Evaluation of synthetic acridones and 4-quinolinones as potent inhibitors of cathepsins L and v

Article abstract of DOI:10.1016/j.ejmech.2012.04.002

Cathepsins, also known as lysosomal cysteine peptidases, are members of the papain-like peptidase family, involved in different physiological processes. In addition, cathepsins are implicated in many pathological conditions. This report describes the synthesis and evaluation of a series of N-arylanthranilic acids, acridones, and 4-quinolinones as inhibitors of cathepsins V and L. The kinetics revealed that compounds of the classes of acridones are reversible competitive inhibitors of the target enzyme with affinities in the low micromolar range. They represent promising lead candidates for the discovery of novel competitive cathepsin inhibitors with enhanced selectivity and potency. On the other hand, 4-quinolinones were noncompetitive inhibitors and N-arylanthranilic acids were uncompetitive inhibitors.

Full text of DOI:10.1016/j.ejmech.2012.04.002

European Journal of Medicinal Chemistry 54 (2012) 10e21
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Evaluation of synthetic acridones and 4-quinolinones as potent inhibitors
of cathepsins L and V
,
Emerson F. Marques a, Mauro A. Bueno a 1, Patrícia D. Duarte a, Larissa R.S.P. Silva a, Ariani M. Martinelli a,
,
a,
*
Caio Y. dos Santos a, Richele P. Severino b, Dieter Brömme c, Paulo C. Vieira a , Arlene G. Corrêa
*
a Departamento de Química, Universidade Federal de São Carlos, 13565-905 São Carlos, SP, Brazil
b Departamento de Química, Universidade Federal de Goiás, 75704-020 Catalão, GO, Brazil
c Faculty of Dentistry and Center for Blood Research at the University of British Columbia, Vancouver, BC, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Cathepsins, also known as lysosomal cysteine peptidases, are members of the papain-like peptidase
family, involved in different physiological processes. In addition, cathepsins are implicated in many
pathological conditions. This report describes the synthesis and evaluation of a series of N-arylanthranilic
acids, acridones, and 4-quinolinones as inhibitors of cathepsins V and L. The kinetics revealed that
compounds of the classes of acridones are reversible competitive inhibitors of the target enzyme with
affinities in the low micromolar range. They represent promising lead candidates for the discovery of
novel competitive cathepsin inhibitors with enhanced selectivity and potency. On the other hand,
4-quinolinones were noncompetitive inhibitors and N-arylanthranilic acids were uncompetitive
inhibitors.
Received 30 December 2011
Received in revised form
3 April 2012
Accepted 4 April 2012
Available online 21 April 2012
Keywords:
Cathepsins V and L
Synthetic inhibitors
Quinolinone and acridone alkaloids
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
quinolinones is low and comparable to that of other commonly
used antimicrobial agents; so they can be considered relatively
Natural Products are a very important source of therapeutic drugs
in medicine due to their ability to modulate many biochemical
pathways [1]. The first products isolated from natural sources in the
19th century were alkaloids. Since then, hundreds of bioactive
compounds have been isolated from plants, animals or microorgan-
isms with different architectures, presenting a very high chemical
diversity. This includes terpenes, coumarins, flavonoids, acetogenins,
and many others. However, we have few examples of “natural drugs”
which are formulated for ready availability as medicine [2]. In the
majority of cases “natural drugs” must be chemically modified in
order to improve their pharmacological profile [3].
Alkaloids are one of the most important classes of natural
products due to their ability to modulate many pharmacological
events. 4-Quinolinones are widely used as antibacterial and anti-
mycobacterial drugs to treat various infectious diseases [4,5], and
potent chemotherapeutic agents of first choice for the treatment of
a broad range of bacterial infections, and are used as templates in
the development of various inhibitors [6,7]. The toxicity of
well-tolerated [8]. Besides the antibacterial activity, several reports
have shown the anti-parasitic activity of 4-quinolinones [9e13].
N-Arylanthranilic acids are an interesting class of compounds
which show antibacterial [14e17] and anti-inflammatory [16]
properties. They are also employed as precursor of biologically
important compounds such as acridone alkaloids [17e25]. Santos
et al. reported the evaluation of 11 acridones, isolated from Swin-
glea glutinosa, toward Plasmodium falciparum, Leishmania donovani
and Trypanosoma brucei [25].
Cathepsins, also known as lysosomal cysteine peptidases, are
members of the papain-like peptidase family, involved in different
physiological processes. In addition, cathepsins are implicated in
many pathological conditions [26].
Cathepsin V (catV) has been associated with the MHC class II
complex in humans [27] and the enzyme has been considered as
a potential diagnostic marker for colon tumors [28]. This enzyme
also is expressed in colorectal and breast carcinomas but not in
normal colon or mammary tissue [29]. In addition, cathepsin V
together with the cathepsins L, K and S, have been implicated in
atherosclerosis and are considered potential drug targets [30e33].
On the other hand, cathepsin L (catL) has been associated in
processes of keratinocyte differentiation, heart functions and
reproduction [34]. Studies on cathepsin expression revealed
increased expression of cathepsins B and L in melanomas and
* Corresponding authors. Tel.: þ55 16 33518206; fax: þ55 16 33518281.
E-mail addresses: paulo@dq.ufscar.br (P.C. Vieira), agcorrea@ufscar.br
(A.G. Corrêa).
1
Present address: Instituto de Ciências Ambientais e Desenvolvimento Susten-
tável, Universidade Federal da Bahia, 47805-100 Barreiras, BA, Brazil.
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.04.002

E.F. Marques et al. / European Journal of Medicinal Chemistry 54 (2012) 10e21
11
carcinomas of breast, lung, colon and prostate [35]. Recently, it has
been demonstrated that non-metastatic melanoma cells are con-
verted into metastatic cells by overexpression of catL [36]. A potent
inhibitor of catL, cystatin C, blocks the mobility and invasion of
melanoma cells [37,38].
positives. None of the compounds exhibited intrinsic fluorescence.
In the first inhibition screen assay, we used an initial inhibitor
concentration of 25
inhibition of catV and L, they were subsequently diluted to 12.5, 2.5
and 0.25 M and screened against cathepsins L and V. In order to
mM. As most of compounds showed 100%
m
Though these enzymes have been intensively studied as valuable
targets for drug discovery and development [34,39], few natural
product-based inhibitors have been described in literature
[30,31,40e42]. Recently, we reported synthetic flavones and natural
acridones as potent inhibitors of catV, with IC50 values varying in
explore the specificityeactivity relationship (SAR) of cathepsins V
and L inhibitors, we determined IC50 values for both cathepsins of
ten 2-aminoacetophenones, ten 2-substituted-4-quinolones,
seventeen N-arylanthranilic acids, and ten acridones, by making
rate measurements for at least seven inhibitor concentration, as
shown in Tables 3e5, respectively. The tested compounds showed
low selectivity toward the enzymes studied.
Compounds 1aej did not show significant inhibition at
a concentration of 25 mM, although quinolinones 2aej (excepting
for 2e which was not evaluated due to the low yield obtained
the range of 0.8e4.1 mM and 1.2e5.2 mM, respectively [43,44].
This report describes the synthesis and inhibitory evaluation of
a series of N-arylanthranilic acids, acridones, and 4-quinolinones.
Most of the compounds are novel and their inhibitory potency
against cathepsins V and L was determined.
during its preparation) presented values of IC50 varying in the range
2. Results and discussion
of 1.6e20
this series, the most potent catL inhibitor was 2b with an IC50 of
1.6 M and the most potent catV inhibitors were 2a and 2c, both
with IC50 values of 4.7 M. In this small series with substitutions at
the benzyl group, compound 2a showed an IC50 value of 2.6 M for
the catL and 4.7 M for catV. This represents a good basis for further
alterations of the substituent at the ring position and to improve
the potency of this class. Small variation of the size of the alkyl
chain in benzyl group (position 4) already affected directly the
inhibitory potency of this class.
mM and 4.7e20 mM for the catL and catV, respectively. In
2.1. Synthesis of 4-quinolinones
m
m
Several synthetic methods have been developed for the prepa-
ration of 2-substituted-4-quinolones from different starting mate-
rials, and among them, acylated 2-aminoacetophenones and
enamines are the most common [45,46]. These methodologies
involve thermal cyclization usually under extremely harsh condi-
tions [47]. In this work, we report the synthesis of a library of 4-
quinolinones under microwave (MW) irradiation [48,49].
After a systematic study, looking for alternatives for the cycli-
zation of 2-aminoacetophenones (1), we found that MW irradiation
in the presence of t-BuOK in THF, for 10e20 min at 100e120 ꢀC and
180 W is the best condition. We have prepared a series of 2-
substituted-4-quinolinones in good yields (Table 1), excepting for
quinolinone 2e (entry 5) due to formation of the 2-quinolinone
isomer as the major product.
m
m
Compounds 5aeq showed significant inhibition with IC50 values
varying in the range of 2e50 mM and 1e50 mM for the catL and catV,
respectively. Although these inhibitors were sensitive for both
cathepsins, the most potent N-arylanthranilic acid was 5i with an
IC50 value of 1.0
mM for catV. In general, N-arylanthranilic acid
exhibited a slight selectivity for catV as can be seen for the selec-
tivity ratio (S) >1. Despite the limited amount of structureeactivity
relationship (SAR) data, it is possible to note the following prefer-
ences: a) when R1 is a nitro group, the compound is five times more
potent than a methoxy group (see compounds 5b and 5c); b)
addition of a benzyl group at R1 in 5e increases the potency twenty-
fold when compared with compounds 5feh; c) addition of
a methoxy group at R3 decreases the potency when comparing
compounds 5b and 5k (10-fold difference) and 5c and 5l (2-fold
difference); and d) the presence of a methoxy group at R4 and R5
in 5n increased the potency when compared with compound 5k.
Acridones 6aeq showed IC50 values varying in the range of
2.2. Synthesis of N-arylanthranilic acids and aridones
The synthesis of N-arylanthranilic acids can be usually achieved
by Ullmann condensation [50] between a 2-halobenzoic acid and an
aryl amine [51,52] or an anthranilic acid and an aryl halide [53,54].
One of the major drawbacks of these methodologies is the need of
heating during long reaction time [55]. Recently, we reported the
synthesis of a series of novel N-arylanthranilic acids 5, with good to
excellent yields, employing MW as heat source to promote the
Ullmann coupling between aryl bromides 3 and anthranilic acids 4
possessing electron donating or withdrawing groups [56].
To complete the synthesis of acridones 6, a series of experiments,
employing different reagents and conditions, was performed and it
was observed that intramolecular FriedeleCrafts acylation was best
performed employing POCl3 as acylating reagent [57,58]. In general,
the acridones were obtained in good to excellent yields, however, N-
arylanthranilic acids bearing strong electron-withdrawing group at
C3 position (aryl bromide 3b) furnished no product (Table 2, entries
2, 9, 11, 14 and 15). Attempts to obtain the desired compounds
employing previously reported methodologies such as PPA [59], PPE
[60], TFAA [61,62] and H2SO4 [23] as acylating reagents also failed,
which we attributed to the rightly deactivating character of nitro
substituent, making the FriedeleCrafts acylation unfavorable.
0.5e19.3
mM and 1.7e18 mM for the catL and catV, respectively.
Compound 6g was the most potent inhibitor for catL, with an IC50 of
0.5. The structural difference between compounds 6feh is the
methoxy group, benzyl group and the hydrogen at the R5 position.
2.4. Mechanism of inhibition
Some 4-quinolinones (2b), N-arylanthranilic acids (5i and 5j)
and acridones (6a and 6g) with low IC50 values were selected to
determinate the type of inhibition of cathepsins L and V. Interest-
ingly, selected compounds from N-arylanthranilic acids showed
uncompetitive inhibition of catL and catV (Fig. 1), from acridones
exhibited competitive inhibition (Fig. 2), whereas a 4-quinolinone
showed noncompetitive inhibition (Fig. 3).
Uncompetitive inhibitors require the prior formation of the ES
complex and bind exclusively to the ES complex. On the other hand,
competitive inhibitors binds to the free enzyme and compete with
the substrate with a mutually exclusive binding mode [63,64].
Noncompetitive inhibitors display binding affinity for free enzyme
(E) and ES complex. These inhibitors do not compete with the
substrate for binding to free enzyme, they bind at a site distinct
from the active site.
2.3. Biochemical evaluation of synthetic compounds
The design of the compounds presented in this work is based on
the results of previous catV inhibition studies on using natural
acridone alkaloids [44]. First we evaluated the potential intrinsic
autofluorescences of the compounds which could lead to false

12
E.F. Marques et al. / European Journal of Medicinal Chemistry 54 (2012) 10e21
Table 1
Synthesis of 2-substituted-4-quinolinones.
H
H
N
R
NH2
O
O
N
R
Et3N
THF
t-BuOK, THF
MW
+
O
Cl
R
O
O
1
2
Entry
1
R
Acetophenone yield (%)a
Quinolinone yield (%)a
1a, 90
2a, 60
2
1b, 91
1c, 72
2b, 67
2c, 73
O
3
4
1d, 74
1e, 75
2d, 55
2e, 20
(CH2)10CH3
5
6
1f, 83
2f, 72
F
7
8
9
1g, 93
1h, 84
2g, 64
2h, 68
1i, 85
1j, 79
2i, 65
2j, 63
O
10
(CH2)5CH3
a
Isolated yields after chromatographic purification.
The Ki values of the competitive and uncompetitive inhibitors
were obtained using Dixon plot analysis, which consist in plotting
the reciprocal of the initial velocity (1/v0) versus a series of inhibitor
concentrations at constant substrate concentration, where the lines
converge above the x-axis, indicating the inhibition constant-Ki.
The Ki values for inhibitors 5i, 5j, 6a and 6g are reported in Table 6.
conventional heating methods, which significantly improved the
reaction times and yields. Newly synthesized acridones and 4-
quinolinones were shown to be potent inhibitors of cathepsins L
and V. The kinetics revealed that compounds of the classes of
acridones are reversible competitive inhibitors of the target
enzyme with affinities in the low micromolar range. They represent
promising lead candidates for the discovery of novel competitive
cathepsin inhibitors with enhanced selectivity and potency. Inter-
estingly, some N-arylanthranilic acid and 4-quinolinones showed
uncompetitive and 4-quinolinones noncompetitive inhibition
mechanisms, respectively.
3. Conclusions
In this work we described the synthesis of 2-substituted-4-
quinolinones employing microwave irradiation, instead of

E.F. Marques et al. / European Journal of Medicinal Chemistry 54 (2012) 10e21
13
Table 2
Synthesis of N-arylanthranilic acids and acridones.
4
R
O
5
3
2
1
5
CO2H
5
2
1
2
R
R
R
R
R
R
R
K2CO3
+
POCl , 100 ºC, 1h;
3
CuI, L-Pro
MW, 1h
solvent
4
NH2
1
N
H
4
R
EtOH/HCl (10%),
aq
R
Br
N
H
R
R
R
3
Reflux, 1h
HO2C
3
R
4
R
5
3
6
Entry
Aryl bromide
Anthranilic acid
N-Arylanthranilic acid, yield (%)a,b
Acridone, yield (%)b
1
2
3
4
5
6
7
8
3a: R1 ¼ BnO; R2 ¼ H
3b: R1 ¼ NO2; R2 ¼ BnO
3c: R1 ¼ MeO; R2 ¼ BnO
3d: R1 ¼ H; R2 ¼ MeO
3a
4a: R3 ¼ R4 ¼ R5 ¼ H
5a, 78
5b, 83
5c, 85
5d, 77
5e, 82
5f, 76
5g, 69
5h, 82
6a, 82
e
4a
4a
4a
6c, 85
6d, 89
6e, 90
6f, 87
6g, 78
6h, 82
4b: R3 ¼ R4 ¼ H; R5 ¼ NO2
3d
4b
4b
4b
3e: R1 ¼ H; R2 ¼ BnO
3f: R1; R2 ¼ H
CO2H
NH2
9
3b
4c
5i, 54
e
10
11
12
13
14
15
16
17
3c
3b
3c
3f
3b
3b
3c
3e
4c
5j, 81
5k, 92
5l, 70
5m, 54
5n, 83
5o, 54
5p, 38
5q, 65
e
4d: R3 ¼ MeO; R4 ¼ R5 ¼ H
e
4d
4d
e
6m, 99
e
4e: R3 ¼ R4 ¼ R5 ¼ MeO
4f: R3 ¼ H; R4 ¼ R5 ¼ F
e
4f
4f
6p, 93
6q, 95
a
All reactions were performed in anhydrous iso-amyl alcohol as solvent at 140 ꢀC, except for anthranilic acid 4b which was performed in DMF/H2O 90% at 160 ꢀC [56].
Isolated yield after purification.
b
4. Material and methods
4.1.1.1. N-(2-Acetylphenyl)benzamide (1a) [65]. Yield 90%. Mp 99 ꢀC
(dec.). IR (nmax, KBr): 701, 764, 963, 1028, 1247, 1260, 1299, 1316,
1449, 1538, 1587, 1608, 1647, 1673, 2331, 2358, 3217 cmꢂ1. 1H NMR
4.1. Synthesis
(200 MHz, CDCl3)
7.47e7.67 (m, 4H); 7.95 (dd, 1H, J 1.4, 1.6 Hz); 8.05e8.10 (m, 2H);
8.98 (dd, 1H, J 1.2, 1.4 Hz), 12.69 (s, 1H). 13C NMR (50 MHz, CDCl3)
d: 2.72 (s, 3H); 7.16 (ddd, 1H, J 1.2, 7.2, 7.9 Hz);
Unless otherwise noted, all commercially available reagents
were purchased from Aldrich Chemical Co. Reagents and solvents
were purified when necessary according to the usual procedures
described in the literature. 1H and 13C NMR spectra were recorded
on a Bruker ARX-200 (200 and 50 MHz respectively). The IR spectra
refer to films and were measured on a Bomem M102 spectrometer.
Mass Spectra were recorded on a Shimadzu GCMS-QP5000 or Mass
Spectrometer QuatroLC-Micromass. Elemental analyses were per-
formed on a Fisons EA 1108 CHNSeO. Analytical thin-layer chro-
d
:
28.5, 120.9, 122.5, 127.5, 128.8, 130.4, 131.8, 131.9, 155.4, 167.9, 203.2.
MS (m/z): 239 (Mþ), 224, 200, 196, 106, 105 (100), 78, 77, 51.
4.1.1.2. N-(2-Acetylphenyl)-4-butylbenzamide (1b). Yield 91%. Mp
120 ꢀC (dec.). IR (nmax, KBr): 758, 850, 897,1246,1651,1682,1785, 2859,
2930, 2957, 3223, 3245 cmꢂ1.1H NMR (200 MHz, CDCl3)
d: 0.93 (t, 3H, J
7.2 Hz); 1.36 (sex, 2H, J 7.2 Hz); 1.63 (qui, 2H, J 7.2 Hz); 2.64e2.72 (m,
5H); 7.13 (ddd,1H, J 1.2,1.3,1.5 Hz); 7.26e7.34 (m, 2H); 7.60 (ddd,1H, J
1.3,1.5,1.6 Hz); 7.92e8.07 (m, 3H); 8.98 (dd,1H, J 1.2 Hz); 12.65 (s,1H).
matography was performed on a 0.25
mm film of silica gel
containing fluorescent indicator UV254 supported on an aluminum
sheet (SigmaeAldrich). Flash column chromatography was per-
formed using silica gel (Kieselgel 60, 230e400 mesh, E. Merck). Gas
chromatography was performed in a Shimadzu GC-17A with H2 as
carrier and using a DB-5 column. Melting points were performed in
Microquimica MQAPF e 301. Reactions were irradiated in a focused
microwave oven CEM Discover.
13C NMR (50 MHz, CDCl3)
d: 13.9, 22.3, 28.5, 33.2, 35.5, 120.8, 122.3,
127.5, 128.8, 130.6, 131.8, 135.3, 141.5, 147.4, 164.5, 216.1.
4.1.1.3. N-(2-Acetylphenyl)furan-2-carboxamide (1c) [66]. Yield
72%. Mp 105 ꢀC (dec.). IR (nmax, KBr): 750, 786, 883, 959, 1010, 1167,
1248, 1315, 1452, 1525, 1582, 1607, 1651, 1677, 2336, 2359, 3113,
3148 cmꢂ1. 1H NMR (200 MHz, CDCl3)
d: 2.72 (s, 3H); 6.56 (dd, 1H, J
4.1.1. General procedure for the preparation of acetophenone
derivatives 1 [49]
1.8,1.9 Hz); 7.16 (ddd,1H, J 0.8,1.2,1.6 Hz); 7.27 (dd,1H, J 0.8,1.2 Hz);
7.56e7.65 (m, 2H); 7.95 (dd, 1H, J 1.4, 1.6 Hz); 8.90 (dd, 1H, J 1.0,
To a mixture of triethylamine (1 mmol), anhydrous dichloro-
methane (2 mL) and 20-aminoacetophenone (1 mmol), acyl chlo-
ride (1 mmol) was added in drop wise under ice bath. The solution
was stirred for 2 h at room temperature. The mixture was extracted
with dichloromethane (3 ꢁ 10 mL), and the combined organic
layers washed with water (3 ꢁ 10 mL), then dried with Na2SO4.
After removal of the solvent the product was purified by column
chromatography (Hex/Acet 80:20).
1.2 Hz); 12.69 (s, 1H). 13C NMR (50 MHz, CDCl3)
d: 28.5, 107.2, 109.8,
120.9, 122.5, 127.5, 128.8, 131.9, 140.0, 141.4, 147.9, 167.9, 203.2. MS
(m/z): 229 (Mþ), 214, 186, 158, 146, 116, 95 (100), 90, 67, 51.
4.1.1.4. N-(2-Acetylphenyl)undecanamide (1d). Yield 74%. Mp 115 ꢀC
(dec.). IR (nmax, KBr): 704, 735, 1164, 1246, 1265, 1451, 1523, 1583,
1606,1654,1694,1775, 2305, 2358, 2854, 2926, 2954, 2984 cmꢂ1. 1H
NMR (200 MHz, CDCl3) d: 0.88 (t, 3H, J 6.5 Hz); 1.26e1.82 (m, 16H);

14
E.F. Marques et al. / European Journal of Medicinal Chemistry 54 (2012) 10e21
Table 3
Structures of 2-substituted-4-quinolinones and values of IC50 on cathepsin L and cathepsin V.
Structure
CatL IC50
M)a
CatV IC50
M)a
Sb
Structure
IC50
CatL (
IC50
Sb
(
m
(
m
m
M)a
CatV (m
M)a
H
N
H
N
2a
2.6 ꢃ 0.1
4.7 ꢃ 0.1
0.6
2b
1.6 ꢃ 0.2
5.2 ꢃ 0.1
0.3
O
O
H
N
H
N
(CH ) CH
2 10
3
O
2c
2.6 ꢃ 0.1
4.7 ꢃ 0.1
0.6
2d
2.9 ꢃ 0.3
9.9 ꢃ 0.2
0.3
O
O
F
H
N
H
N
2e
ND
ND
e
2f
20 ꢃ 0.8
20 ꢃ 0.5
1.0
O
O
H
N
H
N
2g
16 ꢃ 1.3
16 ꢃ 1.5
1.0
0.6
2h
15 ꢃ 1.2
5.0 ꢃ 0.9
3.0
1.0
O
O
O
(CH ) CH3
2 5
H
N
H
N
2j
8.6 ꢃ 0.7
15 ꢃ 1.1
2i
9.0 ꢃ 0.8
8.5 ꢃ 0.7
O
O
ND ¼ not determined.
a
The values represent means of three individual experiments ꢃ SD.
Selectivity (S) ¼ IC50 cathepsin L/IC50 cathepsin V.
b
2.43 (t, 2H, J 7.2 Hz); 2.67 (s, 3H); 7.10 (ddd, 1H, J 0.9,1.1, 1.2 Hz); 7.55
(ddd, 1H, J 1.6, 1.2, 1.4 Hz); 7.98 (dd, 1H, J 1.5, 1.6 Hz); 8.78 (dd, 1H, J
7.17e7.29 (m, 3H); 7.61 (ddd, 1 H, J 1.6, 1.2, 1.7 Hz); 8.01e8.08 (m,
3H); 8.78 (dd, 1H, J 0.8, 1.2 Hz), 12.71 (s, 1H). 13C NMR (50 MHz,
0.9,1.1 Hz); 11.71 (s,1H). 13C NMR (50 MHz, CDCl3)
d
: 14.3, 21.8, 25.2,
CDCl3) d: 28.7, 116.4, 116.8, 121.3, 123.9, 130.6, 130.8, 133.1, 134.3,
26.6, 28.3, 29.6, 30.1, 30.2, 30.3, 32.7, 38.9, 121.3, 122.3, 131.2, 136.0,
137.5, 139.4, 171.2, 203.8. MS (m/z): 225, 211, 199, 183, 169, 155, 135,
127, 109, 95, 85, 71, 57 (100).
135.9, 137.5, 161.9, 163.7, 165.9, 204.8. MS (m/z): 257 (Mþ), 242, 214,
123 (100), 95, 91, 75, 51.
4.1.1.7. N-(2-Acetylphenyl)-4-ethylbenzamide (1g). Yield 93%. Mp
100 ꢀC (dec.). IR (nmax, KBr): 526, 603, 688, 763, 854, 960, 1120, 1188,
1245, 1315, 1357, 1448, 1510, 1535, 1608, 1645, 1670, 2871, 2931,
4.1.1.5. N-(2-Acetylphenyl)pent-4-enamide (1e). Yield 75%. Mp
110 ꢀC (dec.). IR (nmax, KBr): 517, 605, 735, 757, 916, 960, 1163, 1247,
1311,1359,1451,1521,1584,1652,1696, 2915, 2978, 3001, 3078 cmꢂ1
.
2960, 2972, 3217 cmꢂ1. 1H NMR (200 MHz, CDCl3)
d: 1.26 (t, 3H, J
1H NMR (200 MHz, CDCl3)
d
: 2.47e2.56 (m, 4H); 2.66 (s, 3H);
7.5 Hz); 2.65e2.77 (m, 5H); 7.11 (ddd, 1H, J 1.2, 0.8, 1.1 Hz); 7.33 (dt,
2H, J 0.6, 1.9 Hz); 7.58 (ddd, 1H, J 0.6, 1.2, 0.8 Hz); 7.90e8.01 (m, 3H);
4.97e5.15 (m, 2H); 5.77e5.97 (m, 2H); 7.10 (ddd,1H, J 1.4,1.2, 0.8 Hz),
7.54 (ddd,1H, J 1.8,1.6,1.4 Hz); 7.88 (dd,1H, J 1.6,1.8 Hz); 8.75 (dd,1H,
8.51 (dd, 1H, J 0.9, 1.1 Hz); 12.65 (s, 1H). 13C NMR (50 MHz, CDCl3)
d:
J 0.9,1.2 Hz); 11.72 (s,1H).13C NMR (50 MHz, CDCl3)
d: 27.3, 29.2, 37.8,
15.2, 28.8, 29.8, 120.6, 120.7, 122.2, 123.8, 126.6, 127.5, 128.2, 129.2,
131.7, 135.3, 136.7, 141.5, 166.0, 202.5. MS (m/z): 267 (Mþ), 252, 224,
207, 177, 134, 133 (100), 106, 105, 77, 51.
115.5, 120.7, 122.2, 123.8, 131.6, 135.1, 136.6, 173.1, 200.7. MS (m/z):
217 (Mþ), 202, 189, 174, 160, 135, 120 (100), 116, 92, 84, 65, 55.
4.1.1.6. N-(2-Acetylphenyl)-4-fluorobenzamide (1f). Yield 83%. Mp
105 ꢀC (dec.). IR (nmax, KBr): 459, 520, 576, 607, 628, 678, 754, 850,
960,1008,1097,1232,1319,1450,1506,1539,1593,1652,1672, 3076,
4.1.1.8. N-(2-Acetylphenyl)-4-methylbenzamide (1h). Yield 84%. Mp
95 ꢀC (dec.). IR (nmax, KBr): 474, 522, 607, 628, 648, 757, 833, 962,
1020, 1118,1168, 1193, 1247, 1315,1363,1454, 1508, 1643, 1674, 2921,
3166, 3184, 3199 cmꢂ1
.
1H NMR (200 MHz, CDCl3)
d: 2.71 (s, 1H);
2970, 3207 cmꢂ1. 1H NMR (200 MHz, CDCl3)
d: 2.43 (s, 3H); 2.72 (s,

E.F. Marques et al. / European Journal of Medicinal Chemistry 54 (2012) 10e21
15
Table 4
Structures of N-arylanthranilic acids and values of IC50 on cathepsin L and cathepsin V.
Structure
CatL IC50
(m
M)a CatV IC50
(
m
M)a Sb
Structure
IC50 CatL (
m
M)a
IC50 CatV (m
M)a Sb
BnO
O2N
5a
7.2 ꢃ 0.3
2.1 ꢃ 0.2
3.4 5b
5.7 ꢃ 0.1
6.8 ꢃ 0.2
0.8
BnO
N
H
N
H
CO2H
CO2H
BnO
MeO
5c
5e
5g
26 ꢃ 0.3
25 ꢃ 0.3
1.0 5d
>50
>50
e
MeO
N
H
N
H
CO2H
CO2H
CO2H
CO2H
NO2
MeO
NO2
2.5 ꢃ 0.05
1.5 ꢃ 0.04
1.7 5f
45 ꢃ 0.1
43 ꢃ 0.2
15 ꢃ 0.5
10 ꢃ 0.8
3.0
4.3
BnO
BnO
N
H
N
H
CO2H
NO2
NO2
>50
>50
e
5h
N
H
N
H
CO2H
BnO
O2N
BnO
5i
2.9 ꢃ 0.3
1.0 ꢃ 0.1
2.9 5j
2.1 ꢃ 0.005
1.8 ꢃ 0.01
1.2
N
H
MeO
N
H
CO2H
CO2H
BnO
O2N
MeO
BnO
MeO
5k
>50
>50
>50
>50
e
e
5l
>50
>50
e
N
H
MeO
N
H
CO2H
CO2H
OMe
MeO
BnO
O2N
MeO
OME
5m
5n
18.2 ꢃ 0.1
12.3 ꢃ 1.3
1.5
N
H
N
H
CO2H
CO2H
F
F
BnO
O2N
F
BnO
F
5o
7.2 ꢃ 0.2
3.5 ꢃ 0. 1
2.0 5p
11 ꢃ 0.3
9.0 ꢃ 0.4
1.2
N
H
MeO
N
H
CO2H
F
CO2H
BnO
F
5q
3.2 ꢃ 0.4
6.9 ꢃ 0.1
0.5
N
H
CO2H
a
The values represent means of three individual experiments ꢃ SD.
Selectivity (S) ¼ IC50 cathepsin L/IC50 cathepsin V.
b
3H); 7.15 (ddd, 1H, J 1.9, 1.6, 1.2 Hz); 7.32 (d, 2H, J 7.8 Hz); 7.62 (ddd,
1H, J 0.7, 0.9, 1.2 Hz) 7.94e7.98 (m, 3H); 8.98 (dd, 1H, J 0.7, 7.8 Hz);
4.1.1.9. N-(2-Acetylphenyl)-4-butoxybenzamide (1i). Yield 85%. Mp
125 ꢀC (dec.). IR (nmax, KBr): 487, 522, 609, 682, 763, 840, 980, 1002,
1037, 1124, 1178, 1247, 1315, 1359, 1448, 1508, 1533, 1583, 1606,
12.66 (s, 1H). 13C NMR (50 MHz, CDCl3)
d: 21.3, 28.8, 121.6, 123.7,
127.8, 128.8, 130.8, 131.3, 136.9, 137.4, 140.2, 141.6, 164.0, 203.7. MS
1643, 1676, 2867, 2941, 2952, 3249, 3271, 3288 cmꢂ1 1H NMR
.
(m/z): 253 (Mþ), 235, 210, 119 (100), 106, 91, 77, 65, 41.
(200 MHz, CDCl3) d: 0.98 (t, 3H, J 7.3 Hz),1.50 (sex, 2H, J 7.3 Hz); 1.79

16
E.F. Marques et al. / European Journal of Medicinal Chemistry 54 (2012) 10e21
Table 5
Structures of acridones and values of IC50 on cathepsin L and cathepsin V.
Structure
CatL IC50
M)a
CatV IC50
M)a
Sb
Structure
IC50 CatL
M)a
IC50 CatV
(m
M)a
Sb
(
m
(
m
(
m
O
O
6a
3.9 ꢃ 0.1
3.6 ꢃ 0.1
1.1
6c
1.1 ꢃ 0.02
1.7 ꢃ 0.09
0.6
BnO
BnO
N
H
MeO
N
H
O
O
MeO
NO2
6d
NDc
ND
e
6e
6g
13 ꢃ 0.1
8.2 ꢃ 0.2
1.6
0.3
N
H
BnO
BnO
N
H
O
O
MeO
NO2
NO2
6f
1.6 ꢃ 0.02
2.0 ꢃ 0.02
0.8
0.5 ꢃ 0.01
1.7 ꢃ 0.08
N
H
N
H
O
O
NO2
6h
6.8 ꢃ 0.08
19.3 ꢃ 0.1
7.2 ꢃ 0.08
0.9
1.1
6m
6q
ND
ND
e
N
H
N
H
OMe
O
O
BnO
F
F
BnO
F
F
6p
18 ꢃ 0.2
1.5 ꢃ 0.1
4.9 ꢃ 0.3
0.3
MeO
N
H
N
H
a
The values represent means of three individual experiments ꢃ SD.
Selectivity (S) ¼ IC50 cathepsin L/IC50 cathepsin V.
ND ¼ not determined.
b
c
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
18
A
B
16
14
12
10
8
6
4
2
0
-0.4
-0.2
0.0
0.2
0.4
-0.1
0.0
0.1
0.2
0.3
0.4
1/ [ZRFMCA] (µM -1
)
1/ [ZRFMCA] (µM -1
)
Fig. 1. Uncompetitive inhibition profile of compound 5i (A) against catV and 5j (B) against catL. Kinetic measurements were conducted in the presence of increasing concentrations
of inhibitors. Panel A: 5 M ( ), 3 M ( ) and 1 M (B). Panel B: 7 M ( ), 5 M ( ) and 3 M (B). The absence of inhibitor is depicted by C in panels A and B.
m
6
m
m
m
6
m
m
;
;

E.F. Marques et al. / European Journal of Medicinal Chemistry 54 (2012) 10e21
17
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
D
C
5
4
3
2
1
0
-0.10
-0.05
0.00
0.05
0.10
0.15
0.20
-0.2
-0.1
0.0
0.1
0.2
0.3
0.4
1/ [ZRFMCA] (µM -1
)
1/ [ZRFMCA] (µM -1
)
Fig. 2. Competitive inhibition profile of compound 6a (C) against catL and 6g (D) against catV. Kinetic measurements were conducted in the presence of increasing concentrations of
inhibitors. Panel C: 5 M ( ), 3 M ( ) and 1 M (B). Panel D: 5 M ( ), 3 M ( ) and 1 M (B). The absence of inhibitor is depicted by C in panels A and B.
m
6
m
m
m
6
m
m
;
;
(qui, 2H, J 7.3 Hz); 2.70 (s, 3H); 4.02 (t, 2H, J 6.5 Hz); 6.98 (dt, 2H, J
2.1, 2.9 Hz); 7.12 (ddd, 1H, J 1.4, 0.9, 1.2 Hz), 7.59 (ddd, 1H, J 1.6, 1.2,
1.4 Hz); 7.93 (dd, 1H, J 1.6, 1.4 Hz); 8.02 (dt, 2H, J 2.9, 2.1 Hz); 8.96
22.5, 28.6, 28.8, 31.1, 31.6, 35.8, 120.7, 121.8, 122.3, 127.4, 128.4,
128.8, 130.1, 131.7, 132.1, 135.3, 141.5, 147.4, 166.1, 203.1. MS (m/z):
323 (Mþ), 305, 280, 248, 234, 222, 210, 189 (100), 180, 162, 145, 131,
118, 103, 91, 77, 65, 40.
(dd, 1H, J 1.2, 0.9 Hz); 12.61 (s, 1H). 13C NMR (50 MHz, CDCl3)
d: 13.9,
22.3, 28.5, 33.2, 61.0, 120.8, 122.3, 127.5, 128.8, 130.6, 131.8, 135.3,
141.5, 147.4, 164.5, 216.1. MS (m/z): 311(Mþ), 281, 268, 207, 177, 149,
137, 121(100), 107, 94, 91, 65, 55.
4.1.1.11. N-(2-Acetylphenyl)-4-fluoro-3-(trifluoromethyl)benzamide
(1k). Yield 84%. Mp 120 ꢀC (dec.). IR (nmax, KBr): 613, 763, 844, 910,
966, 1056, 1124, 1161, 1245, 1326, 1363, 1452, 1500, 1544, 1593, 1610,
4.1.1.10. N-(2-Acetylphenyl)-4-hexylbenzamide (1j). Yield 79%. Mp
130 ꢀC (dec.). IR (nmax, KBr): 530, 609, 690, 757, 850, 896, 954, 1018,
1118, 1164, 1247, 1315, 1359, 1452, 1508, 1537, 1585, 1639, 1679,
1650, 1679, 3124, 3157 cmꢂ1. 1H NMR (200 MHz, CDCl3)
d: 2.72 (s,
3H); 7.19 (ddd, 1H, J 0.7, 1.2, 6.0 Hz); 7.34 (t, 1H, J 9.3 Hz); 7.63 (ddd,
1H, J 1.2, 1.5, 5.9 Hz); 7.97 (dd, 1H, J 1.5, 6.0 Hz); 8.22e8.26 (m, 1H);
8.36 (dd, 1H, J 2.0, 4.5 Hz); 8.89 (dd, 1H, J 2.0, 7.5 Hz); 12.84 (s, 1H).
2848, 2916, 2927, 2948 cmꢂ1. 1H NMR (200 MHz, CDCl3)
d: 0.89 (t,
3H, J 7.3 Hz); 1.28e1.40 (m, 6H); 1.64 (qui, 2H, J 7.8 Hz); 2.68 (t, 2H, J
7.8 Hz); 2.71 (s, 1H); 7.15 (ddd, 1H, J 1.2, 1.3, 6.0 Hz); 7.31e7.34 (m,
2H); 7.61 (ddd, 1H, J 1.2, 1.5, 5.9 Hz); 7.94e8.00 (m, 3H); 8.98 (dd,
13C NMR (50 MHz, CDCl3)
d: 28.4, 115.9, 119.3, 121.0, 121.8, 122.3,
123.7,124.9,125.4,127.6,128.1,130.5,131.0,136.1,138.3,144.2,158.6,
161.0,164.3, 203.6. MS (m/z): 325 (Mþ), 310, 306, 282, 279, 264, 244,
234, 207, 191 (100), 170, 163, 143, 134, 106, 92, 75, 65, 43, 40.
1H, J 1.2, 7.3 Hz); 12.66 (s, 1H). 13C NMR (50 MHz, CDCl3)
d: 14.0,
8
2,0
E
F
6
1,5
1,0
0,5
0,0
4
2
0
-0,04
-0,02
0,00
0,02
0,04
0,06
0,08
0,10
0,12
0,14
-0,5
0,0
0,5
1,0
1,5
1/ [ZRFMC] (µM-1)
1/ [ZRFMC] (µM-1)
Fig. 3. Noncompetitive inhibition profile of compound 2b (E) against catV and 2b (F), against catL. Kinetic measurements were conducted in the presence of increasing concen-
trations of inhibitors. Panel E: 8.5 M ( ), 7.5 M ( ) and 2.5 M (B). Panel F: 7 M ( ), 5 M ( ) and 3 M (B). The absence of inhibitor is depicted by C in panels A and B.
m
6
m
m
m
6
m
m
;
;

18
E.F. Marques et al. / European Journal of Medicinal Chemistry 54 (2012) 10e21
1502, 1550, 1597, 1639, 1658, 2846, 2880, 2949, 3001 cmꢂ1. 1H NMR
(200 MHz, CD3OD) : 1.54 (t, 2H, J 1.7 Hz); 1.94 (t, 2H, J 1.7 Hz);
Table 6
Ki values of compounds 5i, 5j, 6a and 6g.
d
5.06e5.23 (m, 2H); 5.86e6.06 (m, 1H); 6.72 (s, 1H); 7.18 (ddd, 1H, J
1.4, 1.2, 0.6 Hz), 7.63 (dt, 1H, J 1.6, 1.4 Hz); 7.97 (t, 1H, J 1.6 Hz); 8.85
Compound
Ki (mM)
Cathepsin L
Cathepsin V
(dd, 1H, J 1.2, 0.6 Hz). 13C NMR (50 MHz, CD3OD)
d: 30.7, 34.1, 104.5,
2b
5i
5j
6a
6g
1.5 noncomp
1.9 uncomp
1.9 uncomp
0.9 comp
0.8 noncomp
0.8 uncomp
1.1 uncomp
1.3 comp
111.7, 114.7, 118.6, 125.5, 129.5, 133.1, 136.1, 138.4, 146.6, 153.1, 177.9.
Anal. Calc. for C13H13NO: C 78.39; H 6.53; N 7.03. Found: C 78.74; H
6.40; N 6.85. MS (m/z): 199 (Mþ), 184 (100), 166, 154, 140, 128, 115,
92, 89, 77, 51.
0.2 comp
0.4 comp
4.1.2.6. 2-(4-Fluorophenyl)quinolin-4(1H)-one (2f) [70]. Yield 72%.
Mp ꢄ300ꢀ. IR (nmax, KBr): 519, 592, 749, 830, 1162, 1240, 1400, 1514,
4.1.2. General procedure for the preparation of 4-quinolinones
derivatives 2
1592, 1606, 1635 cmꢂ1. 1H NMR (200 MHz, DMSO-d6)
d: 6.89 (s, 1H);
A 10 mL vial containing a magnetic stirring bar was charged
with amides 1aej (0.3 mmol), t-BuOK (168.3 mg, 1.5 mmol) and
THF (1.5 mL). The vial was sealed and the resulting suspension was
heated at 100e120 ꢀC, and 180 W for 10e20 min. The reaction
mixture was cooled and poured in water (8 mL) and a solution of
HCl 1 mol Lꢂ1 was added to adjusted pH 5e6. The solution was
concentrated under reduced pressure until copious solid appeared.
The solid was collected and washed successively with water and
a cold mixture of acetone and dichloromethane (1:1 ratio) to give
the pure product.
7.41e7.53 (m, 4H), 7.81 (ddd, 1H, J 1.2, 1.0, 0.8 Hz); 8.00e8.07 (m,
1H); 8.15e8.23 (m, 2H). 13C NMR (50 MHz, DMSO-d6)
d
: 104.4,
105.9, 114.6, 115.8, 116.2, 119.6, 123.9, 124.9, 130.5, 130.7, 132.6,
140.5, 150.9, 161.2, 166.2, 191.3.
4.1.2.7. 2-(4-Ethylphenyl)quinolin-4(1H)-one (2g). Yield 64%. Mp
ꢄ300ꢀ. IR (nmax, KBr): 532, 838, 1110, 1240, 1382, 1423, 1487, 1517,
1596, 1641, 2831, 2937, 2964, 3028 cmꢂ1 1H NMR (200 MHz,
.
CD3OD) d: 1.32 (t, 3H, J 7.7 Hz); 2.81 (q, 2 H, J 7.7 Hz); 7.36 (s, 1H);
7.56 (dt, 2H, J 1.9, 0.6 Hz); 7.82 (q, 1H, J 1.2 Hz); 7.93 (t, 2H, J 1.9 Hz);
8.08 (dt, 1H, J 1.6, 1.4 Hz); 8.20 (dd, 1H, J 0.8, 0.6 Hz); 8.46 (dd, 1H, J
4.1.2.1. 2-Phenyl-quinolin-4(1H)-one (2a) [67]. Yield 60%. Mp
255 ꢀC (dec.). IR (nmax, KBr): 690, 755, 772, 799, 839, 1474, 1505,
1547, 1583, 1595, 1610, 1636, 2329, 2357, 2969, 3067, 3090 cmꢂ1. 1H
0.8, 0.6 Hz). 13C NMR (50 MHz, DMSO-d6)
d: 15.2, 28.0, 105.9, 120.0,
120.2, 123.3, 126.9, 128.6, 128.7, 129.2, 133.9, 140.1, 148.4, 154.1,
170.4. Anal. Calc. for C17H15NO: C 81.93; H 6.02; N 5.62. Found: C
82.05; H 5.93; N 5.74.
NMR (200 MHz, CD3OD)
d: 6.54 (s, 1H); 7.32e7.73 (m, 5H); 7.98 (dd,
2H, J 1.4, 1.3 Hz); 8.25 (d, 1H, J 8.2 Hz); 8.83 (d, 1H, J 8.2 Hz). 13C NMR
(50 MHz, CDCl3) d: 105.6, 118.9, 121.3, 125.0, 125.7, 129.4, 129.5,
4.1.2.8. 2-p-Tolylquinolin-4(1H)-one (2h) [47]. Yield 68%. Mp
ꢄ300ꢀ. IR (nmax, KBr): 482, 514, 536, 567, 671, 756, 815, 873, 958,
1024, 1141, 1186, 1245, 1315, 1357, 1440, 1471, 1510, 1542, 1595, 1635,
1652, 1701, 2914, 2964, 3066, 3087, 3116 cmꢂ1. 1H NMR (200 MHz,
131.8, 139.6, 158.0, 176.9. MS (m/z): 221 (Mþ), 204, 193, 189, 161, 135,
119, 95, 91, 67, 55 (100).
4.1.2.2. 2-(4-Butylphenyl)quinolin-4(1H)-one (2b). Yield 67%. Mp
ꢄ300 ꢀC. IR (nmax, KBr): 752, 829, 1257, 1319, 1400, 1440, 1500, 1539,
1573, 1595, 1631, 2858, 2927, 2952, 3074, 3147, 3257 cmꢂ1. 1H NMR
CD3OD)
1H, J 1.6, 1.7, 4.7 Hz), 7.73 (dd, 1H, J 1.7, 8.0 Hz); 7.80e7.90 (m, 2H);
8.31 (dd, 1H, J 0.8, 6.3 Hz). 13C NMR (50 MHz, CD3OD)
: 22.7, 103.7,
d: 2.46 (s, 3H); 6.78 (s, 1H); 7.42 (d, 2H, J 8.0 Hz); 7.52 (ddd,
d
(200 MHz, CD3OD)
1.58 (qui, 2H, J 7.7 Hz); 2.64 (t, 2H, J 7.7 Hz); 6.49 (s, 1H); 7.31e7.67
(m, 7H); 8.18 (d, 1H, J 8.8 Hz). 13C NMR (50 MHz, CD3OD)
: 14.2,
d
: 0.88 (t, 3H, J 7.2 Hz); 1.31 (sex, 2H, J 7.4 Hz);
119.6, 123.7, 125.4, 125.6, 126.7, 127.9, 128.6, 132.8, 140.3, 141.5,
153.6, 176.4.
d
23.3, 33.7, 34.6, 105.2, 119.7, 125.4, 126.0, 128.5, 130.4, 139.7, 147.8,
157.9, 175.4. Anal. Calc. for C19H19NO: C 82.31; H 6.86; N 5.05.
Found: C 81.98; H 6.51; N 4.92.
4.1.2.9. 2-(4-Butoxyphenyl)quinolin-4(1H)-one (2i). Yield 65%. Mp
ꢄ300ꢀ. IR (nmax, KBr): 540, 838, 1006, 1188, 1236, 1271, 1429, 1494,
1515, 1598, 1645, 2871, 2937, 2970, 3365 cmꢂ1. 1H NMR (200 MHz,
CD3OD) d: 1.01 (t, 3H, J 7.2 Hz); 1.55 (sex, 2H, J 7.2 Hz); 1.82 (qui, 2H, J
4.1.2.3. 2-(Furan-2-yl)quinolin-4(1H)-one (2c) [68]. Yield 73%. Mp
248 ꢀC (dec.). IR (nmax, KBr): 747, 762, 831, 839, 1465, 1501, 1569,
5.9 Hz); 4.13 (t, 2H, J 6.2 Hz); 7.22 (dt, 2H, J 2.9, 1.9 Hz); 7.31 (s, 1H);
7.79 (q,1H, J 1.2 Hz); 7.96 (dt, 2H, J 2.2,1.9 Hz), 8.05 (ddd,1H, J 1.7, 1.5,
1.2 Hz); 8.15 (dd,1H, J 1.5,1.4 Hz); 8.43 (dd,1H, J 1.0, 0.9 Hz).13C NMR
1647, 1699, 2336, 2359, 2860, 2916 cmꢂ1 1H NMR (200 MHz,
.
DMSO-d6)
d: 7.27 (s, 1H); 7.58 (dd, 1H, J 1.8, 1.9 Hz); 8.11 (ddd, 1H, J
(50 MHz, CD3OD) d: 12.7, 19.5, 31.2, 67.1, 104.3, 115.0, 118.0, 121.1,
1.5, 1.3, 1.2 Hz); 8.25 (d, 1H, J 0.5 Hz); 8.45 (dt, 1H, J 1.7, 1.5 Hz); 8.57
123.2, 124.9, 125.9, 132.6, 140.0, 154.9, 160.8, 176.7. Anal. Calc. for
C19H19NO2: C 77.81; H 6.48; N 4.77. Found: C 77.68; H 6.15; N 5.09.
(dd, 1H, J 0.8, 0.7 Hz); 8.81e8.89 (m, 2H), 12.46 (s, 1H). 13C NMR
(50 MHz, DMSO-d6) d: 103.8, 111.8, 116.8, 122.5, 124.6, 126.1, 131.9,
139.0, 140.9, 145.6, 154.8, 177.7. MS (m/z): 207 (Mþ), 177, 162, 149,
135 (100), 120, 107, 98, 82, 69, 55.
4.1.2.10. 2-(4-Hexylphenyl)quinolin-4(1H)-one (2j). Yield 63%. Mp
ꢄ300ꢀ. IR (nmax, KBr): 526, 584, 649, 756, 815, 829, 865, 1022, 1143,
1257, 1319, 1440, 1500, 1541, 1595, 1633, 2854, 2927, 2956,
4.1.2.4. 2-Undecylquinolin-4(1H)-one (2d) [69]. Yield 55%. Mp
155 ꢀC (dec.). IR (nmax, KBr): 756, 765, 1246, 1356, 1365, 1441, 1489,
1552, 1593, 1657, 1713, 2331, 2358, 2850, 2870, 2920, 2954 cmꢂ1. 1H
3006 cmꢂ1 1H NMR (200 MHz, CD3OD)
. d: 0.91 (t, 3H, J 6.9 Hz);
1.28e1.46 (m, 6H); 1.70 (qui, 2H, J 8.1 Hz); 2.77 (t, 2H, J 7.3 Hz); 7.37
(s, 1H); 7.53 (d, 2H, J 8.5 Hz); 7.81 (dt, 1H, J 1.4, 1.2 Hz), 7.93 (ddd, 2H,
J 2.0, 1.9, 1.8 Hz); 8.08 (ddd, 1H, J 1.6, 1.5, 1.4 Hz); 8.20 (dd, 1H, J 0.8,
NMR (200 MHz, CD3OD) d: 0.86 (t, 3H, J 6.5 Hz); 1.22e1.52 (m, 16H);
1.71 (qui, 2H, J 7.8 Hz); 2.69 (t, 2H, J 7.9 Hz); 6.19 (s, 1H); 7.35e7.41
0.6 Hz). 13C NMR (50 MHz, CD3OD)
d: 14.2, 22.2, 28.4, 31.1, 31.7, 37.3,
(m, 1H); 7.66e7.70 (m, 2H); 8.11 (d, 1H, J 8.0 Hz). 13C NMR (50 MHz,
104.8,118.1,122.9,125.7,125.8, 125.9,126.5,127.3,127.8,132.6,140.1,
142.6, 153.8, 177.2. Anal. Calc. for C21H23NO: C 82.62; H 7.54; N 4.59.
Found: C 82.34; H 7.24; N 4.27.
CD3OD) d: 22.7, 25.6, 26.9, 28.7, 28.8, 29.4, 29.6, 29.7, 31.7, 34.9,
107.6, 118.13, 123.8, 124.5, 124.7, 131.6, 139.3, 156.7, 179.3. MS (m/z):
274 (Mþ), 177, 162, 135, 121, 120, 106, 92, 69, 55.
4.1.2.11. 2-(4-Fluoro-3-(trifluoromethyl)phenyl)quinolin-4(1H)-one
(2k). Yield 54%. Mp ꢄ300ꢀ. IR (nmax, KBr): 757, 833, 1139, 1232,
4.1.2.5. 2-(But-3-enyl)quinolin-4(1H)-one (2e). Yield 20%. Mp
185 ꢀC (dec.). IR (nmax, KBr): 455, 520, 667, 753, 908, 1387, 1430,
1247, 1504, 1602, 1633 cmꢂ1. 1H NMR (200 MHz, CD3OD)
d: 6.57 (s,

E.F. Marques et al. / European Journal of Medicinal Chemistry 54 (2012) 10e21
19
1H); 6.99 (d, 1H, J 8.6 Hz); 7.42 (ddd, 1H, J 0.8, 1.3, 4.7 Hz); 7.71e7.81
(m, 3H); 8.24 (dd, 1H, J 0.8, 6.8 Hz); 8.32 (d, 1H, J 2.5 Hz). 13C NMR
7.25e7.48 (m, 8H); 7.94 (d, 1H, J 10 Hz); 8.51 (d, 1H, J 10.0 Hz); 8.66
(dd, 1H, J 10.0, 2.0 Hz); 9.47 (d, 1H, J 2.0 Hz). 13C NMR (50 MHz;
(50 MHz, CD3OD)
d: 104.3, 112.9, 114.6, 119.4, 120.4, 120.8, 123.1,
CDCl3/TFA 10%) d: 72.1; 99.2; 111.9; 114.19; 120.2; 121.2; 122.8;
125.7, 125.9, 126.6, 126.8, 127.7, 131.4, 132.7, 133.2, 139.6, 147.0, 156.1,
161.1, 176.2. Anal. Calc. for C16H9NOF4: C 62.54; H 2.93; N 4.56.
Found: C 62.31; H 3.01; N 4.33.
127.3; 127.8 (2C); 129,1 (2C); 129.2; 129.6; 134.2; 142.7; 143.9;
145.2; 168.2; 170.9. Anal. Calc. For C20H14N2O4: C 69.36; H 4.07; N
8.09. Found: C 69.27; H 4.10; N 8.49.
4.1.3. General procedure for the preparation of acridone alkaloids 6
A mixture of N-arylanthranilic acid [56] 5 (0.16 mmol) and POCl3
(1.0 mL) was refluxed for 2 h under dry nitrogen atmosphere. POCl3
excess was removed under reduced pressure and an 8:1 solution of
ethanol: 10% chloridric acid (5.0 mL) was added to the crude
product and the mixture was refluxed during 1 h. After this time,
the reaction mixture was allowed to cool, water (5.0 mL) was added
and the resulting precipitated was removed by filtration, washed
with water and dried in a dissector to give 6 as yellow solid.
4.1.3.7. 2-Nitroacridin-9(10H)-one (6h). Yield 82%. Mp 295 ꢀC (dec.);
spectral data were according to those previously reported [71].
4.1.3.8. 4-Methoxyacridin-9(10H)-one (6m). Yield 99%. Mp 295 ꢀC;
IR (nmax, KBr): 1603, 1331, 1156 cmꢂ1. 1H NMR (200 MHz; CDCl3/TFA
10%)
d
: 4.05 (s, 3H); 7.15e7.47 (m, 4H); 7.84e7.95 (m, 3H); 8.36 (d,
1H, J 8 Hz). 13C NMR (50 MHz; CDCl3/TFA 10%)
d: 56.4; 113.2; 115.8;
116.3; 116.5; 118.5; 124.9; 125.5; 132.2; 136.6; 139.6; 147.4; 171.7.
Anal. Calc. For C14H11NO2: C 74.65; H 4.92; N 6.22. Found: C 74.57; H
5.02; N 6.54.
4.1.3.1. 3-(Benzyloxy)acridin-9(10H)-one (6a). Yield 82%. Mp 327 ꢀC
(dec.); 1H NMR (200 MHz; CDCl3/TFA 10%)
d: 11.98 (s, 1H), 8.25 (d,
4.1.3.9. 2-(Benzyloxy)-6,7-difluoro-3-methoxyacridin-9(10H)-one
(6p). Yield 93%. Mp 294 ꢀC (dec.). IR (nmax, KBr): 3266, 3186, 1649,
2H, J 8.0 Hz), 7.81 (t, 1H, J 8.0 Hz), 7.67 (d, 1H, J 8.0 Hz), 7.43 (m, 6H),
7.14 (dd, 1H, J 10.0, 2.0 Hz), 7.05 (d, 1H, J 2.0 Hz), 5.20 (s, 2H). 13C
1288, 1200, 1093, 873, 807, 748, 698 cmꢂ1 1H NMR (200 MHz;
.
NMR (50 MHz; CDCl3/TFA 10%)
d
: 167.4, 1662.2, 143.6, 140.0, 136.6,
CDCl3/TFA 10%)
d: 4.06 (s, 3H); 5.19 (s, 2H); 7.07 (s, 1H); 7.36e7.69
134.3, 129.0, 127.6, 126.1, 125.6, 124.0, 120.2, 118.1, 114.5, 110.0, 97.8,
71.3. Anal. Calc. for C20H15NO2: C 79.72; H 5.02; N 4.65. Found: C
79.63; H 4.91; 4.54.
(m, 8H); 7.95e8.04 (m, 1H). 13C NMR (50 MHz; CDCl3/TFA 10%)
d:
56.9; 71.57; 97.44; 102.1; 102.9; 105.7 (d, J 22 Hz); 101.1; 110.7 (d, J
22 Hz); 111.8; 127.8 (2C); 128.5 (2C); 134.4; 136.4; 139.8; 149.0;
159.9; 164.7. Anal. Calc. For C21H15F2NO3: C 68.66; H 4.12; N 3.81.
Found: C 68.59; H 4.18; N 3.92.
4.1.3.2. 2-(Benzyloxy)-3-methoxyacridin-9(10H)-one
85%. Mp 297 ꢀC (dec.); IR (nmax, KBr): 3146, 1633, 1591, 1562, 1501,
1276, 1128, 746 cmꢂ1. 1H NMR (200 MHz; CDCl3/TFA 10%)
: 3.98 (s,
3H); 5.08 (s, 2H); 6.90 (s, 1H); 7.26e7.68 (m, 10H); 8.10 (d, 1H, J
8.0 Hz). 13C NMR (50 MHz; CDCl3/TFA 10%)
: 56.7; 70.9; 97.1; 102.0;
(6c). Yield
d
4.1.3.10. 7-(Benzyloxy)-2,3-difluoroacridin-9(10H)-one (6q). Yield
93%. Mp 294 ꢀC (dec.). IR (nmax, KBr): 3100, 2919, 1647, 1599, 1490,
1382,1289,1097, 784, 564 cmꢂ1. 1H NMR (200 MHz; CDCl3/TFA 10%)
d
109.8; 114.8; 117.8; 123.8; 124.9; 127.6 (2C); 128.6; 128.7 (2C);
134.7; 134.8; 138.3; 138.9; 148.0; 159.7; 165.3. Anal. Calc. for
C21H17NO3: C 76.12; H 5.17; N 4.23. Found: C 75.91; H 5.15; N 4.55.
d
: 5.26 (s, 2H); 7.40e7.46 (m, 6H); 7.73e7.91 (m, 4H); 8.33e8.38 (m,
1H). 13C NMR (50 MHz; CDCl3/TFA 10%)
d: 71.5; 101.6; 106.5; 101.7
(d, J 22 Hz); 112.6; 116.2; 120.7; 127.9 (2C); 128.0; 129.0 (2C); 132.6;
135.0; 137.6; 137.7; 157.7; 166.7. Anal. Calc. For C21H15F2NO3: C
68.66; H 4.12; N 3.81. Found: C 68.59; H 4.18; N 3.92.
4.1.3.3. 2-Methoxyacridin-9(10H)-one (6d). Yield 89%. Mp 278 ꢀC
(dec.); IR (nmax, KBr): 2983, 1683, 1591, 1533, 1475, 1364, 1233, 1160,
1033, 757 cmꢂ1. 1H NMR (200 MHz; CDCl3/TFA 10%)
d
: 3.84 (s, 3H);
7.27e7.74 (m, 7H); 8.17 (d, 1H, J 8 Hz). 13C NMR (50 MHz; CDCl3/TFA
10%) : 55.6; 100.4; 115,0; 116.0; 118,2; 119.9; 124.1; 125.2; 130.2;
4.2. Kinetic measurements
d
Recombinant human cathepsins V and L were produced using
the Pichia pastoris expression system as previously described [72].
The molar concentrations of the enzymes were determined by
active site titration using E-64 following the conditions previously
described [73].
All commercially available reagents were purchased from
Aldrich Chemical Co and Sigma. Kinetic measurements were
carried out in a Molecular Devices Spectra MAX GEMINI XS. Stock
solutions of the compounds were prepared at a concentration of
1 mM in DMSO, and the inhibitors were screened against the
135.4; 136.0; 138.8; 156.7; 167.8. Anal. Calc. for C20H17NO3: C 74.65;
H 4.92; N. 6.22. Found: C 74.57; H 4.15; N 7.13.
4.1.3.4. 6-(Benzyloxy)-2-nitroacridin-9(10H)-one (6e). Yield 90%.
Mp 300 ꢀC (dec.); IR (nmax, KBr): 3188, 2912, 2852, 1638, 1603, 1570,
1507, 1471, 1322, 1182, 1122, 817, 552 cmꢂ1 1H NMR (200 MHz;
.
CDCl3/TFA 10%)
1H, J 10 Hz); 8.45 (d,1H, J 8.0 Hz); 8.57 (dd,1H, J 8.0, 2.0 Hz); 9.37 (d,
1H, J 2.0 Hz). 13C NMR (50 MHz; CDCl3/TFA 10%)
: 71.9; 99.1; 103.1;
d: 7.19 (d, 1H, J 2.0 Hz); 7.26e7.68 (m, 7H); 7.87 (d,
d
115.2; 119.1; 120.6; 122.8; 127.5; 127.8 (2C); 129.1 (2C); 129.2;
129.3; 134.2; 142.6; 143.6; 144.8; 167.8; 171.4. Anal. Calc. For
C20H14N2O4: C 69.36; H 4.07; N 8.09. Found: C 69.45; H 4.28; N 8.27.
cathepsins V and L at initial concentration of 25 mM. Inhibition
measurements were carried out in triplicate in 96-well black plates
as previously described [43,44].
The reaction mixture contained 192
buffer (100 mM, 5 mM EDTA, 5 mM DTE, pH 5.5), 2
Phe-Arg-MCA, 5 L of inhibitor, and 1 L of catV (32 nM) or catL
m
L of a sodium acetate
4.1.3.5. 2-Methoxy-7-nitroacridin-9(10H)-one (6f). Yield 87%. Mp
>350 ꢀC; IR (nmax, KBr): 3097, 1639, 1607, 1574, 1494, 1332, 1155,
mL of 1 mM Z-
m
m
826, 749 cmꢂ1. 1H NMR (200 MHz; CDCl3/TFA 10%)
d
: 4.04 (s, 3H);
7.81e7.95 (m, 3H); 8.03 (d,1H, J 10 Hz); 8.66 (d,1H, J 8.0 Hz); 9.54 (s,
1H). 13C NMR (50 MHz; CDCl3/TFA 10%)
: 56.2; 102.0; 115.9; 118.6;
(20 nM). The enzymes were activated with DTE at 27 ꢀC for 5 min.
Z-Phe-Arg-MCA was added to start the reaction, and the fluores-
cence of released 4-methyl-7-coumaryl-amide was measured at Ex
355 nm and Em 460 nm. Control assays were performed without
inhibitor (negative control) and in the presence of the E-64 (posi-
tive control), an irreversible inhibitor for cysteine peptidase. The
percentage of inhibition was calculated according to the equation:
% inhibition ¼ 100 ꢁ (1 ꢂ Vi/V0), where Vi and V0 are initial veloc-
ities (enzyme activities) determined in the presence and in the
absence of inhibitor, respectively. The selectivity ratio of the
d
120.7; 120.8; 122.9; 128.9; 132.6; 137.9; 141.5; 144.0; 158.7; 171.9.
Anal. Calc. For C14H10N2O4: C 62.22; H 3.73; N 10.37. Found: C 62.73;
H 3.92; N 10.56.
4.1.3.6. 2-(Benzyloxy)-7-nitroacridin-9(10H)-one (6g). Yield 78%.
Mp 295 ꢀC (dec.); IR (nmax, KBr): 3269, 3152, 1612, 1604, 1569, 1329,
1181, 748 cmꢂ1. 1H NMR (200 MHz; CDCl3/TFA 10%)
d: 5.34 (s, 2H);

20
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We gratefully acknowledge financial support from the State of
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Scientific and Technological Development (CNPq, Conselho
Nacional de Pesquisa
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