Structural Investigation and Molecular Modeling Studies of Strobilurin-Based Fungicides Active Against the Rice Blast Pathogen Pyricularia Oryzae

International Journal of Molecular Sciences

Article Structural Investigation and Molecular Modeling Studies of Strobilurin-Based Fungicides Active against the Rice Blast Pathogen Pyricularia oryzae

Andrea Kunova 1 , Luca Palazzolo 2, Fabio Forlani 1 , Giorgia Catinella 1 , Loana Musso 1 , Paolo Cortesi 1, Ivano Eberini 2,3,*, Andrea Pinto 1,* and Sabrina Dallavalle 1

1 Department of Food, Environmental and Nutritional Science (DeFENS), University of Milan, 20133 Milan, Italy; [email protected] (A.K.); [email protected] (F.F.); [email protected] (G.C.); [email protected] (L.M.); [email protected] (P.C.); [email protected] (S.D.) 2 Department of Pharmacological and Biomolecular Sciences (DiSFeB), University of Milan, 20133 Milan, Italy; [email protected] 3 Data Science Research Center (DSRC), University of Milan, 20133 Milan, Italy * Correspondence: [email protected] (I.E.); [email protected] (A.P.)

Abstract: The increasing emergence of fungicide-resistant pathogens requires urgent solutions for crop disease management. Here, we describe a structural investigation of new fungicides obtained by combining strobilurin and succinate dehydrogenase inhibitor pharmacophores. We identiﬁed compounds endowed with very good activity against wild-type Pyricularia oryzae, combined in some cases with promising activity against strobilurin-resistant strains. The ﬁrst three-dimensional Citation: Kunova, A.; Palazzolo, L.; model of P. oryzae cytochrome bc1 complex containing azoxystrobin as a ligand was developed. Forlani, F.; Catinella, G.; Musso, L.; The model was validated with a set of commercially available strobilurins, and it well explains Cortesi, P.; Eberini, I.; Pinto, A.; Dallavalle, S. Structural Investigation both the resistance mechanism to strobilurins mediated by the mutation G143A and the activity and Molecular Modeling Studies of of metyltetraprole against strobilurin-resistant strains. The obtained results shed light on the key Strobilurin-Based Fungicides Active recognition determinants of strobilurin-like derivatives in the cytochrome bc1 active site and will against the Rice Blast Pathogen guide the further rational design of new fungicides able to overcome resistance caused by G143A Pyricularia oryzae. Int. J. Mol. Sci. 2021, mutation in the rice blast pathogen. 22, 3731. https://doi.org/10.3390/ ijms22073731 Keywords: rice blast; crop protection; cytochrome bc1 complex; antifungal compounds; homology modeling; molecular docking Academic Editor: Isabelle Callebaut

Received: 22 February 2021 Accepted: 1 April 2021 1. Introduction Published: 2 April 2021 The mitochondrial respiratory chain is one of the most important targets for the

Publisher’s Note: MDPI stays neutral development of agricultural fungicides [1]. Different fungicide classes act on enzymes with regard to jurisdictional claims in within the respiratory chain, the most important being succinate dehydrogenase inhibitors published maps and institutional afﬁl- (SDHI, or complex II) and strobilurins (quinone outside inhibitors, QoI, or complex III) [2–6]. iations. Strobilurins interact with the cytochrome bc1 complex (complex III) involved in electron transfer, oxidation of hydroquinone, and reduction of cytochrome c in the mitochondrial respiratory chain. Strobilurin fungicides have been a milestone in the fungicide market, and they are still among the best-selling agrochemicals worldwide. Succinate dehydrogenase (SDH), on the other side, is a particular enzyme on the interface between the tricarboxylic Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. acid cycle (TCA) and the mitochondrial respiratory chain. It couples the oxidation of This article is an open access article succinate to fumarate in TCA with the reduction of ubiquinone to ubiquinol in the electron distributed under the terms and transfer chain. SDHI are the fastest growing class of fungicides in terms of newly discovered conditions of the Creative Commons products [7]. Attribution (CC BY) license (https:// Notwithstanding their enormous potential, both fungicide classes have a single-site creativecommons.org/licenses/by/ mode of action, which makes them prone to resistance development in fungal pathogen 4.0/). populations. The resistance to strobilurins is most often determined by a single amino

Int. J. Mol. Sci. 2021, 22, 3731. https://doi.org/10.3390/ijms22073731 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 18

Int. J. Mol. Sci. 2021, 22, 3731 2 of 16

populations. The resistance to strobilurins is most often determined by a single amino acid acidsubstitution substitution from from glycine glycine to alanine to alanine (G143A) (G143A) and and until until now now has has been been detected detected in in approximatelyapproximately 5050 differentdifferent pathogens, while diverse mutations in the B, C C,, and and D D subunits subunits ofof the the SDH SDH enzyme enzyme are are known known in ca. in 20 ca. fungal 20 fungal species species [8–10]. Strict [8–10]. anti-resistance Strict anti-resistance measures aremeasures being applied are being particularly applied particularly to strobilurin to use strobilurin to ensure use their to long-lasting ensure their activity long-lasting and to delayactivity the and spread to delay of fungicide the spread resistance, of fungicide e.g., resistance, limited number e.g., limited of treatments, number preferablyof treatments, in a mixturepreferably or in rotation a mixture with or fungicides rotation with having fungicides a different having mode a different of action mode [11]. of action [11]. Rice blast, caused by the fungus Pyricularia oryzae ,, is is one one of of the the most most serious serious fungal fungal diseasesdiseases of of cultivated cultivated rice worldwide, rice worldwide, causing causing each year each 10–30% year yield 10 losses–30% corresponding yield losses tocorre ca.sponding 70 billion to USD ca. 70 of economicbillion USD loss of [economic12–14]. It loss is managed [12–14]. It predominantly is managed predominantly by fungicides, especiallyby fungicides, in regions especially where in regions traditional where rice traditional blast-susceptible rice blast varieties-susceptible are varieties grown, andare breedinggrown, and of resistantbreeding varietiesof resistant has varieties been neglected. has been neglected. During the During years, the diverse years fungicide, diverse classesfungicide have classes been have used been against usedP. oryzaeagainst, among P. oryzae which, amongβ-tubulin which inhibitorsβ-tubulin inhibitors (e.g., benomyl), (e.g., sterolbenomyl), biosynthesis sterol biosynthesis inhibitors (e.g., inhibitors propiconazole), (e.g., propiconazole), melanin biosynthesis melanin inhibitors biosynthesis (e.g., tricyclazole),inhibitors (e.g. antibiotics, tricyclazole), (e.g., kasugamycin), antibiotics (e.g. or, QoI/strobilurins kasugamycin), (e.g.,or QoI/strobilurins azoxystrobin) [ 15 (e.g.,16,]. Inazoxystrobin) Europe and particularly [15,16]. In in Europe Italy, only and strobilurins particularly and in demethylation Italy, only strobilurins inhibitors(DMI) and aredemethylation currently approved inhibitors [17 (DMI)]. Therefore, are currently to augment approved the portfolio [17]. Therefore, of fungicides to augment for rice blast the managementportfolio of fungicides and to delay/overcome for rice blast themanagement resistance developmentand to delay/o againstvercome at-risk the fungicides,resistance newdevelopment solutions against are urgently at-risk needed. fungicides, new solutions are urgently needed. InIn the search search for for innovative innovative antifungal antifungal treatments, treatments, we we reported reported in a in recent a recent paper paper the thedevelopment development of new of new hybrid hybrid fungicides fungicides obtained obtained by by combining combining the the pharmacoppharmacophorehore featuresfeatures ofof QoIQoI andand SDHISDHI [[18].18]. The first ﬁrst-generation-generation hybrid hybrid compounds compounds (Figure (Figure 11,, 1a1a–c–c)) featuredfeatured thethe threethree ring-basedring-based structurestructure ofof azoxystrobinazoxystrobin ( 2(2)) and and contained contained the the methyl-( methyl-E(E)-)β-- methoxyacrylateβ-methoxyacrylate strobilurin strobilurin pharmacophore pharmacophore connected connected by a by proper a proper linker linker to the carbox- to the amidecarboxamide pharmacophore pharmacophore of three of commercial three commercial SDHI inhibitors, SDHI inhibitors, e.g., mepronil e.g., (mepronil3), ﬂuopyram (3), (fluopyram4), and boscalid (4), and (5). The boscalid pilot hybrid (5). The compounds pilot hybrid showed compounds good in showed vitro mycelium good in growth vitro inhibitionmycelium ofgrowth the rice inhibition blast pathogen, of the ricePyricularia blast pathogen, oryzae; however,Pyricularia they oryzae almost; however completely, they lostalmost their completely activity on lost strobilurin-resistant their activity on strobilurin strains. -resistant strains.

Figure 1. First generation of dual compounds 1a–c [18], commercial strobilurins (2), and succinate dehydrogenaseFigure 1. First generation inhibitors of (SDHIs) dual compounds (3–6), chosen 1a– asc [18], templates. commercial strobilurins (2), and succinate dehydrogenase inhibitors (SDHIs) (3–6), chosen as templates. The major goal of this work was to modify the structure of the most promising candidate 1a in order to obtain highly active compounds, possibly capable of overcoming strobilurin resistance in P. oryzae.

Int. J. Mol. Sci. 2021, 22, 3731 3 of 16

The strobilurin pharmacophore was maintained unaltered, as it was previously found to interact very strongly with the enzyme. Instead, attention was focused on the following aspects: 1. Spatial orientation of the two pharmacophores 2. Role and nature of the linker 3. Nature and substitution pattern of the SDHI pharmacophore.

2. Results To address the objectives described above, we synthesized compounds reported in Figure2. Initially, we investigated the inﬂuence of the orientation of the two pharmacophore moieties. For this purpose, we prepared derivatives 7 and 8 with SDHI and QoI pharmacophores positioned in ortho and para relative positions on the central ring, Int. J. Mol. Sci. 2021, 22, x FOR PEERrespectively REVIEW (Figure2). Moreover, we varied the position of the substituent (methyl group)4 of 18

on the benzamide ring (compounds 9 and 10).

FigureFigure 2. 2.Design Design strategystrategy toto optimizeoptimize the structure of of compound compound 1a1a toto obtain obtain new new dual dual SDHI SDHI-QoI-QoI (7(–715-15).).

2.1. Successively,Chemistry we investigated the effect of the introduction of diverse substituents on theCompound benzamide 7 ring. was Thus, prepared we prepared by reacting compounds (E)-methyl11a 2–-(2g-bearing(bromomethyl)phenyl) substituents with-3- differentmethoxyacrylate steric and [18] stereoelectronic with tert-butyl properties. 2-hydroxyphenylcarbamate 16, followed by removal of theAfter Boc this,protecting we changed group and the natureacylation of theof the aromatic resulting ring compound in the SDHI 18 with moiety, 2-methylben- replacing thezoic benzamide acid. The withsame a synthetic heterocyclic strategy amide was (compounds used to obta12 inand the13 isomer). Compound 8 starting13 isfrom featured tert- butyl 4-hydroxyphenylcarbamate 19 (Scheme 1).

Int. J. Mol. Sci. 2021, 22, 3731 4 of 16

with the pyrazole carboxamide found in the commercial SDHI inhibitor ﬂuxapyroxad (6, Figure1). Finally, we focused our attention on the linker between the two pharmacophores. In compounds 14a–c, we increased the distance between the strobilurin and SDHI moieties, using glycine, β-alanine, and γ-aminobutyric acid (GABA) as linkers. In addition, a further aromatic ring was placed between the two active moieties in compound 15 (Figure2).

2.1. Chemistry Compound 7 was prepared by reacting (E)-methyl 2-(2-(bromomethyl)phenyl)-3- methoxyacrylate [18] with tert-butyl 2-hydroxyphenylcarbamate 16, followed by removal of the Boc protecting group and acylation of the resulting compound 18 with 2-methylbenzoic Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 18 acid. The same synthetic strategy was used to obtain the isomer 8 starting from tert-butyl 4-hydroxyphenylcarbamate 19 (Scheme1).

SchemeScheme 1. SynthesisSynthesis of of compounds compounds 7–7–88. Reagents. Reagents and and conditions conditions:: (a) K (2aCO)K3,2 (COE)-3methyl, (E)-methyl 2-(2-(bro- 2-(2- (bromomethyl)phenyl)-3-methoxyacrylate,momethyl)phenyl)-3-methoxyacrylate, 18-crown 18-crown-6,-6, acetone, acetone, 3h, reflux, 3 h, reﬂux, 23% for 23% 17 for, and17, 64%and for 64% 20 for; (b) TFA, CH2Cl2, 3h, 0 °C, ◦98% for 18, and 95% for 21; (c) 2-methylbenzoic acid, EDC·HCl, HOBt, 20;(b) TFA, CH2Cl2, 3 h, 0 C, 98% for 18, and 95% for 21;(c) 2-methylbenzoic acid, EDC·HCl, HOBt, DIPEA, CH2Cl2, 24h, rt, 35% for 7, and 64% for 8. DIPEA, CH2Cl2, 24 h, rt, 35% for 7, and 64% for 8.

The synthesis of compounds 99–13–13 startedstarted from from the the common common precursor precursor 2222 [18], [18] which, which waswas coupled with with the the suitable suitable carboxylic carboxylic acid acid using using EDC·HCl EDC·HCl and andHOBt HOBt as coupling as coupling rea- reagentsgents and and DIPEA DIPEA at 0 at °C 0 (Scheme◦C (Scheme 2). 2). Compounds 14a–c were prepared starting from 2-methylbenzoic acid 23, which was O condensed with glycine, β-alanine, and GABA, respectively, to obtain intermediate acids 24a–c. Coupling with theMe amineO 22OinMe the presenceO of EDC and HOBtO provided the desired compounds 14a–c. Compound 15 wasO obtainedN by reactionMeO of 23OwithMe 3-aminobenzoicO R O H 9 acid to give 25, which was then condensed with 22 following the protocolO describedN above MeO OMe O H (Scheme3). a O N b c 11a R = OCH H 3 11b R = NHCOCH Me 3 11c R = I 10 O 11d R = OBn f MeO OMe 11e R = OH 11f R = NHBoc g O NH2 11g R = NH2 O O 22 F MeO OMe O MeO OMe O F S O N O N e d H H N 13 N 12 Me Me Scheme 2. Synthesis of compounds 9–13. Reagents and conditions: (a) 3-methylbenzoic acid, EDC·HCl, HOBt, DIPEA, CH2Cl2, 0 °C to rt 84%; (b) 4-methylbenzoic acid, EDC·HCl, HOBt, DIPEA, CH2Cl2, 0°C to rt 82%; (c) for 11a: 2-methoxybenzoic acid, EDC·HCl, HOBt, DIPEA, CH2Cl2, 0 °C to rt, 64%; for 11b: 2-acetamidobenzoic acid, EDC·HCl, HOBt, DIPEA, CH2Cl2, 0 °C to rt; 59%; for 11c: 2-iodobenzoic acid, EDC·HCl, HOBt, DIPEA, CH2Cl2, 0 °C to rt, 86%, for 11d: 2-benzyloxybenzoic acid, EDC·HCl, HOBt, DIPEA, CH2Cl2, 0 °C to rt, 71%, for 11f: 2-tert-butoxycarbomylaminobenzoic acid, EDC·HCl, HOBt, DIPEA, CH2Cl2, 0 °C to rt, 41%; (d) 3-methylthiophene-2-carboxylic acid, EDC·HCl, HOBt, DIPEA, CH2Cl2, 0 °C to rt, 62%; (e) 3-(difluoromethyl)-1-methyl-1H-pyrazole-4- carboxylic acid, EDC·HCl, HOBt, DIPEA, CH2Cl2, 0 °C to rt, 57%; (f) 10% Pd/C, H2, ethyl acetate, rt, overnight, 91%; (g) TFA, CH2Cl2, 2h, rt, 52%.

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 18

Scheme 1. Synthesis of compounds 7–8. Reagents and conditions: (a) K2CO3, (E)-methyl 2-(2-(bromomethyl)phenyl)-3-methoxyacrylate, 18-crown-6, acetone, 3h, reflux, 23% for 17, and 64% for 20; (b) TFA, CH2Cl2, 3h, 0 °C, 98% for 18, and 95% for 21; (c) 2-methylbenzoic acid, EDC·HCl, HOBt, DIPEA, CH2Cl2, 24h, rt, 35% for 7, and 64% for 8.

Int. J. Mol. Sci. 2021, 22, 3731The synthesis of compounds 9–13 started from the common precursor 22 [18], which 5 of 16 was coupled with the suitable carboxylic acid using EDC·HCl and HOBt as coupling reagents and DIPEA at 0 °C (Scheme 2).

MeO OMe O O

O N MeO OMe O R O H 9 O N MeO OMe O H a O N b c 11a R = OCH H 3 11b R = NHCOCH Me 3 11c R = I 10 O 11d R = OBn f MeO OMe 11e R = OH 11f R = NHBoc g O NH2 11g R = NH2 O O 22 F MeO OMe O MeO OMe O F S O N O N e d H H N 13 N 12 Me Me Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 6 of 18 Scheme 2. SchemeSynthesis 2. of Synthesis compounds of compounds9–13. Reagents 9–13 and. Reagents conditions: and (conditionsa) 3-methylbenzoic: (a) 3-methylbenzoic acid, EDC·HCl, acid, HOBt, DI- EDC·HCl,◦ HOBt, DIPEA, CH2Cl2, 0 °C to rt 84%; (b) 4-methylbenzoic acid, EDC·HCl,◦ HOBt, DIPEA, PEA, CH2Cl2, 0 C to rt 84%; (b) 4-methylbenzoic acid, EDC·HCl, HOBt, DIPEA, CH2Cl2, 0 C to rt 82%; (c) for 11a: CH2Cl2, 0°C to rt 82%; (c) for 11a: 2-methoxybenzoic◦ acid, EDC·HCl, HOBt, DIPEA, CH2Cl2, 0 °C to 2-methoxybenzoic acid, EDC·HCl, HOBt, DIPEA, CH2Cl2, 0 C to rt, 64%; for 11b: 2-acetamidobenzoic acid, EDC·HCl, rt, 64%;Compounds for 11b◦ : 2-acetamidobenzoic 14a–c were prepared acid, EDC·HCl, starting HOBt,from 2 DIPEA,-methylbenzoic CH2Cl2, 0 °Cacid to 23rt; ,59%; which◦ for was11c: HOBt, DIPEA, CH2Cl2, 0 C to rt; 59%; for 11c: 2-iodobenzoic acid, EDC·HCl, HOBt, DIPEA, CH2Cl2, 0 C to rt, 86%, for 11d: 2-iodobenzoic acid, EDC·HCl, HOBt, DIPEA, CH2Cl2, 0 °C to rt, 86%, for 11d: 2-benzyloxybenzoic 2-benzyloxybenzoiccondensed acid, with EDC ·glycine,HCl, HOBt, β-alanine DIPEA,, and CH ClGABA,, 0 ◦C respectively, to rt, 71%, for 11fto obtain: 2-tert-butoxycarbomylaminobenzoic intermediate acids acid, EDC·HCl, HOBt, DIPEA, CH2Cl2, 0 °C2 to rt,2 71%, for 11f: 2-tert-butoxycarbomylaminobenzoic 24a–c. Coupling with the amine◦ 22 in the presence of EDC and HOBt provided the desired acid, EDC·HCl,acid, HOBt, EDC·HCl, DIPEA, HOBt, CH2 Cl DIPEA,2, 0 C CH to rt,2Cl 41%;2, 0 °C (d ) to 3-methylthiophene-2-carboxylic rt, 41%; (d) 3-methylthiophene acid,-2-carboxylic EDC·HCl, acid, HOBt, DIPEA, ◦ compounds 14a–c. Compound 15 was obtained by reaction of 23 with 3-aminobenzoic CH2Cl2, 0 CEDC·HCl, to rt, 62%; HOBt, (e) 3-(diﬂuoromethyl)-1-methyl-1H-pyrazole-4-carboxylic DIPEA, CH2Cl2, 0 °C to rt, 62%; (e) 3-(difluoromethyl) acid,-1- EDCmethyl·HCl,-1H HOBt,-pyrazole DIPEA,-4- CH2Cl2, ◦ acid to give 25, which was then condensed with 22 following the protocol described above 0 C to rt, 57%;carboxylic (f) 10% acid, Pd/C, EDC·HCl, H2, ethyl HOBt, acetate, DIPEA, rt, overnight, CH2Cl2, 91%; 0 °C (tog) rt, TFA, 57%; CH (f2)Cl 10%2, 2 Pd/C, h, rt, 52%.H2, ethyl acetate, rt, overnight,(Scheme 3). 91%; (g) TFA, CH2Cl2, 2h, rt, 52%.

◦ Scheme 3. SynthesisScheme 3. of Synthesis compounds of compounds14–15. Reagents 14–15 and. Reagents conditions: and conditions (a) (COCl):2 (,a CH) (COCl)2Cl2,2 DMF,, CH2Cl 0 2,C, DMF, rt, 63%; 0 °C (,b ) for 24a: glycine, 2Mrt, NaOH, 63%; ( 8b h,) for rt, 24a 19%;: glycine, for 24b :2Mβ-alanine, NaOH, 8h, 2M rt, NaOH, 19%; for overnight, 24b: β-alanine, rt, 24%; 2M for NaOH,24c: GABA, overnight, 2M NaOH, rt, 24%; 2 h at 0 ◦C for 24c: GABA, 2M NaOH, 2h at 0 °C then overnight0 ◦ at rt, 41%; (c) i. EDC·HCl, HOBt,◦ CH2Cl2, 30′ at then overnight at rt, 41%; (c) i. EDC·HCl, HOBt, CH2Cl2, 30 at 0 C; ii. 22, DIPEA, CH2Cl2, at 0 C then overnight at rt, for 0 °C; ii. 22, DIPEA, CH2Cl2, at 0 °C then overnight at rt, for 14a: 18%, for 14b: 49%, for 14c: 33%; (d) 0 ◦ 14a: 18%, for 14b: 49%, for 14c: 33%; (d) 3-aminobenzoic acid, TEA, CHCl3, 51%; (e) i. EDC·HCl, HOBt, CH2Cl2, 30 at 0 C; 3-aminobenzoic◦ acid, TEA, CHCl3, 51%; (e) i. EDC·HCl, HOBt, CH2Cl2, 30′ at 0 °C; ii. 22, DIPEA, ii. 22, DIPEA, CH2Cl2, at 0 C then overnight at rt, 33%. CH2Cl2, at 0 °C then overnight at rt, 33%. 2.2. Biological Activity 2.2. Biological Activity The inhibitory activity of the novel compounds 7–15 on the mycelium growth of P. The inhibitoryoryzae activitywas evaluated of the novel and compared compounds to the 7–15 activity on the of mycelium the reference growth compound of P. 1a (Figure3, oryzae was evaluatedTable1). and Acetone compared (ACT), to used the asactivity a solvent, of the did reference not show compound any inhibitory 1a (Figure activity on P. oryzae 3, Table 1). Acetonemycelium (ACT), growth. used Theas a commercial solvent, did fungicides not show azoxystrobinany inhibitory (AZX; activity QoI) on and P. ﬂuxapyroxad oryzae mycelium(FXP, growth. SDHI) The and commercial the newly synthesizedfungicides azoxystrobin compounds were(AZX; tested QoI) and at the fluxap- ﬁnal concentration yroxad (FXP, SDHI)of 25 mg/L. and the AZX newly inhibited synthesized > 90% compounds of the growth were of tested wild-type at the (WT)final con- strains, while the centration of 25strobilurin-resistant mg/L. AZX inhibited (RES) > 90% strains of the were growth inhibited of wild to-type < 50%. (WT) On str theains, contrary, while FXP (SDHI) the strobilurinshowed-resistant low (RES) activity strains on WT were strains inhibited (65% inhibition) to < 50%. but On > the 90% contrary, inhibition FXP of RES strains. (SDHI) showed low activity on WT strains (65% inhibition) but > 90% inhibition of RES strains.

5 WT RES 4

2 Growth (cm)

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 6 of 18

Compounds 14a–c were prepared starting from 2-methylbenzoic acid 23, which was condensed with glycine, β-alanine, and GABA, respectively, to obtain intermediate acids 24a–c. Coupling with the amine 22 in the presence of EDC and HOBt provided the desired compounds 14a–c. Compound 15 was obtained by reaction of 23 with 3-aminobenzoic acid to give 25, which was then condensed with 22 following the protocol described above (Scheme 3).

Scheme 3. Synthesis of compounds 14–15. Reagents and conditions: (a) (COCl)2, CH2Cl2, DMF, 0 °C, rt, 63%; (b) for 24a: glycine, 2M NaOH, 8h, rt, 19%; for 24b: β-alanine, 2M NaOH, overnight, rt, 24%; for 24c: GABA, 2M NaOH, 2h at 0 °C then overnight at rt, 41%; (c) i. EDC·HCl, HOBt, CH2Cl2, 30′ at 0 °C; ii. 22, DIPEA, CH2Cl2, at 0 °C then overnight at rt, for 14a: 18%, for 14b: 49%, for 14c: 33%; (d) 3-aminobenzoic acid, TEA, CHCl3, 51%; (e) i. EDC·HCl, HOBt, CH2Cl2, 30′ at 0 °C; ii. 22, DIPEA, CH2Cl2, at 0 °C then overnight at rt, 33%.

2.2. Biological Activity The inhibitory activity of the novel compounds 7–15 on the mycelium growth of P. oryzae was evaluated and compared to the activity of the reference compound 1a (Figure 3, Table 1). Acetone (ACT), used as a solvent, did not show any inhibitory activity on P. oryzae mycelium growth. The commercial fungicides azoxystrobin (AZX; QoI) and fluxapyroxad (FXP, SDHI) and the newly synthesized compounds were tested at the final concentration of 25 mg/L. AZX inhibited > 90% of the growth of wild-type (WT) strains, while Int. J. Mol. Sci. 2021the, 22 strobilurin, 3731 -resistant (RES) strains were inhibited to < 50%. On the contrary, FXP 6 of 16 (SDHI) showed low activity on WT strains (65% inhibition) but > 90% inhibition of RES strains.

5 WT RES 4

2 Growth (cm)

0 C FXP AZX ACT Cpd 7 Cpd 8 Cpd 9 Cpd 1a Cpd 10 Cpd 12 Cpd 13 Cpd 15 Cpd 11f Cpd 11c Cpd 14c Cpd 11g Cpd 11a Cpd 14a Cpd 11e Cpd 11b Cpd 11d Cpd 14b Figure 3. Mycelium growth of wild-type (WT) and QoI-resistant (RES) Pyricularia oryzae strains on Figure 3. Mycelium growth of wild-type (WT) and QoI-resistant (RES) Pyricularia oryzae strains on control malt-extract agar control malt-extract agar media (C = control, ACT = 1% acetone) and media supplemented with media (C = control, ACT = 1% acetone) and media supplemented with azoxystrobin (AZX), ﬂuxapyroxad (FXP), or dual compounds (Cpds 1a, 7–15) at a concentration of 25 mg/L. Error bars represent the standard deviation of the mean.

Table 1. Inhibition of mycelium growth of wild-type (WT) and QoI-resistant (RES) Pyricularia oryzae (Scheme1), acetone (ACT), and media supplemented with azoxystrobin (AZX), ﬂuxapyroxad (FXP), or dual compounds (Cpds 1a, 7–15) at a concentration of 25 mg/L.

WT RES Compound % Inhibition a Tukey HSD b % Inhibition Tukey HSD Control 0 a 0 a ACT 0 a −4 a AZX 92 m 47 c FXP 67 f h i 94 d 1a 72 h i j 20 b 7 25 b c 26 b 8 67 f g h i 26 b 9 63 e f g h 28 b 10 78 j k l 52 c 11a 79 i j k l 30 b 11b 83 k l 84 d 11c 71 h i j 35 b 11d 58 e f g 23 b 11e 84 k l m 50 c 11f 58 e f 23 b 11g 55 e 27 b 12 73 h i j k 22 b 13 87 l m 57 c 14a 42 d 15 b 14b 38 d 20 b 14c 18 b 15 b 15 31 c d 21 b a Inhibition (%) was calculated as I% = (C − T)/C*100, where C = is growth on control medium, and T = is growth on treated medium. b Tukey post hoc test of mycelium growth data. The different letters indicate statistically signiﬁcant differences between mean growth (p > 0.05).

2.3. SDH and Qo Inhibitory Activity of Compound 11b As compound 11b showed very promising biological activity on both WT and RES strains of P. oryzae (>80% inhibition), we hypothesized that it maintained good Qo and SDH inhibitory activity. The measurement of decylubiquinol:Cyt c reductase activity (i.e., the Cyt bc1 complex activity) was performed to evaluate the QoI action of the selected Int. J. Mol. Sci. 2021, 22, 3731 7 of 16

compound. To this purpose, the reduction rate of Cyt c mediated by the mitochondrial fraction of P. oryzae was measured using decylubiquinol (DBH2) as the electron-donor substrate. The DBH2: Cyt c reductase activity was inhibited by 58 ± 18% by the compound 11b (50 µM). A similar inhibition value (61 ± 10%) was observed for azoxystrobin (50 µM), thus conﬁrming that 11b retained the QoI action. SDH enzymatic assay was performed measuring the electron transfer rate mediated by the mitochondrial fraction of P. oryzae in the presence of 2,3-dimethoxy-5-methyl-p- Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 8 of 18 benzoquinone and succinate by a colorimetric method, using the redox dye 2,6-dichlorophen olindophenol (DCPIP, Figure4).

Figure 4. EffectEffect of of compound compound 11b11b onon the the P. oryzaeP. oryzae mitochondrialmitochondrial activity. SDH activity. To evaluate To evaluate the percent the percentof inhibition of inhibition due to the due SDHI to the action, SDHI the action, rates of the the rates succinate:2,3 of the succinate:2,3-dimethoxy-5-methyl--dimethoxy-5-methyl-p-benzoqui-p- benzoquinonenone dehydrogenase dehydrogenase activity activityof the mitochondrial of the mitochondrial fraction fraction were measured were measured at 25 at°C 25(λ◦ =C( 595λ = nm) 595 nm)using using DCPIP DCPIP in the in presence the presence of 11b of and11b theand reference the reference compound, compound, fluxapyroxad ﬂuxapyroxad (FXP) (FXP),, at the at indi- the indicatedcated concentrations. concentrations. Dat Dataa represent represent the the mean mean ± ±standardstandard deviation deviation of of at at least least three three independent trials. Different superscript lettersletters indicateindicate statisticallystatistically signiﬁcantsignificant differencesdifferences (Tukey(Tukey HSD, HSD,p P≤ ≤ 0.01).0.01).

Fluxapyroxad showedshowed >> 90% inhibition of SDH activity at the concentration concentration of 40 µM.µM. Surprisingly, atat thethe samesame concentration,concentration, nono SDHSDH inhibitioninhibition waswas observedobserved forfor compoundcompound 11b.. By increasing the concentration toto 400 µµM,M, approximately 23%23% inhibitioninhibition ofof thethe SDHSDH enzyme activity was observed.

2.4. In Silico Modeling and Docking Since no no experimental experimental structures structures for forP. oryzae P. oryzaecytochrome cytochromebc1 in bc1 complex in complex with azoxys- with trobinazoxystrobin are available, are available, we produced we produced a 3D modela 3D model by homology. by homology. The RamachandranThe Ramachandran plot (Figureplot (Figure5), the 5), side-chain the side packing,-chain packing and the, stereochemicaland the stereochemical quality were quality carefully were checked carefully to verifychecked that to allverify these that parameters all these parameters were suitable were and suitable consistent and with consistent typical with values typical found val- in templateues found crystallographic in template crystallographic structures. structures. The ﬁnal model contained azoxystrobin that was transferred by the selected template

(PDB ID: 1SQB). The selected docking procedure, thoroughly described in the Materials and Methods section, was validated testing well-known strobilurin QoI: azoxystrobin, triﬂoxystrobin, kresoxim-methyl, and metominostrobin, characterized by the presence of a β-methoxyacrylate, α-methoxyimino acetate, or α-methoxyimino-N-methylacetamide pharmacophore, respectively (Figure6). Metyltetraprole, a newly discovered strobilurin derivative insensitive to G143A mutation and thus active also against strobilurin-resistant pathogens [19,20], was added to validate the QoI binding site of the model.

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 9 of 18

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 9 of 18 Int. J. Mol. Sci. 2021, 22, 3731 8 of 16

Figure 5. Ramachandran plot of the three-dimensional model for Pyricularia oryzae cytochrome bc1 complex. Red and yellow regions are respectively favored and allowed regions. Triangles refer to G, squares to P, and dots to the remaining amino acids.

The final model contained azoxystrobin that was transferred by the selected template (PDB ID: 1SQB). The selected docking procedure, thoroughly described in the Materials and Methods section, was validated testing well-known strobilurin QoI: azoxystrobin, trifloxystrobin, kresoxim-methyl, and metominostrobin, characterized by the presence of a β-methoxyacrylate, α-methoxyimino acetate, or α-methoxyimino -N-methylacetamide Figure 5. Ramachandran plot of the three-dimensional model for Pyricularia oryzae cytochrome bc1 Figurepharmaco 5. Ramachandranphore, respectively plot of the(Figure three -6).dimensional Metyltetraprole, model for a Pyricularianewly discovered oryzae cytochrome strobilurin bc1 complex. Red and yellow regions are respectively favored and allowed regions. Triangles refer to G, complex.derivative Red insensitive and yellow to regions G143A are mutation respectively and thusfavored active and also allowed against regions. strobilurin Triangles-resistant refer to squaresG,pathogens squares to P,to [19,20], and P, and dots dotswas to the toadded the remaining remaining to validate amino amino acids.the acids.QoI binding site of the model.

The finalO model contained azoxystrobinO that was transferredMe by the selected template N N N (PDBMeO ID: 1SQB).OMe The selectedCN dockingMeO procedure,OMe thoroughly describedN in the Materials O N and MethodsO section,O was validated testing well-known strobilurin QoI: azoxystrobin,N tri- O Cl floxystrobin, kresoxim-methyl, and metominostrobin, characterized byO theN presence of a N N Me β-methoxyacrylate, α-methoxyimino acetate, or α-methoxyimino-N-methylacetamide Me pharmacophore, respectively (Figure 6). Metyltetraprole, a newly discovered strobilurin derivativeaz oinsensitivexystrobin to G143A mutationkreso xandim-m thusethy lactive also againstme tstrobilurinyltetraprole -resistant pathogens [19,20],M ewas added to validate the QoI binding site of the model.O HN O N N MeMeO OMe MeO O O MeO O N OMe N N N Me O N N O MeO OMe CN MeO OMe Cl N O N O N O O N O Cl O N N N Me CF3 metominostrobin pyraclostrobin tMriefloxystrobin FigureFigure 6.6. ValidationValidationazoxystrob setisetn ofof marketedmarketed strobilurinsstrobilurinskresoxim andand-me metyltetraprole.tmetyltetraprolehyl . metyltetraprole

Me O The top-scoringHN docking solution for all the validation-set compounds showed a The top-scoring docking solutionO for all the validation-setN compounds showed a binding modeN highly similar to the azoxystrobin placement inMe theO bovineO cytochromeMe bc1 bindingMeO mode highlyO similar Mtoe Othe azoxystrobin placement in the bovine cytochrome bc1 N OMe N Me (PDB(PDB ID: 1SQB) 1SQB) [21],O [21], in in which which the the pharmacophore pharmacophoreN was was very very close close to the to theG143O G143 protein protein res- Cl residueidue (Figure (Figure 7A7A–G,–G, Table Table 2).2). The only validationO N compoundcompound withwith a a top-scoring top-scoring solution solution having a completely different orientation was pyraclostrobin; however, the second solution

was similar to the co-crystallized azoxystrobin. The orientation of the methylester andCF the3 bioisostericmetom groupsinostro showedbin an interestingpyrac variability,lostrobin with two differenttriflox solutionsystrobin (ﬁle S1). TheFigure superposition 6. Validation ofset the of marketed top-scoring strobilurins poses for and the metyltetraprole entire validation. set conﬁrms a key role for Glu273 (Figure7G) as observed in other organisms [22]. The top-scoring docking solution for all the validation-set compounds showed a binding mode highly similar to the azoxystrobin placement in the bovine cytochrome bc1 (PDB ID: 1SQB) [21], in which the pharmacophore was very close to the G143 protein residue (Figure 7A–G, Table 2). The only validation compound with a top-scoring solution

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 10 of 18

having a completely different orientation was pyraclostrobin; however, the second solution was similar to the co-crystallized azoxystrobin. The orientation of the methylester and the bioisosteric groups showed an interesting variability, with two different solutions (file S1). The superposition of the top-scoring poses for the entire validation set confirms a key role for Glu273 (Figure 7G) as observed in other organisms [22].

Table 2. Top-scoring docking solutions for all chemicals in the validation set. a Glide XP Score indicates the approx. binding affinity (kcal/mol) of the top-scoring binding pose. b MM-GBSA indicates a more accurate binding affinity (kcal/mol) of the top-scoring binding pose. c Number of poses for each compound obtained by the molecular docking procedure. d Range of the binding affinities (kcal/mol) for each tested chemical of the validation set. e Number of generated poses with an orientation similar to azoxystrobin (as transferred from the template) for each tested compound of the validation set.

Glide XP a MM-GBSA b Number Range d Orientation of the Ligand [kcal/mol] [kcal/mol] of posesc [kcal/mol] Generated Poses e azoxystrobin −10.6 −76.4 5 (−10.6; −9.4) 5/5 trifloxystrobin −9.8 −65.6 2 (−9.8; −9.7) 2/2 kresoxym-methyl −10.4 −68.9 1 (−10.4) 1/1 metominostrobin −9.9 −79.3 1 (−9.9) 1/1 Int. J. Mol. Sci. 2021, 22, 3731 9 of 16 pyraclostrobin −8.4 −61.7 5 (−10.0; −5.7) 3/5 metyltetraprole −10.2 −62.9 2 (−10.2; −9.8) 2/2

Figure 7. Top-scoringFigure 7. Top docking-scoring poses docking within poses the cytochromewithin the cytochromeb binding pocket b binding of the pocket commercial of the commercial strobilurin fungicides included instrobilurin the validation fungicides set: (A included) azoxystrobin, in the validation (B) kresoxim-methyl, set: (A) azoxystrobin, (C) metyltetraprole, (B) kresoxim (D)-methyl, metominostrobin, (C) (E) pyraclostrobinmetyltetraprole, (the second top-scoring (D) metominostrobin, pose), and (F ()E trifloxystrobin.) pyraclostrobin In (the (G), second all validation top-scoring set poses pose), are and reported (F) tri- to highlight their bindingfloxystrobin. mode and theIn ( interactionG), all validation with GLU273. set poses are reported to highlight their binding mode and the interaction with GLU273. Table 2. Top-scoring docking solutions for all chemicals in the validation set. a Glide XP Score indicates the approx. binding After validating the docking procedure, the five most active new compounds (10, affinity (kcal/mol) of the top-scoring binding pose. b MM-GBSA indicates a more accurate binding affinity (kcal/mol) of the 11a, 11b, 11e, and 13) were docked to gain more insights into their binding interactions top-scoring binding pose. c Number of poses for each compound obtained by the molecular docking procedure. d Range (Table 3). All the five selected compounds showed a top-scoring pose with an orientation of the binding affinities (kcal/mol) for each tested chemical of the validation set. e Number of generated poses with an similar to the co-crystallized azoxystrobin in 1SQB and to the validation set, i.e., with the orientation similar to azoxystrobin (as transferred from the template) for each tested compound of the validation set. methoxyacrylate moiety close to G143; some other poses with higher energy values a b d showed anGlide orientation XP not MM-GBSAcompatible with the azoxystrobin/strobilurinc Range binding mode.Orientation of the Ligand Number of Poses e [kcal/mol] [kcal/mol] [kcal/mol] Generated Poses azoxystrobin −10.6 −76.4 5 (−10.6; −9.4) 5/5 trifloxystrobin −9.8 −65.6 2 (−9.8; −9.7) 2/2 kresoxym-methyl −10.4 −68.9 1 (−10.4) 1/1 metominostrobin −9.9 −79.3 1 (−9.9) 1/1 pyraclostrobin −8.4 −61.7 5 (−10.0; −5.7) 3/5 metyltetraprole −10.2 −62.9 2 (−10.2; −9.8) 2/2

After validating the docking procedure, the five most active new compounds (10, 11a, 11b, 11e, and 13) were docked to gain more insights into their binding interactions (Table3). All the five selected compounds showed a top-scoring pose with an orientation similar to the co-crystallized azoxystrobin in 1SQB and to the validation set, i.e., with the methoxyacrylate moiety close to G143; some other poses with higher energy values showed an orientation not compatible with the azoxystrobin/strobilurin binding mode. The top-scoring poses of all five compounds showed a very similar binding mode, characterized by a strong overlap of the common rings and functional groups, as reported in Figure8. Figure9 describes the ligand interaction diagram for azoxystrobin, metyltetraprole, and compound 11b, showing the role of Glu273 in the formation of a stable H-bond between the carbonyl function of the ester group and the amide function of the protein backbone. Moreover, in both azoxystrobin and metyltetraprole, Phe129 plays a stabilizing role through the formation of a π-π stacking interaction with the aromatic ring distal with respect to the methoxyacrylic function. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 11 of 18

Int. J. Mol. Sci. 2021,Table22, 3731 3. Top-scoring docking solutions for the selected five novel compounds showing the highest 10 of 16 biological activity against Pyricularia oryzae. a Glide XP Score indicates the approx. binding affinity (kcal/mol) of the top-scoring binding poses. b MM-GBSA indicates a more accurate binding affinity (kcal/mol) of the top-scoring binding pose. c Number of poses for each compound obtained by the Table 3. Top-scoringmolecular dockingdocking solutionsprocedure. for d theRange selected of the five binding novel affinities compounds (kcal/mol) showing of individual the highest poses biological for activity against Pyriculariaeach tested oryzae . novela Glide compound. XP Score indicatese Number the of approx. generated binding poses affinity with (kcal/mol) an orientation of the similartop-scoring to binding poses. b MM-GBSAazoxystrobin indicates (as tran a moresferred accurate from the binding template) affinity for (kcal/mol)each tested ofnovel the compound. top-scoring binding pose. c Number of poses for each compound obtained by the molecular docking procedure. d Range of the binding affinities (kcal/mol) of Glide XP a MM-GBSA b Number of Range d Orientation of the individual posesLigand for each tested novel compound. e Number of generated poses with an orientation similar to azoxystrobin [kcal/mol] [kcal/mol] Poses c [kcal/mol] Generated Poses e (as transferred from the template) for each tested novel compound. 10 −11.7 −83.9 3 (−11.7; −9.1) 3/3 11aGlide XP a −9.1 MM-GBSA−60.3 b 5 (−9.1; −7.4) Range d 1/5 Orientation of the Ligand Number of Poses c 11b[kcal/mol] −9.4 [kcal/mol]−59.6 5 (−9.4; −7.4) [kcal/mol] 3/5 Generated Poses e 10 11e −11.7 −12.6 −83.9−83.3 5 3(−12.6; −11.6) (− 11.7; −9.1)5/5 3/3 11a 13 −9.1 −12.2 −60.3−78.7 2 5(−12.2; −10.8) (− 9.1; −7.4)2/2 1/5 11b −9.4 −59.6 5 (−9.4; −7.4) 3/5 11e The top−12.6-scoring poses of− all83.3 five compounds showed 5 a very similar (−12.6; −binding11.6) mode, 5/5 13 characterized−12.2 by a strong overlap−78.7 of the common rings 2 and functional (− 12.2;groups,−10.8) as reported 2/2 in Figure 8.

Figure 8. Top-scoringFigure 8. Top docking-scoring poses docking for compounds poses for compounds (A) 10,(B) (11aA) ,(10C, )(B11b) 11a,(D, ()C11e) 11b, and, (D) ( E11e) 13, and. In (FE),) 13 the. In top-scoring docking poses(F) for, the all top the-scoring tested compoundsdocking poses are for reported. all the tested compounds are reported.

Figure 9 describes the ligand interaction diagram for azoxystrobin, metyltetraprole, and compound 11b, showing the role of Glu273 in the formation of a stable H-bond between the carbonyl function of the ester group and the amide function of the protein backbone. Moreover, in both azoxystrobin and metyltetraprole, Phe129 plays a stabilizing role through the formation of a π-π stacking interaction with the aromatic ring distal with respect to the methoxyacrylic function.

Figure 9. Ligand interaction diagram for (A) azoxystrobin, (B) metyltetraprole, and (C) compound 11b. H-bond between the carbonyl function of the ester group and Glu273 is pointed out by the purple arrow, and π-π stacking between the distal

aromatic ring and Phe129 is pointed out by green line. Int. J. Mol. Sci. 2021, 22, 3731 11 of 16

Since compound 11b maintained its biological activity also against strobilurin-resistant strains containing the G143A mutation (Figure3), we tested the impact of this mutation on the cytochrome bc1 complex using a specific residue scan tool. The mutant protein showed a positive unfavorable ∆Affinity (approx. 5.7 kcal/mol), similar to the ∆Affinity value for azoxystrobin (approx. 4.7 kcal/mol) on the same mutant. On the contrary, the impact of the same mutation on the molecular recognition of metyltetraprole was negligible (approx. −0.7 kcal/mol), suggesting that the correctly oriented and less bulky methyltetrazolone ring did not negatively interact with the methyl group of A143.

3. Discussion Rice blast is the main fungal disease of cultivated rice worldwide, causing 10–30% yield losses each year. Its management still relies on the use of fungicides, but the development of resistance augments food insecurity and requires urgent and innovative solutions. In this paper, we report a structural investigation on new fungicides obtained by combining the pharmacophores of two classes of marketed antifungal compounds: strobilurins and SDH inhibitors. The study focused on the structural variations/modifications of a previously identified hit candidate, with the aim to increase the antifungal activity both on wild-type and, more importantly, on strobilurin-resistant strains of P. oryzae. The spatial orientation of the two pharmacophores, the role and nature of the linker, and the substitution patterns of the SDHI pharmacophore were studied to shed light on the key structural determinants of their activity. The obtained results clearly show that the relative orientation of the QoI and SDHI pharmacophores and the entire shape of the fungicide strongly impact the biological activity of the hybrid molecule. In particular, arranging the SDHI and QoI pharmacophores in ortho position on the central ring (Figure2, compound 7) resulted in a significant drop in activity against the wild-type strains. Compound 8, with the two pharmacophores in para position, maintained activity comparable to 1a (meta position), remaining, however, almost inactive on resistant strains. This indicates that the introduction of the SDHI pharmacophore in ortho position probably creates a steric obstacle to the binding of the QoI-pharmacophore in the cytochrome b binding pocket, while meta and para orientations do not interfere with the binding. The position of the substituent (methyl group) on the benzamide ring of the SDHI pharmacophore also affects the biological activity of the compounds. In particular, compound 10 with the methyl group in para on the carboxamide ring had higher activity on the resistant strains (52% inhibition) than compounds 9 (28%) and 1a (20%). Increasing the length of the spacer between the two pharmacophores was deleterious. In fact, compounds 14a–c, with glycine, β-alanine, and γ-aminobutyric acid as linkers, and 15, with an additional aromatic ring between the two active moieties, showed low activity. Interestingly, the activity decreased with the increase of the chain length (14a versus 14b versus 14c). Changing the nature of the aromatic ring in the SDHI moiety by replacing the benzamide with a heterocyclic amide (compounds 12 and 13) maintained a good activity of dual compounds on WT strains (73% and 87%, respectively). Interestingly, compound 13, which was featured with the pyrazole carboxamide found in the commercial SDHI inhibitor fluxapyroxad, maintained some activity also on RES strains (57% inhibition). The introduction of substituents with different steric and stereoelectronic properties on the SDHI benzamide ring had a major effect on the activity of dual compounds. Com- pounds 11a and 11c showed activity comparable to the reference scaffold 1a, while 11d, 11g, and 11f were endowed with a lower efficacy. Compounds 11b and 11e showed excellent inhibition of wild-type strains (>80% inhibition), with compound 11b maintaining a strong activity also on resistant strains. Unexpectedly, 11b showed low activity against SDH enzyme in the succinate:quinone oxidoreductase (SQR) assay. This prompted us to further investigate the binding mode of the compound with cytochrome bc1 by developing a three-dimensional model of P. oryzae Int. J. Mol. Sci. 2021, 22, 3731 12 of 16

cytochrome bc1 supramolecular assembly in complex with azoxystrobin. We simulated the putative binding site of our novel compounds, evidencing that they share the same binding mode of a set of selected commercially available strobilurins. Our in silico analysis suggests that the new compounds have the same molecular recognition mechanism of azoxystrobin for the P. oryzae cytochrome bc1 Qo binding site, as pointed out by their very good approx. binding free energy values computed through Glide XP Score, spanning from −12.6 to −9.1 kcal/mol, and from binding free energy values computed through MM-GBSA, spanning from −83.9 to −59.6 kcal/mol. Interestingly, our ﬁnding suggests that the unique activity of the compound 11b against resistant strains of P. oryzae cannot be easily explained by our simulations, as the G143A mutation negatively impacts the binding of the compound differently from metyltetraprole. We speculate that additional targets other than cytochrome bc1 and SDH could be involved in the observed activity. Another intriguing hypothesis is that 11b could undergo biotransformation in P. oryzae, resulting in a compound able to efﬁciently bypass the resistance. Future efforts will be devoted to elucidating the peculiar activity of this promising compound. Nevertheless, the availability of a new model for P. oryzae cytochrome bc1 in complex with azoxystrobin paves the way for a further rational design of new highly active compounds, possibly able to overcome the well-known strobilurin resistance in P. oryzae.

4. Materials and Methods 4.1. Chemistry All reagents and solvents were reagent grade or were purified by standard methods before use. Melting points were determined in open capillaries on an SMP3 apparatus and are uncorrected. 1H NMR spectra were recorded on 300 and 600 MHz spectrometers; 13C NMR spectra were recorded on 300 and 600 MHz spectrometers. Solvents were routinely distilled prior to use; anhydrous THF and Et2O were obtained by distillation from sodium benzophenone ketyl; anhydrous CH2Cl2 was obtained by distillation from phosphorus pentoxide. All reactions requiring anhydrous conditions were performed under a positive nitro- gen flow, and all glassware was oven-dried. Isolation and purification of the compounds were performed by flash column chromatography on silica gel 60 (230–400 mesh). Analyti- cal TLC was conducted on TLC plates (silica gel 60 F254, aluminum foil). Compounds on TLC plates were detected under UV light at 254 and 365 nm or were revealed by spraying with 10% phosphomolybdic acid (PMA) in EtOH. Compounds 16 [23], 19 [24], (E)-methyl 2-(2-(bromomethyl)phenyl)-3-methoxyacrylate [18], 22 [18], 24a [25], 25 [26], 2-benzyloxybenzoic acid [27], and 2-tert-butoxycarbonylaminobenzoic acid [28] were prepared as reported in literature. The synthetic procedures for the obtainment of all the new compounds are reported in the Supplementary Material.

4.2. Fungal Strains In this study, four strains of Pyricularia oryzae were used; two strains belonging to the Italian population and sensitive to quinone outside inhibitor (QoI) fungicides (WT): A2.5.2 and TA102; and two strains belonging to the Japanese population and resistant to QoI (RES): PO1312 and PO1336. The strains belong to a vast collection of monoconidial isolates maintained at the laboratory of plant pathology, University of Milan [29,30]. The strains were maintained as single-spore isolates on malt-agar medium (MA: 20 g/L malt extract, Oxoid, U.K.; 15 g/L agar, Oxoid, U.K.) at 4 ◦C.

4.3. Fungicides The commercial fungicides azoxystrobin (AZX, Amistar SC—suspension concentrate, 22.9% ai., Syngenta Crop Protection) and ﬂuxapyroxad (FXP, Sercadis EC—emulsiﬁable Int. J. Mol. Sci. 2021, 22, 3731 13 of 16

concentrate, 30% ai., BASF Italia, S.p.A.) were used as standards to evaluate the activity of QoI (azoxystrobin) and SDHI (ﬂuxapyroxad) fungicides.

4.4. Inhibition of Mycelium Growth of Pyricularia oryzae by Novel Dual Compounds The inhibitory activity of the novel dual compounds and commercial fungicides on the mycelium growth of P. oryzae was evaluated as described previously [18]. In short: a mycelium plug (0.5 cm in diameter) obtained from actively growing fungal colonies of P. oryzae A2.5.2, TA102, PO1312, and PO1336 was transferred to MA medium plates supplemented or not with commercial fungicides (AZX, FXP) and tested compounds at the concentration of 25 mg/L in three biological replicates. Due to the low solubility of the tested dual molecules in water, they were dissolved in acetone. Therefore, two controls were included: MA medium (C, control) and MA medium supplemented with acetone at the ﬁnal concentration of 1% v/v (ACT). The plates were incubated at 24 ◦C in the dark. The mycelium growth was measured at 7 days after inoculation (DAI), and the inhibition of mycelium growth (%) was calculated by comparing the mycelium growth on control and fungicide-supplemented plates. The inhibition percentage was calculated as I% = (C−T)/C*100, where C = mycelium growth in the control medium and T = mycelium growth in the medium added with the tested compound. For AZX and FXP, the control was MA medium. For tested compounds, the control was considered ACT.

4.5. Enzyme Inhibition Assay for the Measurement of SDHI and QoI Action P. oryzae mitochondrial fraction from the A2.5.2 strain was prepared as described previously [18]. The SDHI action was evaluated measuring the succinate: quinone oxidoreductase (SQR) activity (i.e., the succinate dehydrogenase activity; EC 1.3.5.1) of the mitochondrial fraction in the presence of 11b compound using a method based on the use of the redox dye, 2,6-dichlorophenolindophenol (DCPIP) [31], with some modifications as described previously [18]. The enzyme reaction was monitored at 595 nm at fixed times using a fixed wavelength microplate reader (iMark™; Bio-Rad Laboratories, Inc., Hercules, CA, USA). To calculate the SQR reaction rate, the absorbance decrease due to the DCPIP reduction during the 2–60 min time interval was considered. Fluxapyroxad (37047, Sigma Aldrich, Milan, Italy) was used as the reference SDHI. The strobilurin-like (QoI) action was evaluated measuring the decylubiquinol: Cyt c reductase activity (i.e., the Cyt bc1 complex activity; EC 1.10.2.2) of the mitochondrial fraction in the presence of the 11b compound, using the method described by Zhu et al. [32] with some modifications [18]. Azoxystrobin (31697, Sigma Aldrich, Milan, Italy) was used as the reference QoI. In both enzyme assays, the enzyme rate in the presence of the tested molecule (ratemolecule) was compared to that achieved in the presence of the compound diluent DMSO (rateDMSO), in order to calculate the percent of inhibition (I %) as follows: rate − rate I% = molecule DMSO × 100 (1) rateDMSO

4.6. Statistical Analysis The mycelium growth data for each treatment were grouped based on the QoI resistance of P. oryzae strains (WT or RES) and were submitted to ANOVA followed by a Tukey’s HSD post hoc test for multiple comparisons (p < 0.05) using the TukeyC package and R software, version R4.0.0 [33,34]. Similarly, ANOVA followed by posthoc Tukey’s HSD test for multiple comparisons was used for enzymatic activity data analysis. Int. J. Mol. Sci. 2021, 22, 3731 14 of 16

4.7. In Silico Modeling 4.7.1. Homology Modeling of Cytochrome bc1 Complex The Pyricularia oryzae cytochrome bc1 complex (complex III) primary structures were downloaded from the UniProt Protein Knowledgebase database (entry: Q85KP9 form P. grisea and G4N4E1 from P. oryzae, respectively [18]). After a protein BLAST search of the Protein Data Bank (RCSB PDB) database for homolog templates, the crystallographic structure of bovine cytochrome bc1 (PDB ID: 1SQB [21]), co-crystallized with azoxystrobin, chains E and N, was selected as a template for both subunits. Two alignments produced by the ClustalΩ software and manually optimized for cytochrome b and 1SQB chain N were used. Comparative model building was carried out by Schrödinger BioLuminate [35] Multiple Sequence Viewer/Editor in the knowledge-based model setting, including azoxystrobin, heme groups, and Fe-S clusters. The geometry of the ﬁnal model was checked by the Ramachandran plot.

4.7.2. Ligand Preparation Available/commercial ligands were downloaded from PubChem, and original ones were built with the Maestro 3D Builder tool. All ligands were prepared for docking with the LigPrep panel, using the OPLS3e [36] force ﬁeld.

4.7.3. Molecular Docking and Affinity Calculations The molecular docking procedure was carried out with the Schrödinger Small-Molecule Drug Discovery Suite [37]. The QoI binding site of the cytochrome bc1 complex was identified by the presence of azoxystrobin transferred from the bovine cytochrome bc1 complex (PDB ID: 1SQB [21]). Molecular docking was carried out via Glide in its extra precision (XP) mode [38–40]. Before the docking procedures, our model of cytochrome bc1 complex was prepared and energy-minimized via the BioLuminate Protein Preparation Wizard with the OPLS3e [36] force field. The same force field was applied in all the molecular docking procedures. The binding free energy of all the complexes produced by our molecular docking pipeline was evaluated via both Glide XP Score and Prime MM-GBSA that combines molecular mechanics with generalized Born and surface area scoring function [41]. The accuracy of the proposed methods is associated with a low level of accuracy. Glide XP Score is an empirical scoring function that approximates the ligand binding free energy, and that is generally good enough to separate ligands putatively from non-ligands. In Eberini et al. [42], we used an empirical scoring function for estimating ligand binding free energy after molecular docking: the comparison between computed and experimental affinities (i.e., dissociation constants, Ki, computed from ∆G values) showed approx. one order of magnitude accuracy for our predictions. An approach associated with a higher level of accuracy than the one based on Glide XP Score is based on MM-GBSA that allows the ligand to relax in the binding site.

4.7.4. Mutant Generation and Evaluation The evaluation of the impact of G143A mutation on the enzyme stability and on the affinity for compound 11b was carried out with the Maestro BioLuminate Residue Scanning Tool, which considers the impact of a mutation on the stability of the protein (∆Stability) and on the affinity (∆Affinity) for the tested ligand(s), expressed in kcal/mol [43].

Supplementary Materials: The Supplementary Materials are available online at https://www.mdpi. com/article/10.3390/ijms22073731/s1. Author Contributions: Conceptualization, S.D., A.P., I.E., and P.C.; methodology, F.F., G.C., L.M., A.K., and L.P.; investigation, F.F., G.C., L.M., A.K., and L.P.; resources, A.P., S.D., A.K., P.C., and I.E.; data curation, F.F., G.C., L.M., A.K., and L.P.; writing—original draft preparation, A.K., A.P., S.D., and I.E.; writing—review and editing, all authors. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Int. J. Mol. Sci. 2021, 22, 3731 15 of 16

Acknowledgments: The research was partly supported by the University of Milan grant SEED2019 “Dual-active hybrid fungicides against Pyricularia oryzae”. L.P. and I.E. were supported by grants from MIUR—Progetto Eccellenza. I.E. was supported by the departmental “Linea 2- Azione A 2019” grant of the University of Milan. A.P. was supported by “Transition Grant 2015–2017—Linea 1A” of the University of Milan. G.C. was supported by Fondazione F.lli Confalonieri (PhD Scholarship). The authors thank Alessandra Casubolo for assistance in the enzyme assays experiments. Conﬂicts of Interest: The authors declare no conﬂict of interest.

References 1. FRAC. FRAC Code List©* 2020: Fungal Control Agents Sorted by Cross Resistance Pattern and Mode of Action. Available online: https://cpb-us-w2.wpmucdn.com/u.osu.edu/dist/b/28945/files/2020/02/frac-code-list-2020-final.pdf (accessed on 26 March 2021). 2. Lamberth, C. Complex III inhibiting strobilurin esters, amides, and carbamates as broad-spectrum fungicides. In Bioactive Carboxylic Compound Classes: Pharmaceuticals and Agrochemicals; Wiley: Weinheim, Germany, 2016; pp. 371–384. 3. Walter, H. Fungicidal succinate-dehydrogenase-inhibiting carboxamides. In Bioactive Carboxylic Compound Classes: Pharmaceuticals and Agrochemicals; Wiley: Weinheim, Germany, 2016; pp. 405–425. 4. Xiong, L.; Shen, Y.-Q.; Jiang, L.-N.; Zhu, X.-L.; Yang, W.-C.; Huang, W.; Yang, G.-F. Succinate Dehydrogenase: An Ideal Target for Fungicide Discovery. In Discovery and Synthesis of Crop Protection Products; ACS Publications: Washington, DC, USA, 2015; pp. 175–194. ISBN 9780841231023. 5. 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] 6. Musso, L.; Fabbrini, A.; Dallavalle, S. Natural compound-derived cytochrome bc1 complex inhibitors as antifungal agents. Molecules 2020, 25, 4582. [CrossRef][PubMed] 7. Steinhauer, D.; Salat, M.; Frey, R.; Mosbach, A.; Luksch, T.; Balmer, D.; Hansen, R.; Widdison, S.; Logan, G.; Dietrich, R.A.; et al. A dispensable paralog of succinate dehydrogenase subunit C mediates standing resistance towards a subclass of SDHI fungicides in Zymoseptoria tritici. PLoS Pathog. 2019, 15, e1007780. [CrossRef][PubMed] 8. FRAC. FRAC List of Plant Pathogenic Organisms Resistant to Disease Control Agents. Available online: https://www.frac.info/ docs/default-source/working-groups/sdhi-fungicides/group/list-of-resistant-plant-pathogens_2012-edition.pdf?sfvrsn=ef1 8469a_2 (accessed on 26 March 2021). 9. Torriani, S.F.F.; Frey, R.; Buitrago, C.; Wullschleger, J.; Waldner, M.; Kuehn, R.; Scalliet, G.; Sierotzki, H. Succinate-dehydrogenase inhibitor (SDHI) resistance evolution in plant pathogens. In Modern Fungicides and Antifungal Compounds; Deising, H.B., Fraaije, B., Mehl, A., Oerke, E.C., Sierotzki, H., Stammler, G., Eds.; Deutsche Phytomedizinische Gesellschaft: Braunschweig, Germany, 2016; Volume VIII, pp. 89–94. ISBN 978-3-941261-15-0. 10. Fernández-Ortuño, D.; Torés, J.A.; de Vicente, A.; Pérez-García, A. The QoI fungicides, the rise and fall of a successful class of agricultural fungicides. In Fungicides; Carisse, O., Ed.; IntechOpen: London, UK, 2010; pp. 203–220. ISBN 978-953-307-266-1. 11. FRAC. FRAC Reccomendations for QoI Fungicides. Available online: https://www.frac.info/frac-teams/working-groups/qol- fungicides/recommendations-for-qoi (accessed on 16 March 2021). 12. Asibi, A.E.; Chai, Q.; Coulter, J.A. Rice blast: A disease with implications for global food security. Agronomy 2019, 9, 451. [CrossRef] 13. Nalley, L.; Tsiboe, F.; Durand-Morat, A.; Shew, A.; Thoma, G. Economic and environmental impact of rice blast pathogen (Magnaporthe oryzae) alleviation in the United States. PLoS ONE 2016, 11, e0167295. [CrossRef][PubMed] 14. Scheuermann, K.K.; Raimondi, J.V.; Marschalek, R.; de Andrade, A.; Wickert, E. Magnaporthe oryzae genetic diversity and its outcomes on the search for durable resistance. In The Molecular Basis of Plant Genetic Diversity; IntechOpen: London, UK, 2012; pp. 331–356. 15. Ishii, H. Rice pathogens in Japan. In Fungicide Resistance in Plant Pathogens; Springer: Tokyo, Japan, 2015; pp. 341–354. 16. Kunova, A.; Cortesi, P. Tricyclazole and azoxystrobin in rice blast management: A review of their activity and pathogen responses. In Fungicides: Classification, Role in Disease Management and Toxicity Effects; Wheeler, M.N., Johnston, B.R., Eds.; Nova Science Publishers, Inc.: New York, NY, USA, 2013; pp. 39–66. ISBN 978-1-62948-045-9. 17. Fitogest®Image Line Fitogest®. Available online: https://fitogest.imagelinenetwork.com/it/ (accessed on 6 November 2020). 18. Zuccolo, M.; Kunova, A.; Musso, L.; Forlani, F.; Pinto, A.; Vistoli, G.; Gervasoni, S.; Cortesi, P.; Dallavalle, S. Dual-active antifungal agents containing strobilurin and SDHI-based pharmacophores. Sci. Rep. 2019, 9, 11377. [CrossRef][PubMed] 19. Suemoto, H.; Matsuzaki, Y.; Iwahashi, F. Metyltetraprole, a novel putative complex III inhibitor, targets known QoI-resistant strains of Zymoseptoria tritici and Pyrenophora teres. Pest Manag. Sci. 2019, 75, 1181–1189. [CrossRef] 20. Matsuzaki, Y.; Yoshimoto, Y.; Arimori, S.; Kiguchi, S.; Harada, T.; Iwahashi, F. Discovery of metyltetraprole: Identification of tetrazolinone pharmacophore to overcome QoI resistance. Bioorg. Med. Chem. 2020, 28, 115211. [CrossRef] 21. 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 bc 1 complex. J. Mol. Biol. 2004, 341, 281–302. [CrossRef] 22. Palsdottir, H.; Lojero, C.G.; Trumpower, B.L.; Hunte, C. Structure of the yeast cytochrome bc1 complex with a hydroxyquinone anion Qo site inhibitor bound. J. Biol. Chem. 2003, 278, 31303–31311. [CrossRef] Int. J. Mol. Sci. 2021, 22, 3731 16 of 16

23. Kim, E.M.; Jung, C.K.; Choi, E.Y.; Gao, C.; Kim, S.W.; Lee, S.H.; Kwon, O.P. Highly conductive polyaniline copolymers with dual-functional hydrophilic dioxyethylene side chains. Polymer 2011, 52, 4451–4455. [CrossRef] 24. Huang, R.; Li, Z.; Ren, P.; Chen, W.; Kuang, Y.; Chen, J.; Zhan, Y.; Chen, H.; Jiang, B. N- Phenyl- N- aceto-vinylsulfonamides as efficient and chemoselective handles for N-terminal modification of peptides and proteins. Eur. J. Org. Chem. 2018, 2018, 829–836. [CrossRef] 25. Zhou, X.; Wang, Q.; Zhao, W.; Xu, S.; Zhang, W.; Chen, J. Palladium-catalyzed ortho-arylation of benzoic acid derivatives via C-H bond activation using an aminoacetic acid bidentate directing group. Tetrahedron Lett. 2015, 56, 851–855. [CrossRef] 26. Crestey, F.; Frederiksen, K.; Jensen, H.S.; Dekermendjian, K.; Larsen, P.H.; Bastlund, J.F.; Lu, D.; Liu, H.; Yang, C.R.; Grunnet, M.; et al. Identification and electrophysiological evaluation of 2-methylbenzamide derivatives as Nav1.1 modulators. ACS Chem. Neurosci. 2015, 6, 1302–1308. [CrossRef][PubMed] 27. Baramov, T.; Schmid, B.; Ryu, H.; Jeong, J.; Keijzer, K.; von Eckardstein, L.; Baik, M.; Süssmuth, R.D. How many O-donor groups in enterobactin does it take to bind a metal cation? Chem. Eur. J. 2019, 25, 6955–6962. [CrossRef] 28. Vilaivan, T. A rate enhancement of tert-butoxycarbonylation of aromatic amines with Boc2O in alcoholic solvents. Tetrahedron Lett. 2006, 47, 6739–6742. [CrossRef] 29. Kunova, A.; Pizzatti, C.; Cortesi, P. Impact of tricyclazole and azoxystrobin on growth, sporulation and secondary infection of the rice blast fungus, Magnaporthe oryzae. Pest Manag. Sci. 2013, 69, 278–284. [CrossRef] 30. Kunova, A.; Pizzatti, C.; Bonaldi, M.; Cortesi, P. Sensitivity of nonexposed and exposed populations of Magnaporthe oryzae from rice to tricyclazole and azoxystrobin. Plant Dis. 2014, 98, 512–518. [CrossRef] 31. Ye, Y.-H.; Ma, L.; Dai, Z.-C.; Xiao, Y.; Zhang, Y.-Y.; Li, D.-D.; Wang, J.-X.; Zhu, H.-L. Synthesis and antifungal activity of nicotinamide derivatives as succinate dehydrogenase inhibitors. J. Agric. Food Chem. 2014, 62, 4063–4071. [CrossRef] 32. 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] 33. R Core Team. R: A Language and Environment for Statistical Computing; R Core Team, The R Foundation for Statistical Computing: Vienna, Austria, 2020. 34. Faria, J.C.; Jelihovschi, E.G.; Allaman, I.B. Conventional Tukey Test; UESC: Ilheus, Brasil, 2020. 35. Schrödinger Release 2020-3; BioLuminate, Schrödinger, LLC: New York, NY, USA, 2020. 36. Harder, E.; Damm, W.; Maple, J.; Wu, C.; Reboul, M.; Xiang, J.Y.; Wang, L.; Lupyan, D.; Dahlgren, M.K.; Knight, J.L.; et al. OPLS3: A force field providing broad coverage of drug-like small molecules and proteins. J. Chem. Theory Comput. 2016, 12, 281–296. [CrossRef][PubMed] 37. Schrödinger Release 2020-3; Schrödinger, LLC: New York, NY, USA, 2020. 38. Friesner, R.A.; Banks, J.L.; Murphy, R.B.; Halgren, T.A.; Klicic, J.J.; Mainz, D.T.; Repasky, M.P.; Knoll, E.H.; Shelley, M.; Perry, J.K.; et al. Glide: A new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. J. Med. Chem. 2004, 47, 1739–1749. [CrossRef] 39. Halgren, T.A.; Murphy, R.B.; Friesner, R.A.; Beard, H.S.; Frye, L.L.; Pollard, W.T.; Banks, J.L. Glide: A new approach for rapid, accurate docking and scoring. 2. Enrichment factors in database screening. J. Med. Chem. 2004, 47, 1750–1759. [CrossRef] 40. Friesner, R.A.; Murphy, R.B.; Repasky, M.P.; Frye, L.L.; Greenwood, J.R.; Halgren, T.A.; Sanschagrin, P.C.; Mainz, D.T. Extra precision glide: Docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes. J. Med. Chem. 2006, 49, 6177–6196. [CrossRef] 41. Li, J.; Abel, R.; Zhu, K.; Cao, Y.; Zhao, S.; Friesner, R.A. The VSGB 2.0 model: A next generation energy model for high resolution protein structure modeling. Proteins Struct. Funct. Bioinform. 2011, 79, 2794–2812. [CrossRef] 42. Eberini, I.; Rocco, A.G.; Mantegazza, M.; Gianazza, E.; Baroni, A.; Vilardo, M.C.; Donghi, D.; Galliano, M.; Beringhelli, T. Computational and experimental approaches assess the interactions between bovine β-lactoglobulin and synthetic compounds of pharmacological interest. J. Mol. Graph. Model. 2008, 26, 1004–1013. [CrossRef] 43. Beard, H.; Cholleti, A.; Pearlman, D.; Sherman, W.; Loving, K.A. Applying physics-based scoring to calculate free energies of binding for single amino acid mutations in protein-protein complexes. PLoS ONE 2013, 8, e82849. [CrossRef]