4-Oxo-1,4-dihydro-quinoline-3-carboxamides as BACE-1 inhibitors: Synthesis, biological evaluation and docking studies

Article abstract of DOI:10.1016/j.ejmech.2014.04.025

In this work, we report a series of new 4-oxo-1,4-dihydro-quinoline-3- carboxamide derivatives as β-secretase (BACE-1) inhibitors. Supported by docking study, a small library of derivatives were designed, synthesized and biologically evaluated in vitro. The studies revealed that the most potent analog 14e (IC₅₀ = 1.89 μM) with low cellular cytotoxicity and high predicted blood brain barrier permeability, could serve as a good structure for further modification.

Full text of DOI:10.1016/j.ejmech.2014.04.025

European Journal of Medicinal Chemistry 79 (2014) 413e421
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
4-Oxo-1,4-dihydro-quinoline-3-carboxamides as BACE-1 inhibitors:
Synthesis, biological evaluation and docking studies
*
Peng Liu, Yan Niu , Chao Wang, Qi Sun, Yaya Zhai, Jiapei Yu, Jing Sun, Fengrong Xu,
*
Gang Yan, Wenjie Huang, Lei Liang, Ping Xu
Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China
a r t i c l e i n f o
a b s t r a c t
Article history:
In this work, we report a series of new 4-oxo-1,4-dihydro-quinoline-3-carboxamide derivatives as b-
Received 26 November 2013
Received in revised form
7 April 2014
Accepted 7 April 2014
Available online 12 April 2014
secretase (BACE-1) inhibitors. Supported by docking study, a small library of derivatives were designed,
synthesized and biologically evaluated in vitro. The studies revealed that the most potent analog 14e
(IC50 ¼ 1.89
mM) with low cellular cytotoxicity and high predicted blood brain barrier permeability, could
serve as a good structure for further modification.
Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
Alzheimer’s disease
BACE-1 inhibitors
4-Oxo-1,4-dihydro-quinoline-3-
carboxamide
Docking study
1. Introduction
BACE-2 expressed mainly in the periphery [5]. Therefore, BACE-1 is
believed to be the most promising therapeutic target for the
treatment of AD [6]. Over a decade ago, medicinal chemists in in-
dustry and academia have developed many inhibitors of BACE-1,
but only few of these compounds have entered into clinic
research [7e9]. The initial work in the design of BACE-1 inhibitors
was concentrated on peptidomimetic inhibitors [10,11]. Although
some of them were very potent in vitro, the majority of the in-
hibitors were not able to demonstrate significant activity in CNS. In
recent years, more research was focused on developing non-
peptidomimetic BACE-1 inhibitors, which were much smaller in
size and more BBB permeable, and therefore, more potent for anti-
Alzheimer’s disease agent development [12,13].
In this paper, we report the design and synthesis of a variety of
non-peptidic BACE-1 inhibitors bearing the scaffold of 4-oxo-1,4-
dihydro-quinoline-3-carboxamide, which was generated from our
previous in silico virtual screening [14]. Their potencies to inhibit
BACE-1 in vitro were evaluated using Fluorescence Resonance En-
ergy Transfer (FRET) assay.
Alzheimer’s disease (AD) is a progressive neurodegenerative
disease of brain that leads to irreversible loss of neurons and de-
mentia. This disease is characterized in clinic by loss of brain
function, affecting memory, language and learning skills, which
inevitably leads to incapacitation and death. The pathological
hallmarks of AD pathology include the deposition of
peptides (A ) in the neural parenchyma and the formation of
intracellular neurofibrillary tangles [1,2]. The amyloid hypothesis
states that A peptides are derived from the -amyloid precursor
-secretase (BACE) produces a
(sAPP ) and a membrane
-secretase cleaves
C99 to release Ab40 and Ab42 peptides. Ab42 is prone to self-
assemble into fibrils and is the major A component in amyloid
plagues. An increased production of A could induce cell death and
b-amyloid
b
b
b
protein (APP). The cleavage of APP by
b
b
soluble amyloid precursor protein-
b
bound C-terminal fragment called C99. Then,
g
b
b
ultimately lead to dementia [3]. Considering the role of BACE in APP
metabolism, inhibition of BACE is considered a very promising
approach for AD treatment [4].
BACE is a transmembrane aspartyl protease that exists in two
isoforms with BACE-1 located in the central nervous system and
2. Design
Using in silico screening and biological test, we had identified a
series of compounds as weak inhibitors against BACE-1 in our
previous research [14]. Among them, compound 1 was selected as
starting structure in this work for further optimization for the
* Corresponding authors.
E-mail addresses: yanniu@bjmu.edu.cn (Y. Niu), pingxu@bjmu.edu.cn (P. Xu).
http://dx.doi.org/10.1016/j.ejmech.2014.04.025
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

414
P. Liu et al. / European Journal of Medicinal Chemistry 79 (2014) 413e421
following reasons: 1) its simple chemical structure allows for
exploiting a series of novel compounds; 2) docking results (Fig. 1B)
revealed the quinolin-4-one moiety occupied S1 pocket and the NH
group interacted with the catalytic residues of Asp32 through
hydrogen bonds (H-bonds); 3) the 4-oxo-1,4-dihydro-quinoline-3-
carboxamide scaffold was considered as a privileged structure for
drug discovery [15], which is found in many biologically active
natural products and synthetic therapeutic agents [16,17].
Docking result suggested there were several key interactions
between 1 and BACE-1, among which two key H-bonds were
formed between the quinolin-4-one ring and the catalytic Asp32
and Thr72, respectively. The other two H-bonds were formed by the
3-substitution with Asp228 in the S1 pocket and Arg235 in the S20
pocket.
The above observations indicated that increased potency might
be achieved by the introduction of proper substituents, which were
expected to extend into S1/S3 pockets, at C-6 position of the qui-
nolin-4-one core. Considering the S3 pocket could accommodate
large hydrophobic ligands [18,19] and the S1 pocket was also highly
hydrophobic and approximately spherical in shape comprising the
aromatic residues like Phe108 and Trp115 [20], a series of the 6-N-
(3-(trifluoromethyl)phenyl)sulfamoyl or 6-o-tolyl 4-oxo-1,4-
dihydro-quinoline-3-carboxamides were designed and prepared
[21e23], supposing that 6-o-tolyl substitution could orient in the
diethyl ethoxymethylene malonate, and the quinolone product 11
was obtained from 10 with the treatment of PPA and POCl3 in a
yield of 77.27%. Then, Suzuki coupling of 11 with o-tolyl boronic
acid was employed to give 12 [28]. Finally, the carboxylic acid 13,
which was generated from the hydrolysis of ester 12 in presence of
NaOH, was condensated with various amines to give 14aeh.
4. Biological evaluations
The BACE-1 inhibitory evaluation was performed using a Fluo-
rescence Resonance Energy Transfer (FRET) assay kit supplied by
Pan Vera (kit P2985, Madison, WI, USA). The kit protocol was fol-
lowed by minor modification that the total assay volume was
reduced to 15 or 20
mL. Compounds 8aeg and 14aeh were tested at
a concentration of 10
mM and their percentages of BACE-1 inhibi-
tion were given in Table 1. The most active compounds (%
inhibition > 50) were selected to test at more concentrations so
that IC50s were determined by using the linear regression param-
eters. IC50 values were calculated using Graph Pad Prism Software.
The cytotoxicity of the discovered active compounds (Table 2)
was also evaluated using Human Embryonic Kidney 293 cells
(HEK293), which were purchased from the cell bank of the
Shanghai Institute of Cell Biology. The cell viability was determined
by the CellTiter GloÔ luminescent cell viability assay (Promega).
The method to determine the number of viable cells in culture was
based on quantitation of ATP present, which signals the presence of
metabolically active cells. Staurosporine (SigmaeAldrich) was used
as a positive control. The IC50 value was calculated from the dosee
response curves generated by plotting the percentage of the viable
cells versus test concentrations on a logarithmic scale using Sigma
Plot 10.0 software.
To evaluate the capabilities of the acquired compounds to
penetrate BBB (blood brain barrier), the ADME/T module within
Discovery Studio 2.5 software [29] was utilized. The log BB (the
logarithm value of brain to plasma concentration ratio) values for
each compound were calculated based on a quantitative linear
regression model derived from over 800 compounds database that
are known to enter the CNS after oral administration.
S1 pocket properly forming
pep stacking interaction with the ar-
omatic residues, and the 6-sulfamoyl group could extend deeper
into the S3 pocket to improve potency [20,24,25].
3. Chemistry
The structures of designed compounds were shown in Table 1
and they were synthesized following the route shown in Scheme
1 and 2.
As shown in Scheme 1, 4-oxo-6-(N-(3-(trifluoromethyl)phenyl)
sulfamoyl)-1,4-dihydro-quinoline-3-carboxamide analogs 8aeg
were prepared in six steps [26]. Starting from commercially avail-
able 3-(trifluoromethyl)aniline and 4-nitrobenzensulfonyl chloride
2, sulfonamide 3 was yielded, followed by reduction of eNO2 under
H2 atmosphere to afford the aniline 4. Condensation of 4 with
diethyl ethoxymethylene malonate gave the diethyl 2-((phenyl-
amino)methylene)malonate derivative 5, which was converted to
ethyl quinolone-3-carboxylate 6 by treatment with polyphosphoric
acid (PPA) and phosphorus oxychloride (POCl3) [27]. After hydro-
lysis of the ester with NaOH, the resulting carboxylic acid 7 was
then treated with N,N,N0,N0-tetramethyl-o-(1H-benzotriazol-1-yl)
uronium hexafluorophosphate (HBTU), N,N-diisopropylethylamine
(DIPEA) and various amine to give the desired amides 8aeg.
In Scheme 2, 4-bromoaniline was transformed to the diethyl 2-
(phenylamino)methylene malonate derivative 10 by heating with
According to the log BB values, the compounds can be classified
into four levels, which include very high penetrants (log BB ꢀ 0.7),
high penetrants (0
(ꢂ0.52 < log BB < 0) and low penetrants (log BB ꢁ ꢂ0.52).
ꢁ
log BB < 0.7), medium penetrants
5. Results and discussion
All synthesized 6-substituted 4-oxo-1,4-dihydro-quinoline-3-
carboxamide derivatives were tested for BACE-1 inhibitory po-
tencies with data given in Table 1.
Fig. 1. Predicted binding pose of compound 1 in the active site of BACE-1 (PDB ID: 1TQF).

P. Liu et al. / European Journal of Medicinal Chemistry 79 (2014) 413e421
415
Table 1
BACE-1 inhibitory activities of 6-substituted 4-oxo-1,4-dihydro-quinoline-3-carboxamide derivatives.
NO.
R
R1
MW
BACE-1
BACE-1
Inhibition (%)a,b
IC50 (m
M)a
1
8a
8b
8c
8d
8e
8f
8g
14a
14b
14c
14d
14e
14f
14g
14h
H
Benzo[d][1,3]dioxol- 5-ylmethyl
4-Fluorobenzyl
3-Fluorobenzyl
4-Methoxybenzyl
3-Methoxybenzyl
Furan-2-ylmethyl
Pyridine-3ylmethyl
Cyclohexyl
322
519
519
531
531
491
502
493
386
386
436
436
446
358
369
360
11.3 ꢄ 0.2
30.4 ꢄ 0.1
84.6 ꢄ 3.2
50.4 ꢄ 1.9
73.5 ꢄ 0.7
71.1 ꢄ 3.9
35.7 ꢄ 1.6
65.6 ꢄ 1.8
ꢂ13.9 ꢄ 0.6
12.3 ꢄ 0.6
38.1 ꢄ 1.8
67.4 ꢄ 2.9
77.6 ꢄ 4.9
32.9 ꢄ 0.1
10.2 ꢄ 0.5
77.8 ꢄ 0.7
n.dc
n.d
1.85 ꢄ 0.13
7.04 ꢄ 0.48
2.20 ꢄ 0.14
1.61 ꢄ 0.09
n.d
N-(3-(trifluoromethyl)-phenyl)sulfamoyl
N-(3-(trifluoromethyl)-phenyl)sulfamoyl
N-(3-(trifluoromethyl)-phenyl)sulfamoyl
N-(3-(trifluoromethyl)-phenyl)sulfamoyl
N-(3-(trifluoromethyl)-phenyl)sulfamoyl
N-(3-(trifluoromethyl)-phenyl)sulfamoyl
N-(3-(trifluoromethyl)-phenyl)sulfamoyl
o-tolyl
o-tolyl
o-tolyl
o-tolyl
o-tolyl
o-tolyl
o-tolyl
o-tolyl
7.18 ꢄ 0.36
4-Fluorobenzyl
3-Fluorobenzyl
n.dc
n.d
n.d
6.18 ꢄ 0.23
1.89 ꢄ 0.09
n.d
4-(Trifluoromethyl)-benzyl
3-(Trifluoromethyl)-benzyl
3-Bromobenzyl
Furan-2-ylmethyl
Pyridine-3ylmethyl
Cyclohexyl
n.d
11.53 ꢄ 0.57
a
values are mean ꢄ S.D. of two independent experiments for BACE-1 inhibition.
b
c
% inhibition of BACE-1 activity at concentration of 10 mM of the tested compound.
n.d. ¼ not determined.
Scheme 1. Synthesis of 4-oxo-6-(N-(3-(trifluoromethyl)phenyl)sulfamoyl)-1,4-dihydro-quinoline-3-carboxamide derivatives. (Reagents and conditions: (i) pyridine, 0 ꢃC, 3e6 h; (ii)
H2, Pd/C, 6 h, rt; (iii) diethyl ethoxymethylene malonate, 130 ꢃC, 2 h; (iv) PPA, POCl3, 70 ꢃC, 12 h; (v) NaOH, 100 ꢃC, 2 h; (vi) HBTU, DIPEA, CH2Cl2, ReNH2, rt, 10 h.)

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P. Liu et al. / European Journal of Medicinal Chemistry 79 (2014) 413e421
Scheme 2. Synthesis of 6-o-tolyl-4-oxo-1,4-dihydro-quinoline-3-carboxamide derivatives. (Reagents and conditions: (i) diethyl ethoxymethylene malonate, 130 ꢃC, 2 h; (ii) PPA,
POCl3, 70 ꢃC, 12 h; (iii) o-tolyl boronic acid, Pd(PPh3)4, K2CO3, H2O, THF, 80 ꢃC, 10 h; (iv) NaOH, 100 ꢃC, 2 h; (v) HBTU, DIPEA, CH2Cl2, ReNH2, rt, 10 h.)
Compared with the starting compound 1, all acquired com-
pounds with sulfamoyl substitution at the C-6 position (8ae8g)
showed significantly improved potencies. As for the various R1,
phenyl ring with a fluorine atom substituted at meta- position (8b)
exhibited better potency than para- position (8a) did. The meta-
substituted phenyl ring with a fluorine atom (8b) or methoxy
group (8d) seemed to contribute concurrently to potency. Different
from the other analogs, 8g with non-aromatic cyclohexyl substi-
tution at R1 also demonstrated good enzymatic potency
with Thr72. Additionally, the two NH groups within the quinolin-4-
one ring and 3-carboxamide moiety respectively formed another
two critical H-bonds with the catalytic Asp32 and Asp228. Just as
expected, the phenyl ring substituted at the 6-position oriented
properly in the S1 pocket and established favorable CeH/
p
con-
tacts with Ile110. Meanwhile, meta-trifluoromethyl group on the
phenyl ring was observed to extend to the S3 pocket and assisted to
reinforce the ligand’s binding by forming multiple Van der Waal’s
interactions which can well explain the great improvement in po-
tency compared to the 6- non-substituted compound 1.
(IC50 ¼ 7.18
mM). To explore the binding modes of the 6-sulfamoyl-
1,4-dihydro-quinoline-3-carboxamides, one of the most active in-
hibitors 8d was selected as representation and docked to the crystal
structure of BACE-1 (PDB: 1TQF). Seven H-bonds were observed in
the binding mode as shown in Fig. 2A, among which two were
formed between the carbonyl oxygen on the quinolin-4-one moiety
and Gln73 in the S10 pocket, and another two were formed by the
carbonyl group substituted at 3-position of quinolin-4-one ring
As for the R1 substitutions on 4-oxo-6-(o-tolyl)-1,4-dihydro-
quinoline-3-carboxamide, the effect of p- or m- substitution on the
phenyl ring appeared to follow the same rule for the 6-sulfamoyl
analogs that m- substituted 14a and 14c were more favorable
than the p- substituted 14b and 14d, although less potent than the
corresponding 6-sulfamoyl substituted analogs (8ae8d). Mean-
while, the more bulky 3-trifluoromethyl substituted 14d
(IC50 ¼ 6.18
m
M) and bromine substituted 14e (IC50 ¼ 1.89
mM)
displayed much better activities than the 3-fluorine substituted
14b, suggesting there was large room in the corresponding pocket
and bulky substitutions at m- position might be more favorable.
Docking result of 14e (Fig. 2B) suggested the 6-tolyl substitution
Table 2
Cytotoxic profiles and predicted BBB permeabilities of selected compounds.
NO.
Molecular weight
IC50
(
m
M)a,b
Predicted log BB
8b
8c
8d
8e
8g
14d
14e
14h
519.09
531.11
531.11
491.08
493.13
436.14
446.06
360.18
e
>50
>50
>50
>50
>50
>50
>50
>50
Undefinedc
Undefined
Undefined
Undefined
Undefined
0.411
could position well in the S1 pocket and form edge-face
pep
stacking interactions with the adjacent Phe108 and Trp115. How-
ever, compared with 8d, fewer H-bonds were formed and the lower
binding score suggested a lower docking affinity, which was
consistent with the higher IC50 value in Table 1.
The cytotoxicities of the eight active compounds were listed in
Table 2 and all the IC50 values were higher than 50 mM, suggesting
0.351
0.205
Staurosporine
0.056
n.dd
low distinct cytotoxic effects, and these BACE-1 inhibitors should
be safe for normal cells.
The predicted log BB values for the eight active compounds in
Table 2 suggested that some of these compounds were high
a
IC50 values are the means of at least two experiments.
Cytotoxicity of tested compound at concentration of 50
Undefined ¼ no predicted result.
b
c
m
M.
d
n.d. ¼ not determined.

P. Liu et al. / European Journal of Medicinal Chemistry 79 (2014) 413e421
417
Fig. 2. Predicted binding pose of 8d (A) and 14e (B) in the active site of BACE-1 (PDB ID: 1TQF).
penetrants of BBB (log BB > 0.2), among which 14d and 14e, were
7.1.2. 4-Amino-N-(3-(trifluoromethyl)phenyl)benzenesulfonamide
(4)
not only high permeable, but also low in molecular weight
(MW < 450). As for compounds 8aeg, since these novel structures
were outside the 95% and 99% confidence area of database, no
reliable prediction could presently be made by the software.
To a stirred solution of 3 (1.06 g, 3 mmol) in ethanol (20 mL) was
added a catalytic amount of Pd/C (100 mg). The mixture was stirred
under a hydrogen atmosphere (60 psi) for 6 h at room temperature
before the catalyst was removed by filtration, and the filtrate was
concentrated under reduced pressure. The residue was purified by
column chromatography on silica gel. (CH2Cl2/CH3OH ¼ 200/1), and
4 was afforded as a white solid (405 mg, 41.83% yield). m.p. 105e
6. Conclusions
Aided by docking study, a series of 6-substituted 4-oxo-1,4-
dihydro-quinoline-3-carboxamides were designed, synthesized
and evaluated as BACE-1 inhibitors in an enzymatic assay. Eight
106 ꢃC; 1H NMR (400 MHz, CDCl3):
d
7.58 (d, 2H, J ¼ 8.8 Hz), 7.30e
7.36 (m, 3H), 7.25e7.29 (m, 1H), 7.20 (s, 1H), 6.60 (d, 2H, J ¼ 8.8 Hz),
4.15 (s, br, 2H); ESI-MS (m/z): 315 (MꢂH)ꢂ
compounds were found to have IC50s at
optimized drug-like profiles with the relatively low IC50 of 1.89
m
M level, and 14e exhibited
M,
m
7.1.3. Diethyl 2-(((4-(N-(3-(trifluoromethyl)phenyl)sulfamoyl)
phenyl)amino)methylene)-malonate (5)
low molecular weight of 446, high BBB penetrability and high
safety to HEK293 cells, making it a good structure for further
modification.
A mixture of diethyl ethoxymethylene malonate (130 mg,
0.63 mmol) and 4 (200 mg, 0.63 mmol) was heated to 120 ꢃC for 2 h.
When cooled to room temperature, the mixture was diluted with
ethyl acetate (30 mL) and washed with water (20 mL ꢅ 3). The
organic layers were combined, dried with Na2SO4 and concentrated
under reduced pressure. The residue was purified using chroma-
tography on silica gel (CH2Cl2/MeOH ¼ 100:1) to afford 5 (281 mg,
91.53% yield) as a white solid. m.p. 143e144 ꢃC; 1H NMR (400 MHz,
7. Experimental section
7.1. General chemical methods
Melting points were measured with an X4 apparatus and were
uncorrected. 1H NMR spectra were recorded on a Bruker (Bruker
BioSpin AG, Fällanden, Switzerland) Avance III 400 MHz system.
Chemical shifts were reported in parts per million (ppm) relative to
tetramethylsilane (TMS) as internal standard. The spin multiplic-
ities were given as s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet) or br (broad). MS and high-resolution mass spectra
(HRMS) were obtained using Electrospray Ionization (ESI) tech-
nique on a Bruker’s Fourier Transform Ion Cyclotron resonance
Mass Spectrometer. Thin layer chromatography (TLC) analysis was
performed on silica gel GF254 purchased from Qingdao Haiyang
Chemical Co. (Qingdao, Shandong Province, China) or Merck
(Darmstadt, Germany).
CDCl3):
d
11.08 (d, 1H, J ¼ 13.2 Hz), 8.46 (d, 1H, J ¼ 13.2 Hz), 7.80 (d,
2H, J ¼ 8.8 Hz), 7.38 (m, 4H), 7.32 (m, 1H), 7.15 (d, 2H, J ¼ 8.8 Hz),
4.23e4.34 (m, 4H), 1.34 (m, 6H); ESI-MS (m/z): 487 (M þ H)þ
7.1.4. Ethyl 4-oxo-6-(N-(3-(trifluoromethyl)phenyl)sulfamoyl)-1,4-
dihydro-quinoline-3-carboxylate (6)
To a three-necked flask, 5 (300 mg, 0.61 mmol), PPA (1.5 g) and
POCl3 (2.5 g) were added at room temperature. The mixture was
heated to 70 ꢃC for 12 h and then cooled to room temperature. The
mixture was treated with aqueous Na2CO3 solution to remove the
remanent acid and filtered, and the solid was washed with water
and dried to afford the product, which could be used in next step.
m.p. 211e212 ꢃC; 1H NMR (400 MHz, DMSO-d6):
d 12.80 (s, 1H, br),
10.92 (s, 1H), 8.60 (s, 1H), 8.56 (d, 1H, J ¼ 2.0 Hz), 8.02 (dd, 1H,
J1 ¼ 8.7 Hz, J2 ¼ 2.0 Hz), 7.80 (d, 1H, J ¼ 8.7 Hz), 7.50 (t, 1H,
J ¼ 7.7 Hz), 7.36e7.45 (m, 3H), 4.24 (q, 2H, J ¼ 7.2 Hz), 1.29 (t, 3H,
J ¼ 7.2 Hz); ESI-MS (m/z): 441 (M þ H)þ.
7.1.1. 4-Nitro-N-(3-(trifluoromethyl)phenyl)benzenesulfonamide
(3)
To a solution of 3-(trifluoromethyl)aniline (0.8 g, 5 mmol) in
pyridine (0.8 g, 10 mmol), 4-nitrobenzenesulfonyl chloride (1.1 g,
5 mmol) dissolved in CH2Cl2 (20 mL) was added dropwise in an ice
bath. After stirring for 1 h, the ice bath was removed and the
mixture was stirred overnight before concentration in vacuum. The
residue was extracted with ethyl acetate, washed with 0.5 N HCl,
brine in sequence, and dried over anhydrous Na2SO4 to give the
crude product, which was purified by column chromatography on
silica gel (CH2Cl2/MeOH ¼ 500/1) to give 1.56 g of 3 in a yield of
90.17% as a white solid. m.p. 135e136 ꢃC; 1H NMR (400 MHz,
7.1.5. 4-Oxo-6-(N-(3-(trifluoromethyl)phenyl)sulfamoyl)-1,4-
dihydro-quinoline-3-carboxylic acid (7)
A mixture of 6 and NaOH (25 mg, 0.61 mmol) in H2O (20 mL)
was stirred under reflux for 2 h. The mixture was filtered and then
cooled to room temperature. The solution was acidified with 2 N
HCl until pH ¼ 3 to afford solid, which was filtered, washed with
water and dried to get product 7 (60 mg, two steps yield: 24%). m.p.
137e138 ꢃC; 1H NMR (400 MHz, DMSO-d6):
d 14.47 (br, 1H), 8.95 (s,
1H), 8.67 (d,1H, J ¼ 2.1 Hz), 8.14 (dd,1H, J1 ¼8.8 Hz, J2 ¼ 2.1 Hz), 7.96
(d, 1H, J ¼ 8.8 Hz), 7.50 (t, 1H, J ¼ 8.0 Hz), 7.39e7.42 (m, 3H); HRMS
(ESI) for C17H11F3N2O5S: calcd. 413.04135 (M þ H)þ, found:
413.04116 (M þ H)þ.
CDCl3):
d
8.32 (d, 2H, J ¼ 8.8 Hz), 7.98 (d, 2H, J ¼ 8.8 Hz), 7.40e7.48
(m, 2H), 7.36 (s, 1H), 7.30 (d, 1H, J ¼ 6.8 Hz), 6.94 (s, 1H); ESI-MS (m/
z): 345 (MꢂH)ꢂ

418
P. Liu et al. / European Journal of Medicinal Chemistry 79 (2014) 413e421
7.1.6. N-(4-fluorobenzyl)-4-oxo-6-(N-(3-(trifluoromethyl)phenyl)
sulfamoyl)-1,4-dihydro-quinoline-3-carboxamide (8a)
7.1.10. N-(furan-2-ylmethyl)-4-oxo-6-(N-(3-(trifluoromethyl)
phenyl)sulfamoyl)-1,4-dihydro-quinoline-3-carboxamide (8e)
The procedure described for 8a was used, starting from 7
(100 mg, 0.24 mmol), HBTU (92 mg, 0.24 mmol), DIPEA (63 mg,
0.48 mmol) and furfurylamine (23 mg, 0.24 mmol), 8e was ob-
tained as a white solid (28 mg, 23.76% yield). m.p. 209e210 ꢃC; 1H
A mixture of 7 (100 mg, 0.24 mmol), HBTU (92 mg, 0.24 mmol)
and DIPEA (63 mg, 0.48 mmol) was dissolved in CH2Cl2 (30 mL)
under N2 atmosphere. The solution was stirred at room tempera-
ture for 15 min before 4-fluorobenzylamine (30 mg, 0.24 mmol)
was added. Then the mixture was stirred for additional 12 h. The
solution was concentrated under reduced pressure and the residue
was extracted with ethyl acetate (30 mL ꢅ 3) and washed with
water (20 mL ꢅ 3). The combined organic layers were concentrated
and purified by chromatography on silica gel (CH2Cl2/
MeOH ¼ 100:1) to afford 8a as a white solid (35 mg, 28.2% yield).
NMR (400 MHz, DMSO-d6): d 13.01 (s, 1H),10.92 (s, 1H), 10.06 (t,1H,
J ¼ 5.6 Hz), 8.81 (s, 1H), 8.64 (d, 1H, J ¼ 2.0 Hz), 8.05 (dd, 1H,
J1 ¼ 8.8 Hz, J2 ¼ 2.4 Hz), 7.86 (d, 1H, J ¼ 8.8 Hz), 7.61 (s, 1H), 7.49 (t,
1H, J ¼ 8.0 Hz), 7.34e7.44 (m, 3H), 6.42 (m, 1H), 6.31 (t, 1H,
J ¼ 2.8 Hz), 4.56 (d, 2H, J ¼ 5.6 Hz); 13C NMR (100 MHz, DMSO-d6):
d
176.01, 164.09, 152.55, 145.63, 142.79, 142.03, 138.82, 135.53,
m.p. 243e244 ꢃC; 1H NMR (400 MHz, DMSO-d6):
d
13.05 (s, 1H),
131.19, 130.11, 125.94, 125.74, 123.80, 121.46, 121.14, 116.33, 112.39,
10.93 (s, 1H), 10.14 (t, 1H, J ¼ 6.0 Hz), 8.82 (s, 1H), 8.63 (d, 1H,
J ¼ 2.0 Hz), 8.05 (dd, 1H, J1 ¼ 8.8 Hz, J2 ¼ 2.0 Hz), 7.86 (d, 1H,
J ¼ 8.8 Hz), 7.46e7.52 (m, 1H), 7.35e7.42 (m, 5H), 7.16 (t, 2H,
J ¼ 8.8 Hz), 4.54 (d, 2H, J ¼ 5.6 Hz); 13C NMR (100 MHz, DMSO-d6):
110.95, 107.38, 35.86; HRMS (ESI) for C22H16F3N3O5S: calcd.
492.08355 (M þ H)þ, found: 492.08316 (M þ H)þ.
7.1.11. 4-Oxo-N-(pyridin-3-ylmethyl)-6-(N-(3-(trifluoromethyl)
phenyl)sulfamoyl)-1,4-dihydro-quinoline-3-carboxamide (8f)
The procedure described for 8a was used, starting from 7
(100 mg, 0.24 mmol), HBTU (92 mg, 0.24 mmol), DIPEA (63 mg,
0.48 mmol) and 3-(aminomethyl)pyridine (26 mg, 0.24 mmol), 8f
was obtained as a white solid (20 mg, 16.6% yield). m.p. 248e
d
176.02, 164.29, 162.86, 160.47, 145.58, 142.03, 138.84, 136.01,
135.49, 131.19, 130.10, 129.80, 125.98, 125.73, 123.80, 121.43, 121.13,
116.30, 115.68, 115.47, 112.55, 41.89; HRMS (ESI) for C24H17F4N3O4S:
calcd. 520.09487 (M þ H)þ, found: 520.09421 (M þ H)þ.
249 ꢃC; 1H NMR (400 MHz, DMSO-d6):
d 13.05 (s, 1H), 10.95 (s, 1H),
7.1.7. N-(3-fluorobenzyl)-4-oxo-6-(N-(3-(trifluoromethyl)phenyl)
sulfamoyl)-1,4-dihydro-quinoline-3-carboxamide (8b)
10.19 (t, 1H, J ¼ 4.0 Hz), 8.83 (s, 1H), 8.65 (d, 1H, J ¼ 2.0 Hz), 8.58 (s,
1H), 8.47 (d, 1H, J ¼ 3.6 Hz), 8.07 (dd, 1H, J1 ¼ 8.8 Hz, J2 ¼ 2.0 Hz),
7.87 (d, 1H, J ¼ 8.8 Hz), 7.75 (d, 1H, J ¼ 7.6 Hz), 7.50 (t, 1H, J ¼ 7.6 Hz),
7.34e7.44 (m, 4H), 4.60 (d, 2H, J ¼ 4.0 Hz); 13C NMR (100 MHz,
The procedure described for 8a was used, starting from 7
(100 mg, 0.24 mmol), HBTU (92 mg, 0.24 mmol), DIPEA (63 mg,
0.48 mmol) and 3-fluorobenzylamine (30 mg, 0.24 mmol), and 8b
was obtained as a white solid (32 mg, 25.8% yield). m.p. 222e223 ꢃC;
DMSO-d6):
d 175.99, 164.49, 149.36, 148.57, 145.63, 142.03, 138.82,
135.66, 135.50, 131.20, 130.12, 125.97, 125.71, 123.99, 123.78, 121.45,
121.13, 116.26, 112.46, 40.29; HRMS (ESI) for C23H17F3N4O4S: calcd.
503.09954 (M þ H)þ, found: 503.09929 (M þ H)þ.
1H NMR (400 MHz, DMSO-d6):
d 13.04 (s, 1H), 10.91 (s, 1H), 10.18 (s,
1H), 8.83 (s, 1H), 8.54 (s, 1H), 8.07 (d, 1H, J ¼ 8.8 Hz), 7.87 (d, 1H,
J ¼ 8.8 Hz), 7.46e7.54 (m, 1H), 7.33e7.44 (m, 4H), 6.97e7.18 (m, 3H),
4.59 (d, 2H, J ¼ 5.2 Hz); 13C NMR (100 MHz, DMSO-d6):
d
176.05,
7.1.12. N-cyclohexyl-4-oxo-6-(N-(3-(trifluoromethyl)phenyl)
sulfamoyl)-1,4-dihydro-quinoline-3-carboxamide (8g)
The procedure described for 8a was used, starting from 7
(100 mg, 0.24 mmol), HBTU (92 mg, 0.24 mmol), DIPEA (63 mg,
0.48 mmol) and cyclohexylamine (24 mg, 0.24 mmol), 8g was ob-
tained as a white solid (42 mg, 35.6% yield). m.p. 245e246 ꢃC; 1H
164.43, 163.93, 161.51, 145.62, 143.01, 142.04, 138.84, 135.50, 131.18,
130.79, 130.12, 126.00, 125.76, 123.78, 121.42, 121.15, 116.32, 114.44,
114.05, 112.51, 110.00, 42.14; HRMS (ESI) for C24H17F4N3O4S: calcd.
520.09487 (M þ H)þ, found: 520.09428 (M þ H)þ
7.1.8. N-(4-methoxybenzyl)-4-oxo-6-(N-(3-(trifluoromethyl)
phenyl)sulfamoyl)-1,4-dihydro-quinoline-3-carboxamide (8c)
According to procedure described for 8a, starting from 7 (100 mg,
0.24 mmol), HBTU (92 mg, 0.24 mmol), DIPEA (63 mg, 0.48 mmol)
and 4-methoxybenzylamine (33 mg, 0.24 mmol), 8c was obtained as
a white solid (26 mg, 20.4% yield). m.p. 250e251 ꢃC; 1H NMR
NMR (400 MHz, DMSO-d6): d 12.97 (s, 1H),10.90 (s, 1H), 9.80 (s, 1H),
8.78 (s, 1H), 8.67 (s, 1H), 8.05 (s, 1H), 7.87 (s, 1H), 7.30e7.60 (m, 4H),
3.85 (s, 1H), 1.11e1.86 (m, 11H); 13C NMR (100 MHz, DMSO-d6):
d
181.12, 168.07, 150.34, 146.97, 143.84, 140.42, 136.17, 135.23,
134.99, 130.93, 130.78, 128.73, 127.79, 126.37, 126.09, 121.31, 117.79,
52.26, 37.87, 30.67, 29.54; HRMS (ESI) for C23H22F3N3O4S: calcd.
494.13559 (M þ H)þ, found: 494.13505 (M þ H)þ.
(400 MHz, DMSO-d6): d 12.99 (s, 1H), 10.91 (s, 1H), 10.07 (s, 1H), 8.83
(s,1H), 8.65 (s,1H), 8.06 (d,1H, J ¼ 8.0 Hz), 7.86 (d,1H, J ¼ 8.4 Hz), 7.48
(d, 1H, J ¼ 7.2 Hz), 7.35e7.45 (m, 3H), 7.28 (d, 2H, J ¼ 7.6 Hz), 6.91 (d,
2H, J ¼ 7.6Hz), 4.50(d, 2H, J ¼ 4.4 Hz), 3.64 (s, 3H); 13C NMR (100 MHz,
7.1.13. Diethyl 2-(((4-bromophenyl)amino)methylene)malonate
(10)
DMSO-d6):
d
181.02, 169.09, 163.77, 150.52, 147.01, 143.83, 140.45,
Following the same synthetic procedure as described to obtain
5, starting from the 4-bromoaniline 9 (6 g, 34.88 mmol) and diethyl
ethoxymethylene malonate (7.53 g, 34.88 mmol), 10 was obtained
as a white solid (5.1 g, 42.9% yield). m.p. 94e95 ꢃC; 1H NMR
136.71, 136.18, 135.52, 135.18, 134.17, 130.96, 128.77, 127.75, 126.12,
121.28, 119.31, 117.63, 60.48, 47.08; HRMS (ESI) for C25H20F3N3O5S:
calcd. 532.11485 (M þ H)þ, found: 532.11419 (M þ H)þ.
7.1.9. N-(3-methoxybenzyl)-4-oxo-6-(N-(3-(trifluoromethyl)phenyl)
sulfamoyl)-1,4-dihydro-quinoline-3-carboxamide (8d)
(400 MHz, CDCl3):
d
11.00 (d, 1H, J ¼ 13.2 Hz), 8.46 (d, 1H,
J ¼ 13.6 Hz), 7.48 (d, 2H, J ¼ 8.8 Hz), 7.02 (d, 2H, J ¼ 8.8 Hz), 4.17e
4.38 (m, 4H), 1.25e1.45 (m, 6H); ESI-MS (m/z): 342 (M þ H)þ.
7.1.14. Ethyl 6-bromo-4-oxo-1,4-dihydro-quinoline-3-carboxylate
(11)
The procedure described for 8a was used, starting from 7
(100 mg, 0.24 mmol), HBTU (92 mg, 0.24 mmol), DIPEA (63 mg,
0.48 mmol) and 3-methoxybenzylamine (33 mg, 0.24 mmol), 8d
was obtained as a white solid (27 mg, 21.2% yield). m.p. 181e182 ꢃC;
The procedure described for 6 was used, starting from 10 (5 g,
14.9 mmol), PPA (10 g) and POCl3 (6.8 g), 11 was obtained as a white
solid (3.4 g, 77.44% yield). m.p. 247e248 ꢃC; 1H NMR (400 MHz,
1H NMR (400 MHz, DMSO-d6):
d 10.13 (s, 1H), 8.82 (s, 1H), 8.64 (d,
1H, J ¼ 2.0 Hz), 8.05 (dd, 1H, J1 ¼ 8.8 Hz, J2 ¼ 2.0 Hz), 7.85 (d, 1H,
J ¼ 8.8 Hz), 7.48 (d, 1H, J ¼ 7.2 Hz), 7.36e7.42 (m, 3H), 7.26 (t, 1H,
J ¼ 8.0 Hz), 6.91 (m, 2H), 6.84 (d, 1H, J ¼ 8.0 Hz), 4.55 (d, 2H,
DMSO-d6):
d
8.60 (s, 1H), 8.17 (d, 1H, J ¼ 8.0 Hz), 7.61e7.71 (m, 2H),
7.39 (t, 1H, J ¼ 7.2 Hz), 4.21 (q, 2H, J ¼ 7.2 Hz), 1.28 (t, 3H, J ¼ 7.2 Hz);
J ¼ 5.6 Hz), 3.74 (s, 3H); 13C NMR (100 MHz, DMSO-d6):
d 176.05,
ESI-MS (m/z): 296 (M þ H)þ.
164.24, 159.84, 145.58, 142.02, 141.41, 138.82, 135.44, 131.20, 130.09,
129.98, 125.97, 125.76, 123.78, 121.43, 121.10, 119.89, 116.26, 113.15,
7.1.15. Ethyl 4-oxo-6-(o-tolyl)-1,4-dihydro-quinoline-3-carboxylate
(12)
112.62, 55.44, 42.57; HRMS (ESI) for
C
25H20F3N3O5S: calcd.
A mixture of o-tolyl boronic acid (760 mg, 5.58 mmol), 11 (1.5 g,
5.08 mmol), K2CO3 (1.05 g, 7.66 mmol), Pd(PPh3)4 (295 mg) and
532.11485 (M þ H)þ, found: 532.11411 (M þ H)þ.

P. Liu et al. / European Journal of Medicinal Chemistry 79 (2014) 413e421
419
H2O (3.8 mL) in THF (30 mL) was heated to reflux under N2 at-
mosphere for 12 h. After the mixture was cooled to room temper-
ature, filtered, the crude solid product 12 was purified by
recrystallization with ethyl acetate. (1.2 g, 76.92% yield). m.p. 208e
0.35 mmol), 14d was obtained as a white solid (110 mg, 72.36%
yield); m.p. 216e217 ꢃC; 1H NMR (400 MHz, DMSO-d6):
12.82 (s,
d
1H), 10.50 (s, 1H), 8.81 (s, 1H), 8.15 (s, 1H), 7.78 (s, 2H), 7.51e7.74 (m,
4H), 7.24e7.37 (m, 4H), 4.66 (d, 2H, J ¼ 5.6 Hz), 2.25 (s, 3H); 13C
209 ꢃC; 1H NMR (400 MHz, DMSO-d6):
d
12.59 (s, 1H), 8.60 (s, 1H),
NMR (100 MHz, DMSO-d6): d 176.55, 165.22, 144.26, 141.82, 140.66,
8.06 (s, 1H), 7.71 (s, 2H), 7.22e7.37 (m, 4H), 4.23 (q, 2H, J ¼ 7.2 Hz),
2.26 (s, 3H), 1.29 (t, 3H, J ¼ 7.2 Hz); HRMS (ESI) for C19H17NO3: calcd.
308.12812 (M þ H)þ, found: 308.12776 (M þ H)þ.
138.54, 138.37, 135.29, 134.26, 132.01, 130.99, 130.02, 128.22, 126.41,
125.55, 124.33, 123.99, 119.53, 111.22, 42.16, 20.58; HRMS (ESI) for
C
25H19F3N2O2: calcd. 437.14714 (M þ H)þ, found: 437.14763
(M þ H)þ
7.1.16. 4-oxo-6-(o-tolyl)-1,4-dihydro-quinoline-3-carboxylic acid
(13)
.
The procedure described for 7 was used, starting from 12 (4.5 g,
14.65 mmol) and NaOH (1.3 g, 32.50 mmol), 13 was obtained as a
white solid (3.12 g, 76.47% yield). m.p. 252e253 ꢃC; 1H NMR
7.1.21. N-(3-bromobenzyl)-4-oxo-6-(o-tolyl)-1,4-dihydro-
quinoline-3-carboxamide (14e)
The procedure described for 8a was used, starting from 13
(100 mg, 0.35 mmol), HBTU (135.6 mg, 0.35 mmol), DIPEA (93 mg,
0.72 mmol) and 3-bromobenzylamine (66 mg, 0.35 mmol), 14e was
obtained as a white solid (89 mg, 57.05% yield); m.p.184e185 ꢃC; 1H
(400 MHz, DMSO-d6):
d
13.87 (br, 1H), 8.92 (d, 1H, J ¼ 6.4 Hz), 8.18
(s, 1H), 7.90e8.00 (m, 2H), 7.27e7.39 (m, 4H), 2.26 (s, 3H); HRMS
(ESI) for C17H13NO3: calcd. 280.09682 (M þ H)þ, found: 280.09642
(M þ H)þ.
NMR (400 MHz, DMSO-d6):
8.81 (s, 1H), 8.16 (s, 1H), 7.78 (s, 2H), 7.54 (s, 1H), 7.45 (d, 1H,
d
12.81 (s, 1H), 10.44 (t, 1H, J ¼ 5.6 Hz),
7.1.17. N-(4-fluorobenzyl)-4-oxo-6-(o-tolyl)-1,4-dihydro-quinoline-
3-carboxamide (14a)
J ¼ 7.6 Hz), 7.23e7.40 (m, 6H), 4.57 (d, 2H, J ¼ 5.6 Hz), 2.25 (s, 3H);
13C NMR (100 MHz, DMSO-d6):
d 176.55, 165.14, 144.24, 143.13,
The procedure described for 8a was used, starting from 13
(100 mg, 0.35 mmol), HBTU (135.6 mg, 0.35 mmol), DIPEA (93 mg)
and 4-fluorobenzylamine (45 mg, 0.35 mmol), 14a was obtained as
a white solid (91 mg, 67.4% yield). m.p. 180e181 ꢃC; 1H NMR
140.66, 138.45, 138.37, 135.30, 134.23, 131.02, 130.55, 130.13, 128.21,
126.92, 126.64, 126.44, 125.57, 122.12, 119.51, 112.25, 42.00, 20.58;
HRMS (ESI) for C24H19BrN2O2: calcd. 447.07027 (M þ H)þ, found:
447.07010 (M þ H)þ.
(400 MHz, DMSO-d6):
d
12.82 (s,1H),10.42 (t,1H, J ¼ 5.6 Hz), 8.82 (s,
7.1.22. N-(furan-2-ylmethyl)-4-oxo-6-(o-tolyl)-1,4-dihydro-quino-
line-3-carboxamide (14f)
1H), 8.15 (s, 1H), 7.78 (s, 2H), 7.20e7.50 (m, 6H), 7.17 (t, 2H,
J ¼ 8.8 Hz), 4.56 (d, 2H, J ¼ 5.6 Hz), 2.24 (s, 3H); 13C NMR (100 MHz,
The procedure described for 8a was used, starting from 13
(100 mg, 0.35 mmol), HBTU (135.6 mg, 0.35 mmol), DIPEA (93 mg,
0.72 mmol) and furfurylamine (35 mg, 0.35 mmol), 14f was ob-
tained as a white solid. (46 mg, 36.8% yield); m.p. 208e209 ꢃC; 1H
DMSO-d6):
d 176.55, 164.99, 162.71, 160.48, 144.17, 140.65, 138.51,
138.33, 136.31, 135.28, 134.21, 130.99, 130.12, 129.83, 128.21, 126.64,
126.43, 125.55, 119.50, 115.47, 115.68, 111.36, 41.86, 20.58; HRMS
(ESI) for C24H19FN2O2: calcd. 387.15033 (M þ H)þ, found: 387.15003
(M þ H)þ.
NMR (400 MHz, CDCl3):
d 12.86 (s, 1H), 11.05 (s, 1H), 8.80 (d, 1H,
J ¼ 5.6 Hz), 8.50 (s, 1H), 8.21 (s, 1H), 7.63 (t, 1H, J ¼ 7.6 Hz), 7.50 (q,
2H, J ¼ 8.4 Hz), 7.35 (d, 1H, J ¼ 7.6 Hz), 7.23e7.26 (m, 1H), 7.08e7.19
7.1.18. N-(3-fluorobenzyl)-4-oxo-6-(o-tolyl)-1,4-dihydro-quinoline-
3-carboxamide (14b)
(m, 2H), 6.77e6.86 (m, 1H), 4.89 (d, 2H, J ¼ 5.2 Hz), 2.18 (s, 3H); 13
C
NMR (100 MHz, CDCl3):
d 177.27, 166.65, 157.91, 148.77, 143.69,
The procedure described for 8a was used, starting from 13
(100 mg, 0.35 mmol), HBTU (135.6 mg, 0.35 mmol), DIPEA (93 mg,
0.72 mmol) and 3-fluorobenzylamine (45 mg, 0.35 mmol), 14b was
obtained as a white solid. (93 mg, 68.8% yield). m.p. 193e194 ꢃC; 1H
140.29, 139.06, 138.11, 137.37, 135.26, 134.03, 130.50, 129.95, 127.71,
125.99, 122.55, 121,89, 118.52, 115.51, 110.94, 44.71, 20.42; HRMS
(ESI) for C22H18N2O3: calcd. 359.13902 (M þ H)þ, found: 359.13880
(M þ H)þ.
NMR (400 MHz, DMSO-d6):
8.15 (s, 1H), 7.79 (s, 2H), 7.01e7.44 (m, 8H), 4.59 (d, 2H, J ¼ 4.0 Hz),
2.25 (s, 3H); 13C NMR (100 MHz, DMSO-d6):
176.55, 165.12,163.85,
d 12.86 (s, 1H),10.47 (s, 1H), 8.82 (s, 1H),
7.1.23. 4-Oxo-N-(pyridin-3-ylmethyl)-6-(o-tolyl)-1,4-dihydro-
quinoline-3-carboxamide (14g)
d
161.50, 144.25, 143.14, 140.63, 138.52, 138.33, 135.29, 134.24, 130.99,
130.81, 130.13, 128.22, 126.65, 126.41, 125.54, 123.76, 119.52, 114.35,
111.25, 40.28, 20.60; HRMS (ESI) for C24H19FN2O2: calcd. 387.15033
(M þ H)þ, found: 387.14985 (M þ H)þ.
The procedure described for 8a was used, starting from 13
(100 mg, 0.35 mmol), HBTU (135.6 mg, 0.35 mmol), DIPEA (93 mg,
0.72 mmol) and 3-(aminomethyl)pyridine (39 mg, 0.35 mmol), 14g
was obtained as a white solid (93 mg, 72.09% yield); m.p. 198e
7.1.19. 4-Oxo-6-(o-tolyl)-N-(4-(trifluoromethyl)benzyl)-1,4-
dihydro-quinoline-3-carboxamide (14c)
199 ꢃC; 1H NMR (400 MHz, DMSO-d6):
d 12.83 (s, 1H), 10.46 (s, 1H),
8.81 (s, 1H), 8.59 (s, 1H), 8.47 (s, 1H), 8.15 (s, 1H), 7.76 (m, 3H), 7.18e
The procedure described for 8a was used, starting from 13
(100 mg, 0.35 mmol), HBTU (135.6 mg, 0.35 mmol), DIPEA (93 mg,
0.72 mmol) and p-trifluoromethylbenzylamine (63 mg,
0.35 mmol),14c was obtained as a white solid. (99 mg, 65.13% yield)
7.42 (m, 5H), 4.60 (s, 2H), 2.24 (s, 3H); 13C NMR (100 MHz, DMSO-
d6):
d 176.52, 165.18, 149.40, 148.58, 144.20, 140.63, 138.51, 138.34,
135.65, 135.28, 134.23, 130.99, 130.12, 128.21, 126.64, 126.42, 125.54,
123.99, 119.51, 111.27, 40.23, 20.58; HRMS (ESI) for C23H19N3O2:
calcd. 370.15500 (M þ H)þ, found: 370.15507 (M þ H)þ.
7.1.24. N-cyclohexyl-4-oxo-6-(o-tolyl)-1,4-dihydro-quinoline-3-
carboxamide (14h)
The procedure described for 8a was used, starting from 13
(100 mg, 0.35 mmol), HBTU (135.6 mg, 0.35 mmol), DIPEA (93 mg,
0.72 mmol) and cyclohexylamine (36 mg, 0.35 mmol), 14h was
obtained as a white solid (83 mg, 65.8% yield); m.p. 253e254 ꢃC; 1H
m.p. 252e253 ꢃC; 1H NMR (400 MHz, DMSO-d6):
d 12.86 (s, 1H),
10.52 (t, 1H, J ¼ 5.6 Hz), 8.82 (s, 1H), 8.16 (s, 1H), 7.79 (s, 2H), 7.71 (d,
2H, J ¼ 8.0 Hz), 7.56 (d, 2H, J ¼ 8.0 Hz), 7.24e7.38 (m, 4H), 4.67 (d,
2H, J ¼ 5.6 Hz), 2.25 (s, 3H); 13C NMR (100 MHz, DMSO-d6):
d
176.55, 165.22, 145.13, 144.26, 140.62, 138.52, 138.35, 135.29,
134.26, 131.00, 130.13, 128.33, 128.23, 126.66, 126.41, 125.73, 125.53,
119.53, 111.22, 42.18, 20.60; HRMS (ESI) for C25H19F3N2O2: calcd.
437.14714 (M þ H)þ, found: 437.14772 (M þ H)þ.
NMR (400 MHz, DMSO-d6):
d
12.79 (s, 1H), 10.09 (d, 1H, J ¼ 7.6 Hz),
7.1.20. 4-Oxo-6-(o-tolyl)-N-(3-(trifluoromethyl)benzyl)-1,4-
dihydro-quinoline-3-carboxamide (14d)
8.77 (s, 1H), 8.16 (s, 1H), 7.77 (s, 2H), 7.18e7.44 (m, 4H), 3.85 (d, 1H,
J ¼ 7.6 Hz), 2.26 (s, 3H), 1.86 (d, 2H, J ¼ 10.8 Hz), 1.67 (s, 2H), 1.54 (s,
The procedure described for 8a was used, starting from 13
(100 mg, 0.35 mmol), HBTU (135.6 mg, 0.35 mmol), DIPEA (93 mg,
0.72 mmol) and m-trifluoromethylbenzylamine (63 mg,
1H), 1.20e1.44 (m, 5H); 13C NMR (100 MHz, DMSO-d6):
d 176.60,
163.79, 143.95, 140.64, 138.48, 138.19, 135.28, 134.15, 131.01, 130.15,
128.20, 126.66, 126.39, 125.56, 119.46, 111.57, 47.18, 32.99, 25.71,

420
P. Liu et al. / European Journal of Medicinal Chemistry 79 (2014) 413e421
[5] Aleister J. Saunders, Tae-Wan Kim, Rudolph E. Tanzi, BACE maps to chromo-
some 11 and a BACE homolog, BACE 2, reside in the obligate down syndrome
region of chromosome 21, Science 286 (1999) 5443, 1255a.
[6] M. Citron, Alzheimer’s disease: strategies for disease modification, Nature
Reviews Drug Discovery 9 (2010) 387e398.
24.62, 20.61; HRMS (ESI) for C23H24N2O2: calcd. 361.19105
(M þ H)þ, found: 361.19119 (M þ H)þ.
7.2. Molecular docking
[7] A.K. Ghosh, M. Brindisi, J. Tang, Developing b-secretase inhibitors for treat-
The model of BACE-1 was constructed by removing all water
molecules from the X-ray structure of human BACE-1 “apo” form
(PDB ID: 1TQF). The protein was prepared in Discovery Studio 2.5,
with the amino acids ionized at pH 7.4, hydrogens added and
charges calculated. The 3D models of ligands were built using
modified CHARMm force field-based structure minimization
implemented in Discovery Studio’s ’prepare ligands’ protocol.
Docking simulations were carried out by means of GOLD 3.0.1
version. The host ligand was redocked into the binding pocket
and the RMSD between the docked pose and the active confor-
mation was 1.031, confirming that the docking protocol was
reliable.
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The assay was performed with the Human Embryonic Kidney
293 cells (HEK293) which were purchased from the cell bank of
the Shanghai Institute of Cell Biology. The HEK293 cell line was
cultured in the DMEM medium. The media was supplemented
with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and
100 units/mL streptomycin (Invitrogen). The cells were maintained
at 37 ꢃC in a humidified environment with 5% CO2. The cell
viability was determined by using the CellTiter GloÔ luminescent
cell viability assay (Promega). Briefly, the cells were seeded into
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medium. Then the cells were treated with compounds at varying
concentrations. The first compound work concentration is 100 M,
mL of
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m
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for library design and drug discovery, Current Opinion in Chemical Biology 14
(2010) 347e361.
[18] Nicolas Charrier, Brian Clarke, Emmanuel Demont, Colin Dingwall,
Rachel Dunsdon, Julie Hawkins, Julia Hubbard, Ishrut Hussain, Graham Maile,
Rosalie Matico, Julie Mosley, Alan Naylor, Alistair O’Brien, Sally Redshaw,
Paul Rowland, Virginie Soleil, Kathrine J. Smith, Sharon Sweitzer,
Pam Theobald, David Vesey, Daryl S. Walter, Gareth Wayne, Second genera-
tion of BACE-1 inhibitors part 2: optimization of the non-prime side substit-
uent, Bioorganic & Medicinal Chemistry Letters 19 (2009) 3669e3673.
[19] Michael S. Malamas, Jim Erdei, Iwan Gunawan, Keith Barnes,
Matthew Johnson, Yu Hui, Jim Turner, Yun Hu, Erik Wagner, Kristi Fan,
Andrea Olland, Jonathan Bard, Albert J. Robichaud, Aminoimidazoles as potent
and then three fold dilution ten more times. Staurosporine
(SigmaeAldrich, catalog No.S4400) was used as a positive control.
After incubation for 72 h, 10% of CellTiter GloÔ reagent was added
and luminescent signals were read on a VeriScan reader (Thermo
Fisher Scientific). The IC50 value was calculated from the curves
generated by plotting the percentage of the viable cells versus test
concentrations on a logarithmic scale using Sigma Plot 10.0
software.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (21002002, 21172012), the National Basic
Research Program of China (2012CB518000) and the Specialized
Research Fund for the Doctoral Program of Higher Education of
China (20120001110010).
and selective human
b-secretase (BACE 1) inhibitors, Journal of Medicinal
Chemistry 52 (2009) 6314e6323.
[20] Georgia B. McGaughey, Dennis Colussi, Samuel L. Graham, Ming-Tain Lai,
Sanjeev K. Munshi, Philippe G. Nantermet, Beth Pietrak, Hemaka A. Rajapakse,
Harold G. Selnick, Shaun R. Stauffer, M. Katharine Holloway,
b-secretase
(BACE-1) inhibitors: accounting for 10s loop flexibility using rigid active sites,
Bioorganic & Medicinal Chemistry Letters 17 (2007) 1117e1121.
Appendix A. Supplementary data
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F. Patel, Kui Chen, Thomas A. Dineen, Oleg Epstein, Russell Graceffa,
Dean Hickman, Y.-H. Kiang, Steven Louie, Yi Luo, Robert C. Wahl, Paul H. Wen,
Stephen Wood, Robert T. Fremeau Jr., Structure- and property-based design of
aminooxazoline xanthenesas selective, orally efficacious, and CNS penetrable
BACE inhibitors for the treatment of Alzheimer’s disease, Journal of Medicinal
Chemistry 55 (2012) 9156e9169.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.04.025.
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