Insect juvenile hormone analogues and their biological activity on sea lice (Lepeophtheirus salmonis)

Article abstract of DOI:10.1021/jf049259h

A number of synthetic compounds that exhibit juvenile hormone activity when tested on the insect species Tenebrio molitor have been shown to inhibit the development of the sea lice species Lepeophtheirus salmonis. The testing was carried out on both isola

Full text of DOI:10.1021/jf049259h

6944 J. Agric. Food Chem. 2004, 52, 6944−6949
Insect Juvenile Hormone Analogues and Their Biological
Activity on Sea Lice (Lepeophtheirus salmonis)
#
LARS SKATTEBØL,*,† YNGVE STENSTRØM,§ AND CHRISTIAN SYVERTSEN
Department of Chemistry, University of Oslo, P.O. Box 1033 Blindern, N-0315 Oslo, Norway;
Department of Chemistry, Biotechnology and Food Science, Agricultural University of Norway,
P.O. Box 5003, N-1432 Aas, Norway; and Animal Health Division, ALPHARMA,
P.O. Box 158 Skøyen, N-0212 Oslo, Norway
A number of synthetic compounds that exhibit juvenile hormone activity when tested on the insect
species Tenebrio molitor have been shown to inhibit the development of the sea lice species
Lepeophtheirus salmonis. The testing was carried out on both isolated nauplius larvae and live Atlantic
salmon (Salmo salar).
KEYWORDS: Fish farming; Atlantic salmon; Salmo salar; sea lice; Lepeophtheirus salmonis; juvenile
hormone analogues
INTRODUCTION
be pregnant when finally molting to adult. Completion of the
life cycle normally takes ∼6 weeks at a water temperature
between 9 and 12 °C.
Fish farming has become a very important source of fish for
human consumption. Because of the high population density,
farmed fish are particularly susceptible to infections, and disease
control is an important part of this activity. In this respect the
frequent occurrence of infestations by ectoparasites constitutes
a serious problem, giving rise to considerable economic loss to
the fish farmers. The ectoparasites affect the health of farmed
fish in both freshwater and seawater environments. The
crustacean species called sea lice are among the most harmful
of the ectoparasites. Thus, infestations by the sea lice Lepeoph-
theirus salmonis and Caligus elongatus are among the most
important disease problems in farming of salmonids, particularly
for the Atlantic salmon (Salmo salar) and the rainbow trout
(Oncorhynchus mykiss) (1). The costs associated with treatment
and lower classification ratings of slaughtered fish as well as
reduced feed intake contribute to the economic loss caused by
sea lice infestation. Furthermore, current research has shown
that sea lice infestations could be the main reason for the decline
in wild salmonid stock that has been observed in Ireland and
Norway in recent years (2, 3).
The life cycle of the sea lice does not involve an intermediate
host in the metamorphosis to adult lice. It commences with the
mature egg hatching to the nauplius I stage larvae that molts
further through the nauplius II into the copepodid, the infestive
stage. The copepodid settles normally to the ventral surface and
on the fins of the fish, where molting continues successively
through four chalimus stages into the pre-adult stage, in which
the parasite moves freely on the skin of the host. Adult males
may mate with pre-adult females, which therefore may already
At present the most common treatment of sea lice infestastions
involves bathing or immersing the fish in solutions of insecti-
cides such as organophosphates or pyrethroids, which are toxic
to the ectoparasites. There are several disadvantages with this
method. Organophosphates are toxic to humans as well and must
therefore be treated with caution; moreover, overdosing has
resulted in fish mortality on several occasions. On the other
hand, pyrethroids are not acutely toxic to humans, but they are
toxic to fish. Another approach involves the use of the chitin
synthesis inhibitor teflubenzuron, which by oral administration
has been found to be effective against sea lice (4). However,
the inhibitors have no effect against adult stages of the sea lice,
and rapid metabolism in the fish requires repeated treatments.
Longer protection against infestation is obtained with in-feed
use of emamectin benzoate (Slice), a second-generation aver-
mectin pesticide (5, 6).
There is clearly a need for improved means of controlling
sea lice infestations on fish. One possible solution would be
compounds that exert antiparasitic activity by interrupting the
metamorphosis of the sea lice. Juvenile hormones (JH) are
among the hormones that control the metamorphosis of arthro-
pods. The effect of JH on the metamorphosis of insects has
been thoroughly studied (8), but less so in crustacean species;
however, methyl farnesate has been identified in several
crustacean species as a compound affecting the metamorphosis
(9-12). Insects treated with an excess of JH at an early stage
of development remain juvenile or develop into sterile adult
insects. Several structurally different compounds have been
shown to exert such an effect, and some of these so-called
juvenoids are used commercially for insect control. Examples
are methoprene (1), hydroprene (2), fenoxycarb (3), and
pyriproxyfen (4) (Figure 1).
* Author to whom correspondence should be addressed (e-mail
lars.skattebol@kjemi.uio.no).
† University of Oslo.
§ Agricultural University of Norway.
# ALPHARMA.
10.1021/jf049259h CCC: $27.50
© 2004 American Chemical Society
Published on Web 10/15/2004

Biological Activity of Insect Juvenile Hormone Analogues
J. Agric. Food Chem., Vol. 52, No. 23, 2004 6945
Figure 1. Examples of commercially available juvenoids.
In the present work we report on several compounds with
juvenile hormone properties in insects that are biologically
highly active against sea lice by interrupting the metamorphosis.
MATERIALS AND METHODS
Preparation of Compounds. The compounds tested in the present
work are compiled in Figure 2. In addition, we tested the commercially
available juvenoids 1 and 3.
The ethers 6, 11, 12, and 14 are known compounds that were
prepared according to the literature (13). The other aromatic ethers
recorded in Figure 2 were prepared in the same way by reaction of
the appropriate alkyl bromide with a DMF solution of the sodium salt
of either a hydroxypyridine or a phenol derivative at room temperature.
The thioethers were prepared according to a similar procedure; in
separate experiments 3-pyridinethiyllithium and 2-thiophenethiyllithium
reacted with 1-bromo-3,7-dimethyloctane to yield the thioethers 7 and
15, respectively. The phenoxy ethers 19 and 20 were prepared from
2-bromo-1-(4-phenoxyphenoxy)ethane (14) by reaction with 2- and
3-hydroxypyridine, respectively. Similarly, 4-phenoxybenzyl bromide
(15) was transformed to the ether 21 with 3-hydroxypyridine. The
products were purified by column chromatography, followed by short
path distillation or recrystallization, and characterized spectroscopically.
The yields of purified ethers ranged from 50 to 90%. The alkylpyridine
derivative 8 was prepared from 3-cyanopyridine in 30% overall yield
as shown in Scheme 1.
The cyclopropane derivatives 13 and 18 were prepared as outlined
in Scheme 2.
Linalool was converted in excellent yield to the dichlorocyclopropane
derivative 22, as reported in the literature (16). Reduction with sodium
in liquid ammonia furnished the cyclopropyl alcohol 23, which was
transformed to the bromide 24 in 94% overall yield. Only one isomer
is formed, and by comparison of the NMR spectra with those of geranyl
bromide and similar compounds, the E-configuration has been assigned
(17). Reaction of the bromide with 3-hydroxy-6-methylpyridine in the
usual way afforded the ether 13, as the E-isomer. The hydroprene
analogue 18 was prepared by first transforming the diethylacetal of
citronellal to the dichlorocyclopropane derivative 25, which was
subsequently reduced and hydrolyzed to the aldehyde 26 in 71% overall
yield. The aldehyde underwent a Wittig reaction with the ylid 27,
generated from (E)-4-bromo-3-methyl-2-butenoic acid (18) and tri-
phenylphosphine. The acid was initially obtained as a mixture of
stereoisomers, which was isomerized by benzenthiol to the 2E,4E-
isomer 28 (19). Finally, esterification with diazomethane afforded the
methyl ester 18.
Figure 2. Compounds tested in this study.
Scheme 1. Synthesis of Compound 8a
Experimental Procedures. General. The NMR spectra were
recorded on Varian Gemini 200 or Varian Mercury-300 instruments
using CDCl3 as solvent and TMS as an internal standard. IR spectra
were recorded on a Perkin-Elmer Paragon 500 FT spectrometer. MS
spectra were recorded on a GC-MS JEOL DX-303. For analytical GLC
a 25 m SP 2100 capillary column was used.
2-Methyl-5-(3,7-dimethyloctyloxy)pyridine (10). General Ether and
Thioether Synthesis. To a stirred MeOH solution of NaOMe (2 M, 10
mL, 20 mmol) was added 2.18 g (20 mmol) of 3-hydroxy-6-
methylpyridine. After 15 min of stirring at room temperature, the solvent
was evaporated under reduced pressure. The sodium salt was dissolved
in dry DMF (20 mL), and 4.42 g (20 mmol) of 1-bromo-3,7-
dimethyloctane was added. The mixture was stirred at room temperature
for 16 h and then diluted with diethyl ether to precipitate NaBr. The
a Reagents and conditions: i, Mg, ether; ii, HBr; iii, (a) 3-cyanopyridine,
(b) 2 N HCl, H2O, D; N2H2, KOH D.
organic layer was separated and washed successively with 10% aqueous
NaOH and water and dried (MgSO4). Evaporation and short path
distillation at 131 °C/0.02 mmHg gave 4.39 g (88%) of the ether 10 as
a colorless oil: 1H NMR δ 0.87 (d, J ) 6.6 Hz, 6 H), 0.94 (d, J ) 6.6
Hz, 3 H), 1.10-1.23 (m, 3H), 1.23-1.38 (m, 3 H), 1.46-1.72 (m, 3
H), 1.77-1.88 (m, 1 H), 2.48 (s, 3H), 3.96-4.04 (m, 2 H), 7.04 (d, J

6946 J. Agric. Food Chem., Vol. 52, No. 23, 2004
Skattebøl et al.
Scheme 2. Synthesis of the Cyclopropane Derivatives 14 and 18a
a Reagents and conditions: i, Na, NH3(l), NH4Cl; ii, HBr; iii, NaH, DMF, 3-hydroxy-6-methylpyridine; iv, 2 N HCl, 2-propanol; v, 27, NaOMe, MeOH; vi, PhSH;
vii, CH2N2, Et2O.
) 8.4 Hz, 1H), 7.10 (dd, J ) 2.6, 8.4 Hz, 6H), 8.18 (d, J ) 2.6 Hz, 1
H); 13C NMR δ 19.72, 22.70, 22.81, and 23.40 (CH3 × 4), 24.76 (CH2)
and 28.06, 29.85 (CH × 2), 36.24, 37.35, 39.32, and 66.86 (CH2 × 4),
122.15, 123.38, and 136.79 (CH × 3), 150.16 and 153.33 (C × 2); IR
1605 (m), 1580 (s), 1505 (s) cm-1; HRMS (ESI) found (M+) 249.2088,
C16H27NO requires 249.2092.
3-(3,7-Dimethyloctyloxy)pyridine (5). Reaction of 3-hydroxypyridine
and 1-bromo-3,7-dimethyloctane according to the general procedure
gave the ether 5 as an oil in 88% yield: 1H NMR δ 0.87 (d, J ) 6.6
Hz, 6H), 0.95 (d, J ) 6.2 Hz, 3H), 1.12-1.38 (m, 6H), 1.46-1.74 (m,
3H), 1.78-1.90 (m, 1H), 4.00-4.07 (m, 2H), 7.20 (m, 2H), 8.20 (dd,
J ) 2.1, 4.0 Hz, 1H), 8.31 (bdd, J ) 1.1, 2.1 Hz, 1H); 13C NMR δ
19.78, 22.75, and 22.85 (CH3 × 3), 24.80 (CH2), 28.13 and 29.95 (CH
× 2), 36.25, 37.41, 39.38, and 66.88 (CH2 × 4), 121.30, 123.96, 138.16,
and 141.97 (CH × 3), 155.45 (C); IR 3047 (m), 1584 (m), 1570 (m),
1467 (s) cm-1; HRMS found (M+) 235.1940, C15H25NO requires
235.1936.
3-(3,7-Dimethyloctylthio)pyridine (7). Reaction of 3-mercaptopyri-
dine (20) and 1-bromo-3,7-dimethyloctane according to the general
procedure gave the thioether 7, as an oil in 80% yield: 1H NMR δ
0.86 (d, J ) 6.6 Hz, 6H), 0.89 (d, J ) 6.6 Hz, 3H), 1.11-1.32 (m,
6H), 1.42-1.72 (m, 4H), 2.84-3.02 (m, 2H), 7.21 (dd, J ) 4.8, 8.0
Hz, 1H), 7.63 (ddd, J ) 1.4, 2.2, 8.0 Hz, 1H), 8.41 (bd, J ) 4.0 Hz,
1H), 8.56 (bd, J ) 1.4 Hz, 1H); 13C NMR δ 19.46, 22.75, and 22.85
(CH3 × 3), 24.78 (CH2), 28.11 (CH), 31.71 (CH2), 32.32 (CH), 36.34,
37.01, and 39.33 (CH2 × 3), 123.72 (CH), 134.39 (C), 136.79, 146.89,
and 149.95 (CH × 3); IR 3020 (m), 1570 (m), 1560 (m), 1475 (s)
cm-1; HRMS found (M+) 251.1703, C15H25NS requires 251.1707.
3-(4,8-Dimethyl-1-nonyl)pyridine (8). 3,7-Dimethyl-1-octylmagne-
sium bromide was prepared from Mg (2.7 g) and 1-bromo-3,7-
dimethyloctane (22.10 g, 0.10 mol) in dry ether (50 mL). The Grignard
reagent was cooled in an ice bath, and 3-cyanopyridine (9.37 g, 0.09
mol) in dry ether (100 mL) was added dropwise with stirring. The slurry
was stirred at room temperature for an additional 17 h and cooled in
ice. A saturated aqueous solution of NH4Cl (100 mL) and 12 M HCl
(25 mL) were added successively, and the mixture was stirred at room
temperature for 7 h. The ether phase was separated and discarded, and
the aqueous solution was heated under reflux for 14 h. The reaction
mixture was made alkaline to litmus with aqueous NaOH, and the
product was isolated with ether in the usual way. Evaporation of ether
and short path distillation, 98 °C (bath)/0.01 mmHg, furnished 4,8-
dimethyl-1-(3-pyridinyl)-1-nonanone (9.10 g, 41%). The solution of the
ketone (1.98 g, 8 mmol), N2H2 (99%, 1.20 g, 24 mmol) and KOH (85%,
1.00 g, 16 mmol) in triethylene glycol (15 mL) was heated to 180-
190 °C for 5 h and left at room temperature overnight. Brine (125 mL)
was added and the product extracted with CH2Cl2. The dried (K2CO3)
extract was evaporated and the residue distilled to give 1.40 g (74%)
of compound 8: bp 70-72 °C/0.002 mmHg; 1H NMR δ 0.85 (d, J )
1.8 Hz, 3H), 0.86 (d, J ) 1.8 Hz, 6H), 1.0-1.7 (m, 12H), 2.59 (t, J )
8.0 Hz, 2H), 7.22 (dd, J ) 4.7, 7.7 Hz, 1H), 7.51 (d, J ) 7.7 Hz, 1H),
8.44 (d, J ) 4.7 Hz, 2H); 13C NMR δ 19.77, 22.77, and 22.87 (CH3 ×
3), 24.91, 28.13, and 28.81 (CH2 × 3), 32.78 (CH), 33.52, 36.72, 37.34,
and 39.47 (CH2 × 4), 123.50 and 136.25 (CH × 2), 138.35 (C), 147.02
and 149.80 (CH × 2); IR (film) 3082, 3025, 1574, 1559, 1463 cm-1
;
HRMS found (M+) 233.2144, C16H27N requires 233.2143.
2-Methyl-5-octyloxypyridine (9). Reaction of 3-hydroxy-6-meth-
ylpyridine and 1-bromooctane according to the general procedure gave
the ether 9, as a liquid in 68% yield: 1H NMR δ 0.88 (t, J ) 6.6 Hz,
3H), 1.24-1.39 (m, 8H), 1.40-1.50 (m, 2H), 1.46-1.72 (m, 3H), 1.77
(b quintet, J ) 6.6 Hz, 2H), 2.47 (s, 3H), 3.96 (t, J 6.6 ) Hz, 2H),
7.04 (d, J ) 8.4 Hz, 1H), 7.10 (dd, J ) 2.9, 8.4 Hz, 1H), 8.17 (d, J )
2.9 Hz, 1H); 13C NMR δ 14.20 (CH3), 22.76 (CH2), 23.40 (CH3), 26.07
(CH2), 29.33 (CH2 × 2), 29.44 (CH2), 31.91 and 68.59 (CH2 × 2),
122.16, 123.41, and 136.76 (CH × 3), 150.16 and 153.38 (C × 2); IR
1605 (m), 1580 (s), 1505 (s) cm-1; HRMS found (M+) 221.1777,
C14H23NO requires 221.1779.
2-(3,7-Dimethyloctylthiol)thiophene (15). Reaction of 2-thiophene-
thiyllithium, from 1.93 g (23 mmol) of thiophene (21), with 5.00 g (23
mmol) of 1-bromo-3,7-dimethyloctane in ether and dry DMF (10 mL,
1:1) gave, after flash chromatography (SiO2) and short path distillation
at 98 °C (bath)/0.02 mmHg, 3.1 g (53%) of the sulfide 15: 1H NMR
δ 0.86 (d, J ) 6.6 Hz, 9H), 1.06-1.32 (m, 6H), 1.38-1.70 (m, 4H),
2.70-2.88 (m, 2H), 6.95 (dd, J ) 3.7, 5.5 Hz, 1H), 7.09 (dd, J ) 1.1,
3.7 Hz, 1H), 7.31 (dd, J ) 1.1, 5.5 Hz, 1H); 13C NMR δ 19.51, 22.77,
and 22.87 (CH3 × 3), 24.78 (CH2), 28.12 and 32.00 (CH × 2), 36.69
(CH2), 37.04 (CH2 × 2), 39.37 (CH2), 127.56, 128.99, and 133.30 (CH
× 3), 135.20 (C); IR 3070 (w), 1464 (m), 1215 (m), 697 (m) cm-l;
HRMS (EMI), found (M+) 256.1319, C14H24S2 requires 256.1319.
(3,7-Dimethyl-1-octyloxy)benzene (16). Reaction of phenol and
1-bromo-3,7-dimethyloctane according to the general procedure gave
the ether 16 as an oil in 86% yield: 1H NMR δ 0.92 (d, J ) 6.6 Hz,
6H), 0.99 (d, J ) 6.2 Hz, 3H), 1.12-1.44 (m, 6H), 1.52-1.78 (m,
3H), 1.80-1.94 (m, 1H), 3.97-4.07 (m, 2H), 6.90-7.00 (m, 3H), 7.26-
7.35 (m, 2H); 13C NMR δ 19.86, 22.79, and 22.89 (CH3 × 3), 24.86
(CH2), 28.17 and 30.08 (CH × 2), 36.45, 37.50, 39.45, and 66.38 (CH2
× 4), 114.71, 120.63, and 129.56 (CH × 3), 159.35 (C); IR (film)
3061, 3040, 1600, 1586, 1497, 1473, 1244, 752, 690 cm-1. Anal. Calcd
for C16H26O: C, 81.99; H, 11.18. Found: C, 81.69; H, 11.12.
4-(3,7-Dimethyl-1-octa-2,6-dienyloxy)-1,2-(methylenedioxy)ben-
zene (17). Reaction of 3,4-methylenedioxyphenol and 1-bromo-3,7-
dimethylocta-2,6-diene according to the general procedure gave the
ether 17 as a liquid in 50% yield: 1H NMR δ 1.60 (s, 3H), 1.68 (s
3H), 1.71 (s, 3H), 2.05-2.15 (m, 4H), 4.46 (d, J ) 6.6 Hz, 2H), 5.09
(t, J ) 6.9 Hz, 1H), 5.46 (t, J ) 6.6 Hz, 1H), 5.90 (s, 2H), 6.34 (dd,
J ) 2.4, 8.4 Hz, 1H), 6.51, (d, J ) 2.4 Hz, 1H), 6.70 (d, J ) 8.4 Hz,
1H); 13C NMR δ 16.63, 17.88, 25.85, 26.51, 39.74, 66.04, 98.53,
101.25, 106.18, 108.09, 119.80, 124.02, 131.99, 141.28, 141.75, 148.38,
154.64; IR 3066 (w), 1665 (m), 1632 (m), 1608 (m), 1500 (s), 1485
(s), 1377 (m), 1179 (s) cm-l; HRMS (EMI), found (M+) 274.1565,
C17H22O3 requires 274.1568.
1-(4-Phenoxyphenoxy)-2-(2-pyridyloxy)ethane (19). Reaction of
2-hydroxypyridine and 2-bromo-1-(4-phenoxyphenoxy)ethane (14) ac-

Biological Activity of Insect Juvenile Hormone Analogues
J. Agric. Food Chem., Vol. 52, No. 23, 2004 6947
cording to the general procedure gave the ether 19 in 80% yield: mp
85-87 °C; IR (KBr) 3032 (w), 3006 (w), 1615 (m), 1583 (s), 1578
with saturated aqueous NaHCO3 and brine and dried (MgSO4).
Evaporation of solvents and distillation gave 52.3 g (84%) of the acetal
25 as a 6:4 mixture of diastereoisomers: bp 104-106 °C/0.01 mmHg;
1H NMR δ 0.91 (d, J ) 6.2 Hz, 3H), 1.08 (t, (J ) 6.6 Hz, 1H), 1.13
(s, 3H), 1.18 (t, J ) 6.9 Hz, 6H), 1.31 (s, 3H), 1.35-1.48 (m, 5H),
1.60-1.68 (m, 2H), 3.46-3.72 (m, 4H), 4.57 (bs, 1H); 13C NMR δ
15.52, 17.26, and 19.94 (CH3 × 3), 23.13 (CH2), 25.10 (CH3), 28.47
(C), 29.15 (CH), 36.11 (CH2), 38.96 (CH), 40.64 (CH2), 60.81 and
61.12 (diasteroisomeric CH2-O), 72.23 (CCl2), 101.64 (CH); IR 1456
(m), 1375 (m), 1129 (s), 1065 (s), 999 (m), 835 (m) cm-l. Anal. Calcd
for C15H28Cl2O2: C, 57.87; H, 9.06. Found: C, 57.81; H, 8.80.
5-(2,2-Dimethylcyclopropyl)-3-methylpentanal (26). Reduction of the
acetal 25 as described for compound 23 gave 5-(2,2-dimethylcyclo-
propyl)-1,1-diethoxy-3-methylpentane in 94% yield as a 6:4 mixture
of diastereoisomers: bp 85-87 °C/9 mmHg; 1H NMR δ -0.13 (dd, J
) 4.0, 4.4 Hz, 1H), 0.32-0.48 (m, 2H), 0.91 (d, J ) 6.6 Hz, 3H), 1.01
(s, 3H), 1.02 (s, 3H), 1.20 (t, J ) 6.9 Hz, 6H), 1.24-1.46 (m, 5H),
1
(s), 1490 (s) cm-l; H NMR (200 MHz) δ 4.22 (t, J ) 4.9 Hz, 2H),
4.58 (t, J ) 4.9 Hz, 2H), 6.69-7.21 (m, 11H), 7.48 (m, 1H), 8.06 (m,
1H); 13C NMR (50 MHz) δ 62.42 and 65.33 (CH2 × 2), 109.13, 113.53,
114.67, 115.36, 118.40, 120.09, 127.15, 136.09, and 144.09 (CH ×
9), 147.50 (C × 2), 152.50 and 160.50 (C × 2); HRMS (EMI), found
(M+) 307.1215, C17H22O3 requires 307.1208.
1-(4-Phenoxyphenoxy)-2-(3-pyridyloxy)ethane (20). Reaction of
3-hydroxypyridine and 2-bromo-1-(4-phenoxyphenoxy)ethane (14) ac-
cording to the general procedure gave the ether 20 in 80% yield: mp
56-57 °C; IR (KBr) 3030 (w), 3004 (w), 1616 (m), 1583 (s), 1579
1
(s), 1490 (s) cm-l; H NMR (300 MHz) δ 4.27-4.31 (m, 2H), 4.32-
4.36 (m, 2H), 6.88-7.16 (m, 7H), 7.18-7.32 (m, 4H), 8.24 (dd, J )
1.5, 4.5 Hz, 1H), 8.37 (dd, J ) 0.6, 2.7 Hz, 1H); 13C NMR (75 MHz)
δ 67.06 (CH2), 115.91, 117.84, 120.84, 121.56, 122.68, 123.94, 129.73,
138.16, and 132.61 (CH × 9), 150.83, 154.81, 154.98, and 158.39 (C
× 4); HRMS (EMI), found (M+) 307.1203, C17H22O3 requires 307.1208.
3-(3-Phenoxybenzyloxy)pyridine (21). Reaction of 3-hydroxypyridine
and 3-phenoxybenzyl bromide (15) according to the general procedure
gave the ether 21 in 80% yield: mp 44-46 °C; IR (KBr) 3029 (w),
1.57-1.72 (m, 2H), 3.44-3.76 (m, 4H), 4.59 (t, J ) 6.2 Hz, 1H); 13
C
NMR δ 15.52 (C), 15.66 (CH3), 20.04 (CH2), 20.15 and 20.21 (CH3 ×
2), 25.21 (CH), 27.25 (CH2), 27.93 (CH3), 29.29 (CH), 37.99 and 40.83
(CH2 × 2), 60.71 and 61.08 (diasteroisomeric CH2-O), 101.77 (CH);
IR 3052 (w), 1456 (m), 1375 (m), 1126 (s), 1065 (s), 1000 (m) cm-l.
Hydrolysis of the acetal with 2 N HCl in 2-propanol gave the aldehyde
1
3004 (w), 1617 (m), 1581 (s), 1577 (s), 1490 (s) cm-l; H NMR (300
MHz) δ 5.09 (s, 2H), 6.86-6.98 (m, 3H), 6.98-7.10 (m, 3H), 7.12-
7.20 (m, 2H), 7.22-7.32 (m, 3H), 8.16 (bs, 1H), 8.29 (bs, 1H); 13C
NMR (75 MHz) δ 70.00 (CH2), 117.67, 118.49, 119.17, 121.80, 122.05,
123.60, 123.96, 129.87, and 130.13 (CH × 9), 138.16 (C), 138.19 and
142.36 (CH × 2), 154.67, 156.83, and 157.73 (C × 3); HRMS (EMI),
found (M+) 277.1104, C18H15NO2 requires 277.1103.
5-(2,2-Dimethylcyclopropyl)-3-methyl-1-penten-3-ol (23). A solution
of 11.9 g (50 mmol) of the alcohol 22 (16) in ether (35 mL) was added
to a stirred solution of 5.0 g (0.22 mol) of Na in liquid ammonia (125
mL) kept at -78 °C. Stirring was continued at this temperature for an
additional 2h followed by addition of solid NH4Cl. Ammonia was
evaporated and the residue extracted with ether and dried (MgSO4).
Evaporation of the solvent gave 8.3 g (98%) of the alcohol 23: bp
82-84 °C/10 mmHg; 1H NMR δ -0.15 (bs, 1 H), 0.37 (bs, 2 H), 1.04
(s, 6 H), 1.23 (s, 3 H), 1.3-1.6 (m, 4 H), 1.67 (bs, 1 H), 4.9-6.2 (m,
3 H); 13C NMR δ 15.65, 20.10, 22.96, 24.46, 25.01, 27.76, 27.98, 42.93,
73.38, 111.71, 145.43; IR 3400 (s), 1650 (m) cm-1. Anal. Calcd for
C11H20O: C, 78.51; H, 11.98. Found: C, 78.42; H, 12.02.
1
26 in 90% yield: bp 85-87 °C/9 mmHg; H NMR δ -0.13 (dd, J )
3.6, 8.4 Hz, 1H), 0.32-0.48 (m, 2H), 0.97 (d, J ) 6.6 Hz, 3H), 1.01
(s, 3H), 1.02 (s, 3H), 1.20-1.48 (m, 4H), 2.02-2.15 (m, 1H), 2.16-
2.30 (m, 1H), 2.35-2.45 (m, 1H), 9.76 (t, J ) 2.3 Hz, 1H); 13C NMR
δ 15.52 (C), 19.79 (CH2), 20.09 and 24.68 (CH3 × 2), 24.73 (CH),
27.31 (CH2), 27.74 (CH3), 28.23 (CH), 37.58 and 51.33 (CH2 × 2),
203.27 (CHdO); IR 3052 (w), 2712 (m), 1727 (s), 1456 (m), 1376
(m) cm-l; HRMS found (M+) 166.1356, C11H18O requires 166.1357.
Methyl 9-(2,2-Dimethylcyclopropyl)-3,7-dimethyl-2E,4E-nonadienoate
(18). Reaction of the aldehyde 26 with the ylid 27 (19) gave the acid
28 as a mixture of stereoisomers. The mixture was isomerized with
thiophenol according to the literature (19), furnishing the 2E,4E-isomer
in 33% overall yield: mp 37-40 °C; IR 3500 (s), 1690 (s), 1635 (m),
1
1610 (m) cm-1; H NMR δ -0.15 (bs, 1H), 0.38 (bs, 2H), 0.92 (d, J
) 5.5 Hz, 3H), 1.03 (s, 6H), 1.35 (s, 5H), 1.9-2.2 (m, 2H), 2.28 (s,
3H), 5.69 (bs, 1H), 6.12 (bs, 2H), 12.23 (s, 1H). Treatment of the acid
with diazomethane in the usual way gave a quantitative yield of the
1
methyl ester 18 as a liquid: H NMR δ -0.14 (dd, J ) 3.6, 8.4 Hz,
1-Bromo-5-(2,2-dimethylcyclopropyl)-3-methyl-2-pentene (24). To
8.4 g (50 mmol) of the alcohol 23, cooled in ice, was added 50 mL of
48% aqueous HBr. The mixture was stirred at this temperature for 30
min and then diluted with water. The product was extracted with CH2-
Cl2, and the extract was washed with saturated NaHCO3 (aqueous) and
dried (MgSO4). Evaporation and short path distillation of the residue
gave 11.1 g (96%) of the bromide 24 as a liquid, which was used
without further purification: 1H NMR δ -0.12 (bs, 1 H), 0.40 (bs, 2
H), 1.03 (s, 6 H), 1.73 (bs, 3 H), 1.2-2.3 (m, 4 H), 3.95 (d, J ) 8.5
1H), 0.32-0.48 (m, 2H), 0.89 (d, J ) 6.6 Hz, 3H), 1.02 (s, 3H), 1.03
(s, 3H), 1.18-1.46 (m, 5H), 1.52-1.66 (m, 1H), 1.95-2.06 (m, 1H),
2.12-2.25 (m, 1H), 2.28 (s, 3H), 3.70 (s, 3H), 5.70 (bs, 1H), 6.11 (bs,
2H); 13C NMR δ 14.08, 15.53, 19.80, 19.88, 19.95, 20.06, 24.97, 27.82,
33.24, 37.34, 40.57, 51.08, 117.30, 134.88, 136.56, 153.08, 167.89 (Cd
O); IR 3042 (w), 1716 (s), 1636 (w), 1612 (m), 1456 (m), 1376 (m),
1356 (m), 1239 (m), 1156 (s) cm-l; HRMS found (M+) 264.2103,
C17H28O2 requires 264.2089.
Hz, 2 H), 5.53 (bt, J ) 8.5 Hz, 1 H); IR 1655 (m) cm-1
.
Biological screening. Screening for JuVenile Hormone ActiVity. The
Tenebrio Test. This test was carried out in the usual way (8). Newly
molted (4-8 h) Tenebrio molitor pupae were held until the following
molt, and juvenile hormone activity was recorded by observing the
immature characters, using methyl farnesate as reference compound
(8).
5-[5-(2,2-Dimethylcyclopropyl)-3-methylpent-2-enyloxy]-2-methylpy-
ridine (13). Reaction of 3-hydroxy-6-methylpyridine and the bromide
24 according to the general procedure gave the ether 13 as a liquid in
80% yield: 1H NMR δ - 0.10 (dd, J ) 4.4, 9.1 Hz, 1 H), 0.34-0.52
(m, 2 H), 1.01 (s, 3 H), 1.04 (s, 3 H), 1.36-1.52 (m, 2 H), 1.74 (s, 3
H), 2.13 (t, J ) 7.6 Hz, 1 H), 2.19 (t, J ) 7.6 Hz, 1 H), 2.49 (s, 3 H),
4.55 (d, J ) 6.6 Hz, 2 H), 5.48 (t, J ) 6.6 Hz, 1 H), 7.05 (d, J ) 8.4
Hz, 1 H), 7.12 (dd, J ) 2.9, 8.4 Hz, 1 H), 8.18 (d, J ) 2.9 Hz, 1 H);
13C NMR δ 15.69 (C), 17.00 (CH3), 19.95 (CH2), 20.18 and 23.48
(CH3 × 2), 24.57 (CH2), 27.82 (CH3), 28.38 and 40.27 (CH2 × 2),
65.54 (C-O), 118.90, 122.68, 123.42, and 136.73 (CH × 4), 142.31,
150.16, and 153.04 (C × 3); IR 3043 (w), 1670 (w), 1600 (w), 1575
(m), 1456 (m), 1375 (m), 1127 (s), 1065 (s) cm-l; HRMS found (M+)
259.1937, C17H25NO requires 259.1936.
5-(2,2-Dichloro-3,3-dimethylcyclopropyl)-1,1-diethoxy-3-methylpen-
tane (25). To a mixture of 45.6 g (0.20 mol) of racemic citronellal
diethyl acetal, 95.6 g (0.80 mol) of CHCl3, and 1.0 g of triethylben-
zylammonium chloride in CH2Cl2 (25 mL), cooled in ice, was added
with stirring 50% aqueous NaOH (100 mL). The mixture was stirred
vigorously for 16 h at room temperature and then diluted with water.
The product was extracted with CH2Cl2, and the extract was washed
Screening against Sea Lice. A. Testing on Isolated Nauplius LarVae.
Adult females of L. salmonis with mature egg strings were collected
from Atlantic salmon reared in seawater. They were kept in 0.5-2 L
tanks with aerated seawater for hatching of the eggs, and 20-119 newly
hatched larvae of the nauplius I stage were collected and distributed
into containers (50-400 mL Petri dishes or beakers). The compounds
to be tested (Figure 2) were applied as acetone solutions at concentra-
tions ranging from 0.14 to 1.0 ppm, and the vitality and mortality of
the nauplii were monitored for up to 7 days. After specific exposure
times, the nauplii were filtered and transferred to containers with clean
seawater. Acetone was added to the control groups at the same
concentration as that used for the group exposed to the highest
compound concentration. After 6-7 days, the nauplii of the control
groups had developed into live and active copepodids. For compounds
with low or no effect the presence of large numbers of empty cuticles
and vital nauplii with upward swimming movement was typically

6948 J. Agric. Food Chem., Vol. 52, No. 23, 2004
Skattebøl et al.
Table 1. Effect of Different Juvenoids on the Development from
Table 2. Bath Treatment of Sea Lice Infested Salmon with Juvenoidsa
Nauplius I to Copepodida
mean no. of sea lice
test
% reduction of sea lice
% of nauplii
developed to
copepodid
% reduction
compd
day 0
day 7
day 14
relative to control, day 14
total no. of
nauplii at
start
in develop-
ment realtive
to control
control
5
11
12
13
15.0
15.0
15.0
15.0
15.0
15.0
15.0
21.4
17.0
14.8
14.8
8.4
30.8
15.8
12.4
18.4
11.6
23.6
21.8
test
compd
exposure
time (h)
49
60
40
62
23
29
(mean
±
SEM)
5
6
7
7
24
24
24
1
92
71
91
66
119
102
89
91
68
88
88
82
93
90
91
90
9
18
0
5
6
4
6
16
10
7
0
0
12
0
0
±
±
4
6
91
81
100
95
94
95
94
83
90
68
100
100
88
100
100
18
20
14.8
11.8
±
±
±
±
±
±
±
3
2
5
2
9
6
6
9
24
24
24
24
24
24
24
24
24
24
1
a Compound concentration, 2.0 ppm. Exposure time, 3 h (compound 20, 1 h).
Reduction of sea lice after 14 days.
10
12
16
17
19
20
21
Table 3. Bath Treatment of Sea Lice Infested Salmon with
Methoprene (1)a
fenoxycarb
deltamethrin
dichlorvos
control
±
±
5
1
mean ± SEM
control
methoprene (1)
day 0 day 14
sea lice
stage
day 0
day 14
96
chalimus
pre-adult
adult
27
±
0
0
2.5
0
±
±
41
±
0
0
7.3
0
±
±
a Compound concentration, 1.0 ppm. Exposure time, 1 and 24 h. Each value is
the average of three parallels. Seawater salinity, 2.3 3.4%. Temperature, 10 16
C.
19
2.6
2
0.7
7.1
2.0
1.8
0.4
−
−
°
a Mean number of sea lice per fish before treatment and after 14 days.
observed. The endpoint in the initial screening was based on reduced
mobility, reduced molting as observed by the amount of empty cuticles,
or mortality of nauplii. In other experiments the fraction of nauplius I
larvae that developed to copepodid was used as endpoint. The
development took 3-7 days depending on the seawater temperature
and salinity.
In the first initial experiment juvenoid compound 8 was tested at a
concentration of 0.7 ppm. After 14 days of exposure, the fraction of
nauplii developed to copepodid was evaluated. Most of the nauplius
larvae in the control group had developed into copepodid, and lots of
empty cuticles from molting were observed. Larvae exposed to
compound 8 were similar to control.
The activity of compounds 5, 6, and 12 was further tested at
concentrations of 0.01, 0.05, and 0.3 ppm and exposure times of 1,5,
and 24 h. Significant reduction of copepodids compared to controls
was observed for all compounds at 1.0 ppm and 1 h of exposure and
even at 50 ppb and 24 h of exposure time for compounds 5 and 12.
B. Testing on Fish. Atlantic salmon, artificially infested with sea
lice (L. salmonis), was distributed to 7 tanks with 17 fish in each. The
seawater temperature was 8-9 °C and salinity, 3.0-3.4%. The day
before treatment the total number of sea lice was counted, with
differentiation between chalimus and pre-adult stages. The sea lice were
present as 79% chalimus I and II stages and 21% pre-adults originating
from an earlier infestation. The mean was 15.0 sea lice per fish [standard
error mean (SEM) ) 0.7], based on counts on 12 fish per group. The
compounds to be tested were dissolved in an emulsifying mixture of
N-methyl-2-pyrrolidinone, N-octyl-2-pyrrolidinone, and polyoxyl 35
castor oil (approximately 1:1:2) and added to the tanks until a
concentration of 2.0 ppm was reached. During treatment the water flow
to the tank was stopped. Exposure time was 3 h except for compound
20, which caused loss of equilibrium in the fish and was stopped after
1 h. The sea lice were counted on days 7 and 14 after treatment. The
mean number of sea lice on days 7 and 14 was based on counts from
five fish per group per time point. The rise in number of sea lice in the
control is probably due to some young chalimus being overlooked
initially because of the small size. At day 7 chalimus III and IV stages
were the most abundant in all groups, whereas at day 14 pre-adults
dominated. The results are recorded in Table 2.
was added continuously by a pump to one of the tanks during all 14
days. The other tank was for control. All stages of sea lice on the salmon
were counted on 5 fish per group before treatment and on 10 fish on
day 14 after treatment. The results are recorded in Table 3.
Statistics. Statistical analysis was performed using NCSS 2000
(Number Cruncher Statistical Systems, Kaysville, UT). The level of
significance was set at R ) 0.05.
RESULTS AND DISCUSSION
The use of free-living nauplii going through two moltings to
copepodid is convenient as a screening system for compounds
with a potential for interrupting the metamorphosis. The
experiments are influenced by the quality of eggs and the
seawater, especially temperature and salinity. A prerequisite for
success is the presence of a high number of live vital copepodids
in the control group at the conclusion of the test.
The compounds screened for inhibitory effect on the meta-
morphosis of the parasite, L. salmonis, all exhibited juvenile
hormone activity in insects as observed from the Tenebrio test
(see above). In addition, the organophosphate dichlorvos, for
many years used in salmon farming for removing sea lice, was
included for comparison, and likewise the pyrethrin analogue
deltamethrine, which presently is the drug of choice used by
most fish farmers in Norway for combating sea lice.
Results of the initial testing are recorded in Table 1. The
compounds inhibited the process of molting and phase trans-
formation from nauplius I to copepodid, resulting in larvae that
were either dead or had reduced mobility. Compounds 7, 20,
and 21, deltamethrin, and dichlorvos inhibited completely the
development to copepodids, but compounds 5, 8, 9, 10, and 12
also exhibited significant activity.
The results from Table 1 do not allow a serious structure-
activity discussion, but they give some indications. It seems
that a pyridine ring alkyl-substituted at the 3-position is a
preferred structural feature. On the other hand, whether the alkyl
group is attached to the pyridine ring directly or through an
oxygen or sulfur atom seems to be less significant. Concerning
the alkyl group we assumed from tests on insects that it should
preferably have a length of 8-10 carbon atoms; otherwise, the
The effect of methoprene (1) on sea lice infested Atlantic salmon
was examined in a different way. The salmon was infested with
copepodids and distributed with 45 fish (60-70 g) in each of two tanks
of 500 L with running seawater (10 °C). The flow of water was
regulated to give a minimum 70% saturation of oxygen at the outlet.
A 1.7 L acetone solution containing 1 at a concentration of 5000 ppm

Biological Activity of Insect Juvenile Hormone Analogues
J. Agric. Food Chem., Vol. 52, No. 23, 2004 6949
(4) Ritchie, G.; Ronsberg, S. S.; Hoff, K. A.; Branson, E. J. Clinical
efficacy of teflubenzuron (Calicide) for the treatment of
Lepeoptheirus salmonis infestations of farmed Atlantic salmon
Salmo salar at low water temperatures. Dis. Aquat. Organ. 2002,
51, 101-6.
(5) Ramstad, A.; Colquhoun, D. J.; Nordmo, R.; Sutherland, I. H.;
Simmons, R. Field trials in Norway with Slice (0.2% emamectin
benzoate) for the oral treatment of sea lice infestation in farmed
Atlantic salmon Salmo salar. Dis. Aquat. Organ. 2002, 50, 29-
33.
(6) Stone, J.; Sutherland, I. H.; Sommerville, C.; Richards, R. H.;
Varma, K. J. Field trials to evaluate the efficacy of emamectin
benzoate in the control of sea lice, Lepeoptheirus salmonis
(Krøyer) and Caligus elongates (Nordmann), infestations in
Atlantic salmon Salmo salar L. Aquaculture 2000, 186, 205-
19.
(7) Fingerman, M. The endocrine mechanisms of crustaceans. J.
Crust. Biol. 1987, 7, 1-24.
(8) Slama, K.; Romanuk, M.; Sorm, F. Insect Hormones and
Bioanalogues; Springer-Verlag: New York, 1974.
(9) Laufer, H.; Sagi, A.; Ahl, J. S. B.; Homola, E. Methyl farnesoate
appears to be a crustacean reproductive hormone. InVertebr.
Reprod. DeV. 1992, 22, 17-20.
(10) Laufer, H.; Ahl, J.; Rotllant, G.; Baclaski, B. Evidence that
ecdysteroids and methyl farnesoate control allometric growth and
differentiation in a crustacean. Insect Biochem. Mol. Biol. 2002,
32, 205-210.
(11) Homola, E.; Chang, E. S. Methyl farnesoate: Crustacean juvenile
hormone in search of functions. Comp. Biochem. Physiol. 1997,
117B, 347-356.
(12) Charmantier, G.; Charmantier-Daures, M.; Aiken, D. E. Larval
development and metamorphosis of the American lobster
Homarus americanus (crustacean, decapoda): effect of eystalk
ablation and juvenile hormone injection. Gen. Comp. Endocrinol.
1988, 70, 319-333.
(13) Solli, H.; Madsen, H. B.; Holst, P. L.; Klemmensen, P. D. Pyridyl
terpenoid ethers with high juvenile hormone activity. Pestic. Sci.
1976, 7, 503-511.
(14) O’Brien, E. M.; Tipton, K. F.; Meroni, M.; Dostert, P. Inhibition
of monoamine oxidase by clorgyline analogs. J. Neural Transm.,
Suppl. 1994, 41, 295-305.
(15) Surman, M. D.; Mulvihill, M. J.; Miller, M. J. Regio- and
stereoselective ring openings of 3-aza-2-oxabicyclo[2.2.1]hept-
5-ene systems with copper catalyst-modified Grignard re-
agents: Application to the synthesis of an inhibitor of 5-lipoxy-
genase. J. Org. Chem. 2002, 67, 4115-4121.
results of Table 1 indicate that the number of double bonds
and branched methyl groups is less important for activity.
However, for a compound to be of use for combating sea
lice it must be efficacious and safe when applied to live salmon.
Testing on live fish infested with sea lice required multigram
quantities of the compounds to be tested. Because the project
had a practical goal, the ease of preparation of the compound
and economical aspects were considered when the candidates
were chosen. Consequently, the thioether 7 was excluded,
whereas compounds 5, 12, and 20 were chosen on the basis of
the initial test results (Table 1). Furthermore, promising results
from testing on two other crustacean species, namely, the
barnacles Ballanus ballanoides and Ballanus improVisus (22),
and the availability of sufficient quantities led us to include
compounds 11, 13, and 18 as well (Figure 2). Note that
compound 11 is structurally very similar to 10, an obvious
candidate for this testing. Furthermore, the commercially
available juvenoid methoprene was included in the testing on
fish.
From the testing on live fish (Table 2) the best results were
recorded for compounds 11 and 13 with 60 and 62% reductions
of sea lice, respectively, compared with the control. ANOVA
analysis of sea lice numbers on day 14 revealed significant
differences (p < 0.05). Using Duncan’s multiple-comparison
test, compounds 5, 11, and 13 were significantly different from
the control; by comparison, the reductions in number of adult
females were 82% for compound 13, 73% for compound 5, and
64% for compound 11. As shown in Table 3, continuously
treating sea lice infested salmon with water containing metho-
prene at low concentrations resulted in a significant reduction
of sea lice numbers (p ) 0.0001) after 14 days, whereas no
significant change was observed in the control group (p ) 0.07).
A number of factors may induce differences in results from the
two testing systems. All stages of the parasite will be present
when sea lice infested fish in an open water system are tested,
and consequently migration and attachment of drifting copepo-
dids throughout the experiment may take place. Furthermore,
the compounds may also be absorbed by the fish to a certain
degree and thereby exert a systemic effect on attached sea lice.
Our results show that many of the compounds tested,
including the juvenoid methoprene, interrupt the metamorphosis
of sea lice. This supports the notion that the parasite is sensitive
to the external application of compounds that are known to affect
the metamorphosis of insects. Moreover, the activities of the
juvenoids compare favorably with those of the organophosphate
dichlorvos and the pyrethroid deltamethrin, which are used
commercially for treatment of sea lice infestations. Hence, it
seems that juvenoids have a potential for being developed into
a practical method for control of parasitic marine crustacean
species. Furthermore, the compounds reported in the present
paper are easily prepared on a large scale for practical use.
(16) Kleveland, K.; Skattebøl, L.; Sydnes, L. K. Reactions of
dihalocarbenes with allylic alcohols. Acta Chem. Scand. 1977,
B31, 463-468.
(17) Gosselin, P.; Maignan, C.; Rouessac, F. Stereospecific synthesis
of homogeranic and homoneric acids. Synthesis 1984, 876-881.
(18) Habbachi, M.; Gaudemar, M. Silicon in organic synthesis: An
improved 4- of 2-alkenoic acids. Synthesis 1983, 745-746.
(19) Henrick, C. A.; Willy, E. W.; Baum, J. W.; Baer, T. A.; Garcia,
B. A.; Mastre, T. A.; Chang, S. M. Stereoselective synthesis of
alkyl (2E,4E)- and (2Z,4E)-3,7,11-trimethyl-2,4-dodecadienoates.
Insect growth regulators with juvenile hormone activity. J. Org.
Chem. 1975, 40, 1-7.
(20) Fuerst, H.; Heltzig, M.; Goebel, W. Preparation of pyridine-3-
sulfonyl chloride and pyridine-3-thiol. J. Prakt. Chem. (Leipzig)
1967, 36, 160-164.
(21) Jones, E.; Moodie, I. M. 2-Thiophenethiol. Org. Synth. 1970,
50, 104-106.
LITERATURE CITED
(1) Pike, A. W.; Wadsworth, S. L. Sealice on salmonids: their
biology and control. AdV. Parasitol. 1999, 233-337.
(2) Grimnes, A.; Jakobsen, P. J. The physiological effects of salmon
lice (Lepeophtheirus salmonis) infection on post smolt of atlantic
salmon (Salmo salar). J. Fish Biol. 1996, 48, 1179-1194.
(3) Tully, O.; Poole, W. R.; Wheland, K. R. Infestation parameters
for Lepeophtheirus salmonis parasite on sea trout, Salmo trutta
L., off the west coast of Ireland during 1990 and 1991. Aquacult.
Fish. Manage. 1993, 24, 545-555.
(22) Kleveland, K.; Nilsen, N. O.; Stenstrøm, Y.; Skattebøl, L.
Unpublished results.
Received for review May 10, 2004. Revised manuscript received August
20, 2004. Accepted September 3, 2004.
JF049259H