Download - Inhibitory pheromonal activity promoted by sulfur analogs of the sex pheromone of the female processionary moth Thaumetopoea pityocampa (Denis and schiff

Journal of Chemical Ecology, Vol. 16, No. 4, 1990

INHIBITORY PHEROMONAL ACTIVITY PROMOTED SULFUR ANALOGS OF THE SEX PHEROMONE

OF THE FEMALE PROCESSIONARY MOTH Thaumetopoea pityocampa (DENIS AND SCHIFF)

BY

FRANCISCO CAMPS, VICENS GASOL, and A N G E L GUERRERO

Department of Biological Organic Chemistry, C.L D. (CS1C) Jordi Girona Salgado, 18-26, 08034-Barcelona, Spain

(Received February 1, 1989; accepted June 22, 1989)

Abstract--New sulfur analogs of the sex pheromone of the female processionary moth Thaumetopoea pityocampa have been found to be effective inhibitors of the natural pheromone activity both in EAG bioassays and field tests. The structures of these analogs have been derived from replacement of the oxygen atom(s) of the acetate group by sulfur (compounds 3-5) and the olefinic moiety of the enyne function by the isosteric SCH~_ group (compounds 6 and 7). The synthesis and biological activity of 3-[(Z)-12-pentadecen- 10-ynylthio]- 1,1,1 -trifluoropropan-2-one (8), a closely related structure to the pheromone is also described.

Key Words--Sex pheromone, inhibition, processionary moth, Thaumeto- poea pityocampa, sulfur analogs, Lepidoptera, Thaumetopoeidae.

INTRODUCTION

The inhibition process of insect olfaction can be approached by irreversible activation of the receptors (hyperagonism) or by blockage of the pheromone recognition by a receptor cell (antagonism) (Prestwich, 1987). In this context, the design of pheromone analogs may lead not only to a better understanding of the binding interactions between the natural pheromone and the receptor proteins, but also to the development of new compounds potentially useful in pest control (Albans et al., 1984; Roelofs and Comeau, 1971; Camps et al., 1988). These analogs have been logically designed either by isosteric replacement of atoms and functions or by sterical modifications o f the parent molecule with

1155

0098-0331/90/0400-1155506.00/0 �9 1990 Plenum Publishing Corporation

1156 CAMPS ET AL.

preservation of the original electronic environment (Priesner, 1979; Liljefors et al., 1985; Subchev et al., 1987).

The only component found so far in the female sex pheromone gland of the processionary moth Thaumetopoea pityocarnpa has been identified as (Z)- 13-hexadecen-11-ynyl acetate 1 (Guerrero et al., 1981). Several efficient syntheses of this compound have been reported (Camps et al., 1981, 1983; Michelot et al., 1982; Shani et al., 1983; Cardillo et al., 1982; Stille and Simp- son, 1987) and its attractant activity confirmed in field tests (Cuevas et al., 1983; Einhorn et al., 1983).

Continuing our efforts in the development of new analogs with synergistic or inhibitory activity of the sex pheromone of the processionary moth (Camps et al., 1986, 1988), we describe herein the synthesis and biological activity, in EAG bioassays and field tests, of sulfur analogs 3-8. These compounds formally proceed from structural modifications on two of the three putative active sites of the parent molecule 1, which could be involved in the interaction process with the antennal receptors, namely, the acetate group and the double bond of the enyne functionality. However, we have preserved the acetylenic moiety at C-11 since its presence has been shown to be essential to elicit a notable pheromonal activity (Camps et al., 1988). Thioesters 3 and 4 and dithioester 5 have resulted from replacement of the oxygen atom(s) in the acetate group by sulfur, whereas sulfides 6 and 7 proceed from isosteric replacement of the olefinic carbons by the CHES group (Scheme 1).

~ I /~ ,~ , ~ ~ ~ j ~ , OA c ~ ~ SCSCH3

1 5

2 6

3

~ X ~ ~ 0 C S C H 3

4

~NzN

7

~f~x~x~x~x~.x ~SCH2 COCF3

SCHEME 1.

INHIBITION BY SULFUR ANALOGS ] 157

On the other hand, in view of the reported inhibitory activity of insect juvenile hormone esterases by trifluoromethylketone derivatives (Hammock et al., 1982), we included 3-[(Z)-12-pentadecen-10-ynylthio]-l,l,l-trifluoropropan-2-one (8) in our pheromonal inhibition studies.

M E T H O D S A N D M A T E R I A L S

Boiling points are uncorrected. Elemental analyses were determined on a Carlo Erba model 1106. IR spectra were recorded in CC14 solution on a Perkin Elmer 399B grating spectrometer. [IH]- and [13C]NMR spectra were determined in CDC13 solution on a Bruker WP80SY spectrometer, operating at 80 and 20.15 MHz, respectively, and absorptions are expressed in 6 scale relative to TMS. [I9F]NMR spectra were recorded in CDC13 solution on the same instru- ment at 75.39 MHz, and the values are expressed in/5 scale relative to trifluo- roacetic acid (1% in CDC13) as external reference. GLC analyses were performed on Carlo Erba models 2350 and 4130, equipped with a FID detector, by using a 3% OV-101 glass column 2 m x 3 mm ID on Chromosorb W (nitrogen as carrier gas), or a fused silica capillary column SE-54 50 m x 0.32 mm ID (hydrogen as carrier gas). EI mass spectra were determined on a Hewlett Packard 5995C model using a OV-101 25-m x 0.25-mm-ID fused silica capillary column.

Reactions requiring anhydrous and oxygen-free conditions were performed under inert atmosphere (N 2 or A). Commercial reagents were from Fluka AG Buchs, (Switzerland) or Aldrich Chemie 1, Steinheim, (West Germany) and were used without further purification. Anhydrous solvents were prepared as follows: tetrahydrofuran (THF) by distillation from Na benzophenone, diethyl ether from lithium aluminum hydride (LAH), methylene chloride from P205, pyridine and diisopropylamine from KOH, and benzene and hexamethylphos- phoric triamide (HMPT) from Call 2.

S-(Z)-13-Hexadecen-ll-ynyl Thioacetate (3). This compound was prepared by a method similar to that described by Volante (1981). Thus, to a solution of 1.15 g (4.40 mmol) of triphenylphosphine in 17 ml of anhydrous THF, previously cooled to 0~ was added 0.94 g (4.64 mmol) of diisopropyl azodicarboxylate and the mixture stirred for 1 hr at the same temperature. Then, a solution of 0.53 g (2.25 mmol) of alcohol 2 and 0.366 g (4.81 mmol) of thioacetic S-acid in 9 ml of anh. THF was added and the mixture stirred at room temperature for 3 hr. The resulting solid was filtered and washed with ether. The combined organic phases were concentrated under reduced pressure to leave a residue, which was purified by column chromatography on silica gel eluting with hexane-ether 4 :1 to yield 0.52 g (89%) of thioester 3 (Scheme 2).

Anal.: Calcd. for C18H3oOS: C, 73.41; H, 10.27; S, 10.89. Found: C, 73.40; H, 10.37; S, 10.86. IR: p 3010, 2920, 1690, 1135, 1105, 730 cm -~.

1158 CAMPS ET AL.

V=N %

( CH2 ) 100H

2

CH3C(O)SH

iPrOOCNNCOOiPr

Ph3P

V=N %

(CH 2 ) IoSCOCH 3 3

v : N \ (CH2)IoOCOCH 3

I

1. LDA 2. CISiMe 3

Lawesson R. i r

v=k

(CH 2 ) 100CSCH3

H2S

% OS• (CH3) 3 I ~ % SH

( CH 2) i00c -- CH2 ( CH 2 ) i00c ---- CH 2

SCHEME 2.

[1H]NMR: 6 5.75 (dt, 1H, J --- 10.5 and J = 7 Hz, CH2CH=CH), 5.40 (d, 1H, J = 10.5 Hz, CHzCH=CH), 2.50 (t, 2H, J = 6.5 Hz, CH2SCO), 2.30 (c, 4H, CH2CH=CHC =-CCH2), 2.05 (s, 3H, SCOCH3), 1.4 (b, 16H, 8CH2), 1.0 (t, 3H, J = 7.0 Hz, CH3). [13C]NMR: ~ 195.7 (CO), 143.8 (C-14), 108.8 (C-13), 94.4 (C-11), 78.8 (C-12), 75.4 (C-I), 30.4 (C-I'), 28.2-24.4 (C-2 to C-9), 23.3 (C-15), 19.7 (C-10), 13.5 (C-16). MS (El) m/z (relative intensity): 294 (M § 10), 265 (10), 251 (98), 183 (14), 135 (11), 161 (16), 107 (18), 93 (48), 79 (82), 67 (15), 55 (18).

O-(Z)-13-Hexadecen-ll-ynyl Thioacetate (4). To a solution of 0.17 ml (1.19 mmol) of diisopropylamine in 2 ml of anh. THF, cooled to 0~ and under A, was slowly added 1.25 ml of a 0.95 M BuLi (1.19 mmol) in hexane. The mixture was stirred at this temperature for 30 rain and cooled to -78~ Then a mixture of 300 mg (1.079 mmol) of acetate 1 in 5 ml of anh. THF and 140 mg (1.28 mmol) of trimethylsilyl chloride were added and the mixture stirred at that temperature during 10 rain (Corey and Wrigth, 1984). After this period of time, the mixture was wanned to - 10~ and a stream of dry HzS was bub-

INHIBITION BY SULFUR ANALOGS 1159

bled through the solution during 15 min, wanned to room temperature and further stirred for 2 hr. The solvent was stripped off and the residue was chromatographed on silica gel eluting with hexane-ether 5 : 1 to obtain 215 mg (69%) of thioester 4 (Scheme 2).

Anal. Calcd. for C18H3oOS: C, 73.41; H, 10.27; S, 10.89. Found: C, 73.28; H, 10.37; S, 10.87. IR: ~ 3010, 2930, 1600, 1240, 640 cm -~. [1H]NMR: 6 5.8 (dt, 1H, J = 10.5 Hz and J = 7 Hz, CH2CH=CH), 5.45 (d, 1H, J = 10.5 Hz, CH2CH=CH), 3.4 (t, 2H, J = 7 Hz, CH2OCS), 2.4 (s, 3H, OCSCH3), 2.25 (c, 4H, CH2CH=CHC-CCH2) , 1.4 (b, 16H, 8CH2), 0.97 (t, 3H, J = 7 Hz, CH3). [13C]NMR: 6 219.0 (CS), 143.9 (C-14), 108.5 (C- 13), 94.5 (C-11), 78.7 (C-12), 64.0 (C-l), 34.5 (C-1'), 28.5-23.4 (C-2 to C-9 and C-15), 19.5 (C-10), 13.4 (C-16). MS (EI) m/z (relative intensity): 294 (M +, 19), 279 (11), 251 (43), 161 (5), 154 (16), 147 (11), 139 (4), 133 (22), 121 (20), 111 (20), 105 (38), 94 (79), 79 (100), 67 (46), 55 (65).

1-Iodo-(Z)-13-hexadecen-11-yne (9). This compound was prepared according to a procedure previously described by us (Camps et al., 1987). Thus, starting from 313 mg (0.904 mmol) of alcohol, 227 mg (1.08 mmol) of trifluo- roacetic anhydride and 268 mg (4.52 mmol) of anhydrous lithium iodide, 244 mg (78%) of compound 9 was obtained.

Anal.: Calcd. for C16H27I: C, 55.33; H, 7.78. Found: C, 55.28; H, 7.73. IR: v 3020, 2920, 2850, 1610, 1460, 740 cm -1. [~H]NMR: 6 5.85 (dt, 1H, J = 10.5 Hz and J = 7 Hz, CH2CH=CH), 5.4 (d, 1H, J = 10.5 Hz, CHzCH=CH), 3.18 (t, 2H, J = 6.3 Hz, CHzI), 2.3 (c, 4H, C H_2CH=CHC-- CCH_2), 1.7 (c, 2H, CHzCH2I), 1.3 (b, 14H, 7CH2), 0.93 (t, 3H, J = Hz, CH3). MS (EI) m/z (relative intensity): 346 (M +, 100), 317 (52), 303 (13), 154 (10), 149 (16), 135 (47), 121 (26), 107 (25), 95 (30), 79 (47), 55 (22).

(Z)-13-Hexadecen-ll-ynyl Dithioacetate (5). In a three-neck 25-ml round- bottomed flask provided with a gas inlet, septum and magnetic stirrer were placed 3 ml of anh. THF and 0.25 ml (4 mmol) of CS2. The mixture was cooled to -40~ and a solution of 0.45 ml of 1.4 M MeLi in ether (0.64 retool) was added under A. The reaction mixture was stirred for 20 min and, after this period of time, a solution of 110 mg (0.32 retool) of iodo derivative 9 in 2 ml of THF-HMPT (1 : 1) was added and the mixture stirred at room temperature for 4 hr. After quenching with brine and extraction with ether, the combined organic phases were washed with brine and dried (MgSO4) to yield an oily residue, which was chromatographed on silica gel eluting with hexane-ether 6 : 1 to obtain 80 mg (81%) of dithioester 5 (Scheme 3).

IR: p3020, 2940, 1589, 1200, 710 cm -1. [IH]NMR: 6 5.78 (dt, 1H, J =

10.5 Hz and J = 7 Hz, CHzCI-I=CH), 5.38 (d, 1H, J = 10.5 Hz, CH2CH=CH), 2.5 (t, 2H, J = 7 Hz, CH2SCS), 2.4 (s, 3H, CH3), 2.25 (c, 4H, CH2CH=CHC=CCH2) , 1.4 (b, 16H, 8CH2), 0.95 (t, 3H, J = 7 Hz,

1160 CAMPS ET AL.

~ / ~ i. TFAA ~ / ~

(CH2)IoOH (CH2)zOI 2 g

1. CS 2 ~ Lawesson R. 11

2. MeLi \ (CH2)IoSCSCH 3

5

SCHEME 3.

CH3). [13C]NMR: 6 217 (CS), 144.0 (C-14), 108.8 (C-13), 94.5 (C-11), 78.6 (C-12), 74.4 (C-l), 40.7 (C-I'), 28.4-24.5 (C-2 to C-9 and C-15), 19.5 (C- 10), 13.5 (C-16). MS (EI) m/z (relative intensity: 310 (M +, 1), 246 (4), 149 (7), 135 (30), 121 (28), 107 (31), 94 (100), 81 (20), 79 (79), 67 (24), 55 (21).

1-tert-Butyldimethylsilyloxydodec-11-yne (10). This compound was prepared by condensation of lithium acetylide with the corresponding 10-bromo- 1-tert-butyldimethylsilyloxydecane using a similar method to that described by us (Camps et al., 1983). The required starting material, in turn, was obtained by protection of 10-bromodecan-1-ol with tert-butyldimethylsilyl chloride in the presence of triethylamine and N,N'-dimethylaminopyridine (Heathcock and Jarvi, 1982).

IR: v 3400, 2920, 1550, 1250, 1100, 1000, 840 cm -1. [1H]NMR: 6 3.68 (t, 2H, J = 6 Hz, CH20), 2.2 (c, 2H, C=CCH2), 1.9 (t, 1H, J = 2.7 Hz, HC=CCH2), 1.35 (b, 16H, 8CH2), 0.9 (s, 9H, (CH3)3CSi), 0.05 (s, 6H, 2CH3Si).

12-Propylthio-1-tert-butyldimethylsilyloxydodec-11-yne (11). In a three- neck round-bottomed flask provided with a nitrogen inlet, septum, and stirring bar, were placed at -70~ 300 mg (1.01 mmol) of protected acetylene 10 and 4 rnl of anh. THF. To the solution was added 0.85 ml of a 1.2 M BuLi (1.02 rnmol) in hexane and the mixture stirred for 30 min. Then, 34 mg (1.06 mat.g.) of $8 was added and the mixture stirred at -70~ for 1 hr and at room temperature for 1 hr more. The resulting thiolate was cooled to -40~ and a solution of 138 mg (1.1 mmol) of n-propylbromide in 1 ml of anh. THF was slowly added. The mixture was stirred for 4 hr, quenched with water and thor- oughly extracted with ether. The organic phases were washed with brine and dried (MgSO4) to leave, after evaporation of the solvent, a residue that was chromatographed on alumina III eluting with hexane-ether 95 : 5 to yield 300 mg (82%) of the expected compound 11.

INHIBITION BY SULFUR ANALOGS 1 161

IR: ~ 2920, 2850, 1460, 1260, 1090, 840 cm -~. [1H]NMR: 8 3.6 (t, 2H, J = 6 Hz, CH2OSi), 2.7 (t, 2H, J = 6.5 Hz, CH2S), 2.25 (t, 2H, J = 6 Hz, SC-=CCH2), 1.4 (b, 18H, 9CH2), 1.0 (t, 3H, J = 7 Hz, CH3).

12-(Propylthio)dodec-11-ynyl Acetate (6). To a solution of 300 mg (0.82 mmol) of 11 in 4 ml of anh. THF, cooled in an ice bath, was added 630 mg (2 mmol) of tetrabutylammonium fluoride trihydrate. The mixture was stirred for 30 rain at 0~ and 1 hr at room temperature. The solvent was stripped off and the residue chromatographed on alumina III eluting with hexane-ether 5 : 1 to yield 180 mg (90%) of the corresponding alcohol. Acetylation was carried out in the presence of 1 ml of acetic anhydride and 1 ml of anh. pyridine at room temperature for 5 hr. After quenching with methanol and evaporation of the solvent, the organic material was taken up in ether and washed with brine and dried. The residue was purified by column chromatography on alumina III eluting with hexane-ether 7 :2 to afford 180 mg (92%) of acetate 6 (Scheme 4).

IR: u 2920, 2850, 1735, 1460, 1250, 1220, 1040 cm -1. [1H]NMR: 8 4.05 (t, 2H, J = 6.5 Hz, CH2OCO), 2.7 (t, 2H, J = 6.5 Hz, CH2S), 2.3 (t, 2H, J --= 6 Hz, SC=CCH2), 2.05 (s, 3H, OCOCH3), 1.4 (b, 18H, 9CH2), 1.0 (t, 3H, J = 7 Hz, CH3). MS (El) m/z (relative intensity): 298 (M +, 1), 189 (11), 173 (13), 149 (12), 143 (18), 135 (15), 119 (13), 105 (18), 94 (100), 79 (46), 66 (14), 55 (19).

13-Tetrahydropyranyloxytridec-2-yn-1-ol (13). In a previously flamed three-neck round-bottomed flask provided with a magnetic bar, A inlet, and septum, was placed at - 7 8 ~ 1.5 g (5.6 mmol) of 1-tetrahydropyranyloxy- dodec-l l-yne (Camps et al., 1983) in 4 ml of anh. THF. To the solution was slowly added 0.52 ml of a 1 M MeLi (0.52 mmol) in hexane, and the mixture stirred at - 7 8 ~ for 2 hr. Then, 0.180 g (6 mmol) of paraformaldehyde was slowly added and the mixture stirred for 3 hr at - 4 0 ~ and 3 hr more at room temperature. After quenching with brine and extraction with ether, the combined organic phases were washed with brine and dried (MgSO4). The solvent

/~/S\ 1. MeLi/$ 8 • X

HC =--- C (CH2) loOSit-BuMe2 2. n-PrBr (CH2) 100Sit-BuMe2

I0 ii

1 ,, TBAF ~ S \

------ ~\(CH2)IoOAC 2. Ac20

6

SCHEME 4.

1162 CAMPS ET AL.

was removed under vacuum to leave the protected alcohol 13 (1.2 g, 80%), pure enough to be used in the next step of the synthesis.

IR: u 3300, 3280, 2920, 1450, 1210, 1140, 1030, 910 cm -1. [1H]NMR: 6 4.55 (b, 1H, OCHO), 4.20 (t, 2H, J = 2.5 Hz, HOCH2C ~ C), 3.6 (c, 4H, 2CH20), 2.15 (c, 3H, C - C C H 2 and OH), 1.55 (b, 22H, llCH2).

2-(13-Bromotridec-ll-ynyloxy)tetrahydropyran (14). This compound was prepared according to a procedure already described by us (Camps et al., 1987). Thus, starting from 0.595 g (2.01 mmol) of compound 13, 490 mg (72%) of bromo derivative 14 was obtained after purification of alumina III.

IR: v 3290, 2920, 2220, 1460, 1030 cm -1. [1H]NMR: 6 4.55 (b, 1H, OCHO), 3.95 (t, 2H, J = 2.5 Hz, BrCH2C ~ C), 3.5 (c, 4H, 2 CH20), 2.2 (c, 2H, C-- CCH2), 1.45 (b, 22H, 11CH2).

13-(Ethylthio)tridec-ll-yn-l-ol (15). A mixture of 450 mg (1.3 mmol) of 14 in 5 ml of anh. benzene, 5 ml of a 20% NaOH solution, 10 mg of tetrabutylammonium bisulfite, and 138 mg (2.22 mmol) of ethanethiol was stirred at room temperature for 4 hr. The phases were decanted and the solvent evaporated off at reduced pressure. The organic material was taken up in 4 ml of methanol and treated with 10 mg ofp-toluenesulfonic acid for 5 hr. The acid was neutralized with saturated solution of NaHCO3, the solvent stripped off, and the crude product dissolved in ether, washed with brine, and dried. After purification by column chromatography on silica gel eluting with hexane-ether 4 :1 ,240 mg (79%) of the expected alcohol 15 was obtained.

IR: u 3300, 2200, 1040 cm -1. [1H]NMR: ~ 3.6 (t, 2H, J = 6 Hz, CH20), 3.35 (t, 2H, J = 2.5 Hz, SCHzC~C ), 2.8 (q, 2H, J = 7 Hz, SCHzCH3) , 2.2 (c, 2H, C=CCH2), 1.5 (b, 19H, 8 C H 2 and CH3).

13-(Ethylthio)tridec-ll-ynyl Acetate (7). To a solution of 200 mg (0.78 mmol) of alcohol 15 in 2 ml of anh. pyridine was added 2 ml of acetic anhydride. The mixture was stirred at room temperature for 5 hr, cooled on an ice bath, and quenched with 2 ml of methanol. After stirring for 15 min, the volatile material was evaporated under vacuum and the residue taken up in ether, washed with brine, and dried. The product was purified by column chromatography on silica gel eluting with hexane-ether 4:1 to yield 210 mg (90%) of acetate 7 (Scheme 5).

IR: ~ 3300, 2920, 2200, 1740, 1450, 1230, t040 cm -1. [1H]NMR: 6 4.05 (t, 2H, J = 6.5 Hz, CH2OCO), 3.3 (t, 2H, J = 2.5 Hz, S C H z C m C ) , 2 .8 (q,

2H, J = 7 Hz, CH3CHzS), 2.25 (c, 2H, C---CCH2) , 2 .05 (s, 3H, OCOCH3), 1.45 (b, 19H, 8CH2 and CH3). MS (EI) m/z (relative intensity): 298 (M + , < 1), 269 (2), 209 (3), 153 (1), 141 (2), 139 (3), 125 (5), 114 (67), 97 (12), 85 (100), 79 (13), 67 (10), 55 (13).

1-Bromo-(Z)-12-pentadecen-lO-yne (17). This compound was prepared according to the method already reported by us (Camps et al., 1987). Thus, starting from 300 mg (1.35 mmol) of alcohol 16, prepared by sequential pal-

INHIBITION BY SULFUR ANALOGS

HC~C(CH2)IoOTHP

12

BrCH2C~-C(CH2)IoOTHP

14

/~'S/~\(CH2)IoOH 15

i. MeLi

2. H2CO

EtSH

TBABS/NaOH/ PhH

HOCH2C~ C(CH2)IoOTHP

13

(CH2)IoOTHP

Ac20 ~--~ ~S/~(CH2)IoOAC py

7

SCHEME 5.

1163

i. TFAA

2. LiBr

H §

ladium-catalyzed coupling reaction of (Z)-l-bromo-l-butene and 2-(10-unde- cynyloxy)tetrahydropyran (Michelot et al., 1982) followed by acid hydrolysis, 330 mg (89 %) of bromide 17 was obtained after purification on silica gel eluting with hexane.

Anal.: Calcd. for CIsH~sBr: C, 63.16; H, 8.77. Found: C, 63.05; H, 8.81. IR: ~ 3020, 2980, 1610, 1450, 720 cm -1. [1H]NMR: 6 5.80 (dt, 1H, J = 10.5 Hz and J = 7 Hz, CH2CH=CH), 5.4 (d, 1H, J = 10.5 Hz, CHzCH=CH__), 3.45 (t, 2H, J = 6 Hz, CH2Br), 2.2 (c, 4H, CH2C=C and CH2C-=C), 1.4 (b, 14H, 7CH2), 1.0 (t, 3H, J = 7 Hz, CH3). MS (EI) m/z (relative intensity): 286 (M + +2, 38), 284 (M § 40), 257 (20), 175 (14), 149 (20), 135 (55), 121 (20), 107 (60), 93 (69), 79 (100), 67 (23), 55 (15).

(Z)-12-Pentadecen-10-ynethiol (19). To a solution of 179 mg (0.268 mmol) of bromide 17 in 2 ml of ethanol was added 50 mg (0.27 mmol) of thiourea. The mixture was heated to reflux for 2 hr, cooled, and the resulting salt 18 subjected to hydrolysis by reaction with 43 mg (1.1 mmol) of NaOH in 1 ml of water at room temperature for 24 hr. After extraction with benzene, the combined organic phases were washed with brine and dried. The solvent was evaporated off and the residue purified on silica gel to afford 120 mg (78%) of thiol 19.

IR: ~ 3020, 2980, 2590, 1450, 720 cm -1. [1H]NMR: 6 5.80 (dt, 1H, J = 10.5 Hz and J = 7 Hz, CH2CH=CH), 5.40 (d, 1H, J = 10.5 Hz, CH2CH=CH__), 2.45 (t, 2H, J = 6.8 Hz, CH2S), 2.15 (c, 4H, CH2CH=CH--C---CCH2), 1.4 (b, 14H, 7CH2), 1.0 (t, 3H, J = 7 Hz, CH3). MS (EI) m/z (relative intensity): 238 (M § 7), 209 (15), 195 (10), 167 (12),

1164 CAMPS ET AL.

135 (18), 121 (13), 106 (22), 94 (85), 80 (22), 79 (100), 67 (40), 61 (12), 55 (29).

3-[(Z)-12-Pentadecen-lO-ynylthio]-l,l,l-trifluoropropan-2-one (8). To a solution of 120 mg (0.5 mmol) of thiol 19 in 4 ml of anh. ClzCHz were added 200 mg (1.88 mmol) of sodium bicarbonate and 144 mg (0.75 mmol) of 3- bromo-l,1,1-trifluoropropan-2-one. The mixture was stirred at room temperature for four days, filtered, and the solvent removed under vacuum. The residue was purified on column chromatography over silica gel to afford 110 mg (65 %) of the expected ketone 8 (Scheme 6).

IR: v 3020, 2980, 1745, 1460, 1175, 1130, 720 cm -1. [IH]NMR: 6 5.8 (dt, 1H, J = 10.5 Hz and J = 7 Hz, CH2CH=CH), 5.45 (d, 1H, J = 10.5 Hz, CHzCH=C__H), 3.48 (s, 2H, SCH2CO), 2.35 (t, 2H, J = 7 Hz, CHzSCHzCO), 2.1 (c, 4H, C H 2 C H = C H C - CCH2), 1.4 (b, 14H, 7CH2), 0.95 (t, 3H, J = 7 Hz, CH3). MS (EI) m/z (relative intensity): 348 (M +, 12), 279 (8), 183 (3), 169 (8), 156 (4), 149 (4), 140 (3), 135 (8), 121 (9), 107 (17), 94 (100), 79 (52), 67 (20), 55 (13). [mF]NMR: 3 -76 .4 (s).

Laboratory Bioassays. Inhibition experiments were carried out by placing 1- to 2-day old males in 10-cm-ID Petri dishes. The dishes contained a 2 x 2-cm piece of Whatman No. 1 filter paper on which several amounts of the test compounds (0.1, 1, 10, 100, and 1000/~g) in hexane had been applied. The solvent was evaporated off immediately before the assay. A similar piece of paper treated with 100 ~1 of hexane and evaporated to dryness was used as control. The males were subjected to the vapors of the compounds for 4 hr in the dark, then taken out and after 5 rain their antennae removed. Electroanten- nogram activity was determined on a EAG set up as previously described (Guer-

1. TFAA ~ H2NC(S)NH2

(CH2)90H (CH2)9BP 16 17

NaOH ~ / ~ BrCH2COCF3 % % -

(CH2)9SCNH.HBr (CH2)oSH NaHC03 18 19

N/~N

\(CH2)9SCH2COCF 3

8

SCHEME 6.


rero et al., 1986). Ten "puffs" with 10 #g of synthetic pheromone were directed over the excised antennae and the depolarizations recorded at 40-s intervals to ensure full recovery of the antennal receptors. The EAG values were normal- ized to obviate the time-dependent changes in antennal responses. Between three and six replicates were carried out for each test. Inhibition values were obtained from the expression:

mean EAG pheromone response - mean

% inhibit. EAG ph. response after presaturation = • 100 mean EAG pheromone response

and the results were analyzed statistically for significance according to the Stu- dent's t test.

Field Tests. The required amount of test compound in each bait was mixed with 2.5 mg of paraffin, in order to slow down the release rate in the field, and dissolved in 1 ml of nanograde hexane. The solutions were transferred into closed polyethylene vials (3 x 1.1 cm ID), which were used as dispensers. Field trials were conducted in Mora de Rubielos (Teruel, Spain) during the 1987-1988 seasons. Traps used for the experiments were " d r y " and specially designed for processionary moth catches (Montoya, 1984). Traps were hung on trees at a height of 1.7-2.0 m and spaced 50 m apart when belonging to the same parcel. They were set out in statistically randomized blocks and revised and rotated every day. Five traps were used for each formulation. Trap catches were subjected to a square-root transformation followed by analysis of variance, and the data were analyzed statistically for significance according to Duncan's multiple-range test.

RESULTS A N D DISCUSSION

As we have mentioned above, we have prepared new sulfur analogs of the pheromone structure 1 and studied their biological activity in laboratory and field assays. Synthesis of thioester 3 was accomplished by the esterification reaction of (Z)-13-hexadecen-11-yn-l-ol (2) with thioacetic S-acid in the presence of triphenylphosphine-diisopropyl azodicarboxylate (Volante, 1981) in 89% yield. Compound 4 was synthesized in a one-step process by preparation of the trimethylsilyl enol ether ofpityolure 1 followed by reaction with dry H2S in 69% overall yield from 1 (Corey and Wrigth, 1984). It must be noted that reaction of 1 with Lawesson reagent, 2,4-bis(p-methoxyphenyl)-l,3-dithiadi- phosphetane-2,4-disulfide, considered one of the most efficient thionation reagents of ketones, amides, and esters (Pedersen et al., 1978), did not give in our hands acceptable yields of the expected thioester 4 (Scheme 2).

Dithioester 5 was obtained by alkylation of iodide 9 with lithium dithioacetate in 81% yield, prepared in situ by the action of methyllithium on carbon

1166 CAMPS ET AL.

disulfide at low temperature (Meijer et al., 1973). Again, direct thionation reaction of thioester 3 with Lawesson reagent to obtain compound 5 did not yield the expected product (Scheme 3). On the other hand, syntheses of compounds 6 and 7 were successfully accomplished starting from the same substrate, dodec- 11-yn-l-ol, which was protected as the t e r t - b u t y l d i r n e t h y l s i l y l ether 10 or the tetrahydropyranyl ether 12. Reaction of the acetylide of 10 with $8 afforded the corresponding thiol, which was alkylated in situ to the corresponding sulfide 11. After hydrolysis and acetylation, the required compound 6 was obtained in 75 % overall yield from 10 (Scheme 4). Similarly, reaction of the acetylide of 12 with pamformaldehyde furnished alcohol 13, which was transformed into bromide 14 (Camps et al., 1987). Reaction of 14 with ethanethiol under phase- transfer catalytic conditions yielded, after hydrolysis, alcohol 15, which was finally acetylated to afford acetate 7 in 41% overall yield from 12 (Scheme 5). Trifluoromethylketone 8 was prepared, according to the procedure outlined in Scheme 6, by reaction of bromide 17 with thiourea to yield, after hydrolysis, thiol 19, which was coupled with 3-bromo-1,1,1-trifluoropropan-2-one in anhydrous THF in the presence of sodium bicarbonate. The overall yield of the process was 45 % from 16.

In laboratory bioassays, inhibition was tested by recording the EAG response of the natural pheromone when the insects were treated with vapors of variable amounts of the synthetic pheromone and analogs. As shown in Fig- ures 1-3, the inhibition of the EAG responses was plotted versus amount of compounds tested. The most effective doses were in the 100 to 1000-/zg range, whereas the lower concentrations (0.1 and 1/zg) were in most cases inefficient. As expected, the highest inhibition effect was shown by the synthetic pheromone 1, which even at 0.1 /zg displayed a high level of activity. On the other hand, higher doses promoted a U-shaped curve with an inflection point of min- imum activity (55%) at 10/zg. Among the pheromone analogs assayed, compounds 3 and 6 were the most effective (70-83%), with a similar inhibition level shown by thioester 3 in comparison with the pheromone 1 at the 10- and 100-/zg doses (Figure 1). Compounds 4, 5, and 8 showed only moderate activity at 1000/zg (18-35%), whereas compound 7 was practically inactive. On the other hand, in agreement with the results of field trials (see below), the intrinsic EAG responses of the different analogs are relatively low compared with that of the pheromone (Figure 4).

It is noteworthy that, whereas replacement of the oxygen atom of the alkoxy group by sulfur in the ester moiety of the pheromone (compound 3) leads to the highest inhibitory activity, only a very modest effect appears when the corresponding oxygen atom of the carbonyl group is exchanged (compound 4). If both modifications are carried out simultaneously, the resulting compound 5 shows an intermediate inhibitory action.

When trifluoromethylketone 8 was assayed as a pheromone perception


100

80

60

40

20

0

Inhibition of EAG response (%)

0,1 1 10 100 1000 ug of analog

Thioester 3 [ ~ Thloester 4 I---7 Pheromone 1

100 -

80-

60-

40-

20-

Inhibition of EAG response (%)

m~

i

0.1 1000

, - - . .

1 i i

10 100 ug of analog

Dlthloester 5 ~ Sulfide 6 I---] Pheromone I

FIG. 1-3. Inhibition of the natural pheromone EAG response promoted by analogs 3-8. Bars represent the mean relative response (%) of six experiments. Responses with an asterisk (*) are statistically significant at P < 0.05 (Student's t test).

1 1 6 8 C A M P S ET AL.

100

80

60

40-

20-

O-

Inhibition of EA6 response (ok)

0.1 10 100 1000 ug of analog

Sulfide 7 ~-~ Ketone 8 [ ~ Pheromone 1

FIG. 1-3. Continued.

I00

QO

~. 8o

tll 70 r

60

5O

4O

"'-' 30

10

I 3 4 5 5 7 8

No. os Compounds Tested

FIc. 4. Relative intrinsic EAG activity showed by 10/~g of compounds 3-8 .


inhibitor in the laboratory bioassay, only moderate activity was found (30 % at 100- to 1000-/~g concentrations). However, in the field, a marked enhancement of this effect (95 % inhibition) was observed when this compound was mixed with the pheromone in a 10:1 ratio (Table 1). This preliminary result may encourage the pursuit of more extensive work on this possible new application of trifluoromethylketones in pest control studies.

Likewise, in field assays, thioester 4 and dithioester 5 showed a very high inhibition activity, ranging from 85 to 97 %, when mixed with the pheromone is 0.1 : 1, 1 : 1, and 10 : 1 ratios (Table 2), whereas, perhaps surprisingly, compound 3 was only moderately active. Sulfides 6 and 7 behaved as good inhibitors also when applied in baits containing a mixture of inhibitor-pheromone in 1 : 1 and 10:1 ratios. In agreement with the above EAG results, these compounds showed a very low attractant activity when used alone.

In summary, sulfur analogs 3-8, which formally proceed from structural modifications on two of the three putative critical molecular active sites of pityolure l , have been prepared for the first time and appeared to be good antagonists of the pheromone action in laboratory and field assays. In the laboratory, compounds 3 and 6 appeared to cause a marked decrease in the EAG response

TABLE 1. INHIBITORY EFFECTS OF COMPOUNDS 6-8 ON PITYOLURE 1 (MORA DE RUBIELOS, 1988)

Bait formulation (mg) Relative Total No. inhibitory

Parcel a Sulfide 6 Sulfide 7 Ketone 8 Pityolure of catches b activity (%)

IV

VI

0.1 1

10

0.1 1

10

0.1 1

10

1 280 b 15 1 137 c 58 1 126 c 62 1 330 a

53 c (16) C

1 379 b 16 1 303 c 33 1 211 c 53 1 453 a

3 d (0.7) c

1 234 a 29 1 86 b 74 1 15 c 95 1 329 a

2 d (0.6) c

Several km of distance between parcels. bFive replicates per trap. Catches followed by the same letter are not significantly different at P = 0.05 (Duncan's multiple-range test).

CRelative intrinsic attractant activity (%) of the analog compared with pityolure.

1170 CAMPS ET AL.

TABLE 2. INHIBITORY EFFECTS OF COMPOUNDS 3-5 ON PITYOLURE 1 (MORA DE RUBIELOS, 1987-1988)

Bait formulation (mg) Relative Total No. inhibitory

Parcel a Thioester 3 Thioester 4 Dithioester 5 Pityolure of catches b activity (%)

III

0,1 1

10

0.1 1

10

0.1 1

10

1 309 a - 10 c 1 214 bc 24 1 169 bcd 40 1 281 ab

0 e (0) a 1 56 b 85 1 24 b 93 1 16 b 96 1 382 a

12 b (3) d 1 43 b 88 1 10 b 97 1 16 b 95 1 365 a

0 c (0) d

aSeveral km of distance between parcels. bFive replicates per trap. Catches followed by the same letter are not significantly different at P = 0.05 (Duncan's multiple-range test).

CAn enhancement of the trap catch was obtained in this case. dRelative intrinsic attractant activity (%) of the analog compared with pityolure.

of the male processionary moth Thaumetopoea pityocampa to the natural pheromone. Compounds 4, 5, and 8 were, in turn, moderately active in the laboratory, whereas in the field they behaved as very good antagonists of the pheromone action amounting the inhibition level to 97 %.

According to our results, replacement of either one of the oxygen atoms of the acetate group in pityolure by sulfur results, in both cases, in a marked inhibitory action of the resulting analog. On the other hand, replacement of a CH group of the ethylenic moiety by S in c~ posit ion to the triple bond (compound 6) causes a much higher inhibition effect at all doses than when substi- tution takes place in ~ posi t ion (compound 7). This finding and the relatively higher intrinsic E A G activity of 6 in relation to 7 seems to suggest that the presence of electron-donating atoms l ike S in ot to the triple bond might lead to compounds with interesting inhibitory pheromonal activity. Further work on this line is in progress in our laboratory.

Although at present we have no evidence of the physiological mode of action of the analogs tested, several mechanisms can be hypothesized. In the


laboratory tests, the LAG results can be explained by a long-lasting adaptation of electrical responses of the receptor cells or by habituation of the central ner- vous system to the incoming signals. In the field, on the other hand, the inhibitory results might be due to an improper activation of other receptor cell types by the corresponding analog. In any case, the possibility of an irreversible binding of the inhibitors to the receptor proteins cannot be ruled out.

Acknowledgments--We thank CAICYT (PR 84-0087), CICYT (PB 87-0290) and CSIC (Project No. 263/85) for financial support and MEC for a predoctoral fellowship to V.G. We are indebted to R. Hern~indez and R. Montoya (ICONA) for field experiments.

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