GABA receptor antagonists and insecticides: Common structural features of 4-alkyl-1-phenylpyrazoles and 4-alkyl-1-phenyltrioxabicyclooctanes

Article abstract of DOI:10.1016/j.bmc.2004.03.069

Fipronil [5-amino-3-cyano-1-(2,6-dichloro-4-trifluoromethylphenyl)-4- trifluoromethylsulfinylpyrazole] is one of the most important insecticides. Structure-activity studies described here reveal that fipronil retains its very high binding potency at the h

Full text of DOI:10.1016/j.bmc.2004.03.069

Bioorganic & Medicinal Chemistry 12 (2004) 3345–3355
GABA receptor antagonists and insecticides: common structural
features of 4-alkyl-1-phenylpyrazoles and 4-alkyl-1-
phenyltrioxabicyclooctanes
Robert E. Sammelson,a, Pierluigi Caboni,a Kathleen A. Durkinb and John E. Casidaa,*
aEnvironmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management,
University of California, Berkeley, CA 94720-3112, USA
bMolecular Graphics Facility, College of Chemistry, University of California, Berkeley, CA 94720-1460, USA
Received 12 December 2003; accepted 19 March 2004
Available online 6 May 2004
Abstract—Fipronil [5-amino-3-cyano-1-(2,6-dichloro-4-trifluoromethylphenyl)-4-trifluoromethylsulfinylpyrazole] is one of the most
important insecticides. Structure–activity studies described here reveal that fipronil retains its very high binding potency at the
human b3 and house fly c-aminobutyric acid (GABA) receptors and toxicity to house flies on replacing the pyrazole tri-
fluoromethylsulfinyl moiety with tert-butyl or isopropyl and the phenyl trifluoromethyl substituent with ethynyl, trifluoromethoxy,
bromo or chloro. Among the compounds studied, those with other alkyl groups at the 4-position of the pyrazole, as well as phenyl
substitution without one or both of the 2,6-dichloro groups, are less effective. 5-Amino-4-tert-butyl-3-cyano-1-(2,6-dichloro-4-
ethynylphenyl)pyrazole is highly effective and almost isosteric with 4-tert-butyl-3-cyano-1-(4-ethynylphenyl)-2,6,7-trioxabicy-
clo[2.2.2]octane (the most potent 4-alkyl-1-phenyltrioxabicyclooctane) as a noncompetitive GABA antagonist and insecticide. These
findings are interpreted as three binding subsites in the GABA receptor: a hydrophobic site undergoing steric interaction with the
tert-butyl or equivalent group; a hydrogen bonding site to pyrazole N-2; a pi bonding site to the face of the phenyl moiety; with
supplemental enhancement by the 3-cyano and 4-ethynyl substituents.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Fipronil (1) (Fig. 1) is one of the most important insect
control chemicals.1–3 Similar insecticidal activity is ob-
served for its sulfide (2) photoproduct, sulfone (3) pho-
toproduct and metabolite, and desulfinyl photoproduct
Figure 1. Fipronil (1), ethiprole (5) and analogs (2–4, 6, and 7).
Abbreviations: Bu, butyl; c, cyclo; DCM, dichloromethane; DIPA,
(4).4;5 Ethiprole (5), with 4-EtSO replacing the 4-CF3SO
of 1, is a newer analog in advanced development.6
Desulfinylethiprole (6) (the 4-Et analog), a 4-alkyl-1-
phenylpyrazole, was prepared as a candidate photo-
product and found not to be formed photochemically
but surprisingly to have high insecticidal activity.7 The
biochemical target of 1–6 in both insects and mammals
is the c-aminobutyric acid (GABA) receptor and more
specifically its noncompetitive blocker site.8–12 To better
understand the interactions between 4-alkyl-1-phen-
ylpyrazoles and the GABA receptor, a series of com-
pounds was synthesized by varying the 4-position of the
diisopropylamine; [3H]EBOB, 4-n-[3H]propyl-1-(4-ethynylphenyl)-2,6,
7-trioxabicyclo[2.2.2]octane, also known as 4-n-[3H]propyl-40-
ethynylbicycloorthobenzoate; EI, electron impact; Et, ethyl; GABA,
c-aminobutyric acid; Hex, hexyl; HRMS, high-resolution MS; i, iso;
IC50, concentration for 50% inhibition; Me, methyl; mp, melting point;
MS, mass spectrometry or mass spectra; n, normal; PB, piperonyl
butoxide; Pen, pentyl; Pr, propyl; s, secondary; SAR, structure–activity
relationship; SE, standard error; t, tertiary; TBO, 2,6,7-trioxabi-
cyclo[2.2.2]octane.
* Corresponding author. Tel.: +1-510-642-5424; fax: +1-510-642-6497;
e-mail: ectl@nature.berkeley.edu
Present address: Department of Chemistry, Ball State University,
Muncie, IN 47306-0445, USA.
0968-0896/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.03.069

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R. E. Sammelson et al. / Bioorg. Med. Chem. 12 (2004) 3345–3355
pyrazole moiety. A second library was prepared to study
the phenyl substituents. The findings show unexpected
common structural features of 4-alkyl-1-phenylpyraz-
oles and 4-alkyl-1-phenyl-2,6,7-trioxabicyclo[2.2.2]oct-
anes (4-alkyl-1-phenylTBOs) in their action as GABA
receptor antagonists and insecticides.
Scheme 2. Preparation of ethyl 2,3-dicyanoalkanoate intermediates
(D).
Potency evaluations are conveniently made with a
binding assay using 4-n-[3H]propyl-1-(4-ethynylphen-
yl)TBO ([3H]EBOB).13 This determination with house
fly head membranes is predictive of insecticidal activity
as topical LD50 in house flies pretreated with the
CYP450 inhibitor piperonyl butoxide (PB) to minimize
oxidative detoxification.14 Mammalian brain mem-
branes also bind [3H]EBOB in a manner sensitive to
many insecticides and convulsants.15 The b subunit
contains the binding site16 and the human recombinant
homooligomeric b3 receptor is very similar to the
housefly receptor in sensitivity and specificity and
therefore provides a very convenient, well defined and
validated binding assay.17 Accordingly, the present
study uses both the house fly and human b3 receptors to
define the structure–activity relationships (SAR) and the
target site conformation. House fly topical toxicity as-
says alone and with PB are used to evaluate organismal
sensitivity and synergistic effects.
Scheme 3. Synthesis of 4-alkyl analogs of fipronil. (a) i. H2SO4,
NaNO2, CH3CO2H; ii. D, CH3CO2H, H2O; (b) NH3, H2O, DCM.
diastereomers were obtained after stirring for 4 h at
room temperature. The yields and purities of each of
these reactions were excellent and these mixtures of
stereoisomers were utilized without separation or puri-
fication after aqueous extraction. Diazotization of 2,6-
dichloro-4-trifluoromethylaniline with sulfuric acid and
sodium nitrite in acetic acid was followed by reaction
with D to give the yellow diazo addition product C
(Scheme 3). This crude mixture of stereoisomers was
extracted with dichloromethane, made basic, and the
resultant orange solution stirred with 50% ammonium
hydroxide overnight. The substituted derivatives, espe-
cially hindered ones like t-Bu, cyclized at a slower rate
than the unsubstituted derivative (R ¼ H) but under
these conditions it did not affect the overall yield.
Extraction with dichloromethane and concentration of
the organic layer were followed by crystallization or
chromatography to provide the desired 4-alkyl-1-phen-
ylpyrazoles (6, 7, and 9–17).
2. Chemistry
2.1. 4-Alkyl-5-amino-3-cyano-1-(2,6-dichloro-4-trifluo-
romethylphenyl)pyrazoles
Retrosynthetically, these 4-alkyl-1-phenylpyrazoles (A,
Scheme 1) can be obtained from base-induced cycliza-
tion of phenylhydrazone B. Intermediate B arises from
hydrolysis and decarboxylation of diazo compound C,
which can be prepared by addition of D to the diazo-
nium salt of 2,6-dichloro-4-trifluoromethylaniline.
Structure D would originate from ethyl cyanoacetate,
potassium cyanide and the corresponding aliphatic
aldehyde via Knoevenagel condensation/Michael addi-
tion protocol.18;19 This route has been employed for the
unsubstituted case (compound 8, R ¼ H) but is novel for
substituted examples.
2.2. 5-Amino-3-cyano-1-(2,6-dichloro-4-trifluoromethyl-
phenyl)-4-halopyrazoles
The synthesis commenced with the preparation of the
ethyl 2,3-dicyanoalkanoates (D) from ethyl cyanoace-
tate, potassium cyanide, and aldehyde in ethanol
(Scheme 2). In all cases, except when R ¼ H, mixtures of
Compound 8 was synthesized as above for biological
evaluation and was used as a precursor for the 4-
halopyrazole derivatives. Halogenation at the 4-position
of 8 with N-chlorosuccinimide at 80 °C, N-bromosuccin-
Scheme 1. Retrosynthetic approach to 4-alkyl analogs of fipronil.

R. E. Sammelson et al. / Bioorg. Med. Chem. 12 (2004) 3345–3355
3347
with PB to inhibit cytochrome P450-catalyzed oxidative
detoxification.5;14 These assays allowed ranking the 4-
alkyl substituent effects (Table 1). Next, 22 isopropyl- or
tert-butylpyrazoles with varied phenyl substituents were
compared for human b3 GABA receptor IC50 and house
fly mortality at the 1.2 lg/g discriminating dose alone
and with PB (Table 2). This permitted selection of two
of the most active compounds reported here (7 and 11)
for direct potency comparisons to 1 and 5 (Table 3).
Scheme 4. Synthesis of 4-halo analogs of fipronil.
imide at 50 °C, and N-iodosuccinimide at 20 °C in ace-
tonitrile provided the 4-Cl (18), 4-Br (19), and 4-I (20)
derivatives, respectively (Scheme 4).
4. Results and discussion
4.1. Effect of 4-(alkyl or halo)pyrazole substituents on
GABA receptor potency and insecticidal activity
2.3. 5-Amino-3-cyano-1-(2,4,6-substituted-phenyl)-4-(iso-
propyl or tert-butyl)pyrazoles
The effect of 4-(alkyl or halo)pyrazole substituents was
studied first in the 5-amino-3-cyano-1-(2,6-dichloro-4-
trifluoromethylphenyl)pyrazole series (Table 1). The
potency order falls into the same five distinct groups
based on house fly and human b3 receptors and toxicity
to house flies alone and with PB (Table 1). The highest
potency is for t-Bu followed by outstanding potency for
the branched chain 3-Pen, i-Bu and i-Pr substituents.
High potency is retained for the n-Pr, s-Bu, n-Bu and Et
substituents and medium potency for neo-Pen, c-Hex and
the halo analogs (I, Br and Cl). Phenylpyrazoles halo-
genated at the 4-position of the pyrazole showed only a
small variation in activity but generally the larger and
more sterically hindered substituent is preferred and I is
marginally better than Br or Cl for receptor binding and
toxicity. There is a considerable drop to low potency for
compounds with H and Me (the smallest substituents).
Analogs 21–39 were prepared as above for 7 and 11 but
in cases where one or both of the 2,6-dichloro substit-
uents were absent 2 N hydrochloric acid was used in-
stead of concentrated sulfuric acid. Some substituents
give slower cyclization (requiring up to 4 d) and the t-Bu
derivatives also called for heating to reflux in several cases.
2.4. 5-Amino-3-cyano-1-(2,6-dichloro-4-ethynylphenyl)-4-
(isopropyl or tert-butyl)pyrazoles
Ethynyl derivatives 40 and 41 were obtained from
Sonagishira coupling of bromo analogs 38 and 39,
respectively. The coupling reaction was carried out with
trimethylsilylacetylene, tetrakistriphenyl phosphine pal-
ladium and cuprous iodide in diisopropylamine and
toluene (Scheme 5). Desilylation was achieved with
potassium carbonate in methanol.
Further comparisons were made of the effect of three
4-alkylpyrazole substituents on potency as inhibitors
of the human b3 receptors in 2,6-dichlorophenyl com-
pounds with 4-CF3 and 4-Cl substituents (Table 2). In
each case, as above, the potency decreases in the order
for pyrazole substituents of 4-t-BuP4-i-Prꢀ4-Et.
3. Biology
Fifteen
5-amino-3-cyano-1-(2,6-dichloro-4-trifluoro-
methylphenyl)-4-(alkyl or halo)pyrazoles were first
assayed as competitors for [3H]EBOB binding to the
house fly GABA receptor with potency expressed as IC50
values (Table 1). Then a discriminating concentration of
10 nM was used to determine possible sensitivity differ-
ences between house fly and human b3 receptors. In
most cases, the inhibition increased slightly from the
house fly to the human b3 receptor, the exceptions being
c-Hex and possibly neo-Pen. House fly percentage
mortality was then determined at 1.2 lg/g alone and
4.2. Effect of phenyl substituents on GABA receptor
potency and insecticidal activity
The position, type and extent of substitution on the phe-
nyl ring have pronounced effects on human b3 receptor
potency and house fly mortality (Table 2). The receptor
potency order for derivatives with mono substitution is 4-
CF3>4-Cl>4-CN and for disubstituted compounds is 2-
Cl, 4-CF3>2,4-Cl2 ¼ 2,6-Cl2. In the trisubstituted series,
receptor potency and toxicity to house flies (judged both
with and without PB) decreases in the order 2,4-Cl2-4-
CF3 ¼ 2,4-Cl2-4-CF3O>2,4-Cl2-4-Br P 2,4,6-Cl3. Impor-
tantly, the 2,6-Cl2-4-CBCH substitution pattern (com-
pounds 40 and 41) confers very good receptor potency
and, for 41, also insecticidal activity with PB.
4.3. Comparisons of the most active 4-alkyl-1-phenyl-
pyrazoles with commercial insecticide standards
It is of interest to compare the activity of the best new 4-
alkyl-1-phenylpyrazoles with commercial insecticide
Scheme 5. Synthesis of two 4-alkyl-5-amino-3-cyano-1-(2,6-dichloro-
4-ethynylphenyl)pyrazoles.

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R. E. Sammelson et al. / Bioorg. Med. Chem. 12 (2004) 3345–3355
Table 1. Effect of alkyl or halo substituent (R in Fig. 1) on the potency of 5-amino-3-cyano-1-(2,6-dichloro-4-trifluoromethylphenyl)-4-(alkyl or
halo)pyrazoles as inhibitors of [3H]EBOB binding to house fly and human b3 GABA receptors and toxicants to house flies alone and with piperonyl
butoxide
GABA receptor
Inhibition at 10 nM (% ꢁ SE)
House fly Human b3
77
House fly mortality at 1.2 lg/g (% SE)
R in Figure 1
House fly
IC50 (nM SE)
Compd
Alone
100
PB
Highest potency
t-Bu
7
2.4 1.1
8
97
8
100
Outstanding potency
3-Pen
i-Bu
i-Pr
15
13
11
3.3 1.4
4.8 0.2
5.3 1.7
75 11
81
85
89
4
7
9
7
11
100
100
100
57
67
7
7
23 21
78 22
High potency
n-Pr
s-Bu
10
14
12
6
9.6 4.4
55 14
88 12
73 12
76 22
23 21
37 23
100
100
10
12
13
1
6
3
51
52
4
6
n-Bu
Et
3
20 22
6
93 12
100
53 19
65
8
Medium potency
neo-Pen
I
16
20
17
19
18
16
8
47
40
20
31
5
6
4
7
37 19
78 15
7
11
0
83 29
90 17
40 35
63 25
53 21
24 16
26
c-Hex
Br
Cl
1
4
68
1
8
0
30 15
31 19
0
25 11
66 13
0
Low potency
H
Me
8
9
123 35
424 58
24
1
9
1
41 16
19 17
0
10 14
2 5
15 19
Table 2. Effect of phenyl substituents on the potency of 5-amino-3-cyano-1-(substituted-phenyl)-4-(isopropyl or tert-butyl)pyrazoles as inhibitors of
[3H]EBOB binding to human b3 GABAA receptors and toxicants to house flies alone and with piperonyl butoxide
House fly mortality at 1.2 lg/g (% ꢁ SE)
Phenyl substituents
Compd
t-Bu
Human b3 GABA receptor IC50
(nM SE)
Alone
PB
2
4
Cl
6
i-Pr
21
23
25
27
29
31
33
36
38
11
40
i-Pr
t-Bu
i-Pr
0
t-Bu
i-Pr
13
t-Bu
7
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
22
24
26
28
30
32
34
37
39
7
10,000 3500
714 22
>10,000a
337 124
2470 500
177 47
>10,000a
74 24
0
0
0
0
0
6
6
H
CF3
CN
Cl
0
3
5
10
8
0
H
0
10 12
0
5
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
0
10 11
97
CF3
H
15
3
5.6 2.1
253, 319
2.2 0.3
6.2 4.0
3.8, 4.3
1.8 0.7
5.4, 3.6
0
13 15
25 17
100
6
101,145
3.4 0.4
6.2 0.3b
6.3, 6.7
0
83 21
5
6
40 20
100
96 6
CF3O
Cl
Br
100
0
6
3
6
5
20 14
48 13
100
18
97
8
5
5
100
100
CF3
CBCH
1.9 1.1b
34 23
0
41
7.1 0.7
13
8
8
94 13
Mortality values at 1.2 lg/g (% ꢁ SE) for 6 and 35 are 5 6 and 0 0, respectively, without PB and 78 17 and 5 6, respectively, with PB.
a Inhibition of 23–29% at 10,000 nM.
b IC50 values (nM SE) with 4-Et substituent are: 2,6-Cl2-4-CF3 (compound 6) 5.1 2.4 nM; 2,4,6-Cl3 (compound 35) 29 4 nM.
standards. The most effective new compounds (both
binding potency and insecticidal activity) are 5-amino-4-
alkyl-3-cyano-1-(2,4-dichloro-6-substituted-phenyl)-pyr-
azoles with i-Pr or t-Bu substituents (compounds 7, 11,
and 33–41) and particularly 7 and 34. The t-Bu (7) and
i-Pr (11) analogs were selected for direct comparison
with fipronil (1) and ethiprole (5) (Table 3). Ethiprole is
less potent than the others on the receptors. Compounds
1 and 7 are not significantly different in activity and are
generally the most potent of the series. The synergist
factor was particularly high for compound 11.
4.4. Common structural requirements for alkyl substitu-
ents of 4-alkyl-1-phenylpyrazoles, 4-alkyl-1-phenylTBOs,
4-alkyl-1-phosphaTBO 1-oxides and related compounds
A similar, and potentially the same, structural specificity
is observed for the alkyl substituents in several series of

R. E. Sammelson et al. / Bioorg. Med. Chem. 12 (2004) 3345–3355
3349
Table 3. Comparison of the potency of fipronil (1), ethiprole (5) and their t-Bu (7) and i-Pr (11) analogs as inhibitors of [3H]EBOB binding to house
fly and human b3 GABA receptors and toxicants to house flies alone and with piperonyl butoxide
GABA receptor IC50 (nM SE)
Fly LD50 (lg/g SE)
R in Figure 1
Compd
House fly
Human b3
Alone
PB
PB Synergism factor
SOCF3
SOEt
t-Bu
1
5
2.3 0.7a
15 3a
3.1 0.6a
12 2a
0.16 0.01a
0.50 0.03a
0.15 0.07
0.74 0.50
0.023 0.004a
0.30 0.02a
7
2
7
11
2.4 1.1
5.3 1.7
1.8 0.7
1.9 1.1
0.019 0.010
0.067 0.052
8
11
i-Pr
a Ref. 7.
Me
(A)
10
3
1
n-Bu
n-Pr
Et
s-Bu
0.3
0.1
i-Pr
i-Bu
t-Bu
r = 0.87
n = 8
1
3
10 30 100 300
Figure 2. Common structural requirements for the optimal alkyl
substituents of 4-alkyl-5-amino-3-cyano-1-(2,6-dichloro-4-trifluoro-
methylphenyl)pyrazoles (a) compared with 4-alkyl-1-(4-ethynylphen-
yl)TBOs (b), 4-alkyl-1-phosphaTBO 1-oxides (c), 4-alkyl-1-
phosphaTBO 1-sulfides (d), and 5-alkyl-2-(4-ethynylphenyl)-1,3-dithi-
anes (e).
(B)
100
Me
30
10
Et
n-Bu
3
1
compounds (Fig. 2). This is evident on comparing 4-
alkyl-1-phenylpyrazoles (a) at the house fly GABA
receptor [3H]EBOB binding site (this study) with 4-
alkyl-1-(4-ethynylphenyl)TBOs (b) as toxicants to mice20
(r ¼ 0:87, n ¼ 8) (Fig. 3A) and 4-alkyl-1-phosphaTBO
1-oxides (c) at the human brain membrane GABA
receptor ([35S]TBPS binding)21 (r ¼ 0:83, n ¼ 6) (Fig.
3B). Similar correlations to that shown in Figure 3B are
obtained with mouse ip LD50 values of 4-alkyl-1-phos-
phaTBO 1-oxides (c) (r ¼ 0:83, n ¼ 6)22;23 and 4-alkyl-1-
phosphaTBO 1-sulfides (d) (r ¼ 0:99, n ¼ 5).23 When
these last three are combined into a single plot (not
shown), an excellent overall correlation is observed
(r ¼ 0:92, n ¼ 19). The same potency order t-Bu>
i-Pr>n-Pr>Et is also observed with 5-alkyl-2-(4-ethyn-
ylphenyl)-1,3-dithanes (e).24;25 Thus, the shorter and
more hindered alkyl moieties are better in the 4-alkyl-
1-phenylpyrazoles as in the 4-alkyl-1-phenylTBOs,
1-phosphaTBO 1-oxides, 1-phosphaTBO 1-sulfides
and 5-alkyl-2-(4-ethynylphenyl)-1,3-dithanes. The t-Bu
substituent is also present in other active phenyl het-
erocyclic analogs.26
n-Pr
i-Pr
0.3
0.1
r = 0.83
n = 6
t-Bu
1
3
10 30 100 300
(nM)
house fly GABA receptor IC50
4-alkyl-1-phenylpyrazoles
Figure 3. Structural specificity of alkyl substituents for three types of
GABA receptor antagonists. IC50 values for house fly [3H]EBOB
binding site of 4-alkyl-5-amino-3-cyano-1-(2,6-dichloro-4-trifluoro-
methyl)pyrazoles (a) are compared with (panel A) mouse ip LD50 values
of 4-alkyl-1-(4-ethynylphenyl)TBOs (b) and (panel B) IC50 values for
human [35S]TBPS binding site of 4-alkyl-1-phosphaTBO 1-oxides (c).
The disubstituted compounds are more potent at the
receptor than the monosubstituted in the order 2-Cl, 4-
CF3>2,4-Cl2 ¼ 2,6-Cl2. The most potent derivatives at
the receptor and as insecticides are 2,6-dichloro-4-
substituted compounds with 2,6-Cl2-4-CF3 ¼ 2,6-Cl2-4-
OCF3>2,4,6-Cl3 ¼ 2,6-Cl2-4-Br. Importantly, 2,6-Cl2-4-
HCBC-phenyl is an effective substitution pattern in the
4-alkyl-1-phenylpyrazoles (compounds 40 and 41) just
as HCBC-phenyl is for the 4-alkyl-1-phenylTBOs.27;28
4.5. Structural requirements for phenyl substituents of
4-alkyl-1-phenylpyrazoles and 4-alkyl-1-phenylTBOs
4-Isopropyl- or 4-tert-butyl-1-phenylpyrazoles were
examined with one, two or three phenyl substituents
relative to optimization for potency (Table 2). With
mono substitution, the receptor potency decreases in the
order 4-CF3>4-Cl>4-CN. The 4-CN moiety confers
high potency in 4-alkyl-1-(4-cyanophenyl)TBOs21 but
not in the 4-alkyl-1-(4-cyanophenyl)pyrazoles, that is
the substituent effect varies with the compound series.
4.6. Common structural features of 4-alkyl-1-phenyl-
pyrazoles and 4-alkyl-1-phenylTBOs
Four aspects of the SARs developed here for 4-alkyl-1-
phenylpyrazoles are similar to those found earlier in
optimization of 4-alkyl-1-phenylTBOs (Fig. 4). The first
is the preference in both cases for tert-butyl among the
4-alkyl groups examined. Other bulky alkyl groups also

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R. E. Sammelson et al. / Bioorg. Med. Chem. 12 (2004) 3345–3355
potential to participate in hydrogen bonds whereas the
tert-butyl moiety does not.
An electronegative atom in the heterocyclic ring (pyr-
azole, TBO) is another feature common to both systems
and superimposed in the overlays. These atoms may
participate in hydrogen bonding relationships. The
configuration of the CN in the TBO may have little or
no significance since the pyrazole N is planar at that
point and while it may pucker on binding, the nature of
the pucker is not obvious. It seems that either enantio-
mer of the phenylTBO may be a suitable fit.
The phenyl rings have reasonably good overlap in the
phenylpyrazole and phenylTBO systems and the avail-
ability of a pi face is probably important to binding.
However there are some plane angle deviations. Con-
sidering a plane defined by the 1,2,6 carbons of the
phenyl rings, the angle defined by the pyrazole 2-N, 1-N,
and the phenyl plane is 55° whereas for the pyrazole 2-N,
1-N, and the 1-C of the phenyl is 125°. The corre-
sponding values in the TBO system are 68° (O (vicinal to
CN)-C-phenyl plane) and 111°. Rotation of the phenyl is
locked orthogonal to the pyrazole by the dichloro sub-
stituents. This orientation is easily achieved by the
phenylTBO. The commonality of these series is in the
availability of the pi face, even though the exact angle of
the pi face differs somewhat between the species. Pi–pi
interactions can occur in a sandwich style stacking con-
figuration, a parallel displaced configuration, or in a T-
shaped configuration. These latter two forms are close to
isoenergetic in simply substituted benzene rings with the
sandwich form being higher in energy. The strength of
the interaction is dependent on the ring substituents.31;32
As a result, there are a variety of pi interactions, which
can occur depending on the amino acids available in the
receptor pocket.
Figure 4. Comparison of 5-amino-4-tert-butyl-3-cyano-1-(2,6-di-
chloro-4-ethynylphenyl)pyrazole (pyrazole) and 4-tert-butyl-3-cyano-
1-(4-ethynylphenyl)-2,6,7-trioxabicyclo[2.2.2]octane (TBO) as mole-
cular structure (A) and wireframe individual (B) and overlay (C)
images.
confer outstanding potency and it seems that a sterically
hindered moiety at this position is significant with
respect to binding. Second, there is a potentially corre-
sponding electronegative atom in both heterocyclic
rings. Third, the cyano substituent enhances activity but
is not absolutely essential in either series.8;26;29;30 Finally,
4-ethynyl confers high activity in the phenylpyrazoles
and phenylTBOs.
A reasonably good overlay is achieved for the two series
by focusing on the tertiary C of the tert-butyl moieties,
the ring heteroatoms 2-N of pyrazole and O vicinal to
CN of TBO, and the phenyl rings (Fig. 4). The two
molecules overall occupy the same volume, that is
3
3
ꢀ
ꢀ
245.48 A for the phenylpyrazole and 238.57 A for the
phenylTBO. The volumes in this case were calculated
using the algorithm in the Search/Compare module of
InsightII (Accelrys Inc, San Diego, CA), each repre-
senting a grid of the van der Waals surface of the mol-
ecule in question. The X-ray structure of 44 has a
Other substitution patterns on the phenyl in the phenyl–
pyrazole series also confer good activity (Table 2). The
5-amino moiety on the pyrazole coupled with any 2 or 6
phenyl substituent acts to make the two rings twist out
of plane, thus making the orthogonal orientation ener-
getically available.
3
ꢀ
volume of 213.44 A , somewhat smaller than key phen-
ylpyrazole 41 due to the CF3 versus t-Bu moieties at the
4 position of the pyrazole rings, even though both have
similar hydrophobic character.
The tert-butyl moieties of phenylpyrazole 41 and the
corresponding TBO (A pyrazole and A TBO of Fig.
4) with the single attached C [(CH3)3C–C] occupy
4.7. Common binding subsites for 4-alkyl-1-phenylpyr-
azoles and 4-alkyl-1-phenylTBOs
3
ꢀ
very much the same space of about 76 A using the
volume algorithm as above. The difference in volumes in
Three binding subsites can be envisioned that recognize
and interact to varying degrees with the common
structural features of the antagonists. Quantitative
SAR-type analyses based on a wide variety of com-
pounds and large amounts of data (Ozoe and Akama-
tsu12, Brooks33 and references cited therein) have lead to
models for the binding site somewhat different than
those proposed here. From the present findings, the first
subsite interacts with the t-Bu or i-Pr substituent but in
the pyrazole series also accepts CF3, CF3S, CF3SO and
CF3SO2 almost equally well and as such appears to be
involved more in a hydrophobic and steric than an
electronic interaction. The second subsite is proposed to
undergo hydrogen bonding with pyrazole N-2 and TBO
3
ꢀ
the overlaid mode is 15.6 A . On comparing the volume/
charge relationship of model systems, the volume of
3
ꢀ
[CF3S(O)–CH3] is 73.22 A whereas that of [(CH3)3
3
ꢀ
C–CH3] is 80.89 A . If these are overlaid as C–S–C
versus C–C–C then the difference in the volumes is
3
ꢀ
15.28 A . Electronically, they both have a balance of
charges. Electron density mapped onto the electrostatic
potential shows the methyl hydrogens bearing a partial
positive charge. There is a partial negative charge on the
F atoms and the O in [CF3S(O)–CH3] and the same is
true for the C atoms in [(CH3)3C–CH3]. Despite these
similarities, there are also obvious differences between
the structural units, for example, the sulfoxide has the

R. E. Sammelson et al. / Bioorg. Med. Chem. 12 (2004) 3345–3355
3351
O-2. Support for pyrazole N-2 comes from the good
activity of 5-tert-butyl-2-(4-ethynylphenyl)pyrimidine
with an equivalent pyrimidine N-2.34 The pi face of the
phenyl moiety is probably important for binding. Finally,
the 3-cyano and 4-ethynyl substituents may be supple-
mental in binding since they enhance potency but are not
absolutely required in either the pyrazole or TBO series.
DCM once. The combined organic layers were dried,
filtered, and concentrated to give the crude al-
kylphenylpyrazoles, which were purified by crystalliza-
tion (DCM/hexane), preparative TLC or flash
chromatography (20% ethyl acetate in hexane). The
yields reported are for isolated product based on starting
aniline.
5.1.4. 5-Amino-3-cyano-1-(2,6-dichloro-4-trifluoromethyl-
phenyl)-4-ethylpyrazole (6).7 1H NMR d 7.78 (s, 2H),
3.49 (br s, 2H), 2.56 (q, J ¼ 7:5 Hz, 2H), 1.29 (t,
J ¼ 7:5 Hz, 2H).
5. Experimental
5.1. Synthetic procedures
5.1.1. General. Melting points (mp) are uncorrected.
Reagents from Aldrich (Milwaukee, WI) and solvents
from Fisher Scientific (Tustin, CA) were used without
5.1.5. 5-Amino-4-tert-butyl-3-cyano-1-(2,6-dichloro-4-tri-
fluoromethylphenyl)pyrazole (7). (72%): mp 159–161 °C;
1
further purification. H and 13C NMR spectra were ob-
1H NMR d 7.78 (s, 2H), 3.57 (br s, 2H), 1.51 (s, 9H); 13
C
tained on a 300 MHz spectrophotometer using deute-
riochloroform as solvent and standard where d ¼ 7:27
and 77.0, respectively. Mass spectra (MS) were obtained
by GC/MS on a Hewlett–Packard Model 5989A mass
spectrometer using electron impact (EI) and are reported
as m=z. Combustion analysis were performed in the ele-
mental analysis laboratory at the University of Califor-
nia, Berkeley. High-resolution mass spectra (HRMS)
were obtained from the University of Notre Dame.
NMR d 142.5, 136.5, 135.8, 134.2 (q, J ¼ 34 Hz), 126.1,
125.6, 121.9 (q, J ¼ 274 Hz), 115.9, 115.0, 30.6, 30.0. MS
m=z (% relative abundance) 361 (100), 376 [M]þ (12).
Anal. Calcd for C15H13Cl2F3N4: C, 47.76; H, 3.47; N,
14.85. Found: C, 47.66; H, 3.52; N, 14.62.
5.1.6. 5-Amino-3-cyano-1-(2,6-dichloro-4-trifluoromethyl-
phenyl)pyrazole (8).18 1H NMR d 7.79 (s, 2H), 6.06 (s,
1H), 3.80 (br s, 2H).
5.1.2. General procedure for synthesis of ethyl 2,3-
dicyanoalkanoates (D in Schemes 1 and 2). To a stirred
solution of ethyl cyanoacetate (10 mmol) and aldehyde
(10–12 mmol) in ethanol at room temperature was
added potassium cyanide (10 mmol). The reaction was
initially exothermic and went to completion within 4 h
but could stand overnight without problems. Water and
dichloromethane (DCM) were added and then it was
acidified with 2 M hydrochloric acid (6 mL). Caution:
traces of hydrogen cyanide can be produced and this
should always be carried out in a well-ventilated fume
hood. The layers were separated and the aqueous phase
was extracted twice more with DCM. Drying with so-
dium sulfate, filtering, and concentrating provided D in
excellent yields (89–100%), which was used without
further purification.
5.1.7. 5-Amino-3-cyano-1-(2,6-dichloro-4-trifluoromethyl-
1
phenyl)-4-methylpyrazole (9). (37%): mp 162–166 °C; H
NMR d 7.74 (s, 2H), 3.75 (br s, 2H), 2.08 (s, 3H); 13C
NMR d 147.5, 137.7, 137.5, 134.4 (q, J ¼ 34 Hz), 128.9,
128.4, 123.5 (q, J ¼ 271 Hz), 114.7, 101.8, 7.4. MS m=z
(% relative abundance) 334 [M]þ (100), 213 (13). Anal.
Calcd for C12H7Cl2F3N4: C, 43.01; H, 2.11; N, 16.72.
Found: C, 43.39; H, 2.41; N, 16.35.
5.1.8. 5-Amino-3-cyano-1-(2,6-dichloro-4-trifluoromethyl-
phenyl)-4-propylpyrazole (10). (55%): mp 132–134 °C;
1H NMR d 7.78 (s, 2H), 3.49 (br s, 2H), 2.51 (t, J ¼
7:4 Hz, 2H), 1.69 (sext, J ¼ 7:4 Hz, 2H), 0.99 (t, J ¼
7:4 Hz, 3H); 13C NMR d 144.2, 136.3, 135.8, 134.0 (q,
J ¼ 34 Hz), 127.2, 125.9, 121.9 (q, J ¼ 274 Hz), 113.5,
108.7, 24.4, 22.6, 13.2. MS m=z (% relative abundance)
333 (100), 362 [M]þ (16). Anal. Calcd for
C14H11Cl2F3N4: C, 46.30; H, 3.05; N, 15.43. Found: C,
46.35; H, 3.09; N, 15.59.
5.1.3. General procedure for synthesis of 4-alkyl-1-phen-
ylpyrazoles (Schemes 1 and 3). Concentrated sulfuric
acid (0.30 mL) or 2 M hydrochloric acid (2 mL) was ad-
ded to sodium nitrite (69 mg, 1 mmol) at room tempera-
ture followed after 10 min by glacial acetic acid
(0.25 mL). This nitrosyl bisulfate or nitrosyl chloride was
added to 2,6-dichloro-4-trifluoromethylaniline (230 mg,
1 mmol) or other anilines in glacial acetic acid (0.25 mL).
After 30 min this mixture was introduced to D (1 mmol)
in 40% aqueous acetic acid (1.67 mL) and after 1 h DCM
and water were added and the aqueous layer extracted a
second time with DCM. The combined organic layers
were made basic with careful addition of 10% ammonium
hydroxide. The aqueous layer was removed and the or-
ganic phase stirred rapidly 12 h–4 days with 50% aqueous
ammonium hydroxide at room temperature. The layers
were separated and the aqueous phase extracted with
5.1.9. 5-Amino-3-cyano-1-(2,6-dichloro-4-trifluoromethyl-
phenyl)-4-isopropylpyrazole (11). (48%): mp 158–160 °C;
1H NMR d 7.76 (s, 2H), 3.56 (br s, 2H), 2.89 (sept,
J ¼ 7:2 Hz, 1H), 1.37 (d, J ¼ 7:2 Hz, 6H); 13C NMR d
142.7, 136.4, 135.9, 134.2 (q, J ¼ 35 Hz), 126.1, 125.7,
121.9 (q, J ¼ 274 Hz), 114.9, 114.1, 24.2, 22.1. MS m=z
(% relative abundance) 347 (100), 362 [M]þ (18). Anal.
Calcd for C14H11Cl2F3N4: C, 46.30; H, 3.05; N, 15.43.
Found: C, 46.35; H, 3.20; N, 15.28.
5.1.10. 5-Amino-4-butyl-3-cyano-1-(2,6-dichloro-4-trifluo-
1
romethylphenyl)pyrazole (12). (44%): mp 83–85 °C; H
NMR d 7.77 (s, 2H), 3.54 (br s, 2H), 2.52 (t, J ¼ 6 Hz,

3352
R. E. Sammelson et al. / Bioorg. Med. Chem. 12 (2004) 3345–3355
2H), 1.62, (quint, J ¼ 7:4 Hz, 2H), 1.38, (sext,
J ¼ 7:4 Hz, 2H), 0.96, (t, J ¼ 7:4 Hz, 3H); 13C NMR d
143.6, 136.4, 134.9, 133.3 (q, J ¼ 34 Hz), 127.2, 126.1,
121.9 (q, J ¼ 274 Hz), 113.5, 109.6, 31.6, 22.5, 22.2, 13.8.
MS m=z (% relative abundance) 333 (100), 376 [M]þ
(17), 246 (11). Anal. Calcd for C15H13Cl2F3N4: C, 47.76;
H, 3.47; N, 14.85. Found: C, 48.09; H, 3.68; N, 14.47.
114.2, 114.0, 34.2, 32.2, 26.6, 25.5. MS m=z (% relative
abundance) 359 (100), 402 [M]þ (44), 333 (36). Anal.
Calcd for C17H15Cl2F3N4: C, 50.64; H, 3.75; N, 13.89.
Found: C, 50.73; H, 3.68; N, 14.04.
5.1.16. General procedure for halogenation of 8 (Scheme
4). Phenylpyrazole 8 (64 mg, 0.20 mmol) was dissolved in
acetonitrile (1 mL) and N-halosuccinimide (0.21 mmol)
was added at room temperature. After reacting for 1 h at
reflux (X ¼ I), 50 °C (X ¼ Br), or room temperature
(X ¼ C1) ethyl acetate and water were added. The sep-
arated organic layer was washed with water and brine,
dried and concentrated to provide the desired products
after purification by preparative TLC.
5.1.11. 5-Amino-4-isobutyl-3-cyano-1-(2,6-dichloro-4-tri-
fluoromethylphenyl)pyrazole (13). (62%): mp 120–121 °C;
1H NMR d 7.78 (s, 2H), 3.48 (br s, 2H), 2.40 (d,
J ¼ 7:2 Hz, 2H), 2.05–1.88 (m, 1H), 0.98 (d, J ¼ 6:7 Hz,
6H); 13C NMR d 144.2, 136.4, 135.9, 134.2 (q,
J ¼ 34 Hz), 127.9, 126.0, 121.9 (q, J ¼ 274 Hz), 113.6,
108.3, 31.7, 29.3, 22.0. MS m=z (% relative abundance)
333 (100), 376 [M]þ (12). Anal. Calcd for
C15H13Cl2F3N4: C, 47.76; H, 3.47; N, 14.85. Found: C,
47.91; H, 3.68; N, 14.60.
5.1.17. 5-Amino-4-chloro-3-cyano-1-(2,6-dichloro-4-tri-
fluoromethylphenyl)pyrazole (18). (94%): mp 187–
1
188 °C; H NMR d 7.80 (s, 2H), 3.92 (br s, 2H); 13C
NMR d 143.3, 136.4, 135.2, 134.8 (q, J ¼ 34 Hz), 127.3,
126.3, 121.9 (q, J ¼ 274 Hz), 111.4, 97.1. MS m=z (%
relative abundance) 354 [M]þ (100), 213 (17), 240 (14),
77 (14). Anal. Calcd for C11H14Cl3F3N4: C, 37.16; H,
1.13; N, 15.76. Found: C, 37.27; H, 1.27; N, 15.81.
5.1.12. 5-Amino-4-sec-butyl-3-cyano-1-(2,6-dichloro-4-
trifluoromethylphenylpyrazole (14). (75%): mp 144–
146 °C; H NMR d 7.78 (s, 2H), 3.44 (br s, 2H), 2.62
(sext, J ¼ 7:3 Hz, 1H), 1.75 (quint, J ¼ 7:4 Hz, 2H), 1.42
(d, J ¼ 7:2 Hz, 3H), 0.92 (t, J ¼ 7:2 Hz, 3H); 13C NMR
d 143.5, 136.4, 135.9, 134.1 (q, J ¼ 34 Hz), 126.1, 126.0,
121.9 (q, J ¼ 274 Hz), 114.0, 113.3, 31.3, 29.5, 19.9, 12.2.
MS m=z (% relative abundance) 347 (100), 376 [M]þ
(11). Anal. Calcd for C15H13Cl2F3N4: C, 47.76; H, 3.47;
N, 14.85. Found: C, 47.37; H, 3.54; N, 14.46.
1
5.1.18. 5-Amino-4-bromo-3-cyano-1-(2,6-dichloro-4-tri-
fluoromethylphenyl)pyrazole (19). (87%): mp 208–
1
210 °C; H NMR d 7.80 (s, 2H), 4.01 (br s, 2H); 13C
NMR d 144.9, 136.4, 135.3, 134.8 (q, J ¼ 34 Hz), 128.1,
126.3, 121.9 (q, J ¼ 274 Hz), 111.9, 81.1. MS m=z
(% relative abundance) 400 [M]þ (100), 213 (19), 77 (12).
Anal. Calcd for C11H14BrCl2F3N4: C, 33.03; H, 1.01; N,
14.01. Found: C, 33.22; H, 1.00; N, 14.11.
5.1.13. 5-Amino-3-cyano-1-(2,6-dichloro-4-trifluorometh-
ylphenyl)-4-(3-pentyl)pyrazole (15). (71%): mp 145–
1
147 °C; H NMR d 7.78 (s, 2H), 3.46 (br s, 2H), 2.41–
2.31 (m, 1H), 1.88–1.66 (m, 4H), 0.90 (t, J ¼ 7:4 Hz,
6H); 13C NMR d 144.1, 136.4, 135.9, 134.2 (q,
J ¼ 34 Hz), 126.5, 126.1, 121.9 (q, J ¼ 274 Hz), 114.0,
111.5, 39.2, 27.5, 12.2. MS m=z (% relative abundance)
361 (100), 333 (15), 390 [M]þ (7). Anal. Calcd for
C16H15Cl2F3N4: C, 49.12; H, 3.86; N, 14.32. Found: C,
49.30; H, 3.99; N, 14.13.
5.1.19. 5-Amino-3-cyano-1-(2,6-dichloro-4-trifluorometh-
ylphenyl)-4-iodopyrazole (20).7 1H NMR 7.80 (s, 2H),
3.98 (br s, 2H).
5.1.20. 5-Amino-1-(4-chlorophenyl)-3-cyano-4-isoprop-
ylpyrazole (21). (42%): 1H NMR 7.52–7.45 (m, 4H),
3.79 (br s, 2H), 2.84 (sept, J ¼ 7 Hz, 1H), 1.36 (d,
J ¼ 7 Hz, 6H); 13C NMR 141.5, 136.0, 134.6, 129.8,
125.5, 123.8, 114.7, 114.5, 24.2, 22.1. HRMS Calcd for
C13H14ClN4: 261.0907, found: 261.0888.
5.1.14. 5-Amino-3-cyano-1-(2,6-dichloro-4-trifluorometh-
ylphenyl)-4-neo-pentylpyrazole (16). (64%): mp 189–
1
191 °C; H NMR d 7.78 (s, 2H), 3.52 (br s, 2H), 2.40 (s,
2H), 1.02 (s, 9H); 13C NMR d 144.7, 136.4, 136.0, 134.2
(q, J ¼ 34 Hz), 128.9, 126.2, 121.9 (q, J ¼ 274 Hz),
114.0, 107.1, 37.0, 34.0, 29.2. MS m=z (% relative
abundance) 333 (100), 57 (28), 299 (21), 247 (18), 390
[M]þ (16), 375 (15). Anal. Calcd for C16H15Cl2F3N4: C,
49.12; H, 3.86; N, 14.32. Found: C, 49.16; H, 3.98; N,
14.16.
5.1.21. 5-Amino-4-tert-butyl-1-(4-chlorophenyl)-3-cyano-
1
pyrazole (22). (13%): H NMR 7.48 (s, 4H), 3.86 (br s,
2H), 1.46 (s, 9H); 13C NMR 141.3, 135.7, 134.9, 129.9,
126.2, 124.0, 115.7, 115.5, 30.7, 30.4. HRMS Calcd for
C14H16ClN4: 275.1063, found 275.1056.
5.1.15. 5-Amino-3-cyano-4-cyclohexyl-1-(2,6-dichloro-4-
trifluoromethylphenyl)pyrazole (17). (66%): mp 162–
5.1.22. 5-Amino-3-cyano-1-(4-trifluoromethylphenyl)-4-
isopropylpyrazole (23). (25%): 1H NMR 7.77 (d,
J ¼ 9 Hz), 7.73 (d, J ¼ 9 Hz), 3.88 (br s, 2H), 2.85 (sept,
J ¼ 7 Hz, 1H), 1.36 (d, J ¼ 7 Hz, 6H); 13C NMR 141.8,
140.5, 130.4 (q, J ¼ 33 Hz), 126.8 (q, J ¼ 5 Hz), 124.3,
124.1, 123.5 (q, J ¼ 270 Hz), 115.2, 114.4, 24.1, 22.0.
1
164 °C; H NMR d 7.77 (s, 2H), 3.48 (br s, 2H), 2.53–
2.43 (m, 1H), 1.91–1.88 (m, 4H), 1.79–1.71 (m, 3H),
1.41–1.33 (m, 3H); 13C NMR d 143.0, 136.3, 135.9, 134.2
(q, J ¼ 34 Hz), 126.0, 125.7, 121.9 (q, J ¼ 274 Hz),

R. E. Sammelson et al. / Bioorg. Med. Chem. 12 (2004) 3345–3355
3353
Anal. Calcd for C14H13F3N4: C, 57.14; H, 4.45; N,
19.04. Found: C, 56.79; H, 4.48; N, 18.79.
C15H14ClF3N4: C, 52.56; H, 4.12; N, 16.35. Found: C,
52.36; H, 4.36; N, 16.01.
5.1.23. 5-Amino-4-tert-butyl-3-cyano-1-(4-trifluorometh-
ylphenyl)pyrazole (24). (16%): 1H NMR 7.79 (d,
J ¼ 9 Hz), 7.72 (d, J ¼ 9 Hz), 3.92 (br s, 2H), 1.48 (s,
9H); 13C NMR 141.5, 140.3, 130.7 (q, J ¼ 33 Hz), 128.9,
125.3, 124.8, 124.5, 116.1, 115.4, 30.7, 30.3. Anal. Calcd
for C15H15F3N4: C, 58.44; H, 4.90; N, 18.17. Found: C,
58.10; H, 5.00; N, 17.85.
5.1.30. 5-Amino-3-cyano-1-(2,6-dichlorophenyl)-4-isoprop-
1
ylpyrazole (31). (9%): H NMR 7.51–7.41 (m, 3H), 3.48
(br s, 2H), 2.91 (sept, J ¼ 7 Hz, 1H), 1.40 (d, J ¼ 7 Hz,
6H); 13C NMR 142.5, 135.4, 132.7, 131.9, 129.0, 125.2,
114.5, 114.3, 24.3, 22.2. HRMS Calcd for C13H13Cl2N4:
295.0517, found 295.0495.
5.1.31. 5-Amino-4-tert-butyl-3-cyano-1-(2,6-dichlorophen-
yl)pyrazole (32). (7%): 1H NMR 7.53–7.44 (m, 3H), 3.59
(br s, 2H), 1.49 (s, 9H); 13C NMR 142.4, 135.4, 132.5,
132.0, 129.0, 125.1, 115.5, 115.4, 30.7, 30.4. HRMS
Calcd for C14H15Cl2N4: 309.0674, found 309.0669.
5.1.24. 5-Amino-3-cyano-1-(4-cyanophenyl)-4-isopropyl-
1
pyrazole (25). (12%): H NMR 7.82 (s, 4H), 3.80 (br s,
2H), 2.86 (sept, J ¼ 7 Hz, 1H), 1.39 (d, J ¼ 7 Hz, 6H);
13C NMR 141.5, 141.3, 133.6, 125.1, 124.0, 117.8, 115.9,
114.1, 112.0, 24.2, 22.1. HRMS Calcd for C14H14N5:
252.1249, found 252.1278.
5.1.32. 5-Amino-3-cyano-1-(2,6-dichloro-4-trifluorometh-
oxyphenyl)-4-isopropylpyrazole (33). (45%): 1H NMR
7.39 (s, 2H), 3.53 (br s, 2H), 2.90 (sept, J ¼ 7 Hz, 1H),
1.39 (d, J ¼ 7 Hz, 6H); 13C NMR 150.1, 142.7, 136.5,
131.4, 125.6, 121.2, 120.0 (q, J ¼ 260 Hz), 114.7, 114.1,
24.3, 22.2. Anal. Calcd for C14H11Cl2F3N4O2: C, 44.35;
H, 2.92; N, 14.78. Found: C, 44.45; H, 2.90; N, 14.62.
5.1.25. 5-Amino-4-tert-butyl-3-cyano-1-(4-cyanophenyl-
pyrazole (26). (15%): H NMR 7.83–7.75 (m, 4H), 3.96
(br s, 2H), 1.47 (s, 9H); 13C NMR 141.5, 141.0, 133.6,
125.0, 124.8, 117.7, 116.5, 115.2, 112.2, 30.7, 30.3. Anal.
Calcd for C15H15N5: C, 67.90; H, 5.70; N, 26.40. Found :
C, 67.77; H, 5.79; N, 26.29.
1
5.1.33. 5-Amino-4-tert-butyl-3-cyano-1-(2,4-dichloro-4-
1
5.1.26. 5-Amino-3-cyano-1-(2,4-dichlorophenyl)-4-isoprop-
ylpyrazole (27). (45%): 1H NMR 7.60 (d, J ¼ 1 Hz, 1H),
7.46–7.39 (m, 2H), 3.56 (br s, 2H), 2.88 (sept, J ¼ 7 Hz,
1H), 1.38 (d, J ¼ 7 Hz, 6H); 13C NMR 142.7, 136.9,
133.3, 132.9, 130.6, 130.5, 128.4, 124.5, 114.3, 114.2,
24.1, 22.1. Anal. Calcd for C13H12Cl2N4: C, 52.90; H,
4.10; N, 18.98. Found: C, 52.94; H, 4.08; N, 18.76.
trifluoromethoxyphenyl)pyrazole (34). (17%): H NMR
7.39 (s, 2H), 3.65 (br s, 2H), 1.48 (s, 9H); 13C NMR
150.1, 142.4, 136.6, 131.3, 125.6, 121.2, 120.0 (q,
J ¼ 260 Hz), 115.7, 115.1, 30.7, 30.3. HRMS Calcd for
C15H14Cl2F3N4O: 393.0497, found 393.0476.
5.1.34. 5-Amino-3-cyano-4-ethyl-1-(2,4,6-trichlorophen-
1
yl)pyrazole (35). (39%): H NMR 7.52 (s, 2H), 3.51 (br
5.1.27. 5-Amino-4-tert-butyl-3-cyano-1-(2,4-dichlorophen-
yl)pyrazole (28). (28%): 1H NMR 7.60 (d, J ¼ 1 Hz, 1H),
7.46–7.39 (m, 2H), 3.65 (br s, 2H), 1.48 (s, 9H); 13C
NMR 142.6, 136.9, 133.2, 133.1, 130.7, 130.5, 128.4,
124.4, 115.3, 115.2, 30.5, 30.2. Anal. Calcd for
C14H14Cl2N4: C, 54.38; H, 4.56; N, 18.12. Found: C,
54.41; H, 4.61; N, 18.06.
s, 2H), 2.54 (q, J ¼ 8 Hz, 2H), 1.27 (t, J ¼ 8 Hz, 3H);
13C NMR 143.5, 137.4, 135.9, 131.4, 129.0, 126.6, 113.7,
110.2, 16.1, 14.0. MS m=z (% relative abundance) 299
(100), 314 [M]þ (33), 212 (12), 264 (11). Anal. Calcd for
C12H9Cl3N4: C, 45.67; H, 2.87; N, 17.75. Found: C,
45.43; H, 2.95; N, 17.59.
5.1.35. 5-Amino-3-cyano-4-isopropyl-1-(2,4,6-trichloro-
phenyl)pyrazole (36). (42%): 1H NMR 7.51 (s, 2H),
3.56 (br s, 2H), 2.88 (sept, J ¼ 7 Hz, 1H), 1.37 (d,
J ¼ 7 Hz, 6H); 13C NMR 142.7, 137.4, 135.9, 131.5,
129.0, 125.3, 114.4, 114.2, 24.2, 22.2. MS m=z (% relative
abundance) 313 (100), 328 [M]þ (17). Anal. Calcd for
C13H11Cl3N4: C, 47.37; H, 3.36; N, 17.00. Found: C,
47.14; H, 3.51; N, 16.88.
5.1.28. 5-Amino-1-(2-chloro-4-trifluoromethylphenyl)-3-
cyano-4-isopropylpyrazole (29). (67%): 1H NMR 7.86
(s, 1H), 7.73 (d, J ¼ 8 Hz, 1H), 7.63 (d, J ¼ 8 Hz, 1H),
3.58 (br s, 2H), 2.90 (sept, J ¼ 7 Hz, 1H), 1.40 (d,
J ¼ 7 Hz, 6H); 13C NMR 143.0, 137.9, 133.4 (q,
J ¼ 34 Hz), 132.7, 130.5, 127.9, 125.0, 124.8, 122.6 (q,
J ¼ 270 Hz), 114.6, 114.2, 24.1, 22.0. HRMS Calcd for
C14H13ClF3N4: 329.0781, found 329.0777.
5.1.36. 5-Amino-4-tert-butyl-3-cyano-1-(2,4,6-trichloro-
phenyl)pyrazole (37). (37%): 1H NMR 7.52 (s, 2H),
3.59 (br s, 2H), 1.49 (s, 9H); 13C NMR 142.3, 137.5,
136.1, 131.4, 129.1, 125.6, 115.8, 115.2, 30.7, 30.4. MS
m=z (% relative abundance) 327 (100), 342 [M]þ (10).
Anal. Calcd for C14H13Cl3N4: C, 48.93; H, 3.81; N,
16.30. Found: C, 48.79; H, 3.86; N, 16.16.
5.1.29. 5-Amino-4-tert-butyl-1-(2-chloro-4-trifluorometh-
ylphenyl)-3-cyanopyrazole (30). (19%): 1H NMR 7.85 (s,
1H), 7.72 (d, J ¼ 8 Hz, 1H), 7.61 (d, J ¼ 8 Hz, 1H), 3.64
(br s, 2H), 1.48 (s, 9H); 13C NMR 142.7, 137.7, 133.4 (q,
J ¼ 33 Hz), 133.1, 130.6, 127.9, 125.0, 124.7, 123.6 (q,
J ¼ 270 Hz), 115.5, 115.2, 30.5, 30.2. Anal. Calcd for

3354
R. E. Sammelson et al. / Bioorg. Med. Chem. 12 (2004) 3345–3355
5.1.37. 5-Amino-1-(4-bromo-2,6-dichlorophenyl)-3-cyano-
1
4-isopropylpyrazole (38). (21%): H NMR 7.67 (s, 2H),
the candidate inhibitor added in dimethyl sulfoxide
(0.5% final concentration). Following incubation for
70 min at 25 °C with shaking, the samples were filtered
through Whatman GF/B paper [presoaked in 0.1%
polyethylenimine (v/v) for 1 h] and rinsed three times
with 5 mL of ice cold rinsing buffer (0.9% sodium
chloride w/v) using a Brandel 24-well harvester. Radio-
activity bound to membranes on the filters was mea-
sured with a liquid scintillation counter. Nonspecific
binding was determined in the presence of 0.5 lM-
endosulfan. Sigma Plot version 4.01 was used for least-
squares regression analysis to determine the concentra-
tion for 50% inhibition (IC50) with three or four deter-
minations for the standard error (SE) values.
3.50 (br s, 2H), 2.89 (sept, J ¼ 7 Hz, 1H), 1.38 (d,
J ¼ 7 Hz, 6H); 13C NMR 142.6, 136.1, 131.93, 131.87,
125.5, 124.9, 114.6, 114.1, 24.3, 22.2. Anal. Calcd for
C13H11BrCl2N4: C, 41.74; H, 2.96; N, 14.98. Found: C,
41.67; H, 2.89; N, 14.62.
5.1.38. 5-Amino-4-tert-butyl-3-cyano-1-(4-bromo-2,6-di-
chlorophenyl)pyrazole (39). (19%): 1H NMR 7.65 (s,
2H), 3.68 (br s, 2H), 1.45 (s, 9H); 13C NMR 142.5, 136.1,
131.8, 131.7, 125.3, 124.9, 115.5, 115.2, 30.6, 30.3.
HRMS Calcd for C14H14BrCl2N4: 386.9779, found
386.9798.
5.3. Toxicity to house flies
5.1.39. General procedure for synthesis of 1-(4-ethynyl-
phenyl)pyrazoles (Scheme 5). The aryl bromide
(100 lmol), tetrakis(triphenylphosphine)palladium (0)
(2.3 mg, 2 mol), and copper (1) iodide were placed in a
round-bottom flask under nitrogen. Toluene (2.3 mL)
and diisopropylamine (DIPA) (700 lL) were added and
the solution stirred for 20 min at room temperature be-
fore addition of (trimethylsilyl)acetylene (24.6 mg,
250 lmol). After heating to 60 °C for 14 h, the reaction
was cooled and the dark mixture was filtered and
evaporated to dryness, redissolved in toluene and fil-
tered through a short silica gel column. The resulting
product was mixed with potassium carbonate (100 mg)
and methanol (6 mL) for 1 h. Water and dichlorome-
thane were added and the organic layer was concen-
trated and purified via preparative TLC.
Topical LD50 values or mortality at a discriminating
dose were determined for adult female house flies 24 h
after topical application of the test compound in 0.5 lL
acetone to the ventrum of the abdomen.14 For synergism
studies, PB was applied topically to the thorax at 250 lg/
g 2 h before the test compound to inhibit most cyto-
chrome P450-catalyzed oxidative detoxification.5;14
Acknowledgements
The project described was supported by grant R01
ES08419 from the National Institute of Environmental
Health Sciences (NIEHS), NIH and its contents are
solely the responsibility of the authors and do not rep-
resent the official views of NIEHS, NIH. We thank
Ligong Chen, Daryl Wong, Motohiro Tomizawa,
Nanjing Zhang and Gary Quistad of this laboratory and
Paolo Cabras of the University of Cagliari, Italy for
helpful suggestions.
5.1.40. 5-Amino-3-cyano-1-(2,6-dichloro-4-ethynylphen-
yl)-4-isopropylpyrazole (40). (89%): 1H NMR 7.60 (s,
2H), 3.47 (br s, 2H), 3.32 (s, 1H), 2.90 (sept, J ¼ 7 Hz,
1H), 1.39 (d, J ¼ 7 Hz, 6H); 13C NMR 142.5, 135.3,
133.0, 132.2, 126.6, 125.5, 114.7, 114.2, 82.0, 79.8, 24.3,
22.2. HRMS Calcd for C15H13Cl2N4: 319.0517, found
319.0528.
5.1.41. 5-Amino-4-tert-butyl-3-cyano-1-(2,6-dichloro-4-
ethynylphenyl)pyrazole (41). (84%): 1H NMR 7.58 (s,
2H), 3.63 (br s, 2H), 3.32 (s, 1H), 1.47 (s, 9H); 13C NMR
142.4, 135.3, 132.8, 132.1, 126.6, 125.3, 115.6, 115.2,
82.1, 79.7, 30.6, 30.3. HRMS Calcd for C16H15Cl2N4:
333.0674, found 333.0667.
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