key: cord-0707211-h1gipfte authors: Mattio, Luce M.; Catinella, Giorgia; Pinto, Andrea; Dallavalle, Sabrina title: Natural and nature-inspired stilbenoids as antiviral agents date: 2020-07-04 journal: Eur J Med Chem DOI: 10.1016/j.ejmech.2020.112541 sha: 83ff7d8f8b7deb4225d4d33649a5767d64818e13 doc_id: 707211 cord_uid: h1gipfte Viruses continue to be a major threat to human health. In the last century, pandemics occurred and resulted in significant mortality and morbidity. Natural products have been largely screened as source of inspiration for new antiviral agents. Within the huge class of plant secondary metabolites, resveratrol-derived stilbenoids present a wide structural diversity and mediate a great number of biological responses relevant for human health. However, whilst the antiviral activity of resveratrol has been extensively studied, little is known about the efficacy of its monomeric and oligomeric derivatives. The purpose of this review is to provide an overview of the achievements in this field, with particular emphasis on the source, chemical structures and the mechanism of action of resveratrol-derived stilbenoids against the most challenging viruses. The collected results highlight the therapeutic versatility of stilbene-containing compounds and provide a prospective insight into their potential development as antiviral agents. Viruses represent a major threat to the global health and economy. Every year emerging and re-emerging viruses from natural reservoirs constantly infect human population causing risks of viral epidemics and pandemics [1] . In 1997, avian influenza A (H5N1) directly spread from poultry to humans. In 1999 a severe encephalitis outbreak, caused by a new paramyxovirus (Nipah virus), According to WHO, it is imperative to find new antiviral agents, including those against drugresistant and vaccine immunity escaping viral strains [3] , or finding new therapeutic indications for existing approved FDA-drugs (the so-called "drug repurposing" approach) in order to reduce time and cost for drug development for infectious diseases [4] . The urgent need of antivirals appeared since 1980s because of HIV (human immunodeficiency virus) spread causing acquired immune deficiency syndrome. Zidovudine (AZT) was the first anti-HIV drug approved in the United States in 1986. Since then, many research efforts to treat HIV led to important improvement in antiviral research and many new classes of drugs targeting a wide variety of human viruses have been introduced. Natural products from marine sponges, sea algae, arthropods and plants were largely screened as source of inspiration for new antiviral agents [5] . Indeed, natural materials, such as herbs, spices, roots, leaves, barks have been used throughout the history as traditional medicines, flavours or food preservatives. Nowadays, many clinically used drugs have been inspired by natural products, which constitute a broad biodiversity of molecules in terms of chemical space and biological properties. In the last 40 years, 185 antiviral agents were introduced. Vaccines account for 47%, but looking at small molecules, 19 were totally synthetic molecules, while 53 were natural derivatives or nature-inspired semisynthetic compounds [6] . Among natural products, stilbenoids represent a class of non-flavonoid polyphenolic compounds largely studied in the last decades because of their many bioactivities. Stilbenoids are phytoalexins, secondary metabolites produced by the plant as means of defence against pathogens or stress factors [7] . The antimicrobial activity of natural stilbenoids [8] [9] [10] and their presence in plants as both constitutive and inducible secondary metabolites suggest that these compounds may play an important role in the resistance to diseases [11] . Stilbenoids can exist as both monomers and oligomers. Monomers such as resveratrol, pterostilbene, piceatannol and oxyresveratrol (Fig. 1) , are characterized by the presence of two aromatic rings linked by an olefin, and the trans isomer (E) is usually the most stable and the most common in nature. Besides the diverse number and different position of the hydroxy groups, the aromatic rings can bear prenyl, geranyl or farnesyl chains. Oligomers derive from the oxidative coupling of monomers. All the stilbenoids can be found as aglycones or as glycosylated forms [12] . The major dietary source of stilbenoids comes from grapes and wine from Vitaceae family (Vitis vinifera L.) but they are also present in peanuts, cocoa, blueberry, bilberry, cowberry, red currant, cranberry, strawberries [11] . Resveratrol has been largely studied in the last decades for its antioxidant, anti-inflammatory, anticancer, antidiabetic, antimicrobial activities [13] . Its antiviral activity has been also widely investigated and it has been exhaustively highlighted in the recent literature [14] [15] [16] [17] [18] . Whilst the antiviral activity of resveratrol has been extensively studied, little is known about the efficacy of its monomeric and oligomeric derivatives. In this review we provide an overview of natural resveratrol-derived stilbenoids investigated as antiviral agents, with emphasis on targets and mechanism of action. The review also highlights the evidence of antiviral activity of synthetic resveratrol analogues designed to improve the stability and increase the potency of the natural precursors. The review is divided into sections according to the target virus. For each virus, the recent advances in the research of potentially active natural and synthetic stilbenoids are summarized. Viruses are obligate intracellular parasites, needing a host cell to exploit cellular biochemical pathways and factors to replicate. Their genome, single or double stranded DNA or RNA, encodes for various structural and regulatory proteins, and it is contained in the protein capsid, forming the nucleocapsid. Some viral species acquire a phospholipid-containing envelope from the host cell membrane during viral budding (Fig. 2) [19] . The viral life cycle begins when the virus binds to a host cell through electrostatic adsorption to specific host cell receptors (i.e. CXCR4 and CD4 on immune system cells), structurally complementary to exterior structures of the viral particle (i.e. the HIV envelope glycoproteins gp120 and gp41). After penetration into the host cell, viral uncoating and release of viral nucleic acids occur. Using host resources, virus starts transcription and synthesis of early viral proteins, like polymerase enzymes, followed by nucleic acids replication, and, in the case of retrovirus, the viral integrase (IN) incorporates viral nucleic acids into the host DNA. Eventually, also late viral proteins undergo transcription and translation, and their assembly leads to new viral particles, named "virions", which can be released to infect other cells (Fig. 2) . In the case of influenza virus, the viral enzyme neuraminidase (NA) is required to cleave residues on virions, allowing their release from the infected host cell. Antiviral agents block one of these steps, or may interfere with host cell functions, which facilitate viral replication [19, 20] . Indeed, since viral genome encodes just for a few structural and regulatory proteins, viruses need to exploit host cell metabolism and biochemical signalling pathways to survive. In particular, NF-κB (nuclear factor-κB) pathway, regulating the expression of several proteins acting in the immune response, has been demonstrated to be an attractive target for viral pathogens because it is rapidly activated during infections and is involved in critical steps of the host cell cycle. Modulating the NF-κB pathway, viruses such as HIV, herpesviruses, and HCV, have been shown to block host cell apoptosis, thus prolonging the host cell survival and gaining time for viral replication and progeny production. Viruses such as HIV and HSV harbour NF-κB binding sites in their promoters, whose activation results in enhanced viral transcription. In these cases, molecules interfering with NF-κB pathway have been shown to have antiviral activity against both HIV-1 and HSV-1 [21, 22] . Depending on the type of virus and on the host cells, several assays are available to investigate the activity of antiviral compounds. In the plaque reduction assay (PRA), infected cells are treated with a potential antiviral agent, which should cause a decrease of the number of pfu (plaque forming units) in comparison with untreated cells, allowing the determination of IC 50 values (the concentration of the compound able to decrease plaque numbers by 50% with respect to untreated cells). Determining compounds cytotoxicity as the concentrations reducing cell viability by 50% (CC 50 ), the selectivity index (SI) can be calculated as the ratio of CC 50 to IC 50 [23, 24] . Cytopathic effect (CPE) reduction assays measure the IC 50 values as the inhibitory concentrations of antivirals needed to lower by 50% the viral induced CPEs [25] , which are morphological changes in host cells caused by viral invasion [26] . Time-of-addition (TOA) assays may be employed to explore which steps of viral cycle life are blocked, by adding an antiviral compound to the virus/host cells at different time points relative to viral inoculation [27] . Other methods, such as immunoassays, flow cytometry, fluorescence and transmission electron microscopy, polymerase chain reaction (PCR) techniques, enzymatic assays (i.e. NA activity assay), are also used to study viral replication, to detect viral products such as DNA, RNA, proteins, and to identify the target of the antiviral agent studied [24, 28] . Influenza viruses are responsible for acute respiratory infections in two billions of people, which may result in hundred thousands of deaths every year worldwide, according to WHO estimations [29] . Influenza viruses belong to the Orthomyxoviridae family. There are four types of seasonal influenza viruses (A-D), but only type A and B are the main responsible for human infections and may cause seasonal epidemics. Influenza A virus is a single-stranded, segmented RNA virus that presents different subtypes based on haemagglutin (HA1-18) and neuraminidase (NA1-11) transmembrane glycoproteins [30] . The infection occurs when the viral HA binds to the host sialic acid receptors, which mediate the virus entry by endocytosis. The moderately acidic pH However, time-of-addition (TOA) assay showed no significant difference in the SI values for any of the tested compounds between addition to the medium at the same time of viral infection and addition immediately after viral infection, suggesting that the active compounds did non inhibit the early stages of viral replication (adsorption and penetration) [33] . In 2010, five stilbenoids were isolated together with resveratrol from the lianas of Gnetum pendulum (Gnetaceae) by Liu and colleagues and were evaluated by neuraminidase (NA) activity assay and CPE reduction assay [34] (Fig. 4) . In the NA activity assay, all the six molecules Influenza A may induce the inflammation of infected airway epithelial cells, which produce several chemotactic cytokines, in particular RANTES (Regulated upon Activation, Normal T Cells Expressed and Secreted), a potent chemoattractant for monocytes and macrophages [35] . RANTES belongs to CC chemokine ligand 5 (CCL5), whose expression is affected by viral infections since its gene promoter regions contain recognition sites for many virus-activated transcription factors. Moreover, influenza A virus activates the phosphatidylinositol 3-kinase (PI3K)/Akt signal pathway, which is involved in CCL5 retinal expression in human pigment epithelial cells after viral infection [36] . Various studies demonstrated the capability of resveratrol to interfere with the function of chemoattractant receptors [37] . In 2008, Huang et al. isolated five oligostilbenes from the roots of Vitis thumbergii and evaluated their activity on influenza A virus (H1N1)-stimulated RANTES production in human alveolar epithelial cell line A549 [38] . Compounds (+)-ε-viniferin, (−)viniferal, ampelopsin C, miyabenol A and (+)-vitisin A (Fig. 5 ) exhibited significant inhibitory effects of RANTES production at noncytotoxic concentration (0.1-1.0 µM) with EC 50 (half maximal effective concentration) values lower than that of resveratrol (Table 1 ). Notably, the tested tetramers and trimers resulted more active than dimers. In particular, (+)-vitisin A was the most active compound with EC 50 value of 0.27 µM and low cytotoxicity (CC 50 value of 22.4 µM). Furthermore, the authors showed that influenza A (H1N1)-induced RANTES secretion was correlated with the activation of the PI3K/Akt and the signal transducer and activator of transcription (STAT1) signaling cascades in the A549 lung epithelial cells. Western blot analysis revealed that (+)-vitisin A reduced H1N1-induced Akt phosphorylation and subsequently STAT1 activation, suggesting a potential use of (+)-vitisin A in inflammatory disorders after virus infection [38] . Fig. 6 ). Thirty-five compounds were found to be active with IC 50 values ranging from 4.95 to 186 µM [39] . The active derivatives were used to develop 3D quantitative structure-activity relationship (3D QSAR) models and molecular docking in order to elucidate the molecular interaction with the target NA (X-ray structure taken from the RSCB Protein Data Bank, ID 1A4G). In the 3D QSAR studies, [39] . Coronaviruses represent a diverse family (Coronaviridae) of enveloped, single-stranded positive-sense RNA viruses usually causing gastrointestinal and respiratory disorders in humans and animals. Their genome accounts for about 30000 nucleotides, making it the largest found in any RNA viruses. Human coronaviruses often lead to respiratory illnesses that may degenerate into pneumonia and severe acute lung injury [41, 42] . SARS-CoV is the coronavirus responsible for the epidemic of the severe acute respiratory syndrome (SARS), emerged in November 2002 and lasted until July 2003 with 9.6% mortality rate [2] . To the best of our knowledge, there are no example in the literature of natural stilbenoids tested as SARS-CoV inhibitors. However, a series of twelve synthetic resveratrol analogues were prepared and evaluated by Li et al. as potential inhibitors of SARS-CoV replication in Vero E6 cells. In particular, compounds 5 and 6 ( Fig. 6 ) did not show cytotoxicity in concentration ≥ 2 mg/mL (8.2 mM) and were able to inhibit the cytopathic effect (CPE) in concentration < 0.5 mg/mL (2.05 mM). Possible lower doses were not investigated [40] . illnesses, with a 34% mortality, firstly identified in Saudi Arabia in 2012 [43] . Up to date, there are still no effective anti-MERS drugs or vaccines approved on the market [44] . In in vitro studies Lin et al. [45] investigated the effect of resveratrol on MERS-CoV infection and showed that the cytotoxicity of MERS-CoV-infected Vero E6 cells (CRL-1586) was reduced by resveratrol treatment (250 µM) to 25%. To determine where the resveratrol action occurred, they found that MERS-CoV RNA replication was suppressed and MERS titers were significantly reduced. Moreover, they showed that resveratrol significantly inhibited MERS nucleocapsid (N) protein translation, fundamental for MERS-CoV replication, in a dose dependent manner, in the concentrations ranging from 125 to 250 µM. Since MERS-CoV is well known to induce cell apoptosis [46] and high levels of cleaved Caspase 3 were reported after MERS-CoV infection as apoptosis indicator [47] , Lin et al. found that resveratrol decreased Caspase 3 cleavage dosedependently, suggesting that resveratrol reduced the MERS-CoV mediated cells apoptosis [45] . The exact mechanism needs further investigations, but it is known that resveratrol may reduce inflammation by interfering with the NF-κB (Nuclear Factor-Kappa B) pathway [48] and it could down-regulate fibroblast growth factor 2 (FGF-2) signalling [49] , involved in MERS-CoV-induced apoptosis [50] . Hepatitis C virus (HCV) is a bloodborne positive single-stranded RNA virus, belonging to the Flaviviridae family in the Hepacivirus genus [51] . HCV causes hepatitis C that can be a mild illness lasting a few weeks (acute) or a lifelong and serious disease (chronic). Hepatitis C may degenerate into cirrhosis and HCV is the major cause of liver cancer. Worldwide 71 million people have chronic HCV infection [52] . HCV exists in seven genotypes and more than 80 subtypes, which differ in the pathogenesis and response to treatments. Because of its high genetic variability, due to a high replication rate and lack of proofreading activity by the HCV RNA-dependent RNA polymerase (vRdRp), so far there is no effective vaccine against hepatitis C, and new antivirals able to overcome the drug-resistance and to reduce the side effects in the long treatment are needed [51, 52] . The effect and the mechanism of action of resveratrol as anti-HCV agent was reported in the review by Abba et al. [14] . More recently, Nguyen et al. [55] studied the anti-HCV and antitumoral activities of the natural compound Z-3,5,4'-trimethoxystilbene (Z-TMS, Fig. 1 ), isolated for the first time from the bark of Virola elongate [56] . The compound was tested on hepatoma cell lines (GS5 and FCA4) expressing HCV-1b subgenomic replicons. Z-TMS was 100 times more potent than resveratrol in downregulating the levels of HCV NS5B polymerase in a dose-dependent manner, without displaying cytotoxicity to human hepatocytes in vitro or in mice liver. The authors demonstrated that Z-TMS interfered with the cancer stem cell (CSC) marker DCLK1, microtubule dynamics and induced autophagy, G 2 -M arrest, and nuclear fragmentation [55] . Other natural stilbenoids were identified as potent anti-HCV. In the last years, Lee et al. published two papers about this topic. In 2016, they observed the inhibitory effect of an extract from the roots of Vitis vinifera on HCV infected-Huh7.5 hepatocarcinoma cells and identified five oligostilbenes as responsible for the suppression of HCV replication exerted by the extract. The compounds were two resveratrol dimers, ampelopsin A ( Fig. 7) and (+)-ɛ-viniferin (Fig. 5) , and three resveratrol tetramers, vitisin A (Fig. 5 ), wilsonol C and vitisin B (Fig. 7) . Also in this case, compounds with more complex structures (tetramers) demonstrated higher potency in comparison to simpler ones (dimers). In particular, vitisin B resulted to be the most potent oligostilbene, displaying its activity by directly binding the NS protein HCV NS3, dramatically decreasing viral replication at nanomolar concentrations (EC 50 value = 6 nM) ( Table 2) . Moreover, the authors verified that vitisin B, combined with an NS5B polymerase inhibitor, sofosbuvir, exhibited a synergistic or at least additive antiviral activity. Eventually, in in vivo pharmacokinetic studies, after intraperitoneal injection in rats, vitisin B showed a preferred tissue distribution in the liver, which is a major organ of HCV replication [57] . Because of the difficulties to obtain the pure compound in substantial amounts for further investigation, studies on infected mice were not included [58] . In 2019, the same authors confirmed the inhibitory capability of (+)-ɛ-viniferin against HCV [58] . The pure enantiomer (+)-ɛ-viniferin ((+)-ɛ-VF) (Fig. 5) was isolated from the extract of roots of Vitis vinifera and, thanks to an efficient synthetic methodology, substantial amounts of (±)-ɛviniferin ((±)-ɛ-VF) and penta-acetylated (±)-ɛ-viniferin ((±)-ɛ VF-5Ac) were made available. All Additionally, the authors confirmed that (+)-ɛ-VF suppressed HCV NS3 helicase activity, using a GO-based NS3 helicase assay described by Jang [59] and comparing the effect to resveratrol activity. The pharmacokinetic profile of (±)-ɛ-VF were studied on mice after oral and intraperitoneal administration. The intraperitoneal dosing resulted in much higher plasma concentrations than those after oral administration, suggesting that the low oral bioavailability of (±)-ɛ-VF is due to poor absorption through the intestinal epithelium and to intestinal first pass effects. [58] . Dengue virus (DENV) is a mosquito-borne virus, causing a wide spectrum of diseases, which can vary from mild fever to acute-flu like illness and life-threatening haemorrhagic fever inhibit RNA viral synthesis in DENV2 replication, but they resulted inactive against the viral RdRp. In a pretreatment cells study, only PNR-5-02 affected the DENV2 replication, implying at least partially interactions with host cell factors required for viral replication. On the other hand, PNR-4-44, inactive in the pretreatment, seemed to directly target the virus. The exact mechanism of action is not known. Interestingly, Dengue virus belong to the same family of HCV, sharing a similar NS3 protein [67] . Since vitisin B and ɛ-viniferin were found to inhibit HCV NS3 [57, 58] , Dengue virus NS3 could be likely one of the possible target. The key molecular features of resveratrol analogues involved in the anti-DENV activity still need to be elucidated and the authors underlined that SAR studies are still needed to improve their activity-cytotoxicity profile [66] . Human immunodeficiency virus type 1 (HIV-1) is an enveloped single-stranded RNA virus of the Lentiviridae family [68] , responsible for the acquired immunodeficiency syndrome (AIDS), which currently affects more than 37 million people [69] . Although resveratrol showed various antiviral activity for several viruses, it did not exhibit any inhibition against wild-type HIV-1 replication in activated T cells or in transformed T cell lines [70] . On the contrary, together with nucleoside analogue reverse transcriptase inhibitors (NRTIs) such as tenofovir, didanosine and zidovudine, resveratrol seemed to improve the inhibition of cellular ribonucleotide reductase (RNR) [71] . In 2017, Chan et al. confirmed the inefficiency of resveratrol as anti-HIV in activated T cells and in transformed T cell line Jurkat [72] . Surprisingly, the authors found that resveratrol prevented productive infection of resting CD4 T cells in a dosedependent manner. Moreover, four natural stilbenoids, pterostilbene, piceid, piceatannol and isorhapontigenin (Fig. 1, 3) , were tested for anti-HIV-1 activity on resting CD 4 T cells. Pterostilbene was active at all concentrations tested, piceid and isorhapontigenin inhibited productive infection at a concentration 30 µM, while piceatannol was less potent at the same concentrations [72] . This finding suggests that in the tested monomers the free hydroxy groups reduce the antiviral potency. Pflieger et al. confirmed this observation in another study. In 2013, they isolated a collection of stilbenoids, consisting of monomers and oligomers (Fig. 1, Fig. 9 Polygonum cuspidatum and Polygonum multiflorum by Lin et al. [74] . The compounds were tested on cell line C8166 as anti-HIV-1 agents. Resveratrol showed the greatest inhibitory activity against HIV replication with EC 50 4.37 µg/mL and therapeutic index value (TI = CC 50 /EC 50 ) of 8.14, while the glycosylated derivatives showed a severe decrease of potency with respect to their corresponding aglycones (Fig. 10, Table 3 ). Among the glycosylated compounds, the position of sulphate group did not influence the antiviral activity. Conversely, the stereochemistry of the double bond seemed to affect the activity, as the cis isomer 14 (EC 50 = 84.77 µg/mL) was more active than the trans isomer 13b (EC 50 > 200 µg/mL), even if more cytotoxic (CC 50 = 98.82 µg/mL and CC 50 = 812.88 µg/mL for 14 and 13b, respectively) [74] . Interestingly, compounds 14 is characterised by a cis configuration at the double bond, which likely favors the interaction with the target, thus increasing the antiviral activity. (Fig. 11) . The isolated compounds were tested in the NCI (National Cancer Institute) primary anti-HIV screen: dibalanocarpol and balanocarpol showed Compound CC 50 [75] . In this case, the dihydrobenzofuran ring of balanocarpol and dibalanocarpol with respect to the benzofuran ring of malibatol A and B seemed to play a key role in the anti-HIV activity. In a more recent study, other stilbene disulfonic acids resulted to inhibit IN by a novel mechanism [77] (Fig. 13) Fig. 13 ), a compound already known for its cytopathic effects on HIV-1 [79] , as the most active among the molecules that showed an inhibitory activity on ST step. Notably, NCS34931 consisted of a chemical scaffold different from that of the known ST inhibitors. SAR studies were performed on the selected compounds and the naphthalene moiety resulted to be fundamental for the activity. Indeed, NSC163 and NSC163175 (Fig. 13) , lacking aromatic rings, were completely inactive at the highest concentrations tested, and replacing the naphthalene ring with an ethoxybenzene caused a 10-fold decrease of potency in NSC47745 (Fig. 13 ). NSC34933, with just some rearrangements in the naphthalene substitution pattern with respect to NSC34931, appeared to maintain the sub-micromolar activity of the parent compound and to be even less cytotoxic (Fig. 13 , Table 4 ). The two compounds were tested against clinically relevant mutants resistant to conventional IN inhibitors and the stilbene derivatives maintain their activity at submicromolar concentration, representing a potential therapeutic alternative. Indeed, they were found to compete with DNA in binding IN, especially involving the C-terminal domain (CTD), at concentrations 10 times lower than that necessary to inhibit 3'P and ST [77] . Tat induces the HIV-coreceptor expression, such as CXCR4 and CCR5, and the release of chemokines, which attract monocytes and CD4 T lymphocytes to perpetuate the infection [81] . In 2000, a synthetic stilbene compound, CGA137053 (Fig. 14) , was found to bind directly Tat protein and to prevent the upregulation of the HIV coreceptor CXCR4. The two negative charged sulfonate groups, the spacer between them, and the pyrazolic nucleus of CGA137053 seemed to be fundamental in the interaction with Tat. The inhibition of Tat protein was demonstrated to work intracellularly and to inhibit HIV-1 replication on HIV-infected, primary human leucocytes (PBL) and macrophages in a dose-dependent manner (EC 90 , 90% effective concentration, ranging from 0.5 to 5 µM depending on the HIV strains) [82] . Lethal mutagenesis is another antiviral approach that takes advantage of the high rate of RNA virus mutation by intentionally further increasing it so that the virus becomes unable to replicate its genome with enough fidelity to maintain its infectiousness [83] . Clouser et al. studied the synergistic effect of resveratrol in combination with decitabine (5-aza-2'-deoxycytidine, 5-aza-dC), a deoxyribonucleoside analogue able to lethally mutagenize HIV-1 in a cell culture system [70] . In a previous study, the same research group demonstrated the synergistic effect between a nucleoside analogue and a ribonucleotide reductase inhibitor (RNRI) in increasing the HIV mutation rate, resulting in HIV infectivity decrease [84] . Indeed, resveratrol has been reported to inhibit HIV-1 replication by interacting with SIRT1 [15] and to be an inhibitor of ribonucleotide reductase, the enzyme involved in the reduction of ribonucleotides into the corresponding deoxyribonucleotides [85] . Therefore, Clouser et al. synthesized fifteen resveratrol derivatives and screened them on HIV-1-infected 293T-cells alone and in combination with decitabine. Piceatannol and 23a showed an improved activity against HIV-1 infection, maintaining a low cytotoxicity (Fig. 15 , Table 5 ). Replacing the double bond with a heterocycle (20-22, 23e-23f) or with an α, βunsaturated ketone (19) did not improve the potency of resveratrol. The 2-hydroxy-naphthalene (23c) and the isoquinoline (23d) moieties were also ineffective in improving the potency. Conversely, hydroxy groups seemed to play a key role in the antiviral activity since piceatannol, bearing four hydroxy functions, was one of the most active compound, whereas compound 7, differing from the most active compound 23a only in the pyridine ring in place of a 4-hydroxy phenyl ring, completely lost potency. The benzofuran and benzothiophene derivatives (10a, b-11a, b) , bearing three hydroxyl groups, demonstrated a better activity than resveratrol, having however a low SI. Overall, the distance between the aromatic rings bearing at least three hydroxy group and the resulting conformation of the compounds seemed to influence the antiviral effects. On the other hand, only resveratrol demonstrated a synergism with decitabine, increasing HIV-1 mutant frequency [70] . However, in 2016 the same authors studied combinations of resveratrol and 5azacytidine (5-aza-C), the decitabine riboside analogue, and they found that the synergistic activity at low concentration of resveratrol as RNRI is mainly due to decreased accumulation of RT products rather than to increased viral mutagenesis [86] . (Table 6 ). M8 seemed to inhibit the viral attachment to host cells. Indeed, firstly in TOA assays, M8 did not show any activity when added 2h after the infection, suggesting that M8 targets an early step in HIV-1 replication. Secondly, quantitative real-time PCR analysis showed a decrease level of early viral reverse transcription products in a dose-dependent manner. Lastly, in a post-attachment assay, authors demonstrated that compound M8 was able to block virus attachment to cells before the fusion step [87] . Table 6 . Antiretroviral activity of M8 against laboratory strains of HIV-1 in different cells from [87] . The Fig. 17 ) isolated from the leaves of Macaranga barteri (Euphorbiaceae) [95] . The pure compounds were tested against echovirus 7, 13 and 19 serotypes and were inactive against echovirus E13, while a good activity was reported on E19. In particular, vadelianin exhibited an IC 50 value of 0.0036 nM and the best selectivity profile with SI value of 216.7 [95] . Herpes simplex viruses (HSVs) belong to Herpesviridae family, Alphaherpesvirinae subfamily. Differently from all the other viruses treated above, they are double-stranded DNA viruses and exist as two types: HSV-1 and HSV-2 [96] . HSV-1 is the most common form of herpes. More than 60% of the human population contract orofacial infections, that can lead to infectious blindness and viral encephalitis in adults [97] . On the other hand, HSV-2 infection affects the genital area and is a major cause of sexually transmitted diseases (STD), such as HIV and human papillomavirus (HPV) [98] . The transmission of herpes viruses occurs from person to person by direct contact with infected secretions [99] . The infections may be latent and appear periodically, due to the virus capability to infect neurons, in particular the sensorial nerve termini, then traveling in a retrograde manner. Therefore, HSV may reactivate a lytic-replication cycle leading to a recurrent infection, viral shedding and transmission to new hosts [17, 96] . Nucleoside analogues, such as acyclovir and pencyclovir, are administered as therapeutic agents against HSV. However, it is necessary to develop new antiherpetic compounds due to the growth of herpes simplex virus strains resistant to acyclovir [100] . An exhaustive overview on resveratrol as novel anti-herpes simplex virus was reported by Annunziata et al. [17] . to the medium during viral infection, and throughout the incubation thereafter, or immediately after infection, whereas ACV exhibited a lower effect. This study suggested that the antiviral activity of these compounds is due to a different mode of action in comparison with that of the current clinically-used drug ACV [33] . In 2012, another collection of dimeric and oligomeric resveratrol derivatives (Fig. 19 ), previously isolated from plants of Hopea genus, was screened as anti-HSV-1 and anti-HSV-2 agents by Chen. et al. [103] . In general, the compounds were more active against HSV-2 infection than that from HSV-1. Vaticaffinol, a tetramer with reported antifungal properties, was one of the most promising compounds, with an IC 50 value of 3.2 µM against HSV-2. The results of this study showed a potent, dose-dependent antiviral effect of the compounds, which promoted ROS production, coinciding with suppression of HSV-1 and HSV-2 replication in treated cells [103] . (Fig. 1) , the major constituent of the heartwood of Artocarpus lakoocha (Moraceae), and its mechanism of action. The activity was determined against HSV-1 (7401H and KOS) and HSV-2 (Baylor 186) on infected Vero cells, with IC 50 values of 19.8 µg/mL, 24.0 µg/mL and 18.7 µg/mL, respectively. In particular, oxyresveratrol showed a better anti-HSV activity than ACV in the plaque reduction assay against thymidine kinase (TK)-deficient (ACV-resistant) strain. Indeed, ACV is a nucleoside analogue, which exerts its antiviral action after TK phosphorylation [104] . This finding suggested that oxyresveratrol displayed a different mechanism of action than ACV, probably inhibiting the late viral protein synthesis, similarly to resveratrol. Moreover, in in vivo studies the authors demonstrated that HSV-1-infected mice orally treated with oxyresveratrol (125 mg/kg/dose), showed a significant delay in herpetic skin lesions development, while the topical administration of 30% oxyresveratrol ointment five times per day significantly retarded skin lesions, preventing mice death. Therefore, oxyresveratrol may be a suitable anti-HSV agent in topical treatment [105] . Nowadays, huge efforts are required to face emerging and re-emerging viruses that constantly infect human population, threating global public health and economy. Viruses easily undergo mutations, leading to drug resistance and increasing the need of new antiviral compounds with new mechanisms of action. In this scenario, targeting viruses with compounds from natural sources represents a promising strategy. Stilbenoids are a class of natural products endowed with several biological activities. Stilbenoids are synthesized by plants as means of protection against pathogens, whereby the potential antiviral properties of this class of natural compounds have attracted interest in the last years. Resveratrol has received massive attention for its potential health benefits, including anticarcinogenesis, anti-aging, antimicrobial and also antiviral properties. In this review we focused on the studies concerning other natural stilbene monomers and oligomers, which in most cases demonstrated to be more active than resveratrol itself. Notably, many compounds were discovered to exploit new mechanisms of action, interacting directly with the virus or modulating different pathways involved in the immune response, which may overcome virus drug resistance. However, though many in vitro studies provided promising results on this wide class of compounds, a limited number of in vivo studies has been performed so far. This is mainly due to the difficulty of obtaining substantial amount of desired pure compounds, necessary for in vivo model biological evaluation, by extraction and purification procedures [13, 106, 107] . To overcome this problem, in the last decade, a number of research groups have focused on the development of versatile synthetic procedures to selectively produce pure derivatives [9, 10, 58, [107] [108] [109] [110] , taking advantage in many cases of chemo-enzymatic approaches. In this respect, biocatalysis has a number of important advantages such as high efficiency, mild reaction conditions, versatility and high selectivity (chemo-, regio-and stereoselectivity). Notably, preliminary in vivo studies reported in the literature have shown that in general, stilbenoids show low bioavailability and undergo extensive metabolism and it has been pointed out that bioconverted forms of polyphenols, (phase I and II metabolism) may probably have more importance than the parent compound found in the diet or administered in therapy. In many cases, synthetic efforts produced stilbene derivatives with greater potency than their parent compounds, allowing to expand knowledge on modes of action and to deepen structure-activity relationship studies of the most active compounds [39, 70, 76, 77, 82, 91] . Despite the promising results reported in the cited studies, future efforts, involving complementary expertise of chemists, nutritionists, molecular biologists, pharmacologists, are still needed to carry out in vivo experiments and remain of primary importance to confirm the antiviral potential of stilbenoids for clinical applications. 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The authors declare no competing financial interest.