Synthesis and GABA receptor potency of 3-thiomethyl-4-(hetero)aryl-5-amino- 1-phenylpyrazoles

Article abstract of DOI:10.1016/j.bmcl.2004.07.033

GABA receptor potency of 3-thiomethyl-4-(hetero)aryl-5-amino-1- phenylpyrazoles 4 and their synthesis is described. A convenient synthetic route to novel 4-arylpyrazoles is described. The potential for insecticidal activity through GABA channel blockage by this series of compounds, as well as their selectivity for insect versus mammalian receptors, are explored through in vitro and in vivo assays.

Full text of DOI:10.1016/j.bmcl.2004.07.033

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4949–4953
Synthesis and GABA receptor potency of
3-thiomethyl-4-(hetero)aryl-5-amino-1-phenylpyrazoles
a
a
a
b
Sanath K. Meegalla,a, Dario Doller, DeYou Sha, Rich Soll, Nancy Wisnewski,
*
Gary M. Silverb and Dale Dhanoaa
a3-Dimensional Pharmaceuticals, Inc., A wholly owned Johnson & Johnson company, 665 Stockton Drive, Exton, PA 1934, USA
bHeska Corporation, 1613 Prospect Parkway, Fort Collins, CO 80525, USA
Received 12 May 2004; revised 9 July 2004; accepted 12 July 2004
Available online 4 August 2004
Abstract—A convenient synthetic route to novel 4-arylpyrazoles is described. The potential for insecticidal activity through GABA
channel blockage by this series of compounds, as well as their selectivity for insect versus mammalian receptors, are explored
through in vitro and in vivo assays.
ꢀ 2004 Elsevier Ltd. All rights reserved.
The 1-phenylpyrazole core has been shown to bestow
pharmacological activity in a number of areas in the
pharmaceutical and agrochemical industries. In the lat-
ter field, select examples of biological activities include
insecticidal, miticidal, and herbicidal. More specifically,
1-phenylpyrazoles with alkyl, acyl, thioalkyl or cyano
substituents at the 4-position exhibit potent insecticidal
activity.1 In particular, 5-amino-1-(2,6-dichloro-4-tri-
fluoromethylphenyl)-4-trifluoromethanesulfinyl-1H-pyr-
azole-3-carbonitrile (Fipronilꢁ, 1) is one of the most
commercially successful insecticides. In fleas, ticks, and
other arthropods it acts as a GABA-gated chloride
channel inhibitor causing neuronal cell hyperexcitability
and eventual death.
class of compounds. Indeed, early reports had shown
that removal of both substituents on C-3 and C-5, lead-
ing to 1-(2,6-dichloro-4-trifluoromethylphenyl)-4-tri-
fluoromethanesulfinyl-1H-pyrazole (3, IC50 = 4.2nM),
had no detrimental effect on the binding affinity for
the housefly GABA receptor.3 We reasoned that these
experimental facts reflected the tolerance of this binding
site of the GABA receptor for lipophilic substituents at
the C-4 position of 1-arylpyrazoles of this type. Thus we
contemplated the possibility of increasing affinity for
this receptor by exploring diverse lipophilic functionali-
ties at C-4, while maintaining the thioalkyl functionality
at the C-3 position (generic structure 4)4 (Fig. 1).
The chemistry employed in our synthesis is depicted in
Scheme 1. The key step in this synthesis is palladium-
catalyzed cross coupling of iodopyrazole 11 and an
appropriate arylboronic acid or arylstannane.5 This
strategy, carried out through a parallel synthesis format,
enabled us to efficiently prepare a number of 4-arylpyr-
azoles with various substitution patterns in a short time.
The reaction of ethylcyanoacetate 5 with CS2 in the
presence of 2equiv of NaH, followed by addition of
3equiv of MeI afforded ketedithioacetal 6 in 58% iso-
lated yield.6 Compound 6 was then cyclocondensed with
2,6-dichloro-4-trifluoromethylphenylhydrazine 7 in re-
fluxing ethanol to obtain the 5-aminopyrazole ester 8
in 73% yield. The ester group of 8 was saponified by
treatment with LiOH in methanol–water to obtain the
corresponding carboxylic acid 9, which contained 15–
20% of decarboxylation product 10. The conversion of
As a part of our own program searching for novel,
orally bioavailable small molecule insecticidal agents,
we learned that we could maintain high binding affinity
for insect GABA receptors by swapping the thioalkyl
and nitrile functionalities on C-3 and C-4 of 1, leading
to 3-thiomethyl-4-cyano-5-amino-1-aylpyrazoles such
as 2a. This observation led us to the discovery of a po-
tent insect GABA radiolabeled ligand (CTOM, 2b), with
500-fold selectivity against the mammalian (mouse
brain) GABA receptor.2 Presence of the C-3 thiomethyl
on 2 was essential to maintain the high affinity of this
Keywords: Aryl pyrazole; GABA; Insecticidal activity.
*
Corresponding author. Tel.: +1-610-458-8959; fax: +1-610-458-
8249; e-mail: smeegalla@prdus.jnj.com
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.07.033

4950
S. K. Meegalla et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4949–4953
O
S
O
NC
R
S
CN
S
MeS
Ar
CF3
CF3
3
4
N
N
N
N
1
NH2
Cl
NH2
Cl
NH2
Cl
N
N
N
N
Cl
Cl
Cl
Cl
Cl
CF3
CF3
CF3
CF3
1, Fipronil
2a, R=CH3
3
4
2b, R=C[3H3]
Housefly IC50= 2.3 nM
Housefly IC50= 4.2 nM
Housefly IC50= 8 nM
Figure 1.
MeS
R
N
N
NH2
Cl
MeS
MeS
CO2Et
CN
CO2Et
a
b
Cl
CN
5
6
CF3
8.R = CO2Et
9.R = CO2H
c
d
e
10. R= H
11. R= I
12. R= Ar
f
Scheme 1. Synthesis of 3-thiomethyl-4-aryl-5-amino-1-arylpyrazoles: (a) (1) NaH, (2) CS2, (3) MeI, 63%; (b) EtOH, 2,6-Cl2-4-CF3-6H2NHNH2 (7),
reflux, 76%; (c) (1) LiOH; (d) 180ꢂC, 53%; (e) NIS, CH2Cl2 64%; (f) ArB(OH)2, Ph(PPh3)4, toluene–NaHCO3 (aq) 12–90%.
9 to 10 was completed by heating the vacuum-dried
crude carboxylic acid 9 at 180–190ꢂC, yielding product
10 in 85% yield from ester 8. The iodo functionality
was then introduced on C-4 by treating pyrazole 10 with
N-iodosuccinimide.7 Similarly, reaction with N-chloro-
or N-bromosuccinimide provided the 4-Cl or 4-Br ana-
logs, respectively.
homozygous serine (resistant, CO fleas), homozygous
alanine (sensitive, ARC fleas), and a mixed homozygous
and heterozygous population (NC fleas).
Table 1 shows affinity data for substituents smaller than
an aryl group on C-4. The high affinity for the nitrile
The cross-coupling reactions of 11 with appropriate
arylboronic acids were carried out using catalytic
Pd(PPh3)4 in a biphasic toluene/aq NaHCO3 system.5
Yields of isolated product under unoptimized reaction
conditions ranged from 12% to 90%. The 5-oxadiazole
analog 43 could only be obtained, albeit in low yield,
through the arylstannane coupling reaction using the
same Pd(0) catalyst in DMF at 75ꢂC; the corresponding
oxadiazoleboronic acid failed to produce any detectable
amounts of product.
Table 1. C-4 small substituents
MeS
R
N
NH2
Cl
N
Cl
CF3
Selectivityb
a
Compound
R
Housefly IC50
The potential for insecticidal activity of the synthesized
compounds was evaluated by determining their ability
to bind to housefly neuronal membrane receptors dis-
placing radioligand [3H]-EBOB.8 Their in vitro selectiv-
ity was determined by measuring their binding affinities
for mouse brain GABA receptors. The in vivo flea-kill-
ing efficacy of these compounds was also evaluated
through a contact assay with house flies and three differ-
ent flea strains.8 Three different flea strains were used to
assess differences in insecticidal activity among the three
genetic variants of the cyclodiene resistant allele-
10
12
13
11
2a
14
8a
8b
9
H
400
740
380
160
8
250
210
Cl
Br
I
2500
43
CN
500
C„CH
CO2Me
CO2Et
CO2H
105
43
>10,000
NA
400
25
700
45
a IC50 in nM.
b Ratio of mammalian receptor affinity to insect affinity.

S. K. Meegalla et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4949–4953
4951
Table 2. C-4 aryl substituents
R4
R3
R2
MeS
R1
NH2
Cl
N
N
Cl
CF3
Entry
R1
H
R2
H
R3
R4
H
Housefly
a
IC50
Selectivityb
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
H
300
350
IA
4
20
F
Cl
––
––
Br
IA
CN
CF3
CH3
OCH3
SCH3
CF3
i-Pr
H
1500
4000
120
170
52
666
2600
66
180
230
3
400
IA
––
CH3
Cl
400
800
26
NA
31
OCH3
i-Pr
CF3
t-Bu
H
346
IA
1000
IA
5
––
CH3
SCH3
OCH3
F
50
400
IA
500
IA
NA
––
CF3
H
90
6000
12
CF3
Cl
CF3
H
700
1000
500
325
124
IA
F
4
OCH3
H
OCH3
OCH3
2000
49
OCH3
H
OCH2O
64
OCH3
OCH3
OCH3
––
a IC50 in nM.
b Ratio of mammalian receptor affinity to insect affinity. IA = inactive. NA = not available.
functionality is notable. Related functionalities, such as
esters 8a and 8b, show fair levels of affinity, but halogens
or the isosteric acetylene group bind less effectively.
analog 44 exhibited 90nM binding affinity for house
fly brain preparations, chloro substitution at 5-position
of thiophene resulted in ꢀ4-fold increase in binding
affinity in the same assay. However, other related thio-
phene analogs 45, 47, and 48 had no activity at all.
Among nitrogen heterocycles, isomeric pyridines 52–54
are all inactive, but pyrazine substitution as in com-
pound 50 and oxadiazole 43 only yielded fair binding
affinity levels.
Analogs obtained by introduction of a substituted phe-
nyl group on C-4 are shown in Table 2. While no analog
shows improved affinity over nitrile 2a, comparable lev-
els of activity are seen with the 3-anisyl analog 28, which
shows selectivity greater than 300-fold. Increasingly low-
er affinity levels were obtained for 2-substituted analogs
32 and 36 (methyl and trifluoromethyl, respectively) and
4-substituted analogs 23, 21, and 22 (thiomethyl,
methyl, and methoxy, respectively). Their selectivities
(66–4000-fold) were comparable or better than that for
28.
The next step was the evaluation of compounds with dif-
ferent levels of in vitro binding activity in the in vivo
contact assay. The results of these experiments on se-
lected analogs are shown in Table 4. Even at the rela-
tively high concentration used in the assay (100mM),
only moderate activity was seen with compounds that
had sub-micromolar affinity for the receptor. While
binding to the receptor is necessary for an insecticide
Table 3 shows binding results for analogs containing an
heterocyclic C-4 substituent. Although 2-thiophenyl

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S. K. Meegalla et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4949–4953
Table 4. In vivo efficacy of selected compounds evaluated by the
contact assay
Table 3. C-4 heterocyclic substituents
MeS
R
Compound Housefly CO
a
IC50
NC
Houseflyb
92.8
fleasb fleasb
N
NH2
Cl
N
Dual acting
compound
12
740
32.3
3.3
Cl
Fly selective 10
160
105
110
124
4.4
2.2
0
0
0
0
0
42.4
80.1
95.4
27.6
14
43
41
CF3
Compound
R
Housefly
a
IC50
Selectivityb
1.4
Flea selective 50
87
90
50
77.4
58.7
60.8
89.9
0
0
36
32
3.9
9.6
N
43
110
9
0
N
O
a Affinity for the housefly receptor, IC50 in nM.
b % Mortality at 100mM.
44
45
90
44
––
S
IA
compounds 32 and 36, both with ortho substituents on
the C-4 aryl group, showed selectivity between the flea
colonies used. Based on these results it seems that the
housefly receptor is not always a good predictor of activ-
ity in contact assays, both against fleas and flies.
S
46
47
48
23
434
––
Cl
S
S
IA
IA
In conclusion, a number of novel 4-substituted 1-aryl-3-
thiomethyl-5-aminopyrazoles were made9 and their
insecticidal activities evaluated. Compounds with a
variety of insecticidal profiles were obtained, including
the desired broad anti-flea activity, as determined
through contact assay models with various strains of
cat fleas.
––
S
S
49
50
150
87
25
45
COCH3
N
Acknowledgements
N
N
We wish to thank Mike Kolpak, Stephen Eisnnagel, He-
idi Ott, Malini Dasgupta, Joely Maddux, Victor Ozols,
and Scott Walmsley for analytical and technical sup-
port.
51
52
53
Negative
inhibition
––
––
––
––
N
IA
IA
IA
N
References and notes
N
N
1. For examples of insecticidal activity, see: (a) Banks, B. J.
Eur. Pat. Appl. EP 846686, 1998; (b) Banks, B. J. U.S.
Patent 6,069,157, 2000; (c) Lankau, H.-J.; Menzer, M.;
Rostock, A.; Arnold, T.; Rundfelt, C.; Unverfeth, K.
Pharmazie 1999, 54, 705.
2. Meegalla, S. K.; Doller, D.; Silver, G. M.; Wisnewski, N.;
Soll, R. M.; Dhanoa, D. Bioorg. Med. Chem. Lett. 2003, 13,
4035.
54
a IC50 in nM.
b Ratio of mammalian receptor affinity to insect affinity. IA = inactive.
NA = not available.
3. Cole, L. M.; Nicholson, R. A.; Casida, J. E. Pestic.
Biochem. Physiol. 1993, 46, 47–54.
4. For other examples of C-4 substituted phenylpyrazole
derivatives, see: Sammelson, R. E.; Caboni, P. C.; Durkin,
K. A.; Casida, J. E. Bioorg. Med. Chem. 2004, 12, 3345.
5. Tsuji, J. Palladium Reagents and Catalysis; John Wiley &
Sons Ltd, 1995.
6. Villemin, D.; Ben Alloum, A. Synthesis 1991, 301.
7. Preliminary experiments showed that the presence of 5-
amino group is necessary for successful iodination. The
amino group could then be removed quantitatively by
treatment with isoamyl nitrite in DMF at 60ꢂC.
to work through this mechanism, the lack of in vivo
activity in contact assays could be due to poor skin per-
meability, inappropriate disposition, and metabolism of
the substrate which would preclude it from reaching the
target site. Nevertheless, different profiles in terms of
selectivity of in vivo insecticidal action emerged from
our studies: dual acting 12, fly selective 10, 14, 43, 41,
or flea selective 32, 36, and 50 compounds. In addition

S. K. Meegalla et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4949–4953
4953
8. Dhanoa, D. S.; Meegalla, S. K.; Doller, D.; Soll, R. M.
U.S. Patent 6,409,988.
9. General synthetic procedures are as follows:
Synthesis of compound 8: A solution of 2,6-dichloro-4-
trifluoromethylphenylhydrazine 7 (245mg, 1mmol) and
NIS (1.2mmol, 270mg) at 0ꢂC. The resulting mixture was
stirred at room temperature for 30min and concentrated
under reduced pressure. The residue was dissolved in
CH2Cl2 (20mL) and washed with water (10mL), aq 10%
Na2S2O3 (10mL), and saturated Na2CO3 (10mL). The
organic layer was separated, dried (Na2SO4), and concen-
trated. The residue was purified on silica (EtOAc–hexanes
15:85) to obtain the desired compound (63%). 1H NMR (d,
CDCl3): 2.54 (3H, s), 7.65 (2H, s).
3,3-(bismethylthio)-2-cyanoacrylic acid ethyl ester
6
(217mg, 1mmol) in isopropanol (15mL) was heated at
reflux for 16h. The solvent was removed under reduced
pressure. The residue was purified on silica EtOAc–hexanes
(1:9) to afford compound 8 (75%). 1H NMR (d, CDCl3): 1.4
(3H, t, J = 7.2Hz), 2.48 (3H, s), 4.13 (2H, q, J = 7.2Hz),
7.76 (2H, s).
General procedure for synthesis of 3-thiomethyl-4-(hetero)-
aryl-5-amino-1-phenylpyrazoles:
(a) A solution of compound 11 (47.8mg, 0.1mmol) and
boronic acid or ester (2equiv, 0.2mmol) in toluene (5mL)
was placed in a N2-flushed vial. Aqueous NaHCO3 (2M,
2mL) ethanol (2mL) and tetrakis(triphenylphosphine)
palladium(0) (12mg, 0.01mmol) was added. The reaction
mixture was then heated at 100ꢂC for 6h. The reaction
mixture was allowed to cool to room temperature and the
organic layer was separated. Solvents were removed and the
residue was purified on silica with an appropriate solvent
system.
(b) A solution of compound 11 (47.8mg, 0.1mmol) and the
corresponding arylstannane (2equiv, 0.2mmol) in DMF
(5mL) was placed in a N2-flushed vial. Tetrakis(triphenyl-
phosphine) palladium(0) (12mg, 0.01mmol) was then added
and the resulting mixture was heated at 75ꢂC for 12h. The
reaction mixture was allowed to cool to room temperature
and poured into water. The water layer was extracted with
CH2Cl2 (3 · 10mL). The organic layers were separated,
combined, dried, and concentrated. The residue obtained
was purified on silica with the appropriate solvent system.
Synthesis of compound 9: Compound 8 (212mg, 0.5mmol)
was dissolved in LiOH (96mg, 4mmol) in methanol/water
(7mL, 9:1 mixture). The resulting solution was stirred at
reflux for 16h and then cooled to room temperature.
Methanol was removed and the reaction mixture was
acidified to pH4 by adding AcOH and extracted with
CH2Cl2 (3 · 20mL). The CH2Cl2 layers were combined and
dried (Na2SO4) to yield compound 9 (90%) contaminated
with compound 10 which was directly used in next step
without further purification. For compound 9: 1H NMR (d,
CDCl3): 2.5 (3H, s), 3.58 (2H, q, J = 7.2Hz), 7.77 (2H, s).
Synthesis of compound 10: Compound 9 (1.16g) was
heated at 180–190ꢂC for 20min under N2. The reaction
mixture was allowed to cool to room temperature and
purified on silica (EtOAc–hexanes 15:85) to yield com-
pound 10 (65%). 1H NMR (d, CDCl3): 2.5 (3H, s), 5.67
(1H, s), 7.75 (2H, s).
Synthesis of compound 11: A solution of compound 10
(352mg, 1mmol) in acetonitrile (5mL) was treated with