Crinipellins a and I, Two Diterpenoids from the Basidiomycete Fungus Crinipellis Rhizomaticola, As Potential Natural Fungicides

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Article Crinipellins A and I, Two Diterpenoids from the Basidiomycete Fungus Crinipellis rhizomaticola, as Potential Natural Fungicides

Jae Woo Han 1,†, Mira Oh 1,2,†, Yu Jeong Lee 1,2, Jaehyuk Choi 3 , Gyung Ja Choi 1,2 and Hun Kim 1,2,*

1 Center for Eco-friendly New Materials, Korea Research Institute of Chemical Technology, Daejeon 34114, Korea; [email protected] (J.W.H.); [email protected] (M.O.); [email protected] (Y.J.L.); [email protected] (G.J.C.) 2 Department of Green Chemistry and Environmental Biotechnology, University of Science and Technology, Daejeon 34113, Korea 3 Division of Life Sciences, Incheon National University, Incheon 22012, Korea; [email protected] * Correspondence: [email protected]; Tel.: +82-42-860-7436 † These authors contributed equally to this work.

Received: 27 August 2018; Accepted: 14 September 2018; Published: 17 September 2018

Abstract: In the course of screening for microbes with antifungal activity, we found that the culture filtrate of the IUM00035 isolate exhibited strong antifungal activity against Magnaporthe oryzae and Colletotrichum coccodes in planta. Based on the phylogenetic analysis with the ITS region, the IUM00035 isolate was identified as Crinipellis rhizomaticola. To identify antifungal compounds from the C. rhizomaticola IUM00035 isolate, the culture filtrate of the isolate was partitioned with ethyl acetate and n-butanol and, consequently, two active compounds were isolated from the ethyl acetate extract. The chemical structures of the isolated compounds were determined as crinipellin A (1) and a new crinipellin derivative, crinipellin I (2), by NMR spectral analyses and a comparison of their NMR and MS data with those reported in the literature. Crinipellin A (1) exhibited a wide range of antifungal activity in vitro against C. coccodes, M. oryzae, Botrytis cinerea, and Phytophthora infestans (MICs = 1, 8, 31, and 31 µg/mL, respectively). Furthermore, when plants were treated with crinipellin A (1) (500 µg/mL) prior to inoculation with fungal pathogens, crinipellin A (1) exhibited disease control values of 88%, 65%, and 60% compared with non-treatment control against tomato late blight, pepper anthracnose, and wheat leaf rust, respectively. In contrast to crinipellin A (1), crinipellin I (2) showed weak or no activity (MICs > 250 µg/mL). Taken together, our results show that the C. rhizomaticola IUM00035 isolate suppresses the development of plant fungal diseases, in part through the production of crinipellin A (1).

Keywords: antifungal; Crinipellis rhizomaticola; crinipellin; biological control; mushroom

1. Introduction Plant-pathogenic fungi cause severe plant diseases, resulting in a loss of crop yields and economic damage to the agricultural industry [1]. Synthetic fungicides have been recognized as a primary method to control plant diseases caused by fungal pathogens, although the overuse of chemical pesticides has caused severe problems, such as toxicity for human health and environmental pollution. Therefore, the development of safer and more effective alternatives is necessary to replace chemical pesticides [2]. Among the various natural resources, antagonistic microbes (or microbial metabolites) have been recognized as an attractive source for new antifungal agents [3].

Molecules 2018, 23, 2377; doi:10.3390/molecules23092377 www.mdpi.com/journal/molecules Molecules 2018, 23, 2377 2 of 11

Mushrooms have been used as a functional food or as active ingredients of folk medicine due to their various bioactive compounds [4]. Currently, over 140,000 species of mushrooms have been reported,Molecules and 2018, more 23, x than 200 species have been known to possess antimicrobial properties2 of 11 [5 ,6]. The fruiting bodies, mycelial biomass, or culture filtrates of mushrooms contain bioactive compounds reported, and more than 200 species have been known to possess antimicrobial properties [5,6]. The with a wide range of antimicrobial activity, including antifungal and antibacterial activities [7–9]. fruiting bodies, mycelial biomass, or culture filtrates of mushrooms contain bioactive compounds Strobilurus tenacellus In particular,with a wide strobilurins, range of antimicrobial produced byactivity, wood-inhabiting including antifungal forest mushroomand antibacterial activities [7–9]. Inand Oudemansiellaparticular, mucidastrobilurins,, are oneproduced of the by greatest wood-inhabiting findings in forest the history mushroom of fungicides Strobilurus [10tenacellus]. Strobilurins and act asOudemansiella inhibitors ofmucida the, respiratoryare one of the chain greatest at findings the quinone in the outerhistory binding of fungicides site of[10]. the Strobilurins cytochrome act bc1 complexas inhibitors [11]. Due of the to the respiratory unique andchain specific at the quinone modes ofouter action, binding a number site of the of strobilurincytochrome analogues bc1 complex have been[11]. synthesized Due to the with unique improved and specific antifungal modes activity, of action, stability, a number and dissemination,of strobilurin analogues of which have several been have beensynthesized developed with as highly improved successful antifungal commercial activity, stability, products and such dissemination, as kresoxim-methyl, of which several pyraclostrobin, have and trifloxystrobinbeen developed [as12 highly]. successful commercial products such as kresoxim-methyl, pyraclostrobin, andIn the trifloxystrobin search for antifungal[12]. agents from mushrooms, we recently found that a culture filtrate of CrinipellisIn the rhizomaticola search for antifungalIUM00035 agents isolate from hasmushroom the potentials, we recently to control found plant that a diseasesculture filtrate caused of by MagnaportheCrinipellis oryzaerhizomaticolaand Colletotrichum IUM00035 isolate coccodes has. Furthermore,the potential to we control isolated plant and diseases identified caused the activeby Magnaporthe oryzae and Colletotrichum coccodes. Furthermore, we isolated and identified the active small molecules from the culture filtrate of C. rhizomaticola IUM00035 and investigated their in vitro small molecules from the culture filtrate of C. rhizomaticola IUM00035 and investigated their in vitro and in vivo antifungal activity. Taken together, our results provide a useful information for the and in vivo antifungal activity. Taken together, our results provide a useful information for the developmentdevelopment of plant-protectingof plant-protecting agents agents using using microbial-derivedmicrobial-derived materials materials to tocontrol control plant plant diseases diseases causedcaused by fungal by fungal pathogens. pathogens.

2. Results2. Results and and Discussion Discussion

2.1. In2.1. Vivo In Vivo Antifungal Antifungal Activity Activity of of the the Culture Culture Filtrate Filtrate ofof thethe IUM00035 Isolate Isolate To findTo find a mushroom a mushroom isolate isolate showing showing a plant a plant disease disease control control activity, activity, we we have have examined examined the thein in vivo antifungalvivo antifungal activity ofactivity the culture of the filtratesculture filtrates of over of 100 over mushroom 100 mushroom isolates isolates against against seven seven plant plant diseases: rice bdiseases:last (RCB, rice caused blast (RCB, by M. caused oryzae ),byr iceM. soryzaeheath), bricelight sheath (RSB, blight caused (RSB, by causedRhizoctonia by Rhizoctonia solani), tomato solani),g ray moldtomato (TGM, gray caused mold by (TGM,Botrytis caused cinerea by), Botrytistomato cinerealate blight), tomato (TLB, late caused blight by (TLB,Phytophthora caused by infestansPhytophthora), barley powderyinfestansmildew), barley (BPM, powdery caused mildew by Blumeria (BPM, caused graminis by Blumeriaf. sp. hordei graminis), wheat f. sp.leaf hordeirust), wheat (WLR, leaf caused rust by Puccinia(WLR, triticina caused), by and Pucciniapepper triticinaanthrac), andnose pepper (PAN, anthracn causedose by (PAN,C. coccodes caused). by Among C. coccodes the). culture Among filtrates the of overculture 100 filtrates mushroom of over isolates, 100 mush theroom treatment isolates, of the undiluted treatment IUM00035 of undiluted culture IUM00035 filtrate culture suppressed filtrate the suppressed the development of RCB, TGM, TLB, WLR, BPM, and PAN; however, there was no effect development of RCB, TGM, TLB, WLR, BPM, and PAN; however, there was no effect on RSB (Figure1). on RSB (Figure 1). In particular, this culture filtrate had strong effects against RCB and PAN with In particular, this culture filtrate had strong effects against RCB and PAN with disease control values of disease control values of 70% and 65% compared with the non-treatment control (distilled water 70%containing and 65% compared 0.025% Tween with 20), the respectively. non-treatment Three-fold control diluted (distilled culture water filtrate containing of the IUM00035 0.025% Tween isolate 20), respectively.further showed Three-fold disease diluted suppression culture filtrate of RCB, of the TLB, IUM00035 WLR, and isolate PAN further (Figure showed 1). Thus, disease our suppression results of RCB,suggest TLB, that WLR, the andIUM00035 PAN (Figure culture1 ).filtrate Thus, may our contai resultsn suggestantifungal that substances the IUM00035 for the culture control filtrate of plant may containdiseases antifungal caused substances by some plant-pathogenic for the control offungi. plant diseases caused by some plant-pathogenic fungi.

FigureFigure 1. Antifungal 1. Antifungal activity activity of the of culturethe culture filtrate filtrate of the of IUM00035 the IUM00035 isolate. isolate. Plant diseasePlant disease control control values of the culturevalues filtrateof the culture of the IUM00035 filtrate of isolate.the IUM00035 RCB, rice isolate. blast; RCB, RSB, rice rice blast; sheath RSB, blight; rice TGM, sheath tomato blight; gray TGM, mold; TLB,tomato tomato gray late mold; blight; TLB, WLR, tomato wheat late leaf blight; rust; WLR, BPM, wheat barley leaf powdery rust; BPM, mildew; barley powdery PAN, pepper mildew; anthracnose. PAN, n.s. indicatespepper anthracnose. not shown, n.s. meaning indicate thats not disease shown, control meaning effects that of thedisease culture control filtrate effects were of not the observed. culture filtrate were not observed.

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2.2. In Vitro Antifungal Activity of the IUM00035 Culture According to Incubation Period To investigate the antifungal activity of the IUM00035 isolate according to the incubation time, Molecules 2018, 23, 2377 3 of 11 the IUM00035 isolate was incubated for five, seven, 10, 14, and 21 days. For this in vitro assay, the model fungus Saccharomyces cerevisiae was used as a target microbe rather than plant-pathogenic 2.2. In Vitro Antifungal Activity of the IUM00035 Culture According to Incubation Period fungi such as M. oryzae and C. coccodes. The reason is that the optical density (OD600) was used to calculate the fungalTo investigate inhibition. the antifungal When activity S. cerevisiae of the IUM00035 was treated isolate accordingwith 5-, to 7-, the and incubation 10-day-old time, culture the IUM00035 isolate was incubated for five, seven, 10, 14, and 21 days. For this in vitro assay, the model filtrates of the IUM00035 isolate with different concentrations, the treatments did not show any fungus Saccharomyces cerevisiae was used as a target microbe rather than plant-pathogenic fungi such as differencesM. in oryzae theand antifungalC. coccodes. Theacti reasonvity regardless is that the optical of concentrations density (OD600) was used. used to In calculate addition, the fungal these culture filtrates exhibitedinhibition. weak When antifungalS. cerevisiae was activity treated ranging with 5-, 7-, from and 10-day-old 10% to 20% culture (Figure filtrates 2). of However, the IUM00035 the 14- and 21-day-oldisolate culture with filtrates different concentrations,showed strong the treatmentsantifungal did activity not show depending any differences on inthe the concentration antifungal used activity regardless of concentrations used. In addition, these culture filtrates exhibited weak antifungal (Figure 2). Similar results have been reported by Imtiaj and Lee (2007) [13] who investigated the activity ranging from 10% to 20% (Figure2). However, the 14- and 21-day-old culture filtrates showed antifungalstrong activity antifungal of mushrooms activity depending according on the to concentration the culture used incubation (Figure2). period Similar resultsand have have beenfound that the 20-day-oldreported culture by Imtiajfiltrates and Leeof (2007)Sterium [13] whoostrea investigated, Pycnoporus the antifungal cinnabarinus activity of, mushroomsP. coccineus according, Oudemansiella mucida andto Cordyceps the culture incubationsobolifera period had an and antifungal have found thatactivity the 20-day-old against cultureplant-pathogenic filtrates of Sterium fungi ostrea B., cinerea, C. Pycnoporus cinnabarinus, P. coccineus, Oudemansiella mucida and Cordyceps sobolifera had an antifungal gloeosporioides and C. miyabeanus. Additionally, Hatvani (2001) noted that culture fluid of Lentinus activity against plant-pathogenic fungi B. cinerea, C. gloeosporioides and C. miyabeanus. Additionally, edodes incubatedHatvani (2001)for more noted than that culture7 weeks fluid has of Lentinusan antimicrobial edodes incubated activity for more against than 7Streptococcus weeks has an pyogenes, Staphylococcusantimicrobial aureus activityand Bacillus against Streptococcus megaterium pyogenes [7]. Thus,, Staphylococcus a long aureusincubationand Bacillus time megaterium of more[7 ].than three weeks seemsThus, to a longbe incubationrequired timefor ofthe more production than three weeksof antifungal seems to be substances. required for the Further production study of will be antifungal substances. Further study will be required to optimize the culture conditions such as the required to optimize the culture conditions such as the temperature, pH, and light. temperature, pH, and light.

Figure 2. Effects of the incubation time of the IUM00035 isolate on the antifungal activity of its culture Figure 2. Effectsfiltrate. Theof the IUM00035 incubation culture time filtrate of was the added IUM00035 to wells is containingolate onSaccharomyces the antifungal cerevisiae activityto reach of its culture filtrate. Thefinal IUM00035 concentrations culture of 1.25%, filtrate 2.50%, was 5%, andadded 10% (tov/ vwells). The containing different small Saccharomyces letters in each bar cerevisiae indicate to reach final concentrationsa significant differenceof 1.25%, at 2.50%,p < 0.05 5%, (Duncan’s and 10% test). (v/v). The different small letters in each bar indicate a significant2.3. Molecular difference Identification at P < of0.05 the (Duncan’s IUM00035 Isolate test). For the molecular identification of the IUM00035 isolate, the internal transcribed spacer (ITS) 2.3. Molecularregion Identificatio was amplifiedn of from the the IUM00035 IUM00035 gDNA,Isolate and the resulting sequences (an amplicon of 616 bp) were searched by the BLASTn program of NCBI (http://www.ncbi.nlm.nih.gov). The ITS region of For thethe molecular IUM00035 isolate identification had a sequence of the similarity IUM00035 of 99.8%, isolate, 96.4%, the and 95.5%internal to those transcribed of Crinipellis spacer (ITS) region wasrhizomaticola amplifiedBRNM from 712570,the IUM00035C. scabella 7154,gDNA, and anC.d scabella the resultingZTPAM, respectively.sequences Comparison(an amplicon of 616 bp) were searchedof the IUM00035 by the ITS BLASTn sequences program with 20 Crinipellisof NCBIspecies (http://www.ncb revealed thati.nlm.nih.gov). the IUM00035 isolate The is ITS region of the IUM00035phylogenetically isolate close had to aC. sequence rhizomaticola similari(Figure3ty). Theof 99.8%, genus Crinipellis 96.4%, isand a mushroom 95.5% to belonging those of to Crinipellis the order Agricales in Basidiomycota and consists of over 65 species [14]. In particular, C. rhizomaticola rhizomaticolaidentified BRNM in this712570, study C. is phylogenetically scabella 7154, closeand toC.C. scabella stipitaria ZTPAM,, which produces respectively. natural tetraquinane Comparison of the IUM00035products ITS sequences such as crinipellins with [1520]. Crinipellis species revealed that the IUM00035 isolate is phylogenetically close to C. rhizomaticola (Figure 3). The genus Crinipellis is a mushroom belonging to the order Agricales in Basidiomycota and consists of over 65 species [14]. In particular, C. rhizomaticola identified in this study is phylogenetically close to C. stipitaria, which produces natural tetraquinane products such as crinipellins [15].

Molecules 2018, 23, 2377 4 of 11 Molecules 2018, 23, x 4 of 11

Figure 3. Phylogenetic analysis of the IUM00035 isolate. Phylogenetic analysis was performed based on Figurethe 3. ITS Phylogenetic region of IUM00035 analysis and of the the other IUM00035 20 Crinipellis isolate.species. Phylogenetic The neighbor-joining analysis was method performed was used based on thefor ITS this analysis,region of and IUM00035 numbers atand the the nodes other indicate 20 Crinipellis the levels of species. bootstrap The support neighbor (%) by-joining 1000 resampled method was useddatasets. for this NCBIanalysis, accession and numbers of at each the sequencenodes in aredicate in parentheses. the levels of Bar, bootstrap 5 substitution support per 1000(%) by nt. 1000 resampled datasets. NCBI accession numbers of each sequence are in parentheses. Bar, 5 substitution 2.4. In Vivo Antifungal Activity of the Solvent Extracts of the C. rhizomaticola Culture Filtrate per 1000 nt. The culture filtrate of the C. rhizomaticola IUM00035 isolate was partitioned by organic solvents, 2.4. Inand Vivo the Antifungal disease control Activity efficacy of the of Solvent the crude Extracts extracts of the was C. investigated. rhizomaticola The Culture culture Filtrate filtrate (10 L) was extracted with ethyl acetate and n-butanol, successively. Each extract was sprayed at three concentrationsThe culture filtrate of 500, 1000,of the and C. 2000rhizomaticolaµg/mL onto IUM00035 plants prior isolate to infection was partitioned with plant-pathogenic by organic fungi. solvents, and Thethe disease ethyl acetate control extract efficacy exhibited of the an crude antifungal extracts activity was investigated. in planta, while The the culturen-butanol filtrate and water(10 L) was extractedextracts with were ethyl not active. acetate When and the plantsn-butanol, were treatedsuccessively. with the Each ethyl acetateextract extract was (2000sprayedµg/mL), at three concentrationsthe disease controlof 500, values 1000, ofand TLB, 2000 WLR, µg/mL RCB, TGM,onto andplants PAN prior were to 100%, infection 100%, with 90%, 71%,plant-pathogenic and 79% fungi.relative The ethyl to the acetate non-treatment extract controls,exhibited respectively an antifungal (Figure activity4). Additionally, in planta, the whilein vivo the antifungaln-butanol and wateractivity extracts of the were ethyl not acetate active. extract When was the decreased plants dependingwere treated on thewith concentration the ethyl acetate of the treatmentextract (2000 µg/mL),(Figure the4). disease Of the control seven plantvalues diseases, of TLB, TLB WLR, was RCB, the mostTGM, sensitive and PAN to thewere ethyl 100%, acetate 100%, extract, 90%, 71%, and which79% relative resulted to in the over non-treatment 96% of the disease controls, control respectively value compared (Figure to the4). non-treatmentAdditionally, control,the in vivo antifungalwhereas activity RSB and of BPM the wereethyl not acetate affected extract by the wa treatments decreased with ethyldepending acetate on extract the (Figureconcentration4). of the treatment2.5. Isolation (Figure and 4). Structure Of the Determination seven plant of diseases, the Antifungal TLB Compounds was the most sensitive to the ethyl acetate extract, which resulted in over 96% of the disease control value compared to the non-treatment Considering the potent antifungal activity described above, the antifungal compounds were control, whereas RSB and BPM were not affected by the treatment with ethyl acetate extract (Figure isolated from the ethyl acetate extract by a series of chromatographic procedures. The ethyl acetate 4). extract was subjected to a silica gel column chromatography to yield 10 fractions (E1–E10). The E2 fraction exhibited an antifungal activity in vitro against M. oryzae and P. infestans (data not shown). Compounds 1 and 2 were finally purified from the E2 fraction by a preparative HPLC system (Figure S1). The spectroscopic data of 1 and 2 are presented in Tables1 and2, as well as in Supplementary Materials. The ESI-MS spectrum of 1 showed a molecular ion at m/z 331 [M + H]+ (Figure S2). The NMR spectroscopic data and ESIMS data of 1 were in agreement with those of crinipellin A (Figure5A; Table1 and Table S1) [ 16–19]. The HREIMS of 2 showed a molecular ion at m/z 332.1985 + [M ] (Figure S3), which is consistent with the molecular formula C20H28O4 (calculated m/z 332.1988). The mass difference of 2 amu compared with crinipellin A may indicate the reduction of a double bond in the molecule. Among the known crinipellins, dihydrocrinipellins A and B and crinipellin

Figure 4. Plant disease control efficacies of the ethyl acetate extract of the Crinipellis rhizomaticola IUM00035 culture filtrate. n.s. indicates not shown, meaning that disease control effects of ethyl acetate extracts were not observed. RCB, rice blast; RSB, rice sheath blight; TGM, tomato gray mold; TLB, tomato late blight; WLR, wheat leaf rust; BPM, barley powdery mildew; PAN, pepper anthracnose.

Molecules 2018, 23, x 4 of 11

Figure 3. Phylogenetic analysis of the IUM00035 isolate. Phylogenetic analysis was performed based onMolecules the ITS2018 region, 23, 2377 of IUM00035 and the other 20 Crinipellis species. The neighbor-joining method5 of 11was used for this analysis, and numbers at the nodes indicate the levels of bootstrap support (%) by 1000

resampledE have the datasets. same molecular NCBI accession formula, numbers i.e., C20H of28 eachO4 [18 sequence–21]. Based are onin parentheses. the comparison Bar, of 5 NMRsubstitution data per(Tables 10001 nt. and 2), the structure of 2 was not the same as those of the other known crinipellins [18–21]. 13 3 The C-NMR spectrum showed two ketone carbonyl (δC 212.4 and 216.6), four quaternary sp (δC 79.4, 3 3 2.4. In 61.5,Vivo 54.6,Antifungal and 51.7), Activity six methine of the sp Solvent(δC 86.1, Extracts 58.4, 52.7, of the 42.0, C. 40.4,rhizom andaticola 29.0), threeCulture methylene Filtrate sp (δC 33.6, 33.2, and 23.5), and five methyl groups (δC 25.2, 19.8, 16.2, 15.4, and 9.5) (Table2). Furthermore, Thethe culture results of filtrate the HSQC of the and C. HMBC rhizomaticola correlations IUM00035 (Figures S9 isolate and S10) was suggested partitioned that 2 wasby organic a diterpene solvents, and theof disease the crinipellin control family efficacy (Figure of 5theB) [crude17–19]. extracts This hypothesis was investigated. was supported The by culture the presence filtrate of (10 the L) was 3 extractedquaternary with spethylcarbon acetate at δC 61.5and which n-butanol, is shared successively. by all three cyclopentane Each extract rings inwas the tetraquinanesprayed at three concentrationsscaffold [16 of–19 500,]. The 1000, proton and signal 2000 of theµg/mL Me-18 onto (δH 0.94) plants showed prior HMBC to infection correlations with to C-2plant-pathogenic (δC 40.4), fungi. C-3The ( δethylC 42.0) acetate and C-4 extract (δC 212.4) exhibited (Figure5 B);an additionally, antifungal correlationsactivity in fromplanta, H-2 while ( δH 2.74) the to n C-4-butanol (δC and 212.4), C-5 (δ 58.4) and C-6 (δ 79.4) revealed that ring A contains a ketone function and an oxirane water extracts wereC not active. WhenC the plants were treated with the ethyl acetate extract (2000 ring. A comparison of the 1H-NMR data (Table1) of 1 and 2 revealed that the exocyclic methylene µg/mL),group the ondisease C-3 in 1controlwas replaced values by of a methylTLB, WLR, group RCB, (Me-18) TGM, in 2. Theand relative PAN were stereochemistry 100%, 100%, of 2 was90%, 71%, and 79%obtained relative from to analysis the non-treatment of ROESY spectrum. controls, The ROESY respectively correlations (Figure among H-14). (Additionally,δH 2.18), H-2 (δH the2.74), in vivo antifungalH-3 ( δactivityH 2.45), andof the Me-20 ethyl (δH acetate1.21) revealed extract that wa theses decreased protons were depending on the same on sidethe inconcentrationα-orientation. of the treatmentHowever, (Figure the ROESY4). Of correlationsthe seven amongplant diseases, H-5 (δH 3.29), TLB H-14 was (δ Hthe1.38), most and sensitive Me-19 (δH to0.92) the indicated ethyl acetate extract,that which H-5, H-14,resulted and in Me-19 over were 96%β -orientedof the dise (Figurease 5controlB and Figure value S11). compared Thus, 2towas the identifiednon-treatment S S R R R R R S R H H control,as whereas rel-(1a ,3 RSB,3a ,4aand,7 BPM,7a ,8were,9a not,9b affected)-8-hydroxy-7-isopropyl-3,7a,9a-trimethylhexahydro-3 by the treatment with ethyl acetate extract,5 (Figure -pentaleno[6a’,1’:5,6]pentaleno[1,6a-b]oxirene-2,9(1aH,9aH)-dione, which was designated as crinipellin I 4). (Figure5).

Figure 4. Plant disease control efﬁcacies of the ethyl acetate extract of the Crinipellis rhizomaticola FigureIUM00035 4. Plant culturedisease ﬁltrate. control n.s. indicatesefficacies not of shown, the ethyl meaning acetate that disease extract control of the effects Crinipellis of ethyl acetaterhizomaticola IUM00035extracts culture were notfiltrate. observed. n.s. RCB,indicates rice blast; not RSB,shown, rice sheathmeaning blight; that TGM, disease tomato control gray mold; effects TLB, of ethyl acetatetomato extracts late were blight; not WLR, observed. wheat leaf RCB, rust; rice BPM, blast; barley RSB, powdery rice sheath mildew; blight; PAN, pepper TGM, anthracnose. tomato gray mold; 1 TLB, tomato late blight; TableWLR, 1. wheatH-NMR leaf data (500rust; MHz) BPM, of 1barleyand 2 in powdery CDCl3. mildew; PAN, pepper anthracnose. 1 2 Position δH,(J in Hz) δH,(J in Hz) 1 2.51, dd (14.3, 7.5); 1.39, dd (14.2, 13.0) 2.23, dd (14.0, 6.6); 1.20 dd (14.4, 13.7) 2 3.08, dd (12.5, 7.7) 2.69 dt (13.7, 7.0) 3 - 2.55 p (7.3) 5 3.46, s 3.29, s 9 4.40, s 4.42, d (2.7) 12 1.87, dt (9.9, 7.1); 1.59, m 1.88, m; 1.63, m 13 1.59, m 1.63, m 14 1.36, m 1.37, dd (9.3, 2.5) 15 2.09, pd (7.0, 2.8) 2.12, qd (6.8, 2.7) 16 0.85, d (7.0) 0.89, d (6.9) 17 0.82, d (6.8) 0.86, d (6.9) 18 5.48, d (1.4); 6.13, d (1.8) 1.03, d (6.3) 19 1.02, s 1.02, s 20 1.31, s 1.30, s Molecules 2018, 23, x 6 of 11

Table 2. 1H- and 13C-NMR data (500 and 125 MHz) a of 2 in CD3CN. Molecules 2018, 23, 2377 6 of 11

Position δC, Type δH, (J in Hz) HMBC

1 133.6, CH213 2.18, dd (13.9, 6.8); 1.21, m C-2,a 3, 6, 7, 10, 11 Table 2. H- and C-NMR data (500 and 125 MHz) of 2 in CD3CN. 2 40.4, CH 2.74, dt (13.9, 7.2) C-1, 4, 5, 6

Position3 δ 42.0,C, Type CH 2.45,δH p,( J(7.4)in Hz) C-2, 4, HMBC 18

14 33.6,212.4, CH CO2 2.18, dd (13.9, - 6.8); 1.21, m C-2, 3, 6, 7, 10, 11 25 40.4, 58.4, CH CH 2.74, 3.29, dt (13.9,s 7.2) C-4, C-1, 6 4, 5, 6 36 42.0,79.4, CH C 2.45, - p (7.4) C-2, 4, 18 4 212.4, CO - 57 58.4,51.7, CH C - 3.29, s C-4, 6 68 216.6, 79.4, C CO - - 79 51.7,86.1, CCH 4.38, d (2.8) - C-8, 10, 20 810 216.6,54.6, CO C - 9 86.1, CH 4.38, d (2.8) C-8, 10, 20 1011 54.6,61.5, C C 1112 33.2, 61.5, CCH2 1.89, ddd (12.3, 8.8, 3.9); 1.59, m C-7, 10, 11, 13 1213 33.2,23.5, CH CH2 2 1.89, ddd1.59, (12.3, m 8.8, 3.9); 1.59, m C-7, 10, 11, 13 1314 23.5, 52.7, CH CH2 1.38, td 1.59,(9.5, m2.5) C-9, 10, 15, 16, 17, 20 14 52.7, CH 1.38, td (9.5, 2.5) C-9, 10, 15, 16, 17, 20 1515 29.0, 29.0, CH CH 2.09, 2.09, pd pd(6.9, (6.9, 2.5) 2.5) C-10, C-10, 13, 14, 13, 16, 14, 17 16, 17 1616 25.2,25.2, CH CH3 3 0.84,0.84, d (5.4) d (5.4) C-14, C-14, 15, 17 15, 17 1717 19.8,19.8, CH CH3 3 0.83,0.83, d (5.6) d (5.6) C-16 C-16 18 9.5, CH3 0.94, d (7.4) C-2, 3, 4 18 9.5, CH3 0.94, d (7.4) C-2, 3, 4 19 15.4, CH3 0.92, s C-6, 7, 8, 11 19 15.4, CH3 0.92, s C-6, 7, 8, 11 20 16.2, CH3 1.21, s C-9, 10, 11, 14 a Assignments20 16.2, (δ CHvalues)3 were conﬁrmed 1.21, bys the HSQC and HMBC C-9, 10, experiments. 11, 14 a Assignments (δ values) were confirmed by the HSQC and HMBC experiments.

FigureFigure 5. 5. A A:. Structures Structures of of1 1and and2 2;.;B B: Key: Key HMBC HMBC (blue (blue arrow) arrow) and and ROESY ROESY (red (red arrow) arrow) correlations correlations ofof2 . 2. Crinipellins are structurally interesting diterpenoids with a unique tetraquinane skeleton and continueCrinipellins to attract are the structurally attention interesting of synthetic diterpenoids chemists [ 16with,17 ].a unique Crinipellin tetraquinane A, O-acetylcrinipellin skeleton and A,continue tetrahydrocrinipellin to attract the attention A, crinipellin of synthetic B, andchemists dihydrocrinipellin [16,17]. Crinipellin B were A, O-acetylcrinipellin first reported from A, C.tetrahydrocrinipellin stipitaria [18,20]. Lately, A, crinipellin dihydrocrinipellin B, and dihydrocrinipellin A, tetrahydrocrinipellin B were first B, reported and crinipellin from C. C–Hstipitaria have been[18,20]. reported Lately, from dihydrocrinipellin two different A,Crinipellis tetrahydrocrinstrainsipellin [19,21 B,]. and In thecrinipellin present C–H study, have we been first reported isolated crinipellinsfrom two different from C. rhizomaticolaCrinipellis strainsand identified[19,21]. In the structurepresent study, of crinipellin we first isolated A and the crinipellins new derivative from crinipellinC. rhizomaticola I. and identified the structure of crinipellin A and the new derivative crinipellin I.

2.6.2.6. Minimum Minimum Inhibitory Inhibitory ConcentrationsConcentrations (MICs)(MICs) of Crinipellins A A and and I I TheThe MICs MICs of of crinipellin crinipellin AA ((11)) werewere determineddetermined against se sevenven plant-pathogenic plant-pathogenic fungi: fungi: AlternariaAlternaria porriporri, B., B. cinerea cinerea, C., C. coccodes coccodes, Fusarium, Fusarium oxysporum oxysporum, M., M. oryzae oryzae, P.,infestans P. infestans, and, andR. solani. R. solani.Of the Of testedthe tested fungi, C.fungi, coccodes C. coccodeswas found was be found the most be the sensitive most sensitive to crinipellin to crinipellin A (1) (MIC A ( =1) 1 (MICµg/mL), = 1 µg/mL), followed followed by M. oryzae by , P.M. infestans oryzae,, andP. infestans,B. cinerea and(MICs B. cinerea = 8, 31, (MICs and 31= 8,µ g/mL)31, and (Table 31 µg/mL)3). Lorenzen (Table and3). Lorenzen Anke demonstrated and Anke thatdemonstrated the high cytotoxic that the andhigh antimicrobialcytotoxic and activitiesantimicrob ofial crinipellins activities of A crinipellins and B are A due and to B the are presence due to ofthe the presence electrophilic of the enone electrophilic moiety enone (α-methylene-conjugated moiety (α-methylene-conjugated ketone group) ketone [10]. group) Other [10]. derivatives Other withoutderivatives the α-methylenewithout the ketone α-methylene group such ketone as dihydrocrinipellins group such as dihydrocrinipellins A and B, tetrahydrocrinipellins A and B, Atetrahydrocrinipellins and B, and crinipellins A Cand and B, D,and were crinipellins devoid ofC and antimicrobial, D, were devoid antitumor, of antimicrobial, and anti-inﬂammatory antitumor, activitiesand anti-inflammatory [18,19,21]. Similarly, activities the α[18,19,21].-methylene-conjugated Similarly, the α ketone-methylene-conjugated was also absent inketone the structure was also of crinipellin I (2) which could explain its a weak antifungal activity (MICs = 250 µg/mL) against A. porri, C. coccodes, M. oryzae, and P. capsici. Additionally, the in vitro antibacterial activity of crinipellins A (1) Molecules 2018, 23, x 7 of 11

absent in the structure of crinipellin I (2) which could explain its a weak antifungal activity (MICs = Molecules 2018, 23, 2377 7 of 11 250 µg/mL) against A. porri, C. coccodes, M. oryzae, and P. capsici. Additionally, the in vitro antibacterial activity of crinipellins A (1) and I (2) against nine plant-pathogenic bacteria was also investigated. andCrinipellin I (2) against A nine(1) exclusively plant-pathogenic exhibited bacteria an antibacterial was also activity investigated. against Crinipellin Acidovorax Aavenae (1) exclusively subsp. exhibitedcattleyae an (MIC antibacterial = 31 µg/mL), activity whereas against crinipellinAcidovorax I (2)avenae had nosubsp. antibacterialcattleyae activity(MIC (Table = 31 µ S1).g/mL), Kupka whereas et al. [20] showed that O-acetylcrinipellin A, identified from C. stipitaria, had an antimicrobial activity crinipellin I (2) had no antibacterial activity (Table S1). Kupka et al. [20] showed that O-acetylcrinipellin against Gram-positive bacteria, such as Bacillus spp., suggesting that O-acetylcrinipellin A had effects A, identiﬁed from C. stipitaria, had an antimicrobial activity against Gram-positive bacteria, such as on Gram-positive bacteria but not to Gram-negative strains [20]. In this study, it was found that most BacillusGram-negativespp., suggesting plant-pathogenic that O-acetylcrinipellin bacteria were not A hadaffected effects by crinipellin on Gram-positive A (1), except bacteria for A. butavenae not to Gram-negativesubsp. cattleyae strains (Table [20 S2).]. In this study, it was found that most Gram-negative plant-pathogenic bacteria were not affected by crinipellin A (1), except for A. avenae subsp. cattleyae (Table S2). Table 3. Minimum inhibitory concentration (MIC) of crinipellins A (1) and I (2) against plant- Table 3. Minimumpathogenic inhibitoryfungi. concentration (MIC) of crinipellins A (1) and I (2) against plant-pathogenic fungi.

MIC (µg/mL) (µg/mL) Plant-PathogenicPlant-Pathogenic Fungi Fungi 1 1 2 2 Alternaria porri 125 250 Alternaria porri 125 250 Botrytis cinereaBotrytis cinerea 31 31 >250 > 250 ColletotrichumColletotrichum coccodes coccodes 1 1 250 250 Fusarium oxysporumFusarium oxysporum 125 125 >250 > 250 MagnaportheMagnaporthe oryzae oryzae 8 8 250 250 Phytophthora infestans 31 250 Phytophthora infestans 31 250 Rhizoctonia solani 125 >250 Rhizoctonia solani 125 > 250

2.7. Suppressive2.7. Suppressive Effects Effects of Crinipellinof Crinipellin A A (1) (1) on on Plant Plant DiseasesDiseases Ca Causedused by by Plant-Pathogenic Plant-Pathogenic Fungi Fungi To investigateTo investigate the thein in vivo vivoantifungal antifungal activity activity ofof crinipellin A A (1 (),1), plants plants were were treated treated with with 125 125 andand 500 500µg/mL µg/mL of of this this compound compound prior prior to to inoculation inoculation with with the fungal the fungal pathogens. pathogens. As crinipellin As crinipellin I (2) I(2)was was isolated isolated in in a avery very small small amount, amount, its in its vivoin vivo antifungalantifungal activity activity assay assaywas not was performed. not performed. In In greenhousegreenhouse conditions,conditions, crinipellin crinipellin A A (1 ()1 exhibited) exhibited an anantifungal antifungal activity activity in plantin plantagainstagainst M. oryzaeM.,oryzae B. , B. cinereacinerea, P., P. infestans infestans,,P. P. triticina triticina,, andand C. coccodes, ,which which are are the the causative causative agents agents of RCB, of RCB, TGM, TGM, TLB, TLB, WLR,WLR, and PAN,and PAN, respectively. respectively. The treatmentThe treatment with with crinipellin crinipellin A (1 )A (500 (1) µ(500g/mL) µg/mL) showed showed disease disease control control values of 88%, 86%, 65%, 60%, and 50%, respectively, against TLB, TGM, PAN, WLR, and values of 88%, 86%, 65%, 60%, and 50%, respectively, against TLB, TGM, PAN, WLR, and RCB, whereas RCB, whereas no effect was observed against RSB and BPM (Figure 6). At a concentration of 125 no effect was observed against RSB and BPM (Figure6). At a concentration of 125 µg/mL, crinipellin µg/mL, crinipellin A (1) had disease control values ranging from 13% to 29% against TGM, TLB, and A(1)WLR had (Figure disease 6). control In addition values to the ranging disease from control 13% efficacy, to 29% no against phytotoxic TGM, symptoms TLB, and were WLR observed (Figure 6). In additionin the crinipellin to the disease A (1)-treated control plants efﬁcacy, (data no not phytotoxic shown). The symptoms disease control were observed spectrum in of the crinipellin crinipellin A A (1)-treated(1) was plants similar (data to that not of shown). the IUM00035 The disease culture control filtrate spectrum and ethyl of acetate crinipellin extract. A ( 1These) was results similar to thatsuggested of the IUM00035 that crinipellin culture A ﬁltrate (1) is anda major ethyl antifungal acetate extract. metabolite These produced results suggestedby the C. rhizomaticola that crinipellin A(1)IUM00035 is a major isolate antifungal and represents metabolite potential produced for by deve thelopmentC. rhizomaticola as a naturalIUM00035 fungicide isolate to control and represents plant- potentialpathogenic for development fungi. as a natural fungicide to control plant-pathogenic fungi.

Figure 6. In vivo antifungal activities of crinipellin A (1). (A) Plant disease inhibitory effects of crinipellin A on plants. RCB, rice blast; RSB, rice sheath blight; TGM, tomato gray mold; TLB, tomato late blight;

WLR, wheat leaf rust; BPM, barley powdery mildew; PAN, pepper anthracnose. n.s. indicates not shown, meaning that disease control effects of ethyl acetate extracts were not observed. (B) Representatives of the plants treated with crinipellin A (1). a, inoculation of pathogens without treatment of crinipellin A; b, treatment with crinipellin A (1) (500 µg/mL); c, treatment with crinipellin A (1) (125 µg/mL). Molecules 2018, 23, 2377 8 of 11

3. Materials and Methods

3.1. General Experimental Procedures The ESIMS was recorded on a single-quadruple mass spectrometer (Acquity QDa; Waters, Manchester, UK), and the HREIMS was measured by a JMS-700 MStation (JEOL, Tokyo, Japan). The IR spectrum was measured on a Smiths IdentifyIR spectrometer (Danbury, CT, USA) with an attenuated total reﬂection (ATR) device. The 1D and 2D NMR spectra were recorded by a Bruker Advance 500 MHz spectrometer (Bruker BioSpin, Rheinstetten, Germany) in CD3CN or CDCl3 (99.8 atom% D, Cambridge Isotope Laboratories, Tewksbury, MA, USA). Chemical shifts were referenced to solvent peaks (δH 1.94 ppm and δC 118.26 ppm for CD3CN; δH 7.26 ppm and δC 77.0 ppm for CDCl3).

3.2. Mushrooms and Culture Conditions The mushroom isolates used in this study were kindly provided by Dr. Jaehyuk Choi of Incheon National University, and the voucher specimen of the IUM00035 isolate was deposited in the “Culture Collection of Mushrooms” at Incheon National University. The isolates were maintained on potato dextrose agar (PDA) medium at 25 ◦C. To test the in vivo and in vitro antifungal activity of the mushroom isolates, 10 agar plugs that were punched with an 8 mm-diameter cork borer from culture plates were inoculated into 500 mL of potato dextrose broth (PDB) and incubated at 25 ◦C for three weeks.

3.3. Genomic DNA Extraction and Phylogenetic Analysis For the isolation of genomic DNA (gDNA), the IUM00035 isolate was grown in 50 mL of PDB medium at 25 ◦C for 4 days on a rotary shaker (150 rpm), and the gDNA was extracted using the cetyltrimethylammonium bromide (CTAB) procedure as previously described [22]. For phylogenetic analysis, the ITS region was amplified by universal primer sets ITS1 (50-TCCGTAGGTGAACCTGC GG-30) and ITS4 (50-TCCTCCGCTTATTGATATGC-30). The resulting amplicons were purified using the GeneAll ExpinTM PCR purification kit (GeneAll, Seoul, Korea) and then analyzed by a sequencing analysis service (Macrogen, Daejoen, Korea). The resulting sequences were analyzed with the BLASTn program of the NCBI (http://www.ncbi.nlm.nih.gov). Phylogenetic trees were constructed by the neighbor-joining method [23] using the program MEGA 6.0 [24]. The robustness at the individual branching points was estimated by bootstrapping with 1000 replications [25].

3.4. Isolation of Antifungal Compounds The culture broth (10 L) of the IUM00035 isolate was filtered with four layers of cheese cloth, and then successively extracted with ethyl acetate (2 × 10 L), and the aqueous phase was re-extracted with n-butanol (2 × 10 L). We obtained 1.4 g of ethyl acetate extract, 1.6 g of n-butanol extract, and 19.2 g of water extract. The ethyl acetate extract (1.4 g), which exhibited in vivo antifungal activity, was dissolved in a small amount of chloroform and subsequently loaded onto silica gel column (Merck, Darmstadt, Germany). The column was eluted with mixtures of chloroform/methanol (95.5:0.5, v/v) to obtain 10 fractions (E1–E10). For the antifungal activity-guided fractionation, all chromatographic fractions were investigated for their antifungal activity against rice blast fungus M. oryzae. Compounds 1 (34 mg) and 2 (7 mg) were finally purified from the active fraction E2 (44 mg) with a LC-6AD HPLC system (Shimadzu, Kyoto, Japan) equipped with a Polaris C18-A column (21.2 × 250 mm, 10 µm; Agilent, Santa Clara, CA, USA). The column was eluted with a linear gradient (80–100% for 50 min) of aqueous methanol at a flow rate of 5 mL/min. The effluent was monitored with the SPD-M10Avp photodiode array detector (Shimadzu). Molecules 2018, 23, 2377 9 of 11

3.4.1. Crinipellin A (1)

1 13 A colorless solid; UV (MeOH) λmax (logε): 235 nm (3.73); H- and C-NMR (CDCl3) data, Table1 and Table S1; ESIMS m/z 331 [M + H]+.

3.4.2. Crinipellin I (2)

20 A colorless solid; [α]D = −98.2 (c = 0.35, MeCN) UV (MeOH): λmax (logε): 235 nm (3.70); IR (ATR) −1 1 1 13 νmax 2956, 2874, 1735, 1458, 1375 cm ; H-NMR (CDCl3) data, Table1; H- and C-NMR (CD3CN) + data, Table2; HREIMS m/z 332.1985 [M ] (calculated for C20H28O4, 332.1988).

3.5. Optimization of the Incubation Time for the Production of Antifungal Substances To investigate the effect of the incubation time on the production of antifungal compounds, IUM00035 was incubated on PDB medium for 5, 7, 10, 14 and 21 days. The antifungal activity of each culture filtrate was measured with the broth microdilution method against S. cerevisiae. Culture filtrates of IUM00035 were recovered by centrifugation (20 min; 10,000× g) and sterilized with a syringe filter (0.2 µm pore size; Advantec, Tokyo, Japan). Each culture filtrate was diluted 10-, 20-, 40-, and 80-fold with YPD medium, and then yeast cells (OD600 = 0.03) were added. The PDB medium was used as a negative control, and azoxystrobin (0.03 µg/mL) was treated as a positive control. After an overnight ◦ incubation at 30 C, OD600 values were measured in an XMark microplate spectro-photometer (Bio-Rad, Hercules, CA, USA). The fungal inhibitory effect was calculated with the following equation: inhibition (%) = 100 × [1 − (OD600 of treatment/OD600 of non-treatment)].

3.6. In Vitro Antifungal Activity Assay against Plant Pathogens The MIC values of crinipellins A (1) and I (2) against plant-pathogenic fungi were determined by the broth microdilution method using two-fold serial dilutions staring with 250 µg/mL as described by the modiﬁed CLSI M38-A method [26]. Spore suspensions (5 × 104 spores/mL) of A. porri, B. cinerea, C. coccodes, F. oxysporum, M. oryzae, P. infestans, and R. solani were added to the wells of a 96-well microtiter plate. Crinipellins dissolved in methanol (25 mg/mL) were added and then serially two-fold diluted to reach the ﬁnal concentrations ranging from 0.5–250 µg/mL. The chemical fungicide azoxystrobin (0.03 µg/mL) and PDB medium containing 1% methanol was used as positive and negative controls, respectively. The microtiter plates were incubated for 2–3 days, and the MIC values were determined by visual inspection of complete growth inhibition [26]. The assay was performed two times with three replicates for each compound at all concentrations investigated.

3.7. Disease Control Efficacy Test against Plant Pathogens To investigate the plant disease control efficacies, solvent extracts (2000, 1000, and 500 µg/mL) and crinipellin A (1) (500 and 125 µg/mL) were adjusted by dissolving in a 5% aqueous methanol solution containing 0.025% Tween 20. The final concentration of the methanol in each treatment did not exceed 5% of the volume. As a control, we used 5% aqueous methanol solution containing 0.025% Tween 20. The 1- and 3-fold-diluent culture filtrates containing 0.025% Tween 20 were directly sprayed onto the plants. After treatment with the culture filtrate, solvent extracts, and crinipellin A (1), the plants were inoculated with a fungal pathogen and incubated as previously described [27,28]. As hosts for the pathogens, we used rice (Oryza sativa L, cv. Nakdong), tomato (Solanum lycopersicum cv. Seokwang), barely (Hordeum sativum cv. Dongbori), wheat (Triticum aestivum cv. Eunpa), and pepper (Capsicum annuum cv. Bugang), which were grown in a greenhouse at 25 ± 5 ◦C for 1–4 weeks. Disease severity was evaluated on days 3 to 11 after inoculation. The experiment was conducted twice with three replicates for each treatment, and the disease control efficacy was calculated with the following equation: control efficacy (%) = 100 × [1 − B/A], where A is the mean lesion area (%) on the leaves or sheaths of the control plants, and B is the mean lesion area (%) on the leaves or sheaths of the treated plants [27,28]. Molecules 2018, 23, 2377 10 of 11

3.8. Statistical Analysis Data were subjected to one-way ANOVA, and the means of the treatments were separated by Duncan’s multiple range test (p < 0.05) using the R-software (Version 3.3.1).

4. Conclusions Regardless of the versatile bioactivity of crinipellins such as antitumor, anti-inﬂammatory, and antimicrobial activities, their antifungal property against plant-pathogenic fungi has not yet been tested. In the present study, crinipellin A (1) and a new derivative (crinipellin I (2)) were identiﬁed in C. rhizomaticola, and these compounds exhibited an antifungal activity against plant-pathogenic fungi. These results suggest that C. rhizomaticola and crinipellins have potential in the development of natural fungicides for the control of various plant diseases.

Supplementary Materials: The following are available online. Figure S1, HPLC data of compounds 1 and 2; Figures S2 and S3, MS data for compounds 1 and 2; Figures S4–S11, NMR spectra of compounds 1 and 2; Table S1, 13 C-NMR data (CDCl3) of compound 1; and Table S2, MIC of crinipellins against plant pathogenic bacteria. Author Contributions: J.W.H., and M.O. performed the experiments, analyzed data, and wrote the paper; Y.J.L. performed the experiments; J.C. collected and identified the mushroom materials. G.J.C. supervised this study and designed experiments. H.K. supervised and financed this study, designed experiments, and wrote the paper. Funding: This research was funded by the Next-generation BioGreen21 program (No. PJ01180201) of the Rural Development Administration and a project BS.K18M504 of the Korea Research Institute of Chemical Technology, Republic of Korea. Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compound 1 and 2 are available from the authors.

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