a reversible and specific manner, the functions of the male gamete. Azorellanone (17), isolated from A. yareta, inhibited sperm motility in a concentration-dependent manner (0.15-3 mM), while sperm viability was inhibited at 3 mM. Assays with 17 significantly inhibited sperm-zona binding, progesterone-induced acrosome reactions, and intracellular Ca 2+ concentration. Additionally, 17 also affected protease activity and inhibited trypsin-and chymotrypsin-like activities. These results suggest that 17 may be a potential candidate as a contraceptive agent used in the manufacture of vaginal jellies or creams [68]. Other diterpenoids, such as mulinenic acid (4), mulinolic acid (5), and azorellan-17,13-(β)olide (22), have been evaluated for their spermatostatic activity. Compounds (5) and (22) demonstrated significant spermatostatic properties [31].

Antidiabetic
Mulinolic acid (5) and azorellanol (9), both isolated from A. compacta, were evaluated for their antidiabetic activity in Streptozotocin-induced diabetic rats and both metabolites decreased glycemia at 180 mg/mL, a similar value to that observed for Chlorpropamide used as positive control. Azorellanol (9) increased the insulin levels in serum; however, with (5) the levels of insulin remained unchanged; these results suggested that while (9) could be acting on the β-cells of pancreatic islets, (5) may be acting on glucose utilization or production in the liver [23].

Anti-Inflammatory
A search for bioactive metabolites with anti-inflammatory and analgesic properties produced by A. compacta, A. yareta, and L. acaulis resulted in the identification of azorellanol (9), azorellanone (17), and 7β-deacetylazorellanol (16); azorellanol (9) showed anti-inflammatory activity when tested on arachidonic acid (AA) and 12-deoxyphorbol-13-Tetradecanoate (TPA)-induced edemas. The fact that (9) showed a higher activity on the TPA than in the AA-induced edema assays (dose: 15 × 10-7 mol/ear) and that the dermal anti-inflammatory activity was of 70.8%, suggests that the mechanism of action of (9) could involve the inhibition of cyclo-oxygenase activity. Alternatively, (17) showed the strongest analgesic activity when the three metabolites were tested in the acetic acid-induced abdominal constriction response in mice model (59% of analgesic effect, dose: 10 × 10 -5 mol/kg) [42]. Additional studies on the anti-inflammatory properties of azorellanol (9) showed its having an effect (25 mg/mL) on the inhibition of the transcription factor Nuclear Factor-kappa Beta (NF-κB), one of the key regulators of the genes involved in the immune/inflammatory response [70], in the NF-κB-dependent luciferase gene reporter assay. Finally, 7β-deacetylazorellanol (16) also demonstrated anti-NF-κB activity [44].

Anti-Alzheimer
Mulinanes have also been evaluated in the inhibition of acetylcholinesterase (AChE). Inhibition of AChE serves as a strategy for the treatment of Alzheimer disease (AD), senile dementia, ataxia, myasthenia gravis, and Parkinson disease, and it has been considered as a potential therapeutic approach to AD. Mulinolic acid (5) and mulin-11,13-dien-20-oic acid (6), both isolated from A. trifurcata, have shown moderate inhibitory activity toward the enzyme AChE in a colorimetric assay with IC 50 of 200 and 180 µg/mL, respectively [69].

Conclusions
This is a report that systematically describes the biosynthesis, occurrence, isolation, structures, and biological activities of mulinane and azorellane diterpenoids. In summary, a total of 95 of these compounds has been reported since 1990. Thirty-seven mulinanes and 12 azorellanes have been isolated from species of Azorella, Laretia, and Mulinum genera. Synthesis, chemical modifications, and biotransformation by Mucor plumbeus and M. circinelloides have produced 4 synthetics and 44 mulinane derivatives. Even though these diterpenoids have been extensively studied because of their biological properties such as antimicrobial, antiprotozoal, spermicidal, gastroprotective, anti-inflammatory, antidiabetic, cytotoxic, and anti-Alzheimer, a large number of mulinanes and azorellanes have shown important anti-M. tuberculosis and gastroprotective activities. The antimycobacterial activity of the semisynthetic n-propyl (74) and n-butyl (75) esters of isomulinic acid (2) and of 13α-hydroxy-mulin-11-en-20-oic-acid n-propyl ester (62) suggest that an increase in the size/length of the substituent could increase the potency of mulinane derivatives.
Currently, only a small amount of research has been involved in the analysis of the structure-activity relationship in mulinanes and azorellanes. Similarly, studies on the target genes, target proteins, and signaling pathways involved in the mechanisms of action of mulinane and azorellane diterpenoids are limited; these studies are necessary in order have a mulinane or azorellane become a potential pharmaceutical. With this review we intended to make a significant contribution to the current knowledge about these interesting diterpenoids, as well as to encourage their continuing study.