Design, synthesis, and biological evaluation of a series of piperazine ureas as fatty acid amide hydrolase inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.12.023

A series of piperazine ureas were designed, synthesized, and evaluated for their potential as novel orally efficacious fatty acid amide hydrolase (FAAH) inhibitors for the treatment of neuropathic and inflammatory pain. We carried out an optimization stud

Full text of DOI:10.1016/j.bmc.2013.12.023

Bioorganic & Medicinal Chemistry 22 (2014) 1468–1478
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and biological evaluation of a series of piperazine
ureas as fatty acid amide hydrolase inhibitors
a
a
a
a
Mitsunori Kono a, , Takahiro Matsumoto , Toshihiro Imaeda , Toru Kawamura , Shinji Fujimoto ,
⇑
Yohei Kosugi a, Tomoyuki Odani a, Yuji Shimizu a, Hideki Matsui a, Masato Shimojo a, Masakuni Kori b
a Pharmaceutical Research Division, Takeda Pharmaceutical Company Ltd, 26-1, Muraokahigashi 2-Chome, Fujisawa, Kanagawa 251-8555, Japan
b 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 were designed, synthesized, and evaluated for their potential as novel orally
efficacious fatty acid amide hydrolase (FAAH) inhibitors for the treatment of neuropathic and inflamma-
tory pain. We carried out an optimization study of compound 5 to improve its in vitro FAAH inhibitory
activity, and identified the 2-pyrimidinylpiperazine derivative 21d with potent inhibitory activity, favor-
able DMPK profile and brain permeability. Compound 21d showed robust and dose-dependent analgesic
efficacy in animal models of both neuropathic and inflammatory pain.
Received 1 November 2013
Revised 5 December 2013
Accepted 7 December 2013
Available online 20 December 2013
Keywords:
Ó 2014 Published by Elsevier Ltd.
Fatty acid amide hydrolase inhibitors
Arachidonoylethanolamide
Piperazine ureas
Neuropathic pain
Inflammatory pain
1. Introduction
the cannabinoid 2 (CB2) receptor is highly expressed in immune
organs such as spleen and implicated in the regulation of inflam-
Fatty acid amide hydrolase (FAAH)1–3 is a membrane-bound
serine hydrolase that catalyzes the intracellular hydrolysis of sev-
eral endogenous lipid amides4–7 such as endogenous cannabinoid
(EC), anandamide (arachidonoylethanolamide: AEA),8–11 sleep-
inducing substance oleamide,12–15 appetite-suppressing agent
oleoyl ethanolamide (OEA),16 and anti-inflammatory compound
palmitoyl ethanolamide (PEA).17 FAAH tightly controls the signal-
ing function of these lipid amides, which have several physiological
effects.18–22 FAAH knockout mice are viable and healthy, have
highly increased endogenous levels of AEA in several brain regions,
and show an analgesic phenotype in several animal models of neu-
ropathic and inflammatory pain.23–25
matory and immune response.29,30 A potential therapeutic ap-
proach utilizing the activation of the CB1 receptor has been
recognized as an attractive and beneficial way to treat pain, as well
as other central nervous system disorders. Although direct activa-
tion of the CB1 receptor with CB1 agonists would exert strong
pharmacological effects, it also causes a variety of undesirable ad-
verse effects such as sedation, dependence, cognitive impairment,
psychosis, and affection of cardiovascular system by their systemic
activation.31,32 The CB1 and CB2 receptors are activated by AEA.
FAAH inhibitors enhance the activation of CB receptors by blocking
AEA degradation. However, FAAH inhibitors may offer site-specific
increase of AEA in tissues in which ECs are being produced through
physiological responses, suggesting that they exhibit the pharma-
cological effects with less adverse effects. Moreover, FAAH inhibi-
tors may provide anti-inflammatory effects by suppressing the
release of inflammatory chemical mediators through stimulation
of the CB2 receptor in immune cells.
The cannabinoid 1 (CB1) receptor is highly expressed in hippo-
campus, striatum, nigra, olfactory bulb, and cerebellum in the cen-
tral nervous system, and suppressively controls the release of
neurotransmitters from sensory nerve.26–28 On the other hand,
Various classes of FAAH inhibitors, including urea derivatives
(e.g., PF-0445784533 and JNJ-4035500334), carbamates (e.g.,
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; PK,
pharmacokinetics; PD, pharmacodynamics; Troc, 2,2,2-trichloroethoxy carbonyl;
CFA, complete Freund’s adjuvant; SNI, sciatic nerve injury.
URB59735 and SA-4736), and keto-heterocycles (e.g., OL-13537
)
have been reported (Fig. 1). Previously, we reported a series of thi-
adiazolylpiperazine urea derivatives including compound 1 and
found that analogues such as 2, having the 3,4-dimethylisoxazolyl
as the terminal heterocycle, showed potent FAAH inhibitory
⇑
Corresponding author. Tel.: +81 466 32 1108; fax: +81 466 29 4455.
E-mail address: mitsunori.kouno@takeda.com (M. Kono).
0968-0896/$ - see front matter Ó 2014 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmc.2013.12.023

M. Kono et al. / Bioorg. Med. Chem. 22 (2014) 1468–1478
1469
activity (Fig. 2).38 Further optimization of 3,4-dimethylisoxazole
compound 2 led to the identification of the thiazole derivative 3,
which orally showed anti-nociceptive effects in the acetic acid-in-
duced writhing test in mice. In addition, we also found that com-
pound 4, which have the pyridazinyl group as the terminal
heterocycle, possessed potent in vitro activity. In order to identify
additional series of novel potent FAAH inhibitors, we focused on
the modification of compound 4. Our structure–activity relation-
ship (SAR) study for the 3,4-dimethylisoxazole compounds38 sug-
gested that the urea moiety (A in Fig. 2) is essential for in vitro
activity by interacting with catalytic residues Ser241 and Ser217,
and that the piperazine moiety (B in Fig. 2) is also important as a
central spacer. On the other hand, replacement of the thiadiazole
ring with other 5-membered heterocycles was practical (C in
Fig. 2). Taking into account this SAR information, we investigated
a new series of compounds by modifying the ring structure of re-
gion C in compound 4. In order to investigate the potential for a
6-membered ring in region C, we initially synthesized the pyrimi-
dine derivative 5 and found the compound exhibited moderate
FAAH inhibitory activity. In addition, replacement of the thiadia-
zole ring with a pyrimidine ring notably improved solubility (6.3
were easily separated by silica gel column chromatography. Alter-
natively, compound 10 was regioselectively synthesized by the
reaction of 2,4-dichloropyrimidine 6 with tert-butyl 4-methylpi-
perazine-1-carboxylate in 73% yield.40 Suzuki coupling of 7 and
10 with appropriate aryl boronic acids provided compounds 8
and 11–15. Acidic deprotection and subsequent urea formation
with the corresponding 2,2,2-trichloroethoxy carbonyl (Troc)
derivative 2341 or phenylcarbamate 24 yielded compounds 5 and
21a–21e.
Compounds 44a–44e were synthesized as shown in Scheme 2.
Buchwald amination of aryl halide 25–27 with N-Boc-piperazine
afforded corresponding aryl piperazine 29–31. Suzuki coupling of
compounds 28–31 with appropriate aryl boronic acids gave biaryl
derivatives 34–36 and 38, respectively. 4-Bromo-2-chloropyridine
32 was converted to biaryl 33 by Suzuki coupling, and subsequent
Buchwald amination yielded compound 37. Compounds 44a–44e
were obtained by acidic deprotection of 34–38 and subsequent
urea formation with the Troc derivatives 23.
3. Results and discussion
and 52 lg/mL in pH 6.8 solution for 4 and 5, respectively). On
The synthesized compounds were evaluated using in vitro and
in vivo biological tests as follows. FAAH inhibitory activity was
measured by a fluorescence-based assay using human and rat
FAAH enzyme fractions and a FAAH substrate, 7-amino-4-methyl-
coumarin arachidonoyl amide (AMCAA: manufactured by CAYMAN
CHEMICAL) (Tables 1 and 2). The acetic acid-induced writhing test
in mice was performed as the first screening of in vivo efficacy (Ta-
ble 2). Further evaluation of analgesic efficacy of the selected com-
pounds was conducted in the rat sciatic nerve injury (SNI)-induced
neuropathic pain model and the rat complete Freund’s adjuvant
(CFA)-induced inflammatory pain model.
the basis of these results, we selected compound 5 as a lead and
performed an optimization study of compounds X to improve
in vitro activity, focusing on the modification of the 6-membered
ring (Y) and substituents (R) on the terminal benzene ring. Here,
we describe the synthesis, SAR and biological evaluation of a series
of novel piperazine-urea FAAH inhibitors having nitrogen-contain-
ing 6-membered heterocycles such as pyridine and pyrimidine
rings.39
2. Chemistry
First, replacement of the pyrimidine of the ring Y with pyridine
or rotation of the pyrimidine ring was examined. As shown in Ta-
ble 1, pyridine derivatives 44a and 44b, in which the nitrogen atom
at the 1-position (Y1) or 3-position (Y2) of the pyrimidine ring in
compound 5 was replaced with CH, showed comparable inhibition
to that of compound 5. Interestingly, compound 44d (Y3 = N)
The general synthetic method for 2-pyrimidinylpiperazine and
4-pyrimidinylpiperazine derivatives (5 and 21a–21e) is shown in
Scheme 1. The coupling reaction of 2,4-dichloropyrimidine 6 with
N-Boc-piperazine gave a mixture of the 4-substituted compound 7
and 2-substituted compound 10 (7:10 = ca. 12:1). These isomers
Figure 1. Anandamide (AEA) and known FAAH inhibitors.

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M. Kono et al. / Bioorg. Med. Chem. 22 (2014) 1468–1478
Figure 2. Structural modification of the hit compound 1.
Scheme 1. Synthesis of compounds 5 and 21a–21e. Reagents and Conditions: (a) N-Boc-piperazine, triethylamine, DMF, room temp, 90%; (b) (2,4-difluorophenyl)boronic
i
acid, Pd(PPh3)4, 2 N aq Na2CO3, toluene, 100 °C, 40%; (c) 4 N HCl in EtOAc, room temp then 1 N NaOH, 85%; (d) 23, Pr2NEt, DMSO, 70 °C, 22% [for 5 and 21a]; (e) 1-Boc-4-
methylpiperazine, toluene, 110 °C, 73%; (f) Ar-B(OH)2, Pd(PPh3)4, 2 N aq Na2CO3, toluene or DME, 71%-quant.; (g) 4 N HCl in EtOAc, room temp., 80%-quant.; (h) 24, Et3N,
acetone, 40 °C, 39–91% [for 21b–21e]; (i) 2,2,2-trichloroethyl chloroformate, pyridine, THF, 0 °C, 46%; (j) phenylchloroformate, pyridine, MeCN, 0 °C, 87%.
exhibited more potent inhibitory activity for both human and rat
FAAH, whereas compound 44c (Y4 = N) showed less potent activity.
These results suggested that the nitrogen atom at Y3 was important
for FAAH inhibitory activity. Therefore, we synthesized pyrimidine
derivatives, having the nitrogen atom at Y3, and found that com-
pounds 44e and 21a showed further increase in FAAH inhibitory
activity.
Next, we investigated the optimal position of the difluoro
groups of compound 21a. The results are shown in Table 2. The
2,3-difluoro group (21b) enhanced human FAAH inhibitory activ-
ity, whereas rat FAAH inhibitory activity was not increased. The
2,5-difluoro and 3,5-difluoro derivatives (21c and 21e) exhibited
very potent activity for both human and rat FAAH. 3,4-Difluoro
derivative 21d was equipotent to compound 21a. It was of our
interest that the solubility of compound 21d was notably im-
proved (3.1 lg/mL in pH 6.8 solution) compared with that of
other derivatives. In vivo analgesic efficacy of compounds 21b–
21e was evaluated using the acetic acid-induced writhing test
in mice. Among them, compound 21d exerted anti-nociceptive
efficacy at a dose of 10 mg/kg, po. From these biological and
physicochemical results, we selected compound 21d for further
evaluation.

M. Kono et al. / Bioorg. Med. Chem. 22 (2014) 1468–1478
1471
Scheme 2. Synthesis of compounds 44a–44e. Reagents and Conditions: (a) N-Boc-piperazine, Pd2(dba)3, xantphos, NaOtBu, toluene, 100 °C, 70–79%; (b) (2,4-
difluorophenyl)boronic acid, Pd(PPh3)4, 2 N aq Na2CO3, toluene or DME, 37–94%; (c) 2,4-difluorophenylboronic acid, Pd(PPh3)4, Na2CO3, MeOH, 50 °C, 91%; (d) N-Boc-
piperazine, Pd(OAc)2, BINAP, NaOtBu, 1,4-dioxane, 85 °C, 76%; (e) TFA, CH2Cl2, room temp, 72% [for 39]; (f) 4 N HCl in EtOAc, room temp then 1 N NaOH, 83–94% [for 40, 41,
i
43]; (g) 4 N HCl in MeOH, room temp, 98% [for 42]; (h) 23, Pr2NEt, DMSO, 70 °C, 10–57%.
We examined the analgesic effects of compound 21d in rat SNI-
induced neuropathic pain model and CFA-induced inflammatory
pain model (Fig. 3). In SNI rats, the pain threshold of the ipsilateral
hind paw to tactile stimuli was dramatically decreased compared
to that in sham-operated rats, indicating that SNI operation evoked
tactile allodynia in rats. Orally administered compound 21d at
doses of 1–10 mg/kg significantly ameliorated tactile allodynia in
a dose-dependent fashion in SNI rats. In CFA rats, the pain thresh-
old of the ipsilateral hind paw to tactile stimuli was dramatically
decreased compared to that in sham-operated rats, indicating that
CFA injection evoked tactile allodynia in rats. Compound 21d also
significantly ameliorated tactile allodynia of the ipsilateral hind
paw at doses of 3–10 mg/kg. In conclusion, these results in well-
established pain models suggest that FAAH inhibitors such as com-
pound 21d have potential to improve abnormal pain involved in
neuropathic and inflammatory pain in the clinic.
In order to confirm the pharmacokinetics (PK) and pharmacody-
namics (PD) relationships, we measured the concentration of com-
pound 21d and AEA in plasma and brain after administration of the
compound (0.03, 0.3, 3 mg/kg, po). Plasma and brain concentra-
tions of compound 21d were elevated in a dose-dependent manner
(Table 3), consistent with the observed efficacy in rat pain models.
Peak concentration levels of 803 ng/mL (plasma) and 1600 ng/g tis-
sue (brain) were observed at 4 h after oral administration of com-
pound 21d at a dose of 3 mg/kg in rats, indicating that compound
21d has high brain permeability (brain/plasma concentration ra-
tio = 2.0). We also examined the effects on the tissue content of
AEA as a PD marker for FAAH inhibition and found that the
concentration of AEA in the brain was elevated at the efficacious
dose of compound 21d. These results indicate that compound
21d exerted analgesic efficacy at doses where compound 21d
inhibited FAAH.
To date, two main classes of FAAH inhibitors with therapeutic
potential have been reported. Urea and carbamate-type FAAH
inhibitors show irreversible inhibition, and form a covalent tight-
binding with the catalytic Ser-241 residue within the active site.42
On the other hand, reversible inhibitors such as
a-keto-heterocy-
cles tend to form a tetrahedral intermediate arising from the inter-
action of the electrophilic carbonyl group with catalytic serine to
give a hemiacetal bond.43,44 Co-crystallization study of our thi-
adiazolylpiperazine urea derivative38 revealed the compound
forms a covalent bond with FAAH to inactivate the enzyme irre-
versibly. To investigate the inhibition mode of compound 21d,
we performed a progress curve analysis as an enzyme kinetic anal-
ysis (Fig. 4). Non-linear progress curve was observed indicating
that the inhibitory activity of compound 21d time-dependently in-
creased. Taken together with previous structural analysis of piper-
azine-urea-type derivatives, the result suggested that compound
21d probably forms a covalent bond with the active site Ser 241
of FAAH, which causes irreversible inhibition in a binding mode
similar to that observed with the series of thiadiazole derivatives.
4. Conclusion
In the course of exploring novel FAAH inhibitors that are thera-
peutically effective against neuropathic and inflammatory pain, we

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M. Kono et al. / Bioorg. Med. Chem. 22 (2014) 1468–1478
Table 1
a 2-pyrimidinylpiperazine led to an increase in FAAH inhibitory
activity. The substitution pattern of the difluoro groups on the phe-
nyl ring affected in vivo efficacy. We identified the 2-pyrimidinyl-
piperazine derivative 21d, which dose-dependently ameliorated
the decrease in pain threshold in animal models of both neuro-
pathic and inflammatory pain. The results suggested that the po-
tent and selective piperazine urea FAAH inhibitor 21d would
become a beneficial analgesic agent for the treatment of neuro-
pathic and inflammatory pain.
Human and rat FAAH activities of derivatives modified at the thiadiazole ring moiety
5. Experimental section
5.1. Chemistry
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
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)
a
Compound
Ring Y
Apparent FAAH IC50 (nM) (human/rat)
5
23/34
44a
44b
36/13
28/12
44c
44d
44e
21a
140/74
2.2/4.3
1.5/1.7
1.1/0.43
through
a
L-column
2
ODS (3.0 ꢀ 50 mm,
2 lm) column at
1.2 mL minꢁ1. Area % purity was measured at 254 nm.
5.1.1. tert-Butyl 4-(2-chloropyrimidin-4-yl)piperazine-1-
carboxylate (7)
A
mixture of 2,4-dichloropyrimidine (1.00 g, 6.71 mmol),
a
IC50 values were determined at a 30 min reaction.
N-Boc-piperazine (1.37 g, 7.38 mmol), triethylamine (1.40 mL,
10.1 mmol), and DMF (10 mL) was stirred at room temperature
for 4.0 h. The mixture was diluted with 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 to give 7 (1.80 g, 90%) as a colorless powder.
designed, synthesized, and evaluated a series of piperazine urea
compounds incorporating nitrogen-containing 6-membered het-
erocycles. Replacement of the 4-pyrimidinylpiperazine ring with
Table 2
Human and rat FAAH inhibitory activities of derivatives modifying the central piperazine ring moiety
a
Compound
R
Apparent FAAH IC50 (nM) (human/rat)
Solubilityb
(l
g/mL)
Acetic acid writing testc % of inhibition
21a
21b
21c
21d
21e
2,4-F
2,3-F
2,5-F
3,4-F
3,5-F
1.1/0.43
0.85
0.16
<0.06
3.1
N.T.
18.9
2.2
0.072/1.2
0.025/0.17
0.72/0.28
0.08/0.34
34.7⁄⁄
0.9
0.1
a
b
IC50 values were determined at a 30 min reaction.
Solubility in pH 6.8 solution.
10 mg/kg, po administration in mice.
c
**
p<0.01 vs. vehicle-treated mice (Student’s t-test).

M. Kono et al. / Bioorg. Med. Chem. 22 (2014) 1468–1478
1473
Figure 3. Analgesic effect of compound 21d in rat neuropathic and inflammatory pain models. (A) Neuropathic pain was induced by sciatic nerve injury (SNI) in rats.
Compound, 21d and vehicle were orally administered 7 days after SNI operation. Tactile allodynia of ipsilateral hind paw was assessed by von Frey filaments 2 h after drug
treatment. Data are shown in mean and SE of 8 rats. ###p <0.001 versus sham-operated rats (Student’s t-test). ⁄p <0.025 versus vehicle-treated SNI rats (Shirley-Williams test).
(B) Inflammatory pain was induced by CFA injection into hind paw. Compound 21d and vehicle were orally administered 5 days after CFA injection. Tactile allodynia of
ipsilateral hind paw was assessed by von Frey filaments 4 h after drug treatment. Data are shown in mean and SE of 8–10 rats. ###p <0.001 versus sham-operated rats
(Student’s t-test). ⁄p <0.025 versus vehicle-treated CFA rats (Shirley-Williams test).
Table 3
Pharmacokinetic and pharmacodynamic parameters in rats
Dose
(mg/kg)
0.03
0.3
3
Plasma
Brain
Plasma
Brain
Plasma
Brain
1600
4.0
15955
Compound 21d
Cmax
Tmax
AUC
(ng/mL or ng/g)
(h)
(ng h/mL or ng h/g)
0
0
0
0
0
0
57
4.0
533
100
4.0
898
803
4.0
8819
AEA
Cmax
Tmax
AUC
(ng/mL or ng/g)
(h)
(ng h/mL or ng h/g)
0.2
8.0
3.4
6.8
4.0
64.3
0.4
8.0
7.9
19.1
4.0
232.9
0.5
12.0
9.5
19.2
4.0
311.6
Mean (n = 3).
Compound 21d was suspended in 0.5% methylcellulose solution for oral administration. The concentrations of compound 21d and AEA in the plasma and brain were
determined using liquid chromatography/tandem mass spectrometry (LC/MS/MS).
1H NMR (300 MHz, CDCl3) d: 1.49 (9H, s), 3.47–3.58 (4H, m), 3.60–
3.71 (4H, m), 6.40 (1H, d, J = 6.2 Hz), 8.07 (1H, d, J = 6.2 Hz). MS
(ESI): m/z 299 [M+H]+.
5.1.2. tert-Butyl 4-[2-(2,4-difluorophenyl)pyrimidin-4-
yl]piperazine-1-carboxylate (8)
A mixture of tert-butyl 4-(2-chloropyrimidin-4-yl)piperazine-1-
carboxylate (1.80 g, 6.02 mmol), (2,4-difluorophenyl)boronic acid
(1.43 g, 9.04 mmol), 1 M aq Na2CO3 (24 mL, 48.2 mmol), tetra-
kis(triphenylphosphine)palladium (0) (835 mg, 0.723 mmol), and
toluene (60 mL) was stirred at 100 °C overnight under N2 atmo-
sphere. The mixture was diluted with water, and extracted with
EtOAc. The organic layer was washed with water, dried over anhy-
drous MgSO4, and concentrated in vacuo. The residue was purified
by silica gel column chromatography (hexane–EtOAc) to give 8
(907 mg, 40%) as a colorless viscous oil. 1H NMR (300 MHz, CDCl3)
d: 1.49 (9H, s), 3.48–3.76 (8H, m), 6.44 (1H, d, J = 6.4 Hz), 6.84–6.99
(2H, m), 8.02–8.12 (1H, m), 8.36 (1H, d, J = 6.4 Hz).
Figure 4. Enzyme kinetic analysis of compound 21d. Kinetic analysis of FAAH
inhibition by compound 21d. Progress curve analysis was performed using AMCAA
method at room temperature in the presence (s) or absence (d) of 10 nM
compound 21d. Data represent the mean S.E.M. from three independent
experiments.
room temperature. After stirring at room temperature for 5.0 h,
the mixture was concentrated in vacuo. The residue was dissolved
in water, neutralized with 1 M NaOH, and extracted with EtOAc.
The organic layer was dried over anhydrous MgSO4, and concen-
trated in vacuo to give 9 (554 mg, 85%) as a pale yellow viscous
oil. 1H NMR (300 MHz, CDCl3) d: 3.01–3.17 (4H, m), 3.69–3.98
5.1.3. 2-(2,4-Difluorophenyl)-4-(piperazin-1-yl)pyrimidine (9)
To a stirred solution of tert-butyl 4-[2-(2,4-difluorophenyl)
pyrimidin-4-yl]piperazine-1-carboxylate (888 mg, 2.36 mmol) in
EtOAc (9.0 mL) was added 4 M HCl in EtOAc (9.0 mL) dropwise at

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M. Kono et al. / Bioorg. Med. Chem. 22 (2014) 1468–1478
(4H, m), 6.37–6.52 (1H, m), 6.81–7.03 (2H, m), 7.93–8.17 (1H, m),
8.29–8.45 (1H, m).
5.1.9. tert-Butyl 4-[4-(3,4-difluorophenyl)pyrimidin-2-
yl]piperazine-1-carboxylate (14)
A mixture of tert-butyl 4-(2-chloropyrimidin-4-yl)piperazine-
1-carboxylate (10.0 g, 33.5 mmol), (3,4-difluorophenyl)boronic
acid (7.90 g, 50.2 mmol), 1 M aq Na2CO3 (45 mL, 90.0 mmol),
tetrakis(triphenylphosphine)palladium (0) (4.6 g, 4.02 mmol),
and DME (300 mL) was stirred at 95 °C overnight under N2
atmosphere. After cooling to room temperature, the mixture
was stirred at room temperature for 15 h, diluted with water,
and extracted with EtOAc. The organic layer was dried over
anhydrous MgSO4, and concentrated in vacuo to give 14 as a
brown oil. This product was used for next reaction without
further purification.
5.1.4. 4-[2-(2,4-Difluorophenyl)pyrimidin-4-yl]-N-(pyridazin-3-
yl)piperazine-1-carboxamide (5)
A mixture of 2-(2,4-difluorophenyl)-4-(piperazin-1-yl)pyrimi-
dine (100 mg, 0.362 mmol), 2,2,2-trichloroethyl pyridazin-3-ylcar-
bamate
(108 mg,
0.398 mmol),
N-ethyldiisopropylamine
(0.126 mL, 0.724 mmol), and DMSO (1.5 mL) was stirred at 70 °C
overnight. The mixture was 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 purified
by silica gel column chromatography (hexane–EtOAc), and recrys-
tallized from EtOAc–hexane to give 5 (31.9 mg, 22%) as colorless
crystals, mp 200–201 °C. 1H NMR (300 MHz, DMSO-d6) d: 3.58–
3.71 (4H, m), 3.72–3.84 (4H, m), 6.88 (1H, d, J = 6.2 Hz), 7.14–
7.25 (1H, m), 7.28–7.39 (1H, m), 7.59 (1H, dd, J = 4.6, 9.1 Hz),
7.96–8.14 (2H, m), 8.36 (1H, d, J = 6.2 Hz), 8.85 (1H, d, J = 4.0 Hz),
9.99 (1H, s). MS (ESI): m/z 398 [M+H]+ Anal. Calcd for C19H17 F2N7-
Oꢂ0.5H2O: C, 56.15; H, 4.46; N, 24.13. Found: C, 56.08; H, 4.21; N,
24.00.
5.1.10. tert-Butyl 4-[4-(3,5-difluorophenyl)pyrimidin-2-
yl]piperazine-1-carboxylate (15)
Compound 15 was prepared in a manner similar to that de-
scribed for 14 as a brown oil. This product was used for next reac-
tion without further purification.
5.1.11. 4-(2,4-Difluorophenyl)-2-(piperazin-1-yl)pyrimidine
(16)
5.1.5. tert-Butyl 4-(4-chloropyrimidin-2-yl)piperazine-1-
carboxylate (10)
Compound 16 was prepared in a manner similar to that de-
scribed for 9 in 80% yield as a pale yellow oil. 1H NMR (300 MHz,
CDCl3) d: 2.94–3.00 (4H, m), 3.83–3.90 (4H, m), 6.83–7.03 (3H,
m), 8.10–8.21 (1H, m), 8.37 (1H, d, J = 4.9 Hz). MS (ESI): m/z 277
[M+H]+.
A mixture of 2,4-dichloropyrimidine (50 g, 336 mmol), tert-bu-
tyl 4-methylpiperazine-1-carboxylate (67.2 g, 336 mmol), and tol-
uene (500 mL) was stirred at 110 °C overnight. The mixture was
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 10 (109 g, 73%) as a colorless
powder. 1H NMR (300 MHz, CDCl3) d: 1.49 (9H, s), 3.44–3.53 (4H,
m), 3.75–3.84 (4H, m), 6.53 (1H, d, J = 5.1 Hz), 8.16 (1H, d,
J = 5.1 Hz). MS (ESI): m/z 299 [M+H]+.
5.1.12. 4-(2,3-Difluorophenyl)-2-(piperazin-1-yl)pyrimidine
dihydrochloride (17)
To a stirred solution of tert-butyl 4-[4-(2,3-difluorophenyl)pyr-
imidin-2-yl]piperazine-1-carboxylate (11.3 g, 30.0 mmol) in EtOAc
(70 mL) and MeOH (45 mL) was added 4 M HCl in EtOAc (38 mL)
dropwise at room temperature. After stirring at room temperature
overnight, the mixture was concentrated in vacuo. To the residue
was added EtOAc (300 mL) and MeOH (60 mL), and the mixture
was stirred at room temperature for 2 h. The solid was collected
by filtration and washed with EtOAc to give 17 (9.90 g, 94%) as a
pale yellow powder. 1H NMR (300 MHz, DMSO-d6) d: 3.19 (4H, br
s), 4.04 (4H, t, J = 4.0 Hz), 7.15–7.17 (1H, m), 7.35–7.41 (1H, m),
7.58–7.65 (1H, m), 7.83–7.86 (1H, m), 8.57 (1H, d, J = 4.0 Hz),
9.21 (2H, br s). MS (ESI): m/z 277 [M+H]+.
5.1.6. tert-Butyl 4-[4-(2,4-difluorophenyl)pyrimidin-2-
yl]piperazine-1-carboxylate (11)
Compound 11 was prepared in a manner similar to that de-
scribed for 8 in 71% yield as a colorless viscous oil. 1H NMR
(300 MHz, CDCl3) d: 1.50 (9H, s), 3.46–3.58 (4H, m), 3.85–3.93
(4H, m), 6.83–7.08 (3H, m), 8.08–8.22 (1H, m), 8.39 (1H, d,
J = 5.3 Hz). MS (ESI): m/z 377 [M+H]+.
5.1.7. tert-Butyl 4-[4-(2,3-difluorophenyl)pyrimidin-2-
yl]piperazine-1-carboxylate (12)
5.1.13. 4-(2,5-Difluorophenyl)-2-(piperazin-1-yl)pyrimidine
dihydrochloride (18)
A mixture of tert-butyl 4-(2-chloropyrimidin-4-yl)piperazine-1-
carboxylate (9.00 g, 30.1 mmol), (2,3-difluorophenyl)boronic acid
(7.10 g, 45.1 mmol), 1 M aq Na2CO3 (41 mL, 82.0 mmol), tetra-
kis(triphenylphosphine)palladium (0) (4.2 g, 3.61 mmol), and
DME (270 mL) was stirred at 95 °C overnight under N2 atmosphere.
After cooling to room temperature, the mixture was diluted with
water, and extracted with EtOAc. The organic layer was washed
with water, dried over anhydrous MgSO4, and concentrated in va-
cuo. The residue was purified by silica gel column chromatography
(hexane–EtOAc) to give 12 (11.3 g, quant.) as pale yellow crystals.
1H NMR (300 MHz, CDCl3) d: 1.50 (9H, s), 3.53 (4H, t, J = 4.0 Hz),
3.89 (4H, t, J = 4.0 Hz), 7.04–7.06 (1H, m), 7.16–7.29 (2H, m),
7.81–7.85 (1H, m), 8.41 (1H, d, J = 4.0 Hz). MS (ESI): m/z 377 [M+H]+.
Compound 18 was prepared in a manner similar to that de-
scribed for 17 in 95% yield as a pale yellow solid. 1H NMR
(300 MHz, DMSO-d6) d: 3.19 (4H, br s), 4.05 (4H, t, J = 4.0 Hz),
7.17–7.19 (1H, m), 7.43–7.47 (2H, m), 7.87–7.92 (1H, m), 8.57
(1H, d, J = 4.0 Hz), 9.29 (2H, br s). MS (ESI): m/z 277 [M+H]+.
5.1.14. 4-(3,4-Difluorophenyl)-2-(piperazin-1-yl)pyrimidine
dihydrochloride (19)
To a stirred solution of tert-butyl 4-[4-(3,4-difluorophenyl)
pyrimidin-2-yl]piperazine-1-carboxylate in EtOAc (70 mL) and
MeOH (100 mL) was added 4 M HCl in EtOAc (42 mL) dropwise
at room temperature. After stirring at room temperature over-
night, the mixture was concentrated in vacuo. To the residue
was added EtOAc (300 mL) and MeOH (60 mL), and the mixture
was stirred at room temperature for 2 h. The solid was collected
by filtration and washed with EtOAc to give 19 (11.7 g, quant.)
as a pale yellow powder. 1H NMR (300 MHz, DMSO-d6) d: 3.19
(4H, br s), 4.07 (4H, t, J = 5.1 Hz), 7.42–7.47 (2H, m), 7.91–7.94
(2H, m), 8.57 (1H, d, J = 5.4 Hz), 9.18 (2H, br s). MS (ESI): m/z
277 [M+H]+.
5.1.8. tert-Butyl 4-[4-(2,5-difluorophenyl)pyrimidin-2-
yl]piperazine-1-carboxylate (13)
Compound 13 was prepared in a manner similar to that de-
scribed for 12 in 96% yield as
a
colorless solid. 1H NMR
(300 MHz, CDCl3) d: 1.52 (9H, s), 3.53 (4H, t, J = 4.0 Hz), 3.88 (4H,
t, J = 4.0 Hz), 7.09–7.14 (3H, m), 7.81–7.86 (1H, m), 8.41 (1H, d,
J = 4.0 Hz). MS (ESI): m/z 377 [M+H]+.

M. Kono et al. / Bioorg. Med. Chem. 22 (2014) 1468–1478
1475
5.1.15. 4-(3,5-Difluorophenyl)-2-(piperazin-1-yl)pyrimidine
dihydrochloride (20)
J = 4.5, 9.1 Hz), 7.82–7.95 (2H, m), 8.02 (1H, dd, J = 1.5, 9.1 Hz),
8.54 (1H, d, J = 5.3 Hz), 8.85 (1H, d, J = 3.4 Hz), 9.97 (1H, s). MS
(ESI): m/z 398 [M+H]+ Anal. Calcd for C19H17 F2N7O: C, 57.43; H,
4.31; N, 24.67. Found: C, 57.24; H, 4.33; N, 24.39.
Compound 20 was prepared in a manner similar to that de-
scribed for 19 in 95% yield as
a
yellow powder. 1H NMR
(300 MHz, DMSO-d6) d: 3.19 (4H, br s), 4.07 (4H, t, J = 4.9 Hz),
7.15 (1H, dd, J = 2.4, 5.1 Hz), 7.35–7.40 (1H, m), 7.57–7.59 (1H,
m), 8.05–8.09 (1H, dt, J = 1.7, 7.8 Hz), 8.55 (1H, d, J = 5.1 Hz), 9.39
(2H, br s).
5.1.21. 2,2,2-Trichloroethyl pyridazin-3-ylcarbamate (23)
To a stirred solution of pyridazin-3-amine (2.9 g, 30.5 mmol)
and pyridine (7.4 mL, 91.5 mmol) in THF (40 mL) and DMA
(20 mL) was added 2,2,2-Trichloroethyl chloroformate (6.31 mL,
45.7 mmol) at 0 °C dropwise. 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
concentrated in vacuo. The residue was recrystallized from
EtOAc–hexane to give 23 (3.76 g, 46%) as an off-white crystals 1H
NMR (300 MHz, CDCl3) d: 4.88 (2H, s), 7.50–7.55 (1H, m), 8.25–
8.28 (1H, m), 8.74 (1H, br s), 8.95–8.97 (1H, m).
5.1.16. 4-[4-(2,4-Difluorophenyl)pyrimidin-2-yl]-N-(pyridazin-
3-yl)piperazine-1-carboxamide (21a)
Compound 21a was prepared in a manner similar to that de-
scribed for 5 in 22% yield as colorless crystals, mp 184–185 °C.
1H NMR (300 MHz, DMSO-d6) d: 3.60–3.68 (4H, m), 3.82–3.91
(4H, m), 7.06 (1H, dd, J = 2.5, 5.1 Hz), 7.23–7.32 (1H, m), 7.38–
7.47 (1H, m), 7.58 (1H, dd, J = 5.1, 9.1 Hz), 8.02 (1H, dd, J = 1.5,
9.1 Hz), 8.11–7.21 (1H, m), 8.50 (1H, d, J = 5.1 Hz), 8.85 (1H, dd,
J = 1.5, 4.5 Hz), 9.97 (1H, s). MS (ESI): m/z 398 [M+H]+ Anal. Calcd
for C19H17 F2N7Oꢂ0.5H2O: C, 56.15; H, 4.46; N, 24.13. Found: C,
55.91; H, 4.43; N, 23.93.
5.1.22. Phenyl pyridazin-3-ylcarbamate (24)
To a stirred suspension of pyridazin-3-amine hydrochloride
(10.0 g, 76.0 mmol) and pyridine (13.6 mL, 167 mmol) in MeCN
(50 mL) was added phenyl chloroformate (11.4 mL, 91.2 mmol)
dropwise at 0 °C. The mixture was stirred at 0 °C for 1 h. To the
mixture was added water (100 mL) and the mixture was stirred
at room temperature for 30 min and 0 °C for 1 h. The solid was col-
lected by filtration and washed with MeCN/water (1:2) (20 mL),
water (20 mL) to give 24 (16.3 g, 87%) as an off-white powder. 1H
NMR (300 MHz, CDCl3) d: 7.17–7.33 (3H, m), 7.37–7.47 (2H, m),
7.50 (1H, dd, J = 4.7, 9.0 Hz), 8.29 (1H, dd, J = 1.4, 9.0 Hz), 8.91
(1H, br s), 8.95 (1H, dd, J = 1.4, 4.7 Hz).
5.1.17. 4-[4-(2,3-Difluorophenyl)pyrimidin-2-yl]-N-(pyridazin-
3-yl)piperazine-1-carboxamide (21b)
A mixture of 4-(2,3-difluorophenyl)-2-(piperazin-1-yl)pyrimi-
dine dihydrochloride (200 mg, 0.573 mmol), phenyl pyridazin-3-
ylcarbamate (136 mg, 0.630 mmol), triethylamine (0.319 mL,
2.29 mmol), and acetone (1.0 mL) was stirred at 45 °C for 3 h.
The mixture was diluted with water, and stirred at room tempera-
ture for 1 h. The solid was collected by filtration, washed with
water, and recrystallized from THF–hexane to give 21b (206 mg,
91%) as colorless crystals, mp 248–249 °C. 1H NMR (300 MHz,
DMSO-d6) d: 3.54–3.72 (4H, m), 3.77–3.98 (4H, m), 7.09 (1H, dd,
J = 2.5, 5.0 Hz), 7.32–7.43 (1H, m), 7.52–7.67 (2H, m), 7.79–7.91
(1H, m), 8.02 (1H, dd, J = 1.3, 9.0 Hz), 8.54 (1H, d, J = 5.0 Hz), 8.85
(1H, dd, J = 1.3, 4.5 Hz), 9.97 (1H, s). MS (ESI): m/z 398 [M+H]+ Anal.
Calcd for C19H17 F2N7O: C, 57.43; H, 4.31; N, 24.67. Found: C, 57.18;
H, 4.45; N, 24.57.
5.1.23. tert-Butyl 4-(2-chloropyridin-4-yl)piperazine-1-
carboxylate (29)
A mixture of 4-bromo-2-chloropyridine (3.87 mL, 34.9 mmol),
N-Boc-piperazine (5.00 g, 26.9 mmol), sodium tert-butoxide
(3.87 g, 40.3 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylx-
anthene(932.2 mg, 1.61 mmol), tris(dibenzylideneacetone)dipalla-
dium (0) (492 mg, 0.537 mmol), and toluene (270 mL) was
stirred at 100 °C overnight under N2 atmosphere. The mixture
was diluted with water, and extracted with EtOAc. The organic
layer was washed with water, dried over anhydrous MgSO4, and
concentrated in vacuo. The residue was purified by silica gel col-
umn chromatography (hexane–EtOAc) to give 29 (5.62 g, 70%) as
a colorless powder. 1H NMR (300 MHz, CDCl3) d: 1.49 (9H, s),
3.30–3.37 (4H, m), 3.52–3.60 (4H, m), 6.57 (1H, dd, J = 2.4,
6.1 Hz), 6.65 (1H, d, J = 2.4 Hz), 8.04 (1H, d, J = 6.1 Hz).
5.1.18. 4-[4-(2,5-Difluorophenyl)pyrimidin-2-yl]-N-(pyridazin-
3-yl)piperazine-1-carboxamide (21c)
Compound 21c was prepared in a manner similar to that de-
scribed for 21b in 44% yield as colorless crystals, mp 271–272 °C.
1H NMR (300 MHz, DMSO-d6) d: 3.55–3.72 (4H, m), 3.79–3.96
(4H, m), 7.11 (1H, dd, J = 2.4, 5.1 Hz), 7.38–7.50 (2H, m), 7.58
(1H, dd, J = 4.7, 9.0 Hz), 7.82–7.93 (1H, m), 8.02 (1H, dd, J = 1.3,
9.0 Hz), 8.53 (1H, d, J = 5.1 Hz), 8.85 (1H, dd, J = 1.3, 4.7 Hz), 9.97
(1H, s). MS (ESI): m/z 398 [M+H]+ Anal. Calcd for C19H17 F2N7O:
C, 57.43; H, 4.31; N, 24.67. Found: C, 57.19; H, 4.41; N, 24.54.
5.1.24. tert-Butyl 4-(5-bromopyridin-3-yl)piperazine-1-
carboxylate (30)
Compound 30 was prepared in a manner similar to that de-
scribed for 29 in 79% yield as a colorless viscous oil. 1H NMR
(300 MHz, CDCl3) d: 1.49 (9H, s), 3.14–3.23 (4H, m), 3.55–3.63
(4H, m), 7.28–7.32 (1H, m), 8.15 (1H, d, J = 1.7 Hz), 8.21 (1H, d,
J = 1.7 Hz).
5.1.19. 4-[4-(3,4-Difluorophenyl)pyrimidin-2-yl]-N-(pyridazin-
3-yl)piperazine-1-carboxamide (21d)
Compound 21d was prepared in a manner similar to that de-
scribed for 21b in 83% yield as colorless crystals, mp 218–219 °C.
1H NMR (300 MHz, DMSO-d6) d: 3.56–3.73 (4H, m), 3.80–3.99
(4H, m), 7.30 (1H, d, J = 5.3 Hz), 7.51–7.66 (2H, m), 7.96–8.11
(2H, m), 8.18–8.28 (1H, m), 8.50 (1H, d, J = 5.3 Hz), 8.85 (1H, d,
J = 3.4 Hz), 9.97 (1H, s). MS (ESI): m/z 398 [M+H]+ Anal. Calcd for
5.1.25. tert-Butyl 4-(6-chloropyrimidin-4-yl)piperazine-1-
carboxylate (31)
Compound 31 was prepared in a manner similar to that de-
C19H17 F2N7O: C, 57.43; H, 4.31; N, 24.67. Found: C, 57.40; H,
scribed for 29 in 72% yield as a
yellow powder. 1H NMR
4.46; N, 24.69.
(300 MHz, CDCl3) d: 1.49 (9H, s), 3.50–3.57 (4H, m), 3.61–3.70
(4H, m), 6.50 (1H, s), 8.39 (1H, s).
5.1.20. 4-[4-(3,5-Difluorophenyl)pyrimidin-2-yl]-N-(pyridazin-
3-yl)piperazine-1-carboxamide (21e)
5.1.26. 2-Chloro-4-(2,4-difluorophenyl)pyridine (33)
Compound 21e was prepared in a manner similar to that de-
scribed for 21b in 39% yield as colorless crystals, mp 270–271 °C.
1H NMR (300 MHz, DMSO-d6) d: 3.53–3.75 (4H, m), 3.78–4.00
(4H, m), 7.35 (1H, d, J = 5.3 Hz), 7.38–7.49 (1H, m), 7.58 (1H, dd,
To a solution of 4-bromo-2-chloropyridine (30.0 g, 156 mmol),
(2,4-difluorophenyl)boronic acid (24.6 g, 156 mmol), and sodium
carbonate (43.1 g, 312 mmol) in MeOH (195 mL) was added tetra-
kis(triphenylphosphine)palladium (0) (9.01 g, 7.79 mmol) at room

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M. Kono et al. / Bioorg. Med. Chem. 22 (2014) 1468–1478
temperature under N2 atmosphere, and the mixture was stirred at
50 °C for 17 h. The reaction mixture was cooled to room tempera-
ture and the resulting solid was filtered. The filtrate was dried over
anhydrous MgSO4, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (hexane–EtOAc) to
give 33 (31.9 g, 91%) as a colorless powder. 1H NMR (300 MHz,
CDCl3) d: 6.95–7.05 (2H, s), 7.37–7.39 (1H, m), 7.43–7.47 (1H, m),
7.49 (1H, br s), 8.47 (1H, d, J = 13.2 Hz).
5.1.32. 1-[6-(2,4-Difluorophenyl)pyridin-2-yl]piperazine (39)
solution of tert-butyl 4-[6-(2,4-difluorophenyl)pyridin-2-
A
yl]piperazine-1-carboxylate (2.84 g, 7.55 mmol) in trifluoroacetic
acid–methylene chloride (1:2) was stirred at room temperature
for 2 h and the mixture was concentrated in vacuo. The residue
was neutralized by adding aqueous saturated sodium hydrogen
carbonate solution, and extracted with methylene chloride. The ex-
tract was dried over anhydrous Na2SO4 and the solvent was dis-
tilled off under reduced pressure. The residue was recrystallized
from hexane and Et2O to give 39 (1.50 g, 72%) as colorless crystals.
1H NMR (300 MHz, CDCl3) d: 2.97–3.05 (4H, m), 3.39–3.41 (4H, m),
6.55 (1H, d, J = 8.4 Hz), 6.79–6.82 (1H, m), 6.86–6.88 (1H, m), 7.08
(1H, dd, J = 2.4, 7.6 Hz), 7.46 (1H, t, J = 8.0 Hz), 7.95 (1H, q,
J = 8.8 Hz).
5.1.27. tert-Butyl 4-[6-(2,4-difluorophenyl)pyridin-2-
yl]piperazine-1-carboxylate (34)
To a solution of tert-butyl 4-(6-bromopyridin-2-yl)piperazine-
1-carboxylate (3.00 g, 8.77 mmol) and (2,4-difluorophenyl)boronic
acid (1.40 g, 12.3 mmol) in 1,2-dimethoxyethane (30 mL) and
water (3.0 mL) was added sodium carbonate (1.25 g, 17.5 mmol)
and
tetrakis(triphenylphosphine)palladium
(0)
(340 mg,
5.1.33. 1-[2-(2,4-Difluorophenyl)pyridin-4-yl]piperazine (40)
Compound 40 was prepared in a manner similar to that de-
scribed for 9 in 91% yield as a pale yellow viscous oil. 1H NMR
(300 MHz, CDCl3) d: 2.94–3.06 (4H, m), 3.26–3.39 (4H, m), 6.65
(1H, dd, J = 2.5, 5.9 Hz), 6.81–7.02 (2H, m), 7.11 (1H, t, J = 2.0 Hz),
7.84–7.99 (1H, m), 8.37 (1H, d, J = 5.9 Hz).
0.440 mmol) at room temperature under N2 atmosphere and
heated under reflux for 1 h. The reaction mixture was cooled to
room temperature, poured into water, and extracted with EtOAc.
The extract was washed with aqueous saturated sodium hydrogen
carbonate solution, dried over anhydrous Na2SO4, and concen-
trated in vacuo. The residue was purified by silica gel column chro-
matography (hexane–EtOAc) to give 34 (2.84 g, 86%) as a colorless
viscous oil. This product was used for next reaction without further
purification.
5.1.34. 1-[5-(2,4-Difluorophenyl)pyridin-3-yl]piperazine (41)
Compound 41 was prepared in a manner similar to that de-
scribed for 9 in 94% yield as a pale yellow viscous oil. 1H NMR
(300 MHz, CDCl3) d: 2.99–3.12 (4H, m), 3.16–3.28 (4H, m), 6.87–
7.04 (2H, m), 7.24–7.33 (1H, m), 7.35–7.47 (1H, m), 8.21 (1H, s),
8.31 (1H, d, J = 2.8 Hz).
5.1.28. tert-Butyl 4-[2-(2,4-difluorophenyl)pyridin-4-
yl]piperazine-1-carboxylate (35)
Compound 35 was prepared in a manner similar to that de-
scribed for 8 in 37% yield as a yellow viscous oil. 1H NMR
(300 MHz, CDCl3) d: 1.49 (9H, s), 3.34–3.41 (4H, m), 3.54–3.63
(4H, m), 6.65 (1H, dd, J = 2.5, 5.9 Hz), 6.83–7.03 (2H, m), 7.08–
7.14 (1H, m), 7.87–8.00 (1H, m), 8.39 (1H, d, J = 5.9 Hz).
5.1.35. 1-[4-(2,4-Difluorophenyl)pyridin-2-yl]piperazine (42)
To a stirred solution of tert-butyl 4-[4-(2,4-difluorophenyl)pyri-
din-2-yl]piperazine-1-carboxylate (19.0 g, 50.6 mmol) in MeOH
(63 mL) was added 4 N HCl in MeOH (50 mL) dropwise at room
temperature. After stirring at room temperature for 16 h, the
resulting solid was collected by filtration and washed with EtOAc
to give 42 (15.5 g, 98%) as an off-white powder. 1H NMR
(300 MHz, DMSO-d6) d: 3.22 (4H, s), 3.91 (4H, s), 7.01–7.02 (1H,
m), 7.23 (1H, br s), 7.26–7.31 (1H, m), 7.44–7.50 (1H, m), 7.72–
7.78 (1H, m), 8.20 (1H, d, J = 5.6 Hz), 9.40 (2H, br s). MS (ESI): m/
z 276 [M+H]+.
5.1.29. tert-Butyl 4-[5-(2,4-difluorophenyl)pyridin-3-
yl]piperazine-1-carboxylate (36)
Compound 36 was prepared in a manner similar to that de-
scribed for 8 in 94% yield as a yellow viscous oil. 1H NMR
(300 MHz, CDCl3) d: 1.49 (9H, s), 3.18–3.26 (4H, m), 3.57–3.65
(4H, m), 6.88–7.04 (2H, m), 7.28–7.32 (1H, m), 7.35–7.46 (1H,
m), 8.24 (1H, s), 8.31 (1H, d, J = 2.8 Hz).
5.1.36. 4-(2,4-Difluorophenyl)-6-(piperazin-1-yl)pyrimidine
(43)
5.1.30. tert-Butyl 4-[4-(2,4-difluorophenyl)pyridin-2-
yl]piperazine-1-carboxylate (37)
Compound 43 was prepared in a manner similar to that de-
scribed for 9 in 83% yield as a pale yellow viscous oil. 1H NMR
(300 MHz, CDCl3) d: 2.91–3.03 (4H, m), 3.63–3.74 (4H, m), 6.83–
7.06 (3H, m), 8.01–8.15 (1H, m), 8.68 (1H, s). MS (ESI): m/z 277
[M+H]+.
A mixture of 2-chloro-4-(2,4-difluorophenyl)pyridine (15.0 g,
66.5 mmol), N-Boc-piperazine (12.4 g, 66.6 mmol), palladium ace-
tate (746 mg, 3.32 mmol), 2,2-bis(diphenylphosphino)-1,1-
binaphthyl (3.31 g, 5.32 mmol), sodium tert-butoxide (13.0 g,
133 mmol), and 1,4-dioxane (133 mL) was stirred at 85 °C for
18 h. The reaction mixture was cooled to room temperature and
the solvent was distilled off under reduced pressure. To the resi-
due was added EtOAc, washed with water, dried over anhydrous
MgSO4, and concentrated in vacuo. The residue was purified by
silica gel column chromatography (hexane–EtOAc) to give 37
(19.0 g, 76%) as a colorless powder. 1H NMR (300 MHz, CDCl3)
d: 1.49 (9H, s), 3.57 (8H, s), 6.75–6.78 (2H, m), 6.90–6.98 (2H,
m), 7.39–7.45 (1H, m), 8.24 (1H, d, J = 4.8 Hz). MS (ESI): m/z
376 [M+H]+.
5.1.37. 4-[6-(2,4-Difluorophenyl)pyridin-2-yl]-N-(pyridazin-3-
yl)piperazine-1-carboxamide (44a)
Compound 44a was prepared in a manner similar to that de-
scribed for 5 in 52% yield as colorless crystals, mp 200–201 °C.
1H NMR (300 MHz, DMSO-d6) d: 3.62–3.64 (8H, m), 6.90 (1H, d,
J = 9.2 Hz), 7.07 (1H, dd, J = 2.4, 4.8 Hz), 7.18–7.22 (1H, m), 7.30–
7.36 (1H, m), 7.56 (1H, dd, J = 4.4, 6.8 Hz), 7.65 (1H, t, J = 8.0 Hz),
7.97–8.05 (2H, m), 8.83 (1H, dd, J = 1.2, 3.0 Hz), 9.94 (1H, br s).
MS (ESI): m/z 397 [M+H]+ analytical HPLC showed 100% purity.
5.1.31. tert-Butyl 4-[6-(2,4-difluorophenyl)pyrimidin-4-
yl]piperazine-1-carboxylate (38)
5.1.38. 4-[2-(2,4-Difluorophenyl)pyridin-4-yl]-N-(pyridazin-3-
yl)piperazine-1-carboxamide (44b)
Compound 38 was prepared in a manner similar to that de-
scribed for 8 in 72% yield as a yellow viscous oil. 1H NMR
(300 MHz, CDCl3) d: 1.50 (9H, s), 3.52–3.60 (4H, m), 3.68–3.75
(4H, m), 6.86–7.06 (3H, m), 8.05–8.16 (1H, m), 8.70 (1H, d,
J = 1.1 Hz). MS (ESI): m/z 377 [M+H]+
Compound 44b was prepared in a manner similar to that de-
scribed for 5 in 57% yield as colorless crystals, mp 189–190 °C.
1H NMR (300 MHz, DMSO-d6) d: 3.40–3.51 (4H, m), 3.62–3.72
(4H, m), 6.90 (1H, dd, J = 2.5, 5.9 Hz), 7.12–7.25 (2H, m), 7.29–
7.40 (1H, m), 7.58 (1H, dd, J = 4.6, 9.1 Hz), 7.82–8.05 (2H, m),

M. Kono et al. / Bioorg. Med. Chem. 22 (2014) 1468–1478
1477
8.30 (1H, d, J = 5.9 Hz), 8.83–8.88 (1H, m), 9.99 (1H, s). MS (ESI): m/
z 397 [M+H]+ analytical HPLC showed 95.8% purity.
centrifugation at 900g, the supernatant was recovered and further
centrifuged at 10,000g. A pellet obtained therefrom was suspended
in M-PER (Catalog No. 78501; Pierce Biotechnology, Inc.) to give an
enzyme fraction.
5.1.39. 4-[5-(2,4-Difluorophenyl)pyridin-3-yl]-N-(pyridazin-3-
yl)piperazine-1-carboxamide (44c)
Compound 44c was prepared in a manner similar to that de-
scribed for 5 in 22% yield as colorless crystals, mp 172–173 °C.
1H NMR (300 MHz, DMSO-d6) d: 3.23–3.42 (4H, m), 3.60–3.76
(4H, m), 7.17–7.29 (1H, m), 7.35–7.51 (2H, m), 7.54–7.74 (2H,
m), 7.97–8.07 (1H, m), 8.16 (1H, s), 8.38 (1H, d, J = 2.6 Hz), 8.80–
8.90 (1H, m), 10.01 (1H, s). MS (ESI): m/z 397 [M+H]+ Anal. Calcd
for C20H18 F2N6Oꢂ0.5H2O: C, 59.25; H, 4.72; N, 20.73. Found: C,
59.36; H, 4.68; N, 20.55.
5.3.2. Enzyme reaction
A test compound of various concentration, the enzymatic frac-
tion (final concentration of 125–250 ng) and AMC substrate arach-
idonoyl amide (AMCAA: manufactured by CAYMAN CHEMICAL:
final concentration of 3 lM) were reacted in reaction buffer
(Tris–HCl (pH 9.0) of 125 mM, EDTA of 1 mM, HEPES of 0.4 mM,
glycerol of 0.2%, Triton X-100 of 0.02% and fatty acid free BSA of
0.3% as final concentrations) at 37 °C for 90 min. After the reaction,
the fluorescent count of the plate was measured by a ARVOSX 1420
MULTILABEL COUNTER (manufactured by WALLAC) or EnVision
(Perkinelmer) under excitation at 355 nm and emission at
460 nm. The count of a sample containing solvent instead of the
test compound was taken as 100%, and the count at zero time
was taken as 0%, to calculate the inhibitory activity of the
compound.
5.1.40. 4-[4-(2,4-Difluorophenyl)pyridin-2-yl]-N-(pyridazin-3-
yl)piperazine-1-carboxamide (44d)
Compound 44d was prepared in a manner similar to that de-
scribed for 5 in 31% yield as a colorless crystals, mp 188–189 °C.
1H NMR (300 MHz, DMSO-d6) d: 3.63 (8H, s), 6.82–6.84 (1H, m),
6.98 (1H, s), 7.21–7.26 (1H, m), 7.38–7.44 (1H, m), 7.56–7.60
(1H, m), 7.66–7.72 (1H, m), 8.00–8.03 (1H, m), 8.21 (1H, d,
J = 5.2 Hz), 8.85 (1 H, d, J = 3.6 Hz), 9.96 (1H, s). MS (ESI): m/z 397
[M+H]+ analytical HPLC showed 98.3% purity.
5.4. Evaluation of analgesic effect in mouse acetic acid-induced
writhing test
5.1.41. 4-[6-(2,4-Difluorophenyl)pyrimidin-4-yl]-N-(pyridazin-
3-yl)piperazine-1-carboxamide (44e)
Male ICR mice (25–40 g) purchased from CLEA Japan Inc. (To-
kyo, Japan) were used in experiments. All of the animals were
housed in cages with free access to food and water. Experiments
were conducted between 9:00 and 17:00 h to minimize the diurnal
variation. The care and use of animals and the experimental proto-
cols used in this study were approved by the Experimental Animal
Care and Use Committee of Takeda Pharmaceutical Company Lim-
ited. Compounds were suspended to 0.5% methylcellulose solution
(Wako Pure Chemical Industries Limited, Osaka, Japan) and orally
administered to mice at a volume of 10 mL/kg. After 60 min of drug
administration, 0.6% (v/v) acetic acid (Wako Pure Chemical Indus-
tries Limited, 10 mL/kg) was intraperitoneally injected and each
mouse was then placed in an individual clear plastic observation
chamber. The number of stretching movements (arching of back,
development of tension in the abdominal muscles, elongation of
the body, and extention of the forelimbs) made by each mouse
was counted for 20 min.
Compound 44e was prepared in a manner similar to that de-
scribed for 5 in 10% yield as colorless crystals, mp 249–250 °C.
1H NMR (300 MHz, DMSO-d6) d: 3.60–3.69 (4H, m), 3.71–3.80
(4H, m), 7.16 (1H, s), 7.20–7.29 (1H, m), 7.36–7.47 (1H, m), 7.59
(1H, dd, J = 4.6, 9.1 Hz), 7.94–8.05 (2H, m), 8.63 (1H, d, J = 1.4 Hz),
8.85 (1H, dd, J = 1.4, 4.6 Hz), 9.98 (1H, s). MS (ESI): m/z 398
[M+H]+ Anal. Calcd for C19H17 F2N7Oꢂ0.5H2O: C, 56.15; H, 4.46; N,
24.13. Found: C, 55.86; H, 4.24; N, 24.17.
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.
5.3. Measurement of FAAH inhibitory activity
5.5. Analgesic effect of compound 21d in rat neuropathic pain
model
5.3.1. Preparation of enzyme fraction
The FAAH gene was cloned by PCR. That is, an amplified frag-
ment was obtained by carrying out the reaction at 95 °C. For 30 s
and at 55 °C. for 30 sec in one cycle and at 72 °C. for 2 min in 45
cycles, using a human brain library as cDNA library, and using 50-
Seven-week-old male Sprague Dawley rats were used in this
experiment. To prepare spared nerve injured rats, the right com-
mon peroneal nerve was exposed and tightly ligated with 4-0 silk
sutures followed by transection at the distal side under anestheti-
zation with pentobarbital. To prepare sham-operated rats, the right
common peroneal nerve was exposed, but left uninjured. The rats
were orally administered with compound, 21d at 1, 3, 10 mg/kg or
0.5% methylcellulose as vehicle 7 days after SNI operation. Then
tactile allodynia of ipsilateral hind paw was assessed by von Frey
filaments in a blinded manner 2 h after drug treatment. Fifty per-
cent threshold (g) of paw withdrawal response to tactile stimula-
tion was determined using the up-down method with starting
the filament of 4.56 g.
AAAAGAATTCGCCACCATGGTGCAGTACGAGCTGTG-30[SEQ
ID
NO:1] and 50-TTTTGTCGACTCAGGATGACTGCTTTT-30 [SEQ ID
NO:2] as primer set, and KOD DNA polymerase (Toyobo Co., Ltd)
as a DNA polymerase. The amplified fragment was cleaved with
restriction enzymes EcoRI and SalI, recovered, and then was in-
serted into a pMSR
restriction enzymes EcoRI and SalI and recovered, thereby to ob-
tain pMSR -human FAAH. A cell line CHO-K1/human was pre-
avector which had been cleaved with the same
a
pared, in which human FAAH was stably expressed in the cell
line CHO-K1 by a method known perse, using the above-obtained
plasmid. The CHO-K1/human FAAH was cultured in a CO2 incuba-
tor at 37 °C, using a medium in which fetal bovine serum (FBS) and
G418 were added to Ham’s F-12 medium to final concentrations of
5.6. Analgesic effect of compound 21d in rat inflammatory pain
model
10% and 800
l
g/mL, respectively, and then the cells were har-
Six-week-old male Sprague Dawley rats were used in this
vested. After washing with PBS, the cells were suspended in a buf-
fer (10 mM Tris, 1 mM EDTA and 10 mM MgCl2, all at final
concentrations) and disrupted with a Polytron homogenizer. After
experiment. To prepare CFA-treated rats, 150 lL of CFA was in-
jected into the plantar surface of the right hind paw. To prepare
sham-operated rats, liquid paraffin, vehicle, was injected instead

1478
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