Synthesis, SAR study, and biological evaluation of novel quinoline derivatives as phosphodiesterase 10A inhibitors with reduced CYP3A4 inhibition

Article abstract of DOI:10.1016/j.bmc.2014.11.039

A novel class of phosphodiesterase 10A inhibitors with potent PDE10A inhibitory activity and reduced CYP3A4 inhibition was designed and synthesized starting from 2-[4-({[1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl]oxy}methyl)phenyl]quinoline (1). Replacement of pyridine ring of 1 with N-methyl pyridone ring drastically improved CYP3A4 inhibition, and further optimization of these quinoline analogues identified 1-methyl-5-(1-methyl-3-{[4-(quinolin-2-yl)phenoxy]methyl}-1H-pyrazol-4-yl)pyridin-2(1H)-one (42b), which showed potent PDE10A inhibitory activity and a good CYP3A4 inhibition profile. A PET study with ¹¹C-labeled 42b indicated that 42b exhibited good brain penetration and specifically accumulated in the rodent striatum. Further, oral administration of 42b dose-dependently attenuated phencyclidine-induced hyperlocomotion in mice with an ED₅₀ value of 2.0 mg/kg and improved visual-recognition memory impairment at 0.1 and 0.3 mg/kg in mice novel object recognition test.

Full text of DOI:10.1016/j.bmc.2014.11.039

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, SAR study, and biological evaluation of novel quinoline
derivatives as phosphodiesterase 10A inhibitors with reduced
CYP3A4 inhibition
⇑
Wataru Hamaguchi , Naoyuki Masuda, Satoshi Miyamoto, Yasuhiro Shiina, Shigetoshi Kikuchi,
Takuma Mihara, Hiroyuki Moriguchi, Hiroshi Fushiki, Yoshihiro Murakami, Yasushi Amano,
Kazuya Honbou, Kouji Hattori
Drug Discovery Research, Astellas Pharma Inc., 21, Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel class of phosphodiesterase 10A inhibitors with potent PDE10A inhibitory activity and reduced
CYP3A4 inhibition was designed and synthesized starting from 2-[4-({[1-methyl-4-(pyridin-4-yl)-1H-
pyrazol-3-yl]oxy}methyl)phenyl]quinoline (1). Replacement of pyridine ring of 1 with N-methyl pyri-
done ring drastically improved CYP3A4 inhibition, and further optimization of these quinoline analogues
identified 1-methyl-5-(1-methyl-3-{[4-(quinolin-2-yl)phenoxy]methyl}-1H-pyrazol-4-yl)pyridin-2(1H)-
one (42b), which showed potent PDE10A inhibitory activity and a good CYP3A4 inhibition profile. A
PET study with 11C-labeled 42b indicated that 42b exhibited good brain penetration and specifically
accumulated in the rodent striatum. Further, oral administration of 42b dose-dependently attenuated
phencyclidine-induced hyperlocomotion in mice with an ED50 value of 2.0 mg/kg and improved visual-
recognition memory impairment at 0.1 and 0.3 mg/kg in mice novel object recognition test.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 10 October 2014
Revised 26 November 2014
Accepted 27 November 2014
Available online xxxx
Keywords:
PDE10A inhibitor
Schizophrenia
CYP3A4 inhibition
Quinoline
1. Introduction
N
N
N
N
O
Schizophrenia is a chronic and debilitating psychiatric disorder
that affects approximately 1% of the world’s population.1 However,
the majority of current therapeutic treatments primarily address
positive symptoms, with only limited efficacy on negative symp-
toms and cognitive dysfunction. In addition, current antipsychotics
frequently cause undesirable side effects such as extrapyramidal
syndrome, weight gain and diabetes,2 highlighting the unmet med-
ical needs for drugs less prone to such side effects.
Cyclic nucleotide phosphodiesterases (PDEs) are enzymes that
regulate intracellular signaling by hydrolyzing cyclic adenosine
monophosphate (cAMP) and cyclic guanosine monophosphate
(cGMP). The PDE superfamily of enzymes is divided into 11 fami-
lies in mammals (PDE1–11). PDE10A enzyme is a dual substrate
(cAMP/cGMP) phosphodiesterase that is highly expressed in the
brain, particularly in the medium spiny neurons of the mammalian
striatum.3 The striatal complex forms the core of the basal ganglia,
a system of interconnected nuclei that process cortical information
in the context of dopaminergic signaling to regulate motoric,
N
N
O
N
N
1
MP-10
PDE10A IC50 : 0.55 nM
mouse CLint : >1000 mL/min/kg
human CLint : 186 mL/min/kg
PDE10A IC50 : 29 nM
mouse CLint : 366 mL/min/kg
human CLint : 124 mL/min/kg
Figure 1. Structures of MP-10 and compound 1.
appetitive, and cognitive processes.4 Inhibition of PDE10A may
enhance the intracellular second messenger signaling and striatal
output suggested to be impaired in schizophrenic patients.5 Fur-
ther, some selective PDE10A inhibitors such as MP-10 (Fig. 1) have
shown potent efficacy in several rodent behavioral models of
schizophrenia.6 Thus, PDE10A inhibitors have garnered attention
as a new therapeutic method for the treatment of schizophrenia.7
We previously reported that the quinoline analogue 1 (Fig. 1)
had moderate PDE10A inhibitory activity with in vivo efficacy in
mice behavioral model for positive symptom and cognitive
⇑
impairment of schizophrenia.7r Compound
improved metabolic stability in human liver microsomes (HLM),
1 also exhibited
Corresponding author. Tel.: +81 29 863 6743; fax: +81 29 852 5387.
E-mail address: wataru.hamaguchi@astellas.com (W. Hamaguchi).
http://dx.doi.org/10.1016/j.bmc.2014.11.039
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Hamaguchi, W.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.11.039

2
W. Hamaguchi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
2. Chemistry
N
O
N
N
N
O
O
c
a, b
HO
The synthesis of target compounds is shown in Schemes 1–8.
The reaction of reagents 2a and 2b with N,N-dimethylformamide
dimethyl acetal followed by cyclization with methylhydrazine
gave 3a and 3b, which were reacted with [4-(quinolin-2-
yl)phenyl]methanol under Mitsunobu-type reaction condition to
give 4a and 4b, respectively. As shown in Scheme 2, compounds
6a and 6b were also synthesized in a similar manner from their
precursors 5a and 5b, respectively. The acetal group of 6a was
deprotected under acidic condition to afford ketone 7.
N
Y
X
Y
X
Y
X
4a (X = N, Y = CH)
4b (X = CH, Y = N)
3a (X = N, Y = CH)
3b (X = CH, Y = N)
2a (X = N, Y = CH)
2b (X = CH, Y = N)
Scheme 1. Reagents and conditions: (a) N,N-dimethylformamide dimethyl acetal,
DMF; (b) methylhydrazine, acetic acid, EtOH; (c) [4-(quinolin-2-yl)phenyl]metha-
nol, ADDP, nBu3P, THF.
but PDE10A inhibitory activity remained less potent than that of
MP-10. In addition, compound 1 was found to have a potent inhi-
bition of human cytochrome P450 3A4 (CYP3A4). As CYP3A4 is
considered to be involved in the metabolism of more than 50% of
drugs approved for human use,8 inhibition of CYP3A4 is a signifi-
cant concern regarding drug–drug interactions in clinical practice.
Unencumbered basic nitrogens of heterocycles, such as 4-pyridyl
ring, bind to the heme portion of CYP enzymes and result in CYP
inhibition.9 The attenuation of CYP3A4 inhibitory activity was
therefore assumed to be achieved by replacing pyridine ring or
introducing substituents into the pyridine ring of 1.
Scheme 3 shows the synthesis of 11 and 13a–g. The Mitsunobu-
type reaction between pyrazole analogue 8 and [4-(quinolin-2-
yl)phenyl]methanol generated 9, which was iodinated to give
intermediate 10. The Suzuki coupling reaction between 10 and
phenylboronic acid afforded 11. Halogen–magnesium exchange
of intermediate 10 followed by the addition of borate gave the
correspondent boronic acid ester 12,10 which was then reacted
with various substituted halides using Suzuki coupling reaction
to yield 13a–g.
The synthesis of compound 20 is shown in Scheme 4. Pyrazole
analogue 8 was alkylated with benzyl bromide to give 14, which
was converted to pyrazole pinacol borate 16 in a manner similar
to that of 12. The Suzuki coupling reaction between 16 and
5-bromo-1-methylpyridone followed by the deprotection of the
benzyl group (Bn) yielded intermediate 18. The pyridone ring of
In this paper, we synthesized analogues of our lead compound 1
and discuss SAR of PDE10A inhibitory activity and CYP3A4 inhibi-
tion. We also describe the successful development of potent
PDE10A inhibitor with reduced CYP3A4 inhibition.
N
N
N
N
)
O
a, b, c
O
O
d
O
N
N
X
X
O
O
O
O
7
6a (X =
5a (X =
)
O
6b (X = O)
5b (X = O)
Scheme 2. Reagents and conditions: (a) LiHMDS, HCO2Me, THF; (b) methyl hydrazine, EtOH; (c) 2-[4-(chloromethyl)phenyl]quinoline hydrochloride, K2CO3, DMF;
(d) p-TsOHꢀH2O, THF, H2O.
N
N
H
N
I
N
N
N
N
N
c
a
O
O
b
O
O
N
N
N
HO
8
10
9
11
d
N
B
N
R
N
N
e
O
N
N
O
O
12
13a-g
Scheme 3. Reagents and conditions: (a) [4-(quinolin-2-yl)phenyl]methanol, ADDP, nBu3P, THF; (b) CAN, I2, MeCN; (c) PhB(OH)2, Pd2(dba)3, Xphos, K3PO4, nBuOH;
(d) 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, iPrMgCl, THF; (e) R-Br or R-Cl, PdCl2(dppf)ꢀCH2Cl2, Na2CO3, DMF, H2O.
N
N
N N
N
N
N
N
N
N
BnO
HO
HO
N
BnO
O
N
N
a
e
b
c
d
g
N
f
N
N
B
N
O
O
BnO
14
BnO
I
HO
O
N
N
N
N
8
15
O
O
O
19
17
18
20
16
Scheme 4. Reagents and conditions: (a) benzyl bromide, K2CO3, DMF; (b) CAN, I2, MeCN; (c) PdCl2(dppf)ꢀCH2Cl2, iPrMgCl, 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-
dioxaborolane, THF; (d) 5-bromo-1-methylpyridin-2(1H)-one, PdCl2(dppf)ꢀCH2Cl2, Na2CO3, DMF, H2O; (e) H2, Pd–C, EtOH; (f) H2, PtO2, AcOH; (g) 2-[4-(chloro-
methyl)phenyl]quinoline hydrochloride, K2CO3, DMF.
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W. Hamaguchi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
N
N
OH
O
N
Cl
OH
b
a
N
N
+
HO
B
OH
N
22
23
21
O
24
Scheme 5. Reagents and conditions: (a) Pd(PPh3)4, Na2CO3, DME, H2O; (b) 18, CMBP, toluene.
H
8
O H
c, d
7
N
Cl
N
O
8
5
b
a
2
2
N
7
R
R
6
2
1
4
2
R
6
R
1
5
R
25a (R2 = 4-CN)
25b (R2 = 2-F)
26a (R1 = Me, R2 = 6-CN)
26b (R1 = Me, R2 = 8-F)
26c (R1 = Me, R2 = 5-F)
26d (R1 = Me, R2 = 6-F)
26e (R1 = Me, R2 = 7-F)
26f (R1 = Et, R2 = H)
27a (R1 = Me, R2 = 6-CN)
27b (R1 = Me, R2 = 8-F)
27c (R1 = Me, R2 = 5-F)
27d (R1 = Me, R2 = 6-F)
27e (R1 = Me, R2 = 7-F)
27f (R1 = Et, R2 = H)
26g (R1 = Me, R2 = 6-Me)
26h (R1 = Me, R2 = 6-OMe)
26i (R1 = -CHO, R2 = H)
27g (R1 = Me, R2 = 6-Me)
27h (R1 = Me, R2 = 6-OMe)
27i (R1 = -CHO, R2 = H)
N
N
N
N
O
e
8
O
7
N
N
2
(R1 = -CHO, R2 = H)
R
1
N
6
R
F
F
N
5
O
O
28a (R1 = Me, R2 = 6-CN)
28b (R1 = Me, R2 = 8-F)
28c (R1 = Me, R2 = 5-F)
28d (R1 = Me, R2 = 6-F)
28e (R1 = Me, R2 = 7-F)
28f (R1 = Et, R2 = H)
29
28g (R1 = Me, R2 = 6-Me)
28h (R1 = Me, R2 = 6-OMe)
28i (R1 = -CHO, R2 = H)
Scheme 6. Reagents and conditions: (a) POCl3, CTAB, DMF; (b) 22, Pd(PPh3)4, Na2CO3, DME, H2O; (c) SOCl2, CH2Cl2; (d) 18, K2CO3, DMF; (e) Deoxo-Fluor,ÒCH2Cl2.
N
N
N
I
N
OH
O
O
c, d
N
a, b
N
N
N
+
N
HO
N
31
O
30
32
33
Scheme 7. Reagents and conditions: (a) CMBP, toluene; (b) CAN, I2, MeCN; (c) isopropoxyboronic acid pinacol ester, iPrMgCl, THF; (d) 5-bromo-1-methylpyridin-2(1H)-one,
PdCl2(dppf)ꢀCH2Cl2, Na2CO3, DMF, H2O.
N
N
N
N
N
N
Cl
HCl
N
O
O
O
N
N
b
c
B
a
+
O
OH
I
O
N
N
N
O
N
N
O
O
f
O
38
O
2
O
N
36
37
35
+
34
2
1
N
O
O
OH
R
R
O
e
N
Cl
d
N
N
42a (R1 = Me, R2 = F)
42b (R1 = H, R2 = H)
1
F
R
F
R
40
39
41a (R1 = Me, R2 = F)
41b (R1 = H, R2 = H)
Scheme 8. Reagents and conditions: (a) Pd(PPh3)4, Cs2CO3, DMF, H2O; (b) LiBH4, EtOH, THF; (c) SOCl2; (d) 4-(2-tetrahydropyranyloxy)phenylboronic acid, Pd(PPh3)4, Na2CO3,
DME, H2O; (e) HCl, THF, H2O; (f) K2CO3, DMF.
intermediate 18 was hydrogenated to give 19, which was alkylated
with 2-[4-(chloromethyl)phenyl]quinoline to afford 20.
Substituted quinoline derivatives 24, 28a–h, and 29 were pre-
pared using the synthesis depicted in Schemes 5 and 6. Chloro-
quinoline analogue 21 and boronic acid 22 underwent Suzuki
coupling reaction to give 23, which was then reacted with com-
pound 18 to give 24. 2-Chloroquinoline analogues 26a and 26b
were prepared from 25a or 25b via cyclization reaction under Vils-
meier–Haack conditions.11 Compounds 26d–i were commercially
available and the synthesis of 26c was as previously reported.12
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4
W. Hamaguchi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
N
N
N
N
N
N
N
N
O
N
N
O
I
O
e
b, c
O
a
d
I
N
HO
I
N
N
OH
44
N
H
N
11 C
43
O
46
45
O
[
11C]42b
Scheme 9. Reagents and conditions: (a) CDI, THF, then NaBH4, H2O; (b) SOCl2, CH2Cl2; (c) 41b, K2CO3, DMF; (d) 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine-2-ol,
PdCl2(dppf)ꢀCH2Cl2, Na2CO3, MeCN, H2O; (e) [11C]CH3OTf, NaH, THF, DMSO.
Suzuki coupling reaction between 26a–i and 22 yielded 27a–i. The
chlorination of hydroxyl group of 27a–i followed by the reaction
with 18 gave 28a–i. The aldehyde moiety of 28i was converted
to difluoromethyl group using Deoxo-FluorÒ to give 29.
Compound 33 was prepared in four steps as outlined in
Scheme 7. Compounds 30 and commercially available alcohol 31
were reacted under Mitsunobu-type reaction condition,13 followed
by iodination of the pyrazole ring to give 32. Compound 32 was
then converted to pyrazole pinacol borate and underwent Suzuki
coupling with 5-bromo-1-methylpyridone to afford 33.
Scheme 8 shows the synthesis of 42a and 42b. Suzuki coupling
of compound 34 with 35 followed by the reduction of ester group
with lithium borohydride gave alcohol 37. Treatment of 37 with
thionyl chloride followed by the etherification with 41a or 41b
gave desired products 42a and 42b, respectively. Compound 41a
was synthesized from its precursor 39 via Suzuki coupling and sub-
sequent deprotection of tetrahydropyranyl group, and the synthe-
sis of 41b was already reported.14
Table 1
PDE10A potency and CYP3A4 inhibition
N
N
O
R
N
Compd
R
PDE10A IC50 (nM)
29
CYP3A4 residual activityb (%)
19
1
N
4a
4b
274
7.5
N
N
>1000
NTa
The synthesis of 11C-labeled 42b ([11C]42b) is shown in
Scheme 9. The reduction of carboxylic acid of 43 yielded alcohol
44. Hydroxyl group of 44 was chlorinated with thionyl chloride fol-
lowed by etherification to give 45, which was converted to 46 via
Suzuki coupling. [11C]42b was synthesized from 46 and 11C-labeled
methyl trifluoromethanesulfonate, and total synthesis time was
36 min from the end of bombardment (EOB). The decay corrected
radiochemical yield was 24–27%, calculated from the end of syn-
thesis, available as: 3.0–3.8 GBq; 95% radiochemically pure and
13a
13b
25
54
31
48
Me
N
Me
N
Me
13c
11
135
NTa
96
OMe
N
>100,000
29–60 GBq/lmol.
N
13e
13d
24
59
65
Me
O
3. Results and discussion
O
N
PDE10A inhibitory potencies of synthesized compounds were
tested using in vitro inhibition of human recombinant PDE10A cat-
alyzed cAMP hydrolysis, and CYP3A4 inhibitory activity was
described as the residual activity of midazolam following 30 min
151
Me
N
13f
73
46
N
Me
of pre-incubation in the presence of compound (5 lM). As shown
O
in Table 1, we first investigated the position of pyridyl nitrogen
atom of our lead compound 1. The rank order of PDE10A inhibitory
activity was 4-pyridyl > 3-pyridyl > 2-pyridyl (1, 4a, and 4b).
Regarding PDE10A inhibitory activities, the introduction of methyl
group into the pyridine ring of 1 retained activity (13a), whereas
one more methyl group led to decreased activity (13b). As for
CYP3A4 inhibition, slight improvement was observed depending
on the introduction of methyl group, which indicated that bulki-
ness of the methyl group hampered the binding of pyridyl nitrogen
atom to the heme portion of CYP3A4 enzyme.9 Substituting the
methyl group of 13a for a methoxy group resulted in a loss of
PDE10A inhibitory activity (13c). We next examined compound
with a phenyl ring at 4-position of the pyrazole ring to confirm
the effect on CYP3A4 inhibition. Although 11 totally lost PDE10A
inhibitory activity, CYP3A4 inhibition was dramatically improved,
indicating that basicity of pyridyl nitrogen atom of 1 contributes
to CYP3A4 inhibition and that loss of pyridyl nitrogen atom
N
N
13g
20
289
27
78
49
Me
Me
O
O
N
7
138
155
NTa
92
O
O
6b
a
Not tested.
b
Residual activities of HLM for metabolism of midazolam in the presence of test
compounds were determined following preincubation for 30 min. See Section 5.
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W. Hamaguchi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
resulted in the loss of PDE10A inhibitory activity. We hypothesized
that a non-basic ring structure with high hydrogen bond acceptor
(HBA) ability can support both potent PDE10A inhibitory activity
and improved CYP3A4 inhibition. The HBA strength was quantified
by calculating the hydrogen bond basicity (pKBHX), and the non-
basic N-methyl pyridone ring was reported to have high pKBHX
value (pKBHX = 2.50).15 We therefore replaced pyridine ring of 1
with N-methyl pyridone ring. Compound 13e showed similar
PDE10A inhibitory activity and improved CYP3A4 inhibition com-
pared with 1, whereas 13d showed reduced CYP3A4 inhibition
but less PDE10A inhibitory activity, suggesting that a position of
the carbonyl group as an HBA is important for PDE10A inhibitory
activity. The location of HBA on the extension line of a bond from
4-positon of the pyrazole ring might be important. Although the
distance between pyrazole ring and HBA of 13e is marginally
longer than that of 1, we consider pyridonyl oxygen atom of 13e
and pyridyl nitrogen atom of 1 to be located at similar position
inside PDE10A catalytic pocket. Additionally, pyridonyl oxygen
atom of 13e might form hydrogen bond with water molecule
inside PDE10A catalytic pocket, as with MP-10.6e We further inves-
tigated non-basic rings which could function as HBA. The introduc-
tion of one more nitrogen atom into pyridone ring of 13e was
detrimental to in vitro activity (13f, 13g). Replacement of pyridone
ring of 13e with saturated piperidone ring retained in vitro activity,
but CYP3A4 inhibition of 20 was a little more potent than that of
13e, which may be due to the higher lipophilicity of 20 (ACD/log
P = 4.9) compared with 13e (ACD/log P = 3.2).9,16 Compounds with
cyclohexanone or tetrahydropyran ring showed weaker PDE10A
inhibitory activity than 13e (7, 6b). We conducted correlation
analyses based on the pIC50 values of PDE10A inhibitory activity
and the reported pKBHX values listed in Table 2.15,17 Scatter plot
Figure 2. Scatter plot of pIC50 against pKBHX values.
Table 3
Substituent effect on PDE10A potency and CYP3A4 inhibition
N
N
O
R
N
O
Compd
R
PDE10A IC50
(nM)
CYP3A4 residual activityc
(%)
N
N
Table 2
13e
24
24
59
30
pIC50 of PDE10A inhibitory activity and pKBHX values
N
N
1.6
Me
Et
O
R
N
N
N
28f
29
22
Insolubleb
86
F
100
Compd
R
pIC50
pKBHX
F
N
1
7.54
7.60
7.27
6.87
1.86
N
N
28c
43
53
Me
F
F
13a
13b
13c
2.03
2.14
0.99
N
N
Me
28d
28e
15
52
66
27
F
F
Me
Me
N
Me
Me
N
OMe
Me
N
28b
37
35
Me
N
N
N
13e
20
7.59
7.57
2.50
2.60
28h
28g
28a
21
157
91
Insolubleb
70
O
O
MeO
Me
Me
N
N
Me
Me
NTa
7
6.86
6.81
1.39
1.23
NC
Me
O
O
a
b
c
Not tested.
Insoluble in assay buffer.
Residual activities of HLM for metabolism of midazolam in the presence of test
6b
compounds were determined following preincubation for 30 min. See Section 5.
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6
W. Hamaguchi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Table 4
Tyr693 of PDE10A enzyme, improving in vitro activity. When
CYP3A4 inhibition of 24 was compared to that of 13e, methyl
group at 3-position of quinoline ring was found to have a negative
impact. The ACD/log P value of 24 and 13e was 3.7 and 3.2,
respectively, and higher lipophilicity of 24 might lead to high
CYP3A4 inhibition. An electron withdrawing difluoromethyl group
improved CYP3A4 inhibition but decreased PDE10A inhibitory
activity (29). We further examined the substituents effect at other
positions on the quinoline ring. The introduction of fluorine group
reportedly can improve CYP3A4 inhibition,18 we therefore intro-
duced fluorine atom into quinoline ring of 24. The fluorine-intro-
duced analogues 28c, 28d, 28e and 28b showed moderate
in vitro activity, with fluorine group at 6-position being optimal
(28d). The fluorine group at 6-position also gave drastically
improved CYP3A4 inhibition (28d). We next replaced fluorine
group of 28d with methoxy, methyl or cyano groups. While 28g
with a methyl group and 28a with a cyano group at the 6-position
showed weak activity, 28h with methoxy group had similar
PDE10A inhibitory activity to 28d. Unfortunately, CYP3A4 inhibi-
tion of 28h was not determined due to its poor solubility in the
assay buffer.
As compounds 13e, 24, and 28d had more potent PDE10A inhib-
itory activity and reduced CYP3A4 inhibition than our lead
compound 1, we finally modified ‘oxy-methyl’ unit of these com-
pounds. As described above, lower ACD/log P value of our quinoline
analogues may result in less CYP3A4 inhibition. We therefore con-
ducted a replacement of ‘oxy-methyl’ unit of 13e, 24, and 28d with
‘methyl-oxy’ unit, which reduced ACD/log P values, as shown in
Table 4. As expected, this conversion resulted in a further improve-
ment of CYP3A4 inhibition in all cases (42b, 33, 42a). Moreover,
this conversion had positive impact on PDE10A inhibitory activity.
Among compounds listed in Table 4, compound 33 showed the
most potent in vitro activity but showed high intrinsic clearance
in HLM. Compounds 24 and 42a also exhibited very potent PDE10A
inhibitory activity but still had higher intrinsic clearance in HLM
than 13e or 42b. These results indicated that a methyl group at
3-position of quinoline ring increased PDE10A inhibitory activity
but made compounds metabolically unstable. The X-ray co-crystal
structure of 42b in the catalytic domain of PDE10A enzyme con-
firmed that the quinolynyl nitrogen atom formed a hydrogen bond
to the Tyr693 of PDE10A enzyme, while the quinoline ring forms
Effect of linker unit on PDE10A potency, CYP3A4 inhibition and intrinsic clearance
N
N
linker
R
N
O
Compd
R
Linker
PDE10A CYP3A4 h CLint
ACD/
IC50
(nM)
residual (mL/min/kg) logP
activitya
(%)
N
N
13e
24
–CH2O– 24
–CH2O– 1.6
59
30
168
239
3.2
3.7
Me
N
28d
42b
33
–CH2O– 15
–OCH2– 5.1
–OCH2– 1.6
66
83
67
263
120
576
4.4
2.0
2.4
F
F
Me
N
N
Me
N
42a
–OCH2– 2.8
82
445
3.2
Me
a
Residual activities of HLM for metabolism of midazolam in the presence of test
compounds were determined following preincubation for 30 min. See Section 5.
of pIC50 against pKBHX is shown in Figure 2, and the coefficient of
determination showed that the value of R2 was 0.779, which would
explain the positive correlation between the pIC50 and the pKBHX
values. This correlation analysis result suggested that strength of
hydrogen bond between compound and the water molecule inside
PDE10A catalytic pocket is important for potent PDE10A inhibitory
activity.
We next investigated the substituent effect on quinoline ring of
13e (Table 3). The introduction of methyl group at 3-position
of quinoline ring increased activity (24), while the replacement
of methyl group of 24 with ethyl group led to decreased PDE10A
inhibitory activity (28f). Electron donation by methyl group of 24
would increase the HBA ability of quinolynyl nitrogen atom, which
may strengthen hydrogen bond between this nitrogen atom and
CH–p interaction with Gly725 and Met713 (Fig. 3). Although water
molecules were not assigned in the cocrystal structure of 42b due
to low resolution, pyridonyl oxygen atom of 42b is located at
Figure 3. Crystal structure of 42b (lime green) bound to the PDE10A (PDB code: 4WN1).
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W. Hamaguchi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
Figure 4. Superimposition of 42b (green) on the crystal structure of MP-10 (purple) bound PDE10A (PDB code: 3HR1). Red spheres indicate water molecules and dashed lines
indicate hydrogen bonds.
nearly the same position as pyridyl nitrogen atom of MP-10 and
likely binds to the water molecule (Fig. 4).6e Figure 4 indicated that
pyrazole ring of 42b is located at a different position from that of
MP-10 to assign pyridonyl oxygen atom to a suitable position for
possible hydrogen bond to the water molecule.
Compound 42b exhibited approximately 6-fold more potent
activity than 1 and reduced CYP3A4 inhibition and improved
metabolic stability in HLM. PDE selectivity of 42b was also
investigated, with results showing a good profile of greater than
1200-fold selectivity over PDE1, 3, 4, 5, 6, 7, 8, 9, and 11. The accu-
mulation of 42b in striatum using positron-labeled [11C]42b was
next investigated. In a distribution study in mice, radioactivity in
the striatum and cerebellum was measured at 60 min after intrave-
nous injection of [11C]42b, which resulted in the higher standard-
ized uptake value (SUV) in striatum than that in cerebellum
(Fig. 5). As shown in Figure 6, PET imaging study in rat also showed
that [11C]42b specifically accumulated in striatum, and blocking
with MP-10 (10 mg/kg, iv) resulted in the inhibition of this accu-
mulation. These results indicate that 42b exhibited good brain
penetration and specifically bound to PDE10A in the striatum.
Figure 5. The standardized uptake value (SUV) after intravenous injection of
[
11C]42b in mice striatum and cerebellum.
Figure 6. PET images of [11C]42b in rat brain. (a) PET image after intravenous administration of [11C]42b. (b) PET image after intravenous administration of [11C]42b in the
presence of MP-10.
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8
W. Hamaguchi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Table 6
Brain concentration of MP-10 and 42b after 3.0 mg/kg oral dosage
Compd
Brain concd (ng/mg)
Fu, braina
Free brain concdb (ng/mg)
MP-10
42b
99
163
0.003
0.006
0.30
1.0
a
Unbound fraction in brain tissue.
Free brain concd was calculated by multiplying brain concd by fu,brain.
b
in vitro properties
N
N
O
PDE10A IC50: 5.1 nM
other PDEs: >1200 fold
N
N
in vivo properties
O
mice PCP-HL ED50: 2.0 mg/kg, po
mice NORT MED: 0.1 mg/kg, po
42b
Pharmacokinetics
CYP inhibition (1A2, 2C9, 2C19, 2D6); IC50: >10uM
CYP3A4 inhibition; residual activity: 83%
CLint (mL/min/kg) (human, mouse, rat, dog, monkey): 120, 285, 484, 148, 299
F (1.0 mg/kg; rat, dog, monkey): 45, 112, 105 %
Figure 7. Effect of oral administration of 42b on PCP-induced hyperlocomotion in
mice. PCP was administered subcutaneously (sc). The data represent the mean
-
CLtotal (mL/min/kg) (i.v.; rat, dog, monkey): 12, 1.1, 1.7 (mL/min/kg)
SEM (n = 8 in each group): ###p < 0.001 versus normal group (Student’s t test);
⁄⁄p < 0.01, ⁄⁄⁄p < 0.001 versus control group (Dunnett’s test).
t1/2 (h) (i.v.; rat, dog, monkey) : 1.3, 4.5, 2.5
Figure 9. Profiles of compound 42b.
impairment in neonatal PCP-treated mice by the oral administra-
tion at 0.1 and 0.3 mg/kg in novel object recognition test (Fig. 8).
Profiles of 42b and MP-10 were shown in Table 5. Compound
42b was more stable in human and mouse liver microsomes than
MP-10, and 42b showed less CYP3A4 inhibition over MP-10. The
in vitro PDE10A inhibitory activity of 42b was 9-fold lower than
that of MP-10, but in vivo potency in PCP-induced hyperlocomo-
tion model was nearly equal between the two compounds. Brain
free concentrations of 42b and MP-10 at 1 h after oral administra-
tion of 3.0 mg/kg to mice were calculated as 1.0 ng/g and 0.3 ng/g,
respectively (Table 6). The higher free concentration of 42b than
MP-10 would contribute to its in vivo potency. Additional profiles
of 42b are shown in Figure 9. Compound 42b exhibited low
CYP1A2, 2C9, 2C19, and 2D6 inhibition in addition to its low
CYP3A4 inhibition. Compound 42b was adequately stable in rat,
dog, and monkey liver microsomes, and showed good PK profiles
in rat, dog, and monkey. These results indicated that 42b would
have a potential to be used for the treatment of schizophrenia with
less concern for drug–drug interactions compared to MP-10.
Figure 8. Effect of 42b on neonatal PCP treatment-induced learning deficit in mice
during novel object recognition test. The data represent the mean SEM (n = 13 or
14 in each group): ##p < 0.01 versus normal group (Student’s t-test); ⁄p < 0.05
versus control group (Dunnett’s test).
Table 5
Profiles of MP-10 and 42b
4. Conclusions
MP-10
42b
In this study, we investigated the SAR of PDE10A inhibitory
activity and CYP3A4 inhibition to obtain potent PDE10A inhibitors
with less concern for drug–drug interactions. Substituting pyridine
ring of 1 with non-basic pyridone ring gave 13e with improved
CYP3A4 inhibition, and the positive correlation was observed
between the pIC50 of PDE10A inhibitory activity and the pKBHX
values of substituent at 4-position of pyrazole ring. Replacement
of ‘oxy-methyl’ unit of 13e with ‘methyl-oxy’ unit to give 42b
resulted in improved PDE10A inhibitory activity and further
reduced CYP3A4 inhibition. PET study indicated that 42b exhibited
good brain penetration and specifically accumulated in rodent
striatum. Further, compound 42b attenuated the locomotor activ-
ity induced by PCP after oral administration with an ED50 value
of 2.0 mg/kg, and 42b significantly improved visual-recognition
memory impairment by the oral administration at 0.1 and
0.3 mg/kg in novel object recognition test.
PDE10A IC50 (nM)
0.55
2.5
5.1
2.0
PCP-induced hyperlocomotion in mouse:
ED50 (mg/kg)
CYP3A4 inhibition: residual activitya (%)
CLint (mL/min/kg) (human, mouse)
35
83
120, 285
186, >1000
a
Residual activities of HLM for metabolism of midazolam in the presence of test
compounds were determined following preincubation for 30 min. See Section 5.
We also assessed the in vivo behavioral effect on hyperlocomo-
tion induced by phencyclidine (PCP) in mice, an animal model for
positive symptom of schizophrenia. As shown in Figure 7, 42b
dose-dependently attenuated the locomotor activity after oral
administration, with an ED50 value of 2.0 mg/kg. Compound 42b
also
significantly
improved
visual-recognition
memory
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W. Hamaguchi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
9
(m, 1H), 8.72 (d, 1H, J = 8.6 Hz), 8.88 (s, 1H); MS (ESI) m/z 393
[M+H]+; Anal. Calcd for C25H20N4Oꢀ2HClꢀ0.4C2H6Oꢀ1.9H2O: C,
59.82; H, 5.49; N, 10.82; Cl, 13.69. Found: C, 59.51; H, 5.81; N,
10.42; Cl, 14.06.
5. Experimental section
5.1. Chemistry
1H NMR spectra were recorded on a Varian VNS-400, JEOL JNM-
LA400, or JEOL JNM-AL400 and the chemical shifts were expressed
in d (ppm) values with tetramethylsilane as an internal reference
(s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet,
dd = doublet of doublets, td = triplet of doublets, and br = broad
peak). Mass spectra (MS) were recorded on Thermo Electron LCQ
Advantage or Agilent 6140. Elemental analyses were performed
using Yanaco MT-6 (C, H, N), Elementar Vario EL III (C, H, X), and
Dionex ICS-3000 (S, halogene) and were within 0.4% of theoretical
values.
5.1.5. 2-[4-({[4-(1,4-Dioxaspiro[4.5]dec-8-yl)-1-methyl-1H-
pyrazol-3-yl]oxy}methyl)phenyl]quinoline (6a)
To a stirred mixture of lithium bis(trimethylsilyl)amide (1 M
solution in THF, 30.1 mL, 30.1 mmol) in THF (45 mL) cooled with
dry ice–acetone bath were dropwisely added a solution of ethyl
1,4-dioxaspiro[4.5]dec-8-ylacetate (5a, 6.55 g, 28.7 mmol) with
keeping the temperature below ꢁ65 °C, and the mixture was stir-
red at the same temperature for 50 min. To the resultant mixture
was dropwisely added methyl formate (3.45 g, 57.4 mmol) with
keeping the temperature below ꢁ65 °C, and the mixture was stir-
red at the same temperature for 10 min. The resultant mixture
was allowed to warm up to room temperature and stirred for
3 h. The reaction was cooled with ice-water bath and quenched
with ca. 40 mL of 1 M HCl aqueous solution. The mixture was
diluted with brine and extracted with CHCl3, The organic layer
was dried over MgSO4, filtered and concentrated in vacuo to give
an orange oil. To this orange oil in EtOH (59 mL) was added meth-
ylhydrazine (2.64 g, 57.4 mmol), and the mixture was stirred at
100 °C for 3 h before cooling at room temperature. The mixture
was concentrated in vacuo, and the residue was purified by silica
gel column chromatography (0–15% MeOH in CHCl3) to give a
white solid (2.91 g). To this white solid (1.48 g) and 2-[4-(chloro-
methyl)phenyl]quinoline hydrochloride (1.8 g, 6.20 mmol) in
DMF (18 mL) was added K2CO3 (2.14 g, 15.5 mmol), and the
mixture was stirred at 60 °C for 1 h before cooling at room
temperature. The mixture was diluted with water and brine, and
the mixture was extracted with CHCl3/MeOH (5:1) for 3 times.
The combined organic layer was dried over MgSO4, filtered and
concentrated in vacuo. The residue was purified by silica gel
column chromatography (0–3% MeOH in CHCl3) to give 6a
(1.10 g, 17%) as a pale yellow oil. 1H NMR (DMSO-d6) d 1.46–1.72
(m, 8H), 2.35–2.44 (m, 1H), 3.56 (s, 3H), 3.83–3.86 (m, 4H), 5.14
(s, 2H), 7.15 (s, 1H), 7.60–7.65 (m, 3H), 7.77–7.83 (m, 1H), 8.02
(d, 1H, J = 8.2 Hz), 8.09 (d, 1H, J = 8.4 Hz), 8.19 (d, 1H, J = 8.7 Hz),
8.33 (d, 2H, J = 8.4 Hz), 8.48 (d, 1H, J = 8.7 Hz); MS (ESI) m/z 456
[M+H]+.
5.1.1. 1-Methyl-4-(pyridin-3-yl)-1H-pyrazol-3-ol (3a)
A mixture of ethyl pyridin-3-ylacetate (2a, 2.00 g, 12.1 mmol)
and N,N-dimethylformamide dimethyl acetal (3.61 g, 30.3 mmol)
in DMF (10 mL) was stirred at 110 °C for 2 h. After cooling at room
temperature, the mixture was concentrated in vacuo to give a red-
dish brown oil. To this reddish brown oil in EtOH (20 mL) were
added acetic acid (5.0 mL) and methylhydrazine (1.12 g,
24.2 mmol), and the mixture was stirred at room temperature for
16 h before the mixture was concentrated in vacuo. The residue
was purified by silica gel column chromatography (0–10% MeOH
in CHCl3) to give 3a (620 mg, 29%) as a beige solid. 1H NMR
(DMSO-d6) d 3.67 (s, 3H), 7.29–7.34 (m, 1H), 7.94–7.99 (m, 2H),
8.30 (dd, 1H, J = 4.7, 1.6 Hz), 8.83 (d, 1H, J = 1.6 Hz), 10.43 (br s,
1H); MS (ESI) m/z 176 [M+H]+.
5.1.2. 1-Methyl-4-(pyridin-2-yl)-1H-pyrazol-3-ol (3b)
Compound 3b was prepared from 2b in a manner similar to that
described for compound 3a, with a yield of 45% as a brown solid.
1H NMR (DMSO-d6) d 3.69 (s, 3H), 7.10–7.15 (m, 1H), 7.65–7.69
(m, 1H), 7.75–7.80 (m, 1H), 8.07 (s, 1H), 8.44–8.46 (m, 1H), 10.94
(br s, 1H); MS (ESI) m/z 176 [M+H]+.
5.1.3. 2-[4-({[1-Methyl-4-(pyridin-3-yl)-1H-pyrazol-3-
yl]oxy}methyl)phenyl]quinoline dihydrochloride (4a)
To a stirred mixture of 3a (263 mg, 1.50 mmol) and [4-(quino-
lin-2-yl)phenyl]methanol (388 mg, 1.65 mmol) in THF (20 mL)
were added 1,10-(azodicarbonyl)-dipiperidine (ADDP, 568 mg,
2.25 mmol) and tributylphosphine (455 mg, 2.25 mmol), and the
mixture was stirred at room temperature for 12 h before the mix-
ture was concentrated in vacuo. The residue was purified by flash
column chromatography (silica gel; 0–5% MeOH in CHCl3, then NH
silica gel; 20–50% EtOAc in hexane) to give a free form of the title
compound, which was diluted with EtOH (20 mL) and treated with
4 M HCl/EtOAc (1.5 mL). After the mixture was stirred at room
temperature for 30 min, the mixture was concentrated in vacuo
and recrystallized from EtOH/EtOAc to give 4a (446 mg, 64%) as a
cream-colored solid. 1H NMR (DMSO-d6) d 3.81 (s, 3H), 5.49 (s,
2H), 7.66–7.77 (m, 3H), 7.85–7.93 (m, 1H), 8.05 (dd, 1H, J = 8.2,
5.7 Hz), 8.11 (d, 1H, J = 7.9 Hz), 8.25 (d, 2H, J = 8.7 Hz), 8.33 (d,
2H, J = 8.4 Hz), 8.45 (s, 1H), 8.62–8.70 (m, 3H), 8.71–8.75 (m,
1H); MS (ESI) m/z 393 [M+H]+; Anal. Calcd for C25H20N4Oꢀ
2HClꢀ3.3H2O: C, 57.21; H, 5.49; N, 10.68; Cl, 13.51. Found: C,
57.09; H, 5.77; N, 10.31; Cl, 13.89.
5.1.6. 2-[4-({[1-Methyl-4-(tetrahydro-2H-pyran-4-yl)-1H-
pyrazol-3-yl]oxy}methyl)phenyl]quinoline hydrochloride (6b)
Free form of title compound was prepared from 5b in a manner
similar to that described for compound 6a, with a yield of 25% as a
colorless oil. To this colorless oil (139 mg, 0.35 mmol) in EtOAc
(9.7 mL) was added 4 M HCl/EtOAc (0.174 mL), and the mixture
was stirred at room temperature for 1 h. The precipitate was col-
lected by filtration to give 6b (76 mg, 50%) as a beige solid. 1H
NMR (DMSO-d6) d 1.46–1.66 (m, 4H), 2.53–2.63 (m, 1H), 3.33 (td,
2H, J = 11.5, 2.5 Hz), 3.58 (s, 3H), 3.79–3.88 (m, 2H), 5.19 (s, 2H),
7.27 (s, 1H), 7.68 (d, 2H, J = 8.4 Hz), 7.72 (d, 1H, J = 7.9 Hz), 7.86–
7.95 (m, 1H), 8.08–8.17 (m, 1H), 8.20–8.39 (m, 4H), 8.69 (d, 1H,
J = 8.4 Hz); MS (ESI) m/z 400 [M+H]+; Anal. Calcd for C25H25N3O2ꢀ
1.55HClꢀ1.2H2O: C, 62.87; H, 6.11; N, 8.80; Cl, 11.51. Found: C,
62.96; H, 6.23; N, 8.90; Cl, 11.45.
5.1.7. 4-(1-Methyl-3-{[4-(quinolin-2-yl)benzyl]oxy}-1H-pyrazol-
4-yl)cyclohexanone dihydrochloride (7)
5.1.4. 2-[4-({[1-Methyl-4-(pyridin-2-yl)-1H-pyrazol-3-
yl]oxy}methyl)phenyl]quinoline dihydrochloride (4b)
To a stirred solution of 6a (1.10 g, 2.40 mmol) in THF (11 mL)
and water (11 mL) was added 4-methylbenzenesulfonic acid
hydrate (pTsOHꢀH2O, 229 mg, 1.20 mmol), and the mixture was
stirred at room temperature for 3 days. The mixture was diluted
with NaHCO3 aqueous solution and brine, and extracted with
CHCl3 for 2 times. The combined organic layer was dried over
Compound 4b was prepared from 3b in a manner similar to that
described for compound 4a, with a yield of 72% as a cream-colored
solid. 1H NMR (DMSO-d6) d 3.86 (s, 3H), 5.53 (s, 2H), 7.67–7.78 (m,
4H), 7.89–7.95 (m, 1H), 8.14 (d, 1H, J = 7.9 Hz), 8.23 (d, 1H,
J = 8.3 Hz), 8.26–8.36 (m, 4H), 8.44–8.50 (m, 1H), 8.64–8.67
Please cite this article in press as: Hamaguchi, W.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.11.039

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W. Hamaguchi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
MgSO4, filtered and concentrated in vacuo. The residue was puri-
fied by silica gel column chromatography (0–3% MeOH in CHCl3)
to give a free form of the title compound (989 mg, quant) as a pale
yellow oil. To this pale yellow oil (117 mg, 0.28 mmol) in EtOAc
(3.5 mL) cooled with ice–water bath was added 4 M HCl/EtOAc
(0.21 mL, 0.84 mmol), and the mixture was stirred at the same
temperature for 15 min. The precipitate was collected by filtration
to give 7 (105 mg, 82%) as a white solid. 1H NMR (DMSO-d6) d
1.65–1.79 (m, 2H), 2.09–2.28 (m, 4H), 2.43–2.57 (m, 2H), 2.83–
2.93 (m, 1H), 3.32 (s, 3H), 5.15 (s, 2H), 7.45 (d, 2H, J = 8.3 Hz),
7.71 (t, 1H, J = 7.3 Hz), 7.90 (t, 1H, J = 7.3 Hz), 8.00 (s, 1H), 8.12 (t,
1H, J = 8.0 Hz), 8.20–8.29 (m, 4H), 8.69 (s, 1H); MS (ESI) m/z 412
[M+H]+; Anal. Calcd for C26H25N3O2ꢀ1.7HClꢀ1.75H2O: C, 61.95; H,
6.02; N, 8.34; Cl, 11.96. Found: C, 62.10; H, 6.35; N, 8.36; Cl, 12.08.
(m, 5H), 7.83–7.89 (m, 1H), 8.03 (s, 1H), 8.08 (d, 1H, J = 7.8 Hz),
8.16 (d, 1H, J = 8.4 Hz), 8.23 (d, 1H, J = 8.7 Hz), 8.31 (d, 2H,
J = 8.4 Hz), 8.61 (d, 1H, J = 8.7 Hz); MS (ESI) m/z 392 [M+H]+; Anal.
Calcd for C26H21N3Oꢀ0.6HClꢀ0.6H2O: C, 73.62; H, 5.42; N, 9.91; Cl,
5.02. Found: C, 73.81; H, 5.42; N, 10.03; Cl, 4.80.
5.1.11. 2-[4-({[1-Methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lan-2-yl)-1H-pyrazol-3-yl]oxy}methyl)phenyl]quinoline (12)
To a mixture of 10 (2.0 g, 4.53 mmol) in THF (40 mL) cooled
with MeOH–ice bath was dropwisely added isopropylmagnesium
chloride (2 M in THF solution, 2.60 mL, 5.20 mmol) with keeping
the temperature below ꢁ10 °C, and the mixture was stirred at tem-
perature between ꢁ18 and 10 °C for 45 min. To the resultant mix-
ture cooled with MeOH–ice bath was added 2-isopropoxy-4,4,5,
5-tetramethyl-1,3,2-dioxaborolane (1.39 mL, 6.80 mmol), and the
mixture was allowed to stir at room temperature for 90 min. To
the resultant mixture was cooled at ꢁ15 °C were added isopropyl-
magnesium chloride (2 M in THF solution, 1.00 mL, 2.00 mmol) and
2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (422 mg,
2.27 mmol), and the mixture was stirred at room temperature for
1 h. The reaction was diluted with EtOAc and washed with satu-
rated NH4Cl aqueous solution and brine, dried over MgSO4, filtered
and concentrated in vacuo. The residue was purified by silica gel
column chromatography (0–40% EtOAc in hexane) to give 12 as a
colorless solid. 1H NMR (CDCl3) d 1.33 (s, 12H), 3.73 (s, 3H), 5.40
(s, 2H), 7.45 (s, 1H), 7.49–7.55 (m, 1H), 7.65 (d, 2H, J = 8.4 Hz),
7.70–7.75 (m, 1H), 7.83 (d, 1H, J = 8.1 Hz), 7.89 (d, 1H, J = 8.6 Hz),
8.13–8.18 (m, 3H), 8.22 (d, 1H, J = 8.6 Hz); MS (ESI) m/z 442
[M+H]+.
5.1.8. 2-(4-{[(1-Methyl-1H-pyrazol-3-yl)oxy]methyl}phenyl)
quinoline (9)
To
a
solution of 1-methyl-1H-pyrazol-3-ol (8, 1.01 g,
10.3 mmol), 4-(quinolin-2-yl)phenyl)methanol (2.43 g, 10.3 mmol)
and ADDP (3.91 g, 15.5 mmol) in THF (122 mL) was added tributyl-
phosphine (3.14 g, 15.5 mmol), and the mixture was stirred at
room temperature for 6 h. The precipitate was filtered off and
the filtrate was concentrated in vacuo. The residue was suspended
in EtOAc, and the insoluble material was removed by filtration and
concentrated in vacuo. The residue was purified by silica gel col-
umn chromatography (0–50% EtOAc in hexane) to give 9 (2.20 g,
68%) as a pale yellow solid. 1H NMR (DMSO-d6) d 3.69 (s, 3H),
5.21 (s, 2H), 5.71 (d, 1H, J = 2.3 Hz), 7.49 (d, 1H, J = 2.3 Hz),
7.58–7.63 (m, 3H), 7.77–7.81 (m, 1H), 8.01 (dd, 1H, J = 8.2,
1.1 Hz), 8.08 (d, 1H, J = 8.2 Hz), 8.16 (d, 1H, J = 8.7 Hz), 8.28–8.31
(m, 2H), 8.47 (d, 1H, J = 8.4 Hz); MS (ESI) m/z 316 [M+H]+.
5.1.12. 2-[4-({[1-Methyl-4-(2-methylpyridin-4-yl)-1H-pyrazol-
3-yl]oxy}methyl)phenyl]quinoline dihydrochloride (13a)
5.1.9. 2-(4-{[(4-Iodo-1-methyl-1H-pyrazol-3-yl)oxy]methyl}
phenyl)quinoline (10)
Under argon gas atmosphere, to the mixture of 12 (200 mg,
0.45 mmol), 4-bromo-2-methylpyridine (156 mg, 0.91 mmol), and
Na2CO3 (144 mg, 1.36 mmol) in DMF (2.0 mL) and water (0.87 mL)
was added [1,10-bis(diphenylphosphino)ferrocene]dichloropalla-
dium(II) dichloromethane complex (PdCl2(dppf)ꢀCH2Cl2, 22 mg,
0.027 mmol), and the mixture was stirred at 100 °C for 1 h. After
cooling at room temperature, the mixture was concentrated in
vacuo, and the residue was purified by silica gel column chromatog-
raphy (0–5% MeOH in CHCl3) to give a free from of the title com-
pound, which was dissolved in MeOH and the mixture was
treated with 4 M HCl/EtOAc. The precipitate formed was collected
by filtration and washed with Et2O to give 13a (91 mg, 42%) as a col-
orless solid. 1H NMR (DMSO-d6) d 2.67 (s, 3H), 3.83 (s, 3H), 5.51 (s,
2H), 7.68 (t, 1H, J = 7.1 Hz), 7.73 (d, 2H, J = 8.4 Hz), 7.84–7.89 (m,
1H), 7.98 (dd, 1H, J = 6.5, 1.8 Hz), 8.03 (br d, 1H, J = 1.8 Hz), 8.08
(d, 1H, J = 7.8 Hz), 8.20 (d, 1H, J = 8.4 Hz), 8.24 (d, 1H, J = 8.7 Hz),
8.34 (d, 2H, J = 8.4 Hz), 8.58–8.65 (m, 3H); MS (ESI) m/z 407
[M+H]+; Anal. Calcd for C26H22N4Oꢀ2.3HClꢀ3H2O: C, 57.36; H, 5.61;
N, 10.29; Cl, 14.98. Found: C, 57.28; H, 5.67; N, 10.15; Cl, 15.22.
To a solution of 9 (2.20 g, 6.97 mmol) in MeCN (44 mL) were
added ceric ammonium nitrate (CAN, 2.29 g, 4.18 mmol) and
iodine (1.06 g, 4.18 mmol), and the mixture was stirred at room
temperature for 1 h. The mixture was concentrated in vacuo, and
to the residue were added CHCl3 and 5% NaHSO3 aqueous solution
during ice-water cooling. The organic layer was washed with brine,
dried over MgSO4, filtered and concentrated in vacuo. The residue
was purified by silica gel column chromatography (0–30% EtOAc in
hexane) to give 10 (893 mg, 29%) as a colorless solid. 1H NMR
(CDCl3) d 3.77 (s, 3H), 5.34 (s, 2H), 7.20 (s, 1H), 7.50–7.55 (m,
1H), 7.62 (d, 2H, J = 8.1 Hz), 7.70–7.75 (m, 1H), 7.83 (d, 1H,
J = 8.1 Hz), 7.89 (d, 1H, J = 8.6 Hz), 8.15–8.20 (m, 3H), 8.23 (d, 1H,
J = 8.6 Hz); MS (ESI) m/z 442 [M+H]+.
5.1.10. 2-(4-{[(1-Methyl-4-phenyl-1H-pyrazol-3-yl)oxy]methyl}
phenyl)quinoline hydrochloride (11)
To a mixture of 10 (300 mg, 0.68 mmol), phenylboronic acid
(124 mg, 1.02 mmol), 2-dicyclohexylphosphino-20,40,60-triisoprop-
ylbiphenyl (Xphos, 65 mg, 0.14 mmol), tris(dibenzylideneace-
tone)dipalladium(0) (Pd2(dba)3; 62 mg, 0.068 mmol), and K3PO4
(289 mg, 1.36 mmol) was added n-butanol (2 mL) under an argon
gas atmosphere, and the mixture was stirred at 80 °C for 90 min.
The mixture was concentrated in vacuo, and the residue was parti-
tioned between EtOAc and water. The organic layer was washed
with brine, dried over MgSO4, filtered and concentrated in vacuo.
The residue was purified by silica gel column chromatography
(0–30% EtOAc in hexane) to give a free form of the title compound,
which was dissolved in a mixture of EtOH and EtOAc. The solution
was treated with 4 M HCl/AcOEt and concentrated in vacuo. The
residue was washed with a mixture of EtOH and Et2O to give 11
(135 mg, 46%) as a yellow solid. 1H NMR (DMSO-d6) d 3.75 (s, 3H),
5.41 (s, 2H), 7.13–7.17 (m, 1H), 7.31–7.36 (m, 2H), 7.65–7.71
5.1.13. 2-[4-({[4-(2,6-Dimethylpyridin-4-yl)-1-methyl-1H-
pyrazol-3-yl]oxy}methyl)phenyl]quinoline dihydrochloride
(13b)
Compound 13b was prepared from 12 and 4-bromo-2,6-
dimethylpyridine hydrobromide in a manner similar to that
described for compound 13a, with a yield of 62% as a colorless
solid. 1H NMR (DMSO-d6) d 2.65 (s, 6H), 3.82 (s, 3H), 5.52 (s, 2H),
7.67 (t, 1H, J = 7.5 Hz), 7.72 (d, 2H, J = 8.4 Hz), 7.85–7.88 (m, 3H),
8.08 (d, 1H, J = 7.9 Hz), 8.19 (d, 1H, J = 8.4 Hz), 8.23 (d, 1H,
J = 8.7 Hz), 8.34 (d, 2H, J = 8.3 Hz), 8.57 (s, 1H), 8.61 (d, 1H,
J = 8.7 Hz); MS (ESI) m/z 421 [M+H]+; Anal. Calcd for
C
27H24N4Oꢀ2.2HClꢀ3.5H2O: C, 57.52; H, 5.94; N, 9.94; Cl, 13.83.
Found: C, 57.87; H, 5.83; N, 9.81; Cl, 13.64.
Please cite this article in press as: Hamaguchi, W.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.11.039

W. Hamaguchi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
11
5.1.14. 2-[4-({[4-(2-Methoxypyridin-4-yl)-1-methyl-1H-pyrazol-
3-yl]oxy}methyl)phenyl]quinoline dihydrochloride (13c)
Compound 13c was prepared from 12 and 4-bromo-2-meth-
oxylpyridine in a manner similar to that described for compound
13a, with a yield of 51% as a colorless solid. 1H NMR (DMSO-d6)
d 3.79 (s, 3H), 3.95 (s, 3H), 5.47 (s, 2H), 7.29 (s, 1H), 7.41 (dd, 1H,
J = 5.9, 1.3 Hz), 7.71–7.76 (m, 3H), 7.89–7.95 (m, 1H), 8.13 (d, 1H,
J = 8.2 Hz), 8.17 (d, 1H, J = 5.9 Hz), 8.28 (d, 2H, J = 8.7 Hz), 8.33 (d,
2H, J = 8.4 Hz), 8.45 (s, 1H), 8.72 (d, 1H, J = 8.6 Hz); MS (ESI) m/z
423 [M+H]+; Anal. Calcd for C26H22N4O2ꢀ2.2HClꢀ3.5H2O: C, 56.10;
H, 5.58; N, 10.06; Cl, 12.74. Found: C, 56.33; H, 5.68; N, 9.88; Cl,
12.52.
501 mg, 3.30 mmol) in 1,2-dimethoxyethane (20 mL) were added
Pd(PPh3)4 (173 mg, 0.15 mmol) and 1 M Na2CO3 aqueous solution
(7.5 mL), and the mixture was stirred at 90 °C for 19 h under argon
gas atmosphere. After cooling at room temperature, the mixture
was partitioned between EtOAc and water. The organic layer was
dried over Na2SO4, filtered and concentrated in vacuo. The residue
was purified by silica gel column chromatography (0–5% MeOH in
CHCl3) to give 23 as a pale yellow oil. 1H NMR (DMSO-d6) d 2.46 (s,
3H), 4.60 (d, 2H, J = 5.7 Hz), 5.27 (t, 1H, J = 5.7 Hz), 7.45 (d, 2H,
J = 8.3 Hz), 7.56–7.65 (m, 3H), 7.67–7.73 (m, 1H), 7.93 (d, 1H,
J = 8.1 Hz), 7.98 (d, 1H, J = 8.2 Hz), 8.25 (s, 1H); MS (ESI) m/z 250
[M+H]+.
5.1.15. 1-Methyl-4-(1-methyl-3-{[4-(quinolin-2-yl)benzyl]oxy}-
1H-pyrazol-4-yl)pyridin-2(1H)-one dihydrochloride (13d)
Compound 13d was prepared from 12 and 4-bromo-1-methyl-
pyridin-2(1H)-one in a manner similar to that described for com-
pound 13a, with a yield of 68% as a colorless solid. 1H NMR
(DMSO-d6) d 3.39 (s, 3H), 3.75 (s, 3H), 5.42 (s, 2H), 6.54 (dd, 1H,
J = 7.1, 2.0 Hz), 6.74 (d, 1H, J = 1.9 Hz), 7.65 (d, 1H, J = 7.1 Hz),
7.68–7.73 (m, 3H), 7.87–7.93 (m, 1H), 8.11 (d, 1H, J = 8.0 Hz),
8.19–8.23 (m, 2H), 8.27 (d, 1H, J = 8.7 Hz), 8.31 (d, 2H, J = 8.4 Hz),
8.68 (br d, 1H, J = 8.7 Hz); MS (ESI) m/z 423 [M+H]+; Anal. Calcd
for C26H22N4O2ꢀ2HClꢀ4H2O: C, 55.03; H, 5.68; N, 9.87; Cl, 12.50.
Found: C, 55.15; H, 5.84; N, 9.58; Cl, 12.46.
5.1.20. 3-(Benzyloxy)-1-methyl-1H-pyrazole (14)
To a suspension of 8 (8.09 g, 82.5 mmol) and K2CO3 (13.7 g,
99.0 mmol) in DMF (100 mL) cooled with ice–water bath was
added benzylbromide (16.9 g, 98.6 mmol), and the mixture was
allowed to stir at room temperature for 1.5 h. The mixture was stir-
red at 50 °C for another 4 h. The resultant mixture was partitioned
between EtOAc and water, and the organic layer was washed with
NaCl aqueous solution, dried over MgSO4, filtered and concen-
trated in vacuo. The residue was purified by silica gel column chro-
matography (10–30% EtOAc in hexane) to give 14 (13.1 g, 84%) as a
colorless oil. 1H NMR (CDCl3) d 3.74 (s, 3H), 5.18 (s, 2H), 5.64 (d, 1H,
J = 2.3 Hz), 7.12 (d, 1H, J = 2.3 Hz), 7.28–7.33 (m, 1H), 7.34–7.39 (m,
2H), 7.43–7.46 (m, 2H); MS (ESI) m/z 189 [M+H]+.
5.1.16. 1-Methyl-5-(1-methyl-3-{[4-(quinolin-2-yl)benzyl]oxy}-
1H-pyrazol-4-yl)pyridin-2(1H)-one dihydrochloride (13e)
Compound 13e was prepared from 12 and 5-bromo-1-methyl-
pyridin-2(1H)-one in a manner similar to that described for com-
pound 13a, with a yield of 39% as a pale yellow solid. 1H NMR
(DMSO-d6) d 3.46 (s, 3H), 3.72 (s, 3H), 5.41 (s, 2H), 6.47 (d, 1H,
J = 9.4 Hz), 7.67–7.78 (m, 4H), 7.87 (s, 1H), 7.92–7.97 (m, 2H),
8.16 (d, 1H, J = 7.9 Hz), 8.28–8.34 (m, 4H), 8.77 (d, 1H, J = 8.7 Hz);
MS (ESI) m/z 423 [M+H]+; Anal. Calcd for C26H22N4O2ꢀ2.2HClꢀ2.7H2O:
C, 56.64; H, 5.41; N, 10.16; Cl, 14.15. Found: C, 56.97; H, 5.78; N,
9.93; Cl, 14.25.
5.1.21. 3-(Benzyloxy)-4-iodo-1-methyl-1H-pyrazole (15)
Compound 15 was prepared from 14 in a manner similar to that
described for compound 10 with a yield of 61% as a brown oil. 1H
NMR (CDCl3) d 3.77 (s, 3H), 5.25 (s, 2H), 7.19 (s, 1H), 7.29–7.33 (m,
1H), 7.35–7.40 (m, 2H), 7.45–7.48 (m, 2H); MS (ESI) m/z 315
[M+H]+.
5.1.22. 3-(Benzyloxy)-1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)-1H-pyrazole (16)
Compound 16 was prepared from 15 in a manner similar to that
described for compound 12 with a yield of 82% as a gum. 1H NMR
(CDCl3) d 1.31 (s, 12H), 3.72 (s, 3H), 5.32 (s, 2H), 7.24–7.30 (m, 1H),
7.32–7.37 (m, 2H), 7.43 (s, 1H), 7.49 (d, 2H, J = 7.5 Hz); MS (ESI) m/z
315 [M+H]+.
5.1.17. 1-Methyl-5-(1-methyl-3-{[4-(quinolin-2-yl)benzyl]oxy}-
1H-pyrazol-4-yl)pyrimidin-2(1H)-one dihydrochloride (13f)
Compound 13f was prepared from 12 and 5-bromo-1-methyl-
pyrimidin-2(1H)-one in a manner similar to that described for
compound 13a, with a yield of 37% as a pale yellow solid. 1H
NMR (DMSO-d6) d 3.55 (s, 3H), 3.75 (s, 3H), 5.40 (s, 2H), 7.64 (m,
3H), 7.83–7.88 (m, 1H), 7.96 (s, 1H), 8.07 (d, 1H, J = 8.1 Hz), 8.16
(d, 1H, J = 8.6 Hz), 8.22 (d, 1H, J = 8.7 Hz), 8.30 (d, 2H, J = 8.4 Hz),
8.60 (d, 1H, J = 8.7 Hz), 8.67 (d, 1H, J = 3.2 Hz), 8.86 (d, 1H,
J = 3.2 Hz); MS (ESI) m/z 424 [M+H]+; Anal. Calcd for C25H21N5O2ꢀ
2HClꢀ3.3H2O: C, 54.02; H, 5.37; N, 12.60; Cl, 12.76. Found: C,
54.03; H, 5.66; N, 12.46; Cl, 12.44.
5.1.23. 5-[3-(Benzyloxy)-1-methyl-1H-pyrazol-4-yl]-1-
methylpyridin-2(1H)-one (17)
Under argon gas atmosphere, to the mixture of 16 (3.5 g,
11.1 mmol),
5-bromo-1-methylpyridin-2(1H)-one
(2.53 g,
13.5 mmol), and Na2CO3 (3.54 g, 33.4 mmol) in DMF (33 mL) and
water (15 mL) was added PdCl2(dppf)ꢀCH2Cl2 (543 mg, 0.67 mmol),
and the mixture was stirred at 100 °C for 2 h. After cooling at room
temperature, the mixture was diluted with EtOAc and water, and
filtered through Celite pad. The organic layer of the filtrate was
washed with NaCl aqueous solution, dried over MgSO4, filtered
and concentrated in vacuo. The residue was purified by silica gel
column chromatography (0–5% MeOH in CHCl3) to give 17
(1.23 g, 37%) as a pale green solid. 1H NMR (CDCl3) d 3.51 (s, 3H),
3.78 (s, 3H), 5.32 (s, 2H), 6.58 (d, 1H, J = 9.4 Hz), 7.30 (s, 1H),
7.32–7.50 (m, 6H), 7.70 (d, 1H, J = 2.5 Hz); MS (ESI) m/z 296
[M+H]+.
5.1.18. 2-Methyl-6-(1-methyl-3-{[4-(quinolin-2-yl)benzyl]oxy}-
1H-pyrazol-4-yl)pyridazin-3(2H)-one hydrochloride (13g)
Compound 13g was prepared from 12 and 6-chloro-2-meth-
ylpyridazin-3(2H)-one in a manner similar to that described for
compound 13a, with a yield of 49% as a pale yellow solid. 1H
NMR (DMSO-d6) d 3.67 (s, 3H), 3.76 (s, 3H), 5.43 (s, 2H), 6.98 (d,
1H, J = 9.6 Hz), 7.73–7.81 (m, 4H), 7.95–8.01 (m, 1H), 8.04 (s, 1H),
8.19 (d, 1H, J = 8.0 Hz), 8.30–8.34 (m, 3H), 8.41 (d, 1H, J = 8.5 Hz),
8.85 (d, 1H, J = 8.5 Hz); MS (ESI) m/z 424 [M+H]+; Anal. Calcd for
5.1.24. 5-(3-Hydroxy-1-methyl-1H-pyrazol-4-yl)-1-methylpyridin-
2(1H)-one (18)
C
25H21N5O2ꢀ1.5HClꢀ1.7H2O: C, 59.02; H, 5.13; N, 13.76; Cl, 10.45.
Found: C, 59.24; H, 5.53; N, 13.79; Cl, 10.33.
The mixture of 17 (1.2 g, 4.06 mmol) and 10% Pd–C (150 mg) in
EtOH (20 mL) was stirred at room temperature for 3 days under
3 atm of H2 atmosphere. The mixture was filtered through Hyflo
pad and washed with MeOH/EtOH/CHCl3, and the filtrate was
concentrated in vacuo to give 18 (678 mg, 81%) as a colorless solid.
5.1.19. [4-(3-Methylquinolin-2-yl)phenyl]methanol (23)
To a suspension of 2-chloro-3-methylquinoline (21, 533 mg,
3.00 mmol) and [4-(hydroxymethyl)phenyl]boronic acid (22,
Please cite this article in press as: Hamaguchi, W.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.11.039

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W. Hamaguchi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
1H NMR (DMSO-d6) d 3.43 (s, 3H), 3.62 (s, 3H), 6.40 (d, 1H,
J = 9.4 Hz), 7.64 (dd, 1H, J = 9.4, 2.6 Hz), 7.69 (s, 1H), 7.83 (d, 1H,
J = 2.6 Hz), 10.26 (br s, 1H); MS (ESI) m/z 206 [M+H]+.
was poured onto ice and diluted with CHCl3. To the mixture was
added silica gel and filtered. The filtrate was extracted with CHCl3
for 2 times. The combined organic layer was dried over Na2SO4, fil-
tered and concentrated in vacuo. The residue was purified by silica
gel column chromatography (0–20% EtOAc in hexane) to give 26a
(595 mg, 14%) as beige solid. 1H NMR (CDCl3) d 2.59 (s, 3H), 7.82
(dd, 1H, J = 8.7, 1.7 Hz), 8.03 (s, 1H), 8.08 (d, 1H, J = 8.5 Hz), 8.16
(d, 1H, J = 1.7 Hz); MS (ESI) m/z 203 [M+H]+.
5.1.25. 1-Methyl-5-(1-methyl-3-{[4-(3-methylquinolin-2-
yl)benzyl]oxy}-1H-pyrazol-4-yl)pyridin-2(1H)-one
dihydrochloride (24)
To a suspension of 18 (200 mg, 0.98 mmol) and 23 (292 mg,
1.17 mmol) in toluene (15 mL) was added (tributylphosphorany-
lidene)acetonitrile (CMBP, 353 mg, 1.46 mmol) and stirred at
100 °C for 10 h before the mixture was concentrated in vacuo.
The residue was purified by flash column chromatography (silica
gel; 0–5% MeOH in CHCl3, then NH silica gel; 50–80% EtOAc in hex-
ane) to give a free form of the title compound, which was dissolved
in a mixture of EtOAc and EtOH, and the mixture was treated with
4 M HCl/EtOAc (0.97 mL). The precipitate was collected by filtra-
tion and washed with Et2O to give 24 (182 mg, 40%) as a pale blue
solid. 1H NMR (DMSO-d6) d 2.52 (s, 3H), 3.46 (s, 3H), 3.73 (s, 3H),
5.44 (s, 2H), 6.47 (d, 1H, J = 9.4 Hz), 7.71 (dd, 1H, J = 9.4, 2.6 Hz),
7.75 (d, 2H, J = 8.3 Hz), 7.84 (d, 2H, J = 8.3 Hz), 7.88–7.94 (m, 3H),
8.04 (t, 1H, J = 7.7 Hz), 8.25 (d, 1H, J = 8.1 Hz), 8.35 (d, 1H,
J = 8.6 Hz), 9.00 (s, 1H); MS (ESI) m/z 437 [M+H]+; Anal. Calcd for
C27H24N4O2ꢀ1.9HClꢀ3H2O: C, 57.87; H, 5.74; N, 9.85; Cl, 12.47.
Found: C, 58.08; H, 5.77; N, 9.88; Cl, 12.19.
5.1.29. 2-Chloro-8-fluoro-3-methylquinoline (26b)
Compound 26b was prepared from 25b in a manner similar to
that described for compound 26a with a yield of 1.5%. 1H NMR
(CDCl3) d 2.56 (d, 3H, J = 1.0 Hz), 7.34–7.39 (m, 1H), 7.44–7.49
(m, 1H), 7.54 (d, 1H, J = 8.1 Hz), 8.01 (s, 1H); MS (ESI) m/z 196
[M+H]+.
5.1.30. [4-(5-Fluoro-3-methylquinolin-2-yl)phenyl]methanol
(27c)
Under argon atmosphere, a mixture of 2-chloro-5-fluoro-3-
methylquinoline (26c, 443 mg, 2.27 mmol) and 22 (379 mg,
2.49 mmol), and Pd(PPh3)4 (130 mg, 0.11 mmol) in 1 M Na2CO3
aqueous solution (5.7 mL) and DME (15 mL) was stirred at 90 °C
for 12 h. The mixture was filtered through Celite pad and concen-
trated in vacuo. The residue was purified by silica gel column chro-
matography (0–10% MeOH in CHCl3) to give 27c (432 mg, 71%) as a
white solid. 1H NMR (CDCl3) d 2.50 (s, 3H), 4.61 (d, 2H, J = 5.7 Hz),
5.29 (t, 1H, J = 5.7 Hz), 7.38–7.49 (m, 3H), 7.59–7.63 (m, 2H),
7.66–7.73 (m, 1H), 7.85 (d, 1H, J = 8.5 Hz), 8.38 (s, 1H); MS (ESI)
m/z 268 [M+H]+.
5.1.26. 5-(3-Hydroxy-1-methyl-1H-pyrazol-4-yl)-1-methylpiper-
idin-2-one (19)
The suspension of 18 (285 mg, 1.39 mmol) and PtO2 (50 mg,
0.22 mmol) in AcOH (10 mL) was stirred at room temperature for
15 h under 3 atm of H2 atmosphere. The mixture was filtered
through Hyflo pad and concentrated in vacuo. The residue was
purified by silica gel column chromatography (0–10% MeOH in
CHCl3) to give 19 (193 mg, 66%) as a colorless solid. 1H NMR
(DMSO-d6) d 1.72–1.92 (m, 2H), 2.19–2.35 (m, 2H), 2.80 (s, 3H),
2.82–2.90 (m, 1H), 3.15–3.22 (m, 1H), 3.33–3.39 (m, 1H), 3.56 (s,
3H), 7.23 (s, 1H), 9.56 (s, 1H); MS (ESI) m/z 210 [M+H]+.
5.1.31. 2-[4-(Hydroxymethyl)phenyl]-3-methylquinoline-6-
carbonitrile (27a)
Compound 27a was prepared from 26a in a manner similar to
that described for compound 27c, with a yield of 76% as a beige
solid. 1H NMR (CDCl3) d 1.93 (t, 1H, J = 5.9 Hz), 2.52 (d, 3H,
J = 0.8 Hz), 4.80 (d, 2H, J = 5.9 Hz), 7.49–7.53 (m, 2H), 7.59–7.63
(m, 2H), 7.80 (dd, 1H, J = 8.8 Hz), 8.07 (s, 1H), 8.18–8.21 (m, 2H);
MS (ESI) m/z 275 [M+H]+.
5.1.27. 1-Methyl-5-(1-methyl-3-{[4-(quinolin-2-yl)benzyl]oxy}-
1H-pyrazol-4-yl)piperidin-2-one dihydrochloride (20)
A mixture of 19 (150 mg, 0.72 mmol), 2-[4-(chloromethyl)
phenyl]quinoline hydrochloride (208 mg, 0.72 mmol), and K2CO3
(248 mg, 1.79 mmol) in DMF (4.2 mL) was stirred at 60 °C for
10 h. After cooling at room temperature, the mixture was parti-
tioned between EtOAc and water. The organic extracts were
washed with NaCl aqueous solution and dried over MgSO4, filtered
and concentrated in vacuo. The residue was purified by silica gel
column chromatography (0–5% MeOH in CHCl3) to give a free form
of the title compound, which was dissolved in a mixture of EtOH
and EtOAc. To the solution was added 4 M HCl/EtOAc. The resulting
precipitate was collected by filtration to give 20 (266 mg, 74%) as a
pale yellow solid. 1H NMR (DMSO-d6) d 1.80–2.00 (m, 2H), 2.22–
2.40 (m, 2H), 2.82 (s, 3H), 2.95–3.03 (m, 1H), 3.24–3.30 (m, 1H),
3.39–3.44 (m, 1H), 3.66 (s, 3H), 5.33 (s, 2H), 7.43 (s, 1H), 7.71 (d,
2H, J = 8.4 Hz), 7.81–7.86 (m, 1H), 8.01–8.06 (m, 1H), 8.25 (d, 1H,
J = 7.8 Hz), 8.32 (d, 2H, J = 8.4 Hz), 8.38 (d, 1H, J = 8.7 Hz), 8.56 (d,
1H, J = 8.5 Hz), 8.97 (d, 1H, J = 8.7 Hz); MS (ESI) m/z 427 [M+H]+;
Anal. Calcd for C26H26N4O2ꢀ2.3HClꢀ1.9H2O: C, 57.34; H, 5.94; N,
10.29; Cl, 14.97. Found: C, 57.36; H, 6.04; N, 10.33; Cl, 15.18.
5.1.32. [4-(8-Fluoro-3-methylquinolin-2-yl)phenyl]methanol
(27b)
Compound 27b was prepared from 26b in a manner similar to
that described for compound 27c with a quantitative yield as a yel-
low amorphous solid. 1H NMR (CDCl3) d 2.04 (br s, 1H), 2.49 (s, 3H),
4.75 (s, 2H), 7.31–7.37 (m, 1H), 7.41–7.48 (m, 3H), 7.56 (d, 1H,
J = 8.2 Hz), 7.61 (d, 2H, J = 8.1 Hz), 8.03 (s, 1H); MS (ESI) m/z 268
[M+H]+.
5.1.33. [4-(6-Fluoro-3-methylquinolin-2-yl)phenyl]methanol
(27d)
Compound 27d was prepared from 2-chloro-6-fluoro-3-meth-
ylquinoline (26d) in a manner similar to that described for com-
pound 27c, with a yield of 93% as a beige solid. 1H NMR (CDCl3)
d 2.13 (t, 1H, J = 6.0 Hz), 2.46 (s, 3H), 4.77 (d, 2H, J = 6.0 Hz),
7.37–7.49 (m, 4H), 7.54–7.58 (m, 2H), 7.97 (s, 1H), 8.12 (dd, 1H,
J = 9.2, 5.4 Hz); MS (ESI) m/z 268 [M+H]+.
5.1.34. [4-(7-Fluoro-3-methylquinolin-2-yl)phenyl]methanol
(27e)
5.1.28. 2-Chloro-3-methylquinoline-6-carbonitrile (26a)
Compound 27e was prepared from 2-chloro-7-fluoro-3-methyl-
quinoline (26e) in a manner similar to that described for com-
pound 27c, with a yield of 87% as a beige solid. 1H NMR (CDCl3)
d 1.95 (br s, 1H), 2.46 (s, 3H), 4.78 (s, 2H), 7.28–7.35 (m, 1H),
7.48 (d, 2H, J = 8.1 Hz), 7.58 (d, 2H, J = 8.1 Hz), 7.64–7.70 (m, 1H),
7.73–7.79 (m, 1H), 8.02 (s, 1H); MS (ESI) m/z 268 [M+H]+.
Under argon atmosphere, to
a mixture of N-(4-cyano-
phenyl)propanamide (25a, 3.61 g, 20.7 mmol) and cetyltrimethyl-
ammonium bromide (CTAB, 754 mg, 2.07 mmol) cooled with
ice–water bath were added POCl3 (9.5 mL, 104 mmol) and DMF
(3.2 mL, 41.5 mmol), and the mixture was stirred at 120 °C for
12 h. The mixture was concentrated in vacuo, and the residue
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W. Hamaguchi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
13
5.1.35. [4-(3-Ethylquinolin-2-yl)phenyl]methanol (27f)
Compound 27f was prepared from 2-chloro-3-ethylquinoline
(26f) in a manner similar to that described for compound 27c, with
a yield of 56% as a beige solid. 1H NMR (CDCl3) d 1.20 (t, 3H,
J = 7.5 Hz), 2.12 (t, 1H, J = 6.1 Hz), 2.76–2.84 (m, 2H), 4.77 (d, 2H,
J = 6.1 Hz), 7.46 (d, 2H, J = 8.3 Hz), 7.51–7.56 (m, 3H), 7.63–7.70
(m, 1H), 7.81 (dd, 1H, J = 8.1, 1.2 Hz), 8.06 (s, 1H), 8.13 (d, 1H,
J = 8.4 Hz); MS (ESI) m/z 264 [M+H]+.
as stirred at room temperature for 2 h. The reaction was diluted
with EtOAc, and the precipitate was collected by filtration to give
2-[4-(chloromethyl)phenyl]-3,6-dimethylquinoline hydrochloride
(245 mg, 81%) as a beige solid. To this beige solid (244 mg,
0.77 mmol) and 18 (173 mg, 0.84 mmol) in DMF was added
K2CO3 (318 mg, 2.30 mmol), and the mixture was stirred at 60 °C
for 12 h. After cooling at room temperature, the mixture was
diluted with water and stirred for a while. The precipitate was col-
lected by filtration and purified by silica gel column chromatogra-
phy (0–10% MeOH in CHCl3) to give a white amorphous solid,
which was dissolved in EtOH (5 mL). To the solution was added
4 M HCl/EtOAc (2 mL), and the precipitate was collected by filtra-
tion to give 28g (360 mg, 90%) as a beige solid. 1H NMR (DMSO-
d6) d 2.51 (s, 3H), 2.59 (s, 3H), 3.45 (s, 3H), 3.72 (s, 3H), 5.44 (s,
2H), 6.46 (d, 1H, J = 9.4 Hz), 7.70 (dd, 1H, J = 9.4, 2.6 Hz), 7.75 (d,
2H, J = 8.2 Hz), 7.82 (d, 2H, J = 8.2 Hz), 7.87 (s, 1H), 7.88–7.93 (m,
2H), 8.01 (s, 1H), 8.21 (d, 1H, J = 8.8 Hz), 8.89 (1H, br s); MS (ESI)
m/z 451 [M+H]+.
5.1.36. [4-(3,6-Dimethylquinolin-2-yl)phenyl]methanol (27g)
Compound 27g was prepared from 26g in a manner similar to
that described for compound 27c, with a yield of 87% as a white
solid. 1H NMR (CDCl3) d 1.94 (t, 1H, J = 5.7 Hz), 2.45 (s, 3H), 2.54
(s, 3H), 4.77 (d, 2H, J = 5.7 Hz), 7.45–7.51 (m, 3H), 7.54 (s, 1H),
7.56–7.60 (m, 2H), 7.92 (s, 1H), 8.01 (d, 1H, J = 8.6 Hz); MS (ESI)
m/z 264 [M+H]+.
5.1.37. [4-(6-Methoxy-3-methylquinolin-2-yl)phenyl]methanol
(27h)
Compound 27h was prepared from 2-chloro-6-methoxy-3-
methylquinoline (26h) in a manner similar to that described for
compound 27c, with a yield of 33% as a beige solid. 1H NMR (CDCl3)
d 2.04 (t, 1H, J = 5.7 Hz), 2.45 (s, 3H), 3.94 (s, 3H), 4.76 (d, 2H,
J = 5.7 Hz), 7.04 (d, 1H, J = 2.8 Hz), 7.32 (dd, 1H, J = 9.2, 2.8 Hz),
7.46 (d, 2H, J = 8.0 Hz), 7.57 (d, 2H, J = 8.0 Hz), 7.92 (s, 1H), 8.02
(d, 1H, J = 9.2 Hz); MS (ESI) m/z 280 [M+H]+.
5.1.41. 5-(3-{[4-(8-Fluoro-3-methylquinolin-2-yl)benzyl]oxy}-
1-methyl-1H-pyrazol-4-yl)-1-methylpyridin-2(1H)-one
trihydrochloride (28b)
Compound 28b was prepared from 27b in a manner similar to
that described for compound 28g, with a yield of 26% as a beige
solid. 1H NMR (DMSO-d6) d 2.48 (s, 3H), 3.46 (s, 3H), 3.73 (s, 3H),
5.39 (s, 2H), 6.48 (d, 1H, J = 9.4 Hz), 7.50–7.80 (m, 8H), 7.87 (s,
1H), 7.93 (d, 1H, J = 2.5 Hz), 8.36 (br s, 1H); ESI+: 455; Anal. Calcd
for C27H23FN4O2ꢀ2.9HClꢀ3.4H2O: C, 52.18; H, 5.30; N, 9.01; Cl,
16.54; F, 3.06. Found: C, 52.19; H, 5.06; N, 9.04; Cl, 16.76; F, 3.31.
5.1.38. 2-[4-(Hydroxymethyl)phenyl]quinoline-3-carbaldehyde
(27i)
Compound 27i was prepared from 2-chloroquinoline-3-carbox-
aldehyde (26i) in a manner similar to that described for compound
27c, with a yield of 85% as a yellow solid. 1H NMR (DMSO-d6) d 4.64
(d, 2H, J = 5.8 Hz), 5.34 (t, 1H, J = 5.8 Hz), 7.53 (d, 2H, J = 8.3 Hz),
7.67–7.75 (m, 3H), 7.94–7.99 (m, 1H), 8.13 (d, 1H, J = 8.4 Hz),
8.28 (d, 1H, J = 8.1 Hz), 8.97 (s, 1H), 10.09 (s, 1H); MS (ESI) m/z
264 [M+H]+.
5.1.42. 5-(3-{[4-(5-Fluoro-3-methylquinolin-2-yl)benzyl]oxy}-
1-methyl-1H-pyrazol-4-yl)-1-methylpyridin-2(1H)-one
dihydrochloride (28c)
Compound 28c was prepared from 27c in a manner similar to
that described for compound 28g, with a yield of 64% as a beige
solid. 1H NMR (DMSO-d6) d 2.52 (d, 3H), 3.45 (s, 3H), 3.73 (s, 3H),
5.40 (s, 2H), 6.46 (d, 1H, J = 9.4 Hz), 7.49 (dd, 1H, J = 9.6, 7.8 Hz),
7.65 (d, 2H, J = 8.3 Hz), 7.68–7.81 (m, 4H), 7.86 (s, 1H), 7.89–7.94
(m, 2H), 8.55 (br s, 1H); MS (ESI) m/z 455 [M+H]+; Anal. Calcd for
5.1.39. 3-Methyl-2-[4-({[1-methyl-4-(1-methyl-6-oxo-1,6-
dihydropyridin-3-yl)-1H-pyrazol-3-
yl]oxy}methyl)phenyl]quinoline-6-carbonitrile (28a)
To a stirred mixture of 27a (591 mg, 2.15 mmol) in CH2Cl2
(12 mL) was added SOCl2 (0.47 mL, 6.44 mmol), and the mixture
was stirred at room temperature for 2 h. The reaction was diluted
with EtOAc, and the precipitate was collected by filtration to give
C
27H23FN4O2ꢀ2HClꢀ2H2O: C, 57.55; H, 5.19; N, 9.94; Cl, 12.58; F,
3.37. Found: C, 57.59; H, 5.32; N, 9.94; Cl, 12.61; F, 3.22.
5.1.43. 5-(3-{[4-(6-Fluoro-3-methylquinolin-2-yl)benzyl]oxy}-
1-methyl-1H-pyrazol-4-yl)-1-methylpyridin-2(1H)-one
dihydrochloride (28d)
a
2-[4-(chloromethyl)phenyl]-3-methylquinoline-6-carbonitrile
hydrochloride (547 mg, 77%) as a beige solid. A mixture of this beige
solid (300 mg, 0.91 mmol), 18 (206 mg, 1.00 mmol) and K2CO3
(378 mg, 2.74 mmol) in DMF (6 mL) was stirred at 60 °C for 12 h.
After cooling at room temperature, the mixture was diluted with
water and stirred for a while. The precipitate was collected by filtra-
tion and purified by silica gel column chromatography (0–10%
MeOH in CHCl3) to give a yellow solid, which was washed with
EtOAc, then EtOH to give 28a (216 mg, 51%) as a yellow solid. 1H
NMR (DMSO-d6) d 2.50 (s, 3H), 3.44 (s, 3H), 3.72 (s, 3H), 5.38 (s,
2H), 6.44 (d, 1H, J = 9.4 Hz), 7.62 (d, 2H, J = 8.2 Hz), 7.66–7.72 (m,
3H), 7.85 (s, 1H), 7.89 (d, 1H, J = 2.5 Hz), 7.99 (dd, 1H, J = 8.7,
1.8 Hz), 8.13 (d, 1H, J = 8.7 Hz), 8.39 (s, 1H), 8.60 (d, 1H,
J = 1.7 Hz); MS (ESI) m/z 462 [M+H]+; Anal. Calcd for C28H23N5O2
ꢀ0.2H2O: C, 72.30; H, 5.07; N, 15.06. Found: C, 72.36; H, 4.92; N,
15.00.
Compound 28d was prepared from 27d in a manner similar to
that described for compound 28g, with a yield of 75% as a beige
solid. 1H NMR (DMSO-d6) d 2.53 (s, 3H), 3.50 (s, 3H), 3.74 (s, 3H),
5.45 (s, 2H), 6.54 (d, 1H, J = 9.4 Hz), 7.73–7.84 (m, 5H), 7.89 (s,
1H), 7.91–8.00 (m, 2H), 8.06 (dd, 1H, J = 9.0, 2.8 Hz), 8.35 (dd, 1H,
J = 9.4, 5.0 Hz), 8.89 (s, 1H); MS (ESI) m/z 455 [M+H]+; Anal. Calcd
for C27H23FN4O2ꢀ2HClꢀ4H2O: C, 54.10; H, 5.55; N, 9.35; Cl, 11.83;
F, 3.17. Found: C, 54.28; H, 5.55; N, 9.30; Cl, 11.78; F, 3.16.
5.1.44. 5-(3-{[4-(7-Fluoro-3-methylquinolin-2-yl)benzyl]oxy}-
1-methyl-1H-pyrazol-4-yl)-1-methylpyridin-2(1H)-one
dihydrochloride (28e)
Compound 28e was prepared from 27e in a manner similar to
that described for compound 28g, with a yield of 42% as a beige
solid. 1H NMR (DMSO-d6) d 2.47 (s, 3H), 3.44 (s, 3H), 3.72 (s, 3H),
5.40 (s, 2H), 6.45 (d, 1H, J = 9.3 Hz), 7.59–7.74 (m, 6H), 7.81 (dd,
1H, J = 9.3, 2.4 Hz), 7.86 (s, 1H), 7.91 (d, 1H, J = 2.4 Hz), 8.14 (dd,
1H, J = 9.1, 6.2 Hz), 8.54 (br s, 1H); MS (ESI) m/z 455 [M+H]+; Anal.
Calcd for C27H23FN4O2ꢀ1.9HClꢀ3.8H2O: C, 54.76; H, 5.53; N, 9.46; Cl,
11.37; F, 3.21. Found: C, 54.98; H, 5.57; N, 9.29; Cl, 11.08; F, 3.16.
5.1.40. 5-(3-{[4-(3,6-Dimethylquinolin-2-yl)benzyl]oxy}-1-
methyl-1H-pyrazol-4-yl)-1-methylpyridin-2(1H)-one
dihydrochloride (28g)
To a stirred mixture of 27g (252 mg, 0.96 mmol) in CH2Cl2
(5 mL) was added SOCl2 (0.21 mL, 2.88 mmol), and the mixture
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W. Hamaguchi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5.1.45. 5-(3-{[4-(3-Ethylquinolin-2-yl)benzyl]oxy}-1-methyl-
1H-pyrazol-4-yl)-1-methylpyridin-2(1H)-one dihydrochloride
(28f)
brown syrup, which was dissolved in MeCN (250 mL). To this
solution were added CAN (16.0 g, 29.3 mmol) and iodine (7.43 g,
29.3 mmol), and the mixture was stirred at room temperature for
15 min. To the mixture was added iodine (7.43 g, 29.3 mmol),
and the mixture was stirred at room temperature for further
30 min. The reaction was concentrated in vacuo, and the residue
was diluted with CHCl3 and washed with saturated sodium thio-
sulfate aqueous solution. The organic layer was dried over Na2SO4,
filtered and concentrated in vacuo, and the residue was purified by
silica gel column chromatography (20–50% EtOAc in hexane) to
give 32 (10.6 mg, 64%) as brown solid. 1H NMR (DMSO-d6) d 2.48
(s, 3H), 3.87 (s, 3H), 5.01 (s, 2H), 7.14–7.18 (m, 2H), 7.54–7.64
(m, 3H), 7.67–7.72 (m, 1H), 7.89–7.93 (m, 2H), 7.97 (d, 1H,
J = 8.6 Hz), 8.23 (s, 1H); MS (ESI) m/z 456 [M+H]+.
Compound 28f was prepared from 27f in a manner similar to
that described for compound 28g, with a yield of 44% as a beige
solid. 1H NMR (DMSO-d6) d 1.20 (t, 3H, J = 7.5 Hz), 2.83 (q, 2H,
J = 7.5 Hz), 3.46 (s, 3H), 3.73 (s, 3H), 5.44 (s, 2H), 6.47 (d, 1H,
J = 9.4 Hz), 7.71 (dd, 1H, J = 9.4, 2.6 Hz), 7.75 (d, 2H, J = 8.3 Hz),
7.79 (d, 2H, J = 8.3 Hz), 7.87–7.95 (m, 3H), 8.06 (t, 1H, J = 7.8 Hz),
8.27–8.36 (m, 2H), 9.06 (br s, 1H); MS (ESI) m/z 451 [M+H]+; Anal.
Calcd for C28H26N4O2ꢀ2.3HClꢀ2H2O: C, 58.96; H, 5.71; N, 9.82; Cl,
14.30. Found: C, 58.83; H, 5.87; N, 9.77; Cl, 14.38.
5.1.46. 5-(3-{[4-(6-Methoxy-3-methylquinolin-2-
yl)benzyl]oxy}-1-methyl-1H-pyrazol-4-yl)-1-methylpyridin-
2(1H)-one dihydrochloride (28h)
5.1.50. 1-Mmethyl-5-(1-methyl-3-{[4-(3-methylquinolin-2-
yl)phenoxy]methyl}-1H-pyrazol-4-yl)pyridin-2(1H)-one
dihydrochloride (33)
Compound 28h was prepared from 27h in a manner similar to
that described for compound 28g, with a yield of 40% as a beige
solid. 1H NMR (DMSO-d6) d 2.50 (s, 3H), 3.45 (s, 3H), 3.72 (s, 3H),
3.98 (s, 3H), 5.43 (s, 2H), 6.46 (d, 1H, J = 9.4 Hz), 7.63 (d, 1H,
J = 2.5 Hz), 7.66–7.77 (m, 4H), 7.80 (d, 2H, J = 8.2 Hz), 7.87 (s, 1H),
7.92 (d, 1H, J = 2.5 Hz), 8.24 (d, 1H, J = 9.4 Hz), 8.83 (br s, 1H); MS
(ESI) m/z 467 [M+H]+; Anal. Calcd for C28H26N4O3ꢀ2HClꢀ2.6H2O: C,
57.36; H, 5.71; N, 9.56; Cl, 12.09. Found: C, 57.72; H, 5.87; N,
9.35; Cl, 11.79.
To a solution of 32 (6.00 g, 13.2 mmol) in THF (120 mL) cooled
with MeOH–ice bath was dropwisely added isopropylmagnesium
chloride (2.0 M THF solution; 8.24 mL, 16.5 mmol), and the
mixture was stirred at the same temperature for 45 min. To the
resultant mixture was added 2-isopropoxy-4,4,5,5-tetramethyl-
1,3,2-dioxaborolane (4.30 mL, 21.1 mmol), and the mixture was
allowed to stir at room temperature for 2 h. The reaction was
quenched with NH4Cl aqueous solution and extracted with EtOAc.
The organic layer was concentrated in vacuo, and the residue was
purified by silica gel column chromatography (10–60% EtOAc in
hexane, then 30–60% EtOAc in hexane) to give 3-methyl-2-
(4-{[1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-
pyrazol-3-yl]methoxy}phenyl)quinoline (2.32 g, 39%) as a brown
solid. To a mixture of this brown solid (500 mg, 1.10 mmol) and
5-bromo-1-methylpyridin-2(1H)-one (413 mg, 2.20 mmol) in
DMF (5 mL) and water (1 mL) were added PdCl2(dppf)ꢀCH2Cl2
(54 mg, 0.066 mmol) and Na2CO3 (349 mg, 3.29 mmol), and the
mixture was stirred at 100 °C for 1 h. The reaction was diluted with
water and extracted with EtOAc. The organic layer was concen-
trated in vacuo, and the residue was purified by silica gel column
chromatography (0–5% MeOH in CHCl3) to give a free form of the
title compound, which was diluted with EtOH (5 mL). This mixture
was treated with 4 M HCl/EtOAc (1.1 mL) and stirred at room tem-
perature for 30 min. The mixture was concentrated in vacuo, and
the residue was washed with EtOAc to give 33 (80 mg, 14%) as a
beige solid. 1H NMR (DMSO-d6) d 2.55 (s, 3H), 3.40 (s, 3H), 3.88
(s, 3H), 5.21 (s, 2H), 6.44 (d, 1H, J = 9.3 Hz), 7.35 (d, 2H,
J = 8.8 Hz), 7.58 (dd, 1H, J = 9.3, 2.6 Hz), 7.76–7.82 (m, 3H), 7.88
(t, 1H, J = 7.5 Hz), 7.94 (s, 1H), 8.04 (t, 1H, J = 7.6 Hz), 8.23 (d, 1H,
J = 8.1 Hz), 8.30 (d, 1H, J = 8.5 Hz), 8.98 (s, 1H); MS (ESI) m/z 437
[M+H]+; Anal. Calcd for C27H24N4O2ꢀ1.95HClꢀ0.1C4H8O2ꢀ1.7H2O: C,
60.16; H, 5.56; N, 10.24; Cl, 12.64. Found: C, 60.28; H, 5.89; N,
9.92; Cl, 12.59.
5.1.47. 2-[4-({[1-Methyl-4-(1-methyl-6-oxo-1,6-dihydropyridin-
3-yl)-1H-pyrazol-3-yl]oxy}methyl)phenyl]quinoline-3-
carbaldehyde (28i)
Compound 28i was prepared from 27i in a manner similar to
that described for compound 28a, with a yield of 24% as a dark yel-
low solid. 1H NMR (CDCl3) d 3.56 (s, 3H), 3.79 (s, 3H), 5.44 (s, 2H),
6.61 (d, 1H, J = 9.4 Hz), 7.31 (s, 1H), 7.49–7.53 (m, 1H), 7.63–7.68
(m, 3H), 7.71–7.74 (m, 3H), 7.86–7.92 (m, 1H), 8.02 (dd, 1H,
J = 8.2, 1.3 Hz), 8.19–8.23 (m, 1H), 8.86 (s, 1H), 10.21 (s, 1H); MS
(ESI) m/z 451 [M+H]+.
5.1.48. 5-[3-({4-[3-(Difluoromethyl)quinolin-2-yl]benzyl}oxy)-
1-methyl-1H-pyrazol-4-yl]-1-methylpyridin-2(1H)-one
dihydrochloride (29)
To a solution of 28i (150 mg, 0.33 mmol) in CH2Cl2 (3 mL) was
added bis(2-methoxyethyl)aminosulfur trifluoride (Deoxo-FluorÒ,
125 mg, 0.57 mmol), and the mixture was stirred at room temper-
ature overnight. The reaction was quenched with water and
extracted with EtOAc. The organic layer was washed with water
and brine, dried over Na2SO4 and concentrated in vacuo. The resi-
due was purified by flash column chromatography (NH silica gel,
(38–70% CHCl3 in hexane) to give a free form of the title compound,
which was dissolved in EtOAc. The solution was treated with 4 M
HCl/EtOAc, and the precipitate was collected by filtration to give
29 (83 mg, 46%) as a pale yellow solid. 1H NMR (DMSO-d6) d 3.47
(s, 3H), 3.73 (s, 3H), 5.40 (s, 2H), 6.50 (d, 1H, J = 9.4 Hz), 7.20 (t,
1H, J = 54 Hz), 7.64–7.79 (m, 6H), 7.88 (s, 1H), 7.93–7.98 (m, 2H),
8.14 (d, 1H, J = 8.5 Hz), 8.26 (d, 1H, J = 7.9 Hz), 8.93 (s, 1H); MS
(ESI) m/z 473 [M+H]+; Anal. Calcd for C27H22F2N4O2ꢀ2.3HClꢀ2H2O:
C, 54.74; H, 4.82; N, 9.46; Cl, 13.77; F, 6.41. Found: C, 54.83; H,
4.93; N, 9.53; Cl, 13.73; F, 6.50.
5.1.51. Methyl 1-methyl-4-(1-methyl-6-oxo-1,6-dihydropyridin-
3-yl)-1H-pyrazole-3-carboxylate (36)
To a mixture of 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxa-
borolan-2-yl)pyridin-2(1H)-one (34, 2.3 g, 9.78 mmol) and methyl
4-iodo-1-methyl-1H-pyrazole-3-carboxylate (35, 1.25 g, 4.70
mmol) in DMF (40 mL) and water (10 mL) were added Pd(PPh3)4
(814 mg, 0.71 mmol) and Cs2CO3 (3.06 g, 9.40 mmol), and the mix-
ture was stirred at 80 °C for 12 h. The mixture was concentrated in
vacuo, and the residue was purified by silica gel column chromatog-
raphy (0–5% MeOH in CHCl3) to give 36 (812 mg, 70%) as colorless
solid. 1H NMR (DMSO-d6) d 3.44 (s, 3H), 3.74 (s, 3H), 3.91 (s, 3H),
6.39 (d, 1H, J = 9.3 Hz), 7.51 (dd, 1H, J = 9.4, 2.6 Hz), 7.86 (d, 1H,
J = 2.6 Hz), 7.94 (s, 1H); MS (ESI) m/z 248 [M+H]+.
5.1.49. 2-{4-[(4-Iodo-1-methyl-1H-pyrazol-3-yl)methoxy]phenyl}-
3-methylquinoline (32)
To
a solution of (1-methyl-1H-pyrazol-3-yl)methanol (31,
4.00 g, 35.7 mmol) and 4-(3-methylquinolin-2-yl)phenol (30,
8.53 g, 36.3 mmol)in toluene (120 mL) was added CMBP (13.1 g,
54.4 mmol), and the mixture was stirred at 100 °C for 8 h. The mix-
ture was concentrated in vacuo. The residue was purified by silica
gel column chromatography (0–5% MeOH in CHCl3) to give a
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W. Hamaguchi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
15
5.1.52. 5-[3-(Hydroxymethyl)-1-methyl-1H-pyrazol-4-yl]-1-
methylpyridin-2(1H)-one (37)
7.16–7.20 (m, 2H), 7.56–7.63 (m, 4H), 7.71 (dd, 1H, J = 9.5,
2.9 Hz), 7.77 (d, 1H, J = 2.5 Hz), 7.83 (s, 1H), 8.03 (dd, 1H, J = 9.2,
5.5 Hz), 8.22 (s, 1H); MS (ESI) m/z 455 [M+H]+; Anal. Calcd for
To a mixture of 36 (8.24 g, 33.3 mmol) in THF (200 mL) were
added LiBH4 (1.45 g, 66.7 mmol) and EtOH (5.83 mL, 100 mmol),
and the mixture was stirred under reflux condition for 2 h. After
cooling at room temperature, to the reaction mixture was added
1 M HCl aqueous solution. The mixture was neutralized with
NaHCO3 and concentrated in vacuo. The residue was purified by
silica gel column chromatography (0–10% MeOH in CHCl3) to give
37 (3.23 g, 44%) as colorless solid. 1H NMR (DMSO-d6) d 3.45 (s,
3H), 3.80 (s, 3H), 4.41 (d, 1H, J = 5.1 Hz), 5.17 (t, 1H, J = 5.1 Hz),
6.43 (d, 1H, J = 9.4 Hz), 7.65 (dd, 1H, J = 9.4, 2.6 Hz), 7.83 (s, 1H),
7.88 (d, 1H, J = 2.6 Hz); MS (ESI) m/z 220 [M+H]+.
C
27H23FN4O2ꢀ0.2H2O: C, 70.79; H, 5.15; N, 12.23; F, 4.15. Found:
C, 70.59; H, 5.11; N, 12.24; F, 4.21.
5.1.57. 1-Methyl-5-(1-methyl-3-{[4-(quinolin-2-yl)phenoxy]
methyl}-1H-pyrazol-4-yl)pyridin-2(1H)-one (42b)
Compound 42b was prepared from 4-(quinolin-2-yl)phenol
(41b) in a manner similar to that described for compound 42a,
with a yield of 53% as a colorless solid. 1H NMR (DMSO-d6) d
3.37 (s, 3H), 3.87 (s, 3H), 5.15 (s, 2H), 6.41–6.45 (d, 1H,
J = 9.3 Hz), 7.20–7.25 (m, 2H), 7.54–7.60 (m, 2H), 7.73–7.79 (m,
2H), 7.93 (s, 1H), 7.95–7.99 (m, 1H), 8.03 (d, 1H, J = 8.4 Hz), 8.11
(d, 1H, J = 8.7 Hz), 8.23–8.28 (m, 2H), 8.41 (d, 1H, J = 8.4 Hz); MS
(ESI) m/z 423 [M+H]+; Anal. Calcd for C26H22N4O2: C, 73.92; H,
5.25; N, 13.26. Found: C, 73.89; H, 5.23; N, 13.27.
5.1.53. 5-[3-(Chloromethyl)-1-methyl-1H-pyrazol-4-yl]-1-
methylpyridin-2(1H)-one hydrochloride (38)
To a mixture of 37 (395 mg, 1.80 mmol) in CH2Cl2 (20 mL) was
added SOCl2 (0.39 mL, 5.40 mmol), and the mixture was stirred at
room temperature for 2 h. The reaction mixture was concentrated
in vacuo to give 38 (493 mg, quant) as a colorless solid. 1H NMR
(DMSO-d6) d 3.47 (s, 3H), 3.83 (s, 3H), 4.80 (s, 2H), 6.47 (d, 1H,
J = 9.3 Hz), 7.58 (dd, 1H, J = 9.3, 2.6 Hz), 7.79 (d, 1H, J = 2.6 Hz),
7.87 (s, 1H); MS (ESI) m/z 238, 240 [M+H]+.
5.1.58. (4-Iodo-1-methyl-1H-pyrazol-3-yl)methanol (44)
To
a solution of 4-iodo-1-methyl-1H-pyrazole-3-carboxylic
acid (43, 719 mg, 2.82 mmol) in THF (7 mL) was added
1,10-carbonyldiimidazole (CDI; 685 mg, 4.23 mmol), and the mix-
ture was stirred at room temperature for 1 h. To the resultant mix-
ture cooled with ice–water bath was added a mixture of NaBH4
(320 mg, 8.46 mmol) in water (7 mL), and the mixture was stirred
at room temperature for 3 h. The reaction was diluted with water
and extracted with EtOAc. The organic layer was dried over Na2-
SO4, filtered and concentrated in vacuo. The residue was purified
by silica gel column chromatography (0–5% MeOH in CHCl3) to give
44 (577 mg, 86%) as a white solid. 1H NMR (DMSO-d6) d 3.80 (s,
3H), 4.30 (d, 2H, J = 5.3 Hz), 4.93 (t, 1H, J = 5.3 Hz), 7.78 (s, 1H);
MS (ESI) m/z 239 [M+H]+.
5.1.54. 6-Fluoro-3-methyl-2-[4-(tetrahydro-2H-pyran-2-
yloxy)phenyl]quinoline (40)
To
a mixture of 2-chloro-6-fluoro-3-methylquinoline (39,
4.23 g, 21.6 mmol), 4-(2-tetrahydropyranyloxy)phenylboronic acid
(5.04 g, 22.7 mmol), and Pd(PPh3)4 (1.44 g, 1.25 mmol) was added
Na2CO3 (5.64 g, 53.2 mmol) in DME (80 mL) and water (25 mL),
and the mixture was stirred at 100 °C for 16 h. After cooling at
room temperature, the mixture was diluted with EtOAc and
washed with water and brine, dried over MgSO4, filtered and con-
centrated in vacuo. The residue was purified by silica gel column
chromatography (0–10% EtOAc in CHCl3) to give a pale brown
solid, which was washed with EtOAc to give 40 (6.29 g, 86%) as a
pale yellow solid. 1H NMR (CDCl3) d 1.58–1.77 (m, 3H), 1.87–1.93
(m, 2H), 1.99–2.11 (m, 1H), 2.49 (d, 3H, J = 0.8 Hz), 3.60–3.66 (m,
1H), 3.90–3.97 (m, 1H), 5.50 (t, 1H, J = 3.2 Hz), 7.15–7.19 (m, 2H),
7.35–7.43 (m, 2H), 7.51–7.55 (m, 2H), 7.94 (s, 1H), 8.09 (dd, 1H,
J = 9.1, 5.4 Hz); MS (ESI) m/z 338 [M+H]+.
5.1.59. 2-{4-[(4-Iodo-1-methyl-1H-pyrazol-3-yl)methoxy]
phenyl}quinoline (45)
To a mixture of 44 (690 mg, 2.90 mmol) in CH2Cl2 (7 mL) cooled
with ice bath was added SOCl2 (0.32 mL, 4.39 mmol), and the mix-
ture was stirred at room temperature for 2 h. The reaction was
concentrated in vacuo. To the residue in DMF (10 mL) were added
4-(quinolin-2-yl)phenol (41b, 488 mg, 2.21 mmol) and K2CO3
(800 mg, 5.79 mmol), and the mixture was stirred at 70 °C for
8 h. After cooling at room temperature, the residue was partitioned
between water and EtOAc. The organic layer was washed with
brine, dried over anhydrous MgSO4, filtered and concentrated in
vacuo. The residue was purified by silica gel column chromatogra-
phy (0–5% MeOH in CHCl3) to give 45 (908 mg, 93%) as a pale yel-
low solid. 1H NMR (DMSO-d6) d 3.87 (s, 3H), 5.02 (s, 2H), 7.17–7.22
(m, 2H), 7.54–7.69 (m, 1H), 7.73–7.79 (m, 1H), 7.91 (s, 1H), 7.97 (br
d, 1H), 8.04 (br d, 1H), 8.11 (d, 1H, J = 8.7 Hz), 8.23–8.28 (m, 2H),
8.41 (br d, 1H); MS (ESI) m/z 442 [M+H]+.
5.1.55. 4-(6-Fluoro-3-methylquinolin-2-yl)phenol (41a)
To a mixture of 40 (6.29 g, 18.6 mmol) in THF (90 mL) was
added 1 M HCl aqueous solution (40 mL, 40 mmol), and the mix-
ture was stirred at room temperature for 1 h. The reaction was
quenched with 1 M NaOH aqueous solution, and the precipitate
was collected by filtration to give 41a (4.72 g, 90%) as pale yellow
solid. 1H NMR (CDCl3) d 2.46 (d, 3H, J = 0.6 Hz), 6.85–6.91 (m, 2H),
7.46–7.51 (m, 2H), 7.54–7.61 (m, 1H), 7.69 (dd, 1H, J = 9.5, 2.9 Hz),
8.01 (dd, 1H, J = 9.2, 5.5 Hz), 8.19 (s, 1H); MS (ESI) m/z 254 [M+H]+.
5.1.60. 5-(1-Methyl-3-{[4-(quinolin-2-yl)phenoxy]methyl}-1H-
pyrazol-4-yl)pyridin-2-ol (46)
5.1.56. 5-(3-{[4-(6-Fluoro-3-methylquinolin-2-yl)phenoxy]
methyl}-1-methyl-1H-pyrazol-4-yl)-1-methylpyridin-2(1H)-one
(42a)
Compound 46 was prepared from 45 in a manner similar to that
described for compound 13a, with a yield of 6.0% as a brown solid.
1H NMR (CDCl3) d 3.94 (s, 3H), 5.12 (s, 2H), 6.60 (d, 1H, J = 9.4 Hz),
7.17 (d, 2H, J = 8.9 Hz), 7.41 (s, 1H), 7.47–7.52 (m, 2H), 7.57 (dd, 1H,
J = 9.4, 2.7 Hz), 7.68–7.73 (m, 1H), 7.78–7.85 (m, 2H), 8.11–8.20 (m,
4H), 11.61 (br s, 1H); MS (ESI) m/z 409 [M+H]+.
To a stirred mixture of 41a (270 mg, 1.07 mmol) and 38
(351 mg, 1.28 mmol) in DMF (7.0 mL) was added K2CO3 (369 mg,
2.67 mmol) and stirred at 70 °C for 12 h. After cooling at room tem-
perature, the mixture was diluted with EtOAc, washed with water.
The aqueous layer was extracted with EtOAc. The combined
organic layer was dried over MgSO4, filtered and concentrated in
vacuo. The residue was purified by flash column chromatography
(NH silica gel; 0–20% EtOAc in CHCl3) to give an off-white solid
which was washed with EtOAc to give 42a (390 mg, 81%) as an
off-white solid. 1H NMR (DMSO-d6) d 2.47 (d, 3H, J = 0.6 Hz), 3.38
(s, 3H), 3.88 (s, 3H), 5.13 (s, 2H), 6.44 (d, 1H, J = 9.2 Hz),
5.1.61. 1-[11C]Methyl-5-(1-methyl-3-{[4-(quinolin-2-yl)
phenoxy]methyl}-1H-pyrazol-4-yl)pyridin-2(1H)-one ([11C]42b)
[
11C]CO2 was produced by the 14N(p, 11C nuclear reaction
a)
using a Cyclone 18/9 cyclotron (IBA) and [11C]CH3I was produced
from [11C]CO2 by its reduction with LiAlH4 and subsequent
reaction with HI. Generated [11C]CH3I was passed through a heated
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16
W. Hamaguchi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
column (200 °C) of silver triflate (AgOTf), and converted to
11C]methyl triflate (CH3OTf). [11C]CH3OTf was introduced into a
relative to an uninhibited control (100%) and IC50 values were cal-
culated using Prism software (GraphPad Software, Inc.).
[
reaction vessel containing 46 (1.2 mg) and 2 mg of NaH (60% oil
dispersion, pre-washed with hexane) in a mixed solution of THF
5.3. CYP3A4 inhibitory assay
(300
l
L) and DMSO (25
l
L) at ꢁ20 °C. The solution was heated at
60 °C for 3 min. After cooling at room temperature, the mixture
was transferred to a semi-preparative reverse phase HPLC system
(YMC-Pack Pro C18 10 ꢂ 250 mm column (YMC Co., Ltd), eluted
with 55:45 [0.05 M NH4OAc–0.1% AcOH]/CH3CN, eluted at
5.0 mL), which included a solution wash of the reaction vessel with
two portions of 70:30 [0.05 M NH4OAc–0.1% AcOH]/CH3CN (800,
Time-dependent inhibition assay for CYP3A4 was performed in
two steps, a pre-incubation step where the test compound was
incubated with human liver microsomes and the secondary incu-
bation period where CYP3A4 substrate, midazolam, was added to
the preincubate to measure residual CYP3A4 activity. Midazolam
10-hydroxylation was used to monitor the CYP3A4 activity.
1000
l
L). The fraction containing [11C]42 was collected in a rotary
L) and
Each test compound (5 lM) was pre-incubated with human
evaporator including 25% ascorbic acid in dist. water (50
l
liver microsomes (0.1 mg/mL) and NADPH (1.5 mM) at 37 °C. The
pre-incubation times used were 0 and 30 min. Following the pre-
incubation step, each compound was co-incubated with midazo-
ethanol (0.5 mL). The solvent was removed under reduced pres-
sure. The residue was dissolved in 10% DMF, 10% PEG400-saline
(3.0 mL) and sterile filtered (Millipore GS) to furnish an injectable
solution of [11C]42b. Analytical chromatography was performed
with an Agilent 1200 HPLC system (Agilent Technologies Japan)
and an Aloka positron detector RLC-700 (Aloka). Reverse phase
chromatography was performed using a YMC-Pack C18 Pro column
lam (2 lM) at 37 °C for 20 min. At the end of the incubation, the
reaction was terminated by the addition of aqueous solution
containing 80% acetonitrile. The concentration of 10-hydroxymi-
dazolam was determined by LC–MS analysis. The inhibition of
CYP3A4 activity was assessed by comparing the amount of
10-hydroxymidazolam formed in the presence of varying concen-
trations of inhibitor to the amount of 10-hydroxymidazolam
formed in the solvent control. In each study, a CYP3A4 potent
and specific inhibitor, verapamil was used as positive control.
(5
[0.05 M NH4OAc–0.1% AcOH]/CH3CN at 1 mL/min (Rt = 8 min).
11C]42b was identified by comparison with authentic 42b.
lm, 4.6 ꢂ 150 mm; YMC, Kyoto, Japan), eluted with 55:45
[
5.2. PDE10A enzyme assay protocol
% Residual activity ¼ 100ꢃðActivity NME; 30 min=Activity vehicle; 30 minÞ
ð1Þ
5.2.1. Cloning and vector construction of PDE10A2
The full-length human PDE10A2 was amplified by PCR using the
1st strand cDNA synthesized from the total RNA isolated from
human neuroblastoma TGW cell line. The PCR products were
cloned into a pCR2.1-TOPO vector (Invitrogen. Inc.) to confirm
sequences. The confirmed plasmid was digested with restricted
enzymes, BamHI/HindIII, and this digested product was inserted
into a pFastBac1 vector (Invitrogen. Inc.).
where Activity NME, 30 min is the activity in the presence of test
compound and without pre-incubation, and Activity vehicle,
30 min is the activity in the absence of test compound and with
pre-incubation.
5.4. Animal experiments
All animal experimental procedures were approved by the Insti-
tutional Animal Care and Use Committee of Astellas Pharma Inc.
Further, the Astellas Pharma Inc. Tsukuba Research Center was
awarded Accreditation Status by the AAALAC International. All
efforts were made to minimize the number of animals used and
to avoid suffering and distress.
5.2.2. Preparation of human PDE10A2 enzyme
Human PDE10A2 enzyme protein was expressed in a Spodoptera
frugiperda Sf9 insect cell using the Bac-to-Bac Baculovirus Expres-
sion System (Invitrogen. Inc.). The infected Sf9 cells were collected
by the centrifuge and removed medium. The collected cells were
lysed by sonication in the lysis buffer (50 mM Tris–HCl (pH 8.0),
150 mM NaCl, 3 mM DTT, 0.1% NP-40, 20% Glycerol with protease
inhibitors). The lysate was centrifuged and supernatant was col-
lected to obtain the PDE10A2 enzyme solution. We confirmed
the PDE10A2 expression by Western blot analysis.
5.4.1. In vivo behavioral assay in mice
Phencyclidine-induced hyperlocomotion: ICR mice aged
5–6 weeks were used to evaluate the effect of PDE10A inhibitor
on hyper-locomotion induced by the NMDA antagonist phencycli-
dine (PCP). Immediately after oral administration of either vehicle
or agent as pre-treatment, mice were placed into individual plastic
test cages (30 ꢂ 35 ꢂ 17.5 cm) of a SUPERMEX system (PAT.P;
Muromachi Kikai Co., Ltd), and measurement of locomotor activity
was started. After 1 h, the mice were injected with a post-
treatment of saline or PCP (2.5 mg/10 mL/kg, sc), and locomotor
activity was measured for a further 60 min. Total locomotor
activity for 60 min post-treatment was calculated.
NORT in neonatally PCP-treated mice: Three-day-old male ddY
mice were housed 10–12 per cage with a stepmother. Saline or
PCP (15 mg/kg) was administered subcutaneously once daily on
days 7, 9, and 11 after birth. The mice were separated from their
mother at 3 weeks of age, and used for NORT at 8–9 weeks old.
Neonatal mice were treated with PCP, and the NORT was con-
ducted as previously described.19
5.2.3. PDE10A2 inhibition assay
Inhibition of compounds on human PDE10A enzyme activity
was assessed by measuring the amount of cAMP by the Homoge-
neous Time-Resolved Fluorescence (HTRF) detection method. The
assay was performed in 12
lL samples containing a optimal
amount of the PDE10A enzyme, a buffer (40 mM Tris–HCl pH
7.5; 5 mM MgCl2), 0.1
compounds (0.1 nM–10
l
l
M cAMP and various concentrations of
M). After compounds were preincubated
for 30 min with the enzyme, the reaction was initiated by adding
the substrate cAMP and the mixture was incubated for 60 min at
room temperature with agitation. The reaction was terminated
by the addition of the fluorescence acceptor (cAMP labeled with
the dye d2) and the fluorescence donor (anti-cAMP antibody
labeled with Cryptate, Cisbio). After 60 min, the fluorescence trans-
fer corresponding to the amount of residual cAMP was measured at
lex. 320 nm, lem. 620 nm and lem. 665 nm using an Envision plate
reader (PerkinElmer) and signal ratio (665/620) was calculated.
The ratio determined in the absence of enzyme was subtracted
from all data. The obtained results were converted to activity
5.4.2. Mouse pharmacokinetic study
The ICR mice were treated orally with compound 42b or MP-10
(3.0 mg/kg) suspended in 0.5% methylcellulose aqueous solution.
Whole brain samples were collected at 1 h after administration,
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W. Hamaguchi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
17
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LC–MS/MS with a ACQUITY UPLC(Waters) and Xevo TQ(Waters).
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16. ACD/logP values were calculated with ACD/PhysChem Batch (version 12.01).
17. The pKBHX values of the pyrimidone ring and the pyridazinone ring were not
available in Ref. 15.
18. Wu, Y.-J.; Davis, C. D.; Dworetzky, S.; Fitzpatrick, W. C.; Harden, D.; He, H.;
Knox, R. J.; Newton, A. E.; Philip, T.; Polson, C.; Sivarao, D. V.; Sun, L.-Q.;
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weighed and measured for radioactivity using a c-counter (Wallac
2480, Perkin Elmer, Waltham, MA, USA). The data obtained from the
brain in units of Bq/g were converted to SUV using the following
equation:
Brain region radioactivity ðBq=gÞ
SUV ¼
Injected radioactivity per body weight ðBq=gÞ
Rat PET imaging study: PET scans were conducted using an Inv-
eon Multimodality system (Siemens, Knoxville, TN, USA). PET
emission scans were performed with 8 weeks old SD rats under
anesthesia induced by 2.5% isoflurane. [11C]42b (approximately
50 MBq) was bolus intravenously injected to rats treated with
MP-10 (10 mg/kg, iv) or saline at 10 min before radioligand
administration. And then, dynamic PET emission data were col-
lected for 60 min. Data from PET acquisition was reconstructed
using ASIproVM software (Siemens).
5.5. Human liver microsomal assays
Pooled human liver microsomes (Xenotech LLC.) were diluted in
100 mM KH2PO4/K2HPO4 buffer (pH 7.4) containing 0.1 mM EDTA.
The incubation mixtures (270
0.2 mg/mL of microsomal proteins, and 1 mM NADPH (30
pre-incubated for 5 min at 37 °C. Reactions were initiated by the
addition of 0.2 M of substrates. After the appropriate incubation
time (0, 15, 30, and 45 min), 50 L of incubation mixture was
transferred into 80% acetonitrile containing internal standard
(50 ng/mL diazepam, 250 L) and centrifuged for 10 min at
2800 rpm. The supernatant (200 L) was prepared and analyzed
lL total volume), which contained
l
L) were
l
l
l
l
via LC–MS/MS with a Surveyor HPCL system (Thermo Fisher Scien-
tific Inc.) and TSQ Quantum ultra tandem triple quadrupole mass
spectrometer (Thermo Fisher Scientific Inc.). The in vitro intrinsic
clearance (CLint, vitro) was calculated using Eq. 2, which is based
on the time course of the residual ratio of the compounds.20
Keð1=minÞꢂMS content ðmg=kgÞ
CLint;vitro ðmL=min=kgÞ ¼
ð2Þ
MS Protein Concdðmg=mLÞ
where Ke is the disappearance rate constant.
Acknowledgments
The authors thank Dr. Takeshi Shimada and Ms. Junko Yarimizu
for performing pharmacological evaluations, and the staff of
Astellas Research Technologies Co., Ltd, for conducting liver micro-
somal assays, elemental analysis, and spectral measurements.
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Please cite this article in press as: Hamaguchi, W.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.11.039