Podophyllotoxin: History, Recent Advances and Future Prospects

Podophyllotoxin, along with its various derivatives and congeners are widely recognized as broad-spectrum pharmacologically active compounds. Etoposide, for instance, is the frontline chemotherapeutic drug used against various cancers due to its superior anticancer activity. It has recently been redeveloped for the purpose of treating cytokine storm in COVID-19 patients. Podophyllotoxin and its naturally occurring congeners have low bioavailability and almost all these initially discovered compounds cause systemic toxicity and development of drug resistance. Moreover, the production of synthetic derivatives that could suffice for the clinical limitations of these naturally occurring compounds is not economically feasible. These challenges demanded continuous devotions towards improving the druggability of these drugs and continue to seek structure-optimization strategies. The discovery of renewable sources including microbial origin for podophyllotoxin is another possible approach. This review focuses on the exigency of innovation and research required in the global R&D and pharmaceutical industry for podophyllotoxin and related compounds based on recent scientific findings and market predictions.


Introduction
Podophyllotoxin is an aryltetralin-type lignan isolated from species of Podophyllum [1,2]. Two most common sources are the rhizomes of Podophyllum peltatum (American mayapple) and Sinopodophyllum hexandrum Royle (Barberry family) [1,3]. These perennial herbs are distributed widely across the Himalayan region and Western China [4][5][6][7]. Plants with high podophyllotoxin or podophyllotoxin-analogues' content have been extensively used in traditional medicine since a long time in different cultures [8][9][10][11]. This remarkable molecule was first isolated in 1880 by Podwyssotzki [12][13][14][15], while it had already been described as early as in 1753 by Linnaeus [16]. Due to its remarkable biological activity, podophyllotoxin has remained a subject of various investigations ever since. It is mainly obtained from the alcohol-soluble fraction of Podophyllum species, called podophyllin-a bitter tasting resin.
The podophyllotoxin extract has been documented for its use as a laxative, and as a remedy for various medical complications such as gonorrhea, tuberculosis, menstrual disorders, psoriasis, dropsy, cough, syphilis and venereal warts [8][9][10][11]. The podophyllotoxinfamily is now confirmed to elicit various curative properties such as mitotoxic, neurotoxic, Figure 1. The sequential discovery of podophyllotoxin-group of drugs. FDA approved anticancer drugs etoposide, teniposide and etopophos were derived from the parent compound podophyllotoxin, which was originally extracted from mayapple plant as a curative for various diseases. As the side-effects for podophyllotoxin and its primary derivatives became evident, less toxic derivatives such as TOP-53, NK611, GL-331, azatoxin and various others were designed and synthesized.

Structural Characteristics of Podophyllotoxin
Podophyllotoxin has a five-ring system (ABCDE) having four chiral centers (C1-C4) in a row- Figure 2. There are five important structural characteristics of most podophyllotoxin species: (1) a tetracyclic group going from dioxole ring (A) to the lactone ring (D); (2) four oxygen atoms located at the functional groups lactone, methoxys, secondary alcohol and oxoles; (3) ring E with alpha-configuration located at position 1; (4) four asymmetrical adjacent centers and (5) the unique stereochemical properties of C4 define the molecules' mechanism of action [31,32]. Studies have revealed, rational modification at C4 can improve the molecule's topoisomerase II inhibitory activity, drug resistance profile, water solubility and antimitotic activities [33]. The lactone ring is known to exhibit transconfiguration, which easily epimerizes in low basic conditions [32].

Derivatives, Analogues and Hybrids of Podophyllotoxin
Derivatives of this multifaceted molecule are synthesized as properties of its four rings. Ring A, for instance is not essential for the exhibition of antimitotic activity, and aromatization of ring C may cause displacement of axially positioned ring E-leading to a loss of cytotoxic property. Structural diversity hence brings many attractive medicinal properties and a comprehensive insight to the molecule's mechanism of action. These derivatives were first introduced by Schreier through selective cleavage of ring A to produce a 6, 7-dihydroxy derivative (compound A), which was further methylated with diazomethane to produce yet another derivative (compound B) shown in Figure 3 [34]. Schreier's methods were later on slightly modified to produce a large group of podophyllotoxin derivativescompound I-XXfrom compound C, Figure 3. However, the structural modifications are not alone to contribute towards the novelty of the derivatives, instead, changes in the chemical skeleton of podophyllotoxin cyclo-lignan add particularity to each individual derivative.    With slight modifications in the podophyllotoxin skeleton, the ring-A-open compounds appeared biologically less active than podophyllotoxin itself [35]; functional studies indicate that an intact A-ring system is important for the compounds' DNA topoisomerase II (dtopII) inhibiting activity. Based on these findings, three crucially significant domains in the parent pharmacophore model were reported [36]. Modifications at these domains further revealed the unexplored potential of novel clinically applicable derivatives (shown in Figure 4)with a mechanism of action distinct to that of the previously well-established derivative; etiposide [37]. The paucity of studies on B-ring modifications in podophyllotoxin led the investigation of α-peltatin and β-peltatin. These two compounds ( Figure 5) exhibited significant antiviral and antitumor activities. In order to investigate the influence of B-ring substitutions and modifications, a series of ester-and ether-derivatives of both structures were prepared by various researchers but were reported to be biologically less active than their parent compounds [38][39][40][41][42][43].    Structures of alpha-peltatin and beta-peltatin. These two B-ring modified podophyllotoxin derivatives are known to exhibit significant antiviral and antitumor activities. The B-ring modified pharmacophore was identified from these compounds and was taken further to synthesize more derivatives-as shown bracketed-none of which elicited any clinical efficacy.
Several C-ring modified podophyllotoxin analogues have also been subjected to extensive research. Unlike the two natural C-aromatized lignans; justicidin A and diphyllin; the many synthetic C-ring aromatized compounds were prepared but reported to have no cytotoxic activities. This observation proposed that the axial E-ring conformation was lost during synthetic preparations. The loss of the axial E-ring conformation is now known to be directly linked to the loss of molecule's cytotoxic and dtopII inhibiting activity [44]. Notably, however, delactonized analogues of podophyllotoxin-apopicropodophyllotoxin; and its beta-isomer-β-apopicropodophyllotoxin give strong antimitotic activities, otherwise indicating the untrammelled antimitotic potential of podophyllotoxin-like compounds in the absence of the lactone ring [45][46][47]. The antitumor activity of podophyllotoxin and its derivatives is mainly due to the ability to inhibit tubulin polymerization [48], which is a property of the axially placed E-ring. The cis-fused lactone rings, however, modulating its selectivity from tubulin to IGF-1 (insulin-like growth factor I) receptor to trigger cell death [49][50][51].
Transfused γ-lactone D ring is also strict requirement for podophyllotoxin's antitumor activity. Since it is susceptible to isomerization, the opening of this lactone ring is undesirable for it limits the physiological lifetime of these compounds and their biological effectiveness as well. A series of delactonized D-ring derivatives were prepared [52][53][54][55][56][57] and studied by various researchers, most of which were found to be less cytotoxic than the parent compound itself. However, an ethyl hydrazide derivative ( Figure 6) was found to retain its clinical efficacy but was discontinued due to severe adverse aftereffects [58]. Studies that later discovered immunosuppressive ability of these derivatives were attempted to revive D-ring modified podophyllotoxin-antitumor drug preparations [59,60]. 4 -d-β-DMEP (4 -d-β-Demethylepipodophyllotoxin) is a natural lignin, which is used in chemotherapy as an aglycon of etoposide [61,62]. 4 -d-α-DMEP is a novel biological derivative of 4 -d-β-DMEP reported by Jia et al. (2020), which has a higher antitumor activity [63].

Biosynthesis of Plant Derived Podophyllotoxin
The understanding of plant pathways is comparatively absolute to the microbial pathways characterized to date. Plant genes that are involved the biosynthetic pathways of various plant-derived clinical drugs remain obscure, hence preventing the transfer of which to heterologous hosts for industrial production. The complete biosynthetic pathway ( Figure 8) for podophyllotoxin was elucidated only recently in 2015, allowing more facile access to its natural and processed, clinically significant derivatives-otherwise difficult to synthesize on an industrial scale [108].

Microbial Sources of Podophyllotoxin
Biotechnological procedures for podophyllotoxin production were introduced as a means to overcome its large scale production challenges, which includes the extinction of various podophyllotoxin-plant sources, slow growth of plant sources, exacting constructs and low yields. Micro-organisms, however, demonstrate an enormity of biodiversity, which overdo those of plants. Fungal sources, for instance, have rendered to be a much fruitful alternative to somatic embryogenesis, tissue culturing or macropropagation techniques. The main fungal sources used for industrial podophyllotoxin production are; Trametes hirsuta [110], Fusarium oxysporum [111], Fungus Alternaria [112], Mucor fragilis [113], Fusarium solani [114], Trametes histuria [110], Sebacina vermifera, Phialocephala fortinii [115] and Aspergillus fumigatus [101,116]. The endophytic sources of podophyllotoxin are also tabulated below Table 2 [111] describes the process of podophyllotoxin production from Fusarium oxysporum, which lives on Juniperus recurva. The outcome suggests F. oxysporum to be a promising contender for industrial podophyllotoxin production. In 2009, the specie Juniperus communis L. Horstmann was also found to produce deoxypodophyllotoxin, which shared remarkable structural relativity to the lignan of podophyllotoxin [117]. No bacterial sources of podophyllotoxin have yet been identified.

Parametric Analysis of Podophyllotoxin Biosynthesis
Studies report five major parameters, which influence the biosynthesis of podophyllotoxin; these include luminosity, chilling units/hours, macro-and micro-nutrients and soil's pH and nutrient availability. Any fluctuation in the strength of which can result in altered podophyllotoxin yields. In the presence of red-light, for instance, increased the overall product formation in comparison to light of other wavelengths [123]; likewise, chilling units set at 4 • C were reported to have caused a 5-folds increase in product yield. The variation in concentrations of macro-and micro-nutrients such as glucose, nitrogen, NO 3 − , PO 4 3− , Na + , Fe 2+ , Mn 2+ , etc., has shown similar correlation with podophyllotoxin production. Moreover, the acidity or alkalinity levels of the soil in cases for podophyllotoxin producing plants also demonstrate yield modifications. These parameters are tabulated and discussed below Table 3.

Pharmacological Significance of Podophyllotoxin and Its Derivatives
Podophyllotoxin and its derivatives possess a wide-spectrum of pharmacological potential. The achievability of broad chemical modifications brings the podophyllotoxin pharmacophore its diverse applicability as a medicinal compound-tabulated in Table 4. Antineoplastic activity is the most outstanding of all its clinical properties. Various studies validate podophyllotoxin derivatives including etopophos, teniposide, etoposide, etoposide phosphate, GL331, NK-611, TOP53 and NPF as anticancer drugs [64,[128][129][130][131][132]. Many clinical studies have reported the efficacy of these compounds against various forms of cancer including lung cancer, Wilmstumours, diverse types of genital tumors such as carcinoma verrucosus and for non-Hodgkin lymphoma, multiform glioblastoma lymphoma and nonlymphocytic leukemia [17]. Podophyllotoxin has also shown activity against various (multi) drug resistant tumor cells. As an example, Hu and coworkers presented a 4-β-anilino-podophyllotoxin derivative as a potential anticancer drug against the KB/VCR cells in both conditions; in vivo and in vitro [133]. For the development of new anticancer drugs, podophyllotoxin containing analogues are of prime focus in recent studies. Ming et al. [134] explained that an endophytic fungus named as Dysosma versipellis has both anticancer and antimicrobial properties. The aqueous extract of Podophyllum peltatum [41] fractionated by reverse-phase chromatography was observingly the most active component that was found to cause the inhibition in the replication process of herpes simplex type 1 virus and measles.
Moreover, picropodophyllotoxin, deoxypodophyllotoxin and peltatins are also determined as useful antiviral compounds [135][136][137]. Activity against Sindbis and cytomegalovirus has also been recorded [138]. They either decrease the capacity of the infected cell to release virus or restrain these viruses in the replication cycle at an essential early stage, following the virus absorption into cells. Not only this, podophyllotoxin can also be used for treating Condyloma acuminatum that is usually caused by HPV (papilloma virus) [139] and for treating other perianal and venereal warts [140]. With a goal to achieve better therapeutic effectiveness, cocktail therapies are currently in use with other registered chemotherapeutic agents, combined with additional techniques that are beneficial in fighting against cancer and other viral infections. Podophyllotoxins with interferon therapy has shown greater effectivity against genital human infections, combination with cisplatin, on the other hand, is useful for treating neuroblastomas. Recently a study reports the study progression of etoposide in phase II clinical trials (ClinicalTrials NTC04356690) as a redeveloped drug to treat COVID-19 patients suffering with cytokine storm complication [141]. Similarly, in recent years podophyllotoxin derivatives have shown some interesting insecticidal activities against the larvae of Brontispa longissima and Mythimna separata [142]. Furthermore, derivatives are also being synthesized and used for the treatment of malaria and psoriasis [143]. Podophyllotoxins also hold dermatological significance and prove to be potential therapeutic agents for psoriasis vulgaris. Podophyllotoxin exhibited considerable ichthyotoxic activity and phyto growth inhibitory activities, even though the effects were weaker as compared to deoxy podophyllotoxin, in all cases observed [144,145].

New hybrids of podophyllotoxin and indirubin
Activity was checked against human leukemia cancer cells as a multifunctional anti-MDR agent Podophyllotoxin-indirubin hybrid (Da-1) showed potential to overcome drug resistance. It is a novel hybrid havingpotent antiproliferative activity [150] Cyclolignans, derived from podophyllotoxin Activity was checked againstA-549 human lung carcinoma, P-388 murine leukemia and HT-29 colon carcinoma A number of substances were active in assay at concentrations below 1 pM; deoxypodophyllotoxin being the most potent compound in all cases [151]

Patents
In the past decade, numerous attempts have been globally to produce derivatives or compositions of podophyllotoxins in order to overcome their natural dose-limiting toxicity, poor biodistribution and low aqueous solubility. These inventions have found extensive application for effective chemotherapeutic purposes. These inventions have succeeded at demonstrating sufficient activity and can considerably contribute to future studies, some of these new derivatives are tabulated below (Table 5).

US-9828386-B2
Elevated antitumor activity than podophyllotoxin or 4 demethylepipodophyllotoxin [159] The great interest of institutional researchers and pharmaceutical companies is also validated by a surge in number of patents protecting various inventions involving podophyllotoxin. Searching the term "podophyllotoxin" gave 93,446 results in the Google patent database while 26,582 in the Patent Lens database with the first patent application filed in 1967-12-06 and granted in 1970-08-18. When "podophyllotoxin" is used as keywords for the the patents and/or patent applications annual filed (Figure 9), key applicant companies (Figure 10), key inventors (Figure 11), and key owners based in US ( Figure 12) [160,161]

Conclusions
Novel approaches towards structure optimization and the finding of alternative sources can help unravel less toxic, more efficient and easy to yield podophyllotoxin and its derivatives. Being known for decades as the main bioactive compound in various traditional medicinal formulas, these compounds were later approved for clinical use against a number of health conditions. In the recent pandemic, various known drugs were repurposed against the novel coronavirus strain 2019. Amongst this struggle of trial and error, etoposide-a podophyllotoxin derivative well cited for its antineoplastic activityhas also been redeveloped to manage cytokine storm complication in COVID-19 patients. This finding opens new potential for podophyllotoxin, its derivatives and its congeners to be explored. For as the growing demand for podophyllotoxin is increasing, plant sources are proving to be an unreliable option for the future, this explains the exigency of utilizing microbial biotransformation as a considerable approach along with the need of devoted effort towards rationally designing the new generation of the group of podophyllotoxinderived compounds. Data Availability Statement: All the data produced here is available and can produced when required.

Conflicts of Interest:
The authors declare no conflict of interest.