Synthesis, SAR study, and biological evaluation of a series of piperazine ureas as fatty acid amide hydrolase (FAAH) inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.11.006

A series of piperazine ureas was designed, synthesized, and evaluated for their potential as novel orally available fatty acid amide hydrolase (FAAH) inhibitors that are therapeutically effective against pain. We carried out an optimization study of the l

Full text of DOI:10.1016/j.bmc.2012.11.006

Bioorganic & Medicinal Chemistry 21 (2013) 28–41
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, SAR study, and biological evaluation of a series of piperazine ureas
as fatty acid amide hydrolase (FAAH) inhibitors
Mitsunori Kono a, , Takahiro Matsumoto a, Toru Kawamura a, Atsushi Nishimura b, Yoshihiro Kiyota a,
⇑
Hideyuki Oki a, Junichi Miyazaki c, Shigeru Igaki a, Craig A. Behnke d, , Masato Shimojo a, Masakuni Kori e
a Pharmaceutical Research Division, Takeda Pharmaceutical Company Ltd, 26-1, Muraokahigashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
b CMSO Office, Takeda Pharmaceutical Company Ltd, 1-1, Doshomachi 4-chome, Chuo-ku, Osaka 540-8645, Japan
c Pharmaceutical Production Division, Takeda Pharmaceutical Company Ltd, 17-85, Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532-8686, Japan
d Takeda California, Inc., 10410 Science Center Drive, San Diego, CA 92121, United States
e CMC Center, Takeda Pharmaceutical Company Ltd, 17-85, Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532-8686, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of piperazine ureas was designed, synthesized, and evaluated for their potential as novel orally
available fatty acid amide hydrolase (FAAH) inhibitors that are therapeutically effective against pain. We
carried out an optimization study of the lead compound 3 to improve its DMPK profile as well as in vitro
potency. We identified the thiazole compound 60j with potent inhibitory activity, high brain permeabil-
ity, and good bioavailability. Compound 60j showed a potent and dose-dependent anti-nociceptive effect
in the acetic acid-induced writhing test in mice.
Received 5 October 2012
Revised 1 November 2012
Accepted 2 November 2012
Available online 15 November 2012
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
Fatty acid amide hydrolase inhibitors
Arachidonoylethanolamide
Piperazine ureas
Thiazole derivatives
Thiadiazole derivatives
1. Introduction
bulb, and cerebellum in the central nervous system, and suppres-
sively controls the release of neurotransmitters from sensory
Fatty acid amide hydrolase (FAAH)1–3 is an intracellular serine
hydrolase that catalyzes the deactivating hydrolysis of several
endogenous lipid amides4–7 such as endogenous cannabinoid
(EC), anandamide (arachidonoylethanolamide: AEA),8–11 olea-
mide,12–15 oleoylethanolamide,16 and palmitoylethanolamide.17
These lipid amides have several physiological effects, including
the alleviation of pain,18–21 promotion of sleep,12,14,15 and regula-
tion of appetite.22 FAAH knockout mice are viable and healthy,
and showed an analgesic phenotype in several neuropathic and
inflammatory pain models associated with increased brain AEA
levels.23–25
nerves.26–28 On the other hand, the CB2 receptor is highly ex-
pressed in immune organs such as the spleen, and is implicated
in the regulation of inflammatory and immune responses.29,30
A
therapeutic approach utilizing the activation of the CB1 receptor
is expected to be beneficial for the treatment of pain, anxiety,
depression, and sleep disorders. Although CB1 agonists have potent
pharmacological effects, there is concern about adverse effects
arising from their systemic activation, such as sedation, depen-
dence, cognitive impairment, psychosis, and affection of the car-
diovascular system.31,32 FAAH inhibitors enhance the activation
of CB receptors by blocking the degradation of AEA, but they may
offer site-specific increase of AEA in tissues where ECs are being
produced by physiological protective mechanisms, suggesting that
they exhibit pharmacological effects with less adverse effects.
Moreover, FAAH inhibitors may have anti-inflammatory effects
by suppressing the release of inflammatory chemical mediators
by stimulating the CB2 receptor in immune cells.
AEA is an endogenous cannabinoid that activates the cannabi-
noid 1 (CB1)/cannabinoid 2 (CB2) receptors. The CB1 receptor is
highly expressed in the hippocampus, striatum, nigra, olfactory
Abbreviations: FAAH, fatty acid amide hydrolase; EC, endogenous cannabinoid;
AEA, arachidonoylethanolamide; CB1, cannabinoid 1; CB2, cannabinoid 2; SAR,
structure–activity relationship; DMPK, drug metabolism and pharmacokinetics;
Troc, 2,2,2-trichloroethoxy carbonyl.
To date, several groups have reported various classes of FAAH
inhibitors including carbamates (e.g., URB59733 and SA-4734), urea
derivatives (e.g., PF-75035 and LY218324036), and keto-heterocy-
cles (e.g., OL-13537) (Fig. 1). Carbamates and urea-type FAAH inhib-
itors are regarded as irreversible inhibitors that form covalent tight
⇑
Corresponding author. Tel.: +81 466 32 1108; fax: +81 466 29 4455.
E-mail address: mitsunori.kouno@takeda.com (M. Kono).
Current address: Sapphire Energy, Inc., 3115 Merryfield Row, San Diego, CA
92121, United States.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.11.006

M. Kono et al. / Bioorg. Med. Chem. 21 (2013) 28–41
29
Figure 1. Anandamide (AEA) and known FAAH inhibitors.
binding with the catalytic Ser241 residue within the active site of
the FAAH enzyme.38 On the other hand, reversible inhibitors such
as potent keto-heterocycles are believed to form a hemiacetal bond
with the catalytic Ser residue.39,40
In the course of our exploration of novel FAAH inhibitors, high-
throughput screening of our chemical library identified pipera-
zine–urea 141 and aryl carbamate 2, and we started to design novel
FAAH inhibitors from these compounds.42 Initially, our design was
based on the hypothesis that the benzoisoxazolyl carbamate struc-
ture of compound 2 (region b) corresponded to the phenyl urea
moiety (region a) of compound 1. If the benzoisoxazolyl group con-
tributed to the FAAH inhibitory activity of compound 2, it was
thought that the replacement of the benzene ring in region a of 1
with a benzoisoxazolyl group would enhance the inhibitory activ-
ity. On the basis of this hypothesis, we synthesized a hybrid com-
pound 3 possessing region c, and found that 3 showed more potent
inhibition than compounds 1 and 2 (Fig. 2). However, compound 3
had poor aqueous solubility (0.09 lg/mL in pH 6.8 solution) and
bioavailability (F = 5.0% in rats). Therefore, we performed an opti-
mization study based on this compound as a lead to improve the
DMPK profile as well as the in vitro potency. First, we established
the putative binding model between the lead compound 3 and hu-
man FAAH using the X-ray crystal structure of rat FAAH protein43
(Fig. 3). This model suggested that the urea moiety was essential
for in vitro activity by interacting with the catalytic residues
Ser241 and Ser217. The terminal benzene ring was assumed to
make a hydrophobic interaction with the lipophilic site. Thr236
and Thr488 are located near the benzoisoxazole and thiadiazole
moieties, respectively, indicating that these residues can interact
with heteroatoms in the heterocycles of the ligand. On the basis
of this modeling information, we investigated the modification of
compound 3 for the thiadiazole (X), central piperazine (Y), termi-
nal heterocycle (Z), and substituents on the other terminal benzene
ring (R). In this paper, we describe the synthesis, biological evalu-
ation, and structure–activity relationship (SAR) study of a series of
piperazine ureas as novel FAAH inhibitors.
Figure 2. Thiadiazolylpiperazine urea derivative as a promising lead compound.
2. Chemistry
The general synthetic method for the key intermediates 8–11
and 23–28, halogenated 5-membered heterocycles, is shown in
Scheme 1. The aryl amidine hydrochlorides 4–7 were treated with
perchloromethyl mercaptan under strongly basic conditions to af-
ford the thiadiazole intermediates 8–11.44 Thiocyanation of the 2-
bromo-acetophenone derivatives 12–16 was carried out to give
17–21, and subsequent cyclization under acidic conditions fur-
nished the thiazole intermediates 23–27.45 The thiophene interme-
diate 28 was obtained by the bromination of 3-phenylthiophene
22.

30
M. Kono et al. / Bioorg. Med. Chem. 21 (2013) 28–41
none 4 with N-Boc-4-aminothiocarbonylpiperidine gave com-
pound 50. Acidic or basic deprotection and subsequent urea
formation with the corresponding Troc derivatives 34–38 afforded
compounds 60a–60l.
The conversion of the central piperazine ring is illustrated in
Scheme 3. The coupling reaction of several amines with 5-chloro-
3-phenyl-1,2,4-thiadiazole 8, followed by deprotection afforded
compounds 62a–62d. Amidation of 62a with 3-aminobenzoisoxaz-
ole and urea formation of 62b–62d with compound 38 provided
compounds 65a–65d. Compound 65e was obtained by the cou-
pling reaction of 8 with amine 64 derived from urea formation of
N-Boc-ethylenediamine with compound 38 and subsequent
deprotection.
3. Results and discussion
The synthesized compounds were evaluated using in vitro and
in vivo biological tests as follows. The FAAH inhibitory activity
was measured by radioisotopic (RI) assay using human and rat
FAAH enzyme fractions and the substrate anandamide [ethanol-
amine 1-3H] (Tables 1–4). The pharmacokinetic (PK) profiles of
the compounds were analyzed by rat cassette-dosing tests (Ta-
ble 4). The brain concentration was measured from homogenized
brain tissue 1 h after administration in rats (0.1 mg, i.v.) (Table 4).
The in vivo therapeutic efficacy of the compounds was evaluated
using the acetic acid-induced writhing test in mice, a representa-
tive pain model for the evaluation of compounds (Fig. 4).
First, the replacement of the thiadiazole ring at X with other
five-membered ring systems was examined, as shown in Table 1.
The exchange of the sulfur atom for an oxygen atom in the thiadi-
azole ring diminished the inhibitory activity (43d). The thiazole
derivative 60e, in which the nitrogen atom at the 2-position of
Figure 3. Putative binding model of human FAAH with compound 3.
Compounds 43a–43d and 60a–60l were synthesized as shown
in Scheme 2. Starting from the heterocyclic amines 29–33, carba-
mylation with 2,2,2-trichloroethyl chloroformate46 gave the
2,2,2-trichloroethoxy carbonyl (Troc) derivatives 34–38. Urea for-
mation of the Troc derivatives 34 and 38 with commercially avail-
able N-Boc-piperazine and subsequent deprotection yielded 41 and
42. Treatment of compound 41 and 42 with 9–11 or 5-chloro-3-
phenyl-1,2,4-oxadiazole generated compounds 43a–43d. Com-
pounds 44–49 and 51 were synthesized from the corresponding
intermediates 8 and 23–28 by the coupling reaction in the pres-
ence of base or by Buchwald amination with the N-protected
piperazine reagent. Thiazole ring formation of 2-bromoacetophe-
Scheme 1. Synthesis of compounds 8–11 and 23–28. Reagents and conditions: (a) KSCN, EtOH, 80 °C, 68–96%; (b) perchloromethyl mercaptan, NaOH, CH2Cl2, H2O, 0 °C–room
temp, 71–87%; (c) HBr, AcOH, 130 °C, 61–99%; (d) Br2, AcOH.

M. Kono et al. / Bioorg. Med. Chem. 21 (2013) 28–41
31
Scheme 2. Synthesis of compounds 43a–43d and 60a–60l. Reagents and conditions: (a) 2,2,2-trichloroethyl chloroformate, pyridine, THF, 0 °C, 9–64%; (b) N-Boc-piperazine,
iPr2NEt, DMSO, 70 °C, 33–41%; (c) TFA, room temp, 95–96%; (d) 9, 10, 11, or 5-chloro-3-phenyl-1,2,4-oxadiazole, Et3N, DMF, room temp, 16–72%; (e) N-Boc-piperazine, Et3N,
DMF, room temp, 57%; (f) N-Boc-piperazine, K2CO3, DMF, 120 °C, 52–86%; (g) N-ethoxycarbonylpiperazine, Pd2(dba)3, BINAP, NaOtBu, toluene, 100 °C, 5%; (h) N-Boc-4-
aminothiocarbonylpiperidine, K2CO3, DMF, 110 °C, 55%; (i) 4 N HCl in EtOAc, room temp then 1 N NaOH, 67–97%; (j) 8 N NaOH, EtOH, 100 °C, 89%; (k) 34, 35, 36, 37, or 38,
iPr2NEt, DMSO, 70 °C, 14–70%.
Scheme 3. Synthesis of compounds 65a–65e. Reagents and conditions: (a) amines (R1–Y–H), Et3N, DMF, room temp, 70–99%; (b) 2 N NaOH, THF–EtOH, 80 °C, 89%; (c) 2 N HCl
in MeOH, room temp then 1 N NaOH, 24–95%; (d) (COCl)2, 3-amino-benzoisoxazole, pyridine, THF–DMF, room temp, 15%; (e) 38, iPr2NEt, DMSO, 70 °C, 31–54%; (f)
i
BocHN(CH2)2NH2, Pr2NEt, DMSO, 70 °C, 87%; (g) 4 N HCl in EtOAc, room temp then 1 N NaOH, 84%; (h) 8, Et3N, NaI, MeCN, 90 °C, 23%.
the thiadiazole ring was replaced with CH, was equipotent to com-
pound 3. From our putative binding model, Thr488 is located near
this area, and the 2-nitrogen atom of the thiadiazole or the 5-
hydrogen of the thiazole ring are assumed to act as a hydrogen-
bond acceptor/donor by interacting with the Thr488 residue.
However, the removal of the nitrogen atom of the thiazole ring

32
M. Kono et al. / Bioorg. Med. Chem. 21 (2013) 28–41
Table 1
replacement of the pyridazinyl group with a 2-pyridyl group
diminished the activity (60c). On the other hand, the 3-pyridyl
analog 60d showed a potency comparable to that of compound
60b. These results indicated that heteroaryl groups having a het-
eroatom at the meta position improved the inhibitory potency
and solubility. A nitrogen atom at that position is assumed to act
as a hydrogen-bond acceptor by interacting with Thr236. We se-
lected the 3,4-dimethylisoxazolyl derivative 60a, which showed
the most promising activity, for further study, and investigated
the substituent R on the phenyl ring.
Human and rat FAAH activities of derivatives modified at the thiadiazole ring moiety
a
Compds
X
Apparent FAAH IC50 (nM)
(Human/rat)
In assessing the substituent R, we investigated whether the
introduction of fluorine could result in increased potency as well
as brain permeability by a strengthening of the interaction with
the lipophilic site of FAAH. In addition to thiadiazole derivatives,
thiazole analogs were evaluated, taking into account the compa-
rable activities of 60e and 3. The results are shown in Table 4.
The introduction of a 4-fluoro group resulted in a slight increase
in activity in the series of thiadiazole derivatives (43c vs 60a,
43a, 43b). Among the thiazole derivatives, an increase in brain
concentration as well as drug exposure was achieved by fluorine
introduction. In addition, the 2-fluoro and 4-fluoro groups (60g
and 60i) exhibited potent inhibitory activities. The thiazole
derivatives showed more potent inhibitory activities along with
higher exposures and bioavailabilities than the thiadiazole deriv-
atives (60a, 43a, 43b, 43c vs 60f, 60g, 60h, 60i). It is noteworthy
that the replacement of thiadiazole into the thiazole ring led to
great improvement of the solubility despite the increasing lipo-
philicity (cLogP 60a/60f = 2.4/3.1). This phenomenon was
thought to be advantageous not only for the inhibitory activity,
but also for brain penetration. Finally, we identified the 2,4-
difluorophenyl derivative 60j with extraordinarily potent activity
for the FAAH enzyme (hFAAH/rFAAH IC50 = 0.43/0.46 nM) and
sufficient drug exposure, brain concentration, and bioavailability.
Compound 60j was orally available, and exhibited a high brain
3
4.8/5.5
87/130
3.7/6.1
110/290
43d
60e
60l
a
IC50 value were determined at a 30 min reaction.
decreased the activity (60l). These results indicated that the sulfur
atom and the nitrogen atom at the 4-position of the thiadiazole
ring were important for the FAAH inhibitory activity, and that
the thiadiazole and thiazole ring were favorable for group X.
Table 2 shows the SAR for the central piperazine ring moiety Y.
Replacement of the left-hand nitrogen atom with carbon resulted
in a decrease in inhibitory activity (65a). Compound 65d, with a
secondary amine outside the piperidine ring, also showed a similar
loss in activity. The thiazolylpiperidine compound 60k, in which
the right-hand nitrogen atom was replaced with carbon, resulted
in a 10-fold loss in activity compared to compound 3. Ring opening,
ring expansion, or replacement with a bicyclic structure were not
tolerated (65e, 65c, 65b). The results indicated that piperazine
was favorable for the central ring Y.
concentration in mice (AUC = 16.7
lg h/mL, MRT = 3.7 h, brain
concentration at 1 h = 5.86 lg/g at a dose of 10 mg/kg, po
(n = 3)).
In the optimization of the terminal heterocycle Z, as shown in
Table 3, the 3,4-dimethylisoxazolyl analog 60a showed strong
in vitro activity. In addition, notably improved solubility was ob-
Compound 60j was evaluated for its in vivo analgesic efficacy
using the acetic acid-induced writhing test in mice. As shown in
Figure 4, oral administration of compound 60j exerted robust
anti-nociceptive efficacy, which is defined as a decreased number
of stretching movements in a dose-dependent manner. Further
studies to evaluate the effects of compound 60j in other various
pain models are worth exploring the potential of compound 60j
as an analgesic agent.
served for 60a (1.6 lg/mL in pH 6.8 solution), presumably because
of its increased hydrophilicity caused by the removal of the ben-
zene ring part from the benzoisoxazole group. Introduction of a
3-pyridazinyl group instead of the 5-membered ring, produced
compound 60b which exhibited good potency and an additional in-
crease in solubility (6.3 lg/mL in pH 6.8 solution). However, the
Table 2
Human and rat FAAH activities of derivatives modified at the central piperazine ring moiety
a
a
Compds
Y
X
Apparent FAAH IC50 (nM)
(Human/rat)
Compds
Y
X
Apparent FAAH IC50 (nM)
(Human/rat)
3
N
N
4.8/5.5
65c
N
>1000/>1000
65a
>1000/>1000
65d
65b
N
N
>1000/>1000
>1000/>1000
60k
65e
CH
N
43/55
>1000/>1000
a
IC50 value were determined at a 30 min reaction.

M. Kono et al. / Bioorg. Med. Chem. 21 (2013) 28–41
33
Table 3
Human and rat FAAH activities of derivatives modified at the terminal heterocycle
Solubilityb
(lg/mL)
a
Compds
Z
Apparent FAAH IC50 (nM)
(Human/rat)
3
4.8/5.5
1.7/1.3
0.09
1.6
60a
Figure 4. Analgesic effect of compound 60j in acetic acid-induced writhing test.
Compound 60j (3, 10, 30 mg/kg) and vehicle were orally administered 60 min prior
to acetic acid injection. Data are shown in mean SEM, n = 10, #P < 0.025 vs vehicle
(Williams test).
60b
60c
60d
2.9/3.3
120/48
2.4/3.8
6.3
NTc
4.9
a
b
c
sized, and evaluated a series of piperazine urea compounds. The re-
moval of the benzene ring in the benzoisoxazole moiety improved
the solubility. Replacement of the thiadiazole ring with thiazole led
to increase of the FAAH inhibitory activity and a further improve-
ment of solubility. The introduction of fluoro group on the phenyl
ring enhanced activity along with increase of drug exposure and
brain permeability to give compound 60j. Oral administration of
compound 60j showed potent analgesic efficacy in the acetic
acid-induced writhing test in mice. The results suggest that potent
and selective FAAH inhibitors have potencial as beneficial analgesic
agents.
IC50 value were determined at a 30 min reaction.
Solubility in pH 6.8 solution.
Not tested.
These piperazine–urea-type derivatives are assumed to form a
covalent bond with FAAH to inactivate the enzyme irreversibly.
To confirm this with our compounds, we performed co-crystalliza-
tion study of compound 60d with rat FAAH. The co-crystal of com-
pound 60d with rat FAAH enzyme indicated the tight binding of
60d with FAAH (Fig. 5). The bound complex is the carbamylated
enzyme resulting from the attack of the catalytic Ser241 residue
at the urea carbonyl carbon within the active site and the loss of
the 3-aminopyridine as a leaving group. The piperazine ring forms
a chair conformation, and the carbonyl oxygen interacts with the
backbone nitrogen atoms in the 238–241 loop.
5. Experimental section
5.1. Chemistry
4. Conclusion
Melting points were determined with a Yanagimoto melting
point apparatus or a Büchi melting point apparatus B-545 and
are uncorrected. 1H NMR spectra were obtained at 300 MHz on a
Varian Ultra-300 or a Bruker DPX-300 spectrometer. Chemical
In the course of our exploration of novel FAAH inhibitors that
are therapeutically effective against pain, we designed, synthe-
Table 4
Profiles of the thiadiazole and thiazole derivatives
X1
Apparent FAAH IC50 (nM)
(Human/rat)
Solubilityb
(
lg/mL)
AUCc (ngꢀh/mL)
Fc (%)
Brain conc.d (ng/g at 1 h)
a
Compds
R
60a
60f
43a
60g
43b
60h
43c
60i
H
H
N
CH
N
CH
N
CH
N
1.7/1.3
0.92/0.96
2.1/1.2
0.67/0.75
3.2/2.0
1.2/1.4
1.3/0.80
0.86/0.71
0.43/0.46
1.6
38
1.9
3.0
0.51
17
0.56
2.7
1.3
645
915
415
398
706
1329
1040
2655
1309
66
45
31
98
42
91
70
118
98
25
23
4.6
103
12
101
17
132
82
2-F
2-F
3-F
3-F
4-F
4-F
2,4-F
CH
CH
60j
a
b
c
IC50 value were determined at a 30 min reaction.
Solubility in pH 6.8 solution.
Rat cassette dosing, 1 mg/kg, po.
d
Rat 0.1 mg/kg, i.v. administration.

34
M. Kono et al. / Bioorg. Med. Chem. 21 (2013) 28–41
Figure 5. Co-crystallization study of compound 60d with rat FAAH protein.
shifts are given in d values (ppm) using tetramethylsilane as the
internal standard. Peak multiplicities are expressed as follows: s,
singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublet;
br, broad; br s, broad singlet; m, multiplet. Elemental analyses
were carried out by Takeda Analytical Research Laboratories Ltd.
Reactions were followed by TLC on Silica gel 60 F 254 precoated
TLC plates (E. Merck) or NH TLC plates (Fuji Silysia Chemical Ltd).
Chromatographic separations were carried out on silica gel 60
(0.063–0.200 or 0.040–0.063 mm, E. Merck) or basic silica gel
(ChromatorexÒ NH, 100–200 mesh, Fuji Silysia Chemical Ltd) using
the indicated eluents. Yields are unoptimized. The HPLC analyses
were performed using a Shimadzu UFLC instrument. Elution was
done with a gradient of 5–90% solvent B in solvent A (solvent A
was 0.1% TFA in water, and solvent B was 0.1% TFA in acetonitrile)
5.1.4. 5-Chloro-3-(4-fluorophenyl)-1,2,4-thiadiazole (11)
Compound 11 was prepared in a manner similar to that de-
scribed for 9 in 85% yield as a colorless oil. 1H NMR (300 MHz,
CDCl3) d: 7.12–7.19 (2H, m), 8.22–8.27 (2H, m).
5.1.5. 1-Phenyl-2-thiocyanatoethanone (17)
A mixture of 2-bromo-1-phenylethanone (10 g, 50.0 mmol),
potassium thiocyanate (4.90 g, 50.0 mmol), and EtOH (80 mL)
was stirred at 80 °C for 3.0 h. After cooling to room temperature,
the mixture was diluted with water. The solid was collected by fil-
tration and washed with 50% EtOH/water to give 17 (5.99 g, 68%)
as a colorless powder. 1H NMR (300 MHz, CDCl3) d: 4.75 (2H, s),
7.51–7.56 (2H, m), 7.65–7.71 (1H, m), 7.93–7.97 (2H, m).
through
a
L-column
2
ODS (3.0 ꢁ 50 mm,
2
lm) column at
5.1.6. 1-(2-Fluorophenyl)-2-thiocyanatoethanone (18)
1.2 mL minꢂ1. Area% purity was measured at 254 nm.
A mixture of 2-bromo-1-(2-fluorophenyl)ethanone (5.0 g,
23.0 mmol), potassium thiocyanate (2.24 g, 23.0 mmol), and EtOH
(50 mL) was stirred at 80 °C for 2.0 h, diluted with water, and ex-
tracted with chloroform. The organic layer was washed with water,
dried over anhydrous MgSO4, and concentrated in vacuo to give 18
(4.29 g, 96%) as a colorless solid. 1H NMR (300 MHz, CDCl3) d: 4.66
(2H, d, J = 3.0 Hz), 7.19–7.25 (1H, m), 7.29–7.35 (1H, m), 7.62–7.69
(1H, m), 7.96–8.02 (1H, m).
5.1.1. 5-Chloro-3-phenyl-1,2,4-thiadiazole (8)
To a stirred solution of benzamidine hydrochloride (25 g,
160 mmol) and perchloromethyl mercaptan (17.2 mL, 160 mmol)
in CH2Cl2 (160 mL) was added
a solution of NaOH (31.9 g,
798 mmol) in H2O (65 mL) dropwise at 0 °C. The mixture was
stirred at 0 °C for 1.0 h and at room temperature for 1.0 h. The or-
ganic layer was washed with water, dried over anhydrous MgSO4,
and concentrated in vacuo to give 8 (31.4 g) as a pale yellow oil.
This product was used for next reaction without further
purification.
5.1.7. 1-(3-Fluorophenyl)-2-thiocyanatoethanone (19)
Compound 19 was prepared in a manner similar to that de-
scribed for 17 in 74% yield as
a
colorless solid. 1H NMR
(300 MHz, CDCl3) d: 4.70 (2H, s), 7.35–7.42 (1H, m), 7.50–7.57
5.1.2. 5-Chloro-3-(2-fluorophenyl)-1,2,4-thiadiazole (9)
(1H, m), 7.62–7.67 (1H, m), 7.71–7.74 (1H, m).
To
a stirred solution of 2-fluorobenzenecarboximidamide
(1.38 g, 10.0 mmol) and perchloromethyl mercaptan (1.07 mL,
10.0 mmol) in CH2Cl2 (10 mL) was added a solution of NaOH
(1.60 g, 40.0 mmol) in H2O (4.0 mL) dropwise at 0 °C. The mixture
was stirred at 0 °C for 1.0 h and at room temperature for 1.0 h. The
organic layer was washed with water, dried over anhydrous
MgSO4, and concentrated in vacuo. The residue was purified by sil-
ica gel column chromatography (hexane–EtOAc) to give 9 (1.88 g,
87%) as a colorless oil. 1H NMR (300 MHz, CDCl3) d: 7.22–7.30
(2H, m), 7.44–7.52 (1H, m), 8.14–8.19 (1H, m).
5.1.8. 1-(4-Fluorophenyl)-2-thiocyanatoethanone (20)
Compound 20 was prepared in a manner similar to that de-
scribed for 17 in 83% yield as
a
colorless solid. 1H NMR
(300 MHz, CDCl3) d: 4.71 (2H, s), 7.18–7.22 (2H, m), 7.96–8.01
(2H, m).
5.1.9. 1-(2,4-Difluorophenyl)-2-thiocyanatoethanone (21)
Compound 21 was prepared in a manner similar to that de-
scribed for 17 in 75% yield as
a
colorless solid. 1H NMR
(300 MHz, CDCl3) d: 4.61 (1H, s), 4.62 (1H, s), 6.92–7.09 (2H, m),
5.1.3. 5-Chloro-3-(3-fluorophenyl)-1,2,4-thiadiazole (10)
Compound 10 was prepared in a manner similar to that de-
scribed for 9 in 71% yield as a colorless solid. 1H NMR (300 MHz,
CDCl3) d: 7.15–7.22 (1H, m), 7.42–7.49 (1H, m), 7.93–7.97 (1H,
m), 8.03–8.06 (1H, m).
8.02–8.10 (1H, m).
5.1.10. 2-Bromo-4-phenylthiazole (23)
To a stirred solution of 1-(2-fluorophenyl)-2-thiocyanatoetha-
none (20.0 g, 113 mmol) in AcOH (200 mL) was added 25% HBr in

M. Kono et al. / Bioorg. Med. Chem. 21 (2013) 28–41
35
AcOH (200 mL) dropwise at room temperature. The mixture was
stirred at 130 °C for 2.0 h and at room temperature for 1.0 h. The
mixture was diluted with water, and extracted with chloroform.
The organic layer was washed with water, dried over anhydrous
MgSO4, and concentrated in vacuo. The residue was purified by sil-
ica gel column chromatography (hexane–EtOAc) to give 23 (15.2 g,
56%) as a pale yellow oil. 1H NMR (300 MHz, CDCl3) d: 7.35–7.45
(4H, m), 7.83–7.87 (2H, m).
5.1.18. 2,2,2-Trichloroethyl pyridin-3-ylcarbamate (37)
Compound 37 was prepared in a manner similar to that de-
scribed for 36 in 64% yield as a colorless powder. 1H NMR
(300 MHz, CDCl3) d: 4.85 (2H, s), 7.31 (1H, dd, J = 4.8, 8.4 Hz),
8.01 (1H, br s), 8.07–8.10 (1H, m), 8.40 (1H, d, J = 4.8 Hz), 8.58–
8.59 (1H, m).
5.1.19. 2,2,2-Trichloroethyl benzo[d]isoxazol-3-ylcarbamate
(38)
Compound 38 was prepared in a manner similar to that de-
scribed for 36 in 58% yield as an off-white solid. 1H NMR
(300 MHz, CDCl3) d: 4.91 (2H, s), 7.29–7.39 (1H, m), 7.47–7.64
(2H, m), 7.88 (1H, br s), 8.16 (1H, d, J = 7.9 Hz).
5.1.11. 2-Bromo-4-(2-fluorophenyl)thiazole (24)
Compound 24 was prepared in a manner similar to that de-
scribed for 23 in 61% yield as a pale yellow oil. 1H NMR
(300 MHz, CDCl3) d: 7.10–7.35 (3H, m), 7.69 (1H, s), 8.14–8.19
(1H, m).
5.1.20. tert-Butyl 4-[(3,4-dimethylisoxazol-5-
yl)carbamoyl]piperazine-1-carboxylate (39)
5.1.12. 2-Bromo-4-(3-fluorophenyl)thiazole (25)
Compound 25 was prepared in a manner similar to that de-
scribed for 23 in 99% yield as a pale yellow oil. 1H NMR
(300 MHz, CDCl3) d: 7.04–7.05 (1H, m), 7.34–7.42 (1H, m), 7.43
(1H, s), 7.56–7.64 (2H, m).
A mixture of N-Boc-piperazine (5.0 g, 26.8 mmol), 2,2,2-trichlo-
roethyl (3,4-dimethylisoxazol-5-yl)carbamate (3.85 g, 13.4 mmol),
N-ethyldiisopropylamine (2.33 mL, 13.4 mmol), and DMSO (50 mL)
was stirred at 70 °C for 2.0 h, poured into water, and extracted with
EtOAc. The organic layer was washed with water, dried over anhy-
drous MgSO4, and concentrated in vacuo. The residue was recrys-
tallized from EtOAc–hexane to give 39 (1.80 g, 41%) as colorless
crystals. 1H NMR (300 MHz, DMSO-d6) d: 1.48 (9H, s), 1.87 (3H,
s), 2.19 (3H, s), 3.49 (8H, s), 6.81 (1H, br s).
5.1.13. 2-Bromo-4-(4-fluorophenyl)thiazole (26)
Compound 26 was prepared in a manner similar to that de-
scribed for 23 in 92% yield as a pale yellow solid. 1H NMR
(300 MHz, CDCl3) d: 7.07–7.14 (2H, m), 7.34 (1H, s), 7.80–7.86
(2H, m).
5.1.21. tert-Butyl 4-(benzo[d]isoxazol-3-
ylcarbamoyl)piperazine-1-carboxylate (40)
Compound 40 was prepared in a manner similar to that de-
scribed for 39 in 33% yield as colorless crystals. 1H NMR
(300 MHz, CDCl3) d: 1.50 (9H, s), 3.56–3.66 (8H, m), 7.27–7.32
(1H, m), 7.46–7.58 (2H, m), 8.05 (1H, d, J = 8.4 Hz), 8.37 (1H, s).
5.1.14. 2-Bromo-4-(2,4-difluorophenyl)thiazole (27)
Compound 27 was prepared in a manner similar to that de-
scribed for 23 in 74% yield as a pale yellow solid. 1H NMR
(300 MHz, CDCl3) d: 6.86–7.00 (2H, m), 7.62–7.63 (1H, m), 8.12–
8.20 (1H, m).
5.1.22. N-(3,4-Dimethylisoxazol-5-yl)piperazine-1-carboxamide
2,2,2-trifluoroacetate (41)
5.1.15. 2,2,2-Trichloroethyl (3,4-dimethylisoxazol-5-
yl)carbamate (34)
A mixture of tert-butyl 4-[(3,4-dimethylisoxazol-5-yl)carbam-
oyl]piperazine-1-carboxylate (1.00 g, 3.08 mmol) and trifluoroace-
tic acid (20 mL) was stirred at room temperature for 2.0 h, and then
the solvent was concentrated in vacuo to give 41 (1.00 g, 96%) as a
yellow oil. 1H NMR (300 MHz, CDCl3) d: 1.76 (3H, s), 2.13 (3H, s),
3.17 (4H, br s), 3.63 (4H, br s), 8.89 (2H, br s), 9.38 (1H, br s).
To a stirred solution of 5-amino-3,4-dimethylisoxazole (1.0 g,
8.92 mmol) and pyridine (0.873 mL, 10.7 mmol) in THF (30 mL)
was added 2,2,2-trichloroethyl chloroformate (1.48 mL, 10.7
mmol) dropwise at 0 °C. The mixture was stirred at 0 °C for 1.0 h,
poured into water, and extracted with EtOAc. The organic layer
was washed with water, dried over anhydrous MgSO4, and concen-
trated in vacuo. The residue was purified by silica gel column chro-
matography (hexane–EtOAc) to give 34 (1.61 g, 63%) as a colorless
powder. 1H NMR (300 MHz, CDCl3) d: 1.92 (3H, s), 2.22 (3H, s), 4.82
(2H, s), 7.40 (1H, br s).
5.1.23. N-(Benzo[d]isoxazol-3-yl)piperazine-1-carboxamide
2,2,2-trifluoroacetate (42)
A
mixture of tert-butyl 4-(benzo[d]isoxazol-3-ylcarba-
moyl)piperazine-1-carboxylate (16.5 g, 47.6 mmol) and trifluoro-
acetic acid (200 mL) was stirred at room temperature for 2.0 h,
and then the solvent was concentrated in vacuo. The residue was
recrystallized from MeOH–Et2O to give 42 (16.3 g, 95%) as a solid.
1H NMR (300 MHz, DMSO-d6) d: 3.18–3.22 (4H, m), 3.72–3.75 (4H,
m), 7.30–7.35 (1H, m), 7.59–7.67 (2H, m), 7.86 (1H, d, J = 8.4 Hz),
8.99 (2H, br s), 10.11 (1H, s).
5.1.16. 2,2,2-Trichloroethyl pyridazin-3-ylcarbamate (35)
Compound 35 was prepared in a manner similar to that de-
scribed for 34 in 9% yield as a yellow powder. 1H NMR (300 MHz,
CDCl3) d: 4.87 (2H, s), 7.52 (1H, dd, J = 8.7, 5.1 Hz), 8.25 (1H, dd,
J = 1.5, 8.7 Hz), 8.31 (1H, br s), 8.95 (1H, dd, J = 1.5, 5.1 Hz).
5.1.24. N-(3,4-Dimethylisoxazol-5-yl)-4-[3-(2-fluorophenyl)-
1,2,4-thiadiazol-5-yl]piperazine-1-carboxamide (43a)
5.1.17. 2,2,2-Trichloroethyl pyridazin-2-ylcarbamate (36)
To a stirred solution of pyridin-2-amine (1.0 g, 10.6 mmol) and
pyridine (1.01 mL, 12.7 mmol) in THF (35 mL) was added 2,2,2-
trichloroethyl chloroformate (1.76 mL, 12.7 mmol) dropwise at
0 °C. The mixture was stirred at 0 °C for 1.5 h, poured into water,
and extracted with EtOAc. The organic layer was washed with
water, dried over anhydrous MgSO4, and concentrated in vacuo.
The residue was triturated with Et2O–hexane to give 36 (1.76 g,
62%) as a colorless powder. 1H NMR (300 MHz, CDCl3) d: 4.88
(2H, s), 7.01–7.05 (1H, m), 7.71–7.76 (1H, m), 8.03 (1H, d,
J = 8.4 Hz), 8.48–8.51 (1H, m).
A
mixture of N-(3,4-dimethylisoxazol-5-yl)piperazine-1-
carboxamide trifluoroacetate (297 mg, 0.707 mmol), 5-chloro-3-
(2-fluorophenyl)-1,2,4-thiadiazole (152 mg, 0.707 mmol),
triethylamine (0.491 ml, 3.53 mmol), and DMF (3.0 mL) was stirred
at room temperature for 3.0 h, poured into water, and extracted
with EtOAc. The organic layer was washed with brine, dried over
anhydrous MgSO4, and concentrated in vacuo. The residue was
recrystallized from EtOAc–hexane to give 43a (44.0 mg, 16%) as
colorless crystals, mp 239–240 °C. 1H NMR (300 MHz, DMSO-d6)
d: 1.76 (3H, s), 2.13 (3H, s), 3.63 (8H, br s), 7.28–7.35 (2H, m),

36
M. Kono et al. / Bioorg. Med. Chem. 21 (2013) 28–41
7.49–7.56 (1H, m), 8.00–8.06 (1H, m), 9.34 (1H, s). Analytical HPLC
showed 100% purity.
CDCl3) d: 1.49 (9H, s), 3.51–3.61 (8H, m), 6.82 (1H, s), 6.94–7.00
(1H, m), 7.29–7.36 (1H,m), 7.53–7.60 (2H,m).
5.1.25. N-(3,4-Dimethylisoxazol-5-yl)-4-[3-(3-fluorophenyl)-
1,2,4-thiadiazol-5-yl]piperazine-1-carboxamide (43b)
Compound 43b was prepared in a manner similar to that de-
scribed for 43a in 45% yield as colorless crystals, mp 219–220 °C.
1H NMR (300 MHz, CDCl3) d: 1.88 (3H, s), 2.20 (3H, s), 3.66–3.75
(8H, m), 7.08–7.15 (1H, m), 7.36–7.44 (1H, m), 7.86–7.91 (1H,
m), 7.96–8.00 (1H, m), 9.05 (1H, s). Analytical HPLC showed 100%
purity.
5.1.32. tert-Butyl 4-[4-(4-fluorophenyl)thiazol-2-yl]piperazine-
1-carboxylate (48)
Compound 48 was prepared in a manner similar to that de-
scribed for 45 in 52% yield as a colorless oil. 1H NMR (300 MHz,
CDCl3) d: 1.49 (9H, s), 3.50–3.60 (8H, m), 6.71 (1H, s), 7.02–7.08
(2H, m), 7.77–7.82 (2H, m).
5.1.33. tert-Butyl 4-[4-(2,4-difluorophenyl)thiazol-2-
yl]piperazine-1-carboxylate (49)
5.1.26. N-(3,4-Dimethylisoxazol-5-yl)-4-[3-(4-fluorophenyl)-
1,2,4-thiadiazol-5-yl]piperazine-1-carboxamide (43c)
Compound 43c was prepared in a manner similar to that de-
scribed for 43a in 20% yield as colorless crystals, mp 220–221 °C.
1H NMR (300 MHz, CDCl3) d: 1.77 (3H, s), 2.13 (3H, s), 3.64 (8H,
s), 7.28–7.34 (2H, m), 8.13–8.18 (2H, m), 9.34 (1H, s). Analytical
HPLC showed 98.2% purity.
Compound 49 was prepared in a manner similar to that de-
scribed for 45 in 86% yield as a colorless powder. 1H NMR
(300 MHz, CDCl3) d: 1.49 (9H, s), 3.48–3.60 (8H, m), 6.81–6.95
(2H, m), 7.02–7.03 (1H, m), 8.08–8.16 (1H, m).
5.1.34. tert-Butyl 4-(4-phenylthiazol-2-yl)piperidine-1-
carboxylate (50)
A mixture of tert-butyl 4-carbamothioylpiperidine-1-carboxyl-
ate (1.0 g, 7.24 mmol), 2-bromo-1-phenylethanone (1.58 g,
7.96 mmol), potassium carbonate (1.0 g, 7.24 mmol), and DMF
(30 mL) was stirred at 110 °C for 1.5 h, poured into water, and ex-
tracted with EtOAc. The organic layer was washed with brine, dried
over anhydrous MgSO4, and concentrated in vacuo. The residue
purified by silica gel column chromatography (hexane–EtOAc) to
give 50 (1.38 g, 55%) as a colorless oil. 1H NMR (300 MHz, CDCl3)
d: 1.46 (9H, s), 1.69–1.86 (2H, m), 2.13–2.18 (2H, m), 2.88–2.96
(2H, m), 3.17–3.27 (1H, m), 4.19 (2H, br s), 7.29–7.51 (4H, m),
7.86–7.89 (2H, m).
5.1.27. N-(Benzo[d]isoxazol-3-yl)-4-(3-phenyl-1,2,4-oxadiazol-
5-yl)piperazine-1-carboxamide (43d)
Compound 43d was prepared in a manner similar to that de-
scribed for 43a in 72% yield as colorless crystals, mp 215–216 °C.
1H NMR (300 MHz, DMSO-d6) d: 3.72 (8H, s), 7.30–7.35 (1H, m),
7.49–7.67 (5H, m), 7.85–7.94 (3H, m), 10.09 (1H, s). Anal. Calcd
for C20H18N6O3: C, 61.53; H, 4.65; N, 21.53. Found: C, 61.68; H,
4.70; N, 21.42.
5.1.28. tert-Butyl 4-(3-phenyl-1,2,4-thiadiazol-5-yl)piperazine-
1-carboxylate (44)
A mixture of N-Boc-piperazine (29.7 g, 160 mmol), 5-chloro-3-
phenyl-1,2,4-thiadiazole (31.4 g, 160 mmol), triethylamine
(89.0 ml, 638 mmol), and DMF (320 mL) was stirred at room tem-
perature for 2.0 h, poured into water, and extracted with EtOAc.
The organic layer was washed with brine, dried over anhydrous
MgSO4, and concentrated in vacuo. The residue was purified by sil-
ica gel column chromatography (hexane–EtOAc) to give 44 (31.6 g,
57% from 8) as a colorless powder. 1H NMR (300 MHz, CDCl3) d:
1.50 (9H, s), 3.60 (8H, s), 7.38–7.49 (3H, m), 8.14–8.24 (2H, m).
5.1.35. Ethyl 4-(4-phenylthiophen-2-yl)piperazine-1-
carboxylate (51)
To a stirred solution of 3-phenylthiophene (5.00 g, 32.0 mmol)
in AcOH (65 mL) was added
a solution of bromine (5.00 g,
32.0 mmol) in AcOH (50 mL) dropwise at room temperature, and
the mixture was refluxed for 5.0 h. After cooling to room tempera-
ture, the mixture was poured into water, and extracted with Et2O.
The organic layer was washed with sat. aq NaHCO3, dried over
anhydrous MgSO4, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (hexane–EtOAc) to
give the mixture of 2-bromo-4-phenylthiophene and 2-bromo-3-
phenylthiophene (6.00 g) as an oil. The obtained compound was
dissolved in toluene (50 mL), and ethyl piperazine-1-carboxylate
(3.96 g, 25.0 mmol), sodium tert-butoxide (3.48 g, 36.2 mmol),
5.1.29. tert-Butyl 4-(4-phenylthiazol-2-yl)piperazine-1-
carboxylate (45)
A mixture of N-Boc-piperazine (25.4 g, 136 mmol), 2-bromo-4-
phenylthiazole (16.4 g, 68.2 mmol), potassium carbonate (9.43 g,
68.2 mmol), and DMF (230 mL) was stirred at 120 °C for 8.0 h,
poured into water, and extracted with EtOAc. The organic layer
was washed with water and brine, dried over anhydrous MgSO4,
and concentrated in vacuo. The residue was purified by silica gel
column chromatography (hexane–EtOAc) to give 45 (14.6 g, 62%)
as a colorless powder. 1H NMR (300 MHz, CDCl3) d: 1.49 (9H, s),
3.51–3.59 (8H, m), 6.79 (1H, s), 7.27–7.39 (3H, m), 7.80– 7.83
(2H, m).
2,20-bis(diphenylphosphino)-1,10-binaphthyl
(468 mg,
0.750
mmol), tris(dibenzylideneacetone)dipalladium (0) (544 mg,
0.594 mmol) were added to the mixture. The mixture was stirred
at 100 °C for 48 h, and solid was removed by filtration, and the fil-
trate was concentrated in vacuo. The residue was purified by silica
gel column chromatography (hexane–EtOAc) to give 51 (365 mg,
5% from 22) as a colorless oil. 1H NMR (300 MHz, CDCl3) d: 1.29
(3H, t, J = 7.2 Hz), 3.14–3.17 (4H, m), 3.63–3.67 (4H, m), 4.17 (2H,
q, J = 7.2 Hz), 6.47 (1H, d, J = 1.8 Hz), 6.78 (1H, d, J = 1.8 Hz),
7.26–7.30 (1H, m), 7.34–7.40 (2H, m), 7.52–7.56 (2H, m).
5.1.30. tert-Butyl 4-[4-(2-fluorophenyl)thiazol-2-yl]piperazine-
1-carboxylate (46)
Compound 46 was prepared in a manner similar to that de-
scribed for 45 in 80% yield as a colorless oil. 1H NMR (300 MHz,
CDCl3) d: 1.49 (9H, s), 3.48–3.61 (8H, m), 7.05–7.27 (4H, m),
8.10–8.16 (1H, m).
5.1.36. 3-Phenyl-5-(piperazin-1-yl)-1,2,4-thiadiazole (52)
To a stirred solution of tert-butyl 4-(4-phenylthiazol-2-yl)piper-
azine-1-carboxylate (31.6 g, 91.3 mmol) in EtOAc (300 mL) was
added 4 N HCl in EtOAc (300 mL) dropwise at room temperature.
After stirring at room temperature overnight, the mixture was di-
luted with hexane (600 mL). The solid was collected by filtration,
and suspended in water, neutralized with sat. aq NaHCO3. The
resulting precipitate was collected by filtration and washed with
water to give 52 (21.7 g, 97%) as a colorless powder. 1H NMR
5.1.31. tert-Butyl 4-[4-(3-fluorophenyl)thiazol-2-yl]piperazine-
1-carboxylate (47)
Compound 47 was prepared in a manner similar to that de-
scribed for 45 in 69% yield as a colorless oil. 1H NMR (300 MHz,

M. Kono et al. / Bioorg. Med. Chem. 21 (2013) 28–41
37
(300 MHz, DMSO-d6) d: 2.77–2.91 (4H, m), 3.41–3.57 (4H, m),
7.42–7.51 (3H, m), 8.06–8.16 (2H, m).
layer was washed with water, dried over anhydrous MgSO4, and
concentrated in vacuo. The residue was recrystallized from THF–
hexane to give 60a (125 mg, 44%) as colorless crystals, mp 233–
234 °C. 1H NMR (300 MHz, DMSO-d6) d: 1.86 (3H, s), 2.19 (3H, s),
3.65–3.68 (4H, m), 3.73–3.76 (4H, m), 7.40–7.44 (3H, m), 8.15–
8.18 (2H, m), 9.24 (1H, s). Anal. Calcd for C18H20N6O2S: C, 56.23;
H, 5.24; N, 21.86. Found: C, 56.02; H, 5.21; N, 21.53.
5.1.37. 4-Phenyl-2-(piperazin-1-yl)thiazole (53)
To a stirred solution of tert-butyl 4-(4-phenylthiazol-2-yl)piper-
azine-1-carboxylate (14.1 g, 40.7 mmol) in EtOAc (220 mL) was
added 4 N HCl in EtOAc (100 mL) dropwise at room temperature.
After stirring at room temperature for 4.0 h, the mixture was con-
centrated in vacuo. The residue was dissolved in water, neutralized
with 1 N NaOH, and extracted with chloroform. The organic layer
was dried over anhydrous MgSO4, and concentrated in vacuo to
give 53 (10.1 g, quant.) as a colorless oil. 1H NMR (300 MHz, CDCl3)
d: 2.99–3.03 (4H, m), 3.51–3.54 (4H, m), 6.77 (1H, s), 7.24–7.40
(3H, m), 7.82–7.85 (2H, m).
5.1.45. 4-(3-Phenyl-1,2,4-thiadiazol-5-yl)-N-(pyridazin-3-
yl)piperazine-1-carboxamide (60b)
A
mixture of 2,2,2-trichloroethyl pyridazin-3-ylcarbamate
(200 mg, 0.793 mmol), 1-(3-phenyl-1,2,4-thiadiazol-5-yl)pipera-
zine (182 mg, 0.739 mmol), N-ethyldiisopropylamine (0.129 ml,
0.739 mmol), and DMSO (3.0 mL) was stirred at 70 °C overnight.
The reaction mixture was poured into water and a solid was col-
lected by filtration. This was recrystallized from EtOAc to give
60b (117 mg, 43%) as off-white crystals, mp 185–186 °C. 1H NMR
(300 MHz, DMSO-d6) d: 3.65–3.73 (8H, m), 7.46–7.50 (3H, m),
7.59 (1H, dd, J = 9.0, 5.4 Hz), 8.01 (1H, dd, J = 1.5, 9.0 Hz), 8.11–
8.14 (2H, m), 8.86 (1H, dd, J = 5.4, 1.5 Hz), 10.11 (1H, br s). Anal.
Calcd for C17H17N7OSꢀ0.2H2O: C, 55.03; H, 4.73; N, 26.43. Found:
C, 55.21; H, 4.67; N, 26.08.
5.1.38. 4-(2-Fluorophenyl)-2-(piperazin-1-yl)thiazole (54)
Compound 54 was prepared in a manner similar to that de-
scribed for 53 in 67% yield as a colorless oil. 1H NMR (300 MHz,
CDCl3) d: 2.99–3.02 (4H, m), 3.50–3.54 (4H, m), 7.05–7.25 (4H,
m), 8.12–8.18 (1H, m).
5.1.39. 4-(3-Fluorophenyl)-2-(piperazin-1-yl)thiazole (55)
Compound 55 was prepared in a manner similar to that de-
scribed for 53 in 86% yield as a colorless powder. 1H NMR
(300 MHz, CDCl3) d: 2.99–3.03 (4H, m), 3.50–3.54 (4H, m), 6.79
(1H, s), 6.90–6.99 (1H, m), 7.28–7.35 (1H, m), 7.53–7.61 (2H, m).
5.1.46. 4-(3-Phenyl-1,2,4-thiadiazol-5-yl)-N-(pyridazin-2-
yl)piperazine-1-carboxamide (60c)
A
mixture of 3-phenyl-5-(piperazin-1-yl)-1,2,4-thiadiazole
(200 mg, 0.738 mmol), 2,2,2-trichloroethyl pyridazin-2-ylcarba-
mate (271 mg, 0.610 mmol), N-ethyldiisopropylamine (0.129 mL,
0.738 mmol), and DMSO (2.5 mL) was stirred at 70 °C for 3.0 h,
poured into water, and extracted with EtOAc. The organic layer
was washed with water, dried over anhydrous MgSO4, and concen-
trated in vacuo. The residue was purified by silica gel column
chromatography (hexane–EtOAc), and recrystallized from EtOAc–
hexane to give 60c (124 mg, 55%) as colorless crystals, mp
162–163 °C. 1H NMR (300 MHz, CDCl3) d: 3.69–3.72 (8H, m),
6.96–7.02 (1H, m), 7.39–7.44 (4H, m), 7.64–7.70 (1H, m), 7.99
(1H, d, J = 8.1 Hz), 8.15–8.22 (3H, m). Anal. Calcd for C18H18N6OS:
C, 59.00; H, 4.95; N, 22.93. Found: C, 58.96; H, 4.95; N, 22.92.
5.1.40. 4-(4-Fluorophenyl)-2-(piperazin-1-yl)thiazole (56)
Compound 56 was prepared in a manner similar to that de-
scribed for 53 in 67% yield as a colorless oil. 1H NMR (300 MHz,
CDCl3) d: 2.94–2.99 (4H, m), 3.45–3.50 (4H, m), 6.64 (1H, s),
6.96–7.05 (2H, m), 7.24–7.80 (2H, m).
5.1.41. 4-(2,4-Difluorophenyl)-2-(piperazin-1-yl)thiazole (57)
Compound 57 was prepared in a manner similar to that de-
scribed for 53 in 74% yield as a colorless powder. 1H NMR
(300 MHz, CDCl3) d: 2.99–3.02 (4H, m), 3.49–3.53 (4H, m), 6.81–
6.94 (2H, m), 6.99–7.00 (1H, m), 8.09–8.17 (1H, m).
5.1.42. 4-Phenyl-2-(piperidin-4-yl)thiazole (58)
Compound 58 was prepared in a manner similar to that de-
scribed for 53 in 98% yield as a pale yellow oil. 1H NMR
(300 MHz, CDCl3) d: 1.70–1.83 (2H, m), 2.14–2.19 (2H, m), 2.74–
2.83 (2H, m), 3.15–3.24 (3H, m), 7.28–7.44 (4H, m), 7.86–7.90
(2H, m).
5.1.47. 4-(3-Phenyl-1,2,4-thiadiazol-5-yl)-N-(pyridin-3-
yl)piperazine-1-carboxamide hydrochloride (60d)
A
mixture of 3-phenyl-5-(piperazin-1-yl)-1,2,4-thiadiazole
(8.0 g, 32.5 mmol), 2,2,2-trichloroethyl pyridin-3-ylcarbamate
(9.63 g, 35.7 mmol), N-ethyldiisopropylamine (6.20 mL,
35.7 mmol), and DMSO (108 mL) was stirred at 70 °C overnight,
poured into water, and extracted with EtOAc. The organic layer
was washed with water, dried over anhydrous MgSO4, and concen-
trated in vacuo. The residue was purified by silica gel column
chromatography (EtOAc), and recrystallized from EtOAc–hexane
to give 4-(3-phenyl-1,2,4-thiadiazol-5-yl)-N-(pyridin-3-yl)pipera-
zine-1-carboxamide. This product (5.10 g) was dissolved in THF
(600 mL), and 4 N HCl in EtOAc (120 mL) was added. The mixture
was stirred at room temperature for 1.0 h and concentrated in va-
cuo. The residue was recrystallized from MeOH–Et2O to give 60d
(5.46 g, 42%) as colorless crystals, mp 198–199 °C. 1H NMR
(300 MHz, DMSO-d6) d: 3.68–3.71 (4H, m), 3.82–3.86 (4H, m),
7.40–7.44 (3H, m), 7.85–7.90 (1H, m), 8.11–8.16 (2H, m), 8.42
(1H, d, J = 5.4 Hz), 8.69–8.73 (1H, m), 9.29 (1H, m), 10.06 (1H, s).
Anal. Calcd for C18H19ClN6OS: C, 53.66; H, 4.75; N, 20.86; Cl,
8.80. Found: C, 53.54; H, 4.76; N, 20.77; Cl, 8.71.
5.1.43. 1-(4-Phenylthiophen-2-yl)piperazine (59)
To a stirred solution of ethyl 4-(4-phenylthiophen-2-yl)pipera-
zine-1-carboxylate (350 mg, 1.11 mmol) in EtOH (10 mL) was
added 8 N NaOH (5.0 mL, 40.0 mmol) dropwise at room tempera-
ture. After stirring at 100 °C for 3.0 h, the mixture was concen-
trated in vacuo. The residue was diluted with water, and
extracted with chloroform. The organic layer was dried over anhy-
drous MgSO4, and concentrated in vacuo to give 59 (240 mg, 89%)
as a pale yellow oil. 1H NMR (300 MHz, CDCl3) d: 3.03–3.06 (4H,
m), 3.15–3.18 (4H, m), 6.42 (1H, d, J = 1.8 Hz), 6.74 (1H, d,
J = 1.8 Hz), 7.26–7.29 (1H, m), 7.34–7.39 (2H, m), 7.54–7.56 (2H,
m).
5.1.44. N-(3,4-Dimethylisoxazol-5-yl)-4-(3-phenyl-1,2,4-
thiadiazol-5-yl)piperazine-1-carboxamide (60a)
A
mixture of 3-phenyl-5-(piperazin-1-yl)-1,2,4-thiadiazole
5.1.48. N-(Benzo[d]isoxazol-3-yl)-4-(4-phenylthiazol-2-
yl)piperazine-1-carboxamide (60e)
Compound 60e was prepared in a manner similar to that de-
scribed for 60a in 70% yield as off-white crystals, mp 213–214 °C.
1H NMR (300 MHz, DMSO-d6) d: 3.58–3.60 (4H, m), 3.62–3.72
(212 mg, 0.738 mmol), 2,2,2-trichloroethyl (3,4-dimethylisoxazol-
5-yl)carbamate (200 mg, 0.812 mmol), N-ethyldiisopropylamine
(0.141 mL, 0.812 mmol), and DMSO (2.5 mL) was stirred at 70 °C
for 3.0 h, poured into water, and extracted with EtOAc. The organic

38
M. Kono et al. / Bioorg. Med. Chem. 21 (2013) 28–41
(4H, m), 7.27–7.43 (5H, m), 7.58–7.66 (2H, m), 7.85–7.89 (3H, m),
10.07 (1H, s). Anal. Calcd for C21H19N5O2S: C, 62.21; H, 4.72; N,
17.27. Found: C, 62.18; H, 4.85; N, 17.02.
7.84 (1H, d, J = 8.1 Hz), 10.04 (1H, s). Anal. Calcd for
C22H20N4SO2ꢀ0.1H2O: C, 65.04; H, 5.01; N, 13.79. Found: C, 65.12;
H, 4.80; N, 13.48.
5.1.49. N-(3,4-Dimethylisoxazol-5-yl)-4-(4-phenylthiazol-2-
yl)piperazine-1-carboxamide (60f)
5.1.56. Ethyl 1-(3-phenyl-1,2,4-thiadiazol-5-yl)piperidine-4-
carboxylate (61a)
Compound 60f was prepared in a manner similar to that de-
scribed for 60c in 14% yield as colorless crystals, mp 185–186 °C.
1H NMR (300 MHz, CDCl3) d: 1.89 (3H, s), 2.21 (3H, s), 3.66 (8H,
s), 6.63 (1H, br s), 6.83 (1H, s), 7.29–7.31 (1H, m), 7.36–7.41 (2H,
m), 7.82–7.84 (2H, m). Anal. Calcd for C19H21N5O2S: C, 59.51; H,
5.52; N, 18.26. Found: C, 59.50; H, 5.52; N, 18.30.
A mixture of ethyl piperidine-4-carboxylate (0.782 mL, 5.08
mmol), 5-chloro-3-phenyl-1,2,4-thiadiazole (1.00 g, 5.08 mmol),
triethylamine (0.708 mL, 5.08 mmol), and DMF (10 mL) was stirred
at room temperature for 30 min, poured into water, and extracted
with EtOAc. The organic layer was washed with brine, dried over
anhydrous MgSO4, and concentrated in vacuo. The residue was
recrystallized from EtOAc–hexane to give 61a (1.22 g, 76%) as a
colorless powder. 1H NMR (300 MHz, CDCl3) d: 1.28 (3H, t,
J = 7.2 Hz), 1.82–1.95 (2H, m), 2.04–2.11 (2H, m), 2.55–2.63 (1H,
m), 3.26–3.35 (2H, m), 3.95–4.00 (2H, m), 4.18 (2H, q, J = 7.2 Hz),
7.39–7.44 (3H, m), 8.17–8.20 (2H, m).
5.1.50. N-(3,4-Dimethylisoxazol-5-yl)-4-[4-(2-
fluorophenyl)thiazol-2-yl]piperazine-1-carboxamide (60g)
Compound 60g was prepared in a manner similar to that de-
scribed for 60c in 38% yield as colorless crystals, mp 179–180 °C.
1H NMR (300 MHz, CDCl3) d: 1.89 (3H, s), 2.20 (3H, s), 3.63–3.69
(8H, m), 6.71 (1H, s), 7.07–7.26 (4H, m), 8.10–8.16 (1H, m). Anal.
Calcd for C19H20FN5O2S: C, 56.84; H, 5.02; N, 17.44. Found: C,
56.53; H, 5.07; N, 17.04.
5.1.57. tert-Butyl 4-(4-phenylthiazol-2-yl)-1,4-diazepane-1-
carboxylate (61b)
Compound 61b was prepared in a manner similar to that de-
scribed for 61a in 89% yield as a colorless oil. 1H NMR (300 MHz,
CDCl3) d: 1.44 (9H, s), 2.01–2.09 (2H, m), 3.37–3.49 (2H, m),
3.66–3.73 (5H, m), 3.90 (1H, br s), 7.39–7.44 (3H, m), 8.16–8.21
(2H, m).
5.1.51. N-(3,4-Dimethylisoxazol-5-yl)-4-[4-(3-
fluorophenyl)thiazol-2-yl]piperazine-1-carboxamide (60h)
Compound 60h was prepared in a manner similar to that de-
scribed for 60c in 43% yield as colorless crystals, mp 144–145 °C.
1H NMR (300 MHz, CDCl3) d: 1.87 (3H, s), 2.19 (3H, s), 3.59–3.69
(8H, m), 6.84 (1H, s), 6.94–7.01 (1H, m), 7.07 (1H, br s), 7.29–
7.37 (1H, m), 7.52–7.60 (2H, m). Anal. Calcd for C19H20FN5O2S: C,
56.84; H, 5.02; N, 17.44. Found: C, 56.85; H, 5.04; N, 17.32.
5.1.58. tert-Butyl 5-(3-phenyl-1,2,4-thiadiazol-5-yl)-2,5-
diazabicyclo[2.2.1]heptane-2-carboxylate (61c)
Compound 61c was prepared in a manner similar to that de-
scribed for 61a in 70% yield as a colorless powder, 1H NMR
(300 MHz, CDCl3) d: 1.44–1.48 (9H, m), 2.05 (2H, br s), 3.44–3.63
(4H, m), 4.06–4.74 (2H, m), 7.40–7.45 (3H, m), 8.18–8.20 (2H, m).
5.1.52. N-(3,4-Dimethylisoxazol-5-yl)-4-[4-(4-
fluorophenyl)thiazol-2-yl]piperazine-1-carboxamide (60i)
Compound 60i was prepared in a manner similar to that de-
scribed for 60c in 50% yield as colorless crystals, mp 183–184 °C.
1H NMR (300 MHz, CDCl3) d: 1.89 (3H, s), 2.20 (3H, s), 3.62–3.69
(8H, m), 6.72 (1H, s), 6.75 (1H, s), 7.03–7.09 (2H, m), 7.77–7.82
(2H, m). Anal. Calcd for C19H20FN5O2Sꢀ0.1H2O: C, 56.33; H, 5.00;
N, 17.29. Found: C, 56.24; H, 5.03; N, 17.01.
5.1.59. tert-Butyl [1-(3-phenyl-1,2,4-thiadiazol-5-yl)piperidin-
4-yl]carbamate (61d)
Compound 61d was prepared in a manner similar to that de-
scribed for 61a in 99% yield as a colorless powder, 1H NMR
(300 MHz, CDCl3) d: 1.46 (9H, s), 1.51–1.61 (2H, m), 2.07–2.12
(2H, m), 3.26–3.35 (2H, m), 3.74 (1H, br s), 3.96–4.00 (2H, m),
4.50 (1H, br s), 7.40–7.45 (3H, m), 8.15–8.19 (2H, m).
5.1.53. 4-[4-(2,4-Difluorophenyl)thiazol-2-yl]-N-(3,4-
dimethylisoxazol-5-yl)piperazine-1-carboxamide (60j)
Compound 60j was prepared in a manner similar to that de-
scribed for 60c in 34% yield as colorless crystals, mp 166–167 °C.
1H NMR (300 MHz, CDCl3) d: 1.89 (3H, s), 2.20 (3H, s), 3.61–3.70
(8H, m), 6.72 (1H, br s), 6.83–6.96 (2H, m), 7.05–7.06 (1H, m),
8.08–8.16 (1H, m). Anal. Calcd for C19H19F2N5O2S: C, 54.41; H,
4.57; N, 16.70. Found: C, 54.40; H, 4.51; N, 16.96.
5.1.60. 1-(3-Phenyl-1,2,4-thiadiazol-5-yl)piperidine-4-
carboxylic acid (62a)
To a stirred solution of ethyl 1-(3-phenyl-1,2,4-thiadiazol-5-
yl)piperidine-4-carboxylate (1.12 g, 3.53 mmol) in THF (20 mL)
and EtOH (12 mL) was added 2 N NaOH (4.0 mL, 8.00 mmol) drop-
wise at room temperature. After stirring at 80 °C for 1 h, 2 N HCl
(4.0 mL) was added, and the solvent was concentrated in vacuo.
The residue was diluted with water, and extracted with EtOAc.
The organic layer was washed with brine, dried over anhydrous
MgSO4, and concentrated in vacuo. The residue was recrystallized
from EtOAc–hexane to give 62a (910 mg, 89%) as colorless crystals.
1H NMR (300 MHz, CDCl3) d: 1.82–1.95 (2H, m), 2.06–2.13 (2H, m),
2.54–2.62 (1H, m), 3.28–3.37 (2H, m), 3.94–3.98 (2H, m), 7.38–7.44
(3H, m), 8.15–8.19 (2H, m).
5.1.54. N-(Benzo[d]isoxazol-3-yl)-4-(4-phenylthiazol-2-
yl)piperidine-1-carboxamide (60k)
Compound 60k was prepared in a manner similar to that de-
scribed for 60c in 59% yield as off-white crystals, mp 129–130 °C.
1H NMR (300 MHz, DMSO-d6) d: 1.71–1.82 (2H, m), 2.14–2.18
(2H, m), 3.09–3.16 (2H, m), 3.34–3.38 (1H, m), 4.24–4.29 (2H,
m), 7.29–7.36 (2H, m), 7.42–7.47 (2H, m), 7.58–7.65 (2H, m),
7.84 (1H, d, J = 7.8 Hz), 7.95–7.98 (2H, m), 8.02 (1H, s), 7.94 (1H,
s). Anal. Calcd for C22H20N4O2S: C, 65.33; H, 4.98; N, 13.85. Found:
C, 65.04; H, 4.93; N, 13.68.
5.1.61. 5-(2,5-Diazabicyclo[2.2.1]heptan-2-yl)-3-phenyl-1,2,4-
thiadiazole (62b)
To a stirred solution of tert-butyl 5-(3-phenyl-1,2,4-thiadiazol-
5-yl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate
(1.16 g,
5.1.55. N-(Benzo[d]isoxazol-3-yl)-4-(4-phenylthiophen-2-
yl)piperazine-1-carboxamide (60l)
Compound 60l was prepared in a manner similar to that de-
scribed for 60c in 21% yield as colorless crystals, mp 179–180 °C.
1H NMR (300 MHz, DMSO-d6) d: 3.23 (4H, br s), 3.71 (4H, br s),
6.72 (1H, s), 7.11 (1H, s), 7.24–7.41 (4H, m), 7.58–7.67 (4H, m),
3.24 mmol) in THF (10 mL) was added 2 N HCl in MeOH (10 mL)
dropwise at room temperature. After stirring at room temperature
for 12 h, the solvent was concentrated in vacuo. The residue was
recrystallized from MeOH–Et2O to give 5-(2,5-diazabicy-
clo[2.2.1]heptan-2-yl)-3-phenyl-1,2,4-thiadiazole dihydrochloride
as a solid. The solid was dissolved in 1 N NaOH, and extracted with

M. Kono et al. / Bioorg. Med. Chem. 21 (2013) 28–41
39
chloroform. The organic layer was dried over anhydrous MgSO4,
5.1.67. N-(Benzo[d]isoxazol-3-yl)-5-(3-phenyl-1,2,4-thiadiazol-
and concentrated in vacuo to give 62b (450 mg, 24%) as a pale
yellow oil. 1H NMR (300 MHz, CDCl3) d: 1.56 (1H, s), 1.91 (1H,
d, J = 9.9 Hz), 2.00 (1H, d, J = 9.9 Hz), 3.11–3.15 (1H, m), 3.21
(1H, d, J = 10.5 Hz), 3.30 (1H, d, J = 9.9 Hz), 3.67 (1H, d,
J = 9.6 Hz), 3.92 (1H, s), 4.58 (1H, br s), 7.41–7.46 (3H, m), 8.17–
8.21 (2H, m).
5-yl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxamide (65b)
Compound 65b was prepared in a manner similar to that de-
scribed for 60c in 45% yield as colorless crystals, mp 166–167 °C.
1H NMR (300 MHz, DMSO-d6) d: 2.19 (2H, s), 3.66–3.89 (4H, m),
4.92 (1H, br s), 5.04 (1H, s), 7.26–7.31 (1H, m), 7.42–7.56 (5H,
m), 8.13–8.22 (4H, m). Anal. Calcd for C21H18N6O2S: C, 60.27; H,
4.34; N, 20.08. Found: C, 60.13; H, 4.21; N, 20.07.
5.1.62. 2-(1,4-Diazepan-1-yl)-4-phenylthiazole (62c)
Compound 62c was prepared in a manner similar to that de-
scribed for 62b in 52% yield as a colorless oil. 1H NMR (300 MHz,
CDCl3) d: 1.94–2.02 (2H, m), 2.93–2.96 (2H, br s), 3.09–3.12 (2H,
br s), 3.74 (4H, br s), 7.39–7.43 (3H, m), 8.17–8.20 (2H, m).
5.1.68. N-(Benzo[d]isoxazol-3-yl)-4-(3-phenyl-1,2,4-thiadiazol-
5-yl)-1,4-diazepane-1-carboxamide (65c)
Compound 65c was prepared in a manner similar to that de-
scribed for 60c in 54% yield as a white powder, mp 80–81 °C. 1H
NMR (300 MHz, CDCl3) d: 2.22–2.28 (2H, m), 3.70–3.74 (2H, m),
3.81 (2H, br s), 3.94 (4H, br s), 7.18–7.21 (1H, m), 7.38–7.53 (5H,
m), 7.96 (1H, d, J = 8.1 Hz), 8.16–8.19 (2H, m). Analytical HPLC
showed 100% purity.
5.1.63. 1-(3-Phenyl-1,2,4-thiadiazol-5-yl)piperidin-4-amine
(62d)
Compound 62d was prepared in a manner similar to that de-
scribed for 62b in 95% yield as a colorless powder. 1H NMR
(300 MHz, CDCl3) d: 1.28 (2H, br s), 1.43–1.56 (2H, m), 1.93–2.00
(2H, m), 2.95–3.05 (1H, m), 3.21–3.31 (2H, m), 3.97–4.01 (2H,
m), 7.39–7.46 (3H, m), 8.16–8.21 (2H, m).
5.1.69. 1-(Benzo[d]isoxazol-3-yl)-3-[1-(3-phenyl-1,2,4-
thiadiazol-5-yl)piperidin-4-yl]urea (65d)
Compound 65d was prepared in a manner similar to that
described for 60a in 64% yield as an off-white crystals, mp
264–265 °C. 1H NMR (300 MHz, DMSO-d6) d: 1.62–1.78 (2H, m),
2.04–2.07 (2H, m), 3.41–3.48 (2H, m), 3.93 (3H, br s), 7.34–7.38
(2H, m), 7.47–7.48 (3H, m), 7.64–7.65 (2H, m), 8.10–8.17 (3H,
m), 10.10 (1H, s). Analytical HPLC showed 100% purity.
5.1.64. tert-Butyl {2-[3-(benzo[d]isoxazol-3-
yl)ureido]ethyl}carbamate (63)
A
mixture of tert-butyl (2-aminoethyl)carbamate (20 mg,
0.125 mmol), 2,2,2-trichloroethyl benzo[d]isoxazol-3-ylcarbamate
(46.4 mg, 0.150 mmol), N-ethyldiisopropylamine (0.026 mL,
0.150 mmol), and DMSO (2.0 mL) was stirred at 70 °C overnight,
poured into water, and extracted with EtOAc. The organic layer
was washed with water, dried over anhydrous MgSO4, and concen-
trated in vacuo. The residue was purified by silica gel column chro-
matography (hexane–EtOAc) to give 63 (34.7 mg, 87%) as a
colorless powder. 1H NMR (300 MHz, CDCl3) d: 1.50 (9H, s), 3.56–
3.66 (8H, m), 7.27–7.32 (1H, m), 7.46–7.58 (2H, m), 8.05 (1H, d,
J = 8.4 Hz), 8.73 (1H, s).
5.1.70. 1-(Benzo[d]isoxazol-3-yl)-3-{2-[(3-phenyl-1,2,4-
thiadiazol-5-yl)amino]ethyl}urea (65e)
A mixture of 1-(2-aminoethyl)-3-(benzo[d]isoxazol-3-yl)urea
hydrochloride (200 mg, 0.778 mmol), 5-chloro-3-phenyl-1,2,4-
thiadiazole (153 mg, 0.778 mmol), triethylamine (0.433 mL,
3.11 mmol), sodium iodide (117 mg, 0.778 mmol), and MeCN
(10 mL) was stirred at 90 °C for 6.0 h, and the solvent was concen-
trated in vacuo. The residue was purified by silica gel column
chromatography (EtOAc). The product was recrystallized from
EtOAc–hexane to give 65e (69 mg, 23%) as colorless crystals, mp
221–222 °C. 1H NMR (300 MHz, DMSO-d6) d: 3.53 (4H, br s),
7.31–3.43 (4H, m), 7.50 (1H, br s), 7.62–7.64 (2H, m), 8.05–8.08
(2H, m), 8.14(1H, d, J = 8.1 Hz), 8.65 (1H, br s), 10.14 (1H, s).
Analytical HPLC showed 99.2% purity.
5.1.65. 1-(2-Aminoethyl)-3-(benzo[d]isoxazol-3-yl)urea
hydrochloride (64)
To a stirred solution of tert-butyl {2-[3-(benzo[d]isoxazol-3-
yl)ureido]ethyl}carbamate (450 mg, 1.40 mmol) in THF (10 mL)
was added 4 N HCl in EtOAc (10 mL) dropwise at room tempera-
ture. After stirring at room temperature for 3.0 h, and the solvent
was concentrated in vacuo. The residue was recrystallized from
MeOH–Et2O to give 64 (300 mg, 84%) as colorless crystals. 1H
NMR (300 MHz, DMSO-d6) d: 2.96 (2H, br s), 3.45–3.51 (2H, m),
7.34–7.39 (1H, m), 7.58–7.66 (3H, m), 7.97 (3H, br s), 8.19 (1H, d,
J = 8.1 Hz), 10.36 (1H, s).
5.2. Solubility determination
Small volumes of the compound DMSO solutions were added to
the aqueous buffer solution (pH 6.8). After incubation, precipitates
were separated by filtration. The solubility was determined by
HPLC analysis of each filtrate.
5.1.66. N-(Benzo[d]isoxazol-3-yl)-1-(3-phenyl-1,2,4-thiadiazol-
5-yl)piperidine-4-carboxamide (65a)
5.3. Measurement of FAAH inhibitory activity
To a stirred solution of 1-(3-phenyl-1,2,4-thiadiazol-5-yl)piper-
idine-4-carboxylic acid (289 mg, 1.00 mmol) and DMF (0.010 mL)
in THF (5.0 mL) was added oxalyl chloride (0.174 mL, 2.00 mmol)
dropwise at 0 °C. After stirring at room temperature for 1 h, the
solvent was concentrated in vacuo. To the residue were added
THF (5.0 mL), benzo[d]isoxazol-3-amine (134 mg, 1.00 mmol),
and pyridine (0.404 mL, 5.00 mmol) at 0 °C. The mixture was stir-
red at room temperature for 1.0 h, and the solvent was concen-
trated in vacuo. The residue was purified by silica gel column
chromatography (hexane–EtOAc), and recrystallized from EtOAc–
hexane to give 65a (60.3 mg, 15%) as colorless crystals, mp 204–
205 °C. 1H NMR (300 MHz, CDCl3) d: 2.05–2.22 (4H, m), 2.88
(1H, bs), 3.36–3.45 (2H, m), 4.11–4.18 (2H, m), 7.32–7.63 (6H,
m), 8.19–8.27 (3H, m), 9.46 (1H, br s). Analytical HPLC showed
97.4% purity.
5.3.1. Preparation of enzyme fraction
The FAAH gene was cloned by PCR. That is, a human brain li-
brary was used as
a
cDNA library, 50-AAAAGAATTCGCCAC-
CATGGTGCAGTACGAGCTGTG-30 [SEQ ID NO:1] and 50-
TTTTGTCGACTCAGGATGACTGCTTTT-30 [SEQ ID NO:2] were used
as a primer set, and KOD DNA polymerase (Toyobo Co., Ltd) was
used as a DNA polymerase. One cycle of the reaction comprises
95 °C for 30 s, 55 °C for 30 s and 72 °C for 2 min, and 45 cycles of
the reaction was carried out to obtain an amplified fragment. The
amplified fragment was cleaved with restriction enzymes EcoRI
and SalI, and then was inserted into a pMSR
been cleaved with the same restriction enzymes EcoRI and SalI to
obtain a pMSR –human FAAH. A cell line CHO-K1 and the
a vector which had
a
above-obtained plasmid were subjected to a method known per
se to prepare the cell line CHO-K1/human FAAH in which human

40
M. Kono et al. / Bioorg. Med. Chem. 21 (2013) 28–41
FAAH was stably expressed. The CHO-K1/human FAAH was cul-
tured in a CO2 incubator at 37 °C, using a medium (Ham’s F-12
medium supplemented with final concentration 10% of fetal bovine
Crystals were grown utilizing Takeda California’s automated
nanovolume crystallization technology platform. Crystals suitable
for data collection were obtained from sitting-drop vapor diffusion
experiments formed from a 50:50 nl mixture of concentrated pro-
tein and mother liquor (40% PEG 400, 0.1 M sodium acetate pH
4.5). Crystals were harvested after seven days, flash frozen and
stored under liquid nitrogen in an ALS compatible crystal mount-
ing cassette. Diffraction data for the co-crystal structure of FAAH
was collected to 2.91 Å from cryo-cooled crystals at the Advanced
Light Source (ALS, Berkeley CA) beamline 5.0.2. Data was reduced
using the HKL2000 software package48 in the P3221 space group
(a = 103.9 Å, b = 103.9 Å, c = 252.0 Å, alpha = 90.0°, beta = 90.0°,
gamma = 120.0°). The structure was determined by molecular
replacement with subsequent refinement and model re-building
conducted utilizing REFMAC49 and XtalView50 software packages.
The structures have been deposited with the RCSB structure data-
base with accession codes 4HBP (compound 60d).
serum (FBS) and final concentration 800 lg/mL of G418), and then
the cells were harvested. After washing with PBS, the cells were
suspended in a buffer (10 mM Tris, 1 mM EDTA and 10 mM MgCl2,
all at final concentrations) and disrupted with a Polytron homoge-
nizer. After centrifugation at 900 g, the supernatant was recovered
and further centrifuged at 10,000 g. A pellet obtained therefrom
was suspended in M-PER (Catalog No. 78501; PIERCE) to give an
enzyme fraction.
5.3.2. Enzyme reaction
Using a white walled 96-well plate (Coster Corp.), the test com-
pound at various concentrations, 300 ng of the enzyme fraction
and the substrate anandamide [ethanolamine 1-3H] (final concen-
tration 2.5 nM) were reacted in 50 ll of a reaction buffer (125 mM
Tris–HCl (pH 9.0), 1 mM EDTA, 0.4 mM HEPES, 0.2% glycerol, 0.02%
Triton X-100 and 0.3% fatty acid-free BSA, all at final concentra-
tions) at 37 °C for 30 minutes. The reaction mixture was trans-
ferred to a 96-well MultiScreen-HA filter plate (Millipore Corp.)
and then was left to stand overnight at room temperature in order
to allow the unreacted substrate to be absorbed on the filter. The
plate was washed with PBS using a MultiScreen Vacuum Manifold
Acknowledgments
We thank Robert Skene for assistance with the preparation of
the manuscript; Mr. Takashi Santou for the excellent technical
assistance for conducting biological assays; Mr. Satoshi Yamamoto
and Dr. Shinji Fujimoto for helpful discussions.
(Millipore Corp.) and dried. To each well, 50 ll of liquid scintilla-
tion cocktail was added and stirred, and then counting was per-
formed with a TopCount (Perkin–Elmer Corp.). The count of a
sample containing a solvent instead of the test compound was ta-
ken as 0%, and the count at zero time was taken as 100%, to calcu-
late the inhibitory activity of the compound.
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