New fluorinated derivatives as esterase inhibitors. Synthesis, hydration and crossed specificity studies

Article abstract of DOI:10.1016/S0968-0896(02)00467-4

A variety of new fluorinated chemicals have been prepared for the first time and tested as inhibitors of esterases, one of the main enzymes involved in pheromone catabolism, in two economically important pests, the Egyptian armyworm Spodoptera littoralis (SL) and the Mediterranean corn borer Sesamia nonagrioides (SN). Using the respective major component of the pheromone as substrate, the K_m and V_max of the antennal esterase of both insects resulted to be 5.66×10^-4 M and 8.47×10^-6 Mmin^-1 for SL and 1.61×10^-7 M and 1.25×10^-7 Mmin^-1 for SN, pointing out that SN esterase has a higher affinity for its corresponding substrate than SL. In general, the trifluoromethyl ketones (TFMKs) exhibited higher inhibitory potency than the corresponding difluoromethyl ketones (DFMKs) or difluoroaldehydes (DFAs). The compounds appeared to hydrate differently in aqueous solution, the extent of hydration following the order: α,α-DFMKs<α,α-difluoro-β-thioalkylmethyl ketoneshyd and the inhibitory potency and no specificity has been found when the chemicals were assayed on extracts of both insects.

Full text of DOI:10.1016/S0968-0896(02)00467-4

Bioorganic & Medicinal Chemistry 11 (2003) 1047–1055
New Fluorinated Derivatives as Esterase Inhibitors. Synthesis,
Hydration and Crossed Specificity Studies
Carmen Quero,a Gloria Rosell,b Oscar Jimenez,a Sergio Rodriguez,a
M. Pilar Boscha and Angel Guerreroa,*
aDepartment of Biological Organic Chemistry, Institute of Chemistry and Environmental Research (CSIC),
Jordi Girona 18-26, 08034 Barcelona, Spain
bDepartment of Pharmacology and Therapeutic Chemistry, Unity Associated to CSIC, Faculty of Pharmacy,
University of Barcelona, Avda. Diagonal, s/n. 08028 Barcelona, Spain
Received 28 June 2002; revised 29 August 2002; accepted 20 September 2002
Abstract—A variety of new fluorinated chemicals have been prepared for the first time and tested as inhibitors of esterases, one of
the main enzymes involved in pheromone catabolism, in two economically important pests, the Egyptian armyworm Spodoptera
littoralis (SL) and the Mediterranean corn borer Sesamia nonagrioides (SN). Using the respective major component of the pher-
omone as substrate, the Km and Vmax of the antennal esterase of both insects resulted to be 5.66ꢀ10ꢁ4 M and 8.47ꢀ10ꢁ6 Mminꢁ1
for SL and 1.61ꢀ10ꢁ7 M and 1.25ꢀ10ꢁ7 Mminꢁ1 for SN, pointing out that SN esterase has a higher affinity for its corresponding
substrate than SL. In general, the trifluoromethyl ketones (TFMKs) exhibited higher inhibitory potency than the corresponding
difluoromethyl ketones (DFMKs) or difluoroaldehydes (DFAs). The compounds appeared to hydrate differently in aqueous solu-
tion, the extent of hydration following the order: a,a-DFMKs<a,a-difluoro-b-thioalkylmethyl ketones<TFMKs<b-thiotri-
fluoromethyl ketones<a,a-DFAs. No clear correlation has been found between the Khyd and the inhibitory potency and no
specificity has been found when the chemicals were assayed on extracts of both insects.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
microsomal carboxylesterases,8 or cytosolic human
phospholipase A2.9 Inhibition studies carried out with
these fluorinated derivatives may be therapeutically sig-
nificant in different areas, f.i. arachidonoyl ethanola-
mide (anandamide) hydrolysis inhibitors in processes
involving analgesia, mood, nausea, memory, and so
on,10 and renin or angiotensin-converting enzyme inhi-
bitors in hypertension phenomena.11 The fluorinated
ketones function as transition-state analogues of the
enzyme, with the inhibition activity arising by formation
of an adduct of tetrahedral geometry between the serine
residue, present at the active site of the enzyme, with the
highly electrophilic carbonyl moiety.12,13 A crystal
structure has been obtained for the covalent complex of
porcine pancreatic elastase and peptidyl a,a-difluoro-b-
keto amide, wherein the inhibitor is bound at the active
site as a hemiketal with a Ser-195.14
The development of potent enzyme inhibitors is an area
of pivotal importance in the pharmaceutical and agro-
chemical fields.1 Illustrative of the significance of these
compounds is, for example, the development of inhibi-
tors of human neutrophil elastase for treatment of pul-
monary emphysema,2 or inhibitors of neuropathy target
esterase, the target site of certain neurotoxic organo-
phosphorus compounds.3 Incorporation of poly-
fluoroketone moieties into inhibitors exhibiting close
structural analogy to the substrates has proven to be a
useful strategy for generating strong inhibitors of
diverse serine hydrolases, many members of this type of
enzymes being pharmacologically attractive targets.
Among others, enzymes inhibited by fluoroketone inhi-
bitors include acetylcholinesterase,4 chymotrypsin,5
trypsin,6 juvenile hormone esterase,7 human liver
We have previously reported that trifluoromethyl
ketones (TFMKs) are good antagonists of the pher-
omone action in insects, as a consequence, at least in
part, of the inhibition of the pheromone catabolism
*Corresponding author. Tel.: +34-93-400-6120; fax: +34-93-204-
5904; e-mail: agpqob@iiqab.csic.es
0968-0896/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00467-4

1048
C. Quero et al. / Bioorg. Med. Chem. 11 (2003) 1047–1055
process.13,15ꢁ20 We now report the synthesis, hydration
and crossed specificity studies of new TFMKs, a,a-
difluoromethyl ketones (DFMKs) and a,a-difluoro-
aldehydes (DFAs) (Scheme 1) as esterase inhibitors of
two economically important pests, the Egyptian army-
worm Spodoptera littoralis (SL) and the Mediterranean
corn borer Sesamia nonagrioides (SN). TFMKs are
known as potent inhibitors of the antennal esterase in
male olfactory tissues.15,21,22 Difluorinated ketones, in
turn, have been reported to be good inhibitors of serine
proteases23 and HIV-1 proteases,24 and difluoro-
aldehydes as inhibitors of acetylcholinesterase4 and
aldehyde-oxidizing enzymes of antennal tissues of Heli-
coverpa (Heliothis) virescens.25
difluoroketones.29 However, in order to prepare alkyl
difluoromethyl ketones, reductive deoxygenation of the
hydroxy group at the 3 position is required but this
process is by no means straightforward. In fact, we
experienced serious drawbacks when we attempted the
related reductive deoxygenations of unsaturated 3-
hydroxy-2,2-difluoroesters by a number of methods30
since this process requires radical reduction conditions,
non-compatible with radical sensitive groups (double
and triple bonds) present in the substrate. Another
approach for the synthesis of DFMKs was developed by
Burton and Qiu,31 who reported the radical addition of
alkenes to iododifluoromethyl ketones in the presence of
Pd(PPh3)4 followed by reductive deiodination with Zn/
NiCl2.6H2O. Here, again, the procedure appears not to
be appropriate for unsaturated substrates. A more ela-
borated approach was described in which difluor-
acylsilane intermediates suffered a Brook rearrangement
when treated with CH2N2 to produce difluorinated tri-
methylsilyl enol ethers, which were converted by HF
treatment to the expected DFMKs in good overall
yields.32,33 In our case, since we had previously devel-
oped an efficient synthesis of long-chain a,a-difluor-
oesters,30 we decided to test the direct reaction of these
compounds with methyllithium (Scheme 2). The process
required 1.5 equivof the nucleophile but a larger excess
of the latter is not detrimental if the reaction is carried
out at ꢁ78 ꢂC in THF. At room temperature the reac-
tion is not selective and proceeds further to give the
corresponding trifluoromethyl alcohol. Surprisingly,
only one precedent has been found in the literature on
this type of reaction.34 The reaction can be extended to
other alkyllithiums (Table 1) resulting in a good, prac-
tical method of synthesis of a,a-difluoroketones 2a–2f.
For preparation of 2f, the corresponding ester precursor
1f was obtained by treatment of 1-octanethiol with
NaH/DMF followed by reaction with ethyl bromodi-
fluoroacetate.
Results and Discussion
Synthesis
Introduction of a gem-difluoromethylene group in
organic molecules has been recently reviewed.26 For the
synthesis of DFMKs many of the processes involve
transformation of the more readily available short syn-
thons chloro- or iododifluoromethyl ketones. Thus,
Ishihara et al.27 described the reaction of chlorodi-
fluoromethyl ketones with Zn/Me3SiCl in CH3CN to
produce the trimethylsilyl difluoroenol ethers inter-
mediates, which were coupled with aldehydes and
ketones to provide 3-hydroxydifluoromethyl ketones in
25–68% overall yields. The enol could be trapped ‘in
situ’ by Lang and Schaub28 with Zn/DMF and reacted
with aldehydes in a one-step process. More recently,
Medebielle et al. described the use of tetra-
kis(dimethylamino)ethylene as an alternative to Zn to
generate the difluoracetyl anion in route to aromatic
Scheme 2. Synthetic approach to DFMKs 2 and DFAs 4.
Table 1. Synthesis of difluoromethyl ketones 2a–2f
Ester
R
R’
Time (h)
Product
Yield (%)
1a
1b
1c
1d
1d
1f
C9H19
C14H29
(Z)-11-C16H31
(Z)-12-C17H33
(Z)-12-C17H33
Me
Me
Me
Me
Ph
1.5
1
4.75
1
1
3
2a
2b
2c
2d
2e
2f
76
44
84
44
47
95
Scheme 1. Structures of compounds 2–9 synthesized as potential inhi-
bitors of esterases.
C8H17
S
Me

C. Quero et al. / Bioorg. Med. Chem. 11 (2003) 1047–1055
1049
Reduction of difluoroesters 1 to the corresponding
alcohols was easily carried out by treatment with
lithium aluminum hydride (2.9 equiv) in ether at 0 ꢂC in
good to excellent yields (Table 2).
hydrolysis in the presence of the inhibitor, with regard
to mean values of hydrolysis obtained in control
experiments (see Experimental). The TFMKs (5, 6, 7)
exhibited higher inhibitory activity than the DFMKs
(2f, 2c, 2d) and DFAs (4b, 4c, 4d) in either insect (Table
3). The inductive effect of the fluoro substituents sub-
stantially contributes to the potency of the inhibitors,
the effect being higher with an increasing number of the
halogen in the vicinity of the carbonyl to stabilize the
hemiacetal adduct with the serine residue.40
Synthesis of DFAs 4 was much more troublesome.
Thus, direct reduction of 1a or 1d with diisobutylalu-
minum hydride in pentane at ꢁ55 ꢂC25 led to mixtures
of the difluorinated alcohols 3a–3d and aldehydes 4a–4d
in low yields. An apparently successful preparation of
a,a-difluorononaldehyde hydrate has been reported by
treatment of the ester with DIBAH in ether but the yield
of the aldehyde or hydrate was not disclosed.35 There-
fore, a study of the oxidation of the fluorinated alcohols
3 was undertaken. Treatment of 3d with PDC in CH2Cl2
yielded a mixture of the starting alcohol (25%) and the
unexpected ester 10 (15%) (Scheme 3), resulting from an
overoxidation to the acid followed by esterification, and
other unidentified products.
The highest inhibitory activity on both insects was
shown by OTFP (5) with an IC50 5.9 mM in SL and 16.3
mM in SN followed by compounds 7 (IC50 64.5 mM in
SL and 54.5 mM in SN) and 6 (IC50 121.0 mM in SL and
123.7 mM in SN). Compound 7 is the TFMK analogue
of (Z,E)-9,11-tetradecadienyl acetate, the major com-
ponent of the pheromone of SL,44 while 6 is the same
analogue of (Z)-11-hexadecenyl acetate, the major
component of the SN pheromone.45 In both instances,
the only modification was replacement of the acetate
group of the parent pheromone by the trifluoroacyl
moiety COCF3. OTFP has been shown to act as a
potent inhibitor of JHE,41 and it is interesting to note
that its closely related ketone 2f elicited no activity. In
this case, the much higher contents of hydrate in aqu-
eous or organic solution of 5 (see below), in equilibrium
with the free ketone, may account for this result. We
have found by 19F NMR that freshly prepared solutions
of 5 in CHCl3 appear to be a mixture of the hydrate and
keto forms in 21:79 ratio, while in 2f the hydrate form is
practically undetectable. In this context, the activity of
certain di- and trifluoro derivatives as inhibitors of
acetylcholinesterase was postulated to be due to the
interaction enzyme-unhydrated form of the inhibitor,4
whereas the activity of difluorinated inhibitors of pepsin
was attributed to binding of the hydrated form of the
inhibitor to the enzyme.43
Treatment of 3c with TEMPO (2,2,6,6-tetramethyl-1-
piperidinyloxy)/N-cholorosuccinimide/TBACl in
a
biphasic CH2Cl2-aqueous pH 8.6 buffer system36 or
with the Dess–Martin periodinane reagent37 under dif-
ferent conditions led to mixtures of products and low
yields of 4c (ca. 15%). The best conditions found for the
preparation of DFAs 4 were through Swern oxidation38
using 3 equivof (COCl) , 6 equivDMSO and 12 equiv
2
of Et3N in CH2Cl2 at room temperature for 30 min
(Scheme 2). Compounds 4 were obtained in 35–66%
isolated yields (Table 2), as mixtures of the hydrate and
aldehyde forms in ca. 4:1 ratio as determined by 19F
NMR spectroscopy.
Inhibition studies
The inhibition tests were carried out using a preincuba-
tion time of 10 min since we assumed that, in a similar
manner to TFMKs, the new DFMKs and DFAs might
exert also their inhibition effect through formation of a
stable hemiacetal with a serine residue of the enzyme.39
The inhibitory potency of the chemicals was determined
by the relative decrease of the substrate (the major
component of the pheromone of either insect species)
However, attribution of the higher activity of 5 with
respect to 2f to the different level of hydration in solu-
tion appears to be too simplistic since DFAs are also
highly hydrated (the hydrate/keto form ratio found for
freshly prepared 4d was 80:20 in CHCl3 soln., see below)
but exert only moderate inhibitory activity. The real
Table 2. Synthesis of difluoroalcohols 3 and DFAs 4
Table 3. Comparative inhibitory activity of compounds 2–9
Ester
R
3 (%)a
4 (%)a
Compd
S. littoralis IC50 (mM)a
S. nonagrioides IC50 (mM)a
1a
1b
1c
1d
C9H19
C14H29
(Z)-11-C16H31
(Z)-12-C17H33
3a (75)
3b (65)
3c (96)
3d (69)
4a (66)
4b (35)
4c (58)
4d (42)
5
5.9 (ꢃ1.2)
N.A.b
16.3 (ꢃ0.4)
N.A.b
2f
2b
4b
2c
4c
2d
4d
6
622.4 (ꢃ109.2)
513.1 (ꢃ95.5)
261.4 (ꢃ45.0)
260.3 (ꢃ22.4)
N.A.b
850 (ꢃ56.4)
376.3 (ꢃ7.8)
264.2 (ꢃ73.3)
254.2 (ꢃ39.1)
N.A.b
164.9 (ꢃ26.3)
123.7 (ꢃ39.0)
54.5 (ꢃ7.8)
—
aDFAs were obtained as mixtures of the hydrate and keto forms.
N.A.b
121.0 (ꢃ26.5)
64.5 (ꢃ4.7)
N.A.b
7
8
9
N.A.b
—
aIC50 values were calculated by least squares regression analyses in at
least triplicate experiments considering five different concentrations of
inhibitor and 2–3 controls for each replicate.
Scheme 3. Structures of compounds 10–12.
bNon active.

1050
C. Quero et al. / Bioorg. Med. Chem. 11 (2003) 1047–1055
active form (keto or hydrate) responsible for esterase
activity remains, therefore, an open question.
containing 0, 0.1, 0.24, 0.37, 0.5 and 0.75 mole fraction
water. Except for compounds 2f and 5, the substrates
were not completely soluble at ꢀH O=0.75 and, there-
fore, Khyd app could not be readily determined at this
molar fraction. On the other hand, for compound 4a at
2
Compounds 8 and 9, showing absolutely different
structures than the pheromone, were completely inac-
tive. Also, compounds with a longer chain than the main
pheromone component, such as 2d and 4d, were inef-
fective as inhibitors except 4d on SN. This compound is
also a close analogue of the major component of the
pheromone, which in addition contains (Z)-11-hex-
adecenol and (Z)-11-hexadecenal.45 The activity of 4d
may be attributed to an additional inhibition on other
oxidizing enzymes (oxidases, aldehyde dehydrogenases,
etc.) required to perform a successful pheromone cata-
bolism. In this context, inhibition of aldehyde dehy-
drogenases in antennal extracts of H. virescens by DFAs
has been reported.25 In SL, on the other hand, the
pheromone complex is a mixture of dienic, monoenic
and saturated acetates,46 and, therefore, we could expect
that in this insect esterases should be the major type of
the catabolic proteins of the pheromone.
ꢀ
H O=0.1 the Khyd app was already 438, so lower mole
2
fractions of water in the mixtures had to be considered.
The keto and hydrate forms were allowed to equilibrate
for 2.5 h, and the apparent Khyd values were determined
by the ratio of the areas of the two forms in the 19F
NMR spectra. The results are shown in Table 4. The
Khyd app and ꢀH O values were plotted and adjusted to a
straight line of equation
2
À
Á
y ¼ 1:07x þ 2:46 r2 ¼ 0:96
The interception point at the Y axis (ꢀH O=0) corre-
2
sponded to the calculated Khyd in pure water. It should
be noted that the Khyd app have only an approximate
value since the study has been carried out following
Linderman’s assumptions for ideal solutions which obey
Henry’s or Raoult’s law.48 Our results show that there is
considerable variation in the Khyd values for the
TFMKs, DFMKs, and DFAs tested. Thus, while
DFMKs (f.i. 2a) do not practically hydrate in solution,
DFAs (f.i. 4a) are completely hydrated. The extent of
hydration follows the order: a,a-DFMKs <a,a-
difluoro-b-thioalkylmethyl ketones <TFMKs <b-thio-
trifluoromethyl ketones <a,a-DFAs. Comparison of
the Khyd with the IC50 values shows that although com-
pounds with very low hydration, such as 2f, are also
inactive as inhibitors, no clear correlation between the
two parameters was apparent among the active chemi-
cals. Thus, the best inhibitor (compound 5) showed a
high Khyd but DFA 4a displayed a Khyd ffi 15Â higher
being ffi 16Â less active. Linderman et al.,48 in contrast,
reported a reasonable correlation of Khyd and the
potency of some TFMKs as inhibitors of JHE. Our
results agree with those of the Raleigh group in that the
most potent inhibitor (5) was also the most hydrated
among the different TFMKs tested (6, 11, 12). In this
context, it is worthy of note that in a molecular model-
ing study we have demonstrated that b-substituted
TFMKs, like 5, show an intramolecular hydrogen bond
between the hydrate and the heteroatom (O, S, SO and
SO2) located in b position to the carbonyl and that the
strength of this hydrogen bond correlates well with the
inhibitory activity.42
The calculated values of Km and Vmax of both esterase
preparations were 5.66ꢀ10ꢁ4 M and 8.47ꢀ10ꢁ6
M
M
minꢁ1 for SL and 1.61ꢀ10ꢁ7 M and 1.25ꢀ10ꢁ7
minꢁ1 for SN, according to the double reciprocal plot of
Lineweaver and Burk (Fig. 1). These results suggest that
SN esterase has higher affinity for the substrate than
that of SL, which agrees with the lower doses of the
pheromone required (100 ng–1 mg) to elicit the complete
behavioral sequence on SN males in comparison with
SL (10 mg).19
Hydration studies
In order to establish a possible correlation between the
hydration constant (Khyd) and the inhibitory potency of
the chemicals, we carried out for the first time studies
directed to determine the Khyd of these analogues by 19
F
NMR. As cited above, several groups have established
by spectrocopic means that the TFMKs inhibit several
types of esterases by binding to the active serine residue
as an ionized kemiketal.12ꢁ14,47
We followed a similar procedure to that reported by
Linderman.48 The selected compounds 2a, 2f, 4a, 5, 6
along with the saturated n-tetradecyl trifluoromethyl
ketone (11) and n-hexadecyl trifluoromethyl ketone (12)
(Scheme 3) were dissolved in H2O/CH3CN mixtures
Conclusions
In summary, new fluorinated derivatives have been
synthesized as inhibitors of antennal esterases of two
economically important pests. With the exception of
compound 4d in SN, the best esterase inhibitors found
in both insects are the TFMKs structurally similar to
the corresponding pheromone, the DFMKs and DFAs
being less efficient inhibitors. The compounds appear to
hydrate to different extent in aqueous solution, but no
clear correlation has been found between the Khyd and
the inhibitory potency. The new type of compounds
Figure 1. Lineweaver–Burk reciprocal plot of 1/[V] versus 1/[S] for
determination of Km and Vmax of the antennal esterase of SL and SN.

C. Quero et al. / Bioorg. Med. Chem. 11 (2003) 1047–1055
1051
Table 4. Equilibrium hydration constants of substrates 2a, 2f, 11, 12, 6, 5 and 4a by 19F NMR spectroscopy in comparison with their inhibitory
activity
Substrate
Mole fraction of H2O in H2O/CH3CN mixtures
Calcd Khyd (r2)
IC50 (mM)
0
0.025
0.05
0.1
0.24
0.37
0.5
0.75
a
2a
2f
11
12
6
0
0
0
0
0
—
—
—
—
—
—
26.6
—
—
—
—
—
—
139.7
0.0
0.1
2.5
3.5
2.3
24.0
438.5
0.1
0.6
9.6
10.0
10.4
72.7
>999
0.2
0.9
0.3
1.0
—
2.0
0.6 (0.97)
2.5 (0.97)
—
N.A.
—
a
13.3
12.4
14.3
91.2
>999
14.2
16.7
17.9
109.7
>999
—
—
31.3 (0.94)
33.8 (0.98)
37.5 (0.96)
299 (0.94)
4499 (0.94)
a
—
a
—
249.0
123.7
16.3
254.2
5
4a
0.3
4.0
a
—
aSubstrate not completely soluble.
described may prove useful for further biochemical and
basic studies in the search for new and more potent
esterase inhibitors as tools in new approaches to pest
control.
3H, CH3) ppm. 19F NMR (282 MHz), d: ꢁ107.49 (t,
J=17.5 Hz) ppm. 13C NMR (75 MHz), d: 199.1 (t,
J=32 Hz, CF2CO), 129.8 (C¼C), 118.1 (t, J=250 Hz,
CF2), 32.3 (t, J=22.8 Hz, CH2CF2), 31.9, 29.7, 29.5,
29.4, 29.3, 29.26, 29.24, 27.1, 26.9, 24.1, 22.3, 21.2 (t,
J=4.3 Hz, CH2CF2), 13.9 (CH3) ppm. MS (EI), m/z
(%): 316 (M+, 0.6), 97 (33), 83 (50), 69 (73), 56 (62), 55
(100), 41 (98). Elem. Anal.: calcd for C19H34OF2: C,
72.11; H, 10.82; F, 12.00. Found: C, 72.34; H, 10.93; F,
12.47.
Experimental
Chemicals
The major components of the pheromone of SL and
SN, (Z,E)-9,11-tetradecadienyl acetate and (Z)-11-hex-
adecenyl acetate, respectively, were purchased from
Sigma (St. Louis, MO, USA) and found to be ꢄ98%
pure by GC analysis. Compound 5 (OTFP) was pre-
pared by alkylation of 1-octanethiol with 3-bromo-
1,1,1-trifluoropropan-2-one in CH2Cl2 using Na2CO3 as
base.17 (Z)-11-hexadecenyl trifluoromethyl ketone (6)
and (E,Z)-9,11-tetradecadienyl trifluoromethyl ketone
(7) were prepared by trans-metallation reaction of the
corresponding iodinated precursors49 with tert-BuLi
followed by treatment with ethyl trifluoroacetate.50
Phenoxyacetyl-Ala-TFMK 8 and phenoxyacetyl-Phe-
TFMK 9 had been prepared as inhibitors of metallo-b-
lactamases51 and were kindly supplied by Prof. C. J.
Schofield.
3,3-Difluorododecan-2-one (2a). Yield 76%. IR (film), n:
2958, 2927, 2856, 1747, 1467, 1361, 1207, 1083, 997,
1
806, 722 cmꢁ1. H NMR (300 MHz), d: 2.29 (t, J=1.5
Hz, 3H, CH3), 1.93 (m, 2H, CH2CF2), 1.23 (bs, 14H,
7CH2), 0.85 (t, J=6.9 Hz, 3H, CH3) ppm. 19F NMR
(282 MHz), d: ꢁ107.52 (t, J=17.2 Hz) ppm. 13C NMR
(75 MHz), d: 199.08 (t, J=32.5 Hz, CO), 118.16 (t,
J=249.6 Hz CF2), 32.68, 32.38, 32.07, 31.83, 29.35,
29.28, 29.25, 29.22, 22.64, 21.25 (t, J=4.3 Hz, CH2CF2),
14.06 (CH3) ppm. MS (EI), m/z (%): 220 (M+,0.2%),
149 (1.5), 95(1.5), 94 (2), 57(8), 55(9), 43 (100). Elem.
Anal.: calcd for C12H22OF2: C, 65.42; H, 10.06; F,
17.25. Found: C, 65.55, H: 9.99, F,17.20.
3,3-Difluoroheptadecan-2-one (2b). (44% yield) IR
(film), n: 2925, 2956, 2854, 1747, 1427, 1467, 721 cmꢁ1
.
General procedure for the synthesis of difluoromethyl ke-
tones 2a–2f: (Z)-3,3-dfluoro-14-nonadecen-2-one (2c).
Into a previously-flamed round-bottomed flask was
introduced under Ar a soln of ethyl (Z)-2,2-difluoro-13-
octadecenoate (21 mg, 0.06 mmol) in 1 mL of anhyd
THF. The solution was cooled to ꢁ78 ꢂC and then 77
mL of a 1.17 M soln of methyllithium in ether (0.09
mmol) were added dropwise. After stirring for 3 h 30
min at this temperature, the reaction was quenched by
adding 1 mL of NH4Cl satd soln. The solvent was
stripped off and the organic material extracted with
hexane (4ꢀ10 mL). The organic phases were washed
with NaHCO3 satd soln, brine and dried (MgSO4).
Evaporation of the solvent left a residue which was
purified by column chromatography on silica gel 60 A
(SDS, 35–70 mm) eluting with hexane/diethyl ether mix-
tures to give 16 mg (84%) of ketone 2c. IR (film), n:
2954, 2925, 2854, 1747, 1433, 1361, 1207, 1068, 968
1H NMR (300 MHz), d: 2.28 (t, J=1.5 Hz, 3H, CH3),
1.96 (m, 2H, CH2CF2), 1.23 (bs, 24H, 12CH2), 0.85 (t,
J=6.9 Hz, 3H, CH3) ppm. 19F NMR (282 MHz), d:
ꢁ107.50 (t, J=18.6 Hz) ppm. 13C NMR (75 MHz), d:
198.9 (t, J=32.2 Hz, CO), 118.1 (t, J=249.6 Hz, CF2),
32.37, 31.92, 29.68, 29.65, 29.62, 29.57, 29.40, 29.35,
29.29, 29.25, 23.99, 22.68, 21.2 (t, J=4.3 Hz, CH2CF2),
14.06 (CH3) ppm. MS (EI), m/z (%): 290 (M+, 0.5), 163
(1.5), 150 (1), 85 (5), 71 (9), 57 (15), 43 (100).
(Z)-3,3-Difluoro-15-eicosen-2-one (2d). (44% yield) IR
(film), n: 3004, 2956, 2925, 2856, 1747, 1465, 1074, 910,
734 cmꢁ1
.
1H NMR (300 MHz), d: 5.32 (2dt,
J1=J2=4.5 Hz, 2H, CH=CH), 2.29 (t, J=1.8 Hz, 3H,
C(O)CH3), 2.0 (m, 6H, CH2CF2, 2CH2CH=CH), 1.24
(bs, 22H, 11CH2), 0.87 (t, J=6.9 Hz, 3H, CH3) ppm.
19F NMR (282 MHz), d: ꢁ107.49 (t, J=17.2 Hz) ppm.
13C NMR (50 MHz), d: 199.1 (t, J=33.5 Hz, C¼O),
129.8 (CH¼CH), 118.1 (t, J=250 Hz, CF2), 32.35,
31.96, 29.76, 29.55, 29.39, 29.28, 27.19, 26.90, 24.10,
22.34, 21.23 (t, J=4.6 Hz, CH2CF2), 13.99 (CH3) ppm.
MS (EI), m/z (%): 330 (M+, 3), 312 (3), 287 (3), 185 (3),
1
cmꢁ1. H NMR (300 MHz), d: 5.32 (m, 2H, CH¼CH),
2.29 (t, J=1.5 Hz, 3H, CH3CO), 2.02–1.85 (c, 6H,
CH2CF2,
CH2CH2CF2), 1.23 (bs, 18H, 9CH2), 0.87 (t, J=6.6 Hz,
CH2CH¼CHCH2),
1.44
(m,
2H,

1052
C. Quero et al. / Bioorg. Med. Chem. 11 (2003) 1047–1055
180 (2), 97 (23), 83 (32), 69 (41), 55 (70), 43 (100). Elem.
Anal.: calcd for C20H36F2O: C: 72.68; H: 10.97; F:
11.49. Found: C: 72.27; H: 11.13.
room temperature for 1 h, again cooled to 0 ꢂC and
quenched with 20 mL of water. After reaching room
temperature, 1 mL of water and 2 mL of 0.5 N HCl
were sequentially added. The organic material was
extracted with ether (4ꢀ10 mL) and washed with brine
and dried (MgSO4). Evaporation of the solvent left a
residue which was purified by column chromatoghraphy
on silica gel eluting with hexane/diethyl ether mixtures
to furnish alcohol 3c (53 mg, 96%). Mp 47–50 ꢂC. IR
(Z)-1,1-Difluoro-13-octadecenyl phenyl ketone (2e).
Yield 47%. IR (film), n: 3002, 2925, 2854, 1703, 1598,
1
1450, 1178, 713 cmꢁ1. H NMR (300 MHz), d: 8.07 (m,
2H, arom.), 7.61 (m, 1H, arom.), 7.47 (m, 2H, arom.),
5.33 (2dt, J1=J2=4.5 Hz, 2H, CH¼CH), 2.01 (m, 6H,
CH2CF2, 2CH2CH¼CH), 1.25 (bs, 22H,–CH2–), 0.88
(t, J=6.8 Hz, 3H, CH3) ppm. 19F NMR (282 MHz), d:
ꢁ100.74 (t, J=17.7 Hz) ppm. 13C NMR (50 MHz), d:
189.5 (t, J=31 Hz, C¼O), 134.1, 130.0, 128.5, 127.1
(C6H5), 129.84, 129.80 (CH¼CH), 119.8 (t, J=251 Hz,
CF2), 31.96, 29.75, 29.56, 29.52, 29.41, 29.31, 29.20,
27.18, 26.90, 22.33, 21.3 (t, J=4.3 Hz, CH2CF2), 13.97
(CH3) ppm. MS (EI), m/z (%): 392 (M+, 1), 374 (3), 219
(2), 206 (4), 156 (3), 105 (100), 77 (15), 55 (11).
(film), n: 3359, 2954, 2925, 2850, 1465, 908, 734 cmꢁ1
.
1H NMR (300 MHz), d: 5.32 (m, 2H, CH¼CH), 3.7 (dt,
J1=13 Hz, J2=6.9 Hz, 2H, CH2OH), 2.0–1.8 (c, 7H,
CH2CF2, CH2CH¼CHCH2, OH), 1.48 (m, 2H,
CH2CH2CF2), 1.24 (bs, 18H, 9CH2), 0.87 (t, J=7.2 Hz,
3H, CH3) ppm. 19F NMR (282 MHz), d: ꢁ109.12 (tt,
J1=17.6 Hz, J2=13 Hz) ppm. 13C NMR (75 MHz), d:
129.8 (C¼C), 123.3 (t, J=240 Hz, CF2), 64.0 (t, J=32
Hz, CF2CH2O), 32.2 (t, J=23.7 Hz, CH2CF2), 31.9,
29.7, 29.5, 29.48, 29.43, 29.36, 29.34, 29.2, 27.1, 26.8,
22.3, 21.7 (t, J=4.5 Hz), 13.9 (CH3) ppm. MS (EI), m/z
(%): 304 (M+, 1.8), 83 (50), 69 (60), 55 (100), 43 (33).
Elem. Anal.: calcd for C18H34OF2: C, 71.01; H, 11.25;
F, 12.48. Found: C, 71.36; H, 11.33; F, 12.31.
Ethyl 2,2-difluoro-2-octylthioacetate (1f). In a three-
necked dry round-bottomed flask, equipped with mag-
netic stirrer and argon inlet, was placed sodium hydride
(37 mg, 0.76 mmol) as a 55% dispersion in oil, which
was washed with pentane. To the dry hydride was added
DMF (0.6 mL) and 1-octanethiol (146 mg, 1 mmol) in
DMF (1 mL) and the slurry was stirred until evolution
of hydrogen ceased. Then, ethyl bromodifluoroacetate
(156 mg, 0.76 mmol) was added and the reaction mix-
ture stirred for 1 h. After the usual work up, the result-
ing reaction mixture was chromatographed on silica gel
60 A (SDS, 35–70 mm) eluting with hexane/ether mix-
tures to give the expected difluorinated ester 1f (134 mg,
65%). IR n: 2958, 2929, 2856, 1770, 1467, 1299, 1101,
987, 723 cmꢁ1. 1H NMR (CDCl3) d: 4.32 (q, J=7.2 Hz,
2H, CH2CH3), 2.83 (t, J=7.5 Hz, 2H, CH2S), 1.63(qt,
J=7.80 Hz, 2H, CH2), 1.33 (t, J=7.2 Hz, 3H, CH2CH3),
2,2-Difluoroundecanol (3a). Yield 75%. IR (film), n:
3278, 2952, 2923, 2854, 1465, 1201, 1082, 908, 736 cmꢁ1
.
1H NMR (300 MHz), d: 3.73 (dt, J1=12.9 Hz, J2=6.3
Hz, 2H, CH2OH), 1.85 (m, 2H, CH2CF2), 1.24 (bs,14H,
–CH2–), 0.85 (t, J=6.6 Hz, 3H, CH3) ppm. 19F NMR
(282 MHz), d: ꢁ109.14 (tt, J1=17.2 Hz, J2=12.4 Hz)
ppm. 13C NMR (75 MHz), d: 123.3 (t, J=240 Hz, CF2),
64.0 (t, J=31 Hz, CH2OH), 33.2 (t, J=24 Hz,
CH2CF2), 31.84, 29.36, 29.25, 22.65, 21.7 (t, J=4.4 Hz,
CH2CH2CF2), 14.06 (CH3) ppm. MS (EI), m/z (%): 208
(M+, <1), 141 (1.6), 110 (5), 99 (13), 85 (43), 69 (27), 55
(45), 43 (100).
1.24 (bs, 14H, 7CH2), 0.84 (t, J=6.30 Hz, 3H, CH3). 19
F
NMR (CDCl3) d: ꢁ83.35 (s). 13C NMR (CDCl3) d:
161.88 (t, J=32.88 Hz, CO), 120.68 (t, J=285.24 Hz,
CF2), 63.47, 31.70, 29.54, 29.04, 28.92, 28.67 (t, J=2.94
Hz, CH2S), 28.61, 22.57, 13.99, 13.80. Elem. Anal.: calcd
for C12H22F2O2S: C: 53.70; H: 8.26; F: 14.16; S: 11.95.
Found: C: 53.68; H: 8.42; F: 14.36; S: 11.77.
2,2-Difluorohexadecanol (3b). (65% yield) IR (film), n:
3278, 2954, 2921, 2848, 1462, 1209, 1024, 908, 734 cmꢁ1
.
1H NMR (300 MHz), d: 3.70 (dt, J1=12.9 Hz, J2=4.2
Hz, 2H, CH2OH), 1.99 (m, 2H, CH2CF2), 1.23 (bs,
24H, 12CH2), 0.85 (t, J=6.8 Hz, 3H, CH3) ppm. 19F
NMR (282 MHz), d: ꢁ109.13 (tt, J1=17.2 Hz, J2=12.7
Hz) ppm. 13C NMR (75 MHz), d: 123.3 (t, J=240 Hz,
CF2), 64.0 (t, J=32 Hz, CH2OH), 32.2 (t, J=24 Hz,
CH2CF2), 31.91, 29.68, 29.66, 29.64, 29.60, 29.45, 29.37,
29.35, 22.68, 21.7 (t, J=4.6 Hz, CH2CH2CF2), 14.10
(CH3) ppm. MS (EI), m/z (%): 278 (M+, <1), 258 (0.2),
240 (0.6), 212 (0.3), 85 (72), 71 (51), 57 (91), 43 (100).
3,3-Difluoro-3-octylthiopropan-2-one (2f). (95% yield)
IR (film), n: 2956, 2927, 2856, 1747, 1467, 1087, 904
1
cmꢁ1. H NMR (300 MHz), d: 2.74 (t, J=7.2 Hz, 2H,
CH2S), 2.35 (t, J=1.5 Hz, 3H, COCH3), 1.61(qt, J=7.8
Hz, 2H, CH2), 1.28 (bs, 12H, 6CH2), 0.85(t, J=6.60 Hz,
3H, CH3). 19F NMR (282 MHz), d: ꢁ87.62 (s). 13C
NMR (50 MHz), d: 193.79 (t, J=30.92 Hz, CO), 123.16
(t, J=288.11 Hz, CF2), 31.72, 29.53, 29.06, 28.93, 28.63,
28.53 (t, J=3.24 Hz, CH2S), 23.35, 22.59, 14.03. Elem.
Anal.: calcd for C11H20F2OS: C, 55.43; H, 8.46; S,
13.45; F, 15.94. Found: C, 55.83; H, 8.22; S, 13.69; F,
15.96.
(Z)-2,2-Difluoro-14-nonadecen-1-ol (3d). (69% yield) IR
(film), n: 3400, 3011, 2954, 2925, 2854, 1465, 1070, 908,
722 cmꢁ1 1H NMR (300 MHz), d: 5.32 (m, 2H,
.
CH¼CH), 3.71 (dt, J1=12.6 Hz, J2=4.8 Hz, 2H,
CH2OH), 1.99 (m, 6H, CH2CF2, 2CH2CH¼CH), 1.24
(bs, 22H, 11CH2), 0.87 (t, J=7.2 Hz, 3H, CH3) ppm.
19F NMR (282 MHz), d: ꢁ109.14 (tt, J1=17.2 Hz,
J2=12.7 Hz) ppm. 13C NMR (75 MHz), d: 129.87,
129.83 (CH¼CH), 123.33 (t, J=241 Hz, CF2), 64.07 (t,
J=32 Hz, CH2OH), 33,28 (t, J=24 Hz, CH2CF2),
31.96, 29.75, 29.58, 29.52, 29.44, 29.37, 29.34, 29.29,
27.18, 26.90, 22.33, 21.77 (t, J=4.6 Hz, CH2CH2CF2),
13.98 (CH3) ppm.
General procedure for the synthesis of difluoroalcohols
3a-3d: (Z)-2,2-Difluoro-13-octadecenol (3c). To a mix-
ture of lithium aluminum hydride (21 mg, 0.52 mmol) in
3 mL of anhyd ether was added, at 0 ꢂC under Ar, ethyl
(Z)-2,2-difluoro-13-octadecenoate (63 mg, 0.18 mmol)
in 1 mL of anhyd ether. The mixture was stirred at

C. Quero et al. / Bioorg. Med. Chem. 11 (2003) 1047–1055
1053
General procedure for the synthesis of difluoroaldehydes
4a–4d: (Z)-2,2-difluoro-13-octadecenal (4c). To a cold
solution (ꢁ60 ꢂC) of oxalyl chloride (25 mL, 0.295
mmol) in 0.2 mL of anhyd methylene chloride were
added, under Ar and with vigorous stirring, anhyd
DMSO (41 mL, 0.591 mmol) and anhyd methylene
chloride (0.3 mL). The reaction mixture was stirred for
2 min and then a soln of (Z)-2,2-difluoro-13-octadece-
nol (3c) (30 mg, 0.098 mmol) in 0.2 mL of anhyd meth-
ylene chloride was added. The mixture was stirred for
30 min at ꢁ60 ꢂC, then anhyd triethylamine (165 mL,
1.18 mmol) was added and the resulting mixture stirred
at 0 ꢂC for 30 min. After dilution with methylene chlo-
ride (5 mL), the reaction was quenched by addition of
1N HCl (2 mL). The aqueous layer was extracted with
methylene chloride (4ꢀ5 mL), washed with NaHCO3
sat. soln, brine and dried (MgSO4). Removal of the sol-
vent left a residue, which was purified by column chro-
matography on silica gel eluting with hexane/ether
mixtures. The expected (Z)-2,2-difluoro-13-octadecenal
(4c) was obtained (17 mg, 58%) as a mixture of the keto
and hydrate forms in a ratio of 1:4. IR (film), n: 3350,
3200, 3004, 2954, 2921, 2852, 1720, 1654, 1465, 1060,
(75 MHz), d: 190.61 (t, J=36 Hz, CHO), 121.38 (t,
J=244 Hz, CF2), 89.72 (t, J=31 Hz, CH(OH)2), 31.92,
29.71, 29.64, 29.60, 29.55, 29.45, 29.27, 29.20, 22.68,
20.85 (t, J=4.5 Hz, CH2CF2), 14.11 (CH3) ppm. MS
(EI), m/z (%): 121(8), 97(20), 85(24), 71(46), 57(92), 43
(100).
(Z)-2,2-Difluoro-14-nonadecenal (4d). (42% yield) IR
(film), n: 3361, 3230, 3011, 2954, 2920, 2920, 2850, 1720,
1
1656, 1471, 1076, 736 cmꢁ1. H NMR (300 MHz), d:
9.48 (s, 1H, CHO), 5.32 (m, 2H, CH¼CH), 4.97 (t,
J=7.0 Hz, 1H, CH(OH)2), 3.14 (bs, 2H, 2OH), 1.99 (m,
6H, CH2CF2, 2CH2CH¼CH), 1.24 (bs, 22H, 11CH2),
0.87 (t, 7.2 Hz, 3H, CH3) ppm. 19F NMR (282 MHz), d:
ꢁ111.21 (t, J=17.5 Hz, CF2CHO), ꢁ118.33 (dt,
J1=18.3 Hz, J2=6.7 Hz, 2F, CF2CH(OH)2) ppm. 13C
NMR (75 MHz), d: 190.6 (t, J=39 Hz, CHO), 129.87,
129.85 (CH¼CH), 121,3 (t, J=333 Hz, CF2), 89.6 (t,
J=31.3 Hz, CF2CH(OH)2), 31.96, 29.75, 29.69, 29.57,
29.52, 29.44, 29.39, 29.35, 29.33, 29.27, 27.18, 26.90,
22.34, 21.11 (t, J=4.5 Hz, CH2CF2), 13.9 (CH3) ppm.
MS (EI), m/z (%): 316 (M+, 24), 296 (1), 288 (1), 260
(2), 203 (8), 189 (7), 125 (8), 111 (16), 97 (29), 83 (32), 69
(41), 55 (100).
1
734 cmꢁ1. H NMR (300 MHz), d: 9.48 (t, J=1.2 Hz,
1H, CHO), 5.32 (m, 2H, CH¼CH), 4.97 (t, J=6.9 Hz,
1H,
CH(OH)2),
2.0–1.8
(c,
6H,
CH2CF2,
Determination of hydration constants
CH2CH¼CHCH2), 1.24 (bs, 20H, 10CH2), 0.87 (t,
J=7.2 Hz, 3H, CH3) ppm. 19F NMR (282 MHz), d:
ꢁ111.2 (t, J=17.4 Hz, CF2CHO), ꢁ118.3 (dt, J1=18
Hz, J2=6.4 Hz, CH2CF2CH(OH)2) ppm. 13C NMR
(75 MHz), d: 190.6 (t, J=39 Hz, CHO), 129.8 (C¼C),
121.3 (t, J=333 Hz, CF2), 89.6 (t, J=31 Hz,
CF2CH(OH)2), 31.9, 29.7, 29.54, 29.50, 29.46, 29.43,
29.38, 29.34, 29.27, 29.25, 27.1, 26.8, 22.3, 21.1 (t,
J=4.2 Hz), 13.9 (CH3) ppm. MS (EI), m/z (%): 302
(M+, 1.6), 97 (50), 83 (59), 69 (82), 57 (57), 56 (77), 55
(100), 41 (57). Exact Mass. calcd for C18H32OF2:
302.2421. Found: 302.2420.
Compounds 2a, 2f, 4a, 5, 6, 11 and 12 were dissolved in
water-acetonitrile mixtures, left for 2.5 h at a constant
20 ꢂC temperature, and the 19F NMR spectrum of the
mixtures was recorded. The apparent Khyd value was
determined by simply integrating the area of the signal
for the hydrate and keto forms and using these values to
obtain the hydrate/keto ratio.48 The actual Khyd in pure
water was obtained by extrapolation of the Khyd app
from a series of known mole fraction water (ꢀH O 0.025,
0.05, 0.1, 0.24, 0.37, 0.5 and 0.75). The experiments were
2
carried out in duplicate. The apparent Khyd measured is
a function of ꢀH O and is related to the actual Khyd by
2
2,2-Difluoroundecanal (4a). Yield 66%. IR (film), n:
3300, 3282, 2956, 2918, 2850, 1714, 1467, 1076, 1226,
the activity of water (aH O) in the solvent mixture. In the
ideal case, the aH O equals to ꢀH O and therefore
2
2
2
1
1049, 736 cmꢁ1. H NMR (300 MHz), d: 9.48 (bs, 1H,
CHO), 4.98 (t, J=6.6 Hz, 1H, CH(OH)2), 3.10 (bs, 2H,
CH(OH)2), 1,90 (m, 2H, CH2CF2), 1.24 (bs, 24H, –
CH2–), 0.85 (t, J=6.6 Hz, 3H, CH3) ppm. 19F NMR
(282 MHz), d: ꢁ111.22 (t, J=17.7 Hz, CF2CHO),
ꢁ118.34 (dt, J1=17.7 Hz, J2=7.0 Hz, 2F,
CF2CH(OH)2) ppm. 13C NMR (75 MHz), d: 190.63 (t,
J=39 Hz, CHO), 121.4 (t, J=244 Hz, CF2), 89.69 (t,
J=31 Hz, CH(OH)2), 31.85, 30.32, 29.41, 29.33, 29.25,
29.21, 22.66, 21.06 (t, J=4.3 Hz, CH2CF2), 14.08 (CH3)
ppm. MS (EI), m/z (%): 206 (M+<1), 186 (1.5), 177
(1.6), 160 (2), 146(6), 135(39), 121 (28), 71 (27), 57 (50),
43(100).
½hydrateꢀequiv
½ketoꢀequiv
Khyd app ¼ Khyd Áꢀ
¼
Áꢀ
H2O
H2O
Insects
SL were reared in our laboratory on a slightly modified
diet as compared to that reported previously.52 Pupae
were sexed and adults maintained at 25ꢃ1 ꢂC on a 16
h:8 h L/D cycle until use. SN pupae proceeded from a
culture maintained at the laboratories of the University
of Lleida-IRTA (Lleida, Spain) following a modified
diet from Poitout and Bues.53 After reception, pupae
were placed in a reverse photoperiod until emergence
under similar conditions than SL.
2,2-Difluorohexadecanal (4b). (35% yield) IR (film), n:
3100, 2956, 2918, 2850, 1714, 1471, 1076 cmꢁ1 1H
.
NMR (300 MHz), d: 9.48 (bs, 1H, CHO), 4.98 (t, J=6.6
Hz, 1H, CH(OH)2), 3.02 (bs, 2H, CH(OH)2), 1.90 (m,
2H, CH2CF2), 1.24 (bs, 24H, 12CH2), 0.85 (t, J=6.3
Hz, 3H, CH3) ppm. 19F NMR (282 MHz), d: ꢁ111.21 (t,
J=18.0 Hz, CF2CHO), ꢁ118.36 (dt, J1=18.6 Hz,
J2=7.0 Hz, 2F, CF2CH(OH)2) ppm. 13C NMR
Inhibition studies
Males of 1–2 days old were anesthetized with CO2 and
their antennae removed. These were immediately frozen

1054
C. Quero et al. / Bioorg. Med. Chem. 11 (2003) 1047–1055
and kept at ꢁ80 ꢂC until use. Antennal esterase pre-
parations of SL were obtained by homogenizing batches
of frozen antennae in 20 mM Tris–HCl buffer (pH=7.4)
on a variable speed Heidolph ZZR-2000 mixer for 5 min
in an ice bath. The same protocol was used for SN
esterase at pH=8.0. Preliminary experiments had
shown optimum enzymatic activity of extracts of the
two insects at these pHs. The homogenate was sonicated
at 40 w for 10 s and centrifuged at 14,000g for 2 min at
4 ꢂC to remove the cuticular debris. The supernatant
was adjusted with the required volume of the corre-
sponding buffer solutions so that an aliquot of 100 mL
was the equivalent of four antennae. For each assay,
this aliquot was added to borosilicate tubes, previously
treated with 1-decanol to prevent adsorption of the
substrate to the glass surface.54 The inhibitor solutions
were prepared by concentration to dryness of an aliquot
of the mother solution in hexane or ethyl acetate, fol-
lowed by addition of ethanol so that 2 mL of the soln in
100 mL of the antennal preparation gave the desired
final concentration of the inhibitor (1–800 mM). The
inhibition tests were conducted by preincubation of 2
mL of the inhibitor solutions with one aliquot of the
extract for 10 min at 28 ꢂC for SL and 32 ꢂC for SN.
Then, the major component of the pheromone (2
mmoles for that of SL and 0.35 mmoles for that of SN)
in 2 mL of ethanol was added to the mixture and incu-
bated for 60 min more at the preincubation temperature
for each insect. Incubation was stopped by addition of
160 mL of hexane. After vortexing for 1 min, the organic
phase was separated and stored at ꢁ80 ꢂC. Just before
analysis, the solution was concentrated to a small
volume (5–10 mL) and injected in GC. The extent of
hydrolysis was calculated by the relative amount of the
pheromone-derived alcohols with regard to the parent
for SL and 32 ꢂC for SN and the process stopped by
addition of 160 mL of hexane. A similar protocol was
applied, and at least six replicates for each concentra-
tion were conducted.
Acknowledgements
This work was supported by grants from CICYT (AGF
2000-1695-C02-01 and 2FD97-1333) and Comissionat
per a Universitats i Recerca (1999SGR 00187). We also
thank J. Casas and A. Cortes (University of Barcelona)
for useful comments to the manuscript, C. J. Schofield
(The Dyson Perrins Laboratory, Oxford) for com-
pounds 8 and 9 and MEC for a contract to C.Q. We are
also indebted to SEDQ S.A. for providing free samples
as starting materials
References and Notes
1. Taylor, P.; Radic, Z. Annu. Rev. Pharmacol. Toxicol. 1994,
34, 281.
2. Trainor, D. A. Trends Pharm. Sci. 1987, 8, 303.
3. Johnson, M. K. J. Neurochem. 1974, 23, 785.
4. Gelb, M. H.; Svaren, J. P.; Abeles, R. H. Biochemistry
1985, 24, 1813.
5. Parisi, M. F.; Abeles, R. H. Biochemistry 1992, 31, 9429.
6. Angliker, H.; Wikstrom, P.; Rauber, P.; Stone, S.; Shaw, E.
Biochem. J. 1988, 256, 481.
7. Abdel-Aal, Y. A. I.; Hammock, B. D. Insect Biochem.
1985, 15, 111.
8. Ashour, M.-B. A.; Hammock, B. D. Biochem. Pharm 1987,
36, 1869.
9. Street, I. P.; Lin, H. K.; Laliberte, F.; Ghomashchi, F.;
Wang, Z.; Perrier, H.; Tremblay, N. M.; Huang, Z.; Weech,
P. K.; Gelb, M. H. Biochemistry 1993, 32, 5935.
10. Koutek, B.; Prestwich, G. D.; Howlett, A. C.; Chin, S. A.;
Salehani, D.; Akhavan, N.; Deutsch, D. G. J. Biol. Chem.
1994, 269, 22937.
11. Patel, D. V.; Rielly-Gauvin, K.; Ryono, D. E. Tetrahedron
Lett. 1988, 29, 4665.
12. Linderman, R. J.; Leazer, J.; Roe, R. M.; Venkatesh, K.;
Selinsky, B. S.; London, R. E. Pest. Biochem. Physiol 1988, 31,
187.
13. Rosell, G.; Herrero, S.; Guerrero, A. Biochem. Biophys.
Res. Comm. 1996, 226, 287.
14. Takahashi, L. H.; Radhakrishnan, R.; Rosenfield, R. E.,
Jr.; Meyer, E. F., Jr.; Trainor, D. A. J. Am. Chem. Soc. 1989,
111, 3368.
15. Duran, I.; Parrilla, A.; Feixas, J.; Guerrero, A. Bioorg.
Med. Chem. Lett. 1993, 3, 2593.
16. Parrilla, A.; Guerrero, A. Chem. Senses 1994, 19, 1.
17. Parrilla, A.; Villuendas, I.; Guerrero, A. Bioorg. Med.
Chem. 1994, 2, 243.
acetates on
a GC fused silica capillary column
(HP-FFAP 25 mꢀ0.25 mm ID). For SL the GC condi-
tions were injection at 80 ꢂC hold for 1 min, followed by
temperature program of 10 ꢂC/min up to 230 ꢂC and
held at this temperature for 10 min. For SN the condi-
tions were injection at 100 ꢂC hold for 1 min, followed
by temperature program of 5 ꢂC/min up to 200 ꢂC and
then at 10 ꢂC/min up to 230 ꢂC and held at this tem-
perature for an additional 10 min. Inhibitory potencies
of the chemicals were determined by the relative
decrease of substrate hydrolysis in the presence or not
of inhibitor. IC50 of each compound was calculated by
least squares regression analyses in at least triplicate
experiments considering 5 different concentrations of
the inhibitor and 2–3 controls for each replicate. An
inhibitor was considered non active when no inhibition
activity was found at 300 mM concentration.
18. Renou, M.; Lucas, P.; Malo, E.; Quero, C.; Guerrero, A.
Chem. Senses 1997, 22, 407.
19. Bau, J.; Martınez, D.; Renou, M.; Guerrero, A. Chem.
Senses 1999, 24, 473.
20. Riba, M.; Sans, A.; Bau, P.; Grolleau, G.; Renou, M.;
Guerrero, A. J. Chem. Ecol 2001, 27, 1879.
21. Vogt, R. G.; Riddiford, L. M.; Prestwich, G. D. Proc.
Natl. Acad. Sci. U.S.A. 1985, 82, 8827.
22. Prestwich, G. D.; Streinz, L. J. Chem. Ecol 1988, 14, 1003.
23. Imperiali, B.; Abeles, R. H. Biochemistry 1986, 25, 3760.
24. Schirlin, D.; Baltzer, S.; Altenburger, J. M.; Tarnus, C.;
Remy, J. M. Tetrahedron 1996, 52, 305.
Determination of kinetic parameters
For the determination of Km and Vmax of the enzyme of
each species, four antennal equivalents in 100 mL of 20
mM Tris–HCl buffer (pH=7.4 for SL or pH=8.0 for
SN) were added to borosilicate tubes, previously treated
with 1-decanol as above. Then, 2 mL of a mother solu-
tion of the substrates in ethanol were added to the tubes
to get a final concentration of the substrates from 1.5 to
50 mM. The mixture was incubated for 60 min at 28 ꢂC

C. Quero et al. / Bioorg. Med. Chem. 11 (2003) 1047–1055
1055
25. Ding, Y.-S.; Prestwich, G. D. J. Chem. Ecol. 1988, 14,
2033.
26. Tozer, M. J.; Herpin, T. F. Tetrahedron 1996, 52, 8619.
27. Yamana, M.; Ishihara, T.; Ando, T. Tetrahedron Lett.
1983, 24, 507.
41. Hammock, B. D.; Abdel-Aal, Y. A. I.; Mullin, C. A.;
Hanzlik, T. N.; Roe, R. M. Pest. Biochem. Physiol 1984, 22,
209.
42. Filizola, M.; Rosell, G.; Guerrero, A.; Perez, J. J. Quant.
Struct.-Act. Relat 1998, 17, 205.
28. Lang, R. W.; Schaub, B. Tetrahedron Lett. 1988, 29, 2943.
29. Burkholder, C.; Dolbier, W. R., Jr.; Medebielle, M.;
Ndedi, A. Tetrahedron Lett. 1998, 39, 8853.
30. Jimenez, O.; Bosch, M. P.; Guerrero, A. Synthesis 2000,
1917.
43. Rich, D. H.; Salituro, F. G.; Holladay, M. W.; Schmidt,
P. S. Conformationally Directed Drug Design; American Che-
mical Society: Washington, DC, 1984; Vol. 251.
44. Nesbitt, B. F.; Bevor, P. S.; Hall, D. R.; Lester, R.; Poppi,
R. G. Nature New Biol. 1973, 244, 208.
31. Qiu, Z.-M.; Burton, D. J. J. Org. Chem. 1995, 60, 5570.
32. Metcalf, B. W.; Jarvi, E. T.; Burkhart, J. P. Tetrahedron
Lett. 1985, 26, 2861.
33. McCarthy, P. A.; Wint, L. T.; Diaz, C. L. Bioorg. Med.
Chem. Lett. 1992, 2, 119.
45. Mazomenos, B. J. Chem. Ecol 1989, 15, 1241.
`
46. Navarro, I.; Mas, E.; Fabrias, G.; Camps, F. Bioorg. Med.
Chem. 1997, 5, 1267.
47. Liang, T. C.; Abeles, R. H. Biochemistry 1987, 26,
7603.
34. Dreyer, G. B.; Metcalf, B. W. Tetrahedron Lett. 1988, 29,
6885.
48. Linderman, R. J.; Jamois, E. A.; Roe, R. M. Rev. Pestic.
Toxicol 1991, 1, 261.
35. Erni, B.; Khorana, H. G. J. Am. Chem. Soc. 1980, 102, 3888.
36. Einhorn, J.; Einhorn, C.; Ratajczak, H.; Pierre, J. J. Org.
Chem. 1996, 61, 7452.
49. Camps, F.; Gasol, V.; Guerrero, A. Synthesis 1987, 511.
50. Villuendas, I.; Parrilla, A.; Guerrero, A. Tetrahedron
1994, 50, 12673.
37. Dess, D. B.; Marin, J. C. J. Org. Chem. 1983, 48.
38. Mancuso, A. J.; Huang, S.-L.; Swern, D. J. Org. Chem.
1978, 43, 2480.
39. Allen, K. N.; Abeles, R. H. Biochemistry 1989, 28, 8466.
40. Hudlicky, M. Chemistry of Organic Fluorine Compounds;
Ellis Horwood: New York, 1992.
51. Walter, M. W.; Adlington, R. M.; Baldwin, J. E.; Scho-
field, C. J. Tetrahedron 1997, 53, 7275.
52. Poitout, S.; Bues, R.; Le Rumeur, C. Ent. exp. Appl 1972,
15, 341.
53. Poitout, S.; Bues, R. Ann. Zool. Ecol. Anim 1974, 6, 431.
54. Du, G.; Prestwich, G. D. Biochemistry 1995, 34, 8726.