Natural Compound-Derived Cytochrome Bc1 Complex Inhibitors As Antifungal Agents

molecules

Review Natural Compound-Derived Cytochrome bc1 Complex Inhibitors as Antifungal Agents

Loana Musso * , Andrea Fabbrini and Sabrina Dallavalle

Department of Food, Environmental and Nutritional Sciences (DeFENS), Division of Chemical and Biomolecular Sciences, Università degli Studi di Milano, Via Celoria 2, 20133 Milan, Italy; [email protected] (A.F.); [email protected] (S.D.) * Correspondence: [email protected]

Received: 4 August 2020; Accepted: 6 October 2020; Published: 7 October 2020

Abstract: The high incidence of fungal pathogens has become a global issue for crop protection. A promising strategy to control fungal plant infections is based on the use of nature-inspired compounds. The cytochrome bc1 complex is an essential component of the cellular respiratory chain and is one of the most important fungicidal targets. Natural products have played a crucial role in the discovery of cytochrome bc1 inhibitors, as proven by the development of strobilurins, one of the most important classes of crop-protection agents, over the past two decades. In this review, we summarize advances in the exploration of natural product scaﬀolds for the design and development of new bc1 complex inhibitors. Particular emphasis is given to molecular modeling-based approaches and structure–activity relationship (SAR) studies performed to improve the stability and increase the potency of natural precursors. The collected results highlight the versatility of natural compounds and provide an insight into the potential development of nature-inspired derivatives as antifungal agents.

Keywords: natural compounds; cytochrome bc1 complex; antifungals; (E)-β-methoxyacrylate; strobilurins

1. Introduction Plant diseases caused by fungal pathogens are a major threat to the agricultural industry worldwide. Controlling plant fungal infections is therefore key to increase food production, as the planet’s continuously growing population demands maximized crop yields [1]. The cytochrome bc1 complex (also known as complex III) is one of the most important fungicidal targets. The complex is an essential component of the cellular respiratory chain. It catalyzes the ubiquinol oxidation (UQH2) to quinone (UQ) and channels the resulting electrons to cytochrome c through the ubiquinone cycle (Qo cycle, Figure1). Furthermore, the ubiquinol oxidation results in a bifurcated electron transfer. One electron is transferred, via the Rieske iron–sulfur protein and cytochrome c1, to cytochrome c, while the second electron passes through two b-type haems (bL and bH) to reduce ubiquinone at the Qi site [2]. This process results in proton translocation and generates the proton motive force required in the production of ATP. Protons are taken up from the mitochondrial matrix when ubiquinone is reduced at the Qi site and they are released into the intermembrane space when ubiquinol is oxidized at the Qo site. Inhibition of the activity of the bc1 complex blocks the generation of ATP, leading to cell death. For this reason, inhibitors of the cytochrome bc1 complex have aroused great interest in controlling fungal diseases. The cyt bc1 inhibitors target either the ubiquinol oxidation site (Qo or QP) or the ubiquinone reduction site (Qi or QN).

Molecules 2020, 25, 4582; doi:10.3390/molecules25194582 www.mdpi.com/journal/molecules Molecules 2020, 25, 4582 2 of 28

Natural products have played a crucial role in the investigation of cytochrome bc1 inhibitors, leading to the discovery of one of the most important classes of crop-protection agents, the strobilurins. These compounds still have a dominant position in the global market. Natural products, with their enormous structural diversity, are an invaluable source of inspiration in the design and development of new biologically active compounds [3]. Having evolved over several millennia to acquire specific ligand–protein binding motifs, these privileged compounds serve as important, biologically pre-validated platforms for the development of new leads in medicinal chemistry and agriculture. However, the structural complexity, toxicity, and Moleculesunfavorable2020, 25 bioavailability, 4582 often associated with natural products can limit their potential, which2 of 28is why as such structural modification is often required.

FigureFigure 1.1. SchematicSchematic modelmodel ofof cytochromecytochrome bc1bc1 complex.complex. TheThe homodimerichomodimeric bc1bc1 complexcomplex presentspresents threethree

catalyticcatalytic subunits:subunits: cytochromecytochrome bb (cyt(cyt b)b) withwith twotwo b-typeb-typehaems haems(b (bHH andand bbLL),), thethe RieskeRieske iron–sulfuriron–sulfur proteinprotein (FeS) (FeS) and and cytochrome cytochrome c1 c1 (cyt (cyt c1) c1) with with one one c-type c-type haem. haem. The The two two binding binding sites sites for inhibitors for inhibitors and ubiquinoneand ubiquinone (UQ), (UQ), Qi and Qi Qo, and are Qo shown., are shown. The bifurcated The bifurcated electron electron transfer transfer pathway pathway from the from Qo the site Qo is shownsite is shown by red by arrows. red arrows.

Natural products have played a crucial role in the investigation of cytochrome bc1 inhibitors, In this review, we summarize the efforts towards the exploration of natural product scaffolds leading to the discovery of one of the most important classes of crop-protection agents, the strobilurins. for the design and development of bc1 complex inhibitors, focusing on what has been observed and These compounds still have a dominant position in the global market. achieved in the past decade. In particular, we concentrate on studies focused on optimization of the Natural products, with their enormous structural diversity, are an invaluable source of inspiration core natural scaffold by simplification, direct substitution, and/or by use of isosteric modifications. in the design and development of new biologically active compounds [3]. Having evolved over Molecular modeling-based approaches and structure–activity relationship (SAR) studies performed several millennia to acquire specific ligand–protein binding motifs, these privileged compounds serve to improve the potency, selectivity, and stability of bioactive natural products are highlighted as as important, biologically pre-validated platforms for the development of new leads in medicinal well. chemistry and agriculture. However, the structural complexity, toxicity, and unfavorable bioavailability often2. Strobilurin associateds with natural products can limit their potential, which is why as such structural modification is often required. InStrobilurins this review, are we natural summarize compounds the efforts isolated towards for the exploration first time in of 1977 natural from product basidiomycetes. scaffolds forThey the are design the andfirst development example of natural of bc1 complex fungicides inhibitors, belonging focusing to the on group what of has QoI been (quinone observed outside and achievedinhibitors, in (FRACthe past group decade. 11 In)). particular, Strobilurin we-based concentrate products, on studies obtained focused by activity on optimization-guided rational of the coredesign, natural have scabeenffold a milestone by simplification, in the fungicidal direct substitution, market worldwide. and/or byIndeed, use of these isosteric compounds modifications. have a Molecularbroad-spectrum modeling-based of activity, approaches being effective and structure–activity against all four relationship major classes (SAR) studies of phytopathogenic performed to improveorganisms the (ascomycetes, potency, selectivity, basidiomycetes, and stability deuteromycetes of bioactive natural, and products oomycetes are highlighted). They show as well. acute toxicity against germinating fungal spores and relatively low toxicity for terrestrial animals. 2. StrobilurinsThe major drawbacks of these fungicides are their toxicity to aquatic organisms and the increased number of fungal pathogens that are showing occurrence of resistance, mainly due to Strobilurins are natural compounds isolated for the first time in 1977 from basidiomycetes. They are mutations on mitochondrial genes encoding for the Qo site [4]. the first example of natural fungicides belonging to the group of QoI (quinone outside inhibitors, Based on this evidence, the development of new strobilurin derivatives is still pressing because (FRAC group 11)). Strobilurin-based products, obtained by activity-guided rational design, have been of their role in the market and the need of overcoming the explosive increase of fungal resistance a milestone in the fungicidal market worldwide. Indeed, these compounds have a broad-spectrum worldwide. of activity, being effective against all four major classes of phytopathogenic organisms (ascomycetes, basidiomycetes, deuteromycetes, and oomycetes). They show acute toxicity against germinating fungal spores and relatively low toxicity for terrestrial animals. The major drawbacks of these fungicides are their toxicity to aquatic organisms and the increased number of fungal pathogens that are showing occurrence of resistance, mainly due to mutations on mitochondrial genes encoding for the Qo site [4]. Based on this evidence, the development of new strobilurin derivatives is still pressing because of their role in the market and the need of overcoming the explosive increase of fungal resistance worldwide. Molecules 2020, 25, 4582 3 of 28

MoleculesVarious2020, 25 , 4582reviews have been published about the design and synthesis of 3 new of 28 strobilurin-derived compounds [5,6]. In the next section, we provide an overview of the efforts made in this field in the last 10 years. Various reviews have been published about the design and synthesis of new strobilurin-derived compoundsFrom Natural [ 5to,6 Synthetic]. In the nextStrobilurins section, we provide an overview of the efforts made in this field in the last 10 years. Strobilurin A (1) was the first compound of this family isolated from Strobilurus tenacellus by FromAnke Naturalet al. [7], to Syntheticfollowed Strobilurinsby strobilurin B (2) and so on (Figure 2). Although isolated from different fungi, the various strobilurins have a very similar structure characterized by a pharmacophore Strobilurin A (1) was the first compound of this family isolated from Strobilurus tenacellus by portion, an unsaturated bridge, and a side chain, varying only in the aromatic ring substituents. The Anke et al. [7], followed by strobilurin B (2) and so on (Figure2). Although isolated from di fferent interest for strobilurins increased following the discovery of their fungicidal activity by Anke et al. fungi, the various strobilurins have a very similar structure characterized by a pharmacophore portion, [7], which led scientists to search for these compounds in various fungi. However, their activity was an unsaturated bridge, and a side chain, varying only in the aromatic ring substituents. The interest for weak and, more importantly, they exhibited stability problems because of their photo lability. strobilurins increased following the discovery of their fungicidal activity by Anke et al. [7], which led During the first 1980s, the ICI and the BASF groups simultaneously, although working scientists to search for these compounds in various fungi. However, their activity was weak and, separately, identified methoxyacrylate stilbene (3, MOAS, Figure 2) as a stable synthetic strobilurin more importantly, they exhibited stability problems because of their photo lability. with a benzene ring in place of the central double bond of the original triene system [6].

Side chain lead structure Bridge development

MeO OMe O O R2 Pharmacophore O O Enol ether stilbene Strobilurin A (1) R1 = H, R2 = H

Strobilurin B (2) R = OCH , R = H 1 3 2 MOAS = methoxyacrylate stilbene (3)

Figure 2. Structure of natural strobilurin A and B, and the methoxyacrylate stilbene (MOAS) Figure 2. Structure of natural strobilurin A and B, and the methoxyacrylate stilbene (MOAS) lead lead structure. structure. During the first 1980s, the ICI and the BASF groups simultaneously, although working separately, Compound 3 immediately became the lead synthetic strobilurin because of its stability and identified methoxyacrylate stilbene (3, MOAS, Figure2) as a stable synthetic strobilurin with a benzene increased efficacy (Figure 2). Successive studies led to the introduction into the market of various ring in place of the central double bond of the original triene system [6]. synthetic analogs, featuring broad structural variations on the side chain portion as well as Compound 3 immediately became the lead synthetic strobilurin because of its stability and containing bioisosteres of the (E)-β-methoxyacrylate pharmacophoric group (Figure 3) [8]. increased efficacy (Figure2). Successive studies led to the introduction into the market of various synthetic analogs, featuring broad structural variations on the side chain portion as well as containing β N N bioisosteres of the (E)- O-methoxyacrylate pharmacophoric group (Figure3)[8]. While the early chemical modificationsO of the strobilurinO scaffoldO addressed the improvement of O O CN O O O O physical characteristics (i.e., volatility,N stability, resistance to UV breakdown) andN resulted in commercial O O NH strobilurins like azoxystrobin, picoxystrobin, pyraclostrobin, and trifloxystrobin (Figure3), in the latest years new strobilurinsKresoxi werem-meth developedyl (4) to increaseAzoxy theirstrobin e ffi(5)cacy as wellMetom asinos totro overcomebin (6) the resistance to their action on QoI-resistant strains. F C O F C N O For this3 purpose,N different approaches3 were used, such as the me-tooO approach, biorational or chemorational design, fragment-basedO O drug designO (FBDD),O intermediateO derivatizationO methods N N based on bioisosteric replacement,O and pharmacophore-linkedO fragment virtualNH screening (PFVS). Hao et al. developed a new molecular design method based on PFVS, by integrating the advantages Trifloxystrobin (7) of FBDD and the advantages of docking methodsPico [x9y].str Throughobin (8) this approach,Dimoxystro Yangbin (9) and coworkers [10] designed and synthesized a series of benzophenoneN /fluorenone-containingN O derivatives (Figure4) to obtain newC strobilurinl N analogsO with higher fungicidal activity. As shownN in FigureO 4, the O-bridged N O O N O N Cl F N O O O derivative 14b (Ki = 3.28 nmol/L)O is more active thanO its correspondingN NS-bridgedN derivative 14a (Ki = 13.95 nmol/L) and the NHO-bridged derivative 14c (Ki >O1000 nmol/L). Interestingly,O NH compound 14d showed an improvedPyraclo bindingstrobin (10 a) ffinity (Ki =F1.89luoxas nmoltrobin (/1L)1) to the porcineOrysastro cytochromebin (12) bc1 complex (porcine SCR, succinate cytochrome c reductase) compared to the commercial inhibitor azoxystrobin. Figure 3. Commercial strobilurins.

Molecules 2020, 25, 4582 3 of 28

Various reviews have been published about the design and synthesis of new strobilurin-derived compounds [5,6]. In the next section, we provide an overview of the efforts made in this field in the last 10 years.

From Natural to Synthetic Strobilurins Strobilurin A (1) was the first compound of this family isolated from Strobilurus tenacellus by Anke et al. [7], followed by strobilurin B (2) and so on (Figure 2). Although isolated from different fungi, the various strobilurins have a very similar structure characterized by a pharmacophore portion, an unsaturated bridge, and a side chain, varying only in the aromatic ring substituents. The interest for strobilurins increased following the discovery of their fungicidal activity by Anke et al. [7], which led scientists to search for these compounds in various fungi. However, their activity was weak and, more importantly, they exhibited stability problems because of their photo lability. During the first 1980s, the ICI and the BASF groups simultaneously, although working separately, identified methoxyacrylate stilbene (3, MOAS, Figure 2) as a stable synthetic strobilurin with a benzene ring in place of the central double bond of the original triene system [6].

Side chain lead structure Bridge development

MeO OMe O O R2 Pharmacophore O O Enol ether stilbene Strobilurin A (1) R1 = H, R2 = H

Strobilurin B (2) R = OCH , R = H 1 3 2 MOAS = methoxyacrylate stilbene (3)

Figure 2. Structure of natural strobilurin A and B, and the methoxyacrylate stilbene (MOAS) lead structure.

Compound 3 immediately became the lead synthetic strobilurin because of its stability and increased efficacy (Figure 2). Successive studies led to the introduction into the market of various Moleculessynthetic2020 analogs,, 25, 4582 featuring broad structural variations on the side chain portion as well4 of 28as containing bioisosteres of the (E)-β-methoxyacrylate pharmacophoric group (Figure 3) [8].

Molecules 2020, 25, 4582 N N 4 of 28 O O O O O O CN O O O O While the early chemical modificationsN of the strobilurin scaffold addressedN the improvement of physical characteristics (i.e.O , volatility, stability, resistanceO to UV breakdown)NH and resulted in commercial strobilurinsKresoxim like-meth azoxystrobin,yl (4) picoxystrobin,Azoxystrobin ( 5pyraclostrobin,) Metomin oandstrob trifloxystrobinin (6) (Figure 3), in the latest years new strobilurins were developed to increase their efficacy as well as to overcome theF C resistanceO to their action onF QoIC -NresistantO strains. 3 N 3 O For this purpose, differentO approachesO were used,O suchO as the me-too approach, biorational or N O O chemorational design, fragment-based drug design (FBDD), intermediate derivatizationN methods O O NH based on bioisosteric replacement, and pharmacophore-linked fragment virtual screening (PFVS). Hao et al. developedTrifloxystro b ai n new(7) molecular P designicoxystro b methodin (8) basedDim onoxy s PFVS,trobin (9 ) by integrating the advantages of FBDD and the advantages of docking methods [9]. Through this approach, Yang and N N O coworkers Cl[10] designedN O and synthesized a series of benzophenone/fluorenoneN O -containing N O O N derivatives (Figure 4) to obtainO newN strobilurinCl analogsF Nwith higherO fungicidal activity.O AsO shown in O O N N N Figure 4, the O-bridged derivativeO 14b (Ki = 3.28 nmol/L)O is more activeO than N itsH corresponding S-bridged derivative 14a (Ki = 13.95 nmol/L) and the NH-bridged derivative 14c (Ki > 1000 nmol/L). Pyraclostrobin (10) Fluoxastrobin (11) Orysastrobin (12) Interestingly, compound 14d showed an improved binding affinity (Ki = 1.89 nmol/L) to the porcine cytochrome bc1 complex (porcineFigureFigure SCR, 3.3. CommercialCommercialsuccinate cytochromestrobilurins.strobilurins. c reductase) compared to the commercial inhibitor azoxystrobin.

NH bioisosteric O substitution S X

MeO OMe MeO OMe

O O 13 14a, X = S Ki = 13.95 nmol/L 14b, X = O Ki = 3.28 nmol/L 14c, X = NH Ki = >1000 nmol/L AZ Ki = 297.6 nmol/L

O O

MeO OMe

O 14d Ki = 1.89 nmol/L Figure 4. Structural optimization of lead compound 13 and inhibitory activities of representative Figure 4. Structural optimization of lead compound 13 and inhibitory activities of representative compounds 14a–14d and azoxystrobin (AZ) against porcine SCR (succinate cytochrome c reductase) compounds 14a–14d and azoxystrobin (AZ) against porcine SCR (succinate cytochrome c reductase) with cytochrome c as substrate. with cytochrome c as substrate. The binding mode of compound 14d in Qo site of bc1 complex demonstrated that the pharmacophoreThe binding of mode this new of inhibitor compound bound 14d inin the Qo same site way of bc1 of typical complex methoxyacrylate demonstrated inhibitors, that the interactingpharmacophore with Phe128,of this new Tyr131, inhibitor Phe274, bound and in Glu271, the same and way forming of typical an H-bond methoxyacrylate between the inhibitors, methoxy groupinteracting of the with methoxyacrylate Phe128, Tyr131, moiety Phe274, and and the Glu271, backbone and forming nitrogen a ofn H Glu271.-bond between The presence the methoxy of the fluorenonegroup of the ring methoxyacrylate in 14d significantly moiety improved and the the backboneπ–π interactions nitrogen with of Glu271. Phe274 The compared presence with of that the observedfluorenone for ring the in azoxystrobin, 14d significantly justifying improved the higher the π potency–π interactions of the compound with Phe274 [10 compared]. with that observedMoreover, for the most azoxystrobin, of the new justifying compounds the higher displayed potency excellent of thein compound vivo fungicidal [10]. activity against SphaerothecaMoreover, fuliginea most atof thethe concentration new compounds of 200 displayedµmol/L. excellent in vivo fungicidal activity against SphaeStartingrotheca fuliginea from the at sca theffold concentration of enoxastrobin of 200 (15 μmol/L., Figure 5) developed by Rohm and Haas Company, Xie etStarting al. introduced from the modifications scaffold of enoxastrobin in the side chain. (15, Figure In order 5) developed to stabilize by the RohmE-styryl and group, Haas theCompany, authors Xie firstly et al. synthesized introduced a small modifications library of in indene-substituted the side chain. In oxime order ethers to stabilize (16, Figure the E5-)[styryl11]. Afterward,group, the authors they prepared firstly synthesized new oximeethers a small featuring library of heterocyclic indene-substituted moieties, oxime which ethers could drive(16, Figure some 5) [11]. Afterward, they prepared new oxime ethers featuring heterocyclic moieties, which could drive some physicochemical properties, such as lipophilicity and solubility, toward the optimal balanced range for uptake and bioavailability. In particular, benzothiophene, benzofuran, and indole analogs were developed (Figure 5) [12–14].

Molecules 2020, 25, 4582 5 of 28 physicochemical properties, such as lipophilicity and solubility, toward the optimal balanced range for uptake and bioavailability. In particular, benzothiophene, benzofuran, and indole analogs were developedMolecules 2020 (Figure, 25, 45825 )[12–14]. 5 of 28

O N MeO OMe MeO OMe Cl O O Strobilurin A (1) Enoxastrobin (15)

O O N N S MeO OMe MeO OMe R Cl 17 O 16 O

O O N N R O MeO OMe 1 N MeO OMe R2 O O 18 19a R1 = H R2 = H 19b R1 = H R2 = CH3 19c R1 = F R2 = H

Figure 5. EnoxastrobinEnoxastrobin-inspired-inspired analogs.

All thethe newnew synthesized synthesized compounds compounds were were tested tested on Pyricularia on Pyricularia oryzae ,oryzaeBotrytis, Botrytis cinerea, Erysiphecinerea, Erysiphe graminis, Colletotrichumgraminis, Colletotrichum lagenarium lagenarium, Pseudoperonospora, Pseudoperonospora cubensis, cubensis and Puccinia, and Puccinia sorghi Schw, sorghiand Schw, some and ofsome them of (e.g.,them17 (e.g., 18,, 17and, 1819b, and) showed 19b) showed fungicidal fungicidal activity similaractivity or similar higher or than higher enoxastrobin than enoxastrobin (Table1). (Table 1). Various research groups synthesized novel strobilurin derivatives containing a triazole moiety. TheseTab studiesle 1. In focused vitro and on in the vivo introduction fungicidal activity of 1,2,4-triazole (inhibition %)group of representative mainly because indene of ( its16a), broad spectrumbenzothiophene of biological (17), activity benzofuran [15– 20(18].), Aand large indole number (19a–c) of -substituted antifungal oxime agents ethers. containing from References 1,2,4-triazole group[11 as–14]. pharmacophore inhibit the formation of the cell membrane by preventing the ergosterol biosynthesis [21].

O O O O Remarkably,N the 1,2,4-triazole analogsN synthesized by ChaudharyN et al. and Liu etN al. showed antifungal activitiesMeO comparableOMe toS or betterMeO thanOMe azoxystrobinO Me [O15,17].OMe R1 N MeO OMe R2 16a O Cl 17 O 18 O O The authors studied the activity of 1,2,4-triazole thiol strobilurin analogs on19a R plant1 = H R2 = pathogens H (Fusarium oxysporum, Magnaporthe grisea, Drechslera oryzae) as well as human pathogens19b R1 = H (RCryptococcus2 = CH3 19c R1 = F R2 = H neoformans NCM3378, CryptococcusIn vitro neoformans NCM3542, Aspergillus fumigatusIn vivo )[15].

The introduction of a25 chlorine mg/L atom in para position on the phenyl6.25 ringmg/L had a positive effect on the fungicidal activityPyricul ofaria these compounds.Botrytis The pErysiphe-chlorophenyl derivativeColletotrichum20 (Figure 6Puccinia) was the sorghi most Cpd effective inhibitor againstorizae all the testedcinerea pathogens withgraminis MIC (minimumlagenarium inhibitory concentration)Schw. values in the16a range of 16-64100µg/ L). The inhibition50 of resazurin100 (RZ) reduction indicated70 that this compound/ has a19a mechanism of action80 similar to azoxystrobin,80 inhibiting50 the mitochondrial0 respiration by100 binding D oryzae to the19b Qo site of cytochrome100 b (EC5080 for inhibition95 of RZ reduction80 in . by azoxystrobin90 and compound 20 were 3.42 0.03 µg/mL and 3.62 0.21 µg/mL, respectively). At the same time, 19c 100 ± 80 99± 75 60 the authors observed that 1,2,3-triazole derivatives (i.e., compound 21) were ineffective in controlling enoxastrobin 50 100 100 85 100 the fungal growth at the highest concentration (512 µg/mL), and concluded that the 1,2,4-triazole In vitro In vivo moiety contributed to the antifungal activity of these derivatives. 6.25 mg/L 6.25 mg/L Pyricularia Botrytis Erysiphe Colletotrichum Puccinia sorghi Cpd orizae cinerea graminis lagenarium Schw. 17 50 100 50 100 70 18 / 100 100 98 40 enoxastrobin 50 100 100 90 98 Various research groups synthesized novel strobilurin derivatives containing a triazole moiety. These studies focused on the introduction of 1,2,4-triazole group mainly because of its broad

Molecules 2020, 25, 4582 5 of 28

O N MeO OMe MeO OMe Cl O O Strobilurin A (1) Enoxastrobin (15)

O O N N S MeO OMe MeO OMe R Cl 17 O 16 O

O O N N R O MeO OMe 1 N MeO OMe R2 O O 18 19a R1 = H R2 = H 19b R1 = H R2 = CH3 19c R1 = F R2 = H

Figure 5. Enoxastrobin-inspired analogs.

All the new synthesized compounds were tested on Pyricularia oryzae, Botrytis cinerea, Erysiphe Molecules 2020, 25, 4582 6 of 28 graminis, Colletotrichum lagenarium, Pseudoperonospora cubensis, and Puccinia sorghi Schw, and some of Molecules 2020, 25, 4582 6 of 28 them (e.g., 17, 18, and 19b) showed fungicidal activity similar or higher than enoxastrobin (Table 1). spectrumTable of 1. biologicalTabInle vitro 1. Inand activity vitro in and vivo [15 in vivo–fungicidal20 fungicidal]. A large activity activity number ( (inhibitioninhibition of antifungal %) %)of representative of agents representative containing indene (16a indene), 1,2,4 (16a-triazole), groupbenzothiophene as pharmacophorebenzothiophene (17), benzofuran ( 17inhibit), benzofuran the (18 ),(formation18 and), and indole indole of ( 19a the––c)c -)cellsubstituted -substituted membrane oxime oxime ethers. by ethers.frompreventing References from References the ergosterol [11–14]. biosynthesis[11–14]. [21]. Remarkably, the 1,2,4-triazole analogs synthesized by Chaudhary et al. and Liu et al. showed

O O O O antifungal activitiesN comparable to or betterN than azoxystrobinN [15,17]. N MeO OMe S MeO OMe O MeO OMe R1 N MeO OMe The authors studied the activity of 1,2,4-triazole thiol strobilurin analogsR2 on plant pathogens 16a O Cl 17 O 18 O O 19a R1 = H R2 = H (Fusarium oxysporum, Magnaporthe grisea, Drechslera oryzae) as well as human19b pathogensR1 = H R2 = CH3 (Cryptococcus 19c R1 = F R2 = H neoformans NCM3378, Cryptococcus neoformans NCM3542, Aspergillus fumigatus) [15]. The introduction of a Inchlorine vitro atom in para position on the phenyl In vivo ring had a positive effect on the fungicidal activity of 25 these mg/L compounds. The p-chlorophenyl 6.25 derivative mg/L 20 (Figure 6) was the Cpd Pyricularia orizae Botrytis cinerea Erysiphe graminis Colletotrichum lagenarium Puccinia sorghi Schw. most effective inhibitor against all the tested pathogens with MIC (minimum inhibitory 16a 100In vitro 50 100In 70vivo / concentration) values in the range of 16-64 µg/L). The inhibition of resazurin (RZ) reduction 19a 8025 mg/L 80 506.25 0mg/L 100 indicated that this compoundPyricularia has aBotrytis mechanism Erysiphe of action similarColletotrichum to azoxystrobin, Puccinia sorghi inhibiting the 19b Cpd 100 80 95 80 90 mitochondrial respirationorizae by bindingcinerea to the Qograminis site of cytochromelagenarium b (EC50 forSchw. inhibition of RZ 19c 100 80 99 75 60 reduction in D16a. oryzae by 100azoxystrobin 50 and compound100 20 were 3.4270 ± 0.03 µg/mL/ and 3.62 ± 0.21 enoxastrobin19a 5080 10080 10050 0 85 100 100 µg/mL, respectively). At the same time, the authors observed that 1,2,3-triazole derivatives (i.e., 19b 100In vitro 80 95 80 In vivo 90 compound 2119c) were ineffective6.25100 mg/ Lin controlling80 the 99fungal growth 6.2575at mgthe/L highest concentration60 (512 µg/mL), Cpdandenoxastrobin concludedPyricularia that orizae50 the Botrytis 1,2,4 cinerea-triazole100 Erysiphe moiety100 graminis contributedColletotrichum 85to the lagenarium antifungalPuccinia100 activity sorghi Schw. of these In vitro In vivo derivatives.17 50 100 50 100 70 6.25 mg/L 6.25 mg/L Liu 18 and coworkers/ [17] obtained100 a small 100 library of strobilurin 98 1,2,4-triazole 40 derivatives Pyricularia Botrytis Erysiphe Colletotrichum Puccinia sorghi Cpd containingenoxastrobin furan or thiophene50orizae rings 100 (cinerea22a–e , Figuregraminis 100 6), which exhibitedlagenarium 90 in vitro fungicidalSchw. 98 activities against Physalospora piricola, Cercospora arachidicola Hori, Rhizoctonia cerealis comparable to or higher than azoxy strobin.

H O N N N S Cl N N N MeO OMe MeO OMe 20 O 21 O

Cl R N Y O

X Y N N MeO OMe N MeO OMe N O O O 22a X = H, Y = O, R = Et R 23a R = H 22b X = H, Y = O, R = iPr 23b R = CH3 22c X = H, Y = O, R = iBu 23c R = CH2CH3 22d X = Br, Y = O, R = nBu 22e X = H, Y = S, R = iBu

Br N S N N N O MeO OMe R N O MeO OMe S O 24 25a R = 4-OCH3 25b R = 3,4,5-tri -F

N N

O O O O 26 O Figure 6.6. Structure of strobilurin derivatives containing a triazole moiety (20–25) andand structurestructure ofof azoxystrobin analoganalog 2626..

Molecules 2020, 25, 4582 7 of 28

Liu and coworkers [17] obtained a small library of strobilurin 1,2,4-triazole derivatives containing furan or thiophene rings (22a–e, Figure6), which exhibited in vitro fungicidal activities against Physalospora piricola, Cercospora arachidicola Hori, Rhizoctonia cerealis comparable to or higher than azoxystrobin. Among the furanyl derivatives, compounds featuring a three- or four-carbon chain (22b, 22c, Table2) linked to the 1,2,4-triazole group had the highest inhibitory e ﬀect (Table2). The EC50 values of compounds 22a (14.82 mg/L) and 22b (18.72 mg/L) against Cercospora arachidicola Hori were much lower than that of the control (40.54 mg/L). Moreover, the introduction of a bromine atom linked to the furan ring allowed to obtain compound 22d with an inhibition rate of 90% against Rhizoctonia cerealis. The EC50 values of compounds 22d (9.89 mg/L) and 22e (8.66 mg/L) against Rhizoctonia cerealis were lower than that of azoxystrobin (10.86 mg/L).

Table 2. Fungicidal activity (growth inhibition rate, %) of compounds 22a–e at 50 mg/L in vitro from Reference [17].

Compd X Y R PP a CH a RC a CO a SS a FG a

22a HOC2H5 60.0 87.0 80.3 38.9 76.3 54.8 22b HO i-C3H7 60.0 84.8 83.1 36.1 80.3 42.9 22c HO i-C4H9 43.3 73.9 87.3 19.4 31.6 38.1 22d Br O n-C4H9 63.3 69.6 90.1 25.0 82.9 61.9 22e HS i-C4H9 56.7 50.0 94.4 33.3 56.6 57.1 AZ 63.3 56.5 81.7 72.2 82.9 73.8 a PP: Physalospora piricola; CH: Cercospora arachidicola Hori; RC: Rhizoctonia cerealis; CO: Colletotrichum orbiculare; SS: Sclerotinia sclerotiorum; FG: Fusarium graminearum; AZ: azoxystrobin.

In 2017 the same research group obtained strobilurin analogs containing the 1,3,4-oxadiazole moiety [22] (Figure6). The antifungal activity of some representative compounds ( 23a–c, 24) is reported in Table3. In general, the e ﬃcacy of these new analogs in controlling the fungal growth of Sclerotinia sclerotiorum was comparable to that of azoxystrobin. Interestingly, some of them were more eﬀective than the control against Rhizoctonia cerealis.

Table 3. Inhibition of fungal growth (%) of compounds 23a-c and 24 at 50 mg/L and EC50 (mg/L) against Sclerotinia sclerotiorum and Rhizoctonia cerealis from Reference [22].

% Inhibition (50 mg/L) EC50 (mg/L) Compd Sclerotinia sclerotiorum Rhizoctonia cerealis Sclerotinia sclerotiorum Rhizoctonia cerealis 23a 100.0 98.8 7.67 15.93 23b 98.8 97.7 7.35 9.35 23c 100.0 87.2 6.15 13.45 24 82.7 95.3 / 9.20 azoxystrobin 96.3 70.9 4.67 22.86

Li and coworkers designed new strobilurin analogs combining the 2-(2-methylphenyl)-3- methoxyacrylate pharmacophore with a series of 4-halo-5-aryl-1,2,3-triazoles, which had previously showed excellent fungicidal activity against Phytophthora capsici and Sclerotinia sclerotiorum [23]. The compounds exhibited moderate to good fungicidal activity against Phytophthora capsici and Alternaria alternata. In particular, compounds 25a and 25b (Figure6) inhibited the fungal growth of Phytophthora capsici up to 73.6%, a signiﬁcantly higher value than the 42.5% observed for difenoconazole. Based on the active substructure combination approach, Liu et al. designed and synthesized new azoxystrobin analogs with various substituted phenyl groups linked to the pyrimidine ring [24]. The analogs exhibited moderate or remarkable antifungal activities against three tested fungi, Botrytis cinerea, Colletotrichum orbiculare, and Phytophthora capsici Leonian. Molecules 2020, 25, 4582 8 of 28

Preliminary structure–activity relationships (SAR) studies showed that the introduction of an electron-withdrawing group on the phenyl ring increased the activity against all the tested fungi with the following trend: 2-substituted phenyl derivative > 4-substituted phenyl derivative > 3-substituted phenyl derivative. Conversely, the eﬀect of the introduction of electron-donating groups was the following (in terms of activity): 4-substituted phenyl derivative > 3-substituted phenyl derivative > 2-substituted phenyl derivative. Interestingly, among the tested compounds, the 2,5-dimethylphenyl derivativeMolecules 202026, 25(Figure, 4582 6) displayed the most promising activity, with a growth inhibition rate8 (%)of 28 comparable (75–78%) to that of azoxystrobin (66–71%) against Colletotrichum orbiculare, Botrytis cinerea Pers,inhibition and Phytophthora rate (%) comparable capsici Leonian. (75–78%) to that of azoxystrobin (66–71%) against Colletotrichum orbiculareThe discovery, Botrytis cinerea of coumoxystrobin Pers, and Phytophthora (27, Figure capsici??)[25 Leonian.], containing the coumarin skeleton, paved the wayTh toe the discovery synthesis of ofcoumoxystrobin new strobilurins. (27, StartingFigure 7) from [25], the containing structure the of coumarin this compound, skeleton, Liu paved et al. investigatedthe way to the the synthesis activity of of new derivatives strobilurins. containing Starting the from quinolin-2(1H)-one the structure of this moiety compound, as a bioisoster Liu et al. of coumarininvestigated (Figure the activity??) and of tested derivatives their eﬃ containingcacy in controlling the quinol thein growth-2(1H)-one of ten moiety plant pathogensas a bioisoster [26]. of coumarin (Figure 7) and tested their efficacy in controlling the growth of ten plant pathogens [26].

O O O

O O n-Bu O Coumoxystrobin (27) Intermediate derivatization method

H H H O N O O N O O N N

O O O O O O

28 O 29 O 30 O Figure 7. Structure 7. Structure of representative of representative coumoxystrobin coumoxystrobin analogs 28–30 containing analogs the quinolin-2(1H)-one28–30 containing moiety. the quinolin-2(1H)-one moiety. The modification of the lactam ring resulted in crucial for the fungicidal activity. The introduction of theThe ethyl m groupodification at N1-position of the lactam gave compounds ring resulted more in eff ectivecrucial against for the all the fungicidal tested fungi activity. compared The tointroduction the N-H-containing of the ethyl compounds group at N1 (e.g.,-position29 vs. gave28). compounds Compounds more featuring effective an against amino all group the tested at C7 positionfungi compared of the quinolinone to the N-H-containing moiety (e.g., compounds30) displayed (e.g., better 29 vs. antifungal28). Compou activitynds featuring against Gibberellaan amino fujikuroigroup at, SclerotiniaC7 position sclerotiorum of the quinolinone, Phytophthora moiety infestans (e.g., 30, )Alternaria displayed solani, betterand antifungalFusarium activity graminearum against thanGibberella compounds fujikuroi containing, Sclerotinia an sclerotiorum ether linkage,, Phytophthora which, on the infestans contrary,, Alternaria was beneficial solani to, theand antifungal Fusarium activitygraminearum against thanFusarium compounds oxysporum containingand Rhizoctonia an ether linkage cerealis,. which, on the contrary, was beneficial to the antifungalIn particular, activity the against growth Fusarium inhibition oxysporum activity and of compound Rhizoctonia29 cerealiswas. comparable and in some casesIn higher particular, than the the growth activity inhibition of the reference activity of coumoxystrobin. compound 29 was The comparable authors reported and in some the cases EC50 valueshigher ofthan compounds the activity29 (ECof the50 3.4646 referenceµg/mL) coumoxystrobin. and 30 (EC50 8.9148The authorsµg/mL) reported against Rhizoctoniathe EC50 values cerealis of. Thecompounds results indicate 29 (EC that50 3.464 compound6 μg/mL)29 is and 4.17- 30 and(EC 5.07-fold50 8.9148 more μg/mL) effective against than Rhizoctonia coumoxystrobin cerealis (EC. The50 14.4593results indicateµg/mL) andthat azoxystrobincompound 29 (EC is 504.1717.5804- and µ5.07g/mL),-fold respectively, more effective against than this coumoxystrobin pathogen. (EC50 14.4593In 2017μg/mL) Chen and et azoxystrobin al. published (EC two50 17.5804 papers μg/mL), about the respectively, synthesis of against novel this fungicides pathoge containingn. isothiazole-,In 2017 thiadiazole-,Chen et al. published and thiazole-based two papers structures about the (Figure synthesis8). of novel fungicides containing isothiazoleUsing- trifloxystrobin, thiadiazole-, and (7) thiazole structure-based as a structures template, the(Figure authors 8). first designed and synthesized 3,4-dichloroisothiazole-containingUsing trifloxystrobin (7) structure strobilurins as a [ template,27]. This heterocyclic the authors sca firstffold designed can be found and in synthesiz numeroused biologically3,4-dichloroisothiazole active molecules-containing [28]. In strobilurins particular, the [27]. 3,4-dichloroisothiazole-5-carboxylic This heterocyclic scaffold can acid be derivativefound in isotianilnumerous was biologically developed asactive a novel molecules fungicide [28]. with In activatingparticular, defense the 3,4 responses-dichloroisothiazole against a wide-5-carboxylic range of plantacid derivative pathogens [isotianil29]. was developed as a novel fungicide with activating defense responses against a wide range of plant pathogens [29]. Compound 31 (Figure 8) exhibited excellent activity with EC50 of 0.07 μg/mL and 0.49 μg/mL against R. cerealis and P. infestans, respectively (Table 4). These values are comparable to that of the positive control azoxystrobin. Moreover, compound 31 was more effective than azoxystrobin in controlling the growth of G. zeae and B. cinerea (Table 4). Similarly, the same authors prepared novel 1,2,3-thiadiazole and thiazole-based strobilurins [30]. The 1,2,3-thiadiazole derivative 32 (Figure 8) exhibited excellent activities against G. zeae, S. sclerotiorum, and R. cerealis with EC50 values of 2.68, 0.44, and 0.01 μg/mL, respectively.

Molecules 2020, 25, 4582 9 of 28 Molecules 2020, 25, 4582 9 of 28

O O Cl Cl N N O N N O N H N H N N N S O S O

31 32

FigureFigure 8.8. StructureStructure ofof 3,4-dichloroisothiazole3,4-dichloroisothiazole andand 1,2,3-thiadiazole-containing1,2,3-thiadiazole-containing strobilurins.strobilurins. Compound 31 (Figure8) exhibited excellent activity with EC of 0.07 µg/mL and 0.49 µg/mL The most active compounds of each series were evaluated50 as fungicide candidates against against R. cerealis and P. infestans, respectively (Table4). These values are comparable to that of Sphaerotheca fuliginea and Pseudoperonospora cubensis in cucumber fields. the positive control azoxystrobin. Moreover, compound 31 was more effective than azoxystrobin in Both the 3,4-dichloroisothiazole derivative 31 and the 1,2,3-thiadiazole derivative 32 showed controlling the growth of G. zeae and B. cinerea (Table4). better efficacy against cucumber S. fuliginea than azoxystrobin and trifloxystrobin at the same application rate (37.5 g ai/ha). Moreover, compound 32 showed significantly better efficacy (p < 0.05) Table 4. EC50 value of compounds 31 and 32 from References [27,30]. against P. cubensis than trifloxystrobin and efficacy comparable to azoxystrobin (at the same application rate of 75 g ai/ha). Compounds 31EC showed50 (µg/mL) efficacy against P. cubensis comparable to pyraclostrobin but significantly betterR.c a than P.ithata of triG.zflaoxystrobinB.c a at S.sthea same application rate (75 g ai/ha.). 31 0.07 0.49 1.75 0.15 / 32 0.01 / 2.68 // Table 4. AZEC50 value 0.06 of compounds 0.040 31 6.92 and 32 from 6.31 References 4.04 [27,30]. a R. c, Rhizoctonia cerealis; P. i, Phytophthora infestans (Mont) de Bary; G. z, Gibberella zeae; B. c, Botrytis cinerea; S. s, Sclerotinia sclerotiorum; AZ, azoxystrobin. EC50 (µg/mL) R.ca P.ia G.za B.ca S.sa Similarly, the same authors prepared31 0.07novel 0.49 1,2,3-thiadiazole 1.75 0.15 and/ thiazole-based strobilurins [30]. The 1,2,3-thiadiazole derivative 3232(Figure 0.018) exhibited/ 2.6 excellent8 / activities/ against G. zeae, S. sclerotiorum, and R. cerealis with EC50 values ofAZ 2.68, 0.06 0.44, and0.040 0.01 6.92µg/ mL,6.31 respectively. 4.04 Thea R. c, most Rhizoctonia active cerealis compounds; P. i, Phytophthora of each seriesinfestans were (Mont) evaluated de Bary; G. as z, fungicide Gibberella zeae candidates; B. c, Botrytis against Sphaerothecacinerea; S. fuliginea s, Sclerotiniaand Pseudoperonosporasclerotiorum; AZ, azoxystrobin cubensis.in cucumber fields. BothAn interesting the 3,4-dichloroisothiazole approach to develop derivative new 31strobiluand therin 1,2,3-thiadiazole-based fungicides derivative was recently32 showed reported better by eSuffi cacyet al. against[31]. The cucumber strategy isS. basedfuliginea on thanthe combination azoxystrobin of andthe pharmacophore trifloxystrobin at moieties the same of applicationstrobilurins rate(β-methoxyacrylate, (37.5 g ai/ha). Moreover, methoxyiminoacetamide compound 32 showed, or significantly methoxy-N better-methylacrylamide) efficacy (p < 0.05) with against a Pmonoterpenic. cubensis than phenol trifloxystrobin typically and found effi cacyin plant comparable essential tooils azoxystrobin (EOs). (at the same application rate of 75Antifungal g ai/ha). Compounds activity of 31EOsshowed has been effi cacyfrequently against associatedP. cubensis withcomparable the presence to pyraclostrobin of monoterpenic but significantlyphenols, such better as thymol, than that carvacrol, of trifloxystrobin and paeonol at the same[32]. However,application EOs rate components (75 g ai/ha.). are generally volatile,An interestingunstable to approach light or heat. to develop Additionally, new strobilurin-based their short-term fungicides fungicidal was efficacy recently and reportedslow action by Suconsiderably et al. [31]. Therestrict strategy their ispossible based onpractical the combination application. of the pharmacophore moieties of strobilurins (β-methoxyacrylate,The authors prepared methoxyiminoacetamide, seventeen new compounds or methoxy-N-methylacrylamide) and tested their potential with fungicidal a monoterpenic activity phenolagainst typically eleven species found inof plantplant essentialpathogen oils fungi. (EOs). The structure of representative analogs containing carvacrolAntifungal and thymol activity moiety of EOs and has EC been50 values frequently against associated Sclerotinia withsclerotiourum the presence are reported of monoterpenic in Figure phenols,9. Compound such as 33 thymol, exhibited carvacrol, a very interesting and paeonol activity [32]. However, against Pestalotiopsis EOs components theae, arePhom generallyopsis adianticola volatile,, unstableSclerotinia to sclerotiorum light or heat., and Additionally, Magnapothe their grisea short-term as well. fungicidal efficacy and slow action considerably restrict their possible practical application. The authors prepared seventeen new compounds and tested their potential fungicidal activity against eleven species of plant pathogen fungi. The structure of representative analogs containing carvacrol and thymol moiety and EC50 values against Sclerotinia sclerotiourum are reported in Figure9. Compound 33 exhibited a very interesting activity against Pestalotiopsis theae, Phomopsis adianticola, Sclerotinia sclerotiorum, and Magnapothe grisea as well.

Molecules 2020, 25, 4582 10 of 28 Molecules 2020, 25, 4582 10 of 28

O O N X O O O Y monoterpenic O phenol O

Kresoximmethyl (4) X = CH or N Y = O or NH carvacrol containing analogues O O

O N O O H O O

33 34

EC50 = 0.003 +/- 0.00 mg/L EC50 = 0.64 +/- 0.06 mg/L

thymol containing analogues O O O N O N O O O O H O O O

35 36 37 EC = 0.10 +/- 0.02 mg/L EC50 = 0.25 +/- 0.05 mg/L EC50 = 6.75 +/- 0.12 mg/L 50

Figure 9. 9. Structure of representative representative compounds compounds and and EC EC5050 values against SclerotiniaSclerotinia sclerotiourumsclerotiourum (azoxystrobin(azoxystrobin ECEC50 = 18.7518.75 ± 1.101.10 mg/L) mg/L) [EC [EC50 valuesvalues from from R Referenceeference 31]. [31 ]]. 50 ± 50 Using pyraclostrobin pyraclostrobin (10 (10) as) a as lead a lead compound, compound, Liu and Liu coworkers and coworkers [33] obtained [33] 44obtained new strobilurin 44 new analogsstrobilurin by introducinganalogs by introducing the methoxyiminoacetate the methoxyiminoacetate or the methoxyacrylate or the methoxyacrylate pharmacophore pharmacophore into halo- or (un)substitutedinto halo- or (un)substituted arylpyrazole sca arylpyrazoleffolds (Figure scaffolds 10). In particular,(Figure 10). the In authors particular, focused the theirauthors attention focused on threetheir attention structural on aspects: three structural the effect ofaspects: different the pharmacophores effect of different and pharmacophores their position; theand e theirffect ofposition; nature andthe effect position of nature of the and substituent position on of thethe terminalsubstituent benzene on the ring; terminal the e benzeneffect of halogen ring; the substituent effect of halogen on the pyrazolesubstituent ring. on the pyrazole ring. The SARs SARs study study highlighted highlighted that the that combination the combination of both methoxyiminoacetate of both methoxyiminoacetate pharmacophore andpharmacophore electron-withdrawing and electron substituent-withdrawing R substituent on phenyl R ring on phenyl improved ring improved the fungicidal the fungicidal activity. Moreover,activity. Moreover, the type the and type size and of the size halogen of the halogenon the pyrazole on the pyrazole ring resulted ring resulted crucialfor crucial the e forfficacy. the Inefficacy. particular, In particular, the presence the presence of a chloro of a substituent chloro substituent had a positive had a positive effect on effect the fungicidal on the fungicidal activity, whetheractivity, whether it was on it thewas pyrazoleon the pyrazole ring or ring on the or on phenyl the phenyl ring. ring. The collectedThe collected biological biological data data and and the comparativethe comparative molecular molecular field analysis field analysis allowed allowed the authors the authors to build to up build a 3D-QSAR up a 3D model-QSAR for model these new for strobilurinthese new strobilurin analogs with analogs high correlativewith high correlative and predictive and abilitiespredictive [33 abilities]. [33]. Among the obtained analogs, compounds 38–41 (Figure 1010)) exhibited 98.94%, 83.40%, 71.40% 71.40%,, and 65.87% 65.87% inhibition inhibition rates rates at at 0.1 0.1µ g μg/mL/mL against againstRhizoctonia Rhizoctonia solani solani, respectively,, respectively, which which are better are better than commercialthan commercial pyraclostrobin pyraclostrobin (35.73%). (35.73%).

Molecules 2020, 25, 4582 11 of 28 Molecules 2020, 25, 4582 11 of 28

O O Z R O O Y N O N N N N O CH2O

X Pyraclostrobin (10) Structure-activity relationship:

1) different pharmacophores 2) different substituents and position on phenyl ring 3) different substituents on pyrazole ring Cl Cl

O O N N O O N N O N N N H O O

Cl 38 39 O O N O O N N O O N N

O O Cl 40 41 FigureFigure 10. 10. StructureStructure of of pyraclostrobin pyraclostrobin analogs analogs from from Reference Reference [ [33].33].

InIn 2018, 2018, Wanget Wang al. et [ al.34] introduced[34] introduced a pyridinyl a pyridinyl group as group bridge asportion bridge and port sideion chain and modifications, side chain modifications,obtaining a library obtaining of N- orthoa librarysubstituted of N-ortho pyridine substituted analogs pyridine of pyraclostrobin analogs of (Figurepyraclostrobin 11). Besides (Figure the 11).in vitro Besidesfungicidal the in activitiesvitro fungicidal against activities five important against plant five pathogens important (Botrytis plant pathogens cinerea, Phytophthora (Botrytis cinerea capsici, , PhytophthoraFusarium sulphureum capsici, ,FusariumGloeosporium sulphureum pestis, ,and GloeosporiumSclerotinia pestis sclerotiorum, and ),Sclerotinia the authors sclerotiorum evaluated), the the authorspercentage evaluated of inhibition, the percentage the IC50 values, of inhibition, and predicted the IC50 the values, total bindingand predicted free energy the total of representative binding free energycompounds of representative against porcine compounds SCR (Table against5). Compound porcine SCR43 exhibited (Table 5). the Compound lowest IC50 43value exhibit (0.95edµ theM). lowestIn contrast, IC50 value compound (0.95 µM).42, withoutIn contrast, the compound methylene 42 linker,, without displayed the methylene only 11% linker inhibitory, displayed activity only at 11%100 µ inhibitoryM concentration, activity suggestingat 100 µM thatconcentration, the flexible suggesting side chain isthat favorable the flexible for the side enzymatic chain is favorable inhibition foractivity. the enzymatic In general, inhibition the presence activity. of a In hydrophobic general, the side presence chain of is criticala hydrophobic for the antifungal side chain activityis critical of forthese the analogs. antifungal Moreover, activity biaryl of these rings analogs at the side. Moreover, chain are biaryl preferable rings andat the certain side flexibilitychain are ispreferable beneficial andto the certain inhibitory flexibility activity is beneficial against SCR to the bc1 inhibitory complex. activity against SCR bc1 complex. InteresInterestingly,tingly, dockingdocking studiesstudies into into the the binding binding pocket pocket of of the the cytochrome cytochrome bc1 bc1 complex complex showed showed that thatthe pyridinyl the pyridinyl group cangroup form can stable form arene-H stable interaction arene-H interaction with residue with proline-271, residue thus proline improving-271, thus the improvingbinding to the cytochrome binding to bc1 cytochrome complex. bc1 complex.

Molecules 2020, 25, 4582 12 of 28 Molecules 2020, 25, 4582 12 of 28 Molecules 2020, 25, 4582 12 of 28

N N O N N O O O O N N O N O O N O O N O O O N N N O N O N O O O O O O O O O

Pyraclostrobin (10) Cl 42 43 Pyraclostrobin (10) 43 Cl 42 Figure 11. Structure of pyraclostrobin analogs from Reference [34]. FigureFigure 11. 11.Structure Structure of of pyraclostrobin pyraclostrobin analogs analogs from from Reference Reference [34 [34]]. . Table 5. Percentage of inhibition, IC50 values, and predicted total binding free energy of selected Table 5. Percentage of inhibition, IC50 values, and predicted total binding free energy of selected compoundscompoundsTable 5. Percentage againstagainst porcineporcine of inhibition, SCRSCR fromfrom IC ReferenceReference50 values, [ and34[34]]. . predicted total binding free energy of selected compounds against porcine SCR from Reference [34]. c % Inhibition IC50IC ± SD SD ΔGpred. b Compd µ 50 ∆Gpred. Compd % Inhibition%(100 Inhibition (100µM) M) IC(µM)50 ± SDa± a (Kcal/mol)ΔGpred. c Compd (µM) (Kcal/mol) (100 µM) (µM) a (Kcal/mol) 42 42 11 11 / / −23.84 23.84 − 43 4342 99.999.911 0.95 0.95 ± /0.012 0.012 −−44.1723.84 44.17 ± − pyraclostrobinpyraclostrobin43 99.999.999.9 1.760.95 1.76 ± 0.17nM± 0.0120.17nM −−35.8544.17 35.85 ± − penthiopyrad 95 1.56 0.12 penthiopyradpyraclostrobin 99.995 1.761.56 ± ± 0.17nM 0.12± −35.85 a b a ValuesValues are are the the mean meanpenthiopyradstandard ± standard deviation deviation (SD) of95 (SD) three of replicates. three1.56 replicates.Predicted ± 0.12 b Predicted binding free binding energies free between energies each compound and cytochrome± bc1 complex (PDB ID: 3TGU, ein = 1.0). betweena Values each are the compound mean ± standard and cytochrome deviation bc1 (SD) complex of three (PDB replicates. ID: 3TGU, b Predicted ein = 1.0). binding free energies between each compound and cytochrome bc1 complex (PDB ID: 3TGU, ein = 1.0). InIn 2016,2016, JiaJia etet al.al. investigatedinvestigated compoundscompounds thatthat sharedshared structuralstructural characteristicscharacteristics ofof strobilurinstrobilurin and N′In -2016nitrohydrazinecarboximidamide,, Jia et al. investigated compounds discover thating shared compound structural 44 characteristics(Figure 12) asof thestrobilurin main and N0-nitrohydrazinecarboximidamide, discovering compound 44 (Figure 12) as the main byproduct.byproduct.and N′-nitrohydrazinecarboximidamide, TheThe compound compound showed showed excellent excellent discover fungicidal fungicidaling compound activity activity against against44 (Figure PhytophthoraPhytophthora 12) as theinfestansinfestans main,, BotryosphaeriaBotryosphaeriabyproduct. The dothidea compound, Botrytis showed cinerea cinerea,, and excellentand several several fungicidal other other plant plant activity pathogenic pathogenic against fungi. fungi.Phytophthora For For this this reason infestans reason,, in , in2019Botryosphaeria 2019 the same dothidea the authors , Botrytis synthesized same cinerea , and (E)-methyl several authors other 2-(2-((1-cyano-2-hydrocarbylidenehydrazinyl)- plant pathogenic synthesized fungi. For this (E) reason-methyl, in methyl)-phenyl)-2-(methoxyimino)acetates22019-(2-((1 -cyano-2-hydroc the arbylidenehydrazinyl) same using-methyl) authors44 as-phenyl) a lead- compound2-(methoxyimino)acetates synthesized [35]. Although (E) using no-methyl new 44 moleculesas2- a(2 lead-((1- cyanocompound resulted-2-hydroc more [35].arbylidenehydrazinyl) Although active than no the new lead molecule compound,-methyl)s resulted-phenyl) they more- isolated2-(methoxyimino)acetates active compound than the lead45 (Figurecompound, using 12 ),44 whichtheyas a isolatedlead showed compound compound a broad [35]. spectrum 45 Although (Figure of activity,12), no newwhich as moleculea showed byproducts resulteda broad of their morespectrum synthetic active of than pathway.activity, the leadas a compound,byproduct ofthey their isolated synthetic compound pathway. 45 (Figure 12), which showed a broad spectrum of activity, as a byproduct of their synthetic pathway. CN NCN O N N N 2 N N O2N N NMeO OMe NMeO OMe Cl N Cl N MeO OMe MeO OMe Cl N O Cl N O 44 O 45 O 44 45 FigureFigure 12.12. Structure of strobilurin analogsanalogs containingcontaining hydrazono-methylhydrazono-methyl moiety.moiety. Figure 12. Structure of strobilurin analogs containing hydrazono-methyl moiety. Recently,Recently Matsuzaki, Matsuzaki and and coworkers coworkers [36] obtained [36] obtained new Qo new inhibitors Qo inhibitorscharacterized characterized by a tetrazolinone by a pharmacophoretetrazolinoneRecently pharmacophore, andMatsuzaki a methyl and group and coworkers a at methyl position [36]group 3 on obtained theat po centralsition new phenyl3 Qoon the inhibitors ring. central These phenyl characterized compounds ring. These were by a designedcompoundstetrazolinone to overcome were pharmacophore designed the resistance to overcomeand developed a methyl the resistancebygroup different at po developed pathogenicsition 3 on by speciesthe different central and pathogenicassociatedphenyl ring. with species These the G143Aandcompounds associated mutation were with at the designed the target G143A site. to mutation overcome The presence at the the resistance of target the methyl site. developed The group presence of by the different of alanine the methyl residuepathogenic group at position speciesof the 143alanineand seems associated residue to cause atwith severepo sitionthe steric G143A 143 hindrance seems mutation toat cause theat the central severe target linking steric site. ringshindranceThe presence of QoIs. at the of thecentral methyl linking group rings of theof QoIs.alanineThrough residue an at initial position random 143 screeningseems to cause on 200 severe QoI-like steric compounds hindrance toat identifythe central the linking key structural rings of moietiesQoIs.Through to retain an theinitial activity random against screening G143A on mutant, 200 QoI the-like authors compounds found that to identify compound the 46key(Figure structural 13), featuringmoietiesThrough theto retain tetrazolinone an initialthe activity random pharmacophore against screening G143A and on mutant, the200 sameQoI -thelike side authors compounds chain offound triﬂoxystrobin, to tha identifyt compound the was key less 46 structural a(Figureﬀected by13moieties), this featuring mutation, to retain the showing tetrazolinone the activity a resistant againstpharmacophore factor G143A RF = mutant,2 and (Figure the the 13same, authors Table side6). chainfound After aof tha careful trifloxystrobin,t compound structure–activity 46 was (Figure less relationshipaffected13), featuring by thisinvestigation, the mutation, tetrazolinone the showing authors pharmacophore a obtained resistant compound factorand the RF same =47 2 (Figure side chain 1413) , of with Table trifloxystrobin, the 6). same After side a was careful chain less ofstructureaffected pyraclostrobin – byactivity this mutation,(4-chlorophenylpyrazolerelationship showing investigation, a resistant structure). the authors factor The RFobtained =EC 250 (Figureof compound compound 13, Table 47 47(Figure 6in). Afterthe 14) wild-type with a careful the samestructure side– chainactivity of relationshippyraclostrobin inv (4estigation,-chlorophenylpyrazole the authors obtained structure). compound The EC 4750 of(Figure compound 14) with 47 thein same side chain of pyraclostrobin (4-chlorophenylpyrazole structure). The EC50 of compound 47 in

Molecules 2020, 25, 4582 13 of 28 the wild-type sensitive strain (WT) and G143A mutant were both 0.02 ppm, showing a 10- to 20-fold increase of activity compared to compound 46 (Table 6). Molecules 2020 25 Finally,, the, 4582 introduction of the 3-methyl group on the central phenyl ring led to obtain13 of 28 methyltetraprole 48, showing a 10-fold higher activity than that of compound 47 (Table 6). The sensitiveauthors hyp strainothesized (WT) and that G143A the presence mutant wereof the both methyl 0.02 groupppm, showing might have a 10- a to role 20-fold in limiting increase the of activityrotation comparedof the side to chain compound and in 46avoiding(Table6 ).steric hindrance between the QoI and the mutated target site.

F C O N O 3 N Cl N N N N O N O 46 N N 47 N N

O Cl N N N N O N N Methyltetraprole ( 48)

MoleculesFigure 2020, 25 13. , 4582 StructureStructure of strobilurins analogs containing the tetrazo tetrazolinonelinone pharmacophore (46–48). 14 of 28

Table 6. In vitro antifungal tests against sensitive wild-type and G143A mutant of Zymoseptoria tritici R O H C H O from Reference [36]. 7 15 H N COOCH3 N N COOCH3 H Sensitive Wild Type G143A MutantN Resistant Cpd O H EC50 (ppm) a EC50 (ppm) OFactor b CH3O strobilurin A 0.02 0.2 CH310O 51 R = -CH2CH(pyraclostrobinCH3)2 Cyrmen in A (49a) 0.001 0.2 O200H C H O R = -CH2CH2CH3 46Cy rmenin B1 (49b) 0.2 7 15 0.4 2.0H R = -CH(CH3)2 Cyrmenin B2 (49c) N COOCH 47 0.02 0.02 N 1.0 3 H methyltetraprole (48) 0.002 0.002 O1.0 52 a TheO values represent the 50% effective concentration (EC50) determined fromCH 3 twoO biological H replicates. The 95% confidence intervals of all EC50 values ranged between 50% (MIN) and 200% N COOCH3 C7H15 O R N b H (MAX) of the representative values. Resistance Factor: ([EC50 of resistant strain]/[EC50 of sensitive H N COOCH3 strain]). O 50a, R = N C7H15 H CH3O O 53 3. Other Natural Products5 0Inhibitingb R = C 1the0H2 Cytochrome1 bc1 Complex

3.1. Cyrmenin A, B1 and B2 50c, R =

Cyrmenins A, B1, and B2 (49a–c, Figure 14) are antifungal metabolites isolated from the culture broth of myxobacteriaFigure Cystobacter 14. Structure armeniaca of natural and cyrmenins Archangium and analogs. gephyra. These modified N-acyldipeptide compoundsFigure contain 14. Structure a dehydroalanine, of natural cyrmenins a 2-amino and- 3analogs.-methoxyacrylate moiety, and a (2E, 4Finally,Z)-undecadienoic the introduction or dodecadienoic of the 3-methyl acid residue. group Cyrmenines on the central exhibit phenyl high ringantifungal led to activity, obtain methyltetraproleshowingThe at bioassay the same48 , results showing time exceptionally clearly a 10-fold indicated higher low toxicitythe activity relevance for than animal that of of eachcell compound cultures portion [37].47 of (Table the parent6). The molecule authors (lipophilic unsaturated chain, dehydroalanine moiety, and β-methoxyacrylate system), leaving very hypothesizedThe first thattotal the synthesis presence of of cyrmenin the methyl B1 group(Figure might 14) was have reported a role in in limiting 2009 [38] the rotation and the of same the sideauthorslittle chainspace reported and for synthetic in avoidingthe synthesis modifications. steric of hindrance some representative between the derivatives QoI and the for mutated structure target–activity site. relationship 1 studiesInt erestingly [39]. The, only antifungal the modification activity of of cyrmenin the conjugated B1 and double analogs bond (50 geometry–53) was (cyrmenin tested against B vs. Saccharomycesits (8E,10E)-geometrical cerevisiae Meyen isomer ex 50a Ec.) wasHansen, tolerated. strain The IPV complete637, Aspergillus lack of niger activity Tegh., of compoundsstrain IPV F303, 50b Botrytisand 50c cinereaunderlined Pers., that strain maintaining IPV F5.2, Cochliobolusthe lipophilicity miyabeanus of the molecu, (S.Ito leand was Kurib) not enough Drechsler to have ex Dastur, active andcompounds. Pyricularia The oryzae exo doubleCavara, bond strain of IPV the A1parent. molecule was also crucial for the antifungal activity. In fact, both compound 51, bearing an alanine residue, and the compound bearing a serine residue (52) were inactive against all the tested strains. The modification of the expected crucial β-methoxyacrylate group was also deleterious (compound 53). All these results evidenced a limitation for the development of new fungicide compounds derived from natural cyrmenins.

3.2. Mixothiazols, Melithiazols and Fulvuthiacens Myxobacteria are a rich source of various biologically active secondary metabolites. These include the antifungal agents myxothiazol and melithiazol, which are very potent inhibitors of the electron transport through the cytochrome bc1 complex of the eukaryotic respiratory chain. In 1980, Reichenbach et al. and Hofle et al. reported the isolation of a novel myxobacterial antibiotic named myxothiazol A (54) [40,41], whose structure is characterized by a central bis-thiazole unit linked to a β-methoxyacrylate moiety and a heptadienyl side-chain bearing a stereogenic center at the α-position of the thiazole ring (Figure 15). Acting as potent inhibitors of the electron transport through the cytochrome bc1 complex, both myxothiazol A (54) and its corresponding methyl ester, myxothiazol Z (55), exhibit broad antifungal activity as well as significant cytotoxicity against different human tumor cell lines with IC50 values reaching as low as 0.01 ng/mL [42,43]. The high mammalian toxicity hampered the development of myxothiazol synthetic derivatives. During the following three decades, more than 20 structurally related fungicides have been isolated from various strains of myxobacteria, including the congeneric melithiazols. Melithiazols A–N contain a β-substituted β-methoxyacrylate pharmacophore and act as inhibitors of the cytochrome bc1 complex as well [44].

Molecules 2020, 25, 4582 14 of 28

Table 6. In vitro antifungal tests against sensitive wild-type and G143A mutant of Zymoseptoria tritici from Reference [36].

Sensitive Wild Type G143A Mutant b Cpd a Resistant Factor EC50 (ppm) EC50 (ppm) strobilurin A 0.02 0.2 10 pyraclostrobin 0.001 0.2 200 46 0.2 0.4 2.0 47 0.02 0.02 1.0 methyltetraprole (48) 0.002 0.002 1.0 a The values represent the 50% eﬀective concentration (EC50) determined from two biological replicates. The 95% conﬁdence intervals of all EC50 values ranged between 50% (MIN) and 200% (MAX) of the representative values. b Resistance Factor: ([EC50 of resistant strain]/[EC50 of sensitive strain]).

3. Other Natural Products Inhibiting the Cytochrome bc1 Complex

3.1. Cyrmenin A, B1 and B2

Cyrmenins A, B1, and B2 (49a–c, Figure 14) are antifungal metabolites isolated from the culture broth of myxobacteria Cystobacter armeniaca and Archangium gephyra. These modified N-acyldipeptide compounds contain a dehydroalanine, a 2-amino-3-methoxyacrylate moiety, and a (2E, 4Z)-undecadienoic or dodecadienoic acid residue. Cyrmenines exhibit high antifungal activity, showing at the same time exceptionally low toxicity for animal cell cultures [37]. The first total synthesis of cyrmenin B1 (Figure 14) was reported in 2009 [38] and the same authors reported the synthesis of some representative derivatives for structure–activity relationship studies [39]. The antifungal activity of cyrmenin B1 and analogs (50–53) was tested against Saccharomyces cerevisiae Meyen ex Ec. Hansen, strain IPV 637, Aspergillus niger Tegh., strain IPV F303, Botrytis cinerea Pers., strain IPV F5.2, Cochliobolus miyabeanus, (S.Ito and Kurib) Drechsler ex Dastur, and Pyricularia oryzae Cavara, strain IPV A1. The bioassay results clearly indicated the relevance of each portion of the parent molecule (lipophilic unsaturated chain, dehydroalanine moiety, and β-methoxyacrylate system), leaving very little space for synthetic modifications. Interestingly, only the modification of the conjugated double bond geometry (cyrmenin B1 vs. its (8E,10E)-geometrical isomer 50a) was tolerated. The complete lack of activity of compounds 50b and 50c underlined that maintaining the lipophilicity of the molecule was not enough to have active compounds. The exo double bond of the parent molecule was also crucial for the antifungal activity. In fact, both compound 51, bearing an alanine residue, and the compound bearing a serine residue (52) were inactive against all the tested strains. The modification of the expected crucial β-methoxyacrylate group was also deleterious (compound 53). All these results evidenced a limitation for the development of new fungicide compounds derived from natural cyrmenins.

3.2. Mixothiazols, Melithiazols and Fulvuthiacens Myxobacteria are a rich source of various biologically active secondary metabolites. These include the antifungal agents myxothiazol and melithiazol, which are very potent inhibitors of the electron transport through the cytochrome bc1 complex of the eukaryotic respiratory chain. In 1980, Reichenbach et al. and Hofle et al. reported the isolation of a novel myxobacterial antibiotic named myxothiazol A (54)[40,41], whose structure is characterized by a central bis-thiazole unit linked to a β-methoxyacrylate moiety and a heptadienyl side-chain bearing a stereogenic center at the α-position of the thiazole ring (Figure 15). Acting as potent inhibitors of the electron transport through the cytochrome bc1 complex, both myxothiazol A (54) and its corresponding methyl ester, myxothiazol Z (55), exhibit broad antifungal activity as well as significant cytotoxicity against different human tumor cell lines with IC50 values reaching as low as 0.01 ng/mL [42,43]. The high mammalian toxicity hampered the development of myxothiazol synthetic derivatives. During the following three Molecules 2020, 25, 4582 15 of 28

Unlike myxothiazols, all melithiazols occur only as methyl esters and lack the lipophilic heptadienyl side chain. Moreover, the greatly reduced mammalian toxicity of these metabolites with respect to the toxicity of myxothiazols makes melithiazols interesting for the development of new fungicides. Unfortunately, due to the poor amounts of melithiazols usually obtained from Molecules 2020, 25, 4582 15 of 28 fermentation, it has not been possible to develop a derivatization program to investigate structure–activity relationships. However, synthetic efforts to obtain melithiazol B (56) [45] and C (decades,57) [46] th morerough than oxidative 20 structurally degradation related of myxothiazol fungicides A have and beenreduct isolatedive cleavage from of various a thiazole strains ring, of respectively,myxobacteria, allowed including to test the the congeneric biological melithiazols. activities of intermediates and derivatives, to obtain some structure–activity correlations.

OCH3 OCH3 7 S N S N 4

6 1 N COR N 10 S 14 S COOCH3 H3CO H3CO

Myxothiazol A (54) R = NH2 Melithiazol B (56) Myxothiazol Z (55) R = OCH3 OCH3 OCH3 O N S N S COOCH3 O H3CO N S COOCH3 H3CO 58 Melithiazol C (57)

OCH3 OCH3 S N N HO N S COOCH3 S COOCH3 H3CO H3CO

59 60

OCH3 OCH3 O N RO N

S CONH2 S COOCH3 H3CO H3CO

61 62a R = H

62b R = Et O OH O OCH OCH N 3 3 R OCH3 N N S S COOCH3 H3CO Fulvuthiacen A (64) R = -(CH2)7CH(CH3)2 63a (6E, 11E) Fulvuthiacen B (65) R = -(CH ) CH(CH ) 2 5 3 2 63b (6E, 11Z) FigureFigure 15 15.. StructuresStructures of of mixothiazols, mixothiazols, melithiazols melithiazols,, and and fulvuthiacens fulvuthiacens..

ConcerningMelithiazols melithiazol A–N contain B ( a56β)-substituted and its derivatives,β-methoxyacrylate the authors pharmacophore highlighted that and compounds act as inhibitors with anof theester cytochrome pharmacophore bc1 complex and a as short well polar [44]. side-chain (e.g., compounds 58 and 59) showed high antifungalUnlike activity myxothiazols, (Table all 7) melithiazols. Moreover, occur in general only as, methylthe cytotoxicity esters and lack decreased the lipophilic significantly heptadienyl by decreasingside chain. the Moreover, lipophilicity the greatlyof these reduced molecules. mammalian toxicity of these metabolites with respect to the toxicityStructure of–activity myxothiazols relationship makes studies melithiazols on melithiazol interesting C ( for57) thederivatives development showed of newthat fungicides.the amide analogUnfortunately, (61) and duethe 14 to- thehydroxy poor amountsderivative of los melithiazolst the antifungal usually activity obtained (62a) from, whereas fermentation, the 14-ethoxy it has derivativenot been possible (62b) showed to develop gooda antifungal derivatization activity program and only to investigate slightly increased structure–activity cytotoxicity. relationships. Moreover, theHowever, (6Z)-isomer synthetic of melithiazol eﬀorts to obtain C was melithiazol essentially B inactive (56)[45] in and all C test (57 )[systems.46] through oxidative degradation of myxothiazol A and reductive cleavage of a thiazole ring, respectively, allowed to test the biological activities of intermediates and derivatives, to obtain some structure–activity correlations. Concerning melithiazol B (56) and its derivatives, the authors highlighted that compounds with an ester pharmacophore and a short polar side-chain (e.g., compounds 58 and 59) showed high antifungal activity (Table7). Moreover, in general, the cytotoxicity decreased signiﬁcantly by decreasing the lipophilicity of these molecules. Molecules 2020, 25, 4582 16 of 28

Table 7. Biological activities and lipophilicity of selected myxothiazol and melithiazol derivatives. [a].

Botrytis cinerea Inibition of Citotoxicity Lipophilicity Cpd Inibition Zone at 2 [b] NADH Oxidation [d] IC50 (ng/mL) [c] Log POW µg/disc (mm) IC50 (ng/mL) 54 [e] 16 1 11 5.29 55 13 2 17 7.17 56 29 20 18 4.75 58 39 55 29 3.37 59 19 220 37 2.36 57 18 700 730 2.92 60 35 2000 250 3.97 63a 42 500 42 3.62 63b 42 400 85 3.81 Kresoxim-methyl [e] 33 400 72 3.70 [a] from References [45,46]. [b] The cytotoxicity was measured by an MTT assay with the mouse ﬁbroblast cell line L929. [c] The inhibition of the NADH oxidation was measured with submitochondrial particles isolated from beef heart. [d] Estimated by RP-18 TLC. [e] Values taken from Reference [44].

Structure–activity relationship studies on melithiazol C (57) derivatives showed that the amide analog (61) and the 14-hydroxy derivative lost the antifungal activity (62a), whereas the 14-ethoxy derivative (62b) showed good antifungal activity and only slightly increased cytotoxicity. Moreover, the (6Z)-isomer of melithiazol C was essentially inactive in all test systems. The vinyl derivative 60 and the (11E) and (11Z) methoximes 63a and 63b showed an exceptionally high antifungal activity and low cytotoxicity, comparable to that of the commercial fungicide kresoxim-methyl. Recently, Panter and coworkers [47] elucidated the structure of two new secondary metabolites (fulvuthiacene A (64) and B (65), Figure 15) isolated from Myxococcus fulvus MCy9280 by combining the statistics-based mining of mass spectrometry approach and multidimensional NMR spectroscopy. These compounds contain a terminal β-methoxymethyl acrylate moiety and their evaluation provided new insights into the overall structure–activity relationship picture of the β- methoxyacrylate class of bc1 complex inhibitors. Indeed, fulvuthiacene A and B showed very poor activity against a panel of fungi and yeast. Comparing the NADH oxidation IC50 values of known naturally occurring methoxymethacrylate type respiratory chain inhibitors, the authors observed that a wide variety of residues are tolerated in C-2 position of the β-methoxymethacrylate pharmacophore (strobilurin A IC50 = 83 ng/mL, oudemansin IC50 = 400 ng/mL, and cyrmenin A IC50 = 27 ng/mL). On the contrary, for compounds bearing a C-3 extended β-methoxyacrylate warhead, the structural requirements are very narrow and a bisthiazol or thiazolinethiazol moiety is needed to produce a strong bc-1 inhibitor (melithiazol B IC50 = 18 ng/mL, melithiazol C IC50 = 1600 ng/mL, fulvuthiacene A no inhibition of NADH oxidation up to 64,000 ng/mL). The results reported in this study might thus serve as starting points for the development of the next generation of β-methoxymethacrylate fungicides.

3.3. Miuraenamides Miuraenamide A–F (66–71, Figure 16) are cyclic hybrid polyketide-peptide antibiotics isolated from Paraliomixa miuraensis, a slightly halophilic myxobacterium discovered by Ojika et al. at the seashore on Miura Peninsula in Kanagawa, Japan [48,49]. The biological properties of these natural products included antimicrobial activity and inhibitory activity against NADH oxidase. Molecules 2020, 25, 4582 17 of 28

Molecules 2020, 25, 4582 17 of 28

X Br Br OH OH OH

O Ph O OCH O Ph H H H H 3 H H N N N N N N N OCH N Ph N O O 3 O O O O O O O O O O O

Miuraenamide A (66) X = Br Miuraenamide D (69) Miuraenamide E (70) Miuraenamide B (67) X = I Miuraenamide C (68) X = Cl Br Br Br

OH OCOCH3 OH

O Ph O Ph O H H H H H H N N N N N N N OCH N OCH N O 3 O 3 O O O O O O O O O O

Miuraenamide F (71) 72 73 Br OH

O Ph H H N N N OCH O 3 O COOR OH 74 R = H 75 R = CH3

Figure 16. StructuresStructures of of miuraenamides miuraenamides A A-F-F ( 66–71)) and related derivatives ( 7272–75).

StructureStructure–activity–activity relationship studies on miuraenamides AA–F–F and related derivatives were conducted on the phytopathogen Phytophthora capsici (Table8 8).). TheThe authorsauthors highlightedhighlighted thatthat thethe typetype of halogen present in the tyrosine residue in in miuraenamide miuraenamidess A A–C–C is is not not important important for for their their activity. activity. Moreover, thethe presencepresence andand the the geometry geometry of of the theβ -methoxyacrylateβ-methoxyacrylate moiety moiety (C12–C14, (C12–C14, compounds compounds69, 6970,, 70and, and74) 74 aﬀ)ects affects the the activity activity of these of these compounds. compounds. The The lipophilicity lipophilicity of the of the polyketide polyketide moiety moiety and and the thefree free phenol phenol group group on the on peptidethe peptide portion portion of the of structure the structure seem seem to be to important be important for the for activity the activity of these of thesecompounds, compounds, as demonstrated as demonstrated by the low by the activity low observed activity observed for miuraenamide for miuraenamide F (71) and F acetate (71) and72. acetateThe macrocyclic 72. Thestructure macrocyclic of the structure miuraenamides of the is essential miuraenamides as well, with is essential the anti- Phytophthora as well, withactivity the antibeing-Phytophthora completely lostactivity in the being case completely of the ring-opened lost in the derivatives case of the74 ringand-75opened, although derivatives these compounds 74 and 75, althoughcontain the theseβ-methoxyacrylate compounds contain moiety. the β-methoxyacrylate moiety.

Table 8. 8. MinimumMinimum doses doses of of miuraenamides miuraenamides and and derivatives derivatives for for anti- anti-PhytophthoraPhytophthora activity from Reference [ [49].49].

CompdCompd 1 12 23 34 45 56 67 78 89 910 10 Dose[µDoseg per disk] 0.025 0.025 0.025 1 10 0.13 5 2 >50 50 0.025 0.025 0.025 1 10 0.13 5 2 >50 50 [µg per disk] 3.4. Crocacins In 1994, crocacins A, B, C, and D (76–79, Figure 17) were isolated from the myxobacterium Chondromyces crocatus. The compounds crocacin A and D inhibited the electron transport chain at

Molecules 2020, 25, 4582 18 of 28

Molecules3.4. Crocacins2020, 25 , 4582 18 of 28 In 1994, crocacins A, B, C, and D (76–79, Figure 17) were isolated from the myxobacterium complexChondromyces III in acrocatus beef heart. The mitochondrial compounds crocacin respiration A and assay D and inhibited inhibited the theelectron growth transport of several chain fungi at incomplex vitro.[50 III]. in Although a beef heart natural mitochondrial crocacins had respiration interesting assay biological and inhibited activity, theirthe growth practical of useseveral would fungi be severelyin vitro. limited[50]. Although by their natural physical crocacins properties, had such interesting as the very biological poor photostability activity, their andpractical their use structural would complexity.be severely However, limited by the their emergence physical of properties, resistance such to di ﬀ aserent the pathogenic very poor photostabilityspecies associated and with their G143Astructural mutation complexity. renewed However, the interest the toward emer crocacingence of A, resistance as it showed to little different or no cross-resistance pathogenic species to a strobilurin-resistantassociated with G143A strain mutation of Plasmopara renewed viticola the interestin small toward vine plants crocacin in the A, glasshouse,as it showed or little against or no a straincross- ofresistance yeast engineered to a strobilurin with the-resistant G143A mutation. strain of Plasmopara viticola in small vine plants in the glasshouse, or against a strain of yeast engineered with the G143A mutation.

CH3 CH3 R = CH Crocacin A (76) NH 3 O N COOR R = H Crocacin B (77) H OCH3OCH3 O R = H Crocacin C (78) CH CH 3 3 H R = N Crocacin D (79) R O N COOCH3 H OCH3OCH3 O

CH3O O

OCH3OCH3

H3CO O OH CH3 CH3

Stigmatellin A (80) Figure 17. Structures of crocacins A–D (76–79) and stigmatellin A (80). Figure 17. Structures of crocacins A–D (76–79) and stigmatellin A (80). Crowley and coworkers [51] studied the binding of crocacins to the active site in order to design structurallyCrowley simpler and coworkers and more stable[51] studied synthetic the analogs.binding of An crocacins inhibitor to binding the active model site to in the order mammalian to design cytochromestructurallybc1 simpler complex and was more constructed, stable synthetic which was analogs. consistent An with inhibitor X-ray crystallographic binding model analysis to the ofmammalian an analog bound cytochrome to the chicken bc1 complex heart cytochrome was constructed, bc1 complex. which The proposed was consistent binding mode with for X- theray crocacinscrystallographic combined analysis some binding of an analog features bound of stigmatellin to the chicken A (80 heart, Figure cytochrome 17, a potent bc1 inhibitor complex. of theThe quinolproposed oxidation binding (Qo) mode site offor the the cytochrome crocacins combined bc1 complex) some and binding some binding features features of stigmatellin of strobilurins. A (80, TheFigure diff 17,erences a potent in binding inhibitor between of the quinol the crocacins oxidation and (Qo) methoxyacrylate site of the cytochrome stilbene couldbc1 complex) explain and the apparentsome binding lack of features cross-resistance of strobilurins. of the crocacins The differences with strobilurins. in binding between the crocacins and methoxyacrylateThe binding sitestilbene model, could coupled explain with the further apparent molecular lack of modeling, cross-resistance was used of the to design crocacins analogs with ofstrobilurins. crocacins A (76) and D (79), with mixed results. To improve the stability of the synthetic analogs, effortsThe were binding made site to replace model, the coupled three doublewith further bonds, molecular which most modeling, likely could was used give riseto design to significant analogs instabilityof crocacins to A sunlight, (76) and withD (79 more), with robust mixed groups, results. suchTo improve as aromatic the stability rings. Thisof the strategy synthetic had analogs, been successfulefforts were in themade development to replace the of stablethree double analogs bonds, of the strobilurins.which most likely could give rise to significant instabilityReplacement to sunlight, of the sidewith chain more with robust simple groups, n-alkyl such chains as aromatic or with side rings. chains This containing strategy hadaromatic been ringssuccessful (e.g., n in-alkoxybenzamide) the development gaveof stable compounds analogs veryof the active strobilurins. in the beef heart mitochondrial respiration assayReplacement but inactive as of fungicides. the side chain Further with modeling simple showed n-alkyl thatchains substituting or with side the benzamide chains containing with a 4-substitutedaromatic rings benzyl (e.g group., n-alkoxybenzamide) gave a very good overlay gave withcompounds the crocacin very side active chain. in Some the compounds beef heart (mitochondrial84–87) showed not respiration only potent assay inhibition but inactive of respiration as fungicides. but also activity Further on modeling vine downy showed mildew that on smallsubstituting vine plants the benzamide similar to or with better a than4-substituted crocacins benzyl (Table 9group). gave a very good overlay with the crocacin side chain. Some compounds (84–87) showed not only potent inhibition of respiration but also activity on vine downy mildew on small vine plants similar to or better than crocacins (Table 9).

Molecules 2020, 25, 4582 19 of 28

Molecules 2020, 25, 4582 19 of 28 Table 9. Activity of crocacin analogues on NADH oxidase and vine downy mildew on plants. Molecules 2020, 25, 4582 19 of 28 TableCompd. 9. Activity of crocacin analogues on NADH oxidase and vine downy mildew on plants. Molecules 2020, 25 , 4582 Breakpoint 19 of 28 Molecules 2020TableCompd., 25 9., 4582 Activity of crocacin analogues on NADH oxidase and vine downyIC50 mildew on plants. 19T of50 28 Vine Table 9. Activity of crocacin analogues on NADH oxidase and vine downyNADH mildew Breakpoint on plants. Glass Molecules 2020Compd., 25, 4582 Side Chain R IC50 Downy 19T of50 28 Table 9. Activity of crocacin analogues on NADH oxidase and vine downyoxidA semildew onVine plants. Slide Compd. NADH BreakpointMildew Glass MoleculesMolecules2020 2020,Table,25 25,, 45824582 9. Activity of crocacin analoguesSide on NADHChain Roxidase and vine downy(nM)IC50 mildewDowny on plants. 19 of(h) T2850 19 of 28 Compd. oxidAse Breakpoint(µM)Vine Slide NADIC50H Mildew GlassT50 Compd. Side Chain R (nM) BreakpointDownyVine (h) Table 9. Activity of crocacin analogues on NADH oxidase and vine downyoxidNADIC Amildew50H se on (µM)plants. GlassSlideT50 MildewVine Side Chain R NAD(nM)H BreakpointDowny Glass(h) TableCompd. 9. Activity of crocacin analogues on NADH oxidaseoxid andICA vine50se downy mildewSlideT on50 plants. Side Chain R MildewDowny(µM)Vine oxidNAD(nM)Ase H SlideGlass(h) BreakpointMildew(µM) Side Chain R IC(nM)50 Downy T50(h) Compd. oxidAse Slide 81 36 VineMildew(µM)25 0.6 NAD(nM)H BreakpointGlass(h) Side Chain R IC NADHDowny T Glass 81 Side Chain R oxidA36se50 (µM)25 VineSlide Downy0.6 50 oxidAse (nM)Mildew Slide (h) (nM) Mildew(h) (µM) (µM) 81 36 25 0.6

82 n-C12H21- 24 >100 0.1 81 36 25 0.6 81 36 25 0.6 82 n-C12H21- 24 >100 0.1 81 36 25 0.6 8183 n-C5H11O 21 36>100 255 0.6 82 n-C12H21- 24 >100 0.1

81 83 n-C H O12 21 36 25 0.6 82 5 n11-C H - 2421 >100 0.15 82 n-C12 H 21- 24 >100 0.1 82 n-C H n-CO12H21- 24 >100 0.1 83 5 11 21 >100 5 82 n-C12H21- 24 >100 0.1 8483 Br n-C5H11OC H 1721 >10010 125 2 8383 n-C5H11O 21 21 >100 >1005 5 82 n-C12H21- 24 >100 0.1 84 Br C H2 17 10 12 83 n-C H O 21 >100 5 5 11 Br C H 84 2 17 10 12 83 n-C H O 21 >100 5 8484 Br 5 11 C H 17 1710 1012 12 85 EtO CH22 9 25 20 84 Br C H 17 10 12 2 85 EtO C H2 9 25 20 84 Br C H2 17 10 12

8585 EtO C H2 9 925 2520 20 84 Br CH 17 10 12 85 EtO C2 H 9 25 20 86 I CH2 2 18 10 NT 85 EtO C H 9 25 20 2 86 86 I C H 2 18 1810 10NT NT 85 EtO CH2 9 25 20

I CH 86 2 18 10 NT 85 EtO CH 9 25 20 86 I CH 2 18 10 NT 8787 Et CH22 16 1610 10NT NT 86 I CH 18 10 NT 2 87 Et C H2 16 10 NT 86 I CH2 18 10 NT

Et CH The fact87 that some of the designed compounds 2 were inactive despite16 good modeling10 intoNT the 86 I CH 18 10 NT The fact that87 some of the designedEt compoundsC2 H were inactive16 despite good10 modelingNT into the active active site highlights the limitations of a rather2 simple model based on shape fit, the The fact87 that some of the designedEt compoundsC H were inactive despite16 good modeling10 intoNT the site highlightscomplementarity the of limitations hydrogen bonding of arather groups, simpleand2 ligand model conformational based on analysis, shape as fit,the authors the complementarity active site highlights the limitations of a rather simple model based on shape fit, the themselvesThe fact suggest. 87that some Actually of the, otherdesigned Eimportantt compounds factorCH s,were such inactive as solvation despite 16and go entropicod modeling10 effects, into could NTthe of hydrogencomplementarity bonding of hydrogen groups, bonding and groups, ligand and conformational2 ligand conformational analysis, analysis, as as thethe authors authors themselves beactive criticalThe site fact in binding highlightsthat some interactions. of the the limitations designed compounds of a rather were simple inactive model despite based good on modeling shape into fit, the suggest.themselves Actually, suggest. other Actually important, other important factors, factor such s, as such solvation as solvation and and entropic entropic eeffects,ffects, could could be critical in complementarityactiveNevertheless,The site fact87 highlightsthat of some the hydrogen designof the the limitations bondingdesigned strategyEt wasgroups,compounds of successfulC aH 2and rather ligandwere insimple inactivethat conformational analogs model despite16 ofbased go theanalysis,od naturally on modeling10 shape as the occurring intoauthors fit,NT the be critical in binding interactions. bindingfungicidethemselvesactivecomplementarity interactions. site crocacin suggest. highlights of D hydrogenActually ( 79 the) were , limitations otherbonding synthesized. important groups, of a These factor and rather compoundss,ligand such simple conformationalas solvation model were active basedand analysis, entropic both on in shapeas effects, a the respiration fit,authors could the Nevertheless,The fact that some the design of the strategydesigned was compounds successful were in thatinactive analogs despite of thegood naturally modeling occurring into the assay,becomplementarityNevertheless,themselves critical on fungi in suggest. binding, and of the on hydrogenActuallyinteractions. plants, design, otherandbondingstrategy were important groups,significantly was factor and successful s, ligandmore such stable conformationalas solvation in than that the and analogsnatural analysis, entropic compounds. ofas effects, the the authors naturallycould occurring fungicideactive site crocacin highlights D (79 the) were limitations synthesized. of Thesea rather compounds simple model were active based both on in shape a respiration fit, the themselvesbe ThecriticalNevertheless, fact in thatsuggest. binding some the Actuallyinteractions. of design the designed, other strategy important compounds was successful factor weres, such ininactive that as solvation analogs despite ofandgo theod entropic modeling naturally effects, into occurring couldthe fungicideassay,complementarity crocacinon fungi, and D of ( on79hydrogen )plants, were andbonding synthesized. were groups,significantly Theseand ligandmore compounds stable conformational than the were natural analysis, active compounds. as both the inauthors a respiration assay, active3.5.fungicidebe critical Neopeltolide site in crocacin highlights binding D interactions. ( the79) were limitations synthesized. of a Theserather compounds simple model were based active on both shape in a respiration fit, the on fungi,themselvesNevertheless, and on suggest. plants, the Actually design and were, strategyother significantlyimportant was successful factor mores, such in that stable as solvation analogs than of and the the entropic natural naturally effects, compounds. occurring could complementarityassay,Nevertheless, on fungi , ofand hydrogen the on designplants, bonding and strategy were groups, wassignificantly successful and ligand more in conformational thatstable analogs than the of analysis,natural the naturally compounds. as the authors occurring fungicide3.5.be criticalNeopeltolideNeopeltolide crocacinin binding ( 88 D, interactions. (Figure79) were 18), synthesized. a marine natural These product compounds isolated were in 2007, active was both found in ato respirationinhibit the themselvesfungicideassay, on fungisuggest. crocacin, and Actually D on (79 plants,) were, other and synthesized. important were significantly factor Theses, compoundssuchmore as stable solvation werethan theand active natural entropic both compounds. ieffects,n a respiration could bovineNevertheless, heart mitochondrial the design cytochrome strategy bc1 was complex successful with in an that IC50 analogs value of of2.0 the nM naturally [52]. However, occurring its 3.5.be Neopeltolideassay,3.5. critical NeopeltolideNeopeltolide on in fungi binding, and (interactions.88 on, Figure plants, 18), and a weremarine significantly natural product more stableisolated than in 2007,the natural was found compounds. to inhibit the complexfungicide structure crocacin prevented D (79) were the synthesized.obtainment of These the compound compounds by werechemical active synthesis. both in SAR a respiration studies bovineNevertheless, heart mitochondrial the design cy strategytochrome was bc1 successful complex with in that an IC analogs50 value of of the 2.0 naturallynM [52]. However, occurring its Neopeltolide3.5.assay, NeopeltolideNeopeltolide on fungi , and ( (8888 ,on, Figure Figure plants, 18), and18 a ),marine were a marine significantly natural natural product more isolated productstable than in 2007, isolated the naturalwas found in compounds. 2007, to inhibit was the found to inhibit fungicide3.5.complex Neopeltolide crocacin structure D prevented (79) were the synthesized. obtainment These of the compounds compound wereby chemical active bothsynthesis. in a respirationSAR studies bovineNeopeltolide heart mitochondrial (88, Figure cy 18),tochrome a marine bc1 natural complex product with an isolated IC50 value in 2007, of 2.0 was nM found [52]. However,to inhibit the its theassay, bovine on fungi heart, and mitochondrialon plants, and were cytochrome significantly more bc1 stable complex than the with natural an compounds. IC50 value of 2.0 nM [52]. complex3.5. Neopeltolide structure (prevented88, Figure 18),the aobtainment marine natural of the product compound isolated by chemicalin 2007, was synthesis. found toSAR inhibit studies the However,bovine itsheart complex mitochondrial structure cytochrome prevented bc1 complex the obtainmentwith an IC50 value of theof 2.0 compound nM [52]. However, by chemical its synthesis. bovine heart mitochondrial cytochrome bc1 complex with an IC50 value of 2.0 nM [52]. However, its 3.5.complex NeopeltolideNeopeltolide structure prevented(88, Figure the18), obtainment a marine natural of the product compound isolated by chemical in 2007, wassynthesis. found SARto inhibit studies the SARcomplex studies structure showed prevented that the the carbamate-containing obtainment of the compound oxazole by chemical moiety synthesis. was the SAR key studies structural feature, bovine heart mitochondrial cytochrome bc1 complex with an IC50 value of 2.0 nM [52]. However, its whereasNeopeltolide the 14-membered (88, Figure macrolactone18), a marine natural moiety product did isolated not make in 2007, a significantwas found to contributioninhibit the to binding. complex structure prevented the obtainment of the compound by chemical synthesis. SAR studies bovine heart mitochondrial cytochrome bc1 complex with an IC50 value of 2.0 nM [52]. However, its Zhu and coworkers [53] determined the binding mode of neopeltolide by integrating molecular complex structure prevented the obtainment of the compound by chemical synthesis. SAR studies docking, molecular dynamics (MD) simulations, and molecular mechanics Poisson-Boltzmann surface area calculations (MM/PBSA), which showed that neopeltolide is a Qo site inhibitor of the bc1 complex.

Based on neopeltolide binding mode, structural modiﬁcation was carried out with the aim to simplify its structure. Thus, a series of new neopeltolide derivatives with much simpler chemical structures were rationally designed and synthesized. Compound 89 bearing a naphthylether fragment in place of the structurally complex 14-membered macrolactone moiety showed high inhibitory activity against porcine SCR, with an IC50 value of 0.047 µM. The introduction of a bromo group on the naphthalene ring (compounds 90a and 90b) further improved the activity against SCR. Compound 90a was the most potent candidate against SCR (IC50 = 12 nM, Table 10)[53]. Molecules 2020, 25, 4582 20 of 28 Molecules 2020, 25, 4582 20 of 28

O O O O H O MeO O N NH H O

O Neopeltolide (88) O O O

O N O N R1 O CH3 N O H R2

O N 89 R1 = R2 = H O 90a R1 = Br, R2 = H 90b R = H, R = Br N O 91 1 2 H Figure 18. Structure of neopeltolide (88) and representative simpliﬁed analogs (89, 90a,b and 91). Figure 18. Structure of neopeltolide (88) and representative simplified analogs (89, 90a,b and 91).

[a] Zhu and coworkersTable 10. [53]IC 50determined(µM) value the of compounds binding mode89–91 ofagainst neopeltolide porcine SCR.by integrating. molecular docking, molecular dynamics (MD) simulations, and molecular mechanics Poisson-Boltzmann Cpd R IC50 (µM) SD surface area calculations (MM/PBSA), which showed that neopeltolide± is a Qo site inhibitor of the 89 H 0.045 0.001 bc1 complex. Based on neopeltolide binding mode, structural± modification was carried out with the 90a 7-Br 0.012 0.002 aim to simplify its structure. Thus, a series of new neopeltolide± derivatives with much simpler 90b 6-Br 0.016 0.001 ± chemical structures were rationally91 designed and synthesized.0.70 0.012 ± Compound 89 bearing a AZ naphthylether[b] fragment0.205 in 0.001 place of the structurally complex ± 14-membered macrolactone moiety[a] from showed Reference high [53 ,54inhibitory]. [b] azoxystrobin. activity against porcine SCR, with an IC50 value of 0.047 μM. The introduction of a bromo group on the naphthalene ring (compounds 90a and 90bMolecular) further docking improved of the the activity newly against synthesized SCR. compoundsCompound 90a was was performed, the most followedpotent candidate by MD againstsimulations SCR (IC and50 = MM 12 nM,/PBSA Table calculations. 10) [53]. Computational simulations revealed that most of the compoundsMolecular inside docking the Qo of site the ofnewly the bc1 synthesized complex formed compounds a hydrogen was performed, bond with Glu271followed and by a MDπ-π simulationsinteraction with and Phe274, MM/PBSA while calculations. the most active Computational compound ( 90a simulations) formed an revealed additional that hydrogen most of bond the compoundswith His161. inside the Qo site of the bc1 complex formed a hydrogen bond with Glu271 and a π-π interactionRecently, with the Phe274, same while authors the most prepared active a compound new set of (90a analogs) formed by an replacing additional the hydrogen 14-membered bond withmacrolactone His161. ring with an indole ring linked to the carbamate moiety by an ester or an amide Recently, the same authors prepared a new set of analogs by replacing the 14-membered bond [54]. The new analogs exhibited IC50 values ranging from 0.70 to 1.75 µM, and compound 91 macrolactone ring with an indole ring linked to the carbamate moiety by an ester or an amide bond showed the highest inhibitory activity against porcine SCR (IC50 0.70 µM). [54]. The new analogs exhibited IC50 values ranging from 0.70 to 1.75 µM, and compound 91 showed the3.6. highest Chromanols inhibitory activity against porcine SCR (IC50 0.70 µM). In the search for non-redox eﬀects of chromanols (vitamin E-related compounds), it was noted Table 10. IC50 (µM) value of compounds 89–91 against porcine SCR. [a]. that chromanols share structural similarity to stigmatellin (80, Figure 17), e.g., the chroman core. Based on this common structuralCpd feature, R MullebnerIC50 (µM) and ± SD coworkers [55] studied the extent and the mechanism of inhibitory eﬀects of89 natural H tocopherols 0.045 ± (0.001α, β, γ-, and δ-Toc, 92–95), their oxidation products tocopheryl quinones (α, β,90aγ-, and7-Brδ-TQ, 0.01296–99 ±), 0.002 and synthetic (low molecular weight TQ analogs and chromanones) compounds90b 1006and-Br 1010.016(Figure ± 0.001 19). 91 0.70 ± 0.012 AZ [b] 0.205 ± 0.001 [a] from Reference [53,54]. [b] azoxystrobin.

Molecules 2020, 25, 4582 21 of 28

3.6. Chromanols In the search for non-redox effects of chromanols (vitamin E-related compounds), it was noted that chromanols share structural similarity to stigmatellin (80, Figure 17), e.g., the chroman core. Based on this common structural feature, Mullebner and coworkers [55] studied the extent and the mechanism of inhibitory effects of natural tocopherols (α, β, γ-, and δ-Toc, 92–95), their oxidation products tocopheryl quinones (α, β, γ-, and δ-TQ, 96–99), and synthetic (low molecular weight TQ analogs and chromanones) compounds 100 and 101 (Figure 19). The authors found by enzymatic experiments that the 6-hydroxychroman structures of Toc-related compounds and the para-benzoquinone structure of TQ-related molecules can modulate the function of the mitochondrial cyt bc1 complex. For both Toc and TQ, an incomplete methyl substitution (β, γ-, and δ-congeners) increased the inhibitory potential. This affinity was further enhanced by the introduction of keto groups into the chroman ring, thus leading to chromanones. For TMC2O (6-hydroxy-4,4,7,8-tetramethylchroman- 2-one, 100), it was shown that it binds to the Qo pocket of the cyt bc1 complex and delays the electron transfer from dUQH2 to cyt c1 in the complex. Molecules 2020, 25, 4582 21 of 28 Docking experiments were performed to study the interaction of 100 with the cyt bc1 complex, which was similar but not identical to that of stigmatellin.

Figure 19. StructuresStructures of of natural natural tocopherols tocopherols (α,( αβ,, γβ-,, andγ-, andδ-Toc,δ-Toc, 92–9592),– of95 ),tocopheryl of tocopheryl quinones quinones (α, β, (γα -,,β and, γ -,δ- andTQ, δ96-TQ,–9996), and–99), low and molecular low molecular weight weight analogs analogs (100 and (100 101and). 101).

3.7. KarrikinolideThe authors found by enzymatic experiments that the 6-hydroxychroman structures of Toc-related compounds and the para-benzoquinone structure of TQ-related molecules can modulate the function Karrikinolide (3-methyl-2H-furo[2 ,3-c]-pyran-2-one, KAR1, 102) is a natural butenolide found of the mitochondrial cyt bc1 complex. in the smoke of burning plant material, which promotes the seed germination of a wide range of For both Toc and TQ, an incomplete methyl substitution (β, γ-, and δ-congeners) increased the plants [56]. The molecular simplicity, structural stability, and the novel skeleton of 102 inspired the inhibitory potential. This affinity was further enhanced by the introduction of keto groups into the synthesis of numerous analogs in order to study their efficacy in promoting seed germination [57]. chroman ring, thus leading to chromanones. For TMC2O (6-hydroxy-4,4,7,8-tetramethylchroman- In 2016, Chen et al. exploited this scaffold with the aim to develop new cytochrome bc1 complex 2-one, 100), it was shown that it binds to the Qo pocket of the cyt bc1 complex and delays the electron inhibitors and reported the synthesis of 20 karrikinolide derivatives by introducing different transfer from dUQH2 to cyt c1 in the complex. functional groups at C3-position (Figure 20) [58]. The inhibitory activity of the newly prepared Docking experiments were performed to study the interaction of 100 with the cyt bc1 complex, compounds was tested against SCR. SCR is composed of respiratory complex II (SQR) and complex which was similar but not identical to that of stigmatellin. III (the bc1 complex), which are believed to form a complex II–complex III supercomplex. Out of the 3.7.tested Karrikinolide derivatives, compound 103a–d exhibited limited activity against SQR at a concentration of 10 mM (Table 11). On the other hand, their inhibitory activities against SCR and the bc1 complex Karrikinolide (3-methyl-2H-furo[2,3-c]-pyran-2-one, KAR1, 102) is a natural butenolide found in the smoke of burning plant material, which promotes the seed germination of a wide range of plants [56]. The molecular simplicity, structural stability, and the novel skeleton of 102 inspired the synthesis of numerous analogs in order to study their efficacy in promoting seed germination [57]. In 2016, Chen et al. exploited this scaffold with the aim to develop new cytochrome bc1 complex inhibitors and reported the synthesis of 20 karrikinolide derivatives by introducing different functional groups at C3-position (Figure 20)[58]. The inhibitory activity of the newly prepared compounds was tested against SCR. SCR is composed of respiratory complex II (SQR) and complex III (the bc1 complex), which are believed to form a complex II–complex III supercomplex. Out of the tested derivatives, compound 103a–d exhibited limited activity against SQR at a concentration of 10 mM (Table 11). On the other hand, their inhibitory activities against SCR and the bc1 complex support the hypothesis that these compounds act as inhibitors of the bc1 complex. In spite of the promising results, further studies will be necessary to confirm the antifungal activity of the compounds. Molecules 2020, 25, 4582 22 of 28

Moleculessupport2020, 25the, 4582 hypothesis that these compounds act as inhibitors of the bc1 complex. In spite of the22 of 28 promising results, further studies will be necessary to confirm the antifungal activity of the compounds. R

O O

karrikinolide (102) 103a R = OCF3 103b R = OiPr 103c R = nBu 103d R = iBu FigureFigure 20. 20.Karrikinolide Karrikinolide (102 (102)) and and selectedselected derivatives derivatives 103a103a–d– fromd from Reference Reference [59].[ 59].

TableTable 11. 11.Inhibitory Inhibitory e ﬀeffectect of of selected selected compounds on on SCR, SCR, SQR SQR,, and and cyt cytbc1. bc1.

IC50 (µM)(µM) I%I% (10 (10 µM)µM) I%I% (10 (10 µM)µM) I% I%(10 (10µM)µ M) CpdCpd 50 SelectivitySelectivity SCR SCRSCR SQRSQR cyt cytbc1 bc1 103a103a 3.55 ± 0.110.11 76% 76% 14% 14% 66% 66% cyt bc1 cyt bc1 ± 103b103b 9.39 ± 0.120.12 50% 50% <10%<10% 46% 46% cyt bc1 cyt bc1 ± 103c103c 0.737 ± 0.010.0111 82% 82% 39% 39% 83% 83% cyt bc1 cyt bc1 ± 103d103d 1.10 ± 0.200.20 73% 73% 26% 26% 67% 67% cyt bc1 cyt bc1 ± 3.8. Picolinamide UK-2A 3.8. Picolinamide UK-2A The natural compound picolinamide UK-2A (104, Figure 21) is a pyridine carboxamide Molecules 2020, 25, 4582 23 of 28 Theoriginally natural isolated compound from fermentation picolinamide broths UK-2A of the (104 actinomycete, Figure 21Streptomyces) is a pyridine sp. 517– carboxamide02 [59]. originallyPyridineAmong isolated carboxamides the fromanalogs fermentationinhibit featuring mitochondrial modifications broths respiration of to the the actinomycete macrocby bindingycle tobenzyl theStreptomyces Qi position, ubiquinone thesp. cyclohexyl site 517–02 of the [59]. Pyridinecytochromeanalog carboxamides (108 )bc1 was complex. the most inhibit Remarkably, active mitochondrial derivative no target on respiration mitochondrial site-based bycross electron binding-resistance transport to with the Qi(MET)Qo ubiquinoneinhibitors assays (ICwas site50 of the cytochromeobserved1.23 nM) forand bc1 these inhibited complex. compounds. the Remarkably, in vitro However, growth no to target of date Zymoseptoria site-basedonly two tritici molecules cross-resistance (EC50 acting 2.8 ppb) as with Qi and site Qo Leptosphaeria inhibitors was observedhavenodorum forbeen these(EC commercialized:50 6.2 compounds. ppb) more cyazofamid strongly However, than and to UK dateamisulbrom-2A only (EC50 two 5.3 as and moleculesoomycete 11.3 ppb-specific acting for Z .substances astritici Qi and site .L inhibitors . nodorum, have beenrespectively). commercialized:Picolinamide On UKthe cyazofamid -2A contrary, was used and the amisulbromas introduction a lead compound ofas oomycete-speciﬁc heterocyclic to design ring new systems substances. macrocyclic and polar fungicidal linker moleculesfunctionalities, and onrecently this position at Dow resulted AgroSciences in substantial LLC the lossnovel of fungicideactivity. fenpicoxamid (105, Inatreq™, Figure 21) was developed by semi-synthetic modification of the natural compound. The acyloxymethyl ether derivative fenpicoxamid showed a broad spectrum of activity in in vitro assays and excellent efficacy on Zymoseptoria tritici (synonym, Mycosphaerella graminicola, wheat leaf blotch), the pathogen of greatest concern for wheat production in Europe. Recently, Owen and coworkers [60–62] described structure–activity relationship (SAR) studies on UK-2A, analyzing the impact of modifications of the macrocycle isobutyryl ester position, ring replacement, and modifications to the macrocycle benzyl position. Moreover, the relative activities of the new analogs were rationalized, based on a homology model constructed for the Z. tritici Qi binding site. In particular, the isobutyryl ester of UK-2A was replaced by a series of ester groups and a set of ether groups, carbonate, and carbamate moieties. The authors reported that linkages other than esters are well tolerated at the 7-position of the macrocycle, with the only exception of the carbamate analogs. The n-butyl ether (106) was the most active analog evaluated, and compound 107 (pivaloate ester) was the most active among the ester derivatives (Table 12). The 3-hydroxy-4-methoxypicolinic acid moiety of UK-2A can be replaced by a variety of o-hydroxy-substituted arylcarboxylic acids, while retaining strong activity against Z. tritici and other relevant fungi.

Figure 21. Structure of natural picolinamide UK-2A (104), of fenpicoxamid (105), and structures of Figure 21. Structure of natural picolinamide UK-2A (104), of fenpicoxamid (105), and structures of representativerepresentative analogs analogs (106 (106–108–108). ).

Table 12. Comparative target site and in vitro antifungal activities of UK-2A and representative compounds 106–108 from References [61–63].

IC50 MET EC50 Compd (nM) (µg/L) UK-2A 1.46 4.4 106 1.08 5.3 107 1.44 6.3 108 1.23 2.8 AZ 11.49 3.5 MET: Mitochondrial electron transport assays.

4. Conclusions There is an urgent need for new compounds that specifically target pathogenic fungi due to the increasing rates of resistance to the drugs currently available on the market. The cytochrome bc1 complex is one of the most important fungicidal targets. Inhibitors of the cytochrome bc1 complex have been broadly studied, in particular for controlling fungal diseases. In this context, natural products have played a crucial role. Commercial analogs of the natural compound strobilurin are among the most successful classes of agricultural fungicides, with a dominant position on the global market. This review provides an outline of advances in the investigation of new bc1 complex inhibitors based on natural products. Emphasis has been given to studies involved in the optimization of the natural scaffold. In fact, despite their enormous potential, natural compounds are often

Molecules 2020, 25, 4582 23 of 28

Picolinamide UK-2A was used as a lead compound to design new macrocyclic fungicidal molecules, and recently at Dow AgroSciences LLC the novel fungicide fenpicoxamid (105, Inatreq™, Figure 21) was developed by semi-synthetic modification of the natural compound. The acyloxymethyl ether derivative fenpicoxamid showed a broad spectrum of activity in in vitro assays and excellent efficacy on Zymoseptoria tritici (synonym, Mycosphaerella graminicola, wheat leaf blotch), the pathogen of greatest concern for wheat production in Europe. Recently, Owen and coworkers [60–62] described structure–activity relationship (SAR) studies on UK-2A, analyzing the impact of modifications of the macrocycle isobutyryl ester position, ring replacement, and modifications to the macrocycle benzyl position. Moreover, the relative activities of the new analogs were rationalized, based on a homology model constructed for the Z. tritici Qi binding site. In particular, the isobutyryl ester of UK-2A was replaced by a series of ester groups and a set of ether groups, carbonate, and carbamate moieties. The authors reported that linkages other than esters are well tolerated at the 7-position of the macrocycle, with the only exception of the carbamate analogs. The n-butyl ether (106) was the most active analog evaluated, and compound 107 (pivaloate ester) was the most active among the ester derivatives (Table 12).

Table 12. Comparative target site and in vitro antifungal activities of UK-2A and representative compounds 106–108 from References [61–63].

IC MET EC Compd 50 50 (nM) (µg/L) UK-2A 1.46 4.4 106 1.08 5.3 107 1.44 6.3 108 1.23 2.8 AZ 11.49 3.5 MET: Mitochondrial electron transport assays.

The 3-hydroxy-4-methoxypicolinic acid moiety of UK-2A can be replaced by a variety of o-hydroxy-substituted arylcarboxylic acids, while retaining strong activity against Z. tritici and other relevant fungi. Among the analogs featuring modiﬁcations to the macrocycle benzyl position, the cyclohexyl analog (108) was the most active derivative on mitochondrial electron transport (MET) assays (IC50 1.23 nM) and inhibited the in vitro growth of Zymoseptoria tritici (EC50 2.8 ppb) and Leptosphaeria nodorum (EC50 6.2 ppb) more strongly than UK-2A (EC50 5.3 and 11.3 ppb for Z. tritici and L. nodorum, respectively). On the contrary, the introduction of heterocyclic ring systems and polar linker functionalities on this position resulted in substantial loss of activity.

4. Conclusions There is an urgent need for new compounds that specifically target pathogenic fungi due to the increasing rates of resistance to the drugs currently available on the market. The cytochrome bc1 complex is one of the most important fungicidal targets. Inhibitors of the cytochrome bc1 complex have been broadly studied, in particular for controlling fungal diseases. In this context, natural products have played a crucial role. Commercial analogs of the natural compound strobilurin are among the most successful classes of agricultural fungicides, with a dominant position on the global market. This review provides an outline of advances in the investigation of new bc1 complex inhibitors based on natural products. Emphasis has been given to studies involved in the optimization of the natural scaffold. In fact, despite their enormous potential, natural compounds are often characterized by structural complexity, toxicity, and unfavorable bioavailability, which can limit their investigation and severely hamper their development. For this reason, this review focuses on those modifications Molecules 2020, 25, 4582 24 of 28 of the natural core that have expanded the potency and stability of analogs compared to the parent compounds, opening the possibility of further development. In this context, it should be stressed that, besides the in vitro antifungal test (percentage of inhibition of fungal growth or EC50 values), a significant number of enzymatic tests to evaluate the cyt bc1 inhibitory activity were performed using as a model system the porcine succinate cytochrome c reductase, a mixture of respiratory complex II, and bc1 complex [10,34,50,52–54,56]. Despite the general lack of data around the fungal cytochrome bc1 complex, those studies were also included in the review in order to obtain a complete overview of the SAR investigations on cytochrome bc1 complex inhibitors. The history of strobilurins clearly demonstrates the value of a detailed structure–activity investigation in the context of a strategy focused on the variation of molecular structures to optimize the biological profile of the parent molecule. From the analysis of all the reported studies, it has emerged that the knowledge of the target binding site is often crucial to define the effect of substituent changes and predict modifications for enhanced potency, safety, and circumvention of resistance. However, a major concern in the development of new cytochrome bc1 complex inhibitors is related to the toxicity to non-target organisms., e.g., plants and animals. In some cases, the evolutionary conservation of sequence and structure of the key functional subunits of the respiratory chain complexes is reflected in a low selectivity of electron transport inhibitors in different species [6]. In fact, it has recently been demonstrated that even strobilurins, which are allegedly non-toxic to humans, can also exert their mode of action in mammalian cells [63]. Despite the improved understanding of the existing targets and their inhibitors given by crystallographic and structural biology studies [64], the design of novel inhibitors of the bc1 complex with appropriate species selectivity still remains a very challenging goal. A few papers report efforts towards this aim. Monzote et al. [65] and Rotsaert et al. [66], found a certain degree of selectivity in the inhibition of the cyt bc1 activity of different species by selected molecules. Nevertheless, it should be stressed that the relevance of the molecular target of a fungicide may change during the life cycle of the treated organism. As a consequence, different development stages may exhibit different sensitivities to a given molecule. The ATP energy-demanding spore germination is, compared to mycelial growth, particularly sensitive to respiration inhibitors such as strobilurins. Since, among eukaryotes, spore germination occurs almost exclusively in fungi, this also contributes, on the physiological level, to selective action against fungal pathogens [6]. It should be also considered that careful optimization of the biological profile of the antifungal compounds—including modulation of their pharmacodynamic and pharmacokinetic properties—can contribute to the reduction of mammalian toxicity. Overall, recently reported results highlight the importance of cytochrome bc1 complex as a fungicidal target and spark a renewed interest in natural products as sources for new antifungal molecules. However, a great effort to increase species selectivity and to reduce the toxicity of fungicides is still needed and remains of primary importance for future antifungal drug discoveries.

Author Contributions: Conceptualization, S.D. and L.M.; supervision, S.D.; writing—original draft preparation, L.M., A.F., and S.D.; writing—review and editing, L.M. and S.D. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: The authors gratefully acknowledge L. Merlini for helpful suggestions and discussions. Conﬂicts of Interest: The authors declare no conﬂict of interest. Molecules 2020, 25, 4582 25 of 28

References

1. Calicioglu, O.; Flammini, A.; Bracco, S.; Bellù, L.; Sims, R. The future challenges of food and agriculture: An integrated analysis of trends and solutions. Sustainability 2019, 11, 222. [CrossRef] 2. Hunte, C.; Palsdottir, H.; Trumpower, B.L. Protonmotive pathways and mechanisms in the cytochrome bc1 complex. FEBS Lett. 2003, 545, 39–46. [CrossRef] 3. Lorsbach, B.A.; Sparks, T.C.; Cicchillo, R.M.; Garizi, N.G.; Hahn, D.R.; Meyer, K.G. Natural products: A strategic lead generation approach in crop protection discovery. Pest Manag. Sci. 2019, 75, 2301–2309. [CrossRef][PubMed] 4. Fisher, N.; Meunier, B.; Biagini, G.A. The cytochrome bc 1 complex as an antipathogenic target. FEBS Lett. 2020, in press. [CrossRef] 5. Balba, H. Review of strobilurin fungicide chemicals. J. Environ. Sci. Health B 2007, 42, 441–451. [CrossRef][PubMed] 6. Sauter, H. Strobilurins and Other Complex III Inhibitors. In Modern Crop Protection Compounds; Kramer, W., Schimer, U., Eds.; Wiley: Weinheim, Germany, 2012; pp. 584–627. 7. Anke, T.; Oberwinkler, F.; Steglich, W.; Schramm, G. The strobilurins—new antifungal antibiotics from the basidiomycete (PERS. Ex FR.) SING. J. Antibiot. 1977, 806–810. [CrossRef][PubMed] 8. Bartlett, D.W.; Clough, J.M.; Godwin, J.R.; Hall, A.A.; Hamer, M.; Parr-Dobrzanski, B. The strobilurin fungicides. Pest Manag. Sci. 2002, 58, 649–662. [CrossRef] 9. Hao, G.-F.; Wang, F.; Li, H.; Zhu, X.-L.; Yang, W.-C.; Huang, L.-S.; Wu, J.-W.; Berry, E.A.; Yang, G.-F. Computational Discovery of Picomolar Qo Site Inhibitors of Cytochrome bc1 Complex. J. Am. Chem. Soc. 2012, 134.[CrossRef] 10. Zhu, X.; Wang, F.; Li, H.; Yang, W.; Chen, Q.; Yang, G. Design, synthesis, and bioevaluation of novel Strobilurin derivatives. Chin. J. Chem. 2012, 30, 1999–2008. [CrossRef] 11. Tu, S.; Xu, L.-H.; Ye, L.-Y.; Wang, X.; Sha, Y.; Xiao, Z.-Y. Synthesis and Fungicidal Activities of Novel Indene-Substituted Oxime Ether Strobilurins. J. Agric. Food Chem. 2008, 56, 5247–5253. [CrossRef] 12. Tu, S.; Xie, Y.-Q.; Gui, S.-Z.; Ye, L.-Y.; Huang, Z.-L.; Huang, Y.-B.; Che, L.-M. Synthesis and fungicidal activities of novel benzothiophene-substituted oxime ether strobilurins. Bioorg. Med. Chem. Lett. 2014, 24, 2173–2176. [CrossRef][PubMed] 13. Xie, Y.-Q.; Huang, Y.-B.; Liu, J.-S.; Ye, L.-Y.; Che, L.-M.; Tu, S.; Liu, C.-L. Design, synthesis and structure–activity relationship of novel oxime ether strobilurin derivatives containing substituted benzofurans. Pest Manag. Sci. 2015, 71, 404–414. [CrossRef][PubMed] 14. Xie, Y.-Q.; Huang, Z.-L.; Yan, H.-D.; Li, J.; Ye, L.-Y.; Che, L.-M.; Tu, S. Design, synthesis, and biological activity of oxime ether strobilurin derivatives containing indole moiety as novel fungicide. Chem. Biol. Drug Des. 2015, 85, 743–755. [CrossRef][PubMed] 15. Chaudhary, P.M.; Tupe, S.G.; Jorwekar, S.U.; Sant, D.G.; Deshpande, S.R.; Maybhate, S.P.; Likhite, A.P.; Deshpande, M.V. Synthesis and antifungal potential of 1,2,3-triazole and 1,2,4- triazole thiol substituted strobilurin derivatives. Indian J. Chem. 2015, 54b, 908–917. 16. Wang, X.; Wang, H.; Chen, P.; Pang, Y.; Zhao, Z.; Wu, G. Synthesis of novel (E)-α-(methoxyimino) benzeneacetate derivatives and their fungicidal activities. J. Chem. Soc. Pak. 2015, 37, 502–510. 17. Liu, Y.; Liu, M.; Zhang, D.; Hua, X.; Wang, B.; Zhou, S.; Li, Z. Design, synthesis and fungicidal activity of novel strobilurin-1,2,4-triazole derivatives containing furan or thiophene rings. Chem. Res. Chin. Univ. 2016, 32, 952–958. [CrossRef] 18. Hong Song, H.; Song, H.-X.; Shi, D.-Q. Synthesis and fungicidal activity of strobilurin analogues containing 1,2,4-triazole oxime ether moiety. J. Heterocyclic Chem. 2014, 51, 1603–1606. [CrossRef] 19. Song, H.-X.; Shi, D.-Q. Synthesis and fungicidal activity of (E)-α-(Methoxyimino)-benzeneacetate derivatives containing 1,2,4-triazole Schiﬀ base side chain. J. Heterocylic Chem. 2014, 51, 1345–1348. [CrossRef] 20. Wang, X.; Wang, H.; Chen, P.; Pang, Y.; Zhao, Z.; Wu, G. Synthesis and biological activities of some novel (E)-α-(methoxyimino-)-benzeneacetate derivatives with modiﬁed 1,2,4-triazole moiety. J. Chem. 2014, 2014, 1–9. [CrossRef] 21. Worthington, P. Sterol biosynthesis inhibiting triazole fungicides. In Bioactive Heterocyclic Compound Classes: Agrochemicals; Lamberth, C., Dinges, J., Eds.; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, Germany, 2012; pp. 129–145. [CrossRef] Molecules 2020, 25, 4582 26 of 28

22. Liu, Y.; Liu, M.; Chen, M.; Wu, C.; Hua, X.; Zhou, S.; Wang, B.; Li, Z. Design, synthesis and bioactivities of novel strobilurin derivatives containing 1,3,4-oxadiazole moiety. Chin. J. Org. Chem. 2017, 37, 403–410. [CrossRef] 23. Li, Y.; Lei, S.; Liu, Y. Design, synthesis and fungicidal activities of novel 1,2,3-triazole functionalized strobilurins. ChemistrySelect 2019, 4, 1015–1018. [CrossRef] 24. Liu, H.-J.; Zhang, X.; Gao, Y.-X.; Li, J.-Z.; Wang, H.-L. Design, synthesis, and antifungal activities of new β-methoxyacrylate analogues. J. Chin. Chem. Soc. 2013, 60, 27–34. [CrossRef] 25. Chai, B.-S.; Liu, C.-L.; Li, H.-C.; Zhang, H.; Liu, S.-W.; Huang, G.; Chang, J.-B. The discovery of SYP-10913 and SYP-11277: Novel strobilurin acaricides. Pest Manag. Sci. 2011, 67, 1141–1146. [CrossRef][PubMed] 26. Liu, M.; Liu, Y.; Zhou, S.; Zhang, X.; Yu, S.; Li, Z. Synthesis and antifungal activities of novel strobilurin derivatives containing quinolin-2(1H)-one moiety. Chem. Res. Chin. Univ. 2016, 32, 600–606. [CrossRef] 27. Chen, L.; Guoa, X.-F.; Fan, Z.-J.; Zhang, N.-L.; Zhu, Y.-J.; Zhang, Z.-M.; Khazhieva, I.; Yurievich, M.Y.; Belskaya, N.P.; Bakulev, V.A. Synthesis and fungicidal activity of 3,4-dichloroisothiazole based strobilurins as potent fungicide candidates. RSC Adv. 2017, 7, 3145–3151. [CrossRef] 28. Clerici, F.; Gelmi, M.L.; Pellegrino, S.; Pocar, D. Chemistry of Biologically Active Isothiazoles. Top. Heterocycl. Chem. 2007, 9, 179–264. [CrossRef] 29. Bektas, Y.; Eulgem, T. Synthetic plant defense elicitors. Front. Plant Sci. 2015, 5, 804. [CrossRef] 30. Chen, L.; Zhu, Y.-J.; Fan, Z.-J.; Guo, X.-F.; Zhang, Z.-M.; Xu, J.-H.; Song, Y.-Q.; Yurievich, M.Y.; Belskaya, N.P.; Bakulev, V.A. Synthesis of 1,2,3-thiadiazole and thiazole-based strobilurins as potent fungicide candidates. J. Agric. Food Chem. 2017, 65, 745–751. [CrossRef] 31. Su, H.; Wang, W.; Bao, L.; Wang, S.; Cao, X. Synthesis and evaluation of essential oil-derived β-methoxyacrylate derivatives as high potential fungicides. Molecules 2017, 22, 763. [CrossRef] 32. Isman, M.B. Plant essential oils for pest and disease management. Crop Prot. 2000, 19, 603–608. [CrossRef] 33. Liu, Y.; Lv, K.; Li, Y.; Nan, Q.; Xu, J. Synthesis, fungicidal activity, structure-activity relationships (SARs) and density functional theory (DFT) studies of novel strobilurin analogues containing arylpyrazole rings. Sci. Rep. 2018, 8, 7822. [CrossRef][PubMed] 34. Wang, L.; Zhao, S.; Kong, X.; Cao, L.; Tian, S.; Ye, Y.; Qiao, C. Design, synthesis and fungicidal evaluation of novel pyraclostrobin analogues. Bioorg. Med. Chem. 2018, 26, 875–883. [CrossRef][PubMed] 35. Jia, C.; Yuan, X.; Liu, X.; Zhang, L.; Xiao, Y.; Fu, B.; Li, J.-Q.; Qin, Z. Synthesis and fungicidal activity of (E)-methyl-2-(2-((1-cyano-2-hydrocarbylidenehydrazinyl)methyl)phenyl)-2-(methoxyimino)acetates. Pest Manag. Sci. 2019, 75, 3160–3166. [CrossRef][PubMed] 36. Matsuzaki, Y.; Yoshimoto, Y.; Arimori, S.; Kiguchi, S.; Harada, T.; Iwahashi, F. Discovery of methyltetraprole: Identification of tetrazolinone pharmacophore to overcome QoI resistance. Bioorg. Med. Chem. 2020, 28, 115211. [CrossRef] 37. Leibold, T.; Sasse, F.; Reichenbach, H.; Höﬂe, G. Cyrmenins, novel antifungal peptides Containing a Nitrogen-Linked β-methoxyacrylate pharmacophore: Isolation and structural elucidation. Eur. J. Org. Chem. 2004, 2004, 431–435. [CrossRef] 38. Chakor, N.S.; Musso, L.; Dallavalle, S. First Total Synthesis of Cyrmenin B1. J. Org. Chem. 2009, 74, 844–849. [CrossRef] 39. Chakor, N.S.; Dallavalle, S.; Musso, L.; Sardi, P. Synthesis and evaluation of structural requirements for

antifungal activity of cyrmenin B1 analogues. Tetrahedron Lett. 2012, 53, 228–231. [CrossRef] 40. Gerth, K.; Irschik, H.; Trowitzsch, W.; Reichenbach, H. Myxothiazol, an antibiotic from Myxococcus fulvus (Myxobacterales). J. Antibiot. 1980, 33, 1474–1479. [CrossRef] 41. Trowitzsch, W.; Hofle, G.; Sheldrick, W.S. The stereochemistry of myxothiazol. Tetrahedron Lett. 1981, 22, 3829–3832. [CrossRef] 42. Ahn, J.-W.; Woo, S.-H.; Lee, C.-O.; Cho, K.-Y.; Kim, B.-S. KR025, a New Cytotoxic Compound from Myxococcus fulvus. J. Nat. Prod. 1999, 62, 495–496. [CrossRef] 43. Steinmetz, H.; Forche, E.; Reichenbach, H.; Hofle, G. Biosynthesis of myxothiazol Z, the ester-analog of myxothiazol A. Myxococcus fulvus Tetrahedron 2000, 56, 1681–1684. [CrossRef] 44. Böhlendorf, B.; Hermann, M.; Hecht, H.-J.; Sasse, F.; Forche, E.; Kunze, B.; Reichenbach, H.; Höfle, G. Antibiotics from gliding bacteria, 85[#] melithiazols A–N: New antifungal β-methoxyacrylates from myxobacteria. Eur. J. Org. Chem. 1999, 1999, 2601–2608. [CrossRef] 45. Söker, U.; Sasse, F.; Kunze, B.; Höfle, G. Synthesis of melithiazol B and related compounds via oxidative degradation of myxothiazol A and Z. Eur. J. Org. Chem. 2000, 2000, 1497–1502. [CrossRef] Molecules 2020, 25, 4582 27 of 28

46. Söker, U.; Sasse, F.; Kunze, B.; Höfle, G. Neighbouring-group assisted thiazole-ring cleavage by DIBAL-H: An expeditious synthesis of melithiazol C from myxothiazol A. Eur. J. Org. Chem. 2000, 2000, 2021–2026. [CrossRef] 47. Panter, F.; Krug, D.; Müller, R. Novel methoxymethacrylate natural products uncovered by statistics-based mining of the Myxococcus fulvus secondary metabolome. ACS Chem. Biol. 2019, 14, 88–98. [CrossRef] 48. Iizuka, T.; Fudou, R.; Ogawa, S.; Yamanaka, S.; Inukai, Y.; Ojika, M. Miuraenamides A and B, Novel Antimicrobial Cyclic Depsipeptides from a New Slightly Halophilic Myxobacterium: Taxonomy, Production, and Biological Properties. J. Antibiot. 2006, 59, 385–391. [CrossRef] 49. Ojika, M.; Inukai, Y.; Kito, Y.; Hirata, M.; Iizuka, T.; Fudou, R.; Miuraenamides, R. Antimicrobial Cyclic Depsipeptides Isolated from a Rare and Slightly Halophilic Myxobacterium. Chem. Asian J. 2008, 3, 126–133. [CrossRef] 50. Kunze, B.; Jansen, R.; Höfle, G.; Reichenbach, H. Crocacin, a new electron transport inhibitor from crocatus (Myxobacteria). Production, isolation, physico-chemical and biological properties. J. Antibiot. 1994, 47, 881–886. [CrossRef] 51. Crowley, P.J.; Berry, E.A.; Cromartie, T.; Daldal, F.; Godfrey, C.R.A.; Lee, D.-W.; Phillips, J.; Taylor, E.A.; Viner, R. The role of molecular modeling in the design of analogues of the fungicidal natural products crocacins A and D. Bioorg. Med. Chem. 2008, 16, 10345–10355. [CrossRef] 52. Wright, A.E.; Botelho, J.C.; Guzmaán, E.; Harmody, D.; Linley, P.; McCarthy, P.J.; Pitts, T.P.; Pomponi, S.A.; Reed, J.K. Neopeltolide, a Macrolide from a Lithistid Sponge of the Family Neopeltidae. J. Nat. Prod. 2007, 70, 412–416. [CrossRef][PubMed] 53. Zhu, X.-L.; Zhang, R.; Wu, Q.-Y.; Song, Y.-J.; Wang, Y.-X.; Yang, J.-F.; Yang, G.-F. Natural product Neopeltolide as a cytochrome bc1 complex inhibitor: Mechanism of action and structural modification. J. Agric. Food Chem. 2019, 67, 2774–2781. [CrossRef] 54. Xiong, M.-Q.; Chen, T.; Wang, Y.-X.; Zhua, X.-L.; Yang, G.-F. Design and synthesis of potent inhibitors of bc1 complex based on natural product neopeltolide. Bioorg. Med. Chem. Lett. 2020, 30, 127324. [CrossRef] [PubMed] 55. Müllebner, A.; Patel, A.; Stamberg, W.; Staniek, K.; Rosenau, T.; Netscher, T.; Gille, L. Modulation of the mitochondrial cytochrome bc1 complex activity by chromanols and related compounds. Chem. Res. Toxicol. 2010, 23, 193–202. [CrossRef][PubMed] 56. Posta,˘ M.; Light, M.E.; Papenfus, H.B.; Van Staden, J.; Kohout, L. Structure–activity relationships of analogs of 3,4,5-trimethylfuran-2(5H)-one with germination inhibitory activities. J. Plant Physiol. 2013, 170, 1235–1242. [CrossRef] 57. Scaffidi, A.; Flematti, G.R.; Nelson, D.C.; Dixon, K.W.; Smith, S.M.; Ghisalberti, E.L. The synthesis and biological evaluation of labelled karrikinolides for the elucidation of the mode of action of the seed germination stimulant. Tetrahedron 2011, 67, 152–157. [CrossRef] 58. Chen, C.; Wu, Q.-Y.; Shan, L.-Y.; Zhang, B.; Verpoort, F.; Yang, G.-F. Discovery of cytochrome bc1 complex inhibitors inspired by the natural product karrikinolide. RSC Adv. 2016, 6, 97580–97586. [CrossRef] 59. Ueki, M.; Abe, K.; Hanafi, M.; Shibata, K.; Tanaka, T.; Taniguchi, M. UK-2A, B, C and D, Novel Antifungal Antibiotics from Streptomyces sp. 517-02. J. Antibiot. 1996, 49, 639–643. [CrossRef] 60. Owen, W.J.; Meyer, K.G.; Slanec, T.J.; Meyer, S.T.; Wang, N.X.; Fitzpatrick, G.M.; Niyaz, N.M.; Nugent, J.; Ricks, M.J.; Rogers, R.B.; et al. Synthesis and biological activity of analogs of the antifungal antibiotic UK-2A. III. Impact of modifications to the macrocycle isobutyryl ester position. Pest Manag. Sci. 2020, 76, 277–286. [CrossRef] 61. Owen, W.J.; Meyer, K.G.; Slanec, T.J.; Wang, N.X.; Meyer, S.T.; Niyaz, N.M.; Rogers, R.B.; Bravo-Altamirano, K.; Herrick, J.L.; Chenglin Yao, C. Synthesis and biological activity of analogs of the antifungal antibiotic UK-2A. I. Impact of picolinamide ring replacement. Pest Manag. Sci. 2019, 75, 413–426. [CrossRef][PubMed] 62. Owen, W.J.; Meyer, K.G.; Meyer, S.T.; Li, F.; Slanec, T.J.; Wang, N.X.; Yao, C. Synthesis and biological activity of analogs of the antifungal antibiotic UK-2A. II. Impact of modifications to the macrocycle benzyl position. Pest Manag. Sci. 2019, 75, 1831–1846. [CrossRef][PubMed] 63. Flampouri, E.; Mavrikou, S.; Mouzaki-Paxinou, A.-C.; Kintzios, S. Alterations of cellular redox homeostasis in cultured fibroblast-like renal cells upon exposure to low doses of cytochrome bc1 complex inhibitor kresoxim-methyl. Biochem. Pharmacol. 2016, 113, 97–109. [CrossRef][PubMed] 64. Esser, L.; Quinn, B.; Li, Y.-F.; Zhang, M.; Elberry, M.; Yu, L.; Yu, C.-A.; Xia, D. Crystallographic Studies of Quinol Oxidation Site Inhibitors: A Modified Classification of Inhibitors for the Cytochrome bc1 Complex. J. Mol. Biol. 2004, 341, 281–302. [CrossRef][PubMed] Molecules 2020, 25, 4582 28 of 28

65. Monzote, L.; Stamberg, W.; Patel, A.; Rosenau, T.; Maes, L.; Cos, P.; Gille, L. Synthetic chromanol derivatives and their iInteraction with complex III in mitochondria from bovine, yeast, and Leishmania. Chem. Res. Toxicol. 2011, 24, 1678–1685. [CrossRef][PubMed] 66. Rotsaert, F.A.J.; Ding, M.G.; Trumpower, B.L. Diﬀerential eﬃcacy of inhibition of mitochondrial and bacterial cytochrome bc1 complexes by center N inhibitors antimycin, ilicicolin H and funiculosin. Biochimica et Biophysica Acta (BBA)—Bioenergetics 2008, 1777, 211–219. [CrossRef][PubMed]

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).