Chemistry and Physics of Lipids, 61 (1992) 157-167 Elsevier Scientific Publishers Ireland Ltd.

157

Synthesis of biosynthetic inhibitors of the sex pheromone of Spodoptera littoralis. Part II: acetylenic and cyclopropane fatty acids* F. C a m p s a, S. H o s p i t a l a, G. R o s e l l b, A. D e l g a d o b a n d A. G u e r r e r o a aDepartment of Biological Organic Chemistry, CID ( CSIC), Jordi Girona Salgado, 18, 08034-Barcelona and bDepartment of Pharmacology and Medicinal Chemistry, Faculty of Pharmacy, Av. Diagonal s/n, 08028-Barcelona (Spain) (Received November 5th, 1991; accepted January 24th, 1992)

The synthesis of new acetylenic and cyclopropane fatty acids, as potential inhibitors of the B-oxidation step in the proposed biosynthesis of the sex pheromone of the Egyptian armyworm Spodoptera littoralis, is reported. The biological activity of the compounds has been determined by in vitro and in vivo bioassays, and among all the compounds tested, dichlorocyclopropane acid has shown the highest inhibition activity displayed so far.

Key words: inhibition; B-oxidation; Spodoptera littoralis; acetylenic fatty acids; cyclopropane fatty acids

Introduction

In recent years it has been established that most biosynthetic pathways of insect sex pheromones in Lepidoptera are regulated by one or more chainshortening steps, along with other key processes, i.e. desaturation, reduction, acetylation and oxidation [2]. Although the chain shortening pathway has not yet been studied at the enzymatic level in insect tissues, it is likely to be similar to that occurring in vertebrates, where it involves a/~-oxidation process localized in the peroxisomes [3]. In insects, the/3-oxidation reaction has been found to be a key step in the proposed biosynthetic scheme of the cabbage looper Trichoplusia ni [4] and the pink bollworm Pectinophora gossypiella [5], among others. Although development of inhibitors of the /3oxidation process has been pursued for over thirty years as potential hypoglycemic drugs or as tools Correspondence to: Angel Guerrero, Department of Biological Organic Chemistry, CID, CSIC, Jordi Girona 18-26, 08034-Barcelona, Spain. *Part I: see Ref. 1.

to study the regulation of fatty acid oxidation [6], inhibitors of this process in the biosynthesis of insect sex pheromones has been only scarcely considered [1], in spite of its potential usefulness in opening new strategies for insect control. The major component of the sex pheromone of the Egyptian armyworm Spodoptera littoralis, (Z,E)-9,11-tetradecadienyl acetate, has been postulated to originate via fl-oxidation of palmitic acid into myristic acid through an acyl-CoA dehydrogenase [7]. In this paper and continuing our efforts in this field, we now report on the synthesis of some acetylenic and cyclopropylsubstituted palmitic acids, as new potential inhibitors of the oxidation process. The compounds have been designed by blockage of the 2 and/or 3 position(s) of the parent fatty acid (acids 1-3) or as irreversible suicide inhibitors (acids 4-7) (Scheme 1), capable of producing in situ a very reactive species which can irreversibly bind an active site of the enzyme [8]. Among these compounds, most of them previously unreported in the literature, dichlorocyclopropyl acid (7) turned out to be the best inhibitor both in vitro and in vivo bioassays.

0009-3084/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

158

R~COOH

R~J~",COOH

Br R--C-C--COOH

2

R---CH2--CEC_.,-.-COOH 3

R--C-C--CH2--COOH 4

.. coo. F F

. coo.

R = C12H15

distilled from P205. Anhydrous diglyme was obtained by distillation from Call2.

Synthesis 1- Tetradecyne (8) Alkylation of l-bromododecane with lithium acetylide in liquid ammonia afforded 1-tetradecyne as previously described by us [9] (68%). IR 3320, 2920 cm -1 lH-NMR 6 2.3-2.0 (c, 2H, CH2C-C), 1.92 (t J = 2.8 Hz, 1H, H C - C ) , 1.7-1.1 (b, 20H, 10CH2), 0.87 (t J = 7.2 Hz, 3H, CH3).

Scheme 1.

1-Pentadecyne (9)

Experimental

By the same procedure, the title compound was obtained in 72% yield. IR v 3314, 2924, 2853, 2108, 1460 cm -1. tH-NMR 6 2.3-2.0 (c, 2H, CH 2 C--C), 1.91 (t J = 2.4 Hz, 1H, HC~-C), 1.7-1.15 (b, 22H, 11CH2), 0.86 (distorted t, 3H, CH3).

General Infrared spectra were recorded in CCI 4 on a Perkin Elmer model 399B. 1H- and 13C-NMR spectra were determined in CDCI 3 on a Bruker WP80SY, a Varian XL200 and a Varian Unity 300 spectrometer operating at 80, 200 and 300 MHz, respectively for 1H and at 20, 50 and 75 MHz for 13C. 19F-NMR spectra were determined on a Bruker WP80SY spectrometer working at 75.39 MHz. Chemical shifts in 1H and I3C spectra are reported in 6 scale (ppm) relative to TMS, whereas trifluoroacetic acid was used as external standard in the 19F spectra. Elemental analyses were determined on a Carlo Erba model 1106. Low resolution mass spectra were run on a HP 5995 mass spectrometer coupled with a gas chromatograph. GLC analyses were performed on Carlo Erba models 2350 and 4130 equipped with a FID detector, using 3% OV-101 glass column 2 m x 1/8" i.d. on Chromosorb W or a fused silica capillary column SPB-5 30 m x 0.32/~m i.d. using hydrogen as carrier gas. Unless otherwise noted, compounds were purified by column chromatography on silica gel eluting with hexane-ether mixtures. Reactions requiring anhydrous conditions were performed under N 2 or A atmosphere. Tetrahydrofuran and dimethoxyethane were distilled from Na/benzophenone under N 2. Anhydrous acetone was

2-Pentadecynoic acid (2) To a solution of 8 (300 mg, 1.55 mmol) in 6 ml of anhydrous THF, cooled to -10°C and under A, was added 1.3 ml of 1.25 M BuLi in hexane (1.6 mmol) and stirred for 30 min. The reaction mixture was warmed to 0°C and a stream of CO2 was bubbled during 35 min. After quenching with water and acidification, the organic material was extracted with ether, washed with brine and dried. The solvent was removed and the residue purified by column chromatography to yield acid 2 (220 mg, 50%) as a white solid, m.p. 40-41°C. Analysis: calculated for C15H2602; C, 75.58; H, 10.99. Found: C, 75.53; H, 11.17. IR p 3340-2400, 2920, 2840, 2240, 1690, 1280 cm -1. IH-NMR & 2.35 (t J = 7.0 Hz, 2H, CH2C--C), 1.70-1.50 (m, 2H, CH_2CH2C=C), 1.50-1.20 (b, 18H, 9CH2), 0.88 (distorted t, 3H, CH3). 13C-NMR t5 158.0 (CO), 92.7 (C-3), 72.5 (C-2), 31.9 (C-13), 29.6-27.3 (C-5 to C-12), 22.6 (C-14), 18.7 (C-4), 14.1 (C-15). MS m/z (%) 239 (M + 1, 1), 140 (6), 109 (14), 97 (16), 96 (20), 95 (44), 86 (17), 83 (23), 82 (43), 81 (100), 80 (13), 79 (16), 71 (12), 70 (12), 69 (35), 68 (30), 67 (78), 57 (49), 56 (20), 55 (73), 54 (32), 53 (13).

2-Hexadecynoic acid (3) The same procedure as for compound 2 was

159 applied. Thus, starting from 1-pentadecyne 9 (300 mg, 1.4 mmol) in 5 ml of THF the expected acid 3 was obtained as a white solid (162 mg, 46% yield), after chromatography on silica gel eluting with CH2CI2/MeOH 96:4, m.p. 51-52°C. Analysis: calculated for C16H2802; C, 76.14; H, 11.18. Found: C, 76.41; H, 11.37. IR p 3600-2375, 2926, 2854, 2236, 1691, 1278 cm -1. IH-NMR t5 2.35 (t J = 7.2 Hz, 2H, CH2CmC), 1.8-1.4 (m, 2H, CH2CH_2C--C), 1.4-1.15 (b, 20H, 10CH2), 0.89 (distorted t, 3H, CH3). 13C-NMR t5 158.1 (CO), 92.8 (C-3), 72.6 (C-2), 31.9 (C-14), 29.6-27.4 (C-5 to C-13), 22.7 (C-15), 18.7 (C-4), 14.1 (C-16). MS m/z (%) 253 (M + 1, 1), 252 (M, 1), 251 (M - 1, 4), 235 (7), 110 (5), 109 (12), 97 (18), 96 (46), 95 (50), 83 (25), 82 (68), 81 (100), 69 (32), 68 (28), 67 (67), 57 (32), 55 (50), 54 (25), 53 (10). 3-Hexadecynol (10) To a solution of 1-tetradecyne 8 (15 g, 0.077 mol) in 4 ml of anhydrous THF was added dropwise a solution of 0.092 mol of EtMgBr in 50 ml of anhydrous THF. The mixture was refluxed for 2.5 h, cooled to room temperature. Then, condensed ethylene oxide (4.23 g, 0.096 mol) was added and the mixture heated at 50°C for 90 min. After pouring into cold NHaC1 saturated solution, the organic material was extracted with pentane and washed with brine and dried. Removal of solvent afforded a residue, which was chromatographed to yield pure 10 (11.53 g, 63%) as a white solid, m.p. 39-40°C. Analysis: calculated for C16H300; C, 80.60; H, 12.68. Found: C, 80.26; H, 12.87. IR ~, 3600, 2920 cm -l. 1H-NMR t5 3.68 (t J = 6.6 Hz, 2H, CH20), 2.41 (m, 2H, CH_2CH20), 2.3-2.0 (c, 2H, CH2CmC), 1.75 (s, 1H, OH), 1.6-1.1 (b, 20H, 10CH2), 0.86 (t J = 5.5 Hz, 3H, CH3). 13C-NMR 82.9 (C-3), 76.3 (C-4), 61.4 (C-l), 31.9 (C-14), 29.6-28.9 (C-6 to C-13), 23.2 (C-2), 22.7 (C-15), 18.7 (C-5), 14.1 (C-16). MS m/z (%): 195 (2), 153 (3), 139 (4), 135 (7), 125 (6), 123 (7), 122 (7), 121 (17), 111 (10), 109 (25), 108 (13), 107 (33), 97 (70), 96 (21), 95 (54), 93 (42), 84 (57), 83 (37), 82 (29), 81 (67), 80 (20), 79 (59), 69 (68), 68 (35), 67 (73), 57 (40), 56 (27), 55 (100), 54 (47), 53 (37). 3-Hexadecynoic acid (4) To a cold solution (0°C) of alcohol 10 in 4 ml of

anhydrous acetone was added 0.5 ml of a 1.7-M solution of chromium trioxide in acetone [10]. The reaction mixture was stirred for 23 h at room temperature and quenched by adding isopropyl alcohol. The mixture was filtered, the solvent evaporated off and the residue taken up in water, thoroughly extracted with ether and dried to afford a crude, which was purified by flash chromatography to obtain 4 as a white solid (50 mg, 47%), m.p. 66-68°C. Analysis: calculated for C16H2802; C, 76.14; H, 11.18. Found: C, 76.04, H, 11.13. IR v 2940, 2920, 1720 cm -J. IH-NMR t5 3.33 (tJ = 2.4 Hz, 2H, CH2COOH), 2.19 (m, 2H, CH2C--C), 1.51 (m, 2H, CH_2CH2C---C), 1.44-1.2 (b, 18H, 9CH2), 0.88 (t J = 6.9 Hz, 3H, CH3) 13CNMR 6 173.9 (CO), 84.7 (C-3), 70.5 (C-4), 31.9 (C-14), 29.6-28.6 (C-6 to C-13), 25.8 (C-2), 22.7 (C-15), 18.7 (C-5), 14.1 (C-16). MS m/z (%): 253 (M + 1, 2), 167 (2), 163 (2), 154 (2), 153 (4), 149 (5), 139 (13), 135 (15), 122 (13), 121 (25), 111 (12), 109 (17), 108 (17), 107 (25), 97 (40), 95 (44), 94 (64), 93 (51), 82 (21), 81 (49), 80 (34), 79 (56), 70 (43), 69 (42), 67 (48), 59 (100), 58 (99), 57 (50), 55 (77), 54 (20), 53 (21). 3-Hexadecynyl acetate (11) To an ice-cooled mixture of alcohol 10 (6 g, 25.2 mmol) in 20 ml of pyridine was added acetic anhydride (4.75 ml, 50.3 mmol). The reaction was stirred for 3 h at room temperature, quenched with water and extracted with hexane. The organic phases were sequentially washed with 2 N HCI, NaHCO a saturated solution, brine, and dried. Evaporation of the solvent left compound 11 as a colorless oil (6.81 g, 97%), pure enough to be used in the next step without further purification. An analytical sample was obtained by column chromatography. Analysis: calculated for C18H3202; C, 77.07; H, 11.52. Found: C, 77.09; H, 11.54. IR v 2920, 1730, 1240, 1040 cm -1. 1H-NMR t5 4.12 (t J = 7.8 Hz, 2H, CH20), 2.46 (m, 2H, CH_2CH20), 2.3-2.0 (c, 2H, CH2C---C), 2.05 (s, 3H, CHACO), 1.7-1.1 (b, 20H, 10CH2), 0.88 (t J = 5.2 Hz, 3H, CH3). 13C-NMR 6 170.5 (CO), 81.9 (C-3), 75.3 (C-4), 62.7 (C-l), 31.8 (C-14), 29.5-28.8 (C-6 to C-13), 22.5 (C-15), 20.6 (C-1 '), 19.1 and 18.5 (C-2 and C-5), 13.9 (C-16). MS rn/z (%): 220 (M-60, 1), 192, 191 (3), 149 (6), 135 (16),

160 122 (ll), 121 (20), 109 (12), 108 (24), 107 (24), 95 (13), 94 (12), 93 (29), 81 (16), 80 (20), 79 (41), 67 (20), 55 (19), 43 (100), 41 (27). (Z)-3-Hexadecenyl acetate (12) A solution of acetylene 11 (3.15 g, 11.3 mmol) and 0.28 ml of quinoline in 35 ml of hexane was hydrogenated in the presence of Lindlar catalyst (280 mg) at room temperature. The mixture was filtered and the organic solution washed with 1 N HCI and water and dried. Removal of the solvent yielded the expected ester 12 (2.6 g, 82%), which was purified by column chromatography. Stereochemical purity Z:E 94:6. Analysis: calculated for CIsH3402; C, 76.54; H, 12.13. Found: C, 76.66; H, 12.48. IR v 2920, 1740, 1235, 1035 cm -1. lH-NMR 8 5.40 (m, 2H, CH=CH), 4.05 (t J = 7.1 Hz, 2H, CH20), 2.35 (dt J = 7.4 Hz and 6.6 Hz, 2H, CH_2CH20), 2.1-1.9 (c, 2H, CH2C=C), 2.02 (s, 3H, CH3CO), 1.5-1.1 (b, 20H, 10CH2), 0.89 (t J = 6.0 Hz, 3H, CH3). 13C-NMR 8 171.0 (CO), 133.0 (C-3), 124.2 (C-4), 63.9 (C-l), 31.9 (C-14), 29.6-29.3 (C-6 to C-13), 27.3 (C-5), 26.8 (C-2), 22.6 (C-15), 20.9 (C-1 '), 14.0 (C-16). MS m/z (%): 222 (M-60, 14), 194 (3), 180 (6), 166 (3), 152 (4), 138 (5), 137 (5), 124 (10), 123 (8), 111 (4), 110 (15), 109 (13), 97 (13), 96 (41), 95 (23), 83 (16), 82 (44), 81 (36), 69 (18), 68 (54), 67 (42), 57 (13), 55 (32), 54 (38), 43 (100), 41 (35). cis-l-(2-Acetoxyethyl)-2-dodecylcyclopropane (13) A suspension of zinc (2.3 g, 36 mmol) in 16 ml of anhydrous dimethoxyethane was placed in an ultrasonic bath at room temperature for 2 h [11]. Then, acetate 12 was added and the bath heated to reflux. Diiodomethane (0.95 g, 38.3 mmol) was added dropwise and the reaction continued for 5 h. The mixture was poured into NH4CI saturated solution and worked-up as usual to afford the expected acetate 13 contaminated with 24% of unreacted material. The compounds turned out to be inseparable, even by AgNO3-impregnated silica gel, so the mixture was subjected to ozonolysis in CH2C12 for 3 h at -78°C. The ozonide was treated with 3% periodic acid in THF at room temperature for 1 h. After work-up, the crude material was purified by flash chromatography to give pure 13

(1.1 g, 35%). Stereochemical purity was Z.'E 97:3 as shown by GC-MS analysis. Analysis: calculated for C19H3602; C, 76.98; H, 12.24. Found: C, 76.97; H, 12.81. IR ~ 3050, 2925, 1725, 1250, 1030 cm -1. IH-NMR 8 4.22 (m, 2H, CH20), 1.81 (s, 3H, CH3CO), 1.72 (m, IH, HA of C_H2CH2OAc), 1.54-1.28 (b, 22H, llCH2), 1.22 (m, 1H, HB of CH2CH2OAc), 1.02 (t J = 6.9 Hz, 3H, CH3) , 0.78-0.58 (c, 3H, H b of the cyclopropane trans to both substituents and CH-CH), -0.23 (m, 1H, H b cis to both substituents). 13C-NMR 8 171.3 (CO), 65.0 (C-I), 31.9 (C-14), 30.0-29.3 (C-6 to C-13), 28.7 (C-5), 27.9 (C-2), 22.7 (C-15), 21.1 (C-I'), 15.4 and 12.2 (C-3 and C-4), 14.1 (C-16), 10.6 (CH 2 of the cyclopropane ring). MS m/z (%): 236 (M-60, 5), 208 (6), 194 (3), 138 (4), 137 (4), 124 (7), 123 (7), 110 (12), 109 (14), 97 (15), 96 (37), 95 (26), 83 (20), 82 (47), 81 (48), 79 (10), 69 (23), 68 (57), 67 (45), 57 (17), 56 (10), 55 (54), 54 (36), 43 (100), 41 (40). cis-2-(2-Acetoxyethyl)-3-dodecyl-1,1-difluorocyclopropane (15) A modified procedure to that described by Taguchi and colleagues was applied [12]. Thus, a solution of 12 (375 mg, 1.32 mmol) in 3 ml of anhydrous diglyme was heated to reflux and then sodium chlorodifluoroacetate (4.04 g, 24 mmol) (dried at 110°C/0.3 torr for 7 h) in 10 ml of diglyme was slowly added under nitrogen. The reaction mixture was stirred for 1 h, diluted with hexane, filtered and the filtrate repeatedly washed with water and brine. Evaporation of the solvent afforded, after purification, the expected difluorinated cyclopropane 15 (222 mg, 51%). Stereochemical purity was Z:E 97:3 by GC-MS analysis. Analysis: calculated for CI9H34F202; C, 68.64; H, I0.31. Found: C, 68.60; H, 10.65. IR p 2920, 2850, 1745, 1465, 1230, 1040 cm -1. IHNMR 8 4.12 (ddt J = 10.8 Hz, 6.6 Hz and 6.6 Hz, 2H, CH20 ), 2.07 (s, 3H, CH3CO), 1.81-1.72 (m, 2H, CH-CH), 1.61-1.22 (c, 24H, 12CH2) , 0.88 (t J = 6.3 Hz, 3H, CH3). 19F-NMR -48.6 (din J = 155.9 Hz), -78.2 (d J = 155.9 Hz). MS m/z (%): 317 (M-15, 1), 233 (3), 221 (4), 193 (3), 177 (9), 146 (8), 144 (13), 132 (7), 118 (7), 115 (12), 101 (47), 97 (13), 95 (10), 69 (13), 57 (17), 56 (10), 55 (24), 43 (100), 41 (33).

161 cis-2-( 2-Dodecylcyclopropyl)ethanol (14) To a solution of 13 (650 mg, 2.19 mmol) in 3.4 ml of anhydrous THF was added 3.4 ml of 10% KOH solution. The reaction mixture was stirred at room temperature for 2.5 h, the solvent stripped off and the residue dissolved in water and extracted with ether. Usual work-up afforded, after purification on silica gel, the expected alcohol 14 (350 mg, 63%). Analysis: calculated for C17H340; C, 80.35; H, 13.47. Found: C, 80.31; H, 13.82. IR p 3600, 3400, 3050, 2920, 2850, 1100-1010 cm -l. IH-NMR 6 3.72 (t J = 6.2 Hz, 2H, CH20), 1.52 (s, 1H, OH), 1.9-1.1 (b, 24H, 12CH2), 0.89 (t J = 6.2 Hz, 3H, CH3) , 0.68 (b, 3H, H b of the cyclopropane trans to both substituents and 2Ha) , -0.20 (m, 1H, Hb cis to both substituents). 13CNMR 6 63.5 (C-I), 31.7 (C-14), 29.9-29.2 (C-2 and C-6 to C-13), 28.7 (C-5), 22.6 (C-15), 15.1 and 12.1 (C-3 and C-4), 14.0 (C-16), 10.5 (CH 2 of the cyclopropane ring). MS m/z (%): 236 (M-18, 2), 208 (4), 138 (5), 137 (7), 125 (5), 124 (10), 123 (12), 112 (5), 111 (17), 110 (18), 109 (19), 97 (37), 96 (55), 95 (41), 85 (15), 84 (15), 83 (55), 82 (78), 81 (72), 79 (14), 71 (33), 70 (29), 69 (56), 68 (80), 67 (75), 57 (61), 56 (37), 55 (100), 54 (33), 53 (12), 43 (84), 42 (23), 41 (99). cis-( 2-Dodecylcyclopropyl)acetic acid (5) To a solution of alcohol 14 (53 mg, 0.20 mmol) in 1.5 ml of freshly distilled DMF was added pyridinium dichromate (274 mg, 0.73 mmol) under N2. The reaction mixture was stirred at room temperature for 7 h, poured into water and repeatedly extracted with ether. Conventional work-up afforded a residue, which was purified by column chromatography on silica gel eluting with CH2Cl2/methanol 98:2 to yield the expected acid (23 mg, 43%) as a white solid, m.p. 37°C. Analysis: calculated for C17H3202; C, 76.07; H, 12.02. Found: C, 76.49; H, 12.35. IR p 3600~-2460, 2920, 2850, 1695, 1455, 1230 cm -l. IH-NMR 6 2.35 (ddd AB of an ABX system JAB = 16.5, JAX = 7.2 and JBx = 7.5 Hz, 2H, CH2COOH), 1.70-1.20 (b, 24H, 12CH2), 1.20-1.02 (m, IH, C_I_I_~CH2 COOH), 0.87 (t J = 6.6 Hz, 3H, CH3) , 0.84-0.70 (c, 2H, CH2CHb (CH2) and CH t of the cyclopropane ring trans to both substituents), -0.11 (dt J = 5.1 and 4.8 Hz, 1H, CH c of the

cyclopropane ring cis to both substituents). 13CNMR /t 179.0 (C-l), 33.5 (C-2), 31.9 (C-14), 29.9-29.3 (C-6 to C-13), 28.8 (C-5), 22.7 (C-15), 15.1 and 11.1 (C-3 and C-4), 14.1 (C-16), 10.8 (CH2 of the cyclopropane ring). MS m/z (%): 250 (M-18, 2), 214 (5), 208 (5), 179 (5), 178 (100), 171 (8), 129 (14), 115 (8), 111 (13), 98 (16), 97 (30), 96 (13), 95 (12), 87 (19), 85 (18), 84 (22), 83 (40), 82 (20), 81 (17), 73 (51), 71 (33), 70 (29), 69 (49), 68 (18), 67 (26), 60 (47), 57 (65), 56 (35), 55 (76), 54 (17), 53 (12).

cis-2- (2, 2-Difluoro-3-dodecylcyclopropyl) ethanol (16) A solution of acetate 15 (177 mg, 0.53 mmol) in 10 ml of a 0.02-M K2CO 3 solution in methanol was stirred at room temperature for 1 h. The solvent was removed, water added and extracted with ether. Usual work-up yielded, after purification, alcohol 16 in 81% yield (127 rag). IR v 3600, 2920, 2850, 1465, cm -1. 1H-NMR/~ 3.71 (t J = 6.3 Hz, 2H, CH2OH), 1.69 (m, IH, CF2CH_CH2), 1.621.45 (c, 3H, CH_2CHCF2 and CH_2CH2OH), 1.45-1.34 (c, 2H, CH_2CHCF2), 1.34-1.05 (c, 20H, 10CH2), 0.88 (t J = 6.3 Hz, 3H, CH3). 13C-NMR 6 114.5 (CF2), 62.1 (C-I), 31.8 (C-14), 29.6-29.2 (C-2 and C-5 to C-13), 24.9 and 21.7 (C-3 and C-4), 22.6 (C-15), 14.0 (C-16). ~9F-NMR -49.3 (dm J = 154.1 Hz), -78.0 (d J = 155.1 Hz). cis-( 2,2-Difluoro-3-dodeeylcyclopropyl)acetic

acid

(6) The same method was applied as for compound 5. Thus, starting from alcohol 16 (32 mg, 0.11 mmol), the expected acid 6 was obtained (11 mg, 32.5%) as a white solid, m.p. 46-47°C, after column chromatography on silica gel eluting with chloroform/methanol 95:5. Analysis: calculated for CI7H30F202; C, 67.07; H, 9.93. Found: C, 66.51; H, 9.96. IR u 2920, 2850, 1710, 1470, 1110 cm -l. 1H-NMR 6 2.52 (m, 2H, CH2COOH), 1.85 (m, IH, CH_CH2COOH), 1.7-1.5 (b, 1H, CHCF2), 1.5-1.1 (b, 20H, 10CH2), 0.88 (t J = 6.6 Hz, 3H, CH3) ~3C-NMR ~5 176.9 (CO), 113.9 (CF2), 31.9 (C-14), 29.6-29.0 (C-6 to C-13), 26.8 and 21.6 (C-2 and C-5), 24.8 (C-3), 22.7 (C-15), 20.0 (C-4), 14.1 (C-16) 19F-NMR 50.7 (dm J = 157.8 Hz), 77.5 (d J = 157.9 Hz). MS m/z (%): 265 (1), 224 (1), 205

162

(2), 177 (8), 113 (10), 111 (12), 109 (10), 99 (10), (12), 97 (23), 96 (12), 95 (15), 93 (12), 90 (ll), (27), 84 (14), 83 (31), 82 (14), 81 (20), 79 (10), (21), 73 (11), 71 (42), 70 (25), 69 (44), 68 (12), (21), 60 (13), 59 (10), 57 (100), 56 (33), 55 (72), (13), 53 (13).

98 85 77 67 54

evaporation of the solvent, a residue which was purified by flash chromatography to afford the expected silyl ether 18 in 73% yield (174 mg). Analysis: calculated for C22H46OSi; C , 74.50; H, 13.07. Found: C, 73.93; H, 13.12. IR v 3005, 2925, 2860, 1260, 1100, 840 cm -~. 1H-NMR (80 MHz) t~ 5.4 (m, 2H, CH=CH), 3.6 (t J = 7.2 Hz, 2H, CH20), 2.25 (art J = 6.0 and 4.8 Hz, 2H, CH_2CH20), 2.15-1.8 (c, 2H, CHEC=C), 1.6-1.1 (b, 20H, 10CH2) , 0.89 (s, 9H, (CH3)3C), 0.85 (t, 3H, C H 3 ) , 0.4 (s, 6 H , (CH3)2Si). M S m/z (%)" 339 (M-15, 2), 299 (5), 288 (23), 297 (100), 143 (10), 129 (13), 101 (12), 89 (15), 75 (37), 73 (14), 43 (ll), 41 (ll).

(Z)-3-Hexadecen-l-ol (17) This compound was prepared as l l , except that the reduction was carried out at -10°C. Stereochemical purity Z.'E 95:5 by GC-MS analysis. Yield: 84%. Analysis: calculated for C16H320; C, 79.52; H, 13.41. Found: C, 79.73; H, 13.46. IR v 3635, 3600-3200, 2930, 2860, 1050 cm -1. IH-NMR/~ 5.45 (m, 2H, CH=CH), 3.65 (t J = 6.6 Hz, 2H, CH20), 2.35 (dt J -- 6.0 and 4.8 Hz, 2H, CH_2CH20), 2.15-1.8 (c, 2H, CH2C=C), 1.5-1.1

(b, 20, 1 0 C H 2 ) , 0.89 (t, 3 H , C H 3 ) .

cis-2- (2-t-Butyldimethylsilyloxyethyl)-l, 1-dichloro3-dodecylcyclopropane (19) The same procedure described by Kenney et al. [13] was used except that the molar ratio NaOEt/ethyl trichloroacetate/substrate was 31:24:1 and the reaction time 18 h. Compound 19 was obtained in 32% yield after column chromatography on silica gel. IR v 2925, 2850, 1255, 1205, l l00, 835 cm -l. 1H-NMR ~ 3.7 (t J = 6.0 Hz, 2H, CH20), 1.8-1.45 (c, 4H, CH_2CH20 and CH-CH), 1.45-1.2 (b, 22H, 11CH2) , 0.9 (m, 12H, C_H3CH2 and (CH3)3C), 0.08 (s, 6H, (CH3)2Si). 13C-NMR fi 61.9 (C-l), 32.7 (C-2), 31.9 (C-14), 29.7-28.4 (C-5 to C-13), 25.9 ((CH3)3) , 25.0 and 17.9 (C-3 and C-4), 22.7 (C-15), 14.1 (C-16), -5.3 (CHaSi). MS m/z (%): 342 (2), 234 (2), 233 (6), 149 (9), 135 (17), 125 (32), 123 (100), 121 (24), I09 (18), 107 (12), 95 (37), 93 (53), 89 (14), 83 (11), 81 (16), 79 (11), 73 (25), 69 (15), 67 (11), 57 (21), 43 (15), 41 (10).

Iac-

N M R t~ 133.6 (C-3), 124.9 (C-4), 62.3 (C-l), 31.9 (C-14), 30.8 (C-2), 29.7-29.3 (C-6 to C-13), 27.4 (C-5), 22.6 (C-15), 14.0 (C-16). MS m/z (%): 222 (M-18, 7), 194 (3), 166 (3), 152 (3), 138 (6), 137 (6), 124 (10), 123 (11), ll0 (17), 109 (18), 82 (81), 81 (65), 71 (21), 70 (15), 69 (44), 68 (100), 67 (74), 57 (48), 56 (22), 55 (89), 54 (35), 43 (80), 42 (20), 41 (99).

1-t-Butyldimethylsilyloxy-3-hexadecene (18) To a cold solution (0°C) of t-butyldimethylsilyl chloride (120 mg, 0.79 mmol) in 0.35 ml of D M F was sequentially added imidazole (114 mg, 1.67 mmol) and alcohol 17 (163 mg, 0.67 mmol). The bath was removed and the reaction mixture stirred at room temperature for 19 h. Pentane was then added and washed with brine to leave, after

R--Br

i

~

R--C-CH

ii

R--C-C--COOH

8 R = C12H25 9 R = C13H27

R--C --CH 8

iii

~-

R--C --C/~'CH20H

2 R = CI2H2s 3 R = C13H27 iv

~'-

1o

8, 10, 4: R = C12H25 Scheme 2. (i) Lithium acetylide/NH3; (ii) BuLi/THF, CO2; (iii) EtMgBr/ethylene oxide; (iv) CrO 3, acetone.

R--C --=C/~'COOH 4

163

eis-2- ( 2,2-Dichloro-3-dodecylcyclopropyl) ethanol

(20) A mixture of silyl ether 19 (32 mg, 0.073 mmol), tetrabutylammonium fluoride trihydrate (70 mg, 0.22 mmol) and 7 ml of THF was stirred at room temperature for 30 min. After quenching with water, the organic material was extracted with ethyl acetate and washed with brine to afford the expected alcohol 20 in 54% yield, after purification on column chromatography eluting with pentane/ethyl acetate 80:20. Stereochemical purity Z:E 84:16 on GC-MS analysis. Analysis: calculated for C17H32C120; C, 63.15; H, 9.98. Found: C, 63.12; H, 10.16. IR v 3630, 3400, 2920, 1050, cm -l. 1H-NMR /~ 3.78 (t J = 6.6 Hz, 2H, CH20), 1.68 (m, 2H, CH2CH20), 1.54 (m, 2H, OH and CH A of the cyclopropane ring), 1.45 (m, 3H, CH_2CH and CHB of the cyclopropane ring), 1.35-1.16 (b, 20H, 10CH2) , 0.88 (t J = 6.9 Hz, CH3). 13C-NMR fi 61.7 (C-l), 32.7 (C-2), 31.9 (C-14), 29.7-29.3 (C-6 to C-13), 28.6, 28.1 and 25.0 (C-3, C-4 and C-5), 22.7 (C-15), 14.1 (C-16). MS m/z (%): 251 (4), 221 (10), 182 (9), 152 (14), 150 (20), 142 (44), 140 (64), 128 (13), 125 (13), 124 (18), 123 (17), 122 (18), 121 (12), 115 (12), 114 (14), 113 (12), ll2 (63), I l l (28), ll0 (100), 97 (22), 95 (El), 83 (24), 81 (20), 69 (25), 57 (30), 55 (33), 43 (46), 41 (45).

cis-( 2,2-Dichloro-3-dodecylcyclopropyl ) acetic acid

(7) The same procedure described for acid 5 was applied. Thus, starting from alcohol 20 (110 mg, 0.4 mmol), pyridinium dichromate (1.05 g, 2.8 mmol) and 3 ml of DMF, the acid 7 was obtained as a colourless oil in 36% yield after purification on silica gel eluting with CH2CI2/MeOH 98:2. Analysis: calculated for C17H30C1202; C, 60.52; H, 8.98. Found: C, 60.40; H, 8.98. IR o 2926, 2854, 1718, 1209, 791 cm -l. IH-NMR ~i 2.61 (dd J = 18.0 and 7.5 Hz, 1H, CHACO2H), 2.46 (ddJ = 17.6 and 7.2 Hz, 1H, CHBCOEH), 1.96 (dr J = 10.8 and 7.2 Hz, 1H, CH_CH2COEH), 1.65 (m, 1H, CHCCI2), 1.50-1.35 (m, 2H, CH2CH_2CH), 1.35-1.20 (b, 20H, 10CH2), 0.88 (t J = 6.6 Hz, 3H, CH3). 13CN M R ~ 177.6 (CO), 64.2 (CC12), 32.6 (C-3), 31.9 (C-14), 30.0-29.3 (C-6 to C-13), 28.5 (C-2), 27.8 (C-4), 24.9 (C-5), 22.7 (C-15), 14.1 (C-16). MS m/z

(%): 265 (2), 256 (3), 221 (3), 205 (4), l ll (25), 109 (27), 102 (28), 101 (29), 97 (46), 95 (52), 94 (22), 93 (26), 91 (26), 88 (48), 85 (20), 84 (27), 83 (71), 82 (44), 81 (58), 80 (22), 79 (62), 77 (43), 71 (44), 70 (58), 69 (92), 68 (33), 67 (83), 66 (25), 65 (100). Results and Discussion

The/3-oxidation process involves the successive oxidative loss of acetyl groups from long chain fatty acids as acetyl-CoA units. Inhibition of the pro, cess can thus occur at any stage between the initial transformation of the fatty acids into their CoA esters and the final conversion into acetyl-CoA [14]. So far, the inhibitors for which their mechanism of action have been reasonably well studied, are inhibitors of acyl-CoA synthetase, carnitine palmitoyltransferase, acyl-CoA dehydrogenase and acyl-CoA thiolase [6]. For the inhibition of the biosynthesis of the major component of the sex pheromone of the Egyptian armyworm Spodoptera littoralis, we have synthesized some acetylenic and cyclopropyl substituted fatty acids, unreported so far in the literature, and tested their biological activity in vitro and in vivo bioassays (Scheme 1). Substituted fatty acids 2-3 have been considered because of their expected good Michael acceptor character, not requiring further activation to interact with the enzyme. In this context, 2-alkynoyl-CoA derivatives have been found to be irreversible inhibitors of the pig kidney general acyl-CoA dehydrogenases [15]. Acetylenic acid 4, in turn, was considered as a potential 'suicide' inhibitor since the initial carbanionic species, produced by the proton abstraction at C-2, could rapidly rearrange to the corresponding allene and this be subjected to a Michael addition reaction at C-3 by a nucleophilic active residue of the enzyme. This mechanism has been proposed by Fendrich and Abeles [16] and Frerman et al. [17] to explain inactivation of acyl-CoA dehydrogenases by acetylenic

~COOH NH2 A

~COOH B

Scheme 3. Structures of hipoglycin 21 and its metabolite 22.

164 X R

--X-

"-~-~

R~CO--SCoA

"CO -- SCoA

X = F , CI

R = C12H25

Scheme 4. Proposed mechanism of action of acids 6 and 7.

thioesters. Preparation of compounds 2-4, was accomplished, respectively, by condensation of CO 2 and ethylene oxide with the corresponding acetylides followed, in the case of 4, by Jones oxidation of the resulting alcohol 10 (Scheme 2). 2-Bromopalmitic acid was also included in our study as a standard reference, since it has already been shown to be a good inhibitor of fatty acid oxidation [1,6]. Cyclopropane acids 5 - 7 have been postulated as potential biosynthetic inhibitors due to their structural similarity to methylenecyclopropylacetic acid (MCPA) B, a metabolite of the hypoglycemic amino acid hypoglycin A, the active and toxic component of the unripe ackee fruit Blighia sapida [18] (Scheme 3). MCPA has been implicated in some of the toxic effects of the fruit in mammals and its CoA ester is believed to inhibit oxidation of fatty acids in vivo [19] and in vitro [20] bioassays. In addition, MCPA-CoA irreversibly inactivates pig kidney general acyl-CoA dehydrogenase [18], through a mechanism involving deprotonation at the a position of the CoA ester, followed by ring opening to generate a

nucleophilic species capable of irreversibly alkylating the flavine. In our case, the same type of mechanism would be foreseen for acid 5 whereas for acids 6 and 7 the generated carbanionic species could eventually open the cyclopropane ring giving rise to an activated halodiene via 1,4-elimination. The resulting diene would be a good Michael acceptor capable of irreversibly alkylating a nucleophilic active site of the enzyme (Scheme 4). In this context, it is known that the ring opening reaction of 1,l-difluorocyclopropane derivatives in the presence of an appropriate base proceeds in a stereospecific manner leading either to E,E or E,Z fluorodienes depending upon the original stereochemistry of the cyclopropane [21,22]. In our case, we decided to prepare the cyclopropyl substituted acids 6-7 with cis stereochemistry, since the ring opening reaction should almost exclusively lead to the E,Z (trans, trans) isomer, the same stereoisomer expected in the a,fldehydrogenation of other acyl-CoA derivatives. Synthesis of acid 5 was based on the

R--C ----C'/~"CH2OH

R ~ O A c

10

i

R--C _~C/'~CH2OAc

ii

ll

12

iii 13

14

$

R = C12H25 Scheme 5. (i) Ac20/pyridine; (ii) H2, Pd/BaSO4, quinoline; (iii) Zn/CH212, DME; dichromate/DMF.

(iv) K2CO3/MeOH;

(v) pyridinium

165

R ~ O A c

R~ , , - . . ~ O A c

i

12

'x F F

R~ j " . ~ O H

i ii

ii

15

~._

F F

R~ , , . ~

COOH

F F

16

R = C12H25 Scheme 6. (i) CICF2COONa/diglyme; (ii) K2CO3/MeOH; (iii) pyridinium dichromate/DMF.

cyclopropanation of unsaturated acetate 12 with CH2I 2 and ultrasonic-activated Zn in DME [11], since the standard Simmons-Smith reaction (ZnCu/CH212 in ether) [23] did not work out satisfactorily with our unactivated olefin. The expected compound 13 was obtained although contaminated with 24% of unreacted material 12. Since separation of both compounds turned out to be extremely troublesome, ozonolysis of the mixture had to be carried out to yield pure, olefin-free, cyclopropane 13 in stereochemical purity Z : E 97:3, although in modest yield (35%). Acetate 12 was obtained in a straightforward manner from acetylenic alcohol 10, an intermediate in the synthesis of 4, in 80% overall yield. Base treatment of 13 followed by pyridinium dichromate oxidation yielded the expected acid 5, which was characterized by its analytical and spectroscopic properties (Scheme 5). The 1H-NMR (300 MHz) showed, as

R--C ~C~'CH2OH 10

i ~

R~tOH 17

ii

the main features, a doublet of triplets (J = 5.1 and 4.8 Hz) at ~5-0.11 which was assigned to the Hc proton of the cyclopropane ring and cis to both substituents, a complex absorption at 0.70-0.84 corresponding to two protons, the H b adjacent to the CH 2 of the cyclopropyl group and the Ht trans to both substituents, and a multiplet at ~t 1.02-1.20 corresponding to C_HaCH2CO2H. The methylene group in ot position to the carboxyl group appeared, as expected, as the AB of an ABX system (/~ 2.35) with JAB = 16.5 Hz, JAX = 7.2 Hz and JBX = 7.5 Hz. In the 13C-NMR spectrum, the CH2 of the cyclopropane ring resonates at 10.8 ppm whereas C-3 and C-4 appear at 15.1 and 11. I ppm (assignments may be interchangeable). Preparation of difluorocyclopropane 6 was accomplished by a similar route to that described for 5. In this case, cyclopropanation of acetate 12 was carried out using sodium chlorodifluoroacetate as

R~OTBS 18

i ii R~./OTBS

iv ~ R,~....~OH

v R,~

CI CI

CI Cl

Cl Cl

19

20

7

COOH

R = C12H25 Scheme 7. (i) H 2, Pd/BaSO 4, quinoline; (ii) tert-butyldimethylsilyl chloride, imidazole/DMF; (iii) CIBCCO2Et , NaOEtYheptane; (iv) T B A F ' 3 H20/THF; (v) pyridinium dichromate/DMF.

166

the carbene source, following a modified procedure to that described by Taguchi et al. [12]. The isolated yield of difluoroacetate 15 (51%) was in the range of the best found in the literature [24,25] for unbranched and unactivated olefinic compounds. It must be noted that the reaction was stereospecific, since only the cis isomer was detected, and no isomerization of the double bond occurred as shown by GC-MS on a SPB-5 fused silica capillary column (Z:E 97:3) (Scheme 6). Preparation of dichlorocyclopropane acid 7, in turn, proceeded through cyclopropanation of tertbutyldimethylsilyl-protected alcohol 18 with CCI3COOEt/NaOEt by a modified procedure of that described by Kenney et al. [13]. Thus, the molar ratio NaOEt/CClaCOOEt/substrate had to be increased to 31:24:1 and the reaction time lengthened to 18 h, since our compound 18 reacted sluggishly under the described reaction conditions. It should be added that previous attempts of cyclopropanation with CHCla/tert-BuOK were unsuccessful in our case, due to the competitive addition reaction of the carbene with the in situ originated tert-BuOH. In the former case, the formation of ethyl carbonate as a non-protic secondary product precluded any subsequent reaction with the carbene. The required large excess of base, however, promoted isomerization (16%) of the double bond as determined by GC-MS analysis, as it has been previously noticed [26] (Scheme 7). The biological activity of compounds 1-7 has been determined by in vitro and in vivo bioassays. In the in vitro experiments radiolabelled [1-14C]palmitic acid and the inhibitor were added to the pheromone gland preparation in the presence of NAD, NADP, ATP and coenzyme A as cofactors. The extent of inhibition was measured by the relative loss of [1-laC]acetic acid, produced in the B-oxidation step, in comparison with the control (no inhibitor present). Dichloroacid 7 displayed the highest activity when mixed with radiolabelled palmitic acid in 10:1, 5:1 and 3:1 ratios, being the inhibition values of 61.9, 51.2 and 42.6%, respectively. Slightly lower activity was shown by the reference compound 1, whereas the remaining acids (compounds 3, 5, 6) were only poorly active (5-9%) or practically inac-

tive (acids 2, 4). In the in vivo bioassays the putative inhibitor, dissolved in DMSO, was topically applied to the sex pheromone gland along with [16,16,16-d3]-palmitic acid. The relative decreased intensity of m/z 245 ion, corresponding to deuterated methyl myristate from the/3-oxidation step, determined the extent of inhibition. Again, compound 7 displayed a good inhibition activity being the dose which inhibits 50% of the acyl-CoA oxidase activity IC50 = 1.3 × 10-3 #mol/insect, practically the same order of magnitude as the standard compound 1. The striking difference in activity found in the cyclopropane acids 5-7 could be rationalized in base of the mechanisms of action proposed in each case. Thus, if unsubstituted cyclopropane 5 gives the expected carbanion by proton abstraction at C-2, in a similar way to that of MCPA-CoA B (see above), the resulting species would not be as stabilized as the one resulting from MCPA, wherein three resonance forms are possible, and therefore one might expect the former to be more prone to decomposition than susceptible to attack the flavine residue of the enzyme. In this context, the unsaturated analog of acid $ with an extra double bond at C-4 would be expected to be active, and provide additional clues to support the proposed mechanism and the above assumption as well. On the other hand, the same carbanionic species in compounds 6-7 would yield, by loss of a neutral HX molecule, the corresponding 3,-halo-o~,/3,3,,& unsaturated esters, potentially good Michael acceptors for a nucleophilic site of the enzyme (Scheme 4). However, according to our experience on fluorinated compounds, fluorine should be considered both as an electron-donating and electron-withdrawing atom when located on a s p 2 carbon. Thus, in the 13C-NMR spectra of fluoroethylenes it exerts a clear shielding effect on the fl and 3' carbons of the double bond when compared with the parent non-fluorinated compound [27] (A6 ~. -20-25 ppm for/3 and -2-9 ppm for 3'). The ~ carbon, in turn, is deshielded (At5~ 20 ppm) as could be expected from the strong inductive effect of the halogen. In dienic systems, the same type of effect has been found on carbons ct,/~, ~', and 3' in the 13C NMR absorptions of methyl

167

4-fluoro-2,4-pentadienoate [27] in comparison with the corresponding non-fluorinated counterpart [28]. Moreover, the IR absorption at 1720 cm -I of the fluorinated double bond in 6-fluorogeraniol [29], also confirms the electrondonating ability of the halogen in fluorinated oleflns. As a consequence, the fluorinated dienic CoA ester (Scheme 4) might not be as good Michael acceptor as could be expected, and probably less than its chlorinated analog, from which only strong inductive effect a priori might be anticipated. Therefore, a much lower inhibition activity might be displayed by difluoroacid 6 in comparison with dichloroacid 7, which is in agreement with our experimental results.

7 8 9 10 11 12

13 14 15 16

Acknowledgements 17

We gratefully acknowledge CICYT (PB 87-0290) for financial support. We are indebted to M. Feliz (University of Barcelona) for recording the 200 MHz N M R spectra, P. Dom~nech (C.I.D.) for elemental analyses and to MEC for a predoctoral fellowship to S.H.

References

18 19 20 21 22 23

l

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