Imine derivatives as new potent and selective CB2 cannabinoid receptor agonists with an analgesic action

Article abstract of DOI:10.1016/j.bmc.2007.10.087

In this study, a novel series of CB₂ receptor agonist imine derivatives, 1-6, was synthesized and evaluated for activity against the CB₂ receptor. In a previous paper we reported the synthesis and SARs of thiazole derivative 1, a pot

Full text of DOI:10.1016/j.bmc.2007.10.087

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 1111–1124
Imine derivatives as new potent and selective CB2
cannabinoid receptor agonists with an analgesic action
Hiroshi Ohta,a,* Tomoko Ishizaka,a Makoto Tatsuzuki,a Mitsukane Yoshinaga,a
Izumi Iida,b Tomomi Yamaguchi,c Yasumitsu Tomishima,c Nobuko Futaki,c
Yoshihisa Todac and Shuji Saitoa
aMedicinal Chemistry Laboratories, Taisho Pharmaceutical Co., Ltd, 1-403 Yoshino-cho, Kita-ku,
Saitama-shi, Saitama 331-9530, Japan
bDrug Safety and Pharmacokinetics Laboratories, Taisho Pharmaceutical Co., Ltd, 1-403 Yoshino-cho,
Kita-ku, Saitama-shi, Saitama 331-9530, Japan
cMolecular Function and Pharmacology Laboratories, Taisho Pharmaceutical Co., Ltd, 1-403 Yoshino-cho,
Kita-ku, Saitama-shi, Saitama 331-9530, Japan
Received 10 September 2007; revised 26 October 2007; accepted 28 October 2007
Available online 1 November 2007
Abstract—In this study, a novel series of CB2 receptor agonist imine derivatives, 1–6, was synthesized and evaluated for activity
against the CB2 receptor. In a previous paper we reported the synthesis and SARs of thiazole derivative 1, a potent CB2 receptor
agonist, but we had not assessed chemical modifications of the 5-membered heteroring of 1. In the present study, we therefore tried
chemically modifying the 5-membered heteroring of 1 in an attempt to further improve binding affinity for the CB2 receptor. In the
course of making the structural modifications, we discovered that a novel pyrazole derivative 6b (CBS0550) had high affinity for the
CB2 receptor (IC50 = 2.9 nM, EC50 = 1.8 nM, Emax = 85%), high selectivity for CB2 (CB1 IC50/CB2 IC50 = 1400), and good physi-
cochemical properties (solubility in water: 5.9 mg/100 mL at 25 ꢀC). Oral administration of 6b to rats at a dose of 10 mg/kg resulted
in significant plasma concentrations, and orally administered compound 6b significantly reversed mechanical hyperalgesia in the
Randall–Selitto model of inflammatory pain in rats.
ꢁ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
The discovery and identification of two distinct cannab-
inoid receptors, the CB1 receptor3 and the CB2 recep-
tor,4 stimulated a rebirth of interest in the medicinal
chemistry of the cannabinoids. The CB1 receptor is
abundant in the CNS and is present to a lesser extent
in other tissues, and the central effects of the cannabi-
noids are related to the CB1 receptor. The CB2 receptor,
on the other hand, is found wholly in the periphery and
is primarily associated with cells of the immune sys-
tem.5–7 Recently, however, the CB2 receptor has also
been found in CNS tissue.8 Both receptors are members
of the G-protein-coupled superfamily of receptors, and
they share 68% homology with one another at the trans-
membrane level and 44% homology overall.4 Although
the development of highly selective CB2 receptor ligands
is important to exploration of some of the physiological
effects of cannabinoids, such as their immunosup-
pressive, anti-inflammatory, and antinociceptive effects,
a few compounds that are selective for the CB2 recep-
tor, such as aminopyrimidine9 (GW842166X, CB2
Although Cannabis sativa L. (marijuana) has a long his-
tory of use for medicinal and recreational purposes,
more knowledge about marijuana has been acquired in
the last three decades than the entire last century. Del-
ta9-tetrahydrocannabinol (delta9-THC), which contains
tricyclic structures derived from the benzopyrane, has
been isolated from marijuana as its major psychoactive
component.1 Cannabinoids, whose definition has now
been extended to include THC analogues, have a wide
range of biological properties, including analgesic,
anti-inflammatory, antiemetic, anticonvulsive, and anti-
cancer properties.2 However, therapeutic use of cannab-
inoids has been limited because of the difficulty of
separating their beneficial and psychotropic activities.
Keywords: CB2 agonist; Imine; Inflammatory; Pain; Analgesic; CB2.
*
Corresponding author. Tel.: +81 48 663 1111; fax: +81 48 652
7254; e-mail: hiroshi.ohta@po.rd.taisho.co.jp
0968-0896/$ - see front matter ꢁ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.10.087

1112
H. Ohta et al. / Bioorg. Med. Chem. 16 (2008) 1111–1124
EC50 = 50 nM, CB1 EC50 > 30,000 nM), 3-carbonylin-
dole10 (A-796260, CB2 IC50 = 0.77 nM, CB1 IC50
no-4-methyl-5-(1-methylethyl)-1,3-oxazole 16. Amino
oxazole 16 was converted to oxazole derivative 3 by
the same procedure as from 7 to 2.
=
330 nM), and aminoalkylindole11 (AM1241, CB2
Ki = 3.4 nM, CB1 Ki = 280 nM), are known (Fig. 1).
Scheme 3 illustrates the synthesis of the isothiazole
derivative (4). 3-Amino-5-tert-butylisothiazole 22 was
obtained from pinacolone 20 by Hackler’s procedure.15
(2Z)-3-chloro-4,4-dimethylpent-2-enenitrile (21) was
prepared from pinacolone 20 in a one-pot procedure,
with a 100% yield, and ring closure of 21 resulted in for-
mation of the 3-aminoisothiazole 22. Aminoisothiazole
22 was converted to the isothiazole derivative 4 by the
same procedure as from 7 to 2.
In this paper, we report the synthesis and SARs of imine
derivatives 1–6, which are novel CB2 receptor ligands. In
a previous paper we described the evaluation of thiazole
derivative 1 (IC50 = 13 nM, EC50 = 10 nM, Emax = 91%,
CB1 IC50/CB2 IC50 = 270) and its analogue as potent
and selective CB2 agonists,12 but we had not assessed
chemical modifications of thiazole ring of 1. In the pres-
ent study, we tried chemically modifying the heteroring
of 1 in attempt to further improve binding affinity and
selectivity for the CB2 receptor.
Scheme 4 shows the synthesis of the isoxazole derivative
(5). Aminoisoxazole 26, a commercially available com-
pound (Wako Pure Chemical Industries, Ltd), was con-
verted to isoxazole derivative 5 by the same procedure as
from 7 to 2.
2. Chemistry
Scheme 1 illustrates the synthesis of the 1,3,4-thiadiazole
derivative (2). 2-Amino-5-tert-butyl-1,3,4-thiadiazole
(7), a commercially available compound (Aldrich Chem-
ical Company, Inc.), was treated with trifluoroacetic
anhydride to obtain 8, with a 91% yield. The 3-position
of the thiadiazole ring of 8 was alkylated, followed by
deprotection under basic conditions, producing the
intermediate 10. Acylation of intermediate 10 by treat-
ment with 3-trifluoromethylbenzoyl chloride and Et3N
converted it to 1,3,4-thiadiazole (2), with a 49% yield.
Scheme 5 illustrates the preparation of pyrazole deriva-
tives 6a–f. Cyclopropylmethyl alcohol 30 was treated
with MsCl and Et3N to yield mesylate 31. Mesylate 31
was transformed into alkylhydrazine 32 by treatment
with hydrazine, with a 53% yield. Cyclization of 32 by
treatment with pivaloylacetonitrile yielded aminopyraz-
ole 33, with a 42% yield. Aminopyrazole 33 was treated
with trifluoroacetic anhydride and pyridine to obtain 34,
with a 100% yield. The 1-position of the pyrazole ring of
34 was alkylated, followed by deprotection under basic
conditions, producing the intermediate 36. The interme-
diate 36 was easily transformed into pyrazole derivatives
6a–f in one step by acylation of the intermediate 36 by
treatment with the corresponding aryl carbonyl chlo-
rides and Et3N.
Scheme 2 shows the synthesis of the oxazole derivative
(3). 2-Amino-4-methyl-1,3-oxazole (12) was obtained
by reaction between hydroxyacetone (11) and cyana-
mide, with a 48% yield.13 Friedel–Crafts acylation at
the 5-position of the oxazole ring was then performed
to obtain 13,14 and protection of the amino group in
the 2-position of oxazole 13 was achieved with a Boc
group. Compound 14 was treated with MeLi to obtain
15, with a 90% yield. Reduction and deprotection of
15 with Et3SiH under acidic conditions yielded 2-ami-
Scheme 6 shows the preparation of the pyrazole deriva-
tives 6g–j. 2-Alkyl-3-amino-5-tert-butylpyrazoles 40g–j
were synthesized from the corresponding alcohols,
37g–j, by the same procedure as from 30 to 33. Acyla-
tion of 40g–j was performed by treatment with
2-fluoro-3-trifluoromethylbenzoyl chloride and Et3N.
Finally, methylation at the 1-position of the pyrazole
ring 41g–j yielded 6g–j.
OH
OH
OH
3. Results and discussion
O
HO
Delta 9-THC
CP 55,940
The affinity of imine derivatives 1–5, 6a–j and CP
55,940,16 a nonselective cannabinoid receptor agonist
for CB2 receptor, was evaluated on the basis of
[3H]CP 55,940 binding to membranes of Chinese ham-
ster ovary (CHO) cells expressing the human CB2 recep-
tor, and the affinity of compounds 1, 6b–e, 6g–j and CP
55,940 for the CB1 receptor was evaluated on the basis
of [3H]CP 55,940 binding to membranes of CHO cells
expressing the human CB1 receptor. The agonist activi-
ties of 1, 6b–e, 6g–j and CP 55,940 were evaluated by
using a functional assay based on [35S]GTPcS binding
to membranes of CHO cells expressing the human CB2
receptor. Emax was compared to the maximal response
to CP 55,940, a full agonist of the human CB2
receptor.
CF3
O
O
Cl
O
N
N
N
H
N
O
N
H
N
Cl
GW842166X
A-796260
O
I
N
N
NO2
AM1241
Figure 1. Structure of Delta9-THC, CP 55,940, GW842166X, A-796260,
and AM1241.

H. Ohta et al. / Bioorg. Med. Chem. 16 (2008) 1111–1124
1113
N
S
N
N
N
NH2
a
NH
O
c
b
d
N
S
N
S
2
N
N
S
CF3
N
O
NH
CF3
7
8
9
10
Scheme 1. Reagents and conditions: (a) (CF3CO)2O, toluene, rt, 91%; (b) cyclopropylmethylbromide, NaI, K2CO3, DMF, rt, 91%; (c) 2 M NaOH,
THF, 65 ꢀC, 99%; (d) 3-trifluoromethylbenzoyl chloride, Et3N, CHCl3, rt, 49%.
N
O
O
N
O
N
N
d
a
b
c
NHBoc
NH2
NHBoc
NH2
OH
O
O
HO
11
12
13
O
14
15
O
N
N
O
e
f
NH
O
h
CF3
g
i
NH2
N
N
O
3
O
CF3
N
NH
O
O
16
17
18
19
Scheme 2. Reagents and conditions: (a) NH2CN, H2O, 45 ꢀC, 48%; (b) Ac2O, AlCl3, CHCl3, 0 ꢀC, 10%; (c) Boc2O, DMAP, THF, 60 ꢀC, 22%; (d)
MeLi, THF, À78 ꢀC, 90%; (e) Et3SiH, TFA, rt, 91%; (f) (CF3CO)2O, pyridine, CHCl3, rt, 82%; (g) cyclopropylmethylbromide, NaI, NaH, DMF,
60 ꢀC, 10%; (h) 2 M NaOH, MeOH, rt, 100%; (i) 3-trifluoromethylbenzoyl chloride, Et3N, CHCl3, rt, 22%.
N
Cl
O
S
N
c
a
NH
O
b
S
CN
NH2
CF3
20
21
22
23
e
CF3
d
f
N
N
4
S
S
N
O
NH
24
25
Scheme 3. Reagents and conditions: (a) POCl3, dichloroethane, (H2NOH)2H2SO4, DMF, 85 ꢀC, 100%; (b) (i) NaS; 1 M HCl, EtOH, rt; (ii) NH3aq,
NaClO, rt, 9%; (c) (CF3CO)2O, pyridine, CHCl3, rt, 82%; (d) cyclopropylmethylbromide, NaI, NaH, DMF, 60 ꢀC, 17%; (e) K2CO3, MeOH, H2O, rt,
95%; (f) 3-trifluoromethylbenzoyl chloride, Et3N, CHCl3, rt, 44%.
N
N
O
O
c
a
b
d
NH2
NH
O
5
N
N
O
O
CF3
N
O
NH
CF3
26
27
28
29
Scheme 4. Reagents and conditions: (a) (CF3CO)2O, pyridine, CHCl3, rt, 98%; (b) cyclopropylmethylbromide, NaI, NaH, DMF, rt, 11%; (c) K2CO3,
MeOH, H2O, rt, 100%; (d) 3-trifluoromethylbenzoyl chloride, Et3N, CHCl3, rt, 52%.
In a previous paper we reported the synthesis and SARs
of thiazole derivative 1 and its analogue, potent CB2
receptor agonists.12 However, we had not assessed
chemical modifications of the 5-membered heteroring
of 1. Since we thought that the 5-membered heteroring
acted as a linker and that fine adjustment of the size
of heteroring would affect affinity for the CB2 receptor,
we tried chemically modifying the 5-membered heteror-
ing of 1 in attempt to further improve binding affinity
for the CB2 receptor. A previous SAR study of thiazole
derivatives suggested (1) that introduction of a cyclopro-
pylmethyl group at the 3-position of the thiazole ring
was suitable to obtain a high affinity for the CB2 recep-
tor, (2) that the presence of a bulky lipophilic group like
tert-butyl at the 5-position of the thiazole ring was
important to obtaining high affinity for the CB2 recep-
tor, and (3) that a 3-trifluoromethylphenyl amide
yielded high affinity and selectivity for the CB2 receptor.
As shown in Table 1, we assessed 5-membered heteror-
ing derivatives which had a cyclopropylmethyl group,
a tert-butyl group, and a 3-trifluoromethylphenyl amide
at corresponding positions, respectively. We synthesized
5-isoproryl oxazole instead of 5-tert-butyl oxazole be-
cause of the difficulty of synthesizing. The oxazole (3),

1114
H. Ohta et al. / Bioorg. Med. Chem. 16 (2008) 1111–1124
N
c
N
d
N
a
OMs
b
NHNH2
OH
N
NH2
NH
O
CF3
30
31
32
33
34
e
f
N
N
g
N
N
6a-f
N
O
NH
CF3
35
36
Scheme 5. Reagents and conditions: (a) MsCl, Et3N, CHCl3, rt, 96%; (b) NH2NH2H2O, EtOH, rt, 53%; (c) pivaloylacetonitrile, EtOH, reflux, 42%;
(d) (CF3CO)2O, pyridine, CHCl3, rt, 100%; (e) Me2SO4, NaHCO3, toluene, 80 ꢀC, 14%; (f) 2 M NaOH, MeOH, rt, 100%; (g) ArCOCl, Et3N,
CHCl3, rt.
R
N
R
N
F
CF3
c
N
d
a
OMs
R
b
NHNH2
OH
R
e
N
NH2
6g-j
NH
O
R
37g-j
38g-j
39g-j
40g-j
41g-j
Scheme 6. Reagents and conditions: (a) MsCl, Et3N, CHCl3, rt; (b) NH2NH2H2O, EtOH, rt; (c) pivaloylacetonitrile, EtOH, reflux; (d) 2-fluoro-3-
trifluoromethylbenzoyl chloride, Et3N, CHCl3, rt; (e) Me2SO4, NaHCO3, toluene, 80 ꢀC.
isothiazole (4), isoxazole (5), and pyrazole (6a) deriva-
tives had good affinity for the CB2 receptor (3:
IC50 = 24 nM, 4: IC50 = 9.5 nM, 5: IC50 = 46 nM, and
6a: IC50 = 51 nM), but the thiadiazole derivative (2)
exhibited lower affinity for the CB2 receptor than thia-
zole derivative (1 vs 2). These findings suggested that
CB2 binding affinity increased as the size of the 5-mem-
bered heteroring increased (ring size: 6a < 3, 5 < 1, 4),
but that too large heteroring was unsuitable for obtain-
ing high affinity for the CB2 receptor. The 5-membered
heteroring acts as a linker, and fine adjustment of the
size of the heteroring affected affinity for the CB2
receptor.
100 mL at 25 ꢀC). These findings suggested that the elec-
tron density on the benzene ring affects the interaction
between the CB2 receptor and its ligand. Next modifica-
tion focused on the cyclopropylmethyl group at the
2-position of the pyrazole ring. Compounds that con-
tained a cycloalkyl group larger than a cyclopropylm-
ethyl group exhibited high CB2 binding affinity (6g–h).
Alkoxyalkyl groups, which were suitable functional
groups at the 3-position of thiazole derivatives for
obtaining high affinity for the CB2 receptor,12 were eval-
uated. Compounds 6i–j exhibited good affinity for the
CB2 receptor, the same as compound 6b, but they exhib-
ited lower selectivity for the CB2 receptor than 6b, and
compounds 6h and 6j were partial agonists (6h:
Emax = 67%, 6j: Emax = 76%). The results of structural
transformations indicated that the most suitable func-
tional group at this site for selectivity and agonist activ-
ity was a cyclopropylmethyl group (6b).
Compounds 1–5 are neutral lipophilic molecules and
have very low solubility in water (solubility in water 1:
<0.01 mg/100 mL at 25 ꢀC). By contrast, pyrazole deriv-
ative 6a is basic molecule and will probably have good
solubility in water. We tried assessing its analogues in
attempt to discover compounds with high affinity for
the CB2 receptor and good physicochemical properties,
using 6a as lead compound. Since a previous SAR study
of thiazole derivatives suggested that the 2-substituted-
5-trifluoromethylphenyl or 2-substituted-3-trifluoro-
methylphenyl amide exhibited high affinity for the CB2
receptor,12 we examined 2,3- or 2,5-disubstituted phenyl
compounds 6a–f (Table 2). The 2,3-disubstituted phenyl
compounds (6b–d) exhibited higher CB2 binding affinity
than 2,5-disubstituted phenyl compounds (6e–f), and
compounds containing electron-withdrawing groups,
such as fluorine and chlorine, had higher affinity than
compounds containing electron-donating groups, such
as methyl group (6b–c vs 6d, 6e vs 6f). 2-Fluoro-3-tri-
fluoromethylphenyl compound 6b exhibited particularly
high affinity and selectivity for the CB2 receptor and had
good solubility in water (solubility in water 6b: 5.9 mg/
Compound 6b, which showed high affinity and selectiv-
ity for the CB2 receptor and had good solubility in
water, exhibited good metabolic stability in human
and rat liver microsomes (human metabolic stability
92% and rat metabolic stability 86%).17 we therefore
evaluated plasma levels of 6b. Table 3 shows the phar-
macokinetic parameters of 6b following oral administra-
tion in rats at a dose of 10 mg/kg.
The maximum plasma concentration (Cmax
)
of
545 ng/mL was reached 1.67 h after the oral adminis-
tration of 6b at a dose of 10 mg/kg. The Cmax of 6b
was about twofold higher than that of thiazole deriv-
ative 1,12 and the corresponding AUC0–24 h value was
2160 ng h/mL. The oral administration of 6b at a
dose of 10 mg/kg (po) in rats resulted in significant
plasma concentration.

H. Ohta et al. / Bioorg. Med. Chem. 16 (2008) 1111–1124
1115
Table 1. Pharmacological profile of imine derivatives
4. Conclusions
In this paper, we reported the synthesis and SARs of no-
vel CB2 receptor agonists. The SAR study showed that
the 5-membered heteroring acted as a linker, and that
the functional groups at the 2-position in the pyrazole
ring and the Ar group in the amide greatly affected affin-
ity and selectivity for the CB2 receptor. It was also found
(1) that CB2 binding affinity increased as the size of
5-membered heteroring increased, but that too large size
of a heteroring was unsuitable for achieving high affinity
for the CB2 receptor, (2) that introduction of a cyclopro-
pylmethyl group at the 2-position of the pyrazole ring is
important to obtaining high selectivity for the CB2
receptor, and (3) that a 2-substituted-3-trifluoromethyl-
phenyl amide yielded high affinity for the CB2 receptor.
These studies led to compound 6b (CBS0550), which
showed combine good potency for the CB2 receptor
and excellent pharmacokinetics in rats. Furthermore,
when administered orally, compound 6b significantly
reversed mechanical hyperalgesia in the Randall–Selitto
model of inflammatory pain in rats. We think that
CBS0550 may be useful for exploring the function of
the CB2 receptor and for the treatment of pain.
X
CF3
O
Compound
X
Binding
affinity IC50
(nM)
Agonist activity
a
CB2
CB1 CB2 EC50 Emax
(nM)
(%)
1
13
3500 10
91
N
S
N
N
N
N
N
N
2
300
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
N
S
N
3
24
N
O
5. Experimental
5.1. Chemistry
4
9.5
N
N
N
Melting points were determined on a Mettler FP-61 or a
Yanaco MP-500D melting point apparatus. NMR spec-
tra were recorded at 200, 300 or 600 MHz using a Var-
ian Instruments Gemini 2000, a Varian Instruments
INOVA 300 or a JEOL ECA600 with tetramethylsilane
as an internal standard. Electron impact (EI) mass spec-
tra and Chemical ionization (CI) were taken on a Micro-
mass Platform GCI mass spectrometer. Electrospray
ionization (ESI) mass spectra were taken on a Micro-
mass Platform LC mass spectrometer. Elemental analy-
ses were performed on EA2400 elemental analyzers, and
the results were within 0.4% of calculated values. Reac-
tions were monitored by TLC analysis using Merck
silica gel 60F-254 thin-layer plates. Column chromato-
graphy was carried out on silica gel Wako Pure Chemical
C-200 and NH silica gel Fuji Silicia chromatorex
DM1020.
S
O
N
5
46
51
6a
a Compared to maximal response of CP 55,940, a full agonist of human
CB2 receptor.
5.1.1. N-(5-tert-Butyl-1,3,4-thiadiazol-2-yl)-2,2,2-trifluo-
roacetamide (8). To a mixture of 7 (3.0 g, 19 mmol) and
toluene (15 mL) was added trifluoroacetic anhydride
(4.8 g, 23 mmol) with ice-cooling and the mixture was
stirred for 1 h. To the reaction mixture was added water
and the mixture was extracted with ethyl acetate. The
organic layer was evaporated in vacuo and purified by
silica gel column chromatography (eluent: hexane/ethyl
acetate = 7:1) to give 4.4 g (yield; 91%) of 8 as colorless
powder: 1H NMR (200 MHz, chloroform-d) d ppm 1.48
(s, 9H); MS(ESI) m/z 254 (M+H).
Compound 6b also showed high affinity for the rat
CB2 receptor, displacing [3H]CP 55,940 binding to
rat spleen membranes with an IC50 value of 1.4 nM,
and thus 6b did not show species differences in affinity
for the CB2 receptor. Thus, the analgesic effect of
compound 6b was assessed in the Randall–Selitto test
in rats (Fig. 2).18 Oral administration of 6b at 10 mg/
kg and 30 mg/kg dose-dependently and significantly
reversed mechanical hyperalgesia in the Randall–Selit-
to test at 1 h after administration, and the antinoci-
ceptive effect lasted at least 3 h after administration.
These results indicate that compound 6b has an anal-
gesic effect in rats.
5.1.2. N-[5-tert-Butyl-3-(cyclopropylmethyl)-1,3,4-thia-
diazol-2(3H)-ylidene]-2,2,2-trifluoroacetamide (9). To a
mixture of 8 (1.0 g, 4.0 mmol), potassium carbonate

1116
H. Ohta et al. / Bioorg. Med. Chem. 16 (2008) 1111–1124
Table 2. Pharmacological profile of pyrazole derivatives
R
N
N
N
O
Ar
Compound
Ar
R
Binding affinity IC50
(nM)
Agonist activity
a
Emax (%)
CB2
51
2.9
3.7
11
CB1
CB2 EC50 (nM)
6a
6b
3-CF3–Ph
CH2-(cyclopropyl)
CH2-(cyclopropyl)
CH2-(cyclopropyl)
CH2-(cyclopropyl)
CH2-(cyclopropyl)
CH2-(cyclopropyl)
CH2-(cyclobutyl)
CH2-(cyclopentyl)
C2H4–OEt
—
—
1.8
—
85
82
73
90
—
96
67
82
76
100
2-F-3-CF3–Ph
2-Cl-3-CF3–Ph
2-Me-3-CF3–Ph
2-F-5-CF3–Ph
2-Me-5-CF3–Ph
2-F-3-CF3–Ph
2-F-3-CF3–Ph
2-F-3-CF3–Ph
2-F-3-CF3–Ph
4000
2200
1300
3700
—
6c
6d
1.5
4.4
6e
23
88
8.8
—
6f
6g
1.7
940
810
870
760
0.32
0.79
1.1
6h
6i
1.1
3.7
2.6
0.82
6j
CP 55,940
C2H4–OiPr
0.85
0.86
2.1
a Compared to maximal response of CP 55,940, a full agonist of human CB2 receptor.
Table 3. Pharmacokinetic parameters of 6b following oral administration to rats at a dose of 10 mg/kg
Compound
Dose (mg/kg)
10
Tmax (h)
Cmax (ng/mL)
t1/2 (h)
2.47 0.88
AUC0–8 h (ng h/mL)
2160 293
6b
1.67 0.58
545 170
Each value represents the mean SD of three animals.
0.42–0.69 (m, 4H), 1.24–1.42 (m, 1H), 1.43 (s, 9H),
4.25 (d, J = 7.5 Hz, 2H); MS(ESI) m/z 308 (M+H).
160
1h
3h
140
120
100
80
5.1.3. 5-tert-Butyl-3-(cyclopropylmethyl)-1,3,4-thiadiazol-
2(3H)-imine (10). To a mixture of 8 (1.1 g, 3.6 mmol)
and THF (10 mL) was added 2 M NaOH (5.0 mL,
10 mmol), and the mixture was stirred at 65 ꢀC for 1 h.
The organic layer was dried over sodium sulfate, evapo-
rated in vacuo, and purified by silica gel column chro-
matography (eluent: hexane/ethyl acetate = 7:1) to give
0.75 g (yield; 99%) of 9 as colorless oil: 1H NMR
(200 MHz, chloroform-d) d ppm 0.30–0.59 (m, 4H),
1.26 (s, 9H), 1.16–1.37 (m, 1H), 3.73 (d, J = 7.0 Hz,
2H); MS(ESI) m/z 212 (M+H).
***
***
**
60
40
20
0
3
10
6b
30
(mg/kg, p.o.)
5.1.4. N-[5-tert-Butyl-3-(cyclopropylmethyl)-1,3,4-thia-
diazol-2(3H)-ylidene]-3-(trifluoromethyl)benzamide (2).
To a mixture of 10 (0.10 g, 0.47 mmol), triethylamine
(0.027 g, 0.47 mmol), and CHCl3 (1.0 mL) was added
3-trifluoromethylbenzoyl chloride (0.098 g, 0.47 mmol)
and the mixture was stirred for 1 h. To the reaction
mixture was added 2 M NaOH and the mixture was
extracted with CHCl3. The organic layer was evapo-
rated in vacuo and purified by silica gel column chro-
matography (eluent: hexane/ethyl acetate = 2:1) to give
0.090 g (yield; 49%) of 2 as colorless oil: 1H NMR
(200 MHz, chloroform-d) d ppm 0.50–0.68 (m, 4H),
1.36–1.52 (m, 1H), 1.44 (s, 9H), 4.32 (d, J = 7.5 Hz,
2H), 7.57 (t, J = 7.7 Hz, 1H), 7.75 (d, J = 7.7 Hz,
1H), 8.47 (d, J = 7.7 Hz, 1H), 8.57 (s, 1H); MS(ESI)
m/z 384 (M+H); Anal. Calcd for C18H20F3N3OS: C,
56.38; H, 5.26; N, 10.96. Found: C, 56.39; H, 5.24;
N, 10.72.
Figure 2. Effect of 6b (3, 10, and 30 mg/kg, po) in the Randall–Selitto
model of inflammatory pain in rats. Data are expressed as means
SEM of seven animals. **p < 0.01, ***p < 0.001 compared to vehicle-
treated animals (Dunnett’s test). %MPE = (nociceptive threshold post
drug À nociceptive threshold control)/(nociceptive threshold base-
line À nociceptive threshold control) · 100.
(0.66 g, 4.8 mmol), sodium iodide (0.060 g, 0.40 mmol),
and DMF (10 mL) was added cyclopropylmethyl bro-
mide (0.64 g, 4.7 mmol) and the mixture was stirred
for overnight. To the reaction mixture was added 2 M
HCl and the mixture was extracted with ethyl acetate.
The organic layer was evaporated in vacuo and purified
by silica gel column chromatography (eluent: hexane/
ethyl acetate = 6:1) to give 1.1 g (yield; 91%) of 9 as col-
1
orless oil: H NMR (200 MHz, chloroform-d) d ppm

H. Ohta et al. / Bioorg. Med. Chem. 16 (2008) 1111–1124
1117
5.1.5. 2-Amino-4-methyl-1,3-oxazole (12). To a mixture
of 11 (180 g, 2.4 mol) and H2O (390 mL) was added
cyanamide (100 g, 2.4 mol) and the mixture was stirred
at 45 ꢀC for 30 min. To the reaction mixture was added
2 M NaOH and the mixture was extracted with Et2O.
The organic layer was dried over sodium sulfate and
evaporated in vacuo to give 110 g (yield; 48%) of 12 as
yellow oil: H NMR (300 MHz, chloroform-d) d ppm
2.03 (s, 3H), 4.52 (br s, 2H), 6.82–6.97 (m, 1H); MS(ESI)
m/z 99 (M+H).
ppm 1.18 (d, J = 7.0 Hz, 6H), 1.97 (s, 3H), 2.87 (quin,
J = 7.0 Hz, 1H), 4.34 (br s, 2H); MS(ESI) m/z 141
(M+H).
5.1.10. 2,2,2-Trifluoro-N-[4-methyl-5-(1-methylethyl)-1,3-
oxazol-2-yl]acetamide (17). To a mixture of 16 (0.65 g,
4.6 mmol), pyridine (0.40 g, 5.1 mmol), and CHCl3
(10 mL) was added trifluoroacetic anhydride (1.1 g,
5.1 mmol) with ice-cooling and the mixture was stirred
at room temperature for 1 h. To the reaction mixture
was added saturated aqueous NH4Cl and the mixture
was extracted with CHCl3. The organic layer was dried
over sodium sulfate, evaporated in vacuo, and washed
with hexane and IPE to give 0.90 g (yield; 82%) of 15
1
5.1.6. 1-(2-Amino-4-methyl-1,3-oxazol-5-yl)ethanone (13).
To a mixture of 12 (50 g, 0.51 mol), aluminum chloride
(140 g, 1.0 mol), and CHCl3 (1.0l) was added dropwise
acetic anhydride (52 g, 0.51 mol) in CHCl3 (500 mL)
with ice-cooling and the mixture was stirred for 6 h.
To the reaction mixture was added saturated aqueous
NaHCO3 with ice-cooling and the precipitate was fil-
tered off through Celite pad, the filtrate was extracted
with ethyl acetate. The organic layer was dried over so-
dium sulfate, evaporated in vacuo, and washed with
Et2O to give 5.7 g (yield; 10%) of 13 as yellow solid:
1H NMR (300 MHz, chloroform-d) d ppm 2.36 (s,
3H), 2.41 (s, 3H), 5.02 (br s, 2H); MS(ESI) m/z 141
(M+H).
1
as colorless solid: H NMR (200 MHz, chloroform-d)
d ppm 1.27 (d, J = 7.0 Hz, 6H), 2.17 (s, 3H), 2.96 (quin,
J = 7.0 Hz, 1H); MS(ESI) m/z 237 (M+H).
5.1.11. N-[3-(Cyclopropylmethyl)-4-methyl-5-(1-methyl-
ethyl)-1,3-oxazol-2(3H)-ylidene]-2,2,2-trifluoroacetamide
(18). To a mixture of 17 (0.77 g, 3.3 mmol) and sodium
iodide (0.049 g 0.33 mmol) in DMF (15 mL) was added
sodium hydride (0.13 g, 3.3 mmol), and the mixture was
stirred for 5 min. To the reaction mixture was added
cyclopropylmethyl bromide (0.66 g, 4.9 mmol), and the
reaction mixture was stirred at 60 ꢀC for 2 h. To the
reaction mixture was added H2O, and the mixture was
extracted with ethyl acetate. The organic layer was dried
over sodium sulfate, evaporated in vacuo, and purified
by silica gel column chromatography (eluent: ethyl ace-
5.1.7. tert-Butyl (5-acetyl-4-methyl-1,3-oxazol-2-yl)car-
bamate (14). To a mixture of 13 (3.6 g, 26 mmol),
dimethylaminopyridine (0.31 g, 2.6 mmol), and THF
(200 mL) was added di-tert-butyldicarbonate (32 g,
150 mmol) and the mixture was stirred at 60 ꢀC for
30 min. The reaction mixture was evaporated in vacuo
and purified by silica gel column chromatography (elu-
ent: hexane/ethyl acetate = 1:1) to give colorless solid.
The solid was washed with IPE to give 1.4 g (yield;
22%) of 14 as colorless solid: 1H NMR (200 MHz, chlo-
roform-d) d ppm 1.55 (s, 9H), 2.45 (s, 3H), 2.49 (s, 3H),
8.98 (br s, 1H); MS(ESI) m/z 239 (MÀH).
1
tate) to give 0.090 g (yield; 10%) of 18 as brown oil: H
NMR (200 MHz, chloroform-d) d ppm 0.38–0.66 (m,
4H), 1.04–1.22 (m, 1H), 1.30 (d, J = 7.0 Hz, 6H), 2.15
(s, 3H), 2.84–3.03 (m, 1H), 3.68 (d, J = 7.0 Hz, 2H);
MS(ESI) m/z 291 (M+H).
5.1.12. 3-(Cyclopropylmethyl)-4-methyl-5-(1-methyleth-
yl)-1,3-oxazol-2(3H)-imine (19). To a mixture of 18
(0.090 g, 0.31 mmol) and MeOH (2.0 mL) was added
2 M NaOH (2.0 mL, 2.0 mmol) and the mixture was
stirred for 3 h. The reaction mixture was evaporated in
vacuo and extracted with Et2O. The organic layer was
dried over sodium sulfate and evaporated in vacuo to
give 0.062 g (yield; 100%) of 19 as yellow oil: 1H
NMR (200 MHz, chloroform-d) d ppm 0.26–0.62 (m,
4H), 1.05–1.27 (m, 1H), 1.16 (d, J = 7.0 Hz, 6H), 1.93
(s, 3H), 2.60–2.83 (m, 1H), 3.39 (d, J = 6.6 Hz, 2H);
MS(ESI) m/z 195 (M+H).
5.1.8. tert-butyl [5-(1-Hydroxy-1-methylethyl)-4-methyl-
1,3-oxazol-2-yl]carbamate (15). To a mixture of 14
(1.4 g, 5.7 mmol) and THF (30 mL) was added dropwise
MeLi (1.0 M Et2O solution, 13 mL, 13 mmol) at À78 ꢀC
and the mixture was stirred for 1 h. To the reaction mix-
ture was added saturated aqueous NH4Cl and the mix-
ture was extracted with ethyl acetate. The organic
layer was dried over sodium sulfate, evaporated in va-
cuo, and purified by silica gel column chromatography
(eluent: hexane/ethyl acetate = 1:1) to give 1.3 g (yield;
90%) of 15 as colorless solid: 1H NMR (200 MHz, chlo-
roform-d) d ppm 1.52 (s, 9H), 1.60 (s, 6H), 2.24 (s, 3H);
MS(ESI) m/z 257 (M+H).
5.1.13. N-[3-(Cyclopropylmethyl)-4-methyl-5-(1-methyl-
ethyl)-1,3-oxazol-2(3H)-ylidene]-3-(trifluoromethyl)benz-
amide (3). To a mixture of 19 (0.020 g, 0.10 mmol),
triethylamine (0.011 g, 0.11 mmol), and CHCl3 (2.0 mL)
was added 3-trifluoromethylbenzoyl chloride (0.023 g,
0.47 mmol) with ice-cooling and the mixture was stirred
at room temperature for overnight. To the reaction mix-
ture was added saturated aqueous NaHCO3 and the mix-
ture was extracted with CHCl3. The organic layer was
dried over sodium sulfate, evaporated in vacuo, and puri-
fied by silica gel column chromatography (eluent: hexane/
ethyl acetate = 3:1) to give 0.0080 g (yield; 22%) of 3 as
colorless solid: mp 65–67 ꢀC; 1H NMR (200 MHz, chlo-
roform-d) d ppm 0.42–0.68 (m, 4H), 1.14–1.36 (m, 1H),
5.1.9. 2-Amino-4-methyl-5-(1-methylethyl)-1,3-oxazole (16).
To a mixture of 15 (1.3 g, 5.1 mmol) and TFA (15 mL)
was added triethylsilane (1.2 g, 7.7 mmol) and the mix-
ture was stirred for 2.5 h. To the reaction mixture was
added saturated aqueous NaHCO3 and the mixture
was extracted with CHCl3. The organic layer was dried
over sodium sulfate, evaporated in vacuo, and purified
by silica gel column chromatography (eluent: CHCl3/
MeOH = 10:1) to give 0.65 g (yield; 91%) of 15 as pale
yellow solid: 1H NMR (200 MHz, chloroform-d) d

1118
H. Ohta et al. / Bioorg. Med. Chem. 16 (2008) 1111–1124
1.31 (d, J = 7.0 Hz, 6H), 2.14 (s, 3H), 2.86–3.05 (m, 1H),
3.74 (d, J = 7.0 Hz, 2H), 7.44–7.57 (m, 1H), 7.64–7.73
(m, 1H), 8.37–8.44 (m, 1H), 8.47–8.55 (m, 1H); MS(ESI)
m/z 367 (M+H); Anal. Calcd for C19H21F3N2O2: C, 62.29;
H, 5.78; N, 7.65. Found: C, 62.17; H, 5.78; N, 7.58.
organic layer was evaporated in vacuo and purified by
silica gel column chromatography (eluent: hexane/ethyl
acetate = 4:1) to give 0.19 g (yield; 17%) of 24 as color-
1
less solid: H NMR (200 MHz, chloroform-d) d ppm
0.44–0.82 (m, 4H), 1.08–1.36 (m, 1H), 1.43 (s, 9H),
4.01 (d, J = 7.5 Hz, 2H), 7.76 (s, 1H); MS(ESI) m/z
307 (M+H).
5.1.14. (2Z)-3-Chloro-4,4-dimethylpent-2-enenitrile (21).
POCl3 (74 g, 480 mmol) was added dropwise to DMF
(38 g, 520 mmol) with ice-cooling. To the reaction mix-
ture was added pinacolone (20 g, 200 mmol) in 1,2-
dichloroethane (32 mL) and the reaction mixture was
stirred at 75 ꢀC for 4 h. To the reaction mixture was
added hydroxylamine sulfate (79 g, 480 mmol) in 1,2-
dichloroethane (150 mL) and the reaction mixture was
stirred at 85 ꢀC for 1 h. The mixture was partitioned be-
tween H2O and hexane. The separated organic phase
was dried over sodium sulfate and evaporated in vacuo
5.1.18. 5-tert-Butyl-2-(cyclopropylmethyl)isothiazol-3(2H)-
imine (25). To a mixture of 24 (0.19 g, 0.62 mmol),
MeOH (5.0 mL), and H2O (2.0 mL) was added potas-
sium carbonate (0.26 g, 1.7 mmol) and the mixture was
stirred for 1 h. The mixture was partitioned between
2 M NaOH and CHCl3. The separated organic phase
was dried over sodium sulfate and evaporated in vacuo
to give 0.12 g (yield; 95%) of 25 as colorless oil: 1H
NMR (200 MHz, chloroform-d) d ppm 0.27–0.74 (m,
4H), 0.99–1.24 (m, 1H), 1.29 (s, 9H), 3.45 (d,
J = 7.5 Hz, 2H), 5.85 (s, 1H); MS(ESI) m/z 211 (M+H).
1
to give 29 g (yield; 100%) of 21 as yellow oil: H NMR
(200 MHz, chloroform-d) d ppm 1.25 (s, 9H), 5.56 (s,
1H); MS(ESI) m/z 144 (M+H).
5.1.19. N-[5-tert-Butyl-2-(cyclopropylmethyl)isothiazol-
3(2H)-ylidene]-3-(trifluoromethyl)benzamide (4). To a
mixture of 25 (0.040 g, 0.19 mmol), triethylamine
(0.019 g, 0.19 mmol), and CHCl3 (1.0 mL) was added
3-trifluoromethylbenzoyl chloride (0.040 g, 0.19 mmol)
and the mixture was stirred for 1 h. To the reaction mix-
ture was added H2O and the mixture was extracted with
CHCl3. The organic layer was washed with 2 M NaOH
and 2 M HCl, dried over sodium sulfate, evaporated in
vacuo, and purified by silica gel column chromatogra-
phy (eluent: hexane/ethyl acetate = 3:1) to give 0.032 g
5.1.15. 3-Amino-5-tert-butylisothiazole (22). To a mix-
ture of 21 (12 g, 80 mmol) and EtOH (63 mL) were
added dropwise sodium sulfide (6.5 g, 83 mmol) and
1 M HCl (84 mL, 84 mmol) with ice-cooling and the
mixture was stirred at room temperature for 2 h. To
the reaction mixture was added dropwise NH2Cl, which
was prepared from 24% NH3 (50 mL) and 5.3% NaClO
(120 mL), with ice-cooling and the reaction mixture was
stirred at room temperature for overnight. To the reac-
tion mixture was added 6 M HCl with ice-cooling and
the mixture was washed with Et2O. The water phase
was added into 6 M NaOH with ice-cooling and the
mixture was extracted with CHCl3. The organic layer
was dried over sodium sulfate, evaporated in vacuo,
and purified by silica gel column chromatography (elu-
ent: hexane/ethyl acetate = 3:1) to give 1.2 g (yield; 9%)
of 22 as yellow solid: 1H NMR (200 MHz, chloro-
form-d) d ppm 1.36 (s, 9H), 4.28 (br s, 2H), 6.28 (s,
1H); MS(ESI) m/z 157 (M+H).
1
(yield; 44%) of 4 as colorless solid: mp 54–55 ꢀC; H
NMR (200 MHz, chloroform-d) d ppm 0.44–0.58 (m,
2H), 0.68–0.83 (m, 2H), 1.21–1.38 (m, 1H), 1.43 (s,
9H), 4.05 (d, J = 7.5 Hz, 2H), 7.52 (t, J = 7.5 Hz, 1H),
7.69 (d, J = 7.5 Hz, 1H), 7.86 (s, 1H), 8.46 (d,
J = 7.5 Hz, 1H), 8.57 (s, 1H); MS(ESI) m/z 383
(M+H); Anal. Calcd for C19H21F3N2OS: C, 59.67; H,
5.53; N, 7.32. Found: C, 59.75; H, 5.55; N, 7.16.
5.1.20. N-(5-tert-Butylisoxazol-3-yl)-2,2,2-trifluoroaceta-
mide (27). To a mixture of 26 (5.0 g, 36 mmol), pyridine
(3.1 g, 39 mmol), and CHCl3 (80 mL) was added trifluo-
roacetic anhydride (8.3 g, 439 mmol) and the mixture
was stirred for 30 min. To the reaction mixture was
added saturated aqueous NH4Cl and the mixture was
extracted with CHCl3. The organic layer was dried over
sodium sulfate, evaporated in vacuo, and purified by sil-
ica gel column chromatography (eluent: hexane/ethyl
acetate = 3:1) to give 8.2 g (yield; 98%) of 27 as colorless
5.1.16. N-(5-tert-Butylisothiazol-3-yl)-2,2,2-trifluoroace-
tamide (23). To a mixture of 22 (0.60 g, 3.8 mmol), pyr-
idine (0.33 g, 4.2 mmol), and CHCl3 (6.0 mL) was added
trifluoroacetic anhydride (0.89 g, 4.2 mmol) and the
mixture was stirred for 30 min. To the reaction mixture
was added saturated aqueous NH4Cl and the mixture
was extracted with CHCl3. The organic layer was dried
over sodium sulfate and evaporated in vacuo to give
1
0.92 g (yield; 82%) of 23 as colorless solid: H NMR
(200 MHz, chloroform-d) d ppm 1.44 (s, 9H), 7.67 (s,
1H), 9.61 (br s, 1H); MS(ESI) m/z 253 (M+H).
1
solid: H NMR (200 MHz, chloroform-d) dppm 1.37 (s,
9H), 6.70 (s, 1H), 9.75 (br s, 1H); MS(ESI) m/z 235
(MÀH).
5.1.17. N-[5-tert-Butyl-2-(cyclopropylmethyl)isothiazol-
3(2H)-ylidene]-2,2,2-trifluoroacetamide (24). To a mix-
ture of 23 (0.92 g, 3.7 mmol) and sodium iodide
(0.055 g 0.37 mmol) in DMF (5.0 mL) was added so-
dium hydride (0.15 g, 3.7 mmol), and the mixture was
stirred for 5 min. To the reaction mixture was added
cyclopropylmethyl bromide (0.74 g, 5.5 mmol), and the
reaction mixture was stirred at 60 ꢀC for 4 h. To the
reaction mixture was added saturated aqueous NH4Cl
and the mixture was extracted with ethyl acetate. The
5.1.21. N-[5-tert-Butyl-2-(cyclopropylmethyl)isoxazol-3(2H)-
ylidene]-2,2,2-trifluoroacetamide (28). To a mixture of
27 (5.0 g, 21 mmol) and sodium iodide (0.32 g 2.1 mmol)
in DMF (45 mL) was added sodium hydride (0.85 g,
21 mmol), and the mixture was stirred for 20 min. To
the reaction mixture was added cyclopropylmethyl bro-
mide (4.3 g, 32 mmol), and the reaction mixture was stir-
red for 4 days. To the reaction mixture was added H2O
and the mixture was extracted with ethyl acetate. The

H. Ohta et al. / Bioorg. Med. Chem. 16 (2008) 1111–1124
1119
organic layer was evaporated in vacuo and purified by
silica gel column chromatography (eluent: hexane/ethyl
acetate = 3:1) to give 0.68 g (yield; 11%) of 28 as color-
5.1.26. 5-Amino-3-tert-Butyl-1-(cyclopropylmethyl)-1H-
pyrazole (33). A mixture of 32 (30 g, 350 mmol) and
pivaloylacetonitrile (44 g, 350 mmol) in EtOH (230 mL)
was heated at reflux for 5 h. The reaction mixture was
evaporated in vacuo and purified by silica gel column
chromatography (eluent: hexane/ethyl acetate = 1:1) to
1
less solid: H NMR (200 MHz, chloroform-d) d ppm
0.40–0.70 (m, 4H), 1.14–1.32 (m, 1H), 1.36 (s, 9H),
4.13 (d, J = 7.0 Hz, 2H), 7.07 (s, 1H); MS(ESI) m/z
291 (M+H).
1
give 22 g (yield; 42%) of 33 as colorless solid: H NMR
(200 MHz, chloroform-d) d ppm 0.27–0.39 (m, 2H),
0.48–0.62 (m, 2H), 1.10–1.31 (m, 1H), 1.26 (s, 9H), 3.42
(br s, 2H), 3.86 (d, J = 6.2 Hz, 2H), 5.42 (s, 1H); MS(ESI)
m/z 194 (M+H).
5.1.22. 5-tert-Butyl-2-(cyclopropylmethyl)isoxazol-3(2H)-
imine (29). To a mixture of 28 (0.15 g, 0.52 mmol),
MeOH (5.0 mL), and H2O (2.0 mL) was added potas-
sium carbonate (0.23 g, 1.6 mmol) and the mixture was
stirred for 30 min. The mixture was partitioned between
2 M NaOH and Et2O. The separated organic phase was
dried over sodium sulfate and evaporated in vacuo to
5.1.27. N-[3-tert-Butyl-1-(cyclopropylmethyl)-1H-pyra-
zol-5-yl]-2,2,2-trifluoroacetamide (34). To a mixture of
33 (1.0 g, 5.2 mmol), pyridine (0.41 g, 5.2 mmol), and
CHCl3 (10 mL) was added dropwise trifluoroacetic
anhydride (1.10 g, 5.17 mmol) with ice-cooling and the
mixture was stirred at room temperature for 2 h. To
the reaction mixture was added saturated aqueous
NH4Cl and the mixture was extracted with CHCl3.
The organic layer was dried over sodium sulfate and
evaporated in vacuo to give 1.6 g (yield; 100%) of 34
1
give 0.12 g (yield; 100%) of 25 as yellow oil: H NMR
(200 MHz, chloroform-d) d ppm 0.25–0.61 (m, 4H),
1.04–1.21 (m, 1H), 1.24 (s, 9H), 3.56 (d, J = 7.0 Hz,
2H), 5.30 (s, 1H); MS(ESI) m/z 195 (M+H).
5.1.23. N-[5-tert-Butyl-2-(cyclopropylmethyl)isoxazol-3(2H)-
ylidene]-3-(trifluoromethyl)benzamide (5). To a mixture
of 29 (0.040 g, 0.21 mmol), triethylamine (0.023 g,
0.23 mmol), and CHCl3 (2.0 mL) was added 3-trifluoro-
methylbenzoyl chloride (0.048 g, 0.23 mmol) and the
mixture was stirred for 1 h. To the reaction mixture
was added saturated aqueous NaHCO3 and the mixture
was extracted with CHCl3. The organic layer was dried
over sodium sulfate, evaporated in vacuo, and purified
by silica gel column chromatography (eluent: hexane/
ethyl acetate = 3:1) to give 0.040 g (yield; 52%) of 5 as
1
as colorless solid: H NMR (200 MHz, chloroform-d)
d ppm 0.28–0.45 (m, 2H), 0.62–0.77 (m, 2H), 1.03–1.30
(m, 1H), 1.29 (s, 9H), 3.96 (d, J = 6.6 Hz, 2H), 6.37 (s,
0H), 8.10 (br s, 1H); MS(ESI) m/z 290 (M+H).
5.1.28. N-[5-tert-Butyl-2-(cyclopropylmethyl)-1-methyl-
1,2-dihydro-3H-pyrazol-3-ylidene]-2,2,2-trifluoroaceta-
mide (35). To a mixture of 34 (1.6 g, 5.2 mmol), sodium
hydrogen carbonate (1.3 g, 16 mmol), and toluene
(30 mL) was added dimethyl sulfate (2.0 g, 16 mmol)
and the mixture was stirred at 80 ꢀC for overnight. To
the reaction mixture was added H2O and the mixture
was extracted with ethyl acetate. The organic layer was
dried over sodium sulfate, evaporated in vacuo, and
purified by silica gel column chromatography (eluent:
CHCl3/MeOH = 3:1) to give 0.72 g (yield; 14%) of 35
1
pale yellow oil: H NMR (200 MHz, chloroform-d) d
ppm 0.41–0.73 (m, 4H), 1.17–1.41 (m, 1H), 1.36 (s,
9H), 4.17 (d, J = 7.0 Hz, 2H), 7.17 (s, 1H), 7.46–7.57
(m, 1H), 7.65–7.73 (m, 1H), 8.38–8.46 (m, 1H), 8.48–
8.57 (m, 1H); MS(ESI) m/z 367 (M+H); Anal. Calcd
for C19H21F3N2O2: C, 62.29; H, 5.78; N, 7.65. Found:
C, 62.03; H, 5.80; N, 7.67.
1
as colorless solid: H NMR (200 MHz, chloroform-d)
5.1.24. Cyclopropylmethyl methanesulfonate (31). To a
mixture of 30 (50 g, 690 mmol), triethylamine (0.023 g,
900 mmol), and CHCl3 (200 mL) was added dropwise
methanesulfonyl chloride (100 g, 900 mmol) with ice-
cooling and the mixture was stirred at room temperature
for 3 h. To the reaction mixture was added H2O and the
mixture was extracted with CHCl3. The organic layer
was dried over sodium sulfate and evaporated in vacuo
dppm 0.50–0.65 (m, 4H), 0.90–1.18 (m, 1H), 1.43 (s,
9H), 3.90 (s, 3H), 4.21 (d, J = 7.0 Hz, 2H), 6.94 (s,
1H); MS(ESI) m/z 304 (M+H).
5.1.29. 5-tert-Butyl-2-(cyclopropylmethyl)-1-methyl-1,2-
dihydro-3H-pyrazol-3-imine (36). To a mixture of 35
(0.21 g, 0.69 mmol) and MeOH (5.0 mL) was added be-
tween 2 M NaOH (2.5 mL, 5.0 mmol) and the mixture
was stirred at 70 ꢀC for 4 h. The mixture was partitioned
2 M NaOH and CHCl3, and separated organic phase
was dried over sodium sulfate and evaporated in vacuo
to give 0.17 g (yield; 100%) of 36 as yellow oil: 1H NMR
(200 MHz, chloroform-d) d ppm 0.26–0.58 (m, 4H),
0.93–1.18 (m, 1H), 1.29 (s, 9H), 3.16 (s, 3H), 3.62 (d,
J = 6.6 Hz, 2H), 5.29 (br s, 1H); MS(ESI) m/z 208
(M+H).
1
to give 100 g (yield; 96%) of 31 as brown oil: H NMR
(200 MHz, chloroform-d) d ppm 0.31–0.78 (m, 4H),
1.11–1.37 (m, 1H), 3.03 (s, 3H), 4.09 (d, J = 7.5 Hz,
2H); MS(ESI) m/z 151 (M+H).
5.1.25. (Cyclopropylmethyl)hydrazine (32). To a mixture
of 31 (100 g, 660 mmol) and EtOH (200 mL) was added
hydrazine hydrate (200 g, 4000 mmol) with ice-cooling
and the mixture was stirred at room temperature for
overnight. The reaction mixture was evaporated in va-
cuo and the mixture was extracted with CHCl3. The or-
ganic layer was dried over sodium sulfate and
evaporated in vacuo to give 30 g (yield; 53%) of 32 as
5.1.30. N-[5-tert-Butyl-2-(cyclopropylmethyl)-1-methyl-
1,2-dihydro-3H-pyrazol-3-ylidene]-3-(trifluoromethyl)benz-
amide (6a). To a mixture of 36 (0.017 g, 0.082 mmol),
triethylamine (0.0091 g, 0.090 mmol), and CHCl3
(1.0 mL) was added 3-trifluoromethylbenzoyl chloride
(0.019 g, 0.0090 mmol) and the mixture was stirred for
1 h. The reaction mixture was evaporated in vacuo
1
yellow oil: H NMR (200 MHz, chloroform-d) d ppm
0.09–0.23 (m, 2H), 0.43–0.61 (m, 2H), 0.82–1.05 (m,
1H), 2.64 (d, J = 6.6 Hz, 2H); MS(ESI) m/z 87 (M+H).

1120
H. Ohta et al. / Bioorg. Med. Chem. 16 (2008) 1111–1124
and purified by silica gel column chromatography (elu-
ent: hexane/ethyl acetate = 1:10) to give 0.010 g (yield;
32%) of 6a as colorless solid: mp 97–99 ꢀC; H NMR
Calcd for C21H26F3N3OÆ1/2H2O: C, 62.67; H, 6.67; N,
10.44. Found: C, 62.42; H, 6.64; N, 10.43.
1
(200 MHz, chloroform-d) d ppm 0.51–0.69 (m, 4H),
1.00–1.29 (m, 1H), 1.45 (s, 9H), 3.85 (s, 3H), 4.30 (d,
J = 7.0 Hz, 2H), 7.10 (s, 1H), 7.40–7.55 (m, 1H), 7.56–
7.68 (m, 1H), 8.35–8.47 (m, 1H), 8.48–8.58 (m, 1H);
MS(ESI) m/z 380 (M+H); Anal. Calcd for
C20H24F3N3OÆ1/4H2O: C, 62.57; H, 6.43; N, 10.94.
Found: C, 62.90; H, 6.36; N, 10.89.
5.1.36. Cyclobutylmethyl methanesulfonate (38g). To a
mixture of 37g (13 g, 150 mmol), triethylamine (20 g,
200 mmol), and CHCl3 (44 mL) was added dropwise
methanesulfonyl chloride (23 g, 200 mmol) with ice-
cooling and the mixture was stirred at room temperature
for 2 h. To the reaction mixture was added H2O and the
mixture was extracted with CHCl3. The organic layer
was dried over sodium sulfate and evaporated in vacuo
to give 27 g (yield; 100%) of 38g as brown oil: 1H NMR
(200 MHz, chloroform-d) d ppm 1.72–2.23 (m, 6H),
2.60–2.84 (m, 1H), 3.01 (s, 3H), 4.19 (d, J = 6.6 Hz,
2H); MS(CI) m/z 165 (M+H).
5.1.31. N-[5-tert-Butyl-2-(cyclopropylmethyl)-1-methyl-
1,2-dihydro-3H-pyrazol-3-ylidene]-2-fluoro-3-(trifluoro-
methyl)benzamide (6b). As colorless solid: mp 104–
106 ꢀC; 1H NMR (200 MHz, chloroform-d) d ppm
0.51–0.65 (m, 4H), 1.01–1.19 (m, 1H), 1.45 (s, 9H),
3.86 (s, 3H), 4.26 (d, J = 7.0 Hz, 2H), 7.07 (s, 1H),
7.13–7.25 (m, 1H), 7.49–7.63 (m, 1H), 8.07–8.22 (m,
1H); MS(ESI) m/z 398 (M+H); Anal. Calcd for
C20H23F4N3O: C, 60.44; H, 5.83; N, 10.57. Found: C,
60.48; H, 5.74; N, 10.59.
5.1.37. (Cyclobutylmethyl)hydrazine (39g). To a mixture
of 38g (27 g, 150 mmol) and EtOH (45 mL) was added
hydrazine hydrate (46 g, 920 mmol) and the mixture
was stirred for over night. The reaction mixture was
evaporated in vacuo and the mixture was extracted with
CHCl3. The organic layer was dried over sodium sulfate
and evaporated in vacuo to give 20 g (yield; 100%) of
5.1.32. N-[5-tert-Butyl-2-(cyclopropylmethyl)-1-methyl-
1,2-dihydro-3H-pyrazol-3-ylidene]-2-chloro-3-(trifluoro-
methyl)benzamide (6c). As colorless solid: mp 145–
146 ꢀC; 1H NMR (200 MHz, chloroform-d) d ppm
0.47–0.62 (m, 4H), 1.00–1.16 (m, 1H), 1.45 (s, 9H),
3.85 (s, 3H), 4.21 (d, J = 7.0 Hz, 2H), 7.07 (s, 1H),
7.27–7.37 (m, 1H), 7.57–7.64 (m, 1H), 7.69–7.77 (m,
1H); MS(ESI) m/z 414 (M+H); Anal. Calcd for
C20H23ClF3N3O: C, 58.04; H, 5.60; N, 10.15. Found:
C, 58.08; H, 5.51; N, 10.20.
1
39g as yellow oil: H NMR (600 MHz, chloroform-d)
d ppm 1.61–1.73 (m, 2H), 1.79–1.98 (m, 2H), 2.00–2.12
(m, 2H), 2.40–2.55 (m, 1H), 2.79 (d, J = 7.3 Hz, 2H);
MS(ESI) m/z 99 (MÀH).
5.1.38. 5-Amino-3-tert-Butyl-1-(cyclobutylmethyl)-1H-
pyrazole (40g). A mixture of 39g (20 g, 150 mmol) and
pivaloylacetonitrile (19 g, 150 mmol) in EtOH (110 mL)
was heated at reflux for 2.5 h. The reaction mixture
was evaporated in vacuo, purified by silica gel column
chromatography (eluent: hexane/ethyl acetate = 1:1),
and washed with hexane and ethyl acetate to give 12 g
(yield; 37%) of 40g as colorless solid: 1H NMR
(200 MHz, chloroform-d) d ppm 1.25 (s, 9H), 1.71–2.13
(m, 6H), 2.67–2.88 (m, 1H), 3.91 (d, J = 6.6 Hz, 2H),
5.40 (s, 1H); MS(ESI) m/z 208 (M+H).
5.1.33. N-[5-tert-Butyl-2-(cyclopropylmethyl)-1-methyl-
1,2-dihydro-3H-pyrazol-3-ylidene]-2-methyl-3-(trifluoro-
methyl)benzamide (6d). As colorless solid: mp 90-91 ꢀC;
1H NMR (200 MHz, chloroform-d) d ppm 0.43–0.65
(m, 4H), 0.98–1.16 (m, 1H), 1.45 (s, 9H), 2.59–2.66 (m,
3H), 3.83 (s, 3H), 4.20 (d, J = 7.0 Hz, 2H), 7.06 (s,
1H), 7.17–7.28 (m, 1H), 7.51–7.58 (m, 1H), 7.68–7.77
(m, 1H); MS(ESI) m/z 394 (M+H); Anal. Calcd for
C21H26F3N3OÆ1/4H2O: C, 63.38; H, 6.71; N, 10.56.
Found: C, 63.24; H, 6.60; N, 10.54.
5.1.39. N-[3-tert-Butyl-1-(cyclobutylmethyl)-1H-pyrazol-
5-yl]-2-fluoro-3-(trifluoromethyl)benzamide (41g). To a
mixture of 40g (0.40 g, 1.9 mmol), triethylamine (0.22 g,
2.1 mmol), and CHCl3 (8.0 mL) was added 2-fluoro-3-tri-
fluoromethylbenzoyl chloride (0.48 g, 2.1 mmol) and the
mixture was stirred for overnight. To the reaction mixture
was added saturated aqueous NH4Cl and the mixture was
extracted with CHCl3. The organic layer was dried over
sodium sulfate, evaporated in vacuo, and purified by silica
gel column chromatography (eluent: hexane/ethyl ace-
tate = 3:1) to give 0.61 g (yield; 79%) of 41g as colorless
solid: 1H NMR (200 MHz, chloroform-d) d ppm 1.31 (s,
9H), 1.71–2.14 (m, 6H), 2.70–2.92 (m, 1H), 4.04 (d,
J = 7.0 Hz, 2H), 6.35 (s, 1H), 7.41–7.54 (m, 1H), 7.79–
7.92 (m, 1H), 8.10–8.28 (m, 1H), 8.34–8.47 (m, 1H);
MS(ESI) m/z 398 (M+H).
5.1.34. N-[5-tert-Butyl-2-(cyclopropylmethyl)-1-methyl-
1,2-dihydro-3H-pyrazol-3-ylidene]-2-fluoro-5-(trifluoro-
methyl)benzamide (6e). As colorless solid: mp 124–
126 ꢀC; 1H NMR (200 MHz, chloroform-d) d ppm
0.53–0.65 (m, 4H), 1.03–1.18 (m, 1H), 1.45 (s, 9H),
3.86 (s, 3H), 4.27 (d, J = 7.0 Hz, 2H), 7.06–7.19 (m,
1H), 7.10 (s, 1H), 7.50–7.60 (m, 1H), 8.27–8.35 (m,
1H); MS(ESI) m/z 398 (M+H); Anal. Calcd for
C20H23F4N3OÆ1/2H2O: C, 59.11; H, 5.95; N, 10.34.
Found: C, 58.96; H, 5.74; N, 10.26.
5.1.35. N-[5-tert-Butyl-2-(cyclopropylmethyl)-1-methyl-
1,2-dihydro-3H-pyrazol-3-ylidene]-2-methyl-5-(trifluoro-
methyl)benzamide (6f). As colorless solid: mp 156–
157 ꢀC; 1H NMR (200 MHz, chloroform-d) d ppm
0.50–0.66 (m, 4H), 1.02–1.17 (m, 1H), 1.45 (s, 9H),
2.68 (s, 3H), 3.84 (s, 3H), 4.24 (d, J = 6.6 Hz, 2H),
7.06 (s, 1H), 7.21–7.29 (m, 1H), 7.40–7.47 (m, 1H),
8.13–8.17 (m, 1H); MS(ESI) m/z 394 (M+H); Anal.
5.1.40.
N-[5-tert-Butyl-2-(cyclobutylmethyl)-1-methyl-
1,2-dihydro-3H-pyrazol-3-ylidene]-2-fluoro-3-(trifluoro-
methyl)benzamide (6g). To a mixture of 41g (0.61 g,
1.5 mmol), sodium hydrogen carbonate (0.38 g,
4.6 mmol), and toluene (6.0 mL) was added dimethyl sul-
fate (0.58 g, 4.6 mmol) and the mixture was stirred at

H. Ohta et al. / Bioorg. Med. Chem. 16 (2008) 1111–1124
1121
80 ꢀC for overnight. To the reaction mixture were added
H2O and saturated aqueous NaHCO3, and the mixture
was extracted with ethyl acetate. The organic layer was
dried over sodium sulfate, evaporated in vacuo, purified
by silica gel column chromatography (eluent: hexane/
ethyl acetate = 1:20), and washed with hexane and ethyl
acetate to give 0.070 g (yield; 11%) of 6g as colorless solid:
5.1.48. 5-Amino-3-tert-Butyl-1-(2-ethoxyethyl)-1H-pyra-
zole (40i). As yellow solid: H NMR (200 MHz, chloro-
form-d) d ppm 1.17 (t, J = 7.0 Hz, 3H), 1.26 (s, 9H), 3.47
(q, J = 7.0 Hz, 2H), 3.68 (t, J = 4.8 Hz, 2H), 4.04
(br s, 2H), 4.13 (t, J = 4.8 Hz, 2H), 5.35 (s, 1H);
MS(ESI) m/z 212 (M+H).
1
1
mp 102–103 ꢀC; H NMR (200 MHz, chloroform-d) d
5.1.49. N-[3-tert-Butyl-1-(2-ethoxyethyl)-1H-pyrazol-5-
yl]-2-fluoro-3-(trifluoromethyl)benzamide (41i). As brown
ppm 1.42 (s, 9H), 1.83–2.11 (m, 6H), 2.55–2.77 (m, 1H)
3.74 (s, 3H), 4.38 (d, J = 7.0 Hz, 2H), 7.07 (s, 1H), 7.15–
7.26 (m, 1H), 7.52–7.62 (m, 1H), 8.12–8.25 (m, 1H);
MS(ESI) m/z 412 (M+H); Anal. Calcd for C21H25F4N3O:
C, 61.30; H, 6.12; N, 10.21. Found: C, 61.35; H, 6.03; N,
10.24.
1
oil: H NMR (200 MHz, chloroform-d) d ppm 1.03 (t,
J = 7.0 Hz, 3H), 1.33 (s, 9H), 3.52 (q, J = 7.0 Hz, 2H),
3.77 (t, J = 4.8 Hz, 2H), 4.31 (t, J = 4.8 Hz, 2H), 6.55
(s, 1H), 7.35–7.48 (m, 1H), 7.74–7.86 (m, 1H), 8.20–
8.35 (m, 1H), 9.82–9.97 (m, 1H); MS(ESI) m/z 402
(M+H).
5.1.41. Cyclopentylmethyl methanesulfonate (38h). As
1
brown oil: H NMR (200 MHz, chloroform-d) d ppm
1.15–1.44 (m, 2H), 1.48–1.92 (m, 6H), 2.18–2.42 (m,
1H), 3.01 (s, 3H), 4.11 (d, J = 7.0 Hz, 2H).
5.1.50. N-[5-tert-Butyl-2-(2-ethoxyethyl)-1-methyl-1,2-
dihydro-3H-pyrazol-3-ylidene]-2-fluoro-3-(trifluoromethyl)-
benzamide (6i). As colorless solid: mp 95–96 ꢀC; 1H
NMR (200 MHz, chloroform-d)
d ppm 1.12 (t,
5.1.42. (Cyclopentylmethyl)hydrazine (39h). As yellow
oil: 1H NMR (200 MHz, chloroform-d) d ppm 1.04–
1.31 (m, 2H), 1.42–1.88 (m, 6H), 1.90–2.15 (m, 1H),
2.72 (d, J = 7.0 Hz, 2H), 3.04 (br s, 3H); MS(ESI) m/z
115 (M+H).
J = 7.0 Hz, 3H), 1.43 (s, 9H), 3.43 (q, J = 7.0 Hz, 2H),
3.73 (t, J = 4.8 Hz, 2H), 3.89 (s, 3H), 4.47 (t,
J = 4.8 Hz, 2H), 7.02 (s, 1H), 7.14–7.25 (m, 1H), 7.51–
7.61 (m, 1H), 8.10–8.23 (m, 1H); MS(ESI) m/z 416
(M+H); Anal. Calcd for C20H25F4N3O2: C, 57.82; H,
6.07; N, 10.11. Found: C, 57.84; H, 5.96; N, 10.10.
5.1.43. 5-Amino-3-tert-Butyl-1-(cyclopentylmethyl)-1H-
pyrazole (40h). As colorless solid: H NMR (200 MHz,
chloroform-d) d ppm 1.14–1.36 (m, 2H), 1.25 (s, 9H),
1.42–1.79 (m, 6H), 2.24–2.51 (m, 1H), 3.36 (br s, 2H),
3.82 (d, J = 7.5 Hz, 2H), 5.40 (s, 1H); MS(ESI) m/z
222 (M+H).
1
5.1.51. 2-(1-Methylethoxy)ethyl methanesulfonate (38j).
As brown oil: 1H NMR (200 MHz, chloroform-d) d
ppm 1.18 (d, J = 6.2 Hz, 6H), 3.07 (s, 3H), 3.58–3.74
(m, 3H), 4.32–4.42 (m, 2H); MS(CI) m/z 183 (M+H).
5.1.52. [2-(1-Methylethoxy)ethyl]hydrazine (39j). As yel-
low oil: 1H NMR (200 MHz, chloroform-d) d ppm
1.16 (d, J = 6.2 Hz, 6H), 2.94 (t, J = 4.8 Hz, 1H), 3.17
(br s, 3H), 3.49–3.68 (m, 3H); MS(ESI) m/z 117 (MÀH).
5.1.44. N-[3-tert-Butyl-1-(cyclopentylmethyl)-1H-pyra-
zol-5-yl]-2-fluoro-3-(trifluoromethyl)benzamide (41h). As
pale brown solid: H NMR (600 MHz, chloroform-d)
d ppm 1.18–1.31 (m, 2H), 1.30 (s, 9H), 1.49–1.74 (m,
6H), 2.35–2.46 (m, 1H), 3.95 (d, J = 7.3 Hz, 2H), 6.34
(s, 1H), 7.42–7.48 (m, 1H), 7.80–7.86 (m, 1H), 8.14–
8.26 (m, 1H), 8.35–8.41 (m, 1H); MS(ESI) m/z 412
(M+H).
1
5.1.53. 5-Amino-3-tert-Butyl-1-[2-(1-methylethoxy)ethyl]-
1H-pyrazole (40j). As colorless solid: 1H NMR
(200 MHz, chloroform-d) d ppm 1.11 (d, J = 6.2 Hz,
6H), 1.25 (s, 9H), 3.47–3.63 (m, 1H), 3.70 (t,
J = 4.8 Hz, 2H), 4.07 (br s, 2H), 4.12 (t, J = 4.8 Hz,
2H), 5.34 (s, 1H); MS(CI) m/z 226 (M+H).
5.1.45. N-[5-tert-Butyl-2-(cyclopentylmethyl)-1-methyl-
1,2-dihydro-3H-pyrazol-3-ylidene]-2-fluoro-3-(trifluoro-
methyl)benzamide (6h). As colorless solid: mp 140.5–
141.5 ꢀC; 1H NMR (200 MHz, chloroform-d) d ppm
1.37–1.80 (m, 8H), 1.39–1.49 (m, 9H), 2.18–2.40 (m,
1H), 3.77 (s, 3H), 4.27 (d, J = 7.47 Hz, 2H), 7.08 (s,
1H), 7.20 (t, J = 7.69 Hz, 1H), 7.50–7.63 (m, 1H),
8.06–8.24 (m, 1H); MS(ESI) m/z 426 (M+H); Anal.
Calcd for C22H27F4N3O: C, 62.11; H, 6.40; N, 9.88.
Found: C, 61.98; H, 6.35; N, 9.86.
5.1.54. N-{3-tert-Butyl-1-[2-(1-methylethoxy)ethyl]-1H-
pyrazol-5-yl}-2-fluoro-3-(trifluoromethyl)benzamide (41j).
As yellow oil: 1H NMR (200 MHz, chloroform-d) d
ppm 1.00 (d, J = 6.2 Hz, 6H), 1.33 (s, 9H), 3.48–3.69
(m, 1H), 3.76 (t, J = 4.4 Hz, 2H), 4.32 (t, J = 4.4 Hz,
2H), 6.55 (s, 1H), 7.35–7.50 (m, 1H), 7.74–7.87 (m,
1H), 8.20–8.33 (m, 1H), 9.83–9.99 (m, 1H); MS(ESI)
m/z 416 (M+H).
5.1.46. 2-Ethoxyethyl methanesulfonate (38i). As brown
oil: H NMR (200 MHz, chloroform-d) d ppm 1.22 (t,
J = 7.0 Hz, 3H), 3.07 (s, 3H), 3.56 (q, J = 7.0 Hz, 2H),
3.71 (t, J = 4.4 Hz, 2H), 4.37 (t, J = 4.4 Hz, 2H); MS(E-
SI) m/z 169 (M+H).
5.1.55.
N-{5-tert-Butyl-1-methyl-2-[2-(1-methylethoxy)-
1
ethyl]-1,2-dihydro-3H-pyrazol-3-ylidene}-2-fluoro-3-(tri-
fluoromethyl)benzamide (6j). As colorless solid: mp 80–
81 ꢀC; H NMR (200 MHz, chloroform-d) d ppm 1.06
(d, J = 6.2 Hz, 6H), 1.43 (s, 9H), 3.42–3.57 (m, 1H),
3.73 (t, J = 4.8 Hz, 2H), 3.90 (s, 3H), 4.46 (t,
J = 4.8 Hz, 2H), 7.03 (s, 1H), 7.13–7.25 (m, 1H), 7.51–
7.62 (m, 1H), 8.12–8.24 (m, 1H); MS(ESI) m/z 430
(M+H); Anal. Calcd for C21H27F4N3O: C, 58.73; H,
6.34; N, 9.78. Found: C, 58.65; H, 6.23; N, 9.76.
1
1
5.1.47. (2-Ethoxyethyl)hydrazine (39i). As yellow oil: H
NMR (200 MHz, chloroform-d) d ppm 1.21 (t,
J = 7.0 Hz, 3H), 3.39–3.69 (m, 6H), 3.87 (br s, 3H);
MS(ESI) m/z 105 (M+H).

1122
H. Ohta et al. / Bioorg. Med. Chem. 16 (2008) 1111–1124
5.2. Solubility
branes of CHO cells expressing hCB1 or hCB2. Prepared
membranes of CHO cells expressing hCB1 or hCB2 were
suspended in assay buffer (50 mM Tris–HCl, 2.5 mM
EDTA, and 5 mM MgCl2, pH 7.4) containing 0.2%
BSA. Membranes at a protein concentration of 7 lg/well
(hCB1) or 2.5 lg/well (hCB2) were incubated with vari-
ous concentrations of test compounds and [3H]CP
55,940 (2.1 nM for hCB1, 0.82 nM for hCB2) at 25 ꢀC
for 120 min. The reaction was terminated by rapid filtra-
tion over a GF/C filter presoaked with 0.1% poly-L-ly-
sine (Sigma–Aldrich, St. Louis, MO, USA). The filter
plates were washed three times with the assay buffer con-
taining 0.1% BSA, using a UniFliter96 harvester (Pack-
ard Instruments, Meriden, CT, USA). Microscinti 0
scintillator (Packard Instruments, Meriden, CT, USA)
was added, and filter-bound activity was counted in a
TopcountTM scintillation counter (Packard Instruments,
Meriden, CT, USA). Nonspecific binding was deter-
mined in the presence of 2 lM CP 55,940. Specific bind-
ing was determined by subtracting nonspecific binding
from total binding.
An excess amount of each compound was added to water
and shaken on a shaker (model SA31, Yamato Kagaku)
at room temperature for 24 h. The suspension was centri-
fuged for 10 min at 25 ꢀC, 11,000 rpm with a centrifugal
separator (model CF 15R, Hitachi). The supernatant was
diluted with 50% aqueous acetonitrile solution, and the
concentrations were measured by HPLC. The HPLC
analysis was performed with a Shimadzu HPLC system
composed of a LC-10AD, SPD-10AV, and SIL-10A.
The conditions for HPLC were: mobile phase, 0.1%
aqueous phosphoric acid solution/acetonitrile = 7:3; flow
rate, 1.0 mL/min; column, reversed-phase (Capcell Pak
UG120, 4.6 mm I.D. · 150 mm; Shiseido) at 40 ꢀC; and
detection wavelength, 230 nm.
5.3. In vitro pharmacological studies
5.3.1. Cell culture and preparation of human CB1 receptor
and human CB2 receptor membranes. Cells expressing
human CB1 (hCB1) receptor or human CB2 (hCB2)
receptor were generated19 and grown in alpha modified
Eagle’s medium supplemented with 10% fetal calf ser-
um, 100 U/mL penicillin, 100 lg/mL streptomycin, and
600 lg/mL G418. Cells expressing hCB1 receptor or
hCB2 receptor were collected by centrifugation and used
immediately or stored at À80 ꢀC. Cell pellets were sus-
pended in homogenization buffer (10 mM Tris–HCl,
5 mM EDTA, and 3 mM EGTA, pH 7.6) containing
protease inhibitor cocktail (Roche Molecular Biochemi-
cals, Indianapolis, IN, USA) and homogenized. Intact
cells and nuclei were removed by centrifugation (270g
for 5 min at 4 ꢀC). The supernatant was centrifuged at
100,000g for 30 min at 4 ꢀC, and the pellet was sus-
pended in a Gly-Gly buffer (20 mM glycylglycine,
1 mM MgCl2, and 250 mM sucrose, pH 7.2) and stored
at À80 ꢀC. Protein concentrations were determined
using BCA protein assay reagent (Pierce, Rockford,
IL, USA), with bovine serum albumin (BSA) as a
standard.
5.3.4. [3H]CP 55,940 binding to rat spleen membranes.
Displacement of [3H]CP 55,940 was measured in rat
spleen membranes of rat. Prepared rat spleen mem-
branes were suspended in assay buffer (50 mM Tris–
HCl, 5 mM EDTA, and 5 mM MgCl2, pH 7.4) contain-
ing 0.2% BSA. Membranes at a protein concentration of
100 lg/well were incubated with various concentrations
of test compounds and [3H]CP 55,940 (0.57 nM) at
25 ꢀC for 120 min. The reaction was terminated by rapid
filtration over a GF/C filter presoaked with 0.1% poly-L-
lysine (Sigma–Aldrich, St. Louis, MO, USA), and the
filter plates were washed three times with the assay buf-
fer containing 0.1% BSA, using a UniFliter96 harvester
(Packard Instruments, Meriden, CT, USA). Microscinti
0 scintillator (Packard Instruments, Meriden, CT, USA)
was added, and filter-bound activity was counted in a
TopcountTM scintillation counter (Packard Instruments,
Meriden, CT, USA). Nonspecific binding was deter-
mined in the presence of 2 lM CP 55,940. Specific bind-
ing was determined by subtracting nonspecific binding
from total binding.
5.3.2. Preparation of rat spleen membranes. Spleens from
male Sprague–Dawley rats (160–200 g, Charles River,
Yokohama, Japan) were dissected and maintained in
ice-cold homogenization buffer (10 mM Tris–HCl,
5 mM EDTA, and 3 mM EGTA, pH 7.6) containing
protease inhibitor cocktail. After removing the connec-
tive tissue, the total tissue weight was determined. The
tissue was then homogenized and centrifuged at 270g
for 10 min at 4 ꢀC, and after centrifuging the superna-
tant at 100,000g for 20 min at 4 ꢀC, the pellet was
washed with the buffer by homogenization and centrifu-
gation again. The final pellet was resuspended in assay
buffer (50 mM Tris–HCl, 5 mM EDTA, and 5 mM
MgCl2, pH 7.4), and protein concentration was deter-
mined with BCA protein assay reagent and with BSA
as a standard.
5.3.5. [35S]GTPcS binding assay to recombinant human
CB2 receptors. Prepared membranes of CHO cells
expressing hCB2 were suspended in assay buffer
(20 mM Hepes, 100 mM NaCl, 3 mM MgCl2, 0.2%
BSA, 3 lM GDP, and 30 lg/mL saponin, pH 7.4).
Membranes at a protein concentration of 5 lg/well were
pre-incubated with various concentrations of test com-
pounds for 30 min at 30 ꢀC. [35S]GTPcS (specific radio-
activity: 46.3 TBq/mmol; PerkinElmer Life Sciences,
Boston, MA, USA, 0.1 nM) was then added, and the
specimens were incubated for 30 min at 30 ꢀC. The reac-
tion was terminated by rapid filtration under a vacuum
through a UniFilter GF/C microplate (PerkinElmer Life
Sciences, Boston, MA) presoaked with the assay buffer.
The filter plates were washed three times with 0.3 mL of
wash buffer (20 mM Hepes buffer, pH 7.4), using a
UniFliter96 harvester (Packard Instruments, Meriden,
CT, USA). Microscinti 0 scintillator (Packard Instru-
ments, Meriden, CT, USA) was added, and filter-bound
5.3.3. [3H]CP 55,940 binding to recombinant human
cannabinoid receptors. Displacement of [3H]CP 55,940
(specific radioactivity, 5942.0 GBq/mmol; PerkinElmer
Life Sciences, Boston, MA, USA) as measured in mem-

H. Ohta et al. / Bioorg. Med. Chem. 16 (2008) 1111–1124
1123
activity was counted in a TopcountTM scintillation coun-
5.5. In vivo pharmacological studies
ter (Packard Instruments, Meriden, CT, USA). Nonspe-
cific binding was determined in the presence of 10 lM
GTPcS. Specific binding was determined by subtracting
nonspecific binding from total binding.
5.5.1. Animals. Male Wistar rats (200–220 g, Charles
River, Yokohama, Japan) were maintained under a
12-h light/dark cycle (light on 7:00 AM) in a tempera-
ture- and humidity-controlled holding room. The ani-
mals were given free access to food and water. All
studies were reviewed by the Taisho Pharmaceutical
Co., Ltd Animal Care Committee and met the Japanese
Experimental Animal Research Association standards,
as defined in the Guidelines for Animal Experiments
(1987).
5.4. Pharmacokinetics
5.4.1. Metabolic stability. Compound 6b (CBS0550) was
incubated at 37 ꢀC for 15 min with 1.0 mg protein/mL of
a human (Xenotech) or rat (Gentest) liver microsomal
fraction in 250 mM phosphate buffer containing
69 mM KCl (pH 7.4) at a final compound concentration
of 5 lM, in the presence of an NADPH-generating sys-
tem (2.4 mM MgCl2, 1.4 mM glucose-6-phosphate,
0.18 U glucose-6-phosphate dehydrogenase). The stabil-
ity was assessed by incubating compounds with micro-
somes, and then the remaining compounds were
analyzed after boiling the microsome reaction mixture.
All experiments were performed in triplicate. After incu-
bation, a twofold volume of DMSO was added to the
incubation medium, and the tube was vortexed and cen-
trifuged at 2000g (4 ꢀC) for 10 min. The supernatant ob-
tained was analyzed with a LC–MS/MS system.
5.5.2. Randall–Selitto test. The rat model of yeast-in-
duced inflammatory pain was produced by the previ-
ously reported method.18 For induction of
inflammation, 0.1 mL of a 20% yeast suspension was in-
jected into the plantar surface of the right hind paw of
the rat. The mechanical nociceptive threshold was mea-
sured using an algesiometer (Ugo Basile, VA, Italy). The
device generated a mechanical force that increased line-
arly over time, and the force was applied to the dorsal
surface of the inflamed hind paw via a cone-shaped sty-
lus with a rounded tip (2 mm2). The nociceptive thresh-
old was defined as the force (in g) at which the rat
withdrew its paw (cut-off force 250 g). The control
paw withdrawal threshold was measured two hours after
the yeast injection, and the rats were given the indicated
doses of 6b (CBS0550) (3, 10, and 30 mg/kg) or vehicle
(5% gum arabic). The paw withdrawal threshold was
determined again 1 and 3 h after administration. The ef-
fect of the compound was expressed as the percent max-
imum possible effect (% MPE) calculated as:
[(nociceptive threshold after administration of the com-
pound À nociceptive threshold in the control)/(baseline
nociceptive threshold À nociceptive threshold in the
control) · 100]. There were seven animals per group.
5.4.2. Animals. Male Sprague–Dawley rats (Charles Riv-
er, Japan) 7–8 weeks old were used in the experiments.
All animals were used after acclimation for at least 4
days. The rats were given free access to water and stan-
dard laboratory diet (MF, Oriental yeast Co., Japan)
during the acclimation period. Environmental condi-
tions were controlled at
a relative humidity of
50 20% and temperature of 23 3 ꢀC.
5.4.3. Plasma concentrations. Compound 6b (CBS0550)
was orally administered to male Sprague–Dawley rats
(Charles River, Japan). Blood was collected from the
tail vein, and plasma was separated by centrifugation
(11,200g, 4 ꢀC, 3 min) and stored at À80 ꢀC until
the bioanalysis. The plasma concentration of 6b
(CBS0550) was determined by liquid chromatography-
tandem mass spectrometry (LC–MS/MS) with electro-
spray ionization (ESI). A 50 lL aliquot of plasma
was added with 200 lL of organic solvent (acetoni-
trile/methanol (9:1, v/v)) containing an internal stan-
dard and vortex-mixed. After centrifugation, the
supernatant (5 lL) obtained was directly injected into
a LC–MS/MS system composed of a CTC HTS-PAL
autosampler (CTC Analytics AG, Zwingen, Switzer-
land), HP1100 binary pump system (Agilent Technolo-
gies, CA, US), and an API 3000 tandem mass
spectrometer equipped with a Turbo Ionspray interface
(Applied Biosystems, CA, US). Chromatographic sepa-
ration with linear gradient elution was performed on a
Zorbax SB C18 (3.5 lm, 30 mm · 4.6 mm ID, Agilent
Technologies) using a 0.1% aqueous solution of formic
acid and acetonitrile. The column temperature and mo-
bile phase flow rate were set at 40 ꢀC and 1.0 mL/min,
respectively. Approximately 1/5 of the column effluent
was directed to the ion source, and multiple reaction
monitoring (MRM) with positive ion detection was per-
formed. The lower limit of quantification (LLOQ) for
rat plasma was 1 ng/mL.
References and notes
1. Gaoni, Y.; Mechoulam, R. J. Am. Chem. Soc. 1964, 86, 1646.
2. Watson, S. J.; Benson, J. A., Jr.; Joy, J. E. Arch. Gen.
Psychiatry 2000, 57, 547.
3. Devane, W. A.; Dysarz, F. A.; Johnson, M. R.; Melvin, L.
S.; Howlett, A. C. Mol. Pharmacol. 1988, 34, 605.
4. Munro, S.; Thomas, K. L.; Abu-Shaar, M. Nature 1993,
365, 61.
5. Howlett, A. C.; Barth, F.; Bonner, T. I.; Cabral, G.;
Casellas, W. A.; Devane, W. A.; Felder, C. C.; Hereken-
ham, M.; Mackie, K.; Martin, B. R.; Mechoulam, R.;
Pertwee, R. G. Pharmacol. Rev. 2002, 54, 161.
6. Klein, T. W.; Friedman, H.; Specter, S. J. Neuroimmunol.
1998, 83, 102.
7. Buckley, N. E.; McCoy, K. L.; Mezey, E.; Bonner, T.;
Zimmer, A.; Felder, C. C.; Glass, M.; Zimmer, A. Eur. J.
Pharmacol. 2000, 396, 141.
8. Fernandez-Ruiz, J.; Romero, J.; Velasco, G.; Tolon, R.
M.; Ramos, J. A.; Guzman, M. Trends Pharmacol. Sci.
2007, 28, 39.
9. Giblin, G. Abstracts of Papers, 229th National Meeting of
the American Chemical Society, Sandiego, CA, March 13–
17, 2005; Giblin, G. American Chemical Society: Wash-
ington, DC, 2005; MEDI 5; Eatherton, A. J.; Giblin, G.
M. P.; Green, R. H.; Mitchell, W. L.; Naylor, A.;

1124
H. Ohta et al. / Bioorg. Med. Chem. 16 (2008) 1111–1124
Rawlings, D. A.; Slingsby, B. P.; Whittington, A. R. 13. Crank, G.; Foulis, M. J. J. Med. Chem. 1998, 41,
WO2004/018433; Giblin, G. M. P.; O’Shaughnessy, C. T.;
Naylor, A.; Mitchell, W. L.; Eatherton, A. J.; Slingsby, B.
P.; Rawlings, D. A.; Goldsmith, P.; Brown, A. J.; Haslam,
C. P.; Clayton, N. M.; Wilson, A. W.; Chessell, I. P.;
Wittington, A. R. J. Med. Chem. 2007, 50, 2597.
1075.
14. Mekonnen, B.; Crank, G. J. Heterocycl. Chem. 1997, 34,
567.
15. Hackler, R. E.; Burow, K. W.; Kaster, S. V., Jr.;
Wickiser, D. I. J. Heterocycl. Chem. 1989, 26,
1575.
16. Johnson, M. R.; Melvin, L. S., Jr.; U.S. Patent 4,371,720,
1981.
17. In vitro metabolic stability expressed as a residual
percentage of parent compound (5 lM) after 15 min.
18. Randall, L. O.; Selitto, J. J. Arch. Int. Pharmacodyn. Ther.
1957, 1, 409.
10. Meyer, M. Abstracts of Papers, 16th Annual Symposium
on the Cannabinoids, Tihany, Hungary, June 24–28, 2006;
International Cannabinoid Research Society, Buring-
ton,VT, 2006; p 147.
11. Ibrahim, M. M.; Deng, H.; Zvonok, A.; Cockayne, D. A.;
Kwan, J.; Mata, H. P.; Vanderah, T. W.; Lai, J.; Porreca,
F.; Makriyannis, A.; Malan, T. P., Jr. Proc. Natl. Acad.
Sci. U.S.A. 2003, 100, 10529.
19. Shire, D.; Calandra, B.; Rinaldi-Carmona, M.; Oustric,
`
D.; Pessegue, B.; Bonnin-Cabanne, O.; Le Fur, G.; Caput,
D.; Ferrara, P. Biochim. Biophys. Acta 1996, 1307, 132–
136.
12. Ohta, H.; Ishizaka, T.; Tatuzuki, M.; Yoshinaga, M.; Iida,
I.; Tomishima, Y.; Toda, Y.; Saito, S. Bioorg. Med. Chem.
Lett. 2007, 17, 6299.