Structure-activity relationship study of non-steroidal NPC1L1 ligands identified through cell-based assay using pharmacological chaperone effect as a readout

Article abstract of DOI:10.1016/j.bmc.2014.05.022

Niemann-Pick type C1-like 1 (NPC1L1) is an intestinal cholesterol transporter that is known to be the target of the cholesterol absorption inhibitor ezetimibe. We previously discovered steroidal NPC1L1 ligands by using a novel cell-based assay that employs pharmacological chaperone effect as a readout. Those steroid derivatives bound to a site different from both the sterol-binding domain and the ezetimibe-binding site, implying that they may be a novel class of NPC1L1 inhibitors with a distinct mode of action. As an extension of that work, we aimed here to find non-steroidal NPC1L1 ligands, which may be better candidates for clinical application than steroidal ligands, by using the same assay to screen our focused library of ligands for liver X receptor (LXR), a nuclear receptor that recognizes oxysterols as endogenous ligands. Here we describe identification of a novel class of NPC1L1 ligands with a ring-fused quinolinone scaffold, and an analysis of the structure-activity relationships of their derivatives as NPC1L1 ligands.

Full text of DOI:10.1016/j.bmc.2014.05.022

Bioorganic & Medicinal Chemistry 22 (2014) 3587–3609
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure–activity relationship study of non-steroidal NPC1L1
ligands identified through cell-based assay using pharmacological
chaperone effect as a readout
a
a
b
Fumika Karaki a, Kenji Ohgane a,b, , Hiromitsu Fukuda , Masahiko Nakamura , Kosuke Dodo ,
⇑
Yuichi Hashimoto a
a Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
b RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Niemann-Pick type C1-like 1 (NPC1L1) is an intestinal cholesterol transporter that is known to be the tar-
get of the cholesterol absorption inhibitor ezetimibe. We previously discovered steroidal NPC1L1 ligands
by using a novel cell-based assay that employs pharmacological chaperone effect as a readout. Those ste-
roid derivatives bound to a site different from both the sterol-binding domain and the ezetimibe-binding
site, implying that they may be a novel class of NPC1L1 inhibitors with a distinct mode of action. As an
extension of that work, we aimed here to find non-steroidal NPC1L1 ligands, which may be better can-
didates for clinical application than steroidal ligands, by using the same assay to screen our focused
library of ligands for liver X receptor (LXR), a nuclear receptor that recognizes oxysterols as endogenous
ligands. Here we describe identification of a novel class of NPC1L1 ligands with a ring-fused quinolinone
scaffold, and an analysis of the structure–activity relationships of their derivatives as NPC1L1 ligands.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 16 April 2014
Revised 12 May 2014
Accepted 12 May 2014
Available online 21 May 2014
Keywords:
Niemann-Pick type C1-like 1
Cholesterol
Pharmacological chaperone
Non-steroidal ligands
Inhibitors
Structure–activity relationship
1. Introduction
provided NPC1L1 inhibitors of several structural classes (Fig. 1,
compound 2).13–15 However, these inhibitors were found by means
Hypercholesterolemia is one of the key risk factors for coronary
heart disease, a major cause of death in developed countries.
Plasma cholesterol is derived from a combination of de novo bio-
synthesis and dietary intake. Statins, a class of HMG-CoA reductase
inhibitors that reduce plasma LDL cholesterol level by inhibiting
the rate-limiting step of the de novo cholesterol synthetic pathway,
and ezetimibe (Zetia, Fig. 1), a cholesterol absorption inhibitor that
inhibits intestinal cholesterol transporter NPC1L1 (Niemann-Pick
type C1-like 1), have been shown to reduce plasma LDL cholesterol
level.1–6
NPC1L1 is a 13-pass transmembrane protein expressed on the
apical membrane of enterocytes and hepatocytes, and plays a crit-
ical role in the intestinal uptake of dietary cholesterol.7–9 NPC1L1
has three large extracellular loops. The first N-terminal loop (N-
terminal domain, NTD) is a cholesterol-binding domain and is
required for cholesterol absorption.10,11 The second loop connect-
ing transmembrane domains 2 and 3 has been proposed to be
important for binding of ezetimibe.12 Recent efforts directed
toward discovery of novel cholesterol absorption inhibitors have
of competitive binding assay with fluorescence- or radioisotope-
labeled ezetimibe analogs, and they are considered to bind at the
same site as ezetimibe.
We previously reported the identification of steroidal NPC1L1
ligands by means of a cell-based assay that employs the pharmaco-
logical chaperone effect as a readout.16 Pharmacological chaper-
ones are small-molecular ligands that correct trafficking defects
of target proteins with reduced folding efficiency, such as point
mutants or proteins with small deletions.17–22 The mechanism of
the pharmacological chaperone effect is thought to involve direct
binding of the ligands to the destabilized folding intermediate of
the mutant protein.17 While folding-defective proteins are usually
retained in the endoplasmic reticulum (ER) by the cellular quality
control system, pharmacological chaperones added to the cells sta-
bilize the mutant proteins and promote their escape from the ER to
the proper locations. Thus, the trafficking defects of mutant pro-
teins can be reversed by small-molecular ligands, and this can be
visualized as a change in the localization pattern of the mutant
proteins. We considered that this phenomenon could be applied
for ligand screening. Thus, we generated folding-defective ER-
retained NPC1L1 mutants and used them to screen for NPC1L1
ligands by monitoring correction of localization. This enabled us
⇑
Corresponding author. Tel.: +81 3 5841 7848.
E-mail address: ohgane@me.com (K. Ohgane).
http://dx.doi.org/10.1016/j.bmc.2014.05.022
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

3588
F. Karaki et al. / Bioorg. Med. Chem. 22 (2014) 3587–3609
O
OH
N
O
N
H
OH
O
N
H
N
N
O
N
O
H
H
HO
HO
F
N
O
O
F
N
N
ezetimibe (1)
2
3
Figure 1. Chemical structures of reported NPC1L1 ligands [compound 2: Ref. 13, compound 3: Ref. 16].
to identify a new class of steroidal NPC1L1 ligands (Fig. 1).16 We
further established that the binding sites of these steroidal ligands
are likely to be different from both the NTD and the ezetimibe-
binding site.16,23 This implies that these steroidal ligands might
inhibit NPC1L1 via a different mode of action from that of ezetim-
ibe. However, the metabolic instability and rather low selectivity of
steroids might make them ineligible as antihyperlipidemic agents.
Thus, we decided to search for non-steroidal counterparts of these
ligands using our pharmacological chaperone effect-based assay.
In order to identify non-steroidal NPC1L1 ligands, we focused
on our library of liver X receptor (LXR) ligands. Liver X receptor
(LXR) is a member of the nuclear receptor family, and its endoge-
nous ligands include oxysterols. As a subset of oxysterols was
active toward NPC1L1 in our pharmacological chaperone-based
assay,16 we hypothesized that non-steroidal compounds that bind
to NPC1L1 protein might be present in our library of non-steroidal
LXR ligands and their analogs.
chloride gave the amide (64). n-Butylation on the amide nitrogen
and subsequent intramolecular Heck reaction gave the phenanthri-
din-6-one (65). Hydrolysis of the 2-ester and condensation with
various amines afforded the desired compounds.
HEK293 cells stably expressing NPC1L1L1072T/L1168I were treated
with test compounds and the % plasma membrane localization val-
ues at 30 lM and 10 l
M were determined as previously reported.16
As shown in Table 1, an alkyl chain on the nitrogen atom, either lin-
ear or cyclic, was required for the activity. Among amides with linear
alkyl groups, the activity increased as the chain was elongated (8, 9,
10, 11), which may indicate that hydrophobicity of this moiety is
important for interaction with NPC1L1. This idea is consistent with
the fact that compounds with free hydroxyl groups (12) and an ether
moiety (19) were inactive. Nevertheless, amide NH proton did not
diminish the activity (7 vs 8). Of the seven secondary amides, cyclo-
propyl amide (15) was the most potent. Isopropyl amide (16) and
tert-butyl amide (17) were slightly less potent than the cyclopropyl
derivative (15), and cyclopentyl amide (13), cyclohexyl amide (14)
and benzyl amide (20) were almost inactive. This may indicate that
the hydrophobic pocket cannot accommodate a group larger than a
cyclopropyl moiety.
Here, we report the identification of ring-fused quinolinone
derivatives as pharmacological chaperone effect-based assay hits
from our LXR-related library, together with the results of struc-
ture–activity relationship studies of these compounds as NPC1L1
ligands.
2.3. Structure–activity relationship for the 5-alkyl moiety of
phenanthridin-6-one
2. Results and discussion
We next investigated the effect of the 5-alkyl moiety of phenan-
thridin-6-one, after fixing the 2-substituent as diethylamide. The
synthetic route to the 5-alkylated phenanthridin-6-ones is illus-
trated in Scheme 2. 4-Aminobenzoic acid (67) was converted to
diethylamide and then condensed with 2-bromobenzoyl chloride.
The amide nitrogen of 68 was protected with a p-methoxybenzyl
(PMB) group, and the phenanthridinon-6-one scaffold was con-
structed by intramolecular Heck reaction. Then the PMB group of
(25) was deprotected under acidic conditions, and various 5-alkyl-
ated phenanthridin-6-ones were obtained by means of alkylation
reaction. The ester moieties of 70 and 71 were hydrolyzed to give
26 and 27, and then condensation with dimethyamine afforded 28
and 29, respectively.
2.1. Identification of phenanthridin-6-one derivatives that
correct the cellular localization of mutant NPC1L1
Using pharmacological chaperone effect-based assay, we
screened our LXR-related library, which includes well-known LXR
agonists such as T0901317 (4) and GW3965 (5) (Fig. 2) and conform-
ationally restricted T0901317 analogs with a phenanthridin-6-one
scaffold.24–27 We found that some 2-amide derivatives of phenan-
thridin-6-ones with no LXR activity could correct the localization
of mutant NPC1L1. Other derivatives with alkyl, hydroxyl, carbonyl
and other groups were ineffective (data not shown). As amide at the
2-position was necessary for the activity, we first performed struc-
ture–activity relationship studies focused on this position.
As shown in Table 2, a sufficiently long alkyl chain at the 5-posi-
tion is essential for activity (21, 22, 23, 9). Considering that polar
substituents (acids and amides) on the alkyl chain diminished
the activity (26, 27, 28, 29), it appears that the hydrophobicity of
the alkyl group is important. The potency of the compound with
a benzyl group (24), which is also hydrophobic, could not be deter-
mined precisely due to its low solubility. Since we considered that
the poor solubility might be attributable to high planarity of the
phenanthridin-6-one scaffold, we set out to evaluate less planar
scaffolds.28
2.2. Structure–activity relationship for the 2-amide moiety of
phenanthridin-6-one
Several 2-amide derivatives of phenanthridin-6-one were syn-
thesized according to Scheme 1. Schotten–Baumann reaction
between methyl 4-aminobenzoate (63) and 2-bromobenzoyl
F3C
N
S
2.4. Structure development to obtain less planar scaffolds
N
O
O
F3C
HO
F3C
Cl
O
To design less planar scaffolds than phenanthridin-6-ones, we
adopted three strategies: (1) replace the C-ring of phenanthridin-
6-ones with saturated rings, (2) replace the pyridone moiety of
phenanthridin-6-ones with a seven-membered oxazepinone ring
CF3
T0901317 (4)
CO2H
GW3965 (5)
Figue 2. Chemical structures of LXR agonists T0901317 and GW3965.

F. Karaki et al. / Bioorg. Med. Chem. 22 (2014) 3587–3609
3589
nBu
nBu
a
b
c
H
NH2
N
O
N
O
N
MeO2C
O
Br
HO2C
MeO2C
MeO2C
63
64
65
66
6
R=R'=H
nBu
7
R=Me, R'=H
R=R'=Me
R=R'=Et
d, e or f
N
O
8
9
R
N
10 R=R'=nPr
11 R=R'=nBu
R'
O
12 R=R'=CH2CH2OH
13 R=c-pentyl, R'=H
14 R=c-hexyl, R'=H
15 R=cPr, R'=H
16 R=i-Pr, R'=H
17 R=t-Bu, R'=H
18 NRR'=piperazyl
19 NRR'=morpholine
20 R=Bn, R'=H
Scheme 1. Synthetic route to 2-amide derivatives of phenanthridin-6-one. Reagents and conditions: (a) 2-bromobenzoyl chloride, saturated aqueous NaHCO3, CH2Cl2, rt; (b)
NaH, n-butyl iodide, DMF, rt then Pd(OAc)2, PCy3ꢀHBF4, Cs2CO3, DMA, 130 °C; (c) LiOHꢀH2O, THF, H2O, rt; (d) (COCl)2, DMF, CH2Cl2, RR0NH (for 6 28% aqueous ammonia
solution), Et3N, rt; (e) (COCl)2, DMF, CH2Cl2, diethanolamine, saturated aqueous NaHCO3, rt; (f) isobutyl chloroformate, RR0NH (9, 19) or 40% aqueous RR0NH solution (7, 8),
Et3N, THF, rt.
Table 1
Pharmacological chaperone effect of 2-amide derivatives of phenanthridin-6-one
N
O
R
No.
R
% PM localization
10
No.
R
% PM localization
10 lM
30
0
lM
l
M
30
11
l
M
H
N
H2N
6
7
8
N.D.
1
14
15
16
3
O
O
O
H
N
H
N
24
4
55
49
14
0
O
O
O
H
N
N
N.D.
O
N
H
N
9
45
50
1
17
18
41*
47
11
0
O
N
N
10
14
O
O
O
N
O
N
11
Toxic
18
19
20
0
0
N.D.
N.D.
O
OH
H
N
12
13
0
N.D.
N.D.
N
O
HO
O
H
N
0*
O
N.D.: not determined.
*
Precipitation was observed.

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F. Karaki et al. / Bioorg. Med. Chem. 22 (2014) 3587–3609
PMB
N
PMB
N
H
a
NH2
O
b
c
N
Et2N
Et2N
O
Br
HO2C
Et2N
O
Br
O
O
68
67
69
O
25
R
H
N
O
d
O
O
f
n
Et2N
N
O
Et2N
21
O
e
70 n=1 R=Et
71 n=3 R=Et
g
h
26 n=1 R=OH
27 n=3 R=OH
R
N
O
28 n=1 R=NMe2
29 n=3 R=NMe2
Et2N
O
22 R=Me
23 R=Et
24 R=Bn
Scheme 2. Synthetic route to 5-alkylated derivatives of phenanthridin-6-one. Reagents and conditions: (a) SOCl2, DMF, CH2Cl2, rt then 2-bromobenzoyl chloride, saturated
aqueous NaHCO3, CH2Cl2 rt; (b) NaH, 4-methoxybenzyl chloride, DMF, rt; (c) Pd(OAc)2, PCy3ꢀHBF4, Cs2CO3, DMA, 130 °C; (d) TFA (neat), reflux; (e) R-I (R = Me, Et) or R-Br
(R = Bn), Cs2CO3, DMF, 90 °C; (f) Br(CH2)nCO2Et, Cs2CO3, TBAI, DMF, 90 °C; (g) 10 M aqueous NaOH, MeOH, H2O, rt; (h) isobutyl chloroformate, 40% aqueous dimethylamine
solution, Et3N, THF, CH2Cl2, rt.
Table 2
Pharmacological chaperone effect of 5-alkyl derivatives of phenanthridin-6-one
R
N
O
Et2N
O
No.
R
% PM localization
10
No.
R
% PM localization
10 lM
30
0
lM
lM
30
18
l
M
H
21
22
23
N.D.
N.D.
N.D.
25
26
27
0
OMe
O
O
0
0*
0*
0
N.D.
N.D.
N.D.
N.D.
OH
O
4
OH
9
45
0*
1
28
29
NMe2
O
24
N.D.
0
NMe2
N.D.: not determined.
*
Precipitation was observed.
and (3) remove the C-ring of phenanthridin-6-ones. The designed
compounds are shown in Table 3.
The synthetic route to the 5,6,7,8,9,10-hexahydrophenanthri-
dine scaffold (34) is illustrated in Scheme 4. Methyl 4-butyr-
ylamidobenzoate (80) was synthesized by Schotten–Baumann
reaction between methyl 4-aminobenzoate (63) and butyryl chlo-
ride. (2-Bromocyclohex-1-en-1-yl)methanol (81) was obtained by
Luche reduction of the aldehyde (73b). O-Mesylation of 81 and
subsequent SN2 reaction gave 82. Intramolecular Heck reaction fol-
lowed by hydrolysis of the ester and condensation with diethyl-
amine afforded the desired compound.
The synthetic route to dibenzoxazepinone (35) is illustrated in
Scheme 5. Schotten–Baumann reaction between methyl 4-amino-
3-hydroxybenzoate and 2-fluorobenzoyl chloride and subsequent
SNAr reaction gave 86. N-n-Butylation, hydrolysis of the ester,
and condensation with diethylamine gave the desired compound.
The synthetic route to the cycloalkaquinolinones (30, 31, 32,
33) is illustrated in Scheme 3. 2-Bromocycloalkene-1-carboxylic
acids (74a–d) to serve as C-ring units were synthesized from
corresponding cycloalkanones according to the literature proce-
dure.29 Methyl 4-(butylamino)benzoate (76), which corresponds
to the A-ring, was synthesized in two steps from methyl 4-amino-
benzoate (63). Condensation between methyl 4-(butylamino)
benzoate (76) and 2-bromocycloalkene-1-carboxylic acids
(74a–d), followed by intramolecular Heck cyclization afforded
cycloalkaquinolinones (78a–d). Hydrolysis of the methyl esters
under basic conditions and condensation with diethylamine gave
the desired compounds.

F. Karaki et al. / Bioorg. Med. Chem. 22 (2014) 3587–3609
3591
Table 3
Pharmacological chaperone effect of compounds with various scaffolds
Compound
% PM localization
Compound
% PM localization
10 lM
30
15
l
M
10
lM
30
1
lM
O
N
N
N
O
O
O
N
30
31
32
1
34
35
36
N.D.
N.D.
N.D.
Et2N
O
Et2N
O
O
N
O
42
42
2
3
1
Et2N
O
Et2N
O
N
O
14
Et2N
O
Et2N
O
N
O
33
44
5
Et2N
O
N.D.: not determined.
R
NH2
CO2H
CHO
Br
NH
O
a
b
c
Br
MeO2C
MeO2C
n
n
n
63
72a n=1
72b n=2
72c n=3
72d n=4
75 R=COCF3
76 R=nBu
73a n=1
73b n=2
73c n=3
73d n=4
74a n=1
d
74b n=2
74c n=3
74d n=4
78a n=1, R=Me
78b n=2, R=Me
78c n=3, R=Me
78d n=4, R=Me
CO2H
74a n=1
74b n=2
74c n=3
74d n=4
Br
n
nBu
nBu
nBu
g
n
N
O
79a n=1, R=OH
79b n=2, R=OH
79c n=3, R=OH
79d n=4, R=OH
e
NH
N
f
R
O
O
Br
n
MeO2C
MeO2C
h
77a n=1
77b n=2
77c n=3
77d n=4
30 n=1,R=NEt2
31 n=2, R=NEt2
32 n=3, R=NEt2
33 n=4, R=NEt2
76
Scheme 3. Synthetic route to the cycloalkaquinolinones. Reagents and conditions: (a) DMF, PBr3, CHCl3, reflux or rt; (b) NaH2PO4, NaOClO, H2O2, CH3CN, H2O, rt; (c)
(CF3CO)2O, Et3N, CH2Cl2, rt; (d) n-butyl iodide, Cs2CO3, DMF, 90 °C then K2CO3, MeOH, 70 °C; (e) (COCl)2, DMF, DMAP, CH2Cl2, 60 °C; (f) Pd(OAc)2, PCy3ꢀHBF4, Cs2CO3, DMA,
130 °C; (g) NaOH, THF, MeOH, H2O, rt; (i) (COCl)2, DMF, Et2NH, Et3N, CH2Cl2, rt.
The synthetic route to 2-oxo-1,2,3,4-tetrahydroquinoline (36) is
illustrated in Scheme 6. Bromination at the 6-position of 3,4-dihy-
dro-2(1H)-quinolinone (88)30 and subsequent coupling with phe-
nyl formate31 were conducted by employing reported reaction
conditions. N-Alkylation, hydrolysis using hydrogen peroxide32,33
and condensation gave the desired compound.
is the planarity or aromaticity of the B-rings. Thus, the properties
of the B-ring may be important for the activity. Among the cyclo-
alkaquinolinones, cycloheptaquinolinone (32) was the most
potent. Thus, the protein appears to contain a hydrophobic pocket
large enough to interact with a cycloheptane ring.
Although cycloalkaquinolinones corrected the localization of
mutant NPC1L1, bicyclic compound (36) was inactive (Table 3).
This may imply that a tricyclic scaffold is needed to serve as a ste-
roid mimic for interaction with NPC1L1. However, tricyclic com-
pounds 34 and 35 were almost inactive. One of the differences
between cycloalkaquinolinones and the other tricyclic compounds
2.5. Structure–activity relationship for the 2-amide moiety of
cycloheptaquinolinone
We next optimized the amide substituent at the 2-position of
cycloheptaquinolinone. Various amides were synthesized from
acid 79c as illustrated in Scheme 7. Again, an alkyl group on amide

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F. Karaki et al. / Bioorg. Med. Chem. 22 (2014) 3587–3609
H
N
NH2
a
O
MeO2C
MeO2C
63
80
O
O
CHO
Br
b
c
d
OH
N
N
Br
R
Br
MeO2C
73b
81
O
82
83 R=OMe
84 R=OH
34 R=NEt2
e
f
Scheme 4. Synthetic route to 5,6,7,8,9,10-hexahydrophenanthridine derivative. Reagents and conditions: (a) butyryl chloride, saturated aqueous NaHCO3, CH2Cl2, rt; (b)
NaBH4, CeCl3ꢀ7H2O, THF, MeOH, rt; (c) methanesulfonyl chloride, Et3N, CH2Cl2, 0 °C then NaH, 80, TBAI, DMF, 90 °C; (d) Pd(OAc)2, PCy3ꢀHBF4, Cs2CO3, DMA, 150 °C; (e) NaOH,
THF, MeOH, H2O, rt; (f) EDCI, DMAP, Et2NH, CH2Cl2, rt.
H
N
O
NH2
OH
O
a
b
N
O
MeO
MeO
O
R
O
O
86
O
85
87 R=OMe
35 R=NEt2
c
Scheme 5. Synthetic route to the dibenzoxazepinone. Reagents and conditions: (a) 2-fluorobenzoyl chloride, saturated aqueous NaHCO3, CH2Cl2, rt then K2CO3, DMF, 150 °C;
(b) n-butyl iodide, Cs2CO3, DMF, 90 °C; (c) NaOH, MeOH, H2O, 70 °C then EDCI, HOBt, Et2NH, DMF, rt.
H
H
H
N
O
a
N
O
b
c
N
O
N
O
R
Br
PhO2C
O
90
88
89
91 R=OPh
92 R=OH
36 R=NEt2
d
e
Scheme 6. Synthetic route to 2-oxo-1,2,3,4-tetrahydroquinoline derivative. Reagents and conditions: (a) NBS, DMF, 0 °C to rt; (b) phenyl formate, nBu3N, Pd(OAc)2,
P(tBu3)ꢀHBF4, NMP, 150 °C; (c) n-butyl iodide, Cs2CO3, DMF, rt; (d) NaOH, H2O2, 1,4-dioxane, H2O, rt; (e) (COCl)2, DMF, Et2NH, Et3N, CH2Cl2, rt.
nBu
nBu
nBu
nBu
N
O
N
O
N
O
N
O
a or b
R
N
R
N
c
H
N
R'
HO2C
O
O
O
79c
51 R=Me
52 R=Et
37 R=R'=H
38 R=Me, R'=H
39 R=R'=Me
50
40 R=Et, R'=H
41 NRR'=pirrolidyl
42 R=nPr, R'=H
43 R=nBu, R'=H
44 R=iPr, R'=H
45 R=tBu, R'=H
46 R=Ph, R'=H
47 R=CH2CH2OH, R'=H
48 R=c-pentyl, R'=H
49 R=c-hexyl, R2=H
50 R=cPr, R'=H
Scheme 7. Synthetic route to the amide derivatives of cycloheptaquinolinones. Reagents and conditions: (a) (COCl)2, DMF, RR0NH, Et3N, CH2Cl2, rt; (b) (COCl)2, DMF, aqueous
amine solution, CH2Cl2, rt; (c) NaH, R-I, DMF, rt.
nitrogen is required for activity (compare 37 with alkylated
amides) and cyclopropyl amide (50) is relatively potent (Table 4).
Cycloheptaquinolinones with isopropyl and tert-butyl groups
(44 and 45, respectively) were less potent, even though
phenanthridin-6-ones with these alkyl groups are quite potent.
This may be attributable to the difference in the size of the C
rings between the phenanthridin-6-ones and the cycloheptaquin-
olinones. As the C ring of the cycloheptaquinolinones is larger,

F. Karaki et al. / Bioorg. Med. Chem. 22 (2014) 3587–3609
3593
Table 4
Pharmacological chaperone effect of amide derivatives of cycloheptaquinolinone
N
O
R
O
No.
R
% PM localization
10
No.
R
% PM localization
10 lM
30
0
l
M
lM
30
25
l
M
H
N
H2N
37
38
39
40
N.D.
0
44
45
46
47
0
O
O
O
H
N
H
N
23
2
1
0
0
N.D.
N.D.
N.D.
O
O
H
N
N
N.D.
0
O
H
N
H
N
HO
28
O
O
O
H
N
N
N
32
42
14
48
16
6
O
O
H
N
41
42
43
33
36
18
1
0
0
49
50
51
5
2
3
1
O
H
H
N
N
49
21
O
O
H
N
N
O
O
O
N
52
45
8
N.D.: not determined.
the position of the scaffold might be shifted slightly so that the
amide group interacts less effectively with the binding pocket.
Longer or more bulky substituents (43, 46, 48 and 49) and a hydro-
philic group (47) diminished the activity. Amide NH proton did not
diminish the activity, as was the case in the phenanthridin-6-ones.
A methyl group diminished the activity (38 vs 39 and 50 vs 51), but
an ethyl group did not (40 vs 32 and 50 vs 52). From these results it
can be assumed that the pocket can accommodate both hydrogen
bond-mediated interaction with NH proton and hydrophobic inter-
action with an ethyl group. The lower potency of 39 and 51 might
be due to the absence of these two interactions.
2,4-dimethoxybenzyl group of 98 was deprotected under acidic
conditions to give 53, and 53 was alkylated with various alkyl
halides. The compound with a free hydroxyl group (60) was
obtained by deprotecting the TBS group of 99 with TBAF.
In the case of cycloheptaquinolinones, no precipitation was
observed under our assay conditions, indicating improved solubil-
ity of these compounds compared to the phenanthridin-6-ones
(Table 5). The effects of the 5-alkyl group were nearly the same
as in phenanthridin-6-ones (Table 2). An alkyl chain at the
5-position was necessary for the activity and the optimum length
was n-butyl; shorter or longer derivatives showed diminished
activity (53–56, 52, 57). As the compounds with n-hexyl (58) and
isopropyl (61) groups were cytotoxic, we could not evaluate their
activity. In contrast to the N-benzyl derivative of phenanthridin-
6-one (24), the N-benzyl counterpart of cycloheptaquinolinone
(62) was sufficiently soluble to evaluate, but was only weakly
active. Hydrophilic hydroxyl and ether groups (59, 60) diminished
the activity, as in the case of phenanthridin-6-ones.
So far, we could obtain potent non-steroidal NPC1L1 ligand 52 via
structure–activity relationship study, starting from a hit compound
in our library of LXR ligands, which mainly contains ring-fused
derivatives of a LXR agonist T0901317. To examine selectivity of
the ring-fused quinolinone 52 over LXR, we tested whether the
ring-fused quinolinone 52 activate LXR by using reporter gene
assay.27 While T0901317 activate a LXR even at as low as 100 nM,
2.6. Structure–activity relationship for the 5-alkyl moiety of
cycloheptaquinolinone
Finally, we investigated the effect of 5-alkyl groups of cycloh-
eptaquinolinones, with the 2-substituent fixed as cyclopropyleth-
ylamide. The synthetic route to the 5-alkylated derivatives is
illustrated in Scheme 8. 4-Iodobenzoic acid (93) was condensed
with cyclopropylamine and then alkylated with ethyl iodide to
afford 95. Then copper-catalyzed coupling of 95 and 2,4-dime-
thoxybenzylamine was performed according to the literature
procedure.34 The amine (96) was condensed with 74c, and the
resultant amide (97) was cyclized by intramolecular Heck reac-
tion to afford 98 with
a cycloalkaquinolinone scaffold. The

3594
F. Karaki et al. / Bioorg. Med. Chem. 22 (2014) 3587–3609
OMe
Br
OMe
O
I
I
NH
N
a
c
d
MeO
MeO
CO2H
O
N
R
93
O
N
O
N
94 R=H
95 R=Et
96
97
Et
Et
b
OH
OTBS
N
R
e
N
O
i
N
O
O
N
N
N
O
O
O
98 R=2,4-dimethoxybenzyl
53 R=H
99
60
f
54 R-Me
g or h
55 R=Et
56 R=nPr
57 R=n-Pentyl
58 R=n-Hexyl
59 R=CH2CH2OMe
61 R=iPr
62 R=Bn
99 R=CH2CH2OTBS
Scheme 8. Synthetic route to 5-alkyl derivatives of cycloheptaquinolinones. Reagents and conditions: (a) (COCl)2, DMF, cyclopropylamine, CH2Cl2, rt; (b) NaH, ethyl iodide,
DMF, 90 °C; (c) 2,4-dimethoxybenzylamine, CuI, K3PO4, ethylene glycol, nBuOH, 100 °C; (d) 74c, (COCl)2, DMAP, DMF, CH2Cl2, 50 °C; (e) Pd(OAc)2, PCy3ꢀHBF4, Cs2CO3, DMA,
130 °C; (f) TFA (neat), 80 °C; (g) R-I, Cs2CO3, DMF, 90 °C; (h) R-Br, TBAI, Cs2CO3, DMF, 90 °C; (i) TBAF, THF, 50 °C.
Table 5
Pharmacological chaperone effect of 5-alkylated derivatives of cycloheptaquinolinone
R
N
O
N
O
No.
R
% PM localization
10
No.
R
% PM localization
10 lM
30
lM
lM
30
35
l
M
H
53
54
0
0
N.D.
N.D.
57
58
3
1
Toxic
0
55
56
7
N.D.
0
59
60
N.D.
N.D.
OMe
OH
39
0
52
45
8
61
62
Toxic
0
0
14
N.D.: not determined.
the ring-fused quinolinone 52 was totally inactive at 30
not shown). Thus, the NPC1L1 ligand 52 was found to be selective
l
M (data
idea that the ring-fused quinolinones indeed act as pharmacologi-
cal chaperones and not as proteostasis regulators.
over LXR by at least two orders of magnitude.
It has been reported that chemical perturbation of cellular fold-
ing machineries can also correct mislocalization of folding-defec-
tive proteins, and small molecules with such activity are called
proteostasis regulators.35–37 In order to exclude the possibility that
the ring-fused quinolinone derivatives act as proteostasis regula-
tors, we examined their effect on folding-defective NPC1I1061T
mutant,38 a close homologue of NPC1L1. While mo56AZK, a ste-
rol-derived pharmacological chaperone for NPC1,23 corrected the
localization of NPC1I1061T, the ring-fused quinolinone 52, one of
the most potent compounds towards NPC1L1L1072T/L1168I mutant,
had no effect on NPC1I1061T (Fig. 3a). This specificity supports the
2.7. Cycloheptaquinolinone derivatives act independently of the
N-terminal sterol-binding site
We previously proposed the existence of a non-NTD sterol-
binding site on NPC1L1, and suggested that our steroidal NPC1L1
ligands were bound at this second sterol-binding site.16 Accordingly,
we tested whether or not the ligand-mediated rescue of mutant
NPC1L1 by our newly discovered non-steroidal ligands requires
the NTD. As shown in Figure 3b, cycloheptaquinolinone compound
(52) corrected the localization defect of NTD-deleted NPC1L1L1072T/
(D
NTD-NPC1L1L1072T/L1168I). Further, cycloheptaquinolinone
L1168I

F. Karaki et al. / Bioorg. Med. Chem. 22 (2014) 3587–3609
3595
a
b
vehicle
30µM 52
10µM mo56AZK
O
N
H
H
H
N3
O
H
H
N
O
OH
OH
O
mo56AZK
(a pharmacological chaperone for NPC1)
c
vehicle
30µM 52
Full-length NPC1L1
(L1072T/L1168I)
full-length
delNTD
160
140
120
100
80
NTD NPC1L1
(L1072T/L1168I)
160
140
120
100
80
52
55
53
1 (ezetimibe)
1 3
10
30
Concentration (µM)
Figure 3. Characterization of cycloheptaquinolinones as pharmacological chaperones, dispensability of NTD for their activity, and selectivity for NPC1L1L1072T/L1168I over
another folding-defective protein, NPC1I1061T mutant. (a) HEK293 cells stably expressing folding-defective NPC1I1061T-tGFP were treated as indicated for 20 h, and the
localization of the NPC1 proteins was examined. The compound mo56AZK is a pharmacological chaperone for NPC1 proteins. (b) The N-terminal domain is dispensable for the
pharmacological chaperone effect of the cycloheptaquinolinones. HEK293 cells stably expressing full-length NPC1L1L1072T/L1168I-EGFP or
D
NTD-NPC1L1L1072T/L1168I-EGFP were
m. (c) Ligand-mediated stabilization of
NTD-NPC1L1
treated as indicated for 20 h and the localization of NPC1L1 was examined. Green, NPC1L1-GFPs. Blue, Hoechst-33342. Scale bar, 20
l
NPC1L1 mutant proteins is independent of the presence of NTD. Upper, dose-dependent up-regulation of full-length NPC1L1 mutant. Lower, up-regulation of
D
mutant. The steady-state expression levels of full-length or NTD-deleted NPC1L1L1072T/L1168I-GFP were quantified by measuring GFP fluorescence in the lysate of cells treated
with or without the indicated compounds. The GFP fluorescence was normalized with respect to total protein concentration determined by bicinchonic acid assay, and is
presented as% of that of vehicle-treated cells.
derivatives stabilized both full-length and NTD-deleted
NPC1L1L1072T/L1168I mutant proteins, and increased steady-state lev-
els ofthe proteins in a similar manner tothat observedwith steroidal
ligands16,39 (Fig. 3c). Moreover, the order of the potency among the
representative derivatives (52 > 55 > 53 and ezetimibe, which had
no effect) was also conserved between full-length and NTD-deleted
NPC1L1 mutant proteins. These results strongly suggest that our
non-steroidal ligands act independently of the NTD.
4. Experimental
4.1. Biology
4.1.1. General
Cell culture, NPC1L1L1072T/L1168I localization assay and quantifi-
cation of GFP fluorescence in lysates were performed according to
our previous report.16
4.1.2. Localization of NPC1L1-EGFP mutants and NPC1-tGFP in
stably transfected cells16,23
3. Conclusion
HEK293 cells stably expressing NPC1L1 or NPC1 constructs
were grown in EzView 96-well glass-bottomed plates coated with
poly-D-lysine (20 lg/mL in PBS). After incubation for 24 h, cells
were treated with test compounds (solutions contained 0.1% final
concentration of DMSO) and further incubated for the indicated
times. Cells were fixed with 3.7% formaldehyde-PBS containing
In the present work, we used our previously developed pharma-
cological chaperone effect screening assay to identify novel non-ste-
roidal ligands targeting the non-NTD sterol-binding site of NPC1L1.
Although the identified non-steroidal compounds, even after struc-
ture development based on structure–activity relationship studies,
were not as potent as our steroidal ligands, our results confirm the
effectiveness of the pharmacological chaperone-based assay as a
screening tool. Screening of larger libraries may yield more potent
compounds that would serve as better starting points for optimiza-
tion. We are planning to examine the inhibitory activity of the com-
pounds identified here on NPC1L1-mediated cholesterol absorption.
Hoechst 33342 (1 lg/mL), and images were obtained on a Zeiss
LSM710 (63ꢁ oil-immersion objective lens with NA 1.40). Image
processing (noise reduction with median filter, contrast enhance-
ment, and merging channels) was performed using ImageJ soft-
ware with the Bio-Formats plugin.

3596
F. Karaki et al. / Bioorg. Med. Chem. 22 (2014) 3587–3609
4.2. Chemistry
to pH 2 with 2 N aqueous HCl and extracted with ethyl acetate.
The organic layer was washed with water and brine, dried over
Na2SO4 and concentrated to afford the title compound (609.3 mg,
2.063 mmol, quant.) as a colorless solid. 1H NMR (DMSO-d6) d:
8.91 (1H, d, J = 2.3 Hz), 8.50 (1H, d, J = 8.6 Hz), 8.34 (1H, dd,
J = 8.0, 1.7 Hz), 8.10 (1H, dd, J = 9.2, 2.3 Hz), 7.86 (1H, ddd, J = 8.0,
8.0, 1.2 Hz), 7.68–7.65 (2H, m), 4.33 (2H, t, J = 7.5 Hz), 1.65–1.62
(2H, m), 1.43–1.39 (2H, m), 0.93 (3H, t, J = 7.5 Hz). 13C NMR
(DMSO-d6) d: 166.85, 160.30, 139.75, 133.19, 132.61, 130.52,
128.67, 128.09, 124.99, 124.75, 124.53, 122.39, 118.25, 115.81,
41.96, 29.11, 19.64, 13.75. FAB-MS m/z: 296 (MH+).
4.2.1. General
Proton and carbon nuclear magnetic resonance spectra (1H and
13C NMR) were recorded on a JEOL JMM-ECA 500 (500 MHz) spec-
trometer in the indicated solvent. ‘CD3OD/CDCl’3 indicates that 10%
CDCl3 in CD3OD. ‘CDCl3/CD3OD’ indicates that 10% CD3OD in CDCl3.
Chemical shifts (d) are reported in parts per million relative to the
internal standard tetramethylsilane. Fast atom bombardment
(FAB) mass spectra were recorded on a JEOL JMA-HX110 mass
spectrometer. Reagents were purchased from Aldrich, Tokyo Kasei
Kogyo, Wako Pure Chemical Industry, and Kanto Kagaku and used
without purification. Open and flash column chromatographies
were performed using silica gel 60 (particle size 0.060–
0.210 mm) supplied by Kanto Kagaku.
4.2.2.3.1. General procedure for the synthesis of 2-amide deriva-
tives of phenanthridin-6-one (A). To a solution of 66 (40 mg,
0.15 mmol) and DMF (100
chloride (30 L, 0.35 mmol) at 0 °C. The mixture was stirred at this
temperature for 10 min, then triethylamine (100 L, 0.721 mmol)
and amine (100 L) were added at 0 °C. Stirring was continued at
lL) in CH2Cl2 (1 mL) was added oxalyl
l
l
4.2.2. Synthesis of 2-amide derivatives of phenanthridin-6-ones
4.2.2.1. Methyl 4-[(2-bromobenzoyl) butylamino] benzoate
l
ambient temperature for 20 min, and then the mixture was diluted
with ethyl acetate. The organic layer was washed with water and
brine, dried over Na2SO4 and concentrated. The product was
purified as indicated below.
(64).
To a vigorously stirred mixture of methyl 4-aminoben-
zoate (1581 mg, 10.46 mmol) in CH2Cl2 (50 mL) and saturated
aqueous NaHCO3 (50 mL) at 0 °C was added 2-bromobenzoyl chlo-
ride (1800
l
L, 13.78 mmol). Stirring was continued for 9 h at ambi-
4.2.2.3.2. General procedure for the synthesis of 2-amide deriva-
ent temperature. The biphasic mixture was separated and the
organic phase was washed with saturated aqueous NaHCO3, satu-
rated aqueous NH4Cl and brine, then dried over Na2SO4 and con-
centrated. The residue was recrystallized from CH2Cl2/n-hexane
to afford the title compound (3060 mg, 9.157 mmol, 88% yield) as
colorless needles. 1H NMR (CDCl3) d: 8.05 (2H, d, J = 8.6 Hz), 7.93
(1H, br s), 7.73 (2H, d, J = 8.6 Hz), 7.66–7.64 (2H, m), 7.42 (1H,
ddd, J = 6.9, 6.9, 1.2 Hz), 7.34 (1H, ddd, J = 8.0, 8.0, 1.7 Hz), 3.91
(3H, s). 13C NMR (CDCl3) d: 166.52, 165.58, 141.60, 137.28,
133.64, 132.01, 130.93 (2C), 129.93, 127.86, 126.18, 119.19,
119.11 (2C), 52.11. FAB-MS m/z: 336, 334 (MH+).
tives of phenanthridin-6-one (B).
0.18 mmol) and Et3N (65 L, 0.50 mmol) in THF (6 mL) was treated
with isobutyl chloroformate (35 L, 0.27 mmol) at 0 °C, and stirred
at the same temperature for 10 min. To the resulting mixture was
added a suitable amine or 40% aqueous amine solution (100 L) in
A solution of 66 (54 mg,
l
l
l
one portion. The reaction mixture was stirred for 3 h at ambient
temperature, then partitioned between water and AcOEt. The
organic phase was washed with water and brine, dried, and
concentrated. The product was purified as indicated below.
4.2.2.3.3. 5-Butyl-6-oxo-5,6-dihydrophenanthridine-2-carboxam-
ide (6). This compound was prepared according to the general
procedure A on 0.9 mmol scale. Column chromatography (CH2Cl2/
MeOH = 20/1) gave the title compound (186 mg, 0.632 mmol, 72%)
as a colorless solid. 1H NMR (CDCl3) d 8.86 (d, 1H, J = 1.8 Hz), 8.56
(dd, 1H, J = 8.5, 1.5 Hz), 8.39 (d, 1H, J = 7.9 Hz), 7.91 (dd, 1H, J = 8.5,
1.8 Hz), 7.81 (ddd, 1H, J = 7.9, 6.4, 1.5 Hz), 7.64 (td, 1H, J = 7.9,
1.5 Hz), 7.45 (d, 1H, J = 8.5 Hz), 4.42 (t, 2H, J = 7.6 Hz), 1.83–1.76
(m, 2H), 1.56–1.51 (m, 2H), 1.03 (t, 3H, J = 7.3 Hz). MS (FAB,
[M+H]+) m/z 295. Anal. Calcd for C18H18N2O2: C, 73.45; H, 6.16;
N, 9.52. Found: C, 73.22; H, 6.31; N, 9.52.
4.2.2.3.4. N-Methyl-5,6-dihydro-5-n-butyl-6-oxo-phenanthridine-
2-carboxamide (7). This compound was prepared according to the
general procedure B. Flash column chromatography (n-hexane/
AcOEt/CH2Cl2 = 1/1/1 to 0/1/1) afforded the title compound
(39.7 mg, 0.129 mmol, 70%) as colorless needles (recrystallized
from CH2Cl2/n-hexane, mp 196.2–198.3 °C). 1H NMR (CD3OD) d:
8.91 (1H, d, J = 1.7 Hz), 8.52 (1H, d, J = 8.0 Hz), 8.43 (1H, dd,
J = 8.0, 1.7 Hz), 8.05 (1H, dd, J = 8.6, 2.3 Hz), 7.87 (1H, ddd J = 8.6,
8.6, 1.2 Hz), 7.66 (1H, ddd, J = 8.0, 8.0, 1.2 Hz), 7.64 (1H, d
J = 9.2 Hz), 4.43 (2H, t, J = 7.5 Hz), 2.99 (3H, s), 1.81–1.73 (2H, m),
1.56–1.47 (2H, m), 1.02 (3H, t, J = 7.5 Hz). 13C NMR (CD3OD) d:
169.64, 163.10, 140.19, 134.84, 134.32, 129.68, 129.65, 129.61,
129.44, 126.38, 124.05, 123.34, 120.53, 116.87, 43.78, 30.72,
27.02, 21.18, 14.19. FAB-MS m/z: 309 (MH+). Anal. Calcd for
4.2.2.2. Methyl 5,6-dihydro-5-n-butyl-6-oxo-phenanthridine-2-
carboxylate (65).
To a solution of 64 (1026 mg, 3.072 mmol)
in DMF (6 mL) was added NaH (210.1 mg, 5.253 mmol, ca. 60%) at
0 °C. The mixture was stirred for 1 h at ambient temperature, then
1-iodobutane (750 lL, 6.60 mmol) was added and stirring was con-
tinued at ambient temperature overnight. Water was added and
the whole was extracted with ethyl acetate. The organic layer
was washed with water and brine, dried over Na2SO4 and concen-
trated. The resultant oil was subjected to the next reaction without
further purification. A de-aerated mixture of the oil, Pd(OAc)2
(81.8 mg, 0.364 mmol), PCy3ꢀHBF4 (143.6 mg, 0.3718 mmol) and
Cs2CO3 (821.6 mg, 2.523 mmol) in DMA (10 mL) was stirred at
130 °C for 4 h, then cooled to ambient temperature and filtered
through a Celite pad. The pad was washed with ethyl acetate.
The filtrate and washing were combined, washed with water and
brine, dried over Na2SO4 and concentrated. Open column chroma-
tography (n-hexane/ethyl acetate = 8/1 to 4/1) gave the title com-
pound (639.1 mg, 2.066 mmol, 67% yield for
2 steps) as a
colorless solid. 1H NMR (CDCl3) d: 8.97 (1H, d, J = 2.3 Hz), 8.53
(1H, dd, J = 8.1, 1.2 Hz), 8.35 (1H, d, J = 8.0 Hz), 8.16 (1H, dd,
J = 9.2, 2.3 Hz), 7.79 (1H, ddd, J = 6.9, 6.9, 1.1 Hz), 7.62 (1H, ddd,
J = 8.1, 8.1, 1.2 Hz), 7.42 (1H, d, J = 9.2 Hz), 4.39 (2H, t, J = 8.0 Hz),
3.99 (3H, s), 1.81–1.75 (2H, m), 1.57–1.49 (2H, m), 1.02 (3H, t,
J = 7.5 Hz). 13C NMR (CDCl3) d: 166.55, 161.44, 140.40, 133.10,
132.75, 130.33, 128.86, 128.49, 125.53, 125.49, 123.79, 121.85,
119.19, 114.99, 52.28, 42.84, 29.48, 20.33, 13.87. FAB-MS m/z:
310 (MH+).
C
19H20N2O2: C, 74.00; H, 6.54; N, 9.08. Found: C, 74.22; H, 6.67;
N, 8.98.
4.2.2.3.5. N,N0-Dimethyl-5,6-dihydro-5-n-butyl-6-oxo-phenanthri-
dine-2-carboxamide (8). This compound was prepared according
to the general procedure B. Flash column chromatography (n-hex-
ane/AcOEt/CH2Cl2 = 1:1:1 to 0:1:1) afforded the title compound
(22.3 mg, 0.0692 mmol, 44%) as a colorless oil. 1H NMR (CDCl3) d:
8.55 (1H, dd, J = 8.0, 1.0 Hz), 8.41 (1H, d, J = 2.0 Hz), 8.27 (1H, d,
J = 8.0 Hz), 7.76 (1H, td, J = 8.0, 1.0 Hz), 7.62–7.59 (2H, m), 7.41
(1H, d, J = 8.5 Hz), 4.40 (2H, t, J = 8.0 Hz), 3.13 (6H, s), 1.82–1.76
4.2.2.3. 5,6-Dihydro-5-n-butyl-6-oxo-phenanthridine-2-carbox-
ylic acid (66).
A solution of 65 (616.3 mg, 1.992 mmol) and
LiOHꢀH2O (278.4 mg, 6.635 mmol) in THF (12 mL) and water
(8 mL) was stirred at ambient temperature for 4 h, then adjusted

F. Karaki et al. / Bioorg. Med. Chem. 22 (2014) 3587–3609
3597
(2H, m), 1.55–1.50 (2H, m), 1.02 (3H, t, J = 7.5 Hz). 13C NMR
(CD3OD) d: 173.07, 163.02, 138.96, 134.65, 134.31, 131.58, 129.71,
129.69, 129.42, 126.41, 124.11, 123.39, 120.67, 116.87, 43.73,
40.27, 35.90, 30.72, 21.19, 14.20. FAB-MS m/z: 323 (MH+). HRMS
(FAB, [M+H]+) calcd for C20H23N2O2 323.1760, found 323.1739.
4.2.2.3.6. N,N0-Diethyl-5,6-dihydro-5-n-butyl-6-oxo-phenanthri-
dine-2-carboxamide (9). This compound was prepared according
to the general procedure B. Flash column chromatography (n-hex-
ane/AcOEt/CH2Cl2 = 1:1:1) afforded the title compound (13.3 mg,
0.038 mmol, 24%) as a colorless oil. 1H NMR (CDCl3) d: 8.55 (1H,
dd, J = 8.0, 1.0 Hz), 8.34 (1H, d, J = 2.0 Hz), 8.26 (1H, d, J = 8.0 Hz),
7.77 (1H, td, J = 7.0, 1.0 Hz), 7.61 (1H, t, J = 7.5 Hz), 7.56 (1H, dd,
J = 8.5, 1.5 Hz), 7.41 (1H, d, J = 9.0 Hz), 4.40 (2H, t, J = 7.5 Hz),
3.60–3.35 (4H, br m), 1.82–1.75 (2H, m), 1.56–1.48 (2H, m),
1.31–1.20 (6H, br m), 1.02 (3H, t, J = 7.5 Hz). 13C NMR (CD3OD/
CDCl3) d: 172.83, 162.93, 138.68, 134.54, 134.24, 132.33, 129.67,
129.41, 128.85, 126.38, 123.28, 123.16, 120.74, 116.92, 45.14,
43.73, 41.08, 30.68, 21.17, 14.52, 14.21, 13.12. HRMS (FAB,
[M+H]+) calcd for C22H27N2O2 351.2073, found 351.2073.
127.58, 125.40, 122.95, 121.78, 119.13, 114.80, 51.92, 42.65,
33.21 (2C), 29.44, 23.91 (2C), 20.28, 13.85. FAB-MS m/z: 363
(MH+). Anal. Calcd for C23H26N2O2: C, 76.21; H, 7.23; N, 7.73.
Found: C, 76.19; H, 7.27; N, 7.70.
4.2.2.3.10.
N-c-Hexyl-5,6-dihydro-5-n-butyl-6-oxo-phenanthri-
dine-2-carboxamide (14). This compound was prepared according
to the general procedure A. Open column chromatography (n-hex-
ane/ethyl acetate = 12/5 to 2/1) gave the title compound (39.3 mg,
0.104 mmol, 70% yield) as colorless needles (recrystallized from
CH2Cl2/n-hexane, mp 173.5–174.1 °C). 1H NMR (CDCl3) d: 8.66
(1H, d, J = 2.3 Hz), 8.46 (1H, dd, J = 6.9, 1.2 Hz), 8.22 (1H, d,
J = 8.6 Hz), 7.83 (1H, dd, J = 8.6, 1.8 Hz), 7.68 (1H, ddd, J = 8.1, 8.1,
1.2 Hz), 7.55 (1H, ddd, J = 8.0, 8.0, 1.2 Hz), 7.29 (1H, d, J = 9.2 Hz),
6.54 (1H, d, J = 8.0 Hz), 4.29 (2H, t, J = 8.1 Hz), 4.07–4.02 (1H, m),
2.11–2.08 (2H, m), 1.83–1.77 (2H, m), 1.74–1.36 (3H, m),
1.50–1.41 (4H, m), 1.40–1.33 (2H, m), 1.26–1.21 (1H, m), 0.98
(3H, t, J = 7.5 Hz). 13C NMR (CDCl3) d: 166.05, 161.28, 138.88,
133.01, 132.54, 128.82, 128.73, 128.32, 127.56, 125.41, 122.95,
121.78, 119.14, 114.81, 48.98, 42.64, 33.27 (2C), 29.44, 25.57,
25.04 (2C), 20.27, 13.84. FAB-MS m/z: 377 (MH+). Anal. Calcd for
4.2.2.3.7.
N,N0-(Di-n-propyl)-5,6-dihydro-5-n-butyl-6-oxo-phe-
nanthridine-2-carboxamide (10). This compound was prepared
according to the general procedure A. Open column chromatogra-
phy (n-hexane/ethyl acetate = 2/1) gave the title compound
(45.0 mg, 0.119 mmol, 83% yield) as colorless cubes (recrystallized
from CH2Cl2/n-hexane, mp 80.1–80.7 °C). 1H NMR (CDCl3) d: 8.56
(1H, dd, J = 8.0, 1.1 Hz), 8.32 (1H, d, J = 1.7 Hz), 8.26 (1H, d,
J = 8.0 Hz), 7.77 (1H, ddd, J = 7.7, 7.7, 1.8 Hz), 7.61 (1H, dd,
J = 7.0 Hz), 7.56 (1H, dd, J = 8.6, 1.8 Hz), 7.42 (1H, d, J = 8.6 Hz),
4.40 (2H, t, J = 8.0 Hz), 3.51 (2H, br s), 3.28 (2H, br s), 1.83–1.76
(2H, m), 1.75 (2H, br s), 1.62 (2H, br s), 1.57–1.49 (2H, m), 1.02
(3H, t, J = 7.5 Hz), 1.02 (3H, br s), 0.79 (3H, br s). 13C NMR (CDCl3)
d: 171.10, 161.28, 137.62, 133.12, 132.59, 131.11, 128.90, 128.35,
127.80, 125.68, 122.28, 121.65, 119.37, 114.99, 50.97, 46.62,
42.67, 29.49, 22.08, 20.80, 20.34, 13.89, 11.52, 11.18. FAB-MS m/
z: 379 (MH+). HRMS (FAB, [M+H]+) calcd for C24H31N2O2
379.2386, found 379.2426.
C
24H28N2O2ꢀ2/3H2O: C, 74.20; H, 7.61; N, 7.21. Found: C, 74.21;
H, 7.32; N, 7.26.
4.2.2.3.11. N-c-Propyl-5,6-dihydro-5-n-butyl-6-oxo-phenanthri-
dine-2-carboxamide (15). This compound was prepared according
to the general procedure A. Open column chromatography (n-hex-
ane/ethyl acetate = 1/1) gave the title compound (44.7 mg,
0.134 mmol, 92% yield) as a colorless powder (recrystallized
from CH2Cl2/MeOH/n-hexane, mp 203.3–204.9 °C). 1H NMR
(CDCl3/CD3OD) d: 8.67 (1H, d, J = 2.3 Hz), 8.39 (1H, d, J = 8.0 Hz),
8.23 (1H, d, J = 8.6 Hz), 7.89 (1H, dd, J = 8.6, 1.8 Hz), 7.66 (1H,
ddd, J = 7.8, 7.8, 1.2 Hz), 7.61 (1H, br s), 7.52 (1H, dd, J = 7.5,
7.5 Hz), 7.28 (1H, d, J = 9.2 Hz), 4.25 (2H, t, J = 8.0 Hz), 2.98–2.95
(1H, m), 1.72–1.66 (2H, m), 1.49–1.46 (2H, m), 0.98 (3H, t,
J = 7.5 Hz), 0.89 (2H, dd, J = 12.6, 6.9 Hz), 0.75–0.73 (2H, m). 13C
NMR (CDCl3/CD3OD) d: 168.81, 161.55, 138.89, 133.13, 132.72,
128.58, 128.40, 128.11, 128.03, 125.19, 122.95, 121.83, 119.15,
114.99, 42.78, 29.46, 23.25, 20.28, 13.83, 6.50 (2C). FAB-MS m/z:
335 (MH+). Anal. Calcd for C21H22N2O2: C, 75.42; H, 6.63; N, 8.38.
Found: C, 75.14; H, 6.66; N, 8.25.
4.2.2.3.12. N-i-Propyl-5,6-dihydro-5-n-butyl-6-oxo-phenanthri-
dine-2-carboxamide (16). Open column chromatography (n-hex-
ane/ethyl acetate = 2/1) gave the title compound (39.8 mg,
0.118 mmol, 85% yield) as colorless cubes (recrystallized from
CH2Cl2/n-hexane, mp 181.1–183.9 °C). 1H NMR (CDCl3) d: 8.62
(1H, d, J = 2.3 Hz), 8.41 (1H, dd, J = 8.1, 1.2 Hz), 8.17 (1H, d,
J = 8.0 Hz), 7.80 (1H, dd, J = 8.6, 1.7 Hz), 7.62 (1H, ddd, J = 7.4, 7.4,
1.2 Hz), 7.50 (1H, ddd, J = 8.0, 8.0, 1.2 Hz), 7.23 (1H, d, J = 9.7 Hz),
6.59 (1H, br d, J = 6.9 Hz), 4.37–4.30 (1H, m), 4.24 (2H, t,
J = 7.5 Hz), 1.66 (2H, tt, J = 7.5, 7.5 Hz), 1.44 (2H, tt, J = 7.5, 7.5 Hz),
1.31 (6H, d, J = 6.9 Hz), 0.94 (3H, t, J = 7.4 Hz). 13C NMR (CDCl3) d:
166.15, 161.21, 138.85, 132.95, 132.47, 128.64, 128.54, 128.26,
127.55, 125.33, 122.89, 121.71, 119.06, 114.74, 42.59, 42.12,
29.38, 22.78 (2C), 20.20, 13.76. FAB-MS m/z: 337 (MH+). HRMS
(FAB, [M+H]+) calcd for C21H25N2O2 337.1916, found 337.1909.
4.2.2.3.8. N,N0-(Di-n-butyl)-5,6-dihydro-5-n-butyl-6-oxo-phenan-
thridine-2-carboxamide (11). This compound was prepared
according to the general procedure A. Open column chromatogra-
phy (n-hexane/ethyl acetate = 3/1) gave the title compound
(54.3 mg, 0.134 mmol, 95% yield) as a colorless oil. 1H NMR (CDCl3)
d: 8.55 (1H, dd, J = 8.0, 1.7 Hz), 8.33 (1H, d, J = 2.3 Hz), 8.26 (1H, d,
J = 8.1 Hz), 7.77 (1H, ddd, J = 7.5, 7.5, 1.8 Hz), 7.61 (1H, ddd, J = 7.5,
7.5, 1.2 Hz), 7.56 (1H, dd, J = 8.6, 1.8 Hz), 7.42 (1H, d, J = 8.6 Hz),
4.41 (2H, t, J = 8.1 Hz), 3.55 (2H, br s), 3.30 (2H, br s), 1.82–1.76
(2H, m), 1.70 (2H, br s), 1.57 (2H, br s), 1.57–1.49 (2H, m), 1.45
(2H, br s), 1.18 (2H, br s), 1.02 (3H, t, J = 7.5 Hz), 1.02 (3H, br s),
0.81 (3H, br s). 13C NMR (CDCl3) d: 170.96, 161.29, 137.63,
133.12, 132.58, 131.09, 128.90, 128.35, 127.83, 125.69, 122.29,
121.65, 119.33, 114.98, 49.09, 44.84, 42.67, 31.02, 29.70, 29.49,
20.34, 20.17, 19.85, 13.90, 13.82, 13.75. FAB-MS m/z: 407 (MH+).
HRMS (FAB, [M+H]+) calcd for C26H35N2O2 407.2699, found
407.2701.
4.2.2.3.9.
N-c-Pentyl-5,6-dihydro-5-n-butyl-6-oxo-phenanthri-
dine-2-carboxamide (13). This compound was prepared according
to the general procedure A. Open column chromatography (n-hex-
ane/ethyl acetate = 2/1) gave the title compound (35.7 mg,
0.0985 mmol, 70% yield) as a colorless powder (recrystallized from
CH2Cl2/n-hexane, mp 179.8–180.8 °C). 1H NMR (CDCl3) d: 8.65 (1H,
d, J = 1.7 Hz), 8.45 (1H, d, J = 8.0 Hz), 8.21 (1H, d, J = 8.1 Hz), 7.82
(1H, dd, J = 9.2, 2.3 Hz), 7.67 (1H, ddd, J = 6.9, 6.9, 1.1 Hz), 7.54
(1H, dd, J = 8.0, 8.0 Hz), 7.27 (1H, d, J = 8.6 Hz), 6.66 (1H, d,
J = 7.5 Hz), 4.51–4.42 (1H, m), 4.28 (2H, t, J = 7.5 Hz), 2.18–2.11
(2H, m), 1.84–1.73 (2H, m), 1.73–1.66 (4H, m), 1.66–1.57 (2H,
m), 1.52–1.42 (2H, m), 0.98 (3H, t, J = 7.5 Hz). 13C NMR (CDCl3) d:
166.65, 161.29, 138.88, 133.02, 132.54, 128.72, 128.70, 128.33,
4.2.2.3.13.
N-t-Butyl-5,6-dihydro-5-n-butyl-6-oxo-phenanthri-
dine-2-carboxamide (17). Open column chromatography (n-hex-
ane/ethyl acetate = 3/1) gave the title compound (40.3 mg,
0.115 mmol, 82% yield) as a colorless amorphous solid. 1H NMR
(CDCl3) d: 8.59 (1H, d, J = 1.7 Hz), 8.43 (1H, dd, J = 8.0, 1.2 Hz),
8.21 (1H, d, J = 8.1 Hz), 7.72 (1H, dd, J = 8.6, 1.7 Hz), 7.67 (1H,
ddd, J = 8.0, 8.0, 1.2 Hz), 7.53 (1H, ddd, J = 7.5, 7.5, 1.2 Hz), 7.23
(1H, d, J = 8.6 Hz), 6.31 (1H, br s), 4.25 (2H, t, J = 7.5 Hz), 1.68
(2H, ttt, J = 7.4, 7.4 Hz), 1.52 (9H, s), 1.48–1.41 (2H, m), 0.96 (3H,
t, J = 7.5 Hz). 13C NMR (CDCl3) d: 166.36, 161.23, 138.76, 132.99,
132.51, 129.44, 128.68, 128.28, 127.29, 125.40, 122.82, 121.73,
119.09, 114.68, 51.85, 42.59, 29.39, 28.89 (3C), 20.23, 13.79.

3598
F. Karaki et al. / Bioorg. Med. Chem. 22 (2014) 3587–3609
FAB-MS m/z: 351 (MH+). HRMS (FAB, [M+H]+) calcd for C22H27N2O2
351.2073, found 351.2040.
29.40, 20.25, 13.82. FAB-MS m/z: 383 (MH+). Anal. Calcd for
C22H26N2O4ꢀ4/5H2O: C, 66.58; H, 7.01; N, 7.06. Found: C, 66.48;
H, 6.99; N, 6.67.
4.2.2.3.14.
5,6-Dihydro-5-n-butyl-6-oxo-phenanthridine-2-car-
boxylic acid piperazine amide (18). Open column chromatography
(n-hexane/ethyl acetate = 2/1 to 1/1) gave the title compound
(44.1 mg, 0.122 mmol, 86% yield) as colorless needles (recrystal-
lized from CH2Cl2/n-hexane, mp 100.5–102.3 °C). 1H NMR (CDCl3)
d: 8.50 (1H, dd, J = 8.0, 1.7 Hz), 8.34 (1H, d, J = 1.7 Hz), 8.22 (1H,
d, J = 8.0 Hz), 7.72 (1H, ddd, J = 6.9, 6.9, 1.2 Hz), 7.58–7.53 (2H,
m), 7.38 (1H, d, J = 8.6 Hz), 4.36 (2H, t, J = 7.5 Hz), 3.58 (4H, br s),
1.78–1.72 (2H, m), 1.71–1.66 (2H, m), 1.61 (4H, br s), 1.52–1.45
(2H, m), 0.98 (3H, t, J = 6.9 Hz). 13C NMR (CDCl3, 50 °C) d: 169.73,
161.24, 138.04, 133.14, 132.48, 130.05, 128.91, 128.28, 128.14,
125.78, 122.88, 121.65, 119.41, 114.86, 46.29 (2C), 42.64, 29.55,
26.15 (2C), 24.59, 20.27, 13.73. FAB-MS m/z: 363 (MH+). Anal. Calcd
for C23H26N2O2: C, 76.21; H, 7.23; N, 7.73. Found: C, 76.38; H, 7.31;
N, 7.71.
4.2.3. Synthesis of 5-alkyl derivatives of phenanthridin-6-ones
4.2.3.1. N,N0-(Diethyl)-4-(2-bromobenzoyl)aminobenzoic amide
(68).
To a suspension of 4-aminobenzoic acid (1379 mg,
10.05 mmol) and DMF (2 drops) in CH2Cl2 (10 mL) was added thio-
nyl chloride (3 mL, 40 mmol) at 0 °C. The mixture was stirred at
40 °C for 1 h and 60 °C for 1 h, and then cooled to ambient temper-
ature. Diethylamine (10 mL) was added and stirring was continued
overnight at ambient temperature, then the mixture was poured
into water and extracted with CH2Cl2. The organic layer was
washed with water and brine, dried over Na2SO4 and concentrated.
The residue was subjected to the next reaction without further
purification. To a vigorously stirred mixture of the crude amide
in CH2Cl2 (50 mL) and saturated aqueous NaHCO3 (50 mL) was
added 2-bromobenzoyl chloride (1.8 mL, 13.78 mmol) at 0 °C.
Stirring was continued for 15 h at ambient temperature. The bipha-
sic mixture was separated and the organic phase was washed with
saturated aqueous NaHCO3 and brine, dried over Na2SO4 and
concentrated. Open column chromatography (n-hexane/ethyl ace-
tate = 1/1 to 1/2) gave the title compound (1621 mg, 4.320 mmol,
43% yield for 2 steps) as a brown solid. 1H NMR (CDCl3/CD3OD)
d: 7.68 (2H, d, J = 8.6 Hz), 7.64 (1H, d, J = 8.0 Hz), 7.61 (1H, br s),
7.60 (1H, dd, J = 7.5, 1.2 Hz), 7.43–7.40 (1H, m), 7.36 (1H, d,
J = 8.0 Hz), 7.39–7.31 (2H, m), 3.52 (2H, br s), 3.31 (2H, br s), 1.23
(3H, br s), 1.14 (3H, br s). 13C NMR (CDCl3/CD3OD) d: 171.23,
166.20, 138.72, 137.91, 133.44, 132.95, 131.60, 129.47, 127.67,
127.36 (2C), 119.86 (2C), 119.42, 43.51, 38.54, 14.26, 12.82. FAB-
MS m/z: 377, 375 (MH+).
4.2.2.3.15.
5,6-Dihydro-5-n-butyl-6-oxo-phenanthridine-2-car-
boxylic acid morpholine amide (19). This compound was prepared
according to the general procedure B. Flash column chromatogra-
phy (n-hexane/AcOEt = 1:1 to 1:3) afforded the title compound
(22.2 mg, 0.061 mmol, 32%) as colorless cubes (recrystallized from
CH2Cl2/n-hexane, mp 165.9–166.9 °C). 1H NMR (CD3OD) d: 8.55
(1H, d, J = 1.2 Hz), 8.49 (1H, d, J = 8.6 Hz), 8.44 (1H, dd, J = 8.6,
1.2 Hz), 7.86 (1H, ddd, J = 6.9, 6.9, 1.2 Hz), 7.69–7.65 (3H, m),
4.44 (2H, t, J = 8.0 Hz), 3.99–3.45 (8H, m), 1.81–1.74 (2H, m),
1.56–1.48 (2H, m), 1.02 (3H, t, J = 7.5 Hz). FAB- MS m/z: 365
(MH+). Anal. Calcd for C22H24N2O3: C, 72.51; H, 6.64; N, 7.69.
Found: C, 72.31; H, 6.77; N, 7.48.
4.2.2.3.16.
N-Benzyl-5,6-dihydro-5-n-butyl-6-oxo-phenanthri-
dine-2-carboxamide (20). Open column chromatography (n-hex-
ane/ethyl acetate = 3/1) gave the title compound (42.0 mg,
0.109 mmol, 78% yield) as colorless needles (recrystallized from
CH2Cl2/n-hexane, mp 152.0–154.2 °C). 1H NMR (CDCl3) d: 8.73
(1H, d, J = 1.7 Hz), 8.41 (1H, dd, J = 8.0, 1.1 Hz), 8.18 (1H, d,
J = 8.0 Hz), 7.87 (1H, dd, J = 9.2, 2.3 Hz), 7.61 (1H, ddd, J = 7.8, 7.8,
1.2 Hz), 7.50 (1H, ddd, J = 7.5, 7.5, 1.2 Hz), 7.36–7.22 (7H, m),
4.66 (1H, d, J = 4.6 Hz), 4.24 (2H, t, J = 8.0 Hz), 1.66 (2H, tt, J = 7.5,
7.5 Hz), 1.43 (2H, tt, J = 7.5, 7.5 Hz), 0.94 (3H, t, J = 7.5 Hz). 13C
NMR (CDCl3) d: 166.82, 161.25, 139.09, 138.20, 132.92, 132.53,
128.65 (4C), 128.32, 127.86 (2C), 127.70, 127.48, 125.33, 123.12,
121.74, 119.18, 114.85, 44.18, 42.62, 29.38, 20.19, 13.77. FAB-MS
m/z: 385 (MH+). Anal. Calcd for C25H24N2O2: C, 78.10; H, 6.29; N,
7.29. Found: C, 78.25; H, 6.31; N, 7.20.
4.2.3.2.
zyl]aminobenzoic amide (69).
N,N0-(Diethyl)-4-[(2-bromobenzoyl)-p-methoxyben-
To solution of 68
a
(1621 mg, 4.320 mmol) in DMF (15 mL) was added NaH
(237.5 mg, 5.938 mmol, ca. 60%) at 0 °C. The mixture was stirred
for 1 h at ambient temperature, then 4-methoxybenzyl chloride
(750 lL, 5.56 mmol) was added and stirring was continued at
ambient temperature overnight. Water was added and the whole
was extracted with ethyl acetate. The organic layer was washed
with water and brine, dried over Na2SO4 and concentrated. Open
column chromatography (n-hexane/ethyl acetate = 1/1) gave the
title compound (2137 mg, 4.314 mmol, 100%) as a yellow amor-
phous solid. 1H NMR (DMSO-d6) d: 7.45 (1H, d, J = 8.0 Hz), 7.28–
7.21 (3H, m), 7.17 (1H, dd, J = 8.1 Hz), 7.13–7.04 (5H, m), 6.85
(2H, d, J = 8.6 Hz), 3.70 (3H, s), 3.36–3.26 (2H, m), 2.88–2.78 (2H,
m), 1.04 (3H, br s), 0.81 (3H, br s). 13C NMR (DMSO-d6) d: 169.03,
167.48, 158.49, 141.35, 138.14, 135.93, 132.17, 130.17, 129.60
(2C), 129.15, 128.68, 128.03 (2C), 126.94, 126.39 (2C), 119.05,
113.76 (2C), 54.95, 50.85, 40.00, 39.00, 13.80, 12.73. FAB-MS m/z:
497, 495 (MH+).
4.2.2.4. N,N0-Bis(2-hydroxyethyl)-5,6-dihydro-5-n-butyl-6-oxo-
phenanthridine-2-carboxamide (12).
To a solution of 66
(43.8 mg, 0.148 mmol) and DMF (100
lL) in CH2Cl2 (1 mL) was
added oxalyl chloride (30
was stirred at this temperature for 10 min, then added to a vigor-
ously stirred solution of diethanolamine (30 L, 0.31 mmol) in
lL, 0.35 mmol) at 0 °C. The mixture
l
saturated aqueous NaHCO3 (1 mL). Stirring was continued for 5 h
at ambient temperature, then the biphasic mixture was separated.
The organic phase was washed with saturated aqueous NH4Cl and
brine, dried over Na2SO4 and concentrated. Open column chroma-
tography (ethyl acetate then CHCl3/MeOH = 9/1) gave the title
compound (15.8 mg, 0.0413 mmol, 28% yield) as colorless cubes
(recrystallized from CH2Cl2/n-hexane, mp 80.8–82.1 °C). 1H NMR
(CDCl3) d: 8.60 (1H, d, J = 1.2 Hz), 8.40 (1H, dd, J = 8.0, 1.2 Hz),
8.17 (1H, d, J = 8.0 Hz), 7.72 (1H, d, J = 8.6 Hz), 7.63 (1H, ddd,
J = 7.5, 7.5, 1.2 Hz), 7.50 (1H, dd, J = 8.0, 8.0 Hz), 7.31 (1H, d,
J = 8.6 Hz), 4.25 (2H, t, J = 8.0 Hz), 4.02 (2H, br s), 3.81 (2H, br s),
3.74 (2H, br s), 3.55 (2H, br s), 1.72–1.66 (2H, m), 1.47–1.42 (2H,
m), 0.96 (3H, t, J = 7.5 Hz). 13C NMR (CDCl3) d: 173.15, 161.22,
137.90, 133.04, 132.62, 129.12, 129.07, 128.59, 128.29, 125.32,
123.94, 121.87, 119.03, 115.05, 60.46, 59.49, 53.49, 49.29, 42.62,
4.2.3.3. N,N0-(Diethyl)-5,6-dihydro-5-p-methoxybenzyl-6-oxo-
phenanthridine-2-carboxamide (25).
A de-aerated mixture
of 69 (1974 mg, 3.984 mmol), Pd(OAc)2 (97.5 mg, 0.434 mmol),
PCy3ꢀHBF4 (192.2 mg, 0.4976 mmol) and Cs2CO3 (1005 mg,
3.085 mmol) in DMA (15 mL) was stirred at 130 °C for 4 h, then
cooled to ambient temperature and filtered through a Celite pad.
The pad was washed with ethyl acetate. The filtrate and washing
were combined, washed with water and brine, dried over Na2SO4
and concentrated. Open column chromatography (n-hexane/ethyl
acetate = 1/1) gave the title compound (1565 mg, 3.774 mmol,
95% yield) as a pale yellow amorphous solid. 1H NMR (CDCl3) d:
8.62 (1H, dd, J = 8.0, 1.2 Hz), 8.34 (1H, d, J = 2.3 Hz), 8.30 (1H, d,
J = 8.0 Hz), 7.81 (1H, ddd, J = 7.5, 7.5, 1.7 Hz), 7.65 (1H, ddd,
J = 7.5, 7.5, 1.2 Hz), 7.43 (1H, dd, J = 8.6, 2.3 Hz), 7.36 (1H, d,

F. Karaki et al. / Bioorg. Med. Chem. 22 (2014) 3587–3609
3599
J = 8.6 Hz), 7.20 (2H, d, J = 9.2 Hz), 6.83 (2H, dt, J = 8.6, 2.9 Hz), 5.61
(2H, br s), 3.76 (3H, s), 3.58 (2H, br s), 3.33 (2H, br s), 1.34–1.23
(3H, m), 1.23–1.11 (3H, m). 13C NMR (CDCl3) d: 170.52, 161.80,
158.79, 137.88, 133.31, 132.86, 131.24, 129.21, 128.45, 128.23,
127.83 (2C), 127.52, 125.57, 122.01, 121.77, 119.45, 115.90,
114.22 (2C), 55.24, 46.00, 43.41, 39.51, 14.34, 12.88. FAB-MS m/z:
415 (MH+). HRMS (FAB, [M+H]+) calcd for C26H27N2O3 415.2022,
found 415.2004.
Open column chromatography (n-hexane/ethyl acetate = 1/1) gave
the title compound (11.0 mg, 0.0286 mmol, 27% yield) as colorless
cubes (recrystallized from CH2Cl2/n-hexane, mp 221.5–223.0 °C).
1H NMR (CDCl3) d: 8.63 (1H, dd, J = 8.0, 1.2 Hz), 8.35 (1H, d,
J = 1.7 Hz), 8.31 (1H, d, J = 8.0 Hz), 7.82 (1H, ddd, J = 7.4, 7.4,
1.2 Hz), 7.65 (1H, ddd, J = 7.7, 7.7, 1.2 Hz), 7.41 (1H, dd, J = 8.6,
2.3 Hz), 7.32–7.23 (6H, m), 5.68 (2H, br s), 3.58 (2H, br s), 3.33
(2H, br s), 1.26 (3H, br s), 1.19 (3H, br s). 13C NMR (CDCl3) d:
170.53, 161.85, 137.90, 136.22, 133.37, 132.95, 131.33, 129.27,
128.86 (2C), 128.52, 127.58, 127.33, 126.49 (2C), 125.55, 122.06,
121.83, 119.50, 115.95, 46.57, 43.48, 39.52, 14.40, 12.87. FAB-MS
m/z: 385 (MH+). Anal. Calcd for C25H24N2O2: C, 78.10; H, 6.29; N,
7.29. Found: C, 77.99; H, 6.37; N, 7.20.
4.2.3.4. N,N0-(Diethyl)-5,6-dihydro-6-oxo-phenanthridine-2-car-
boxamide (21).
A solution of 25 (1531 mg, 3.694 mmol) in
TFA (4 mL) was refluxed for 8 h, then cooled to ambient tempera-
ture and diluted with ethyl acetate. The organic layer was washed
with water, saturated aqueous NaHCO3 and brine, dried over
Na2SO4 and concentrated. Open column chromatography (n-hexane/
ethyl acetate = 1/2 to 1/3) gave the title compound (681.4 mg,
2.314 mmol, 63%) as colorless cubes (recrystallized from CH2Cl2/
MeOH/n-hexane, mp 233.2–236.0 °C). 1H NMR (CDCl3) d: 10.82
(1H, br s), 8.59 (1H, dd, J = 8.0, 1.2 Hz), 8.33–8.29 (2H, m), 7.84
(1H, ddd, J = 8.0, 8.0, 1.2 Hz), 7.66 (1H, ddd, J = 7.4, 7.4, 1.2 Hz),
7.53 (1H, dd, J = 8.6, 1.8 Hz), 7.40 (1H, d, J = 8.0 Hz), 3.60 (2H, br
s), 3.36 (2H, br s), 1.28 (3H, br s), 1.21 (3H, br s). 13C NMR (CDCl3/
CD3OD) d: 175.53, 166.70, 140.77, 138.22, 137.23, 135.06, 132.44,
132.09, 131.40, 129.64, 126.54, 125.38, 122.51, 120.57, 47.88,
43.90, 18.12, 16.63. FAB-MS m/z: 295 (MH+). HRMS (FAB,
[M+ H]+) calcd for C18H19N2O2 295.1447, found 295.1418.
4.2.3.6. General procedure for synthesis of 5-(ethoxycar-
bonyl)alkyl derivatives of N,N0-(diethyl)-5,6-dihydro-6-oxo-
phenanthridine-2-carboxamide.
A solution of 21 (120 mg,
0.39 mmol), Cs2CO3 (240 mg, 0.74 mmol), TBAI (20 mg, 0.05 mmol)
and alkyl bromide (1.9 mmol) in DMF (2 mL) was stirred at 90 °C
for 1 h, then diluted with ethyl acetate. The organic layer was
washed with water and brine, dried (Na2SO4) and concentrated.
The product was purified as indicated below.
4.2.3.6.1. N,N0-(Diethyl)-5,6-dihydro-5-(ethoxycarbonyl)methyl-6-
oxo-phenanthridine-2-carboxamide (70). Open column chroma-
tography (n-hexane/ethyl acetate = 1/1 to 1/2) gave the title
compound (114.0 mg, 0.2996 mmol, 77% yield) as a colorless solid.
1H NMR (CDCl3) d: 8.55 (1H, dd, J = 8.0, 1.7 Hz), 8.36 (1H, d,
J = 1.7 Hz), 8.29 (1H, d, J = 8.6 Hz), 7.81 (1H, ddd, J = 7.5, 7.5,
1.7 Hz), 7.63 (1H, ddd, J = 7.5, 7.5, 1.2 Hz), 7.53 (1H, dd, J = 8.6,
2.3 Hz), 7.16 (1H, d, J = 8.6 Hz), 5.21 (2H, s), 4.26 (2H, q,
J = 7.2 Hz), 3.60 (2H, br s), 3.35 (2H, br s), 1.28 (3H, br s), 1.27
(3H, t, J = 7.2 Hz), 1.21 (3H, br s). 13C NMR (CDCl3) d: 170.47,
168.23, 131.49, 137.73, 133.39, 133.13, 131.65, 129.19, 128.57,
127.73, 125.28, 122.33, 121.90, 119.46, 114.43, 61.86, 44.44,
43.49, 39.56, 14.33, 14.16, 12.95. FAB-MS m/z: 381 (MH+).
4.2.3.6.2. N,N0-(Diethyl)-5,6-dihydro-5-(3-ethoxycarbonyl)propyl-
6-oxo-phenanthridine-2-carboxamide (71). Open column chroma-
tography (n-hexane/ethyl acetate = 2/1 to 1/2) gave the title
compound (103.6 mg, 0.2538 mmol, 66% yield) as a colorless solid.
1H NMR (CDCl3) d: 8.55 (1H, dd, J = 8.1, 1.2 Hz), 8.36 (1H, d,
J = 1.8 Hz), 8.28 (1H, d, J = 8.1 Hz), 7.78 (1H, ddd, J = 6.9, 6.9,
1.1 Hz), 7.67–7.58 (3H, m), 4.47 (2H, t, J = 6.9 Hz), 4.18 (2H, q,
J = 7.5 Hz), 3.60 (2H, br s), 3.38 (2H, br s), 2.55 (2H, t, J = 6.9 Hz),
2.13 (2H, tt, J = 6.9 Hz), 1.29 (3H, t, J = 7.5 Hz), 1.34–1.16 (6H, m).
13C NMR (CDCl3) d: 173.18, 170.64, 161.39, 137.65, 133.21,
132.72, 131.21, 128.90, 128.39, 127.78, 125.55, 122.24, 121.77,
119.41, 115.17, 60.64, 43.52, 42.12, 39.62, 31.34, 22.40, 14.40,
14.25, 12.97. FAB-MS m/z: 409 (MH+).
4.2.3.5. General procedure for synthesis of 5-alkyl derivatives of
N,N0-(diethyl)-5,6-dihydro-6-oxo-phenanthridine-2-carboxam-
ide.
0.2 mmol) and alkyl iodide or alkyl bromide (0.5 mmol) in DMF
(500 L) was stirred at 90 °C for 1 h, then diluted with ethyl ace-
A solution of 21 (30 mg, 0.1 mmol), Cs2CO3 (70 mg,
l
tate. The organic layer was washed with water and brine, dried
over Na2SO4 and concentrated. The product was purified as indi-
cated below.
4.2.3.5.1. N,N0-(Diethyl)-5,6-dihydro-5-methyl-6-oxo-phenanthri-
dine-2-carboxamide (22). Methyl iodide was used for the reaction.
Open column chromatography (n-hexane/ethyl acetate = 1/2) gave
the title compound (20.9 mg, 0.0678 mmol, 73% yield) as colorless
cubes (recrystallized from CH2Cl2/n-hexane, mp 122.1–123.4 °C).
1H NMR (CDCl3) d: 8.55 (1H, dd, J = 8.0, 1.1 Hz), 8.34 (1H, d,
J = 1.7 Hz), 8.27 (1H, d, J = 8.0 Hz), 7.77 (1H, ddd, J = 6.9, 6.9,
1.2 Hz), 7.61 (1H, ddd, J = 7.4, 7.4, 1.2 Hz), 7.58 (1H, dd, J = 8.6,
1.8 Hz), 7.43 (1H, d, J = 8.6 Hz), 3.83 (3H, s), 3.60 (2H, br s), 3.36
(2H, br s), 1.29 (3H, br s), 1.22 (3H, br s). 13C NMR (CDCl3) d:
170.65, 161.58, 138.54, 133.07, 132.65, 131.23, 128.97, 128.41,
127.62, 125.71, 121.99, 121.76, 119.21, 114.93, 43.52, 39.60,
30.11, 14.43, 13.02. FAB-MS m/z: 309 (MH+). Anal. Calcd for
C
19H20N2O2: C, 74.00; H, 6.54; N, 9.08. Found: C, 74.14; H, 6.64;
N, 8.99.
4.2.3.5.2. N,N0-(Diethyl)-5,6-dihydro-5-ethyl-6-oxo-phenanthri-
4.2.3.7. General procedure for synthesis of 5-(carboxy)alkyl
derivatives of N,N0-(diethyl)-5,6-dihydro-6-oxo-phenanthri-
dine-2-carboxamide (23). Ethyl iodide was used for the reaction.
Open column chromatography (n-hexane/ethyl acetate = 1/2) gave
the title compound (27.2 mg, 0.0844 mmol, 86% yield) as a color-
less oil. 1H NMR (CDCl3) d: 8.56 (1H, dd, J = 8.0, 1.1 Hz), 8.36 (1H,
d, J = 1.8 Hz), 8.28 (1H, d, J = 8.0 Hz), 7.78 (1H, ddd, J = 6.9, 6.9,
1.2 Hz), 7.62 (1H, ddd, J = 8.1, 8.1, 1.2 Hz), 7.58 (1H, dd, J = 8.6,
1.7 Hz), 7.45 (1H, d, J = 8.6 Hz), 4.48 (2H, q, J = 7.5 Hz), 3.60 (2H,
br s), 3.38 (2H, br s), 1.42 (3H, t, J = 6.9 Hz), 1.37–1.13 (6H, m).
13C NMR (CDCl3) d: 170.66, 161.09, 137.47, 133.12, 132.61,
131.02, 128.86, 128.38, 127.67, 125.73, 122.22, 121.70, 119.44,
114.86, 43.49, 39.57, 37.85, 14.42, 13.03, 12.71. FAB-MS m/z: 323
(MH+). HRMS (FAB, [M+H]+) calcd for C20H23N2O2 323.1760, found
323.1796.
dine-2-carboxamide.
in MeOH (15 mL) and water (10 mL) was added a 10 N aqueous
solution of NaOH (200 L). The mixture was stirred at ambient
To a solution of ethyl ester (0.15 mmol)
l
temperature for 16 h, then adjusted to pH 2 with 2 N aqueous
HCl and extracted with ethyl acetate. The organic layer was
washed with brine, dried over Na2SO4 and concentrated to give
the desired compound.
4.2.3.7.1.
N,N0-(Diethyl)-5,6-dihydro-5-(carboxy)methyl-6-oxo-
phenanthridine-2-carboxamide (26). Colorless powder (recrystal-
lized from CH2Cl2/MeOH/n-hexane, mp 283.5–285.1 °C). 60.3 mg,
0.171 mmol, 94%. 1H NMR (CDCl3/CD3OD) d: 8.53 (1H, d,
J = 8.0 Hz), 8.36 (1H, d, J = 1.7 Hz), 8.32 (1H, d, J = 8.0 Hz), 7.83
(1H, ddd, J = 7.7, 7.7, 1.8 Hz), 7.65 (1H, dd, J = 8.0, 8.0 Hz), 7.54
(1H, dd, J = 8.0, 1.8 Hz), 7.28 (1H, d, J = 8.6 Hz), 5.19 (2H, s), 3.60
(2H, br s), 3.36 (2H, br s), 1.31 (3H, br s), 1.21 (3H, br s). 13C
4.2.3.5.3. N,N0-(Diethyl)-5,6-dihydro-5-benzyl-6-oxo-phenanthri-
dine-2-carboxamide (24). Benzyl bromide was used for the reaction.

3600
F. Karaki et al. / Bioorg. Med. Chem. 22 (2014) 3587–3609
NMR (CDCl3/CD3OD) d: 171.49, 170.32, 162.26, 138.04, 133.70 (2C),
131.53, 129.13, 129.04, 127.91, 125.32, 122.35, 122.28, 119.85,
115.34, 44.67, 44.16, 40.21, 14.43, 12.91. FAB-MS m/z: 353
(MH+). HRMS (FAB, [M+H]+) calcd for C20H21N2O4 353.1501, found
353.1541.
stirred at ambient temperature for 2 h until a pale-yellow precipi-
tate was observed. Then cycloalkanone (20 mmol) in CHCl3
(1.6 mL) was added and the whole was refluxed for 2 h. n-Hexane
(20 mL) and 1 N aqueous NaOH (20 mL) were added and the whole
was extracted with AcOEt/n-hexane = 1/1. The organic layer was
washed with water and brine, dried and concentrated. The product
was purified as indicated below.
4.2.4.1.1. 2-Bromo-1-cyclopentene-1-carbaldehyde (73a). Col-
umn chromatography (n-hexane/AcOEt = 99/1 to 91/9) gave the
title compound (894.4 mg, 5.110 mmol, 26% yield) as a brown oil.
1H NMR (CDCl3) d: 9.88 (1H, s), 2.88 (2H, tt, J = 7.5, 2.3 Hz), 2.51
(2H, tt, J = 7.5, 2.3 Hz), 2.00 (2H, tt, J = 7.5, 7.5 Hz). 13C NMR (CDCl3)
d: 189.23, 141.41, 139.97, 42.52, 29.23, 21.34.
4.2.3.7.2. N,N0-(Diethyl)-5,6-dihydro-5-(3-carboxy)propyl-6-oxo-
phenanthridine-2-carboxamide (27). Colorless cubes (recrystal-
lized from CH2Cl2/n-hexane, mp 181.1–184.7 °C). 51.3 mg,
0.135 mmol, quant. 1H NMR (CDCl3) d: 8.54 (1H, dd, J = 8.0,
1.1 Hz), 8.36 (1H, br s), 8.29 (1H, d, J = 8.0 Hz), 7.99 (1H, ddd,
J = 8.3, 8.3, 1.1 Hz), 7.64–7.63 (3H, m), 4.37 (2H, t, J = 7.5 Hz), 3.62
(2H, br s), 3.38 (2H, br s), 2.52 (2H, t, J = 6.9 Hz), 2.10 (2H, tt,
J = 7.5, 7.5 Hz), 1.31 (3H, br s), 1.22 (3H, br s). 13C NMR (CDCl3) d:
176.38, 171.21, 161.46, 137.62, 133.16, 132.83, 130.73, 128.94,
128.49, 127.92, 125.41, 122.18, 121.75, 119.40, 115.39, 43.68,
42.08, 39.81, 30.94, 22.20, 14.37, 12.94. FAB-MS m/z: 381 (MH+).
Anal. Calcd for C22H24N2O2ꢀ1/3H2O: C, 68.38; H, 6.43; N, 7.25.
Found: C, 68.18; H, 6.26; N, 7.13.
4.2.4.1.2. 2-Bromo-1-cyclohexene-1-carbaldehyde (73b). Col-
umn chromatography (n-hexane/AcOEt = 99/1 to 92/8) gave the
title compound (1233 mg, 6.522 mmol, 33% yield) as a pale-yellow
oil. 1H NMR (CDCl3) d: 10.03 (1H, s), 2.76–2.73 (2H, m), 2.29–2.27
(2H, m), 1.78–1.76 (2H, m), 1.70–1.68 (2H, m).
4.2.4.1.3. 2-Bromo-1-cycloheptene-1-carbaldehyde (73c). Col-
umn chromatography (n-hexane/AcOEt = 99/1 to 91/9) gave the
title compound (1738 mg, 8.557 mmol, 43% yield) as a pale-yellow
oil. 1H NMR (CDCl3) d: 9.90 (1H, s), 3.03–2.97 (2H, m), 2.49–2.45
(2H, m), 1.81–1.75 (2H, m), 1.67–1.61 (2H, m), 1.46–1.40 (2H,
m). 13C NMR (CDCl3) d: 193.28, 148.35, 140.51, 44.23, 31.38,
25.64, 25.15, 24.79.
4.2.3.8. General procedure for synthesis of 5-(N,N0,-dimethyl-
carbamoyl)alkyl derivatives of N,N0-(diethyl)-5,6-dihydro-6-
oxo-phenanthridine-2-carboxamide.
acid (0.1 mmol) and triethylamine (20
To a solution of the
lL, 0.14 mmol) in THF
(10 mL) and CH2Cl2 (4 mL) was added isobutyl chloroformate
(18 L, 0.14 mmol) at 0 °C. The mixture was stirred at this temper-
l
ature for 20 min, then dimethylamine (40% in water, 0.5 mL) was
added and stirring was continued at ambient temperature for
30 min. The whole was diluted with ethyl acetate. The organic
layer was washed with water and brine, dried over Na2SO4 and
concentrated. The product was purified as indicated below.
4.2.4.1.4. 2-Bromo-1-cyclooctene-1-carbaldehyde (73d). To a
mixture of DMF (2.4 mL) and CHCl3 (8 mL) was added PBr3
(2.4 mL, 25 mmol) dropwise at 0 °C. Then cyclooctanone
(1258 mg, 9.968 mmol) in CHCl3 (3 mL) was added at 0 °C, and
the mixture was stirred at ambient temperature for 6 h. n-Hexane
(10 mL) and water (20 mL) were added and the whole was
extracted with AcOEt. The organic layer was washed with water
and brine, dried and concentrated. Column chromatography
(n-hexane/AcOEt = 99/1 to 91/9) gave the title compound
(127.4 mg, 0.5868 mmol, 6% yield) as a colorless oil. 1H NMR
(CDCl3) d: 9.96 (1H, s), 2.92 (2H, t, J = 6.3 Hz), 2.45 (2H, t,
J = 5.7 Hz), 1.81–1.73 (2H, m), 1.54–1.40 (6H, m). 13C NMR (CDCl3)
d: 193.46, 146.61, 138.20, 39.67, 29.83, 28.24, 26.52, 26.16, 25.51.
4.2.3.8.1.
N,N0-(Diethyl)-5,6-dihydro-5-(N,N0,-dimethylcarba-
moyl)methyl-6-oxo-phenanthridine-2-carboxamide (28). Open col-
umn chromatography (ethyl acetate then CHCl3/MeOH = 40/1)
gave the title compound (22.0 mg, 0.0580 mmol, 53% yield) as col-
orless plates (recrystallized from CH2Cl2/n-hexane, mp 227.4–
230.0 °C). 1H NMR (CDCl3) d: 8.52 (1H, dd, J = 8.0, 1.2 Hz), 8.33
(1H, d, J = 1.7 Hz), 8.27 (1H, d, J = 8.0 Hz), 7.78 (1H, ddd, J = 7.7,
7.7, 1.7 Hz), 7.60 (1H, ddd, J = 8.0, 8.0, 1.1 Hz), 7.50 (1H, dd,
J = 8.6, 1.7 Hz), 7.19 (1H, d, J = 8.6 Hz), 5.24 (2H, s), 3.59 (2H, br
s), 3.36 (2H, br s), 3.36 (3H, s), 3.04 (3H, s), 1.28 (3H, br s), 1.20
(3H, br s). 13C NMR (CDCl3) d: 170.65, 166.41, 161.64, 138.24,
133.48, 132.93, 131.34, 129.04, 128.35, 127.55, 125.32, 122.09,
121.81, 119.38, 115.15, 44.52, 43.42, 39.55, 36.61, 35.97, 14.30,
12.96. FAB-MS m/z: 380 (MH+). Anal. Calcd for C22H25N3O3ꢀ1/
5H2O: C, 68.98; H, 6.68; N, 10.97. Found: C, 69.16; H, 6.67; N, 10.80.
4.2.4.2. General procedure for synthesis of 2-bromocycloalkene-
1-carboxylic acid.
The oxidation was conducted under
reported reaction conditions [Ohe 2000]. To a mixture of the alde-
hyde (0.59–8.5 mmol), NaH2PO4 (19.6–318 mg, 0.16–2.6 mmol,
0.3 equiv) and 30% aqueous H2O2 (0.06–1.1 mL) in CH3CN (0.5–
9 mL) and water (0.25–9 mL) was added NaClO2 (92–1500 mg,
0.81–13 mmol, 1.4 equiv ca. 80%) in water (0.7–14 mL) at ambient
temperature. The mixture was stirred at this temperature for 6–
16 h, alkalified by the addition of 10 N aqueous NaOH, and then
washed with AcOEt. The whole was acidified with 2 N aqueous
HCl and extracted with CHCl3/iPrOH = 3/1. The organic layer was
washed with water and brine, dried and concentrated to give the
desired compound.
4.2.4.2.1. 2-Bromo-1-cyclopentene-1-carboxylic acid (74a). Off-
white solid. 828.0 mg, 4.335 mmol, 85% yield. 1H NMR (CDCl3) d:
7.99 (1H, br s), 2.84 (2H, tt, J = 8.0, 2.3 Hz), 2.66 (2H, tt, J = 8.0,
2.3 Hz), 1.97 (2H, tt, J = 8.0, 8.0 Hz). 13C NMR (CDCl3) d: 168.96,
135.60, 131.37, 43.53, 32.86, 21.56. FAB-MS m/z: 193, 191 (MH+).
4.2.4.2.2. 2-Bromo-1-cyclohexene-1-carboxylic acid (74b). Col-
orless solid. 1567 mg, 7.642 mmol, 95% yield. 1H NMR (CDCl3) d:
2.68–2.61 (2H, m), 2.46–2.39 (2H, m), 1.76–1.65 (4H, m).
4.2.4.2.3. 2-Bromo-1-cycloheptene-1-carboxylic acid (74c). Col-
orless solid. 1681 mg, 7.671 mmol, 90% yield. 1H NMR (CDCl3)
d: 2.90–2.84 (2H, m), 2.53–2.47 (2H, m), 1.81–1.74 (2H, m),
1.65–1.56 (4H, m). 13C NMR (CDCl3) d: 173.37, 135.17,
132.10, 42.92, 31.19, 31.05, 25.56, 24.58. FAB-MS m/z: 221, 219
(MH+).
4.2.3.8.2.
N,N0-(Diethyl)-5,6-dihydro-5-(N,N0-dimethylcarba-
moyl)propyl-6-oxo-phenanthridine-2-carboxamide (29). Open col-
umn chromatography (ethyl acetate then CHCl3/MeOH = 40/1)
gave the title compound (26.9 mg, 0.0660 mmol, 74% yield) as col-
orless plates (recrystallized from CH2Cl2/n-hexane, mp 112.9–
115.5 °C). 1H NMR (CDCl3) d: 8.54 (1H, dd, J = 8.0, 1.2 Hz), 8.36
(1H, d, J = 2.3 Hz), 8.29 (1H, d, J = 8.0 Hz), 7.93 (1H, d, J = 8.6 Hz),
7.77 (1H, ddd, J = 7.7, 7.7, 1.2 Hz), 7.63–7.59 (2H, m), 4.48 (2H, t,
J = 7.5 Hz), 3.60 (2H, br s), 3.37 (2H, br s), 3.03 (3H, s), 3.00 (3H,
s), 2.55 (2H, t, J = 6.3 Hz), 2.14 (2H, tt, J = 7.5, 6.3 Hz), 1.29 (3H, br
s), 1.22 (3H, br s). 13C NMR (CDCl3) d: 172.16, 170.76, 161.42,
137.85, 133.34, 132.61, 131.13, 128.79, 128.24, 127.86, 125.59,
122.19, 121.80, 119.32, 115.80, 43.51, 42.74, 39.55, 37.23, 35.53,
30.35, 22.54, 14.39, 13.04. FAB-MS m/z: 408 (MH+). HRMS (FAB,
[M+H]+) calcd for C24H30N3O3 408.2287, found 408.2238.
4.2.4. Synthesis of cycloalkaquinolinones
4.2.4.1. General procedure for synthesis of 2-bromocycloalkene-
1-carbaldehydes.
Formylation was conducted as reported
[Ohe 2000]. To a mixture of DMF (9 mL) and CHCl3 (18 mL) was
added dropwise PBr3 (2.2 mL, 23 mmol) at 0 °C. The mixture was

F. Karaki et al. / Bioorg. Med. Chem. 22 (2014) 3587–3609
3601
4.2.4.2.4. 2-Bromo-1-cyclooctene-1-carboxylic acid (74d). Col-
orless solid. 121.6 mg, 0.5217 mmol, 89% yield. 1H NMR (CDCl3)
d: 2.81–2.75 (2H, m), 2.51–2.44 (2H, m), 1.76–1.67 (4H, m),
1.55–1.48 (4H, m). 13C NMR (CDCl3) d: 172.35, 132.47, 131.01,
38.64, 30.72, 30.00, 28.09, 26.32, 25.66. FAB-MS m/z: 233, 235
(MH+).
(3H,s), 3.74 (2H, t, J = 7.5 Hz), 2.28 (2H, br s), 2.12 (2H, br s),
1.54–1.35 (6H, brm), 1.34–1.25 (2H, m), 0.85 (3H, t, J = 7.5 Hz).
FAB-MS m/z: 396, 394 (MH+).
4.2.4.5.3. Methyl 4-(2-bromo-N-butylcyclohept-1-ene-1-carboxa-
mido)benzoate (77c). Column chromatography (n-hexane/
AcOEt = 94/6 to 52/48) gave the title compound (184.0 mg,
0.4506 mmol, 97% yield) as a colorless oil. 1H NMR (DMSO-d6,
100 °C) d: 7.98 (2H, d, J = 8.6 Hz), 7.45 (2H, d, J = 8.6 Hz), 3.88
(3H, s), 3.72 (2H, t, J = 7.5 Hz), 2.55 (2H, br s), 2.18 (2H, br s),
1.54 (2H, br s), 1.49–1.42 (2H, m), 1.39 (2H, br s), 1.34–1.25 (4H,
m), 0.85 (3H, t, J = 7.5 Hz). FAB-MS m/z: 410, 408 (MH+).
4.2.4.5.4. Methyl 4-(2-bromo-N-butylcyclooct-1-ene-1-carboxa-
mido)benzoate (77d). Column chromatography (n-hexane/
AcOEt = 98/2 to 84/16) gave the title compound (63.6 mg,
0.151 mmol, 56% yield) as a colorless oil. 1H NMR (DMSO-d6,
100 °C) d: 7.95 (2H, d, J = 8.0 Hz), 7.45 (2H, d, J = 8.0 Hz), 3.87
(3H, s), 3.74 (2H, t, J = 7.2 Hz), 2.46 (2H, br s), 2.21 (2H, br s),
1.62 (2H, br s), 1.56 (2H, br s), 1.51–1.40 (6H, m), 1.34–1.25 (2H,
m), 0.85 (3H, t, J = 7.2 Hz). FAB-MS m/z: 424, 422 (MH+).
4.2.4.3.
(75).
Methyl
4-(2,2,2-trifluoroacetamido)benzoate
To a solution of methyl 4-aminobenzoate (616.7 mg,
4.080 mmol) and triethylamine (2.0 mL, 14 mmol) in CH2Cl2
(30 mL) was added trifluoroacetic anhydride (1.0 mL, 7.1 mmol)
at 0 °C. The mixture was stirred at ambient temperature for 14 h,
then saturated aqueous NaHCO3 was added dropwise to it. The
whole was extracted with CH2Cl2 and the organic layer was
washed with saturated aqueous NaHCO3 and brine, dried and con-
centrated. Column chromatography (n-hexane/AcOEt = 96/4 to 68/
32) gave the title compound (974.0 mg, 3.941 mmol, 97% yield) as
an off-white solid. 1H NMR (CDCl3) d: 8.74 (1H, br s), 8.05 (2H, d,
J = 8.6 Hz), 7.72 (2H, d, J = 8.6 Hz), 3.92 (3H, s). 13C NMR (CDCl3)
d: 166.45, 155.16 (q, J = 38.4 Hz), 139.45, 130.89 (2C), 127.52,
119.98 (2C), 115.52 (q, J = 287.9 Hz), 52.28. FAB-MS m/z: 248
(MH+).
4.2.4.6. General procedure for intramolecular Heck cycliza-
tion.
A de-aerated mixture of the amide 77a–d (0.14–
0.44 mmol), Pd(OAc)2 (3.4–11 mg, 0.015–0.049 mmol, 10 mol%),
PCy3-HBF4 (5.6–19 mg, 0.015–0.049 mmol, 10 mol%) and Cs2CO3
(51–160 mg, 0.16–0.48 mmol, 1.1 equiv) in DMA (0.5–1.5 mL)
was stirred at 130 °C for 4 h, then cooled to ambient temperature
and diluted with AcOEt. The organic layer was washed with water
and brine, dried and concentrated. The product was purified as
indicated below.
4.2.4.4. Methyl 4-(butylamino)benzoate (76).
sion of 75 (1497 mg, 6.057 mmol) and Cs2CO3 (2792 mg,
7.914 mmol) in dry DMF (2 mL) was added n-butyl iodide
To a suspen-
(1350 lL, 12.0 mmol). The mixture was stirred at 90 °C for 2 h
and diluted with AcOEt. The organic layer was washed with water
and brine, dried and concentrated. To a solution of the crude triflu-
oroacetylamide in MeOH (8 mL) was added K2CO3 (2500 mg,
18.09 mmol). The whole was stirred at 70 °C for 1 h and diluted
with AcOEt. The organic layer was washed with water and brine,
dried and concentrated. Column chromatography (n-hexane/
AcOEt = 98/2 to 84/16) gave the title compound (702.6 mg,
3.390 mmol, 56% for 2 steps) as a colorless solid. 1H NMR (CDCl3)
d: 7.85 (2H, d, J = 8.6 Hz), 6.54 (2H, d, J = 8.6 Hz), 4.08 (1H, br s),
3.85 (3H, s), 3.19–3.14 (2H, m), 1.65–1.58 (2H, m), 1.47–1.39
(2H, m), 0.96 (3H, t, J = 7.5 Hz). 13C NMR (CDCl3) d: 167.35,
152.11, 131.53 (2C), 117.98, 111.27 (2C), 51.49, 43.04, 31.39,
20.19, 13.84. FAB-MS m/z: 208 (MH+).
4.2.4.6.1.
Methyl
5-butyl-4-oxo-2,3,4,5-tetrahydro-1H-cyclo-
penta[c]quinoline-8-carboxylate (78a). Column chromatography
(n-hexane/AcOEt = 94/6 to 52/48) gave the title compound
(91.0 mg, 0.304 mmol, 72% yield) as a colorless solid. 1H NMR
(CDCl3) d: 8.12 (1H, d, J = 1.7 Hz), 8.07 (1H, dd, J = 8.6, 1.7 Hz),
7.30 (1H, d, J = 8.6 Hz), 4.24 (2H, t, J = 7.4 Hz), 3.89 (3H, s), 3.11
(2H, t, J = 6.9 Hz), 2.92 (2H, t, J = 6.9 Hz), 2.14 (2H, tt, J = 6.9,
6.9 Hz), 1.69–1.61 (2H, m), 1.47–1.39 (2H, m), 0.94 (3H, t,
J = 7.5 Hz). 13C NMR (CDCl3) d: 166.36, 160.61, 150.16, 141.99,
133.81, 129.96, 127.52, 123.04, 119.23, 114.23, 52.08, 42.01,
32.19, 31.23, 29.66, 22.43, 20.15, 13.72. FAB-MS m/z: 300 (MH+).
4.2.4.6.2. Methyl 5-butyl-6-oxo-5,6,7,8,9,10-hexahydrophenan-
4.2.4.5. General procedure for synthesis of methyl 4-(2-bromo-
thridine-2-carboxylate
(78b). Column
chromatography
N-butylcycloalk-1-ene-1-carboxamido)benzoate.
tion of acid 74a–d (0.50–1.0 mmol, 2 equiv) and DMF (2 drop) in
dry CH2Cl2 (500 L) was added oxalyl chloride (45–85 L,
To a solu-
(n-hexane/AcOEt = 97/3 to 68/32) gave the title compound
(74.5 mg, 0.238 mmol, 83% yield) as a colorless solid. 1H NMR
(CDCl3) d: 8.43 (1H, d, J = 1.7 Hz), 8.13 (1H, dd, J = 9.2, 1.7 Hz), 7.37
(1H, d, J = 9.2 Hz), 4.32 (2H, t, J = 8.0 Hz), 3.95 (3H, s), 2.92 (2H, tt,
J = 6.3, 1.7 Hz), 2.67 (2H, tt, J = 5.7, 1.7 Hz), 1.92–1.86 (2H, m),
1.85–1.79 (2H, m), 1.75–1.69 (2H, m), 1.53–1.46 (2H, m), 1.00 (3H,
t, J = 6.9 Hz). 13C NMR (CDCl3) d: 166.73, 162.00, 142.04, 140.49,
129.75, 129.38, 126.08, 123.08, 121.03, 114.03, 52.19, 42.53, 29.66,
25.56, 24.54, 21.88, 21.86, 20.33, 13.85. FAB-MS m/z: 314 (MH+).
l
l
0.52–0.99 mmol, 2 equiv) at ambient temperature. The mixture
was stirred at this temperature for 10 min, then FK273 (56–
98 mg, 0.27–0.47 mmol) and DMAP (68–130 mg, 0.55–1.0 mmol,
2 equiv) were added and stirring was continued at 60 °C for 2 h.
The whole was diluted with CH2Cl2. The organic layer was washed
with water, 2 N HCl aq, 2 N NaOH aq, and brine, dried over Na2SO4
and concentrated. The product was purified as indicated below.
4.2.4.5.1. Methyl 4-(2-bromo-N-butylcyclopent-1-ene-1-carboxa-
mido)benzoate (77a). Column chromatography (n-hexane/
AcOEt = 96/4 to 68/32) gave the title compound (165.0 mg,
0.4339 mmol, 92% yield) as a pale-yellow oil. 1H NMR (DMSO-d6,
100 °C) d: 7.95 (2H, d, J = 8.6 Hz), 7.38 (2H, d, J = 8.6 Hz), 3.87
(3H, s), 3.77 (2H, d, J = 6.9 Hz), 2.44 (2H, tt, J = 7.5, 2.3 Hz), 2.36
(2H, tt, J = 7.5, 2.3 Hz), 1.76 (2H, tt, J = 7.5, 7.5 Hz), 1.51–1.44 (2H,
m), 1.34–1.26 (2H, m), 0.86 (3H, t, J = 7.5 Hz). FAB-MS m/z: 382,
380 (MH+).
4.2.4.6.3.
Methyl
5-butyl-6-oxo-6,7,8,9,10,11-hexahydro-5H-
cyclohepta[c]quinoline-2-carboxylate (78c). Column chromatogra-
phy (n-hexane/AcOEt = 97/3 to 76/24) gave the title compound
(91.8 mg, 0.280 mmol, 64% yield) as a colorless oil. 1H NMR (CDCl3)
d: 8.49 (1H, d, J = 1.7 Hz), 8.08 (1H, dd, J = 8.6, 1.7 Hz), 7.32 (1H, d,
J = 8.6 Hz), 4.27 (2H, t, J = 8.0 Hz), 3.92 (3H, s), 3.07–2.98 (4H, m),
1.90–1.84 (2H, m), 1.72–1.62 (4H, m), 1.59–1.53 (2H, m), 1.50–
1.42 (2H, m), 0.96 (3H, t, J = 7.5 Hz). 13C NMR (CDCl3) d: 166.64,
161.73, 148.64, 141.06, 134.31, 129.85, 126.82, 123.05, 120.19,
114.22, 52.11, 43.08, 32.07, 29.48, 28.02, 26.53, 25.76, 25.31,
20.25, 13.75. FAB-MS m/z: 328 (MH+).
4.2.4.5.2. Methyl 4-(2-bromo-N-butylcyclohex-1-ene-1-carboxa-
mido)benzoate (77b). Column chromatography (n-hexane/
AcOEt = 97/3 to 76/24) gave the title compound (122.2 mg,
0.3099 mmol, 84% yield) as a colorless oil. 1H NMR (DMSO-d6,
100 °C) d: 7.97 (2H, d, J = 8.6 Hz), 7.45 (2H, d, J = 8.6 Hz), 3.87
4.2.4.6.4. Methyl 5-butyl-6-oxo-5,6,7,8,9,10,11,12-octahydrocyclo-
octa[c]quinoline-2-carboxylate (78d). Column chromatography
(n-hexane/AcOEt = 97/3 to 76/24) gave the title compound
(27.4 mg, 0.0803 mmol, 56% yield) as a colorless oil. 1H NMR

3602
F. Karaki et al. / Bioorg. Med. Chem. 22 (2014) 3587–3609
(CDCl3) d: 8.44 (1H, d, J = 1.7 Hz), 8.09 (1H, dd, J = 8.6, 1.7 Hz), 7.33
(1H, d, J = 8.6 Hz), 4.29 (2H, t, J = 8.0 Hz), 3.92 (3H, s), 3.10 (2H, t,
J = 6.3 Hz), 2.91 (2H, t, J = 5.7 Hz), 1.85–1.78 (2H, m), 1.74–1.66
(4H, m), 1.51–1.41 (6H, m), 1.38–1.22 (2H, m), 0.97 (3H, t,
J = 7.5 Hz). 13C NMR (CDCl3) d: 166.67, 161.40, 145.22, 141.25,
132.56, 129.77, 127.18, 123.14, 119.93, 114.26, 52.15, 42.82,
29.88, 29.61, 29.57, 27.24, 26.58, 26.54, 25.91, 20.26, 13.78.
FAB-MS m/z: 342 (MH+).
for 10 min, then diethylamine (33–67
lL, 0.32–0.65 mmol,
5 equiv) and triethylamine (44–90 L, 0.32–0.65 mmol, 5 equiv)
l
were added. The mixture was stirred at ambient temperature for
30 min, then diluted with ethyl acetate. The organic layer was
washed with water and brine, dried over Na2SO4, and concen-
trated. The product was purified as indicated below.
4.2.4.8.1. 5-Butyl-N,N-diethyl-4-oxo-2,3,4,5-tetrahydro-1H-cyclo-
penta[c]quinoline-8-carboxamide (30). Column chromatography
(n-hexane/AcOEt = 88/12 to 4/96) gave the title compound
(32.9 mg, 0.0966 mmol, 89% yield) as a colorless oil. 1H NMR
(CDCl3) d: 7.55 (1H, d, J = 2.3 Hz), 7.50 (1H, dd, J = 8.6, 2.3 Hz),
7.33 (1H, d, J = 8.6 Hz), 4.27 (2H, t, J = 8.0 Hz), 3.42 (4H, br s),
3.09 (2H, t, J = 7.5 Hz), 2.95 (2H, t, J = 7.5 Hz), 2.16 (2H, tt, J = 7.5,
7.5 Hz), 1.71–1.64 (2H, m), 1.49–1.40 (2H, m), 1.19 (6H, br s),
0.96 (3H, t, J = 7.5 Hz). 13C NMR (CDCl3) d: 170.55, 160.64,
149.82, 139.50, 133.96, 130.23, 127.50, 123.99, 119.50, 114.37,
43.26, 41.96, 39.47, 32.15, 31.29, 29.75, 22.56, 20.22, 14.01,
13.79, 13.21. FAB-MS m/z: 341 (MH+). HRMS (FAB, [M+H]+) calcd
for C21H29N2O2 341.2229, found 341.2258.
4.2.4.7. General procedure for the ester hydrolysis of cycloalk-
aquinolinones.
A solution of ester 78a–d (0.080–0.30 mmol)
in THF (2 or 4 mL) and MeOH (1 or 2 mL) was treated with 0.5 M
aqueous NaOH (0.5 or 1 mL). The mixture was stirred at ambient
temperature overnight, acidified by the addition of hydrochloric
acid, and then extracted with CHCl3/iPrOH = 3/1. The organic layer
was washed with water and brine, dried, and concentrated. The
product was purified as indicated below.
4.2.4.7.1. 5-Butyl-4-oxo-2,3,4,5-tetrahydro-1H-cyclopenta[c]quin-
oline-8-carboxylic acid (79a). Recrystallization (CH2Cl2/MeOH)
gave the title compound (69.0 mg, 0.248 mmol, 80% yield) as a col-
orless solid. 1H NMR (CDCl3/CD3OD) d: 8.26 (1H, d, J = 1.7 Hz), 8.19
(1H, dd, J = 9.2, 1.7 Hz), 7.42 (1H, d, J = 9.2 Hz), 4.34 (2H, t,
J = 8.0 Hz), 3.20 (2H, t, J = 7.5 Hz), 3.00 (2H, t, J = 7.5 Hz), 2.22 (2H,
tt, J = 7.5, 7.5 Hz), 1.77–1.69 (2H, m), 1.54–1.46 (2H, m), 1.01 (3H,
t, J = 7.5 Hz). 13C NMR (CDCl3/CD3OD) d: 168.30, 161.06, 150.80,
141.99, 133.65, 130.48, 128.06, 123.42, 119.37, 114.45, 42.24,
32.23, 31.17, 29.71, 22.50, 20.16, 13.72. FAB-MS m/z: 286 (MH+).
4.2.4.7.2. 5-Butyl-6-oxo-5,6,7,8,9,10-hexahydrophenanthridine-2-
carboxylic acid (79b). The acid, obtained as a colorless solid
(71.7 mg, 0.240 mmol, 100% yield), was subjected to the next reac-
tion without further purification. 1H NMR (CDCl3) d: 8.51 (1H, d,
J = 1.7 Hz), 8.20 (1H, dd, J = 8.6, 1.7 Hz), 7.41 (1H, d, J = 8.6 Hz),
4.34 (2H, t, J = 7.5 Hz), 2.94 (2H, t, J = 6.3 Hz), 2.68 (2H, t,
J = 6.3 Hz), 1.93–1.87 (2H, m), 1.87–1.79 (2H, m), 1.77–1.70 (2H,
m), 1.55–1.47 (2H, m), 1.01 (3H, t, J = 7.5 Hz). 13C NMR (CDCl3) d:
170.21, 162.03, 142.05, 141.06, 130.26, 129.54, 126.85, 121.98,
121.11, 114.18, 42.60 29.65, 25.52, 24.53, 21.85, 21.82, 20.31,
13.84. FAB-MS m/z: 300 (MH+).
4.2.4.8.2. 5-Butyl-N,N-diethyl-6-oxo-5,6,7,8,9,10-hexahydrophe-
nanthridine-2-carboxamide
(31). Column
chromatography
(n-hexane/AcOEt = 88/12 to 4/96) gave the title compound
(31.4 mg, 0.0886 mmol, 91% yield) as a colorless oil. 1H NMR
(CDCl3) d: 7.77 (1H, d, J = 1.7 Hz), 7.53 (1H, dd, J = 8.6, 1.7 Hz),
7.36 (1H, d, J = 8.6 Hz), 4.31 (2H, t, J = 8.0 Hz), 3.57 (2H, br s),
3.34 (2H, br s), 2.86 (2H, t, J = 6.3 Hz), 2.67 (2H, t, J = 6.3 Hz),
1.90–1.85 (2H, m), 1.82–1.80 (2H, m), 1.75–1.69 (2H, m), 1.53–
1.47 (2H, m), 1.40–1.07 (6H, brm), 1.00 (3H, t, J = 7.5 Hz). 13C
NMR (CDCl3) d: 170.96, 161.91, 141.63, 137.89, 130.30, 129.50,
127.25, 122.42, 121.23, 114.15, 43.65, 42.43, 39.61, 29.75, 25.61,
24.66, 22.01, 21.96, 20.41, 14.50, 13.94, 12.98. FAB-MS m/z: 355
(MH+). FAB-MS m/z: 341 (MH+). HRMS (FAB, [M+H]+) calcd for
C22H31N2O2 355.2386, found 355.2383.
4.2.4.8.3. 5-Butyl-N,N-diethyl-6-oxo-6,7,8,9,10,11-hexahydro-5H-
cyclohepta[c]quinoline-2-carboxamide (32). Column chromatogra-
phy (n-hexane/AcOEt = 88/12 to 4/96) gave the title compound
(39.3 mg, 0.107 mmol, 91% yield) as a colorless oil. 1H NMR (CDCl3)
d: 7.85 (1H, d, J = 1.7 Hz), 7.48 (1H, dd, J = 8.6, 1.7 Hz), 7.31 (1H, d,
J = 8.6 Hz), 4.27 (2H, t, J = 7.5 Hz), 3.41 (4H, br s), 3.04–2.97 (4H, m),
1.89–1.83 (2H, m), 1.73–1.60 (4H, m), 1.59–1.53 (2H, m), 1.50–1.41
(2H, m), 1.19 (6H, br s), 0.96 (3H, t, J = 7.5 Hz). 13C NMR (CDCl3) d:
170.85, 161.60, 148.21, 138.47, 134.36, 130.10, 127.25, 123.26,
120.42, 114.18, 43.11, 42.92, 39.94, 32.04, 29.50, 28.01, 26.50,
25.81, 25.24, 20.27, 13.78, 13.61 (2C). FAB-MS m/z: 369 (MH+).
FAB-MS m/z: 341 (MH+). HRMS (FAB, [M+H]+) calcd for
4.2.4.7.3.
5-Butyl-6-oxo-6,7,8,9,10,11-hexahydro-5H-cyclohep-
ta[c]quinoline-2-carboxylic acid (79c). Reprecipitation (CHCl3/
MeOH) gave the title compound (78.2 mg, 0.250 mmol, 90% yield)
as a colorless solid. 1H NMR (DMSO-d6) d: 8.44 (1H, d, J = 1.7 Hz),
8.07 (1H, dd, J = 9.2, 1.7 Hz), 7.62 (1H, d, J = 9.2 Hz), 4.27 (2H, t,
J = 7.5 Hz), 3.31 (3H, s), 3.08–3.02 (2H, m), 2.97–2.92 (2H, m),
1.88–1.81 (2H, m), 1.65–1.54 (4H, m), 1.51–1.44 (2H, m), 1.43–
1.35 (2H, m), 0.93 (3H, t, J = 6.9 Hz). 13C NMR (DMSO-d6) d:
171.01, 160.70, 148.26, 140.54, 133.43, 130.12, 126.12, 123.78,
119.31, 115.07, 42.22, 31.52, 29.22, 27.31, 25.90, 25.48, 24.92,
19.62, 13.72. FAB-MS m/z: 314 (MH+).
C23H33N2O2 369.2542, found 369.2532.
4.2.4.8.4. 5-Butyl-N,N-diethyl-6-oxo-5,6,7,8,9,10,11,12-octahydro-
cyclooctaycloocta[c]quinoline-2-carboxamide (33). Column chro-
matography (n-hexane/AcOEt = 92/8 to 36/64) gave the title
compound (23.4 mg, 0.0612 mmol, 95% yield) as colorless cubes
(recrystallized from CH2Cl2/n-hexane, mp 91.1–92.0 °C). 1H NMR
(CDCl3) d: 7.82 (1H, d, J = 1.7 Hz), 7.53 (1H, dd, J = 8.6, 1.7 Hz),
7.36 (1H, d, J = 8.6 Hz), 4.32 (2H, t, J = 7.5 Hz), 3.56 (2H, br s),
3.36 (2H, br s), 3.08 (2H, t, J = 6.3 Hz), 2.95 (2H, t, J = 6.3 Hz),
1.84–1.78 (2H, m), 1.77–1.49 (4H, m), 1.53–1.46 (4H, m), 1.42–
1.35 (2H, m), 1.24 (6H, br s), 1.01 (3H, t, J = 6.9 Hz). 13C NMR
(CDCl3) d: 170.81, 161.28, 144.76, 138.66, 132.60, 130.21, 127.33,
123.48, 120.08, 114.31, 43.40, 42.67, 39.58, 29.88, 29.59, 29.52,
27.20, 26.58 (2C), 26.00, 20.29, 14.29, 13.81, 12.89. FAB-MS m/z:
383 (MH+). Anal. Calcd for C24H34N2O2: C, 75.35; H, 8.96; N, 7.32.
Found: C, 75.30; H, 8.89; N, 7.26.
4.2.4.7.4.
5-Butyl-6-oxo-5,6,7,8,9,10,11,12-octahydrocyclooc-
ta[c]quinoline-2-carboxylic acid (79d). Recrystallization (CH2Cl2/
n-hexane) gave the title compound (21.1 mg, 0.0644 mmol, 80%
yield) as a colorless solid. 1H NMR (CDCl3/CD3OD) d: 8.53 (1H, br
s), 8.17 (1H, d, J = 9.2 Hz), 7.40 (1H, d, J = 9.2 Hz), 4.34 (2H, t,
J = 7.5 Hz), 3.15 (2H, t, J = 6.3 Hz), 2.95 (2H, t, J = 5.2 Hz), 1.89–
1.81 (2H, m), 1.78–1.70 (4H, m), 1.55–1.44 (4H, m), 1.42–1.36
(2H, m), 1.01 (3H, t, J = 7.5 Hz). 13C NMR (CDCl3/CD3OD) d:
168.72, 161.60, 145.63, 141.23, 132.36, 130.19, 127.65, 123.24,
119.96, 114.36, 42.91, 29.80, 29.58, 29.53, 27.17, 26.55, 26.50,
25.87, 20.19, 13.72. FAB-MS m/z: 328 (MH+).
4.2.4.8. General procedure for the synthesis of cycloalkaquino-
4.2.5. Synthesis of the 5,6,7,8,9,10-hexahydrophenanthridine
derivative
4.2.5.1. Methyl 4-butyramidobenzoate (80). To a vigorously stirred
mixture of methyl 4-aminobenzoate (3037 mg, 20.09 mmol) in
line carboxamide derivatives.
(0.064–0.12 mmol) and DMF (50–100
added oxalyl chloride (16–33 L, 0.19–0.38 mmol, 3 equiv) at
ambient temperature. The whole was stirred at this temperature
To a solution of acid 79a–d
lL) in CH2Cl2 (1 mL) was
l

F. Karaki et al. / Bioorg. Med. Chem. 22 (2014) 3587–3609
3603
CH2Cl2 (50 mL) and saturated aqueous NaHCO3 (50 mL) at 0 °C
was added butyryl chloride (2700 L, 25.85 mmol). Stirring was
123.49, 123.16, 52.12, 45.92, 36.09, 28.12, 24.27, 22.41, 22.08,
l
19.34, 13.79. FAB-MS m/z: 314 (MH+).
continued for 11 h at ambient temperature. The biphasic mixture
was separated and the organic phase was washed with saturated
aqueous NaHCO3 and brine, dried over Na2SO4 and concentrated.
The residue was recrystallized from CH2Cl2/n-hexane to afford
the title compound (3190 mg, 14.42 mmol, 72% yield) as colorless
needles. 1H NMR (CDCl3) d: 7.96 (2H, d, J = 8.9 Hz), 7.65 (1H, br
s), 7.59 (2H, d, J = 8.9 Hz), 3.87 (3H, s), 2.34 (2H, t, J = 7.5 Hz),
1.73 (2H, tq, J = 7.5, 7.5 Hz), 0.97 (3H, t, J = 7.5 Hz). 13C NMR (CDCl3)
d: 171.66, 166.65, 142.20, 130.77 (2C), 125.36, 118.75 (2C), 52.00,
39.67, 18.88, 13.68. FAB-MS m/z: 222 (MH+).
4.2.5.5. 5-Butyryl-5,6,7,8,9,10-hexahydrophenanthridine-2-car-
boxylic acid (84).
A solution of 83 (51.1 mg, 0.163 mmol) in
THF (2 mL) and MeOH (1 mL) was treated with 0.5 M aqueous
NaOH (0.5 mL). The mixture was stirred at ambient temperature
for 6 h and diluted with water and 0.5 M aqueous NaOH. The whole
was washed with CH2Cl2, acidified with hydrochloric acid, and
extracted with CHCl3/iPrOH = 3/1. The organic layer was washed
with water and brine, dried, and concentrated. Recrystallization
(CH2Cl2/n-hexane) gave the title compound (28.3 mg, 0.0945
mmol, 58%) as a pale-yellow solid. 1H NMR (CDCl3) d: 7.94 (1H,
d, J = 2.0 Hz), 7.90 (1H, dd, J = 8.6, 2.0 Hz), 7.31–7.19 (1H, m), 4.23
(2H, s), 2.46 (2H, t, J = 7.5 Hz), 2.44–2.39 (2H, m), 2.19–2.15 (2H,
m), 1.82–1.76 (2H, m), 1.73–1.65 (4H, m), 0.90 (3H, t, J = 6.9 Hz).
FAB-MS m/z: 300 (MH+).
4.2.5.2. (2-Bromocyclohex-1-en-1-yl)methanol (81).
To a
solution of 73b (1148 mg, 6.074 mmol) in THF (20 mL) and MeOH
(10 mL) was added CeCl3ꢀ7H2O (3409 mg, 9.151 mmol) and NaBH4
(711.4 mg, 18.81 mmol). The whole was stirred at ambient temper-
ature for 1 h, then partitioned between ethyl acetate and water,
and the water layer was extracted with ethyl acetate. The com-
bined organic layer was washed with water and brine, dried and
concentrated. Column chromatography (n-hexane/AcOEt = 98/2
to 84/16) of the residue gave the title compound (1097 mg,
5.740 mmol, 95% yield) as a colorless oil. 1H NMR (CDCl3) d: 4.21
(2H, s), 2.52–2.46 (2H, m), 2.27–2.21 (2H, m), 1.72–1.64 (4H,
m).13C NMR (CDCl3) d: 135.30, 121.49, 66.31, 36.62, 29.13, 24.62,
22.24. FAB-MS m/z: 192 (MH+).
4.2.5.6.
5-Butyryl-N,N-diethyl-5,6,7,8,9,10-hexahydrophenan-
thridine-2-carboxamide (34).
A mixture of 84 (26.1 mg,
0.0872 mmol), diethylamine (28
l
L, 0.27 mmol) DMAP (11.9 mg,
0.0974 mmol) and EDC (40.5 mg, 0.211 mmol) in CH2Cl2 (1.5 mL)
was stirred at ambient temperature for 14 h, then diluted with
ethyl acetate. The organic layer was washed with water, 2 M aque-
ous HCl, saturated aqueous NaHCO3, and brine, then dried, and
concentrated. Column chromatography (n-hexane/AcOEt = 92/8
to 36/64) of the residue gave the title compound (18.4 mg,
0.0519 mmol, 60% yield) as a colorless oil. 1H NMR (CD3OD) d:
7.42 (1H, br s), 7.27 (1H, d, J = 1.7 Hz), 7.23 (1H, dd, J = 8.0,
1.7 Hz), 4.21 (2H, br s), 3.54 (2H, br s), 3.33 (2H, br s), 2.51 (2H,
t, J = 7.5 Hz), 2.42–2.37 (2H, m), 2.25–2.19 (2H, m), 1.84–1.78
(2H, m), 1.75–1.69 (2H, m), 1.66–1.57 (2H, m), 1.25 (3H, br s),
1.16 (3H, br s), 0.88 (3H, t, J = 6.6 Hz). 13C NMR (CD3OD) d:
174.79, 173.23, 138.00, 135.30, 133.44, 128.60, 127.23, 125.21,
125.03, 121.33, 45.02 (2C), 40.97, 36.71, 29.06, 25.31, 23.62,
23.27, 20.20, 14.48, 13.95, 13.04. FAB-MS m/z: 355 (MH+). HRMS
(FAB, [M+H]+) calcd for C22H31N2O2 355.2386, found 355.2376.
4.2.5.3. Methyl 4-(N-((2-bromocyclohex-1-en-1-yl)methyl)buty-
ramido)benzoate (82).
0.9923 mmol) and triethylamine (210
(1 mL) was added methanesulfonyl chloride (120
To
a
solution of 81 (189.6 mg,
L, 1.52 mmol) in CH2Cl2
L, 1.55 mmol)
l
l
at 0 °C. The mixture was stirred at this temperature for 6 h, then
water was added and the whole was extracted with ethyl acetate.
The organic layer was washed with water and brine, dried and
concentrated. The mixture of the mesylate and the chloride was sub-
jected to the next reaction without further purification. To a solution
of the crude product in dry DMF (300 lL) was added NaH (42.3 mg,
1.06 mmol, ca. 60%) and the whole was stirred at ambient tempera-
ture for 1 h. Then TBAI (39.2 mg, 0.106 mmol) and the mixture of the
4.2.6. Synthesis of dibenzoxazepinone derivatives
mesylate and the chloride in dry DMF (600
lL) were added and stir-
4.2.6.1. Methyl 11-oxo-10,11-dihydrodibenzo[b,f][1,4]oxaze-
ring was continued at 90 °C for 2 h. The whole was diluted with ethyl
acetate. The organic layer was washed with water and brine, dried
and concentrated. Column chromatography (n-hexane/AcOEt = 96/
4 to 68/32) of the residue gave the title compound (196.7 mg,
0.4988 mmol, 50% for 2 steps) as a pale-yellow oil. 1H NMR (CDCl3)
d: 8.08 (2H, d, J = 8.0 Hz), 7.21 (2H, d, J = 8.0 Hz), 4.63 (2H, s), 3.94
(3H, s), 2.37 (2H, br s), 2.14 (2H, br s), 2.06 (2H, t, J = 7.5 Hz), 1.66–
1.56 (6H, m), 0.83 (3H, t, J = 6.9 Hz).13C NMR (CDCl3) d: 172.69,
166.25, 145.88, 131.14, 130.71 (2C), 129.43, 128.23 (2C), 123.41,
52.61, 52.27, 36.65, 36.26, 29.05, 24.55, 22.17, 18.84, 13.74. FAB-
MS m/z: 396, 394 (MH+).
pine-7-carboxylate (86).
2-Fluorobenzoyl chloride (533 lL,
4.50 mmol) was added to a solution of methyl 4-amino-3-hydroxy-
benzoate (502 mg, 3.00 mmol) in CH2Cl2 (15 mL) and saturated
aqueous NaHCO3 at 0 °C. The reaction mixture was stirred for
20 h at rt, then acidified with hydrochloric acid, and extracted with
CHCl3 + MeOH. The organic phase was washed with brine, dried
over MgSO4 and concentrated. The crude amide was used for the
next reaction without further purification. In the next step, DMF
(15 mL) was added to the crude amide (1.07 g), K2CO3 (1.24 g,
9.00 mmol). The reaction mixture was stirred for 2 h at 150 °C,
then cooled to rt, washed with H2O and brine, dried over MgSO4
and concentrated. The resulting residue was purified by silica gel
chromatography (AcOEt/hexane = 1/13) and recrystallization
4.2.5.4. Methyl 5-butyryl-5,6,7,8,9,10-hexahydrophenanthri-
dine-2-carboxylate (83).
A de-aerated mixture of 82 (104.8
(CHCl3 + MeOH) to afford 13 (287 mg, 269 lmol, 36%) as colorless
mg, 0.2658 mmol), Pd(OAc)2 (6.4 mg, 0.029 mmol), PCy3ꢀHBF4
(11.0 mg, 0.0285 mmol) and Cs2CO3 (91.7 mg, 0.281 mmol) in
DMA (1 mL) was stirred at 150 °C for 7 h, then cooled to ambient
temperature and diluted with AcOEt. The organic layer was
washed with water and brine, dried and concentrated. Column
chromatography (n-hexane/AcOEt = 96/4 to 68/32) of the residue
gave the title compound (52.6 mg, 0.168 mmol, 63% yield) as a col-
orless solid. 1H NMR (CDCl3) d: 7.88 (1H, d, J = 1.7 Hz), 7.83 (1H, dd,
J = 8.6, 1.7 Hz), 7.21 (1H, br s), 4.21 (2H, s), 3.90 (3H, s), 2.44 (2H, t,
J = 7.5 Hz), 2.42–2.38 (2H, m), 2.18–2.13 (2H, m), 1.81–1.72 (2H,
m), 1.71–1.63 (4H, m), 0.89 (3H, t, J = 7.5 Hz). 13C NMR (CDCl3) d:
172.72, 166.79, 139.99, 135.00, 131.59, 127.22, 126.70, 125.87,
needles. 1H NMR (CDCl3) d: 8.03 (1H, s), 7.96–7.94 (2H, m), 7.83
(1H, dd, J = 8.3, 2.0 Hz), 7.58–7.54 (1H, m), 7.29–7.27 (2H, m),
7.05 (1H, d, J = 7.4 Hz), 3.92 (3H, s). FAB-MS m/z: 270 (M+H)+.
4.2.6.2. Methyl 10-butyl-11-oxo-10,11-dihydrodibenzo[b,f]-
[1,4]oxazepine-7-carboxylate (87).
Butyl iodide (66.0
lL,
585 mol) was added to a suspension of 86 (105 mg, 390
l
lmol),
Cs2CO3 (389 mg, 1.19 mmol) in DMF (4.0 mL) at ambient tempera-
ture. The reaction mixture was stirred for 1.5 h at 90 °C. The reac-
tion was quenched with H2O diluted with AcOEt. The organic phase
was separated, washed with H2O and brine, dried over Mg2SO4,
and concentrated. The resulting residue was purified by silica gel

3604
F. Karaki et al. / Bioorg. Med. Chem. 22 (2014) 3587–3609
chromatography (AcOEt/hexane = 1/10 to 1/5) to afford the title
organic layer was washed with water and brine, dried and concen-
trated. Column chromatography (n-hexane/AcOEt = 95/5 to 60/40)
of the residue gave the title compound (30.6 mg, 0.0946 mmol, 89%
yield) as a colorless oil. 1H NMR (CDCl3) d: 8.10 (1H, dd, J = 8.0,
2.3 Hz), 8.01 (1H, d, J = 2.3 Hz), 7.44 (2H, dd, J = 7.5, 7.5 Hz), 7.31–
7.26 (1H, m), 7.21 (2H, d, J = 7.5 Hz), 7.09 (1H, d, J = 8.0 Hz), 3.99
(2H, t, J = 7.5 Hz), 2.99 (2H, t, J = 7.5 Hz), 2.70 (2H, t, J = 7.5 Hz),
1.69–1.61 (2H, m), 1.46–1.37 (2H, m), 0.98 (3H, t, J = 7.5 Hz). 13C
NMR (CDCl3) d: 170.05, 164.65, 150.92, 144.20, 130.05, 129.88,
129.50 (2C), 126.48, 125.87, 123.42, 121.71 (2C), 114.64, 42.09,
31.60, 29.19, 25.38, 20.11, 13.81. FAB-MS m/z: 324 (MH+).
compound (111 mg, 341 lmol, 87%) as a colorless oil.
1H NMR (500 MHz, CDCl3) d: 7.92 (1H, d, J = 1.7 Hz), 7.86 (1H,
dd, J = 8.6, 2.3 Hz), 7.84 (1H, dd, J = 7.4, 1.1 Hz), 7.47–7.44 (1H,
m), 7.34 (1H, d, J = 8.6 Hz), 7.24–7.21 (2H, m), 4.15 (2H, br s),
3.92 (3H, s), 1.73–1.67 (2H, m), 1.42–1.33 (2H, m), 0.91 (3H, t,
J = 7.4 Hz). FAB-MS m/z: 325 (M)+, 326 (M+H)+.
4.2.6.3. 10-Butyl-N,N-diethyl-11-oxo-10,11-dihydrodibenzo[b,f]-
[1,4]oxazepine-7-carboxamide (35).
(320 L, 1.60 mmol) was added to a solution of 87 (63.5 mg,
195 mol) in MeOH (0.8 mL) at ambient temperature. The reaction
Aqueous 5 N NaOH
l
l
mixture was stirred for 3 h at 70 °C, then acidified with hydrochloric
acid, extracted with CHCl3, dried over Na2SO4 and concentrated. The
crude acid was used for the next reaction without further purifica-
4.2.7.4. 1-Butyl-2-oxo-1,2,3,4-tetrahydroquinoline-6-carboxylic
acid (92).
(1.5 mL) was treated with 30% aqueous H2O2 (400
aqueous NaOH (400 L). The mixture was stirred at ambient tem-
A solution of 91 (47.8 mg, 0.148 mmol) in dioxane
lL) and 2 M
tion. In the next step, HNEt2 (51
tion of the crude acid, EDC (47.0 mg, 246
246 mol) in DMF (1.0 mL) at ambient temperature. The reaction
l
L, 492
lmol) was added to a solu-
l
lmol) and HOBt (33.0 mg,
perature for 1 h, then 2 M aqueous Na2S2O3 was added. The whole
was acidified with hydrochloric acid, and extracted with CHCl3/
iPrOH = 4/1. The organic layer was washed with water and brine,
dried, and concentrated. Recrystallization (CH2Cl2/n-hexane) gave
the title compound (36.3 mg, 0.147 mmol, 99% yield) as a colorless
solid. 1H NMR (CDCl3) d: 7.98 (1H, d, J = 8.6 Hz), 7.89 (1H, s), 7.04
(1H, d, J = 8.6 Hz), 3.95 (2H, t, J = 8.0 Hz), 2.94 (2H, t, J = 6.9 Hz),
2.67 (2H, t, J = 6.9 Hz), 1.65–1.57 (2H, m), 1.42–1.35 (2H, m), 0.94
(3H, t, J = 7.5 Hz). 13C NMR (CDCl3) d: 170.18, 144.33, 130.11,
129.91, 129.63, 126.39, 115.26, 114.60, 42.16, 31.54, 29.19, 25.32,
20.10, 13.80. FAB-MS m/z: 248 (MH+).
l
mixture was stirred for 19 h at this temperature, then quenched
with H2O and diluted with ethyl acetate. The organic layer was
washed with saturated aqueous NaHCO3 and brine, dried (MgSO4)
and concentrated. The resulting residue was purified by column
chromatography (AcOEt/hexane = 1/1) to afford 35 (70.0 mg,
191 l
mol, 98%) as a white oil. 1H NMR (CDCl3) d: 7.86 (1H, dd,
J = 7.4, 1.7 Hz), 7.45–7.42 (1H, m), 7.32–7.30 (2H, m), 7.24–7.20
(2H, m), 7.17 (1H, d, J = 7.4 Hz), 4.14 (2H, t, J = 7.2 Hz), 3.41 (4H,
hrs), 1.76–1.70 (2H, m), 1.44–1.37 (2H, m), 1.20 (6H, s), 0.94 (3H, t,
J = 7.4 Hz). FAB-MS m/z: 367 (MH)+. HRMS (FAB, [M+H]+) calcd for
C22H27N2O3 367.2022, found 367.2028.
4.2.7.5. 1-Butyl-N,N-diethyl-2-oxo-1,2,3,4-tetrahydroquinoline-
6-carboxamide (36).
0.133 mmol) and DMF (2 drops) in CH2Cl2 (1.5 mL) was added oxalyl
chloride (40 L, 0.47 mmol) at ambient temperature. The mixture
was stirred at this temperature for 10 min, then diethylamine
(80 L, 0.78 mmol) and triethylamine (100 L, 0.721 mmol) were
added. The whole was stirred at ambient temperature for 30 min,
then diluted with CH2Cl2. The organic layer was washed with water
and brine, dried and concentrated. Column chromatography (n-hex-
ane/AcOEt = 88/12 to 0/100) of the residue gave the title compound
(30.8 mg, 0.102 mmol, 76% yield) as a colorless oil. 1H NMR (CDCl3)
d: 7.24 (1H, dd, J = 8.6, 1.7 Hz), 7.20 (1H, d, J = 1.7 Hz), 6.96 (1H, d,
J = 8.6 Hz), 3.90 (2H, t, J = 8.0 Hz), 3.42 (4H, br s), 2.87 (2H, t,
J = 7.5 Hz), 2.62 (2H, t, J = 7.5 Hz), 1.62–1.55 (2H, m), 1.40–1.32
(2H, m), 1.78 (6H, t. J = 6.9 Hz), 0.93 (3H, t, J = 7.5 Hz). 13C NMR
(CDCl3, 60 °C) d: 170.78, 169.79, 140.68, 131.40, 126.69, 126.62,
125.81, 114.45, 41.95, 41.52 (2C), 31.75, 29.36, 25.62, 20.09, 13.67,
13.54 (2C). FAB-MS m/z: 303 (MH+). HRMS (FAB, [M+H]+) calcd for
To
a
solution of 92 (33.0 mg,
4.2.7. Synthesis of 2-oxo-1,2,3,4-tetrahydroquinoline derivatives
4.2.7.1. 6-Bromo-3,4-dihydroquinolin-2(1H)-one (89).
solution of 3,4-dihydro-2(1H)-quinolinone
To a
(454.8 mg,
l
3.090 mmol) in DMF (5 mL) was added N-bromosuccinimide
(587.1 mg, 3.299 mmol) in DMF (5 mL) at 0 °C. The mixture was
then allowed to warm to ambient temperature, stirred for 9 h,
and diluted with ethyl acetate. The organic layer was washed with
water and brine, dried, and concentrated. The residue was recrys-
tallized from ethanol/water to yield the title compound (385.5 mg,
1.705 mmol) as an off-white solid. 1H NMR (CDCl3) d: 8.56 (1H, br
s), 7.30–7.26 (2H, m), 6.68 (1H, d, J = 8.0 Hz), 2.96 (2H, t, J = 7.5 Hz),
2.63 (2H, t, J = 8.0 Hz). 13C NMR (CDCl3) d: 171.37, 136.34, 130.85,
130.39, 125.71, 116.84, 115.45, 30.32, 25.14.
l
l
4.2.7.2. Phenyl 2-oxo-1,2,3,4-tetrahydroquinoline-6-carboxyl-
ate (90).
phenyl formate (51.3 mg, 0.420 mmol), tributylamine (95
0.40 mmol), Pd(OAc)2 (5.2 mg, 0.023 mmol) and P(tBu)3ꢀHBF4
(26.6 mg, 0.0917 mmol) in NMP (600 L) was stirred at 150 °C
A de-aerated mixture of 89 (49.2 mg, 0.218 mmol),
lL,
C18H27N2O2 303.2073, found 303.2058.
l
4.2.8. Synthesis of amide derivatives of 5-butyl-6-oxo-
6,7,8,9,10,11-hexahydro-5H-cyclohepta[c]quinoline-2-
carboxylic acid
4.2.8.1. General procedure for synthesis of amide derivatives of
5-butyl-6-oxo-6,7,8,9,10,11-hexahydro-5H-cyclohepta[c]quino-
for 4 h, then cooled to ambient temperature and diluted with
AcOEt. The organic layer was washed with water, 2 N aqueous
HCl and brine, then dried and concentrated. Column chromatogra-
phy (n-hexane/AcOEt = 94/6 to 52/48) of the residue gave the title
compound (28.0 mg, 0.105 mmol, 48% yield) as a colorless solid. 1H
NMR (CD3OD/CDCl3) d: 8.01 (1H, d, J = 1.7 Hz), 7.99 (1H, dd, J = 8.6,
1.7 Hz), 7.43 (2H, dd, J = 8.0, 7.5 Hz), 7.27 (1H, t, J = 7.5 Hz), 7.19
(2H, d, J = 8.0 Hz), 6.99 (1H, d, J = 8.6 Hz), 3.05 (2H, t, J = 6.9 Hz),
2.63 (2H, t, J = 6.9 Hz). 13C NMR (CD3OD/CDCl3) d: 173.79, 166.30,
152.46, 144.11, 130.97 (2C), 130.51 (2C), 126.91, 125.39, 124.88,
122.84 (2C), 116.43, 31.26, 25.96. FAB-MS m/z: 268 (MH+).
line-2-carboxylic acid (A).
0.10 mmol) and DMF (100
chloride (20
was stirred at this temperature for 10 min, then triethylamine
(70 L, 0.51 mmol) and amine (0.50 mmol) were added. The whole
To a solution of 79c (30 mg,
lL) in CH2Cl2 (1 mL) was added oxalyl
lL, 0.23 mmol) at ambient temperature. The mixture
l
was stirred at ambient temperature for 30 min, then diluted with
ethyl acetate. The organic layer was washed with water and brine,
dried over Na2SO4 and concentrated. The product was purified as
indicated below.
4.2.7.3. Phenyl 1-butyl-2-oxo-1,2,3,4-tetrahydroquinoline-6-
carboxylate (91).
0.106 mmol) and Cs2CO3 (54.8 mg, 0.155 mmol) in DMF (500
was added butyl iodide (23 L, 0.20 mmol). The whole was stirred
at ambient temperature for 3 h and diluted with AcOEt. The
To
a
suspension of 90 (28.3 mg,
l
L)
4.2.8.2. General procedure for synthesis of amide derivatives of
5-butyl-6-oxo-6,7,8,9,10,11-hexahydro-5H-cyclohepta[c]quino-
l
line-2-carboxylic acid (B).
To a solution of 79c (30 mg,

F. Karaki et al. / Bioorg. Med. Chem. 22 (2014) 3587–3609
3605
0.10 mmol) and DMF (100
l
L) in CH2Cl2 (1 mL) was added oxalyl
120.54, 114.28, 43.09, 35.08, 32.15, 29.58, 28.08, 26.65, 25.85,
25.45, 20.33, 14.99, 13.84. FAB-MS m/z: 341 (MH+). HRMS (FAB,
[M+H]+) calcd for C21H29N2O2 341.2229, found 341.2180.
chloride (20 L, 0.23 mmol) at ambient temperature. The mixture
l
was stirred at this temperature for 10 min, then aqueous amine
solution (1 mL) was added. The whole was stirred at ambient tem-
perature for 60 min, then diluted with ethyl acetate. The organic
layer was washed with water and brine, dried over Na2SO4 and
concentrated. The product was purified as indicated below.
4.2.8.2.5.
5-Butyl-6-oxo-6,7,8,9,10,11-hexahydro-5H-cyclohep-
ta[c]quinoline-2-carboxylic acid pyrrolidine amide (41). This com-
pound was synthesized according to the general procedure A.
Column chromatography (n-hexane/AcOEt = 83/17 to 0/100) gave
the title compound (29.5 mg, 0.0806 mmol, 90%) as a colorless
amorphous solid. 1H NMR (CDCl3) d: 8.05 (1H, d, J = 1.7 Hz), 7.65
(1H, dd, J = 8.6, 1.7 Hz), 7.32 (1H, d, J = 8.6 Hz), 4.29 (2H, t,
J = 7.5 Hz), 3.67 (2H, t, J = 6.9 Hz), 3.49 (2H, t, J = 6.3 Hz), 3.05–
3.00 (4H, m), 2.01–1.93 (2H, m), 1.92–1.84 (4H, m), 1.75–1.55
(6H, m), 1.52–1.44 (2H, m), 0.98 (3H, t, J = 7.5 Hz). 13C NMR (CDCl3)
d: 169.17, 161.72, 148.40, 138.94, 134.37, 130.16, 127.96, 124.45,
120.36, 113.94, 49.86, 46.46, 43.01, 32.11, 29.58, 28.10, 26.58,
26.54, 25.88, 25.36, 24.46, 20.34, 13.84. FAB-MS m/z: 367 (MH+).
HRMS (FAB, [M+H]+) calcd for C23H31N2O2 367.2386, found
367.2381.
4.2.8.2.6. N-n-Propyl-5-butyl-6-oxo-6,7,8,9,10,11-hexahydro-5H-
cyclohepta[c]quinoline-2-carboxamide (42). This compound was
synthesized according to the general procedure A. Column chroma-
tography (n-hexane/AcOEt = 92/8 to 36/64) gave the title compound
(27.7 mg, 0.0781 mmol, 83%) as colorless needles (recrystallized
from CH2Cl2/n-hexane, mp 223.6–224.8 °C).1H NMR (CDCl3) d:
8.33 (1H, d, J = 2.3 Hz), 7.78 (1H, dd, J = 9.2, 2.3 Hz), 7.33 (1H, d,
J = 9.2 Hz), 6.16 (1H, br s), 4.29 (2H, t, J = 8.0 Hz), 3.48–3.42 (2H,
m), 3.10–3.05 (2H, m), 3.05–3.01 (2H, m), 1.92–1.86 (2H, m), 1.74–
1.55 (8H, m), 1.51–1.43 (2H, m), 0.99 (3H, t, J = 7.5 Hz), 0.98 (3H, t,
J = 7.5 Hz). 13C NMR (CDCl3) d: 166.87, 161.73, 148.62, 140.02,
134.59, 127.68, 126.74, 124.49, 120.56, 114.27, 43.08, 41.89, 32.15,
29.58, 28.09, 26.65, 25.85, 25.44, 23.01, 20.33, 13.84, 11.47. FAB-
MS m/z: 355 (MH+). Anal. Calcd for C22H30N2O2: C, 74.54; H, 8.53;
N, 7.90. Found: C, 74.25; H, 8.26; N, 7.78.
4.2.8.2.7. N-n-Butyl-5-butyl-6-oxo-6,7,8,9,10,11-hexahydro-5H-
cyclohepta[c]quinoline-2-carboxamide (43). This compound was
synthesized according to the general procedure A. Column chroma-
tography (n-hexane/AcOEt = 92/8 to 36/64) gave the title com-
pound (30.0 mg, 0.0815 mmol, 94%) as colorless needles
(recrystallized from CH2Cl2/n-hexane, mp 129.3–131.0 °C).1H
NMR (CDCl3) d: 8.33 (1H, d, J = 1.7 Hz), 7.77 (1H, dd, J = 8.6,
1.7 Hz), 7.33 (1H, d, J = 8.6 Hz), 6.13 (1H, br s), 4.29 (2H, t,
J = 8.0 Hz), 3.51–3.46 (2H, m), 3.09–3.05 (2H, m), 3.05–3.01 (2H,
m), 1.92–1.86 (2H, m), 1.74–1.55 (8H, m), 1.51–1.38 (4H, m),
0.98 (3H, t, J = 7.5 Hz), 0.96 (3H, t, J = 6.9 Hz). 13C NMR (CDCl3) d:
166.82, 161.73, 148.61, 140.02, 134.59, 127.67, 126.71, 124.50,
120.57, 114.26, 43.08, 39.94, 32.15, 31.84, 29.58, 28.09, 26.65,
25.86, 25.45, 20.33, 20.19, 13.84, 13.81. FAB-MS m/z: 369 (MH+).
Anal. Calcd for C23H32N2O2: C, 74.96; H, 8.75; N, 7.60. Found: C,
74.61; H, 8.54; N, 7.51.
4.2.8.2.1.
5-Butyl-6-oxo-6,7,8,9,10,11-hexahydro-5H-cyclohep-
ta[c]quinoline-2-carboxamide (37). This compound was synthe-
sized according to the general procedure B using 28% aqueous
ammonia
solution.
Column
chromatography
(n-hexane/
AcOEt = 83/17 to 0/100) gave the title compound (39.3 mg,
0.126 mmol, 99% yield) as colorless cubes (recrystallized from
CH2Cl2/MeOH/n-hexane, mp 222.9–224.0 °C). 1H NMR (DMSO-d6)
d: 8.42 (1H, d, J = 1.7 Hz), 8.16 (1H, br s), 8.06 (1H, dd, J = 9.2,
1.7 Hz), 7.57 (1H, d, J = 9.2 Hz), 7.40 (1H, br s), 4.27 (2H, t,
J = 8.0 Hz), 3.13–3.08 (2H, m), 2.97–2.91 (2H, m), 1.89–1.81 (2H,
m), 1.64–1.54 (4H, m), 1.51–1.44 (2H, m), 1.43–1.35 (2H, m),
0.92 (3H, t, J = 7.5 Hz). 13C NMR (CDCl3/CD3OD) d: 169.54, 161.83,
149.27, 139.95, 133.94, 127.93, 126.24, 124.87, 120.27, 114.34,
43.02, 31.84, 29.35, 27.77, 26.35, 25.51, 25.13, 20.03, 13.50.
FAB-MS m/z: 313 (MH+). Anal. Calcd for C19H24N2O2ꢀ1/4H2O: C,
72.01; H, 7.79; N, 8.84. Found: C, 72.29; H, 7.63; N, 8.82.
4.2.8.2.2.
N-Methyl-5-butyl-6-oxo-6,7,8,9,10,11-hexahydro-5H-
cyclohepta[c]quinoline-2-carboxamide (38). This compound was
synthesized according to the general procedure B using 40% aque-
ous methylamine solution. Column chromatography (n-hexane/
AcOEt = 83/17 to 0/100) gave the title compound (32.7 mg,
0.100 mmol, quant.) as colorless needles (recrystallized from
CH2Cl2/n-hexane, mp 147.1–148.8 °C). 1H NMR (CDCl3) d: 8.31
(1H, d, J = 2.3 Hz), 7.79 (1H, dd, J = 9.2, 2.3 Hz), 7.33 (1H, d,
J = 9.2 Hz), 6.19 (1H, br s), 4.29 (2H, t, J = 7.5 Hz), 3.04 (3H, d,
J = 4.6 Hz), 3.08–3.01 (4H, m), 1.92–1.86 (2H, m), 1.74–1.63 (4H,
m), 1.61–1.55 (2H, m), 1.51–1.43 (2H, m), 0.98 (3H, t, J = 7.5 Hz).
13C NMR (CDCl3) d: 167.57, 161.73, 148.59, 140.05, 134.60,
127.51, 126.90, 124.32, 120.51, 114.33, 43.10, 32.14, 29.58, 28.08,
26.96, 26.64, 25.85, 25.42, 20.33, 13.84. FAB-MS m/z: 327 (MH+).
Anal. Calcd for C20H26N2O2: C, 73.59; H, 8.03; N, 8.58. Found: C,
73.34; H, 7.89; N, 8.58.
4.2.8.2.3. N,N0-Dimethyl-5-butyl-6-oxo-6,7,8,9,10,11-hexahydro-
5H-cyclohepta[c]quinoline-2-carboxamide (39). This compound
was synthesized according to the general procedure B using 40%
aqueous dimethylamine solution. Column chromatography
(n-hexane/AcOEt = 83/17 to 0/100) gave the title compound
(31.7 mg, 0.0931 mmol, quant.) as a colorless oil. 1H NMR (CDCl3)
d: 7.94 (1H, d, J = 1.7 Hz), 7.54 (1H, dd, J = 8.6, 1.7 Hz), 7.33 (1H,
d, J = 8.6 Hz), 4.30 (2H, t, J = 7.5 Hz), 3.12 (3H, br s), 3.19–2.99
(7H, br m), 1.91–1.85 (2H, m), 1.75–1.62 (4H, m), 1.61–1.55 (2H,
m), 1.52–1.44 (2H, m), 0.99 (3H, t, J = 7.5 Hz). 13C NMR (DMSO-
d6) d: 169.64, 160.55, 148.21, 138.03, 133.34, 129.60, 128.44,
123.75, 119.29, 114.53, 42.01, 34.95 (2C), 31.55, 29.26, 27.21,
25.87, 25.56, 25.00, 19.65, 13.74. FAB-MS m/z: 341 (MH+). HRMS
(FAB, [M+H]+) calcd for C21H29N2O2 341.2229, found 341.2210.
4.2.8.2.8. N-i-Propyl-5-butyl-6-oxo-6,7,8,9,10,11-hexahydro-5H-
cyclohepta[c]quinoline-2-carboxamide (44). This compound was
synthesized according to the general procedure A. Column chroma-
tography (n-hexane/AcOEt = 92/8 to 36/64) gave the title com-
4.2.8.2.4.
N-Ethyl-5-butyl-6-oxo-6,7,8,9,10,11-hexahydro-5H-
pound (22.1 mg, 0.0623 mmol, 74%) as a colorless powder
cyclohepta[c]quinoline-2-carboxamide (40). This compound was
synthesized according to the general procedure B using 70% aque-
ous ethylamine solution. Column chromatography (n-hexane/
AcOEt = 88/12 to 0/100) gave the title compound (31.8 mg,
0.0934 mmol, 100%) as colorless needles (recrystallized from
CH2Cl2/n-hexane, mp 127.2–129.3 °C). 1H NMR (CDCl3) d: 8.33
(1H, d, J = 2.3 Hz), 7.79 (1H, dd, J = 8.6, 2.3 Hz), 7.33 (1H, d,
J = 8.6 Hz), 6.15 (1H, br s), 4.29 (2H, t, J = 8.0 Hz), 3.56–3.49 (2H,
m), 3.09–3.05 (2H, m), 3.04–3.02 (2H, m), 1.92–1.86 (2H, m),
1.74–1.63 (4H, m), 1.61–1.55 (2H, m), 1.51–1.44 (2H, m), 1.27
(3H, t, J = 7.5 Hz), 0.98 (3H, t, J = 7.5 Hz). 13C NMR (CDCl3) d:
166.77, 161.73, 148.62, 140.02, 134.58, 127.62, 126.81, 124.42,
(recrystallized from CH2Cl2/n-hexane, mp 121.1–124.1 °C). 1H
NMR (CDCl3) d: 8.33 (1H, d, J = 1.7 Hz), 7.76 (1H, dd, J = 9.2,
1.7 Hz), 7.32 (1H, d, J = 9.2 Hz), 5.92 (1H, d, J = 7.5 Hz), 4.35–4.26
(3H, m), 3.10–3.05 (2H, m), 3.05–3.01 (2H, m), 1.92–1.86 (2H, m),
1.74–1.64 (4H, m), 1.62–1.55 (2H, m), 1.51–1.43 (2H, m), 1.29
(6H, d, J = 6.3 Hz), 0.98 (3H, t, J = 7.5 Hz). 13C NMR (CDCl3) d:
166.01, 161.73, 148.64, 139.99, 134.57, 127.75, 126.66, 124.54,
120.57, 114.20, 43.07, 42.08, 32.15, 29.57, 28.09, 26.66, 25.85,
25.46, 22.93 (2C), 20.33, 13.84. FAB-MS m/z: 355 (MH+). HRMS
(FAB, [M+H]+) calcd for C22H31N2O2 355.2386, found 355.2397.
4.2.8.2.9.
N-t-Butyl-5-butyl-6-oxo-6,7,8,9,10,11-hexahydro-5H-
cyclohepta[c]quinoline-2-carboxamide (45). This compound was

3606
F. Karaki et al. / Bioorg. Med. Chem. 22 (2014) 3587–3609
synthesized according to the general procedure A. Column chroma-
tography (n-hexane/AcOEt = 95/5 to 60/40) gave the title com-
pound (31.8 mg, 0.0863 mmol, 93%) as a colorless oil. 1H NMR
(CDCl3) d: 8.34 (1H, d, J = 1.7 Hz), 7.71 (1H, dd, J = 8.6, 1.7 Hz),
7.31 (1H, d, J = 8.6 Hz), 5.96 (1H, br s), 4.29 (2H, t, J = 8.0 Hz),
3.09–3.05 (2H, m), 3.05–3.01 (2H, m), 1.91–1.85 (2H, m), 1.73–
1.63 (4H, m), 1.61–1.55 (2H, m), 1.49 (9H, s), 1.50–1.44 (2H, m),
0.98 (3H, t, J = 7.5 Hz). 13C NMR (CDCl3) d: 166.16, 161.73,
148.70, 139.87, 134.54, 128.55, 126.46, 124.57, 120.59, 114.08,
51.78, 43.05, 32.16, 29.57, 28.95 (3C), 28.06, 26.65, 25.87, 25.45,
20.33, 13.84. FAB-MS m/z: 369 (MH+). HRMS (FAB, [M+H]+) calcd
for C23H33N2O2 369.2542, found 369.2549.
(27.1 mg, 0.0687 mmol, 78%) as a colorless powder (recrystallized
from CH2Cl2/n-hexane, mp 184.1–187.8 °C). 1H NMR (CDCl3)
d: 8.32 (1H, d, J = 1.7 Hz), 7.76 (1H, dd, J = 8.6, 1.7 Hz), 7.32
(1H, d, J = 8.6 Hz), 5.96 (1H, d, J = 7.5 Hz), 4.29 (2H, t, J = 8.0 Hz),
4.04–3.94 (1H, m), 3.09–3.05 (2H, m), 3.05–3.01 (2H, m),
2.08–2.01 (2H, m), 1.92–1.85 (2H, m), 1.79–1.63 (8H, m),
1.62–1.55 (2H, m), 1.51–1.39 (4H, m), 1.30–1.19 (2H, m), 0.98
(3H, t, J = 7.5 Hz). 13C NMR (CDCl3) d: 165.95, 161.72, 148.64,
139.96, 134.55, 127.90, 126.69, 124.52, 120.56, 114.20, 48.87,
43.06, 33.31 (2C), 32.15, 29.57, 28.10, 26.65, 25.85, 25.58, 25.46,
24.94 (2C), 20.33, 13.84. FAB-MS m/z: 395 (MH+). HRMS (FAB,
[M+H]+) calcd for C25H35N2O2 395.2699, found 395.2735.
4.2.8.2.10. N-Phenyl-5-butyl-6-oxo-6,7,8,9,10,11-hexahydro-5H-
cyclohepta[c]quinoline-2-carboxamide (46). This compound was
synthesized according to the general procedure A. Column chroma-
tography (n-hexane/AcOEt = 95/5 to 60/40) gave the title com-
pound (37.5 mg, 0.0965 mmol, 100%) as colorless cubes
(recrystallized from CH2Cl2/MeOH, mp 199.5–200.6 °C). 1H NMR
(CDCl3) d: 8.41 (1H, d, J = 2.3 Hz), 7.91 (1H, dd, J = 8.6, 2.3 Hz),
7.66 (2H, d, J = 7.5 Hz), 7.40–7.36 (3H, m), 7.16 (1H, d, J = 7.5 Hz),
4.31 (2H, t, J = 8.0 Hz), 3.11–3.07 (2H, m), 3.06–3.02 (2H, m),
1.93–1.87 (2H, m), 1.76–1.65 (4H, m), 1.63–1.56 (2H, m), 1.52–
1.45 (2H, m), 0.99 (3H, t, J = 7.5 Hz). 13C NMR (CDCl3) d: 165.11,
161.73, 148.52, 140.36, 137.90, 134.86, 129.17 (3C), 127.77,
126.89, 124.75, 124.66, 120.22 (2C), 114.50, 43.14, 32.12, 29.59,
28.13, 26.68, 25.82, 25.41, 20.34, 13.85. FAB-MS m/z: 389 (MH+).
HRMS (FAB, [M+H]+) calcd for C25H29N2O2 389.2229, found
389.2209.
4.2.8.2.14. N-c-Propyl-5-butyl-6-oxo-6,7,8,9,10,11-hexahydro-5H-
cyclohepta[c]quinoline-2-carboxamide (50). This compound was
synthesized according to the general procedure A. Column chroma-
tography (n-hexane/AcOEt = 88/12 to 4/96) gave the title com-
pound (20.5 mg, 0.0582 mmol, 69%) as
a colorless powder
(recrystallized from CH2Cl2/n-hexane, mp 177.4–180.5 °C). 1H
NMR (CDCl3) d: 8.30 (1H, d, J = 1.7 Hz), 7.75 (1H, dd, J = 8.6,
1.7 Hz), 7.32 (1H, d, J = 8.6 Hz), 6.26 (1H, br s), 4.29 (2H, t,
J = 8.0 Hz), 3.09–3.05 (2H, m), 3.04–3.00 (2H, m), 2.95–2.89 (1H,
m), 1.92–1.86 (2H, m), 1.73–1.64 (4H, m), 1.61–1.55 (2H, m),
1.51–1.43 (2H, m), 0.98 (3H, t, J = 7.5 Hz), 0.91–0.86 (2H, m),
0.66–0.62 (2H, m). 13C NMR (CDCl3) d: 168.20, 161.71, 148.58,
140.12, 134.62, 127.25, 126.83, 124.39, 120.51, 114.27, 43.08,
32.14, 29.57, 28.08, 26.65, 25.84, 25.44, 23.26, 20.32, 13.83, 6.86
(2C). FAB-MS m/z: 353 (MH+). Anal. Calcd for C22H28N2O2ꢀ2/3
H2O: C, 72.50; H, 8.11; N, 7.69. Found: C, 72.29; H, 7.75; N, 7.50.
4.2.8.2.11. N-(2-Hydroxy)ethyl-5-butyl-6-oxo-6,7,8,9,10,11-hexa-
hydro-5H-cyclohepta[c]quinoline-2-carboxamide (47). This com-
pound was synthesized according to the general procedure A.
Column chromatography (n-hexane/AcOEt = 50/50 to 0/100) gave
the title compound (26.2 mg, 0.0735 mmol, 76%) as a colorless
powder (recrystallized from CH2Cl2/n-hexane, mp 182.5–
184.0 °C). 1H NMR (CDCl3) d: 8.33 (1H, d, J = 1.7 Hz), 7.82 (1H, dd,
J = 8.6, 1.7 Hz), 7.33 (1H, d, J = 8.6 Hz), 6.65 (1H, t, J = 5.7 Hz), 4.29
(2H, t, J = 8.0 Hz), 3.87 (2H, dt, J = 5.7, 5.7 Hz), 3.67 (2H, dt, J = 4.6,
5.7 Hz), 3.08–3.04 (2H, m), 3.04–3.01 (2H, m), 2.54 (1H, t,
J = 4.6 Hz), 1.92–1.86 (2H, m), 1.74–1.63 (4H, m), 1.61–1.55 (2H,
m), 1.51–1.44 (2H, m), 0.98 (3H, t, J = 7.5 Hz). 13C NMR (CDCl3) d:
167.88, 161.73, 148.59, 140.23, 134.67, 126.98 (2C), 124.57,
120.54, 114.34, 62.56, 43.11, 42.96, 32.13, 29.58, 28.08, 26.66,
25.82, 25.44, 20.33, 13.84. FAB-MS m/z: 357 (MH+). Anal. Calcd
for C21H28N2O3ꢀ1/5H2O: C, 70.05; H, 7.95; N, 7.78. Found: C,
70.25; H, 7.81; N, 7.73.
4.2.8.2.12. N-c-Pentyl-5-butyl-6-oxo-6,7,8,9,10,11-hexahydro-5H-
cyclohepta[c]quinoline-2-carboxamide (48). This compound was
synthesized according to the general procedure A. Column chroma-
tography (n-hexane/AcOEt = 92/8 to 36/64) gave the title
compound (27.6 mg, 0.0725 mmol, 81%) as a colorless powder
(recrystallized from CH2Cl2/n-hexane, mp 164.1–166.2 °C). 1H
NMR (CDCl3) d: 8.33 (1H, d, J = 1.7 Hz), 7.45 (1H, dd, J = 9.2,
1.7 Hz), 7.32 (1H, d, J = 9.2 Hz), 6.04 (1H, d, J = 7.5 Hz), 4.42 (1H,
dt, J = 7.5, 6.9 Hz), 4.29 (2H, t, J = 8.0 Hz), 3.09–3.05 (2H, m),
3.05–3.01 (2H, m), 2.16–2.08 (2H, m), 1.92–1.86 (2H, m), 1.78–
1.63 (8H, m), 1.61–1.55 (2H, m), 1.53–1.43 (4H, m), 0.98 (3H, t,
J = 6.9 Hz). 13C NMR (CDCl3) d: 166.49, 161.72, 148.65, 139.98,
134.57, 127.72, 126.65, 124.59, 120.58, 114.19, 51.88, 43.07,
33.31 (2C), 32.15, 29.57, 28.09, 26.65, 25.85, 25.46, 23.86 (2C),
20.33, 13.84. FAB-MS m/z: 381 (MH+). Anal. Calcd for
4.2.8.3. N-Alkylation of 50. 4.2.8.3.1. N-Methyl-N0-c-propyl-5-
butyl-6-oxo-6,7,8,9,10,11-hexahydro-5H-cyclohepta[c]quinoline-2-
carboxamide (51). To a solution of 50 (32.0 mg, 0.0908 mmol) in
DMF (0.5 mL) was added sodium hydride (8.9 mg, 0.22 mmol, ca.
60%) at ambient temperature. The mixture was stirred at this tem-
perature for 1 h, then methyl iodide (13 lL, 0.21 mmol) was added.
The whole was stirred at ambient temperature for 3 h, then diluted
with ethyl acetate. The organic layer was washed with water and
brine, dried over Na2SO4 and concentrated. Column chromatogra-
phy (n-hexane/AcOEt = 83/17 to 0/100) gave the title compound
(32.0 mg, 0.0873 mmol, 96%) as a colorless oil. 1H NMR (CDCl3) d:
8.05 (1H, d, J = 1.7 Hz), 7.67 (1H, d, J = 8.6 Hz), 7.31 (1H, d,
J = 8.6 Hz), 4.30 (2H, t, J = 8.0 Hz), 3.11 (3H, s), 3.06–3.00 (4H, m),
2.90–2.85 (1H, m), 1.91–1.85 (2H, m), 1.75–1.56 (6H, m), 1.52–
1.44 (2H, m), 0.99 (3H, t, J = 7.5 Hz), 0.63 (2H, br s), 0.49 (2H, br
s). 13C NMR (CD3OD, 60 °C) d: 174.20, 163.44, 150.89, 140.01,
134.88, 131.99, 129.93, 125.65, 121.51, 115.73, 44.09, 35.98,
34.15, 32.82, 30.82, 28.93, 27.41, 26.89, 26.52, 21.12, 14.05, 9.76
(2C). FAB-MS m/z: 367 (MH+). HRMS (FAB, [M+H]+) calcd for
C
23H31N2O2 367.2386, found 367.2407.
4.2.8.3.2. N-Ethyl-N0-c-propyl-5-butyl-6-oxo-6,7,8,9,10,11-hexa-
hydro-5H-cyclohepta[c]quinoline-2-carboxamide (52). To a solu-
tion of 50 (33.6 mg, 0.0953 mmol) in DMF (0.5 mL) was added
sodium hydride (8.5 mg, 0.21 mmol, ca. 60%) at ambient tempera-
ture. The mixture was stirred at this temperature for 1 h, then ethyl
iodide (16 lL, 0.20 mmol) was added. The whole was stirred at
ambient temperature for 3 h, then diluted with ethyl acetate. The
organic layer was washed with water and brine, dried over Na2SO4
and concentrated. Column chromatography (n-hexane/AcOEt = 88/
12 to 4/96) gave the title compound (21.3 mg, 0.0560 mmol, 59%)
as a colorless oil. 1H NMR (CDCl3) d: 8.01 (1H, d, J = 1.8 Hz), 7.64
(1H, d, J = 8.3 Hz), 7.30 (1H, d, J = 8.3 Hz), 4.30 (2H, t, J = 7.5 Hz),
3.58 (2H, q, J = 6.6 Hz), 3.06–2.99 (4H, m), 2.84–2.79 (1H, m),
1.91–1.85 (2H, m), 1.75–1.55 (6H, m), 1.51–1.44 (2H, m), 1.28
(3H, t, J = 6.6 Hz), 0.99 (3H, t, J = 7.5 Hz), 0.65 (2H, br s), 0.50 (2H,
br s). 13C NMR (CD3OD, 60 °C) d: 174.10, 163.45, 150.87, 139.89,
134.89, 132.42, 129.71, 125.32, 121.54, 115.74, 44.09, 44.00,
C
24H32N2O2: C, 75.75; H, 8.48; N, 7.36. Found: C, 75.47; H, 8.28;
N, 7.28.
4.2.8.2.13. N-c-Hexyl-5-butyl-6-oxo-6,7,8,9,10,11-hexahydro-5H-
cyclohepta[c]quinoline-2-carboxamide (49). This compound was
synthesized according to the general procedure A. Column chroma-
tography (n-hexane/AcOEt = 92/8 to 36/64) gave the title compound

F. Karaki et al. / Bioorg. Med. Chem. 22 (2014) 3587–3609
3607
32.83, 32.14, 30.82, 28.93, 27.41, 26.89, 26.53, 21.12, 14.04, 13.57,
a colorless amorphous solid. 1H NMR (DMSO-d6, 100 °C) d: 7.34
(2H,d, J = 8.0 Hz), 7.20 (2H, d, J = 8.0 Hz), 7.12 (1H, br s), 6.44 (1H,
d, J = 2.3 Hz), 6.41 (1H, dd, J = 8.6, 2.3 Hz), 4.84 (2H, br s), 3.71
(3H, s), 3.63 (3H, br s), 3.40 (2H, q, J = 6.9 Hz), 2.77–2.72 (1H, m),
2.51 (2H, br s), 2.17 (2H, br s), 1.59–1.21 (6H, br m), 1.46 (3H, t,
J = 6.9 Hz), 0.55–0.50 (2H, m), 0.41–0.37 (2H, m). FAB-MS m/z:
557, 555 (MH+).
10.01 (2C). FAB-MS m/z: 381 (MH+). HRMS (FAB, [M+H]+) calcd for
C24H33N2O2 381.2542, found 381.2506.
4.2.9. Synthesis of the 5-alkyl derivatives of cycloheptaquin-
olinones
4.2.9.1. N-Cyclopropyl-4-iodobenzamide (94).
To a suspen-
sion of 4-iodobenzoic acid (996.6 mg, 4.018 mmol) and DMF (3
drops) in CH2Cl2 (30 mL) was added oxalyl chloride (550
lL,
4.2.9.5. N-Cyclopropyl-N0-ethyl-5-(2,4-dimethoxybenzyl)-6-oxo-
6,7,8,9,10,11-hexahydro-5H-cyclohepta[c]quinoline-2-carbox-
6.41 mmol) at ambient temperature. The mixture was stirred at
this temperature for 40 min, and then cyclopropylamine
amide (98).
A suspension of 97 (120.0 mg, 0.2160 mmol),
(1390
l
L, 19.96 mmol) was added. Stirring was continued at ambi-
Pd(OAc)2 (5.4 mg, 0.024 mmol), PCy3ꢀHBF4 (9.3 mg, 0.024 mmol)
and Cs2CO3 (77.2 mg, 0.237 mmol) in DMA (1 mL) was stirred
under argon at 130 °C for 4 h, and then diluted with ethyl acetate.
The organic layer was washed with water and brine, dried over
Na2SO4 and concentrated. Column chromatography (n-hexane/
AcOEt = 88/12 to 4/96) of the residue gave the title compound
(72.1 mg, 0.152 mmol, 70%) as a colorless amorphous solid. 1H
NMR (CD3OD, 60 °C) d: 8.05 (1H, d, J = 1.2 Hz), 7.54 (1H, dd,
J = 8.9, 1.2 Hz), 7.32 (1H, d, J = 8.9 Hz), 6.58–6.55 (2H, m), 6.30
(1H, dd, J = 8.6, 2.3 Hz), 5.49 (2H, s), 3.91 (3H, s), 3.70 (3H, s),
3.54 (2H, q, J = 7.5 Hz), 3.16–3.12 (2H, m), 3.09–3.05 (2H, m),
2.89–2.84 (1H, m), 1.95–1.89 (2H, m), 1.74–1.68 (2H, m), 1.65–
1.59 (2H, m), 1.24 (3H, t, J = 7.5 Hz), 0.64–0.58 (2H, m), 0.51–0.45
(2H, m). 13C NMR (CD3OD, 60 °C) d: 173.99, 163.97, 161.91,
159.23, 151.26, 140.12, 134.94, 132.58, 129.54, 128.43, 125.11,
121.54, 117.69, 116.50, 106.20, 99.67, 56.24, 55.90, 43.94, 42.41,
32.84, 32.06, 29.00, 27.57, 26.94, 26.54, 13.57, 9.98 (2C). FAB-MS
m/z: 475 (MH+).
ent temperature for 40 min, then water was added. Extraction with
ethyl acetate provided an organic layer, which was washed with
water and brine, dried over Na2SO4 and concentrated. Recrystalli-
zation of the residue from CH2Cl2/n-hexane gave the title
compound (1058 mg, 3.686 mmol, 92%) as a colorless solid. 1H
NMR (CD3OD) d: 7.81 (2H, d, J = 8.6 Hz), 7.53 (2H, d, J = 8.6 Hz),
2.85–2.79 (1H, m), 0.82–0.75 (2H, m), 0.63–0.60 (2H, m). 13C
NMR (CD3OD) d: 170.94, 138.85 (2C), 135.06, 130.00 (2C), 99.13,
24.02, 6.51 (2C). FAB-MS m/z: 288 (MH+).
4.2.9.2. N-Cyclopropyl-N0-ethyl-4-iodobenzamide (95).
To a
solution of 94 (315.3 mg, 1.098 mmol) and DMF (2 mL) was added
sodium hydride (93.7 mg, 2.34 mmol, ca. 60%) at ambient temper-
ature. The mixture was stirred at this temperature for 1 h, then
ethyl iodide was added. The whole was stirred at 90 °C for 2 h, then
diluted with ethyl acetate. The organic layer was washed with
water and brine, dried over Na2SO4 and concentrated. Column
chromatography (n-hexane/AcOEt = 94/6 to 52/48) of the residue
gave the title compound (299.5 mg, 0.9503 mmol, 87%) as a color-
less oil.1H NMR (CD3OD) d: 7.80 (2H, d, J = 8.0 Hz), 7.25 (2H, d,
J = 8.0 Hz), 3.57 (2H, br s), 2.86 (1H, br s), 1.25 (3H, br s), 0.63
(2H, br s), 0.48 (2H, br s). 13C NMR (CD3OD, 60 °C) d: 174.01,
138.58 (2C), 138.41, 129.89 (2C), 96.21, 43.72, 31.91, 13.42, 9.91
(2C). FAB-MS m/z: 316 (MH+).
4.2.9.6. N-Cyclopropyl-N0-ethyl-6-oxo-6,7,8,9,10,11-hexahydro-
5H-cyclohepta[c]quinoline-2-carboxamide (53).
A solution
of 98 (431.4 mg, 0.9090 mmol) in TFA (3 mL) was stirred at 80 °C
for 90 min, and then diluted with ethyl acetate. The organic layer
was washed with water, saturated aqueous NaHCO3 and brine,
dried over Na2SO4 and concentrated. Column chromatography (n-
hexane/AcOEt = 1/2 then AcOEt) of the residue gave the title com-
pound (259.1 mg, 0.7987 mmol, 88%) as a brown solid. 1H NMR
(DMSO-d6) d: 11.79 (1H, s), 7.91 (1H, s), 7.57 (1H, d, J = 8.3 Hz),
7.27 (1H, d, J = 8.3 Hz), 3.44 (2H, q, J = 6.9 Hz), 3.04–2.99 (2H, m),
2.91 (1H, br s), 2.94–2.85 (2H, m), 1.87–1.81 (2H, m), 1.60–1.54
(2H, m), 1.49–1.43 (2H, m), 1.18 (3H, t, J = 6.9 Hz), 0.58–0.50 (2H,
m), 0.39 (2H, br s). 13C NMR (DMSO-d6) d: 170.58, 161.43,
149.63, 138.04, 133.89, 130.95, 128.33, 123.24, 118.12, 114.63,
41.83, 31.75 (2C), 27.27, 25.61, 25.16, 24.95, 13.30, 9.41 (2C).
FAB-MS m/z: 325 (MH+). HRMS (FAB, [M+H]+) calcd for
4.2.9.3.
amino)benzamide (96).
0.8837 mmol), 2,4-dimethoxybenzylamine (290
copper(I) iodide (19.2 mg, 0.101 mmol), tripotassium phosphate
420.7 mg, 1.936 mmol) and ethylene glycol (220 L, 3.93 mmol)
in n-butanol (1 mL) was stirred under argon at 100 °C for 12 h,
and then diluted with ethyl acetate. The organic layer was washed
with water and brine, dried over Na2SO4 and concentrated. Column
chromatography (n-hexane/AcOEt = 88/12 to 4/96) of the residue
gave the title compound (268.4 mg, 0.7572 mmol, 86%) as a
colorless oil. 1H NMR (CD3OD, 60 °C) d: 7.28 (2H, d, J = 8.8 Hz),
7.15 (1H, d, J = 8.0 Hz), 6.61 (2H, d, J = 8.8 Hz), 6.53 (1H, d,
J = 2.3 Hz), 6.43 (1H, dd, J = 8.0, 2.3 Hz), 4.25 (2H, s), 3.83 (3H, s),
3.76 (3H, s), 3.52 (2H, q, J = 6.9 Hz), 2.87–2.82 (1H, m), 1.21 (3H,
t, J = 6.9 Hz), 0.71–0.66 (2H, m), 0.51–0.46 (2H, m). FAB-MS m/z:
355 (MH+).
N-Cyclopropyl-N0-ethyl-4-((2,4-dimethoxybenzyl)-
suspension of 95 (278.5 mg,
L, 1.93 mmol),
A
l
l
C20H25N2O2 325.1916, found 325.1899.
4.2.9.7. N-Alkylation of a cycloheptaquinolinone 53. 4.2.9.7.1.
General procedure for the synthesis of N-cyclopropyl-N0-ethyl-6-oxo-
5-alkyl-6,7,8,9,10,11-hexahydro-5H-cyclohepta[c]quinoline-2-car-
boxamide (A). To a suspension of 53 (30 mg, 0.1 mmol) and
Cs2CO3 (65 mg, 0.2 mmol) in DMF (0.5 mL) was added alkyl iodide
(0.5 mmol) at ambient temperature. The mixture was stirred at
90 °C for 1 h, then diluted with ethyl acetate. The organic layer
was washed with water and brine, dried over Na2SO4 and concen-
trated. The product was purified as indicated below.
4.2.9.4. 2-Bromo-N-(4-(cyclopropyl(ethyl)carbamoyl)phenyl)-
N0-(2,4-dimethoxybenzyl)cyclohept-1-ene-1-carboxamide
(97).
To a solution of 96 (562.7 mg, 2.568 mmol) and DMF (3
L,
drops) in CH2Cl2 (3 mL) was added oxalyl chloride (220
l
4.2.9.7.2. General procedure for the synthesis of N-cyclopropyl-N0-
ethyl-6-oxo-5-alkyl-6,7,8,9,10,11-hexahydro-5H-cyclohepta[c]quino-
line-2-carboxamide (B). To a suspension of 53 (30 mg, 0.1 mmol),
Cs2CO3 (65 mg, 0.2 mmol) and TBAI (10 mg, 0.03 mmol) in DMF
(0.5 mL) was added alkyl bromide (0.5 mmol) at ambient temper-
ature. The mixture was stirred at 90 °C for 1 h, and then diluted
with ethyl acetate. The organic layer was washed with water and
brine, dried over Na2SO4 and concentrated. The product was puri-
fied as indicated below.
2.57 mmol) at ambient temperature. The mixture was stirred at
this temperature for 10 min, then 74c (605.9 mg, 1.709 mmol) in
CH2Cl2 (4.5 mL) and DMAP (315.5 mg, 2.582 mmol) were added.
The whole was stirred at 60 °C for 2 h, then diluted with ethyl ace-
tate. The organic layer was washed with water, 2 N aqueous HCl,
2 N aqueous NaOH and brine, dried over Na2SO4 and concentrated.
Column chromatography (n-hexane/AcOEt = 88/12 to 4/96) of the
residue gave the title compound (857.9 mg, 1.544 mmol, 90%) as

3608
F. Karaki et al. / Bioorg. Med. Chem. 22 (2014) 3587–3609
4.2.9.7.3. N-Cyclopropyl-N0-ethyl-6-oxo-5-methyl-6,7,8,9,10,11-
hexahydro-5H-cyclohepta[c]quinoline-2-carboxamide (54). This
dd, J = 8.6, 1.7 Hz), 7.55 (1H, d, J = 8.6 Hz), 4.36 (2H, t, J = 8.0 Hz),
3.59 (2H, q, J = 6.9 Hz), 3.14–3.10 (2H, m), 3.05–3.01 (2H, m),
2.96–2.90 (1H, m), 1.94–1.88 (2H, m), 1.77–1.66 (4H, m), 1.62–
1.56 (2H, m), 1.49–1.42 (2H, m), 1.40–1.31 (4H, m), 1.28 (3H, t,
J = 7.5 Hz), 0.90 (3H, t, J = 6.9 Hz), 0.70–0.63 (2H, m), 0.56–0.50
(2H, m). 13C NMR (CD3OD, 60 °C) d: 174.11, 163.46, 150.88,
139.90, 134.90, 132.43, 129.71, 125.33, 121.55, 115.76, 44.31,
43.97, 32.83, 32.60, 32.10, 28.93, 28.62, 27.57, 27.42, 26.90,
26.54, 23.51, 14.16, 13.57, 10.01 (2C). FAB-MS m/z: 409 (MH+).
HRMS (FAB, [M+H]+) calcd for C26H37N2O2 409.2855, found
409.2837.
compound was synthesized according to the general procedure
A. Column chromatography (n-hexane/AcOEt = 83/17 to 0/100)
gave the title compound (26.4 mg, 0.0780 mmol, 88%) as a pale-
yellow oil. 1H NMR (DMSO-d6) d: 8.01 (1H, br s), 7.69 (1H, d,
J = 8.3 Hz), 7.50 (1H, d, J = 8.3 Hz), 3.66 (3H, s), 3.52–3.41 (2H, m),
3.08–3.02 (2H, m), 2.99–2.91 (3H, m), 1.88–1.80 (2H, m), 1.61–
1.55 (2H, m), 1.50–1.44 (2H, m), 1.28–1.14 (3H, m), 0.54 (2H, br
s), 0.41 (2H, br s). 13C NMR (DMSO-d6) d: 175.76, 160.89, 148.37,
138.86, 133.20, 131.13, 128.64, 123.60, 118.80, 114.30, 41.82,
30.97, 30.31, 30.01, 27.19, 25.97, 25.56, 25.13, 13.30, 9.41 (2C).
FAB-MS m/z: 339 (MH+). HRMS (FAB, [M+H]+) calcd for
C21H27N2O2 339.2073, found 339.2076.
4.2.9.7.8.
N-Cyclopropyl-N0-ethyl-6-oxo-5-(2-methoxy)ethyl-
6,7,8,9,10,11-hexahydro-5H-cyclohepta[c]quinoline-2-carboxamide
(59). This compound was synthesized according to the general
procedure B. Column chromatography (n-hexane/AcOEt = 81/19
to 0/100) gave the title compound (21.3 mg, 0.0557 mmol, 65%)
as a pale-yellow oil. 1H NMR (CD3OD, 60 °C) d: 8.05 (1H, s), 7.69
(1H, s), 7.69 (1H, s), 4.57 (2H, t, J = 5.7 Hz), 3.73 (2H, t, J = 5.7 Hz),
3.59 (2H, q, J = 6.9 Hz), 3.32 (3H, s), 3.14–3.10 (2H, m), 3.05–3.01
(2H, m), 2.95–2.90 (1H, m), 1.94–1.88 (2H, m), 1.72–1.66 (2H,
m), 1.62–1.57 (2H, m), 1.28 (3H, t, J = 6.9 Hz), 0.69–0.64 (2H, m),
0.55–0.51 (2H, m). 13C NMR (CD3OD, 60 °C) d: 172.78, 162.36,
149.87, 139.21, 133.47, 131.22, 128.21, 123.80, 120.14, 115.00,
69.73, 57.96, 42.76, 42.66, 31.49, 30.77, 27.64, 26.05, 25.52,
25.15, 12.23, 8.67 (2C). FAB-MS m/z: 383 (MH+). HRMS (FAB,
[M+H]+) calcd for C23H31N2O3 383.2335, found 383.2306.
4.2.9.7.4.
hexahydro-5H-cyclohepta[c]quinoline-2-carboxamide
N-Cyclopropyl-N0-ethyl-6-oxo-5-ethyl-6,7,8,9,10,11-
(55). This
compound was synthesized according to the general procedure
A. Column chromatography (n-hexane/AcOEt = 80/20 to 0/100)
gave the title compound (23.0 mg, 0.0653 mmol, 70%) as a color-
less oil. 1H NMR (DMSO-d6) d: 8.02 (1H, s), 7.48 (1H, d,
J = 8.9 Hz), 7.55 (1H, d, J = 8.9 Hz), 4.31 (2H, q, J = 6.9 Hz), 3.46
(2H, q, J = 6.3 Hz), 3.07–3.01 (2H, m), 2.98–2.90 (3H, m), 1.87–
1.80 (2H, m), 1.62–1.55 (2H, m), 1.51–1.44 (2H, m), 1.25–1.16
(6H, m), 0.56 (2H, br s), 0.42 (2H, br s). 13C NMR (DMSO-d6) d:
174.77, 160.37, 148.38, 137.74, 133.17, 130.94, 128.70, 123.85,
119.03, 113.98, 54.92, 41.67, 37.43, 31.60, 27.24, 25.83, 25.56,
25.06, 13.31, 12.71, 9.24 (2C). FAB-MS m/z: 353 (MH+). HRMS
(FAB, [M+H]+) calcd for C22H29N2O2 353.2229, found 353.2211.
4.2.9.7.5. N-Cyclopropyl-N0-ethyl-6-oxo-5-n-propyl-6,7,8,9,10,11-
4.2.9.7.9. N-Cyclopropyl-N0-ethyl-6-oxo-5-i-propyl-6,7,8,9,10,11-
hexahydro-5H-cyclohepta[c]quinoline-2-carboxamide
(61). This
compound was synthesized according to the general procedure
A. Column chromatography (n-hexane/AcOEt = 94/6 to 52/48) gave
the title compound (16.5 mg, 0.0450 mmol, 63%) as a pale-yellow
oil. 1H NMR (CD3OD, 60 °C) d: 8.11 (1H, d, J = 1.7 Hz), 7.75 (1H, d,
J = 8.0 Hz), 7.62 (1H, dd, J = 8.0, 1.7 Hz), 5.55 (1H, sep, J = 6.3 Hz),
3.59 (2H, q, J = 6.9 Hz), 3.24–3.20 (2H, m), 3.06–3.03 (2H, m),
2.94–2.89 (1H, m), 1.94–1.89 (2H, m), 1.71–1.65 (2H, m),
1.63–1.57 (2H, m), 1.38 (6H, d, J = 6.3 Hz), 1.28 (3H, t, J = 6.9 Hz),
0.67–0.59 (2H, m), 0.56–0.49 (2H, m). 13C NMR (CD3OD, 60 °C) d:
175.14, 161.59, 152.10, 147.39, 133.67, 129.13, 128.32, 127.76,
124.64, 123.73, 69.44, 43.98, 33.10, 32.10, 28.73, 27.24, 27.17,
26.64, 22.43 (2C), 13.58, 9.88 (2C). FAB-MS m/z: 367 (MH+). HRMS
(FAB, [M+H]+) calcd for C23H31N2O2 367.2386, found 367.2402.
4.2.9.7.10. N-Cyclopropyl-N0-ethyl-6-oxo-5-benzyl-6,7,8,9,10,11-
hexahydro-5H-cyclohepta[c]quinoline-2-carboxamide
(56). This
compound was synthesized according to the general procedure
A. Column chromatography (n-hexane/AcOEt = 92/8 to 36/64) gave
the title compound (14.6 mg, 0.0398 mmol, 51%) as a pale-yellow
oil. 1H NMR (CD3OD, 60 °C) d: 8.06 (1H, d, J = 1.7 Hz), 7.71 (1H,
dd, J = 9.2, 1.7 Hz), 7.56 (1H, d, J = 9.2 Hz), 4.33 (2H, t, J = 7.5 Hz),
3.59 (2H, q, J = 7.2 Hz), 3.15–3.10 (2H, m), 3.06–3.01 (2H, m),
2.95–2.90 (1H, m), 1.94–1.88 (2H, m), 1.82–1.73 (2H, m), 1.72–
1.66 (2H, m), 1.62–1.56 (2H, m), 1.28 (3H, t, J = 7.2 Hz), 1.03 (3H,
t, J = 7.5 Hz), 0.70–0.64 (2H, m), 0.56–0.50 (2H, m). 13C NMR
(CD3OD, 60 °C) d: 174.11, 163.51, 150.90, 139.93, 134.90, 132.44,
129.72, 125.31, 121.53, 115.79, 45.77, 43.99, 32.83, 32.12, 28.94,
27.41, 26.89, 26.53, 21.94, 13.56, 11.35, 10.00 (2C). FAB-MS m/z:
367 (MH+). HRMS (FAB, [M+H]+) calcd for C23H31N2O2 367.2386,
found 367.2345.
hexahydro-5H-cyclohepta[c]quinoline-2-carboxamide
(62). This
compound was synthesized according to the general procedure B.
Column chromatography (n-hexane/AcOEt = 88/12 to 4/96) gave
the title compound (27.3 mg, 0.0656 mmol, 84%) as a colorless
amorphous solid. 1H NMR (CD3OD, 60 °C) d: 8.06 (1H, d,
J = 1.7 Hz), 7.56 (1H, dd, J = 8.6, 1.7 Hz), 7.41 (1H, d, J = 8.6 H),
7.28–7.23 (2H, m), 7.21–7.14 (3H, m), 5.63 (2H, s), 3.55 (2H, q,
J = 7.5 Hz), 3.18–3.14 (2H, m), 3.11–3.07 (2H, m), 2.89–2.84 (1H,
m), 1.96–1.91 (2H, m), 1.75–1.69 (2H, m), 1.66–1.60 (2H, m),
1.25 (3H, t, J = 7.5 Hz), 0.64–0.58 (2H, m), 0.51–0.46 (2H, m). 13C
NMR (CD3OD, 60 °C) d: 173.98, 163.86, 151.48, 140.11, 137.89,
134.95, 132.71, 129.77 (2C), 129.57, 128.26, 127.53 (2C), 125.19,
121.62, 116.53, 47.68, 43.94, 32.82, 32.06, 29.03, 27.58, 26.92,
26.51, 13.54, 9.95 (2C). FAB-MS m/z: 415 (MH+). HRMS (FAB,
[M+H]+) calcd for C27H31N2O2 415.2386, found 415.2402.
4.2.9.7.6. N-Cyclopropyl-N0-ethyl-6-oxo-5-n-pentyl-6,7,8,9,10,11-
hexahydro-5H-cyclohepta[c]quinoline-2-carboxamide
(57). This
compound was synthesized according to the general procedure B.
Column chromatography (n-hexane/AcOEt = 92/8 to 36/64) gave
the title compound (17.2 mg, 0.0436 mmol, 48%) as a pale-yellow
oil. 1H NMR (CD3OD, 60 °C) d: 8.06 (1H, d, J = 1.7 Hz), 7.72 (1H,
dd, J = 8.6, 1.7 Hz), 7.55 (1H, d, J = 8.6 Hz), 4.35 (2H, t, J = 8.0 Hz),
3.59 (2H, q, J = 6.9 Hz), 3.14–3.10 (2H, m), 3.05–3.01 (2H, m),
2.95–2.90 (1H, m), 1.94–1.88 (2H, m), 1.78–1.66 (4H, m), 1.62–
1.56 (2H, m), 1.48–1.37 (4H, m), 1.28 (3H, t, J = 7.5 Hz), 0.92 (3H,
t, J = 6.9 Hz), 0.70–0.63 (2H, m), 0.55–0.50 (2H, m). 13C NMR
(CD3OD, 60 °C) d: 174.11, 163.45, 150.88, 139.90, 134.90, 132.43,
129.72, 125.33, 121.55, 115.75, 44.30, 44.01, 32.83, 32.14, 30.12,
28.94, 28.35, 27.42, 26.90, 26.54, 23.38, 14.19, 13.57, 10.01 (2C).
FAB-MS m/z: 395 (MH+). HRMS (FAB, [M+H]+) calcd for
C25H35N2O2 395.2699, found 395.2744.
4.2.9.7.11. N-Cyclopropyl-N0-ethyl-6-oxo-5-(2-(tert-butyldimeth-
ylsilyl)oxy)ethyl-6,7,8,9,10,11-hexahydro-5H-cyclohepta[c]quinoline-
2-carboxamide (99). This compound was synthesized according
to the general procedure B. Column chromatography (n-hexane/
AcOEt = 92/8 to 36/64) gave the title compound (31.1 mg,
0.0664 mmol, 54%) as a pale-yellow oil. 1H NMR (CD3OD, 60 °C)
d: 8.05 (1H, d, J = 1.7 Hz), 7.75 (1H, d, J = 8.6 Hz), 7.67 (1H, d,
J = 8.6, 1.7 Hz), 4.54 (2H, t, J = 5.7 Hz), 4.03 (2H, t, J = 5.7 Hz), 3.58
(2H, q, J = 7.5 Hz), 3.14–3.10 (2H, m), 3.05–3.01 (2H, m),
4.2.9.7.7. N-Cyclopropyl-N0-ethyl-6-oxo-5-n-hexyl-6,7,8,9,10,11-
hexahydro-5H-cyclohepta[c]quinoline-2-carboxamide
(58). This
compound was synthesized according to the general procedure
A. Column chromatography (n-hexane/AcOEt = 92/8 to 36/64) gave
the title compound (19.4 mg, 0.0475 mmol, 59%) as a pale-yellow
oil. 1H NMR (CD3OD, 60 °C) d: 8.06 (1H, d, J = 1.7 Hz), 7.72 (1H,

F. Karaki et al. / Bioorg. Med. Chem. 22 (2014) 3587–3609
3609
6. Yamanashi, Y.; Takada, T.; Suzuki, H. J. Pharmacol. Exp. Ther. 2007, 320, 559.
2.95–2.90 (1H, m), 1.94–1.88 (2H, m), 1.71–1.65 (2H, m), 1.62–1.56
(2H, m), 1.28 (3H, t, J = 7.5 Hz), 0.74 (9H, s), 0.70–0.63 (2H, m),
0.56–0.51 (2H, m), ꢂ0.14 (6H, s). 13C NMR (CD3OD, 60 °C) d:
174.07, 163.71, 151.12, 141.01, 134.80, 132.40, 129.35, 125.05,
121.35, 116.97, 61.91, 48.66, 46.41, 32.82, 32.05, 28.94, 27.36,
26.85, 26.54, 26.27 (3C), 18.94, 13.59, 10.04 (2C), ꢂ5.44 (2C).
FAB-MS m/z: 483 (MH+).
7. Altmann, S. W.; Davis, H. R.; Zhu, L.-J.; Yao, X.; Hoos, L. M.; Tetzloff, G.; Iyer, S. P.
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4.2.9.8.
6,7,8,9,10,11-hexahydro-5H-cyclohepta[c]quinoline-2-carbox-
amide (60). To a solution of 99 (25.7 mg, 0.0532 mmol) in
THF (2 mL) was added TBAF (1 M in THF, 200 L, 0.200 mmol) at
N-Cyclopropyl-N0-ethyl-6-oxo-5-(2-hydroxy)ethyl-
l
ambient temperature. The solution was stirred at 50 °C for 3 h,
and then diluted with ethyl acetate. The organic layer was washed
with water and brine, dried over Na2SO4 and concentrated. Column
chromatography (CHCl3/MeOH = 50/1) of the residue gave the title
compound (21.1 mg, 0.0573 mmol, quant.) as a pale-yellow amor-
phous solid. 1H NMR (CD3OD, 60 °C) d: 8.05 (1H, br s), 7.81 (1H, br
s), 7.70 (1H, br s), 4.51 (2H, t, J = 6.3 Hz), 3.87 (2H, t, J = 6.3 Hz), 3.59
(2H, q, J = 7.5 Hz), 3.14–3.09 (2H, m), 3.05–3.00 (2H, m), 2.95–2.90
(1H, m), 1.94–1.88 (2H, m), 1.72–1.65 (2H, m), 1.62–1.56 (2H, m),
1.28 (3H, t, J = 7.5 Hz), 0.70–0.64 (2H, m), 0.55–0.50 (2H, m). 13C
NMR (CD3OD, 60 °C) d: 174.05, 163.83, 151.14, 140.60, 134.82,
132.50, 129.58, 125.16, 121.48, 116.19, 60.42, 48.66, 46.50, 32.80,
32.07, 28.96, 27.38, 26.83, 26.47, 13.57, 10.01 (2C). FAB-MS m/z:
369 (MH+). HRMS (FAB, [M+H]+) calcd for C22H29N2O3 369.2178,
found 369.2223.
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Acknowledgements
We thank Dr. Tappei Takada, Dr. Yoshihide Yamanashi
(Department of Pharmacy, The University of Tokyo Hospital), and
Dr. Minoru Ishikawa (Institute of Molecular and Cellular Biosci-
ences, The University of Tokyo) for fruitful discussions and helpful
comments. We are grateful to Prof. Mikiko Sodeoka (RIKEN) for mak-
ing available some of her facilities, and to Prof. Makoto Makishima
(Nihon University School of Medicine) for the LXR reporter plasmids.
This work was supported in part by Grants-in-Aid for JSPS Fellows
and for Scientific Research (A) (JSPS KAKENHI Grant Numbers
2210583, 25670052, 256522, and 22249006).
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