Synthesis and insecticidal activity of fluorinated 2-(2,6-dichloro-4- trifluoromethylphenyl)-2,4,5,6-tetrahydrocyclopentapyrazoles

Article abstract of DOI:10.1016/j.bmcl.2005.12.012

A number of fluorinated 1-aryl-tetrahydrocyclopentapyrazoles were synthesized and their insecticidal activity was evaluated. Some of the fluorinated compounds had significant insecticidal properties.

Full text of DOI:10.1016/j.bmcl.2005.12.012

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1702–1706
Synthesis and insecticidal activity of fluorinated
2-(2,6-dichloro-4-trifluoromethylphenyl)-2,4,5,6-
tetrahydrocyclopentapyrazoles
Sanath K. Meegalla,a,* Dario Doller,a Ruiping Liu,a DeYou Sha,a YuKai Lee,a
Richard M. Soll,a Nancy Wisnewski,b Gary M. Silverb and Dale Dhanoaa
aJohnson & Johnson Pharmaceutical Research and Development, 665 Stockton Drive, Suite 104, Exton, PA 19341, USA
bHeska Corporation, 1613 Prospect Parkway, Fort Collins, CO 80525, USA
Received 9 September 2005; revised 2 December 2005; accepted 2 December 2005
Available online 28 December 2005
Abstract—A number of fluorinated 1-aryl-tetrahydrocyclopentapyrazoles were synthesized and their insecticidal activity was eval-
uated. Some of the fluorinated compounds had significant insecticidal properties.
Ó 2005 Elsevier Ltd. All rights reserved.
Prior to 1995, a large proportion of market sales in the
area of animal flea-killing agents came from generic,
‘over-the-counter’ drugs. In the late 1990s’ three new
anti-flea products drove market sales to veterinarians.
These products are: ProgramTM (Novartis, 1995),
AdvantageTM (Bayer, 1996), and FrontlineTM (Rhone-
Merieux Merial, 1996).
More specifically, we became interested in 2-(2,6-
dichloro-4-trifluoromethylphenyl)-2,4,5,6-tetrahydrocy-
clopentapyrazoles (5) and 2-(2,6-dichloro-4-trifluorom-
ethylphenyl)-4-methyl-2,4,5,6-tetrahydrocyclopentapy-
razoles (6), which showed good binding affinity for the
GABA receptor in housefly brain preparations (83
and 30 nM, respectively), with 19- and 126-fold selec-
tivity against mammalian GABA receptors, respective-
ly. Although 5 and 6 caused only 30% and 10%
mortality in a housefly contact assay, respectively,
both compounds produced high mortality in three flea
strains tested (Table 1).
The active pharmaceutical ingredient in each of these
three products has a different mechanism of action.
Lufenuron (1), the active ingredient of ProgramTM, is
a chitin synthesis/polymerization inhibitor. Bayer’s
anti-flea agent AdvantageTM contains imidacloprid (2),
which is a nicotinic acetylcholine receptor agonist. The
active ingredient of FrontlineTM is fipronil (3), a phen-
ylpyrazole which acts as a GABA-gated chloride chan-
nel inhibitor which causes hyperactivity in neurons,
leading to eventual death.
Unfortunately, compounds 5 and 6 were ineffective as
anti-flea agents in in vivo efficacy studies in dogs after
either topical (10 mg/kg) or oral (20 mg/kg) administra-
tion. Although dogs orally dosed with compound 5 had
quantifiable levels of compound in plasma for as long as
3 h post-dosing, only trace plasma levels of 5 were
detected after topical dosing over a comparable period
of time. In vivo metabolite identification studies, carried
out by LC/MS/MS analysis of plasma samples from
dogs that had been orally dosed with compound 5, re-
vealed that the test article was extensively hydroxylated
at benzylic positions 4 and 6. In agreement with this
observation, in vitro metabolic stability studies on 5
showed it had a short half-life (t1/2 = 2.5 min) in dog
liver microsomal preparations (unpublished data).
Recently, we described1 the synthesis and structure–in-
secticidal activity relationships of a series of 3,4-fused-
cycloalkyl-1-arylpyrazoles (generic structure 4). These
could be formally considered as rotationally constrained
analogs of fipronil (3).
Keywords: Phenylpyrazole; GABA; Fipronil; Anti-flea; Anti-Tick.
*
Thestructure–insecticidalbindingaffinitystudies(unpub-
lished data) in this series of compounds demonstrated
Corresponding author. Tel.: +1 610 458 8959; e-mail: smeegall@
prdus.jnj.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.12.012

S. K. Meegalla et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1702–1706
1703
Cl
Cl
Cl
O
Cl
X
Y
N
F
O
F
O
CF3
CN
N
Cl
F3C
N
F
Cl
F
N
NO2
F3C
N
N
N
N
N
H
H
SO2CF3
NH2
N
H
Cl
NH2
(2)
(3)
(1)
(4)
Table 1. Insecticidal activity of fused cyclopentylpyrazoles (5) and (6)
1
N
6
F3C
N
5
4
R
3
Cl
5: R = H, 6: R = CH3
Compound
Binding assay IC50 (nM)
Contact assay LC50 (mM) or % mortality at 100 mM4
Fly
Mouse
Selectivity
Fly (%)
S/S fleas
S/A fleas
A/A fleas (%)
5
6
83
30
1600
3800
19
126
30
10
8
10
7
3
100
100
no tolerance for bulky substituents at the C-6 position,
while the C-4 position tolerated the presence of small sub-
stituents, which, in addition, increased the selectivity
against mammalian GABA receptors. Thus, it appeared
that the selective fluorination of the alicyclic portion of
this series of compounds could be a viable option to ad-
dress their metabolic liabilities while retaining insecticidal
activity. Herein we report the results from our efforts on
the synthesis of novel fluorinated 2-(2,6-dichloro-4-triflu-
oromethylphenyl)-2,4,5,6-tetrahydrocyclopentapyrazoles
and their insecticidal properties.
4. Conversion of the ketone to alkene, vicinal bis
hydroxylation, and vicinal bis fluorination with
DAST.
Reduction of ketone 8 with NaBH4 followed by acid
catalyzed dehydration of the resulting alcohol yielded
cyclopentene 20 and its regioisomer (6:1 ratio). Since
these two compounds could not be separated by chro-
matography, they were converted to the corresponding
vicinal diols, which were easily separable.
In order to evaluate the insecticidal activity and selec-
tivity of the test compounds prepared, the following
assays were used. Binding affinity for the housefly
neuronal membrane GABA receptor was measured
by displacing radioligand [3H]EBOB. Selectivity against
the mammalian receptor was determined by binding
affinities to mouse brain GABA receptors. The com-
pounds were also assayed in ‘contact assays’ with
houseflies and two different flea strains as ‘in vivo’
screens. The two flea strains were used to assess
insecticidal activity differences among the genetic vari-
ants of the cyclodiene resistant allele-homozygous
serine (resistant, CO fleas) and homozygous alanine
(sensitive, ARC fleas).3 (Table 2)
The reaction sequence leading to the synthesis of fluori-
nated analogs of compound 5 is shown in Scheme 1. Tet-
rahydrocyclopentylpyrazole 5, synthesized in two steps
from commercially available cylopentanone and 2,6-
dichloro-4-trifluoromethylpyrazole, served as key start-
ing material for the preparation of all analogs studied.
After some experimentation, compound 5 was oxidized
to a mixture of benzylic ketones 7 and 8 in 86% com-
bined yield (10:1 ratio). These could be easily separated
by flash chromatography on silica gel. Introduction of
fluorine atoms on the fused cyclopentane rings of 7
and 8 was carried out following four different synthetic
protocols.2
1. Reduction of the ketone to alcohol or reaction of the
ketone with a Grignard reagent to obtain the corre-
sponding tertiary alcohol, followed by conversion of
the alcohol to fluoride by the action of diethylamino-
sulfurtrifluoride (DAST).
2. Conversion of the ketone to the corresponding silyle-
nol ethers, followed by a-fluorination with
SELECTFLUOR.
3. Direct gem difluorination of the ketone with DAST
or conversion of the ketone to the corresponding
dithioketal, followed by gem difluorination with
PPHF/NIS.
In general, most compounds induced some degree of
mortality in sensitive ARC fleas. The notable excep-
tions are the ketones 10 and 28, which showed poor
activity in contact assays. As is evident from the data,
high activity in housefly binding assays did not neces-
sarily translate into high anti-flea activity. Although
monofluorination at C-6 slightly decreased the LD50
in CO flea contact assay, it caused a 3-fold decrease
in binding affinity in housefly brain preparations. In
contrast, the presence of fluorine at C-4 position in
compound 18 caused a very significant decrease
in both binding affinity and activity in the contact

1704
S. K. Meegalla et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1702–1706
F
F
F
F
N
O
F
F
N
F
F
a 70%
b 64%
Ar
F
F
F
N
N
(19)
N
N
N
N
Ar
Ar
N
Ar
e 23%
N
(9)
(10)
f 83%
(11)
Ar
b 48%
O
g,f 33%
a 86%
F
(18)
OMe
d,f 28%
F
O
N
+
F
R
F
N
N
N
N
Ar
N
N
N
F
Ar
Ar
Ar
(12)
N
g,f 42%
F
N
(5)
(7)
(8)
Ar
(13)
c 35%
F
F
F
OMe
F
O
N
N
d,j 56%
b 38%
F
N
Ar
N
N
N
N
N
(14)
Ar
Ar
Ar
(17)
(16)
(15)
F
OH
F
F
F
F
O
F
HO
F
f
b
a 76
F
N
N
N
N
N
N
N
N
N
N
Ar
(23)
Ar
Ar
Ar
Ar
(20)
(21)
(22)
(24)
i 65
F
R
F
F
F
O
F
F
F F
F
OH
O
f
a
b 46
F
N
N
N
N
N
N
Ar
N
N
N
N
(25)
Ar
(29)
Ar
Ar
(27)
Ar
(28)
d. 70
F
F
(26)
OH
N
N
Ar
(30)
Scheme 1. Synthesis of fluorinated tetrahydrocyclopentapyrazoles. Reagents and conditions: (a) CrO3/HOAc/50 °C; (b) 1. HSCH2CH2SH/BF3ÆOEt/
CH2Cl2, 2. PPHF/NIS/CH2Cl2/ꢀ30 °C; (c) 1. TMSOTf, Et3N, 2. SELECTFLUOR/CH3CN/12 h; (d) RMgX/ether/0 °C; (e) 1. NaBH4/ether; 2. K-O-
tBu/MeI; (f) DAST/CH2Cl2; (g) NaBH4/ether; (h) cat. OsO4/NMO/t-BuOH/H2O, (i) p-TSA/benzene/reflux/15 min; (j) K-O-tBu/MeI
assay. However, addition of an extra fluorine atom at
C-4 (19) led to improved binding as well as activity in
the contact assay. Compound 9 containing a gem-
difluoro substituent at C-6 maintained the activity of
monofluoro compound 14. Compounds with apical
gem-difluoro substitution (e.g., 27) showed a 4-fold in-
crease in anti-flea activity in the CO flea contact assay
with no selectivity against mammalian GABA recep-
tors. The 5,6-vicinal difluoro compound (22) also
showed an increase in anti-CO flea activity compared
to that of the parent cyclopentane analog 5. Trifluoro
compound 15, 4,4,5,6-tetrafluoro compound 24, and
4,4,5,5-tetrafluoro compound 29 exhibited excellent
anti-flea activity in the CO flea contact assay.
Although both tetrafluoro compounds showed LD50
values near 500 nM in the CO flea contact assay, they
showed a relatively high affinity for mouse GABA
receptors as well. The observation that the presence
of small substituents at C-4 yields compounds with
appropriate selectivity prompted us to study the

S. K. Meegalla et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1702–1706
Table 2. Insecticidal activities of fluorinated tetrahydrocyclopentapyrazoles
1705
Compound
Binding assay IC50 (nM)
Mouse
Contact assay LD50 (mM) or % mortality at 100 mM4
Fly
Selectivity
Fly
S/S fleas
A/A fleas (%)
7
470
20,000
2,800
ND
42
81
NA
0
15
16.5
100
40
9
10
ND
ND
74
NA
NA
4
6%
1%
60.7%
3%
3%
11
11
12
300
93.9%
22%
16.7%
85%
100%
57.5%
ND
7%
100
100
100
95
ND
ND
290
3,700
>10,000
2900
NA
NA
10
13 (R = Me)
14
15
16
<100
320
100
1,000
3,200
12,000
>10,000
1,000
1,000
175
>10
10
1
100
95.4
100
ND
100
100
100
100
100
40
10.8%
1.8
36%
1
17
18
120
>5
1700
>100
200
19
22
<10
5
88
79
1.5
<0.5
5
24
25 (R = Me)
ND
350
ND
> 28
1
0.4
>10,000
175
ND
100%
87.9%
6%
27
28
29
30
200
2
ND
ꢁ100
100
NA
ꢂ0.5
23
1%
<0.5
10
50
2,300
ND
72%
100
100
Table 3. Metabolic stability data
Acknowledgments
Compound
Microsomal t1/2
(min)
Clearance (pmol/
min/1 mg/protein)
We thank Mike Kolpak, Stephen Eisnnagel, Heidi Ott,
Malini Dasgupta, Joely Maddux, Victor Ozols, and
Scott Walmsley for analytical and technical support.
Mouse
Dog
Mouse
Dog
15
17
22
30
64.7
145
Stablea
Stablea
Stablea
506.7
109.2
34.1
Stablea
Stablea
Stablea
296.9
22.4
Stablea
255.1
Stablea
References and notes
a 80% of test substance was detected after 30 min of incubation.
1. Meegalla, S. K.; Doller, D.; Liu; RuiPing; Sha; DeYou;
Soll, R. M.; Dhanoa, D. Tetrahedron Lett. 2002, 43, 8639.
2. Olah, G. A.; Chambers, R. D.; Prakash, G. K. Synthetic
Fluorine Chemistry; John Wiley & Sons Inc: USA, 1992.
3. Dhanoa, D. S.; Meegalla, S.; Soll, R. M.; Doller, D.; Sha;
DeYou; Liu; RuiPing; Silver, G.; Lee, Y.; U.S. Patent
6,545,033.
particular structure–activity relationship at this posi-
tion. Among this type of compounds, compound 17
showed the highest anti-flea activity with CO flea,
with an LD50 of 1.8 mM and 120-fold selectivity
against the insect GABA receptors. The other two
compounds in this subgroup, 25 and 30, were about
25-fold selective for insect GABA receptors with 5
and 10 mM LD50s in CO flea contact assays.3
4. In vitro binding assay. Test compounds were dissolved in
DMSO at concentrations ranging from about 2 nM to
100 mM. One microliter of dissolved test compound was
dispensed into the well of a 96-well polystyrene plate.
100 lL of ice-cold assay buffer (10 mM phosphate, 300 mM
NaCl, pH 7.5) containing 5.2 nM 400-ethynyl-4-n-
[2,3-3H2]propyl-bicycloorthobenzoate ([3H]EBOB, 38 Ci/
mmol) was added followed by 100 lL of ice-cold assay
buffer containing 0.5–1.0 mg/mL neuronal membranes.
Control wells were prepared the same way, except the
neuronal membranes were omitted. The samples were
incubated for 45 min at 24 °C and filtered on a 0.1% (wt/
v) polyethylenimine-soaked glass fiber Filtermat A rinse of
cold assay buffer using Harvester 96-cell harvester. The
Filtermat was air-dried and radioactivity bound to the
Filtermat was detected. Compounds that displaced
[3H]EBOB specifically bound to the housefly were then
tested at 24–48 different final concentrations, varying from
about 0.1 nm to about 125 mM, in order to determine the
concentration at which 50% of the maximum inhibition due
to the addition of that compound was observed (IC50).
In vivo housefly assay. Newly emerged houseflies were
sedated with CO2 gas, collected in 50 mL polypropylene
conical tubes containing filter paper saturated with 10% (wt/
wt) sucrose in water, and allowed to feed at room temper-
Limited in vitro metabolic stability studies of promising
analogs were undertaken for determining half-lives in
mouse and dog liver microsomal preparations (Table
3). These studies revealed that, in general, compounds
containing a C-5 apical fluorine substitution were more
stable than unsubstituted analogs. Unfortunately,
although the introduced substituents OH and/or CH3
at C-4 improved the selectivity of the test compounds
against insect GABA receptors, they also became more
vulnerable to microsomal metabolism.
In conclusion, we have successfully employed selective
fluorination as a tool to address observed metabolic
instabilities of parent compound (5). Some of the
fluorinated compounds exhibited a several-fold in-
crease in activity compared to that of the parent
compound (5).

1706
S. K. Meegalla et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1702–1706
ature for about 2–4 h. Test compounds were dissolved in
In vivo flea assay. Test compounds were dissolved in
DMSO at concentrations ranging from 0.05 to 100 mM.
One microliter of dissolved test compound was dispensed
onto a 6 mm GF/C filter disk in the bottom of a 4 mL
screw-top glass vial and allowed to air-dry for 24–48 h.
Positive control was prepared in the same manner, except
that no test compounds were dissolved in DMSO. Twenty
newly emerged cat fleas were sedated by refrigeration at 0–
4 °C and transferred to each vial. Each vial was sealed with
thin, perforated Teflon septum secured by an open top
screw cap and held vertically in dark. After 24 h, the
healthy, moribund, and dead houseflies in each tube were
counted. The percentage of dead fleas in each vial was then
calculated.
DMSO at concentrations ranging from 0.05 to 100 mM. One
microliter of dissolved test compound and 100 mL of
isopropanol were dispensed into the bottom of a 9 mL
screw-top glass test tube. Positive control was prepared in the
same manner, except that no test compounds were dissolved
in DMSO. Each test tube was rolled to coat the sides with the
chemical solution and allowed to air-dry for 24–48 h. Twenty
houseflies were sedated by refrigeration at 0–4 °C and
transferred to each test tube. Each tube was sealed with
organdy cloth secured by an open top screw cap and laid
horizontally in dark. After 24 h the healthy, moribund, and
dead houseflies in each tube were counted. The percentage of
dead houseflies in each tube was then calculated.