Appl Biol Chem (2016) 59(3):385–389 Online ISSN 2468-0842 DOI 10.1007/s13765-016-0178-3 Print ISSN 2468-0834

ARTICLE

Antifungal of modified neolignans from Hu

Saksri Sanyacharernkul1 . Sirinapa Nantapap1 . Kallaya Sangrueng2 . Narong Nuntasaen3 . Wilart Pompimon4 . Puttinan Meepowpan1

Received: 24 August 2015 / Accepted: 28 January 2016 / Published online: 15 February 2016 Ó The Korean Society for Applied Biological Chemistry 2016

Abstract Chromatographic separation of leaves and Introduction twigs from M. wangii had led to the isolation of cono- carpan (1) and 30-methoxyconocarpan (2). These structures The Mitrephora belongs to the family were assigned on the basis of spectroscopic methods. and comprises approximately 48 distributed in Compound 1 was modified by organic reactions and the (Deepralard et al. 2007; Rayanil et al. 2013)suchas modified compound 5 exhibited potent antifungal, Pyric- , Indonesia, Malaysia, Thailand, , and ularia oryzae. The method used poisoned food technique Southern . M. wangii is a small to medium size . with mycelial growth inhibition for 14 days, 57 % It has a smooth bark, devoid of patterns. The oblong (100 ppm). Compound 5 was stronger than mancozeb leaves are glossy from above, with the undersides cov- which the fungi toxicity drug used as a positive control. ered by fine hairs. The flowers possess a creamy yellow shade. The leaves and twigs of M. wangii were collected Keywords Annonaceae Á Conocarpan Á Mitrephora from Chiang Mai province, Thailand, in 2012. BKF no. wangii Á Neolignans Á Pyricularia oryzae 99291. It was identified by Mr. Narong Nuntasaen, The Forest Herbarium, Department of National Park Wildlife and Conservation, Ministry of Natural Resources and Environment. The previous phytochemical investi- gations of Mitrephora species discovered diterpenoids (Zgoda-Pols et al. 2002;Deepralardetal.2007;Meng et al. 2007; Rayanil et al. 2013), polyacetylene car- boxylic acids/esters (Zgoda-Pols et al. 2001;Lietal. & Wilart Pompimon [email protected] 2009), lignans (Deepralard et al. 2007;Geetal.2008), sesquiterpenes (Deepralard et al. 2007;Geetal.2008), & Puttinan Meepowpan [email protected] and alkaloids (Deepralard et al. 2007). Among these compounds, the diterpenoids and alkaloids showed sig- 1 Department of Chemistry and Center for Innovation in nificant antimicrobial, antimalarial, and cytotoxic activ- Chemistry, Faculty of Science, Chiang Mai University, ities (Zgoda-Pols et al. 2002;Lietal.2005;Mueller Chiang Mai 50200, Thailand et al. 2009). M. wangii has revealed the presence of 2 Department of Chemistry, Faculty of Science and several alkaloids, phenolic amide, steroids, neolignans, Technology, Phranakhon Rajabhat University, Bangkok 10220, Thailand and allantoin. The neolignan derivative compounds, namely conocarpan (1)and30-methoxyconocarpan (2), 3 The Forest Herbarium, Department of National Park, Wildlife and Plant Conservation, Ministry of Natural Resources and exhibited significant cytotoxicities (Tanamatayara, 2011). Environment, Bangkok 10900, Thailand However, there has been no reported either phytochem- 4 istry or biological activities of M. wangii prior to our Laboratory of Natural Products, Department of Chemistry, Faculty of Science, Center for Innovation in Chemistry, work. Lampang Rajabhat University, Lampang 52100, Thailand 123 386 Appl Biol Chem (2016) 59(3):385–389

K CO CH3 2 3 H C excess MeI 3 OCH3 acetone, room temp. O (3) (99%)

3'' 1'' CH3 3 eq. PhCOCl CH3 O H 4 2' 3' 3 3C 5 3a 3 H3C 4' 1.3 eq. NEt3 2'' 2 OH O 1' 6 O DCM, room temp. O 7 7a 6' 5' (54%) Conocarpan (1) (4) 3 eq. Ac2O CH3 O H C CH 1.3 eq. NEt3 3 3 O DCM, room temp. O (5) (93%)

Fig. 1 Structural modification products of compounds 3–5

Materials and methods 24 h. The resulting mixture was filtrated, evaporated to dryness, and purified to methylation product by preparative Extraction and isolation thin-layer chromatography (EtOAc:hexane (2:8) as a developing solvent). Air-dried leaves and twigs of M. wangii (3 kg) (BKF no. Compound 4 (77.6 mg, 54 % yield) was prepared by 99291) were successively macerated with three organic benzoylation of conocarpan (1) (99.9 mg) which was dis- solvents such as hexane (7 L 9 5 time), ethyl acetate (7 L solved in CH2Cl2 and adding benzoyl chloride to the 9 7 time), and methanol (6 L 9 4 time), followed by fil- solution mixture. Then, Et3N was added to the reaction tration. The filtrates were evaporated to dryness under mixture and then stirred at room temperature for 24 h. The reduced pressure to afford 307.15, 296.83, and 190.00 g of resulting mixture was quenched with an aqueous saturated crude extracts, respectively. The hexane extract was sepa- solution of NaHCO3 (10 mL 9 3 times) and extracted with rated by silica gel column (No. 7734, 600 g), eluting with CH2Cl2 (10 mL 9 3 times). The combined organic layer gradient hexane and ethyl acetate (100:0 to 0: 100). Frac- was dried (MgSO4), filtered, and concentrated in vacuo. tions with similar pattern on TLC (10 % ethyl acetate in Purification of the residue by preparative thin-layer chro- hexane) were combined to yield 5 fractions (HA1-HA5). matography (EtOAc:hexane (2:8) as a developing solvent) The fraction HA3 was rechromatographed (silica gel) fol- gave benzoylation product. lowed by crystallization with EtOH to obtain a compound 1 Compound 5 (113.7 mg, in 93 % yield) was obtained by viz. conocarpan (1) (0.4959 g). Ethyl acetate extract was acetylation of conocarpan (1) (104.5 mg) when anhydrous isolated by column chromatography (CC) technique to give Ac2O was employed in the benzoylation mentioned above. 8 fractions (EA1-EA8). The fraction EA2 was rechro- matographed 3 times to achieve compound 2, to be precise 0 3 -methoxyconocarpan (2) (0.0042 g), and fraction EA5 Antifungal activity assay was also rechromatographed 3 times to afford compound 1 (0.9700 g). Identification of the isolated compounds was The antifungal activity of pure and modified compounds done by means of spectroscopic methods (1H, 13CNMR was evaluated against P. oryzae separately by the poisoned and 2D NMR, IR, EI-MS) and comparison with those food technique (Grover and Moore 1962). Treatment sets reports. comprise the concentration at 100, 200, and 300 ppm. Each concentration was aseptically poured into the petri plate Structural modification followed by the addition of melted PDA and was swirled gently to achieve thorough mixing of the contents. In Compound 3 (94.1 mg, 99 % yield) was prepared by control sets, requisite amounts of sterile water were added methylation of conocarpan (1) (101.1 mg) which was dis- to the medium. After the solidification of the media, the solved in acetone (20 mL) and adding K2CO3 to the mycelia of 10 mm diameter of fungus (taken from a 7-day- solution mixture. Then, the excess MeI was added to the old culture) were put into the center of each plate. The reaction mixture and then stirred at room temperature for treatment and control sets were incubated at 25 ± 2 °C for

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1 13 Table 1 H (400 MHz) and C 100 (MHz) NMR spectroscopic data for 3–5 in CDCl3 3'' 1'' CH3 CH3 CH O 6''' 5''' CH3 O H C 4 2' 3' 3 3 5 3a 3 H3C H C H3C CH 4' 3 1''' 4''' 3 2'' 2 OH OCH O 1''' 1' 3 O 6 O O 2''' 3''' O 7 7a 6' 5' O Conocarpan (1) Compound 3 Compound 4 Compound 5

Position Conocarpan (1) Compound (3) Compound (4) Compound (5) d13C (DEPT) d1H(mult, J Hz) d13C (DEPT) d1H(mult, J Hz) d13C (DEPT) d1H(mult, J Hz) d13C (DEPT) d1H(mult, J Hz)

1 –– –– –– –– 2 92.70 (CH) 5.11 (1H, d, 8.9) 92.82(CH) 5.08 (1H, d, 8.9) 92.13 (CH) 5.17 (1H, d, 8.6) 92.07 (CH) 5.12 (1H, d, 8.6) 3 45.01 (CH) 3.43 (1H, m) 45.15(CH) 3.39 (1H, m) 45.57 (CH) 3.41 (1H, m) 45.49 (CH) 3.36 (1H, m) 3a 132.31 (C) – 132.53 (C) – 133.66 (C) – 133.47 (C) – 4 120.73 (CH) 7.17 (1H, s) 120.73 (CH) 7.12 (1H, obsc.) 120.81 (CH) 7.22 (1H, obsc.) 120.79 (CH) 7.09 (1H, obsc.) 5 131.34 (C) – 131.25 (C) – 131.44 (C) – 131.42 (C) – 6 126.26 (CH) 7.16 (1H, d, 8.2) 126.28 (CH) 7.12 (1H, obsc.) 126.37 (CH) 7.22 (1H, obsc.) 126.35 (CH) 7.09 (1H, obsc) 7 109.24 (CH) 6.81 (1H) 109.21 (CH) 6.76 (1H) 109.33 (CH) 6.79 (1H) 109.30 (CH) 6.76 (1H) 7a 157.96 (C) – 159.73 (C) – 158.25 (C) – 158.21 (C) –

8 17.68 (CH3) 1.41 (3H, d, 6.8) 17.92(CH3) 1.38 (3H, d, 6.8) 18.13 (CH3) 1.43 (3H, d, 6.8) 18.10 (CH3) 1.39 (3H, d, 6.8) 10 132.43 (C) – 132.53 (C) – 132.09 (C) – 132.09(C) – 20,60 127.86 (2CH) 7.29 (2H, d, 8.5) 127.60 (2CH) 7.34 (2H, obsc.) 127.14 (2CH) 7.49 (2H, obsc.) 127.14 (2CH) 7.40 (2H, obsc.) 30,50 115.49 (2CH) 6.81 (2H, d, 6.6) 114.06 (2CH) 6.90 (2H, obsc.) 121.76 (2CH) 7.13 (2H, obsc.) 121.76 (2CH) 7.09 (2H, obsc.) 40 155.66 (C) – 158.35(C) – 150.51 (C) – 150.51 (C) – 40-O-Sub - (OH) 5.85 (1H, s) 55.32 (C) 3.80 (3H, s) 165.15 (C) – 169.48 (C) –

1’’’ – – – – 138.52 (C) – 21.16 (CH3) 2.26 (3H, s) 2’’’, 6’’’ – – – – 130.20 (CH) 8.20 (1H, obsc.) – – 3’’’, 5’’’ – – – – 128.59 (CH) 7.49 (1H, obsc.) – – 4’’’, – – – – 129.44 (CH) 7.62 (1H, obsc.)– – 100 130.68 (CH) 6.41 (1H, dd, 15.7, 1.5) 130.79 (CH) 6.36 (1H, dd, 15.7, 1.6) 130.69 (CH) 6.36 (1H, dd, 15.7, 1.6) 130.67(CH) 6.35 (1H, dd, 15.7, 1.6) 200 123.08 (CH) 6.13 (1H, dq, 15.7, 6.6) 124.56 (CH) 6.09 (1H, m) 123.17 (CH) 6.09 (1H, m) 123.17 (CH) 6.08 (1H, m) 00 3 18.34 (CH3) 1.90 (3H, dd, 6,6, 1.6) 18.88 (CH3) 1.85 (3H, dd, 6.6, 1.6) 18.43 (CH3) 1.85 (3H, dd, 6.6, 1.6) 18.43 (CH3) 1.84 (3H, dd, 6.6, 1.6) Sub substituent group, obsc obscure signal 123 387 388 Appl Biol Chem (2016) 59(3):385–389

Table 2 Mycelial growth Compounds Concentrations (ppm) Percentage inhibition of mycelial growth inhibition (P. oryzae)of conocarpan (1) and modified 7 days 14 days compounds 3-5 at different concentrations in comparison 1 100 00.00 ± 0.00 00.00 ± 0.00 with mancozeb for 7 and 14 200 3.95 ± 0.14 2.32 ± 0.06 periods 300 23.03 ± 0.09 23.84 ± 0.78 3 100 1.50 ± 0.14 6.30 ± 0.08 200 8.95 ± 0.56 15.83 ± 0.60 300 18.95 ± 0.44 30.79 ± 0.20 4 100 7.50 ± 0.14 13.50 ± 0.96 200 11.00 ± 0.13 26.05 ± 0.52 300 22.75 ± 0.55 37.69 ± 0.09 5 100 37.50 ± 0.37 57.75 ± 0.32 200 45.41 ± 0.18 59.20 ± 0.21 300 51.82 ± 0.13 67.54 ± 0.10 Mancozeb (positive control) 100 10.53 ± 0.25 18.54 ± 0.13 200 53.95 ± 0.29 63.91 ± 0.90 300 60.53 ± 0.01 81.46 ± 0.03 The values are means of three replicates ± standard error. All compounds were dissolved in 5 % acetone

7 and 14 days. Assessment of the fungitoxicity was carried methyl singlet at d 3.80 (s, 3H) and 13CNMR spectrum out in terms of the percentage of mycelial growth inhibition appeared at d 55.32 ppm (CH3), which was the chemical (Dixit et al. 1978). shift value of methoxy group. On the other hand, hydroxyl d at 5.85 ppm disappeared after the methylation reaction dc À dt Mycelial growth inhibition ðÞ¼% Â 100; occured. Compound 4 was produced from the benzoylation dc reaction of conocarpan, which was a yellow solid. The where d is the average increase in mycelial growth in c molecular formula was established as C25H22O3 consistent 1 control set (mm) and dt is the average increase in mycelial with molecular ion peak at m/z 370.16 (EI-MS). The H growth in treatment set (mm). NMR and 13C NMR spectra of 4 were closely related to those of conocarpa. However, the hydroxyl group was substituted by the benzoyl group, the which has the 1H Results and discussion NMR spectrum appeared at d 8.20 ppm (obsc.,1H), 7.49 ppm (obsc., 1H), and 7.62 ppm (obsc., 1H) and 13C Chemical structure elucidation NMR spectrum appeared at d 165.15 ppm (C=O), 138.52 ppm (C), 130.20 ppm (2CH), 128.59 ppm (2CH), The present study reported the isolation of conocarpan (1) and 129.44 ppm (CH). Compound 5 was obtained as a and 30-methoxyconocarpan (2) from leaves and twigs of white colorless solid and its molecular formula was

M. wangii. Compounds 1 and 2 have previously been determined to be C20H20O3 from its molecular ion peak at isolated from Krameria cytisoides (Achenbach et al. 1987). m/z 308.12 (EI-MS). In addition, similar to the compounds Conocarpan (1) was shown to exhibit anticytotoxic (Ri- 3 and 4, the compound 5 also has the 1H NMR and 13C mando et al. 1994), antibacterial (Arnone et al. 1988), and NMR spectra closely related to those of conocarpan, antiyeast (Freixa et al. 2001) properties. Moreover, the including the hydroxyl substitution at 40-O-sub position. 1H conocarpan (1) was partially synthesized by methylation, NMR spectrum of the substituent appeared at d 2.36 (s, 3H) benzoylation, and acetylation (Fig. 1). and 13C NMR spectrum appeared at d 169.48 ppm (C=O)

Compound 3 was obtained as a white colorless solid. and 21.16 ppm (CH3), which was the chemical shift value The molecular formula was valid as C19H20O2 from the EI- of acetyl group. MS ion at m/z 280.02, while the molecular weight of 1 was Conocarpan (1) and its modified compounds 3–5 were 266.34 m/z, suggesting that one methyl group was sub- tested for rice blast disease. The modified compound 5, one stituent on the molecule of precursor. Its spectroscopic data of the important agricultural fungicides against P. oryzae, (1H NMR and 13CMNR) were closely related to those of 1 is a fungal pathogen that causes rice blast disease (Tanaka (Table 1). However, the 1H NMR spectrum showed the et al. 2009).

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Antifungal activity test Ge F, Tang CP, Ye Y (2008) Lignanamides and sesquiterpenoids from stems of Mitrephora thorelii. Helv Chim Acta 91:1023–1030 The neolignan, conocarpan (1), and its derivatives revealed Grover RK, Moore JD (1962) Toximetric study of fungicides against several biological activities such as anti-inflammatory brown rot organism Sclerotinia fructicola and S. laxa. Phy- (Baumgartner et al. 2011a, b), antifungal (Pessini et al. topathology 52:876–880 2005; Lemos et al. 2013), antinociceptive (Silva et al. Lemos COT, Svidzinsk TIE, Baeza LC, Miranda N, Nakamura CV, Cortez DAG, Cardozo-Filho L, Cabral VF (2013) Characteriza- 2010) and anti-tuberculosis activities (Scodro et al. 2013). tion and anti-inflammatory effects of iodinated acetylenic acids Therefore, we modified structure 1 to the active compounds isolated from the Marine Sponges Suberites mammilaris and 3–5 for antifungal test. Compound 1 and modified com- Suberites japonicas. Nat Prod Res 27:2355–2359 pounds 3–5 induced inhibition of the mycelial growth of Li C, Lee D, Graf TN, Phifer SS, Nakanishi Y, Burgess JP, Riswan S, Setyowati FM, Saribi AM, Soejarto DD, Farnsworth NR, the test fungus P. oryzae in Table 1. Fungicide (mancozeb) Falkinham JO III, Kroll DJ, Kinghorn AD, Wani MC, Oberlies was used as a positive control. Compound 5 showed highly NH (2005) A hexacyclic ent-trachylobane diterpenoids possess- significant fungitoxicity of about three-fold at 100 ppm for ing an oxetane ring from Mitrephora glabra. Org Lett 7: 14 days (57.75 %) in comparison with mancozeb 5709–5712 Li C, Lee D, Graf TN, Phifer SS, Nakanishi Y, RiswanS Setyowati (18.54 %), while other concentrations showed lower inhi- FM, Saribi AM, Soejarto DD, Farnsworth NR, Falkinham JO III, bitions than positive control. Alternatively, conocarpan (1) Kroll DJ, Kinghorn AD, Wani MC, Oberlies NH (2009) and compounds 3 and 4 showed less inhibitory potency Bioactive constituents of the stem bark of Mitrephora glabra. compared with compound 5 and fungicide at the same J Nat Prod 72:1949–1953 Meng DH, Xu YP, Chen WL, Zou J, Lou LG, Zhao WM (2007) Anti- concentrations (Table 2). tumor clerodane-type diterpenes from Mitrephora thorelii. J Asian Nat Prod Res 9:679–684 Acknowledgments The authors thank the Center for Innovation in Mueller D, Davis RA, Duffy S, Avery VM, Camp D, Quinn RJ (2009) Chemistry (PERCH-CIC) and Commission on Higher Education, Antimalarial activity of azafluorenone Alkaloids from the Ministry of Education, and the grant of Thailand’s Office of Higher Australian Tree Mitrephora diversifolia. J Nat Prod Education Commission for the Project on Higher Education and 72:1538–1540 Research Promotion (HERP) 002/2014 for generous financial support. Pessini GL, Dias-Filho BP, Nakamura CVN, Cortez DAG (2005) In addition, we thank the Department of Chemistry, Faculty of Sci- Antifungal activity of the extracts and neolignans from Piper ence, Chiang Mai University, for facilities supporting this research. regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck. J Braz Chem Soc 16:1130–1133 Rayanil KO, Limpanawisut S, Tuntiwachwuttikul P (2013) Ent- References pimarane and ent-trachylobane diterpenoids from Mitrephora alba and their cytotoxicity against three human cancer cell lines. Phytochemistry 89:125–130 Achenbach H, Grob J, Dominguez XA, Cano G, Star JV, Brus- Rimando A, Pezzuto J, Farnsworth N, Santisuk T, Reutrakul V (1994) soloLDC Munoz G, Salgado F, Lopez L (1987) Lignans Revision of the NMR assignments of pterostilbene and of neolignans and norneolignans from Krameria cystisoides. Phy- dihydrodehydrodiconieferyl alcohol: cytotoxic constituents from tochemistry 26:1159–1166 Anogeissus acuminata. Nat Prod Lett 4:267–272 Arnone A, Modugno DV, Nasini G, Venturini I (1988) Isolation and Scodro RBL, Pires CTA, Carrara VS, Lemos COT, Cardozo-Filho L, structure determination of neiv active neolignans and norneolig- Souza VA, Correˆa AG, Siqueira VLD, Lonardoni MVC, Cardoso nans from Ratanhia. Gazz Chim Ital 118:675–682 RF, Cortez DAG (2013) Anti-tuberculosis neolignans from Piper Baumgartner L, Sosa S, Atanasov AG, Bodensieck A, Fakhrudin N, regnellii. Phytomedicine 20:600–604 Bauer J, Favero GD, Ponti C, Heiss EH, Schwaiger S, Ladurner Silva RZD, Yunes RA, Souza MMD, Monache FD, Cechinel-Filho V A, Widowitz U, Logoa RD, Rollinger JM, Werz O, Bauer R, (2010) Antinociceptive properties of conocarpan and orientin Dirsch VM, Tubaro A, Stuppner H (2011a) Lignan derivatives obtained from Piper solmsianum C. DC. var. solmsianum from Krameria lappacea roots inhibit acute inflammation in vivo (Piperaceae). J Nat Med 64:402–408 and pro-inflammatory mediators in vitro. J Nat Prod Tanaka M, Nakayashiki H, Tosa Y (2009) Population structure of 74:1779–1786 eleusine isolates of Pyricularia oryzae and its evolutionary Baumgartner L, Schwaiger S, Stuppner H (2011b) Quantitative implications. J Gen Plant Pathol 75:173–180 analysis of anti-inflammatory lignan derivatives in Ratanhiae Tanamatayara P (2011) Bioactive compounds from Pterospermum radix and its tincture by HPLC–PDA and HPLC–MS. J Pharma- grande Craib and Mitrephora wangii Hu. Silpakorn University, ceut Biomed 56:546–552 Thailand Deepralard K, Pengsuparp T, Moriyasu M, Kawanishi K, Suttisri R Zgoda-Pols JR, Freyer AJ, Killmer LB, Porter JR (2001) Poly- (2007) Chemical constituents of Mitrephora maingayi. Biochem acetylene carboxylic acids from Mitrephora celebica. J Nat Prod Syst Ecol 35:696–699 64:1348–1349 Dixit SN, Tripathi NN, Tripathi SC (1978) Fungitoxicity of some seed Zgoda-Pols JR, Freyer AJ, Killmer LB, Porter JR (2002) Antimicro- extract. Natl Acad Sci Letters 1:287–288 bial diterpenes from the stem bark of Mitrephora celebica. Freixa B, Vila R, Ferro E, Adzet T, Cangueral S (2001) Antifungal Fitoterapia 73:434–438 principles from Piper fulvens. Planta Medica 67:873–875

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