BIOLOGICALLY ACTIVE NATURAL PRODUCTS: AGROCHEMICALS - CHAPTER 11 pps

11 Potent Mosquito Repellents from the Leaves of Eucalyptus and Vitex Plants Hiroyuki Nishimura and Atsushi Satoh CONTENT 11.1 Mosquito Repellent from Eucalyptus Oils 11.2 Mosquito Repellent from E. citriodora Oil 11.3 Mosquito Repellent from E. camaldulensis Oil 11.4 New Natural Mosquito Repellent from Vitex rotundifolia References ABSTRACT N,N-Diethyl-m-toluamide (DEET), which is a commercially available repel- lent against mosquitoes, has many disadvantages such as an unpleasant odor, skin pene- tration, and carcinogenicity. We have explored alternative repellents without such drawbacks. New repellents, p-menthane-3,8-diols (cis and trans), were isolated from the leaves of Eucalyptus citriodora. Especially the pentanoyl (C 5 ) and caproyl (C 6 ) esters of the diols exhibited much higher activity than DEET in terms of repellency and repellent dura- bility against mosquitoes, Aedes albopictus and Culex pipiens. In addition, (+)-eucamalol, (1R, 6R)-(+)-3-formyl-6-isopropyl-2-cyclohexene-1-ol, which is a new mosquito repellent, was isolated from E. camaldulensis leaves. The absolute configuration was determined by synthesizing from (S)-(–)-perillaldehyde. While, from information of the traditional usage of Vitex rotundifolia for repelling mosquitoes at the southern part of Japan, we tried to iso- late a mosquito repellent from the leaves. As a result, a cyclopentene dialdehyde, named rotundial, was identified. 11.1 Mosquito Repellent from Eucalyptus 0ils As a postdoctoral fellow at the University of California, Berkeley (1975 to 1977), I became interested in research on Eucalyptus oil (Nishimura, 1995). One day, I noticed that mosqui- toes never appeared at the staff barbecues held near the Eucalyptus groves surrounding the campus. While enjoying the barbecues, I could smell Eucalyptus volatiles in the air and I observed that the volatile chemicals from Eucalyptus leaves repelled mosquitoes quite a dis- tance from the groves. N,N-Diethyl-m-toluamide (DEET) has been used as a repellent against blood-sucking insects such as mosquitoes all over the world. However, DEET has many drawbacks such © 1999 by CRC Press LLC as an unpleasant odor and its skin penetration (Moody et al., 1986). Furthermore, DEET also reacts with certain plastics and synthetic rubber, resulting in considerable damage to eyeglasses and watchbands, pens, and other plastic items. Therefore, a search for new repellents lacking these undesirable properties has been undertaken. A bioassay of repellent activity against mosquitoes was carried out according to the fol- lowing protocol. Pupae of Aedes albopictus (Hatoyama race) were obtained from Laboratory of Parasitology at Teikyo University and incubated at 25°C for 3 weeks. The hatched adults were released into a cage (25 × 25 × 25 cm) made of stainless steel and nylon gauze, and the bioassay was performed in the cage. Female Wistar mice (Nippon SLC Ltd.), 6 to 7 weeks old were used. Test samples were diluted with acetone at concentrations of 1.0, 5.0, or 10 mg/ml. The acetone solution of each test sample was applied to a wire gauze bag (7 cm I.D. × 12 cm) at rate of 50 ml/m 2 (50, 250, and 500 mg/m 2 ) and the bag was air-dried at room temperature. A mouse was then put into the bag and the bag placed in the cage of mosquitoes. Each test was run using 20 female mosquitoes which were 7 days old follow- ing emergence from pupae. The total number of mosquitoes landing on the mouse was counted immediately. The mouse was then taken out of the cage after 10 min, and the mos- quitoes killed in a drying oven at 160°C. Each dead mosquito was crushed and the mosqui- toes that had sucked blood were counted. Repellency (%) was calculated as and feed inhibition as Leaves of several species of Eucalyptus which had been collected in Australia were cut into small pieces and extracted with acetone in a glass bottle at ambient temperature. Ace- tone extracts were steam-distilled to obtain essential oils. The repellent activities of the essential oils against mosquito (Aedes albopictus) are shown in Table 11.1. The larger number indicates the greater repellency. From this result, the essential oils from E.citriodora (lemon- scented gum tree) and E.camaldulensis (river red gum tree) had relatively high activities against mosquitoes. TABLE 11.1 Repellent Activities of Several Eucalyptus Oils Against Mosquito, Aedes albopictus Repellency (%) Essential oils 2.5 0 0.25 g/m 2 Mouse Skin Area Eucalyptus radiata 54 24 Eucalyptus citriodora 90 43 Eucalyptus viminalis 27 – Eucalyptus camaldulensis 93 60 Eucalyptus pulverulenta 16 – Eucalyptus globulus –30 Cinnamomum camphora 00 Total mosquitoes a mosquitoes Total mosquitoes − × ttracted 100% Total mosquitoes bloodsucking mosquitoes Total mosquitoes − × 100% © 1999 by CRC Press LLC 11.2 Mosquito Repellent from E.citriodora Oil Silica gel column chromatography of the E.citriodora essential oil was guided by the bioas- say of repellent activity against mosquitoes. Subsequently, two active crystalline com- pounds were isolated. The spectral interpretation of the chemicals isolated from E. citriodora oil gave rise to the identification of p-menthane-3,8-diols (cis type 1; 4.5 mg/g fresh weight leaves and trans type 2; 2.2 mg/g fresh weight leaves) as shown in Figure 11.1 (Nishimura et al., 1986; Nish- imura and Mizutani, 1989). The physicochemical data were as follows: p-Menthane-3.8-cis-diol (1). Mp 81.0-82.5° (crystallized from Et 2 O-hexane), [α] 23 D ±0° (CHCl 3 ; c = 0.2), IRν KBr max cm –1 3240, 2930, 2900, 1450, 1420, 1250, 1160, 930. High resolution FIMS m/z(rel.int.):173.1532 [M+H] + (16), 157 [M-CH 3 ] + (37), 154 [M-H 2 O] + .(100), 114(9), 96 [M-OH-hydroxyisopropyl] + (83), 77(41), 59(85). EIMS(probe)70eV, (rel.int.): no.M + . peak, 157 [M-CH 3 ] + (1), 154 [M-H 2 O] + . (2), 139 [M-H 2 O-CH 3 ] + (3), 121(2), 111(2), 96(40), 81 [M-OH- hydroxyisopropyl-CH 3 ] + (100), 68(6), 59(79), 55(21), 54(21), 43(42), 41(34). 1 H NMR(200MHz, CDCl 3 , TMS): 0.87(3H,d,J = 6.4Hz, H-7), 1.22(3H,s,H-9), 1.36(3H,s,H-10), 4.41(1H,q,J = 2.4Hz, H-3). 13 CNMR(50MHz, CDCl 3 , TMS): δ20.4 (t,C-5), 22.3 (q,C-7), 25.7 (d,C-1), 28.8(q,C-9), 29.0(q,C-10), 35.0(t,C-6), 42.6(t,C-2), 48.4(d,C-4), 68.1(d,C-3), 73.3(s,C-8). p-Menthane-3,8-trans-diol(2). Mp 77.3-78.3° (from Et 2 O-hexane), [α] 23 D ± 0° (CHCl 3 ; c = 0.1). IRν KBr max cm –1 : 3250, 2960, 2920, 1450, 1420, 1220, 1180, 1000, 910, 870. High resolution FIMS m/z (rel.int.):173.1546 [M+H] + (47), 157 [M-CH 3 ] + (11), 154 [M-H 2 O] + . (30), 114(10), 113(15), 96 [M-OH-hydroxyisopropyl] + (54), 77(87), 59(100). EIMS(probe) 70eV, (rel. int.):no.M + . peak, 157 [M-CH 3 ] + (1),154 [M-H 2 O] + . (1), 139[M-H 2 O-CH 3 ] + (3), 121(2), 111(1), 96(38), 81(100), 68(10), 59(90), 55(13), 54(20),43(34), 41(19). 1 H NMR(200MHz,CDCl 3 , TMS): δ0.92(3H,d,J = 6.4Hz, H-7), 1.22(6H,s,H-9 and H-10), 3.72(1H,dt,J = 10.4,4.3Hz,H-3). 13 C NMR(50MHz,CDCl 3 , TMS): δ22.0(q,C-9), 23.8(q,C-10), 27.1(t,C-5), 30.1(q,C-7), 31.4(d,C-1), 34.6(t,C-6), 44.7(t,C-2), 53.5(C-4), 72.9(d,C-3), 75.0(s,C-8). To explore repellents with higher activity against mosquitoes, several esters of p-men- thane-3,8-diols were prepared. The repellent activities of the esters are shown in Table 11.2. From this result, the pentanoyl (C 5 ) and caproyl (C 6 ) esters of p-menthane-3,8-cis-diol had FIGURE 11.1 Chemical structures of p-menthane-3,8-diols (cis and trans) from E. citriodora leaves. © 1999 by CRC Press LLC higher activity than DEET which is a commercially available repellent. Interestingly enough, the caproyl ester had higher repellent durability than DEET (unpublished data). 11.3 Mosquito Repellent from E. camaldulensis Oil As shown in Table 11.1, the essential oil from E. camaldulensis leaves had significant repel- lent activity against Aedes albopictus (Nishimura et al., 1986). These observations prompted us to purify the mosquito repellent in the E. camaldulensis essential oil. The leaves of E. camaldulensis (1.5 kg) were collected in Matsudo, Chiba prefecture, Japan. They were cut into small pieces and steam-distilled to yield 1.6 g of the essential oil. The oil was purified by successive preparative TLC to give two repellents against mosquito. Bioassay of repellent activity against mosquitoes was carried out according to the follow- ing methods: A chick whose abdominal feathers were removed with a haircutter was fixed on a wood board (7 × 15 cm). The shaved abdominal skin of the chick (2.5 × 4 cm) was exposed and an ethanol solution of a test compound was applied to the skin (1.5 g/m 2 ). About 500 adult mosquitoes which were 6 to 8 days old after emergence (Aedes aegypti, approximately equal numbers of females and males) were released in a cage (21 × 21 × 30 cm) made of stainless steel and nylon gauze. Two chicks (one was treated and the other untreated) were put in the cage for 2 min. Then, the total number of landed mosquitoes on each chick was counted. Repellency (%) was calculated according to following equation. The repellency was evaluated every hour after treatment until the repellency was reduced to less than 80%. TABLE 11.2 Repellent Activities of p-Menthane-3,8-diols and their Derivatives Against Mosquito, Aedes albopictus cis trans Repellency (%) R Repellency (%) 59 H 52 22 COCH 3 15 38 COCH 2 CH 3 26 30 CO(CH 2 ) 2 CH 3 – 72 CO(CH 2 ) 3 CH 3 – 67 CO(CH 2 ) 4 CH 3 – 65 DEET Note: Sample concentration: 30 mg/m 2 mouse skin area. Repellency % Number of landed mosquitoes on treated chicks Number of landed mosquitoes on untreated chicks () =− ×1 100 © 1999 by CRC Press LLC The bioassay-guided chromatography of the essential oil from E. camaldulensis leaves gave rise to the isolation of two active principles. From the spectral interpretation, two repellents were identified as 4-isopropylbenzyl alcohol (3) and a new compound, (+)-euca- malol (4) (3-formyl-6-isopropyl-2-cyclohexen-1-ol) (Watanabe et al., 1993) as shown in Figure 11.2. The mosquito-repelling activity of 4-isopropylbenzyl alcohol (3) and eucamalol (4) were examined against A. aegypti (Table 11.3) in comparison with that of DEET. All tested com- pounds exhibited potent mosquito repelling activities against A. aegypti immediately after the application. In 1 h after treatment, the effectiveness of 3 was lost. Although the duration of the effectiveness for DEET was within 2 h after the treatment by our test method, euca- malol (4) showed 75% repellency even 3 h after the treatment. This result indicates that the repellency of eucamalol against A. aegypti is superior to that of DEET. 4-Isopropylbenzyl alcohol(3):MS,m/z 150 [M] + , 132(M-H 2 O) + (base peak), 107 [M-C 3 H 7 ] + ; 1 H NMRδ TMS CDC1 , 7.05(2H,d,J = 7.0 Hz), 6.95(2H,d,J = 7.0 Hz), 4.00(2H,s,CH 2 OH), 2.85(1H,m,CH), 1.00 (6H, d,J = 6.8Hz). FIGURE 11.2 Chemical structures of 4-isopropylbenzyl alcohol (3) and (+)-eucamalol (4) from E. camaldulensis leaves. TABLE 11.3 Repellent Activities of 4-Isopropylbenzyl alcohol (3), (+)-Eucamalol (4) and DEET against the Yellow Fever Mosquito, Aedes aegypti Repellency (%) Compounds 0 h a 1 h2 h3 h 3 100 23 – – 4 100 92 87 75 DEET 100 84 55 – Note: Sample concentration: 1.5 g/m 2 chick skin area. a Immediately after treatment. © 1999 by CRC Press LLC (+)-Eucamalol(4):[α] 25 D +13.5° (c = 0.80, CH 3 OH); HRMS m/z 168.1151 (C 10 H 16 O 2 , Calcd. 168.1150); MS, m/z 168[M] + , 139[M-CHO] + , 125[M-C 3 H 7 ] + , 69(base peak); IRν KBr max 3400(0H group), 2980, 1680(conjugate aldehyde)cm –1 ; 1 H NMRδ TMS CDC1 a , 9.45(1H,s), 6.63(1H,d,J = 2.2Hz), 4.28(1H,dd,J = 2.2&9.3Hz), 2.37(1H,m), 2.08(1H,m), 2.04(1H,m), 1.78(1H, m), 1.39(1H,m), 1.24(1H,m), 0.98(1H,d,J = 6.8Hz), 0.84(1H,d,J = 6.8Hz). However, it was difficult to determine the absolute configuration of (+)-eucamalol from these results. Synthesis of (+)-eucamalol (4) and its 1-epimer (5) from (S)-(–)-perillaldehyde (6) was carried out to determine the absolute configuration and compare their repellent activities against Aedes albopictus. The synthetic scheme is shown in Figure 11.3. (S)-(–)-Perillaldehyde (6) was converted to 8,9-dihydro-perillaldehyde (7) by homoge- nous hydrogenation with tris(triphenylphosphine)rhodium chloride as a catalyst in a 73% yield. Conversion of 7 to 3-bromo-8,9-dihydroperillaldehyde (9) was performed by the pro- cedure of Ishihara et al. (1990). Enol acetylation of 7 with isopropenyl acetate gave an enol acetate (8) in a 38% yield. This enol acetate (8) was brominated by N-bromo-succinimide. Since 3-bromo-8, 9-dihydro-perillaldehyde (9) was unstable, nucleophilic substitution of bromide (9) was subsequently carried out by treating with potassium hydroxide to give two alcohols, (+)-eucamalol (4) and (–)-1-epi-eucamalol (5) in yields of 7.7 and 8.4%, respec- tively (Satoh et al., 1995). The J 1,6 value (9.2Hz) of synthetic (+)-eucamalol (4) shows axial–axial coupling, while the smaller J 1,6 value (<2.0 Hz) of synthetic (–)-1-epi-eucamalol (5) shows axial-equatorial cou- pling. Thus, the J 1,6 value of synthetic (+)-eucamalol (4) indicates that the relative configu- ration at C-1 and C-6 was, like that of natural (+)-eucamalol, of trans-form. The optical rotation of synthetic (+)-eucamalol was +14.1° in methanol, and was very close to the opti- cal rotation of natural eucamalol, [α] = +13.5° (c = 0.80, MeOH) (Watanabe et al., 1993). Consequently, the absolute configuration of (+)-eucamalol was determined to be (1R,6R)- (+)-3-formyl-6-isopropyl-2-cyclohexen-1-ol (Satoh et al., 1995). The repellent activity of synthetic eucamalol and its epimer were evaluated by using Aedes albopictus as the test mosquito strain (Table 11.4). (+)-Eucamalol and its epimer had FIGURE 11.3 Synthesis of (+)-eucamalol (4) and its 1-epimer (5) from (S)-(–)-perillaldehyde (6). © 1999 by CRC Press LLC repellent and feeding inhibitory activities against A. albopictus to the same degree as DEET. In addition, both the repellent and feeding inhibitory activities of (+)-eucamalol were the same as that of its epimer. Structure–activity relationships will be presented elsewhere. 11.4 New Natural Mosquito Repellent from Vitex rotundifolia Vitex rotundifolia has long been used as a medicinal plant for a headache and a cold, and various compounds such as flavonoid, iridoid glycosides, diterpenoids, and sesquiterpe- noids have been identified in this plant (Kimura et al., 1967; Asaka et al., 1973; Tada and Yasuda, 1984). It also has been reported that the leaves and twigs of this plant can be used for repelling mosquitoes (Okuda, 1967). However, the principle responsible for its activity has not been previously studied. Hence, we investigated the mosquito-repelling principle of V. rotundifolia and isolated a new natural cyclopentene dialdehyde named rotundial (10) as shown in Figure 11.4. In this report, we describe the identification and mosquito repel- ling activity of rotundial. The volatile constituents of fresh leaves (5 kg) from V. rotundifolia were subjected to silica gel column and preparative thin-layer chromatography, eluting with hexane-EtOAc (2:1, v/v), to afford 250 mg of rotundial (10, 0.005%) as a colorless and odorless oil; C 6 H 12 O 2 [high resolution MS (HRMS), M + , m/z 152.0780, [calcd. as 152.0837], [αl 25 D +39.3°] (c = 1.0, CHCl 3 ). The existence of both a simple aldehyde and an α,β-unsaturated aldehyde in 10 was con- firmed by UV (λmax 250nm, e = 11,000), IR (νmax 1720 and 1665 cm –1 ) and NMR spectral TABLE 11.4 Repellent and Feeding Inhibition Activities of (+)-Eucamalol and its (–)-1-Epimer Against Aedes albopictus Repellent Activity (RA) Compounds RA(%) 500 250 50 g/m 2 Mouse Skin Area (+)-Eucamalol 100.0 100.0 84.2 (–)-epi-Eucamalol 100.0 100.0 75.0 DEET 100.0 100.0 80.0 Feeding Inhibitory Activity (FIA) Compounds FIA(%) 500 250 50 g/m 2 Mouse Skin Area (+)-Eucamalol 100.0 100.0 74.5 (–)-epi-Eucamalol 100.0 100.0 65.0 DEET 100.0 100.0 85.0 RA = Total mosquitoes attracted mosquitoes Total mosquitoes − × 100% FIA = Total mosquitoes b mosquitoes Total mosquitoes − × loodsucking 100% © 1999 by CRC Press LLC data [ 1 Hδ: 9.93(1H,s) and 9.70(1H,dd, J = 2.0 and 2.0 Hz); 13 Cδ: 201.9(d) and 187.9(d)]. The 1 H-NMR spectrum of 10 showed a broad singlet at δ2.15(3H,-C = C-CH 3 ), two double dou- ble doublets at δ2.88 (1H,J = 2.0, 4.4 and 17.0Hz) and 2.32 (1H,J = 2.0, 9.1 and 17.0 Hz) assignable to the methylene protons adjacent to the simple aldehyde (CH 2 CHO). The 13 C- NMR signals at δ164.2(s) and 139.0(s) indicated full substitution of the double bond in 10. The 1 H-NMR signals indicated the existence of a CH 2 CH 2 CH moiety which formed a cyclo- pentene ring. The long-range correlations from its COLOC spectrum (Kessler et al., 1984) established the structure of rotundial as 10 as shown in Figure 11.4 (Watanabe, 1995). The absolute stereochemistry of 10 was then determined. The LiAlH 4 reduction of 10 gave rotundiol (11;[αl 25 D -16.6°]). The optical rotation data ([α] D ) for its methylated (at C-8) derivatives, (3R,8S)-isodehydroiridodiol (12) and (3R,8R)-isodehydroiridodiol (13) (Figure 11.5) were –20.7° and –15.3°, respectively, suggesting the 3R configuration of 11 (Sakai et al., 1980). Accordingly, the configuration of 10 at C-3 was deduced to be R. This is FIGURE 11.4 Chemical structure of rotundial (10) and correlations of its COLOC spectrum (→). FIGURE 11.5 Chemical structures of rotundiol (11), (3R,8S)-dehydroiridodiol (12) and (3R,8R)-isodehydroiridodiol (13). © 1999 by CRC Press LLC the first isolation of rotundial (10) from a natural source, compared with enzymatic or acid hydrolysis of the aucubin-terated iridoid giving compound 10 (Bianco et al., 1977). The mosquito repelling activity of rotundial (10) was examined against Aedes aegypti in comparison with that of DEET (Table 11.5). Both compounds had potent repellent activity against A. aegypti immediately after the application, while 1 h after treatment, the effective- ness of 10 was almost equivalent to that of DEET. Two h after treatment, the repellency of 10 became higher than that of DEET, indicating that the mosquito repelling activity of rotundial (10) was superior to that of DEET in respect to its long-lasting effectiveness. A monoterpene dialdehyde, chrysomedial, has been isolated from larvae of the chry- somelid beetle, Plagiodra versicolora, as its defensive substance (Meinwald et al., 1977; Mein- wald and Jones, 1978). The structural similarity between 10 and chrysomedial leads us to speculate that rotundial (10) might serve the plant as a defensive principle against insect attack which will be the subject of further investigation. References Asaka, Y., Kamikawa, T., and Kubota, T., Chem. Lett., 937-940, 1973. Bianco, A., Guiso, M., Iavarome, C., Pasacantilli, P., and Trogolo, C., Tetrahedron, 33, 851-854, 1977. Ishihara, M., Kakiuti, H., Tsuneya, T., and Shiga, M., 34th Symposium on the Chemistry of Terpenes, Essential Oil, and Aromatics, Abstract, 1990, 45-47. Kessler, H., Griesinger, C., Zarbock, J., and Loosli, H.R., J. Mag. Res., 57, 331-336, 1986. (The COLOC spectrum of 1 was recorded with an 8k × 256 matrix and a mixing delay of 45 and 22.5 ms.) Kimura, Y., Takido, M., and Hiwatashi, Y., Yakugaku Zasshi (in Japanese), 87, 1429-1430, 1967. Meinwald, J., Jones, T.H., Eisner, T., and Hocks, K., Proc. Natl. Acad. Sci., 74, 2189-2193, 1977. Meinwald, J. and Jones, T.H., J. Am. Chem. Soc., 100, 1883-1886, 1978. Moody, R.P., Sidon, E., and Franklin, C.A., 6th Intl. Congress of Pesticide Chemistry, Ottawa, Symposium Paper 8A/7E-06, 1986. Nishimura, H., Mizutani, J., Umino, T., and Kurihara, T., Intl. Congress of Pesticide Chemistry, Ottawa, Symposium Paper 2D/E-07, 1986. Nishimura, H. and Mizutani, J., Economically useful ingredients and the clonal propagation of Eucalyptus citriodora plant, the Proc. Hokkaido Tokai University, Science and Engineering, no. 2, 1989, 57-65. Nishimura, H., Repellents against mosquito in Eucalyptus oil, Aromatopia (in Japanese), Fragrance Journal LTD. (Japan), no. 11, 1995, 32-34. Okuda, O., “Kouryou Kagaku Soran” (in Japanese), Hirokawa Shoten, Tokyo, Japan, 1967, 310-311. Sakai, T., Nakajima, K., Yoshihara, K., and Sakan, T., Tetrahedron, 36, 3115-3119, 1980. TABLE 11.5 Repellent Activities of Rotundial (10) and DEET Against the Yellow Fever Mosquito, Aedes aegypti Repellency (%) Compounds 0h a 1h 2h 3h Rotundial 100 90 85 70 DEET 100 90 50 35 Note: Sample concentration: 1.5 g/m 2 chick skin area. a Immediately after treatment. © 1999 by CRC Press LLC Satoh, A., Utamura, H., Nakade, T., and Nishimura, H., Absolute Configuration of a new mosquito repellent, (+)-eucamalol and the repellent activity of its epimer, Biosci. Biotech. Biochem., 59, 1139-1141, 1995. Tada, H. and Yasuda, F., Heterocycles, 22, 2203-2205, 1984. Watanabe, K., Shono, Y., Kakimizu, A., Okada, A., Matsuo, N., Satoh, A., and Nishimura, H., J. Agric. Food Chem., 41, 2164-2166, 1993. Watanabe, K., Takada, Y., Matsuo, N., and Nishimura, H., Biosci. Biotech. Biochem., 59, 1979-1980, 1995. Wright, R.H., Sci. Am., 233, 104-111, 1975. © 1999 by CRC Press LLC . identified as 4-isopropylbenzyl alcohol (3) and a new compound, (+)-euca- malol (4) (3-formyl-6-isopropyl-2-cyclohexen-1-ol) (Watanabe et al., 1993) as shown in Figure 11. 2. The mosquito-repelling. 6.4Hz, H-7), 1.22(3H,s,H-9), 1.36(3H,s,H-10), 4.41(1H,q,J = 2.4Hz, H-3). 13 CNMR(50MHz, CDCl 3 , TMS): δ20.4 (t,C-5), 22.3 (q,C-7), 25.7 (d,C-1), 28.8(q,C-9), 29.0(q,C-10), 35.0(t,C-6), 42.6(t,C-2),. 3.72(1H,dt,J = 10.4,4.3Hz,H-3). 13 C NMR(50MHz,CDCl 3 , TMS): δ22.0(q,C-9), 23.8(q,C-10), 27.1(t,C-5), 30.1(q,C-7), 31.4(d,C-1), 34.6(t,C-6), 44.7(t,C-2), 53.5(C-4), 72.9(d,C-3), 75.0(s,C-8). To explore