Arylnaphthalene lactones: structures and pharmacological potentials

Natural arylnaphthalene lactones are representative lignans that are found in various dietary and medicinal plants. Their unique structural features and significant pharmacological activity have attracted considerable attention from both synthetic and medicinal chemists. Owing to their unique structural features such as relative rigid tetracyclic skeleton, structural diversity of more than five substituents, and no chiral center, arylnaphthalene lactones are recognized as a valuable scaffold for drug discovery, in addition to their significant pharmacological activities. This review covers the structures and isolation of all naturally occurring arylnaphthalene lactone congeners reported. Based on the aryl substituents, they were categorized as Type I and Type II and further classified according to the oxidation state of the ring and glycosylation level. Special attention has been paid to natural arylnaphthalene lactones owing to their broad spectrum of biological activities such as cytotoxic, antiplatelet, antiviral, anti-HIV, antifungal, neuroprotective, and anti-inflammatory properties. All the products were reorganized based on their biological activities, and selected data are presented.


Introduction
Arylnaphthalene lignan lactones are naturally occurring fused tricyclic naphthalene lactones with aryl substituents. Structurally, arylnaphthalene lignan lactones consist of two arylpropanoid units and are classified as Type I and Type II ( Fig. 1) based on the relative position of lactone and the aryl substituents (Teponno et al. 2016). Approximately 59 natural arylnaphthalene lignan lactones and their glycosylated congeners have been isolated from various dietary and medicinal plants and structurally elucidated. The broad spectrum of their pharmacological benefits has also been reported such as antiproliferative, antiplatelet aggregation, antiviral, antifungal, neuroprotective, and anti-inflammatory activities.
The unique structural features as well as promising bioactivities of arylnaphthalene lactones have drawn considerable attention from synthetic chemists. Since the first synthesis of an arylnaphthalene lignan lactone skeleton in 1895 by the Bucher group (Michael and Bucher 1895) via the condensation of arylpropiolic acids, various synthetic approaches for arylnaphthalene lignan lactones have been designed and applied successfully. Major synthetic approaches include the intramolecular Diels-Alder reaction for the construction of an arylnaphthalene lactone from arylpropiolic anhydride Stevenson 1964, 1965;Maclean and Stevenson 1966;Block and Stevenson 1971;Stevenson 1970, 1971;Stevenson and Holmes 1971;Stevenson and Block 1971;Block and Stevenson 1973;Weber 1989, 1991;Anastas and Stevenson 1991;Park et al. 2014). Intermolecular Diels-Alder approaches were also investigated using isobenzofurans and acetylenedicarboxylate (de Silva et al. 1980;Plaumann et al. 1980). Other valuable synthetic methodologies utilizing key reactions such as the Blaise reaction-intramolecular [4 ? 2] reaction (He et al. 2012), Garratt-Braverman cyclization Stevenson, 1971, 1973;Arnold et al. 1973;Yamamoto et al. 2015), benzoin condensation-thermal cyclization (Hayat et al. 2015a, b), and transitionmetal catalyzed synthesis (Park et al. 2020) have been reported.
Although the isolation and chemistry of natural lignan products has been broadly reviewed (Teponno et al. 2016;Li et al. 2020), a focused and comprehensive review on the structures and beneficial biological activities of natural arylnaphthalene lignan lactones has not been published. The purpose of this review is to provide a compilation of naturally occurring arylnaphthalene lignan lactones in terms of structure, isolation, and pharmacological activity.

Cytotoxic activities
The reported antiproliferative activities of natural arylnaphthalene lactones are presented in Fig. 4. Significant cytotoxic activity was observed with justicidin A (26) and tuberculatin (41) against human hepatoma cellular carcinoma (Hep3B and HepG2), human breast cancer (MCF-7 and MCF-7-ras), human cervical carcinoma (SiHa), and other cancer cell lines. In addition, these two compounds strongly enhanced tumor-necrosis factor a (TNF-a) generation in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells (Day et al. 2002). Later, 6 0 -hydroxyjusticidin A (27), which was isolated from Justicia procumbens, was evaluated for its cytotoxicity against human cancer cell lines. It showed remarkable inhibitory activity in human bladder cancer cells (EJ) with 50% inhibitory concentration (IC 50 ) values of 57.1 lM and enhanced the generation of reactive oxygen species and induced apoptosis through the caspase pathway (He et al. 2012). Similar results of the mechanism of action were reported by Luo and Hu et al. in 2014. They isolated five lignans, 6 0 -hydroxyjusticidin A (27), justicidin H (3), justicidin B (1), chinensinaphthol methyl ether (30), and taiwanin E methyl ether (32) from J. procumbens and tested their cytotoxic activities. Justicidin H (3) exhibited the best inhibitory activity against human promyelocytic leukemia (HL-60) and mouse lymphocytic leukemia (L1210 and P3881D1) cells with an IC 50 ranging from 3.9 to 26.2 lM (Luo et al. 2014). To investigate the mechanism of action of justicidin H (3), these authors also evaluated its effects on human leukemia K562 cells. The IC 50 of justicidin H (3) was 15.07 lM for K562 cells and reduced mitochondria membrane potential (deltapsi(m)). It also increased the expression of TRPC6 related to regulating calcium homeostasis in cell signaling and induced apoptosis through the caspase pathway (Luo et al. 2018).
Diphyllin (18) was tested to investigate whether it could act as a vacuolar-ATPase (V-ATPase) inhibitor against human gastric cancer cells (SGC7901) and esophageal cancer cells (TE-1 and ECA-109). The IC 50 for SGC7901 was demonstrated to be 7.8 lM. Diphyllin (18) also inhibited the expression of V-ATPases in a dose-dependent manner. In addition, the transmembrane pH gradient was reversed, thereby causing tumor microenvironment acidification (Shen et al. 2011). It also showed significant inhibition against TE-1 and ECA-109 cells with IC 50 values of 0.3 and 0.2 lM, respectively, with S-phase arrest and reduced V-ATPase activity. Reportedly, diphyllin inhibited mammalian target of rapamycin complex 1 (mTORC1), hypoxia-inducible factor-1a (HIF-1a), and vascular endothelial growth factor (VEGF) mRNA expression (Chen et al. 2018). Three diphyllin glycosides cleistanthin A (45), cleistanthoside A (86), and cleistanthoside A tetraacetate were also evaluated for their effect as V-ATPases and their cytotoxicity against human cell lines. Apart from cleistanthoside A (86), cleistanthin A (45) and cleistanthoside A tetraacetate were more potent than paclitaxel against HepG2 cells with the IC 50 values of 36 and 39 nM, respectively. They also inhibited V-ATPase activity, which is critical to tumor invasion and metastasis development. At nanomolar concentrations, they neutralize the pH of lysosomes .
A bioassay-guided fractionation of the stems and roots of Phyllanthus oligospermus resulted in the Fig. 4 Arylnaphthalene lactones with cytotoxic activity against different cell lines. Hep3B, human cervical carcinoma; HepG2, human hepatoma cell; MCF-7, human breast cancer cell; MCF-7 ras, Ha-ras oncogene transformed from MCF-7; EJ, human bladder cell; K562, human leukemia cell; SGC7901, human gastric cancer cell; TE-1 and ECA-109, human esophageal cancer cells; SK-OV-3, human ovarian carcinoma; SK-MEL-5, melanoma; PLC/PRF/5, human hepatoma; HT-3, SiHa, and CaSki, human cervical carcinoma isolation of three arylnaphthalene lignan lactones, phyllanthusmin A-C (31, 49, 50). The most active compound was phyllanthusmin A (31) showing a marked cytotoxic effect against mouse leukemia (P-388) and human epidermoid carcinoma (KB) cells with IC 50 values of 0.13 and 2.24 lg/mL, respectively . Phyllanthusmin A-E (31, 49-52), diphyllin (18), and cleistanthin B (47) were also evaluated for cytotoxicity against colon cancer cells (HT-29). Phyllanthusmin D (51) was the most potent with IC 50 values at 170 nM; however, cleistanthin B (47) and phyllanthusmin A (31) were inactive. These results suggest that the presence of more lipophilic acetyl groups results in higher cytotoxicity. In this connection, mechanistic studies of phyllanthusmin D (51) were also evaluated. It was found that unlike etoposide, phyllanthusmin D (51) did not mediate its cytotoxic effects by inhibiting DNA topoisomerase IIa but did so by inducing HT-29 apoptosis through caspase-3 activation (Ren et al. 2014). However, daurinol (4) acts as a catalytic human topoisomerase IIa inhibitor and demonstrated significant cytotoxic activity against human colorectal cancer cells (HCT116) with an IC 50 of 2.03 lM. It induced S-phase arrest through the increased expression of cyclin E and A (Kang et al. 2011). In a further investigation, Woo et al. (2017) evaluated daurinol (4) for anti-metastatic activity against human breast cancer cells (MDA-MB-231) and human lung cancer cells (A549). Daurinol (3) decreased the expression of focal adhesion kinase, which is hyper-activated and overexpressed in most solid tumors, but did not block the AKT pathway in both cell lines. Using a trans-well assay, daurinol (3) was found to inhibit migration and invasion (Woo et al. 2017).

Antiviral activities
A series of lignans isolated from J. procumbens were tested for activities against the vesicular stomatitis virus. Justicidin A-B (26, 1), diphyllin (18), diphyllin apioside (41), and diphyllin apioside-5-acetate exhibited strong antiviral activities. Their minimum inhibitory concentration (MIC) values were less than 0.25 lg/mL whereas 6 0 -glucosides justicidinoside A-C (106, 35, 9) and Type II justicidin C and D (103, 105) exhibited lower antiviral activity (the MICs ranged from 16 to 125 lg/mL). It is tempting to suggest that the weak activity of justicidinoside A-C (106, 35, 9) is because of the steric bulk of their sugar moiety and that Type I arylnaphthalene lactones were more effective than Type II (Luo et al. 2018). Using the standard plaque reduction assay against human cytomegalovirus, only taiwanin C (2) and retrojusticidin B (91) showed clear antiviral activity with half maximal effective concentration (EC 50 ) values at 1.2 and 7.2 lM, respectively (Chen et al. 1996). Similar antiviral activities were reported for helioxanthin (98) in HepG2.2.15 cells using Southern blot hybridization with the EC 50 value at 1 lM. Helioxanthin (98) reduced 3.5 kb of hepatitis B virus mRNA in a dosedependent manner with EC 50 values at 0.09 lM ). In addition, helioxanthin (98) also exhibited strong antiviral activities against hepatitis C virus and herpes simplex virus type 1 with EC 50 values at 3 and 2 lM, respectively, but showed weak activity against herpes simplex virus type 2 and Epstein-Barr virus with EC 50 values at 35 and above 20 lM, respectively ) (Table1).

Anti-HIV activities
Anti-HIV bioassays with six lignans, phyllamyricin A (93), phyllamyricin B, phyllamyricin C (12), retrojusticidin B (91), justicidin A (26), and justicidin B (1) isolated from Phyllanthus myrtifolius were first   Chang et al. (1995) using human immunodeficiency virus-1 reverse transcriptase assay (HIV-RT). Among these, phyllamyricin B and retrojusticidin B (91) were shown to contribute to the selective inhibitory effect against HIV-RT with IC 50 values at 3.5 and 5.5 lM, respectively, whereas they exhibited much lower activity against human DNA polymerase-a (hDNAP-a) with IC 50 values at 289 and 989 lM (Chang et al. 1995) (Table 2). In a subsequent study in 1996, Lee et al. identified additional lignans from P. myrtifolius and evaluated their anti-HIV activities. Phyllamyricin B and C were inactive and phyllamyricin E (14) exhibited very low anti-HIV-RT activity; however, phyllamyricin A (93) showed an increase in HIV-RT activity by 65% at 1.89 lM (Lee et al. 1996). From the stems and barks of Justicia gendarussa justiprocumin A and B (65, 66) were isolated and justiprocumin B (66) was assayed for its anti-HIV activity against four HIV-1 isolates using a standardized human peripheral blood mononuclear cell culture assay. The HIV-1 isolates BAL, SF162, LAV0.04, and 89.6 were used. Justiprocumin B exhibited IC 50 values at 15, 15, 14, and 21 nM, respectively, whereas the clinically used drug for HIV-1 zidovudine (AZT) showed less activity with the IC 50 value ranging from 77 to 95 nM (Zhang et al. 2017).

Antifungal activities
Antifungal activities of arylnaphthalene lactones are summarized in Table 3. In 2003, the Gertsch group validated antifungal properties of water, dichloromethane, and MeOH extracts of Phyllanthus piscatorum. While the extracts did not exhibit an inhibitory effect against gram-positive bacterial strains of Pseudomonas aeruginosa, Bacillus cereus, Staphylococcus aureus, and Staphylococcus epidermis, they showed significant activity against Aspergillus fumigatus, Aspergillus flavus, and Candida albicans (Gertsch et al. 2004). In a subsequent study, the dichloromethane extract of P. piscatorum resulted in the activity of arylnaphthalene lactone justicidin B (1) and piscatorin (10) when tested against A. flavus, A. fumigatus, and C. albicans. The most active compound was justicidin B (1) with MIC values ranging from 1 to 16 lg/mL; however, showing a higher concentration of 128 lg/mL against Blastoschizomyces capitatus and Cryptococcus neoformans neoformans (Gertsch et al. 2003). Bioassayguided fractionation of the leaf extract of P. myrtifolius led to the isolation of seven lignans, namely, phyllamyricin C (12), retrojusticidin B (91), phyllamyricin A (93), phyllamyricin F, justicidin B (1),   (14), and piscatorin (10). Their activities were validated using the susceptibility test and conidial germination inhibition assay. Phyllamyricin A (93), phyllamyricin E (14), justicidin B (1), and phyllamyricin F exhibited strong inhibition against Fusarium oxysporum ATCC 44,187 with an average inhibition zone of 62-68% (1000 lg/mL). In addition, phyllamyricin C (12) showed the most significant antifungal activity with MIC and minimum fungicidal concentration values of 4.0 and 62.5 lg/ mL, respectively, and the seven lignans inhibited conidia germination of F. oxysporum in a concentration-dependent manner (Windayani et al. 2014).

Neuroprotective activities
Justicidin A (26) was investigated for neuroprotective activities in a cellular model of Alzheimer's disease induced by amyloid beta (Ab) 25-35 in SH-SY5Y cells. Ab 25-35 -induced hyperphosphorylation of tau and okadaic acid-induced hyperphosphorylation were significantly inhibited by pre-treatment with justicidin A at 62.5, 125, and 250 nM in a dose-dependent manner. At the same concentration, justicidin A produced a significant level of decrease in the phosphorylation of glycogen synthase kinase-3beta (GSK-3b) and stimulated the phosphorylation of AMP-activated protein kinase (AMPK). In addition, treatment with justicidin A, resulted in an increase in the level of the LC3 II/I ratio. These results show that justicidin A induced autophagy and inhibited neuronal cell death through reducing hyperphosphorylation of tau (Gu et al. 2016).
Anti-inflammatory activities Prieto et al. (1996) reported for the first time the antiinflammatory activity of an MeOH extract of Haplophyllum hispanicum. The edema of carrageenaninduced paw and TPA-induced ear in mice showed 50% and 37% inhibition at 0.5 mg/ear. Following the guided bioassay, the active compound diphyllin acetylapioside (43) was isolated and showed a significant inhibitory effect against TPA-induced inflammation in mice with a 50% inhibitory dose (ID 50 ) value at 0.27 lMol/ear (Prieto et al. 1996). In a further investigation, the same authors validated the antiinflammatory effects on eicosanoid metabolism using an HPLC-DAD-based method. Diphyllin acetylapioside showed complete inhibition of 5-lipoxygenase activity at 50 lM and exhibited strong inhibitory effects against LTB 4 and 5-hydroxy-6,8,11,14eicosateraenoic acid with IC 50 values of 0.6 and 0.7 lM, respectively; however, diphyllin apioside (41) did not exhibit any effect on 5-lipoxygenase (Prieto et al. 2002). Five lignans isolated from the root of Acanthopanax chiisanenesis were examined for their effect on the production of TPA-induced PGE 2 in rat peritoneal macrophages to elucidate their mechanism of action. Taiwanin C (2) exhibited the most significant inhibitory effect with an IC 50 value at 0.12 lM but showed no effect on the expression of TPA-induced COX-2 protein. However, with IC 50 values at 1.06 and 9.3 1 lM, taiwanin C inhibited the activities of separated COX-1 and COX-2. These results suggest that taiwanin C (2) inhibits PGE 2 production by directly inhibiting COX enzymatic activity (Ban et al. 2002). Three arylnaphthalide lignans from Phyllanthus polyphyllus displayed antiinflammatory effects as measured by NO, TNF-a, and interleukin (IL-12). Justicidin B (1) exhibited the highest IC 50 values of NO production from LPS/IFNc-stimulated peritoneal macrophages at 12.5 lM followed by phyllamyricin C (12) at 25 lM, and dipyllin (18) at 50 lM and 100 lM showing inhibition percentages of 99%, 99%, and 64%, respectively. In addition, they showed significant inhibition of IL-12 and TNF-a production with IC 50 values ranging from 12.5 to 100 lM (Rao et al. 2006).

Conclusion
Natural arylnaphthalene lactones have a 7 0 -phenyl naphthalene lactone skeleton in which the phenyl ring and naphthalene ring are polyhydroxylated, which are further transformed to methyl ethers or dioxolane. The hydroxy group, especially that at the C7 position is commonly conjugated with a variety of sugars to present mono-, di-, and triglycoside metabolites. Structurally, they can be classified into Type I and Type II arylnaphthalene lactones by the cis and trans relationship of lactone carbonyl and the aryl substituents. More than a hundred natural arylnaphthalene lactones have been reported from a wide range of natural sources such as Acanthaceae, Phyllanthaceae, and Schisandraceae. Arylnaphthalene lactones exhibit various significant biological activities, which have been summarized here based on their pharmacological activity. Although all the natural compounds were not fully evaluated, some results such as antiproliferative and antiviral activity could give insights for drug discovery. In fact, several arylnaphthalene lactones such as diphyllin and daurinol have been investigated as anticancer drug candidates with impressive in vitro and in vivo antiproliferative activity. More recently, daurinol was investigated extensively as an antiautoimmune arthritis drug candidate. In the realm of medicinal chemistry, identifying new and valuable scaffolds is always of great interest. Thus, arylnaphthalene lactones attract considerable attention owing to their unique structural features, which include a relative rigid structure, no stereogenic center, and more than nine potential derivatizable sites. The unique structural features and promising pharmacological activities of arylnaphthalene lactones provide great prospects for future drug discovery.
Author contributions DS planned this manuscript. SP and SK searched the reported publications related to this review article. DS and SP wrote the draft of the manuscript, and SP prepared all figures. All authors approved the manuscript in its final form for publication.
Funding This work was supported by grants from the Science Research Program through the National Research Foundation of Korea (NRF), Grant No. NRF-2018M3A9C8024360 and NRF-2020R1A6A1A03043708.

Compliance with ethical standards
Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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