Alkylide derivatives of diphyllin: synthesis and preliminary anticancer evaluation.

Fourteen diphyllin 4-C-substituted alkylide derivatives were designed and synthesized using a Heck coupling and subsequent hydrogenation reaction. Olefins 3g and 3i exhibited the highest cytotoxicity on breast cancer cell lines MCF-7 with IC50 values of 0.08 and 0.07 µM, and they showed weaker V-ATPase inhibitory potency compared to diphyllin.

Bioactivities and Mechanisms of Action of Diphyllin and Its Derivatives: A Comprehensive Systematic Review.

Natural products are treasure houses for modern drug discovery. Diphyllin is a natural arylnaphthalene lignan lactone isolated from the leaf of Astilboides tabularis. Studies have found that it possesses plenty of bioactivity characteristics. In this paper, we reviewed the structure, bioactivity, and mechanism of action of diphyllin and its derivatives. The references were obtained from PubMed, Web of Science, and Science Direct databases up to August 2023. Papers without a bio-evaluation were excluded. Diphyllin and its derivatives have demonstrated V-ATPase inhibition, anti-tumor, anti-virus, anti-biofilm, anti-inflammatory, and anti-oxidant activities. The most studied activities of diphyllin and its derivatives are V-ATPase inhibition, anti-tumor activities, and anti-virus activities. Furthermore, V-ATPase inhibition activity is the mechanism of many bioactivities, including anti-tumor, anti-virus, and anti-inflammatory activities. We also found that the galactosylated modification of diphyllin is a common phenomenon in plants, and therefore, galactosylated modification is applied by researchers in the laboratory to obtain more excellent diphyllin derivatives. This review will provide useful information for the development of diphyllin-based anti-tumor and anti-virus compounds.

Antibacterial and antibiofilm activities of diphyllin against fish pathogens.

Annihilation of biofilm forming bacterial pathogens is a challenging aspect in seafood and aquaculture industries. Microbes growing as biofilms cause deleterious effects on food products leading to food spoilage or loss of shelf life. As a measure to fight biofilms, agents that prevent/disrupt biofilms are recurrently screened. The study exemplifies the bactericidal and biofilm disruption potentials of a plant derived compound, diphyllin, against fish pathogens that colonizes Oreochromis mossambicus and Oreochromis niloticus. Precisely, diphyllin disrupted Salmonella typhi biofilms by triggering reactive oxidative species (ROS). Diphyllin-induced ROS had satisfactory correlation with S. typhi cell membrane damage and intracellular DNA degradation profiles providing a putative mechanistic model. In conclusion, the study identifies diphyllin as a therapeutic and dispersal agent aimed at biofilms formed by food-borne pathogens that persistently plague food processing and aquaculture settings.

Evaluation of potency and metabolic stability of diphyllin-derived Vacuolar-ATPase inhibitors.

Diphyllin is a naturally occurring lignan comprised of an aryl naphthalene lactone scaffold that demonstrates beneficial biological activities in disease models of cancer, obesity, and viral infection. A target of diphyllin and naturally occurring derivatives is the vacuolar ATPase (V-ATPase) complex. Although diphyllin-related natural products are active with in vitro models for viral entry, the potencies and unknown pharmacokinetic properties limit well-designed in vivo evaluations. Previous studies demonstrated that diphyllin derivatives have the utility of blocking the Ebola virus cell entry pathway. However, diphyllin shows limited potency and poor oral bioavailability in mice. An avenue to improve the potency was used in a new library of synthetic derivatives of diphyllin. Diphyllin derivatives exploiting ether linkages at the 4-position with one-to-three carbon spacers to an oxygen or nitrogen atom provided compounds with EC50 values ranging from 7 to 600 nM potency and selectivity up to >500 against Ebola virus in infection assays. These relative potencies are reflected in the Ebola virus infection of primary macrophages, a cell type involved in early pathogenesis. A target engagement study reveals that reducing the ATPV0a2 protein expression enhanced the potency of diphyllin derivatives to block EBOV entry, consistent with effects on the endosomal V-ATPase function. Despite the substantial enhancement of antiviral potencies, limitations were identified, including rapid clearance predicted by in vitro microsome stability assays. However, compounds with similar or improved half-lives relative to diphyllin demonstrated improved pharmacokinetic profiles in vivo. Importantly, these derivatives displayed suitable plasma levels using oral administration, establishing the feasibility of in vivo antiviral testing.

Antiviral, Anticancer and Hypotensive Potential of Diphyllin Glycosides and their Mechanisms of Action.

Diphyllin glycosides (DG) are a type of arylnaphthalene lignans isolated from different plants, and their synthetic derivatives have shown effective antiviral, cytotoxic, hypotensive and diuretic effects at very low concentrations similar to standard drugs that are under clinical use. The biological activities of the DG interfere with signaling pathways of viral infection and cancer induction. The sugar moieties of DG enhance bioavailability and pharmacological activities. The promising results of DG at nanomolar concentrations under in vitro and in vivo conditions should be explored further with clinical trials to determine its toxic effects, pharmacokinetics and pharmacodynamics. This may help identify suitable antiviral and anticancer drugs in the near future. Considering all these activities, the present review is focused on the chemical aspects of DG with a detailed account of the mechanisms of action of DG. An attempt is also made to comment on the status of clinical trials involving DG along with the possible limitations in studies based on available literature till September 2020.

Evaluation of Phenol-Substituted Diphyllin Derivatives as Selective Antagonists for Ebola Virus Entry.

Ebola virus (EBOV) is an aggressive filoviral pathogen that can induce severe hemorrhagic fever in humans with up to 90% fatality rate. To date, there are no clinically effective small-molecule drugs for postexposure therapies to treat filoviral infections. EBOV cellular entry and infection involve uptake via macropinocytosis, navigation through the endocytic pathway, and pH-dependent escape into the cytoplasm. We report the inhibition of EBOV cell entry via selective inhibition of vacuolar (V)-ATPase by a new series of phenol-substituted derivatives of the natural product scaffold diphyllin. In cells challenged with Ebola virus, the diphyllin derivatives inhibit viral entry dependent upon structural variations to low nanomolar potencies. Mechanistically, the diphyllin derivatives had no effect on uptake and colocalization of viral particles with endocytic marker LAMP1 but directly modulated endosomal pH. The most potent effects were reversible exhibiting higher selectivity than bafilomycin or the parent diphyllin. Unlike general lysosomotrophic agents, the diphyllin derivatives showed no major disruptions of endocytic populations or morphology when examined with Rab5 and LAMP1 markers. The dilated vacuole phenotype induced by apilimod treatment or in constitutively active Rab5 mutant Q79L-expressing cells was both blocked and reversed by the diphyllin derivatives. The results are consistent with the action of the diphyllin scaffold as a selective pH-dependent viral entry block in late endosomes. Overall, the compounds show improved selectivity and minimal cytotoxicity relative to classical endosomal acidification blocking agents.

Synthesis and anti-tumor activity of nitrogen-containing derivatives of the natural product diphyllin.

The natural product diphyllin has demonstrated great potential in the treatment of various human cancers, especially pancreatic cancer. However, its relative weak potency, low aqueous solubility, and poor metabolic stability limits its development ability. In this study, we designed and synthesized two series of novel nitrogen-containing diphyllin derivatives with the aim to improve both antitumor efficacy and drug-like properties. Among them, the amino derivative 15 showed an IC50 value of 3 nM against pancreatic cancer CFPAC-1 cells and is about 69-fold more potent than diphyllin. In addition, compound 15 possesses improved aqueous solubility and metabolic stability in liver microsomes. This compound not only significantly induced cell cycle arrest at G0/G1 phase with down-regulation of CDK4 and cyclinD1 in a dose-dependent manner, but also blocked the later stage of autophagy in CFPAC-1 cells. In pancreatic cancer xenograft model, treatment of 15 with 10 mg/kg exhibited much more potent efficacy in suppressing the growth of transplanted PANC02 tumors than diphyllin without obvious safety concern.

2, 4, 5-Trideoxyhexopyranosides derivatives of diphyllin: Synthesis and anticancer activity.

Diphyllin and its natural derivatives were identified as potent vacuolar H+ -ATPase (V-ATPase) inhibitors. In this study, twelve 2, 4, 5-trideoxyhexopyranosides derivatives of diphyllin were synthesized. Most of these compounds showed potent abilities to inhibit the growth of HT-29, MCF-7, HepG2 cancer cells with IC50 values at submicromolar concentration. The compounds 5c3 and 5c4 showed the best inhibitory activity on breast cancer cell lines MCF-7 with IC50 values of 0.09 and 0.10 µM. Compounds 5c3 and 5c4 showed similar V-ATPase inhibitory potency to diphyllin. Molecular docking showed that a hydrogen bond was found between the hydroxyl of 5c3 and SerA534 in the pocket of the V-ATPase receptor.

Diphyllin Shows a Broad-Spectrum Antiviral Activity against Multiple Medically Important Enveloped RNA and DNA Viruses.

Diphyllin is a natural arylnaphtalide lignan extracted from tropical plants of particular importance in traditional Chinese medicine. This compound has been described as a potent inhibitor of vacuolar (H+)ATPases and hence of the endosomal acidification process that is required by numerous enveloped viruses to trigger their respective viral infection cascades after entering host cells by receptor-mediated endocytosis. Accordingly, we report here a revised, updated, and improved synthesis of diphyllin, and demonstrate its antiviral activities against a panel of enveloped viruses from Flaviviridae, Phenuiviridae, Rhabdoviridae, and Herpesviridae families. Diphyllin is not cytotoxic for Vero and BHK-21 cells up to 100 µM and exerts a sub-micromolar or low-micromolar antiviral activity against tick-borne encephalitis virus, West Nile virus, Zika virus, Rift Valley fever virus, rabies virus, and herpes-simplex virus type 1. Our study shows that diphyllin is a broad-spectrum host cell-targeting antiviral agent that blocks the replication of multiple phylogenetically unrelated enveloped RNA and DNA viruses. In support of this, we also demonstrate that diphyllin is more than just a vacuolar (H+)ATPase inhibitor but may employ other antiviral mechanisms of action to inhibit the replication cycles of those viruses that do not enter host cells by endocytosis followed by low pH-dependent membrane fusion.

Synthesis, Cytotoxicity, Anti-Migration and Anti-Invasion Activity of Diphyllin Heterocyclic Derivatives.

BACKGROUND: Diphyllin, an arylnaphthalene lignan lactone, isolated from many traditional medicinal plants, has been reported to possess anticancer and antiviral activities. Natural diphyllin and its glycosides were identified as potent vacuolar H+-ATPase (V-ATPase) inhibitors. OBJECTIVE: The aim of this study was to design and synthesize a series of heterocyclic derivatives of diphyllin as novel anticancer agents. METHODS: The targeted heterocyclic derivatives of diphyllin were synthesized from diphyllin employing etherification reaction and N-substitution reaction. Cytotoxicity of these compounds on four cancer cells was assessed by MTT assay. The inhibitory activity of V-ATPase of compound 3n was measured on MGC-803 cells. Anti-migration and anti-invasion abilities were assessed by transwell invasion assay and scratch wound assay. RESULTS: Most of these derivatives displayed potent cytotoxicity on four cancer cells at submicromolar concentrations. The most potent derivative 3n has been shown to inhibit V-ATPase activity, migration and invasion abilities on MGC-803 cells at 0.75 µM. CONCLUSION: The collective results clearly indicate that heterocyclic derivatives of diphyllin inhibit the viability, V-ATPase activity, migration and invasion of the MGC803 cells. The current findings provide valuable insights for the future development of novel diphyllin derivatives as anticancer agents.

Synthesis and evaluation of diphyllin ß-hydroxyl amino derivatives as novel V-ATPase inhibitors.

Natural diphyllin glycosides were identified as potent vacuolar H+ -ATPase (V-ATPase) inhibitors. A series of diphyllin ß-hydroxyl amino derivatives were designed and synthesized as novel diphyllin derivatives. Most of these derivatives displayed potent cytotoxicity against six cancer cell lines with IC50 values in the submicromolar to nanomolar concentration range. Compounds 2b, 2c, 2l, 2m, and 2n showed similar V-ATPase inhibitory potency to Bafilomycin A1. Compound 2l exhibited potent activity of modulation of lysosomal pH and cytoplasmic pH.

Antiviral Activity of Vacuolar ATPase Blocker Diphyllin against SARS-CoV-2.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a causative agent of the pandemic coronavirus disease 2019 (COVID-19), which has resulted in over two million deaths worldwide to date. Diphyllin and diphyllinosides are known as natural blockers of cellular vacuolar ATPases, and so can act as inhibitors of the pH-dependent fusion of viral envelopes with host cell endosomal membranes. Such pH-dependent fusion is a critical early step during the SARS-CoV-2 replication cycle. Accordingly, the anti-SARS-CoV-2 profiles and cytotoxicities of diphyllin, diphyllinoside cleistanthin B, and two structurally related compounds, helioxanthin 8-1 and helioxanthin 5-4-2, are evaluated here using in vitro cell-based assay systems. Neither helioxanthin exhibits any obvious anti-SARS-CoV-2 effects in vitro. By contrast diphyllin and cleistanthin B do exhibit anti-SARS-CoV-2 effects in Vero cells, with respective 50% effective concentrations (EC50) values of 1.92 and 6.51 µM. Diphyllin displays anti-SARS-CoV-2 effect also in colorectal adenocarcinoma (CaCo-2) cells. Moreover, when diphyllin is added at various times post infection, a significant decrease in viral titer is observed in SARS-CoV-2-infected Vero cells, even at high viral multiplicities of infection. Importantly, neither diphyllin nor cleistanthin B are found cytotoxic to Vero cells in concentrations up to 100 µM. However, the cytotoxic effect of diphyllin is more pronounced in Vero E6 and CaCo-2 cells. Overall, our data demonstrate that diphyllin and diphyllin analogues might be perfected as anti-SARS-CoV-2 agents in future preclinical studies, most especially if nanomedicine approaches may be invoked to optimize functional drug delivery to virus infected cells.

Diphyllin Improves High-Fat Diet-Induced Obesity in Mice Through Brown and Beige Adipocytes.

Brown adipose tissue (BAT) and beige adipose tissue dissipate metabolic energy and mediate nonshivering thermogenesis, thereby boosting energy expenditure. Increasing the browning of BAT and beige adipose tissue is expected to be a promising strategy for combatting obesity. Through phenotype screening of C3H10-T1/2 mesenchymal stem cells, diphyllin was identified as a promising molecule in promoting brown adipocyte differentiation. In vitro studies revealed that diphyllin promoted C3H10-T1/2 cell and primary brown/beige preadipocyte differentiation and thermogenesis, which resulted increased energy consumption. We synthesized the compound and evaluated its effect on metabolism in vivo. Chronic experiments revealed that mice fed a high-fat diet (HFD) with 100 mg/kg diphyllin had ameliorated oral glucose tolerance and insulin sensitivity and decreased body weight and fat content ratio. Adaptive thermogenesis in HFD-fed mice under cold stimulation and whole-body energy expenditure were augmented after chronic diphyllin treatment. Diphyllin may be involved in regulating the development of brown and beige adipocytes by inhibiting V-ATPase and reducing intracellular autophagy. This study provides new clues for the discovery of anti-obesity molecules from natural products.

Comparative Metabolite Profiling of Wild and Cultivated Justicia procumbens L. Based on 1H-NMR Spectroscopy and HPLC-DAD Analysis.

Justicia procumbens L. is known across Korea, India, China, and Taiwan as a remedy against fever, cough, sore throat, and cirrhosis of ascites. J. procumbens provides the raw material for a candidate anti-asthma drug (DW2008S) currently completing phase I clinical trials sponsored by Dong Wha Pharmaceutical Company. HPLC-DAD was used to quantify phytochemical constituents of J. procumbens, and HPLC and 1H-NMR results were assessed by multivariate analysis. This is the first time a comparative study using HPLC-DAD and NMR fingerprints has been applied to identify chemical differences between wild and cultivated J. procumbens. The amount of justicidin B as the marker compound was higher in cultivated samples (0.80 ± 0.25 mg/g) than in wild ones (0.63 ± 0.30 mg/g). Orthogonal partial least squares discriminant analysis (OPLS-DA) from HPLC and NMR data revealed that there were clear differences between wild and cultivated types and identified five secondary metabolites, which could help distinguish between wild and cultivated plants. Among these five lignans, diphyllin showed the most potent discrimination between two types and was significantly detected higher in cultivated ones than in wild ones. A combination of 1H-NMR and HPLC-DAD analysis is effective for J. procumbens standardization and metabolomics studies.

Synthesis, cytotoxicity and anti-invasion activity of three natural diphyllin L-arabinopyranosides.

The concise syntheses of three new natural diphyllin L-arabinopyranosides, Phyllanthusmin D (1), Phyllanthusmin B (4), Phyllanthusmin C (6) and a known analogue 7-O-[(2,3,4-tri-O-acetyl)-α-Larabinopyranosyl)] diphyllin (7) have been achieved employing phase transfer catalysis glycosylation and orthoester rearrangement. In biological assays, 4 showed the best cytotoxicity against human gastric carcinoma MGC803 Cells with the IC50 value 39 µg/mL. Transwell invasion assay showed that glycosides 1, 4, and 7 significantly suppressed MGC-803 cell invasion compared with control.

Glycosylated diphyllin as a broad-spectrum antiviral agent against Zika virus.

BACKGROUND: Flaviviruses such as Zika cause sporadic pandemic outbreaks worldwide. There is an urgent need for anti-Zika virus (ZIKV) drugs to prevent mother-to-child transmission of ZIKV, new infections in high-risk populations, and the infection of medical personnel in ZIKV-affected areas. METHODS: Here, we showed that the small molecule 6-deoxyglucose-diphyllin (DGP) exhibited anti-ZIKV activity both in vitro and in vivo. DGP potently blocked ZIKV infection across all human and monkey cell lines tested. DGP also displayed broad-spectrum antiviral activity against other flaviviruses. Remarkably, DGP prevented ZIKV-induced mortality in mice lacking the type I interferon receptor (Ifnar1-/-). Cellular and virological experiments showed that DGP blocked ZIKV at a pre-fusion step or during fusion, which prevented the delivery of viral contents into the cytosol of the target cell. Mechanistic studies revealed that DGP prevented the acidification of endosomal/lysosomal compartments in target cells, thus inhibiting ZIKV fusion with cellular membranes and infection. FINDINGS: These investigations revealed that DGP inhibits ZIKV infection in vitro and in vivo. INTERPRETATION: The small molecule DGP has great potential for preclinical studies and the ability to inhibit ZIKV infection in humans.

Phenotypic Prioritization of Diphyllin Derivatives That Block Filoviral Cell Entry by Vacuolar (H+ )-ATPase Inhibition.

Many viruses use endosomal pathways to gain entry into cells and propagate infection. Sensing of endosomal acidification is a trigger for the release of many virus cores into the cell cytosol. Previous efforts with inhibitors of vacuolar ATPase have been shown to block endosomal acidification and affect viral entry, albeit with limited potential for therapeutic selectivity. In this study, four novel series of derivatives of the vacuolar ATPase inhibitor diphyllin were synthesized to assess their potential for enhancing potency and anti-filoviral activity over cytotoxicity. Derivatives that suitably blocked cellular entry of Ebola pseudotyped virus were further evaluated as inhibitors of endosomal acidification and isolated human vacuolar ATPase activity. Several compounds with significant increases in potency over diphyllin in these assays also separated from cytotoxic doses in human cell models by >100-fold. Finally, three derivatives were shown to be inhibitors of replication-competent Ebola viral entry into primary macrophages with similar potencies and enhanced selectivity toward antiviral activity.

A Synthetic Disaccharide Derivative of Diphyllin, TAARD, Activates Human Natural Killer Cells to Secrete Interferon-Gamma via Toll-Like Receptor-Mediated NF-κB and STAT3 Signaling Pathways.

Natural products and their derivatives have long been used as pharmacological agents in the fight against cancer. Human natural killer (NK) cells are critical in our immune system in that they are capable of destroying tumor cells directly. However, there are few reports that elucidate the role of natural products in activating NK cells. In this study, we discovered that a synthetic disaccharide derivative of diphyllin, 4-O-{[2'',3'',4''-tri-O-acetyl-α-D-arabinopyranosyl-(1''→4')]-2',3'-di-O-acetyl-α-L-rhamnopyranosyl}diphyllin (TAARD), can alone stimulate interferon (IFN)-γ secretion in primary human NK cells and the NKL cell line. Additionally, it had an additive effect with IL-12 or IL-15 on IFN-γ production, but little adverse effects on NK cells. Mechanistically, TAARD induced the phosphorylation of NF-κB and STAT3, resulting in their binding on the IFNG promoter, which was dependent on TLR1 and TLR3 signaling, respectively. STAT3 and NF-κB knockdown with lentivirus shRNA as well as the NF-κB-specific inhibitor, N-tosyl-l-phenylalaninechloromethyl ketone, significantly suppressed TAARD-induced IFN-γ generation in primary NK cells. Blockade of TLR1 and TLR3 with neutralizing antibodies considerably decreased TAARD-induced activation of NF-κB and STAT3, respectively, as well as IFN-γ generation in NK cells. Collectively, our data suggest that TAARD can induce NK cell IFN-γ production through TLR1-NF-κB and TLR3-STAT3 signaling pathways, rendering its potential use as an agent for cancer prevention or treatment.

Antiviral efficacy of nanoparticulate vacuolar ATPase inhibitors against influenza virus infection.

BACKGROUND: Influenza virus infections are a major public health concern worldwide. Conventional treatments against the disease are designed to target viral proteins. However, the emergence of viral variants carrying drug-resistant mutations can outpace the development of pathogen-targeting antivirals. Diphyllin and bafilomycin are potent vacuolar ATPase (V-ATPase) inhibitors previously shown to have broad-spectrum antiviral activity. However, their poor water solubility and potential off-target effect limit their clinical application. METHODS: In this study, we report that nanoparticle encapsulation of diphyllin and bafilomycin improves the drugs' anti-influenza applicability. RESULTS: Using PEG-PLGA diblock copolymers, sub-200 nm diphyllin and bafilomycin nanoparticles were prepared, with encapsulation efficiency of 42% and 100%, respectively. The drug-loaded nanoparticles have sustained drug release kinetics beyond 72 hours and facilitate intracellular drug delivery to two different influenza virus-permissive cell lines. As compared to free drugs, the nanoparticulate V-ATPase inhibitors exhibited lower cytotoxicity and greater in vitro antiviral activity, improving the therapeutic index of diphyllin and bafilomycin by approximately 3 and 5-fold, respectively. In a mouse model of sublethal influenza challenge, treatment with diphyllin nanoparticles resulted in reduced body weight loss and viral titer in the lungs. In addition, following a lethal influenza viral challenge, diphyllin nanoparticle treatment conferred a survival advantage of 33%. CONCLUSIONS: These results demonstrate the potential of the nanoparticulate V-ATPase inhibitors for host-targeted treatment against influenza.

Differential modulation of Bax/Bcl-2 ratio and onset of caspase-3/7 activation induced by derivatives of Justicidin B in human melanoma cells A375.

Diphyllin and its derivatives are well known cytotoxic natural products structurally related to the anti-cancer drug podophyllotoxin. We here study their structure-activity relationship upon human melanoma cells for first time. To this end, human melanoma A375 cells were incubated with Justicidin B and its 4-methoxylated or 4-glycosylated derivatives to evaluate their selective cytotoxicity and study their effects on cell cycle distribution, caspase activation, apoptosis induction using Annexin V-FITC/PI staining, cell morphology and western blot analysis. Diphyllin methyl ether (GI50 = 3.66 µM) and Justicidin B (GI50 = 1.70 µM) caused an elevation of both early and late apoptosis populations whereas Diphyllin apioside (GI50 = 0.84 µM) and its acetate (GI50= 0.39 µM) enhanced late apoptosis population only (Annexin V-positive/PI-positive). All induced cell cycle arrest at S phase and classic morphological indicators of apoptosis (blebbing, apoptotic bodies, and nuclear fragmentation) accompanied with an elevation of cells with low DNA content (sub-G1). All compounds increased the Bax/Bcl-2 ratio by enhancing Bax expression which evidences the involvement of the mitochondria (intrinsic pathway) in the apoptotic process. These caspase-3/7 results evidence that 4-methoxylation or 4-O-glycosylation of Justicidin B -a caspase independent mitochondrial apoptosis-inducer- triggers caspase-3/7 activation at different times (24h vs. 48h, respectively). Interestingly, the methoxylation causes attenuation of Bcl-2 protein expression contrarily to Diphyllin methyl ether or the O-glycosylated derivatives. Finally, the compounds exhibited significantly less toxicity when tested in adult human dermal fibroblasts and their GI50 in melanoma Sk-Mel-5 cells was not influenced by MDR1/Pgp inhibitors. This study may inform the synthesis of future Diphyllin derivatives with different apoptosis mechanism of action towards human melanoma cells.