Synthesis and evaluation of 2-(1H-indol-3-yl)-4-phenylquinolines as inhibitors of cholesterol esterase

Article abstract of DOI:10.1016/j.bmcl.2014.01.081

A series of 2-(substituted) phenyl and 2-indolyl quinoline derivatives (10a-l) was synthesized by an efficient microwave-assisted, trifluoroacetic acid-catalyzed, solvent-free method. Evaluation of the inhibitory activity led to the identification of two

Full text of DOI:10.1016/j.bmcl.2014.01.081

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1545–1549
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of 2-(1H-indol-3-yl)-4-phenylquinolines
as inhibitors of cholesterol esterase
Gisela C. Muscia a, Stephanie Hautmann b, Graciela Y. Buldain a, Silvia E. Asís a, Michael Gütschow b,
⇑
a Departamento de Química Orgánica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, Ciudad Autónoma de Buenos Aires C1113AAB, Argentina
b Pharmaceutical Institute, University of Bonn, An der Immenburg 4, Bonn D-53115, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2-(substituted) phenyl and 2-indolyl quinoline derivatives (10a–l) was synthesized by an
efficient microwave-assisted, trifluoroacetic acid-catalyzed, solvent-free method. Evaluation of the
inhibitory activity led to the identification of two quinoline inhibitors of cholesterol esterase. 2-(1H-
Received 6 January 2014
Revised 23 January 2014
Accepted 27 January 2014
Available online 7 February 2014
Indol-3-yl)-6-nitro-4-phenylquinoline (10l; IC50 = 1.98
l
M) was characterized as a mixed-type inhibitor
with a pronounced competitive binding mode.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Friedländer reaction
Cholesterol esterase
Quinolines
Mixed-type inhibition
The quinoline scaffold is prevalent in a variety of biologically
active compounds. This heterocyclic class became well known for
the antimalarial activities of several of its representatives, such
as chloroquine (1, Fig. 1).1 Quinolines also possess outstanding
activity against leishmaniasis, and the 6-methoxy-8-alkylamino-
quinoline derivative sitamaquine has been evaluated in clinical
trials with a positive outcome.2 The introduction of 2-alkylami-
nomethyl, 2-aryl, 6-halo, or 8-nitro groups has recently been
examined with respect to the quinolines’ trypanocidal or
antileishmanial activities.3,4 Bedaquiline (2), an inhibitor of the
mycobacterial ATP synthase, was approved to treat multi-drug-
resistant tuberculosis.5 Quinolines, mainly 5-hydroxy derivatives,
have been known for years as iron chelating therapeutics and anti-
fungals.6,7 Several quinoline-based compounds also show effective
anticancer activity,8,9 and cabozantinib (3), a multitargeted recep-
tor tyrosine kinase inhibitor, has reached the market for the treat-
ment of medullary thyroid cancer.10 The antibiotic and cytotoxic
drug nitroxoline (8-hydroxy-5-nitroquinoline) and some if its
derivatives, for example 4, were shown to inhibit the human cys-
teine protease cathepsin B.11 Quinolines have been explored as
anti-inflammatory agents interacting with different targets,12 as
well as inhibitors of monoamine oxidase A and B,13 or phosphodi-
esterase type 4B, as shown, for example, for the 1H-indol-3-yl
derivative 5.14
Moreover, quinolines have been described as inhibitors of the
cholesteryl ester transfer protein (CETP). It mediates the transfer
of neutral lipids such as triglycerides, free cholesterol, and cho-
lesteryl esters in the blood among various lipoprotein particles.
CETP facilitates the transfer of cholesteryl esters packaged in
high-density lipoproteins (HDL) to low-density lipoproteins (LDL)
and very-low-density lipoproteins (VLDL). CETP inhibitors, such
as 6, are expected to provide a potential therapeutic benefit for pa-
tients with coronary artery disease.15
This broad spectrum of biological and biochemical activities of
quinolines has attracted much attention in drug research. The facile
synthetic accessibility and the versatile possibilities to modify the
quinoline core have further contributed to the wide utilization of
this heterocycle. It became obvious that the quinoline scaffold can
be regarded as a ‘privileged structure’ in medicinal chemistry.9,16
In the present study, a series of 2,4-diarylquinolines was evalu-
ated with respect to their inhibitory activity against a panel of pro-
teases and esterases, that is the serine esterases cholesterol
esterase (CEase) and acetylcholinesterase, the serine proteases
thrombin, trypsin, chymotrypsin, and human leukocyte elastase
(HLE), as well as the cysteine protease cathepsin L. The selected en-
zymes share an acyl transfer mechanism by which the reaction is
catalyzed. This mechanism involves the nucleophilic attack of the
active site serine (or cysteine) at the substrate’s carbonyl carbon,
the release of the first cleavage product and the hydrolysis of the
(thio)ester bond to form the second product. Although an impres-
sive number of reports underlie the manifold bioactivities of
quinoline derivatives, an investigation to survey the putative
⇑
Corresponding author. Tel.: +49 228 73 2317; fax: +49 228 73 2567.
E-mail address: guetschow@uni-bonn.de (M. Gütschow).
http://dx.doi.org/10.1016/j.bmcl.2014.01.081
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

1546
G. C. Muscia et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1545–1549
catalysts. Several transition-metal-catalyzed or metal-triflate-cata-
lyzed Friedländer reaction processes have been developed. In addi-
tion, microwave (MW)-assisted or ionic liquid-promoted
Friedländer annulations have been employed.20
N
N
NH
OH
Thus, 2-amino-5-chloro-benzophenone or 2-amino-5-nitro-
benzophenone (7) was reacted under microwave irradiation with
a set of substituted acetophenones 8 in order to obtain 2-phenyl-
quinolines 10a–e and 10g–k, and the acetylindole 9 was employed
to prepare the 2-(3-indolyl)quinolines 10f and 10l (Scheme 1 and
Table 1). In these syntheses, trifluoroacetic acid was used as cata-
lyst, exhibiting a more suitable and efficient performance com-
pared with hydrochloric acid. The final products 10a–l were
afforded in high yields and the reaction times were between 2
and 14 min.21–33
Br
Cl
N
chloroquine (1)
N
O
bedaquiline (2)
H
N
H
N
O
O
O
F
O
O
cabozantinib (3)
The two indolyl-substituted quinolines 10f and 10l were iden-
tified as inhibitors of porcine CEase with IC50 values of 1.33 and
N
NO2
1.98 l
M, respectively (Fig. 2, Table 1).36 Most of the inhibitors of
H
N
CEases bear an electrophilic warhead structure, prone to covalently
interact with the enzyme upon being attacked by the active site
serine.37 Noteworthy, the two identified quinoline derivatives lack
such an electrophilic site. Whereas CEase inhibition was attained
with both, the 6-chloro and the 6-nitro derivative, the 2-indolyl
moiety was essential for inhibitory activity; the presence of
(substituted) monocyclic aryl groups at 2-position led to inactive
compounds.
NC
N
CN
OH
O
F
4
N
N
H
5
OH
F
CF3
Further characterization of the CEase inhibition was exemplari-
ly carried out with 10l by performing the assay in the presence of
different substrate concentrations (Figs. 3 and 4). A Ki value of
N
6
660 nM and a Ki0 value of around 7
lM were obtained, where Ki
Figure 1. Examples of bioactive quinoline derivatives.
and Ki0 represent the equilibrium constants for competitive and
uncompetitive inhibition, respectively. Thus, the analysis revealed
mixed-type inhibition with a pronounced competitive component.
Future investigations are planned to extend the variability of the
quinolines’ substitution pattern. Such envisaged new derivatives
might be designed with respect to a closer similarity to the struc-
ture of cholesterol (esters) and to confirm their putative accommo-
dation within the active site of CEase in a substrate like manner.
CEase catalyses the hydrolysis of dietary cholesteryl esters, be-
sides a variety of other substrates, and plays an important role in
cholesterol absorption. The inhibition of CEase may therefore ren-
der a way to limit the bioavailability of dietary cholesterol. Circu-
lating CEase accumulates in atherosclerotic lesions and is
supposed to have deleterious effects in atherosclerosis. Accord-
ingly, CEase was considered to be a potential target for the devel-
opment of inhibitors for the treatment for diseases such as
hypercholesterolemia and coronary heart disease.37
O
X
Ph
N
Ph
8
X = H, OH,
R
R
O
CH3, OCH3, Br
MW
TFA
or
Ar
NH2
R = Cl, NO2
O
7
10a-l
N
H
9
Scheme 1. Synthesis of quinolines 10.
inhibition of serine and cysteine hydrolases has not been
undertaken so far.
Evaluation of the enzyme-inhibiting properties of 10a–l showed
no inhibitory activity against acetylcholinesterase from Electropho-
rus electricus, human thrombin, bovine trypsin, bovine chymotryp-
sin and human cathepsin L for any of the quinolines (Table 1).38–42
Three derivatives, that is 10f, 10i and 10l, exhibited inhibitory
activity against HLE. The kinetic analysis revealed some deviations
from noncooperative binding. The underlying mode of the elas-
tase-inhibitor interactions was not further investigated in the
course of this study.
In summary, we have employed a MW-assisted, solvent-free
method to afford 2,4-diarylquinoline derivatives in high yields
and short reaction times under eco-friendly conditions. As an out-
come of a screening against selected hydrolases, we identified rep-
resentatives of the ‘privileged’ quinoline structure as inhibitors of
CEase. It would be attractive to combine their structural feature
with those of quinoline-type CETP inhibitors. Such an approach
might lead to the development of bifunctional compounds
addressing two different, therapeutically relevant targets of the
cholesterol homeostasis.
We envisaged the preparation of the target compounds by
Friedländer reaction under microwave conditions, in continuation
of previous studies.3,17,18 In position 2 of the target 4-phenylquin-
olines, several aromatic groups should be introduced, that is
(subst.) phenyl and indol-3-yl residues. The latter group was cho-
sen on the basis of the structure of corresponding bioactive com-
pounds, such as
5 (Fig. 1), and the occurrence of indole
substructures in alkaloids found in the leaves and stem bark ex-
tracts of the Bolivian plant Peschiera van heurkii (Muell. Arg.) which
have been screened for antileishmanial and antibacterial proper-
ties.19 Position 6 of the target quinolines should be occupied by a
halo (i.e. chloro) or nitro group in due consideration of correspond-
ing bioactive representatives.3–5
The Friedländer annulation of o-aminobenzophenones with ke-
tones is a particularly straightforward approach for the synthesis of
polysubstituted quinolines. Friedländer reactions afford high tem-
peratures in the absence of catalyst, but catalytic systems have
been continuously explored in search of improved efficiencies
showing that acid catalysts are mostly more effective than base

G. C. Muscia et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1545–1549
1547
Table 1
2,4-Diarylquinolines prepared and enzyme-inhibitory properties
Ph
N
R
Ar
Compd
R
Ar
IC50 valuesa,b,c,d
CEase
Acetylcholin-esterase
Thrombin
Trypsin
Chymo-trypsin
HLE
10a
10b
10c
10d
10e
10f
10g
10h
10i
Cl
Cl
Cl
Cl
Cl
Cl
NO2
NO2
NO2
NO2
NO2
NO2
Ph
P20
P20
P20
P20
P20
l
M
M
M
M
M
n.i.
n.i.
n.i.
n.i.
n.i.
P37
P37
n.i.
n.i.
n.i.
P30
P30
n.i.
P30
n.i.
P30
P30
P30
n.i.
n.i
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
P28
P28
n.i.
P28
P28
4.22 0.02
n.i.
P28 lM
2.91 0.14
n.i.
P28
0.63 0.02
l
l
M
M
C6H4(4-CH3)
C6H4(4-OH)
C6H4(4-OCH3)
C6H4(3-Br)
Indol-3-yl
Ph
C6H4(4-CH3)
C6H4(4-OH)
C6H4(4-OCH3)
C6H4(3-Br)
Indol-3-yl
l
l
l
l
P51
P51
n.i.
P51
n.i.
P51
n.i.
n.i.
l
l
M
M
l
l
M
M
l
l
M
M
l
M
M
1.33 0.12
l
l
M
M
l
l
M
M
l
M
l
l
l
Me
Me
Me
P20
P20
P20
n.i.
P20
1.98 0.11
lM
lM
lM
l
l
l
l
M
M
M
n.i.
P37
n.i.
10j
10k
10l
lM
n.i.
n.i.
n.i.
lM
lM
22.9
lM
P30
l
M
P30 lM
a
n.i. no inhibition. More than 90% residual activity determined in duplicate experiments in the presence of the inhibitor at a single concentration (25
CEase, 5 M in the case of chymotrypsin, 50 M in the case of cathepsin L, 10 M in the case of the other enzymes).
lM in the case of
l
l
l
b
Limits without standard error were determined from duplicate experiments at a single inhibitor concentration (as noted above).
IC50 values were determined in duplicate experiments in the presence of five different inhibitor concentrations.
None of the compounds inhibited human cathepsin L.
c
d
e
IC50 values were obtained by linear regression according to the equation v = v0/(1+([I]/IC50)x).
100
intercepts
6
0.01
80
0.005
4
2
0
60
40
20
0
0
-8 -6 -4 -2
[I] (µM)
0
2
4
0
2
4
6
8
10
-1
0
1
2
3
4
5
[I] (µM)
[I] (µM)
Figure 2. Inhibition of porcine CEase by compounds 10f (open circles) and 10l
(closed squares). Data are means of duplicate measurements. Non-linear regression
Figure 4. Replots of the data obtained for the inhibition of CEase by 10l as shown in
Figure 3. Slopes and intercepts (inset) were plotted versus concentrations of 10l.
according to the equation v = v0/(1+[I]/IC50) gave values IC50 = 1.33 0.12
IC50 = 1.98 0.11 M, respectively.
lM and
Linear regression gave values Ki = 0.66
l
M and Ki0 ꢀ 7
lM.
l
0.12
0.1
Acknowledgments
This work was supported by a visiting grant of the German Aca-
demic Exchange Service (DAAD) to S.E.A.
0.08
0.06
0.04
0.02
0
References and notes
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Figure 3. Inhibition of porcine CEase by compounds 10l. Lineweaver-Burk plots
from measurements in the presence of six different concentrations of the substrate
para-nitrophenyl butyrate (50, 100, 150, 200, 250, and 300
10l were 0 M (closed triangles), 1 M (open triangles), 2
M (open circles), and 4 M (closed circles). Data are means of sextuplicate
measurements. Linear regression gave values for slopes and intercepts, respectively.
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M (closed squares),
l
l
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3
l
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G. C. Muscia et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1545–1549
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27. 6-Chloro-2-(1H-indol-3-yl)-4-phenylquinoline (10f): Reaction time 3.5 min;
yield 84%; mp 155–158 °C. 1H NMR (CDCl3) d 7.16–7.25 (m, 2H), 7.46–7.47
(m, 1H), 7.48–7.56 (m, 2H), 7.64–7.66 (m, 3H), 7.81–7.87 (m, 3H), 8.02 (s, 1H),
8.55 (d, J = 9.1 Hz, 1H), 8.90 (d, J = 3.2 Hz, 1H), 11.30 (s, 1H, NH); 1H NMR
(DMSO-d6) d 7.22–7.24 (m, 2H), 7.48–7.50 (m, 1H), 7.64–7.68 (m, 6H), 7.79 (d,
J = 8.9 Hz, 1H), 8.07 (s, 1H), 8.17 (d, J = 8.9 Hz, 1H), 8.51 (s, 1H), 8.78 (s, 1H)
11.88 (s, 1H, NH); 13C NMR (DMSO-d6) d 155.11, 137.28, 136.86, 130.33,
129.86, 129.48, 128.91, 125.41, 124.99, 123.95, 123.04, 122.50, 122.32, 120.82,
120.32, 114.16, 112.03; FT-IR (KBr) cmꢁ1 699, 745, 1204, 1602,1634,1675,
2873; GC/MS m/z 354 (M+); Anal. Calcd for C23H15ClN2: C 77.85, H 4.26, N 7.89.
Found: C 77.89, H 4.22, N 7.86.
28. 6-Nitro-2,4-diphenylquinoline (10g): Reaction time 10 min; yield 71%; mp 206–
208 °C. 1H NMR (DMSO-d6) d 7.54–7.75 (m, 8H), 8.26 (s, 1H), 8.33 (d, J = 9.3 Hz,
1H), 8.41 (m, 2H), 8.51 (dd, J = 2.6 Hz, 9.1 Hz, 1H), 8.68 (d, J = 2.6 Hz, 1H); 13C
NMR (DMSO-d6) d 159.66, 151.19, 150.79, 145.45, 138.11, 136.77, 132.05,
131.17, 130.22, 129.84, 129.52, 128.34, 124.61, 123.71, 122.85,120.91; FT-IR
(KBr) cmꢁ1 685, 752, 1338, 1594,2953. Anal. Calcd for C21H14N2O2: C 77.29, H
4.32, N 8.58. Found: C 77.26, H 4.36, N 8.54. Analytical data are in accordance
with the literature.34,35
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ˇ
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´
yield 72%; mp 207–208 °C. 1H NMR (DMSO-d6)
d 2.37 (s, 3H), 7.38 (d,
ˇ
J = 8.1 Hz, 2H), 7.59–7.76 (m, 5H), 8.24 (s, 1H), 8.28 (d, J = 2.5 Hz, 1H), 8.35–
8.37 (m, 2H), 8.48 (dd, J = 2.6 Hz, 9.1 Hz, 1H), 8.68 (d, J = 2.5 Hz, 1H); 13C NMR
(DMSO-d6) d 159.56, 151.04, 150.84, 145.29, 141.13, 136.83, 135.34, 131.91,
130.20, 130.09, 129.79, 129.50, 128.26, 124.50, 123.64, 122.83, 120.67, 21.44;
FT-IR (KBr) cmꢁ1 699, 820, 1337, 1593, 2931. Anal. Calcd for C22H16N2O2: C
77.63, H 4.74, N 8.23. Found: C 77.67, H 4.72, N 8.26.
30. 2-(4-Hydroxyphenyl)-6-nitro-4-phenylquinoline (10i): reaction time 2.5 min;
yield 60%; mp 292–295 °C. 1H NMR (DMSO-d6) d 6.92 (d, J = 8.8 Hz, 2H), 7.41–
7.85 (m, 5H), 8.13 (s, 1H), 8.21 (d, J = 9.3 Hz, 1H), 8.26 (d, J = 8.8 Hz, 2H), 8.43
(dd, J = 2.6 Hz, 9.3 Hz, 1H), 8.64 (d, J = 2.4 Hz, 1H), 10.07 (s, 1H); 13C NMR
(DMSO-d6) d 160.65, 159.50, 150.94, 150.68, 144.86, 136.92, 131.56, 130.13,
129.71, 129.47, 128.90, 124.09, 123.52, 122.77, 120.19, 116.26,115.60; FT-IR
(KBr) cm-1; 701, 833, 1172, 1336, 1590, 3415. Anal. Calcd. for C21H14N2O3: C
73.68, H 4.12, N 8.18. Found: C 73.72, H 4.09, N 8.21.
18. Muscia, G. C.; Carnevale, J. P.; Bollini, M.; Asís, S. E. J. Heterocycl. Chem. 2008, 45,
611.
19. de Carvalho, P. B.; Ferreira, E. I. Fitoterapia 2001, 72, 599.
20. For a review, see: Marco-Contelles, J.; Pérez-Mayoral, E.; Samadi, A.; Carreiras,
M. C.; Soriano, E. Chem. Rev. 2009, 109, 2653.
21. Melting points were determined in a capillary Electrothermal 9100 SERIES-
Digital apparatus. 1H and 13C NMR spectra were recorded on a Bruker 300 MHz
spectrometer at 300.13 and 75.46 MHz for 1H and 13C NMR, respectively. IR
spectra (KBr) were recorded on a FT Perkin Elmer Spectrum One. Elemental
analyses were performed on an Exeter CE 440 apparatus. Microwave-assisted
reactions were carried out in a CEM Discover oven.
31. 2-(4-Methoxyphenyl)-6-nitro-4-phenylquinoline (10j): Reaction time 14 min;
yield 56%; mp 217–218 °C. 1H NMR (DMSO-d6) d 3.02 (s, 3H), 7.12 (d, J = 8.8 Hz,
2H), 7.58–7.75 (m, 5H), 8.23 (s, 1H), 8.28 (d, J = 9.2 Hz, 2H), 8.41 (d, J = 9.1 Hz,
1H), 8.48 (dd, J = 2.6 Hz, 9.3 Hz, 1H), 8.66 (d, J = 2.6 Hz, 1H); 13C NMR (DMSO-
d6) d 163.72, 159.27, 159.91, 150.91, 145.09, 136.90, 134.42, 131.73, 130.48,
130.19, 130.01, 129.76, 129.49, 124.29, 123.64, 122.84, 120.40, 114.8, 55.89;
FT-IR (KBr) cmꢁ1 702, 836, 1028, 1172, 1336, 1589,2920; GC/MS m/z 356(M+).
Anal. Calcd for C22H16N2O3: C 74.15, H 4.53, N 7.86. Found: C 74.18, H 4.56, N
7.83.
22. General procedure for the preparation of compounds 10:
A
mixture of
7
32. 2-(3-Bromophenyl)-6-nitro-4-phenylquinoline (10k): Reaction time 12 min;
(1.0 mmol), or (1.50 mmol) and TFA (0.1 mL) was subjected to MW
8
9
yield 40%; mp 232–233 °C. 1H NMR (DMSO-d6)
d 7.54 (t, J = 8.9 Hz, 1H),
irradiation, at 300 W and 250 °C. After reaction completion (TLC), the mixture
was diluted with CH2Cl2, washed with water, HCl (5%, 10 mL) and brine, dried
(Na2SO4) and concentrated under reduced pressure to give a solid which was
triturated with hot EtOH. The product was isolated by suction filtration. 6-
Chloro-2,4-diphenylquinoline (10a) was prepared as described.17
7.65–7.77 (m, 6H), 8.35 (d, J = 2.6 Hz, 1H), 8.39 (s, 1H), 8.42 (d, J = 9.1, 1H), 8.52
(dd, J = 2.6 Hz, 9.3 Hz, 1H), 8.62 (m, 1H), 8.69 (d, J = 2.4, 1H); 13C NMR (DMSO-
d6) d 157.9, 151.5, 150.6, 145.7, 140.4, 136.6, 133.8, 132.2, 131.6, 130.8, 130.3,
129.9, 129.5, 127.4, 124.8, 123.8,123.0, 122.8, 121.0; FT-IR (KBr) cmꢁ1; 697,
701, 1352, 1536, 2988. Anal. Calcd for C21H13BrN2O2: C 62.24, H 3.23, N 6.91.
Found: C 62.27, H 3.19, N 6.94.
23. 6-Chloro-2-(4-methylphenyl)-4-phenylquinoline (10b): Reaction time 5 min;
yield 67%; mp 129–131 °C. 1H NMR (CDCl3) d 2.44 (s, 3H), 7.33 (d, J = 7.9 Hz,
2H), 7.54–7.58 (m, 5H), 7.66 (dd, J = 2.3 Hz, 9.0 z, 1H), 7.82 (s, 1H), 7.85 (d,
J = 2.3 Hz, 1H), 8.09 (d, J = 8.2 Hz, 2H), 8.17 (d, J = 9.2 Hz, 1H); 13C NMR (CDCl3)
33. 2-(1H-Indol-3-yl)-6-nitro-4-phenylquinoline (10l): Reaction time 6 min; yield
60%; mp 276–279 °C. 1H NMR (DMSO-d6) d 7.17–7.29 (m, 2H), 7.40–7.47 (m,
1H), 7.63–7.66 (m, 5H), 8.16 (s, 1H), 8.23 (d, J = 9.4 Hz, 1H), 8.42 (dd, J = 2.5 Hz,
9.1 Hz, 1H), 8.54–8.61 (m, 2H), 8.90–8.92 (m, 1H), 11.91 (s, 1H); 13C NMR
(DMSO-d6) d 159.15, 151.62, 149.14, 144.05, 137.84, 137.07, 130.93, 130.85,
130.06, 129.50, 129.42, 126.11, 123.48, 123.36, 123.28, 123.05, 122.55, 121.46,
121.10, 115.55, 112.46; FT-IR (KBr) cmꢁ1 699, 745, 1371, 1531, 1634, 2895,
3103; Anal. Calcd. for C23H15N3O2: C 75.60, H 4.14, N 11.50. Found: C 75.57, H
4.17, N 11.46.
34. Leandri, R.; Nanni, D.; Tundo, A.; Zanardi, G.; Ruggiero, F. J. Org. Chem. 1842,
1992, 57.
35. Nedeltchev, A. K.; Han, H.; Bhowmik, P. K. Tetrahedron 2010, 66, 9319.
36. Porcine cholesterol esterase inhibition was assayed spectrophotometrically at
405 nm at 25 °C. Assay buffer was 100 mM sodium phosphate, 100 mM NaCl,
d
164.42, 156.66, 148.38, 139.78, 137.76, 136.26, 131.98, 131.52, 130.38,
129.62, 129.42, 128.78, 128.66, 127.40, 126.38, 124.45, 119.88, 21.5; FT-IR
(KBr) cmꢁ1 699, 771, 835, 1590, 3120. Anal. Calcd for C22H16ClN: C 80.11, H
4.89, N 4.25. Found: C 80.07, H 4.92, N 4.29.
24. 6-Chloro-2-(4-hydroxyphenyl)-4-phenylquinoline (10c): Reaction time 5 min;
yield 67%; mp 221–224 °C. 1H NMR (DMSO-d6): d 6.90 (d, J = 8.5 Hz, 2H), 7.56–
7.59 (m, 5H), 7.71 (s, 1H), 7.77 (d, J = 9.0 Hz, 1H), 7.98 (s, 1H), 8.10 (d, J = 8.9 Hz,
1H), 8.20 (d, J = 8.5 Hz, 2H), 9.94 (s, 1H); 13C NMR (DMSO-d6) d 159.43, 156.24,
146.58, 137.01, 131.56, 130.50, 130.20, 129.48, 129.03, 128.88, 125.50, 123.80,
119.09, 115.67; FT-IR (KBr) cmꢁ1 699, 751, 834, 1172, 1283, 1594, 3320; GC/
MS m/z 331(M+). Anal. Calcd for C21H14ClNO: C 76.02, H 4.25, N 4.22. Found: C
76.05, H 4.22, N 4.25.
pH 7.0. A stock solution of CEase (122 lg/mL) was prepared in 100 mM sodium
25. 6-Chloro-2-(4-methoxyphenyl)-4-phenylquinoline (10d): Reaction time 7 min;
yield 94%; mp 138–141 °C. 1H NMR (CDCl3) d 3.91 (s, 3H), 7.09 (d, J = 8.9 Hz,
2H), 7.55–7.65 (m, 5H), 7.84–7.88 (m, 2H), 7.94 (d, J = 2.0 Hz, 1H), 8.14 (d,
J = 8.9 Hz, 2H), 8.55 (d, J = 9.2 Hz, 1H); 13C NMR (CDCl3) d 163.21, 154.99,
154.28, 140.73, 135.76, 134.45, 133.66, 130.66, 130.17, 129.29, 129.21, 126.29,
125.94, 125.24, 125.15, 121.15, 115.07, 55.61; FT-IR (KBr) cmꢁ1 702, 755, 836,
1187, 1597, 1671, 3250; GC/MS m/z 345 (M+). Anal. Calcd for C22H16ClNO: C
76.41, H 4.66, N 4.05. Found: C 76.45, H 4.70, N 4.01.
26. 2-(3-Bromophenyl)-6-chloro-4-phenylquinoline (10e): Reaction time 7 min;
yield 79%; mp 129–131 °C. 1H NMR (CDCl3) d 7.38 (t, J = 7.8 Hz, 1H), 7.50–
7.60 (m, 6H), 7.66 (dd, J = 2.1 Hz, 9.2 Hz, 1H), 7.78 (s, 1H), 7.85 (d, J = 2.3 Hz,
1H), 8.0 (d, J = 7.7 Hz, 1H), 8.15 (d, J = 9.0 Hz, 1H), 8.35 (s, 1H); 1H NMR (DMSO-
d6) d 7.51 (t, J = 7.9 Hz, 1H), 7.59–7.65 (m, 5H), 7.71 (d, J = 7.7 Hz, 1H), 7.78 (d,
J = 2.0 Hz, 1H), 7.84 (dd, J = 2.2 Hz, 8.9 Hz, 1H), 8.19 (t, J = 8.9 Hz, 2H), 8.35 (d,
J = 7.8 Hz, 1H), 8.55 (s, 1H); 13C NMR (DMSO-d6) d 154.63, 148.29, 146.48,
140.41, 136.70, 132.63, 132.04, 131.67, 131.05, 130.60, 129.93, 129.63, 128.99,
128.90, 126.47, 126.14, 123.91, 122.49, 119.79; FT-IR (KBr) cmꢁ1 697, 709, 760,
phosphate buffer, pH 7.0 and kept at 0 °C. A 1:122 dilution was done with the
same buffer immediately before starting the measurement. Sodium
taurocholate (12 mM) was dissolved in assay buffer and kept at 25 °C. A
stock solution of para-nitrophenyl butyrate (20 mM) was prepared in
acetonitrile. The final concentration of acetonitrile was 3%, of DMSO 3%, of
the substrate para-nitrophenyl butyrate 200
6 mM. Assays were performed with a final concentration of 10 ng/mL of CEase.
Into cuvette containing 430 assay buffer, 500 of the sodium
taurocholate solution, 20 acetonitrile, 10 of the para-nitrophenyl
butyrate solution, and 30 L of an inhibitor solution in DMSO were added
and thoroughly mixed. After incubation for 5 min at 25 °C, the reaction was
initiated by adding 10 L of the enzyme solution (1 g/mL).
lM, and of sodium taurocholate
a
l
L
lL
lL
lL
l
l
l
37. (a) Deck, L. M.; Baca, M. L.; Salas, S. L.; Hunsaker, L. A.; Vander Jagt, D. L. J. Med.
Chem. 1999, 42, 4250; (b) Lin, G.; Liao, W. C.; Chiou, S. Y. Bioorg. Med. Chem.
2000, 8, 2601; (c) Pietsch, M.; Gütschow, M. J. Biol. Chem. 2002, 277, 24006; (d)
Heidrich, J. E.; Contos, L. M.; Hunsaker, L. A.; Deck, L. M.; Vander Jagt, D. L. BMC
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1549
8270; (f) Heynekamp, J. J.; Hunsaker, L. A.; Vander Jagt, T. A.; Royer, R. E.; Deck,
L. M.; Vander Jagt, D. L. Bioorg. Med. Chem. 2008, 16, 5285; (g) Lin, M. C.; Yeh, S.
J.; Chen, I. R.; Lin, G. Protein J. 2011, 30, 220; (h) Lin, M. C.; Lin, G. Z.; Hwang, C.
I.; Jian, S. Y.; Lin, J.; Shen, Y. F.; Lin, G. Protein Sci. 2012, 21, 1344.
38. Enzyme assays were performed as described.39–42
40. HLE: Gütschow, M.; Pietsch, M.; Themann, A.; Fahrig, J.; Schulze, B. J. Enzyme
Inhib. Med. Chem. 2005, 20, 341.
41. Human thrombin, bovine trypsin: Sisay, M. T.; Steinmetzer, T.; Stirnberg, M.;
Maurer, E.; Hammami, M.; Bajorath, J.; Gütschow, M. J. Med. Chem. 2010, 53,
5523.
39. Acetylcholinesterase from Electrophorus electricus, bovine chymotrypsin: Sisay, M.
T.; Hautmann, S.; Mehner, C.; König, G. M.; Bajorath, J.; Gütschow, M.
ChemMedChem 2009, 4, 1425.
42. Human cathepsin B: Frizler, M.; Lohr, F.; Furtmann, N.; Kläß, J.; Gütschow, M. J.
Med. Chem. 2011, 54, 369.