Structure-Activity Relationships of Cyclopropene Compounds, Inhibitors of Pheromone Biosynthesis in Bombyx mori

Article abstract of DOI:10.1021/jf950757n

According to the synthetic route for 11,12-methylenehexadec-11-enoic acid [10-(2-butyl-1-cyclopropenyl)decanoic acid] and the amide, their related cyclopropene compounds, which possessed a propene ring at the 7,8-, 9,10-, or 13,14-position in a C₁₆ chain and the 11,12-position in a C₁₄ or C₁₈ chain, were synthesized via the corresponding 1-alkyl-1,2,2-tribromocyclopropane. Their activities as biosynthetic inhibitors of bombykol [(10E, 12Z)-10,12-hexadecadien-1-ol; sex pheromone of the silkworm moth Bombyx mori L.] were measured with virgin female silkworm moths in vivo. The 7,8-methylene compounds were inactive even at the dose of 10 μg/gland, but other compounds at 1 μg/gland inhibited the conversion of [16,16,16-²H₃]hexadecanoic acid to bombykol to some extent. Each amide showed stronger inhibitory activity than the corresponding acid, and the 11,12-methylene amide with a C₁₆ chain was the strongest (I₅₀ = 0.016 μg/gland) among the tested compounds. Furthermore, experiments comparing the incorporation of [1-¹⁴C]hexadecanoic acid into bombykol and another alcohol component in the pheromone gland, (Z)-11-hexadecen-1-ol, suggested that the Δ11-desaturation was blocked by 9,10- and 11,12-methylene compounds and the subsequent Δ10,-12-desaturation by 11,12- and 13,14-methylene compounds.

Full text of DOI:10.1021/jf950757n

3350
J. Agric. Food Chem. 1996, 44, 3350−3354
Str u ctu r e-Activity Rela tion sh ip s of Cyclop r op en e Com p ou n d s,
In h ibitor s of P h er om on e Biosyn th esis in Bom byx m or i
Tetsu Ando,* Ryuta Ohno, Kazuhisa Ikemoto, and Masanobu Yamamoto
Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and
Technology, Fuchu, Tokyo 183, J apan
According to the synthetic route for 11,12-methylenehexadec-11-enoic acid [10-(2-butyl-1-cyclopro-
penyl)decanoic acid] and the amide, their related cyclopropene compounds, which possessed a propene
ring at the 7,8-, 9,10-, or 13,14-position in a C16 chain and the 11,12-position in a C14 or C18 chain,
were synthesized via the corresponding 1-alkyl-1,2,2-tribromocyclopropane. Their activities as
biosynthetic inhibitors of bombykol [(10E,12Z)-10,12-hexadecadien-1-ol; sex pheromone of the
silkworm moth Bombyx mori L.] were measured with virgin female silkworm moths in vivo. The
7,8-methylene compounds were inactive even at the dose of 10 µg/gland, but other compounds at 1
µg/gland inhibited the conversion of [16,16,16-2H3]hexadecanoic acid to bombykol to some extent.
Each amide showed stronger inhibitory activity than the corresponding acid, and the 11,12-methylene
amide with a C16 chain was the strongest (I50 ) 0.016 µg/gland) among the tested compounds.
Furthermore, experiments comparing the incorporation of [1-14C]hexadecanoic acid into bombykol
and another alcohol component in the pheromone gland, (Z)-11-hexadecen-1-ol, suggested that the
∆11-desaturation was blocked by 9,10- and 11,12-methylene compounds and the subsequent ∆10,-
12-desaturation by 11,12- and 13,14-methylene compounds.
Keyw or d s: Cyclopropene; desaturase pheromone biosynthesis; cyclopropenes structure-activity
relationships; bombykol biosynthesis; ∆11-desaturation
INTRODUCTION
MATERIALS AND METHODS
The female moth of the silkworm Bombyx mori L.
secretes sex pheromone bombykol from its pheromone
gland in the abdominal tip to communicate with the
male moth. Biosynthesis of bombykol starts from acetyl
CoA with the construction of a saturated acyl compound
with a C16 straight chain (maybe CoA of 16:Acid), into
which a CdC bond with (Z)-configuration is introduced
at the 11-position by a ∆11-desaturase. The (Z)-11-
hexadecenoic intermediate is converted into a conju-
gated dienic acyl compound by a ∆10,12-desaturase, and
the acyl moiety is finally reduced to a primary hydroxyl
group to produce the fatty alcohol pheromone (Ando et
al.1988b).
If pheromone biosynthesis can be blocked by an
inhibitor, chemical communication between female and
male moths will be interrupted and their chance of
mating is expected to be very low. It appears promising
that a chemical that prevents the desaturation step can
be found because many lepidopterous pheromones in-
clude the CdC bond at a characteristic position and an
inhibitor might specifically attack the insect enzyme
system. Sterculic acid, 9,10-methyleneoctadec-9-enoic
acid [9-(2-octyl-1-cyclopropenyl)octadecanoic acid], is
known to inhibit the ∆9-desaturation of octadecanoic
acid in vertebrates (Raju and Reiser, 1967; Fogerty et
al., 1972). Recently, we reported that 11,12-methylene-
hexadec-11-enoic acid [10-(2-butyl-1-cyclopropenyl)de-
canoic acid, 1c] and the amide derivative (2c) acted as
inhibitors of bombykol biosynthesis (Ando et al., 1995).
In this study we investigated the syntheses and inhibi-
tory activities of several cyclopropanes to define their
structure-activity relationships.
An a lytica l In str u m en ts. The NMR spectra of each com-
pound in CDCl3 were measured with J EOL EX 270 Fourier
transform spectrometer (270.2 MHz for 1H, and 67.9 MHz for
13C) using tetramethylsilane (TMS) as an internal standard.
Electron-impact GC-MS was accomplished with a J EOL SX-
102A mass spectrometer, with ionization voltage of 70 eV and
a TC-FFAP capillary column (0.25 mm i.d. × 30 m; GL
Sciences, Tokyo, J apan). The ion source temperature was 240
°C, and the column temperature program was 40 °C for 1 min,
50 °C/min to 140 °C, and finally 8 °C/min to 220 °C. The
Shimadzu LC-6A system used for HPLC analyses was equipped
with a UV spectrometric detector (SPD-6A) and either a
normal-phase column (Nucleosil 5 NO2; 8 mm i.d. × 15 cm) or
a reversed-phase column (Nucleosil 5 ODS; 8 mm i.d. × 15
cm) packed by Senshu Kagaku Company (Tokyo, J apan). The
solvent for the NO2 column was 2% 2-propanol in n-hexane
(flow rate, 1.5 mL/min). The retention time (Rt) of bombykol
detected at UV 235 nm was 11.72 min. For the ODS column,
the solvent was 7% water in methanol (flow rate, 1.5 mL/min),
and bombykol (9.0-10.5 min), Z11-16:OH (10.5-12.5 min), and
16:OH (15.0-17.0 min) were collected separately.
Cyclop r op en e Com p ou n d s. In our previous paper (Ando
et al., 1995), syntheses of 11,12-methylenehexadec-11-enoic
acid (1c) and 11,12-methylenehexadec-11-enoamide (2c) were
described in detail. According to the method for these com-
pounds, their ring positional isomers were synthesized as
shown in Figure 1. Namely, 2-bromo-1-decyne (4a ) was
prepared by the addition of dry HBr to 1-decyne (3a ) in the
presence of tetraethylammonium bromide (Cousseau, 1980)
and converted into 1,2,2-tribromo-1-octylcyclopropane (5a ) by
the addition of dibromocarbene (Baird et al., 1985). The
reaction of 5a with 2 mol equivalents of n-butyllithium
followed by 1 mol equivalent of 1-bromo-5-iodopentane yielded
1-bromo-6,7-methylenepentadec-6-ene (6a ). An alkyl chain of
the bromide (6a ) was elongated by one carbon to yield 7,8-
methylenehexadec-7-enenitrile (7a ), which was hydrated to
yield 7,8-methylenehexadec-7-enoamide (2a ) and further hy-
drolyzed to yield 7,8-methylenehexadec-7-enoic acid (1a ). 9,-
10-Methylenes with a C16 chain (1b and 2b ) and 11,12-
* Author to whom correspondence should be ad-
dressed [telephone (0423) 67-5698; fax (0423) 60-8830;
e-mail antetsu@cc.tuat.ac.jp].
S0021-8561(95)00757-6 CCC: $12.00
© 1996 American Chemical Society

Cyclopropenes: Inhibitors of Pheromone Biosynthesis
J. Agric. Food Chem., Vol. 44, No. 10, 1996 3351
h dark cycles at 25 °C. Female pupae and moths were
incubated under the same conditions.
Bioa ssa y w ith 2H3-16:Acid (Exp er im en t A). The phero-
mone gland was topically treated with one of the cyclopropenes
[1a -f, 2a -f, and 8c (10-0.005 µg)] in 1 µL of DMSO, 25-27
h after eclosion (2 h after onset of the second photophase).
Three groups of three virgin females were used per treatment.
Two hours after the treatment, another DMSO solution (1 µL)
of 2H3-16:Acid (10 µg) was topically applied to the glands. After
a further 3 h of incubation, the glands of each group were
excised and extracted with ethyl acetate for 30 min. The
extract was injected into a normal-phase column of HPLC, and
pentadecan-1-ol (10 ng) was added to the recovered bombykol
fraction as the internal standard. An aliquot of this fraction
was analyzed by GC-MS in a selected-ion-monitoring (SIM)
mode. Peak areas of the [M - H2O]+ ion of pentadecan-1-ol
at m/ z 210 (Rt, 12.5 min) and M+ ion of 2H3-bombykol at m/ z
241 (Rt, 16.2 min) were measured to quantitatively analyze
F igu r e 1. Synthetic route for cyclopropene fatty acids (1a -
f), the amide derivatives (2a -f), and methyl ester (8c): (i) HBr/
Et4N+‚Br-/CH2Cl2; (ii) CHBr3/C16H33N+(CH3)3‚Br-/aq. NaOH;
(iii) 2eq. BuLi/HMPA/THF; (iv) ICH2(CH2)n-1CH2Br; (v) NaCN/
acetone-H2O; (vi) NaOH/EtOH; (vii) HCl; (viii) CH2N2/EtOEt.
2
the bombykol incorporating H3-16:Acid. The inhibitory activ-
ity (I50 value) of each cyclopropene was calculated by compar-
ing its incorporation in the females treated with the inhibitor
to that in females treated only with DMSO before the applica-
tion of 2H3-16:Acid. The average amount of 2H3-bombykol
extracted from the female glands untreated with the cyclo-
propene compounds was 26 ( 5 ng/gland.
methylenes with a C18 chain (1e and 2e) were synthesized by
the same manner starting from 1-octyne (3b), and 13,14-
methylenes with a C16 chain (1d and 2d ) and 11,12-methylenes
with a C14 chain (1f and 2f) were synthesized from 1-butyne
(3d ). Treatment of 1c with diazomethane yielded methyl 11,-
12-methylenehexadec-11-enoate (8c). After confirming the
Bioa ssa y w ith 14C-16:Acid (Exp er im en t B). The inhibi-
1
chemical structures by H and 13C NMR (see Table 2), these
tory activities of 1a -f, 2a -f, and 8c were also examined with
synthetic cyclopropenes were dissolved in dimethyl sulfoxide
(DMSO) to bioassay their inhibitory activities against bom-
bykol biosynthesis.
2
14C-16:Acid (50 000 dpm in 1 µL of DMSO) instead of H3-16:
Acid, as in Experiment A. After treatment of three pheromone
glands with an inhibitor and 14C-16:Acid, fatty alcohols
incorporating 14C were extracted with ethyl acetate for 30 min
and purified by the same method established for the research
of bombykol biosynthesis by two types of chromatography,
normal-phase TLC (silica gel 60F254, Merck) and reversed-
phase HPLC with an ODS column (Ando et al., 1988a, 1995).
Radioactivities incorporated into bombykol, Z11-16:OH, and
16:OH were measured by liquid scintillation counting on a
Packard model TRI-CARB 300C.
La beled Ch em ica ls. [16,16,16-2H3]16:Acid (99.7 atom%
2H), supplied by ISOTEC Inc. (Miamisburg, OH), was dissolved
in DMSO at a concentration of ∼10 µg/µL. [1-14C]16:Acid (2.07
GBq/mmol), supplied by American Radiolabeled Chemical Inc.
(St. Louis, MO), was dissolved in the same solvent at a
concentration of ∼50 000 dpm/µL.
In sects. Larvae of B. mori [hybrids of Shunrei (female) ×
Shogetsu (male)] were reared on a semisynthetic diet (Nippon
Nosan-Kogyo Company, Yokohama, J apan) under 16 h light-8
Ta ble 1. NMR Assign m en ts of Syn th etic Com p ou n d s 4a -d a n d 5a -d
NMR chemical shifts (ppm) for indicated position
a
compd
1
2
3
4
5
6
7
8
9
10
CH2
[A] 1H NMR
4a b
4bc
4cd
4d e
5a f
5bg
5ch
5d i
5.38 (d)
5.55 (d)
5.38 (d)
5.55 (d)
5.38 (d)
5.54 (d)
5.37 (d)
5.56 (d)
2.41 (t)
2.41 (t)
2.42 (t)
2.46 (q)
2.0 (m)
2.0 (m)
2.0 (m)
1.55 (tt)
1.53 (tt)
1.54 (tt)
1.13 (t)
1.7 (m)
1.7 (m)
1.7 (m)
1.25 (t)
∼1.3
∼1.3
∼1.3
∼1.3
∼1.3
∼1.3
0.88 (t)
∼1.3
∼1.3
0.89 (t)
1.34 (tq)
0.92 (t)
∼1.3
∼1.3
∼1.3
∼1.3
∼1.3
∼1.3
0.89 (t)
1.84 (d)
1.94 (d)
1.82 (d)
1.95 (d)
1.83 (d)
1.96 (d)
1.83 (d)
1.96 (d)
∼1.3
∼1.3
0.90 (t)
1.39 (tq)
0.95 (t)
2.02 (dq)
2.16 (dq)
[B] 13C NMR
4a
4b
4c
4d
5a
5b
5c
5d
116.2
116.2
116.2
115.2
45.9
45.9
45.8
47.0
135.0
135.0
134.9
136.3
33.2
33.2
33.2
33.0
41.5
41.5
41.2
35.0
41.7
41.7
41.4
35.4
28.4
27.9j
30.1
13.2
29.0j
28.6
29.8
12.0
27.9
29.2j
29.3j
22.6
31.9
14.1
22.7
22.6
14.1
14.1
28.1j
21.6
31.5
13.8
29.2j
27.7
22.1
29.4j
31.7
14.0
27.7
22.6
31.8
14.1
38.1
38.1
38.0
37.9
a
b
c
CH2 of propane ring. J 1,1 ) 1.5 Hz, J 3,4 ) 7.5 Hz, J 4,5 ) 7 Hz, J 9,10 ) 6.5 Hz. J 1,1 ) 1.5 Hz, J 3,4 ) 7.5 Hz, J 4,5 ) 7 Hz, J 7,8 ) 6.5 Hz.
d
e
f
g
J 1,1 ) 1.5 Hz, J 3,4 ) 7.5 Hz, J 4,5 ) 7 Hz, J 5,6 ) 7 Hz. J 1,1 ) 1.5 Hz, J 3,4 ) 7.5 Hz. J 9,10 ) 6.5 Hz, J a,a ) 9 Hz. J 7,8 ) 6.5 Hz, J a,a ) 9
h
i
j
Hz. J 4,5 ) 7 Hz, J 5,6 ) 7.5 Hz, J a,a ) 9 Hz. J 3,3 ) 14.5 Hz, J 3,4 ) 7 Hz, J a,a ) 9 Hz. Assignments for these carbons are not unambiguous
and may be reversed in the same row.

3352 J. Agric. Food Chem., Vol. 44, No. 10, 1996
Ando et al.
Ta ble 2. NMR Assign m en ts of Syn th etic Cyclop r op en e Com p ou n d s 1a -d a n d 2a -d
NMR chemical shifts (ppm) for indicated position
a
compd
1
2
3
dC
-----CH2-----
CH2
14
15
16
CH2
[A] 1H NMR
2.37 (t)
2.37 (t)
2.38 (t)
2.38 (t)
2.37 (t)
2.37 (t)
2.38 (t)
2.38 (t)
1a
1b
1c
1d
2a
2b
2c
2d
2.36 (t)
2.36 (t)
2.35 (t)
2.35 (t)
2.23 (t)
2.23 (t)
2.22 (t)
2.22 (t)
1.64 (tt)
1.64 (tt)
1.63 (tt)
1.63 (tt)
1.64 (tt)
1.64 (tt)
1.63 (tt)
1.64 (tt)
∼1.5
∼1.5
∼1.5
∼1.5
∼1.5
∼1.5
∼1.5
∼1.5
∼1.3
∼1.3
∼1.3
∼1.3
0.88 (t)
0.89 (t)
0.91 (t)
1.13 (t)
0.88 (t)
0.89 (t)
0.92 (t)
1.13 (t)
0.78
0.77
0.77
0.78
0.78
0.77
0.77
0.78
∼1.3
∼1.3
2.39 (q)
∼1.3
∼1.3
∼1.3
∼1.3
∼1.3
∼1.3
2.39 (q)
[B] 13C NMR
25.8
1a
1b
1c
1d
2a
2b
2c
2d
179.2
179.9
179.6
179.2
175.4
175.8
176.1
175.6
33.9
34.0
34.0
33.9
35.8
35.9
36.0
35.9
24.5
24.6
24.7
24.7
25.3
25.5
25.5
25.5
108.9
109.7
109.2
109.5
109.3
109.3
109.1
110.6
109.0
109.7
109.2
109.5
109.3
109.3
109.1
110.6
27.0
27.4
27.3
27.3
27.4
31.9
31.7
22.7
22.6
22.5
19.7
22.7
22.6
22.5
19.7
14.1
14.1
13.9
12.0
14.1
14.1
13.9
12.0
7.4
7.4
7.4
7.4
7.4
7.4
7.4
7.4
26.0
26.0
26.1
25.7
26.0
26.0
∼29.5
27.5
25.8
26.0
26.0
26.1
25.7
26.0
26.0
27.1
27.4
27.3
27.3
27.4
31.9
31.7
∼29.5
27.5
a
CH2 of propene ring.
Ta ble 3. I50 (µg/Gla n d ) Va lu es of Cyclop r op en e
RESULTS
Com p ou n d sa (1a -f a n d 2a -f) Block in g th e In cor p or a tion
of 2H3-16:Acid in to Bom byk ol
Syn th esis of Cyclop r op en e Com p ou n d s. Cyclo-
propene acids (1a -f) and amides (2a -f) were synthe-
sized by a modification of the method reported by Baird
et al. (1992), as shown in Figure 1. Chemical structures
I50 (µg/gland)
inhibitor; carbon length
and position of cyclopropene
acid (1)
amide (2)
C16
C16
C16
C16
C18
C14
7,8-methylene (a )
9,10-methylene (b)
11,12-methylene (c)
13,14-methylene (d )
11,12-methylene (e)
11,12-methylene (f)
> 10
0.73
0.040
2.2
0.32
0.21
> 10
0.23
0.016
0.50
0.17
0.10
1
of all the synthetic compounds were confirmed by H
and 13C NMR. The NMR assignments of synthetic
intermediates 4a -d and 5a -d are shown in Table 1,
and those of the characteristic signals of the final
products 1a -d and 2a -d , which included the unique
methylene signals of the propene ring (δ 0.77-0.78 ppm
a
The I50 value of methyl 11,12-methylenehexadec-11-enoate (8c)
1
is 0.15 µg/gland.
in H NMR and δ 7.4 ppm in 13C NMR) caused by the
diamagnetic anisotropy of the CdC bond, are shown in
Table 2. The NMR spectra of 11,12-methylenes with a
C18 chain (1e and 2e) and 9,10-methylenes with a C16
chain (1b and 2b) are quite similar because these
compounds include the same terminal alkyl structure.
Furthermore, 11,12-methylenes with a C14 chain (1f and
2f) showed almost the same NMR data as those of 13,14-
methylenes with a C16 chain (1d and 2d ), exceptfor CH2
signals of the long chains in a side of the acyl group.
Yields of the acids (1a -f) and amides (2a -f) were ∼0.7
and 0.5%, respectively, from the corresponding 1-alkynes
(3a -d ).
inhibited the incorporation, whereas activities of 13,14-
methylenes (1d and 2d ) were very weak. Although
11,12-methylenes with a C18 chain (1e and 2e) and a
C14 chain (1f and 2f) showed rather strong activities,
I50 values of 1c and 2c with a C16 chain were much
smaller than those of the compounds. Each amide more
potently blocked bombykol biosynthesis than the cor-
responding acid. The activity of methyl ester (8c) was
weaker than the parent acid 1c. 7,8-Methylenes (1a
and 2a ) were inactive at the dose of 10 µg/gland.
Exp er im en t B. The inhibitory activities of the
cyclopropene compounds were also compared in the
experiments using 14C-16:Acid. In the pheromone gland
of B. mori, the 14C-labeled acid was converted into
bombykol and two other fatty alcohols, Z11-16:OH and
16:OH, occurring specifically in the gland. Their 14C-
incorporation, which can be easily counted because of
their good separation by HPLC, is ∼1% in the normal
virgin female (Ando et al., 1988a). The incorporation
ratios (%) into the three alcohols in glands pretreated
with 1 µg of each cyclopropene compound (1a -f, 2a -f,
and 8c) before the 14C-16:Acid application are shown
in Figure 2. The effect of these cyclopropenes on the
14C-incorporation into bombykol is similar to that on the
2H3-incorporation examined in Experiment A. Their
inhibitory activities decreased in the following order:
11,12-methylenes (1c and 2c), 9,10-methylenes (1b and
Exp er im en t A. 2H3-Labeled bombykol in the phero-
2
mone gland treated with H3-16:Acid was quantitatively
measured by GC-SIM analyses with pentadecan-1-ol as
2
an internal standard. When the H3-16:Acid was ap-
plied at 10 µg/gland in the most fertile period of
bombykol biosynthesis (Ando et al., 1988a), 0.26% of it
was incorporated into bombykol. Inhibitory activities
of the synthetic cyclopropene compounds were evaluated
based on this rate, and I50 (µg/gland) values are shown
in Table 3 (doses at these levels block half of the
incorporation observed for untreated females). Among
the cyclopropene compounds with a C16 chain tested in
this study, 11,12-methylenes [1c (I50 ) 0.040 µg/gland)
and 2c (I50 ) 0.016 µg/gland)] were the strongest
inhibitors. 9,10-Methylenes (1b and 2b ) moderately

Cyclopropenes: Inhibitors of Pheromone Biosynthesis
J. Agric. Food Chem., Vol. 44, No. 10, 1996 3353
F igu r e 2. Incorporation (%) of 14C-16:Acid into fatty alcohols [bombykol (A), Z11-16:OH (B), and 16:OH (C)] in the pheromone
glands of B. mori that were pretreated with cyclopropene compounds (1a -f, 2a -f, and 8c; 1 µg).
2b) and 13,14-methylenes (1d and 2d ) among C16 chain
compounds, and C16 (1c and 2c), C14 (1f and 2f), and
C18 (1e and 2e) chain compounds among 11,12-meth-
ylenes. Inhibitions by the amides were stronger than
those by the corresponding acids. The 7,8-methylene
compounds (1a and 2a ) were inactive toward inhibition.
The 14C-incorporation of Z11-16:OH was also blocked
by the 11,12- and 9,10-methylenes, whereas the 7,8- and
13,14-methylenes showed no effect. On the contrary,
the 14C-incorporation of 16:OH increased by more than
three times because of the pretreatment of the 11,12-
methylenes. This incorporation was moderately in-
creased by the 9,10- and 13,14-methylenes, whereas 7,8-
methylenes had no effect.
penetration into the target site and affinity for it. Our
previous paper showed that 7c, the cyano analog of 1c,
was inactive (Ando et al., 1995). This experiment
revealed that esterification of 1c decreased the activity.
The functional group, which optimizes the activity,
should be dynamically studied in the future.
Based on the incorporation experiments with several
precursors, it has been estimated that there are two
desaturation steps, ∆11- and ∆10,12-desaturation, in
the bombykol biosynthesis (Ando et al., 1988b). The C16
saturated acyl compound, which is constructed with
acetyl CoA, is converted into a monounsaturated acyl
compound (derivative of Z11-16:Acid) by ∆11-desatu-
rase, and successively into a diunsaturated acyl com-
pound (derivative of E10,Z12-16:Acid) by ∆10,12-
desaturase. Finally, the acyl moiety was transformed
into alcohol by reductase to accomplish the bombykol
production. The pheromone gland of B. mori possesses
not only bombykol but also two other alcohols, Z11-16:
OH, which are produced from the acyl intermediates by
reductase. Although their roles in the mating com-
munication are unknown, progress in the desaturation
steps can be estimated by counting their 14C-incorpora-
tion from 14C-16:Acid. 11,12-Methylene compounds
with a C16 chain (1c and 2c) significantly diminished
the 14C-incorporation of Z11-16:OH and that of bom-
bykol much more, as shown in Figure 2. This result
indicates that the 11,12-methylenes blocked both of the
two desaturation steps. This blocking of the unsatura-
tion steps caused high level 14C-incorporation into 16:
OH. It is interesting that C18 and C14 11,12-methylenes
(1e, 2e, 1f and 2f) also showed rather strong inhibitory
activities against the enzymes acting in the biosynthesis
of the pheromone with a C16 chain. The steps inhibited
by other cyclopropene compounds were also suggested
from the results of Experiment B. The 9,10-methylenes
(1b and 2b) might selectively inhibit ∆11-desaturation,
because the 14C-incorporation of bombykol and Z11-16:
OH decreased to a similar level by the treatment of
these cyclopropenes. On the contrary, the 13,14-meth-
ylenes (1d and 2d ) selectively inhibited ∆10,12-desatu-
ration because only the 14C-incorporation of bombykol
decreased. The biosynthetic pathway of bombykol and
the steps that are expected to be inhibited by cyclopro-
pene compounds are summarized in Figure 3. We plan
to examine the effect of cyclopropenes on the incorpora-
DISCUSSION
It has been reported that the desaturation of octade-
canoic acid is inhibited by naturally occurring cyclopro-
pene fatty acid, sterculic acid, in the livers of verte-
brates (Raju and Reiser, 1967; Fogerty et al., 1972), and
desaturation of 16:Acid is inhibited by some synthetic
analogs in the pheromone glands of a noctuid moth,
Spodoptera littoralis (Arsequell et al., 1989; Gosalbo et
al., 1992). Our study revealed that the desaturation in
pheromone biosynthesis of B. mori was also inhibited
by some cyclopropene acids, which prevented both the
2
incorporation of H3- and 14C-labeled 16:Acid into the
pheromone bombykol. Furthermore, the incorporation
was blocked by the amide derivatives; their inhibitory
activities being stronger than those of the parent acids.
Among the tested cyclopropene compounds, C16 11,12-
methylene amide (2c) showed the strongest inhibitory
activities against bombykol biosynthesis. This amide
(2c) blocked half of the conversion of 14C-16:Acid into
bombykol at a dose of <0.1 nmol/gland, and the I50
values in Table 3 show that 2c is 2.5 times more active
than the corresponding acid 1c. The I50 values of C16
9,10-methylene amide (2b) and C18 and C14 11,12-
methylene amides (2e and 2f), with moderate inhibitory
activity, are from one-half to one-third of the values of
the corresponding acids 1b, 1e, and 1f, respectively. The
amides seem to prevent the metabolism by â-oxidation,
which easily metabolizes the acids applied to the
pheromone gland. Also some other reasons for the
stronger activity of amides can be postulated; for
example the aminocarbonyl group might enhance its

3354 J. Agric. Food Chem., Vol. 44, No. 10, 1996
Ando et al.
C
14 chain unsaturated at the 9-position and a C12 chain
unsaturated at the 7-position are produced via a chain
shortening reaction after the ∆11-desaturation (Bjostad
et al., 1987). Further comparative studies of inhibitors
against many lepidopterous species will help to define
the ∆11-desaturase.
ABBREVIATIONS USED
16:Acid, hexadecanoic acid (palmitic acid); Z11-16:
Acid, (Z)-11-hexadecenoic acid; 10E,Z12-16:Acid, (10E,-
12Z)-10,12-hexadecadienoic acid; 16:OH, hexadecan-1-
ol; Z11-16:OH, (Z)-11-hexadecen-1-ol; bombykol
(10E,12Z)-10,12-hexadecadien-1-ol.
LITERATURE CITED
F igu r e 3. Biosynthetic pathway of bombykol and the pro-
spective steps that were inhibited by cyclopropene compounds.
The biosynthetic pathway for bombykol is shown by the thick
arrows, and some of the related metabolic reactions by the thin
arrows. The desaturation steps in this pathway proceed on
fatty acyl groups in a lipid or an activated derivative. The
acyl carrier, which has not been identified, is represented by
-OR or -SR.
Ando, T.; Hase, T.; Funayoshi, A.; Arima, R.; Uchiyama, M.
Sex pheromone biosynthesis from 14C-hexadecanoic acid in
the silkworm moth. Agric. Biol. Chem. 1988a , 52, 141-
147.
Ando, T.; Hase, T.; Arima, R.; Uchiyama, M. Biosynthetic
pathway of bombykol, the sex pheromone of the female
silkworm moth. Agric. Biol. Chem. 1988b, 52, 473-478.
Ando, T.; Ohno, R.; Ikemoto, K.; Yajima, Y. Cyclopropene fatty
acid and amide: inhibitors of the pheromone biosynthesis
in Bombyx mori. J . Pestic. Sci. 1995, 20, 25-32.
Arsequell, G.; Fabria`s, G.; Camps, F. Inhibition of a ∆-11
desaturase in Spodoptera littoralis by 12,13-methylenehexa-
dec-12-enoic acid. Insect Biochem. 1989, 19, 623-627.
Baird, M. S.; Nethercott, W.; Slowey, P. D. Preparation of
polyhalogenocyclopropanes by addition of halogenocarbenes
to halogenoalkenes under phase-transfer conditions. J .
Chem. Res. (S) 1985, 370-371.
tion of labeled Z11-16:Acid into bombykol to reveal their
critical target steps. The ∆10,12-desaturation is the
novel step that is recognized as a retro-1,4-addition, and
the mechanism has not been estimated by any experi-
ments. We hope to obtain some information about the
mechanism by the additional investigations with these
inhibitors.
The structure-activity relationships of cyclopropene
fatty acids were studied by Fogerty et al. (1972) by first
using the desaturase enzyme system of hen liver. In
this system, ∆9-desaturation of octadecanoic acid was
strongly inhibited by 9,10-methyleneoctadec-9-enoic acid
(sterculic acid) and 10,11-methylenenonadec-10-enoic
acid, and moderately by 8,9-methyleneheptadec-8-enoic
acid (malvalic acid). Inhibition was not observed by the
treatment with the 11,12-methylene derivative. Re-
cently, Gosalbo et al. (1992) reported the effect of three
synthetic cyclopropene acids on the ∆11-desaturation
of 16:Acid in the pheromone gland of a noctuid moth
Spodoptera littoralis. This ∆11-desaturation was in-
hibited by all of the tested cyclopropenes, 1c, and the
10,11- and 12,13-methylene analogs. The latter two
compounds possess the propene ring at the adjoining
position of the 11,12-bond. These results indicate that
the position of a propene ring in the active compounds
is not strictly coincident with the position of the CdC
bond introduced by desaturase. It is noteworthy that
∆11-desaturase in the pheromone gland of B. mori was
inhibited by even the 9,10-methylene compounds (1b
and 2b), which had the ring at the position removed by
one bond from the CdC bond. The ∆11-desaturation is
one of the key steps in pheromone biosynthesis of
lepidopterous insects. Pheromonal components with a
Baird, M. S.; Dale, C. M.; Lytollis, W.; Simpson, M. J . A new
approach to cyclopropene fatty acids. Tetrahedron Lett.
1992, 33, 1521-1522.
Bjostad, L. B.; Wolf, W. A.; Roelofs, W. L. Pheromone Bio-
chemistry; Prestwitch, G. D., Blomquist, G. J ., Eds.; Aca-
demic: New York, 1987; pp 77-120.
Cousseau, J . A new route to 2-bromo-1-alkenes by hydrobro-
mination of 1-alkynes with tetraethylammonium hydrogen
dibromide. Synthesis 1980, 805-806.
Fogerty, A. C.; J ohnson, A. R.; Pearson, J . A. Ring position in
cyclopropene fatty acids and stearic acid desaturation in hen
liver. Lipids 1972, 7, 335-338.
Gosalbo, L.; Fabrias, G.; Arsequell G.; Camps, F. Inhibition of
sex pheromone biosynthesis in Spodoptera littoralis by
cyclopropene fatty acids. Insect Biochem. Mol. Biol. 1992,
22, 687-690.
Raju, P. K.; Reiser, R. Inhibition of fatty acyl desaturase by
cyclopropene fatty acids. J . Biol. Chem. 1967, 242, 379-
384.
Received for review November 20, 1995. Revised manuscript
received J uly 17, 1996. Accepted J uly 31, 1996.X
J F950757N
X Abstract published in Advance ACS Abstracts, Sep-
tember 15, 1996.