Chemistry and Biological Activities of Naturally Occurring and Structurally Modified Podophyllotoxins

Plants containing podophyllotoxin and its analogues have been used as folk medicines for centuries. The characteristic chemical structures and strong biological activities of this class of compounds attracted attention worldwide. Currently, more than ninety natural podophyllotoxins were isolated, and structure modifications of these molecules were performed to afford a variety of derivatives, which offered optimized anti-tumor activity. This review summarized up to date reports on natural occurring podophyllotoxins and their sources, structural modification and biological activities. Special attention was paid to both structural modification and optimized antitumor activity. It was noteworthy that etoposide, a derivative of podophyllotoxin, could prevent cytokine storm caused by the recent SARS-CoV-2 viral infection.

This review focused on up-to-date studies on the natural podophyllotoxins and their natural sources, structural modification on rings A, B, C, D and E, biological activities including antitumor, antiviral anti-inflammation, miscellaneous effects and toxicity. In addition, total chemical synthesis, biosynthesis and ADME were also included in this review. groups at both B and E rings, and was identified as a cytoxic agent from Haplophyllum ptilostylum. Compound 52 was the 4α isomer of 51, which was obtained from Bursera simaruba. Erlangerin A-D (53)(54)(55)(56) were four ligands isolated from Commiphora erlangeriana. Compounds 53 and 54 belonged to the polygamatin-type; while 55 and 56 were related to podophyllotoxin (1). Compound 57 was a polygamatin-type ligand from Justicia heterocarpa whose structure was confirmed by X-ray diffraction analysis. The chemical structures of natural podophyllotoxins aglycones are shown in Figure 1.

Podophyllotoxin Glycosides
The sugar units usually attached at C-4 consisted of one to several monosaccarides, glucose or apiose ( Figure 3). Compounds 72-85 were monoglycosides. Among these compounds, 72-77 and 79 were podophyllotoxin-type glucosides, 80-81 were apioside of diphyllin-type anologues, and 78 and 82 were featured with acetyl substitution attached at the sugar unit. In these structures, the sugar units located at C-4. Compounds 83-86 were also monoglycosides, but the sugar unit attached at C-4′ or C-5 position. Compounds 87-93 were diglucosides with the sugar side chain linked at C-4. Bispicropodophyllin glucoside (94) was an unique dimeric lignan from Withania coagulans, in which the C ring of both units were opened to form two esters linking the two units [55]. Ciliatoside A (95) and B (96) were lignan glycosides possessing potent anti-inflammatory effect from Justicia ciliate, with a three and four sugar side chain, respectively [56].
The main podophyllotoxins and their natural sources were summarized (Table 1 and Figures 1-3).

Podophyllotoxin Glycosides
The sugar units usually attached at C-4 consisted of one to several monosaccarides, glucose or apiose ( Figure 3). Compounds 72-85 were monoglycosides. Among these compounds, 72-77 and 79 were podophyllotoxin-type glucosides, 80-81 were apioside of diphyllin-type anologues, and 78 and 82 were featured with acetyl substitution attached at the sugar unit. In these structures, the sugar units located at C-4. Compounds 83-86 were also monoglycosides, but the sugar unit attached at C-4 or C-5 position. Compounds 87-93 were diglucosides with the sugar side chain linked at C-4. Bispicropodophyllin glucoside (94) was an unique dimeric lignan from Withania coagulans, in which the C ring of both units were opened to form two esters linking the two units [55]. Ciliatoside A (95) and B (96) were lignan glycosides possessing potent anti-inflammatory effect from Justicia ciliate, with a three and four sugar side chain, respectively [56].
The main podophyllotoxins and their natural sources were summarized (Table 1 and Figures 1-3).   Figure 3. Chemical structures of natural podophyllotoxin glycosides.

Plant Compounds Biological Activities References
Podophyllum peltatum Antitumor (inhibition against P-388 lymphocytic leukemia and human epidermoid carcinoma) [124] Sinopodophyllum emodi Note: "-" means bioactivity was not reported in the references.

Structural Modification in Podophyllotoxins
Most of the natural occurring podophyllotoxins were limited in applications either by their insufficient resources or prohibitive toxicity. In the mid of nineteenth century, investigations on the synthesis or semisynthesis of podophyllotoxins were undertaken to construct new molecules with optimized antineoplastic activity and less toxicity [128], which led to the generation of two widely used anticancer drugs, etoposide and teniposide [129]. Compared to the parent compounds, etoposide and teniposide showed moderate toxicity, improved therapeutic index (TI) and acceptable efficiency in the treatment of many cancers, especially small cell lung carcinoma and testicular cancer [130]. Nevertheless, limitations such as poor solubility and growing drug resistance still existed during their applications [14,131]. So, podophyllotoxin and its derivatives were still hotspots of modifications for novel anticancer agents. Many previous reviews summarized the synthesis or semisynthesis of podophyllotoxin derivatives including simple esterification, demethylation, oxidation, etc. Recently, many researchers were interested in introducing hereronuclears into podophyllotoxins according to the bioisostere theory, as well as the synthesis of spin-labeled derivatives or conjugates with anticancer drugs, e.g., 5-fluorouracil (5-Fu) [27,132].

Introducing Heteronuclears into Podophyllotoxins
Bioisosterism was a rational strategy in molecular modifications [133]. Recently, substitution of carbon atoms with heteronuclears was carried out to synthesize podophyllotoxin analogues. Pharmacological studies revealed that some nitrogen-containing derivatives, such as GL-331 (97) and TOP-53 (98), exhibited more potent cytotoxic activities than their parent compounds ( Figure 4). Additionally, their abilities to reverse multidrug resistance and inhibit P-glycoprotein induced drug efflux were improved.

Ring A
Ring A was reported to be important for the cytotoxicity [3]. Cleavage or changes of A-ring ( Figure 5), such as replacing it with a pyridazine ring (99), will decrease the cytotoxicity, as well as TOPO-II inhibitory activity [134]. But many A-ring modifications were still performed to improve their inhibitory activity on reverse transcriptase (RT) in HIV, and minimize the side-effects. A series of A-ring opened compounds 100-103 were synthesized, and were tested to possess potent anti-HIV activities with the average EC50 less than 0.001 μg/mL and the therapeutic index (TI) value more than 120 (against HIV) [135].

Ring B
Modification in ring B was relatively rare. Introducing a hydroxyl group into ring B (104) could remarkably improve the TOPO-II inhibitory activity of epipodophyllotoxin [3]. It was reported that the alkoxy-substituted benzene ring was replaced by a pyrazole moiety (105)(106)(107)(108)(109)(110). The antiproliferative properties of these heterocyclic compounds were comparable with the currently used anticancer drug etoposide [136].

Ring C
A variety of ring C modified podophyllotoxins have been synthesized, and their diverse biological profiles were attractive. TOPO-II is the major target of podophyllotoxins in cancer therapy, since C-4 position in ring C was identified to be the TOPO-II binding site [3]. It was reported that a bulky at C-4 could enhance their cytotoxic activities [137]. Some researches indicated that replacing the saccharide chain with a non-saccharide moi-

Ring A
Ring A was reported to be important for the cytotoxicity [3]. Cleavage or changes of A-ring ( Figure 5), such as replacing it with a pyridazine ring (99), will decrease the cytotoxicity, as well as TOPO-II inhibitory activity [134]. But many A-ring modifications were still performed to improve their inhibitory activity on reverse transcriptase (RT) in HIV, and minimize the side-effects. A series of A-ring opened compounds 100-103 were synthesized, and were tested to possess potent anti-HIV activities with the average EC 50 less than 0.001 µg/mL and the therapeutic index (TI) value more than 120 (against HIV) [135].

Ring A
Ring A was reported to be important for the cytotoxicity [3]. Cleavage or changes of A-ring ( Figure 5), such as replacing it with a pyridazine ring (99), will decrease the cytotoxicity, as well as TOPO-II inhibitory activity [134]. But many A-ring modifications were still performed to improve their inhibitory activity on reverse transcriptase (RT) in HIV, and minimize the side-effects. A series of A-ring opened compounds 100-103 were synthesized, and were tested to possess potent anti-HIV activities with the average EC50 less than 0.001 μg/mL and the therapeutic index (TI) value more than 120 (against HIV) [135].

Ring B
Modification in ring B was relatively rare. Introducing a hydroxyl group into ring B (104) could remarkably improve the TOPO-II inhibitory activity of epipodophyllotoxin [3]. It was reported that the alkoxy-substituted benzene ring was replaced by a pyrazole moiety (105)(106)(107)(108)(109)(110). The antiproliferative properties of these heterocyclic compounds were comparable with the currently used anticancer drug etoposide [136].

Ring C
A variety of ring C modified podophyllotoxins have been synthesized, and their diverse biological profiles were attractive. TOPO-II is the major target of podophyllotoxins in cancer therapy, since C-4 position in ring C was identified to be the TOPO-II binding site [3]. It was reported that a bulky at C-4 could enhance their cytotoxic activities [137]. Some researches indicated that replacing the saccharide chain with a non-saccharide moi-

Ring B
Modification in ring B was relatively rare. Introducing a hydroxyl group into ring B (104) could remarkably improve the TOPO-II inhibitory activity of epipodophyllotoxin [3]. It was reported that the alkoxy-substituted benzene ring was replaced by a pyrazole moiety (105)(106)(107)(108)(109)(110). The antiproliferative properties of these heterocyclic compounds were comparable with the currently used anticancer drug etoposide [136].

Ring C
A variety of ring C modified podophyllotoxins have been synthesized, and their diverse biological profiles were attractive. TOPO-II is the major target of podophyllotoxins in cancer therapy, since C-4 position in ring C was identified to be the TOPO-II binding site [3]. It was reported that a bulky at C-4 could enhance their cytotoxic activities [137]. Some researches indicated that replacing the saccharide chain with a non-saccharide moiety can remarkably reverse the drug resistance of etoposide [138].
4β-hydroxyl group was the key position of the structural modification on podophyllotoxins. These semisynthetic derivatives showed distinct TOPO-II inhibitory activities. Most of them exceeded their parent compounds, indicating the side chain at C-4 also played a key role in their bioactivity besides the skeleton. Morever, some compounds did not even obey the established structure-active relationship. For example, compounds 142, 144 and 150 without a bulky side chain at C-4 also exhibit potent TOPO-II inhibitory activity; while compound 147, a sulfamide with a long aliphatic side chain showed no TOPO-II binding affinity. Among some polyaromatic substituted 4β-amino podophyllotoxins, 4 -methoxyl derivatives are more cytotoxic than the 4 -hydroxyl compounds. The above structure-activity relationships indicated the existence of some new binding sites for podophyllotoxins on DNA TOPO-II. Furthermore, some compounds are specific for certain cancer cell lines, e.g., colon and prostate, revealing the involvement of some other novel mechanisms.  Instead of substituting the C-4 hydroxyl group with an amino group, nitrogen atom could also be inserted into podophylltoxin skeleton as a part of the ring C. These derivatives could be subdivided into 2-aza-podophyllotoxins and 4-aza-podophyllotoxins. Compound 177, one of the 2-aza-podophyllotoxin was found to exhibit significant activity against several human cancer cell lines [149], but the mechanism was still unclear. 2-azapodophyllotoxins could inhibit TOPO-II in malignant cells. Different from some natural occurring or semi-synthesized podophyllotoxins, an oxidized E-ring would be an essential motif of 2-aza-podophyllotoxins analogues (178) [150]. 4-aza-4-deoxypodophyllotoxin showed potent cytotoxicity against P388 leukemia cells [151]. Another group of dehydropodophyllotoxins were synthesized. A series of 4-aza-2,3-dehydro-4-deoxypodophyllotoxins (180)(181)(182)(183)(184)(185)(186)(187)(188) [152] showed two fold potent cytotoxicity against P-388 leukemia cells than podophyllotoxin. However, a planar 4-aza-C-ring (179) is not favorable with IC50 > 20 μM. The author also proposed an in silico model to predict IC50 of different compounds. Some A-ring removed or replaced 4-aza-podophyllotoxins compounds 193-196 exhibited strong anticancer activity. Their structures are showed in Figure 6. But the exact mechanisms are still under investigation [153]. What is more, SAR of these 4-aza-podophyllotoxins was different from that of some natural or semisynthetic derivatives. It was reported that transfused D-ring was an essential motif for binding microtublin or TOPO-II, Structural modification was also performed to introduce other elements into podophyllotoxins, such as Se or metals. Compounds 175 and 176 with 4β-Se showed enhancement of cell death in a time-and dose-dependent manners, and the mechanism involved the translocation of Bax, the activation of the mitochondrial pathway and apoptosis through the release of proapoptotic factors [147]. Forming complex was an alternative method to introduce metal ions into podophyllotoxins. Hydrazide-podophyllic metal complexes could interact with DNA in different ways. The complexes of Ni and Co-HDPP interacted with DNA mainly by insertion; while the interaction of Zn-HDPP with DNA by partial insertion [148].
Instead of substituting the C-4 hydroxyl group with an amino group, nitrogen atom could also be inserted into podophylltoxin skeleton as a part of the ring C. These derivatives could be subdivided into 2-aza-podophyllotoxins and 4-aza-podophyllotoxins. Compound 177, one of the 2-aza-podophyllotoxin was found to exhibit significant activity against several human cancer cell lines [149], but the mechanism was still unclear. 2-azapodophyllotoxins could inhibit TOPO-II in malignant cells. Different from some natural occurring or semi-synthesized podophyllotoxins, an oxidized E-ring would be an essential motif of 2-aza-podophyllotoxins analogues (178) [150]. 4-aza-4-deoxypodophyllotoxin showed potent cytotoxicity against P388 leukemia cells [151]. Another group of dehydropodophyllotoxins were synthesized. A series of 4-aza-2,3-dehydro-4-deoxypodophyllotoxins (180)(181)(182)(183)(184)(185)(186)(187)(188) [152] showed two fold potent cytotoxicity against P-388 leukemia cells than podophyllotoxin. However, a planar 4-aza-C-ring (179) is not favorable with IC 50 > 20 µM. The author also proposed an in silico model to predict IC 50 of different compounds. Some A-ring removed or replaced 4-aza-podophyllotoxins compounds 193-196 exhibited strong anticancer activity. Their structures are showed in Figure 6. But the exact mechanisms are still under investigation [153]. What is more, SAR of these 4-aza-podophyllotoxins was different from that of some natural or semisynthetic derivatives. It was reported that transfused D-ring was an essential motif for binding microtublin or TOPO-II, and a dioxymethene A-ring has a positive impact on its cytoxic effect. But pharmaceutical results of these 4-aza derivatives revealed that some 4-aza 2,3-dehyro-podophyllotoxins also exhibited promising cytotoxicity. Other 4-aza derivatives like compound 197 and 198, with different linker between ring C and E ring were synthesized, which possessed inhibitory activity on tubulin polymerization, as well as promising antitumor activities [154].
Forming lactone or lactam motif in ring C led to the synthesis of compounds 199-205, which possessed moderate cytotocixities in several cancer cell lines excepted that the C-lactone derivatives showed potency on colon cancer cell line [155].
Besides those ring D opened derivatives, compound 267 with a 1,5-disubstituted triazole ring instead of the lactone motif was synthetized, which showed moderate cytotoxicity with similar mechanism to podophyllotoxin [160]. Another derivative (268) with a substituted cyclosulfite ring exhibited significantly cytotoxicity [161].

Ring E
Several 4 -ester derivatives of GL-331 (269-271) (Figure 8), which were 4β-amino derivative of epipodophyllotoxin under Phase II clinical evaluation [162] were synthetized and showed inhibitory activity on KB and resistant KB-7d tumor cells. The molecular target was confirmed to be DNA topo II. These findings challenged the long-standing premise that a free 4 -hydroxy group was essential for the topo II inhibition [4,163]. Subsequently, the same research group introduced some solubility enhancing moieties to the 4 -hydroxyl position and synthesized eight novel 4 -ester 4β-arylamino analogues (272-279) with improved activity profiles and water-solubility compared with etoposide. Based on the above results, the authors proposed a SAR of these analogues: the pendent E ring and the variable 4β-substitution were respectively defined as the enzyme and DNA interacting domains, and the latter was critical to DNA cleavage specificity and drug-resistance [162]. Other E ring modification led to the synthesis of a N-alkyl-4-amino-1,2-dihydroquinoline-lactone (280) whose pendent E ring could not rotate freely, and its bioactivity was still under testing [164].
Bioisosteric replacement of the phenolic ring with nitrogen-containing heterocycles, such as pyrazoles and triazoles could overcome the reduced drug bioavailability caused by oxidation and glucuronidation of phenolic hydroxyl groups [165,166] Based on compound 180, a dihydropyridopyrazole analogue of podophyllotoxin, a series of E ring modified derivatives (281-304) were synthetized, as substituted E ring with aliphatic, aromatic or heteroaromatic groups. Among these derivatives, those with bromine at meta-position of the aromatic ring E (285, 291-294) showed potent cytoxicities, but the mechanism still needed further investigation [167]. Besides those ring D opened derivatives, compound 267 with a 1,5-disubstituted triazole ring instead of the lactone motif was synthetized, which showed moderate cytotoxicity with similar mechanism to podophyllotoxin [160]. Another derivative (268) with a substituted cyclosulfite ring exhibited significantly cytotoxicity [161] (Figure 8), which were 4β-amino derivative of epipodophyllotoxin under Phase II clinical evaluation [162] were synthetized and showed inhibitory activity on KB and resistant KB-7d tumor cells. The molecular target was confirmed to be DNA topo II. These findings challenged the long-standing prem-  Figure 7. D-ring modified podophyllotoxins.

Spin Labeled Podophyllotoxins
Stable nitroxyl radicals could be used to improve anti-cancer profiles of drugs [168]. Tian's group initiated their synthesis work of spin-labled podophyllotoxins in early 1980th [169]. Subsequently, a series of nitroxyl spin-labeled ester derivatives were synthesized, and the modification positions varied from 4-hydroxyl group, 4-amino group, 4 -hydroxyl to the carboxyl group in the open lactone ring (Figure 9) [170][171][172]. Introduction of nitroxyl radical moieties into 4β-amino-4 -demethylepipodophyllotoxin (305-312) greatly enhanced the antioxidative effect, antitumor and anti-drug resistance activities [173,174]. Besides, a series of 4 -spin-labeled compounds 313-320 were designed and synthesized, and pharmacological experiments showed that most of these molecules exhibited more potent cytotoxicities against HL-60, RPMI-8226 and A549 than the parent compounds. In addition, the synthesized derivatives showed either similar or better antioxidative activities than etoposide [175]. the variable 4β-substitution were respectively defined as the enzyme and DNA interacting domains, and the latter was critical to DNA cleavage specificity and drug-resistance [162]. Other E ring modification led to the synthesis of a N-alkyl-4-amino-1,2-dihydroquinoline-lactone (280) whose pendent E ring could not rotate freely, and its bioactivity was still under testing [164]. Bioisosteric replacement of the phenolic ring with nitrogen-containing heterocycles, such as pyrazoles and triazoles could overcome the reduced drug bioavailability caused by oxidation and glucuronidation of phenolic hydroxyl groups [165,166] Based on compound 180, a dihydropyridopyrazole analogue of podophyllotoxin, a series of E ring modified derivatives (281-304) were synthetized, as substituted E ring with aliphatic, aromatic or heteroaromatic groups. Among these derivatives, those with bromine at meta-position of the aromatic ring E (285, 291-294) showed potent cytoxicities, but the mechanism still needed further investigation [167].

Spin Labeled Podophyllotoxins
Stable nitroxyl radicals could be used to improve anti-cancer profiles of drugs [168]. Tian's group initiated their synthesis work of spin-labled podophyllotoxins in early 1980 th [169]. Subsequently, a series of nitroxyl spin-labeled ester derivatives were synthesized, and the modification positions varied from 4-hydroxyl group, 4-amino group, 4′-hydroxyl to the carboxyl group in the open lactone ring (Figure 9) [170][171][172]. Introduction of nitroxyl radical moieties into 4β-amino-4′-demethylepipodophyllotoxin (305-312) greatly enhanced the antioxidative effect, antitumor and anti-drug resistance activities [173,174]. Besides, a series of 4′-spin-labeled compounds 313-320 were designed and synthesized, and pharmacological experiments showed that most of these molecules exhibited more potent cytotoxicities against HL-60, RPMI-8226 and A549 than the parent compounds. In addition, the synthesized derivatives showed either similar or better antioxidative activities than etoposide [175].   It was well known that cancer formation and development was closely linked to inflammation [176][177][178][179]. In addition, after an inflammatory stimulus, reactive oxygen species (ROS) produced, which could cause cell or DNA damage and eventually mediate carcinogenesis [180]. These cytotoxic podophyllotoxins combined with an antioxidative property was able to reduce tissue damage induced by ROS and prevent tumorigenesis.

Conjugates of Podophyllotoxins
Anticancer drugs were usually joined together for the synergistic treatment of cancer since few tumors are sensitive enough to be cured by single drugs. The anticancer drugs could be connected directly or by means of a linker [181]. The combination of podophyllotoxins and other anticancer drugs led to the synthesis of a series of conjugates.
The connection of podophyllotoxins with other anticancer drugs by various linkers resulted in the construct of many combined agents (Figure 10). Compounds 321-329 were conjugates consisted of podophyllotoxin and antimetabolite 5-FU using different spacers. Among them, 4β-N-substituted-phenylalanine 5-Fu pentyl ester-4′-demethylepipodo- It was well known that cancer formation and development was closely linked to inflammation [176][177][178][179]. In addition, after an inflammatory stimulus, reactive oxygen species (ROS) produced, which could cause cell or DNA damage and eventually mediate carcinogenesis [180]. These cytotoxic podophyllotoxins combined with an antioxidative property was able to reduce tissue damage induced by ROS and prevent tumorigenesis.

Conjugates of Podophyllotoxins
Anticancer drugs were usually joined together for the synergistic treatment of cancer since few tumors are sensitive enough to be cured by single drugs. The anticancer drugs could be connected directly or by means of a linker [181]. The combination of podophyllotoxins and other anticancer drugs led to the synthesis of a series of conjugates.
The connection of podophyllotoxins with other anticancer drugs by various linkers resulted in the construct of many combined agents ( Figure 10). Compounds 321-329 were conjugates consisted of podophyllotoxin and antimetabolite 5-FU using different spacers. Among them, 4β-N-substituted-phenylalanine 5-Fu pentyl ester-4 -demethylepipodophyllo toxin (321-329) was tested to be the most potent cytotoxic activity against HL-60 and A-549 cell, which was stable in plasma [182]. Besides, another series of derivatives (330-339) were synthesized via combining demethyepipodophyllotoxin and 5-FU through a peptide bond derived from natural L-amino acids. These compounds displayed more potent anticancer activity in vitro than etoposide, and showed synergistic effects [183]. A series of thiocolchicine podophyllotoxin derivatives (340-343) connected by the disulfide bond were constructed based on a combinatorial chemistry method. The biological evaluation demonstrated that divalent compounds were not merely the sum of the single compound's activities, thus reflecting a different interaction with the biological target [184]. Inspired by the pharmaceutical results mentioned above, the same research group synthesized hybrids of naturally occurring antimitotic compounds. One of these molecules, the hybrid of vinorelbine and podophyllotoxin (344) linked by succinic anhydride showed good cytotoxicity but with a low efficacy for the inhibition of tubulin assembly, suggesting a different biological target [185]. Furthermore, another group of condensed dimeric compounds 345-349 of thiocolchicine and/or podophyllotoxin with six different dicarboxylic acids were synthesized. Among them, three compounds showed a significant inhibitory activity on the polymerization of tubulin in vitro and causing obvious disruption to the microtubule network in vivo, indicating the spacer unit played an important role on their biological activity [186].
Another example was the hybrid of etoposide and amsacrine, both of which are inhibitors of TOPO-II. The pharmaceutical results indicated that the linkers were highly important for their biological profiles. Compound 351 was more potent than both etoposide and amsacrine according to its DNA cleavage assay, whereas 350 without an ethylene spacer was less potent. Nevertheless, 350 targeted on tubulin polymerization other than its effect on topoisomerase II suggesting the etoposide-amsacrine hybrids might lead to the discovery of dual inhibitors targeting both topoisomerase II and tubulin [187]. An- A series of thiocolchicine podophyllotoxin derivatives (340-343) connected by the disulfide bond were constructed based on a combinatorial chemistry method. The biological evaluation demonstrated that divalent compounds were not merely the sum of the single compound's activities, thus reflecting a different interaction with the biological target [184]. Inspired by the pharmaceutical results mentioned above, the same research group synthesized hybrids of naturally occurring antimitotic compounds. One of these molecules, the hybrid of vinorelbine and podophyllotoxin (344) linked by succinic anhydride showed good cytotoxicity but with a low efficacy for the inhibition of tubulin assembly, suggesting a different biological target [185]. Furthermore, another group of condensed dimeric compounds 345-349 of thiocolchicine and/or podophyllotoxin with six different dicarboxylic acids were synthesized. Among them, three compounds showed a significant inhibitory activity on the polymerization of tubulin in vitro and causing obvious disruption to the microtubule network in vivo, indicating the spacer unit played an important role on their biological activity [186].
Another example was the hybrid of etoposide and amsacrine, both of which are inhibitors of TOPO-II. The pharmaceutical results indicated that the linkers were highly important for their biological profiles. Compound 351 was more potent than both etoposide and amsacrine according to its DNA cleavage assay, whereas 350 without an ethylene spacer was less potent. Nevertheless, 350 targeted on tubulin polymerization other than its effect on topoisomerase II suggesting the etoposide-amsacrine hybrids might lead to the discovery of dual inhibitors targeting both topoisomerase II and tubulin [187]. Another example was the combination of podophyllotoxin and indibulin, which was also a potent microtubulin inhibitor. Further modification of this conjugate led to the synthesis of a series of 4α-O-and 4β-N-indol-3-yl-glyoxyl-substituted derivatives (352-361) of podophyllotoxin [188]. Among them, 354 was tested to be more potent than etoposide. Moreover, YB-1EPN (356) and L1EPO (362) were investigated to have the activities to overcome P-glycoprotein-mediated multidrug resistance in the KBV200 and K562/A02 cell lines, respectively [189,190].
Besides the hybrids of podophyllotoxins with anticancer agents to improve their pharmacological profiles, similar conjugates were also synthesized to optimized their antiviral activities. Conjugates containing stavudine which was a nucleoside reverse inhibitor and podophyllotoxin analogues (363-367) showed increasing bioactivities. Subsequent SAR research showed 7β-amide, cyano group and an opened A-ring or 4 -demethylation are favorable for the anti-HIV activity [191].

Biological Activities of Natural Occurring Podophyllotoxins
Podophyllotoxins were a group of highly bioactive compounds. Historically, podophyllotoxin and its analogues were extracted from plants and directly used as a mixture mainly for external applications [3]. Later, scientists found these compounds could be used in viral infections, such as HPV and HIV diseases [75,192,193]. With the development of pharmacological investigations, the neutrophil activation [194], abnormal vascular vessels destroying [195], radioprotection [196][197][198], antioxidation [199,200], skin pigmentation reduction [33], anti-inflammation, anti-hyperplasia [201] and allergic reaction regulation [202] were extensively studied. Besides, these compounds were found to affect sodium and calcium concentrations in neuron [203]. Besides, podophyllotoxins showed insecticidal activities, for example, podophyllotoxin analogs showed antifeedant activity [204,205]. Similar to the anti-tumor activity, the transfused lactone ring was essential [206].

Antiviral Activities
Natural products were one of the most important sources of antiviral agents and lead compounds [216]. Podophyllotoxin (1) solution and cream could be clinically used in HPV infection patients [10,217], the mechanism involved directly binding a hinge domain E2 in the HPV virus and inhibited the E2/E7 interaction [218]. Some structurally modified podophyllotoxins were found to be effective against HIV.
The pandemic coronavirus disease 2019, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was mainly transmitted via the inhalation system and characterized by fever, cough and difficulty in breathing. Some natural products were found to exhibit useful effects against the COVID-19 [219]. Especially, etoposide, a derivative of podophyllotoxin, could prevent cytokine storm caused by SARS-CoV-2 viral infection [220,221]. Furthermore, molecular docking based on RMSD and RMSF data supported the use of etoposide as an inhibitor of COVID-19 [222].

Anti-Inflammation Activities
Deoxypodophyllotoxin (14) could interfere with many inflammation processes and exhibited potent anti-inflammation activity in pharmacological researches. In inflammation initiating phase, deoxypodophyllotoxin (14) could abolished LPS-induced iNOS expression by inhibiting NF-kappa B [223]; Deoxypodophyllotoxin (14) could decrease the mRNA levels of Th2 cytokines [34]; and it could inhibit TNF-alpha-induced ICAM-1 expression through nuclear factor-kappa B (NF-kappa B) in a dose-dependent manner [224]. It could also inhibit inflammatory cell migration and MMP-2/9 activities, and the MMP-9 transcription [225]. Besides, podophyllotoxins (1) showed antioxidative effect. It would help to clean the reactive oxygen species (ROS), and decrease the inflammation induced tissue damage.

Miscellaneous Activities
Podophyllotoxin was used as medical cream and applied to genital warts and molluscum contagiosum [226]. Podophyllotoxin (1) exposure could affect mouse oocyte maturation by disturbing microtubule dynamics and meiotic spindle formation [3]. Acetylpodophy llotoxin (51) displayed direct antigiardial killing activity on Caco-2 cells [227]. In addition, some podophyllotoxin derivatives exhibited insecticidal activity with final mortality rates of 70%. Especially, a chlorine or bromine atom introduced at the C2 or C2 and C6 positions on the E ring of podophyllotoxin could increase the insecticidal activity [228,229].

Toxicity and Protection
However, podophyllotoxin was well known for its potent cytotoxic properties because of its poor selectivity against tumor cells and narrow therapeutic window. A young woman presented with podophyllin intoxication following topical application of podophyllin resin to genital condylomata acuminata. The disorder was marked by hallucinatory psychosis, bone marrow depression, and mild hepatic dysfunction [230]. A 22 years old man developed a severe sensorimotor neuropathy following ingestion of podophyllin, which had been prescribed for genital condylomata. The initial toxic symptoms were vomiting and diarrhea, followed by peripheral neuropathy. The neuropathy was still present after 18 months [231]. It was noteworthy that most podophyllotoxin intoxication usually results from the accidental ingestion or topical application of podophyllum resin [232]. In addition, etoposide was reported to show immunosuppression, which deserved attention in chemoimmunotherapy [233].
In order to alleviate the toxicity of podophyllotoxin, scientists used some polyphenols e.g., curcumin [234], quercetin [235] and kaempferol [235] to prevent the toxic effect. The protective mechanisms were due to the antioxidant activity of those polyphenols against the oxidative stress induced by podophyllotoxin and the competitive binding of polyphenols against podophyllotoxin in the same colchicines-binding sites.

Total Chemical Synthesis, Biosynthesis and ADME
The total chemical synthesis protocol of podophyllotoxin was introduced in 1996 [236]; however it is time-consuming with low yield. Another efficient and stereoselective strategy for the total synthesis of podophyllotoxin included 12 steps with 29% overall yield [237]. Further investigation showed that this approach can be simplified to an eight step approach with an equal overall yield [238]. The main steps of these syntheses are shown in Figure 11. Later, Ting et al. reported a short total synthesis of podophyllotoxin which could be finished in five steps with 41% overall yield [239]. Xiao et al. reported a nickel-catalyzed approach for the construction of diastereodivergent cores embedded in podophyllum lignans [240]. Besides, an enantioselective total synthesis of (−)-podophyllotoxin was accomplished by organocatalytic Heck cyclization [241]. To date, several elegant strategies have been developed for the synthesis of podophyllotoxin; however, more concise with high yield total synthesis had so far remained an unmet challenge. lotoxin was accomplished by organocatalytic Heck cyclization [241]. To date, several elegant strategies have been developed for the synthesis of podophyllotoxin; however, more concise with high yield total synthesis had so far remained an unmet challenge. Figure 11. Total chemical synthesis of podophyllotoxin.
In order to produce more podophyllotoxins, many experiments focused on biosynthesis of podophyllotoxins in cultures of plant cell lines [242][243][244][245] and endophytic fungus [246]. The biosynthesis of podophyllotoxin was considered to be an attractive alternative because of the much simpler and greener steps and relatively higher yield. The current biosynthesis pathway of podophyllotoxins in plants involved the process of L-phenylalanine/L-tyrosin→coniferyl alcohol→pinoresinol→(-)-secoisolariciresinol→(-)-matairesinol → (-)-pluviatolide → podophyllotoxin → glycosylation modification of podophyllotoxin [247]. Furthermore, chemoenzymatic synthesis had led to the asymmetric configuration of podophyllotoxin. For example, milligram-level synthesis of (−)-deoxypodophyllotoxin has been achieved in tobacco. At the same time, part of the biosynthetic pathway of podophyllotoxin had been expressed in Escherichia coli and Saccharomyces cerevisiae, and different podophyllotoxin intermediates have been obtained. However, limitation still existed. For example, enzymes were characterized by their high selectivity, and thus, the substrates were limited, and not all desired podophyllotoxin-type products can be produced using this method.
In addition, microbial transformation of natural products is an important approach to synthesize derivatives with improved pharmacological properties. Many podophyllotoxin derivative with higher activity and water-solubility were produced via biotransformation by microorganisms, such as Penicillium purpurogenum [248], Pseudomonas aeruginosa [249], Cunninghamella echinulata [250] and Bacillus fusiformis [251]. Microbial transformations can not only obtain new derivatives, but also provide a natural enzyme library with various catalytic types, which has gradually become a choice for biosynthesis because of the high stereoselectivity and regioselectivity, mild reaction conditions and simple operation steps. In order to produce more podophyllotoxins, many experiments focused on biosynthesis of podophyllotoxins in cultures of plant cell lines [242][243][244][245] and endophytic fungus [246]. The biosynthesis of podophyllotoxin was considered to be an attractive alternative because of the much simpler and greener steps and relatively higher yield. The current biosynthesis pathway of podophyllotoxins in plants involved the process of L-phenylalanine/L-tyrosin→coniferyl alcohol→pinoresinol→(-)-secoisolariciresinol→(-)-matairesinol→(-)-pluviatolide→podophyllotoxin→glycosylation modification of podophyllotoxin [247]. Furthermore, chemoenzymatic synthesis had led to the asymmetric configuration of podophyllotoxin. For example, milligram-level synthesis of (−)-deoxypodophyllotoxin has been achieved in tobacco. At the same time, part of the biosynthetic pathway of podophyllotoxin had been expressed in Escherichia coli and Saccharomyces cerevisiae, and different podophyllotoxin intermediates have been obtained. However, limitation still existed. For example, enzymes were characterized by their high selectivity, and thus, the substrates were limited, and not all desired podophyllotoxin-type products can be produced using this method.
In addition, microbial transformation of natural products is an important approach to synthesize derivatives with improved pharmacological properties. Many podophyllotoxin derivative with higher activity and water-solubility were produced via biotransformation by microorganisms, such as Penicillium purpurogenum [248], Pseudomonas aeruginosa [249], Cunninghamella echinulata [250] and Bacillus fusiformis [251]. Microbial transformations can not only obtain new derivatives, but also provide a natural enzyme library with various catalytic types, which has gradually become a choice for biosynthesis because of the high stereoselectivity and regioselectivity, mild reaction conditions and simple operation steps.
The ADME processes of podophyllotoxins in animals were not clearly evaluated, especially for some new derivates. Experiments using enzyme to predict the metabolic pathway were performed. The results showed that CYP3A4, the main human metabolizing enzyme, had the ability to transform deoxypodophyllotoxin into epipodophyllotoxin [252,253]; while CYP1A2 and CYP2C9 could not accomplish this biotransformation. Furthermorde, etoposie and related semi-synthesized podophyllotoxins could be degraded (3-O-demethylation) [254].

Conclusions and Remarks
As described above, podophyllotoxins are widely distributed in nature. Slight structurally modified podophyllotoxins showed different bioactivities from the parent compounds. Their cytotoxicity, safety, pharmacological activity against MDR cell line or selectivity against certain cancer cell lines varied with the structural changes. Limitations on the podophyllotoxin studies existed in several aspects.
Firstly, the mechanism of some compounds was still unknown. So, their targets and pharmacophore of these molecules were still uncertain. Meanwhile, this information was vital to understand the mechanism and further development of these compounds. Secondly, previously established SAR was facing challenge. Some compounds without defined essential motif still showed remarkable cytotoxicity. This could be the result of modification changing the 3-D structures of these molecules. So, previously established SAR seemed to be no longer as comprehensive as before, especially when it was used to predict the ester or ether of these compounds. Thirdly, for many compounds, cytotoxity as the unique activity of this kind of compounds was only tested in limited cell lines, so the cytotoxity cannot be predicted on other cancer cells. Fourthly, for most compounds, the ADME parameters and in vivo activity were not studied. However, cancer was a very complex malignant disease. Different kind of cells were anchored in cancer tissues. So, inhibiting the quick dividing cancer cells did not mean to the cure of this disease [255][256][257][258][259][260]. Finally, the microenvironment [261][262][263] also played very important role in the generation, development and metastasis of a tumor; however, microenvironment was less studied in podophyllotoxins. In order to find a potent drug with high therapeutics, more experiments should be conducted.
In summary, podophyllotoxins were very promising compounds because of their unique chemical structures and diverse bioactivities. Structure modifications make them more suitable for clinical use. A slight change in these chemical structures lead to a remarkable change in their activity. So, the establishment of a comprehensive SARs, which was more suitable for the natural and modified podophyllotoxins, was necessary.