Design and synthesis of novel quinolinone-3-aminoamides and their α-lipoic acid adducts as antioxidant and anti-inflammatory agents

Article abstract of DOI:10.1021/jm061173n

A series of N-substituted-quinolinone-3-aminoamides and their hybrids containing the α-lipoic acid functionality were designed and synthesized as potential bifunctional agents combining antioxidant and anti-inflammatory activity. The new compounds were ev

Full text of DOI:10.1021/jm061173n

2450
J. Med. Chem. 2007, 50, 2450-2458
Design and Synthesis of Novel Quinolinone-3-aminoamides and Their r-Lipoic Acid Adducts as
Antioxidant and Anti-inflammatory Agents
Anastasia Detsi,*.† Dionysia Bouloumbasi,† Kyriakos C. Prousis,† Maria Koufaki,‡ Giorgos Athanasellis,† Georgia Melagraki,†
Antreas Afantitis,† Olga Igglessi-Markopoulou,† Christos Kontogiorgis,§ and Dimitra J. Hadjipavlou-Litina§
Laboratory of Organic Chemistry, School of Chemical Engineering, National Technical UniVersity of Athens, Zorgafou Campus,
GR 157 73, Athens, Greece, Institute of Organic and Pharmaceutical Chemistry, National Hellenic Research Foundation, 48, Vas.
Constantinou AVenue, Athens 116 35, Greece, and Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle
UniVersity Thessaloniki, Thessaloniki 54124, Greece
ReceiVed October 10, 2006
A series of N-substituted-quinolinone-3-aminoamides and their hybrids containing the R-lipoic acid
functionality were designed and synthesized as potential bifunctional agents combining antioxidant and
anti-inflammatory activity. The new compounds were evaluated for their antioxidant activity and for their
ability to inhibit in vitro lipoxygenase as well as for their anti-inflammatory activity in vivo. In general, the
derivatives were found to be potent antioxidant or anti-inflammatory agents. The results are discussed in
terms of structure-activity relationships and an attempt is made to define the structural features required
for activity.
Introduction
moiety as a key structural feature have attracted the interest of
several research groups.7,8
The formation of reactive oxygen species (ROS) is charac-
teristic of aerobic organisms that can normally defend them-
selves against these highly reactive species using enzymes, like
superoxide dismutase and glutathione peroxidase, and naturally
occurring antioxidants, such as R-tocopherol (vitamin E),
ascorbic acid (vitamin C), and â-carotene. However, in many
pathophysiological conditions, the excessive production of ROS
overwhelms the natural antioxidant defense mechanisms. This
imbalance is termed oxidative stress.
Linomide (N-phenylmethyl-1,2-dihydro-4-hydroxy-1-methyl-
2-oxo-3-quinolinecarboxamide, Figure 1),9 is a synthetic im-
munomodulator, which is effective against various types of
cancers and autoimmune disorders, such as multiple sclerosis
(MS), rheumatoid arthritis, systemic lupus erythematosis, and
experimental autoimmune encephalomyelitis. The structure of
linomide has served as the prototype for the synthesis of a
variety of analogues in an effort to optimize this lead
compound.10-14
Oxidative stress has been associated with the inflammation
process. ROS, like superoxide radical anion, hydrogen peroxide,
and hydroxyl radical, are produced during the inflammation
process by phagocytic leukocytes (e.g., neutrophils, monocytes,
macrophages, eosinophils) that invade the tissue. Moreover,
these reactive species are involved in the biosynthesis of
prostaglandins and in the cycloxygenase- and lipoxygenase-
mediated conversion of arachidonic acid into proinflammatory
intermediates.1,2 In addition, ROS may initiate inflammation via
upregulating several different genes involved in this process by
activating certain redox-sensitive transcription factors as the
nuclear factor kB (NFkB) and activator protein-1 (AP-1).3,4
Another synthetic quinolinone derivative, rebamipide15 (2-
(4-chlorobenzoylamino)-3-[2-(1H)-quinolinon-4-yl]propionic acid,
Figure 1) is an effective antioxidant and antiulcer agent,
mediating its pharmacological activity mainly by increasing
endogenous prostaglandin synthesis and by ROS scavenging.16
The structural characteristics of rebamipide that have been
shown to be important for scavenging the hydroxyl radical are
the 3,4-double bond and the 2-oxo functionality of the quino-
linone moiety and the carbonyl part of the amido group in
conjunction with a p-chlorobenzyl function.17
The research group of Ishiwara et al. has recently developed
a novel quinolinone derivative, TA 270 (4-hydroxy-1-methyl-
3-octyloxy-7-sinapinoylamino-2(1H)-quinolinone, Figure 1).18
This compound was designed as an antioxidant, and further
studies showed that it is as well a potent inhibitor of immediate-
and late-airway responses, pulmonary inflammatory cell ac-
cumulation and, airway hyper-responsiveness, therefore, it may
be of therapeutic use for the treatment of bronchial asthma.
Studies of the action mechanisms of TA 270 in cellular level
in vitro revealed that it specifically suppresses the antigen-
induced leukotriene production, probably by inhibiting 5-li-
poxygenase in inflammatory cells.19
The rates of ROS production are increased in most patho-
physiological conditions,5 therefore, it is evident that the
treatment of various diseases could benefit from the use of drugs
that combine antioxidant and anti-inflammatory activity. This
has already been proven for a number of commercially available
nonsteroidal anti-inflammatory drugs (NSAIDs), which simul-
taneously possess radical scavenging properties.6
The quinolinone structure is characteristic of numerous natural
products and synthetic analogues that exhibit a wide variety of
biological activities. The antioxidant and anti-inflammatory
properties of various compounds possessing the quinolinone
A naturally occurring quinolinone alkaloid has been recently
isolated from the aleurone layer of Oryza satiVa cv Hengjinmi.
This compound exhibited moderate antioxidant activity in a
DPPH free radical scavenging assay.20
The potential use of quinolinone derivatives as therapeutic
agents to treat a wide range of inflammation-mediated diseases
* To whom correspondence should be addressed. Tel.: +302107723072.
Fax: +302107723072. E-mail: adetsi@chemeng.ntua.gr.
† National Technical University of Athens.
‡ National Hellenic Research Foundation.
§ Aristotle University Thessaloniki.
10.1021/jm061173n CCC: $37.00 © 2007 American Chemical Society
Published on Web 04/20/2007

Quinolinone-3-aminoamides and R-Lipoic Acid Adducts
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10 2451
According to Method A, quinolinone 3 or 4 was dissolved in
1,2-ethylenediamine or 1,6-hexamethylenediamine, and the
mixture was heated at 100-110 °C for 5-24 h38,46 to afford
aminoamides 5-8 in good yields (62-65%). In the case of
aromatic diamines, anhydrous toluene was used as a solvent,
and the mixture of quinolinone (1 equiv) and diamine (2 equiv)
was heated overnight at 100 °C (Method B). The aminoamides
formed were insoluble in toluene and precipitated upon forma-
tion, therefore, the isolation procedure involved only filtration
and washing with diethylether. Compounds 9-12 were obtained
in high yields (85-99%), and we were gratified to find out that
no further purification was needed, because they produced
excellent elemental analyses data without recrystallization (see
Experimental Section). Because Method B was more advanta-
geous than Method A in isolation, yields, and purity of the
products, we have employed it also for the synthesis of the
aliphatic aminoamides and succeeded in improving the yields
considerably (75-80%) and obtaining analytically pure com-
pounds in a less tedious way. The use of a solvent in which the
resulting amide has low solubility has been previously reported
to increase the yields and purities of quinolinecarboxamides.13,47
The 1H NMR spectra of aminoamides 5-9, containing
aliphatic amines, were obtained in a mixture of CDCl3/CD3OD
or in CDCl3 containing drops of CF3COOD and are character-
ized by the presence of a broad signal at 10-10.45 ppm, owed
to the proton of the amide group (-CONH-). Compounds 9
and 10, produced from 1,2-phenylenediamine, were soluble in
CDCl3, and their spectra revealed the presence of a strongly
hydrogen-bonded proton at 16.6-16.8 ppm, the hydroxyl proton
on position 4 of the heterocyclic ring. This signal, appears at a
lower field than in the corresponding 1-methyl 4-hydroxy-2-
oxo-1,2-dihydro-quinoline-3-carboxylic acid methyl ester (3),
indicating that the electron density of the 3-carbonyl group has
been intensified. In the case of the 1,4-phenylenediamine-
containing aminocarboxamides 11 and 12, CF3COOD had to
be added in CDCl3 to transform the insoluble compounds to
their corresponding soluble TFA salts.
Figure 1.
is also depicted in several patents, which describe the synthesis
and anti-inflammatory activity of a variety of quinolinone
analogues.21
The aim of the present study was to synthesize novel
quinolinone-3-aminoamides bearing a methyl or phenyl group
on the nitrogen heteroatom and possessing a free amino
functionality, which could allow further chemical modifications.
The synthesis of the 4-hydroxy-2-quinolinone pharmacophoric
moiety has already been reported by the research group of
Igglessi-Markopoulou et al., as part of our long-standing interest
in the development of novel methodologies for the synthesis of
this molecular scaffold and analogous fused heterocyclic
systems.22,23 Moreover, because the combination of two phar-
macophores on the same scaffold is a well established approach
to the synthesis of more potent drugs with dual activity,24-32
we decided to also incorporate the R-lipoic acid (LA) moiety
and examine the influence of this modification on the activity
of the aminoamides.
The remarkable antioxidant properties of LA33-35 have been
shown to be coupled with interesting anti-inflammatory activ-
ity: LA possesses skin anti-inflammatory activity when admin-
istered orally36 and can effectively suppress allergic inflamma-
tion and airway hyper-responsiveness in a mouse model of
asthma, therefore, it is a possible candidate for adjuvant
antiasthmatic therapy.37 Moreover, thiazolidinedione-LA adducts
can be potentially useful as oral and topical agents for treating
inflammatory skin conditions.38 LA has been widely used for
the synthesis of various conjugates, possessing multifunctional
activity.25,27,31,32,39-45
To synthesize the hybrids of quinolinone-3-aminoamides with
LA (Scheme 2), racemic R-LA was converted to its N-
hydroxysuccinimidyl-ester 13.48 This activation was chosen
instead of, for example, transformation to the acyl-chloride,
because the ester is easily prepared in high yields, can be stored
for a long time, and the N-hydroxysuccinimide formed as a
byproduct is a water-soluble compound and can be efficiently
removed from the reaction mixture.
The synthesis of conjugates 14-18 was effected by dissolving
equimolar amounts of compounds 5-8 or 10 with the active
ester 13 in dichloromethane containing a few drops of dimeth-
Chemistry
1
ylformamide to facilitate dissolution. Their H NMR spectra
The synthesis of the starting 1-methyl and 1-phenyl-4-
hydroxy-2-oxo-1,2-dihydro-quinoline-3-carboxylic acid methyl
esters 3 and 4 (Scheme 1) was accomplished using our
previously developed methodology.22 The appropriate com-
mercially available N-substituted anthranilic acids 1 and 2 were
treated with equimolar amounts of N-hydroxybenzotriazole and
dicyclohexylcarbodiimide (DCC) in anhydrous THF, and the
nonisolated active ester was used as an acylating agent. The
C-acylation of dimethyl malonate was performed using NaH
in THF to generate the malonate anion, which attacked the
carbonyl carbon of the active ester to produce the intermediate
C-acylation products 3a and 4a. The latter spontaneously
cyclized in the basic reaction conditions to provide compounds
3 and 4 in good yields.
were characterized by the signal of the intramolecular hydrogen-
bonded hydroxyl proton at low field (16.7-17.5 ppm) and the
signals of the amide protons at 10-11.9 ppm for the 3-CONH-
group and at 5.6-6.2 ppm for the -CONH- connected to
R-LA.
The predominance of the enol tautomeric form in the
3-aminoamides and the LA conjugates guarantees the existence
of a 3,4-double bond of the 2-quinolinone moiety.
Results and Discussion
In Vivo Anti-Inflammatory Activity. In this work, we
designed and synthesized a series of novel quinolinone deriva-
tives that were expected to offer protection against inflammation
and radical attack by application of standard synthetic methods
summarized in Schemes 1 and 2.
The preparation of the quinolinone-3-aminoamides 5-12 was
succeeded via two methods, A and B, as depicted in Scheme 1.

2452 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Detsi et al.
Scheme 1a
a Reagents and conditions: (i) HOBt, DCC, THF; (ii) NaH, dimethyl malonate, THF; Method A (for 1,2-ethylenediamine and 1,6-hexamethylenedi-
amine): 3 or 4, diamine, 100-110 °C; Method B: 3 or 4, diamine, toluene, 100 °C.
Scheme 2a
Table 1. Inhibition % LOX of Soybean Lipoxygenase; Inhibition % of
Induced Carrageenin Rat Paw Edema (CPE %) at 0.01 mmol/Kg Body
Weight; and Calculated Lipophilicity Clog P62
LOX %a
(0.1 mM)
cmpd
ClogP
CPE %b
3
4
5
6
7
8
9
10
11
12
14
15
16
17
18
1.74
3.74
0.61
2.60
1.86
3.82
1.66
3.65
1.66
3.65
3.08
5.07
4.02
5.18
5.42
2.39
72.4
100
99.5
82.3
92.1
17.6
71.3
nod
50.8
58.9
66.5
nod
79.1
64.0
100
29.1
nte
58.8**
37.1**
67*
68.1**
68.5**
68.8*
85.3*
48.5*
69.5*
56.4*
48.2*
45.5**
53*
50*
63*
lipoic acid
indomethacin
NDGAc
29.6**
47*
83.7
nte
a SD < 10%. b Statistical studies were done with student’s T-test, ** p
< 0.01, *p < 0.05. c Nordihydroguaiaretic acid. d no ) no action under
the reported experimental conditions. e nt ) not tested.
of diverse mediators.49 Because oedemas of this type are highly
sensitive to NSAIDs, carrageenin has been accepted as a useful
agent for studying new anti-inflammatory drugs.50 This model
reliably predicts the anti-inflammatory efficacy of the NSAIDs,
and during the second phase, it detects compounds that are anti-
inflammatory agents as a result of inhibition of prostaglandin
amplification.51 As shown in Table 1, all the investigated
compounds inhibited carrageenin-induced paw oedema. The
inhibition ranged from 37.1-85.3%, while the reference drug,
indomethacin, induced 47% inhibition at an equivalent concen-
tration. Compound 9 was the most potent (85.3%), followed
a Reagents and conditions: (i) CH2Cl2, DMF, rt.
In acute toxicity experiments, the in vivo examined com-
pounds did not present toxic effects in doses up to 0.5 mmol/
kg body weight.
The in vivo anti-inflammatory effects of the tested quinoli-
none-3-aminocarboxamides and their adducts with R-LA were
assessed by using the functional model of carrageenin-induced
rat paw oedema and are presented in Table 1 as percent
inhibition of induced rat paw oedema. Carrageenin-induced
oedema is a nonspecific inflammation resulting from a complex

Quinolinone-3-aminoamides and R-Lipoic Acid Adducts
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10 2453
Table 2. Interaction % with DPPH (RA %); Competition % with
by compounds 8, 6, 11, 5, 7, 3, and 16, whereas compound 4
had the lowest effect (37.1%).
DMSO for Hydroxyl Radical (•OH %)
0.1 mMa
RA%
20 min
0.1 mMa
RA%
60 min
0.5 mMa 0.1 mMa
For the starting quinolinones 3 and 4, as well as for the
aminoamides 9 and 10, 11 and 12, which contain an aromatic
diamine moiety, the nature of the substituent on the nitrogen of
the heterocyclic ring seems to have a distinct influence on the
inhibition values. Apparently, the presence of the small methyl
group (lower lipophilic contribution, minor steric effects, and
electron-donating nature) instead of the large phenyl group is
correlated with higher inhibition values. However, for com-
pounds 5 and 6, 7 and 8, which contain an aliphatic diamine,
the nature of the substituent on the nitrogen of the ring did not
affect the activity significantly. For the same compounds, the
number of methylene fragments did not alter the activity either.
RA%
RA%
0.1 mMa
•OH%
cmpd
20 min
60 min
3
4
5
6
7
8
9
10
11
12
14
15
16
17
18
8.3
9.1
5.6
7.2
3.1
11.6
6.0
9.4
90.2
89.0
10.9
6.4
9.1
6.2
9.7
20.3
81
7.4
9.1
17.5
16.8
20.9
1.4
13.7
19.3
28.8
21.3
85.1
94.4
28.8
noc
18.4
19.3
37.1
7.9
45
95
11.5
10.0
8.3
17.5
13.5
16.3
88.1
88.3
3.4
40.1
78.6
59.3
27.1
60.5
98
20.7
10
67.9
0
28.6
noc
noc
38.6
ntb
21.9
28.1
41.4
34.7
83.7
89.2
40.1
14.1
38.8
29.5
28.5
6.8
7.8
The presence of the amine group in the ortho- or para-
position of the 3-aminoamide phenyl ring (compounds 9-12)
influences biological response. Thus, compound 9, a derivative
of 1,2-phenylenediamine, exhibits 85.3% inhibition, whereas the
corresponding compound 11, containing 1,4-phenylenediamine,
shows lower inhibition value (69.5%). The opposite phenom-
enon was observed for the N-phenyl-substituted compounds 10
and 12, in which the 1,2-phenylenediamine derivative 10 is less
active than the 1,4-phenylediamine analogue 12.
13.0
10.2
16.1
27.1
82.6
ntb
28.4
23.9
11.0
5.2
lipoic acid
NDGA
trolox
96.5
98
ntb
ntb
ntb
88.2
a SD < 10%. b nt ) not tested. c no ) no action under the reported
experimental conditions.
activity. Lipophilicity does not seem to play a significant role
under the reported experimental conditions.
LA-quinolinone hybrids showed higher anti-inflammatory
activity than LA alone (29.6% inhibition in the carragennin-
induced rat paw edema). However, these hybrids did not show
enhanced anti-inflammatory activity compared to that of the
quinolinone aminoamides, with the exception of compound 18.
Hydroxyl radicals are among the most reactive oxygen species
and are considered to be responsible for some of the tissue
damage occurring in inflammation. It has been claimed that
hydroxyl radical scavengers could serve as protectors, thus
increasing prostaglandin synthesis.
The results show that, in the case of the synthesized
quinolinone derivatives, overall lipophilicity does not increase
in parallel to inhibition (Table 1). On the contrary, compounds
with significantly high in vivo anti-inflammatory activity present
lower clogP values, for example, 9, 11, 5, 7, and 3.
The competition of the quinolinone analogues with DMSO
for HO•, generated by the Fe3+/ascorbic acid system, expressed
as percent inhibition of formaldehyde production, was used for
the evaluation of their hydroxyl radical scavenging activity. In
these experiments (Table 2), compounds 15, 17, and 18 did not
show any inhibition at 0.1 mM. N-phenyl-quinolinones 4, 10,
and 6 were the most active under these experimental conditions.
LA presents 38.6% competition at 0.1 mM. Among the
quinolinone-LA hybrids, compound 14 showed the highest
inhibitory activity (67.9%), whereas compound 16 was slightly
active. The overall low lipophilicity is correlated with higher
competition values (compounds 7 and 16, compounds 6 and
15, compounds 18 and 10, and compounds 17 and 8), with the
exception of compounds 5 (clogP ) 0.61) and 14 (clogP )
3.08).
Leukotrienes play an important role as mediators of a variety
of inflammatory and allergic reactions and are derived from the
biotransformation of arachidonic acid catalyzed by 5-lipoxy-
genase (5-LOX). Inhibitors of 5-LOX have attracted attention
initially as potential agents for the treatment of inflammatory
and allergic diseases, but their therapeutic potential has now
been expanded to certain types of cancer and cardiovascular
diseases.54 Several quinoline derivatives have been synthesized
as inhibitors of human 5-LOX.55-57
In this context, we decided to further evaluate the synthesized
compounds for inhibition of soybean lipoxygenase LOX by the
UV absorbance-based enzyme assay (Table 1).58 While one may
not extrapolate the quantitative results of this assay to the
inhibition of mammalian 5-LOX, it has been shown that
inhibition of plant LOX activity by NSAIDs is qualitatively
similar to their inhibition of the rat mast cell LOX and may be
used as a simple qualitative screen for such activity. Most of
the LOX inhibitors are antioxidants or free radical scavengers,59
because lipoxygenation occurs via a carbon-centered radical.
Some studies suggest a relationship between LOX inhibition
In Vitro Antioxidant Activity and LOX Inhibition. It is
well-known that free radicals play an important role in the
inflammatory process.52 Many nonsteroidal anti-inflammatory
drugs have been reported to act either as inhibitors of free radical
production or as radical scavengers.6,53 Consequently, com-
pounds with antioxidant properties could be expected to offer
protection in rheumatoid arthritis and inflammation and to lead
to potentially effective drugs. Thus, we tested the novel
quinolinone derivatives with regard to their antioxidant ability
and in comparison to well-known antioxidant agents, for
example, nordihydroguaiaretic acid and trolox (Tables 1 and
2).
The interaction of the examined compounds with the stable
free radical DPPH is shown in Table 2. This interaction indicates
their radical scavenging ability in an iron-free system. It is
evident that the presence of an amine group in a position para
on the 3-carboxamide phenyl ring is correlated with higher
values, for example, compounds 11 and 12, which exhibited
the highest interactions (90.2 and 89%). The main reason for
this might be that the free amine group is at the 4-position far
from the carbonyl group and no intermolecular interactions could
happen.
All the other analogues were found to have very low activity
at 0.1 mM. LA exhibits a mild interaction. For the majority of
the compounds, the interaction was time and concentration
dependent.
The number of CH2 groups, the nature of the substituent on
the nitrogen of the heterocyclic ring, and the ester functionality
do not correlate with higher interaction values. Moreover,
introduction of the LA moiety did not improve scavenging

2454 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Detsi et al.
and the ability of the inhibitors to reduce Fe3+ at the active site
to the catalytically inactive Fe2+. LOXs contain a “non-heme”
iron per molecule in the enzyme active site as high-spin Fe2+
in the native state and the high spin Fe3+ in the activated state.
Several LOX inhibitors are excellent ligands for Fe3+. It is
possible that our compounds follow a mechanism of inhibition
acting as chelators for iron at the active site of LOX.
Perusal of % inhibition values shows that compounds 4, 5,
6, 7, and 18 are active, while compounds 10 and 15 do not
inhibit LOX. Compounds 3, 9, 14, 16, and 17 also show
inhibitory activity, followed by derivatives 11 and 12, whereas
analogue 8 slightly inhibits LOX. No inhibition was observed
by lowering the concentration to 0.01 mM. Thus, inhibition
vanishes in 0.01 mM, with the exception of quinolinone 4, which
presents 7.5% inhibition at 0.01 mM. LA presents moderate
inhibitory activity (29.1%). In the case of 3-aminoamide 10,
which shows no inhibitory activity, incorporation of the LA
moiety resulted to an impressive increase of activity (compound
18, inhibition 100%). The presence of an amine group in a
position para to the 3-carboxamide phenyl ring (compound 12)
also enhances inhibitory activity. A preliminary QSAR study
reveals that higher molar volume values are correlated with a
decrease in biological activities. More research is in progress
to delineate the role of structural characteristics.
However, incorporation of the LA moiety to the quinolinone
aminoamide scaffold did not provide compounds with enhanced
dual antioxidant and anti-inflammatory activity. More specifi-
cally, in this series of compounds, the presence of LA increased
the antioxidant activity of compound 5 (compare 5 with the
LA adduct 14) and the anti-inflammatory activity of compound
10 (compare 10 with 18). It seems that, even though LA
possesses all the characteristics required for enhancing antioxi-
dant and anti-inflammatory activity, its hybrids do not always
possess improved activity compared to that of the parent
compounds (the quinolinone aminoamides in this case). This
observation is not unique because other LA adducts have been
synthesized and reported to exhibit lower activity than the parent
compounds.27,31
The results of this study show that quinolinone aminoamides
5-12 exhibit satisfactory anti-inflammatory activity in vivo. It
is noteworthy that the most satisfactory combined antioxidant-
anti-inflammatory activity was exhibited by compounds 6 and
9, which show the most appealing pharmacological profile. It
is, therefore, evident that the design of this type of dual-acting
molecules should be further explored based on the structural
features of these compounds.
Experimental Section
Melting points were determined on a Gallenkamp MFB-595
melting point apparatus and are uncorrected. IR spectra were
recorded on a Nicolet Magna IR 560. 1H NMR spectra (300 MHz)
and 13C NMR spectra (75 MHz) were recorded on a Varian Gemini
2000 300 MHz spectrometer. The numbering of carbon atoms is
shown on the structure in Chart 1. Elemental analyses were obtained
on a Euro EA3000 Series Euro Vector CHNS Elemental Analyzer.
The chemical stability of selected aminoamides and quino-
linone-LA adducts has been tested to investigate the integrity
of the molecules under pH and temperature conditions that
mimic those of the biological experiments. Thus, we have tested
the stability of compounds 5, 6, 9, 10, 15, and 18 in pH 9 (borax
buffer) at rt for 1 h and of compounds 5, 6, 9, 14, and 15 at pH
7.4 (phosphate buffer) at 37 °C for 4 h: a 0.1 M solution of the
tested compound in DMSO was diluted with the appropriate
aqueous buffer solution to 0.01 M, and the slurry was stirred
vigorously. TLC of the compounds showed that no degradation
had taken place. To verify this finding, after extraction with
Chart 1
1
CH2Cl2, drying, and evaporation of the solvent, the H NMR
spectrum of each compound was obtained. The spectra were
identical with the spectra of the untreated compounds.
Conclusion
Materials. All the chemicals used were of analytical grade and
commercially available by Merck, 1,1-diphenyl-2-picrylhydrazyl
(DPPH), nordihydroguairetic acid (NDGA) were purchased from
the Aldrich Chemical Co., Milwaukee, WI (U.S.A.). Soybean
lipoxygenase, linoleic acid sodium salt, and indomethacin were
obtained from Sigma Chemical Co. (St. Louis, MO), and carrag-
eenin, type K, was commercially available. For the in vivo
experiments, male and female Fischer-344 rats (180-240 g) were
used.
General Procedure for the Synthesis of Quinolinone-3-
aminoamides 5-12. Method A: A mixture of 1-methyl- or
1-phenyl-4-hydroxy-2-oxo-1,2-dihydro-quinoline-3-carboxylic acid
methyl ester (1.9 mmol) and the appropriate diamine (5.7mmol)
was heated at 100-110 °C for 24 h. The reaction mixture was
diluted with CHCl3 and washed with saturated aqueous NaHCO3.
The organic layer was dried (Na2SO4) and evaporated in vacuo.
The residue was triturated with diethylether, and the solid product
was filtered off.
In the present study, we have synthesized and biologically
evaluated novel quinolinone 3-aminoamides and their R-LA
hybrids. The synthesis is straightforward and efficient and
provides the desired compounds in satisfactory yields, without
demanding tedious workup and purifying procedures. The
compounds are stable under basic and neutral conditions. We
have shown that the synthesized compounds possess significant
anti-inflammatory activity in vivo, whereas most of them are
potent •OH scavengers and inhibit in vitro soybean lipoxygenase.
Our studies confirm that the presence of a free hydroxyl group
at position 4, a 3,4-double bond, and an aminoamide functional-
ity at position 3 are important structural features for the
antioxidant/anti-inflammatory activity, as it is already known
for NSAIDs containing a â-ketocarboxamide functionality
(“acidic amide”)51 and has been recently shown for the
quinolinone antioxidant rebamipide.17
An overview of the results of the in vivo experiment reveals
that all the synthesized quinolinone-LA hybrids (compounds
14-18) exhibit significantly higher activity than LA. The
chemical stability of the compounds shows that they constitute
new, promising, anti-inflammatory agents as intact molecules
and are not LA prodrugs. This provides an impetus for designing
new anti-inflammatory agents using the quinolinone-LA scaffold
as the starting point.
Method B: 1-methyl- or 1-phenyl-4-hydroxy-2-oxo-1,2-dihydro-
quinoline-3-carboxylic acid methyl ester (1.4 mmol) and the
appropriate diamine (2.7mmol) were added to 7 mL of dry toluene,
and the mixture was heated at 100 °C for 4 h. The product
precipitated in the reaction mixture and, after cooling at ambient
temperature, the solid was filtered off and washed with diethylether.
N-Ethylamino-1,2-dihydro-4-hydroxy-2-oxo-1-phenyl-3-quin-
olinecarboxamide (6). Compound 6 was prepared from quinolinone
4 and 1,2-ethylenediamine: yield 62% (Method A) and 89%
1
(Method B), yellowish solid, mp 163-165 °C. H NMR (CDCl3/

Quinolinone-3-aminoamides and R-Lipoic Acid Adducts
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10 2455
CF3COOD): δ 10.47 (br s, 0.4H, CONH), 8.30 (dd, 1H, J5,6 ) 8.1
Hz, J5,7 ) 1.2 Hz, 5-H), 7.68-7.61 (m, 3H), 7.60-7.56 (m, 1H),
7.41 (pseudotriplet, 1H), 7.28-7.23 (m, 2H), 6.72 (d, 1H, J ) 8.7
Hz), 3.79 (br s, 2H, NH-CH2-), 3.39 (br s, 2H, -CH2NH2). 13C
NMR (CDCl3/CF3COOD): δ 173.4 (9-C), 172.6 (4-C), 163.3 (2-
C), 140.6 (8a-C), 136.0 (1′-C), 134.8 (7-C), 130.9, 130.2, 128.7,
125.6, 124.3, 116.8 (4a-C), 113.0 (8-C), 109.2 (3-C), 41.6 (10-C),
37.5 (11-C). Anal. (C18H17N3O3) C, H, N.
3.68 (s, 3H, N-CH3), 3.65-3.59 (m, 2H, -CONHCH2CH2-
NHCO-), 3.51-3.44 (m, 3H, -CONHCH2CH2NHCO- and 17-
H), 3.14-3.04 (m, 2H), 2.41 (sextet, 1H, J ) 6.3 Hz), 2.20 (m,
2H, -NHCOCH2CH2CH2CH2-), 1.86 (sextet, 1H, J ) 6.4 Hz),
1.65-1.61 (m, 4H, -NHCOCH2CH2CH2CH2-), 1.46-1.43 (m,
2H, -NHCOCH2CH2CH2CH2-). 13C NMR (CDCl3): δ 173.3 (9-
C), 172.3 (4-C), 171.9, 162.8 (2-C), 140.1 (8a-C), 134.1 (7-C), 125.6
(5-C), 122.6 (6-C), 116.2 (8-C), 114.4 (4a-C), 96.8 (3-C), 56.3,
40.3, 40.1, 38.5, 38.3, 36.4, 34.5, 29.1 (N-CH3), 28.7, 25.2. Anal.
(C21H27N3O4S2) C, H, N, S.
N-Hexylamino-1,2-dihydro-4-hydroxy-1-methyl-2-oxo-3-quin-
olinecarboxamide (7). Compound 7 was prepared from quinolinone
3 and 1,6-hexamethylenediamine: yield 46% (Method A) and 69%
(Method B), white solid, mp 156-158 °C. 1H NMR (CDCl3/CD3-
N-(1,2-Dihydro-4-hydroxy-2-oxo-1-phenyl-3-quinolinecarbo-
nyl)-N′-(1,2-dithiolane-3-pentanoyl)-ethylenediamine (15). Com-
pound 15 was obtained by reaction of 6 with 13, as a white solid,
after purification with flash column chromatography (CH2Cl2/
MeOH, 97:3) and recrystallization from CHCl3/petroleum ether:
OD): δ 10.27 (br s, 1H, CONH), 8.12 (dd, 1H, J5,6 ) 8.1, J5,7
)
1.2 Hz, 5-H), 7.62 (pseudotriplet, 1H, 7-H), 7.32-7.21 (m, 2H,
6-H, 8-H), 3.59 (s, 3H, N-CH3), 3.37-3.30 (m, 2H, NH-CH2-),
2.56 (pseudotriplet, 2H, -CH2NH2), 1.58-1.29 (m, 8H, -(CH2)4-
). Anal. (C17H23N3O3) C, H, N.
1
yield 70%, mp 170-172 °C. H NMR (CDCl3): δ 16.92 (s, 1H,
OH), 10.38 (br, 1H, CONH-(CH2)2-), 8.24 (dd, 1H, J5,6 ) 8.1
Hz, J5,7 ) 1.5 Hz, 5-H), 7.66-7.53 (m, 3H), 7.48 (dtd, 1H, J )
1.8, 8.7, 7.2 Hz), 7.32-7.26 (m, 3H), 6.66 (d, 1H, J ) 8.4 Hz),
6.14 (br, 1H, -(CH2)2-NHCO), 3.60-3.42 (m, 5H), 3.15-3.02
(m, 2H), 2.42 (sextet, 1H, J ) 6.3 Hz), 2.17 (m, 2H, -NHCOCH2-
CH2CH2CH2-), 1.88 (sextet, 1H, J ) 6.4 Hz), 1.70-1.61 (m, 4H,
-NHCOCH2CH2CH2CH2-), 1.48-1.40 (m, 2H, -NHCOCH2-
CH2CH2CH2-). 13C NMR (CDCl3): δ 173.2 (9-C), 172.9 (4-C),
172.2, 163.1 (2-C), 141.0 (8a-C), 137.1, 133.7 (7-C), 130.5, 129.4,
129.0, 125.3 (5-C), 122.9 (6-C), 116.2 (8-C), 116.0 (4a-C), 96.7
(3-C), 56.3, 40.2, 40.1, 38.6, 38.3, 36.3, 34.5, 28.7, 25.2. Anal.
(C26H29N3O4S2) C, H, N, S.
N-o-Phenylamino-1,2-dihydro-4-hydroxy-1-methyl-2-oxo-3-
quinolinecarboxamide (9). Compound 9 was prepared from
quinolinone 3 and 1,2-phenylenediamine, according to Method B:
yield 65%, light green solid, mp 230-232 °C (lit.60 mp 230-232
°C). 1H NMR (CDCl3): δ 16.61 (s, 1H, OH), 12.17 (s, 1H, CONH),
8.26 (dd, 1H, J5,6 ) 8.1, J5,7 ) 1.5 Hz, 5-H), 7.73 (dtd, 1H, J7,8
)
8.7 Hz, J7,6 ) 6.9 Hz, J7,5 ) 1.5 Hz, 7-H), 7.55 (dd, 1H, J ) 8.4,
1.8 Hz), 7.09 (ddd, 1H, J ) 9.3, 7.8, 1.5 Hz), 6.85 (pseudotriplet,
2H), 7.41 (d, J8,7 ) 9.0 Hz, 1H, 8-H), 7.35 (ddd, 1H, J6,5 ) 8.1
Hz, J6,7 ) 7.2 Hz, J6,8 ) 1.2 Hz, 6-H), 3.93 (br s, 2H, -NH2), 3.75
(s, 3H, N-CH3). 13C NMR (CDCl3): δ 169.7 (4-C), 159.9 (CONH),
155.0 (2-C), 140.3, 140.2 (8a-C), 134.2 (7-C), 127.1, 125.7, 125.2
(5-C), 122.7, 119.3, 117.3 (4a-C), 114.4 (8-C), 100.0 (3-C), 29.1
(N-CH3). Anal. (C17H15N3O3) C, H, N.
N-(1,2-Dihydro-4-hydroxy-1-methyl-2-oxo-3-quinolinecarbo-
nyl)-N′-(1,2-dithiolane-3-pentanoyl)-hexamethylenediamine (16).
Compound 16 was obtained by reaction of 7 with 13, as a yellowish
solid, after purification with flash column chromatography (CH2-
N-p-Phenylamino-1,2-dihydro-4-hydroxy-1-methyl-2-oxo-3-
quinolinecarboxamide (11). Compound 11 was prepared from
quinolinone 3 and 1,4-phenylenediamine, according to Method B:
1
Cl2/MeOH, 97:3): yield 60%, mp 93-95 °C. H NMR (CDCl3):
δ 17.19 (s, 1H, OH), 10.30 (br, 1H, CONH-(CH2)6-), 8.18 (dd,
1H, J5,6 ) 8.1 Hz, J5,7 ) 1.5 Hz, 5-H), 7.71 (ddd, 1H, J7,8 ) 8.4
Hz, J7,6 ) 7.5 Hz, J7,5 ) 1.8 Hz, 7-H), 7.35-7.25 (m, 2H, 6-H,
8-H), 5.68 (br, 1H, -(CH2)6-NHCO), 3.65 (s, 3H, N-CH3), 3.54
(quintet, 1H, J ) 6.6 Hz), 3.42 (q, 2H, J ) 6.6 Hz, CONH-
CH2(CH2)4CH2-NHCO-), 3.22 (q, 2H, J ) 6.9 Hz, CONH-CH2-
(CH2)4CH2-NHCO-), 3.17-3.30 (m, 2H), 2.42 (sextet, 1H, J )
6.6 Hz), 2.15 (m, 2H, -NHCOCH2CH2CH2CH2-), 1.87 (sextet,
1H, J ) 6.0 Hz), 1.70-1.30 (m, 14H). 13C NMR (CDCl3): δ 172.8
(9-C), 172.0 (4-C), 171.1, 162.8 (2-C), 139.9 (8a-C), 133.7 (7-C),
125.5 (5-C), 122.4 (6-C), 116.3 (8-C), 114.2 (4a-C), 96.7 (3-C),
56.3, 40.1, 39.2, 38.7, 38.3, 36.4, 34.5, 29.3 (N-CH3), 29.0, 28.8,
26.4, 26.3, 25.3. Anal. (C25H35N3O4S2) C, H, N, S.
1
yield 97%, green solid, mp 228 °C (dec). H NMR (CDCl3/CF3-
COOD): δ 8.34 (dd, 1H, J5,6 ) 7.8, J5,7 ) 1.5 Hz, 5-H), 7.90-
7.77 (m, 3H), 7.63-7.43 (m, 5H), 3.82 (s, 3H, N-CH3). 13C NMR
(CDCl3/CF3COOD): δ 172.7 (9-C), 169.8 (4-C), 164.0 (2-C), 139.7,
138.2 (8a-C), 135.8, 130.6 (7-C), 126.3 (5-C), 123.9 (6-C), 120.2,
116.5, 115.4 (4a-C), 112.7 (8-C), 108.9 (3-C), 30.8 (N-CH3). Anal.
(C17H15N3O3) C, H, N.
N-p-Phenylamino-1,2-dihydro-4-hydroxy-2-oxo-1-phenyl-3-
quinolinecarboxamide (12). Compound 12 was prepared from
quinolinone 4 and 1,4-phenylenediamine, according to Method B:
yield 99%, light green solid, mp > 280 °C (dec). 1H NMR (CDCl3/
CF3COOD): δ 8.37 (dd, 1H, J5,6 ) 8.1, J5,7 ) 1.5 Hz, 5-H), 7.75-
7.62 (m, 6H), 7.45 (pseudotriplet, 3H), 7.34-7.31 (m, 2H), 6.78
(d, 1H, J ) 8.7 Hz). 13C NMR (CDCl3/CF3COOD): δ 173.8 (9-
C), 169.8 (4-C), 163.9 (2-C), 140.8, 138.2 (8a-C), 135.9, 135.5,
131.3 (7-C), 130.8, 128.9, 125.8, 125.7 (5-C), 124.5, 124.0 (6-C),
120.3, 117.3 (4a-C), 116.5, 112.8 (8-C), 109.0 (3-C). Anal.
(C22H17N3O3) C, H, N.
General Procedure for the Synthesis of Compounds 14-18.
Quinolinone 3-aminocarboxamides 5-8 or 10 (0.46 mmol) were
mixed with N-(lipoyloxy)succinimide (13; 0.14 g, 0.46 mmol) in
dichloromethane (3 mL), and drops of DMF were added to facilitate
dissolution of the reactants. The mixture was stirred and light-
protected overnight. H2O (10 mL) was added to the mixture and
afterward it was extracted with dichloromethane (3 × 10 mL) and
washed repeatedly with H2O. The organic extracts were combined,
dried (Na2SO4), and concentrated in vacuo to afford the final
compounds, which were triturated with diethylether and filtered
off.
N-(1,2-Dihydro-4-hydroxy-1-methyl-2-oxo-3-quinolinecarbo-
nyl)-N′-(1,2-dithiolane-3-pentanoyl)-ethylenediamine (14). Com-
pound 14 was obtained by reaction of 5 with 13, as a yellowish
solid, after purification with flash column chromatography (CH2-
Cl2/MeOH, 97:3): yield 69%, mp 146-148 °C. 1H NMR
(CDCl3): δ 16.71 (s, 1H, OH), 10.57 (br, 1H, CONH-(CH2)2-),
8.22 (d, 1H, J5,6 ) 8.1 Hz, 5-H), 7.71 (pseudotriplet, 1H, 7-H),
7.39-7.26 (m, 2H, 6-H, 8-H), 6.19 (br, 1H, -(CH2)2-NHCO),
N-(1,2-Dihydro-4-hydroxy-2-oxo-1-phenyl-3-quinolinecarbo-
nyl)-N′-(1,2-dithiolane-3-pentanoyl)-hexamethylenediamine (17).
Compound 17 was obtained by reaction of 8 with 13, as a yellowish
solid, after purification with flash column chromatography (CH2-
Cl2/MeOH, 97:3): yield 72%, mp 110-112 °C. 1H NMR
(CDCl3): δ 17.47 (s, 1H, OH), 10.11 (br, 1H, CONH-(CH2)6-),
8.22 (dd, 1H, J5,6 ) 8.1 Hz, J5,7 ) 1.5 Hz, 5-H), 7.64-7.54 (m,
3H), 7.44 (dtd, 1H, J ) 1.5, 8.4, 7.5 Hz), 7.28-7.24 (m, 3H), 6.62
(d, 1H, J ) 8.4 Hz), 5.63 (br, 1H, -(CH2)6-NHCO), 3.54 (quintet,
1H, J ) 6.6 Hz), 3.39 (q, 2H, J ) 6.9 Hz, CONH-CH2(CH2)4-
CH2-NHCO-), 3.19 (q, 2H, J ) 6.6 Hz, CONH-CH2(CH2)4CH2-
NHCO-), 3.16-3.04 (m, 2H), 2.43 (sextet, 1H, J ) 6.6 Hz), 2.12
(m, 2H, -NHCOCH2CH2CH2CH2-), 1.88 (sextet, 1H, J ) 6.9 Hz),
1.71-1.31 (m, 14H). 13C NMR (CDCl3): δ 173.2 (9-C), 172.9 (4-
C), 171.1, 163.3 (2-C), 141.0 (8a-C), 137.3, 133.4 (7-C), 130.5,
129.3, 129.1, 125.3 (5-C), 122.7 (6-C), 116.1 (8-C), 100.0 (4a-C),
96.7 (3-C), 56.4, 40.2, 39.2, 38.8, 38.4, 36.4, 34.5, 32.5, 29.3, 28.8,
26.3, 25.3. Anal. (C30H37N3O4S2) C, H, N, S.
N-(1,2-Dihydro-4-hydroxy-2-oxo-1-phenyl-3-quinolinecarbo-
nyl)-N′-(1,2-dithiolane-3-pentanoyl)-1,2-phenylenediamine (18).
Compound 18 was obtained by reaction of 10 with 13, as a green
solid, after purification with flash column chromatography (CH2-
Cl2/MeOH, 99:1): yield 68%, mp 196-198 °C. 1H NMR
(CDCl3): δ 16.81 (s, 1H, OH), 11.88 (s, 1H, CONHC6H4-), 8.27
(dd, 1H, J5,6 ) 7.9 Hz, J5,7 ) 1.2 Hz, 5-H), 7.66-7.57 (m, 3H),

2456 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Detsi et al.
7.52-7.47 (m, 1H), 7.39 (d, 1H, J ) 8.1 Hz), 7.33-7.26 (m, 3H),
7.07 (pseudotriplet, 1H), 6.82-6.78 (m, 2H), 6.67 (d, 1H, J ) 8.4
Hz), 3.58 (quintet, 1H, J ) 7.2 Hz), 3.20-3.09 (m, 2H), 2.47
(sextet, 1H, J ) 6.7 Hz), 2.62 (t, 2H, J ) 6.9 Hz, -NHCOCH2-
CH2CH2CH2-), 1.93 (sextet, 1H, J ) 6.7 Hz), 1.84-1.52 (m, 6H).
13C NMR (CDCl3): δ 173.1 (9-C), 169.7 (4-C), 169.3, 163.3 (2-
C), 141.0 (8a-C), 140.7, 137.1, 133.7, 130.5, 129.4, 129.1, 127.4,
125.7, 125.4, 123.0, 122.8, 119.1, 117.3, 116.2, 116.0 (4a-C), 97.2
(3-C), 56.0, 40.1, 38.4, 34.3, 30.9, 28.2, 25.2. Anal. (C30H29N3O4S2)
C, H, N, S.
Biological Experiments. Experiments In Vivo. Toxicity of the
Examined Compounds. Toxicity experiments were carried out
using both male and female Fischer 344 rats. Pregnant females were
excluded. The tested compounds were dissolved in water (salts) or
suspended with a few drops of Tween 80, and ground in water,
and administered by intraperitoneal injection at various concentra-
tions. Mortality was recorded after 24 h.
Inhibition of the Carrageenin-Induced Edema.61 Edema was
induced in the right hind paw of Fisher 344 rats (150-200 g) by
the intradermal injection of 0.1 mL of 2% carrageenin in water.
Both sexes were used. Pregnant females were excluded. Each group
was composed of 6-15 animals. The animals, which have been
bred in our laboratory, were housed under standard conditions and
received a diet of commercial food pellets and water ad libitum
during the maintenance, but they were entirely fasted during the
experiment period. Our studies were in accordance with recognized
guidelines on animal experimentation.
The tested compounds, 0.01 mmol/kg body weight, were
suspended in water with a few drops of Tween 80 and ground in
a mortar before use and were given intraperitoneally simultaneously.
The rats were euthanized 3.5 h after carrageenin injection. The
difference between the weight of the injected and uninjected paws
was calculated for each animal. The change in paw weight was
compared with that in control animals (treated with water) and
expressed as a percent inhibition of the edema CPE % values, Table
1, indomethacin in 0.01 mmol/kg (47%). Values CPE % are the
mean from two different experiments, with a standard error of the
mean less than 10%.
Experiments In Vitro. In the in vitro assays, each experiment
was performed at least in triplicate, and the standard deviation of
absorbance was less than 10% of the mean.
Determination of the Reducing Activity of the Stable Radical
1,1-Diphenyl-picrylhydrazyl (DPPH).61 To a solution of DPPH
in absolute ethanol an equal volume of the compounds (0.1-0.5
mM) dissolved in ethanol was added. As control solution, ethanol
was used. The concentrations of the solutions of the compounds
were 0.1 and 0.5 mM. After 20 and 60 min at room temperature,
the absorbance was recorded at 517 nm (Table 2).
Effect of the Tested Compounds on the •OH Radical-
Mediated Oxidation of DMSO.61 The hydroxyl radicals generated
by the Fe3+/ascorbic acid system, were detected according to Nash,
by the determination of formaldehyde produced from the oxidation
of DMSO. The reaction mixture contained EDTA (0.1 mM), Fe3+
(167 µM), DMSO (33 mM) in phosphate buffer (50 mM, pH 7.4),
the tested compounds (0.1 mM), and ascorbic acid (10 mM). After
30 min of incubation (37 °C), the reaction was stopped with CCl3-
COOH (17% w/v), and the absorbance was recorded at λ 425 nm.
The results are shown in Table 1.
Soybean Lipoxygenase Inhibition Study In Vitro.61 In vitro
study was evaluated as reported previously. The tested compounds
dissolved in ethanol were incubated at room temperature with
sodium linoleate (0.1 mM) and 0.2 mL of enzyme solution (1/9 ×
104 w/v in saline). The conversion of sodium linoleate to 13-
hydroperoxylinoleic acid at 234 nm was recorded and compared
with the appropriate standard inhibitor (nordihydroguaiaretic acid
0.1 mM 83.7%).
Supporting Information Available: Full experimental details,
analytical and spectroscopic data for compounds 5, 8, and 10,
additional H NMR and IR spectroscopic data for all compounds,
and combustion analysis data for all new compounds. This material
is available free of charge via the Internet at http://pubs.acs.org.
1
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Acknowledgment. We would like to thank Dr. C. Hansch
and Biobyte Corp., 201 West Fourth Str., Suite 204, Claremont,
CA, 91711, U.S.A. for free access to the C-QSAR program.

Quinolinone-3-aminoamides and R-Lipoic Acid Adducts
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10 2457
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