Dimeric phenanthrenoids: possible biogenetic pathway and missing compounds

Abstract Secondary metabolites extracted from plants have historically been critical for drug discovery, but their isolation involves expensive and complicated procedures in terms of time and labor resources. Thus, the biogenetic pathway offers the possibility of identifying specific compounds that have not yet been isolated and predicting their isolation from specific natural sources. In plants, biphenanthrenes represent a relatively small group of aromatic secondary metabolites that are considered as important taxonomic markers with promising biological activities. To date, 38 mixed phenanthrenoid dimers have been identified, the biosynthesis of which involves the radical coupling of the two subunits, namely, a phenanthrene and a dihydrophenanthrene. For each of the compounds, it is possible to identify the single phenanthrenic and dihydrophenanthrenic units constituting the considered dimer. Based on the biogenetic pathway, it is possible to identify 19 phenanthrenes and 17 dihydrophenanthrenes, and to distinguish those already known from those not yet isolated. By comparing the results of the possible biosynthetic pathway for each compound with the data in the literature, it is possible to identify three known phenanthrenes and seven known dihydrophenanthrenes, as well as eleven new phenanthrenes and five new dihydrophenanthrenes, and to identify from which plant it is possible to isolate them. This could direct the work of researchers seeking to identify known or new molecules useful for their possible biological properties, and ultimately, to confirm the veracity of the proposed and generally accepted biosynthetic pathway. Graphical Abstract


Introduction
Historically, many diverse natural products have been used extensively in drug discovery, but several resources have yet to be explored in modern research.To date, the Dictionary of Natural Products (https://dnp … ) has recorded approximately 200,000 plant secondary metabolites, including about170,000 unique structures.About 15% of drug trials in the Clinical Trials.govdatabase (https://clinicaltrials.gov) are based on plant-derived content, with about 60% of these drugs coming from 10 taxonomic 'families'.Despite this progress, there remains a wide range of plant species that have not yet been systematically studied.Furthermore, traditional plant-based medicines used by different cultures still need to be explored.In particular, biogenetic pathways offer the possibility of identifying specific compounds that have not yet been isolated from natural sources, and even more interestingly, they have the ability to predict the isolation of new metabolites with well-known structures in specific natural sources.This investigation will be crucial to further revitalize the exploration of new natural products as starting points for drug discovery.
Among plant metabolites, biphenanthrenes represent a relatively small group of aromatic secondary metabolites, which can be divided into three main subgroups consisting of i) two phenanthrenes, ii) two dihydrophenanthrenes, and iii) a phenanthrene and a dihydrophenanthrene (mixed).The mixed ones are estimated to number about forty, and are considered the most important taxonomic markers with promising biological activities.To date, the known ones have been isolated from four Juncaceae species (J.acutus, J. effusus, J. inflexus, J. gerardii), as well as from Appendiculata reflexa, Bletilla formosana and B. striata, Bulbophyllum emarginatum, Cremastra appendiculata, Cyrtopodium paniculatum, Monomeria barbata, Pleione bulbocodioides and Pholidota chinensis.Among these compounds, several possess different biological effects, e.g.antialgal (DellaGreca et al. 2003(DellaGreca et al. , 2004(DellaGreca et al. , 2005)), antimicrobial (DellaGreca et al. 1995(DellaGreca et al. , 2002)), antiproliferative (Sun et al. 2021;Wang et al. 2019;Ma et al. 2015;Stefk o et al. 2020;Ishiuchi et al. 2015), antiviral (Shi et al. 2017;Apel et al. 2012;Li et al. 2019), anti-inflammatory (Sun et al. 2021;Ma et al. 2015;Zhou et al. 2019;Lin et al. 2016), antioxidant (Wang et al. 2007), spasmolytic, anxiolytic (Wang et al. 2014), etc.Thus, the aim of this work is to summarize the occurrence of phenanthrenes and dihydrophenanthrenes, known or new, that have not yet been isolated from specific natural sources, combining the knowledge of the biosynthetic mechanisms proposed for the achievement of their corresponding dimers with the literature data on plants from which the known monomeric compounds have already been isolated.

Results and discussion
To date, 38 compounds have been found with mixed dimeric skeletons, generically represented as shown in Figure 1, indicating the phenanthrenic monomer on the left and the 9,10-didrophenanthrenic monomer on the right, which are linked together in the positions marked with a black circle and numbered as follows.
Below, the structures of the isolated compounds (Figures S1-S4), the corresponding IUPAC names, and the plants from which they were extracted and identified, are reported (Table S1).Compounds 1-38 are though to result from the coupling of two radicals corresponding to the single phenanthrenic and dihydrophenanthrenic units constituting the considered dimer (DellaGreca et al. 2002;Ishiuchi et al. 2015;T oth et al. 2016).Figure 2 shows the possible mechanism of the formation of dimer 30.
Based on this mechanism, it is possible to identify 19 phenanthrenes (Table S2) and 17 dihydrophenanthrenes (Table S3) that differ for compounds 1-38.Obviously, some dimers, even if they are different, will consist of the same basic phenanthrenes and/or dihydrophenanthrenes, as indicated in Table S4.For compounds 29-36, FXa indicates the phenanthrene with the carbon atom C-14 (for compound 29) or the oxygen O-11 (for compounds 30-36), with which it binds to the corresponding dihydrophenanthrene unit.FXb identifies a phenanthrene without this carbon or oxygen, the latter being assigned to the corresponding dihydrophenanthrene unit (Figures 2 and S3 and S4 and Table S4).
For compounds 29-36, DFXa indicates the dihydrophenanthrene without the carbon atom C-14 (for compound 29) or the oxygen O-11 (for compounds 30-36), while DFXb indicates the phenanthrene with this carbon or oxygen, with which it binds to the corresponding phenanthrene unit (Figures 2 and S3 and S4 and Table S4).Once the phenanthrenes and dihydrophenanthrenes constituting the dimeric compounds 1-38 were identified, we proceeded to identify those already known (for which the CAS number is reported) and those not yet isolated.For those isolated, the reference relative to the isolation of the considered phenanthrene (Table S5) or dihydrophenanthrene (Table S6) from the same plant that the corresponding dimer was isolated from is reported.This allowed us to identify three known phenanthrenes and eleven new phenanthrenes, and from which plant it is possible to isolate them (Table S7).Similarly, we identified seven known dihydrophenanthrenes and five new dihydrophenanthrenes, and from which plant it is possible to isolate them (Table S8).

Conclusions
To date, 38 mixed phenanthrenoid dimers have been identified, the biosynthesis of which is hypothesized by means of a pathway that considers the radical coupling of the two subunits, phenanthrenic and dihydrophenanthrenic, respectively.Thus, for each of these dimers, it was possible to identify one phenanthrene and one dihydrophenanthrene.By comparing the results of the possible biosynthetic pathway for each compound with the data in the literature, it was possible i) to identify three known phenanthrenes and seven known dihydrophenanthrenes, as well as eleven new phenanthrenes and five new dihydrophenanthrenes, and ii) to identify from which plant it is possible to isolate them (obviously new ones, never previously identified, and those known but not isolated from the same plant in which the corresponding dimer was isolated).This could direct the work of researchers seeking to identify known or new molecules, which will be useful for determining their possible biological properties and ultimately confirming the veracity of the generally proposed and accepted biosynthetic pathway.

Figure 1 .
Figure 1.Generic structure of a mixed dimer with a phenanthrene-dihydrophenanthrene skeleton.

Figure 2 .
Figure 2. Hypothesized mechanism of formation of compound 30, that serves as an example.