J. Antibiot. 58(1): 50–55, 2005

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1 Antibiot. 58(1): 50–55, 2005 J. THE JOURNAL OF ORIGINAL ARTICLE ANTIBIOTICS g g g g -Lactone Form Nafuredin, Nafuredin- , also Inhibits A Helminth Complex I Kazuro Shiomi, Hideaki Ui, Hideaki Suzuki, Hiroko Hatano, Tohru Nagamitsu, Daisuke Takano, Hiroko Miyadera, Tetsuo Yamashita, Kiyoshi Kita, ̄ Hideto Miyoshi, Achim Harder, Hiroshi Tomoda, Satoshi O mura Received: September 21, 2004 / Accepted: December 10, 2004 © Japan Antibiotics Research Association Nafuredin, a d attractive targets for treatment of helminthiasis. NFRD is a Abstract -lactone antibiotic, is a fungal part of electron transport system of a unique energy metabolite showing selective helminth NADH-fumarate metabolism found in many anaerobic organisms such as reductase inhibition, and whose target had been revealed as helminths, and it is composed of complex I and complex II. complex I. We found that nafuredin is easily converted to nafuredin- g Electrons from NADH are accepted by rhodoquinone by eak alkaline treatment. The structure of w through complex I (NADH-rhodoquinone oxidoreductase), g -lactone form of g as elucidated as a w nafuredin- nafuredin with keto-enol tautomerism. Nafuredin- g shows and then transferred to fumarate through complex II similar complex I inhibitory activity as nafuredin, and it (rhodoquinol-fumarate reductase). This electron transport is used to generate ATP in the absence of oxygen, which is also possesses anthelmintic activity in vivo . different from aerobic one. Keyw ords nafuredin, complex I, NADH-fumarate Therefore, we have screened NFRD inhibitors using reductase, anthelmintic antibiotic mitochondria of Ascaris suum (pig roundworm), and 1 obtained a novel inhibitor, nafuredin ( ), from the cultured FT-0554 isolated from a marine Aspergillus niger broth of sponge [6, 7]. Compound 1 inhibited NFRD of A. suum at Introduction nanomolar levels, while it showed very weak inhibition to Microorganisms produce many useful antiparasitic agents the mammalian enzyme [6]. Its target was revealed as  [1 complex I, and it also showed anthelmintic activity against 5]. We have carried out screening for inhibitors of ADH-fumarate reductase (NFRD) from microbial Haemonchus contortus (barber pole worm) in in vivo trials N is a new potential lead compound as a 1 using sheep. Thus, metabolites to find new class of anthelmintics. Differences -lactone with in energy metabolisms between hosts and helminths are d novel anthelmintic. Its structure is an epoxy- H. Miyoshi: Division of Applied Life Sciences, Graduate School K. Shiomi (Corresponding author) , H. Ui, T. Nagamitsu, D. of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, T School of Pharmaceutical Sciences, Kitasato University, akano: Sakyo-ku, Kyoto 606-8502, Japan 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan, E-mail: Bayer HealthCare AG, Animal Health-Research & A. Harder: [email protected] ̄ Development-Parasiticides, Agricultural Center Monheim, D- S. O mura (Correspoding author) , H. Suzuki, H. Hatano, H. 40789 Monheim, Germany omoda: T The Kitasato Institute, 5-9-1 Shirokane, Minato-ku, ̄ H. Tomoda, S. O o 108-8641, Japan, E-mail: [email protected] ky To Kitasato Institute for Life Sciences, mura: H. Miyadera, T. Yamashita, K. Kita: Department of Biomedical Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108- Chemistry, Graduate School of Medicine, The University of 8641, Japan ky To o, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

2 51 ). 2 ( g ) and nafuredin- 1 Structures of nafuredin ( Fig. 1 a methylated olefinic side chain [7]. The absolute -lactone. Thus, the structure of tautomer g xygen to form o w 2 w configuration of as elucidated by comparing ozonolysis B of as elucidated as 5-hydroxy-4,10,12,16-tetramethyl- 1 and their corresponding synthetic compounds 2-oxo-6,8,12,14-octadecatetraeno-4-lactone. 1 products of 1 has been also achieved [9]. The structure of lactone moiety of tautomer A was [8], and total synthesis of similar to that of tautomer B. Long-range couplings by 1 , we found that 1 In the course of total synthesis of is by  CH–C(CH 2 )–CHOH easily converted to another compound w eak alkaline HMBC indicated the alignment of 3 also showed NFRD inhibitory activity. So 2 treatment, and d as shown in Fig. 2. C-3 ( 121.5) of tautomer A is a C studied its structure and elucidated it as g -lactone methine instead of a methylene of tautomer B. A long- we d tautomers. The compound as named nafuredin- g . Here, range coupling was observed between H-3 ( 2 6.17) and C- w H 1 ( we report the structure elucidation, enzyme inhibitory d 144.2) was very broad 169.0). Though C-2 signal ( d C C . and no correlation was observed, the chemical shifts of C-1 activity, and anthelmintic activity of 2 -lactone. Thus, the to C-4 suggested that they form g as elucidated as 2,5- w 2 structure of tautomer A of dihydroxy-4,10,12,16-tetramethyl-2,6,8,12,14-octadeca- Results and Discussion pentaeno-4-lactone. Therefore, as deduced to have keto- w 2 as added to methanol solution of 1 2 w as confirmed When CaCO w , 1 enol tautomerism at C-2. The structure of was 3 its total synthesis [10]. by revealed that its 2 . HR-FAB-MS of 2 readily converted to H 1 molecular formula is C The postulated conversion mechanism from is , which was the same as that 2 to O 22 4 32 of shown in Fig. 3. An enol ( a ) would be formed under basic . The structure of 2 w as elucidated by NMR study 1 (Table 1 and Fig. 2). The side chain signals of (from C-6 1 condition at first, and then the opening reaction of the b 2 , and it was confirmed by -alcohol ( t epoxide would occur. The resulting to C-18) were also observed in ) that ) T OCSY and HMBC experiments. Many proton signals 2 -lactone ( g cannot be isolated would successively afford (from 3-H to 9-H) and carbon signals (from C-1 to C-13 by translactonization. 2 and C-15) of we re duplicated, which suggested that the lactone moiety of Biological Activities 2 may have tautomerism. We elucidated the structure of one tautomer (tautomer B) v 1 and 2 against A. suum The IC NFRD were 9.7 first. The TOCSY result revealed that a hydroxymethine alues of 50 and 6.4 nM, respectively. Since 5.11) is connected to C-6. w d 4.22, and as shown to inhibit d 77.0, 1 d ( C-5 5-OH 5-H 1 13 C long-range couplings of 3-H H- ( d helminth complex I selectively [6], we evaluated the 2.55, 2.88)/C-4 H 2 23.3), 3-H inhibition of /C-5, 4-CH 84.9), 3-H ( d 2 . Compound 2 complex I by A. suum /4-CH ( ( d d C 2 3 C 2 3 H d /C-5, and 5-H/C-4 inhibited about two and ten times more potently than 1 1.48)/C-3 ( /C-4, 4-CH 40.6), 4-CH C 3 3 w ere observed by HMBC, which indicated the alignment of against NADH-ubiquinone reductase and NADH- CH )–CHOH. Long-range couplings from 3-H rhodoquinone reductase, respectively (Table 2). The –C(CH to 3 2 2 against bovine liver complex I was very 193.7) proved that C-1, C-2, inhibition of 2 d 161.5) and C-2 ( C-1 ( d C C 13 did not inhibit A. suum complex II (succinate- and C-3 are aligned. Chemical shifts in C NMR indicated w eak, and 2 . Therefore 1 ubiquinone reductase) at 1,000 nM as same as that C-1 is an ester carbonyl carbon and C-4 is an against complex I was similar or 2 inhibitory activity of xycarbon, and they are suggested to be connected o via

3 52 52 13 1 able 1 2 C NMR data of H and T automer A (enol form) T Tautomer B (keto form) P osition 1 1 13 a a 13 a a H C H C 161.5 s 11 69.0 s 21 44.2 s 193.7 s 8.7 Hz) J 21.5 d  6.17 s (1H) 40.6 d 31 1 2.55 d (1H,  8.7 Hz) 1 J 2.88 d (1H, 4 86.2 s 84.9 s 4-CH 23.3 q 21.3 q 1.32 s (3H) 1.48 s (3H) 3 57 77.0 d 4.22 m (1H) 4.08 m (1H) 6.6 d 4.32 br d (1H, J 3.6 Hz) 4.0 Hz) 5.11 br d (1H, J  5-OH  61 30.0 d 5.58 dd (1H, J  7 .2, 15.1 Hz) 128.7 d 5.61 dd (1H, J  7 .2, 15.1 Hz) 0.5, 15.1 Hz) 71 33.8 d 6.25 dd (1H, J  1 0.5, 15.1 Hz) 135.0 d 6.34 dd (1H, J  1 J 81 1 0.5, 15.4 Hz)  J 6.05 dd (1H, 128.3 d 0.5, 15.4 Hz) 1  6.02 dd (1H, 28.6 d 142.4 d J 41.4 d 91  7 .2, 15.4 Hz) 5.61 dd (1H, 5.67 dd (1H, J  7 .2, 15.4 Hz) b b d 2.40 m (1H) 35.5 d 2.40 m (1H) 35.6 10 b b  q 6.9 Hz) 20.1 0.91 d (3H, J 1 6.9 Hz) 20.0 0-CH q 0.91 d (3H, J  3 b b t1 .93 m (1H), 2.05 m (1H) 48.1 11 t1 48.2 .93 m (1H), 2.05 m (1H) b b 12 34.74 s1 34.66 1 s 1.67 s (3H) 1.67 s (3H) 16.5 q .5 q 16 2-CH 1 3 b b 1  0.7 Hz) J 5.74 d (1H, d 127.6 0.7 Hz) 13 d 5.74 d (1H, J  1 127.7 6.18 dd (1H, 25.9 d 1 14 J 1 0.7, 15.1 Hz) 125.9 d 0.7, 15.1 Hz) 1   J 6.18 dd (1H, b b 38.8 8.0, 15.1 Hz) d 5.39 dd (1H, J 15 8.0, 15.1 Hz) 138.9 1 d 5.39 dd (1H, J   2.01 m (1H) 39.3 d 2.01 m (1H) 39.3 d 16 J 6.6 Hz) 6-CH  20.5 q 0.93 d (3H, 1  20.5 q 0.93 d (3H, J 6.6 Hz) 3 1.25 m (2H) 1.25 m (2H) 17 30.4 t 30.4 t 4 7. J  J 0.80 t (3H, .0 q 12 18 7. 4  Hz) 12.0 q Hz) 0.80 t (3H, a 1 13 T he acetone- C) were used as references. d signals (2.00 ppm of H and 29.725 ppm of 6 b Each chemical shift is exchangeable for the one of the corresponding carbon of tautomer. As showed anthelmintic activity in vivo after a single 1 oral treatment with 2 mg/kg [6], we analyzed the efficacy of 2 infected sheep. Here we used two H. contortus using 1 treatments with 2 had no activity during because 2 and the single treatment schedule. The first treatment was performed with an oral dosage of 2 mg/kg, given once. The second treatment with as conducted one week after the w 1 was 2 first treatment, and the second treatment with performed at the same dosage after an interval of three (two sheep treated w eeks. Anthelmintic activity of 1 2 and each) against is shown in Table 3. Although 2 H. contortus did not suppress the egg output of female worms after the (reference 6 and Table 3), it first treatment in contrast to 1 Fig. 2 . 2 OCSY and selected HMBC correlations of T reduced the number of faecal eggs about 92% eleven days after the second treatment. The result after the second , and treatment is nearly similar to that of is also 2 1 e , and the inhibition selectivity 1 xpected to be an anthelmintic. more potent than that of is converted into We are interested in studying whether . 1 as also similar to w 1

4 53 he postulated conversion T Fig. 3 . 2 to 1 mechanism from T 2 and able 2 Inhibition of complex I by 1 IC (nM) 50 Complex I 12 4.0 NADH-ubiquinone reductase ( A. suum ) 8.0 2 ( g ) into nafuredin- 1 Conversion of nafuredin ( ) in Fig. 4 2.3 A. suum )24 NADH-rhodoquinone reductase ( neutral buffer. 0,000 1  100,000 NADH ubiquinone reductase (bovine liver) 2 is converted into 1 likely that and shows inhibitory 2 activity. Therefore, both 1 and may inhibit complex I 2 on the faecal egg T able 3 Effects of treatment with 1 or directly. However, it is not certain whether is converted to 1 Haemonchus contortus counts in sheep infected with hen is bound to complex I. It is necessary to continue 2 w 1 the studies to know the inhibition mechanism. Number of eggs per g Annonins are -lactone compounds isolated from seeds a gram of faeces of Annona squamosa and known as complex I inhibitors [11]. Annonin group compounds are called acetogenins, 12 and their inhibitory activity against complex I, cytotoxicity, and insecticidal activity are well studied [12, 13]. Be 791 5400 50  6692 ore treatment f 1  b , a g Acetogenins are characterized by an -unsaturated - 2348  325 After the first treatment 1667  1 5676 b position. The a lactone with long aliphatic side chain at After the second treatment 11. 1 935  27 433   3 tetrahydrofuran rings and some side chain contains 0 a V alues are means of faecal egg counts  S.D. h ydroxyl moieties. Some acetogenins are rearranged to b Second treatment with 1 w as conducted one week after the first g position, which are the form g -lactones with side chain at three weeks after the first treatment. treatment and with 2 . However, shows selective inhibition against 2 same as 2 is very weak helminth complex I, and the cytotoxicity of 2 compared to acetogenins. Therefore, structure difference 2 and acetogenins may affect largely to their 1 is hardly soluble in water, 2 between during enzyme assay. As (17 m M) in the assay buffer was 1 biological activities. highly dilute solution of incubated at 37°C. The buffer was used for the screening of NFRD inhibitors. After 30 minutes, the solution was e xtracted with ethyl acetate, concentrated, and analyzed by Experimental HPLC. No electron transport enzyme was added in this 2 General 1 and solution, because w ere not extracted when the solution contained the enzyme. They were suggested to be adsorbed by the enzyme. NMR spectra were recorded on a Varian Inova 600 2-3 as converted to w 1 About 10% of spectrometer (  Hz in HMBC). Chemical shifts are J as shown in Fig. 4. 2 8 CH shown in 1 w as converted in this study, it is not at 2.00 ppm Since only a part of d v alues (ppm) relative to acetone- d 6

5 54 54 13 1 C NMR. Mass H NMR and at 29.725 ppm for Incubation of Compound 1 in Assay Buffer for spectrometry was conducted on a JEOL JMS-AX505 HA 1 spectrometer. UV and IR spectra were measured with a 3 l of DMSO, and m m g of (8.3 nmole) was dissolved in 50 Shimadzu UV-240 spectrophotometer and a Horiba FT-210 l of the assay buffer (120 mM sodium phosphate 450 m F ourier transform infrared spectrometer, respectively. buffer, pH 7.0) was added. It was incubated for 30 minutes at 37°C, and then extracted with EtOAc. The EtOAc layer Optical rotation was recorded on a JASCO model DIP-181 SO as dried over anhydrous Na w in and concentrated polarimeter. Melting point was measured with a Yanaco 2 4 micro melting point apparatus MP-S3. l of 75% acetonitrile and m . It was dissolved in 50 vacuo m l of the solution was injected to HPLC: Column, 10  gasil ODS (Senshu Scientific Co.), i.d. 4.6 Conversion of 1 to 2 Pe 250 mm; mobile phase, 75% acetonitrile; temperature, 30°C; flow and (10.0 mg) was dissolved in 0.28 ml of MeOH, rate, 1 ml/minute; detection, UV 240 nm. Compounds 1 Compound 1 and 2.0 mg of CaCO ere eluted at 17.4 and 13.7 minutes, respectively. as added in the solution. After the w w 2 3 solution was stirred at room temperature for 30 minutes, We Acknowledgements are grateful to Ms. Akiko Nakagawa 10 ml of EtOAc and 10 ml of saturated NaCl aq soln was and Ms. Chikako Sakabe of School of Pharmaceutical Sciences, added and mixed. The organic layer was dried over Kitasato University for measurements of mass spectra. This work and concentrated to yield a white SO anhydrous Na 4 2 w as supported by a Grant-in-Aid for Scientific Research 2 powder of (9.3 mg). (14593006 to K.S. and 13854011 to K.K.) and a Grant-in-Aid for 26 Nafuredin- 4.6° ( ] g ( c 2 1.0, a  ): white powder; [ D Encouragement of Young Scientists (12771373 to H.U.) from the CHCl ); mp 101  103°; molecular formula C H O ; HR- 32 3 4 22 Japan Society for the Promotion of Science. T.N. acknowledges a  AB-MS ( F m , calcd 383.2198 / Na)  ) found 383.2201 (M z Kitasato University Research Grant for Young Researchers. This 1  3375, 2960, 2926, Na); IR O (for C n H (KBr) cm max 22 32 4 ork was also supported in part by the Grant of the 21st Century w 1755, 1738, 1660, 1456, 1261, 1074, 1024, 800. COE Program, Ministry of Education, Culture, Sports, Science, and Technology. Biological Studies References NFRD activity was assayed using mitochondrial fraction of A. suum A. suum muscle (11 g) was homogenized in 35 ml . ̄ Shiomi K, O mura S. Antiparasitic agents produced by 1. of homogenize buffer (0.14 M sucrose, 5 mM EDTA, microorganisms. Proc Jpn Acad, Ser B 80: 245–258 (2004) 2.5 mM dithiothreitol, 0.15% bovine serum albumin, pH Otoguro K, Ui H, Ishiyama A, Arai N, Kobayashi M, 2. g for 10 minutes to remove 7.4) and centrifuged at 1,000 ̄ T akahashi Y, Masumura R, Shiomi K, Yamada H, O ura S. In cell debris. The supernatant was further centrifuged at vitro antimalarial activities of the microbial metabolites. J 10,000 for 30 minutes and resulted mitochondrial g Antibiot 56: 322–324 (2003) precipitate was resuspended in assay buffer (120 mM Otoguro K, Ui H, Ishiyama A, Kobayashi M, Togashi H, 3. sodium phosphate, pH 7.0). The assay buffer (80 m l) akahashi Y, Masumura R, Tanaka H, Tomoda H, Yamada T containing 0.35 mM NADH, 7.2 mM disodium fumarate, ̄ H, O In vitro and in vivo antimalarial activities of a mura S. m and 10 l of DMSO solution of test compound was non-glycosidic 18-membered macrolide antibiotic, preincubated for 5 minutes at 37°C. The reaction was . J borrelidin, against drug-resistant strains of Plasmodium m l of the mitochondrial initiated by the addition of 10 Antibiot 56: 727–729 (2003) 4. Takatsu T, Horiuchi N, Ishikawa M, Wanibuchi K, fraction (0.3 mg protein/ml) and the incubation was carried Moriguchi T, Takahashi S. 1100-50, a novel nematocide out for 10 minutes at 37°C. The NFRD activity was from Streptomyces lavendulae SANK 64297. J Antibiot 56: measured spectrophotometrically by monitoring the 306–309 (2003) o xidation of NADH at 340 nm. 5. Kumazawa S, Kanda M, Utagawa M, Chiba N, Ohtani H, ADH-ubiquinone and NADH-rhodoquinone reductase N Mikawa T. MK7924, a novel metabolite with nematocidal assays were performed as described previously [6]. The activity from Coronophora gregaria . J Antibiot 56: 652–654 following quinones were used for the assays: ubiquinone 1 (2003) N ADH-ubiquinone reductase, ubiquinone 2 for A. suum ̄ 6. O mura, S, Miyadera H, Ui H, Shiomi K, Yamaguchi Y, for bovine liver NADH-ubiquinone reductase, and Masuma R, Nagamitsu T, Takano D, Sunazuka T, Harder A, ADH-rhodoquinone N decylrhodoquinone for A. suum Kölbl H, Namikoshi M, Miyoshi H, Sakamoto K, Kita K. reductase. An anthelmintic compound, nafuredin, shows selective inhibition of complex I in helminth mitochondria. Proc Natl Method for sheep study was reported previously [6].

6 55 Takano D, Nagamitsu T, Shiomi K, Ui H, Yamaguchi Y, 10. Acad Sci USA 98: 60–62 (2001) ̄ Masuma R, Harigaya Y, Kuwajima I, O Ui H, Shiomi K, Yamaguchi Y, Masuma R, Nagamitsu T, 7. mura S. Total ̄ T akano D, Sunazuka T, Namikoshi M, O synthesis of nafuredin- mura S. Nafuredin, g . Tetrahedron Lett 44: 6441–6444 (2003) a novel inhibitor of NADH-fumarate reductase, produced by 11. Londershausen M, Leicht W, Lieb F, Moeschler H. FT-0554. J Antibiot 54: 234–238 (2001) Aspergillus niger Molecular mode of action of annonins. Pestic Sci 33: 427– Takano D, Nagamitsu T, Ui H, Shiomi K, Yamaguchi Y, 8. ̄ 438 (1991) Masuma R, Kuwajima I, O mura S. Absolute configuration 12. Zafra-Polo MC, Figadère B, Gallardo T, Tormo JR, Cortes of nafuredin, a new specific NADH-fumarate reductase D. Natural acetogenins from Annonaceae, synthesis and inhibitor. Tetrahedron Lett 42: 3017–3020 (2001) mechanism of action. Phytochemistry 48: 1087–1117 (1998) Takano D, Nagamitsu T, Ui H, Shiomi K, Yamaguchi Y, 9. ̄ 13. Alali FQ, Liu XX, Mclaughlin JL. Annonaceous Masuma R, Kuwajima I, O mura S. Total synthesis of acetogenins: recent progress. J Nat Prod 62: 504–540 (1999) nafuredin, a selective NADH-fumarate reductase inhibitor. Org Lett 3: 2289–2291 (2001)

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