Exploring Flavone-8-Acetic Acid and Derivatives in the New Century

molecules

Review Flavonoid-Inspired Vascular Disrupting Agents: Exploring Flavone-8-Acetic Acid and Derivatives in the New Century

Silvia Gobbi * , Federica Belluti , Angela Rampa and Alessandra Bisi

Department of Pharmacy and Biotechnology, Alma Mater Studiorum-University of Bologna, Via Belmeloro 6, I-40126 Bologna, Italy; [email protected] (F.B.); [email protected] (A.R.); [email protected] (A.B.) * Correspondence: [email protected]

Abstract: Naturally occurring flavonoids are found as secondary metabolites in a wide number of plants exploited for both medicine and food and have long been known to be endowed with multiple biological activities, making them useful tools for the treatment of different pathologies. Due to the versatility of the scaffolds and the vast possibilities of appropriate decoration, they have also been regarded as fruitful sources of lead compounds and excellent chemical platforms for the development of bioactive synthetic compounds. Flavone-8-acetic acid (FAA) and 5,6-dimethylxanthone acetic acid (DMXAA) emerged for their antitumour potential due to the induction of cytokines and consequent rapid haemorrhagic necrosis of murine tumour vasculature, and different series of derivatives have been designed thereafter. Although the promising DMXAA failed in phase III clinical trials because of strict species-specificity, a boost in research came from the recent identification of the stimulator of interferon genes (STING), responsible for supporting tumoural innate immune

responses, as a possible biological target. Consequently, in the last decade a renewal of interest for these ﬂavonoid-based structures was noticed, and novel derivatives have been synthesised and

Citation: Gobbi, S.; Belluti, F.; evaluated for a deeper understanding of the molecular features needed for affecting human cells. Un- Rampa, A.; Bisi, A. Flavonoid- doubtedly, these natural-derived molecules deserve further investigation and still appear attractive Inspired Vascular Disrupting Agents: in an anticancer perspective. Exploring Flavone-8-Acetic Acid and Derivatives in the New Century. Keywords: angiogenesis; VDAs; natural products; ﬂavone-8-acetic acid; DMXAA; vadimezan Molecules 2021, 26, 4228. https:// doi.org/10.3390/molecules26144228

Academic Editor: Lucia Morbidelli 1. Introduction The term angiogenesis refers to the development of new capillaries from existing blood Received: 3 June 2021 vessels, and it plays a pivotal role in different physio-pathological processes, being involved Accepted: 9 July 2021 Published: 12 July 2021 in embryo development, in wound healing but also in various diseases. In particular, when dysregulated, angiogenesis has a major impact on human health, becoming involved in

Publisher’s Note: MDPI stays neutral the onset of ischemic, inflammatory and malignant disorders [1]. In normal physiological with regard to jurisdictional claims in conditions, angiogenesis is regulated by a tight network of growth factors and cytokines, published maps and institutional affil- including vascular endothelial growth factor (VEGF), fibroblast growth factors (FGFs), iations. tumour necrosis factor alpha (TNF-α), epidermal growth factor (EGF), interleukin 8 (IL-8) and erythropoietin (EPO), produced by various types of cells, which include endothelial, smooth muscle, inflammatory and cancer cells [2]. Undoubtedly, the fast proliferation of cancer cells requires constant feeding, ensured by a continuous blood flow into the tumour microenvironment. Therefore, one of the key Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. steps in cancer progression and formation of metastases is the development of new blood This article is an open access article vessels, endowed with peculiar features with respect to normal vascular tissue. Indeed, tu- distributed under the terms and mour microvasculature is usually defined as immature, characterized by poorly structured conditions of the Creative Commons walls, layered by irregularly shaped endothelial cells, defectively connected with pericytes Attribution (CC BY) license (https:// and endowed with a high intrinsic permeability to macromolecules, consistent with the creativecommons.org/licenses/by/ imbalance between pro-angiogenic factors, such as VEGF, and anti-angiogenic ones. It has 4.0/). been widely reported that tumours are unable to properly respond to vasoactive agents

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acting on specific vascular smooth muscle cell receptors, mainly due to the deficiency of vascular smooth muscle cells [3]. The heterogeneity of blood flow, together with the unusually elevated levels of deoxygenated blood, are to be held primarily responsible for chronic and cycling hypoxia, decreased cellular pH and increased interstitial pressure found in tumours. Hypoxia, in turn, through the activation of hypoxia-inducible factor (HIF), activates pro-angiogenic factors, among which is VEGF itself [4]. This abnormal vascular network impairs the ability of conventional anticancer drugs to enter cancer cells, reducing their therapeutic efficacy, and promotes cancer metastasis, drug resistance progression and immune evasion. Moreover, recent studies suggest that vascular homeostasis could be directly affected by a series of individual differences, related to genetic polymor- phisms, specific mutations and individual cancer types, paving the way for personalized medicine, thus enabling the selection of the best therapeutic treatment according to the peculiar variability of tumours [5]. Therefore, it is not surprising that angiogenesis is still considered a promising and effective target in anti-cancer therapy. Currently, two strategies are followed for attaining this goal: anti-angiogenic therapy, aimed at inhibiting new blood vessels formation with anti-angiogenic agents (AAs) and anti-vascular therapy, in which preformed tumour blood vessels are destroyed by means of vascular disrupting agents (VDAs). The anti-angiogenic therapy acts by significantly reducing cancer cells nutrient and oxygen supply. AAs belongs to different main groups, among which small molecule inhibitors of angiogenesis, targeting different pathways involved in the process, and monoclonal antibodies are the prevailing ones for cancer treatment. The main drawbacks for clinical use of AAs remain the off-target effects of this drugs in healthy tissues and the development of drug resistance [2]. On the other hand, VDAs can be grouped into three subclasses, namely microtubule destabilizing agents, anti-vascular- acting flavonoids and drugs directly targeting endothelial cells, all causing destruction of tumour vasculature through apoptosis or necrosis of endothelial cells. Comparing the antitumour profile of classic anticancer drugs acting on tumour cells and compounds targeting tumour blood vessels, VDAs appear the most promising, mainly due to their low induction of resistance, and their ability to reach the target blood vessels and to induce substantial necrosis of the tumour core [6]. Unfortunately, one of the main disadvantages associated with VDAs is the incomplete eradication of the tumour, due to the induction of a central necrosis that is surrounded by preserved vascularized cells, able to survive and proliferate. This so-called “narrow rim” of peripheral cancer cells could then induce rapid tumour regrowth. Accordingly, most VDA-based therapeutic approaches still entail combination treatments, acting with multiple mechanisms and able to both inhibit tumour growth and its vasculature [7]. Notably, it is of pivotal importance to follow a proper combined treatment schedule, considering that the reduced blood flow and the altered permeability of blood vessels induced by VDAs in the tumour environment may induce a deep modification in the distribution of simultaneously administered chemotherapeutic agent. In recent years, an increasing number of natural products (NPs) or NP-derived compounds appeared on the scene as VDAs, mainly acting as microtubule destabilizers or apoptosis inducers selectively on cancer endothelial cells. In particular, combretastatins, stilbene derivatives extracted from the bark of the Combretum caffrum tree, are the most known VDAs in the microtubule destabilizing group. These compounds act by binding to the colchicine binding site of tubulin in endothelial cells, inhibiting tubulin polymer- ization and inducing G2/M cell cycle arrest. The resulting activation of the apoptosis machinery leads to endothelial cells death, blood vessels disruption and finally tumour necrosis. A number of combretastatin-based derivatives were submitted to clinical trials, both as single agents and in combination with other anticancer drugs, giving better clinical outcomes when administrated as combinations. On the other hand, flavonoids, plant secondary metabolites found in food, mainly vegetables, fruits and cereals, showed a myriad of beneficial properties, among which were antioxidant, anti-inflammatory, anti-mutagenic and anti-carcinogenic [6,7] properties, and different mechanisms of action were proposed to elucidate their activity in several stages of cancer progression. In particular, they are known Molecules 2021, 26, x FOR PEER REVIEW 3 of 15

Molecules 2021, 26, 4228 and anti-carcinogenic [6,7] properties, and different mechanisms of action were proposed3 of 14 to elucidate their activity in several stages of cancer progression. In particular, they are known to play a relevant role as VDAs by inducing apoptosis. The flavonoid core struc- toture play can a also relevant be regarded role as VDAs as a validated by inducing synthetic apoptosis. platform, The flavonoid which upon core appropriate structure can func- also betionalisation regarded ascan a validatedprovide a synthetic huge number platform, of structurally-related which upon appropriate compounds functionalisation endowed canwith provide distinct a biological huge number prof ofiles. structurally-related In particular, flavonoid- compoundsrelated endowed compounds with distinct have been bio- logicalscreened profiles. in clinical In particular, trials for cancer flavonoid-related treatment, leading compounds in 1985 have to beenthe identification screened in clinical of fla- trialsvone foracetic cancer acid treatment, (FAA, Figure leading 1) [8], in 1985acting to by the promoting identification haemorrhagic of flavone aceticnecrosis acid of (FAA, sub- Figurecutaneous1)[ 8 murine], acting colon by promoting tumours. haemorrhagic Following experiments necrosis of subcutaneousindicated that murine FAA might colon in- tu- mours.duce a cytotoxic Following effect experiments due to its indicated ability to that selectively FAA might impair induce tumour a cytotoxic blood flow. effect Although due to its abilitythis compound to selectively failed impair in clinical tumour studies blood in flow.humans, Although this result this compoundstimulated failedthe synthesis in clinical of studiesmany FAA in humans, analogues, this aimed result at stimulated better clarifying the synthesis the mechanism of many FAAof action analogues, and at overcom- aimed at bettering the clarifying observed the species mechanism specificity of action issue. andThe attricyclic overcoming analogue the observedof FAA, xanthenone-4- species speci- ficityacetic issue.acid (XAA, The tricyclic Figure analogue1) [9], proved of FAA, to be xanthenone-4-acetic as active as FAA and acid suitable (XAA, for Figure structural1)[ 9], provedmodifications, to be as which active allowed as FAA the and structure suitable acti forvity structural relationships modifications, (SARs) for which this allowedpromis- theing structureclass of VDAs activity to expand. relationships With (SARs)the disc forovery this of promising the highly class potent of VDAs5,6-dimethylxan- to expand. Withthenone-4-acetic the discovery acid of (DMXAA, the highly potentASA404, 5,6-dimethylxanthenone-4-acetic Vadimezan, Figure 1) [10,11], aacid safe (DMXAA,and well- ASA404,tolerated Vadimezan,VDA targeting Figure immature1)[10,11 and], a unstable safe and vasculature well-tolerated of solid VDA tumours targeting [7], immature the goal andseemed unstable to have vasculature been reached. of solid tumours [7], the goal seemed to have been reached.

Figure 1. Structures of prototypes ﬂavone-8-aceticflavone-8-acetic acid (FAA), xanthone-4-aceticxanthone-4-acetic acidacid (XAA)(XAA) andand 5,6-dimethylxanthone-4-acetic5,6-dimethylxanthone-4-acetic acid (DMXAA).

A dualdual mechanismmechanism of of antitumour antitumour effect effect has has been been proposed proposed for DMXAA. for DMXAA. A direct A direct effect iseffect related is related to its abilityto its ability to induce to induce apoptosis apoptosis in tumour in tumour endothelial endothelial cells, cells, with with concurrent concur- haemorrhagicrent haemorrhagic necrosis necrosis in tumour in tumour tissues tissues [12]. This[12]. outcomeThis outcome appears appears shortly shortly after adminis-after ad- trationministration and is and followed is followed by the by activation the activation of the of innate the innate immune immune system, system, representing representing the indirectthe indirect effect effect of DMXAA, of DMXAA, not usually not usually found found in other in VDAs.other VDAs. This activation This activation leads to leads inflam- to matoryinflammatory cytokine cytokine production production and tumour-specific and tumour-specific inflammatory inflammatory responses. responses. Once more, Once de- spitemore, the despite exciting the antitumour exciting antitumour effects observed effects in observed mouse models, in mouse DMXAA models, also DMXAA failed when also combinedfailed when with combined standard-of-care with standard-of-care chemotherapy chemotherapy in a phase III in human a phase efficacy III human trial. efficacy In 2012, furthertrial. In mechanistic2012, further studies mechanistic led to thestudies identification led to the of identification the possible targetof the ofpossible this compound, target of thethis stimulatorcompound, of the interferon stimulator genes of interferon (STING, alsogenes known (STING, as ERIS,also known MITA, as TMEM173 ERIS, MITA, and MPYS),TMEM173 an endoplasmicand MPYS), reticuluman endoplasmic adaptor reticulum transmembrane adaptor protein transmembrane involved in protein the innate in- immunevolved in pathway the innate [13 immune]. Recently, pathway the pivotal [13]. Recently, role of STING the pivotal in sustaining role of STING innate in immune sustain- responses in the tumour microenvironment has been validated [14]; and structure–function ing innate immune responses in the tumour microenvironment has been validated [14]; studies of mouse STING (mSTING) and human STING (hSTING) suggested that DMXAA and structure–function studies of mouse STING (mSTING) and human STING (hSTING) is a potent agonist of mSTING, while polymorphism in hSTING makes it unable to bind suggested that DMXAA is a potent agonist of mSTING, while polymorphism in hSTING the compound, and this probably explains its failure in clinical trials [15]. Taken together, makes it unable to bind the compound, and this probably explains its failure in clinical all these findings provide a mechanistic insight for the lack of efficacy of DMXAA in human trials [15]. Taken together, all these findings provide a mechanistic insight for the lack of tumours and give the rationale for carrying out further SAR studies on the structures of efficacy of DMXAA in human tumours and give the rationale for carrying out further SAR FAA and XAA, in order to overcome this drawback and obtain effective new VDAs. studies on the structures of FAA and XAA, in order to overcome this drawback and obtain This review aims to give an overview of the last 20 years of medicinal chemistry effective new VDAs. studies on FAA and XAA, endowed with a peculiar and intriguing antitumour profile, This review aims to give an overview of the last 20 years of medicinal chemistry stud- involving both vascular disrupting activity and immunomodulation. The first decade ies on FAA and XAA, endowed with a peculiar and intriguing antitumour profile, involv- marks the end of intense years of medicinal chemistry efforts, leading to a plethora of derivativesing both vascular with no disrupting major improvement activity and in immunomodulation anticancer activity. In. theThe last first decade, decade the marks revived the interestend of intense in these years flavonoid-related of medicinal moleculeschemistry allowedefforts, leading the tangle to ofa plethora the targets of involvedderivatives in their action to be partially unravelled and for more promising compounds to be identified, in part due to more focused biological testing.

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with no major improvement in anticancer activity. In the last decade, the revived interest in these flavonoid-related molecules allowed the tangle of the targets involved in their action to be partially unravelled and for more promising compounds to be identified, in Molecules 2021, 26, 4228 part due to more focused biological testing. 4 of 14

2. SAR Studies 2001–2009 2. SARDifferent Studies investigations 2001–2009 on FAA and XAA were performed in the last 20 years, and our research group was deeply involved in establishing appropriate SARs for the two re- Different investigations on FAA and XAA were performed in the last 20 years, and our researchlated compounds group was aimed deeply at identifying involved in an establishing effective VDA appropriate and gaining SARs more for theinsight two into related the compoundspeculiar mechanism aimed at of identifying action of these an effective drugs. VDAIndeed, and the gaining biological more targets insight involved into the pecu-were liarstill mechanismunknown, and of action studies of mainly these drugs. focused Indeed, on the the definition biological of structural targets involved requirements were still for unknown,both immune and modulation studies mainly and focused induction on theof cytokines deﬁnition such of structural as TNFα requirements, also involved for inboth vas- immunecular disrupting modulation properties and induction of the parent of cytokines compounds such [6], as TNFto gainα, also enhanced involved anticancer in vascular po- disruptingtential. properties of the parent compounds [6], to gain enhanced anticancer potential.

2.1. FAA AnaloguesAnalogues Continuing ourour previousprevious studiesstudies andand consideringconsidering thethe limitedlimited SARsSARs availableavailable toto datedate forfor thethe leadlead FAA,FAA, inin 20032003 newnew analoguesanalogues werewere synthesized,synthesized, inin whichwhich anan alkoxyalkoxy groupgroup (mainly(mainly methoxy, then then modified modiﬁed to to ethoxy ethoxy and and isopropoxy) isopropoxy) was was inserted inserted in position in position 3 and 3 anda functionalised a functionalised benzene benzene ring ringor a orfuran a furan moie moietyty were were introduced introduced in position in position 2 [16]. 2 [The16]. Thecompounds compounds (1 and (1 and2, Figure2, Figure 2) were2) were tested tested to toevaluate evaluate both both their their direct direct cytotoxicity cytotoxicity on ondifferent different adenocarcinoma adenocarcinoma cell celllines lines and and their their ability ability to induce to induce an indirect an indirect toxic toxic effect effect me- mediateddiated by bymurine murine macrophages macrophages and and human human monocytes monocytes co-cultured co-cultured with with them. them.

Figure 2.2. 3-Alkoxy FAA derivatives.derivatives.

All compoundscompounds were endowed with very low directdirect cytotoxicity,cytotoxicity, butbut mostmost ofof themthem enhanced thethe antitumourantitumour effecteffect ofof immuneimmune cells,cells, showingshowing remarkableremarkable activityactivity onon humanhuman monocytes, which was was not not observed observed before before with with FAA FAA derivatives, derivatives, and and a certain a certain degree degree of ofspecies species specificity specificity could could also also be benoted noted among among the the different different compounds. compounds. Notably, Notably, activity activ- itywas was lost lost with with the introduction the introduction of a ofmethoxy a methoxy group group in position in position 3 of the 3 offlavone the flavone core, while core, whilecompounds compounds bearing bearing ethoxy ethoxy or isopropoxy or isopropoxy functions functions showed showed activity activity on murine on murinemacro- macrophages,phages, but not but on nothuman on human monocytes. monocytes. The intr Theoduction introduction of substituents of substituents on the 2-phenyl on the 2-phenylring allowed ring activity allowed to be activity restored, tobe even restored, if neither even their if position neither theirnor their position chemical nor prop- their chemicalerties seemed properties to play seemed a significant to play role. a significant The most role.interesting The most compounds interesting proved compounds to be 1a proved to be 1a and 1b (Figure2), carrying electron-withdrawing groups in position 3 0 and 1b (Figure 2), carrying electron-withdrawing groups in position 3′ more potent than more potent than the reference compound. In particular, 1a showed higher activity than the reference compound. In particular, 1a showed higher activity than DMXAA in the DMXAA in the human model, while it had no effect on murine macrophages. The two com- human model, while it had no effect on murine macrophages. The two compounds also pounds also proved to induce TNF-α production by human peripheral blood mononuclear proved to induce TNF-α production by human peripheral blood mononuclear cells cells (HPBMCs). (HPBMCs). As a follow-up of a previous study in which the carboxy function of FAA was moved As a follow-up of a previous study in which the carboxy function of FAA was moved to position 20 on the 2-benzene ring, thus maintaining the spatial relationship with the to position 2′ on the 2-benzene ring, thus maintaining the spatial relationship with the flavone core, and the most interesting compound carried a fluorine atom in position 7 (3, flavone core, and the most interesting compound carried a fluorine atom in position 7 (3, Figure3)[ 17], in a subsequent paper the synthesis of other mono- or difluorinated flavones (4, Figure3)[18] was reported.

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Molecules 2021, 26, 4228 5 of 14 Figure 3) [17], in a subsequent paper the synthesis of other mono- or difluorinated flavones (4, Figure 3) [18] was reported.

Figure 3. Fluorinated FAA derivatives.derivatives.

Direct cytotoxicity for the new compounds was seen only at very high concentrations. When When tested tested for for the the potential potential enhancem enhancementent of antitumour of antitumour activity activity mediated mediated by mac- by macrophagesrophages and andhuman human monocytes, monocytes, it was it was seen seen that that the introduction the introduction of fluorine of fluorine atoms atoms in the in theflavone flavone nucleus nucleus led to led an to increase an increase in activity in activity of murine of murine macrophages, macrophages, while while no effect no effect was wasproduced produced in human in human models models with with any any of the of thenew new derivatives, derivatives, showing showing a similar a similar profile profile to tothe the parent parent compound compound FAA. FAA. In aa moremore recentrecent study,study, somesome previouslypreviously synthesizedsynthesized naturally-inspirednaturally-inspired coumarin,coumarin, flavanoneflavanone and flavonolflavonol derivatives structurally-related to FAA (5, 6,, 7,, FigureFigure4 4)) and and for for whichwhich cytotoxiccytotoxic activityactivity on on a a human human adenocarcinoma adenocarcinoma cell cell line line was was described described [19 [19],], were were re- consideredreconsidered for for further further evaluation. evaluation. In particular, In particular, the potential the potential to activate to activate the immune the immune system α bysystem inducing by inducing lytic properties, lytic properties, TNF- TNF-and nitricα and oxide nitric inoxide a cell in linea cell of line human of human monocytes mon- (Monoocytes (Mono Mac 6, MM6)Mac 6, wasMM6) investigated was investigated [20]. [20].

Figure 4. Figure 4. Naturally-inspired coumarin-, flavanone-flavanone- andand flavonol-basedflavonol-based FAAFAA derivatives.derivatives. Despite the very low cytotoxicity observed, some compounds showed a remarkable Despite the very low cytotoxicity observed, some compounds showed a remarkable indirect toxic effect on tumour cells co-cultured with human MM6 cells, comparable to indirect toxic effect on tumour cells co-cultured with human MM6 cells, comparable to DMXAA for the coumarin-based 5a and 5b and flavonol-based 7 (Figure4). To note, the lack DMXAA for the coumarin-based 5a and 5b and flavonol-based 7 (Figure 4). To note, the of cytotoxic effect for the unsubstituted coumarin (5, R = H) highlighted the role played by lack of cytotoxic effect for the unsubstituted coumarin (5, R = H) highlighted the role theplayed 3-phenyl by the ring 3-phenyl on the ring core on structure, the core and structure, the potency and the of 5b potencyindicated of 5b a positive indicated influence a posi- ofplayed a fluorine by the compared 3-phenyl to ring other on substituentsthe core structure, that led and to inactivethe potency compounds. of 5b indicated The reduction a positive influence of a fluorine compared to other substituents that led to inactive compounds. ofThe the reduction 2–3 double of the bond 2–3 of double flavone bond to give of flavone a flavanone to give core a flavanone (6) seemed core not ( to6) seemed lead to anynot improvementThe reduction inof activitythe 2–3 double with respect bond of to flavone FAA. Coumarins to give a flavanone5a and 5b corewere (6) alsoseemed able not to to lead to any improvement in activity with respect to FAA. Coumarins 5a and 5b were induceto lead TNF-to anyα andimprovement nitric oxide in release.activity with respect to FAA. Coumarins 5a and 5b were also able to induce TNF-α and nitric oxide release. 2.2. DMXAA Analogues Moving from FAA to XAA-analogues and considering the structure of the potent 5,6-DMXAA, different series of molecules were designed and synthetized, in which the substituents in positions 5 and 6 were included in different cyclic structures (8, Figure5) to investigate structural requirements and steric limitations in this part of the molecule [21].

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2.2. DMXAA Analogues Moving from FAA to XAA-analogues and considering the structure of the potent 5,6- Molecules 2021, 26, 4228 6 of 14 DMXAA, different series of molecules were designed and synthetized, in which the substituents in positions 5 and 6 were included in different cyclic structures (8, Figure 5) to investigate structural requirements and steric limitations in this part of the molecule [21]. InIn particular,particular, differentdifferent easily easily synthetically synthetically accessible accessible 5-5- oror 6-membered6-membered oxygenated oxygenated ringsrings werewere introducedintroduced inin thethe structure,structure, sincesince substitutionsubstitution withwith methoxymethoxy groupsgroups hadhad provedproved toto maintainmaintain highhigh activityactivity inin XAAXAA derivativesderivatives [[22].22]. TheThe syntheticsynthetic intermediatesintermediates ((99,, FigureFigure5 5),), carryingcarrying bulkybulky alkoxyalkoxy groupsgroups inin thethe favouredfavoured positionposition 66 ofof thethe xanthonexanthone core,core, werewere tested tested togethertogether withwith thethe cyclic compounds and and their their es estersters for for direct direct cytotoxicity cytotoxicity on on adenocarci- adenocar- cinomanoma cell cell lines lines and and for for indirect indirect toxicity, toxicity, evalua evaluatingting the the increase increase in lytic in lytic properties properties for mu- for murinerine macrophages macrophages and and human human monocytes monocytes co-cultured co-cultured with with tumour tumour cells. cells.

FigureFigure 5.5. CyclicCyclic DMXAADMXAA analoguesanalogues andand alkoxyalkoxy derivatives.derivatives.

DespiteDespite lowlow direct toxicity, the compounds pr provedoved to to induce induce an an increase increase in in the the cyto- cy- totoxictoxic properties properties of ofmurine murine macrophages macrophages comparable comparable to or to higher or higher than than the reference the reference com- compoundpound 5,6-DMXAA. 5,6-DMXAA. Notably, Notably, most most of them of them showed showed activity activity on human on human monocytes, monocytes, and andsome some proved proved to be to more be more potent potent than than the thelead, lead, the theester ester of the of thecyclic cyclic compound compound 8a and8a andboth both prenyloxy prenyloxy derivatives derivatives 9a (Figure9a (Figure 5) 5being) being the the most most interesting interesting derivatives. derivatives. In In addi- ad- α dition,tion, these these compounds compounds were were able able toto induce induce TNF- TNF-α productionproduction in inHPBMCs HPBMCs after after 4 h 4 hof ofincubation. incubation. In a following paper, the xanthone scaffold was decorated in position 6 with saturated In a following paper, the xanthone scaffold was decorated in position 6 with satu- alkoxy chains of different lengths bearing a terminal amino moiety (piperidine or mor- rated alkoxy chains of different lengths bearing a terminal amino moiety (piperidine or pholine), to evaluate the effect of a basic function [23]. The same substitution pattern was morpholine), to evaluate the effect of a basic function [23]. The same substitution pattern also inserted in position 3 of the core structure, to obtain a more complete SAR picture. was also inserted in position 3 of the core structure, to obtain a more complete SAR pic- Molecules 2021, 26, x FOR PEER REVIEWThe compounds (10, Figure6) were tested for direct cytotoxicity and for HPBMCs-mediated7 of 15 ture. The compounds (10, Figure 6) were tested for direct cytotoxicity and for HPBMCs- toxicity, and the induction of TNF-α and nitric oxide was evaluated. mediated toxicity, and the induction of TNF-α and nitric oxide was evaluated.

Figure 6.6. XAA analogues with basicbasic sideside chains.chains.

Results of biologicalbiological evaluation showed, apart from the expected low cytotoxicity, thatthat the introduction of of the the selected selected basic basic si sidede chain chain did did not not lead lead to toan an enhancement enhancement of ofthe the indirect indirect antitumour antitumour effects effects of the of thenew new compounds compounds on human on human monocytes monocytes but allowed but al- in some cases the activity of the reference DMXAA to be retained, confirming that bulky substituents in the appropriate positions on the scaffold can be somewhat tolerated. In particular, 10a, 10b and 10c (Figure 6) proved to be the most potent compounds. In addition, 10a, being able to induce the release of TNF-α and NO, seemed to possess the same biological profile of DMXAA. Later, two XAA-analogues from the abovementioned series (9b and 10c, Figure 7) were selected for further investigations to have a complete picture of their antitumour potential in comparison to the reference DMXAA [24]. For these molecules, the in vitro immune-modulating activity on a human monocyte cell line (MonoMac6 cells, MM6) and in vitro and in vivo antivascular properties were evaluated.

Figure 7. XAA derivatives selected for in vivo evaluation of antivascular properties.

The selected compounds were shown to be able to induce immune modulation comparable to DMXAA; in particular, 10c could stimulate TNF-α production to a higher extent. Notably, the release of TNF-α proved to be inversely dose-dependent, as seen in previous studies [25,26]. DMXAA proved to activate nuclear factor-kappa B (NF-κB) by inactivating its inhibitory factor IκB-α [27] through the allosteric interaction with the IKK kinase protein, triggering its phosphorylation and destruction. The selected compounds and especially 10c significantly induced IκB-phosphorylation. The anti-vascular activity of the compounds was then studied in vitro by measuring the induction of apoptosis in human vein umbilical (HUVEC) cells and in vivo by evaluating growth inhibition of murine sarcoma F (SaF) cells. While DMXAA proved to be able to target and damage the tumour vascular network and to inhibit tumour progression, no such activity was seen for 9b when tested at the same concentrations, and only low induction of apoptosis was observed for 10c.

2.3. Miscellaneous In the same years, other research groups reported new flavonoid-based compounds as derivatives of FAA or XAA. In 2003, some analogues of FAA were designed and tested

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Figure 6. XAA analogues with basic side chains.

Molecules 2021, 26, 4228 7 of 14 Results of biological evaluation showed, apart from the expected low cytotoxicity, that the introduction of the selected basic side chain did not lead to an enhancement of the indirect antitumour effects of the new compounds on human monocytes but allowed lowedin some in cases some the cases activity the activity of the ofreference the reference DMXAA DMXAA to be retained, to be retained, confirming conﬁrming that bulky that bulkysubstituents substituents in the in appropriate the appropriate positions positions on the on scaffold the scaffold can canbe somewhat be somewhat tolerated. tolerated. In Inparticular, particular, 10a10a, 10b, 10b andand 10c10c (Figure(Figure 6)6) proved proved to to be be the the most potent compounds.compounds. InIn addi-addition,tion, 10a10a,, beingbeing ableable toto induceinduce thethe releaserelease ofof TNF-TNF-αα andand NO,NO, seemedseemed toto possesspossess thethe samesame biologicalbiological proﬁleprofile ofof DMXAA.DMXAA. Later,Later, twotwo XAA-analoguesXAA-analogues fromfrom thethe abovementionedabovementioned series (9b and 10c10c,, FigureFigure7 7)) werewere selectedselected forfor furtherfurther investigationsinvestigations toto havehave aa completecomplete picturepicture ofof theirtheir antitumourantitumour potentialpotential inin comparisoncomparison toto thethe referencereference DMXAADMXAA [[24].24]. ForFor thesethese molecules,molecules, thethe inin vitrovitro immune-modulatingimmune-modulating activityactivity onon aa humanhuman monocytemonocyte cellcell lineline (MonoMac6(MonoMac6 cells,cells, MM6)MM6) andand inin vitrovitro andand inin vivovivo antivascularantivascular propertiesproperties werewere evaluated.evaluated.

FigureFigure 7.7. XAA derivativesderivatives selectedselected forfor inin vivovivo evaluationevaluation ofof antivascularantivascular properties.properties.

TheThe selectedselected compoundscompounds werewere shownshown toto bebe able to induce immuneimmune modulation com- parableparable toto DMXAA;DMXAA; in in particular, particular,10c 10ccould could stimulate stimulate TNF- TNF-α productionα production to ato higher a higher extent. ex- Notably,tent. Notably, the release the release of TNF- of αTNF-provedα proved to be to inversely be inversely dose-dependent, dose-dependent, as seen as inseen previous in pre- studiesvious studies [25,26]. [25,26]. DMXAA DMXAA proved proved to activate to activate nuclear nuclear factor-kappa factor-kappa B (NF-κ BB) (NF- by inactivatingκB) by inac- itstivating inhibitory its inhibitory factor IκB- factorα [27 ]I throughκB-α [27] the through allosteric the interaction allosteric interaction with the IKK with kinase the protein,IKK ki- triggeringnase protein, its phosphorylationtriggering its phosphorylation and destruction. and The destruction. selected compounds The selected and compounds especially and10c κ significantlyespecially 10c induced significantly I B-phosphorylation. induced IκB-phosphorylation. The anti-vascular The anti-vascular activity of the activity compounds of the wascompounds then studied was inthen vitro studiedby measuring in vitro by the measuring induction ofthe apoptosis induction in of human apoptosis vein in umbilical human (HUVEC)vein umbilical cells and(HUVEC)in vivo cellsby evaluating and in vivo growth by evaluating inhibition growth of murine inhibition sarcoma of Fmurine (SaF) cells. sar- While DMXAA proved to be able to target and damage the tumour vascular network and coma F (SaF) cells. While DMXAA proved to be able to target and damage the tumour to inhibit tumour progression, no such activity was seen for 9b when tested at the same vascular network and to inhibit tumour progression, no such activity was seen for 9b concentrations, and only low induction of apoptosis was observed for 10c. when tested at the same concentrations, and only low induction of apoptosis was ob- Molecules 2021, 26, x FOR PEER REVIEWserved for 10c. 8 of 15 2.3. Miscellaneous In the same years, other research groups reported new flavonoid-based compounds as 2.3. Miscellaneous derivatives of FAA or XAA. In 2003, some analogues of FAA were designed and tested as inhibitorsas inhibitorsIn the of same aminopeptidaseof aminopeptidase years, other Nresearch (APN/CD13),N (APN/CD1 groups 3), overexpressedreported overexpressed new flavonoid-based in somein some tumours, tumours, compounds located located in bothasin bothderivatives human human and of and FAA murine murine or XAA. endothelial endothelial In 2003, vasculature somevasculat analoguesure but but not notof inFAA in normal normal were vessels. designedvessels. ToTo and thisthis tested aim,aim, 3-nitroFAA3-nitroFAA derivativesderivatives ((1111,, FigureFigure8 8)) bearing bearing different different substituents substituents on on the the 2-phenyl 2-phenyl ring ring (mainly(mainly nitronitro oror methoxymethoxy groups),groups), alongalong withwith structurallystructurally constrainedconstrained analoguesanalogues ((1212––1313,, FigureFigure8 8)) were were designed designed and and synthesized synthesized [ [28].28]. In particular, compound 11a (Figure(Figure8 8),), carryingcarrying nitronitro groupsgroups inin positionspositions 33 andand 220′,, provedproved toto efficientlyefficiently andand selectivelyselectively inhibitinhibit APN/CD13,APN/CD13, while while modifications modifications in the position of the substituent, its removal or replace- mentment withwith aa methoxy,methoxy, ledled toto reductionreduction ofof thethe activity.activity.

FigureFigure 8.8. FAA analoguesanalogues asas inhibitorsinhibitors ofof aminopeptidaseaminopeptidase N.N.

InIn orderorder toto obtainobtain moremore informationinformation aboutabout thethe mechanismmechanism ofof actionaction ofof FAAFAA andand toto identifyidentify itsits biologicalbiological target,target, inin 20052005 aa newnew analogueanalogue waswas designed,designed, carryingcarrying anan additionaladditional functional group that could react with a specific peptide tag as an affinity label, allowing the drug-bound protein to be located [29]. In detail, an azide moiety was introduced in the para position on the 2-phenyl ring giving compound 14 (Figure 9), which proved to maintain the biological activity observed with the parent compound and could be activated using the Staudinger reaction, by reacting it with a FLAG peptide–phosphine in mild conditions, similar to those used for standard biological assays.

Figure 9. FAA analogue as affinity label.

The potential use of the azide moiety to develop analogues of the potent DMXAA as photoaffinity labels, that would covalently bind target proteins, was investigated by Ching and coworkers [30]. Here, this function was selected due to synthetic accessibility and lack of steric issues with respect to other photoreactive moieties, and it was introduced on the xanthone core in the most favourable positions for substitution, identified with previous SAR studies, giving compounds 15a–c (Figure 10). While 15c proved to be chemically unstable and was not studied further, the activities of 15a and 15b were evaluated, and both were shown to retain the biological profile of the parent compound, although more toxic and less potent. Moreover, 15b proved to undergo photoreaction to specifically bind to proteins extracted from murine splenocytes.

Figure 10. DMXAA analogues as photoaffinity labels.

Molecules 2021, 26, x FOR PEER REVIEW 8 of 15 Molecules 2021, 26, x FOR PEER REVIEW 8 of 15

as inhibitors of aminopeptidase N (APN/CD13), overexpressed in some tumours, located inas bothinhibitors human of andaminopeptidase murine endothelial N (APN/CD1 vasculat3),ure overexpressed but not in normal in some vessels. tumours, To this located aim, 3-nitroFAAin both human derivatives and murine (11, endothelialFigure 8) bearing vasculat differenture but notsubstituents in normal on vessels. the 2-phenyl To this aim,ring (mainly3-nitroFAA nitro derivatives or methoxy (11 grou, Figureps), along8) bearing with differentstructurally substituents constrained on analoguesthe 2-phenyl (12 –ring13, Figure(mainly 8) nitro were or designed methoxy andgrou syntheps), alongsized with [28]. structurally In particular, constrained compound analogues 11a (Figure (12– 138),, carryingFigure 8) nitro were groups designed in positionsand synthe 3 sizedand 2 ′[28]., proved In particular, to efficiently compound and selectively 11a (Figure inhibit 8), APN/CD13,carrying nitro while groups modifications in positions in the3 and position 2′, proved of the to substituent, efficiently itsand removal selectively or replace- inhibit mentAPN/CD13, with a methoxy,while modifications led to reduction in the ofposition the activity. of the substituent, its removal or replace- ment with a methoxy, led to reduction of the activity.

Figure 8. FAA analogues as inhibitors of aminopeptidase N. Figure 8. FAA analogues as inhibitors of aminopeptidase N. Molecules 2021, 26, 4228 8 of 14 In order to obtain more information about the mechanism of action of FAA and to identifyIn order its biological to obtain target, more in information 2005 a new analogueabout the wasmechanism designed, of carrying action of an FAA additional and to functionalidentify its group biological that target,could reactin 2005 with a new a specific analogue peptide was designed,tag as an affi carryingnity label, an additional allowing thefunctional drug-bound group protein thatthat couldcould to be react located withwith [29]. aa speciﬁcspecific In detail, peptidepeptide an azide tagtag asas moiety anan afﬁnityaffi nitywas label,label, introduced allowingallowing in thethe drug-boundparadrug-bound position proteinon the 2-phenyl toto bebe locatedlocated ring [[29].giving29]. InIn compound detail,detail, anan azideazide 14 (Figure moietymoiety 9), waswas which introducedintroduced proved intoin maintainthe para position the biological on thethe activity 2-phenyl2-phenyl observed ringring givinggiving with compoundcompound the parent 14 compound (Figure(Figure9 9),), and which which could proved proved be acti- to to vatedmaintain using thethe the biologicalbiological Staudinger activity activity reaction, observed observed by with reacting with the the parent it parentwith compound a compoundFLAG peptide–phosphine and and could could be activated be acti- in mildusingvated conditions, theusing Staudinger the Staudingersimilar reaction, to those reaction, by used reacting byfor reactingstandard it with it biological awith FLAG a FLAG peptide–phosphineassays. peptide–phosphine in mild in conditions,mild conditions, similar similar to those to those used forused standard for standard biological biological assays. assays.

Figure 9. FAA analogue as affinity label. Figure 9. FAA analogue asas affinityaffinity label.label. The potential use of the azide moiety to develop analogues of the potent DMXAA as The potential use of the azide moiety to develop analoguesanalogues ofof thethe potentpotent DMXAADMXAA asas photoaffinityphotoaffinity labels,labels, that that would would covalently covalently bind bind target target proteins, proteins, was investigatedwas investigated by Ching by photoaffinity labels, that would covalently bind target proteins, was investigated by Chingand coworkers and coworkers [30]. Here, [30]. this Here, function this function was selected was selected due to synthetic due to synthetic accessibility accessibility and lack Ching and coworkers [30]. Here, this function was selected due to synthetic accessibility andof steric lack issuesof steric with issues respect with to otherrespect photoreactive to other photoreactive moieties, and moieties, it was introducedand it was onintro- the and lack of steric issues with respect to other photoreactive moieties, and it was intro- xanthoneduced on corethe xanthone in the most core favourable in the most positions favourable for substitution, positions for identified substitution, with identified previous duced on the xanthone core in the most favourable positions for substitution, identified withSAR previous studies, givingSAR studies, compounds giving15a compounds–c (Figure 15a 10).–c While(Figure15c 10).proved While to15c be proved chemically to be with previous SAR studies, giving compounds 15a–c (Figure 10). While 15c proved to be chemicallyunstable and unstable was not and studied was further,not studied the activities further, ofthe15a activitiesand 15b ofwere 15a evaluated,and 15b were and eval- both chemically unstable and was not studied further, the activities of 15a and 15b were eval- wereuated, shown and both to retain were theshown biological to retain profile the bi ofological the parent profile compound, of the parent although compound, more toxic alt- uated, and both were shown to retain the biological profile of the parent compound, alt- houghand less more potent. toxic Moreover, and less 15bpotent.proved Moreover, to undergo 15b proved photoreaction to undergo to specifically photoreaction bind to hough more toxic and less potent. Moreover, 15b proved to undergo photoreaction to proteinsspecifically extracted bind to from proteins murine extracted splenocytes. from murine splenocytes. specifically bind to proteins extracted from murine splenocytes.

Figure 10. DMXAA analogues as ph photoaffinityotoaffinity labels. Figure 10. DMXAA analogues as photoaffinity labels. 3. Targeted SAR Studies 2013–2021 In the first twenty years following DMXAA discovery, despite extensive medicinal chemistry efforts, no significant improvement of its biological profile nor better comprehen-

sion of the molecular basis of its mechanism of action were achieved. In 2012, the discovery of the possible involvement of the STING pathway in DMXAA anticancer action paved the way for a renaissance of the studies on this molecule [13]. STING is a transmembrane protein involved in sustaining innate immune responses in the tumour microenvironment. The formation of cytosolic chromatin fragments is often related to malignant transformation, and DNA leakage in cancer cells cytosol may occur, triggering the innate immunity response; the cyclic GMP–AMP synthase-stimulator of interferon genes (cGAS-STING) pathway gets activated, leading to the expression of type I interferon (IFN) in cancer cells, initiating innate anti-cancer immunity. The acknowledged role of cGAS-STING pathway in sustaining anti-tumour innate and adaptive immunity makes its pharmacological activation a valuable strategy to be exploited for anticancer therapy [31]. The ability of DMXAA to directly bind STING was proved in multiple murine models and was associated with a consistent tumour regression. Unfortunately, it was found that the affinity of this drug for STING was limited to murine protein (mSTING), since in human cells this interaction was unable to induce type I IFN expression [14]. Despite these disappointing results, probably due to STING polymorphism, the possibility to focus research on a specific target has led to a revival of studies on DMXAA, its structure and its derivatives. Therefore, different research groups returned to the flavonoid-related structures of FAA and XAA to Molecules 2021, 26, x FOR PEER REVIEW 9 of 15

3. Targeted SAR Studies 2013–2021 In the first twenty years following DMXAA discovery, despite extensive medicinal chemistry efforts, no significant improvement of its biological profile nor better compre- hension of the molecular basis of its mechanism of action were achieved. In 2012, the discovery of the possible involvement of the STING pathway in DMXAA anticancer action paved the way for a renaissance of the studies on this molecule [13]. STING is a transmembrane protein involved in sustaining innate immune responses in the tumour microenvironment. The formation of cytosolic chromatin fragments is often related to malignant transformation, and DNA leakage in cancer cells cytosol may occur, triggering the innate immunity response; the cyclic GMP–AMP synthase-stimulator of interferon genes (cGAS-STING) pathway gets activated, leading to the expression of type I interferon (IFN) in cancer cells, initiating innate anti-cancer immunity. The acknowledged role of cGAS- STING pathway in sustaining anti-tumour innate and adaptive immunity makes its pharmacological activation a valuable strategy to be exploited for anticancer therapy [31]. The ability of DMXAA to directly bind STING was proved in multiple murine models and was associated with a consistent tumour regression. Unfortunately, it was found that the affinity of this drug for STING was limited to murine protein (mSTING), since in human cells this interaction was unable to induce type I IFN expression [14]. Despite these disap- Molecules 2021, 26, 4228 pointing results, probably due to STING polymorphism, the possibility to focus research9 of 14 on a specific target has led to a revival of studies on DMXAA, its structure and its derivatives. Therefore, different research groups returned to the flavonoid-related structures of developFAA and new XAA analogues, to develop for new which analogues, both antivascular for which both and immunomodulatingantivascular and immunomod- properties wereulating evaluated. properties were evaluated.

3.1. Recently Developed FAA DerivativesDerivatives In 2014,2014, aa novel novel series series of of FAA FAA derivatives derivatives was was synthesized synthesized bearing bearing a 2,3-diarylchromone a 2,3-diarylchro- scaffoldmone scaffold (16, Figure (16, Figure11), designed 11), designed by considering by considering the structures the structures and antitumour and antitumour activities ac- of literaturetivities of analoguesliterature andanalogues known and isoﬂavones known [isoflavones32]. The new [32]. derivatives The new were derivatives characterized were bycharacterized a methoxy by group a methoxy in position group 7 andin position methoxy- 7 and or halogen-substituentsmethoxy- or halogen-substituents on the benzene on rings.the benzene Thecompounds rings. The compounds showed moderate showed cytotoxicitymoderate cytotoxicity and, more and, interesting, more interesting,16a and 16b16a (Figureand 16b 11 (Figure) were endowed11) were endowed with indirect with toxicity indirect on toxicity A549 lung on A549 adenocarcinoma lung adenocarci- cells thatnoma proved cells that to beproved higher to whenbe higher cells when were cells co-cultured were co-cultured with HPBMCs with HPBMCs than with than murine with macrophages,murine macrophages, in line within line the with reference the reference DMXAA. DMXAA. In addition, In addition,16a was 16a able was to induceable to TNF-induceα productionTNF-α production to a higher to a extenthigher with extent respect with torespect DMXAA. to DMXAA.

Figure 11. 2,3-Diarylchromones as FAA derivatives.derivatives.

Later, the same same research research group group synthesized synthesized another another series series of of1616-based-based analogues, analogues, in inwhich which the the 7-methoxy 7-methoxy group group was was removed removed (17 (, 17Figure, Figure 11), 11 which), which was was more more potent potent than than the theprevious previous ones ones [33]. [In33 particular,]. In particular, while whilecompounds compounds carrying carrying electron electron withdrawing withdrawing groups groups on the phenyl rings showed moderate direct cytotoxic activity, methoxy-substituted analogues proved to be endowed with indirect toxicity comparable or higher than the reference DMXAA and to induce TNF-α production, 17a and 17b (Figure 11) being the most interesting compounds. FAA is known to be metabolized by murine microsomes to give different hydroxy- and epoxy-derivatives, but it undergoes very low metabolism in human microsomes. Recently, the two main metabolites, 40-OH-FAA and 6-OH-FAA (Figure 12), extensively found in mice but not in humans, were tested for their antivascular effects on EA.hy926 cells, derived from the fusion of human umbilical vein endothelial cells with the permanent human A549 cell line [34]. Notably, 6-OH-FAA was able to induce morphological changes at signiﬁcantly lower concentrations with respect to 40-OH-FAA and FAA itself, and to disrupt preformed capillary networks. In addition, it inhibited microtubules formation by activating the RhoA/Rho signalling pathway. Based on these ﬁndings, it was suggested that the potent antivascular activity of FAA in mice, and the lack of activity in human tumour cells, could be mediated by its metabolite 6-OH-FAA.

3.2. Recently Developed DMXAA Derivatives To gain more insight into the peculiar selectivity for murine tumour cells and the consequent lack of antitumour effect on human cells, demonstrated by DMXAA and with the aim of identifying effective human antitumour agents, the previously synthesized analogues carrying methyl groups in different positions on the xanthone scaffold (18, Figure 13) were Molecules 2021, 26, x FOR PEER REVIEW 10 of 15

on the phenyl rings showed moderate direct cytotoxic activity, methoxy-substituted analogues proved to be endowed with indirect toxicity comparable or higher than the reference DMXAA and to induce TNF-α production, 17a and 17b (Figure 11) being the most interesting compounds. FAA is known to be metabolized by murine microsomes to give different hydroxy- and epoxy-derivatives, but it undergoes very low metabolism in human microsomes. Re- Molecules 2021, 26, x FOR PEER REVIEWcently, the two main metabolites, 4′-OH-FAA and 6-OH-FAA (Figure 12), extensively10 of 15

found in mice but not in humans, were tested for their antivascular effects on EA.hy926 cells, derived from the fusion of human umbilical vein endothelial cells with the perma- nenton the human phenyl A549 rings cell showed line [34]. moderate Notably, direct 6-OH-FAA cytotoxic wasactivity, able methoxy-substitutedto induce morphological ana- changeslogues proved at significantly to be endowed lower cowithncentrations indirect to withxicity respect comparable to 4′-OH-FAA or higher and than FAA the itself,refer- andence to DMXAA disrupt andpreformed to induce capillary TNF-α networks. production, In 17aaddition, and 17b it inhibited (Figure 11) microtubules being the most for- mationinteresting by activatingcompounds. the RhoA/Rho signalling pathway. Based on these findings, it was suggestedFAA isthat known the potent to be metabolizedantivascular activityby murine of FAAmicrosomes in mice, toand give the different lack of activity hydroxy- in humanand epoxy-derivatives, tumour cells, could but beit undergoes mediated byvery its low metabolite metabolism 6-OH-FAA. in human microsomes. Re- cently, the two main metabolites, 4′-OH-FAA and 6-OH-FAA (Figure 12), extensively found in mice but not in humans, were tested for their antivascular effects on EA.hy926 cells, derived from the fusion of human umbilical vein endothelial cells with the permanent human A549 cell line [34]. Notably, 6-OH-FAA was able to induce morphological Molecules 2021, 26, 4228 10 of 14 changes at significantly lower concentrations with respect to 4′-OH-FAA and FAA itself, and to disrupt preformed capillary networks. In addition, it inhibited microtubules formation by activating the RhoA/Rho signalling pathway. Based on these findings, it was considered,Figuresuggested 12. Representative that in 2013,the potent by FAA Ching antivascular metabolites and coworkers activityin murine [ 35of cells.], FAA and in the mice, pattern and of the cytokine lack of inductionactivity in andhuman the tumour anti-vascular cells, could effects be were medi evaluatedated by its in metabolite both human 6-OH-FAA. and murine leukocytes. 3.2. Recently Developed DMXAA Derivatives To gain more insight into the peculiar selectivity for murine tumour cells and the consequent lack of antitumour effect on human cells, demonstrated by DMXAA and with the aim of identifying effective human antitumour agents, the previously synthesized analogues carrying methyl groups in different positions on the xanthone scaffold (18, Figure 13) were considered, in 2013, by Ching and coworkers [35], and the pattern of cytokine induction and the anti-vascular effects were evaluated in both human and murine leuko- Figurecytes. 12.12. Representative FAA metabolitesmetabolites inin murinemurine cells.cells.

3.2. Recently Developed DMXAA Derivatives To gain more insight into the peculiar selectivity for murine tumour cells and the consequent lack of antitumour effect on human cells, demonstrated by DMXAA and with the aim of identifying effective human antitumour agents, the previously synthesized analogues carrying methyl groups in different positions on the xanthone scaffold (18, Figure 13) were considered, in 2013, by Ching and coworkers [35], and the pattern of cytokine Figure 13. Methyl-substituted DMXAA analogues. induction and the anti-vascular effects were evaluated in both human and murine leukocytes. Results clearlyclearly indicatedindicated distinct distinct biological biological effects effects for for the the different different monomethyl monomethyl deriva- de- tives,rivatives, since since substitution substitution in positions in positions 3, 5 3, and 5 and 6 led 6 led to compoundsto compounds with with significant significant activ- ac- ity on murine cells, as the disubstituted 5,6-DMXAA, whereas 2-, 7- and 8-methylXAA tivity on murine cells, as the disubstituted 5,6-DMXAA, whereas 2-, 7- and 8-methylXAA showed remarkable selectivity for human cells and proved to be inactive in the murine showed remarkable selectivity for human cells and proved to be inactive in the murine model. In particular, 8-methylXAA (18a, Figure 13) and the disubstituted 7,8-DMXAA model. In particular, 8-methylXAA (18a, Figure 13) and the disubstituted 7,8-DMXAA (18b, Figure 13) proved to efficiently induce a panel of cytokines (IL-6 and IL-8) in human (18b, Figure 13) proved to efficiently induce a panel of cytokines (IL-6 and IL-8) in human leukocytes as well as a significant antivascular effect, through inhibition of tube forma- Molecules 2021, 26, x FOR PEER REVIEWleukocytes as well as a significant antivascular effect, through inhibition of tube11 offor- 15 tion. Notably, the lack of biological effects seen in murine systems for these compounds mation. Notably, the lack of biological effects seen in murine systems for these compounds calledFigure attention13. Methyl-substituted to the need DMXAA for appropriate analogues. models for a more appropriate evaluation of called attention to the need for appropriate models for a more appropriate evaluation of the potential antitumour activity of this class of molecules. Considering the very recent the potential antitumour activity of this class of molecules. Considering the very recent thatidentification differencesResults clearly of in STING murine indicated as and a potential humandistinct proteinmolecularbiological could targeteffects be responsible of for DMXAA, the different for it wasthe monomethyl peculiar also suggested species de- identification of STING as a potential molecular target of DMXAA, it was also suggested specificitiesthatrivatives, differences since observed insubstitution murine in the and SARin humanpositions of these protein 3, XAA 5 and could derivatives. 6 led be responsibleto compounds for thewith peculiar significant species ac- specificitiestivityDifferent on murine observed series cells, of in asxanthone the the SAR disubstitute analogues of thesed XAA 5,6-DMXAA,were derivatives. also synthesized, whereas 2-, in 7-which and the8-methylXAA carboxylic acidshowed Differentwas remarkable modified series to ofselectivity obtain xanthone esters, for analogues humanamides, werecells arylidene and also proved synthesized, hydrazides, to be in inactivediacylhydrazides which thein the carboxylic murine and acylacidmodel. thiosemicarbazides was In modified particular, to obtain8-methylXAA (19, esters,Figure amides, 14)(18a [36]., Figurearylidene The compounds13) and hydrazides, the weredisubstituteddiacylhydrazides tested for 7,8-DMXAA both direct and cytotoxicityacyl(18b thiosemicarbazides, Figure against13) proved a panel (to19 efficiently, Figureof tumour 14 induce)[ 36cells].The anda panel compounds antiangiogen of cytokines wereic properties(IL-6 tested and for IL-8) bothby evaluating in direct human cy-

tubetotoxicityleukocytes formation against as well and a panel migrationas a ofsignificant tumour in human cells antivascular andvein antiangiogenic umbilical effect, (HUVEC) through properties cells.inhibition by Among evaluating of thesetubetube for-derivatives,formationmation. Notably, some and migration amides the lack and in of human biological arylidene vein effect umbilicalhydrazs seenides (HUVEC) in showed murine cells. systemscytotoxic Among for activity these these compounds derivatives, in the mi- cromolarsomecalled amides attention range and and,to arylidene the remarkably, need hydrazidesfor appropriate were showedproven models cytotoxicto induce for a activitymoremorphological appropriate in the micromolar changes evaluation in range HU- of VECand,the potential remarkably,cells and antitumour reduce were provencell activitymigration to induce of (whereasthis morphological class noof molecules.such changes effect Consideringwas in HUVEC shown by cellsthe the very and reference reducerecent DMXAA).cellidentification migration In particular,of (whereas STING no as19a sucha potential(Figure effect 14) wasmolecular emerged shown target byas thethe of referencemost DMXAA, interesting DMXAA). it was compound also In particular,suggested and 19awas(Figure the subject 14) emerged of docking as thestudies most that interesting suggested compound its potential and wasinteraction the subject with of VEGFR2 docking andstudies EGFR. that suggested its potential interaction with VEGFR2 and EGFR.

Figure 14. Carboxylic acid derivatives of DMXAA.

The same same research research group group synthesized synthesized three three DMXAA DMXAA ester ester or oramide amide derivatives, derivatives, in whichin which the thecarbonyl carbonyl function function in position in position 9 was 9 removed was removed via the via formation the formation of a 2,4-dinitro- of a 2,4- phenylhydrazone function (20, Figure 15) [37]. For the new compounds (together with their xanthone counterpart) only preliminary cytotoxicity studies on different cell lines were performed, suggesting activity in the micromolar range for the two xanthone amides and for the hydrazone 20a (Figure 15) in some of the tested cell lines.

Figure 15. DMXAA hydrazone derivatives.

Considering the identification of mSTING as the molecular target of DMXAA and the evidence of species specificity, new analogues carrying different H-bond acceptor or donor groups in position 7 of the xanthone core were recently designed (21, Figure 16) based on the X-ray crystal structure of DMXAA bound to a mutated human STING (hSTING) that suggested a potential additional H-bond interaction for 7-substituted derivatives [38]. The compounds and their ester synthetic intermediates were tested to assess their binding to both the murine and human proteins and, while bromo- and hydroxy- xanthones showed promising affinity for mSTING, none of the synthesized molecules proved to efficiently bind to the human counterpart.

Molecules 2021, 26, x FOR PEER REVIEW 11 of 15

that differences in murine and human protein could be responsible for the peculiar species specificities observed in the SAR of these XAA derivatives. Different series of xanthone analogues were also synthesized, in which the carboxylic acid was modified to obtain esters, amides, arylidene hydrazides, diacylhydrazides and acyl thiosemicarbazides (19, Figure 14) [36]. The compounds were tested for both direct cytotoxicity against a panel of tumour cells and antiangiogenic properties by evaluating tube formation and migration in human vein umbilical (HUVEC) cells. Among these derivatives, some amides and arylidene hydrazides showed cytotoxic activity in the micromolar range and, remarkably, were proven to induce morphological changes in HU- VEC cells and reduce cell migration (whereas no such effect was shown by the reference DMXAA). In particular, 19a (Figure 14) emerged as the most interesting compound and was the subject of docking studies that suggested its potential interaction with VEGFR2 and EGFR.

Figure 14. Carboxylic acid derivatives of DMXAA. Molecules 2021, 26, 4228 11 of 14 The same research group synthesized three DMXAA ester or amide derivatives, in which the carbonyl function in position 9 was removed via the formation of a 2,4-dinitro- dinitrophenylhydrazonephenylhydrazone function function (20, Figure (20, Figure 15) 15[37].) [37 For]. For the the ne neww compounds compounds (together (together with theirtheir xanthonexanthone counterpart)counterpart) onlyonly preliminarypreliminary cytotoxicitycytotoxicity studiesstudies onon differentdifferent cellcell lineslines werewere performed,performed, suggestingsuggesting activityactivity inin thethe micromolarmicromolar rangerange forfor thethe twotwo xanthonexanthone amidesamides and forfor thethe hydrazonehydrazone 20a20a (Figure(Figure 1515)) inin somesome ofof thethe testedtested cellcell lines.lines.

Figure 15.15. DMXAA hydrazone derivatives.

Considering thethe identificationidentification of of mSTING mSTING as as the the molecular molecular target target of DMXAAof DMXAA and and the evidencethe evidence of species of species specificity, specificity, new analoguesnew analogues carrying carrying different different H-bond H-bond acceptor acceptor or donor or groupsdonor groups in position in position 7 of the 7 xanthoneof the xanthone core were core recently were recently designed designed (21, Figure (21, Figure16) based 16) onbased the X-rayon the crystal X-ray structurecrystal structure of DMXAA of DMXAA bound to bound a mutated to a humanmutated STING human (hSTING) STING that(hSTING) suggested that asuggested potential additionala potential H-bondaddition interactional H-bond forinteraction 7-substituted for 7-substituted derivatives [ 38de-]. Therivatives compounds [38]. The and compounds their ester and synthetic their ester intermediates synthetic intermediates were tested to were assess tested their to binding assess to both the murine and human proteins and, while bromo- and hydroxy-xanthones showed their binding to both the murine and human proteins and, while bromo- and hydroxy- Molecules 2021, 26, x FOR PEER REVIEWpromising affinity for mSTING, none of the synthesized molecules proved to efficiently12 of 15 xanthones showed promising affinity for mSTING, none of the synthesized molecules bind to the human counterpart. proved to efficiently bind to the human counterpart.

Figure 16. 7-Substituted DMXAA analogues.

3.3. DMXAA Combinations/Hybrids Recently, DMXAADMXAA waswas studiedstudied asas partpart ofof aa drugdrug combinationcombination involvinginvolving variouslyvariously decorated xanthones (22, FigureFigure 1717),), aimedaimed atat evaluatingevaluating thethe possiblepossible synergisticsynergistic effectseffects of the two structurally-related moleculesmolecules [[39]39].. TheThe synthesizedsynthesized xanthonesxanthones werewere testedtested forfor direct cytotoxicity toward different humanhuman tumourtumour cellcell lines,lines, andand onlyonly thethe 1,3-dihydroxy1,3-dihydroxy substitution pattern,pattern, togethertogether with with electron electron withdrawing withdrawing groups, groups, seemed seemed to to grant grant activity activity in thein the low low micromolar micromolar range, range, as anyas any modiﬁcation modificati provedon proved to be to detrimental.be detrimental. The The most most potent po- compounds were then tested in combination with DMXAA (1:1 molar ratio), which proved tent compounds were then tested in combination with DMXAA (1:1 molar ratio), which to enhance their cytotoxicity, especially for compound 22a (Figure 17) on MDA-MB-231 proved to enhance their cytotoxicity, especially for compound 22a (Figure 17) on MDA- cells. Furthermore, for this compound and its combination with DMXAA, the induction of MB-231 cells. Furthermore, for this compound and its combination with DMXAA, the in- apoptosis and the regulation of p53/MDM2 were observed. duction of apoptosis and the regulation of p53/MDM2 were observed. In a following study, the combination of DMXAA and pyranoxanthone (Figure 18), a novel xanthone-based inhibitor of p53-MDM2 interaction, was evaluated, and four hybrid molecules were synthesized by coupling the two synthons through an alkyl tether (23, Figure 18)[40]. Both the combination of the two molecules and the hybrids showed higher cytotoxic activity than the single agents, particularly on MCF-7 and HepG-2 cells, the hybrids with longer linker chains being the most interesting compounds, with activities

Figure 17. Novel antitumour xanthones for DMXAA combinations.

In a following study, the combination of DMXAA and pyranoxanthone (Figure 18), a novel xanthone-based inhibitor of p53-MDM2 interaction, was evaluated, and four hybrid molecules were synthesized by coupling the two synthons through an alkyl tether (23, Figure 18) [40]. Both the combination of the two molecules and the hybrids showed higher cytotoxic activity than the single agents, particularly on MCF-7 and HepG-2 cells, the hybrids with longer linker chains being the most interesting compounds, with activities in the submicromolar range. Moreover, the hybrid with a 6-carbon tether proved to induce apoptosis and to regulate p53/MDM2 to a higher extent with respect to the single synthons and the combination.

Figure 18. Pyranoxanthone and hybrid derivatives.

The vascular shutdown caused by treatment with VDAs such as DMXAA has been shown to be associated with the establishment of hypoxic and necrotic areas in the tumour, which would induce the activation of HIF-1α protein, subsequently leading to the

Molecules 2021, 26, x FOR PEER REVIEW 12 of 15

Molecules 2021, 26, x FOR PEER REVIEW 12 of 15 Figure 16. 7-Substituted DMXAA analogues.

3.3. DMXAA Combinations/Hybrids Recently, DMXAA was studied as part of a drug combination involving variously decorated xanthones (22, Figure 17), aimed at evaluating the possible synergistic effects of the two structurally-related molecules [39]. The synthesized xanthones were tested for direct cytotoxicity toward different human tumour cell lines, and only the 1,3-dihydroxy substitution pattern, together with electron withdrawing groups, seemed to grant activity in the low micromolar range, as any modification proved to be detrimental. The most potent compounds were then tested in combination with DMXAA (1:1 molar ratio), which provedFigure 16. to 7-Substituted enhance their DMXAA cytotoxi analogues.city, especially for compound 22a (Figure 17) on MDA- MB-231 cells. Furthermore, for this compound and its combination with DMXAA, the in- duction3.3. DMXAA of apoptosis Combinations/Hybrids and the regula tion of p53/MDM2 were observed. Recently, DMXAA was studied as part of a drug combination involving variously decorated xanthones (22, Figure 17), aimed at evaluating the possible synergistic effects of the two structurally-related molecules [39]. The synthesized xanthones were tested for direct cytotoxicity toward different human tumour cell lines, and only the 1,3-dihydroxy Molecules 2021, 26, 4228 substitution pattern, together with electron withdrawing groups, seemed to grant activity12 of 14 in the low micromolar range, as any modification proved to be detrimental. The most potent compounds were then tested in combination with DMXAA (1:1 molar ratio), which inFigureproved the submicromolar17. to Novel enhance antitumour their range. cytotoxi xanthones Moreover,city, for especially theDMXAA hybrid combinations. for with compound a 6-carbon 22a tether (Figure proved 17) toon induceMDA- apoptosisMB-231 cells. and Furthermore, to regulate p53/MDM2 for this compound to a higher and extent its combination with respect towith the DMXAA, single synthons the in- andduction theIn a combination.of following apoptosis study, and the the regula combinationtion of p53/MDM2 of DMXAA were and pyranoxanthoneobserved. (Figure 18), a novel xanthone-based inhibitor of p53-MDM2 interaction, was evaluated, and four hybrid molecules were synthesized by coupling the two synthons through an alkyl tether (23, Figure 18) [40]. Both the combination of the two molecules and the hybrids showed higher cytotoxic activity than the single agents, particularly on MCF-7 and HepG-2 cells, the hybrids with longer linker chains being the most interesting compounds, with activities in the submicromolar range. Moreover, the hybrid with a 6-carbon tether proved to induce apoptosis and to regulate p53/MDM2 to a higher extent with respect to the single FiguresynthonsFigure 17.17. Noveland the antitumourantitumour combination. xanthonesxanthones forfor DMXAADMXAA combinations.combinations.

induce apoptosis and to regulate p53/MDM2 to a higher extent with respect to the single Figuresynthons 18. Pyranoxanthoneand the combination. and hybrid derivatives. The vascular shutdown caused by treatmenttreatment with VDAsVDAs suchsuch asas DMXAADMXAA hashas beenbeen shown toto bebe associated associated with with the the establishment establishment of hypoxicof hypoxic and and necrotic necrotic areas areas in the in tumour, the tu- whichmour, wouldwhich inducewould theinduce activation the activation of HIF-1 ofα protein,HIF-1α protein, subsequently subsequently leading to leading the develop- to the ment of new blood vessels and immunosuppression, promoting tumour regrowth. Thus, the potential synergistic effect that could be obtained with the combination of DMXAA and digoxin, a known inhibitor of HIF-1α expression [41], was recently considered [42]. The administration of these agents in B16-F10 murine melanoma showed remarkable reduction of tumour blood vessels and enhanced induction of immune cells, together with a

reduction of the expression of HIF-1α, increasing treatment efficacy. Figure 18. Pyranoxanthone and hybrid derivatives. 4. Conclusions and Future Directions The vascular shutdown caused by treatment with VDAs such as DMXAA has been Cancer treatment undoubtedly represents one of the greatest challenges for medicinal shown to be associated with the establishment of hypoxic and necrotic areas in the tu- chemistry and pharmacology, and exploring new therapeutic strategies and targets is mour, which would induce the activation of HIF-1α protein, subsequently leading to the always a valuable research approach. Angiogenesis is essential for tumour progression and metastases development, and VDAs are a peculiar class of anticancer compounds aimed at disrupting established tumour vasculature. A number of NPs were found to act as VDAs and constitute a source of structural templates to be properly modified in order to increase the therapeutic potential and combine different anticancer mechanisms. The synthetic flavone-based FAA and its derivatives, mostly DMXAA and other xanthone-related compounds, seemed to have achieved this goal, but finally gave disappointing results in clinical trials, revealing a species-specificity in their mechanism of action that made them ineffective in humans. Despite a number of chemical modifications that expanded the SAR of these scaffolds and the ability of some derivatives to target human cells, no significant improvement was observed up to 2012, when the discovery of STING as a possible dis- tinctive target for DMXAA gave a second chance to these structures. New encouraging medicinal chemistry studies are now underway, also taking advantage of a more focused target selection to evaluate both antivascular effects and immunomodulation. Indeed, promising VDA candidates were recently identified, worthy of further evaluation. Furthermore, several beneficial features of DMXAA must be taken into account, in particular its low toxicity and well-characterized and predictable pharmacokinetics. Moreover, this compound showed a good potential for combination therapy with a wide number of antitumour drugs, acting with different molecular mechanisms. Considering future medicinal chemistry perspectives, this allows to keep the spotlight on this structural core, Molecules 2021, 26, 4228 13 of 14

aiming at performing further modifications to obtain more effective new analogues and multipotent ligands, able to engage different targets involved in cancer. The multitarget approach should, however, be carefully considered, as it can indeed allow to improve the compliance of a patient that is often overloaded by different drugs, but it can reasonably lead to the development of molecules endowed with worse pharmacokinetic properties with respect to the single entities. On the other hand, the identification of a potential target and the related latest findings could bring back attention to previously reported FAA and XAA derivatives that could undergo further investigations. More focused biological evaluation could open the way for the design and synthesis of novel libraries of variously decorated compounds, leading to a better definition of the structural requirements to obtain human tumour active VDAs. A suitable model for in vivo testing appears then as an imperative requirement to exploit this huge potential.

Author Contributions: Conceptualization, S.G. and A.B.; writing—original draft preparation, S.G. and A.B.; writing—review and editing, F.B. and A.R.; supervision, S.G. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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