(+)-LARIXOL AND LARIXYL ACETATE: SYNTHESES, PHYTOCHEMICAL STUDIES AND BIOLOGICAL ACTIVITY ASSESSMENTS

. (+)-Larixol and larixyl acetate are well known labdane-type diterpenoids widely used in organic synthesis. The chemistry of (+)-larixol had a slower evolution compared to other diterpenoids and the peak of its heyday can be considered the 2000s. During this period, the most important works describing the syntheses based on (+)-larixol and its acetate appeared, some of them being mentioned in reviews devoted to diterpenes. So far, however, no review has been published dedicated exclusively to chemistry of (+)-larixol and larixyl acetate, neither phytochemical investigation of sources containing these compounds nor their biological activity study. The present review seeks to cover and fill in the gaps regarding these topics based on available scientific data published after the 2000s

The review is designed for interested researchers in the field, aiming to present them recent scientific achievements, those partially cited or omitted previously in the fields of chemistry, phytochemistry and biological activity of (+)-larixol and larixyl acetate.

Recent progress in chemistry and phytochemistry of (+)-larixol and larixyl acetate
Most of the syntheses mentioned above are based on the oxidative breakdown of the C-9 side chain of (+)-larixol (1) or its acetate 2 leading to a wide variety of synthons, including 14,15-bisnorlabdene-13-ones 3-5.The next transformations into target drimanes consist of multi-step procedures and this strongly influences their overall yields.For this reason, Vlad, P. et al. used the Norrish II type photochemical degradation of methyl ketones 3-5 (Figure 1) [27].The results regarding the major reaction products were published in some reviews, whilst those regarding minor products were omitted [37].
In such a way, minor compounds 7, 9 and 11 with unexpected bi-and tricyclic structure were isolated from the Norrish II reaction products (Scheme 1).Their structures were confirmed by 1D and 2D NMR analysis and single crystal X-rays diffraction.The compound 7 had a 6α-acetoxy-9,13β-epoxy-13β-methylpodocarpane skeleton and was obtained in ~4% yield, calculated on converted 6-acetoxy ketone 3. Vlad, P. et al. proposed the probable way of its formation as a product of ketone 3 cyclization under UV-irradiation via intermediate state 6 (Scheme 1) [27].
Unlike that, compound 9 (~3%) is a product of the photochemical rearrangement and cyclization, as depicted in Scheme 1, which led to a tricyclic skeleton with a condensed cyclopentane ring (Scheme 1).
The most abundant was compound 11, which was obtained in a 17% yield.Its 6α-acetoxy-8β,13,13,17-diepoxy-14,15dinorlabdane skeleton was a result of ketalization, via intermediate state 10, which occured in molecule of ketone 3 as result of photoexcitation and also of the addition of the residual oxygen from the reaction medium.This fact was confirmed by additional reactions carried out under oxygen atmosphere, when compound 11 was obtained in 40% yield (Scheme 1).Their spectral data were in accordance with those of some related diastereomeric ketoketals reported before [7,14].
The minor compound 12 was isolated from the photochemical degradation product of methyl ketone 4 in a ~18% yield.The spectral data of liquid cyclobuto(18→6)-14,15-bisnorlabd-8( 17)ene-6-ol-13-one (12) were not enough for its structure characterization, for this reason an attempt was made to obtain the 2,4-dinitrophenylhydrazone derivative for the X-ray analysis.Surprisingly, a mixture of two 2,4-DNPH derivatives 13 and 14 was obtained, isolated and characterized due to acidic reaction conditions (Figure 2).
To note that, the meso-tetraphenylporphyrin or eosine sensitized photooxidation was widely and efficient used by Vlad, P. et al. as a green chemistry method for the preparation of some intermediate endoperoxides, such as 21 and 22, or anhydride 20 (Figure 3).Herein, the synthetic schemes were omitted because these were wellpresented in a review published by Mahji, S.  A successful attempt to synthesize fragrolide (24), a sesquiterpene lactone previously isolated from Cinnamosma fragrance [24], was described earlier [38].To achieve this, the authors used the ketoperoxide 22, which was reduced into triol 23 in depicted conditions (Scheme 2).Further oxidation of triol 23 with chromium trioxide led to mixture of ketoeuryfuran 19 and desired fragrolide (24), in ~32% and ~21% overall yield, recalculated for initial acetoxy diene 15, that was much better than those previously published [39,40].
Herein, triole 36 was obtained by saponification of diacetate 35, then it was oxidized with manganese dioxide into lactone 37 (Scheme 6).The transformation of lactone 37 into pereniporin B (38) was reported by Burke, S. et al. and included its reduction with DIBAL-H, followed by Fetizon's oxidation [40].Cinnamosmolide (39) was prepared for the first time by acetylation of pereniporin B (38) in standard conditions [43].Some of the intermediate from the Scheme 4 were synthesized from other sources and previously reported, e.g.triole 36 [44,45] and lactone 37 [46].
In contrast to the syntheses mentioned above, only several syntheses based on (+)-larixol (1) with conservation of the side chain are known [37-39], one of them being the synthesis of (+)-crotonadiol (42).
Later a similar compound was isolated from the bark of African specie of Croton zambesicus Muell.by Ngadjui, B. et al. [48] and named crotonadiol with [α]D= -28°.Five years later, a compound corresponding to crotonadiol (42) was isolated by Yang, B. et al. from the bark of Larix olgensis Henry var.koreana Nakai and its structure was proved by spectral analysis and X-ray diffraction [49].
To note, that by comparing the physicochemical constants of the final compounds 42 and 50, it can be concluded that compound 51 ([α]D= -28.0°) reported by Ngadjui, B. et al. belongs to the ent-labdane series of diterpenes [48] (Figure 4).
Unfortunately, the last published synthesis based on (+)-larixol (1) dates from 2014 [14], the scientific papers that followed, referred to phytochemical analysis of Larch spp.and other conifer species of different geographic origin or their hybrids, and described wood analysis and biological activity assessments of the extracts from mentioned conifer against different pests.Along with other metabolites, the presence of compounds 1 and 2 were reported for the first time in the sources under research.
Other scientific data on this topic are discussed in chronological order.The results of GC-MS analysis of oil extractives components (OECs) from wood and bark of Pinus sylvestris, Abies alba, Picea abies and Larix decidua growing in Czech Republic were reported by Salem, M. et al. [57].Epi-manool (56) (6.31%) was detected in n-hexane extract from the bark of Abies alba and larixyl acetate (2) (0.82%) in that from Picea abies (Figure 7).The highest content of 13-epi-manool (56) (2.77% and 15.40%) and (+)-larixol (1) (4.85% and 33.29%) was found in the wood and bark extracts of Larix decidua.
In order to develop an efficient procedure of larch wood capitalization, the analysis of the wood extractives obtained with an accelerated solvent extractor (ASE) from three different tissues, sapwood, sound knotwood and dead knotwood was performed [62].Using different methods of analysis (GC-MS, FT-RAMAN, FT-IR and FT-NIR), Wagner, K. et al. found that the n-hexane extracts from dead knotwood samples yielded more (+)-larixol (1) and resin acids, e.g.isopimaric acid (62) than the other samples (Figure 5).Most of the recently published bibliographical sources are referring to phytochemical analysis of essential oils and/or extracts obtained from some species of angiosperms, where (+)-larixol (1) and larixyl acetate (2) which normally are chemomarkers specific for coniferous species have been identified for the first time.As mentioned above, scientific results are discussed chronologically.
The comparative study of essential oils obtained by hydro-distillation of stem and aerial parts of Origanum majorana Linn.(Lamiaceae) was reported by Prerna, G. et al. [63].A total number of seventy-eight compounds were identified in the stem oil and eighty-seven in the oil from the aerial part of which linalool (63) and estragole (64) were found as main components (41.31-45.05%and 14.14-25.62%,respectively) and a trace amount of (+)-larixol (1) (0.04%) was detected (Figure 6).
The methanolic extract from Acalypha indica L. leaves, a species originated from India, has the capacity to act as a radical scavenger and also showed potential cytotoxic activity [64].Ravi, S. et al. have mentioned that this was due to a high content of polyphenols.However, the HR-LC/Q-TOF/MS analysis of Acalypha indica extract for the first time confirmed, among the eighty-seven components, there was a low content of larixyl acetate (2) (~0.26%) [64].
As stated by Li, X. et al., after fractionation of the methanol extract of Hypericum longistylum with petroleum ether, (+)-larixol (1) was isolated by column chromatography, together with other compounds, and its structure was proved by instrumental analysis and comparison of spectroscopic data with previously reported [69].

Recent biological investigations of (+)-larixol and larixyl acetate
This subchapter includes the results of recent biological tests, which have highlighted some new properties of vegetal extracts containing (+)-larixol (1) and larixyl acetate (2), or of their pure forms.
In addition to the chemical composition analysis of methanolic stem wood and bark extracts from Picea abies L. Karst.and Larix decidua Mill., reported in the previous subchapter, Salem, M. et al. also performed the antimicrobial assays [58].The methanol (95%) extract from P. abies/L.decidua wood and bark showed good MIC and MFC against Aspergillus flavus (0.13 and 0.25 mg/mL), Candida albicans (1.74 and 3.52 mg/mL), Penicillium funiculosum (0.29 and 0.72 mg/mL) and Penicillium ochrochloron (0.19 and 0.42 mg/mL), comparable with Fluconazole and Ketoconazole standards.Unlike other sources, the high activity of the methanolic extract of L. decidua bark can be explained by the high summary content ~60% of diterpenoids (2,9-dihydroxyverrucosane, abietic acid and (+)-larixol).The results suggest that the P. abies and L. decidua extracts have a potential use in food and/or pharmaceutical industries [58].
In another paper, Thuerig, B. et al. mention that the ethanolic extract of Larch bark with a combined concentration of (+)-larixol/larixyl acetate (~66%) showed promising in vitro antifungal activity against P. viticola at MIC100= 6-23 μg/mL in planta semi-controlled conditions at EC50= 0.2-0.4mg/mL, that means a comparable efficacies of larch extracts reached up to 68% in a stand-alone strategy and 84% in lowcopper strategies.This approach can allow the copper reduction in organic vineyards [60].
The methanolic extract of Acalypha indica L. leaves, a specie originated from India, has the capacity to act as a scavenger of DPPH radical (at IC50= 28.330 μg/mL), H2O2 (at IC50= 84.415 μg/mL), hydroxyl radicals (at IC50= 35.933-84.775μg/mL) and week metal ions reducer.Also, it showed potential cytotoxic activity (LC50= 140.02 μg/mL) against brine shrimp.Ravi, S. et al. mention that all these activities of MEAIL are due to the high content of polyphenolics, flavonoids and saponins [64].
As mentioned above, the essential oil of Origanum majorana is one of the new sources where (+)-larixol (1) was detected.According to Sharma, V. et al., this EO revealed excellent inhibition activity against the test fungal organisms, with presence of maximum inhibition zone (MIZ= 37 mm) against Trichophyton mentagrophytes, (MIZ= 31.67 mm) against Microsporum gypsium and (MIZ= 28.33 mm) against Microsporum nannum, which is mainly due to mono-and sesquiterpene constituents [65].As in other cases, the OECs from Dracocephalum spp.growing in Kazakhstan are new sources where, among others constituents, (+)-larixol (1) was detected, as well.Samples obtained from all three species exhibited acute lethal toxicity towards the larvae of the Artemia salina aquatic crustaceans at all tested concentrations (1-10 mg/mL) and low antiradical activity (at 3.51-8.70%) compared with the standard drugbutylhydroxyanisole at ~80%.Likewise, in this case, Suleimen, Y. et al. mention that the activity is due mono-(C10) and sesqui-(C15) terpenic fraction [66].
Fifteen compounds isolated by Li, X. et al. from Hypericum longistylum were subjected to the separate MMT assay on fibroblast cytotoxicity.According to the test results (+)-larixol (1) had no deleterious effects on normal mouse lung fibroblasts and no significant inhibition of vitality [69].
The assessments of biological activities of the essential oil from Taiwania flousiana Gaussen.performed by Liu, H. et al. showed a wide range of strong algicidal, antifungal, antibacterial and promising antioxidant activities [70].
Surprisingly, Croton matourensis has proven to be a new source of (+)-larixol (1).Bezerra, F. et al. performed biological activity tests of hydrodistillation, hexane and SC-CO2 extracts from leaves and proved that all of them have exhibited antioxidant activity at 14 mg/mL (IC50= 1531.34,2680.11,614.73 g extract/g DPPH).It was mentioned that the SC-CO2 extract showed potential anti-inflammatory and neuroprotective effects on experimental cerebral ischemia in rats.All these activities of extracts from Croton matourensis leaves can be related to the presence of oxygenated diterpenes (37.15-85.62%),especially to (+)-larixol (1) and manoyl oxide (84) [71].
In the last decades, many efforts have been put in identifying chemical entities that may control TRPC6 activity, a nonselective and Ca 2+ -permeable cation channel, which mediates pathophysiological responses within pulmonary and renal diseases.Within several preparations of plant extracts, a strong TRPC6-inhibitory activity was found in Larch balsam.By testing, its main constituents (+)-larixol (1) and larixyl acetate (2) were identified as blockers of Ca 2+ entry and ionic currents through diacylglycerol-or receptoractivated recombinant TRPC6 channels exhibiting approximately 12-and 5-fold selectivity compared with its closest relatives TRPC3 and TRPC7, respectively.The potent inhibition of recombinant TRPC6 by larixyl acetate (IC50= 0.1-0.6 µM) was confirmed for native TRPC6-like [Ca 2+ ]i signals in diacylglycerolstimulated rat pulmonary artery smooth muscle cells [72].
Urban, N. et al. synthesized new TRPC6inhibiting modulators from (+)-larixol (1), and tested the potency and selectivity in cell lines stably expressing various TRP channel isoforms [73].The most promising compound was larixyl N-methylcarbamate (89) which displayed a favourable potency with an IC50 to inhibit wildtype TRPC6 of 0.48 μM that is an about 30-fold higher versus TRPC3 and 5-fold higher versus TRPC7, respectively.
It was proved that (+)-larixol (1) and its acetylated congeners possess selective inhibition of the second-messenger-gated cation channel transient receptor potential canonical 6 (TRPC6) over its close isoforms TRPC3 and TRPC7.Haefner, S. et al. expanded these findings by chemical diversification of (+)-larixol (1) at position C-6, C-9 side chain, C-8 exomethylene group and mixed.As a result of series of screening assays, Haefner, S. et al. reported the larixyl N-methylcarbamate 89 as an efficient TRPC6 blocker with an IC50 value of 5.8 nM that holds promise for the translational treatment of lung ischemia-reperfusion edema (LIRE) [74].
Chen, X. et al. reported improving of endothelial function in wild type control mice subjected to mTBI after 7-days of in vivo treatment with larixyl acetate (2), an inhibitor of TRPC6 channels with an apparent IC50 value of about 0.65 μM, which is 10-100 times lower than that for other members of the TRPC channel subfamily [75].

(+)-Larixol, larixyl acetate and intellectual property protection
As a logical continuation of the research conducted, a number of results found their application in practice and were patented by some of the above cited authors.In continuation, several of the recently published patents will be discussed.
A group of inventors, Meyer, I. et al., was granted with patents [76,77] for an invention which relates to a cosmetic composition and comprises (+)-larixol (1) and optionally some components like a tyrosinase inhibitor, a sun protection factor; an antioxidant, an antiinflammatory agent and a desquamating agent.
Mulholland, D. et al. [78] obtained the title of protection on use of extracts from Larix spp.(Larix decidua, Larix gmelinii, Larix kaempferi, Larix sukaczewii and Larix sibirica) for treating, preventing or reducing an oomycete pathogen infection.They mention that these properties are due to components such as (+)-larixol (1) or lariciresinol (59) or an active derivative thereof, such as larixyl acetate (2) or lariciresinol acetate (60).They mention, that in particular dichloromethane or methanol extracts may treat or prevent an infection caused by Plasmopara spp. or Phytophthora spp., in particular Plasmopara viticola or Phytophthora infestans.An infection to treat can be selected from downy mildew, late blight of potato, sudden oak death, rhodendron root rot, ink diseases of European chestnut, pythium damping off or white blister rust infection.
Next inventions belong to microbiology, more exactly to microbial transformations and microbial production of terpenes [79,80].Schrader, J. et al. claimed a process for de novo microbial synthesis of sesquiterpenes or diterpenes, including (+)-larixol (1), using genetically modified methylotrophic bacteria (Myxococcus xanthus, Methylobacterium extorquens) and alternative carbon sources like methanol and/or ethanol.
The list of recently published patents concludes with a patent for a method of extraction, purification, testing and application of Leonurus pseudomacranthus essential oil claimed by Lai, P. et al. [81].It comprises the condition for the essential oil extraction, the procedures of its purification and GC-MS analysis.According to Lai, P. et al., the Leonurus pseudomacranthus essential oil can be used as a bacteriostatic agent against strains of Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa.

Conclusions
This review presents scientific data regarding the syntheses made based on (+)-larixol and larixyl acetate, published since 2000.Currently, one of the major concerns of researchers in the field are phytochemical studies, resulting in the identification of, new natural sources that contain compounds from the title.
Another current concern of researchers, which has provided excellent practical results, is the in vitro or in vivo testing of (+)-larixol and larixyl acetate or their derivatives, or plant extracts, especially those obtained from some species of conifers.
Based on the above, it can be concluded that in recent decades in the field of (+)-larixol chemistry, the balance has shifted from fundamental studies in the direction of practical applications.However, it can be stated with all certainty that (+)-larixol, larixyl acetate, their derivatives and the products that contain them are still an interesting object for research.

Funding
This work is part of the project PLANTERAS 20.80009.8007.03"New substances with preventive and therapeutic potential based on natural compounds of plant origin and modern methods of organic synthesis" within the State Program (2020-2023) financed by the National Agency for Research and Development.

Figure 6 .
Figure 6.The main constituents of essential oils and extracts from some medicinal species [62-65].