Synthesis and structure-activity relationships of 8-substituted-2-aryl-5- alkylaminoquinolines: Potent, orally active corticotropin-releasing factor-1 receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2012.09.028

We previously reported a series of 8-methyl-2-aryl-5-alkylaminoquinolines as a novel class of corticotropin-releasing factor-1 (CRF₁) receptor antagonists. A critical issue encountered for this series of compounds was low aqueous solubility at physiological pH (pH 7.4). To address this issue, derivatization at key sites (R², R³, R⁵, R ^5′, and R⁸) was performed and the relationships between structure and solubility were examined. As a result, it was revealed that introduction of a methoxy substituent at the C₈ position had a positive impact on the solubility of the derivatives. Consequently, through in vivo and in vitro biological studies, compound 21d was identified as a potent, orally active CRF₁ receptor antagonist with improved physicochemical properties.

Full text of DOI:10.1016/j.bmc.2012.09.028

Bioorganic & Medicinal Chemistry 20 (2012) 6559–6578
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and structure–activity relationships of 8-substituted-2-aryl-5-
alkylaminoquinolines: Potent, orally active corticotropin-releasing factor-1
receptor antagonists
Kunitoshi Takeda a, , Taro Terauchi a, Minako Hashizume a, Kohdoh Shikata b, Ryota Taguchi b,
⇑
Kaoru Murata-Tai c, Masae Fujisawa d, Yoshinori Takahashi a, Kogyoku Shin a, Mitsuhiro Ino b,
Hisashi Shibata b, Masahiro Yonaga a
a Medicinal Chemistry, Tsukuba Research Laboratories, Eisai Co., Ltd, 5-1-3 Tokodai, Tsukuba-shi, Ibaraki 300-2635, Japan
b Biopharmacology, Tsukuba Research Laboratories, Eisai Co., Ltd, 5-1-3 Tokodai, Tsukuba-shi, Ibaraki 300-2635, Japan
c Physical Chemistry, Tsukuba Research Laboratories, Eisai Co., Ltd, 5-1-3 Tokodai, Tsukuba-shi, Ibaraki 300-2635, Japan
d Drug Metabolism and Pharmacokinetics, Tsukuba Research Laboratories, Eisai Co., Ltd, 5-1-3 Tokodai, Tsukuba-shi, Ibaraki 300-2635, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We previously reported a series of 8-methyl-2-aryl-5-alkylaminoquinolines as a novel class of corticotro-
pin-releasing factor-1 (CRF1) receptor antagonists. A critical issue encountered for this series of com-
Received 29 August 2012
Revised 14 September 2012
Accepted 15 September 2012
Available online 23 September 2012
pounds was low aqueous solubility at physiological pH (pH 7.4). To address this issue, derivatization
0
at key sites (R2, R3, R5, R5 , and R8) was performed and the relationships between structure and solubility
were examined. As a result, it was revealed that introduction of a methoxy substituent at the C8 position
had a positive impact on the solubility of the derivatives. Consequently, through in vivo and in vitro bio-
logical studies, compound 21d was identified as a potent, orally active CRF1 receptor antagonist with
improved physicochemical properties.
Keywords:
Corticotropin-releasing factor
Pharmacophore
Ó 2012 Elsevier Ltd. All rights reserved.
Solubility
Structure–activity relationships
Depression
1. Introduction
of depression or other stress-related disorders. Meanwhile, the
benefits of blocking the CRF2 receptor remain uncertain. To date,
Corticotropin releasing factor (CRF), isolated by Vale et al. in
1981 as a 41 amino acid peptide, is the primary regulator of hypo-
thalamic-pituitary-adrenal (HPA) stress response.1 It is known that
CRF exerts its biological functions through binding to two GPCR
subfamily receptors, the CRF1 and CRF2 receptors.2 The CRF1 recep-
tor is abundantly found in the pituitary and is involved in the reg-
ulation of adrenocorticotropic hormone (ACTH), a key mediator of
stress response.3 There is much evidence that CRF and CRF1 recep-
tors are heavily involved in stress-related disorders such as depres-
sion and anxiety. In fact, it was shown that intracerebroventricular
administration of CRF in rodents elicits behavioral and physiologi-
cal effects identical to those caused by natural stressors.4 Further-
more, elevated CRF concentration in CSF has been observed in
patients with depression5 and decreased expression of CRF recep-
tor in the frontal cortex has been observed in suicide victims.6
Therefore, several pharmaceutical research groups have focused
on the discovery of CRF1 receptor antagonists for the treatment
several CRF1 receptor antagonists have been reported and proto-
typical antagonists are illustrated in Figure 1. CRF1 receptor
antagonists 1 (R121919),7 2 (CP-154526),8 3 (DMP696),9 4 (NBI-
27914),10 and 5 (CP-316311)11 exhibited high in vitro affinity to
the receptor and significant activity in animal models. However,
clinical studies remain ambiguous. Antagonist 1 demonstrated effi-
cacy in treating depressed patients in an open-label phase 2 clini-
cal trial,12 but 5 was unsuccessful in a double-blind study for
depression.13 From these results, it is apparent that the discovery
of structurally diverse CRF1 receptor antagonists and the accumu-
lation of clinical studies for clarifying the role of CRF in humans are
essential.
Known CRF1 receptor antagonists have common structural fea-
tures as illustrated in Figure 2. Each has a top region occupied by
an alkyl chain, a central ring system occupied by a mono- or bihe-
teroaromatic core, a small pocket occupied by a methyl group, and
a bottom region occupied by a substituted phenyl or heteroaro-
matic moiety. Accordingly, we hypothesized that these four struc-
tural features were essential for CRF1 receptor antagonism. As a
result of compound design based on this hypothesis, we previously
⇑
Corresponding author. Tel.: +81 (0) 29 847 5842; fax: +81 (0) 29 847 4952.
E-mail address: k5-takeda@hhc.eisai.co.jp (K. Takeda).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.09.028

6560
K. Takeda et al. / Bioorg. Med. Chem. 20 (2012) 6559–6578
To easily prepare compounds derivatized at each site of the quin-
O
N
oline template, two different synthetic approaches were investi-
gated. Derivatization at R3 and R8 was achieved as shown in
Scheme 2. Compound 12d was synthesized by the previously
reported method using 3-chloroquinoline.14 Nitration of 2-halo-3-
substituted quinolines 12a–e afforded corresponding 5-nitroquino-
lines 13a–e, as confirmed by NMR analysis ( Fig. 3). Subsequent
Suzuki–Miyaura cross-coupling reaction with [2,6-dimethoxy-4-
(methoxymethyl)phenyl]boronic acid afforded 2-aryl-quinolines
14a–e. Reduction of the nitro group followed by reductive amina-
tion with acetaldehyde afforded intermediates 16a–e. Halogenation
of intermediate 16a using N-chlorosuccinimide (NCS) and N-
iodosuccinimide (NIS) afforded 8-haloquinolines 17 and 18, respec-
tively, and the positions of the halogen atoms were determined by
NMR analysis (Fig. 3). The iodide of 18 was lithiated and trans-
formed to aldehyde 19 and fluoride 21a. Trifluoromethyl derivative
21b, cyanide 21c, and methoxide 21d were also synthesized from
iodide 18. Aldehyde 19 was subsequently converted to difluoro-
methyl derivative 20a in the presence of [bis(2-methoxyethyl)
amino]sulfur trifluoride. Methoxymethyl derivative 20b was also
synthesized from aldehyde 19 using decaborane in methanol. Tar-
get compounds 23ba–ea were synthesized from intermediates
16b–e in a manner similar to that described for the synthesis of
21d. The positions of the iodine atoms of 22b, 22c, 22d, and 22e
were determined by NMR analysis (Fig. 3). Derivatives with methyl
substituents at the C8 position (23bb–23db) were synthesized from
22b–d using Me2Zn/(tBu3P)2Pd.
N
N
N
N
O
N
N
N
HN
N
N
N
N
N
Cl
HCl
N
Cl
1
2
3
(DMP696)
(R121919)
(CP-154526)
N
O
Cl
N
N
Cl
NH
Cl
N
O
Cl
4
5
(CP-316311)
(NBI-27914)
Figure 1. Known CRF1 receptor antagonists.
Compounds derivatized at R2, R5, and R5’ were synthesized as
shown in Scheme 3. First, 2-chloro-8-methoxy-3-methylquinoline
25 was synthesized by the method reported by Meth-Cohn
et al.15 starting with amide 24. Nitration of 25 and the subsequent
Suzuki–Miyaura cross-coupling reaction were conducted in a man-
ner similar to that described for the synthesis of 14b to afford 2-
aryl-5-nitroquinline 27. The position of the nitro group of 26 was
determined by NMR analysis (Fig. 3). Reduction of the nitro group
of 27 using Fe followed by reductive amination with aldehydes in
the presence of reducing agent NaBH(OAc)3 afforded target com-
pound 29. Compound 21d, which was synthesized by the method
described in Scheme 2, was also conveniently synthesized by this
route. Boc protection of 28 followed by methoxyethylation affor-
ded intermediate 31. Deprotection of the Boc group followed by
reductive amination with aldehydes afforded target compounds
33a–b. Target compounds 36a–c were synthesized from 26 in a
manner similar to that described for the synthesis of 29.
Top region
R
R
N
X
Ar
Central ring system
Small pocket
R
N
O
O
Bottom region
O
Known antagonists
6
Figure 2. Design of a novel CRF1 receptor antagonist.
reported compound 6 as a novel CRF1 receptor antagonist (Fig. 2).14
This compound exhibited potent in vitro activity and in vivo effi-
cacy by oral administration. However, the unacceptable physico-
chemical profile (e.g., low aqueous solubility at pH 7.4) rendered
this compound unsuitable for further development. Herein, we de-
scribe the results of our efforts to resolve this issue. Key sites in 6
were derivatized to generate a new series of compounds and the
relationships between structure and solubility were examined.
Studies of the structure–activity relationships, physicochemical
properties, and electrostatic potentials of the derivatives are dis-
cussed. The biological activities of some of the new compounds
were also examined, revealing a new analog with well-balanced
solubility and in vitro efficacy.
3. Results and discussions
The compounds in this study were first screened for their ability
to inhibit [125I] CRF binding to membranes of cells expressing the
human CRF1 receptor. Compounds with high binding affinity were
then subjected to an in vitro functional assay to evaluate their
antagonistic functions in the inhibition of CRF-induced cAMP (cyc-
lic adenosine monophosphate) production in human CRF1-recep-
tor-expressed HEK293 cells.
2. Chemistry
The explorations initially performed were aimed at investigat-
ing the effects of substituents at the C8 position on activity and
aqueous solubility (pH 7.4). In order to clarify the effects of
lipophilicity on aqueous solubility, ClogP values were calculated
(using Daylight software, version 4.94) and the results are shown
in Table 1. Replacement of methyl with fluorine, difluoromethyl,
trifluoromethyl, or methoxymethyl reduced binding affinity and
antagonistic activity (21a, 20a, 21b, and 20b, respectively). On
the other hand, replacement with chlorine, cyano, or methoxy re-
sulted in retained binding affinity (17, 21c, and 21d, respectively)
compared with parent compound 6. There seemed to be a strict
Derivatives with a methyl substituent at the C8 position were
synthesized as shown in Scheme 1. Nitration of commercially
available compound 7 afforded 5-nitroquinoline 8. The position
of the nitro group was determined by NMR analysis (Fig. 3). All
of the proton and carbon signals of compound 8 were assigned
by HMBC, COSY, and NOESY analysis. Next, the Suzuki–Miyaura
cross-coupling reaction of 8 with [2,6-dimethoxy-4-(methoxy-
methyl)phenyl]boronic acid gave compound 9. Reduction of the ni-
tro group followed by reductive amination with aldehydes or
substitution reaction with alkyl halide afforded derivatives 6 and
11a–c.

K. Takeda et al. / Bioorg. Med. Chem. 20 (2012) 6559–6578
6561
NO2
NH2
NO2
N
N
a
b
c
O
O
O
O
N
N
Cl
Cl
O
O
7
8
9
10
R5
N
R5'
6: (R5, R5') = (Et, Et)
11a: (R5, R5') = (n-Pr, n-Pr)
d
N
O
O
5
5'
11b
: (R , R ) = (H, methoxyethyl)
e
11c: (R5, R5') = (Et, methoxyethyl)
O
Scheme 1. Reagents and conditions: (a) HNO3, H2SO4, ꢀ10 °C to RT; (b) [2,6-dimethoxy-4-(methoxymethyl)phenyl]boronic acid, Pd(PPh3)4, K2CO3, DME, H2O, reflux; (c) Fe,
saturated aqueous NH4Cl, EtOH, reflux; (d) for 6, acetaldehyde, NaBH(OAc)3, AcOH, THF, RT; for 11a, propionaldehyde, NaBH(OAc)3, AcOH, THF, RT; for 11b, 2-bromoethyl
methyl ether, K2CO3, DMF, 100 °C; (e) acetaldehyde, NaBH(OAc)3, AcOH, THF, RT.
NO2
NO2
NO2
NO2
NO2
F
N
N
N
N
N
Cl
I
Cl
Cl
I
Cl
8
13a
13c
13d
13e
N
N
N
N
Cl
I
I
I
N
N
N
N
F
O
O
O
O
O
O
O
O
O
O
N
O
O
17
18
22b
22c
N
NO2
I
I
N
N
O
Cl
O
N
O
O
O
Cl
HMBC
NOESY
26
O
O
COSY
22d
22e
Figure 3. Results of the NMR analysis of derivatives of 6.

6562
K. Takeda et al. / Bioorg. Med. Chem. 20 (2012) 6559–6578
NO2
NH2
N
NO2
a
b
c
d
N
N
R3
O
R3
O
N
R3
O
O
O
N
N
R3
O
R3
X
X
O
O
O
12a-e
13a-e
14a-e
15a-e
16a-e
a : (X, R3) = (Cl, Me)
b : (X, R3) = (Cl, H)
c : (X, R3) = (I, F)
d : (X, R3) = (I, Cl)
e : (X, R3) = (Cl, Et)
a : (X, R3) = (Cl, Me)
b : (X, R3) = (Cl, H)
c : (X, R3) = (I, F)
d : (X, R3) = (I, Cl)
e : (X, R3) = (Cl, Et)
a : R3 = Me
b : R3 = H
c : R3 = F
d : R3 = Cl
e : R3 = Et
a : R3 = Me
b : R3 = H
c : R3 = F
d : R3 = Cl
e : R3 = Et
a : R3 = Me
b : R3 = H
c : R3 = F
d : R3 = Cl
e : R3 = Et
N
Cl
N
O
O
e
N
N
N
H
O
O
O
R8
h
17
N
N
N
O
O
O
O
O
g
f
N
O
O
O
I
16a
19
20a-b
N
a : R8 = CF2H
b : R8 = Methoxymethyl
O
O
N
i
R8
O
21a-d
18
N
a : R8 = F
b : R8 = CF3
c : R8 = CN
d : R8 = OMe
O
O
O
N
N
N
I
R8
j
k
N
N
N
R3
O
R3
R3
O
O
O
O
O
O
O
O
16b-e
22b-e
23
b : R3 = H
c : R3 = F
d : R3 = Cl
e : R3 = Et
b : R3 = H
c : R3 = F
d : R3 = Cl
e : R3 = Et
ba : R3 = H, R8 = OMe
ca : R3 = F, R8 = OMe
da : R3 = Cl, R8 = OMe
ea : R3 = Et, R8 = OMe
bb : R3 = H, R8 = Me
cb : R3 = F, R8 = Me
db : R3 = Cl, R8 = Me
Scheme 2. Reagents and conditions: (a) for 13a, 13b, and 13e, HNO3, H2SO4, ꢀ10 °C to RT; for 13c and 13d, HNO3, fuming HNO3, H2SO4, 0 °C; (b) for 14a, [2,6-dimethoxy-4-
(methoxymethyl)phenyl]boronic acid, Pd(OAc)2, PPh3, K2CO3, DME, H2O, reflux; for 14b, 14c, and 14e, [2,6-dimethoxy-4-(methoxymethyl)phenyl]boronic acid, Pd(PPh3)4,
Na2CO3, DME, H2O, reflux; for 14d, [2,6-dimethoxy-4-(methoxymethyl)phenyl]boronic acid, Pd(PPh3)4, 1 M aqueous Na2CO3, PhMe, EtOH, reflux; (c) for 15a, H2, Pd/C, ethyl
acetate, RT; for 15b, 15c, 15d, and 15e, Fe, saturated aqueous NH4Cl, EtOH, reflux; (d) acetaldehyde, NaBH(OAc)3, AcOH, THF, RT; (e) NCS, DMF, 60 °C; (f) NIS, DMF, RT; (g)
DMF, nBuLi, THF, ꢀ78 °C; (h) for 20a, [bis(2-methoxyethyl)amino]sulfur trifluoride, DCM, RT; for 20b, decaborane, MeOH, RT; (i) for 21a, N-fluorobenzenesulfonimide, nBuLi,
THF, ꢀ78 °C; for 21b, methyl fluorosulfonyldifluoroacetate, Cu, DMF, 90 °C; for 21c, CuCN, DMF, 80 °C; for 21d, CuBr, NaOMe/MeOH, ethyl acetate, reflux; (j) NIS, DMF, RT; (k)
for 23ba, 23ca, 23da, and 23ea, CuBr, NaOMe/MeOH, ethyl acetate, reflux; for 23bb, 23cb, and 23db, Me2Zn, (tBu3P)2Pd, 1,4-dioxane, 80 °C.
requirement for the size of the substituent rather than its elec-
tronic effect at the C8 position. No correlation between ClogP
and aqueous solubility was observed ( Fig. 4). Compounds that
had ClogP values higher than 6 exhibited decreased solubility
(17 and 21b). However, not all compounds that had ClogP values
lower than
6 exhibited improved solubility. For example,

K. Takeda et al. / Bioorg. Med. Chem. 20 (2012) 6559–6578
6563
R5
N
NO2
NH2
R5'
O
NO2
O
O
O
a
b
c
N
d
N
e
N
O
O
O
O
O
O
O
O
HN
N
N
O
Cl
Cl
O
O
O
24
25
26
27
28
21d : (R5, R5') = (Et, Et)
: (R5, R5') = (n-Pr, n-Pr)
29
O
O
O
H
N
Boc
O
N
NH
N
Boc
R5
O
i
h
g
f
N
O
O
O
N
28
N
N
O
O
O
O
O
O
O
O
O
O
O
30
31
32
33a-b
a : R5 = Ethyl
b : R5 = Isobutyl
NO2
NH2
N
j
k
l
O
O
26
O
N
N
N
R2
R2
R2
34a-c
35a-c
36a-c
2
a
: R = 2-chloro-4-methoxymethyl-6-methoxyphenyl
b : R2 = 2,6-dimethoxy-4-cyanophenyl
c : R2 = 2,6-dimethoxy-4-methylphenyl
Scheme 3. Reagents and conditions: (a) POCl3, DMF, 70 °C; (b) HNO3, H2SO4, ꢀ10 °C to RT; (c) [2,6-dimethoxy-4-(methoxymethyl)phenyl]boronic acid, Pd(PPh3)4, K2CO3, 1,4-
dioxane, H2O, reflux; (d) Fe, saturated aqueous NH4Cl, EtOH, reflux; (e) for 21d, acetaldehyde, NaBH(OAc)3, AcOH, MeOH, THF, RT; for 29, propionaldehyde, NaBH(OAc)3,
AcOH, THF, RT; (f) Boc2O, Et3N, DCM, RT; (g) 2-bromoethyl methyl ether, NaH, DMF, 40 °C; (h) TFA, DCM, RT; (i) for 33a, acetaldehyde, NaBH(OAc)3, AcOH, THF, RT; for 33b,
isobutyraldehyde, NaBH(OAc)3, AcOH, THF, RT; (j) for 34a, [2-chloro-6-methoxy-4-(methoxymethyl)phenyl]boronic acid, Pd(PPh3)4, K2CO3, 1,4-dioxane, H2O, reflux; for 34b,
(4-cyano-2,6-dimethoxyphenyl)boronic acid, Pd(PPh3)4, K2CO3, 1,4-dioxane, H2O, reflux; for 34c, (2,6-dimethoxy-4-methylphenyl)boronic acid, Pd(PPh3)4, K2CO3, 1,4-
dioxane, H2O, reflux; (k) Fe, saturated aqueous NH4Cl, EtOH, reflux; (l) acetaldehyde, NaBH(OAc)3, AcOH, THF, RT.
compound 21c, which had the lowest ClogP value of all the deriv-
atives, did not exhibit much improved solubility compared with 6.
On the other hand, compound 21d, which exhibited a slight
decrease in ClogP value compared to 6, showed remarkably im-
proved solubility.
21d were calculated because we speculated that the electronic
state of the quinoline core could be influenced by substituents at
the C8 position. The calculations of electrostatic potential were per-
formed using MOPAC (version 7.01, AM1 method) and the results
are shown in Figure 5. Compared with 6, compound 21d possessed
an area with higher electron density around the 8-methoxy moiety
and the nitrogen atom of the quinoline core (Area A). The anionic
area was thought to have a high affinity for water molecules in
aqueous solutions, which may have led to the improved solubility
observed for 21d. On the other hand, the electrostatic potential of
21c indicated that the electron density of the quinoline core was
lower compared with 6 and 21d owing to introduction of the
cyano substituent. The electrostatic potential of 20b, which has a
methoxymethyl substituent at the C8 position, was also calculated.
An area with high electron density was not observed in this com-
pound, unlike compound 21d. Replacing methyl with methoxy-
methyl generally has a positive impact on aqueous solubility.
However, 20b did not exhibit improved solubility compared with
6. This result indicates that simply introducing polar substituents
at the C8 position would not have a positive impact on aqueous
solubility. These studies of electrostatic potential suggest that the
good solubility of 21d might result from the area with high elec-
tron density around the 8-methoxy moiety and the nitrogen atom
To gain further insight into these results, pKa values were deter-
mined for compounds 6, 21c, and 21d (Table 2). Compound 21c
exhibited a remarkable decrease in pKa relative to 6 (pKa <3 for
21c, pKa = 5.86 for 6), which might be caused by the introduction
of the electron withdrawing cyano moiety. On the other hand,
21d exhibited a slight increase in pKa (pKa = 6.01) relative to 6. In
order to confirm the effects of pKa on aqueous solubility, the solu-
bilities of 6, 21c, and 21d were determined at various pH values
(1.2, 5.0, and 9.0) and the results are shown in Table 2. The results
were reasonable, indicating that the solubility of each compound
increased below pH values approximately equal to their pKa values.
This indicates that for these three compounds, the effect of disso-
ciation on solubility at pH 7.4 was negligible. In short, the differ-
ence in solubility observed at pH 7.4 in these compounds could
not be explained by their lipophilicity (i.e., ClogP) and their basi-
city (i.e., pKa).
Next, to gain further insight into the relationships between
structure and solubility, electrostatic potentials of 6, 21c, and

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K. Takeda et al. / Bioorg. Med. Chem. 20 (2012) 6559–6578
Table 1
Effects of substitution of R8 on biological activity and physicochemical properties
N
8
R8
N
O
O
O
No.
R8
Binding IC50 (nM)
Functional IC50 (nM)
Solubility at pH 7.4a
(lM)
ClogP
6
21a
17
20a
21b
21c
20b
21d
Me
F
Cl
CF2H
CF3
CN
79
226
62
414
288
102
>1000
110
24
67
28
NT
259
36
NT
91
1.7
5.22
4.94
5.51
4.92
5.74
4.33
4.52
5.02
10.5
<0.2
<0.2
<0.2
3.2
Methoxymethyl
OMe
2.9
74.0
NT = not tested.
a
pH 7.4: Dulbecco’s phosphate-buffered saline (PBS).
ethyl (23ea) at R3 retained binding affinity. In functional assays,
23ca, 23da, and 23ea exhibited a tendency for slightly improved
antagonistic activity. This result indicated that introducing any
substituent at the C3 position effectively resulted in high binding
affinity and potent antagonistic activity, although this position
did not seem to be related to the pharmacophores shown in Figure
2. The dihedral angle between the quinoline and aromatic group in
the bottom region might be important for activity because substit-
uents at this position should have a significant impact on the angle.
Furthermore, judging from the enhanced binding affinity and
antagonistic activity observed for substituted derivatives (21d,
23ca, 23da, and 23ea) relative to unsubstituted derivative 23ba,
a twisted conformation might be favored for the aryl moiety in
the bottom region. The same tendency was observed for the 8-
methylquinoline derivatives; substituted derivatives (6, 23cb,
and 23db) displayed more potent activity than the unsubstituted
derivative (23bb). The 8-methoxy derivatives (21d, 23ba, 23ca,
and 23da) exhibited comparable or slightly less activity than the
8-methyl derivatives (6, 23bb, 23cb, and 23db). In terms of solubil-
ity, compound 23ba, which exhibited a slightly lower ClogP value
than 21d, had decreased solubility. We speculated that removing
the methyl group from the C3 position might increase the planarity
of the molecule, which led to the decrease in solubility.16 On the
other hand, compounds 23ca and 23da, which had halogen atoms,
exhibited remarkably lower solubility. In particular, the decreased
solubility of compound 23ca, which exhibited a ClogP value com-
parable to that of 21d, could not be explained by lipophilicity.
Therefore, the electrostatic potentials of these two compounds
were calculated ( Fig. 6). Compared with 21d ( Fig. 5), electron
density of Area A in 23ca and 23da was diffused by the electron-
withdrawing effect of the halogen atoms at the C3 position. We
speculated that the lowered electron density weakened affinity to-
ward water molecules, which led to decreased solubility. As for
23da, both increased lipophilicity and electron density lowered
by chloride atom might cause the decreased solubility. Compound
23ea also exhibited lower solubility, which could be explained by
its increased lipophilicity. On the other hand, solubility of 8-meth-
ylquinoline derivatives (23bb, 23cb, and 23db) did not seem to im-
prove as a result of substitution at C3. In summation, from
comparison of the solubilities of 8-methoxy derivatives with those
Figure 4. Correlation between ClogP and solubility for compounds derivatized at
C8.
Table 2
Solubility and pKa for 6, 21c, and 21d at various pH values
No.
pKa
pH 1.2 (
l
M)a
pH 5 (
l
M)b
pH 7.4 (
l
M)c
pH 9b
(lM)
6
21c
21d
5.86
<3
6.01
>100
>100
>100
22.0
2.1
98.2
1.7
3.2
74.0
2.0
4.2
71.0
a
pH 1.2: HCl/NaCl.
b
pH 5 and pH 9: Britton–Robinson buffer, ionic strength 3 (acetate/phosphate/
borate buffer containing KCl and adjusted with NaOH).
c
pH 7.4: Dulbecco’s phosphate-buffered saline (PBS).
of the quinoline core (Area A). As a result of derivatization at the C8
position, it was determined that introduction of the methoxy sub-
stituent at C8 resulted in well-balanced compatibility of activity
and solubility. Thus, further derivatization was conducted with
the methoxy substituent retained at the C8 position in order to
grasp the SAR and the relationship among structure, lipophilicity,
and solubility.
The effect of R3 was examined and the results are shown in
Table 3. Derivatives with a methyl substituent at C8 were also pre-
pared to further clarify the effects of the methoxy substituent at C8.
Removing the methyl substituent resulted in lower binding affinity
(23ba) and compounds with fluorine (23ca), chlorine (23da), and

K. Takeda et al. / Bioorg. Med. Chem. 20 (2012) 6559–6578
6565
Figure 5. Electrostatic potential of compounds 6, 20b, 21c, and 21d.
Table 3
Effects of substitution of R3 on biological activity and physicochemical properties
N
R8
3
N
R3
O
O
O
No.
R8
R3
Binding IC50 (nM)
Functional IC50 (nM)
Solubility at pH 7.4a
(
lM)
ClogP
21d
OMe
OMe
OMe
OMe
OMe
Me
Me
Me
Me
Me
H
F
Cl
Et
Me
H
110
202
118
144
108
79
184
144
81
91
332
75
57
32
74
38.2
4.6
1.9
12.1
1.7
<0.2
<0.2
<0.2
5.02
4.82
5.00
5.32
5.55
5.22
5.02
5.23
5.55
23ba
23ca
23da
23ea
6
23bb
23cb
23db
24
381
131
45
F
Cl
a
pH 7.4: Dulbecco’s phosphate-buffered saline (PBS).
ether substituents were favored. Accordingly, a few derivatives
0
with different R5 and R5 substituents were prepared and SAR
was briefly examined for the present 8-methoxyquinoline series.
Di-n-propyl derivative 29 retained binding affinity and antagonis-
tic activity compared with diethyl derivative 21d. Replacement of
ethyl with methoxyethyl resulted in fourfold lower binding affinity
(33a), but the compound with isobutyl groups retained binding
affinity and antagonistic activity (33b). In terms of solubility, a
good correlation between solubility and lipophilicity was observed
for these four derivatives as shown in Table 4. These results indi-
cate that well-balanced compatibility of activity and solubility
was achieved for compounds with a diethylamino substituent at
the C5 position. Comparing the solubilities of 8-methoxy deriva-
tives with those of 8-methyl derivatives, it was again clear that
introduction of the methoxy substituent at the C8 position effec-
tively enhanced the solubility of the compounds (21d vs 6 and
33a vs 11c). In terms of activity, derivatives with a methyl substi-
tuent at the C8 position exhibited slightly more potent activity than
those with a methoxy substituent, especially in the functional
assay.
Figure 6. Electrostatic potential of compounds 23ca and 23da.
of 8-methyl derivatives, it was clear that introducing a methoxy
substituent at the C8 position had a significant impact on the solu-
bility of the compounds.
0
The effects of R5 and R5 were also examined, and the results are
shown in Table 4. Compounds with a methyl substituent at the C8
position were also prepared to clarify the effects of the methoxy
substituent at C8. The SAR study of our previously reported 8-
methylquinoline series suggested that secondary amines and
amines with saturated heterocycles such as tetrahydropyran were
not tolerated, and that tertiary amines with chain-like alkyl or
Table 5 summarizes the effects of various R2 substituents.
Replacement of methoxy at the ortho-position of the phenyl ring

6566
K. Takeda et al. / Bioorg. Med. Chem. 20 (2012) 6559–6578
Table 4
0
Effects of substitution of R5 and R5 on biological activity and physicochemical properties
R5
5 N
R5'
O
R8
N
O
O
0
No.
R8
R5
R5
Et
Binding IC50 (nM)
Functional IC50 (nM)
Solubility at pH 7.4a
(l
M)
ClogP
21d
29
33a
33b
6
OMe
OMe
OMe
OMe
Me
Et
nPr
Et
Isobutyl
Et
nPr
Et
110
105
392
156
79
91
99
NT
65
24
70
23
74
5.02
6.08
4.52
5.44
5.22
6.23
4.71
nPr
<0.2
89.4
8.7
1.7
2
Methoxyethyl
Methoxyethyl
Et
11a
11c
Me
Me
nPr
88
109
Methoxyethyl
7.2
NT = not tested.
a
pH 7.4: Dulbecco’s phosphate-buffered saline (PBS).
with chloride (36a) resulted in slightly higher binding affinity and
fourfold more potent antagonistic activity than 21d; however, its
aqueous solubility was lower. The SAR of the para-position of the
phenyl ring was also examined. Replacement of methoxymethyl
with cyano or methyl resulted in retained binding affinity and
slightly improved antagonistic activity (36b and 36c, respectively),
but the aqueous solubilities of these compounds were lower. To
determine the cause of the lower solubility of 36b, which exhibited
a ClogP value comparable to that of 21d, the electrostatic potential
of 36b was calculated (Fig. 7). The results indicated that the elec-
tron density of the phenyl ring in the bottom region was remark-
ably lower and the electron density of Area A was also slightly
influenced. As mentioned before, lower electron density in Area
A might have a negative impact on the solubility of these com-
pounds. Judging from the results of the electrostatic potential cal-
culations for 36b, the electronic state of the phenyl ring in the
bottom region might also be important for better solubility.
To clarify the effects of lipophilicity on solubility in the 8-meth-
oxyquinoline series, the correlation between ClogP and solubility
was analyzed (Fig. 8A). This figure shows the results for all of the
8-methoxyquinoline derivatives (eleven compounds). From the
figure, it can be seen that coefficient determination was low
(least-squares method, R2 = 0.44) and no clear correlation was
Figure 7. Electrostatic potential of compound 36b.
found. As mentioned before, electronic density of aromatic groups
might be a key factor for better solubility. Accordingly, compounds
possessing electron-withdrawing groups (23ca, 23da, 36a, and
36b) were excluded from the correlated calculation. Furthermore,
compound 23ba was also excluded because its lower solubility
might be caused by molecular planarity. The result of the recalcu-
lation is shown in Figure 8B. This result indicates that there was a
clear correlation between ClogP and solubility in these six com-
pounds (least-squares method, R2 = 0.86). In short, these results
confirm that the solubilities of the 8-methoxyquinoline derivatives
were basically proportional to their ClogP values and the lower
solubility observed in the excluded compounds was caused by
Table 5
Effects of R2 on biological activity and physicochemical properties
N
O
N
R2
No.
R2
Binding IC50 (nM)
Functional IC50 (nM)
Solubility at pH 7.4a
(lM)
ClogP
21d
36a
36b
36c
2,6-Dimethoxy-4-methoxymethylphenyl
2-Chloro-4-methoxymethyl-6-methoxyphenyl
2,6-Dimethoxy-4-cyanophenyl
110
67
132
99
91
22
63
43
74.0
3.7
11
5.02
6.06
4.92
5.72
2,6-Dimethoxy-4-methylphenyl
17.3
a
pH 7.4: Dulbecco’s phosphate-buffered saline (PBS).

K. Takeda et al. / Bioorg. Med. Chem. 20 (2012) 6559–6578
6567
Figure 8. Correlation between ClogP and solubility in 8-methoxyquinoline derivatives.
Table 6
Pharmacokinetic parameters for 21d in rats
a
b
iv (3 mg kgꢀ1
)
p.o. (10 mg kgꢀ1
)
T1/2 (h)
Cmax
Tmax (h)
1.73 0.29
(l
g mLꢀ1
)
0.305 0.012
0.500 0.000
0.845 0.036
AUC inf. (
l
g mLꢀ1 hꢀ1
)
1.542 0.063
2147.09 449.80
2637.40 105.17
VDss (mL kgꢀ1
)
Cl tot. b (mL hꢀ1 kgꢀ1
BA (%)
)
16.4
0.61
B/P (1 h after p.o.)
Standard errors of the mean are reported (n = 3 rats).
a
The vehicle for iv studies was 5% EtOH-0.02 mol/L HCl/5% glucose.
b
Figure 9. Effects of 21d on the CRF-induced elevation of plasma ACTH concentra-
tion in F344 rats. The compound was administrated orally 60 min before subcu-
taneous injection of CRF. Plasma ACTH concentration was measured 30 min after
CRF injection. Each value represents the mean S.E.M. of 5–6 rats. #P <0.05 versus
non-CRF control (Welch’s t-test), ⁄P <0.05 versus CRF control (unpaired t-test).
The vehicle for p.o. studies was H2O containing 5% DMSO, 5% CremophorÒ EL
and equivalent HCl.
the apparatus (i.e., the illuminated box) and locomotor activity
(i.e., the number of tunnel crossings) were evaluated in BALB/c
mice. Compound 21d (10 and 30 mg kgꢀ1) dose-dependently in-
creased the time and the number of tunnel crossings compared
with vehicle groups (⁄P <0.05, Dunnett multiple comparison test).
Compound 36a also exhibited dose-dependent increases of both
the time and number of crossings, and statistically significant in-
crease was observed at a 30 mg kgꢀ1 dose (⁄P <0.05, Dunnett multi-
ple comparison test). These results indicate that these compounds
were effective at reducing anxiety-related responses in the light-
dark test.
factors other than lipophilicity, such as electron density. Further-
more, it was revealed that lowering lipophilicity without lowering
the electron density of the aromatic groups was crucial for obtain-
ing highly soluble compounds in the 8-methoxyquinoline series.
Among the 8-methoxyquinoline derivatives, compound 21d
was tested in the CRF-induced ACTH release model. In this model,
the effects of the compounds on CRF-induced ACTH release were
evaluated. Figure 9 shows that subcutaneous CRF administration
in rats (10 l
g kgꢀ1) produced a robust increase in the plasma levels
of ACTH. Orally administered, compound 21d attenuated this effect
at a 30 mg kgꢀ1 dose and statistically significant reduction of the
ACTH levels was observed (⁄P <0.05, unpaired t-test). These results
indicate that in vivo functional activity of this compound con-
firmed that it is a CRF receptor antagonist.
Additionally, the anxiolytic efficacy of compound 21d was eval-
uated in the light-dark test (Fig. 10). Compound 36a was also as-
sessed in this behavior model because this compound exhibited
high binding affinity and potent antagonistic activity in the
in vitro assays. In this model, time spent in the aversive parts of
The pharmacokinetic (PK) profile of compound 21d was studied
in rats (Table 6). The rats were dosed intravenously at 3 mg kgꢀ1
and orally at 10 mg kgꢀ1. Total blood clearance (Cl tot. b) of this
compound was high (2637.40 105.17 mL hꢀ1 kgꢀ1), which was
approximately 80% of hepatic blood flow (55 mL minꢀ1 kgꢀ1). Thus,
the hepatic clearance was thought to be the main cause of the
modest bioavailability (16.4%). Additionally, compound concentra-
tion in the brain was measured 1 h after p.o. administration. As a
result, the brain-to-plasma (B/P) concentration ratio was 0.61. This
PK study suggested that 21d was a brain penetrant compound.
Figure 10. Effects of 21d and 36a on anxiety-related behavior in the light/dark test in BALB/c mice (n = 10). Compounds were administrated orally 60 min before the test and
the behavior of the mice was observed for 5 min. Each value represents the mean S.E.M. ⁄P <0.05 versus vehicle groups (Dunnett multiple comparison test).

6568
K. Takeda et al. / Bioorg. Med. Chem. 20 (2012) 6559–6578
was extracted into ethyl acetate and the organic layer was concen-
4. Conclusions
trated under reduced pressure. The residue was purified by silica
gel column chromatography (9:1–5:5 n-heptane/ethyl acetate gra-
dient) to afford the title compound 9 as a light yellow solid (2.41 g,
6.31 mmol, 85.0%).
Through the preparation and evaluation of compounds deriva-
tized at each site (R2, R3, R5, R5’, and R8), SAR and the relationships
between structure and aqueous solubility were investigated. In the
study of derivatization at the C8 position, it was revealed that
introduction of a methoxy substituent overcame the issue of low
aqueous solubility observed in previously reported compound 6,
without loss of in vitro activity. In the 8-methoxyquinoline series,
it was revealed that lowering lipophilicity without lowering the
electron density of the aromatic groups was crucial for obtaining
highly soluble compounds. Among the derivatives, compound
21d exhibited activity in the CRF-induced ACTH release model with
statistical significance by oral administration (30 mg kgꢀ1). Addi-
tionally, orally administered compounds 21d and 36a exhibited
statistically significant anxiolytic effects in the light-dark test
(10 mg kgꢀ1 for 21d and 30 mg kgꢀ1 for 36a). A PK study of 21d
was conducted and the results suggested that this compound
was a brain penetrant. From these results, 21d was identified as
a promising new lead compound for clinical candidates for the
treatment of stress-related disorders.
1H NMR (600 MHz, CDCl3): d = 2.30 (s, 3H), 2.86 (s, 3H), 3.51
(s, 3H), 3.74 (s, 6H), 4.55 (s, 2H), 6.72 (s, 2H), 7.54 (d, J = 7.9 Hz,
1H), 8.26 (d, J = 7.9 Hz, 1H), 8.87 (s, 1H); 13C NMR (151 MHz,
CDCl3): d = 19.3, 19.6, 56.1, 58.5, 75.0, 103.7, 117.5, 120.7, 124.1,
126.1, 130.9, 135.1, 140.7, 143.3, 145.6, 146.4, 156.6, 158.0;
HRMS (ESI+): m/z [M+H]+ calcd for C21H23N2O5+: 383.1601, found:
383.1600.
5.1.4. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-3,8-
dimethylquinolin-5-amine (10)
A mixture of 9 (2.36 g, 6.17 mmol) and Fe (1.64 g, 29.4 mmol) in
a mixture of EtOH (26 mL) and saturated aqueous NH4Cl (2.60 mL)
was stirred at reflux temperature for 1 h. The reaction mixture was
cooled to room temperature and filtered through a pad of NH-silica
gel with ethyl acetate as the eluent to yield the crude title com-
pound 10 as an orange solid (2.16 g, 6.13 mmol, 99.0%), which
was used in the following reaction without further purification.
1H NMR (600 MHz, CDCl3): d = 2.24 (s, 3H), 2.67 (s, 3H), 3.50 (s,
3H), 3.73 (s, 6H), 4.54 (s, 2H), 6.67–6.74 (m, 3H), 7.26 (d, J = 7.6 Hz,
1H), 8.01 (br s, 1H); 13C NMR (151 MHz, CDCl3): d = 18.0, 19.3, 56.2,
58.4, 75.1, 104.1, 109.8, 118.7, 127.7, 128.3, 129.1, 129.7, 139.6,
140.1, 146.3, 154.7, 158.2; HRMS (ESI+): m/z [M+H]+ calcd for
5. Experimental sections
5.1. Chemistry
5.1.1. General methods
C
21H25N2O3+: 353.1860, found: 353.1868.
1H and 13C NMR spectra were recorded on a Bruker Avance spec-
trometer (operating at 600 MHz for 1H and 151 MHz for 13C). Chem-
ical shifts were expressed in ppm (d) from the residual CHCl3 signal
at dH 7.26 ppm and dC 77.0 ppm in CDCl3 (s = singlet, d = doublet,
t = triplet, q = quadruplet, m = multiplet, and b = broad). Coupling
constants (J) are given in Hertz (Hz). IR spectra were obtained using
the KBr method and the attenuated total reflectance (ATR) method
with an FT/IR-620 spectrometer (JASCO). Only the most significant
absorption bands have been reported. High resolution mass spectra
(HRMS) were recorded on a Thermo Fisher LTQ Orbitrap XL spec-
trometer using electrospray ionization (ESI) and a Waters GCT Pre-
mier using electron ionization (EI). LC–MS analyses were performed
using a Shimadzu LCMS-2010 EV to determine the purities of the
compounds tested. Column chromatography was carried out using
5.1.5. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-N,N-
diethyl-3,8-dimethylquinolin-5-amine (6)
A mixture of 10 (150 mg, 426 lmol), acetaldehyde (119 lL,
2.13 mmol), and NaBH(OAc)3 (451 mg, 2.13 mmol) in a mixture
of AcOH (0.2 mL) and THF (2 mL) was stirred at room temperature
for 12 h. The reaction mixture was concentrated under reduced
pressure and the residue was quenched with saturated aqueous
NaHCO3 and extracted into ethyl acetate. The organic layer was
concentrated under reduced pressure and the residue was purified
by NH-silica gel column chromatography (10:0–5:5 n-heptane/
ethyl acetate gradient) to afford the title compound 6 as a colorless
oil (60.8 mg, 149 lmol, 35.0%). Compound purity: >99%.
1H NMR (600 MHz, CDCl3): d = 1.07 (t, J = 7.1 Hz, 6H), 2.23 (s,
3H), 2.72 (s, 3H), 3.19 (q, J = 7.1 Hz, 4H), 3.50 (s, 3H), 3.74 (s, 6H),
4.54 (s, 2H), 6.71 (s, 2H), 7.05 (d, J = 7.6 Hz, 1H), 7.37 (d,
J = 7.6 Hz, 1H), 8.40 (s, 1H); 13C NMR (151 MHz, CDCl3): d = 12.3,
18.2, 19.4, 48.0, 56.2, 58.4, 75.1, 104.1, 117.7, 119.1, 125.8, 127.3,
a Hi-FlashTM column (40
lm, silica gel and NH-silica gel, Yamazen
Corporation). Chemicals and solvents used in the study were
commercially available.
5.1.2. 2-Chloro-3,8-dimethyl-5-nitroquinoline (8)
130.1, 131.8, 132.3, 139.9, 145.1, 147.0, 154.5, 158.2; HRMS
+
To a solution of 7 (7.00 g, 36.5 mmol) in H2SO4 (35 mL) was
added HNO3 at ꢀ10 °C and the reaction mixture was stirred at
room temperature for 1 h. The reaction mixture was poured into
crushed-ice, filtered, and washed with water to afford the title
compound 8 as a light yellow solid (2.35 g, 9.93 mmol, 27.2%).
1H NMR (600 MHz, CDCl3): d = 2.63 (s, 3H), 2.86 (s, 3H), 7.61 (d,
J = 7.9 Hz, 1H), 8.30 (d, J = 7.9 Hz, 1H), 8.92 (s, 1H); 13C NMR
(151 MHz, CDCl3): d = 18.8, 20.6, 120.6, 124.8, 127.7, 133.7, 134.0,
143.1, 145.3, 145.4, 152.5; HRMS (ESI+): m/z [M+H]+ calcd for
(ESI+): m/z [M+H]+ calcd for C25H33N2O3
: 409.2486, found:
409.2487.
5.1.6. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-3,8-
dimethyl-N,N-dipropylquinolin-5-amine (11a)
A mixture of 10 (150 mg, 426 lmol), propionaldehyde (154 lL,
2.13 mmol), and NaBH(OAc)3 (451 mg, 2.13 mmol) in a mixture of
AcOH (0.2 mL) and THF (2 mL) was stirred at room temperature for
12 h. The reaction mixture was concentrated under reduced pres-
sure and the residue was quenched with saturated aqueous NaH-
CO3 and extracted into ethyl acetate. The organic layer was
concentrated under reduced pressure and the residue was purified
by NH-silica gel column chromatography (10:0–5:5 n-heptane/
ethyl acetate gradient) to afford the title compound 11a as a white
C
11H10ClN2O2+: 237.0425, found: 237.0427.
5.1.3. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-3,8-
dimethyl-5-nitroquinoline (9)
A mixture of 8 (1.75 g, 7.40 mmol), [2,6-dimethoxy-4-(methoxy-
methyl)phenyl]boronic acid (2.17 g, 9.61 mmol), Pd(PPh3)4 (427 mg,
solid (34.6 mg, 79.3 lmol, 18.6%). Compound purity: >99%.
1H NMR (600 MHz, CDCl3): d = 0.89 (t, J = 7.0 Hz, 6H), 1.47–1.57
(m, 4H), 2.23 (s, 3H), 2.71 (s, 3H), 3.08 (t, J = 6.8 Hz, 4H), 3.50 (s,
3H), 3.74 (s, 6H), 4.54 (s, 2H), 6.71 (s, 2H), 7.08 (d, J = 7.2 Hz, 1H),
7.36 (d, J = 7.2 Hz, 1H), 8.42 (s, 1H); 13C NMR (151 MHz, CDCl3):
370 lmol), and K2CO3 (3.07 g, 22.2 mmol) in a mixture of 1,2-
dimethoxyethane (40 mL) and water (10 mL) was stirred at reflux
temperature for 4 h. The reaction mixture was cooled to room tem-
perature and concentrated under reduced pressure. The residue

K. Takeda et al. / Bioorg. Med. Chem. 20 (2012) 6559–6578
6569
d = 11.8, 18.2, 19.4, 20.3, 56.3, 56.5, 58.4, 75.1, 104.2, 117.9, 119.1,
125.7, 127.3, 130.0, 131.8, 132.2, 139.9, 145.9, 147.0, 154.5, 158.2;
HRMS (ESI+): m/z [M+H]+ calcd for C27H37N2O3+: 437.2799, found:
437.2798.
759 cmꢀ1, HRMS-ESI m/z [M+H]+ calcd for C9H6ClIN+: 289.9228,
found: 289.9229.
5.1.10. 3-Chloro-2-iodoquinoline (12d)
A solution of diisopropylamine (322
(10 mL) was cooled to ꢀ30 °C. To the solution was added n-BuLi
(2.77 M in n-hexane, 651 L, 1.80 mmol) and the mixture was stir-
lL, 2.30 mmol) in THF
5.1.7. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-N-(2-
methoxyethyl)-3,8-dimethylquinolin-5-amine (11b)
A mixture of 10 (400 mg, 1.14 mmol), 2-bromoethyl methyl
l
red at ꢀ30 °C for 15 min. To the mixture was added a solution of 3-
chloro-4-iodoquinoline (475 mg, 1.64 mmol) in THF (5 mL) at
ꢀ78 °C and the resultant mixture was stirred at ꢀ78 °C for 2 h.
ether (533 lL, 5.68 mmol), and K2CO3 (1.57 g, 11.4 mmol) in DMF
(8 mL) was stirred at 100 °C for 12 h. The reaction mixture was
cooled to room temperature, quenched with water, and extracted
into ethyl acetate. The organic layer was concentrated under re-
duced pressure and the residue was purified by NH-silica gel col-
umn chromatography (10:0–5:5 n-heptane/ethyl acetate
gradient) to afford the title compound 11b as a light yellow solid
Then, water (443 lL, 24.6 mmol) was added to the mixture at
ꢀ78 °C and stirring was continued at ꢀ78 °C for 10 min. The reac-
tion was quenched with AcOH and concentrated under reduced
pressure. The residue was extracted into ethyl acetate and the or-
ganic layer was concentrated under reduced pressure. The residue
was purified by silica gel column chromatography (10:0–9:1 n-
heptane/ethyl acetate gradient) to afford the title compound 12d
(302 mg, 736 lmol, 64.8%).
1H NMR (600 MHz, CDCl3): d = 2.24 (s, 3H), 2.66 (s, 3H), 3.42–
3.47 (m, 5H), 3.49 (s, 3H), 3.73 (s, 6H), 3.77 (t, J = 5.1 Hz, 2H),
4.53 (s, 2H), 6.56 (d, J = 7.8 Hz, 1H), 6.70 (s, 2H), 7.33 (d,
J = 7.8 Hz, 1H), 8.09 (s, 1H); 13C NMR (151 MHz, C5D5N): d = 17.5,
18.5, 43.5, 55.3, 57.4, 57.8, 70.4, 74.2, 103.5, 103.6, 118.5, 118.5,
as a white solid (267 mg, 922 lmol, 56.2%).
1H NMR (600 MHz, CDCl3): d = 7.54–7.66 (m, 1H), 7.67–7.79 (m,
2H), 8.06 (d, J = 7.9 Hz, 1H), 8.12 (s, 1H); 13C NMR (151 MHz,
CDCl3): d = 122.4, 126.8, 127.7, 128.1, 128.8, 130.4, 133.5, 134.1,
~
147.3; IR (KBr):
778, 756 cmꢀ1
m = 1544, 1484, 1359, 1315, 1293, 1114, 961,
124.3, 128.7, 128.9, 129.2, 140.4, 142.3, 146.7, 154.7, 157.9; HRMS
HRMS-ESI m/z [M+H]+ calcd for C9H6ClIN+:
+
;
(ESI+): m/z [M+H]+ calcd for C24H31N2O4
: 411.2278, found:
289.9228, found: 289.9228.
411.2279.
5.1.11. 2-Chloro-3-methyl-5-nitroquinoline (13a)
5.1.8. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-N-ethyl-N-
To a solution of 12a (19.5 g, 110 mmol) in H2SO4 (100 mL) was
added HNO3 (14.3 mL, 226 mmol) at ꢀ10 °C and the reaction mix-
ture was stirred at ꢀ10 °C for 20 min and at room temperature for
1 h. The mixture was poured into crushed-ice, filtered, and washed
with water. The obtained crude product was dissolved in DCM and
silica gel (40 g) was added to the solution. The solution was con-
centrated under reduced pressure and the residue was purified
by silica gel column chromatography (6:1–4:1 n-heptane/ethyl
acetate gradient) to afford the title compound 13a as a yellow solid
(11.3 g, 50.8 mmol, 46.1%).
(2-methoxyethyl)-3,8-dimethylquinolin-5-amine (11c)
A mixture of 11b (120 mg, 292
l
mol), acetaldehyde (33.0
lL,
585 mol), and NaBH(OAc)3 (124 mg, 585
l
lmol) in a mixture of
AcOH (0.2 mL) and THF (2 mL) was stirred at room temperature
for 12 h. The reaction mixture was concentrated under reduced
pressure and the residue was quenched with saturated aqueous
NaHCO3 and extracted into ethyl acetate. The organic layer was
concentrated under reduced pressure and the residue was purified
by silica gel column chromatography (10:0–7:3 n-heptane/ethyl
acetate gradient) to afford the title compound 11c as a colorless
1H NMR (600 MHz, CDCl3): d = 2.63 (s, 3H), 7.76 (dd, J = 8.3,
7.6 Hz, 1H), 8.30 (d, J = 8.3 Hz, 1H), 8.36 (d, J = 7.6 Hz, 1H), 8.87
(s, 1H); 13C NMR (151 MHz, CDCl3): d = 20.7, 120.6, 124.9, 127.7,
~
m = 3096,
oil (95.7 mg, 218 lmol, 74.6%). Compound purity: >99%.
1H NMR (600 MHz, CDCl3): d = 1.07 (t, J = 7.0 Hz, 3H), 2.23 (s,
3H), 2.72 (s, 3H), 3.25 (q, J = 7.0 Hz, 2H), 3.31 (s, 3H), 3.35 (t,
J = 6.0 Hz, 2H), 3.47 (t, J = 6.0 Hz, 2H), 3.50 (s, 3H), 3.74 (s, 6H),
4.54 (s, 2H), 6.71 (s, 2H), 7.13 (d, J = 7.4 Hz, 1H), 7.38 (d,
J = 7.4 Hz, 1H), 8.45 (s, 1H); 13C NMR (151 MHz, CDCl3): d = 12.4,
18.3, 19.3, 49.6, 53.5, 56.2, 58.4, 58.8, 70.8, 75.1, 104.1, 118.1,
133.7, 134.2, 135.2, 144.9, 146.6, 153.8; IR (KBr):
3044, 1515, 1484, 1392, 1333, 1176, 1061, 937, 832, 740 cmꢀ1
;
HRMS-ESI m/z [M+H]+ calcd for C10H8ClN2O2+: 223.0269, found:
223.0264.
119.1, 125.8, 127.3, 130.3, 131.7, 132.8, 139.9, 144.9, 147.0,
5.1.12. 2-Chloro-5-nitroquinoline (13b)
+
154.7, 158.2; HRMS (ESI+): m/z [M+H]+ calcd for C26H35N2O4
:
To a solution of 12b (5.00 g, 30.6 mmol) in H2SO4 (15 mL) was
added HNO3 (2.04 mL, 45.9 mmol) at ꢀ10 °C and the reaction mix-
ture was stirred at ꢀ10 °C for 20 min and at room temperature for
1 h. The mixture was poured into crushed-ice, filtered and, washed
with water. The obtained crude title compound (5.64 g), which was
used in the following reaction without further purification. (CAS
Registry Number: 13067-94-2).
439.2591, found: 439.2590.
5.1.9. 3-Chloro-4-iodoquinoline
A solution of diisopropylamine (720 lL, 5.14 mmol) in THF
(6 mL) was cooled to ꢀ30 °C. To the solution was added n-BuLi
(2.77 M in n-hexane, 1.46 mL, 4.04 mmol) and the mixture was
stirred at ꢀ30 °C for 15 min. To the mixture was added a solution
of 3-chloroquinoline (600 mg, 3.67 mmol) in THF (3 mL) at
ꢀ78 °C and the mixture was stirred at ꢀ78 °C for 2 h. Then, a solu-
tion of I2 (1.40 g, 5.51 mmol) in THF (3 mL) was added to the mix-
ture at ꢀ78 °C and the mixture was stirred at ꢀ78 °C for 2.15 h. The
reaction mixture was quenched with AcOH and concentrated un-
der reduced pressure. The residue was extracted into ethyl acetate.
The organic layer was purified by silica gel column chromatogra-
phy (n-heptane) to afford the title compound as a white solid
(515 mg, 1.78 mmol, 48.5%).
5.1.13. 3-Fluoro-2-iodo-5-nitroquinoline (13c)
To a solution of 12c (5.00 g, 18.3 mmol) in H2SO4 (40 mL) was
added HNO3 (1.22 mL, 27.5 mmol) at 0 °C and the reaction mixture
was stirred at 0 °C for 20 min. Fuming HNO3 (1.14 mL, 27.5 mmol)
was added and the mixture was stirred at the same temperature
for 2 h. The mixture was poured into crushed ice and extracted into
ethyl acetate. The organic layer was concentrated and the residue
was purified by silica gel column chromatography (9:1–7:3 n-hep-
tane/ethyl acetate gradient) to afford the title compound 13c as a
white solid (2.76 g, 8.68 mmol, 47.4%).
1H NMR (600 MHz, CDCl3): d = 7.63–7.68 (m, 1H), 7.71–7.77 (m,
1H), 8.06 (d, J = 8.3 Hz, 1H), 8.10 (d, J = 8.3 Hz, 1H), 8.73 (s, 1H); 13
C
1H NMR (600 MHz, CDCl3): d = 7.80 (dd, J = 8.3, 7.9 Hz, 1H), 8.41
(d, J = 8.3 Hz, 1H), 8.52 (d, J = 7.9 Hz, 1H), 8.68 (d, J = 8.7 Hz, 1H);
13C NMR (151 MHz, CDCl3): d = 113.1, 114.8, 121.7, 126.4, 127.5,
NMR (151 MHz, CDCl3): d = 114.8, 129.2, 130.0, 130.0, 131.6, 132.5,
135.3, 145.6, 148.2; IR (KBr):
~
m
= 1549, 1483, 1334, 1115, 866,

6570
K. Takeda et al. / Bioorg. Med. Chem. 20 (2012) 6559–6578
~
136.0, 144.6, 146.6, 156.8; IR (KBr):
m
= 3092, 1516, 1396, 1335,
1H), 8.35 (d, J = 7.6 Hz, 1H), 8.49 (d, J = 8.3 Hz, 1H), 8.98 (d,
1212, 1054, 823 cmꢀ1; HRMS-EI: m/z [M]+ calcd for C9H4FIN2O2
:
J = 9.1 Hz, 1H); 13C NMR (151 MHz, CDCl3): d = 56.0, 58.2, 74.8,
+
317.9296, found: 317.9334.
103.3, 117.3, 120.1, 124.1, 127.0, 127.6, 131.1, 136.7, 141.4,
~
145.6, 148.4, 157.1, 158.1; IR (KBr):
m
= 2942, 2842, 1611, 1578,
5.1.14. 3-Chloro-2-iodo-5-nitroquinoline (13d)
1524, 1457, 1417, 1332, 1229, 1116, 1098, 825 cmꢀ1; HRMS-ESI
The title compound was prepared from 12d using a method
analogous to that described for 13c in 42.3% yield as a light yellow
solid.
m/z [M+H]+ calcd for C19H19N2O5+: 355.1289, found: 355.1291.
5.1.18. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-3-fluoro-
5-nitroquinoline (14c)
1H NMR (600 MHz, CDCl3): d = 7.81 (dd, J = 8.3, 7.9 Hz, 1H), 8.37
(d, J = 8.3 Hz, 1H), 8.48 (d, J = 7.9 Hz, 1H), 9.06 (s, 1H); 13C NMR
(151 MHz, CDCl3): d = 120.8, 124.9, 126.1, 128.5, 130.3, 135.9,
~
The title compound was prepared from 13c using a method
analogous to that described for 14b in 99.0% yield as a white solid.
1H NMR (600 MHz, CDCl3): d = 3.45 (s, 3H), 3.75 (s, 6H), 4.53
(s, 2H), 6.69 (s, 2H), 7.75 (dd, J = 8.3, 7.9 Hz, 1H), 8.47 (d,
J = 7.9 Hz, 1H), 8.51 (d, J = 8.3 Hz, 1H), 8.78 (d, J = 10.6 Hz, 1H);
13C NMR (151 MHz, CDCl3): d = 56.0, 58.3, 74.7, 103.1, 111.4,
115.4, 122.1, 125.5, 126.1, 136.9, 142.4, 144.8, 145.3, 149.1,
~
137.8, 144.2, 146.9; IR (KBr):
m = 3077, 1522, 1351, 1327, 1282,
1128, 903, 822, 738 cmꢀ1; HRMS-ESI m/z [M+H]+ calcd for C9H5ClI-
N2O2+: 334.9079, found: 334.9087.
5.1.15. 2-Chloro-3-ethyl-5-nitroquinoline (13e)
The title compound was prepared from 12e using a method
analogous to that described for 13a in 40.4% yield as a white solid.
1H NMR (600 MHz, CDCl3): d = 1.40 (t, J = 7.6 Hz, 3H), 2.99 (q,
J = 7.6 Hz, 2H), 7.77 (dd, J = 8.3, 7.6 Hz, 1H), 8.31 (d, J = 8.3 Hz,
1H), 8.37 (d, J = 7.6 Hz, 1H), 8.86 (s, 1H); 13C NMR (151 MHz,
CDCl3): d = 13.4, 27.1, 120.7, 124.9, 127.7, 132.4, 135.2, 139.4,
~
157.5, 158.5; IR (KBr):
m = 2935, 1582, 1527, 1461, 1413, 1340,
1231, 1129, 1105, 817 cmꢀ1; HRMS-ESI m/z [M+H]+ calcd for
19H18FN2O5+: 373.1194, found: 373.1183.
C
5.1.19. 3-Chloro-2-[2,6-dimethoxy-4-(methoxymethyl)phenyl]-
5-nitroquinoline (14d)
145.0, 146.5, 153.5; IR (KBr):
m = 3089, 2966, 1518, 1395, 1335,
A
mixture of 13d (1.55 g, 4.63 mmol), [2,6-dimethoxy-4-
1172, 1064, 932, 914, 824, 737 cmꢀ1; HRMS-ESI m/z [M+H]+ calcd
(methoxymethyl)phenyl]boronic acid (1.57 g, 6.95 mmol), 1 M
aqueous Na2CO3 (9.26 mL, 9.26 mmol), and Pd(PPh3)4 (535 mg,
0.463 mmol) in a mixture of toluene (20 mL) and ethanol (10 mL)
was stirred at 100 °C for 2 h. The reaction mixture was cooled to
room temperature and extracted into ethyl acetate. The organic
layer was washed with brine, dried over MgSO4 and concentrated
under reduced pressure. The residue was purified by silica gel col-
umn chromatography (3:1–1:2 n-heptane/ethyl acetate gradient)
to afford the title compound 14d as a light yellow solid (772 mg,
1.99 mmol, 42.9%).
for C11H10ClN2O2+: 237.0426, found: 237.0426.
5.1.16. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-3-methyl-
5-nitroquinoline (14a)
A
mixture of 13a (1.77 g, 7.95 mmol), [2,6-dimethoxy-4-
(methoxymethyl)phenyl]boronic acid (2.70 g, 11.9 mmol), Pd(OAc)2
(178 mg, 0.795 mmol), PPh3 (1.04 g, 3.98 mmol), and K2CO3 (3.30 g,
23.9 mmol) in a mixture of 1,2-dimethoxyethane (60 mL) and H2O
(15 mL) was stirred at reflux temperature for 2 h. The mixture was
cooled to room temperature, followed by addition of ethyl acetate.
After thoroughly shaking the mixture, the organic layer was sepa-
rated and the aqueous layer was extracted into ethyl acetate. The
combined organic layers were concentrated under reduced pres-
sure and the residue was purified by NH-silica gel column chroma-
tography (9:1–1:1 n-heptane/ethyl acetate gradient) to afford the
title compound 14a as a light yellow solid (2.56 g, 6.95 mmol,
87.4%).
1H NMR (600 MHz, CDCl3): d = 3.46 (s, 3H), 3.74 (s, 6H), 4.54 (s,
2H), 6.68 (s, 2H), 7.77 (dd, J = 8.3, 7.6 Hz, 1H), 8.44 (d, J = 7.6 Hz,
1H), 8.48 (d, J = 8.3 Hz, 1H), 9.16 (s, 1H); 13C NMR (151 MHz,
CDCl3): d = 56.0, 58.3, 74.8, 103.2, 114.8, 121.1, 125.3, 127.2,
131.1, 134.3, 136.9, 141.9, 144.4, 146.5, 156.2, 158.1; IR (KBr):
~
m
= 2926, 2850, 1583, 1528, 1417, 1348, 1232, 1123, 1094, 1079,
818, 736 cmꢀ1; HRMS-ESI m/z [M+H]+ calcd for C19H18ClN2O5
:
+
389.0899, found: 389.0886.
1H NMR (600 MHz, CDCl3): d = 2.29 (s, 3H), 3.46 (s, 3H), 3.72 (s,
6H), 4.53 (s, 2H), 6.68 (s, 2H), 7.69 (dd, J = 8.3, 7.6 Hz, 1H), 8.32 (d,
J = 7.6 Hz, 1H), 8.45 (d, J = 8.3 Hz, 1H), 8.81 (s, 1H); 13C NMR
(151 MHz, CDCl3): d = 19.8, 55.9, 58.3, 74.9, 103.3, 116.3, 120.9,
124.1, 126.0, 130.8, 135.7, 136.5, 141.1, 145.1, 147.0, 157.8,
~
5.1.20. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-3-ethyl-5-
nitroquinoline (14e)
The title compound was prepared from 13e using a method
analogous to that described for 14b in 53.0% yield as a light brown
oil.
158.5; IR (KBr):
m = 2943, 2841, 1582, 1523, 1415, 1335, 1232,
1121, 822 cmꢀ1; HRMS-ESI m/z [M+H]+ calcd for C20H21N2O5
:
+
1H NMR (600 MHz, CDCl3): d = 1.18 (t, J = 7.5 Hz, 3H), 2.60 (q,
J = 7.5 Hz, 2H), 3.47 (s, 3H), 3.71 (s, 6H), 4.53 (s, 2H), 6.68 (s, 2H),
7.69 (dd, J = 8.3, 7.6 Hz, 1H), 8.33 (d, J = 7.6 Hz, 1H), 8.45 (d,
J = 8.3 Hz, 1H), 8.83 (s, 1H); 13C NMR (151 MHz, CDCl3):
d = 13.8, 26.0, 55.8, 58.3, 74.9, 103.3, 116.3, 121.0, 124.1, 126.0,
129.3, 136.5, 141.0, 141.2, 145.2, 146.9, 157.9, 158.2; IR (KBr):
~
369.1445, found: 369.1435.
5.1.17. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-5-
nitroquinoline (14b)
A mixture of crude 13b (5.64 g, 27 mmol), [2,6-dimethoxy-4-
(methoxymethyl)phenyl]boronic acid (7.93 g, 35.1 mmol), Pd(PPh3)4
(1.56 g, 1.35 mmol), and Na2CO3 (7.15 g, 67.5 mmol) in a mixture
of 1,2-dimethoxyethane (100 mL) and H2O (20 mL) was stirred at
reflux temperature for 4 h. The mixture was cooled to room tem-
perature and concentrated under reduced pressure. The residue
was extracted into ethyl acetate and the organic layer was concen-
trated under reduced pressure. The residue was purified by silica
gel column chromatography (9:1–7:3 n-heptane/ethyl acetate gra-
dient) to afford the title compound 14b as a white solid (1.43 g,
4.04 mmol, 13.2% in two steps).
m
= 2963, 2933, 2838, 1609, 1581, 1522, 1456, 1414, 1336,
1238, 1123, 1092, 824 cmꢀ1; HRMS-ESI m/z [M+H]+ calcd for
C
21H23N2O5+: 383.1607, found: 383.1602.
5.1.21. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-3-
methylquinolin-5-amine (15a)
To a solution of 14a (2.56 g, 6.95 mmol) in ethyl acetate (70 mL)
was added 10% Pd-C (2.00 g) and the mixture was stirred under an
atmosphere of hydrogen at room temperature for 2 h. The reaction
mixture was filtered through Celite and the filtrate was concen-
trated under reduced pressure to afford the crude title compound
1H NMR (600 MHz, CDCl3): d = 3.45 (s, 3H), 3.74 (s, 6H), 4.52 (s,
2H), 6.68 (s, 2H), 7.64 (d, J = 9.1 Hz, 1H), 7.77 (dd, J = 8.3, 7.6 Hz,

K. Takeda et al. / Bioorg. Med. Chem. 20 (2012) 6559–6578
6571
15a as a light yellow solid (2.33 g, 6.89 mmol, 99.1%), which was
5.1.26. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-N,N-
used in the following reaction without further purification.
1H NMR (600 MHz, CDCl3): d = 2.22 (s, 3H), 3.45 (s, 3H), 3.70 (s,
6H), 4.51 (s, 2H), 6.66 (s, 2H), 6.77 (d, J = 7.2 Hz, 1H), 7.41 (dd,
J = 8.3, 7.2 Hz, 1H), 7.62 (d, J = 8.3 Hz, 1H), 7.97 (s, 1H); 13C NMR
(151 MHz, CDCl3): d = 19.4, 55.9, 58.2, 75.0, 103.5, 109.8, 117.6,
118.3, 120.4, 128.5, 128.8, 130.1, 140.3, 141.5, 147.6, 156.3,
157.9; HRMS-ESI m/z [M+H]+ calcd for C20H23N2O3+: 339.1703,
found: 339.1694.
diethyl-3-methylquinolin-5-amine (16a)
A mixture of 15a (4.00 g, 11.8 mmol), acetaldehyde (1.99 mL,
35.4 mmol), and NaBH(OAc)3 (7.50 g, 35.4 mmol) in a mixture of
THF (100 mL) and AcOH (10 mL) was stirred at room temperature
for 4.5 h. The mixture was concentrated under reduced pressure.
The residue was quenched with saturated aqueous NaHCO3 and
extracted into ethyl acetate. The organic layer was concentrated
under reduced pressure and the residue was purified by silica gel
column chromatography (9:1–0:10 n-heptane/ethyl acetate gradi-
ent) to afford the title compound 16a as a light yellow oil (4.08 g,
10.3 mmol, 87.6%).
5.1.22. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]quinolin-
5-amine (15b)
A mixture of 14b (1.30 g, 3.67 mmol) and Fe (1.64 g, 29.4 mmol)
in a mixture of EtOH (26 mL) and saturated aqueous NH4Cl
(2.60 mL) was stirred at reflux temperature for 1 h. The reaction
mixture was cooled to room temperature and filtered through a
pad of NH-silica gel with ethyl acetate as the eluent to yield the
crude title compound 15b as a brown solid (1.18 g, 3.64 mmol,
99.1%), which was used in the following reaction without further
purification.
1H NMR (600 MHz, CDCl3): d = 1.08 (t, J = 6.9 Hz, 6H), 2.23 (s,
3H), 3.22 (q, J = 6.9 Hz, 4H), 3.45 (s, 3H), 3.71 (s, 6H), 4.52 (s,
2H), 6.66 (s, 2H), 7.12 (d, J = 7.2 Hz, 1H), 7.52 (dd, J = 8.3, 7.2 Hz,
1H), 7.84 (d, J = 8.3 Hz, 1H), 8.39 (s, 1H); 13C NMR (151 MHz,
CDCl3): d = 12.2, 19.4, 47.8, 55.9, 58.2, 75.0, 103.5, 117.8, 124.7,
125.7, 127.4, 130.5, 131.9, 140.2, 147.2, 148.2, 156.0, 158.0; IR
~
(KBr):
m = 2963, 2807, 1582, 1454, 1415, 1379, 1240, 1123,
1096, 823 cmꢀ1; HRMS-ESI m/z [M+H]+ calcd for C24H31N2O3
:
+
1H NMR (600 MHz, CDCl3): d = 3.43 (s, 3H), 3.72 (s, 6H), 4.17 (br
s, 2H), 4.51 (s, 2H), 6.65 (s, 2H), 6.80 (d, J = 7.2 Hz, 1H), 7.34 (d,
J = 8.7 Hz, 1H), 7.48 (dd, J = 8.3, 7.2 Hz, 1H), 7.65 (d, J = 8.3 Hz,
1H), 8.17 (d, J = 8.7 Hz, 1H); 13C NMR (151 MHz, CDCl3): d = 56.0,
58.1, 74.9, 103.5, 109.8, 117.4, 118.7, 120.6, 122.8, 128.9, 129.5,
140.5, 142.2, 149.1, 155.2, 158.3; HRMS-ESI m/z [M+H]+ calcd for
395.2329, found: 395.2315.
5.1.27. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-N,N-
diethylquinolin-5-amine (16b)
The title compound was prepared from 15b using a method
analogous to that described for 16a in 71.7% yield as a light yellow
oil.
C
19H21N2O3+: 325.1547, found: 325.1547.
1H NMR (600 MHz, CDCl3): d = 1.08 (t, J = 7.2 Hz, 6H), 3.22 (q,
J = 7.2 Hz, 4H), 3.44 (s, 3H), 3.73 (s, 6H), 4.51 (s, 2H), 6.66 (s, 2H),
7.16 (d, J = 7.6 Hz, 1H), 7.35 (d, J = 8.3 Hz, 1H), 7.59 (dd, J = 8.7,
7.6 Hz, 1H), 7.88 (d, J = 8.3 Hz, 1H), 8.60 (d, J = 8.7 Hz, 1H); 13C
NMR (151 MHz, CDCl3): d = 12.3, 47.8, 56.0, 58.1, 74.9, 103.5,
117.9, 118.8, 123.3, 124.8, 125.0, 128.5, 132.2, 140.4, 148.0,
~
5.1.23. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-3-
fluoroquinolin-5-amine (15c)
The crude title compound was prepared from 14c using a meth-
od analogous to that described for 15b in quantitative yield as a
light yellow solid, which was used in the following reaction with-
out further purification.
149.6, 154.9, 158.3; IR (KBr):
m = 2970, 2933, 2838, 1607, 1582,
1H NMR (600 MHz, CDCl3): d = 3.43 (s, 3H), 3.74 (s, 6H), 4.52 (s,
2H), 6.66 (s, 2H), 6.83 (d, J = 7.6 Hz, 1H), 7.44 (dd, J = 8.3, 7.6 Hz,
1H), 7.66 (d, J = 8.3 Hz, 1H), 7.80 (d, J = 9.8 Hz, 1H); HRMS-ESI m/
z [M+H]+ calcd for C19H20FN2O3+: 343.1453, found: 343.1445.
1456, 1415, 1230, 1124, 823 cmꢀ1; HRMS-ESI m/z [M+H]+ calcd
for C23H29N2O3+: 381.2173, found: 381.2174.
5.1.28. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-N,N-
diethyl-3-fluoroquinolin-5-amine (16c)
5.1.24. 3-Chloro-2-[2,6-dimethoxy-4-
The title compound was prepared from 15c using a method
analogous to that described for 16a in 71.5% yield as a light yellow
solid.
(methoxymethyl)phenyl]quinolin-5-amine (15d)
The title compound was prepared from 14d using a method
analogous to that described for 15b in 96.8% yield as a light yellow
foam, which was used in the following reaction without further
purification.
1H NMR (600 MHz, CDCl3): d = 1.07 (t, J = 6.9 Hz, 6H), 3.20 (q,
J = 6.9 Hz, 4H), 3.44 (s, 3H), 3.75 (s, 6H), 4.53 (s, 2H), 6.67 (s, 2H),
7.21 (d, J = 7.2 Hz, 1H), 7.57 (dd, J = 8.7, 7.2 Hz, 1H), 7.90 (d,
J = 8.7 Hz, 1H), 8.24 (d, J = 10.6 Hz, 1H); 13C NMR (151 MHz, CDCl3):
d = 12.3, 47.9, 56.0, 58.1, 74.8, 103.3, 112.9, 115.5, 119.0, 124.9,
127.2, 127.5, 141.4, 146.2, 146.5, 147.6, 155.4, 158.7; IR (KBr):
~
1H NMR (600 MHz, CDCl3): d = 3.44 (s, 3H), 3.73 (s, 6H), 4.13 (br
s, 2H), 4.53 (s, 2H), 6.66 (s, 2H), 6.81 (d, J = 7.6 Hz, 1H), 7.47 (dd,
J = 8.7, 7.6 Hz, 1H), 7.62 (d, J = 8.7 Hz, 1H), 8.22 (s, 1H); 13C NMR
(151 MHz, CDCl3): d = 56.0, 58.1, 74.9, 103.3, 110.6, 116.0, 118.7,
120.4, 128.6, 128.9, 129.7, 141.1, 141.4, 147.3, 153.7, 158.2;
HRMS-ESI m/z [M+H]+ calcd for C19H20ClN2O3+: 359.1157, found:
359.1146.
m
= 2964, 2934, 2822, 1583, 1459, 1409, 1359, 1230, 1128, 1101,
819 cmꢀ1
;
HRMS-ESI m/z [M+H]+ calcd for C23H28FN2O3
:
+
399.2079, found: 399.2066.
5.1.29. 3-Chloro-2-[2,6-dimethoxy-4-(methoxymethyl)phenyl]-
N,N-diethylquinolin-5-amine (16d)
The title compound was prepared from 15d using a method
analogous to that described for 16a in 68.7% yield as a light yellow
oil.
5.1.25. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-3-
ethylquinolin-5-amine (15e)
The title compound was prepared from 14e using a method
analogous to that described for 15b in 97.6% yield as a yellow solid,
which was used in the following reaction without further
purification.
1H NMR (600 MHz, CDCl3): d = 1.08 (t, J = 6.9 Hz, 6H), 3.21 (q,
J = 6.9 Hz, 4H), 3.44 (s, 3H), 3.74 (s, 6H), 4.53 (s, 2H), 6.67 (s, 2H),
7.19 (d, J = 7.2 Hz, 1H), 7.59 (dd, J = 8.3, 7.2 Hz, 1H), 7.86 (d,
J = 8.3 Hz, 1H), 8.65 (s, 1H); 13C NMR (151 MHz, CDCl3): d = 12.2,
47.9, 56.0, 58.1, 74.9, 103.3, 116.1, 119.0, 124.7, 126.3, 128.7,
~
m = 2928,
1H NMR (600 MHz, CDCl3): d = 1.14 (t, J = 7.4 Hz, 3H), 2.54 (q,
J = 7.4 Hz, 2H), 3.45 (s, 3H), 3.69 (s, 6H), 4.51 (s, 2H), 6.65 (s, 2H),
6.78 (d, J = 7.2 Hz, 1H), 7.41 (dd, J = 7.9, 7.2 Hz, 1H), 7.63 (d,
J = 7.9 Hz, 1H), 7.99 (s, 1H); 13C NMR (151 MHz, CDCl3): d = 14.1,
25.7, 55.8, 58.2, 75.0, 103.4, 109.8, 117.5, 118.4, 120.5, 127.2,
128.5, 135.8, 140.2, 141.6, 147.6, 156.0, 158.1; HRMS-ESI m/z
[M+H]+ calcd for C21H25N2O3+: 353.1860, found: 353.1866.
129.4, 131.8, 141.0, 147.3, 147.8, 153.5, 158.2; IR (KBr):
2837, 1582, 1461, 1415, 1361, 1228, 1126, 1103, 1069, 818 cmꢀ1
;
HRMS-ESI m/z [M+H]+ calcd for C23H28ClN2O3+: 415.1783, found:
415.1766.

6572
K. Takeda et al. / Bioorg. Med. Chem. 20 (2012) 6559–6578
5.1.30. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-N,N,3-
triethylquinolin-5-amine (16e)
The title compound was prepared from 15e using a method
analogous to that described for 16a in 69.6% yield as a light yellow
oil.
ethyl acetate gradient) to afford the title compound 19 as a yellow
oil (199 mg, 471 mol, 34.9%).
l
1H NMR (600 MHz, CDCl3): d = 1.15 (t, J = 6.9 Hz, 6H), 2.26 (s,
3H), 3.37 (q, J = 6.9 Hz, 4H), 3.49 (s, 3H), 3.74 (s, 6H), 4.54 (s, 2H),
6.71 (s, 2H), 7.11 (d, J = 7.9 Hz, 1H), 8.19 (d, J = 7.9 Hz, 1H), 8.29
(s, 1H), 11.25 (s, 1H); 13C NMR (151 MHz, CDCl3): d = 12.2, 19.5,
47.1, 56.2, 58.4, 75.0, 103.8, 115.9, 118.0, 123.7, 126.3, 128.2,
130.8, 132.2, 140.5, 148.1, 153.9, 156.8, 158.0, 193.1; IR (KBr):
~
1H NMR (600 MHz, CDCl3): d = 1.10 (t, J = 7.1 Hz, 6H), 1.16 (t,
J = 7.5 Hz, 3H), 2.54 (q, J = 7.5 Hz, 2H), 3.23 (q, J = 7.1 Hz, 4H),
3.45 (s, 3H), 3.70 (s, 6H), 4.52 (s, 2H), 6.65 (s, 2H), 7.12 (d,
J = 7.6 Hz, 1H), 7.52 (dd, J = 8.3, 7.6 Hz, 1H), 7.84 (d, J = 8.3 Hz,
1H), 8.42 (s, 1H); 13C NMR (151 MHz, CDCl3): d = 12.3, 14.1, 25.6,
47.8, 55.8, 58.2, 75.0, 103.5, 117.6, 117.7, 124.5, 125.7, 127.5,
~
m
= 2958, 2932, 2837, 1667, 1563, 1453, 1414, 1225, 1121, 1097,
1068, 807 cmꢀ1; HRMS-ESI m/z [M+H]+ calcd for C25H31N2O4
:
+
423.2279, found: 423.2263.
130.2, 136.2, 140.1, 147.4, 148.1, 155.6, 158.1; IR (KBr):
m = 2967,
2933, 2871, 2838, 1610, 1582, 1456, 1415, 1230, 1123, 824 cmꢀ1
;
5.1.34. 8-(Difluoromethyl)-2-[2,6-dimethoxy-4-
(methoxymethyl)phenyl]-N,N,-diethyl-3-methylquinolin-5-
amine (20a)
HRMS-ESI m/z [M+H]+ calcd for C25H33N2O3+: 409.2486, found:
409.2487.
A mixture of 19 (90 mg, 0.213 mmol) and [bis(2-methoxy-
ethyl)amino]sulfur trifluoride (0.118 mL, 0.639 mmol) in DCM
(3 mL) was stirred at room temperature for overnight. The reaction
mixture was quenched with saturated aqueous NaHCO3 and ex-
tracted into DCM. The organic layer was concentrated under re-
duced pressure and the residue was purified by silica gel column
chromatography (10:0–6:4 n-heptane/ethyl acetate gradient) to
afford the title compound 20a as a light yellow oil (33.1 mg,
5.1.31. 8-Chloro-2-[2,6-dimethoxy-4-(methoxymethyl)phenyl]-
N,N-diethyl-3-methylquinolin-5-amine (17)
To a solution of 16a (155 mg, 0.391 mmol) in DMF (3 mL) was
added NCS (52.2 mg, 0.391 mmol) and the mixture was stirred at
60 °C for 2 h. The reaction mixture was quenched with H2O and ex-
tracted into ethyl acetate. The organic layer was concentrated un-
der reduced pressure and the residue was purified by silica gel
column chromatography (9:1–6:4 n-heptane/ethyl acetate gradi-
ent) to afford the title compound 17 as a colorless foam (4.1 mg,
74.5 lmol, 35.0%). Compound purity: >99%.
1H NMR (600 MHz, CDCl3): d = 1.10 (t, J = 7.0 Hz, 6H), 2.24 (s,
3H), 3.27 (q, J = 7.0 Hz, 4H), 3.49 (s, 3H), 3.73 (s, 6H), 4.53 (s, 2H),
6.70 (s, 2H), 7.14 (d, J = 7.8 Hz, 1H), 7.80 (t, J = 36.0 Hz, 1H), 7.85
(d, J = 7.8 Hz, 1H), 8.34 (s, 1H); 13C NMR (151 MHz, CDCl3):
d = d = 12.1, 19.4, 47.4, 56.2, 58.4, 75.0, 104.0, 112.9, 116.7, 118.4,
124.7, 124.8, 126.2, 131.0, 131.9, 140.3, 145.4, 149.7, 156.0,
~
m = 2971, 2933, 2840, 1612, 1579, 1456, 1415,
9.56 lmol, 2.44%). Compound purity: 95%.
1H NMR (600 MHz, CDCl3): d = 1.07 (t, J = 7.2 Hz, 6H), 2.23 (s,
3H), 3.20 (q, J = 7.2 Hz, 4H), 3.47 (s, 3H), 3.73 (s, 6H), 4.51 (s, 2H),
6.67 (s, 2H), 7.03 (d, J = 8.1 Hz, 1H), 7.62 (d, J = 8.1 Hz, 1H), 8.39
(s, 1H); 13C NMR (151 MHz, CDCl3): d = 12.2, 19.4, 47.8, 56.3,
58.3, 75.0, 104.1, 117.9, 118.2, 127.1, 127.4, 127.9, 131.6, 132.2,
~
158.1; IR (KBr):
140.3, 144.1, 146.4, 156.7, 158.2; IR (KBr):
m
= 2970, 2840, 1583,
1376, 1228, 1123, 1093, 1014, 823 cmꢀ1; HRMS-ESI m/z [M+H]+
1444, 1416, 1233, 1121, 1062, 828 cmꢀ1; HRMS-ESI m/z [M+H]+
calcd for C25H31F2N2O3+: 445.2297, found: 445.2282.
calcd for C24H30ClN2O3+: 429.1940, found: 429.1926.
5.1.35. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-N,N-
diethyl-8-(methoxymethyl)-3-methylquinolin-5-amine (20b)
A mixture of 19 (90 mg, 0.213 mmol) and decaborane (23 mg,
0.213 mmol) in MeOH (3 mL) was stirred at room temperature
for overnight. The reaction mixture was concentrated under re-
duced pressure and the residue was extracted into ethyl acetate.
The organic layer was concentrated and the residue was purified
by silica gel column chromatography (10:0–7:3 n-heptane/ethyl
acetate gradient) to afford the title compound 20b as a light yellow
5.1.32. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-N,N-
diethyl-8-iodo-3-methylquinolin-5-amine (18)
To a solution of 16a (4.00 g, 10.1 mmol) in DMF (80 mL) was
added NIS (2.50 g, 11.1 mmol) at 0 °C and the mixture was stirred
at room temperature for overnight. The reaction mixture was
quenched with H2O and extracted into ethyl acetate. The organic
layer was concentrated under reduced pressure and the residue
was purified by silica gel column chromatography (9:1–6:4 n-hep-
tane/ethyl acetate gradient) to afford the title compound 18 as a
light red oil (3.44 g, 6.61 mmol, 65.4%).
oil (33.1 mg, 75.5 lmol, 35.4%). Compound purity: >99%.
1H NMR (600 MHz, CDCl3): d = 1.07 (t, J = 7.1 Hz, 6H), 2.22 (s,
3H), 3.20 (q, J = 7.1 Hz, 4H), 3.49 (s, 3H), 3.50 (s, 3H), 3.72 (s, 6H),
4.53 (s, 2H), 5.13 (s, 2H), 6.69 (s, 2H), 7.13 (d, J = 7.8 Hz, 1H), 7.62
(d, J = 7.8 Hz, 1H), 8.37 (s, 1H); 13C NMR (151 MHz, CDCl3):
d = 12.2, 19.4, 47.8, 56.2, 58.4, 58.6, 70.7, 75.1, 104.0, 117.7,
118.9, 125.2, 125.3, 130.3, 131.7, 131.8, 140.0, 145.7, 146.3,
~
154.6, 158.2; IR (KBr): m = 2969, 2930, 2818, 1580, 1455, 1415,
1H NMR (600 MHz, CDCl3): d = 1.07 (t, J = 7.0 Hz, 6H), 2.25 (s,
3H), 3.21 (q, J = 7.0 Hz, 4H), 3.49 (s, 3H), 3.76 (s, 6H), 4.52 (s, 2H),
6.70 (s, 2H), 6.86 (d, J = 7.9 Hz, 1H), 8.12 (d, J = 7.9 Hz, 1H), 8.31
(s, 1H); 13C NMR (151 MHz, CDCl3): d = 12.1, 19.1, 47.7, 56.7,
58.4, 75.0, 96.9, 104.6, 118.7, 119.3, 126.3, 131.6, 132.2, 137.6,
~
140.2, 146.6, 148.3, 156.8, 158.3; IR (KBr):
m
= 2968, 2813, 1581,
1433, 1415, 1234, 1120, 1091, 1061, 819 cmꢀ1; HRMS-ESI m/z
[M+H]+ calcd for C24H30IN2O3+: 521.1296, found: 521.1281.
1375, 1228, 1122, 1066, 825 cmꢀ1; HRMS-ESI m/z [M+H]+ calcd
for C26H35N2O4+: 439.2592, found: 439.2579.
5.1.33. 5-(Diethylamino)-2-[2,6-dimethoxy-4-
(methoxymethyl)phenyl]-3-methylquinoline-8-carbaldehyde
(19)
5.1.36. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-N,N-
diethyl-8-fluoro-3-methylquinolin-5-amine (21a)
To a solution of 18 (250 mg, 0.481 mmol) in THF was added n-
BuLi (0.260 mL, 0.722 mmol, 2.77 M in n-hexane) at ꢀ78 °C and the
mixture was stirred at the same temperature for 1 h. After a solu-
tion of N-fluorobenzenesulfonimide (303 mg, 0.962 mmol) in THF
(5 mL) was added to the reaction mixture, the mixture was stirred
at the same temperature for 2 h. The mixture was quenched with
AcOH and concentrated under reduced pressure. The residue was
extracted into ethyl acetate and the organic layer was concentrated
under reduced pressure. The residue was purified by NH-silica
gel column chromatography (9:1–5:5 n-heptane/ethyl acetate
To a solution of 18 (700 mg, 1.35 mmol) in THF was added n-
BuLi (0.665 mL, 1.76 mmol, 2.64 M in n-hexane) at ꢀ78 °C and
the mixture was stirred at the same temperature for 1 h. Next,
DMF (0.178 mL, 2.29 mmol) was added at ꢀ78 °C and the mixture
was stirred at the same temperature for 1 h. The reaction was
quenched with AcOH and concentrated under reduced pressure.
The residue was extracted into ethyl acetate and the organic layer
was concentrated under reduced pressure. The residue was puri-
fied by silica gel column chromatography (9:1–5:5 n-heptane/

K. Takeda et al. / Bioorg. Med. Chem. 20 (2012) 6559–6578
6573
gradient) to afford the title compound 21a as a colorless oil
(3.2 mg, 7.76 mol, 1.6%). Compound purity: >99%.
acetate gradient) to afford the title compound 21d as a light yellow
solid (81.0 mg, 191 mol, 83.0%). Compound purity: >99%.
l
l
1H NMR (600 MHz, CDCl3): d = 1.06 (t, J = 7.0 Hz, 6H), 2.24 (s,
3H), 3.28 (q, J = 7.0 Hz, 4H), 3.47 (s, 3H), 3.73 (s, 6H), 4.53 (s,
2H), 6.68 (s, 2H), 7.33 (dd, J = 11.5, 9.2 Hz, 1H), 7.94 (dd,
J = 9.2, 4.5 Hz, 1H), 8.51 (s, 1H); 13C NMR (151 MHz, CDCl3):
d = 13.5, 19.4, 48.1, 55.9, 58.2, 75.0, 103.5, 117.5, 118.4, 128.1,
129.2, 130.1, 131.7, 132.3, 140.3, 144.5, 155.4, 158.0, 158.4; IR
~
1H NMR (600 MHz, CDCl3): d = 1.03 (t, J = 7.0 Hz, 6H), 2.20 (s, 3H),
3.13 (q, J = 7.0 Hz, 4H), 3.44 (s, 3H), 3.69 (s, 6H), 3.99 (s, 3H), 4.50 (s,
2H), 6.62 (s, 2H), 6.89 (d, J = 8.1 Hz, 1H), 7.09 (d, J = 8.1 Hz, 1H), 8.42
(s, 1H); 13C NMR (151 MHz, CDCl3): d = 12.4, 19.5, 48.4, 55.9, 56.0,
58.1, 75.0, 103.6, 105.9, 118.3, 118.4, 127.4, 131.3, 131.8, 139.5,
~
139.6, 139.9, 152.3, 154.9, 158.3; IR (KBr):
m = 2962, 2835, 1582,
(KBr):
m
= 2975, 2925, 2850, 1609, 1580, 1463, 1415, 1382,
1462, 1415, 1240, 1124, 1090, 1065, 825 cmꢀ1; HRMS-ESI m/z
[M+H]+ calcd for C25H33N2O4+: 425.2435, found: 425.2415.
1225, 1123, 1097, 990, 823 cmꢀ1; HRMS-ESI m/z [M+H]+ calcd
for C24H30FN2O3+: 413.2235, found: 413.2233.
5.1.40. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-N,N-
diethyl-8-iodoquinolin-5-amine (22b)
The title compound was prepared from 16b using a method
analogous to that described for 18 in 79.2% yield as a light yellow
oil.
5.1.37. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-N,N-
diethyl-3-methyl-8-(trifluoromethyl)quinolin-5-amine (21b)
A mixture of 18 (100 mg, 0.192 mmol), methyl fluorosulphonyldi-
fluoroacetate (0.073 mL, 0.577 mmol), and copper (36.7 mg,
0.577 mmol) in DMF (2.14 mL) was stirred at 90 °C for 1.5 h. The
reaction mixture was cooled to room temperature and filtered
through Celite. The filtrate was extracted into ethyl acetate and
the organic layer was concentrated under reduced pressure. The
residue was purified by silica gel column chromatography (9:1–
7:3 n-heptane/ethyl acetate gradient) to afford the title compound
1H NMR (600 MHz, CDCl3): d = 1.07 (t, J = 7.1 Hz, 6H), 3.21 (q,
J = 7.1 Hz, 4H), 3.47 (s, 3H), 3.81 (s, 6H), 4.52 (s, 2H), 6.71 (s, 2H),
6.90 (d, J = 7.9 Hz, 1H), 7.44 (d, J = 8.5 Hz, 1H), 8.20 (d, J = 7.9 Hz,
1H), 8.50 (d, J = 8.5 Hz, 1H); 13C NMR (151 MHz, CDCl3): d = 12.2,
47.8, 56.8, 58.3, 74.9, 97.1, 104.6, 119.3, 119.4, 124.4, 125.8,
~
m = 2975,
132.3, 138.6, 140.6, 147.9, 149.0, 155.6, 158.6; IR (KBr):
2932, 2824, 1579, 1455, 1415, 1401, 1355, 1232, 1127, 1099,
21b as a light yellow foam (68.0 mg, 147 lmol, 76.6%). Compound
purity: >99%.
1065, 822 cmꢀ1; HRMS-ESI m/z [M+H]+ calcd for C23H28IN2O3
:
+
1H NMR (600 MHz, CDCl3): d = 1.11 (t, J = 6.9 Hz, 6H), 2.26 (s,
3H), 3.28 (q, J = 6.9 Hz, 4H), 3.49 (s, 3H), 3.73 (s, 6H), 4.52 (s, 2H),
6.70 (s, 2H), 7.03 (d, J = 7.9 Hz, 1H), 7.84 (d, J = 7.9 Hz, 1H), 8.33
(s, 1H); 13C NMR (151 MHz, CDCl3): d = 12.0, 19.4, 47.3, 56.5,
58.4, 75.0, 104.6, 115.2, 118.9, 122.0, 124.6, 125.2, 126.2, 131.3,
~
507.1139, found: 507.1137.
5.1.41. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-N,N-
diethyl-3-fluoro-8-iodoquinolin-5-amine (22c)
The title compound was prepared from 16c using a method
analogous to that described for 18 in 75.3% yield as a light yellow
foam.
131.8, 140.2, 144.8, 151.3, 156.3, 158.2; IR (KBr):
m = 2977, 2929,
2844, 1614, 1578, 1457, 1417, 1376, 1328, 1237, 1120, 1106,
1H NMR (600 MHz, CDCl3): d = 1.06 (t, J = 6.6 Hz, 6H), 3.18 (q,
J = 6.6 Hz, 4H), 3.47 (s, 3H), 3.80 (s, 6H), 4.53 (s, 2H), 6.70 (s, 2H),
6.86–6.99 (m, 1H), 8.12–8.27 (m, 2H); 13C NMR (151 MHz, CDCl3):
d = 12.2, 47.8, 56.6, 58.4, 74.9, 96.6, 104.2, 113.5, 115.7, 120.3,
127.5, 137.8, 141.5, 144.8, 146.9, 148.7, 155.9, 159.0; IR (KBr):
~
822 cmꢀ1
; :
HRMS-ESI m/z [M+H]+ calcd for C25H30F3N2O3
+
463.2203, found: 463.2189.
5.1.38. 5-(Diethylamino)-2-[2,6-dimethoxy-4-
(methoxymethyl)phenyl]-3-methylquinoline-8-carbonitrile
(21c)
A mixture of 18 (100 mg, 0.192 mmol) and CuCN (22.4 mg,
0.25 mmol) in DMF (2 mL) was stirred at 80 °C for overnight. The
reaction mixture was cooled to room temperature, quenched with
H2O, and extracted into ethyl acetate. The organic layer was con-
centrated under reduced pressure and the residue was purified
by silica gel column chromatography (9:1–7:3 n-heptane/ethyl
acetate gradient) to afford the title compound 21c as a light yellow
m
= 2972, 2936, 2843, 1584, 1455, 1408, 1235, 1123, 1090, 908,
824 cmꢀ1
;
HRMS-ESI m/z [M+H]+ calcd for C23H27FIN2O3
:
+
525.1045, found: 525.1056.
5.1.42. 3-Chloro-2-[2,6-dimethoxy-4-(methoxymethyl)phenyl]-
N,N-diethyl-8-iodoquinolin-5-amine (22d)
The title compound was prepared from 16d using a method
analogous to that described for 18 in 76.7% yield as a light yellow
solid.
foam (39.3 mg, 93.7 lmol, 48.8%). Compound purity: >99%.
1H NMR (600 MHz, CDCl3): d = 1.07 (t, J = 7.2 Hz, 6H), 3.20 (q,
J = 7.2 Hz, 4H), 3.48 (s, 3H), 3.78 (s, 6H), 4.53 (s, 2H), 6.70 (s, 2H),
6.90 (d, J = 7.9 Hz, 1H), 8.19 (d, J = 7.9 Hz, 1H), 8.58 (s, 1H); 13C
NMR (151 MHz, CDCl3): d = 12.1, 47.8, 56.6, 58.4, 75.0, 96.4,
104.2, 116.7, 120.3, 126.7, 130.5, 132.0, 138.9, 141.0, 146.3,
~
1H NMR (600 MHz, CDCl3): d = 1.13 (t, J = 7.1 Hz, 6H), 2.26 (s,
3H), 3.33 (q, J = 7.1 Hz, 4H), 3.48 (s, 3H), 3.75 (s, 6H), 4.52 (s, 2H),
6.69 (s, 2H), 7.01 (d, J = 7.9 Hz, 1H), 7.89 (d, J = 7.9 Hz, 1H), 8.27
(s, 1H); 13C NMR (151 MHz, CDCl3): d = 12.1, 19.5, 47.2, 56.5,
58.3, 74.9, 104.2, 106.1, 115.6, 118.0, 118.8, 124.3, 132.1, 132.1,
~
148.3, 154.2, 158.5; IR (KBr):
m = 2970, 2932, 2838, 1579, 1453,
134.3, 140.5, 147.8, 152.4, 158.1, 158.1; IR (KBr):
m = 2970, 2871,
1415, 1381, 1235, 1125, 1091, 1070, 824 cmꢀ1; HRMS-ESI m/z
[M+H]+ calcd for C23H27ClIN2O3+: 541.0749, found: 541.0732.
2220, 1569, 1463, 1416, 1378, 1244, 1223, 1120, 1101, 1066,
821 cmꢀ1
;
HRMS-ESI m/z [M+H]+ calcd for C25H30N3O3
:
+
420.2282, found: 420.2267.
5.1.43. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-N,N,3-
triethyl-8-iodoquinolin-5-amine (22e)
5.1.39. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-N,N-
diethyl-8-methoxy-3-methylquinolin-5-amine (21d)
The title compound was prepared from 16e using a method
analogous to that described for 18 in 76.7% yield as a light yellow
foam.
A
mixture of 18 (120 mg, 0.230 mmol), CuBr (16.6 mg,
1H NMR (600 MHz, CDCl3): d = 1.10 (t, J = 6.8 Hz, 6H), 1.16 (t,
J = 7.2 Hz, 3H), 2.58 (q, J = 7.2 Hz, 2H), 3.23 (q, J = 6.8 Hz, 4H),
3.51 (s, 3H), 3.76 (s, 6H), 4.54 (s, 2H), 6.71 (s, 2H), 6.87 (d,
J = 7.6 Hz, 1H), 8.13 (d, J = 7.6 Hz, 1H), 8.35 (s, 1H); 13C NMR
(151 MHz, CDCl3): d = 12.2, 14.2, 25.3, 47.7, 56.6, 58.5, 75.1, 96.8,
104.6, 118.6, 119.0, 126.4, 130.5, 137.2, 137.6, 140.1, 146.5,
~
0.115 mmol), NaOMe (0.922 mL, 4.61 mmol, 28% methanol solu-
tion), and ethyl acetate (0.0227 mL, 0.23 mmol) was stirred at re-
flux temperature for 3 h. The reaction mixture was quenched
with saturated aqueous NH4Cl and extracted into ethyl acetate.
The organic layer was washed with brine, dried over MgSO4, and
concentrated under reduced pressure. The residue was purified
by NH-silica gel column chromatography (9:1–4:6 n-heptane/ethyl
148.5, 156.4, 158.4; IR (KBr):
m = 2962, 2933, 2814, 1579, 1454,

6574
K. Takeda et al. / Bioorg. Med. Chem. 20 (2012) 6559–6578
1412, 1230, 1120, 817 cmꢀ1; HRMS-ESI m/z [M+H]+ calcd for
25H32IN2O3+: 535.1452, found: 535.1445.
1,4-dioxane (2.5 mL) was stired at 80 °C for 1 h. The reaction mix-
ture was cooled to room temperature and concentrated under
reduced pressure. The residue was extracted into DCM and the or-
ganic layer was concentrated under reduced pressure. The residue
was purified by silica gel column chromatography (9:1–5:5 n-hep-
tane/ethyl acetate gradient) to afford the title compound 23bb as a
C
5.1.44. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-N,N-
diethyl-8-methoxyquinolin-5-amine (23ba)
The title compound was prepared from 22b using a method
analogous to that described for 21d in 95.2% yield as a light yellow
solid. Compound purity: >99%.
yellow oil (73.7 mg, 187 lmol, 94.8%). Compound purity: >99%.
1H NMR (600 MHz, CDCl3): d = 1.05 (t, J = 7.0 Hz, 6H), 2.74 (s,
3H), 3.17 (q, J = 7.0 Hz, 4H), 3.46 (s, 3H), 3.76 (s, 6H), 4.52 (s, 2H),
6.69 (s, 2H), 7.07 (d, J = 7.6 Hz, 1H), 7.37 (d, J = 8.5 Hz, 1H), 7.44
(d, J = 7.6 Hz, 1H), 8.60 (d, J = 8.5 Hz, 1H); 13C NMR (151 MHz,
CDCl3): d = 12.4, 18.2, 48.1, 56.4, 58.3, 74.9, 104.2, 117.8, 119.9,
123.2, 125.1, 128.3, 132.0, 132.4, 140.2, 145.9, 148.4, 153.4,
158.5; HRMS (ESI+): m/z [M+H]+ calcd for C24H31N2O3+: 395.2329,
found: 395.2329.
1H NMR (600 MHz, CDCl3): d = 1.03 (t, J = 6.7 Hz, 6H), 3.13 (q,
J = 6.7 Hz, 4H), 3.43 (s, 3H), 3.72 (s, 6H), 4.02 (s, 3H), 4.50 (s, 2H),
6.63 (s, 2H), 6.97 (d, J = 8.1 Hz, 1H), 7.13 (d, J = 8.1 Hz, 1H), 7.38
(d, J = 8.7 Hz, 1H), 8.64 (d, J = 8.7 Hz, 1H); 13C NMR (151 MHz,
CDCl3): d = 12.5, 48.5, 56.0, 56.1, 58.1, 74.9, 103.7, 107.1, 118.4,
119.6, 124.2, 126.7, 132.1, 140.1, 140.2, 141.0, 152.3, 153.8,
~
158.6; IR (KBr):
m = 2961, 2932, 2809, 1608, 1579, 1455, 1414,
1378, 1254, 1238, 1225, 1122, 1105, 984, 820 cmꢀ1; HRMS-ESI
m/z [M+H]+ calcd for C24H31N2O4+: 411.2279, found: 411.2277.
5.1.49. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-N,N-
diethyl-3-fluoro-8-methylquinolin-5-amine (23cb)
5.1.45. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-N,N-
diethyl-3-fluoro-8-methoxyquinolin-5-amine (23ca)
The title compound was prepared from 22c using a method
analogous to that described for 21d in 61.3% yield as a light yellow
solid. Compound purity: >99%.
The title compound was prepared from 22c using a method
analogous to that described for 23bb in 84.6% yield as a yellow
oil. Compound purity: >99%.
1H NMR (600 MHz, CDCl3): d = 1.05 (t, J = 7.1 Hz, 6H), 2.73 (s,
3H), 3.15 (q, J = 7.1 Hz, 4H), 3.47 (s, 3H), 3.77 (s, 6H), 4.53 (s, 2H),
6.70 (s, 2H), 7.12 (d, J = 7.6 Hz, 1H), 7.40 (d, J = 7.6 Hz, 1H), 8.25
(d, J = 10.6 Hz, 1H); 13C NMR (151 MHz, CDCl3): d = 12.4, 18.2,
48.1, 56.3, 58.3, 74.9, 103.8, 114.0, 115.3, 119.2, 127.4, 127.5,
132.8, 141.1, 144.5, 145.3, 145.5, 155.4, 158.9; HRMS (ESI+): m/z
[M+H]+ calcd for C24H30FN2O3+: 413.2235, found: 413.2220.
1H NMR (600 MHz, CDCl3): d = 1.02 (t, J = 7.1 Hz, 6H), 3.10 (q,
J = 7.1 Hz, 4H), 3.43 (s, 3H), 3.73 (s, 6H), 4.01 (s, 3H), 4.51 (s, 2H),
6.63 (s, 2H), 6.92 (d, J = 8.1 Hz, 1H), 7.18 (d, J = 8.1 Hz, 1H), 8.28
(d, J = 10.2 Hz, 1H); 13C NMR (151 MHz, CDCl3): d = 12.5, 48.6,
56.0, 56.0, 58.1, 74.9, 103.4, 106.1, 113.3, 115.4, 119.9, 129.4,
~
137.8, 139.7, 141.1, 144.9, 152.5, 156.1, 159.0; IR (KBr):
m = 2971,
2819, 1586, 1454, 1407, 1321, 1127, 1097, 826 cmꢀ1; HRMS-ESI
5.1.50. 3-Chloro-2-[2,6-dimethoxy-4-(methoxymethyl)phenyl]-
N,N-diethyl-8-methylquinolin-5-amine (23db)
m/z [M+H]+ calcd for C24H30FN2O4+: 429.2184, found: 429.2168.
The title compound was prepared from 22d using a method
analogous to that described for 23bb in 98.3% yield as a yellow
oil. Compound purity: >99%.
5.1.46. 3-Chloro-2-[2,6-dimethoxy-4-(methoxymethyl)phenyl]-
N,N-diethyl-8-methoxyquinolin-5-amine (23da)
The title compound was prepared from 22d using a method
analogous to that described for 21d in 76.2% yield as a light yellow
solid. Compound purity: >99%.
1H NMR (600 MHz, CDCl3): d = 1.05 (t, J = 7.0 Hz, 6H), 2.70 (s,
3H), 3.16 (q, J = 7.0 Hz, 4H), 3.48 (s, 3H), 3.75 (s, 6H), 4.54 (s, 2H),
6.69 (s, 2H), 7.10 (d, J = 7.6 Hz, 1H), 7.43 (d, J = 7.6 Hz, 1H), 8.65
(s, 1H); 13C NMR (151 MHz, CDCl3): d = 12.3, 18.1, 48.1, 56.3,
58.3, 75.0, 103.8, 117.3, 119.0, 126.6, 128.6, 129.3, 131.7, 132.6,
140.7, 145.2, 146.6, 151.9, 158.5; HRMS (ESI+): m/z [M+H]+ calcd
for C24H30ClN2O3+: 429.1940, found: 429.1924.
1H NMR (600 MHz, CDCl3): d = 1.02 (t, J = 7.1 Hz, 6H), 3.11 (q,
J = 7.1 Hz, 4H), 3.43 (s, 3H), 3.72 (s, 6H), 4.00 (s, 3H), 4.51 (s, 2H),
6.63 (s, 2H), 6.96 (d, J = 8.1 Hz, 1H), 7.16 (d, J = 8.1 Hz, 1H), 8.69
(s, 1H); 13C NMR (151 MHz, CDCl3): d = 12.5, 48.6, 56.0, 58.1,
74.9, 103.4, 107.2, 116.7, 119.8, 128.2, 130.7, 131.7, 139.1, 139.4,
~
140.7, 152.3, 152.3, 158.5; IR (KBr):
m
= 2970, 2926, 2837, 1607,
5.1.51. 2-Chloro-8-methoxy-3-methylquinoline (25)
1583, 1453, 1416, 1358, 1310, 1236, 1125, 1108, 1093, 907,
To POCl3 (227 mL, 2.44 mol) was slowly added DMF (37.7 mL,
487 mmol) at 0 °C and the mixture was stirred at the same temper-
ature for 15 min. To the mixture was added 24 (72.8 g, 0.406 mol)
and the mixture was stirred at 70 °C for 2.5 h. The reaction mixture
was cooled to room temperature, quenched with crushed-ice, neu-
tralized with 5 M aqueous NaOH, and extracted into ethyl acetate.
The organic layer was concentrated under reduced pressure and
the residue was purified by NH-silica gel column chromatography
(8:2–7:3 n-heptane/ethyl acetate gradient) to afford the title com-
pound 25 as a white solid (54.8 g, 264 mmol, 65.0%).
825 cmꢀ1
; :
HRMS-ESI m/z [M+H]+ calcd for C24H30ClN2O4
+
445.1889, found: 445.1875.
5.1.47. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-N,N,3-
triethyl-8-methoxyquinolin-5-amine (23ea)
The title compound was prepared from 22e using a method
analogous to that described for 21d in 65.4% yield as a white solid.
Compound purity: >99%.
1H NMR (600 MHz, CDCl3): d = 1.04 (t, J = 6.7 Hz, 6H), 1.14 (t,
J = 7.4 Hz, 3H), 2.51 (q, J = 7.4 Hz, 2H), 3.14 (d, J = 6.7 Hz, 4H),
3.45 (s, 3H), 3.68 (s, 6H), 3.99 (s, 3H), 4.50 (s, 2H), 6.62 (s, 2H),
6.89 (d, J = 8.1 Hz, 1H), 7.08 (d, J = 8.1 Hz, 1H), 8.46 (s, 1H); 13C
NMR (151 MHz, CDCl3): d = 12.5, 14.1, 25.6, 48.4, 55.9, 55.9, 58.2,
75.1, 103.6, 106.0, 118.0, 118.3, 127.5, 130.2, 136.9, 139.5, 139.7,
~
1H NMR (600 MHz, CDCl3): d = 2.53 (s, 3H), 4.05 (s, 3H), 7.01 (d,
J = 7.6 Hz, 1H), 7.31 (d, J = 7.9 Hz, 1H), 7.43 (dd, J = 7.9, 7.6 Hz, 1H),
7.94 (s, 1H); 13C NMR (151 MHz, CDCl3): d = 20.1, 56.0, 107.8,
118.5, 127.1, 128.8, 130.9, 137.8, 138.2, 151.1, 154.5; IR (KBr):
~
m
= 3006, 2841, 1569, 1486, 1466, 1435, 1342, 1268, 1198, 1184,
139.8, 152.2, 154.5, 158.4; IR (KBr):
m
= 2968, 2927, 2835, 1580,
1114, 1031, 761 cmꢀ1
;
HRMS-ESI m/z [M+H]+ calcd for
1455, 1416, 1320, 1261, 1236, 1123, 1097, 826 cmꢀ1; HRMS-ESI
C11H11ClNO+: 208.0524, found: 208.0518.
m/z [M+H]+ calcd for C26H35N2O4+: 439.2592, found: 439.2587.
5.1.52. 2-Chloro-8-methoxy-3-methyl-5-nitroquinoline (26)
To a solution of 25 (16.5 g, 79.5 mmol) in H2SO4 (80 mL) was
added HNO3 (5.29 mL, 119 mmol) at ꢀ10 °C and the mixture was
stirred at room temperature for 3.5 h. The mixture was quenched
with crushed-ice, filtered, and washed with water. The obtained
5.1.48. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-N,N-
diethyl-8-methylquinolin-5-amine (23bb)
A mixture of 22b (100 mg, 197
l
mol), Me2Zn (394
l
L, 394
lmol,
1.0 M in n-hexane), and (t-Bu3P)2Pd (5.03 mg, 9.90
l
mol) in

K. Takeda et al. / Bioorg. Med. Chem. 20 (2012) 6559–6578
6575
crude product was purified by NH-silica gel column chromatogra-
phy (ethyl acetate) to afford the title compound 26 as a light yel-
low solid (6.90 g, 27.3 mmol, 34.4%).
aqueous NaHCO3 and extracted into ethyl acetate. The organic
layer was concentrated under reduced pressure and the residue
was purified by silica gel column chromatography (9:1–0:10 n-
heptane/ethyl acetate gradient) to afford the title compound 29
1H NMR (600 MHz, CDCl3): d = 2.63 (s, 3H), 4.17 (s, 3H), 7.04
(d, J = 8.7 Hz, 1H), 8.51 (d, J = 8.7 Hz, 1H), 9.08 (s, 1H); 13C NMR
(151 MHz, CDCl3): d = 20.7, 56.9, 105.6, 122.4, 127.8, 134.5,
~
as a white solid (18 mg, 39.8
>99%.
lmol, 12.2%). Compound purity:
134.9, 137.1, 137.6, 152.6, 160.0; IR (KBr):
m
= 1557, 1487, 1311,
1H NMR (600 MHz, CDCl3): d = 0.87 (t, J = 7.3 Hz, 6H), 1.43–1.54
(m, 4H), 2.20 (s, 3H), 3.02 (t, J = 7.3 Hz, 4H), 3.44 (s, 3H), 3.70 (s,
6H), 3.99 (s, 3H), 4.50 (s, 2H), 6.63 (s, 2H), 6.88 (d, J = 7.9 Hz, 1H),
7.11 (d, J = 7.9 Hz, 1H), 8.44 (s, 1H); 13C NMR (151 MHz, CDCl3):
d = 11.8, 19.6, 20.4, 55.9, 56.0, 56.9, 58.2, 75.0, 103.7, 106.0,
118.4, 127.3, 131.3, 131.8, 139.5, 139.9, 140.3, 152.2, 154.8,
~
1266, 1108, 1060, 937, 822 cmꢀ1; HRMS-ESI m/z [M+H]+ calcd
for C11H10ClN2O3+: 253.0375, found: 253.0361.
5.1.53. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-8-
methoxy-3-methyl-5-nitroquinoline (27)
A
mixture of 26 (860 mg, 3.4 mmol), [2,6-Dimethoxy-4-
158.3; IR (KBr):
1123, 1097, 824 cmꢀ1
27H37N2O4+: 453.2748, found: 453.2743.
m = 2956, 2927, 1583, 1454, 1417, 1319, 1237,
(methoxymethyl)phenyl]boronic acid (1.15 g, 5.1 mmol), Pd(PPh3)4
(196 mg, 0.17 mmol), and K2CO3 (1.41 g, 10.2 mmol) in a mixture of
1,4-dioxane (20 mL) and H2O (5 mL) was stirred at reflux tempera-
ture for 1.5 h. The reaction mixture was cooled to room temperature
and concentrated under reduced pressure. The residue was ex-
tracted into ethyl acetate and the organic layer was concentrated
under reduced pressure. The residue was purified by silica gel col-
umn chromatography (8:2–0:10 n-heptane/ethyl acetate gradient)
to afford the title compound 27 as a light yellow solid (1.18 g,
2.96 mmol, 87.1%).
;
HRMS-ESI m/z [M+H]+ calcd for
C
5.1.57. Tert-butyl N-{2-[2,6-dimethoxy-4-
(methoxymethyl)phenyl]-8-methoxy-3-methylquinolin-5-
yl}carbamate (30)
A
mixture of 28 (500 mg, 1.36 mmol), Boc2O (1.04 g,
4.76 mmol), and Et3N (1.32 mL, 9.52 mmol) in DCM (10 mL) was
stirred at room temperature for 16 h. The reaction mixture was
concentrated under reduced pressure and the residue was purified
by silica gel column chromatography (9:1–0:10 n-heptane/ethyl
acetate gradient) to afford the title compound 30 as a white solid
1H NMR (600 MHz, CDCl3): d = 2.26 (s, 3H), 3.45 (s, 3H), 3.70 (s,
6H), 4.13 (s, 3H), 4.50 (s, 2H), 6.64 (s, 2H), 6.98 (d, J = 9.1 Hz, 1H),
8.47 (d, J = 9.1 Hz, 1H), 9.02 (s, 1H); 13C NMR (151 MHz, CDCl3):
d = 19.8, 55.9, 56.8, 58.3, 74.9, 103.4, 104.4, 116.8, 122.7, 127.0,
131.4, 136.2, 137.4, 138.0, 140.8, 156.8, 158.1, 161.2; IR (KBr):
~
(390 mg, 832 lmol, 61.2%).
1H NMR (600 MHz, CDCl3): d = 1.54 (br s, 9H), 2.20 (s, 3H), 3.44
(s, 3H), 3.67 (s, 6H), 4.01 (s, 3H), 4.49 (s, 2H), 6.40–6.56 (m, 1H),
6.61 (s, 2H), 6.92 (d, J = 8.7 Hz, 1H), 7.44–7.66 (m, 1H), 8.01 (br s,
1H); 13C NMR (151 MHz, CDCl3): d = 19.5, 28.4, 55.9, 56.1, 58.2,
75.0, 80.4, 103.5, 106.1, 117.8, 124.5, 129.4, 132.3, 138.8, 140.1,
~
m = 3228, 2928, 2839, 1675,
m
= 2926, 2837, 1583, 1550, 1504, 1454, 1308, 1238, 1192, 1126,
1094, 822 cmꢀ1; HRMS-ESI m/z [M+H]+ calcd for C21H23N2O6
:
+
399.1551, found: 399.1559.
153.8, 154.4, 155.3, 158.2; IR (KBr):
5.1.54. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-8-
methoxy-3-methylquinolin-5-amine (28)
1584, 1533, 1456, 1269, 1239, 1171, 1126, 1098, 1023, 827 cmꢀ1
;
HRMS-ESI m/z [M+H]+ calcd for C26H33N2O6+: 469.2333, found:
A mixture of 27 (1.10 g, 2.75 mmol) and Fe (1.23 g, 22.0 mmol)
in a mixture of EtOH (20 mL) and saturated aqueous NH4Cl (2 mL)
was stirred at reflux temperature for 1 h. The reaction mixture was
cooled to room temperature and filtered through a pad of NH-silica
gel with ethyl acetate as the eluent to yield the crude title com-
pound 28 as a light yellow solid (956 mg, 2.59 mmol, 94.4%), which
was used in the following reaction without further purification.
1H NMR (600 MHz, CDCl3): d = 2.21 (s, 3H), 3.44 (s, 3H), 3.68 (s,
6H), 3.96 (s, 3H), 4.50 (s, 2H), 6.62 (s, 2H), 6.72 (d, J = 8.1 Hz, 1H),
6.81 (d, J = 8.1 Hz, 1H), 8.01 (s, 1H); HRMS-ESI m/z [M+H]+ calcd
for C21H25N2O4+: 369.1809, found: 369.1798.
469.2289.
5.1.58. Tert-butyl N-{2-[2,6-dimethoxy-4-
(methoxymethyl)phenyl]-8-methoxy-3-methylquinolin-5-yl}-
N-(2-methoxyethyl)carbamate (31)
To a solution of 30 (340 mg, 0.726 mmol) in DMF (7 mL) was
added NaH (60% oil dispersion, 43.6 mg, 1.09 mmol) at room
temperature and the reaction mixture was stirred at the same tem-
perature for 15 min. To the mixture was added 2-bromoethyl-
methyl-ether (0.102 mL, 1.09 mmol) and the resultant mixture
was stirred at 40 °C for 1 h. The reaction mixture was cooled to
room temperature, quenched with H2O, and extracted into diethyl-
ether. The organic layer was concentrated under reduced pressure
and the residue was purified by NH-silica gel column chromatogra-
phy (9:1–0:10 n-heptane/ethyl acetate gradient) to afford the title
5.1.55. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-N,N-
diethyl-8-methoxy-3-methylquinolin-5-amine (21d)
A mixture of 28 (100 mg, 0.271 mmol), acetaldehyde (0.0456 mL,
0.813 mmol), and NaBH(OAc)3 (172 mg, 0.813 mmol) in a mixture
of THF (2 mL), AcOH (0.2 mL), and MeOH (0.5 mL) was stirred at
room temperature for 14.5 h. The reaction mixture was concen-
trated under reduced pressure and the residue was quenched with
saturated aqueous NaHCO3 and extracted into ethyl acetate. The
organic layer was concentrated under reduced pressure and the
residue was purified by silica gel column chromatography (9:1–
0:10 n-heptane/ethyl acetate gradient) to afford the title compound
compound 31 as a colorless oil (374 mg, 710 lmol, 97.8%). The
obtained product contained some impurities but was used in
the following reaction without further purification.
1H NMR (600 MHz, CDCl3): d = 1.26 (br s, 9H), 2.18 (s, 3H), 3.30
(s, 3H), 3.44 (s, 3H), 3.49–3.56 (m, 2H), 3.67 (s, 3H), 3.69 (s, 3H),
3.73–3.84 (m, 1H), 3.95–4.08 (m, 4H), 4.50 (s, 2H), 6.62 (br s,
2H), 6.91 (br s, 1H), 7.21–7.38 (m, 1H), 7.89 (br s, 1H); HRMS-ESI
m/z [M+H]+ calcd for C29H39N2O7+: 527.2752, found: 527.2739.
21d as a light yellow solid (88 mg, 207 lmol, 76.5%).
5.1.59. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-8-
5.1.56. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-8-
methoxy-3-methyl-N,N-dipropylquinolin-5-amine (29)
methoxy-N-(2-methoxyethyl)-3-methylquinolin-5-amine (32)
A mixture of 31 (324 mg, 0.615 mmol) in a mixture of TFA
(3 mL) and DCM (3 mL) was stirred at room temperature for 1 h.
The reaction mixture was concentrated under reduced pressure
and the residue was quenched with saturated aqueous NaHCO3
and extracted into ethyl acetate. The organic layer was concen-
trated under reduced pressure and the residue was purified by
A
mixture of 28 (120 mg, 0.325 mmol), propionaldehyde
(0.0938 mL, 1.3 mmol), and NaBH(OAc)3 (276 mg, 1.3 mmol) in a
mixture of THF (3 mL) and AcOH (0.3 mL) was stirred at room tem-
perature for 14.5 h. The reaction mixture was concentrated under
reduced pressure and the residue was quenched with saturated

6576
K. Takeda et al. / Bioorg. Med. Chem. 20 (2012) 6559–6578
silica gel column chromatography (7:3–0:10 n-heptane/ethyl ace-
tate gradient) to afford the title compound 32 as a yellow solid
Pd(PPh3)4 (45.6 mg, 0.0395 mmol), and K2CO3 (328 mg,
2.37 mmol) in a mixture of 1,4-dioxane (6 mL) and H2O (1.5 mL)
was stirred at reflux temperature for 2.5 h. The reaction mixture
was cooled to room temperature and concentrated under reduced
pressure. The residue was extracted into ethyl acetate and the or-
ganic layer was concentrated under reduced pressure. The residue
was purified by silica gel column chromatography (8:2–0:10 n-
heptane/ethyl acetate gradient) to afford the title compound 34a
(192 mg, 450 lmol, 73.2%).
1H NMR (600 MHz, CDCl3): d = 2.22 (s, 3H), 3.40 (t, J = 4.9 Hz, 2H),
3.44 (s, 6H), 3.68 (s, 6H), 3.75 (t, J = 4.9 Hz, 2H), 3.96 (s, 3H), 4.50 (s,
2H), 6.57 (d, J = 8.3 Hz, 1H), 6.62 (s, 2H), 6.89 (d, J = 8.3 Hz, 1H), 8.17
(br s, 1H); 13C NMR (151 MHz, CDCl3): d = 19.4, 44.5, 55.9, 56.3, 58.2,
58.8, 71.0, 75.0, 103.5, 105.3, 107.9, 117.6, 120.3, 129.6, 130.9,
~
136.9, 138.8, 140.3, 148.2, 154.6, 158.2; IR (KBr):
m
= 3268, 2924,
as a light yellow oil (280 mg, 695 lmol, 87.7%).
2836, 1613, 1583, 1456, 1415, 1376, 1271, 1230, 1187, 1126,
1H NMR (600 MHz, CDCl3): d = 2.29 (s, 3H), 3.45 (s, 3H), 3.72 (s,
3H), 4.15 (s, 3H), 4.49 (s, 2H), 6.91 (s, 1H), 7.02 (d, J = 8.9 Hz, 1H),
7.08 (s, 1H), 8.51 (d, J = 8.9 Hz, 1H), 9.08 (s, 1H); 13C NMR
(151 MHz, CDCl3): d = 19.5, 56.0, 56.9, 58.4, 73.9, 104.8, 108.4,
120.8, 122.9, 127.0, 127.4, 132.1, 133.8, 135.4, 137.4, 137.9,
~
1096, 790 cmꢀ1; HRMS-ESI m/z [M+H]+ calcd for C24H31N2O5
:
+
427.2228, found: 427.2213.
5.1.60. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-N-ethyl-
8-methoxy-N-(2-methoxyethyl)-3-methylquinolin-5-amine
(33a)
141.2, 156.6, 158.3, 161.2; IR (KBr):
m = 2840, 1608, 1555, 1507,
1462, 1403, 1312, 1261, 1194, 1092, 1053, 834, 821 cmꢀ1
;
A mixture of 32 (70 mg, 0.164 mmol), acetaldehyde (0.0184 mL,
0.328 mmol), and NaBH(OAc)3 (104 mg, 0.492 mmol) in a mixture
of THF (2 mL) and AcOH (0.2 mL) was stirred at room temperature
for 15 h. The reaction mixture was concentrated under reduced
pressure and the residue was quenched with saturated aqueous
NaHCO3 and extracted into ethyl acetate. The organic layer was
concentrated under reduced pressure and the residue was purified
by NH-silica gel column chromatography (9:1–0:10 n-heptane/
ethyl acetate gradient) to afford the title compound 33a as a white
HRMS-ESI m/z [M+H]+ calcd for C20H20ClN2O5+: 403.1055, found:
403.1053.
5.1.63. 3,5-Dimethoxy-4-(8-methoxy-3-methyl-5-nitroquinolin-
2-yl)benzonitrile (34b)
A mixture of 26 (300 mg, 1.19 mmol), (4-cyano-2,6-dimethoxy-
phenyl)boronic acid (296 mg, 1.43 mmol), Pd(PPh3)4 (68.4 mg,
0.0593 mmol), and K2CO3 (492 mg, 3.56 mmol) in a mixture of
1,4-dioxane (8 mL) and H2O (2 mL) was stirred at reflux tempera-
ture for 1.5 h. The reaction mixture was cooled to room tempera-
ture and concentrated under reduced pressure. The residue was
extracted into ethyl acetate and the organic layer was concentrated
under reduced pressure. The residue was purified by silica gel col-
umn chromatography (8:2–0:10 n-heptane/ethyl acetate gradient)
to afford the title compound 34b as a light yellow solid (392 mg,
1.03 mmol, 86.8%).
solid (41.8 mg, 92.0 lmol, 56.1%). Compound purity: >99%.
1H NMR (600 MHz, CDCl3): d = 1.03 (t, J = 6.8 Hz, 3H), 2.20 (s,
3H), 3.19 (q, J = 6.8 Hz, 2H), 3.28 (s, 3H), 3.29 (t, J = 5.9 Hz, 2H),
3.41 (t, J = 5.9 Hz, 2H), 3.44 (s, 3H), 3.69 (s, 6H), 4.00 (s, 3H), 4.50
(s, 2H), 6.63 (s, 2H), 6.89 (d, J = 8.3 Hz, 1H), 7.16 (d, J = 8.3 Hz,
1H), 8.47 (s, 1H); 13C NMR (151 MHz, CDCl3): d = 12.5, 19.5, 50.0,
54.1, 55.9, 56.0, 58.2, 58.7, 70.8, 75.0, 103.6, 106.0, 118.3, 118.8,
127.5, 131.5, 131.8, 139.3, 139.4, 139.9, 152.6, 155.0, 158.3; IR
~
1H NMR (600 MHz, CDCl3): d = 2.25 (s, 3H), 3.73 (s, 6H), 4.14 (s,
3H), 6.94 (s, 2H), 7.02 (d, J = 8.7 Hz, 1H), 8.51 (d, J = 8.7 Hz, 1H),
9.07 (s, 1H); 13C NMR (151 MHz, CDCl3): d = 19.5, 56.2, 56.9,
104.8, 108.2, 113.5, 118.8, 122.4, 122.9, 127.4, 132.0, 135.3,
~
m = 2227, 1574, 1555,
(KBr):
m = 2925, 2839, 1582, 1455, 1418, 1374, 1319, 1237, 1121,
1094, 824 cmꢀ1; HRMS-ESI m/z [M+H]+ calcd for C26H35N2O5
:
+
455.2541, found: 455.2527.
137.4, 138.0, 154.7, 158.4, 161.1; IR (KBr):
1506, 1468, 1413, 1308, 1241, 1194, 1123, 1093, 823 cmꢀ1
;
5.1.61. 2-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-8-
methoxy-N-(2-methoxyethyl)-3-methyl-N-(2-
methylpropyl)quinolin-5-amine (33b)
HRMS-ESI m/z [M+H]+ calcd for C20H18N3O5+: 380.1241, found:
380.1245.
A
mixture of 32 (50 mg, 0.117 mmol), isobutyraldehyde
5.1.64. 2-(2,6-Dimethoxy-4-methylphenyl)-8-methoxy-3-
methyl-5-nitroquinoline (34c)
(12.7 mg, 0.176 mmol), and NaBH(OAc)3 (37.2 mg, 0.176 mmol)
in a mixture of THF (0.8 mL) and AcOH (0.08 mL) was stirred at
room temperature for 2 h. The reaction mixture was concentrated
under reduced pressure and the residue was quenched with
saturated aqueous NaHCO3 and extracted into ethyl acetate. The
organic layer was concentrated under reduced pressure and the
residue was purified by NH-silica gel column chromatography
(9:1–2:8 n-heptane/ethyl acetate gradient) to afford the title
A mixture of 26 (300 mg, 1.19 mmol), (2,6-dimethoxy-4-meth-
ylphenyl)boronic acid (280 mg, 1.43 mmol), Pd(PPh3)4 (68.4 mg,
0.0593 mmol), and K2CO3 (492 mg, 3.56 mmol) in a mixture of
1,4-dioxane (8 mL) and H2O (2 mL) was stirred at reflux tempera-
ture for 2.5 h. The reaction mixture was cooled to room tempera-
ture and concentrated under reduced pressure. The residue was
extracted into ethyl acetate and the organic layer was concentrated
under reduced pressure. The residue was purified by silica gel col-
umn chromatography (8:2–0:10 n-heptane/ethyl acetate gradient)
to afford the title compound 34c as a light yellow foam (380 mg,
1.03 mmol, 86.7%).
compound 33b as a light yellow oil (29.6 mg, 61.3 lmol, 52.4%).
Compound purity: >99%.
1H NMR (600 MHz, CDCl3): d = 0.93 (d, J = 6.8 Hz, 6H), 1.72–1.81
(m, 1H), 2.20 (s, 3H), 2.95 (d, J = 6.8 Hz, 2H), 3.23 (t, J = 6.0 Hz, 2H),
3.28 (s, 3H), 3.43 (t, J = 6.0 Hz, 2H), 3.44 (s, 3H), 3.69 (s, 6H), 3.99 (s,
3H), 4.50 (s, 2H), 6.63 (s, 2H), 6.89 (d, J = 7.9 Hz, 1H), 7.18 (d,
J = 7.9 Hz, 1H), 8.51 (s, 1H); 13C NMR (151 MHz, CDCl3): d = 19.5,
21.0, 26.7, 55.9, 55.9, 56.0, 58.2, 58.7, 62.8, 70.7, 75.0, 103.6,
106.1, 118.3, 119.0, 127.2, 131.4, 131.8, 139.4, 140.0, 140.1,
~
1H NMR (600 MHz, CDCl3): d = 2.28 (s, 3H), 2.41 (s, 3H), 3.67
(s, 6H), 4.12 (s, 3H), 6.47 (s, 2H), 6.97 (d, J = 9.1 Hz, 1H), 8.46
(d, J = 9.1 Hz, 1H), 9.01 (s, 1H); 13C NMR (151 MHz, CDCl3):
d = 19.9, 22.3, 55.8, 56.7, 104.4, 105.2, 115.0, 122.7, 126.9,
131.3, 136.4, 137.4, 138.1, 140.3, 157.1, 157.8, 161.2; IR (KBr):
~
152.4, 155.0, 158.3; IR (KBr):
m = 2951, 2869, 2836, 1610, 1582,
m
= 3004, 2838, 1612, 1554, 1504, 1466, 1403, 1306, 1265,
1458, 1415, 1374, 1319, 1230, 1121, 1094, 823 cmꢀ1; HRMS-ESI
1243, 1194, 1123, 1093, 822, 810 cmꢀ1; HRMS-ESI m/z [M+H]+
m/z [M+H]+ calcd for C28H39N2O5+: 483.2854, found: 483.2838.
calcd for C20H21N2O5+: 369.1445, found: 369.1445.
5.1.62. 2-[2-Chloro-6-methoxy-4-(methoxymethyl)phenyl]-8-
methoxy-3-methyl-5-nitroquinoline (34a)
5.1.65. 2-[2-Chloro-6-methoxy-4-(methoxymethyl)phenyl]-8-
methoxy-3-methylquinolin-5-amine (35a)
A mixture of 26 (200 mg, 0.793 mmol), [2-chloro-6-methoxy-
4-(methoxymethyl)phenyl]boronic acid (219 mg, 0.952 mmol),
The title compound was prepared from 34a using a method
analogous to that described for 15b in 83.4% yield as a yellow solid.

K. Takeda et al. / Bioorg. Med. Chem. 20 (2012) 6559–6578
6577
1H NMR (600 MHz, CDCl3): d = 2.23 (s, 3H), 3.45 (s, 3H), 3.71 (s,
3H), 3.99 (s, 3H), 4.49 (s, 2H), 6.80 (d, J = 8.3 Hz, 1H), 6.86 (d,
J = 8.3 Hz, 1H), 6.89 (s, 1H), 7.06 (s, 1H), 8.08 (s, 1H); HRMS-ESI
m/z [M+H]+ calcd for C20H22ClN2O3+: 373.1313, found: 373.1312.
(br s, 1H); 13C NMR (151 MHz, CDCl3): d = 12.4, 19.6, 22.3, 48.4,
55.9, 55.9, 105.3, 105.9, 116.4, 118.2, 127.4, 131.4, 131.7, 139.5,
~
139.5, 152.3, 155.1, 158.0; IR (KBr):
m
= 2968, 2814, 1611, 1580,
1460, 1401, 1318, 1241, 1124, 1092, 813 cmꢀ1; HRMS-ESI m/z
[M+H]+ calcd for C24H31N2O3+: 395.2329, found: 395.2331.
5.1.66. 4-(5-Amino-8-methoxy-3-methylquinolin-2-yl)-3,5-
dimethoxybenzonitrile (35b)
5.2. Biology
The title compound was prepared from 34b using a method
analogous to that described for 15b in 61.3% yield as a light yellow
solid.
5.2.1. In vitro binding assay
For the binding assay and functional assay, HEK293 cells
expressing the human CRF1 receptor were cloned.2a Screening of
CRF1 receptor binding was performed using a scintillation proxim-
ity assay (SPA, Amersham Pharmacia, UK) using 96-well plates. Cell
1H NMR (600 MHz, CDCl3): d = 2.18 (s, 3H), 3.70 (s, 6H), 3.96 (s,
3H), 6.76 (d, J = 8.1 Hz, 1H), 6.84 (d, J = 8.1 Hz, 1H), 6.91 (s, 2H),
8.02 (s, 1H); 13C NMR (151 MHz, CDCl3): d = 19.2, 56.2, 56.2,
107.6, 108.2, 110.4, 112.9, 119.1, 120.4, 123.8, 129.7, 130.1,
134.3, 139.2, 149.5, 153.0, 158.5; HRMS-ESI m/z [M+H]+ calcd for
membrane (5
(1 mg/well), [125I] human/rat CRF (0.1 nM), and a diluted test com-
pound solution were suspended in 150 L of assay buffer (137 mM
lg/well), wheat germ agglutinin coated SPA beads
C
20H20N3O3+: 350.1499, found: 350.1500.
l
NaCl, 8.1 mM Na2HPO4, 2.7 mM KCl, 1.5 mM KH2PO4, 10 mM
MgCl2, 2 mM EGTA, 1.5% bovine serum albumin (BSA), protease
inhibitor cocktail (Roche Diagnostics GmbH), pH 7.0). Total binding
and nonspecific binding were measured in the absence and pres-
5.1.67. 2-(2,6-Dimethoxy-4-methylphenyl)-8-methoxy-3-
methylquinolin-5-amine (35c)
The title compound was prepared from 34c using a method
analogous to that described for 15b in 94.6% yield as a yellow solid.
1H NMR (600 MHz, CDCl3): d = 2.22 (s, 3H), 2.40 (s, 3H), 3.66 (s,
6H), 3.96 (s, 3H), 6.45 (s, 2H), 6.71 (d, J = 7.8 Hz, 1H), 6.82 (d,
J = 7.8 Hz, 1H), 8.06 (s, 1H); 13C NMR (151 MHz, CDCl3): d = 19.5,
22.3, 55.9, 56.2, 105.3, 107.6, 109.9, 115.8, 120.2, 129.8, 131.1,
134.5, 138.9, 139.8, 149.2, 155.2, 158.0; HRMS-ESI m/z [M+H]+
calcd for C20H23N2O3+: 339.1703, found: 339.1709.
ence of 0.4 lM unlabeled sauvagine, respectively. Plates were sha-
ken gently and incubated for over 2 h at room temperature. The
plates were centrifuged (260ꢁg, 5 min, room temperature), and
the radioactivity was detected using a TopCount scintillation coun-
ter (Perkin Elmer, MA, USA) with 1 min counting time per well.
Each count was corrected by subtracting the non-specific binding
and was represented as a percentage of total binding. The IC50
value of each compound was calculated using a concentration-
response curve.
5.1.68. 2-[2-Chloro-6-methoxy-4-(methoxymethyl)phenyl]-N,N-
diethyl-8-methoxy-3-methylquinolin-5-amine (36a)
The title compound was prepared from 35a using a method
analogous to that described for 16a in 79.6% yield as a light yellow
foam. Compound purity: >99%.
5.2.2. In vitro functional assay
To determine the activities of the antagonists, their effects on
CRF-stimulated intracellular cyclic AMP (cAMP) accumulation
were examined for HEK293 cells expressing the human CRF1 recep-
tor, as described previously.2a The accumulation of cAMP was mea-
sured using an enzyme immunoassay (EIA) kit (Amersham
Pharmacia, UK). HEK293 cells expressing the human CRF1 receptor
were seeded in 96-well plates (5 ꢁ 104 cells/well) in Dulbecco’s
modified eagle medium (DMEM) containing 0.1% fetal bovine ser-
um and 1 mM 3-isobutyl-1-methylxanthine, which is a phosphodi-
esterase inhibitor. After 30 min of preincubation, the diluted test
compounds were added to the wells and incubated for another
30 min at 37 °C. The cells were then stimulated with 1 nM hu-
man/rat CRF for 30 min at 37 °C and collected by centrifugation
(630ꢁg, 5 min, 4 °C). After aspiration of the medium, the cells were
lysed with the EIA kit lysis buffer. The amount of intracellular
cAMP was measured according to the manufacturer’s instructions.
Basal levels of cAMP (i.e., in the absence of CRF) were subtracted
from the measured values and these were then expressed as a per-
centage of total production. The IC50 value of each compound was
calculated using a concentration-response curve.
1H NMR (600 MHz, CDCl3): d = 1.04 (t, J = 6.9 Hz, 6H), 2.21 (s, 3H),
3.14 (q, J = 6.9 Hz, 4H), 3.44 (s, 3H), 3.71 (s, 3H), 4.01 (s, 3H), 4.44–
4.52 (m, 2H), 6.88 (s, 1H), 6.93 (d, J = 8.3 Hz, 1H), 7.06 (s, 1H), 7.12
(d, J = 8.3 Hz, 1H), 8.47 (s, 1H); 13C NMR (151 MHz, CDCl3):
d = 12.4, 19.2, 48.4, 56.0, 56.1, 58.3, 74.1, 106.3, 108.5, 118.7,
120.7, 127.6, 128.5, 130.6, 132.3, 134.2, 139.5, 139.6, 140.4, 152.3,
~
154.9, 158.5; IR (KBr):
m = 2965, 2923, 2817, 1604, 1567, 1466,
1402, 1368, 1258, 1187, 1101, 1045, 822 cmꢀ1; HRMS-ESI m/z
[M+H]+ calcd for C24H30ClN2O3+: 429.1940, found: 429.1937.
5.1.69. 4-[5-(Diethylamino)-8-methoxy-3-methylquinolin-2-
yl]-3,5-dimethoxybenzonitrile (36b)
The title compound was prepared from 35b using a method
analogous to that described for 16a in 77.3% yield as a light yellow
foam. Compound purity: >99%.
1H NMR (600 MHz, CDCl3): d = 1.03 (t, J = 6.3 Hz, 6H), 2.18 (s,
3H), 3.13 (q, J = 6.3 Hz, 4H), 3.71 (s, 6H), 4.00 (s, 3H), 6.83–7.00
(m, 3H), 7.13 (d, J = 7.6 Hz, 1H), 8.46 (s, 1H); 13C NMR (151 MHz,
CDCl3): d = 12.4, 19.2, 48.4, 56.0, 56.2, 106.2, 108.3, 112.7, 118.8,
119.1, 124.1, 127.6, 130.4, 132.2, 139.5, 139.6, 152.2, 152.8,
~
5.2.3. CRF-induced ACTH release model
158.6; IR (KBr):
m
= 2970, 2833, 2225, 1601, 1572, 1465, 1412,
For CRF-induced ACTH elevation studies, male Fischer 344 rats
weighing 152–177 g were purchased from Charles River Laborato-
ries Japan, Inc. (Kanagawa, Japan). Test compounds were orally
administered to the rats 1 h before subcutaneous injection of CRF
1243, 1122, 1096, 1064, 820 cmꢀ1; HRMS-ESI m/z [M+H]+ calcd
for C24H28N3O3+: 406.2125, found: 406.2125.
5.1.70. 2-(2,6-Dimethoxy-4-methylphenyl)-N,N-diethyl-8-
methoxy-3-methylquinolin-5-amine (36c)
(10 l
g kgꢀ1). Non-CRF control and CRF control groups received
an equivalent volume of vehicle (5% DMSO/5% CremophorÒ/90%
saline). Thirty minutes after subcutaneous injection of CRF, blood
samples of over 2 mL were obtained from the decapitated animals,
The title compound was prepared from 35c using a method
analogous to that described for 16a in 76.1% yield as a light yellow
foam. Compound purity: >99%.
stirred with 100
The blood samples were centrifuged (1000ꢁg, 4 C, 5 min), and
200 L of plasma samples per animal were prepared in duplicate.
Plasma ACTH concentrations were determined by using a radioim-
l
L of EDTA 2Na (100 mg mLꢀ1), and kept on ice.
1H NMR (600 MHz, CDCl3): d = 1.03 (t, J = 6.7 Hz, 6H), 2.21 (s,
3H), 2.40 (s, 3H), 3.13 (q, J = 6.7 Hz, 4H), 3.67 (s, 6H), 3.99 (s, 3H),
6.46 (s, 2H), 6.88 (d, J = 8.1 Hz, 1H), 7.08 (d, J = 8.1 Hz, 1H), 8.41
l

6578
K. Takeda et al. / Bioorg. Med. Chem. 20 (2012) 6559–6578
munoassay (RIA) kit (ACTH IRMA, Mitsubishi Chemical Corpora-
tion). The radioactivity of the bead was measured using a scintilla-
tion counter (ARC-1000M, Aloka Corp., Tokyo, Japan). ACTH
concentration was calculated from a standard curve prepared
using standard ACTH solutions.
solution containing an internal standard (imipramine) was added
to plasma or brain homogenate (0.05 mL), centrifuged
(12,000 rpm, 5 min), and filtered. Then, the samples were exam-
ined using an LC–MS/MS methodology. Mobile phase A (distilled
water containing 0.1% HCOOH) and mobile phase B (MeCN con-
taining 0.1% HCOOH) were pumped at 0.5 mL minꢀ1 using a linear
gradient program [B%: 5% (0–1 min), 5–90% (1–4 min), 90% (4–
6.5 min)]. Ionization was initiated using electrospray (positive
mode) with monitoring of parent–daughter peaks of 425.25 >
381.2 for 21d and 281.1 > 86.1 for the internal standard.
5.2.4. Light–dark exploration tests
The apparatus consisted of a dark compartment (10 cm width
(W), 15 cm depth (D), 20 cm height (H)) and a light compartment
(20 cm W, 15 cm D, 20 cm H). The dark compartment had a lid
on top and consisted of black PlexiglasÒ while the light compart-
ment was open at the top and consisted of white PlexiglasÒ. A
black PlexiglasÒ tunnel (7 cm W, 10 cm D, 4.5 cm H) separated
the dark box from the light box. Light intensity in the experimental
room was 150 lx. Mice were transferred and habituated to 10 lx
light intensity at least 2 h before the test. Male BALB/c mice
(n = 10/group) were p.o. administered 1 h before the test and kept
in the 10-lx area until the experiment was started by placing the
mouse in the dark compartment. Behavior was videotaped for a
5-min period, and the time spent in the light compartment and
the number of tunnel crossings was measured after the experi-
ments. A mouse with all four paws in the light compartment was
considered to be in the light compartment fully. The vehicles of
21d and 36a were (0.5% MC containing HCl) and (5% DMSO, 5%
CremophorÒ EL, 90% saline, containing HCl), respectively.
Acknowledgements
The authors would like to thank the colleagues who helped to
generate all of the data reported in this manuscript. In particular,
special thanks are owed to Eri Ena, Satoko Sasaki, Mami Gomibu-
chi, Nao Shibuguchi, and Yumi Yokoyama for NMR, HRMS, and IR
analyses.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.09.028.
References and notes
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15. Meth-Cohn, O.; Rhouati, S.; Tarnowski, B. Tetrahedron Lett. 1979, 20, 4885.
16. Ishikawa, M.; Hashimoto, Y. J. Med. Chem. 2011, 54, 1539.
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5.3.2. Determination of pKa
The pKa values of the test compounds were determined using
the same method reported by Ishihama et al.18
5.4. DMPK study
5.4.1. Rat pharmacokinetic studies
Fasted F344 rats (n = 3 at each dose, 189.4–205.9 g, obtained
from Charles River Laboratories Japan, Inc.) were used for the
study. The dosing solution containing the test compound
(3 mg mLꢀ1 for 3 mg kgꢀ1 iv, 2 mg mLꢀ1 for 10 mg kgꢀ1 p.o.) was
administered (dosing solution: 5% ethanol-0.02 mol Lꢀ1 HCl/5%
glucose for iv; 5% DMSO/5% CremophorÒ EL/equivalent HCl/H2O
for p.o.). Blood samples were collected at designated times: 0.083
(for iv), 0.25, 0.5, 1, 2, 4, 6, and 8 h (for iv and p.o.). Brain samples
were collected 1 h after p.o. administration and homogenized in
distilled water (1:4, v/v). A MeOH/MeCN (50:50, v/v, 0.25 mL)