pathogens

Article Inhibitory Properties of Aldehydes and Related Compounds against Phytophthora infestans—Identification of a New Lead

John J. Mackrill 1,*, Roberta A. Kehoe 2, Limian Zheng 1, Mary L. McKee 2, Elaine C. O’Sullivan 2, Barbara M. Doyle Prestwich 3 and Florence O. McCarthy 2,*

1 Department of Physiology, School of Medicine, University College Cork, T12 K8AF Cork, Ireland; [email protected] 2 School of Chemistry and Analytical and Biological Chemistry Research Facility, University College Cork, Western Road, T12 K8AF Cork, Ireland; [email protected] (R.A.K.); [email protected] (M.L.M.); [email protected] (E.C.O.) 3 School of Biological, Earth and Environmental Sciences, University College Cork, T12 K8AF Cork, Ireland; [email protected] * Correspondence: [email protected] (J.J.M.); [email protected] (F.O.M.); Tel.: +353-21-4901400 (J.J.M.); +353-21-4901695 (F.O.M.)


Received: 5 June 2020; Accepted: 3 July 2020; Published: 7 July 2020


Abstract: The pathogen Phytophthora infestans is responsible for catastrophic crop damage on a global scale which totals billions of euros annually. The discovery of new inhibitors of this organism is of paramount agricultural importance and of critical relevance to food security. Current strategies for crop treatment are inadequate with the emergence of resistant strains and problematic toxicity. Natural products such as cinnamaldehyde have been reported to have fungicidal properties and are the seed for many new discovery research programmes. We report a probe of the cinnamaldehyde framework to investigate the aldehyde subunit and its role in a subset of aromatic aldehydes in order to identify new lead compounds to act against P. infestans. An ellipticine derivative which incorporates an aldehyde (9-formyl-6-methyl ellipticine, 34) has been identified with exceptional activity versus P. infestans with limited toxicity and potential for use as a fungicide.

Keywords: Phytophthora infestans; cinnamaldehyde; thiosemicarbazone; ellipticine; fungicide

1. Introduction One of the most significant threats to global food production and agriculture is the pathogenic oomycete Phytophthora infestans (P. infestans)[1]. It was first described by Berkeley in 1846 and subsequently named Phytophthora infestans by deBary in 1876, which aptly translates as “plant destroyer” [2,3]. Most infamously known for the Irish potato famine in 1845–1849, the pathogen caused incessant, devastating outbreaks of late potato blight resulting in the deaths of one million people and a further one million emigrating [4]. The current global impact of P. infestans is in excess of $6.2 billion per annum due to crop loss and the use of fungicides [5]. The necessity of fungicides to combat the spread of P. infestans is evident and with resistant strains continually developing, designing novel fungicides to target this pathogen is of high importance. The Irish Famine of the 1840s spurred the development of the first-ever pesticide to be adopted worldwide. Developed by French scientists, Pierre Millardet and Ulysse Gayon, the Bordeaux mixture was used to combat the P. infestans blight. This fungicide is a mixture of copper sulphate and lime, which forms the active compound copper(II) hydroxide, stabilised by calcium sulphate. The emergence of this fungicidal treatment was imperative to the conservation of crops and averting hunger, therefore

Pathogens 2020, 9, 542; doi:10.3390/pathogens9070542 www.mdpi.com/journal/pathogens Pathogens 2019, 8, x FOR PEER REVIEW 2 of 24 Pathogens 2020, 9, 542 2 of 24 Pathogens 2019, 8, x FOR PEER REVIEW 2 of 24 hunger, therefore saving lives [6]. The use of the Bordeaux mixture diminished through the hunger,savingemergence lives therefore of [6 less]. The toxicsaving use and of thelives more Bordeaux [6]. targeted The mixture usefungicides of diminishedthe [7].Bordeaux through mixture the emergence diminished of lessthrough toxic andthe emergencemoreThe targeted Bordeaux of less fungicides toxic mixture and [7 ].more was targeted broadly fungicides used from [7]. the 1890s until it was superseded by the The Bordeaux mixture was broadly used from the 1890s until it was superseded by the dithiocarbamateThe Bordeaux class mixture of fungicides was broadly in the 1960s. used fromThe broad-spectrum the 1890s until fungicides it was superseded were used bydue the to dithiocarbamate class of fungicides in the 1960s. The broad-spectrum fungicides were used due to dithiocarbamatetheir high biological class and of chemical fungicides activity, in the with 1960s. the Thebenefit broad-spectrum of low production fungicides cost [8]. were Mancozeb used due (1) their high biological and chemical activity, with the benefit of low production cost [8]. Mancozeb (1) toand their Propineb high biological (3) were andtwo chemicalof the most activity, widely with used the dithio benefitcarbamate of low production fungicides cost [9,10]. [8]. MancozebBoth of these (1) and Propineb (3) were two of the most widely used dithiocarbamate fungicides [9,10]. Both of these andorganosulfur Propineb fungicides (3) were two have of thebeen most found widely to break used down dithiocarbamate into toxic metabolites fungicides as [ 9degradation,10]. Both of begins these organosulfur fungicides have been found to break down into toxic metabolites as degradation begins organosulfurduring storage fungicides to form the have respective been found toxic to metabolites break down (Figure into toxic 1) [11,12]. metabolites Ethylene as degradation thiourea 2 (R begins = H), during storage to form the respective toxic metabolites (Figure 1) [11,12]. Ethylene thiourea 2 (R = H), duringone of the storage main to metabolites form the respective of Mancozeb, toxic metaboliteshas been shown (Figure to 1have)[ 11 many,12]. Ethylene toxic effects thiourea in rats2 (suchR = H as), one of the main metabolites of Mancozeb, has been shown to have many toxic effects in rats such as oneteratogenicity, of the main developmental metabolites of toxicity Mancozeb, and haspromoting been shown thyroid to have neoplasms many toxic[11]. ePropyleneffects in rats thiourea such as 4 teratogenicity, developmental toxicity and promoting thyroid neoplasms [11]. Propylene thiourea 4 teratogenicity,(R = CH3) from developmental Proprineb has toxicity also been and shown promoting to affect thyroid the thyroid neoplasms and [ 11the]. nervous Propylene system thiourea [8]. (R = CH3) from Proprineb has also been shown to affect the thyroid and the nervous system [8]. 4Propineb(R = CH (32)) fromhas been Proprineb banned has by the also European been shown Commission to affect theand thyroid Mancozeb and (1 the) is nervousnow under system scrutiny [8]. Propineb (2) has been banned by the European Commission and Mancozeb (1) is now under scrutiny Propinebfor its negative (2) has effects. been banned by the European Commission and Mancozeb (1) is now under scrutiny for its negative effects. for its negative effects.

Figure 1. Mancozeb ( 1) (and Proprineb 3) metabolising to toxic thioureas 22 and 44.. Figure 1. Mancozeb (1) (and Proprineb 3) metabolising to toxic thioureas 2 and 4. Following dithiocarbamates, phenylamidephenylamide fungicidesfungicides werewere developeddeveloped inin thethe 1970s.1970s. A significant significant Following dithiocarbamates, phenylamide fungicides were developed in the 1970s. A significant advance in in the the control control of of P.P. infestans infestans is attributedis attributed to the to thephenylamides. phenylamides. Metalaxyl Metalaxyl (5) (Figure (5) (Figure 2) is one2) advance in the control of P. infestans is attributed to the phenylamides. Metalaxyl (5) (Figure 2) is one isof onethe ofmost the mostpopular popular phenylam phenylamideide fungicides, fungicides, however, however, seven seven years years after after the the product product waswas of the most popular phenylamide fungicides, however, seven years after the product was commercialised, thethe firstfirst resistantresistant strainsstrains ofof P. infestansinfestans werewere identifiedidentified inin IsraelIsrael [[13].13]. By 1980, resistant commercialised, the first resistant strains of P. infestans were identified in Israel [13]. By 1980, resistant isolatesisolates of P. infestansinfestans were discovered outside of Israel, in Ireland, Switzerland and The Netherlands, isolates of P. infestans were discovered outside of Israel, in Ireland, Switzerland and The Netherlands, demonstrating a need need for for further further fungicidal fungicidal deve development.lopment. To To combat combat the the growing growing resistance resistance of ofP. demonstrating a need for further fungicidal development. To combat the growing resistance of P. P.infestans infestans forfor phenylamides, phenylamides, a mixture a mixture was wasformulated formulated with withother otherfungicides fungicides (e.g., Mancozeb, (e.g., Mancozeb, 1) which1) infestans for phenylamides, a mixture was formulated with other fungicides (e.g., Mancozeb, 1) which whichresulted resulted in the reduction in the reduction of resistant of resistant strains strains emerging emerging [14]. [14]. resulted in the reduction of resistant strains emerging [14].

Figure 2. Chemical structure of fungicide Metalaxyl (5) and Oxathiapiprolin (6). Figure 2. Chemical structure of fungicide Metalaxyl (5) and Oxathiapiprolin (6). Figure 2. Chemical structure of fungicide Metalaxyl (5) and Oxathiapiprolin (6). Approved by the EU in 2017, oxathiapiprolin (6) has been used in more recent years as a targeted Approved by the EU in 2017, oxathiapiprolin (6) has been used in more recent years as a targeted antioomycete fungicide (Figure2). It was designed and synthesised by Pasteris et al. in 2016 [ 15] antioomyceteApproved fungicide by the EU (Figure in 2017, 2). oxathiapiprolin It was designed (6) hasand been synthesised used in more by Pasteris recent yearset al. as in a 2016 targeted [15] basing the structure around a piperidine-thiazole-carbonyl core. After numerous derivatives were antioomycetebasing the structure fungicide around (Figure a piperidine-thiazole-carbonyl 2). It was designed and synthesised core. After by numerous Pasteris et derivatives al. in 2016 were [15] synthesised and tested, the chemical structure of oxathiapiprolin (6) exhibited the greatest control over basingsynthesised the structure and tested, around the chemicala piperidine-thiazole-carbonyl structure of oxathiapiprolin core. After (6) exhibited numerous the derivatives greatest control were P. infestans with low toxicity in humans, birds and fish. The authors also investigated the mechanism synthesisedover P. infestans and tested,with low the toxicitychemical in structure humans, of birds oxathiapiprolin and fish. The (6) exhibitedauthors also the investigatedgreatest control the of action of oxathiapiprolin and uncovered a novel target for oomycete inhibition. The compound overmechanism P. infestans of action with of low oxathiapiprolin toxicity in humans, and uncovered birds and a novel fish. targetThe authors for oomycete also investigated inhibition. Thethe binds to an oxysterol binding protein, although the role for this family is not very well understood. mechanismcompound bindsof action to an of oxysteroloxathiapiprolin binding and protein, uncovered although a novel the targetrole for for this oomycete family isinhibition. not very wellThe Oxathiapiprolin (6) has been approved in many countries around the world and requires a smaller compoundunderstood. binds Oxathiapiprolin to an oxysterol (6) has binding been approved protein, although in many countriesthe role for around this family the world is not and very requires well quantity to be used on crops [16]. However, similarly to the phenylamides, oxathiapiprolin has already understood.a smaller quantity Oxathiapiprolin to be used (on6) hascrops been [16]. approved However, in manysimilarly countries to the phenylamides,around the world oxathiapiprolin and requires a smaller quantity to be used on crops [16]. However, similarly to the phenylamides, oxathiapiprolin

Pathogens 2020, 9, 542 3 of 24 Pathogens 2019, 8, x FOR PEER REVIEW 3 of 24 beenhas already found to been be ine foundffective to against be ineffective emerging against resistant emerging strains of resistantPhytophthora strains capsici of Phytophthora[15,17]. The current capsici focus[15,17]. has The turned current towards focus has the turned use of natural towards products the use of as natural fungicides products to replace as fungicides the previous to replace synthetic the alternatives.previous synthetic Although alternatives. the first report Although of antimicrobial the first report natural of productsantimicrobial was innatural 1676, developmentproducts was has in been1676, slowdevelopment for these compoundshas been slow [18 for]. Of these prime compou interestnds for [18]. natural Of prime source interest fungicides for isnatural the essential source oil,fungicides cinnamaldehyde. is the essential oil, cinnamaldehyde. Cinnamaldehyde ( 7, Figure 33)) isis aa naturalnatural productproduct extractedextracted fromfrom thethe barkbark ofofthe the plant plant genus genus Cinnamona. This This conjugated conjugated aromatic aromatic compound compound has has shown shown to to inhibit inhibit the the growth growth of bacteria, filamentousfilamentous moulds and yeast [[19].19]. There are over 250 species of cinnamon plants and trees and are native to SriSri LankaLanka andand SouthSouth IndiaIndia [[20].20]. Cinnamaldehyde was firstfirst isolatedisolated from cinnamon oil inin 18341834 by Dumas and Peligot [21] [21] and and was was first first synthe synthesisedsised in in 1854 1854 by by Chiozza Chiozza [22]. [22 ].The The essential essential oil oil is isgenerally generally recognised recognised as safe as safe by bythe theUnited United States States Food Food and andDrug Drug Administration Administration and has and been has beenused usedin foods in foods and andmedicines medicines as flavourings as flavourings for formany many years years [23]. [23 ].It Itis isalso also reported reported to to possess many pharmacological activitiesactivities such such as antidiabeticas antidiabetic and anti-inflammatoryand anti-inflammatory effects [effects24,25]. Extensive[24,25]. Extensive research hasresearch also beenhas also carried been outcarried on theout antibacterialon the antibact propertieserial properties of cinnamaldehyde. of cinnamaldehyde. The compoundThe compound has demonstratedhas demonstrated strong strong activity activity against against many many food-borne food-borne bacteria bacteria such such as Bacillus, as Bacillus, Staphylococcus Staphylococcusand Enterobacterand Enterobacter spp. andspp. canand thereforecan therefore be used be used as a foodas a food preservative preservative [26–28 [26–28].].

Figure 3. Chemical structure of cinnamaldehyde (7) and hydrocinnamaldehyde ((88).).

Studies by Quintanilla and Soylu [29,30] were some of the first to demonstrate the effect essential Studies by Quintanilla and Soylu [29,30] were some of the first to demonstrate the effect essential oils have on P. infestans and a follow up study by Ferhout et al. found that cinnamaldehyde oils have on P. infestans and a follow up study by Ferhout et al. found that cinnamaldehyde (7) (7) expressed a greater inhibitory effect on fungal strains than thyme oil [31]. Research by Raut expressed a greater inhibitory effect on fungal strains than thyme oil [31]. Research by Raut determined cinnamaldehyde to be the most effective phenylpropanoid from plant origin against determined cinnamaldehyde to be the most effective phenylpropanoid from plant origin against Candida albicans biofilm formation, being effective at completely inhibiting mature biofilms and Candida albicans biofilm formation, being effective at completely inhibiting mature biofilms and identifying its potential [32]. Jantan et al. tested 14 essential oils against six dermatophytes, identifying its potential [32]. Jantan et al. tested 14 essential oils against six dermatophytes, one one filamentous fungus and five strains of yeasts, finding again that cinnamaldehyde possessed filamentous fungus and five strains of yeasts, finding again that cinnamaldehyde possessed the the strongest activity against all the different strains of fungi studied [33]. strongest activity against all the different strains of fungi studied [33]. From experimental evidence, it appears cinnamaldehyde (7) inhibits cell wall biosynthesis, From experimental evidence, it appears cinnamaldehyde (7) inhibits cell wall biosynthesis, membrane function and some specific enzyme activities. However, specific cinnamaldehyde targets are membrane function and some specific enzyme activities. However, specific cinnamaldehyde targets still not well established. It is known that at lethal concentrations, the cell membrane is disrupted by are still not well established. It is known that at lethal concentrations, the cell membrane is disrupted cinnamaldehyde whereas at below lethal concentrations, it can inhibit ATPase and at low concentrations, by cinnamaldehyde whereas at below lethal concentrations, it can inhibit ATPase and at low it disrupts enzymes involved in cytokine action. It has been demonstrated in many studies that concentrations, it disrupts enzymes involved in cytokine action. It has been demonstrated in many cinnamaldehyde (7) interacts with microbial cell membranes and that it is capable of altering the studies that cinnamaldehyde (7) interacts with microbial cell membranes and that it is capable of membrane lipid profile [34]. It is thought that the lipophilicity of cinnamaldehyde allows the molecule altering the membrane lipid profile [34]. It is thought that the lipophilicity of cinnamaldehyde allows to partition through the lipid membrane of the cells and thus, disrupts bilayer structure. This renders the molecule to partition through the lipid membrane of the cells and thus, disrupts bilayer structure. the cell more permeable and prone to leakage of intracellular material causing cell death [26]. At lethal This renders the cell more permeable and prone to leakage of intracellular material causing cell death concentrations, cinnamaldehyde acts as a noncompetitive inhibitor of cell wall synthesising enzymes [26]. At lethal concentrations, cinnamaldehyde acts as a noncompetitive inhibitor of cell wall leading to the prevention of cell wall formation [35]. synthesising enzymes leading to the prevention of cell wall formation [35]. At sub-lethal concentrations, cinnamaldehyde inhibits transmembrane ATPase activity in E. coli At sub-lethal concentrations, cinnamaldehyde inhibits transmembrane ATPase activity in E. coli and Listeria monocytogenes by entering into the cellular periplasm [36]. This enzyme is essential for and Listeria monocytogenes by entering into the cellular periplasm [36]. This enzyme is essential for maintaining transmembrane electrochemical proton gradient, necessary for the maintenance of cellular maintaining transmembrane electrochemical proton gradient, necessary for the maintenance of pH and uptake of nutrients [37]. At low concentration, cinnamaldehyde has been demonstrated to cellular pH and uptake of nutrients [37]. At low concentration, cinnamaldehyde has been inhibit GDP dependent polymerisation, preventing cell division [38]. In P.parasitica var. nicotianae, demonstrated to inhibit GDP dependent polymerisation, preventing cell division [38]. In P.parasitica Lu et al. found that cinnamaldehyde completely inhibited mycelial growth, sporangial formation, var. nicotianae, Lu et al. found that cinnamaldehyde completely inhibited mycelial growth, sporangial zoospore production and germination at concentrations ranging from about 10 µM to 50 µM[39]. formation, zoospore production and germination at concentrations ranging from about 10 μM to 50 μM [39].

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InIn aa studystudy ofof resistanceresistance of Capsicum annuum (pepper plant) plant) against against root root rot rot caused caused by by P.capsiciP.capsici, Li etet al.al. discovered that resistant plants displayeddisplayed increased transcription of genes involved in thethe phenylpropanoidphenylpropanoid biosynthesisbiosynthesis pathway, pathway, especially especially those those involved involved in cinnamaldehydein cinnamaldehyde production production [40]. This[40]. suggestsThis suggests that cinnamaldehyde that cinnamaldehyde is a naturally is a occurringnaturally protectiveoccurring compound, protective whosecompound, production whose is enhancedproduction in is plant enhanced strains in that plant are strains resistant that to are oomycete resistant infections. to oomycete infections. Transient ReceptorReceptor PotentialPotential (TRP) ion channelschannels play an importantimportant role inin sensingsensing damagedamage to cells. Members Members of of this this channel superfamily, most closely related to thethe polycysticpolycystic kidneykidney diseasedisease (PKD)(PKD) family,family, areare encodedencoded withinwithin thethe genomesgenomes ofof allall oomycetesoomycetes examinedexamined [[41].41]. Compounds that are susceptible toto nucleophilic nucleophilic attack attack from from cystine cystine can can cause cause TRP TRP channel channel activation, activation, disrupting disrupting Ca2+ levels Ca2+ inlevels the cell.in the Hu cell. et al. Hu demonstrated et al. demonstrated that the α ,thatβ-unsaturated the α, β-unsaturated bond in cinnamaldehyde bond in cinnamaldehyde is susceptible tois nucleophilicsusceptible to attack nucleophilic from cystine, attack and from therefore, cystine, modulates and therefore, Ca2+ homeostasis modulates inCaPhytophthora2+ homeostasis capsici in (PhytophthoraP. capsici)[42 ].capsici Intracellular (P. capsici free) Ca[42].2+ isIntracellular a universal free second Ca2+ messenger is a universal in eukaryotic second cells,messenger regulates in cellulareukaryotic processes cells, regulates such as sporulation, cellular processes germination such as and sporulation, growth and germination is important and in everygrowth life-cycle and is stageimportant [41,43 in]. Theevery covalently life-cycle boundstage [41,43]. cinnamaldehyde The covalently to cystine bound was cinnamaldehyde postulated to activate to cystine the TRPwas channelpostulated leading to activate to an ethefflux TRP of Cachannel2+ out leading of the cell to andan efflux subsequently of Ca2+ out leads of the to cell cell death. and subsequently To test their theoryleads to that cell the death.α, β-unsaturated To test their bond theory in cinnamaldehyde that the α, β is-unsaturated important, Hu bond monitored in cinnamaldehyde Ca2+ levels using is aimportant, cinnamaldehyde Hu monitored derivative Ca without2+ levels the αusing, β-unsaturated a cinnamaldehyde bond, hydrocinnamaldehyde derivative without ( 8,the Figure α, 3β).- Treatmentunsaturated of P.bond, capsici withhydrocinnamaldehyde hydrocinnamaldehyde (8, (8)Figure demonstrated 3). Treatment no inhibitory of effP.ects capsici of zoospore with growthhydrocinnamaldehyde indicating that the(8) demonstrated Michael addition no inhibitory through the effectsα, β -unsaturatedof zoospore growth bond of indicating cinnamaldehyde that the wasMichael crucial addition for stimulating through the Ca 2α+, eβffl-unsaturatedux and growth bond inhibition of cinnamalde of P. capsici.hyde was crucial for stimulating Ca2+ effluxThus, and although growth many inhibition studies of exist P. capsici. on antimicrobial activity against several different groups of microorganisms,Thus, although there many is much studies to be exist learned on antimicrobial about the mode activity of action against of cinnamaldehydeseveral different groups7 and the of scopemicroorganisms, of its effects there on P. is infestans much .to As be we learned set out about to identify the mode new of agents action against of cinnamaldehydeP. infestans, we 7 initiated and the ascope broad ofstudy its effects on the on importanceP. infestans. As ofthe we aldehydeset out to aspectidentify of new cinnamaldehyde. agents against P. infestans, we initiated a broadPrevious study cinnamaldehydeon the importance analogues of the aldehyde that have aspect been subjectedof cinnamaldehyde. to P. infestans testing have primarily retainedPrevious their aldehydecinnamaldehyde functionality analogues and are that substituted have been on the subjected aromatic to ring, P. examplesinfestans testing of which have are representedprimarily retained in Figure their4( aldehyde9–13)[44 functionality]. Separately, and a recent are substituted study by Watamotoon the aromatic found ring, a diverse examples set ofof compoundswhich are represented to demonstrate in Figure inhibitory 4 (9-13 e)ff [44].ects Separately, on fungal strains, a recent amongst study by which Watamoto some offound the structural a diverse featuresset of compounds of our targets to demonstrate align [45]. Twoinhibitory hit compounds effects on fromfungal this strains, screen amongst (14 and which15, Figure some4) of have the shownstructural strong features fungicidal of our eff targetsects and align to inhibit [45]. Two the metabolic hit compounds activity from of C. this albicans screen. Of ( these,14 and Bay 15, 11-7082 Figure (4)14 have) possesses shown an strongα, β-unsaturated fungicidal effects bond and similar to inhibit to cinnamaldehyde the metabolic activity and may of act C. throughalbicans. aOf similar these, mechanism.Bay 11-7082 ( Ellipticine14) possesses (15 )an has α, knownβ-unsaturated antimicrobial bond similar and especially to cinnamaldehyde anticancer properties:and may act testing through at bacteriala similar planktonicmechanism. mode Ellipticine showed (15 ()15 has) had known averaged antimicrobial a minimal and inhibitory especially concentration anticancer properties: (MIC) of 8.45testingµM at across bacterial all six planktonicCandida strains mode andshowed hence (15 is) worthyhad averaged of further a minimal investigation. inhibitory concentration (MIC) of 8.45 μM across all six Candida strains and hence is worthy of further investigation.

Figure 4. Cinnamaldehyde analogues previously tested against fungal strains and ellipticine (15). Figure 4. Cinnamaldehyde analogues previously tested against fungal strains and ellipticine (15). The aims of this study are to synthesise and evaluate cinnamaldehyde and aromatic aldehyde The aims of this study are to synthesise and evaluate cinnamaldehyde and aromatic aldehyde derivatives in assays of P. infestans growth and zoospore production. Cinnamaldehyde derivatives derivatives in assays of P. infestans growth and zoospore production. Cinnamaldehyde derivatives will be accessed by specific synthetic modification at positions R1, R2 and R3 represented in Figure 5.

Pathogens 2020, 9, 542 5 of 24 willPathogens be accessed2019, 8, x FOR by specificPEER REVIEW synthetic modification at positions R1,R2 and R3 represented in Figure5 of 245. 4-Fluorocinnamaldehyde (R1 = F, 17) was chosen to probe the effect on electron density withdrawal on 1 4-Fluorocinnamaldehyde (R 2= F, 17) was chosen to probe the effect2 on electron density withdrawal the Michael acceptor. At the R position α-methylcinnamaldehyde (R = CH3, 18) will test steric effects on the Michael acceptor. At the R2 position α-methylcinnamaldehyde (R2 = CH3, 18) will test steric as the methyl group can influence attack on this reactive element. Finally, the rationale is provided effects as the methyl group can influence attack on this reactive element. Finally, the rationale is by the structure of Mancozeb (1, Figure5) for the generation of a hybrid structure 16 and the related provided by the structure of Mancozeb (1, Figure 5) for the generation of a hybrid structure 16 and thiosemicarbazone at the R3 position. Extension of the conjugated aldehyde will also be assessed to the related thiosemicarbazone at the R3 position. Extension of the conjugated aldehyde will also be identify optimal chain length. assessed to identify optimal chain length.

Figure 5. Structure of Mancozeb (1), a cinnamaldehyde thiosemicarbazone hybrid 16 and Figure 5. Structure of Mancozeb (1), a cinnamaldehyde thiosemicarbazone hybrid 16 and screening screening targets. targets. In order to probe the aldehyde functionality, a series of aromatic aldehydes and ellipticine In order to probe the aldehyde functionality, a series of aromatic aldehydes and ellipticine aldehydes will also be assessed in the P. infestans assay to screen for potential lead compounds for future aldehydes will also be assessed in the P. infestans assay to screen for potential lead compounds for development (Figure5). Given the experience of the group, of particular interest are the ellipticines future development (Figure 5). Given the experience of the group, of particular interest are the which would offer a new framework for the inhibition of P. infestans growth and the potential for ellipticines which would offer a new framework for the inhibition of P. infestans growth and the overcoming resistance. potential for overcoming resistance. 2. Results 2. Results Synthesis of novel cinnamaldehyde and ellipticine derivatives was initially undertaken and in additionSynthesis to some of commercialnovel cinnamaldehyde aldehydes were and assessedellipticine in derivatives a parallel approach, was initially via the undertaken determination and in of additioninhibitory to e ffsomeects oncommercial the mycelial aldehydes growth andwere zoosporogenesis assessed in a parallel of Phytophthora approach, infestans via theand determination cytotoxicity ofagainst inhibitory a human effects cell line.on the mycelial growth and zoosporogenesis of Phytophthora infestans and cytotoxicity against a human cell line. 2.1. Effects of Mancozeb, Cinnamaldehyde and Hydrocinnamaldehyde on P. infestans Mycelial Growth 2.1. Effects of Mancozeb, Cinnamaldehyde and Hydrocinnamaldehyde on P. Infestans Mycelial Growth There is substantial published evidence indicating that Mancozeb (1)[9] and cinnamaldehyde (7)[39There,42] is inhibit substantial the mycelial published growth evidence of Phytophthoraindicating thatand Mancozeb of other (1) oomycetes. [9] and cinnamaldehyde In contrast, (at7) concentrations[39,42] inhibit upthe to mycelial 2 mM, hydrocinnamaldehydegrowth of Phytophthora (8 )and is reportedof other tooomycetes. have no detectable In contrast, eff ectat concentrationson the growth up of to mycelia 2 mM, fromhydrocinnamaldehyde the zoospores of P.capsici(8) is reported[42]. Weto have sought no detectable to test the effect potency on the of growththese molecules of mycelia in inhibitingfrom the thezoospores mycelial of growth P.capsici in our[42]. experimental We sought to system test the (Figure potency6). After of these five moleculesdays of incubation, in inhibiting cinnamaldehyde the mycelial growth displayed in our a minimal experimental inhibitory system concentration (Figure 6). After (MIC, five the days lowest of incubation,concentration cinnamaldehyde resulting in no visibledisplayed growth) a minimal for P. infestans inhibitorymycelial concentration growth of approximately (MIC, the lowest 1 mM concentrationand a half-maximal resulting inhibitory in no visible concentration growth) (IC for50 P.) ofinfestans about 0.5mycelial mM (see growth Supplementary of approximately Information). 1 mM andIn contrast a half-maximal to the results inhibitory of Hu concentration et al. [42], we (IC found50) of that about hydrocinnamaldehyde 0.5 mM (see Supplementary inhibited Information).Phytophthora Inmycelial contrast growth to the withresults an of MIC Hu of et 2 al. mM [42] and, we an found IC50 ofthat between hydrocinnamaldehyde 0.25 and 0.5 mM. inhibited However, Phytophthora it should be mycelialnoted that growth the data with reported an MIC by of Hu 2 mM et al. and are an from IC50 a of di ffbetweenerent species 0.25 and (P.capsici 0.5 mM.versus However,P. infestans it should) and bemycelia noted were that generatedthe data reported from a dibyff erentHu et source al. are (germinatedfrom a different from species zoospores (P.capsici versus versus subcultured P. infestans from) andgrowing mycelia mycelia). were generated Mancozeb from inhibited a differentP. infestans sourcegrowth (germinated most from effectively, zoospores with versus an MIC subcultured of 100 µM fromand an growing IC50 of mycelia). between 25Mancozeb and 50 µ inhibitedM. These P. MIC infestans and IC growth50 values most for effectively, Mancozeb with are similar an MIC to of those 100 μreportedM and an previously IC50 of between for P. infestans 25 and, other 50 μ oomycetesM. These MIC and inand the IC fungus50 valuesCylindrocarpon for Mancozeb destructans are similar[10,46 to]. those reported previously for P. infestans, other oomycetes and in the fungus Cylindrocarpon destructans [10,46]. Based on these findings, we selected a fixed concentration of 25 μM to screen compounds for their ability to inhibit P. infestans growth (in order to identify molecules that are more

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Basedeffective on these than findings, Mancozeb), we selected with 1 amM fixed cinnamaldehyde concentration of 25andµM 100 to screenμM Mancozeb compounds in positive for their abilitycontrol toexperiments. inhibit P. infestans growth (in order to identify molecules that are more effective than Mancozeb), with 1 mM cinnamaldehyde and 100 µM Mancozeb in positive control experiments.

Figure 6. Concentration dependency of the effects of cinnamaldehyde, hydrocinnamaldehyde Figure 6. Concentration dependency of the effects of cinnamaldehyde, hydrocinnamaldehyde and and Mancozeb on P. infestans mycelial growth. Different concentrations of (A) cinnamaldehyde, Mancozeb on P. infestans mycelial growth. Different concentrations of (A) cinnamaldehyde, (B) (B) hydrocinnamaldehyde or (C) Mancozeb were incorporated into RyeB agar plates. These were hydrocinnamaldehyde or (C) Mancozeb were incorporated into RyeB agar plates. These were inoculated with plugs of P. infestans cultures and the effects on mycelial growth were assayed after inoculated with plugs of P. infestans cultures and the effects on mycelial growth were assayed after 5 5 days. Values are expressed relative to vehicle (solvent)-only controls (V) and represent the mean days. Values are expressed relative to vehicle (solvent)-only controls (V) and represent the mean values of three independent experiments conducted in duplicate. Error bars represent the standard values of three independent experiments conducted in duplicate. Error bars represent the standard error of the mean; n.d., no growth detected; and levels of statistical significance from V, determined usingerror ANOVA of the mean; are: *n.d.,= p < no0.05; growth ** = pdetected;< 0.01; and and *** leve= pls

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Figure 7.7. transtrans-Cinnamaldehyde-Cinnamaldehyde ( 7(),7), 4-fluorocinnamaldehyde 4-fluorocinnamaldehyde17 17and andα-methylcinnamaldehyde α-methylcinnamaldehyde18 and 18 Figure 7. trans-Cinnamaldehyde (7), 4-fluorocinnamaldehyde 17 and α-methylcinnamaldehyde 18 andstructures structures of test of Ar-CHOtest Ar-CHO compounds compounds19–24 19for-24 screening. for screening. and structures of test Ar-CHO compounds 19-24 for screening. 2.2.1. Synthesis Synthesis of of Extended Cinnamaldehyde Derivatives 2.2.1. Synthesis of Extended Cinnamaldehyde Derivatives To probe the eeffectffect of thethe aldehyde group of cinnamaldehyde on the inhibition of of P.P. infestans growth,To trans-probetrans-cinnamaldehyde cinnamaldehydethe effect of the (aldehyde7 (7) )was was treated treated group with withof cinnamaldehyde(carbethoxym (carbethoxymethylene)triphenylphosphoraneethylene)triphenylphosphorane on the inhibition of P. infestans 25 (Scheme25growth,(Scheme trans-1) 1yielding)cinnamaldehyde yielding product product as (7 asa) was acolourless colourless treated oilwith oil 26 (carbethoxym.26 The. The same same procedureethylene)triphenylphosphorane procedure was was followed followed for forthe the 254- fluorocinnamaldehyde4-fluorocinnamaldehyde(Scheme 1) yielding product 1717 startingstarting as a materialcolourless material however howeveroil 26. Theafter after same 2 2h h thprocedure thereere was was still stillwas evidence evidencefollowed of for starting the 4- material.fluorocinnamaldehyde An An additional additional 0.117 0.1 equivalentstarting equivalent material of of the the ylide ylidehowever 2525 waswas after added added 2 h andth andere stirred stirred was stillat at room room evidence temperature of starting for anmaterial. additional An additional 16 h. The The 0.1crude equivalent material of was was the isolated ylide 25 with withwas addedthe product and stirred 27 isolated at room as temperaturea colourless foroil whichan additional solidifiedsolidified 16 toh. colourlessThe crude crystals material overnight. was isolated with the product 27 isolated as a colourless oil which solidified to colourless crystals overnight.

Scheme 1: 1. SynthesisSynthesis of cinnamaldehyde ex extendedtended conjugated esters. Scheme 1: Synthesis of cinnamaldehyde extended conjugated esters. α-Methylcinnamaldehyde 18 was also used used in in this this reaction reaction however however it it proved proved more more problematic. problematic. Treatmentα-Methylcinnamaldehyde withwith25 25and and stirring stirring in 18 dichloromethanein was dichloromethane also used in for this 48 for reaction h showed48 h showedhowever no consumption no it provedconsumption ofmore starting problematic. of materialstarting materialbyTreatment TLC analysis. by withTLC analysis. A25 second and stirring A equivalent second in equivalentdichloromethane of the ylide of25 thewas ylide for added 48 25 hwasand showed added the reaction no and consumption the was reaction let stir was atof35 starting let◦C stir for atamaterial further35 °C for by 48 a h.TLC further NMR analysis. analysis48 h. ANMR second of the analysis crudeequivalent reactionof the of crude the mixture ylide reaction showed25 was mixture added evidence showed and of the product evidence reaction formation ofwas product let butstir formationalsoat 35 the °C characteristicfor but a furtheralso the 48 aldehydecharacteristic h. NMR starting analysis aldehyde material of the starting crude peak material atreaction 9.59 ppm peakmixture with at 9.59 isolationshowed ppm withevidence of the isolation pure of product of the pureprovingformation product problematic. but proving also the problematic.characteristic aldehyde starting material peak at 9.59 ppm with isolation of the pure product proving problematic. 2.2.2. Synthesis Synthesis of of Cinnamaldehyde Thiosemicarbazone Derivatives 2.2.2. Synthesis of Cinnamaldehyde Thiosemicarbazone Derivatives Given thethe structural structural distinction distinction between between cinnamaldehyde cinnamaldehyde (7) and (7 Mancozeb) and Mancozeb (1), it was (1 envisaged), it was envisagedthat synthesisGiven that the of synthesis astructural hybrid thiosemicarbazoneof distincta hybridion thiosemica between derivative rbazonecinnamaldehyde of the derivative cinnamaldehyde ( 7of) andthe cinnamaldehyde couldMancozeb proff er(1 improved), itcould was profferactivityenvisaged improved based that on synthesis theactivity individual of based a hybrid components. on thethiosemica individualtransrbazone-Cinnamaldehyde components. derivative trans of (7 )the-Cinnamaldehyde was cinnamaldehyde dissolved in absolute(7 )could was dissolvedethanolproffer andimproved in treated absolute withactivity ethanol thiosemicarbazide based and ontreated the andindividualwith a catalyticthiosemicarbazide components. amount ofacetic andtrans a acid -Cinnamaldehydecatalytic (Scheme amount2). The of starting(7 acetic) was acidmaterialdissolved (Scheme was in absolute consumed2). The starting ethanol within materialand 45 mintreated was and withconsumed after th isolationiosemicarbazide within resulted 45 min and in and pure a catalyticafter product isolation amount28 in resulted 34% of acetic yield. in pureTheacid 4-fluorocinnamaldehyde (Schemeproduct 282). inThe 34% starting yield. reaction materialThe 4-fluorocinnamaldehyde proceeded was consumed to completion within reaction 45 after min 16proceededand h atafter 80 ◦ isolationCtoand completion the resultedα-methyl after in pure product 28 in 34% yield. The 4-fluorocinnamaldehyde reaction proceeded to completion after

Pathogens 2019, 8, x FOR PEER REVIEW 8 of 24 Pathogens 2020, 9, 542 8 of 24 Pathogens 2019, 8, x FOR PEER REVIEW 8 of 24 16 h at 80 °C and the α-methyl derivative 30 was formed after 48 h at 80 °C. Both 28 and 29 crystallised as16derivative yellow h at 80 needles °C30 andwas whereasthe formed α-methyl 30 after was derivative 48 an h atamorphous 80 30◦C. was Both formedwhite28 and solid. after29 crystallised 48 h at 80 °C. as Both yellow 28 needlesand 29 crystallised whereas 30 aswas yellow an amorphous needles whereas white solid. 30 was an amorphous white solid.

Scheme 2: Synthesis of cinnamaldehyde thiosemicarbazone derivatives. Scheme 2: 2. SynthesisSynthesis of of cinnamaldehyde cinnamaldehyde thiosemicarbazone thiosemicarbazone derivatives. 2.3. Synthesis of Ellipticines and 9-Formylellipticines 2.3. Synthesis Synthesis of of Elliptici Ellipticinesnes and and 9-Formylellipticines 9-Formylellipticines The Gribble and Saulnier route to ellipticine (15) was chosen to probe the potential of ellipticines as it isThe a versatile Gribble and and high SaulnierSaulnier yielding routeroute synthesis.[48,49]to to ellipticine ellipticine ( 15(15) ) wasThe was chosenamenable chosen to to probenature probe the theof potential the potential ellipticine of of ellipticines ellipticines scaffold as allowsasit isit ais versatilefor a versatile derivatisation and and high high yielding at yieldingnumerous synthesis synthesis.[48,49] positions [48,49]. The(Scheme amenableThe amenable3). The nature N nature-6 of position the of ellipticine the of ellipticine ellipticine scaffold scaffold allowswas substitutedallowsfor derivatisation for by derivatisation the addition at numerous ofat sodiumnu positionsmerous hydride (Schemepositions to deprotonate3). (Scheme The N-6 3).the position The indole N of-6 nitrogen ellipticine position (Scheme wasof ellipticine substituted 3). Due was to by differentsubstitutedthe addition alkyl by of iodidesthe sodium addition possessing hydride of sodium to different deprotonate hydride reactivi to the deprotonate indoleties, the nitrogen time the indoletaken (Scheme nitrogenfor3 ).the Due reaction(Scheme to di ff erent to3). goDue alkyl to to completiondifferentiodides possessing alkyl varied. iodides For diff theerentpossessing methyl reactivities, derivativedifferent the time reactivi31, 16 taken ties,h was for the thesufficient time reaction taken to to achievefor go the to completion reactionfull conversion, to varied.go to whereascompletionFor the methylthe ethylvaried. derivative derivative For the31 , 32 16methyl took h was 72derivative su hffi tocient go toto31 achievecompletion, 16 h fullwasconversion, andsufficient the isopropyl to whereas achieve derivative the full ethyl conversion, derivative 33 48 h albeitwhereas32 took with 72 the significantly h toethyl go toderivative completion reduced 32 yield. andtook the 72The isopropylh quantityto go to derivative completionof alkylating33 and48 agent h the albeit could isopropyl with only significantly derivativebe used in reduced 33slight 48 h excessalbeityield. as with The the quantitysignificantly addition of of alkylating excessreduced or agentyield.extended couldThe reactionquantity only be times of used alkylating can in slight lead agent excessto quater could asnary the only addition salt be formation used of excessin slight on or theexcessextended pyridine as the reaction nitrogen. addition times of canexcess lead or to extended quaternary reaction salt formation times can on lead the to pyridine quaternary nitrogen. salt formation on the pyridine nitrogen.

SchemeScheme 3: 3.SynthesisSynthesis of of6-substituted 6-substituted ellipticines ellipticines and and corresponding corresponding aldehyde aldehyde derivatives. derivatives. Scheme 3: Synthesis of 6-substituted ellipticines and corresponding aldehyde derivatives. EllipticineEllipticine was was then then formylated formylated at at the the nine-position nine-position to to form form the the aldehyde aldehyde by by a method a method described described byby Plug PlugEllipticine et etal. al.[50] [was50 and] then and as first asformylated firstdescribed described at bythe Duff nine-position by Du(Schemeff (Scheme 3)to [51,52].form3)[ the51 This aldehyde,52]. reaction This by reaction was a method carried was described out carried on 6-substitutedbyout Plug on 6-substitutedet al. ellipticine [50] and ellipticineas derivatives first described derivatives to formby Duff to9-formylellipticine form(Scheme 9-formylellipticine 3) [51,52]. derivatives This reaction derivatives 34, was35 andcarried34, 3536. andout The on36 . formylation6-substitutedThe formylation step ellipticine was step generally was derivatives generally high yielding high to yieldingform with 9-formylellipticine good with goodpurity. purity. derivatives 34, 35 and 36. The formylation step was generally high yielding with good purity. 2.4. Screening of Cinnamaldehydes, Aromatic Aldehydes and Ellipticines against P. infestans 2.4. Screening of Cinnamaldehydes, Aromatic Aldehydes and Ellipticines against P. Infestans 2.4. Screening of Cinnamaldehydes, Aromatic Aldehydes and Ellipticines against P. Infestans CompoundsCompounds were were tested tested for for their their ability ability to inhibit to inhibit the themycelial mycelial growth growth and andthe production the production of of zoospores by P. infestans [42]. The cytotoxicity of compounds against human cells was zoosporesCompounds by P. infestans were [42].tested The for cytotoxicity their ability of tocompounds inhibit the against mycelial human growth cells and was the also production evaluated, of also evaluated, in the human embryonic kidney 293T (HEK-293T) cell line, using a sodium inzoospores the human by P. infestansembryonic [42]. Thekidney cytotoxicity 293T of(HEK-293T) compounds cellagainst line, human using cells awas sodium also evaluated, 3’-[1- 3’-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate (phenylaminocarbonyl)-3,4-tetrazolium]-bisin the human embryonic kidney 293T (4-m (HEK-293T)ethoxy-6-nitro) cell benzeneline, usingsulfonic a acidsodium hydrate 3’-[1- (XTT) reduction assay [53,54]. For all assays, a fixed concentration of 25 µM was selected for screening (XTT)(phenylaminocarbonyl)-3,4-tetrazolium]-bis reduction assay [53,54]. For all assays, a fixed(4-m concentrationethoxy-6-nitro) of 25benzene μM was sulfonic selected foracid screening hydrate 1 7 purposes.(XTT)purposes. reduction As As described described assay [53,54].in in Section Section For 2.1,all 2.1 assays, Mancozeb, Mancozeb a fixed (1 () concentration and) and cinnamaldehyde cinnamaldehyde of 25 μM ( was7 () were) wereselected used used for in in screeningpositive positive µ controlpurposes.control experiments experiments As described at at their theirin Section MIC MIC values, values,2.1, Mancozeb of of 100 100 μM (M1 )and and and 1 cinnamaldehyde 1mM, mM, respectively. respectively. (7 )The were The vehicle vehicleused infor forpositive these these compounds,controlcompounds, experiments DMSO, DMSO, was at was theirused used MICat at a a finalvalues, final concentrat concentration of 100 μionM ofand of 0.1% 0.1% 1 mM, in in negative negativerespectively. control control The experiments. experiments. vehicle for these compounds, DMSO, was used at a final concentration of 0.1% in negative control experiments.

Pathogens 2019, 8, x FOR PEER REVIEW 9 of 24

2.4.1. Phytophthora Infestans Mycelial Growth Assay In total, 17 compounds were evaluated for relative inhibitory effects on P. infestans mycelial growth at a concentration 25 μM after 5, 9 and 13 days.

Day 5 Cinnamaldehyde (7) and 9-formyl-6-methylellipticine 34 exhibit the best inhibition of P. infestans with no detectable growth for both compounds (Figure 8). All cinnamaldehyde derivatives 17, 18, 26- 30 prove not as potent as the parent cinnamaldehyde (7), although this is tested at a much higher concentration. It is interesting that thiosemicarbazone cinnamaldehyde derivatives 28-30 show the slowest mycelial growth rate of all the test cinnamaldehyde series. As an example, the α-methyl thiosemicarbazone derivative 30 demonstrates only 38% growth which suggests the cinnamaldehyde/Mancozeb hybrid approach warrants further exploration. Unexpectedly, derivative 26Pathogens (ethyl-(22020E)-5-phenylpenta-2,4-dienoate), 9, 542 caused a small but significant increase in P. infestans9 of 24 mycelial growth, of 113% relative to the vehicle control. As expected, cinnamaldehyde (7) shows good inhibition of P. infestans which given its simple structure2.4.1. Phytophthora has been postulated infestans to Mycelial arise from Growth the presence Assay of an α,β-unsaturated aldehyde group and its potentialIn total, to interact 17 compounds with cysteines were evaluated in target for proteins. relative Compounds inhibitory eff ects(7), 17 on, P.18 infestans, 20-24 andmycelial 34-36 growtharise fromata different concentration chemical 25 µ classesM after but 5, 9 all and contain 13 days. an aldehyde and in comparing their activity, there is no clear relationship between this functional group and mycelial growth. DayThe 5 functionalised cinnamaldehydes 17 and 18 which contain the α,β-unsaturated aldehyde are identified with relatively low mycelial growth inhibition within this screen. Each of the proprietary Cinnamaldehyde (7) and 9-formyl-6-methylellipticine 34 exhibit the best inhibition of P. infestans aromatic aldehydes tested (20-24) show moderate to low inhibition of P. infestans with 5- with no detectable growth for both compounds (Figure8). All cinnamaldehyde derivatives 17, bromoindole-3-carbaldehyde (21) seen as the most promising. 18, 26–30 prove not as potent as the parent cinnamaldehyde (7), although this is tested at a much The most effective compounds in the screen are cinnamaldehyde (7) and 9-formyl-6- higher concentration. It is interesting that thiosemicarbazone cinnamaldehyde derivatives 28–30 methylellipticine 34 and structurally these comprise an α,β-unsaturated aldehyde and an aromatic show the slowest mycelial growth rate of all the test cinnamaldehyde series. As an example, aldehyde. While it is evidently important for potency, this suggests that the aldehyde group is not the α-methyl thiosemicarbazone derivative 30 demonstrates only 38% growth which suggests the solely responsible for inhibition of mycelial growth and that other factors must be explored. cinnamaldehyde/Mancozeb hybrid approach warrants further exploration. Unexpectedly, derivative 26 (ethyl-(2E)-5-phenylpenta-2,4-dienoate) caused a small but significant increase in P. infestans mycelial growth, of 113% relative to the vehicle control.

Figure 8. Screen of the effects of compounds on P. infestans mycelial growth after 5 days of incubation.

Compounds were incorporated into RyeB agar at a concentration of 25 µM and their effects were measured after 5 days. Mancozeb was used at 100 µM and cinnamaldehyde at 1 mM. Untreated Figure 8. Screen of the effects of compounds on P. infestans mycelial growth after 5 days of incubation. cultures were grown on RyeB only. Values are the mean of three independent experiments, expressed Compounds were incorporated into RyeB agar at a concentration of 25 μM and their effects were relative to vehicle-treated growth (V, 0.02% ethanol). Error bars indicate the standard error in the mean;

n.d., no growth detected; and levels of statistical significance from V, determined using ANOVA are: * = p < 0.05; ** = p < 0.01; and *** = p < 0.001.

As expected, cinnamaldehyde (7) shows good inhibition of P. infestans which given its simple structure has been postulated to arise from the presence of an α,β-unsaturated aldehyde group and its potential to interact with cysteines in target proteins. Compounds (7), 17, 18, 20–24 and 34–36 arise from different chemical classes but all contain an aldehyde and in comparing their activity, there is no clear relationship between this functional group and mycelial growth. The functionalised cinnamaldehydes 17 and 18 which contain the α,β-unsaturated aldehyde are identified with relatively low mycelial growth inhibition within this screen. Each of the proprietary aromatic aldehydes tested (20–24) show moderate to low inhibition of P. infestans with 5-bromoindole-3-carbaldehyde (21) seen as the most promising. Pathogens 2019, 8, x FOR PEER REVIEW 10 of 24

measured after 5 days. Mancozeb was used at 100 μM and cinnamaldehyde at 1 mM. Untreated cultures were grown on RyeB only. Values are the mean of three independent experiments, expressed Pathogens 2020, 9, 542 10 of 24 relative to vehicle-treated growth (V, 0.02% ethanol). Error bars indicate the standard error in the mean; n.d., no growth detected; and levels of statistical significance from V, determined using ANOVAThe most are: * = e pff ective< 0.05; ** compounds = p < 0.01; and in*** = the p < 0.001. screen are cinnamaldehyde (7) and 9-formyl-6- methylellipticine 34 and structurally these comprise an α,β-unsaturated aldehyde and an aromatic aldehyde.Of the ellipticines, While it is evidentlyall six-alkylated important compounds for potency, 31, 34, this 35 suggests and 36 exerted that the a aldehyde significant group growth is not inhibitorysolely responsible effect, with for 34 inhibition having a ofC-9 mycelial aldehyde growth installed and on that the other A-ring factors which must may be contribute explored. to the activityOf of thethe ellipticines,compound. allAlthough six-alkylated Mancozeb compounds (1) is four31 ,times34, 35 theand concentration36 exerted a of significant the remaining growth testinhibitory compounds, effect, it withcauses34 havinga relatively a C-9 low aldehyde inhibition installed of growth, on the A-ring with six which other may test contribute compounds to the demonstratingactivity of the more compound. potent effects. Although Mancozeb (1) is four times the concentration of the remaining test compounds, it causes a relatively low inhibition of growth, with six other test compounds Daydemonstrating 9 more potent effects. After nine days, it can be seen once again that cinnamaldehyde (7) and ellipticine aldehyde Day 9 derivative 34 still demonstrate 100% inhibition of mycelial growth (Figure 9). Mancozeb (1) shows a decreaseAfter of 13% nine in days, the inhibition it can be seenof mycelial once again growth that since cinnamaldehyde Day 5. The growth-promoting (7) and ellipticine effect aldehyde of compoundderivative 2634 is stillmaintained, demonstrate at 6% 100% greater inhibition than the of vehicle mycelial control. growth Again, (Figure of 9particular). Mancozeb note ( are1) shows the ellipticinesa decrease 31, of 34 13% and in 35 the which inhibition show ofpromising mycelial potency growth at since thisDay extended 5. The stage. growth-promoting effect of compound 26 is maintained, at 6% greater than the vehicle control. Again, of particular note are the ellipticines 31, 34 and 35 which show promising potency at this extended stage.

Figure 9. Screen of the effects of compounds on P. infestans mycelial growth after 9 days of incubation. For details, please refer to the legend of Figure8. Levels of statistical significance from V, determined Figureusing 9. ANOVA Screen of are: the *effects= p < of0.05; compounds ** = p < 0.01; on P. and infestans *** = p mycelial< 0.001. growth after 9 days of incubation. For details, please refer to the legend of Figure 8. Levels of statistical significance from V, determined Dayusing 13 ANOVA are: * = p < 0.05; ** = p < 0.01; and *** = p < 0.001. The data in Figure 10 demonstrate that there is a high rate of mycelial growth in P. infestans Day 13 incubated with many of the test compounds after 13 days. However, there is still a consistent undetectableThe data in growth Figure for 10 cinnamaldehyde demonstrate that (7) there and only is a 6% high growth rate forof mycelial34. Mancozeb growth (1) in has P. an infestans increased incubatedrate of mycelium with many growth of the of test 14% compounds since Day 9.after Aside 13 fromdays. theHowever, ellipticines there31 ,is34 still–36 athe consistent consistent undetectableactivity of cinnamaldehyde growth for cinnamaldehyde/Mancozeb hybrid (7) 30andis ofonly note. 6% The growth growth-promoting for 34. Mancozeb effect of(1)26 haswas an not increasedstatistically rate significant of mycelium after growth 13 days of (the 14% same since as V,Day 100% 9. Aside growth). from the ellipticines 31, 34-36 the consistent activity of cinnamaldehyde/Mancozeb hybrid 30 is of note. The growth-promoting effect of 2.4.2.26 wasP. not infestans statisticallyZoosporogenesis significant andafter Sporangiogenesis 13 days (the same as V, 100% growth). The test compounds were investigated for their effects on zoosporogenesis. The number of zoospores produced after two weeks’ culture under each condition was counted using a haemocytometer.

All compounds with the exception of the aldehydes 20, 21, 23 and 24, and ellipticine (15), significantly Pathogens 2019, 8, x FOR PEER REVIEW 11 of 24

Pathogens 2020, 9, 542 11 of 24

reduced the production of zoospores. Several compounds demonstrated complete inhibition of zoospore formation with the ellipticines 31, 34 and 35 identified as comparable with cinnamaldehyde Pathogens 2019, 8, x FOR PEER REVIEW 11 of 24 (Figure 11).

Figure 10. Screen of the effects of compounds on P. infestans mycelial growth after 13 days of incubation. For details, please refer to the legend of Figure 8. Levels of statistical significance from V, determined using ANOVA are: * = p < 0.05; ** = p < 0.01; and *** = p < 0.001.

2.4.2. P. Infestans Zoosporogenesis and Sporangiogenesis The test compounds were investigated for their effects on zoosporogenesis. The number of zoospores produced after two weeks’ culture under each condition was counted using a haemocytometer. All compounds with the exception of the aldehydes 20, 21, 23 and 24, and ellipticine (15), significantly reduced the production of zoospores. Several compounds demonstrated complete inhibition of zoospore formation with the ellipticines 31, 34 and 35 identified as comparable with cinnamaldehydeFigure 10. Screen (Figure of the 11). eff ects of compounds on P. infestans mycelial growth after 13 days of incubation. Figure 10. Screen of the effects of compounds on P. infestans mycelial growth after 13 days of For details, please refer to the legend of Figure8. Levels of statistical significance from V, determined incubation. For details, please refer to the legend of Figure 8. Levels of statistical significance from V, using ANOVA are: * = p < 0.05; ** = p < 0.01; and *** = p < 0.001. determined using ANOVA are: * = p < 0.05; ** = p < 0.01; and *** = p < 0.001.

2.4.2. P. Infestans Zoosporogenesis and Sporangiogenesis The test compounds were investigated for their effects on zoosporogenesis. The number of zoospores produced after two weeks’ culture under each condition was counted using a haemocytometer. All compounds with the exception of the aldehydes 20, 21, 23 and 24, and ellipticine (15), significantly reduced the production of zoospores. Several compounds demonstrated complete inhibition of zoospore formation with the ellipticines 31, 34 and 35 identified as comparable with cinnamaldehyde (Figure 11).

Figure 11. Effect of test compounds on P. infestans zoosporogenesis. P. infestans was cultured for 14 days on RyeB containing 25 µM of test compounds, 100 µM Mancozeb, 1 mM cinnamaldehyde, or vehicle (0.02% ethanol). Zoospores were then released from sporangia and counted using a haemocytometer. Data represent the absolute number of zoospores produced from each plate and are the mean of three independent experiments performed in duplicate. Error bars show the standard error in the mean values; n.d., no growth detected; and levels of statistical significance relative to V, determined using ANOVA are: * = p < 0.05; and ** = p < 0.01.

Pathogens 2019, 8, x FOR PEER REVIEW 12 of 24

Figure 11. Effect of test compounds on P. infestans zoosporogenesis. P. infestans was cultured for 14 days on RyeB containing 25 μM of test compounds, 100 μM Mancozeb, 1 mM cinnamaldehyde, or vehicle (0.02% ethanol). Zoospores were then released from sporangia and counted using a haemocytometer. Data represent the absolute number of zoospores produced from each plate and are the mean of three independent experiments performed in duplicate. Error bars show the standard error in the mean values; n.d., no growth detected; and levels of statistical significance relative to V, determined using ANOVA are: * = p < 0.05; and ** = p < 0.01. Pathogens 2020, 9, 542 12 of 24

Decreased zoosporogenesis could result from decreased production of the sporangia, or from the defectiveDecreased generation zoosporogenesis of zoospores could within result these from fruiting decreased bodies. productionTo test this, ofsporangiogenesis the sporangia, orwas from measuredthe defective in P. infestans generation cultured of zoospores in the presence withinthese of aldehyde fruiting or bodies. cinnamaldehyde To test this, test sporangiogenesis compounds for was a periodmeasured of two in P.weeks infestans (Figurecultured 12). Both in the Mancozeb, presence ofaldehyde aldehyde 22 or and cinnamaldehyde ellipticine derivative test compounds 36 all causedfor a significant period of tworeductions weeks (Figurein sporangiogenesis. 12). Both Mancozeb, These fruiting aldehyde structures22 and ellipticinewere undetectable derivative in 36P. all infestanscaused cultured significant in the reductions presence inof sporangiogenesis.1 mM cinnamaldehyde These or fruiting 25 μM structuresof ellipticines were 31 undetectable, 34 and 35, in despiteP. infestans the lattercultured three incompounds the presence permitting of 1 mM some cinnamaldehyde mycelial growth or 25 afterµM of13 ellipticinesdays (see Figure31, 34 and10). 35, Thedespite parent thecompound latter three ellipticine compounds (15) permittingcaused a significant some mycelial increase growth in sporangiogenesis, after 13 days (see Figuredespite 10 ). havingThe parentno significant compound effects ellipticine on mycelial (15) caused growth a or significant zoosporo increasegenesis in(Figures sporangiogenesis, 10 and 11). despite having no significant effects on mycelial growth or zoosporogenesis (Figures 10 and 11).

Figure 12. Effect of aldehyde and ellipticine test compounds on P. infestans sporangiogenesis. P. infestans Figurewas 12. cultured Effect forof aldehyde 14 days on and RyeB ellipticine containing test 25compoundsµM of test on compounds, P. infestans 100 sporangiogenesis.µM Mancozeb, 1P. mM infestanscinnamaldehyde, was cultured or for vehicle 14 days (0.02% on RyeB ethanol). containing Sporangia 25 μ wereM of thentest compounds, detached from 100 mycelia μM Mancozeb, and counted 1 mMusing cinnamaldehyde, a haemocytometer. or vehicle Data represent(0.02% ethanol). the absolute Sporangia number were of then sporangia detached produced from mycelia from each and plate countedand areusing the a mean haemocytometer. of three independent Data represent experiments the absolute performed number in duplicate. of sporangia Error produced bars indicate from the eachstandard plate and error are in the the mean mean of values; three n.d., independent no growth experiments detected; and performed levels of statistical in duplicate. significance Error bars relative indicateto V, determinedthe standard using error ANOVA in the mean are: * =values;p < 0.05; n.d., and no ** growth= p < 0.01. detected; and levels of statistical significance relative to V, determined using ANOVA are: * = p < 0.05; and ** = p < 0.01. 2.4.3. Human Embryonic Kidney Cell XTT Assay 2.4.3. HumanTo determine Embryonic the toxicityKidney of Cell our XTT screening Assay compounds towards mammalian cells, an XTT reduction assayTo determine was performed. the toxicity This assay of our is anscreening improved compounds form of the towards MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- mammalian cells, an XTT reductiondiphenyltetrazolium assay was performed. bromide) This method, assay is in an which improved XTT form is reduced of the MTT to an (3-(4,5-dimethylthiazol- orange formazan dye by 2-yl)-2,5-diphenyltetrazoliummitochondrial dehydrogenases bromide) [53,54 method,]. The levelsin which of thisXTT productis reduced in theto an extracellular orange formazan medium dye can by mitochondrialbe measured using dehydrogen spectrophotometry.ases [53,54]. The The levels activity of this of pr mitochondrialoduct in the extracellular dehydrogenases medium is partially can be determinedmeasured using by the spectrophotometry. number of metabolically The activity active of cells mitochondrial in the assay, dehydrogenases and so XTT reduction is partially can be determinedused as anby indirectthe number indicator of metabolically of cell viability. active This cells XTT in the cytotoxicity assay, and assay so XTT was reduction applied tocan human be embryonic kidney 293T cells (HEK-293T) to determine the toxicity of the compounds against a cell

type of mammalian origin. In total, seven cinnamaldehyde, five ellipticines in addition to five stock aldehydes were tested in triplicate at 25 µM (Figure 13) and incubated for 24 h. Cinnamaldehyde and Mancozeb were tested at concentrations of 1 mM and 100 µM, respectively. Pathogens 2019, 8, x FOR PEER REVIEW 13 of 24 used as an indirect indicator of cell viability. This XTT cytotoxicity assay was applied to human embryonic kidney 293T cells (HEK-293T) to determine the toxicity of the compounds against a cell type of mammalian origin. In total, seven cinnamaldehyde, five ellipticines in addition to five stock aldehydes were tested in triplicate at 25 μM (Figure 13) and incubated for 24 h. Cinnamaldehyde and MancozebPathogens 2020 were, 9, 542 tested at concentrations of 1 mM and 100 μM, respectively. 13 of 24

Figure 13. Figure 13. EffectEffect of of test test compounds compounds on on reduction reduction of of XT XTTT by by a ahuman human cell cell line, line, HEK-293T. HEK-293T. Cells Cells were were cultured in 96-well plates for 24 h in the presence of test compounds, then with XTT reagent for cultured in 96-well plates for 24 h in the presence of test compounds, then with XTT reagent for an an additional 4 h. The absorbance of the orange formazan product was measured at a wavelength additional 4 h. The absorbance of the orange formazan product was measured at a wavelength of 490 of 490 nm, with a reference wavelength of 675 nm. Data are from three independent experiments, nm, with a reference wavelength of 675 nm. Data are from three independent experiments, performed performed in triplicate and normalised to the vehicle control (V, 0.02% ethanol). The exceptions to this in triplicate and normalised to the vehicle control (V, 0.02% ethanol). The exceptions to this are the are the experiments from “cinnamaldehyde derivatives”, which were performed once in triplicate. experiments from “cinnamaldehyde derivatives”, which were performed once in triplicate. Error bars Error bars indicate the standard error in the mean; n.d., no growth detected; and levels of statistical indicate the standard error in the mean; n.d., no growth detected; and levels of statistical significance significance from V, determined using ANOVA are: ** = p <0.01; and *** = p < 0.001. from V, determined using ANOVA are: ** = p <0.01; and *** = p < 0.001. Results from XTT Bioassay Results from XTT Bioassay There is a large variance in toxicity values throughout the library of compounds tested in this studyThere (Figure is a 13 large). Given variance the electrophilic in toxicity values nature thro of theughout screening the panellibrary of of compounds compounds and tested potential in this for studybioadduct (Figure formation, 13). Given the the establishment electrophilic ofnature relative of the toxicity screening is an panel important of compounds step in the and development potential fortowards bioadduct lead compoundformation, choice the establishment and potential forof fieldrelative trials. toxicity The most is toxican important compound step with 5%in XTTthe developmentreduction relative towards to thelead vehicle compound control, choice is cinnamaldehyde and potential for (7 field). Mancozeb trials. The (1) most shows toxic high compound toxicity at with12% 5% of control. XTT reduction There is arelative variance to between the vehicle the cinnamaldehydecontrol, is cinnamaldehyde derivatives with(7). Mancozeb both α, β-unsaturated (1) shows highesters toxicity26 and at 2712%demonstrating of control. There the is lowest a variance toxicity. between Of the cinnamaldehyde aldehydes, compound derivatives23 displays with both the αgreatest, β-unsaturated inhibition esters of XTT 26 and reduction. 27 demonstrating All of the ellipticines, the lowest with toxicity. the exception Of the aldehydes, of compound compound36, display 23 displayssignificant the toxicity greatest to thisinhibition human of cell XTT line. reduction. All of the ellipticines, with the exception of compound 36, display significant toxicity to this human cell line. 3. Discussion 3. Discussion Considering data from Day 5 mycelial growth inhibition and the XTT assay, relationships can be drawnConsidering from the data data from relating Day 5 the mycelial compound growth structure inhibition to the and inhibitory the XTT eassay,ffects againstrelationshipsP. infestans. can beFrom drawn the from outset, the cinnamaldehydedata relating the (compound7) proved verystructure effective to the at inhibitory limiting P. effects infestans againstgrowth P. infestans. albeit at Fromelevated the concentrationsoutset, cinnamaldehyde while interestingly (7) proved the very standard effective Mancozeb at limiting (1) was P. infestans much less growth effective albeit across at elevatedthe evaluations. concentrations The synthetic while inte modificationsrestingly the standard of cinnamaldehyde Mancozeb ( (117) was–18, much26–30 )less proved effective instructive across thewith evaluations. overall less The inhibitory synthetic ability modifications than the parentof cinnamaldehyde (7). In light of (17 the-18 relative, 26-30) concentrationsproved instructive used with(1 mM overall versus less 25 inhibitoryµM), the promisingability than data the forparent28 and (7). 30In shouldlight of be the explored relative furtherconcentrations especially used given (1 mMtheir versus limited 25 μ cytotoxicityM), the promising against data human for 28 cells. and Both30 should compounds be explored arise furt fromher the especially thiosemicarbazone given their cinnamaldehyde Mancozeb hybrid and have significant growth inhibition of P. infestans (56% and 38%, respectively) so it is evident that aldehyde conversion can have an effect on growth. Substitution on the aromatic ring, at the two-position in the chain or extension to a conjugated ester, appears to have little beneficial effect. In relation to the toxicities of the cinnamaldehyde thiosemicarbazone derivatives, Pathogens 2020, 9, 542 14 of 24

4-fluoro 29 proves to be the most toxic at 65% cell viability. In addition, 4-fluorocinnamaldehyde 17 is also more toxic than α-methylcinnamaldehyde 18 with 47% viability in comparison to 96%. Conversion of 4-fluorocinnamaldehyde 17 to the extended unsaturated ester 27 increases the cell viability by 53% indicating that the aldehyde induces a greater cytotoxic effect. It is possible that the 4-fluoro derivatives have higher cytotoxic effects due to the electron-withdrawing influence the fluorine atom has on the Michael acceptor. The commercial aromatic aldehydes had little effect at limiting P. infestans growth although the 5-bromoindole-3-carbaldehyde 21 stands out at 52% growth versus control. This needs to be balanced against the 15% decrease in XTT reduction which limits the further use of this specific framework. Indole derivatives and indole-3-carbaldehydes are natural products derived from the amino acid tryptophan and have been shown to provide innate plant resistance against pathogens so this deserves consideration for future investigations [47]. It is also of interest to note that the indole is a component of the subsequent ellipticines screened. On Day 5, the parent compound ellipticine (15) had little effect on mycelial growth, 106% relative to the control. When this compound is methylated at the N-6 position to generate 31, the growth rate drops to 21%, which decreases to no detectable growth when 31 is formylated at the nine-position to form 34. It appears that not only is substituting the N-6 site important, the alkyl group that is substituted also plays a role. 9-Formyl-6-methylellipticine 34 shows 0% growth of P. infestans whereas increasing to 6-ethyl 35 gives 69% growth and 6-isopropyl 36 demonstrates a 56% growth. Evidently, the size of the substituent at the N-6 position impacts the potency of ellipticine derivatives [52,55]. It is postulated that protecting the N-6 position plays a vital role in lowering the growth rate of P. infestans and the addition of an aldehyde group at the C-9 position results in complete inhibition of growth. Compound 34 is as effective as cinnamaldehyde (7) at a dosage 40 times less and suggests that there is real merit in further exploration of this structure. On extension of the assay to Day 9 and Day 13, a consistent pattern emerges and only after Day 13 do we record any growth with the ellipticine 34 (6%). Evaluation of zoosporogenesis identified that compounds 31, 34, 35 and cinnamaldehyde (7) completely eradicated the formation of zoospores which is an essential mechanism of growth. This is the first time an ellipticine has recorded such activity. There is no strong structural correlation between toxicity toward a human cell line and inhibition of P. infestans mycelial derived from this study (Figure 14). The commercial pesticide Mancozeb (1) expressed a low cell viability value of 12% and a midrange level of P. infestans inhibition at 50% even at four times the concentration of the other test compounds. From the data collected, Mancozeb (1) does not represent the strongest candidate in this study. In addition, cinnamaldehyde (7) is at a greater concentration than the test compounds and has also been shown by Hu et al. [42] to have an effective concentration of 2 mM and therefore, also appears to have limitations. The viability percentage for cinnamaldehyde of 5% represents a serious concern for its potential use as a fungicidal agent. With respect to the cinnamaldehyde derivatives, all are less cytotoxic than cinnamaldehyde (7) (albeit at lower concentrations) and of special note is compound 30 (methyl-substituted cinnamaldehyde thiosemicarbazone) with excellent cytotoxicity to mycelial growth inhibition profile worthy of further investigation. The parent compound ellipticine (15) has a low cell viability of 21%. Substitution at the N-6 position, for example, 6-methylellipticine 31 lowers the toxicity to 37% which lowers further to 40% upon addition of a formyl group at the nine-position to form 9-formyl-6-methylellipticine 34. In addition to substitution playing a vital role in toxicity, the size of the substituted group also has an effect. In comparison to 9-formyl-6-methylellipticine 34, 9-formyl-6-ethylellipticine 35 and 9-formyl-6-isopropylellipticine 36 show cell viabilities of 14% and 82%, respectively. It is evident that the isopropyl substituent expresses the highest cell viability for these compounds. Of the ellipticines, 34 provides the best cytotoxicity to P. infestans growth inhibition profile (Figure 14) and merits further exploration of its activity. It is significant to note that the screening concentration of 25 µM is most Pathogens 2020, 9, 542 15 of 24

Pathogenslikely not 2019 the, 8, optimalx FOR PEER concentration REVIEW for Phytophthora growth inhibition and hence this profile15 has ofthe 24 potential to be significantly improved.

Figure 14. Comparison of the effects of test compounds on XTT reduction by human cells versus Figuremycelial 14. growth Comparison inhibition of the in P. effects infestans of .test Graph compounds showing XTTon XTT reduction reduction by HEK-293Tby human cells cells (a versus proxy mycelialfor viability) growth versus inhibition inhibition in P. of infestansP. infestans. Graphmycelial showing growth XTT afterreduction 5 days. by InHEK-293T both cases, cells values (a proxy are fornormalised viability) to versus the vehicle-treated inhibition of controlP. infestans (V = mycelial100%). The growth dashed after line 5 indicatesdays. In both the best-fit cases, forvalues a linear are 2 normalisedrelationship to between the vehicle-treated these parameters control and (V the= 100%). R the The correlation dashed line coe ffiindicatescient of the this best-fit fit. for a linear 2 4. Materialsrelationship and between Methods these parameters and the R the correlation coefficient of this fit.

TheSolvents parent were compound distilled ellipticine prior to use (15 by) has the followinga low cell methods:viability of ethyl 21%. acetate Substitution was distilled at the fromN-6 position,potassium for carbonate; example, 6-methylellipticine THF was freshly distilled 31 lowers from the sodium toxicity andto 37% benzophenone which lowers and further hexane to 40% was upondistilled addition prior to of use. a formyl Organic grou phasesp at werethe nine-position dried using anhydrous to form 9-formyl-6-methylellipticine magnesium sulphate. Where 34 room. In additiontemperature to substitution is quoted in playing a method, a vital this role is within in toxi thecity, temperature the size of rangethe substituted 15–20 ◦C. group also has an effect.All In comparison commercial to reagents 9-formyl-6-methylellipticine were used without 34 further, 9-formyl-6-ethylellipticine purification unless otherwise35 and 9-formyl- stated. 6-isopropylellipticineAlkyllithium reagents 36 were show titrated cell viabilities prior to useof 14% using and the 82%, Gilman respectively. double titrationIt is evident procedure that the as isopropylfollows; Two substituent 100 mL conical expresses flasks the were highest prepared, cell viability the first for one these containing compounds. water (25 Of mL)the andellipticines, the second 34 providesone containing the best dibromoethane cytotoxicity to (2P. mL,infestans stoppered growth with inhibition a SubaSeal profile under (Figure nitrogen 14) and with merits a provision further explorationfor pressure of release). its activity. The It alkyllithium is significant reagent to note (1 that mL) the was screening added viaconcentration syringe to eachof 25 flaskμM is [56 most,57]. likelyThe reaction not the withoptimal dibromoethane concentration was for more Phytophthora vigorous growth than that inhibition with water, and andhence the this flask profile was swirledhas the potentialduring addition. to be significantly Two drops improved. of phenolphthalein were added to both flasks. The first flask was titrated against 0.1 M HCl until the purple colour disappeared. The SubaSeal was removed from the 4.second Materials flask and and Methods water (25 mL) was added, forming a biphasic mixture and was titrated as before. (Note: constant stirring was maintained to ensure mixing of both phases during titration). Titre value Solvents were distilled prior to use by the following methods: ethyl acetate was distilled from 1 represents the total base (alkyllithium and inorganic base) while titre 2 represents the free base potassium carbonate; THF was freshly distilled from sodium and benzophenone and hexane was (inorganic base not resulting from the alkyllithium). Thus, the molarity of the alkyllithium reagent was distilled prior to use. Organic phases were dried using anhydrous magnesium sulphate. Where room calculated using the following equation: molarity of alkyllithium (M) = [(titre 1 titre 2) 0.1]. temperature is quoted in a method, this is within the temperature range 15–20 °C.− × In reactions utilising alkyllithium and sodium hydride, all glassware was flame dried under All commercial reagents were used without further purification unless otherwise stated. nitrogen prior to use. All low-temperature reactions were carried out in a three-necked round-bottomed Alkyllithium reagents were titrated prior to use using the Gilman double titration procedure as flask equipped with a low-temperature thermometer and the internal temperatures are quoted. Syringes follows; Two 100 mL conical flasks were prepared, the first one containing water (25 mL) and the were used to transfer small volumes of alkyllithium reagents while cannulation from the reagent bottle second one containing dibromoethane (2 mL, stoppered with a SubaSeal under nitrogen with a into a precalibrated addition funnel was used for larger volumes (>15 mL). Low-temperature reactions provision for pressure release). The alkyllithium reagent (1 mL) was added via syringe to each flask used the following cooling mixture: 100 C, absolute ethanol and liquid nitrogen. [56,57]. The reaction with dibromoethane− was◦ more vigorous than that with water, and the flask was 1H (300 MHz) and 13C (75.5 MHz) NMR spectra were recorded on a Bruker AVANCE 300 NMR swirled during addition. Two drops of phenolphthalein were added to both flasks. The first flask was spectrometer. 1H (600 MHz) and 13C (150.9 MHz) NMR spectra were recorded on a Bruker AVANCE III titrated against 0.1 M HCl until the purple colour disappeared. The SubaSeal was removed from the 600 NMR spectrometer equipped with a Bruker Dual C/H cryoprobe or a Bruker Broadband Observe second flask and water (25 mL) was added, forming a biphasic mixture and was titrated as before. (Note: constant stirring was maintained to ensure mixing of both phases during titration). Titre value 1 represents the total base (alkyllithium and inorganic base) while titre 2 represents the free base

Pathogens 2020, 9, 542 16 of 24

H&F cryoprobe. All spectra were recorded at 300 K (26.9 ◦C) in deuterated dimethylsulfoxide (DMSO-d6) using DMSO-d6 as the reference peak or in deuterated chloroform (CDCl3) using trimethylsilane as an internal standard unless otherwise specified. Chemical shifts (δH and δC) are reported in parts per million (ppm) relative to the reference peak. Coupling constants (J) are expressed in Hertz (Hz). Splitting patterns in 1H spectra are designated as s (singlet), br s (broad singlet), d (doublet), t (triplet), br t (broad triplet), q (quartet), sept (septet), dd (doublet of doublets), ddd (doublet of doublet of doublets) and m (multiplet). Signal assignments were supported by COSY (correlation spectroscopy) or HMBC (Heteronuclear Multiple-Bond Correlation spectroscopy) experiments where necessary. 13 C NMR spectra were assigned (aromatic C, CH, CH2, CH3) with the aid of DEPT (Distortionless Enhancement by Polarisation Transfer) experiments run in DEPT-90, DEPT-135 and DEPT-q modes. Specific assignments were made using HSQC (Heteronuclear Single Quantum Correlation) and HMBC (Heteronuclear Multiple-Bond Correlation) experiments. All spectroscopic data for known compounds were in agreement with those previously reported unless otherwise stated. Samples used for comparison (stacked plots) were run at equal concentrations (6–8 mg per 0.65 mL solvent). Carbon analysis is given for compounds that are novel or where full analysis has not been published in the literature. Infrared spectra were recorded on a Bruker Tensor 37 FT-IR spectrophotometer interfaced with 1 Opus version 7.2.139.1294 over a range of 400–4000 cm− . An average of 16 scans was taken for each 1 spectrum obtained with a resolution of 4 cm− . Melting points were measured on a Uni-Melt Thomas Hoover capillary melting point apparatus and are uncorrected. Melting points or boiling points were not obtained for semi-solids or oils. Thin Layer Chromatography (TLC) was carried out on precoated silica gel plates (Merck 60 F254). Visualisation was achieved by UV light detection (254 nm or 366 nm), Wet flash chromatography was carried out using Kieselgel silica gel 60, 0.040–0.063 mm (Merck). Low-resolution mass spectra were recorded on a Waters Quattro Micro triple quadrupole spectrometer (QAA1202) in electron spray ionisation mode (ESI) using acetonitrile/water (1:1) containing 0.1% Formic acid as eluent. High-resolution mass spectrometry (HRMS) spectra were recorded on Waters Vion IMS (model no. SAA055K) in electron spray ionisation mode (ESI) using acetonitrile/water (1:1) containing 0.1% Formic acid as eluent. Ethyl-(2E)-5-phenylpenta-2,4-dienoate 26 trans-Cinnamaldehyde 7 (0.100 g, 0.76 mmol) in dichloromethane (5 mL) was treated with (carbethoxymethylene)triphenylphosphorane 25 (0.267 g, 0.76 mmol) and allowed stir at room temperature for 1 h. The solvent was removed under reduced pressure and hexane was added. The triphenylphosphine oxide byproduct precipitated out as a pale pink solid and was isolated by vacuum filtration. The mother liquor was transferred into a round-bottomed flask and solvent was removed under reduced pressure. The crude product was purified by column chromatography eluting in hexane: ethyl acetate (100:0–99:1). The product eluted as a colourless oil (0.128 g, 83.5%). 1 IR νmax/cm− : 3062, 3019, 2982, 1723, 1178; δH (300 MHz, CDCl3): 1.31 (3H, t, J 7.1, CH2CH3), 4.22 (2H, q, J 7.1, CH2CH3), 5.98 (1H, d, J 15.2, H-2), 6.80-6.94 (2H, m, H-5, H-4), 7.24–7.50 (6H, m, H-3, ArH); m/z (ESI+) 202 [(M + H)+, 50%]. Ethyl-(2E,4E)-5-(4-fluorophenyl)penta-2,4-dienoate 27 4-Fuorocinnamaldehyde 17 (0.100 g, 0.33 mmol) in dichloromethane (5 mL) was treated with (carbethoxymethylene)triphenylphosphorane 25 (0.115 g, 0.33 mmol) and allowed stir at room temperature for 2 h. Analysis by TLC showed residual starting material so an additional portion of (carbethoxymethylene)triphenylphosphorane (0.010 mg, 0.03 mmol) was added and allowed stir overnight. The solvent was removed under reduced pressure and hexane was added. The white triphenylphosphine oxide byproduct precipitated out and was isolated by vacuum filtration. The mother liquor was transferred into a round-bottomed flask and solvent was removed under reduced pressure. The crude product was purified by column chromatography eluting in hexane: ethyl acetate (100:0–97:3). The product eluted as a colourless oil but solidified overnight to a white crystalline solid. (0.43 g, Pathogens 2020, 9, 542 17 of 24

1 64.5%). m.p. 41-43 ◦C (Lit. m.p. 44–46 ◦C) [58]; IR νmax/cm− : 2986, 1696, 1238, 1176; δH (300 MHz, CDCl3): 1.31 (3H, t, J 6.9, CH2CH3), 4.23 (2H, q, J 6.9, CH2CH3), 5.98 (1H, d, J 15.3, H-2), 6.77 (1H, dd, J 15.3, 9.9, H-4), 6.86 (1H, d, J 15.3, H-5), 7.04 (2H, t, J 8.6, H-60, H-20), 7.36-7.47 (3H, m, H-50, H-30, H-3); m/z (ESI+) 221 [(M + H)+, 30%]. 2-((E)-3-Phenylallylidene)hydrazine-1-carbothioamide 28 To a solution of trans-cinnamaldehyde 7 (0.100 g, 0.76 mmol) in absolute ethanol (5 mL) was added thiosemicarbazide (0.069 mg, 0.76 mmol) and acetic acid (2 drops). The reaction mixture was heated to 80 ◦C and allowed stir for 45 min. The reaction mixture was cooled to 0 ◦C for 3 h without agitation. A precipitate formed and was isolated by vacuum filtration. The crude product was recrystallised from ethyl acetate: hexane to form pale yellow crystals (54 mg, 34.3%) m.p. 115–117 ◦C (Lit. m.p. 1 110–113 ◦C) [59]; IR νmax/cm− : 3413, 3255, 3155, 3028, 2981, 1610, 1590, 1536, 1372, 1291, 818; δH (300 MHz, CDCl3): 6.47 (1H, br s, NH2) 6.78 (1H, dd, J 16.0, 9.0, H-2), 6.95 (1H, d, J 16.0, H-3), 7.15 (1H, br s, NH2), 7.30–7.40 (3H, m, H-20, H-30, H-40), 7.45 (2H, dd, J 8.1, 2.0, H-10, H-50), 7.75 (1H, d, J 9.0, H-1), 10.08 (1H, s, CNNH); m/z (ESI+) 206 ((M + H)+, 100%]. 2-((E)-3-(4-Fluorophenyl)allylidene)hydrazine-1-carbothioamide 29 To a solution of 4-fluorocinnamaldehyde 17 (0.100 g, 0.67 mmol) in absolute ethanol (5 mL) was added thiosemicarbazide (0.060 g, 0.67 mmol) and acetic acid (2 drops). The reaction mixture was heated to 80 ◦C and allowed stir for 16 h. The reaction mixture was cooled to 0 ◦C for 3 h without agitation. A precipitate formed and was isolated by vacuum filtration. The crude product was recrystallised from ethyl acetate: hexane to form pure, pale yellow crystals (0.89 g, 59.7%) m.p. 1 144–146 ◦C; IR νmax/cm− : 3386, 3249, 3149, 2985, 1610, 1600, 1528, 1505, 1475, 1286, 1156, 1082, 814; δH (300 MHz, CDCl3): 6.40 (1H, br s, NH2), 6.72 (1H, dd, J 16.0, 9.0, H-3), 6.90 (1H, d, J 16.0, H-2), 7.05 (2H, t, J 8.6, H-20, H-60), 7.13 (1H, br s, NH2), 7.43 (2H, dd, J 8.6, 5.4, H-50, H-30), 7.72 (1H, d, J 9.0, H-1), 9.95 (1H, br s, CNNH); δC (75.5 MHz, DMSO-d6): 79.63 (CH), 116.27 (CH, JC-F 21.7, aromatic CH), 125.41 (CH, aromatic CH), 125.44 (CH, aromatic CH), 129.47 (d, CH JC-F 8.6, aromatic CH), 133.02 (d, C, + JC-F 3.3, aromatic C), 138.09 (CH), 145.11 (CH), 162.75 (d, C, JC-F 247.1, CF), 178.19 (C, CS); m/z (ESI ) 224 + + + [(M + H) , 100%]; HRMS (ESI ): Exact mass calculated for [C10H11FN3S] 224.0652. Found 224.0651. 2-Methyl-3-phenylallylidene hydrazine-1-carbothioamide 30 To a solution of α-methylcinnamaldehyde 18 (0.100 g, 0.68 mmol) in absolute ethanol (5 mL) was added thiosemicarbazide (0.062 g, 0.68 mmol) and acetic acid (2 drops). The reaction mixture was heated to 80 ◦C and allowed stir for 48 h. The reaction mixture was cooled to 0 ◦C for 3 h without agitation. A precipitate formed and was isolated by vacuum filtration. The crude product was recrystallised from ethyl acetate: hexane to form a paper-like white solid (0.76 g, 51.0%) m.p. 173–175 ◦C 1 (Lit. m.p. 170–172 ◦C) [60]; IR νmax/cm− : 3423, 3241, 3149, 2965, 1602, 1508, 819; δH (300 MHz, CDCl3): 2.12 (3H, s, CH3-2), 6.39 (1H, br s, NH2), 6.80 (1H, s, H-3), 7.14 (1H, br s, NH2), 7.27-7.41 (5H, m, ArH), 7.69 (1H, s, H-1), 9.73 (1H, br s, CNNH); m/z (ESI+) 220 [(M + H)+, 100%]. 5,6,11-Trimethyl-6H-pyrido [4,3-b]carbazole 31 5,11-Dimethyl-6H-pyrido[4,3b]carbazole 15 (1.50 g, 6.10 mmol) in anhydrous DMF (20 mL) was treated with NaH (0.375 g, 60% dispersion in mineral oil 9.4 mmol,) at 0 ◦C under nitrogen and allowed stir for 30 min, forming a deep red suspension. Iodomethane (0.39 mL, 6.20 mmol) was added and the reaction was allowed warm to room temperature and stir overnight. The reaction mixture was cooled to 0 ◦C and water (100 mL) was added producing a pale orange precipitate. The yellow product was isolated by vacuum filtration and washed with water (150 mL) and did not require further purification 1 (1.46 g, 91.8%). m.p 205–206 ◦C (Lit. m.p 202–204 ◦C) [61]; IR νmax/cm− : 2921, 2852, 1588, 1476, 1449, 1241, 1102, 806; δH (300 MHz, DMSO-d6): 3.03 (3H, s, CH3-5), 3.19 (3H, s, CH3-11), 4.13 (3H, s, NCH3), 7.31 (1H, overlapping ddd, J 8.0, 6.4, 1.5, H-9), 7.58 (1H, dd, J 8.0, 1.5, H-8), 7.62 (1H, d, J 1.5, H-7), 7.99 (1H, d, J 6.0, H-4), 8.37 (1H, d, J 8.0, H-10), 8.45 (1H, d, J 6.0, H-3), 9.68 (1H, s, H-1); m/z (ESI+) 261 [(M + H)+, 100%]. 6-Ethyl-5,11-dimethyl-6H-pyrido[4,3-b]carbazole 32 Pathogens 2020, 9, 542 18 of 24

5,11-Dimethyl-6H-pyrido[4,3-b]carbazole 15 (3.33 g, 13.5 mmol) in anhydrous DMF (60 mL), at 0 ◦C under nitrogen, was treated with sodium hydride (0.812 g, 20.3 mmol, 60% dispersion in mineral oil) and left to stir for 30 min forming a red suspension. Iodoethane (1.14 mL, 2.22 g, 14.2 mmol) was added and the reaction was allowed to warm to room temperature and was left to stir for a total of 72 h. The reaction mixture was cooled on ice and water (200 mL) was added. The aqueous layer was extracted with dichloromethane-methanol 90:10 (5 50 mL). The combined organic extracts were × washed with water (5 50 mL) and brine (1 50 mL), dried over magnesium sulphate, filtered and × × the solvent removed under reduced pressure. Purification by column chromatography eluting with dichloromethane-methanol (99:1) gave the product as a yellow solid (2.35 g, 63.4%). m.p. 164–165 ◦C; 1 νmax/cm− (KBr): 3020, 2966, 2926, 1596, 1588, 1475, 1395, 1382, 1346, 1232, 741; δH (600 MHz, DMSO-d6): 1.33 [3H, t, J 7.1, N(6)CH2CH3], 2.84 [3H, s, C(5)CH3], 3.03 [3H, s, C(11)CH3], 4.50 [2H, q, J 7.1, N(6)CH2CH3], 7.26 [1H, t, J 7.4, C(9)H], 7.53–7.58 [2H, m, C(7)H, C(8)H], 7.92 [1H, d, J 6.1, C(4)H], 8.26 [1H, d, J 7.8, C(10)H], 8.41 [1H, d, J 6.0, C(3)H], 9.58 [1H, s, C(1)H]; δC (150.9 MHz, DMSO-d6): 13.4 [CH3, C(5)CH3], 14.7 [CH3, C(11)CH3], 15.4 [CH3, N(6)CH2CH3], 40.2 [CH2, N(6)CH2CH3], 108.7 (C, aromatic C), 109.4 (CH, aromatic CH), 116.3 (CH, aromatic CH), 120.1 (CH, aromatic CH), 122.4 (C, aromatic C), 123.3 (C, aromatic C), 124.2 (CH, aromatic CH), 124.5 (C, aromatic C), 127.7 (CH, aromatic CH), 128.8 (C, aromatic C), 134.0 (C, aromatic C), 140.3 (C, aromatic C), 141.2 (CH, aromatic CH), 144.1 (C, aromatic C), 149.9 (CH, aromatic CH); m/z (ESI+): 275 [(M + H)+ 100%]; HRMS (ESI+): Exact mass + calculated for C19H19N2 275.1548. Found 275.1547. 6-Isopropyl-5,11-dimethyl-6H-pyrido[4,3-b]carbazole 33 5,11-Dimethyl-6H-pyrido[4,3-b]carbazole 15 (2.31 g, 9.37 mmol) was added portion-wise to a suspension of sodium hydride (0.75 g, 60% dispersion in mineral oil, equivalent to 0.45 g sodium hydride, 18.8 mmol) in dimethylformamide (10 mL) forming a deep red suspension which was stirred at room temperature for 45 min under nitrogen. 2-Iodopropane (1.10 mL, 1.90 g, 11.2 mmol) in dimethylformamide (5 mL) was added dropwise and the reaction was stirred for 48 h. The reaction mixture was cooled on ice and water (50 mL) added. The aqueous layer was extracted with dichloromethane—Methanol 90:10 (3 50 mL). A precipitate in the aqueous layer which would × not extract was filtered and found to be unreacted ellipticine (402 mg). Combined organic layers were washed with water (5 20 mL) and brine (1 30 mL), dried over magnesium sulphate and × × solvent removed under reduced pressure. Purification by column chromatography eluting with dichloromethane – methanol 99:1 gave the product as a yellow solid (0.96 g, 35.5%). m.p. 168–170 ◦C; 1 νmax/cm− (KBr): 3435, 3079, 2970, 2933, 1598, 1587, 1469, 1390, 1367, 1345, 1288, 1236, 1104, 804, 741; δH (600 MHz, DMSO-d6): 1.59 [6H, d, J 6.9, N(6)CH(CH3)2], 2.87 [3H, s, C(5)CH3], 3.15 [3H, s, C(11)CH3], 5.32 [1H, sept, J 7.0, N(6)CH(CH3)2], 7.28 [1H, overlapping ddd, J 7.8, 7.2, 0.5, C(9)H], 7.51 [1H, overlapping ddd, J 8.2, 7.2, 1.0, C(8)H], 7.77 [1H, d, J 8.2, C(7)H], 7.92 [1H, d, J 6.0, C(4)H], 8.34 [1H, d, J 7.8, C(10)H], 8.43 [1H, d, J 5.8, C(3)H], 9.66 [1H, s, C(1)H]; δH (600 MHz, DMSO-d6): 14.8 [CH3, C(11)CH ], 15.3 [CH , C(5)CH ], 21.4 [2 CH , N(6)CH(CH ) ], 49.6 [CH, N(6)CH(CH ) ], 109.6 (C, 3 3 3 × 3 3 2 3 2 aromatic C), 113.6 (CH, aromatic CH), 116.6 (CH, aromatic CH), 120.2 (CH, aromatic CH), 122.9 (C, aromatic C), 124.5 (CH, aromatic CH), 124.9 (C, aromatic C), 125.3 (C, aromatic C), 127.0 (CH, aromatic CH), 128.3 (C, aromatic C), 134.6 (C, aromatic C), 141.4 (CH, aromatic CH), 143.1 (C, aromatic C), 143.2 (C, aromatic C), 149.9 (CH, aromatic CH); m/z (ESI+): 289 [(M + H)+, 100%]; HRMS (ESI+): Exact mass + calculated for C20H21N2 289.1705. Found 289.1707. 5,6,11-Trimethyl-6H-pyrido[4,3-b]carbazole-9-carbaldehyde 34 5,6,11-Trimethyl-6H-pyrido[4,3-b]carbazole 31 (0.600 g, 2.33 mmol) was dissolved in trifluoroacetic acid (30 mL) and hexamethylenetetramine (3.24 g, 23.41 mmol) added portion-wise. The reaction was heated to reflux for 1 h. Upon cooling to room temperature, the mixture was cooled to 0 ◦C and poured into water (200 mL). Solid sodium bicarbonate was added until neutralised and the mixture was extracted with dichloromethane-methanol (150 mL 4, 9:1). The combined organic layers were × washed with water (2 100 mL) and brine (2 100 mL), dried, filtered and solvent removed under × × reduced pressure to yield an orange solid (0.53 g, 81.0%). m.p. 219–222 ◦C (Lit. m.p. 222-223 ◦C) [55,62]; Pathogens 2020, 9, 542 19 of 24

1 IR νmax/cm− : 3214, 2922, 2852, 1674, 1586, 1461, 1380, 1356, 1099, 983; δH (300 MHz, DMSO-d6): 3.01 (3H, s, CH3-5), 3.21 (3H, s, CH3-11), 4.16 (3H, s, NCH3), 7.75 (1H, d, J 8.7, H-7), 8.01 (1H, d, J 5.8, H-4), 8.07 (1H, dd, J 8.7, 1.5, H-8), 8.49 (1H, d, J 5.8, H-3), 8.78 (1H, d, J 1.5, H-10), 9.70 (1H, s, H-1), 10.10 (1H, s, CHO); m/z (ESI+) 289 [(M + H)+, 100%]. 6-Ethyl-5,11-dimethyl-6H-pyrido[4,3-b]carbazole-9-carbaldehyde 35 6-Ethyl-5,11-dimethyl-6H-pyrido[4,3-b]carbazole 32 (1.00 g, 3.65 mmol) was stirred in trifluoroacetic acid (40 mL) and hexamethylenetetramine (5.11 g, 36.5 mmol) was added portion-wise. The reaction was heated to reflux for 40 min. Upon cooling the reaction mixture was cooled on ice and poured into water (250 mL). The reaction mixture was neutralised using solid sodium bicarbonate [Caution: vigorous reaction] and extracted with dichloromethane-methanol 90:10 (3 100 mL). × The combined organic extracts were washed with water (2 100 mL) and brine (1 100 mL), dried over × × magnesium sulphate, filtered and the solvent removed under reduced pressure to give the product as an 1 orange solid which was used without further purification (1.00 g, 91.0%). m.p. 192–196 ◦C; νmax/cm− (KBr): 2970, 2929, 2720, 1682, 1589, 1452, 1381, 1315, 1235, 1100, 809; δH (500 MHz, DMSO-d6): 1.42 [3H, t, J 6.9, N(6)CH2CH3], 2.95 [3H, s, C(5)CH3], 3.19 [3H, s, C(11)CH3], 4.66 [2H, q, J 6.8, N(6)CH2CH3], 7.78 [1H, d, J 8.5, C(7)H], 8.03 [1H, d, J 5.9, C(4)H], 8.08 [1H, d, J 8.4, C(8)H], 8.49 [1H, d, J 5.6, C(3)H], 8.77 [1H, s, C(10)H], 9.79 [1H, s, C(1)H], 10.10 [1H, s, C(9)CHO]; δC (125.8 MHz, DMSO-d6): 13.4 [CH3, C(5)CH3], 14.9 [CH3, C(11)CH3], 15.5 [CH3, N(6)CH2CH3], 40.7 [CH2, N(6)CH2CH3], 109.9 (CH, aromatic CH), 110.3 (C, aromatic C), 116.6 (CH, aromatic CH), 122.9 (C, aromatic C), 123.3 (C, aromatic C), 123.9 (C, aromatic C), 127.1 (CH, aromatic CH), 128.9 (CH, aromatic CH), 129.3 (C, aromatic C), 129.7 (C, aromatic C), 134.5 (C, aromatic C), 140.6 (C, aromatic C), 141.9 (CH, aromatic CH), 147.9 (C, aromatic C), 150.2 (CH, aromatic CH), 192.4 [C, C(9)CHO]; m/z (ESI+): 303 [(M + H)+, 100%]; HRMS + + (ESI ): Exact mass calculated for C20H19N2O 303.1497. Found 303.1485. 6-Isopropyl-5,11-dimethyl-6H-pyrido[4,3-b]carbazole-9-carbaldehyde 36 6-Isopropyl-5,11-dimethyl-6H-pyrido[4,3-b]carbazole 33 (0.81 g, 2.82 mmol) was stirred in trifluoroacetic acid (40 mL) and hexamethylenetetramine (3.96 g, 28.2 mmol) was added portion-wise. The reaction was heated to reflux for 40 min. Upon cooling the reaction mixture was cooled on ice and poured into water (250 mL). The reaction mixture was neutralised using solid sodium bicarbonate [Caution: vigorous reaction] and extracted with dichloromethane-methanol 90:10 (3 100 mL). × The combined organic extracts were washed with water (2 100 mL) and brine (1 100 mL), dried over × × magnesium sulphate, filtered and the solvent removed under reduced pressure to give the product as an orange solid which was used without further purification (0.836 g, 93.7%). m.p. 200–205 ◦C; 1 νmax/cm− (KBr): 3412, 2969, 2929, 1683, 1588, 1574, 1386, 1321, 1286, 1240, 1192, 1140, 1104, 812; δH (600 MHz, DMSO-d6): 1.67 [6H, d, J 7.0, N(6)CH(CH3)2], 2.94 [3H, s, C(5)CH3], 3.24 [3H, s, C(11)CH3], 5.45 [1H, sept, J 6.9, N(6)CH(CH3)2], 7.97 [1H, d, J 8.6, C(7)H], 8.01 [1H, d, J 6.1, C(4)H], 8.04 [1H, dd, J 8.6, 1.4, C(8)H], 8.50 [1H, d, J 6.0, C(3)H], 8.84 [1H, d, J 1.1, C(10)H], 9.73 [1H, s, C(1)H], 10.11 [1H, s, C(9)CHO]; δ (150.9 MHz, DMSO-d ): 14.9 [CH , C(11)CH ], 15.3 [CH , C(5)CH ], 21.3 [2 CH , C 6 3 3 3 3 × 3 N(6)CH(CH3)2], 50.0 [CH, N(6)CH(CH3)2], 110.8 (C, aromatic C), 113.6 (CH, aromatic CH), 116.9 (CH, aromatic CH), 123.2 (C, aromatic C), 124.1 (C, aromatic C), 125.2 (C, aromatic C), 127.2 (CH, aromatic CH), 128.0 (CH, aromatic CH), 129.1 (C, aromatic C), 129.3 (C, aromatic C), 135.0 (C, aromatic C), 141.9 (CH, aromatic CH), 143.0 (C, aromatic C), 147.1 (C, aromatic C), 150.1 (CH, aromatic CH), 192.6 [C, + + + + C(9)CHO]; m/z (ESI ): 317 [(M + H) , 100%]; HRMS (ESI ): Exact mass calculated for C21H21N2O 317.1654. Found 317.1651. Phytophthora infestans Mycelial Growth Assays Phytophthora infestans strain 88,069 (mating type A1) was originally obtained from Professor Sophien Kamoun (The Sainsbury Laboratory, Norwich, UK) and maintained on RyeA agar at 20 ◦C in the dark [63]. For mycelial growth assay, compounds were dissolved in DMSO in a vial and 15 mL Rye B agar added, at a temperature of approximately 50 ◦C (the final concentration of DMSO was 0.1%). The vial was shaken to distribute the contents and the mixture was then poured into a clean, sterile 10 cm Petri dish. A 10 mm plug of P. infestans mycelium was placed in the centre of the dish, which was Pathogens 2020, 9, 542 20 of 24 then sealed with parafilm. The experiments were carried out in triplicate at 25 µM (unless otherwise stated), stored at 20 ◦C in the dark and checked periodically for mycelial growth. Phytophthora infestans Zoosporogenesis and Sporangiogenesis Assays These were performed essentially as described by Lu et al. [39]. Following two weeks of culture on Rye B agar, sporangia were collected from plates in 5 mL of modified Petri’s solution (MPS, 5 mM CaCl2, 1 mM MgSO4, 1 mM KH2PO4 and 0.8 mM KCl), by scraping with a scalpel blade and were transferred to a 50 mL sterile tube. Plates were washed with another 5 mL of MPS and this was combined with the first 5 mL. To initiate zoospore release, sporangia were place on ice, in the dark, for 3h. Sporangia or zoospores were counted by brightfield microscopy using a 10 objective and a × haemocytometer, in duplicate. XTT reduction assay Human embryonic kidney-293T (HEK-293T) cells were obtained from LGC Standards (Middlesex, UK) and were maintained in Dulbecco’s Modified Eagle’s Medium containing 10% foetal bovine serum, 100 units/mL penicillin and 100 µg/mL streptomycin, at 37 ◦C in a humidified atmosphere of 5% CO2/95% air. For assays, cells were subcultured on 96-well microtitre plates at a density of 5 104 cells/well, in a volume of 50 mL. Following overnight culture, 50 mL of each test compound × at twice their final concentration (50 mM for all apart from cinnamaldehyde (2 mM) and Mancozeb (200 µM)) was added and incubated for another 24 h. Cells were incubated with 100 mL XTT reagents (Sigma-Aldrich, Ireland) for an additional 4 h, after which, the absorbance of the extracellular formazan reduction product was measured at a wavelength of of 490 nm, with a reference wavelength of 675 nm [53,54], using a microtitre plate spectrophotometer. Statistical Methods Numerical data were statistically compared using one-way analysis of variance (ANOVA) and Tukey’s post hoc test (using ezANOVA software, available at: https://people.cas.sc.edu/rorden/ezanova/ index.html.) A probability threshold of p < 0.05 was taken as statistically significant. Generation of histograms and linear regression analyses were performed using Microsoft Excel.

5. Conclusions It is evident from the synthesis and biological evaluation that both cell viability and inhibitory effects are impacted by discreet modifications of the cinnamaldehyde and ellipticine scaffolds. The mycelium growth assay provided information at Days 5, 9 and 13 of P. infestans incubation. From an initial focus on the cinnamaldehyde derivatives, the cinnamaldehyde Mancozeb hybrid proved fruitful and is a template for further discovery given its excellent P. infestans growth inhibition/toxicity profile. While the effects of cinnamaldehyde derivatives were somewhat predicted, uniquely in the screen of aldehydes the ellipticine aldehydes have emerged with excellent Phytophthora growth inhibitory properties. The aldehyde functionality on ellipticine has been shown to increase significantly the inhibitory effects of the compound and limit the toxicity to an extent though this needs to be tempered further before progressing. In contrast, the stock aldehydes did not demonstrate strong inhibition which suggests that there are additional factors affecting the oomycete growth. The effects of ellipticine substitution have identified that alkylation of the six-position considerably improves mycelial growth inhibition. Ellipticines 31 and 34 have significant growth inhibitory properties on mycelia and inhibit zoosporogenesis and sporangiogenesis in comparison to ellipticine (15). It is evident from the biological data that 34 demonstrates the greatest inhibitory effects on P. infestans with a mycelium growth of just 6% after 13 days of incubation and the comparison with cinnamaldehyde is favourable given the respective dosages. The XTT cytotoxicity assay has highlighted that cinnamaldehyde and ellipticine compounds were amongst the most highly toxic of all the compounds tested and currently limits their potential use as fungicides. However, the discovery of 9-formyl-6-methylellipticine 34 as a potent growth inhibitor is the subject of our investigation into the influence of 2- and 6-alkylated 9-formylellipticines on P. infestans growth [64]. This study has Pathogens 2020, 9, 542 21 of 24 resulted in promising new cinnamaldehyde and ellipticine candidates for P. infestans control that warrant further study to examine the role of substituents and mechanism of action.

Supplementary Materials: The Supplementary Information is available online at http://www.mdpi.com/2076- 0817/9/7/542/s1. Experimental: Synthesis of Ellipticine Framework, NMR data: NMR data for compounds 26–33 and 35–36, Figure S1: Mycelial Growth Inhibition of cinnamaldehyde (7), Figure S2: Mycelial Growth Inhibition of hydrocinnamaldehyde 6, Figure S3: Mycelial Growth Inhibition of Mancozeb 1. Author Contributions: B.M.D.P., F.O.M. and J.J.M. conceived and designed the experiments. F.O.M. and J.J.M. wrote the manuscript. E.C.O., L.Z., M.L.M. and R.A.K. performed the experiments, characterised the synthetic products and analysed the data. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by the Department of Agriculture, Food and the Marine (Government of Ireland), Research Stimulus Fund grant number 15S669, awarded to J.J.M., and funded L.Z. and R.A.K. E.C.O. and M.L.M. were both funded under the Irish Research Council Postgraduate Scholarship Scheme. Acknowledgments: The authors thank Don Kelleher and Eileen Dillane for valuable technical assistance. Conflicts of Interest: The authors declare no conflicts of interest.

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