Evolution from a natural flavones nucleus to obtain 2-(4-propoxyphenyl) quinoline derivatives as potent inhibitors of the S. aureus NorA efflux pump

Article abstract of DOI:10.1021/jm200370y

Overexpression of efflux pumps is an important mechanism by which bacteria evade the effects of substrate antimicrobial agents. Inhibition of such pumps is a promising strategy to circumvent this resistance mechanism. NorA is a Staphylococcus aureus efflux pump that confers reduced susceptibility to many structurally unrelated agents, including fluoroquinolones, resulting in a multidrug resistant phenotype. In this work, a series of 2-phenyl-4(1H)- quinolone and 2-phenyl-4-hydroxyquinoline derivatives, obtained by modifying the flavone nucleus of known efflux pump inhibitors (EPIs), were synthesized in an effort to identify more potent S. aureus NorA EPIs. The 2-phenyl-4- hydroxyquinoline derivatives 28f and 29f display potent EPI activity against SA-1199B, a strain that overexpresses norA, in an ethidium bromide efflux inhibition assay. The same compounds, in combination with ciprofloxacin, were able to completely restore its antibacterial activity against both S. aureus SA-K2378 and SA-1199B, norA-overexpressing strains.

Full text of DOI:10.1021/jm200370y

ARTICLE
pubs.acs.org/jmc
Evolution from a Natural Flavones Nucleus to Obtain
2-(4-Propoxyphenyl)quinoline Derivatives As Potent Inhibitors
of the S. aureus NorA Efflux Pump
Stefano Sabatini,*,† Francesca Gosetto,† Giuseppe Manfroni,† Oriana Tabarrini,† Glenn W. Kaatz,‡
Diixa Patel,‡ and Violetta Cecchetti†
†Dipartimento di Chimica e Tecnologia del Farmaco, Universitꢀa degli Studi di Perugia, 06123 Perugia, Italy
‡Department of Internal Medicine, Division of Infectious Diseases, School of Medicine, Wayne State University, and the John D. Dingell
Department of Veteran Affairs Medical Center, Detroit, Michigan 48201, United States
S Supporting Information
b
ABSTRACT:
Overexpression of efflux pumps is an important mechanism by which bacteria evade the effects of substrate antimicrobial agents.
Inhibition of such pumps is a promising strategy to circumvent this resistance mechanism. NorA is a Staphylococcus aureus efflux
pump that confers reduced susceptibility to many structurally unrelated agents, including fluoroquinolones, resulting in a multidrug
resistant phenotype. In this work, a series of 2-phenyl-4(1H)-quinolone and 2-phenyl-4-hydroxyquinoline derivatives, obtained by
modifying the flavone nucleus of known efflux pump inhibitors (EPIs), were synthesized in an effort to identify more potent S. aureus
NorA EPIs. The 2-phenyl-4-hydroxyquinoline derivatives 28f and 29f display potent EPI activity against SA-1199B, a strain that
overexpresses norA, in an ethidium bromide efflux inhibition assay. The same compounds, in combination with ciprofloxacin, were
able to completely restore its antibacterial activity against both S. aureus SA-K2378 and SA-1199B, norA-overexpressing strains.
’ INTRODUCTION
acquire resistance to many antibacterials such as tetracyclines,
macrolides, aminoglycosides, and fluoroquinolones.8 It is, how-
ever, the ability of MRSA to acquire resistance to vancomycin,
the main therapeutic agent for treatment of infections caused by
this organism, which is a major source of concern. Fully
vancomycin-resistant strains of S. aureus (MIC g16 μg/mL)
were first isolated in 2002.9,10
Bacterial resistance that contributes to shorter drug life cycles
is achieved by three main mechanisms: enzymatic inactivation,11
modification of the drug target(s),12,13 and reduction of intra-
cellular drug concentration either by changes in membrane
permeability14 or overexpression of efflux pumps.15ꢀ17 In recent
years, many efforts aimed at overcoming antibacterial drug
resistance have followed different approaches: (i) research on
new antibacterials with novel mechanisms of action,18,19 (ii)
structural manipulation of existing antibacterials to increase the
affinity with the target, even if mutated,20,21 and/or to reduce the
potential for the efflux without compromising antibacterial
activity,22 and (iii) identification of synthetic or natural non-
antibiotic compounds that work as efflux pump inhibitors (EPIs),
The emergence and spread of pathogens that have evolved
mechanisms of resistance to multiple antibiotics is becoming a
major threat to public health in the 21st century.1 The serious-
ness of antibiotic resistance lies in the fact that today bacterial
strains are not only resistant to commonly available antibacterials
but also may have acquired augmented virulence.2 Therefore, the
discovery and development of new antibiotics is of crucial
importance to counter the explosive growth of multidrug resis-
tant pathogens.
Of particular concern among resistant microorganisms is the
alarming rise of methicillin-resistant Staphylococcus aureus
(MRSA) strains that are highly virulent.3 The proportion of
healthcare-associated staphylococcal infections that are due to
MRSA has been increasing: 2% of S. aureus infections in U.S.
intensive-care units were MRSA in 1974, 22% in 1995, and 64%
in 2004.4 Invasive MRSA infections occur in approximately
94000 persons each year and are associated with about 19000
deaths annually in U.S. Approximately 86% of these infections
are healthcare-associated, and the remainder are community-
associated.5
Although MRSA are characterized by the presence of β-lactam
Received: March 30, 2011
Published: July 13, 2011
resistance,6,7 these organisms also have the exceptional ability to
r
2011 American Chemical Society
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Journal of Medicinal Chemistry
ARTICLE
which can restore the susceptibility of resistant strains to
coadministered antibacterials that are efflux pump substrates.22
Efflux pumps may provide a self-defense mechanism by which
antimicrobial substances made by other microbes, which fight for
dominance in an environmental niche, or antibacterial drugs are
actively removed from the cell. This activity results in sublethal
drug concentrations at the active site that in turn may predispose
the organism to the development of high-level target-based
resistance.23 Drug efflux is of major concern because of its key
role in the selection of high level resistant strains.24,25
Efflux pumps fall into two categories based on their substrate
specificity. Some pumps are specific for certain classes of
substrates (e.g., the TetK system which extrudes certain tetra-
cyclines), whereas others are multidrug resistance (MDR)
pumps as they have a broader spectrum of substrate specificity
and are capable of removing structurally unrelated antibacterials
from the cell.26 The proteins which make up these systems are
divided into five distinct families primarily based on amino acid
sequence homology.27 These include the major facilitator super-
family (MFS),28 the resistance-nodulation-division (RND),29
small multidrug-resistance (SMR),30 ATP-binding cassette
(ABC),31 and the multiple antibiotic and toxin extrusion
(MATE) families.32 Efflux of drugs from Gram-positive bacteria
may be mediated by members of any of these families with the
possible exception of RND proteins.26 The most studied efflux
pump of S. aureus is NorA, a transporter belonging to the MFS.
MFS pumps such as NorA are capable of extruding multiple,
structurally dissimilar substrates such as hydrophilic fluoroqui-
nolones, various biocides, and dyes.15
Figure 1. From the 2-phenyl-4H-chromen-4-ones to 2-(4-propoxyphenyl)-
quinoline EPIs.
Therefore, efflux pumps are viable antibacterial targets and
identification and development of potent EPIs is a promising and
valid strategy.33 There are a number of reasons to pursue this
area, including: (i) the EPI restores susceptibility to the anti-
bacterial and an EPIꢀantibacterial combination has been shown
to reduce the rate of emergence of antibiotic-resistant variants,
(ii) this is a natural approach, as there are examples in which
plants produce both antibacterials and EPIs that improve their
antibacterial activity (e.g., berberine and the methoxyhydnocar-
pin, isoflavones and R-linolenic acid, and dalversinol A and a
methoxychalcone),34ꢀ36 (iii) EPIs used in combination with
antibiotics may not only increase antibacterial potency37 but also
may expand the antibacterial spectrum and reduce the frequency
of the emergence of target-based resistance,38 (iv) EPIs have
been shown to reduce biofilm formation and block the anti-
bacterial tolerance of biofilms.39
In recent years, many EPIs capable of potentiating the activity
of antimicrobial substrates have been identified. Early EPIs
included reserpine and verapamil, but concentrations required
for pump-inhibitory activity are too high to be clinically
relevant.40,41 Several other nonantibiotic compounds, such as
omeprazole, paroxetine, chlorpromazine, and respective deriva-
tives, and the synthetic acridone derivative GG918, have been
shown to increase the antibacterial potency of pump substrates
by inhibiting NorA of S. aureus.42ꢀ46 The main efforts to find
EPIs has been made by phytochemists, who have reported an
extremely varied series of natural compounds in terms of their
chemical class and shape including flavones, isoflavones, por-
phyrin phaeophorbide A, and acylated glycosides.47,48
the clinical setting, this approach holds promise for improving
the efficacy and/or extending the clinical utility of existing
antibacterials, giving new life to old drugs37 with secure economic
benefit.
To our knowledge, the NorA binding pocket has not been
defined; sequence homology and the sharing of a wide range of
substrates and/or inhibitors with the B. subtilis Bmr MDR pump
and the plasmid-encoded S. aureus QacA MDR pump have led to
the hypothesis that NorA may have a large hydrophobic binding
site. Such a binding region would permit substrates to associate
with the protein through a combination of hydrophobic effects
and electrostatic attraction rather than by establishing a precise
network of hydrogen bonds. This structural peculiarity could
explain the broad substrate specificity of MDR pumps.54 The lack
of information regarding the mechanism(s) of interaction be-
tween substrates or EPIs and NorA makes it difficult to design
new inhibitors via computational methods. In addition, the
structural heterogeneity of known NorA EPIs do not allow a
clear and unequivocal SAR for this category of compounds. For
these reasons, we have chosen to undertake a classical medicinal
chemistry approach to design new structures which could be
more powerful EPIs with the goal being the identification of a
small molecule chemotype capable of restoring ciprofloxacin
(CPX) activity on S. aureus strains by inhibition of NorA.
Design Rationale. Our previous work in this field led to the
identification of substituted 3-phenyl-1,4-benzothiazine EPIs
by a minimization of the phenothiazine moiety, which were
capable of completely restoring the antibacterial activity of CPX
against NorA overexpressing strains.51 We also identified 6-ami-
no-8-methylquinolone esters, strong inhibitors of the S. aureus
efflux pumps NorA (MFS) and MepA (MATE), by structural
To date, there are only a few examples of rationally designed
inhibitors, and very little work has been done with respect to the
structureꢀactivity relationship (SAR) of NorA inhibitors.49ꢀ53
Although the therapeutic utility of EPIs has yet to be validated in
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Scheme 1a
a Reagents and conditions: (i) dry pyridine, rt; (ii) K2CO3, acetone, reflux; (iii) Amberlist 15, i-PrOH/(i-Pr)2O, reflux.
Scheme 2a
a Reagents and conditions: (i) NaH, (EtO)2CO, rt; (ii) PPA, 90 °C.
modifications of the 6-aminoquinolone antibacterial core.52 In an
attempt to obtain chemotypes with potent inhibitory activity
against NorA, we have chosen to modify the 2-phenyl-4H-
chromen-4-one moiety, a common feature of flavone and natural
flavolignane EPIs.47ꢀ49 This was performed by exchanging the
endocyclic oxygen with an nitrogen atom to obtain the 2-phe-
nylquinolone nucleus and then, after suitable substitutions, the
2-(4-propoxyphenyl)quinoline derivatives which are able to
reduce transport of antibacterial quinolones by NorA (Figure 1).
The 2-phenylquinolone nucleus has all of the known requisites
to provide favorable EPI activity, including a suitable large
hydrophobic area (the two phenyl rings) and the capability of
establishing an electrostatic interaction (by the N-1 and the
ketone in C-4 position).55 Moreover, in addition to being a
mimic of the quinolone antibacterial core, possibly allowing for
an positive interaction with NorA binding site(s), the 2-phenyl-
quinolone nucleus is also a versatile skeleton suitable for
relatively simple chemical modifications that could provide a
large number of structurally different derivatives.
base-catalyzed transposition of the acyl group to the R-methylk-
etone provided the diphenyl-1,3-ketoenol 3 in 60% yield that was
then cyclized, in acid condition, with Amberlist 15, to the 2-(4-
propoxyphenyl)-4H-chromen-4-one 449 (yield 32%) (Scheme 1).
The synthetic route giving rise to the 2-(4-propoxyphenyl)-
4H-thiochromen-4-one 6 (Scheme 2) entailed the synthesis of
the (2Z)-3-hydroxy-3-(4-propoxyphenyl)prop-2-enoate 5,58 ob-
tained by reaction of 1-(4-propoxyphenyl)ethanone59 with
(EtO)2CO and NaH in good yields (76%), which in turn reacted
with thiophenol in polyphosphoric acid (PPA) to give the target
compound 6 in 46%yield.
Attempting to synthesize the 2-[4-(propyloxy)phenyl]quino-
lin-4(1H)-one analogue of flavone 4,49 we observed that if the
N-1 nitrogen atom of the quinolone nucleus was unalkylated, the
only tautomer that we have obtained and detected by 1H NMR
was the 2-(4-propoxyphenyl)-4-hydroxyquinoline 20f instead of
the desired C-4 ketone form. To resolve this limitation, we
decided to make a direct methylation of compound 20f to obtain
the desired N-alkylated 1-methyl-2-(4-propoxyphenyl)quinolin-
4(1H)-one 22f as well as the O-methyl derivative 4-methoxy-
2-(4-propoxyphenyl)quinoline 21f as a byproduct (Scheme 3).
Flavone compound 449 and its strict sulfur-analogue 6 were
preliminarily compared with 2-(4-propoxyphenyl)-4-hydroxy-
quinoline 20f and 1-methyl-2-(4-propoxyphenyl)quinolin-
4(1H)-one 22f for their ability to reduce the efflux of the well-
known NorA substrate ethidium bromide (EtBr) against the
well-described norA-overexpressing strain SA-1199B.60,61 Data
confirm that 2-phenylquinolone 22f displays the same, even if
weak (about 30%), inhibition of EtBr efflux with respect to the
reference flavone compound 4.49 This weak but interesting
activity led us to deepen our study subjecting compound 22f
to a series of chemical manipulations with the goal of obtaining
more powerful derivatives (Table 1).
Chemistry. To investigate if the quinolone nucleus was a good
replacement of the chromen-4-one on the inhibition of NorA, we
have resynthesized the flavone compound 449 (Scheme 1) and its
2-(4-propoxyphenyl)-4H-thiochromen-4-one strict analogue 6
(Scheme 2) to compare with the corresponding 2-(4-propox-
yphenyl)quinolin-4(1H)-one derivatives obtained, introducing a
nitrogen atom instead of the endocyclic oxygen of the 2-(4-
propoxyphenyl)-4H-chromen-4-one 449 (Figure 1).
2-(4-Propoxyphenyl)-4H-chromen-4-one 449 was obtained
starting from the 1-(2-hydroxyphenyl)ethanone following the
procedure reported from Patonay et al.56 for the synthesis of the
flavone nucleus (Scheme 1).
The initial acylation of the 2-phenol group with 4-propoxyben-
zoyl chloride 1f,57 obtained by treating the corresponding 4-pro-
poxy benzoic acid with SOCl2, in dry pyridine at rt, provided the
2-acetylphenyl 4-propoxybenzoate 2 in good yield (72%). Then, a
Modifications initially involved the propoxy group of the C-
2 phenyl ring of compound 22f, in which the propyl chain
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Scheme 3a
a Reagents and conditions: (i) N,N-diethylethane-1,2-diamine or 2-(1-piperidinyl)ethanamine, DMF, 100 °C; (ii) MeI, toluene, reflux;(iii) 1-(2-chloroethyl)-
piperidine hydrochloride, K2CO3, dry DMF, 100 °C; (iv) NaOH 5N, MeOH, rt; (v) SOCl2, reflux; (vi) Et3N, THF, 70 °C; (vii) t-BuOK, t-BuOH, heating;
(viii) MeI, K2CO3, dry DMF, 100 °C or (2-chloroethyl)diethylamine hydrochloride, K2CO3, dry DMF, reflux or 1-(2-chloroethyl)piperidine hydro-
chloride, t-BuOK, dry DMF, 100 °C; (ix) NaH, dry DMF, rt or t-BuOK/t-BuOH, 60 °C; (x) R-X, K2CO3, dry DMF, 70 °C; (xi) CH2Cl2, BBr3, rt;
(xii) LiCl, dry DMF, reflux.
was deleted (22c),62 replaced with shorter alkyl chains (22d,63
22e, and 22g) or with O-ethylamino chains (22hꢀk). Further
modifications involved the C-6 and C-7 positions of the quinolone
nucleus of compound 22f by introducing 6,7-dimethoxy (23a,64
23c, and 23f), 6,7-dihydroxy (26a and 26c), or 6,7-methoxy-
hydroxy (27f) groups, also present on natural and synthetic EPIs
(Chart 1).40ꢀ42,46ꢀ48 Encouraging results were obtained by in-
troducing an ethyl-2-diethylamine or an ethyl-2-piperidine at the
N-1 position of the 2-phenylquinolone nucleus (compounds 24f
and 25f) (Scheme 3).
Chart 1. R4 Substituents
0
It was then decided to explore the chemical space around the C-4
position of the quinolone nucleus by modifying the hydroxyl group
in the C-4 position of the 2-phenyl-4-hydroxyquinoline nucleus of
compound 20f through a simple alkylation reaction, and this
permitted us to obtain compounds 28f,j and 29f (Table 1).
The synthetic strategy was established to obtain both the 2-phenyl-
quinoline (20, 21, 28, 29) and 2-pheniquinolone (22ꢀ27)
nuclei (Scheme 3) needed to provide the appropriate starting
1-(2-aminophenyl)ethanones (7ꢀ12) and the suitable benzoyl
chlorides (1a, 1b, 1f,57 and 1j) to give the key intermediates N-(2-
acetylphenyl)benzamides (15ꢀ19). Starting from the 1-(2-fluoro-
phenyl)ethanone, the 1-{2-[(2-aminoethyl)amino]phenyl}ethanones
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Table 1. EtBr Efflux Inhibition (%) on SA-1199B of Synthesized Compounds at 50 μM Concentration
MIC
0
compd
X
R1
R4
H
R6
R7
R4
EtBr efflux inhib (%) μM μg/mL
4
6
O
S
H
H
H
H
H
H
H
H
On-Pr
On-Pr
On-Pr
On-Pr
32.4
6.3
a
a
20f
22f
N
N
0.0
Me
33.9
22c62
22d63
22e
N
N
N
N
N
N
N
N
Me
Me
Me
Me
Me
Me
Me
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
0.0
0.0
OMe
OEt
10.8
10.3
4.4
22g
22h
22i
Oi-Pr
O(CH2)2N(Me)2
O(CH2)2N(Et)2
O(CH2)2-(piperidin-1-yl)
O(CH2)2-(morpholin-4-yl)
1.7
22j
19.8
0.0
22k
23a64
23c
N
N
N
Me
Me
Me
OMe OMe
H
9.4
0.0
0.0
OMe OMe OH
23f
OMe OMe On-Pr
26a
26c
27f
N
N
N
Me
Me
Me
OH
OH
OH
OH
H
0.0
0.0
9.1
OH
On-Pr
OMe OH
24f
25f
N
N
ꢀ(CH2)2N(Et)2
ꢀ(CH2)2-(piperidin-1-yl)
H
H
H
H
On-Pr
On-Pr
57.3
75.1
>256
>100
21f
28f
28j
29f
N
N
N
N
Me
H
H
H
H
H
H
H
H
On-Pr
63.7
93.4
65.6
88.5
ꢀ(CH2)2N(Et)2
ꢀ(CH2)2N(Et)2
ꢀ(CH2)2-(piperidin-1-yl)
On-Pr
>241
>223
256
>100
>100
100
O(CH2)2-(piperidin-1-yl)
On-Pr
reserpine
84.8
89.7
>164
>303
>100
>100
paroxetine
a Not determined for those compounds that have shown an EtBr inhibition efflux less than 65%.
1165 and 12 were obtained by aromatic nucleophilic substitution
of the fluorine atom with N,N-diethylethane-1,2-diamine and
(2-piperidin-1-ylethyl)amine, in dry DMF at 100 °C, with yields
of 70 and 46%, respectively. The 1-[2-(Methylamino)phenyl]-
ethanones 966 and 1067 were synthesized by methylation with
MeI in toluene at reflux of 1-(2-aminophenyl)ethanone 7 and
1-(2-amino-4,5-dimethoxyphenyl)ethanone 8 in 42 and 48%
yield, respectively.
14j68 was obtained from ethyl 4-hydroxybenzoate by alkylation
with 1-(2-chloroethyl)piperidine hydrochloride in dry DMF and
K2CO3 at 100 °C to give the ethyl 4-(2-piperidin-1-ylethoxy)-
benzoate 13j,68 which was hydrolyzed by NaOH 5% to furnish
the corresponding acid in good yields.
The 1-(2-aminophenyl)ethanones 7, 8, 9,66 10,67 11,65 and 12
gave a nucleophilic substitution to the suitable benzoyl chlorides
1a, 1b, 1f,57 and 1j in dry THF at 70 °C, using Et3N as HCl
scavenger, to obtain the corresponding N-(2-acetylphenyl)ben-
zamides (15ꢀ19). The N-(2-acetylphenyl)benzamides 15f and
15j were then cyclized with t-BuOK in t-BuOH to obtain the
corresponding 40-substituted 2-phenyl-4-hydroxyquinolines 20f
With the exception of 1a, the benzoyl chlorides 1b, 1f,57 and 1j
were obtained in quantitative yields from the respective car-
boxylic acids 14b, 14f, and 14j,68 which were treated with SOCl2
at reflux for 1 h. The 4-(2-piperidin-1-ylethoxy)benzoic acid
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For the more promising compounds (25f, 28f, 28j, and 29f)
demonstrating inhibition of EtBr efflux by SA-1199B by >65% at
a 50 μM concentration, doseꢀresponse curves were built to
assess their activity at lower concentrations in comparison with
the reference compounds reserpine and paroxetine (Figure 2).
Moreover, the same active compounds (25f, 28f, 28j, and 29f)
were tested for their synergism with CPX against two pairs of
S. aureus strains, SA-K1902 (norAꢀ)/SA-K2378 (norA++) and
SA-1199 (norA wild-type)/SA-1199B (norA++ and A116E
GrlA) as well as against S. aureus ATCC 25923 (wild-type
strain), using checkerboard assays,69 evaluating their ability to
reduce the MICs of the fluoroquinolone in comparison with the
above cited reference compounds (Figure 3).
To validate the hypothesis that the quinolone nucleus could
be a suitable scaffold for a new class of EPIs able to confer
a good inhibitory activity against NorA, preliminary screen-
ing was performed by assessing inhibition of EtBr efflux by
SA-1199B. Compounds evaluated included the 2-(4-prop-
oxyphenyl)-4-hydroxyquinoline 20f, the 1-methyl-2-(4-propo-
xyphenyl)quinolin-4(1H)-one 22f, the reference compound
2-(4-propoxyphenyl)-4H-chromen-4-one 4,49 and 2-(4-propo-
xyphenyl)-4H-thiochromen-4-one 6, its strict sulfur-analogue.
The screening concentration employed was 50 μM. The data in
Table 1 confirm that 1-methyl-2-(4-propoxyphenyl)quinolin-
4(1H)-one 22f displays the same, even if low (about to 30%),
inhibitory activity as the reference flavone compound 4.49 This
activity was too weak to permit the quinolone compound 22f to
display a synergistic activity with CPX against norA-overexpres-
sing strains of S. aureus, but it was enough to make this compound
a hit to subject to a series of chemical manipulations in an effort to
obtain more powerful derivatives (Table 1).
Initially, we modified the propoxy group of the phenyl ring in
the C-2 position of the quinolone scaffold of compound 22f by
removing the propyl chain (compound 22c),62 replacing that
with shorter alky chains (compounds 22d,63 22e, and 22g) or
different ethylamino chains (compounds 22hꢀk). None of these
modifications gave more active compounds in terms of EtBr
efflux inhibition activity, suggesting that the best substituent in
this position is the propoxy group (Table 1). The same un-
satisfactory results were obtained by introducing 6,7-dimethoxy
Figure 2. Effect of compounds 25f, 28f, 28j, 29f, reserpine, and
paroxetine on EtBr efflux of SA-1199B.
and 20j. Methylation of 20f with MeI in dry DMF/K2CO3 gave
the O-Me derivative 21f and N-Me derivative 22f. Alkylation of
hydroxyquinolines 20f and 20j with the appropriate 2-chlor-
oethylamines gave only the O-substituted compounds 28f, 28j,
and 29f.
Cyclization of the N-substituted-(2-acetylphenyl)benzamides
16ꢀ19 in NaH/dry DMF or t-BuOK/t-BuOH gave the corre-
sponding 1-alkyl-2-phenyl-4(1H)-quinolinones 22ꢀ25. Under
these conditions, the 40-acethoxy group of compounds 16b
and 17b were also hydrolyzed, resulting in 2-(40-hydroxyphenyl)-
1-methyl-4(1H)-quinolinone 22c62 and its 6,7-dimethoxy
analogue 23c. Starting from 2-(40-hydroxyphenyl)-1-methyl-
4(1H)-quinolinones 22c62 and 23c, the 40-substituted com-
pounds 22d63ꢀk and 23f were obtained in moderate to quite
good yields (14ꢀ65%) by an alkylation procedure in dry DMF/
K2CO3 using the appropriate alkyl halide.
6,7-Dihydroxy derivatives 26a,c were obtained in good yields
(82 and 99%, respectively) by O-demethylation of the corre-
sponding 6,7-dimethoxy derivatives 23a,64c with 1 M BBr3 in
CH2Cl2 at rt. Under the same conditions, or in 48% HBr, the 6,7-
dimethoxy-1-methyl-2-(4-propoxyphenyl)quinolin-4(1H)-one
23f does not give the corresponding 6,7-dihydroxy derivative but
only the trihydroxy compound 26c. Milder conditions such as
LiCl in dry DMF at reflux were used to avoid C-40 O-depropyla-
tion, and only 7-hydroxy-6-methoxy-1-methyl-2-(4-propoxyphe-
nyl)quinolin-4(1H)-one 27f was obtained in low yields (13%)
after 4 days. A confirmation that O-demethylation was carried
Table 2. Evaluation of Intrinsic Antibacterial Activity
(MICs, μg/mL) of Compounds 25f, 28f, 28j, 29f, Reserpine,
Paroxetine, and CPX against the Five S. aureus Strains
Included in the Test of Synergism with CPX
1
out to the OMe in the C-7 position was achieved by an 1Hꢀ H
2D-NOESY NMR experiment (see Supporting Information).
This clearly showed that the OMe group in C-6 position got a
NOE interaction with H-5 and thus indicated that the demethy-
lation happened to the OMe group in position C-7.
modified strains
MIC (μg/mL)
S. aureus
SA-1199B
’ RESULTS AND DISCUSSION
ATCC
SA-K1902 SA-K2378 SA-1199
(norA++/
compd 25923 (WT) (norAꢀ) (norA++) (norA WT) A116E GrlA)
In this study, a series of 2-phenyl-4(1H)-quinolone (22ꢀ27)
and 2-phenyl-4-hydroxyquinoline derivatives (20, 21, 28, 29)
were designed by exchanging the endocyclic oxygen of the
2-phenyl-4H-chromen-4-one nucleus, a common feature of
flavone and natural flavolignane EPIs,47ꢀ49 with a nitrogen atom,
to obtain more effective S. aureus NorA EPIs. A microbiological
approach was used to assess EPI function by determining their
EtBr efflux inhibition using SA-1199B, a well characterized strain
that overexpresses norA,60,61 at 50 μM concentration (Table 1).
25f
>100
100
>100
50
>100
50
>100
>100
>100
100
>100
>100
>100
100
28f
28j
100
100
50
100
50
29f
100
reserpine
>100
>100
>100
>100
100
>100
>100
>100
>100
10
paroxetine >100
CPX 0.31
0.63
2.50
0.63
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Figure 3. Effect of compounds 25f, 28f, 28j, 29f, reserpine, and paroxetine on the MIC of ciprofloxacin against S. aureus ATCC25923, SA-K1902, SA-
K2378, SA-1199, and SA-1199B.
(23a,64 23c, and 23f), 6,7-dihydroxy (26a and 26c), or 6,7-
methoxy-hydroxy (27f) groups, also present on natural and
synthetic EPIs,40ꢀ42,46ꢀ48 in the C-6 and C-7 positions of the
quinolone nucleus of compound 22f (Table 1).
Modification of the substituent in the N-1 position of the
2-phenylquinolone nucleus of compound 22f provided im-
proved results. By introducing an ethyl-2-diethylamine or an
ethyl-2-piperidine in that position, compounds 24f and 25f were
obtained which display EtBr efflux inhibitory activity of 57.3 and
75.1% at 50 μM concentration, respectively (Table 1).
A turning point in this work is represented from the test of
4-methoxy-2-(4-propoxyphenyl)quinoline 21f, previously ob-
tained as a byproduct, that displays reasonably potent inhibition
of EtBr efflux (63.7%). These results led us to switch our
attention to the O-substituted 2-phenyl-4-hydroxyquinoline nu-
cleus of compound 21f instead of the N-substituted 2-phenylqui-
nolone moiety of compound 22f, synthesizing the corresponding
C-4 O-ethyl-2-diethylamine and O-ethyl-2-piperidine derivatives
(compounds 28f and 29f). These compounds were the most
potent NorA inhibitors, demonstrating 93.4 and 88.5% inhibi-
tion of EtBr efflux, respectively. These results were slightly better
than that of the reference compound reserpine (84.8%) and
comparable to that of paroxetine (89.7%) (Table 1). To deepen
our studies on the SAR of this new class of EPIs and to better
comprehend the key role played by the ethylamino substituent, at
least for compounds 24f, 25f, 28f, and 29f, a further ethylpiper-
idine chain was introduced instead of the propoxy group of the
phenyl ring in the C-2 position of the quinoline scaffold of
compound 28f to obtain the derivative 28j. This modification
produces, for compound 28j, a decreased EtBr efflux inhibition
activity (65.6%) when compared with its strict analogue 28f
(Table 1).
For compounds 25f, 28f, 28j, and 29f, which overcome the
threshold of 65% EtBr efflux inhibition against SA-1199B at a
concentration of 50 μM, a doseꢀresponse curve was built to
investigate their potency at different concentrations in compar-
ison with the reference compounds reserpine and paroxetine
(Figure 2).
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The doseꢀresponse curves shown in Figure 2 confirm that
compounds 25f and 28j are inferior to both reference compounds.
The activity of compound 28f is approximately equipotent to
reserpine and paroxetine, whereas compound 29f seems to be
slightly better than the reference compounds at concentrations
less than 50 μM. These results were confirmed by comparing the
IC50 values, which were between 7 and 10 μM for both reference
compounds as well as 28f and 29f.
To establish whether the efflux inhibitory activity of com-
pounds 25f, 28f, 28j, and 29f results in a synergistic interaction
with ciprofloxacin (CPX), a fluoroquinolone antibacterial sub-
strate of the NorA efflux pump, it was necessary to initially
evaluate the intrinsic antibacterial activity of the new EPIs against
the S. aureus strains included in the test (S. aureus ATCC 25923,
SA-K1902, SA-K2378, SA-1199, and SA-1199B), to avoid a
misleading interpretation in assessing NorA inhibitory activity.
Reserpine, paroxetine, and CPX were included for comparative
purposes (Table 2).
Data collected in Table 2 display that compounds 25f, 28f, 28j,
and 29f show weak (50ꢀ100 μg/mL) or no antibacterial activity
up to the top concentration tested, a stark contrast in comparison
with the marketed antibacterial quinolone CPX. The effect of
combining 25f, 28f, 28j, 29f, reserpine, and paroxetine on CPX
MICs was assessed by checkerboard assays,69 which were
performed using two pairs of S. aureus strains, SA-K1902
(norAꢀ)/SA-K2378 (norA++) and SA-1199 (norA wild-type)/
SA-1199B (norA++ and A116E GrlA), as well as S. aureus
ATCC25923 (control, wild-type strain) (Figure 3).
GrlA mutation, all tested compounds display a different level of
synergism with CPX. For the least active compounds 25f, 28j,
and paroxetine, this effect was observed only at high concentra-
tions (50, 100, and 25 μg/mL, respectively). Compound 28f
displays good synergism with CPX, resulting in an 8-fold reduc-
tion in MIC at concentrations above 12.5 μg/mL. These results
were superior to the reference compound reserpine. Compound
29f was most active, showing a synergism with CPX superior to
reserpine at all concentrations above 6.25 μg/mL. MIC reduc-
tions of 16-fold (10 to 0.63 μg/mL) were seen at concentrations
g12.5 μg/mL. Compound 29f completely restored the anti-
bacterial activity of CPX against this highly resistant strain
(Figure 3). This observation suggests that efflux pump inhibition
by 29f is so efficacious as to block all NorA activity in this strain.
Considering that the MIC of compound 29f against SA-1199B
was 100 μg/mL and the best synergistic activity with CPX was
achieved at 12.5 μg/mL, it is likely that there was minimal
interaction between the intrinsic antibacterial activities of the two
compounds. On the other hand, we cannot exclude the involve-
ment of some off-target activities (i.e., inhibition of some non
NorA efflux pumps which have a basal expression in this strain)
both for compound 29f and reserpine.
Data collected in this study highlight that 2-{[2-(4-prop-
oxyphenyl)-4-quinolinyl]oxy}ethanamine derivatives 28f and
29f, obtained by modification of the simplest flavone EPI 4,49
can be effective inhibitors of NorA. For the more active com-
pounds, a good agreement between the inhibitory activity of EtBr
efflux against SA-1199B and the synergistic activity with CPX
against NorA overexpressing S. aureus strains was observed. In an
attempt to delineate a preliminary SAR for this new class of EPIs,
it is possible to state that the best activities were displayed by
compounds carrying the 2-phenyl-4-hydroxyquinoline nucleus
instead of the quinolone core, but only if the C-4 hydroxyl group
was alkylated. A key role was played by the 2-ethylamino chains,
which when inserted at the N-1 position of the quinolone nucleus
or at the C-4 hydroxyl of the quinoline moiety, gave compounds
with activity better than those carrying the same chains in the C-2
phenyl ring. The C-40 propoxy group appears to be the best
substituent for the C-2 phenyl ring, while neither the methoxy
nor the hydroxyl groups are tolerated in the C-6 and C-7
positions of the quinolone nucleus.
Isobolograms shown in Figure 3 reveal no synergistic activity
between any of the tested compounds and CPX against wild-type
strain S. aureus ATCC25923. These data are in agreement with a
low level of expression of the NorA efflux pump in this strain. For
SA-K1902, in which the norA gene is deleted, compounds 25f
and 28j, as well as reserpine and paroxetine, do not show any
significant synergistic activity with CPX. However, compounds
28f and 29f demonstrate a modest synergistic effect manifest by a
4-fold reduction in CPX MIC at 0.78 μg/mL concentration to an
8-fold reduction for concentrations above 25 μg/mL. This synergistic
activity may be the result of weak antibacterial activity of both
compounds that may be observed at concentrations greater than
1/4 of the MIC of the EPIs for this strain (50 μg/mL) or with an
interaction with some undefined off target (Figure 3 and Table 2).
With respect to SA-K2378, which overexpresses norA from a
multicopy plasmid, there is a gradual reduction of CPX MICs
across all tested concentrations of compounds 25f, 28f, 28j, and
29f. Although 25f and 28j were the least active compounds, with
a 4-fold reduction of CPX MICs at 1.56 and 12.5 μg/mL
concentrations respectively, compounds 28f and 29f display a
strong synergistic activity with CPX in that they are able to
reduce MICs 16-fold at 3.13 and 6.25 μg/mL concentrations,
’ CONCLUSION
In conclusion, the 2-{[2-(4-propoxyphenyl)-4-quinolinyl]-
oxy}ethanamine derivatives 28f and 29f show modest or no
intrinsic antistaphylococcal activity up to the highest concentra-
tion tested (100 ug/mL) and were able to restore, in a concen-
tration-dependent manner, the antibacterial activity of CPX
against norA-overexpressing S. aureus strains. Although the EPI
activity of these derivatives in the EtBr efflux inhibition assay
provide results similar to that of reserpine, their synergistic
activity with CPX was superior to that shown by the reference
compound against the norA-overexpressing strain SA-1199B.
Actually, we cannot exclude the involvement of some off target
activities, which will be the subject of further studies.
1
respectively, which are below of /4 of their MICs against this
strain (50 μg/mL). Against SA-K2378, the synergistic activity
with CPX of the compounds 28f and 29f results are better than
that of paroxetine and comparable to that of reserpine. Compar-
ing the synergistic activities with CPX against strains SA-K1902
(norAꢀ) and SA-K2378 (norA++), compounds 28f, 29f, and
reserpine are able to completely restore the antibacterial activity
of CPX against the resistant strain (Figure 3).
For SA-1199 (norA wild-type), none of the tested compounds
nor reserpine or paroxetine have any significant synergistic
activity with CPX (Figure 3). For SA-1199B (CPX MIC
10 μg/mL), which overexpresses norA and also has a A116E
’ EXPERIMENTAL SECTION
Bacterial Strains. The strains of S. aureus employed were
ATCC 25923 (wild-type), SA-K1902 (norA-deleted), SA-1199B (over-
expressing norA and also possesses an A116E GrlA substitution), and its
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Journal of Medicinal Chemistry
ARTICLE
isogenic parent SA-1199.60,61 In addition, SA-K2378, which overex-
presses norA from a multicopy plasmid, was also used. This strain was
produced by cloning norA and its promoter into plasmid pCU1 and then
introducing the construct into SA-K1902.70
Microbiologic Procedures. MICs were determined by micro-
dilution techniques according to CLSI guidelines.71 The effect of
combining reserpine or various test compounds, with scalar dilutions
of freshly prepared solutions of each selected compound, on the MICs of
CPX was also determined. Checkerboard combination studies using
CPX and 25f, 28f, 28j, and 29f were performed as described
previously.69
EtBr Efflux. The loss of EtBr from S. aureus SA-1199B was
determined fluorometrically as previously described.72 Experiments
were performed in duplicate, and the results were expressed as mean
total efflux over a 5 min time course. The effect of increasing concentra-
tions of reserpine, 25f, 28f, 28j, and 29f on the EtBr efflux of SA-1199B
was compared to that in their absence, allowing the calculation of the
percentage reduction in efflux.
title compound 3 as a yellow solid (yield 60%, mp 100.9ꢀ102.2 °C). 1H
NMR (CDCl3): δ 1.07 (3H, t, J = 7.43 Hz, OCH2CH2CH3), 1.80ꢀ1.91
(2H, m, OCH2CH2CH3), 4.01 (2H, t J = 6.54 Hz, OCH2CH2CH3), 6.77
(1H, s, vinyl H, H-30, H-50), 6.88ꢀ7.02 (4H, m, H-30, H-50, H-3, H-5),
7.45 (1H, t, J = 7.68 Hz, H-4), 7.77 (1H, d, J = 7.98 Hz, H-6), 7.92 (2H, d,
J = 8.28 Hz, H-20, H-60), 12.20 (1H, s, OH phenolic), 15.80 (1H, s, OH
enol).
2-(4-Propoxyphenyl)-4H-chromen-4-one (4).49 To a solu-
tion of (2Z)-3-hydroxy-3-(2-hydroxyphenyl)-1-(4-propoxyphenyl)prop-
2-en-1-one 3 (0.10 g, 0.34 mmol) in a mixture of i-PrOH (5 mL) and
(i-Pr)2O (5 mL), 0.10 g of Amberlist 15 was added. The mixture was
maintained at reflux under magnetic stirring for 7 h, then the Amberlist
15 was filtered, and after cooling from the solution, a white solid was
obtained and this was filtrated and purified by column chromatography
with a mixture of EP:Et2O 80:20 to give 0.03 g of the title compound 449
as a white solid (yield 32%, mp 131.1ꢀ132.9 °C). 1H NMR (CDCl3): δ
1.06 (3H, t, J = 7.39 Hz, OCH2CH2CH3), 1.81ꢀ1.91 (2H, m,
OCH2CH2CH3), 4.02 (2H, t J = 6.56 Hz, OCH2CH2CH3), 6.86 (1H,
s, H-3), 7.02 (2H, d, J = 6.01 Hz, H ꢀ30, H-50), 7.44 (1H, dt, J = 7.45 and
1.25 Hz, H-6), 7.58 (1H, dd, J = 8.88 and 1.15 Hz, H-5), 7.71 (1H, dt, J =
7.71 and 1.73 Hz, H-7), 7.91 (1H, d, J = 6.88 Hz, H-2,H-6), 9.30 (1H, dd,
J = 7.94 and 1.31 Hz, H-8). Anal. (C18H16O3) C, H, N.
Synthesis. All reactions were routinely checked by thin-layer
chromatography (TLC) on silica gel 60F254 (Merck) and visualized
using UV illumination. Flash column chromatography was performed
on Merck Silica Gel 60 (mesh 230ꢀ400) using the indicated solvents.
Yields were of purified product and were not optimized. Melting points
were determined in capillary tubes (Mettler PF62 apparatus) and are
uncorrected. Elemental analyses were performed by a Fisons elemental
analyzer (model EA1108CHN), and the data for C, H, and N are within
Ethyl (2Z)-3-hydroxy-3-(4-propoxyphenyl)acrylate (5).58
To a suspension of NaH (0.24 g, 10.0 mmol) in (EtO)2CO (10 mL),
1-(4-propoxyphenyl)ethanone59 (1.50 g, 8.43 mmol) diluted in
(EtO)2CO (3 mL) was added dropwise. The mixture was maintained
under magnetic stirring for 1 h, at rt and then was poured in water and
extracted with EtOAc. The organic solvent was removed under reduced
pressure to obtain 1.60 g of the title compound 558 as a yellow oil (yield
76%). 1H NMR (CDCl3): δ 1.10 (3H, t, J = 7.35 Hz, OCH2CH2CH3),
1.30 (3H, t, J = 7.15 Hz, OCH2CH3), 1.75ꢀ1.90 (2H, m, OCH2CH2-
CH3), 3.90 (2H, s, COCH2CO), 4.00 (2H, t, J = 6.53 Hz, OCH2CH2-
CH3), 4.19 (2H, q, J = 7.15 Hz, OCH2CH3), 6.85 (2H, d, J = 6.85 Hz,
H-3, H-5), 7.90 (2H, d, J = 6.85 Hz, H-2, H-6).
2-(4-Propoxyphenyl)-4H-thiochromen-4-one (6). To 15.0 g
of poliphosphoric acid (PPA) kept at 90 °C in an open vessel under
manual stirring, thiophenol (0.44 g, 4.0 mmol) and ethyl (2Z)-3-
hydroxy-3-(4-propoxyphenyl)acrylate 558 (1.0 g, 4.0 mmol) were slowly
added. The mixture was maintained to that temperature for 3 h and then
was poured in ice/water to obtain a greenish solid, that was filtrated,
solubilized in EtOAc, and washed with Na2CO3 (solution 20%) to
remove the unreacted thiophenol. The organic solution was evaporated
to dryness under reduced pressure, and the residue was crystallized by a
mixture of Et2O/cyclohexane to obtain 0.54 g of the title compound 6 as
a greenish solid (yield 46%, mp 105.3ꢀ106.0 °C). 1H NMR (CDCl3):
δ 1.20 (3H, t, J = 7.40 Hz, OCH2CH2CH3), 1.80ꢀ2.00 (2H, m,
OCH2CH2CH3), 4.00 (2H, t, J = 6.57 Hz, OCH2CH2CH3), 7.00
(2H, d, J = 8.83 Hz H-30,H-50), 7.20 (1H, s, H-3), 7.50ꢀ7.70 (5H, m,
H-6, H-7, H-8, H-20, H-60), 8.55 (1H, d, J = 7.00 Hz, H-5). Anal.
(C18H16O2S) C, H, N.
1
0.4% of the theoretical values. H NMR and 13C NMR spectra were
recorded at 400 and 100.62 MHz, respectively, with a Bruker Advance-
DRX 400 instrument and with Me4Si as the internal standard. The
chemical shift (δ) values are reported in ppm, and the coupling
constants (J) are given in Hz. The abbreviations used are as follows: s,
singlet; bs, broad singlet; d, doublet; dd, double doublet; t, triplet; m,
multiplet. The spectral data are consistent with the assigned structures.
Reagents and solvents were purchased from common commercial
suppliers and were used as received. For routine aqueous workup, the
reaction mixture was extracted with CH2Cl2 or EtOAc. The organic layer
was washed with brine, dried over anhydrous Na2SO4, and concentrated
with a B€uchi rotary evaporator at low pressure. All starting materials were
commercially available unless otherwise indicated.
2-Acetylphenyl 4-propoxybenzoate (2). 4-Propoxybenzoyl
chloride 1f57 was obtained by adding SOCl2 (2 mL) to 4-propoxy
benzoic acid (0.50 g, 2.78 mmol) at reflux under magnetic stirring for 1 h,
distilling the exceeding SOCl2, and washing with dry toluene for three
times to obtain a yellow semisolid, to which was added dry pyridine
(3 mL) and 1-(2-hydroxyphenyl)ethanone (0.38 g, 2.78 mmol) at 0 °C.
The reaction mixture was maintained at rt for 40 h under magnetic
stirring and then was poured in water, basified with NaOH 2N and
extracted with EtOAc (3 ꢁ 20 mL). The collected organic extracts were
washed with HCl 2N and then dried with Na2SO4. The solvent was
removed under reduced pressure to obtain a white semisolid that was
purified by column chormatography with a mixture of EP:Et2O 90:10 to
have 0.60 g of the title compound 2 as a white solid (yield 72%, mp
67.2ꢀ68.4 °C). 1H NMR (CDCl3): δ 1.06 (3H, t, J = 7.40 Hz,
OCH2CH2CH3), 1.80ꢀ1.90 (2H, m, OCH2CH2CH3), 2.54 (3H, s,
COCH3), 4.01 (2H, t, J = 6.55 Hz, OCH2CH2CH3), 7.00 (2H, d, J = 9.10
Hz, H-30 H-50), 7.24 (1H, dd, J = 6.86 and 1.21 Hz, H-5), 7.35 (1H, dt,
J = 6.51 and 1.15 Hz, H-4), 7.57 (1H, dt, J = 6,87 and 1.74 Hz, H-3), 7.85
(1H, dd, J = 7.76 and 2.05 Hz, H-2), 8.16 (2H, d, J = 9.93 Hz, H-20, H-60).
(2Z)-3-Hydroxy-3-(2-hydroxyphenyl)-1-(4-propoxyphe-
nyl)prop-2-en-1-one (3). To a suspension of K2CO3 (2.76 g,
20.0 mmol) in acetone (3 mL), a solution of 2-acetylphenyl 4-propox-
ybenzoate 2 (0.30 g, 1.0 mmol) dissolved in acetone (3 mL) was added
dropwise. The mixture was maintained at reflux under magnetic stirring
for 40 h and then poured in ice/water and filtrated to obtain 0.18 g of the
1-[2-(Methylamino)phenyl]ethanone (9).66 To a solution of
1-(2-aminophenyl)ethanone 7 (5.0 g, 37.0 mmol) in dry toluene
(35 mL), MeI (10.53 g, 74.0 mmol) was added. The reaction mixture
was maintained under magnetic stirring for 48 h at reflux, then it was
filtrated and evaporated to dryness to obtain a residue that was purified
by flash column chromatography (EP:Et2O 98:2) to obtain 2.30 g of the
title compound 966 as a colorless oil (yield 42%). 1H NMR (CDCl3): δ
2.58 (3H, s, COCH3), 2.90 (3H, d, NCH3), 6.59 (1H, dd, J = 6.98 and
1.08 Hz, H-3), 7.39 (1H, dt, J = 8.57 and 1.15 Hz, H-5), 7.39 (2H, d, J =
7.80 and 1.01 Hz, H-4), 7.74 (1H, dt, J = 8.06 and 1.52 Hz, H-6), 8.80
(1H, s, NH).
1-[4,5-Dimethoxy-2-(methylamino)phenyl]ethanone (10).67
With the same procedure described reported for compound 9,66
the title compound 1067 was obtained starting from 1-(2-amino-4,5
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-dimethoxyphenyl)ethanone 8 as a yellow solid (yield 48%, mp
J = 7.60 Hz, H-5), 7.90 (1 H, d, J = 7.60 Hz, H-3), 8.10 (2 H, d, J = 7.90
Hz, H-20, H-60), 9.00 (1 H, d, J = 7.60 Hz, H-6), 11.50 (1 H, bs, NH).
N-(2-Acetylphenyl)-4-(2-piperidin-1-ylethoxy)benzamide
(15j). With the same procedure reported for compound 15f using 4-(2-
piperidin-1-ylethoxy)benzoyl chloride 1j, obtained from the respective
acid as previously reported for 4-propoxybenzoyl chloride 1f,57 the title
1
126.8ꢀ127.7 °C). H NMR (CDCl3): δ 2.51 (3H, s, COCH3), 2.91
(3H, d, NCH3), 3.83 (3H, s, OCH3), 3.93 (3H, s, OCH3), 6.11 (1H, s,
H-3), 7.16 (1H, s, H-6), 8.90 (1H, s, NH).
1-(2-{[2-(Diethylamino)ethyl]amino}phenyl)ethanone
(11).65 1-(2-Fluorophenyl)ethanone (2.0 g, 14.5 mmol) and N,N-
diethylethane-1,2-diamine (2.11 g, 18.1 mmol) were solubilized in dry
DMF (7.5 mL), and the resulting mixture was kept at 100 °C under
magnetic stirring for 33 h. The mixture was poured in water, acidified
with HCl 2N until pH = 3, and washed with Et2O (2 ꢁ 50 mL). The
water layer was then basified with NaOH 2.5N until pH = 12 and
extracted with Et2O (2 ꢁ 50 mL) to obtain, after the evaporation of the
organic solvent under reduced pressure, 2.40 g of the title compound
1165 as an orange oil (yield 70%). 1H NMR (CDCl3): δ 1.08 (6H, t, J =
14.03 Hz, NCH2CH3), 2.52 (3H, s, CH3), 2.65 (4H, q, J = 13.6 Hz,
NCH2CH3), 3.34 (2H, t, J = 9.71 Hz, NCH2CH2), 4.01ꢀ4.12 (2H, m,
NCH2CH2), 6.54 (1H, t, J = 15.06 Hz, NH), 6.70 (1H, d, J = 8.64 Hz,
H-2), 7.31 (2H, t, J = 15.49 Hz, H-3, H-4), 7.69 (1H, d, J = 8.06 Hz, H-5).
1-{2-[(2-Piperidin-1-ylethyl)amino]phenyl}ethanone (12).
With the same procedure reported for compound 1165 using (2-piper-
idin-1-ylethyl)amine instead of N,N-diethylethane-1,2-diamine, the title
compound 12 was obtained as a brown oil (yield 46%). 1H NMR
(CDCl3): δ 1.39ꢀ1.45 (2H, m, NCH2CH2CH2), 1.52ꢀ1.59 (4H, m,
NCH2CH2CH2), 1.78 (4H, t, J = 9.67 Hz, NCH2CH2CH2), 2.53 (3H, s,
CH3), 2.57 (2H, t, J = 9.93 Hz, NCH2CH2), 3.22ꢀ3.32 (2H, m,
NCH2CH2), 6.52 (1H, t, J = 16.21 Hz, NH), 6.66 (1H, d, J = 8.36 Hz,
H-2), 7.30 (2H, t, J = 17.07 Hz, H-3, H-4), 7.69 (1H, d, J = 9.68 Hz, H-5).
Ethyl 4-(2-Piperidin-1-ylethoxy)benzoate (13j).68 A mixture
of ethyl 4-hydroxybenzoate (2.00 g, 12.05 mmol) and K2CO3 (5.82 g,
42.18 mmol) in dry DMF (15 mL) was added dropwise of a solution of
1-(2-chloroethyl)piperidine hydrochloride (3.75 g, 20.49 mmol) in dry
DMF (10 mL) and maintained at 100 °C under magnetic stirring for 17
h. The reaction mixture was poured in water, and the solid obtained was
filtered, solubilized with EtOAc, and anhydrified with Na2SO4. From the
evaporation of the organic solvent under reduced pressure were
obtained 2.30 g of the title compound 13j68 as brownish oil (yield
68%). 1H NMR (CDCl3): δ 1.32 (3H, t, J = 6.97 Hz, CH2CH3),
1.36ꢀ1.46 (2H, m, NCH2CH2CH2), 1.47ꢀ1.69 (4H, m, NCH2CH2-
CH2), 2.58ꢀ2.75 (4H, m, NCH2CH2CH2), 2.76ꢀ2.98 (2H, m, NCH2-
CH2O), 4.24ꢀ4.34 (4H, m, NCH2CH2O, OCH2CH3), 6.86 (2H, d, J =
8.63 Hz, H-2, H-6), 7.94 (2H, d, J = 8.59 Hz, H-3, H-5).
1
compound 15j was obtained as a yellow oil (yield 84%). H NMR
(CDCl3): δ 1.40ꢀ1.55 (2H, m, piperidinic CH2), 1.72ꢀ2.07 (4H, m,
piperidinic CH2), 2.48 (3H, s, CH3), 3.05ꢀ3.25 (4H, m, piperidinic
CH2), 3.30ꢀ3.45 (2H, m, NCH2), 4.32ꢀ4.45 (2H, m, OCH2CH2N),
6.60 (2H, d, J = 8.40 Hz, H-30, H-50), 7.12 (2H, d, J = 8.40 Hz, H-20,
H-60), 7.18ꢀ7.25 (2H, m, ArꢀH), 7.40ꢀ7.55 (2H, m, ArꢀH).
4-{[(2-Acetylphenyl)(methyl)amino]carbonyl}phenyl
Acetate (16b). With the same procedure described for compound 15f
using 4-acetyl benzoyl chloride 1b, obtained from the respective acid as
previously reported for 4-propoxybenzoyl chloride 1f57 and 1-[2-(meth-
ylamino)phenyl]ethanone 9,66 the title compound 16b was obtained as
a brownish solid (yield 90%, mp 137.2ꢀ137.9 °C). 1H NMR (CDCl3):
δ 2.20 (6H, s, OCOCH3, COCH3), 3.40 (3H, s, NꢀCH3), 6.85 (2H, d,
J = 8.50 Hz, H-30, H-50), 7.15ꢀ7.30 (4H, m, H-20, H-60, H-5, H-4),
7.45ꢀ7.55 (2H, m, H-3, H-6).
N-(2-Acetyl-4,5-dimethoxyphenyl)-N-methylbenzamide
(17a). With the same procedure described for compound 15f using
benzoyl chloride 1a and 1-[4,5-dimethoxy-2-(methylamino)phenyl]-
ethanone 10,67 the title compound 17a was obtained as a white solid
1
(yield 60%, mp 134.2ꢀ134.5 °C). H NMR (CDCl3): δ 2.30 (3H, s,
COCH3), 3.50 (3H, s, NCH3), 3.85 (6H, s, OCH3), 6.65 (1H, s, H-3),
7.00 (1H, s, H-6), 7.10ꢀ7.25 (5H, m, H-20, H-30, H-40, H-50, H-60).
4-{[(2-Acetyl-4,5-dimethoxyphenyl)(methyl)amino]car-
bonyl}phenyl Acetate (17b). With the same procedure described
for compound 15f using 4-acety benzoyl chloride 1b, obtained from the
respective acid as previously reported for 4-propoxybenzoyl chloride
1f57 and 1-[4,5-dimethoxy-2-(methylamino)phenyl]ethanone 10,67 the
title compound 17b was obtained, after crystallization with EtOH, as a
white solid (yield 59%, mp 176.2ꢀ177.1 °C). 1H NMR (CDCl3): δ 2.30
(3H, s, OCOCH3), 2.40 (3H, s, COCH3), 3.50 (3H, s, NCH3), 4.00
(6H, s, OCH3), 6.75 (1H, s, H-3), 7.00 (2H, d, J = 8.70 Hz, H-30, H-50),
7.10 (1H, s, H-6), 7.35 (2H, d, J = 8.70 Hz, H-20, H-60).
N-(2-Acetylphenyl)-N-[2-(diethylamino)ethyl]-4-propox-
ybenzamide (18f). Under nitrogen atmosphere, a solution of 1-(2-
{[2-(diethylamino)ethyl]amino}phenyl)ethanone 1165 (0.65 g, 2.77
mmol) and Et3N (1.40 g, 1.90 mL, 13.85 mmol) in 10 mL of THF
dry was added dropwise of 4-propoxybenzoyl chloride 1f57 (0.55 g,
2.77 mmol), prepared as reported above, and was maintained for 3 h, at
rt, under magnetic stirring. The reaction mixture was poured in water,
basified in NaOH 2.5N, and extracted with EtOAc (3 ꢁ 50 mL). After
anhydrification with Na2SO4, from the evaporation of the organic
extracts, a residue was obtained, and this was purified by flash chroma-
tography with CHCl3:MeOH (97:3) to give 0.39 g of the title
compound 18f as a yellowish oil (yield 40%). 1H NMR (CDCl3):
δ 0.88 (3H, t, J = 7.32 Hz, OCH2CH2CH3), 1.30ꢀ1.50 (6H, m,
NCH2CH3), 1.54ꢀ1.68 (2H, m, OCH2CH2CH3), 1.97 (3H, s, CH3),
3.03ꢀ3.70 (6H, m, NCH2), 3.75 (2H, t, J = 7.27 Hz, OCH2CH2CH3),
4.06ꢀ4.45 (2H, m, NCH2CH2N), 6.78 (2H, d, J = 8.81 Hz, H-30, H-50),
7.09 (2H, d, J = 8.84 Hz, H-20, H-60), 7.30ꢀ7.40 (1H, m, H-3),
7.35ꢀ7.65 (3H, m, ArꢀH).
4-(2-Piperidin-1-ylethoxy)benzoic Acid (14j).68 A solution
of ethyl 4-(2-piperidin-1-ylethoxy)benzoate 13j68 (0.77 g, 2.78 mmol)
in MeOH (12 mL) was added of 5% NaOH (4 mL) and maintained for
15 h under magnetic stirring at rt. The mixture was poured in water and
acidified with HCl 6N to obtain a white precipitate that was filtered to
give 0.58 g of the title compound 14j68 as a white solid (yield 84%, mp
1
275.5ꢀ277.1 °C). H NMR (CDCl3): δ 1.30ꢀ1.90 (6H, m, NCH2-
CH2CH2 and NCH2CH2CH2), 3.02ꢀ3.05 (2H, m, NCH2CH2O), 3.50
(4H, t, J = 4.63 Hz, NCH2CH2O), 7.11 (2H, d, J = 8.89 Hz, H-2, H-6),
7.95 (2H, d, J = 8.85 Hz, H-3, H-5), 10.7 (1H, s, OH).
N-(2-Acetylphenyl)-4-propoxybenzamide (15f). A solution
of 1-(2-aminophenyl)ethanone 7 (1.52 g, 12.59 mmol) and Et3N
(6.37 g, 8.76 mL, 62.95 mmol) in 50 mL of dry THF was added
dropwise to 4-propoxybenzoyl chloride 1f57 (2.0 g, 9.60 mmol),
prepared as reported above, and maintained at 70 °C for 3 h. The
mixture was poured in water and extracted with EtOAc (3 ꢁ 50 mL).
From the evaporation of the organic solvent under reduced pressure, a
solid was obtained, which after crystallization with a mixture of Et2O/
EtOH, gave 2.70 g of the title compound 15f as a white solid (yield 72%,
mp 105.7ꢀ106.2 °C). 1H NMR (CDCl3): δ 1.10 (3 H, t, J = 7.41 Hz,
OCH2CH2CH3), 1.75ꢀ2.00 (2 H, m, OCH2CH2CH3), 2.75 (3 H, s,
COCH3), 4.00 (2 H, t, J = 6.56 Hz, OCH2CH2CH3), 7.00 (2 H, d,
J = 7.90 Hz, H-30, H-50), 7.20 (1 H, t, J = 7.60 Hz, H-4), 7.60 (1 H, t,
N-(2-Acetylphenyl)-N-(2-piperidin-1-ylethyl)-4-propox-
ybenzamide (19f). With the same procedure described for com-
pound 18f using 4-propoxybenzoyl chloride 1f,57 obtained from the
respective acid as reported above, and 1-{2-[(2-piperidin-1-ylethyl)-
amino]phenyl}ethanone 12, the title compound 19f was obtained, after
purification by flash chromatography column CH2Cl2:MeOH (85:15),
as a brown oil (yield 28%). 1H NMR (CDCl3): δ 0.93 (3H, t, J = 7.32 Hz,
OCH2CH2CH3), 1.32ꢀ1.47 (6H, m, piperidinic CH2), 1.51ꢀ1.63
5731
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Journal of Medicinal Chemistry
ARTICLE
(2H, m, OCH2CH2CH3), 1.94 (3H, s, CH3), 3.05ꢀ3.68 (6H, m,
piperidinic CH2 and NCH2), 3.75 (2H, t, J = 7.27 Hz, OCH2CH2CH3),
4.13ꢀ4.38 (2H, m, NCH2CH2N), 6.91 (2H, d, J = 8.81 Hz, H-30, H-50),
7.14 (2H, d, J = 8.84 Hz, H-20, H-60), 7.35ꢀ7.43 (1H, m, H-3),
7.48ꢀ7.69 (3H, m, ArꢀH).
7.70ꢀ7.80 (2H, m, H-7, H-5), 8.20 (2H, d, J = 7.86 Hz, H-8), 9.50ꢀ10.50
(1H, bs, OH). Anal. (C16H13NO2) C, H, N.
1-[2-(Diethylamino)ethyl]-2-(4-propoxyphenyl)quinolin-
4(1H)-one (24f). With the same procedure described for compound
22c62 starting from N-(2-acetylphenyl)-N-[2-(diethylamino)ethyl]-4-
propoxybenzamide 18f, the title compound 24f was obtained, after
purification with column chromatography CH2Cl2:MeOH (99:1), as a
white solid (yield 85%, mp 212.2ꢀ213.2 °C). 1H NMR (CDCl3): δ 0.88
(6H, t, J = 7.14 Hz, NCH2CH3), 1.11 (3H, t, J = 7.45 Hz, OCH2-
CH2CH3), 1.78ꢀ2.00 (2H, m, OCH2CH2CH3), 2.39 (4H, q, J = 7.06
Hz, NCH2CH3), 2.66 (2H, t, J = 7.27 Hz, NCH2CH2N), 4.06 (2H, t, J =
6.58 Hz, OCH2CH2CH3), 4.27 (2H, t, J = 7.28 Hz, NCH2CH2N), 6.28
(1H, s, H-3), 7.04 (2H, d, J = 11.45 Hz, H-30, H-50), 7.39 (2H, d, J = 8.75
Hz, H-20, H-60), 7.40ꢀ7.50 (1H, m, H-6), 7.72 (1H, d, J = 10.23 Hz,
H-5), 7.70ꢀ7.77 (1H, m, H-7), 8.53 (1H, dd, J = 10.26 and 3.29 Hz,
H-8). Anal. (C24H30N2O2) C, H, N.
1-(2-Piperidin-1-ylethyl)-2-(4-propoxyphenyl)quinolin-
4(1H)-one (25f). With the same procedure described for compound
22c starting from N-(2-acetylphenyl)-N-(2-piperidin-1-ylethyl)-4-pro-
poxybenzamide 19f, the title compound 25f was obtained, after purifica-
tion by flash column chromatography CHCl3:MeOH (99:1), as a yellow
solid (yield 55%, mp 83.7ꢀ84.5 °C). 1H NMR (CDCl3): δ 1.10 (3H, t,
J = 7.35 Hz, OCH2CH2CH3), 1.30ꢀ1.60 (6H, m, piperidinic CH2),
1.87ꢀ2.00 (2H, m, OCH2CH2CH3), 2.10ꢀ2.29 (4H, m, piperidinic
CH2), 2.55 (2H, t, J = 7,40 Hz, NCH2CH2N), 4.06 (2H, t, J = 6.58 Hz,
OCH2CH2CH3), 4.24 (2H, t, J = 6.51 Hz, NCH2CH2N), 6.27 (1H, s,
H-3),7.03 (2H, d, J = 8.13 Hz, H-30, H-50), 7.32 (2H, d, J = 5.67 Hz, H-20,
H-60), 7.37ꢀ7.49 (1H, m, H-6), 7.68 (1H, d, J = 8.74 Hz, H-5),
7.70ꢀ7.79 (1H, m, H-7), 8.54 (1-H, dd, J = 8.77 and 1.29 Hz, H-8).
Anal. (C25H30N2O2) C, H, N.
6,7-Dimethoxy-1-methyl-2-phenylquinolin-4(1H)-one
(23a).64 To a solution of N-(2-acetyl-4,5-dimethoxyphenyl)-N-methyl-
benzamide 17a (0.30 g, 1.10 mmol) in t-BuOH (15 mL), t-BuOK was
added (0.58 g, 4.80 mmol) and warmed to 60 °C for 3 h. After cooling,
the reaction mixture was filtered, and the resulting filtrate was evapo-
rated under reduced pressure to obtain a crude product that was
crystallized by EtOH to give 0.11 g of the title compound 23a64 as a
white solid (yield 38%, mp 263.3ꢀ265.4 °C). 1H NMR (CDCl3): δ 3.65
(3H, s, NCH3), 4.00 (6H, s, OCH3), 6.25 (1H, s, H-3), 6.85 (1H, s,
H-8), 7.35ꢀ7.55 (5H, m, H-20, H-30, H-40, H-50, H-60), 7.85 (1 H, s,
H-5). Anal. (C18H17NO3) C, H, N.
2-(4-Hydroxyphenyl)-6,7-dimethoxy-1-methylquinolin-
4(1H)-one (23c). With the same procedure described for compound
23a64 starting from 4-{[(2-acetyl-4,5-dimethoxyphenyl)(methyl)-
amino]carbonyl}phenyl acetate 17b, the title compound 23c was
obtained, after crystallization with EtOH/DMF, as a white solid (yield
35%, mp 337.3ꢀ337.9 °C). 1H NMR (DMSO-d6): δ 3.60 (3H, s,
NCH3), 3.85 (3H, s, OCH3), 3.90 (3H, s, OCH3), 5.85 (1H, s, H-3),
6.85 (2H, d, J = 8.55 Hz, H-30, H-50), 7.10 (1 H, s, H-8), 7.30 (2H, d, J =
8.55 Hz, H-20, H-60), 7.55 (1H, s, H-5), 10.00 (1H, bs, OH). Anal.
(C18H17NO4) C, H, N.
2-(4-Propoxyphenyl)quinolin-4-ol (20f). A solution of N-
(2-acetylphenyl)-4-propoxybenzamide 15f (2.70 g, 9.09 mmol) in t-
BuOH (30 mL) was added of t-BuOK (5.10 g, 45.41 mmol) and warmed
at 60 °C for 3 h under magnetic stirring. The mixture was poured in
water, acidified with HCl 2N to obtain a solid that was filtrated and
purified by a flash chromatographic column CHCl3:MeOH (80:20) to
give 1.96 g of the title compound 20f as a white solid (yield 77%, mp
271.0ꢀ273.0 °C). 1H NMR (DMSO-d6): δ 1.00 (3H, t, J = 7.38 OCH2-
CH2CH3), 1.65ꢀ1.75 (2H, m, OCH2CH2CH3), 4.00 (2H, t, J = 6.60
Hz, OCH2CH2CH3), 6.20 (1H, s, H-3), 7.10 (2H, d, J = 8.76 Hz, H-30,
H-50), 7.30 (1H, t, J = 7.20 Hz, H-6), 7.65 (1H, t, J = 7.20 Hz, H-7), 7.75
(3H, m, H-8, H-20, H-60), 8.10 (1H, d, J = 7.82 Hz, H-5), 11.50 (1H, bs,
OH). Anal. (C18H17NO2) C, H, N.
2-[4-(2-Piperidin-1-ylethoxy)phenyl]quinolin-4-ol (20j).
With the same procedure described for compound 20f starting from
N-(2-acetylphenyl)-4-(2-piperidin-1-ylethoxy)benzamide 15j, stirring
for 72 h at 100 °C, the title compound 20j was obtained as a brownish
1
solid (yield 81%, mp 215.0ꢀ217.2 °C). H NMR (CDCl3): δ 1.45ꢀ
1.65 (6H, m, piperidinic CH2), 2.55ꢀ2.70 (4H, m, piperidinic CH2),
2.87 (2H, t, J = 5.99 Hz, NCH2), 4.21 (2H, t, J = 5.46 Hz, OCH2CH2N),
6.53 (1H, s, H-3), 7.04 (2H, d, J = 8.92 Hz, H-30, H-50), 7.38ꢀ7.42 (2H,
m, ArꢀH), 7.45ꢀ7.55 (2H, m, ArꢀH), 7.58ꢀ7.70 (4H, m, H-20, H-60
and ArꢀH).
4-Methoxy-2-(4-propoxyphenyl)quinoline (21f) and 1-Met-
hyl-2-(4-propoxyphenyl)quinolin-4(1H)-one (22f). To a mix-
ture of 2-(4-propoxyphenyl)quinolin-4-ol 20f (0.20 g, 0.72 mmol) and
K2CO3 (0.30 g, 2.16 mmol) in dry DMF (5 mL) was added MeI (0.21 g,
0.09 mL, 1.44 mmol) and maintained at 60 °C for 2 h under magnetic
stirring. The mixture was poured in ice/water and extracted with EtOAc
(3 ꢁ 50 mL). From the evaporation of the organic extracts, a crude
product was obtained, and this was purified by flash chromatography
with CHCl3:MeOH (90:10) to give 0.03 g of the compound 21f as a
white solid (yield 14%, mp 115.8ꢀ117.1 °C) and 0.04 g of the
compound 22f as a white solid (yield 19%, mp 147.7ꢀ148.5 °C).
21f: 1H NMR (CDCl3): δ 1.11 (3H, t, J = 7.34 Hz, OCH2CH2CH3),
1.70ꢀ1.95 (2H, m, OCH2CH2CH3), 4.05 (2H, t, J = 6.57 Hz, OCH2-
CH2CH3), 4.17 (3H, s, OCH3), 7.08 (2H, d, J = 8.69 Hz, H-30, H-50),
7.20 (1H, s, H-3), 7.51 (1H, t, J = 7.15 Hz, H-6), 7.75 (1H, t, J = 6.86 Hz,
H-7), 8.05ꢀ8.25 (4H, m, H-20, H-60, H-5, H-8). Anal. (C19H19NO2)
C, H, N.
22f: 1H NMR (CDCl3): δ 1.12 (3H, t, J = 7.37 Hz, OCH2CH2CH3),
1.75ꢀ2.00 (2H, m, OCH2CH2CH3), 3.86 (3H, s, NCH3), 4.04
(2H, t, J = 6.62 Hz, OCH2CH2CH3), 6.30 (1H, s, H-3), 7.05 (2H, d,
J = 8.53 Hz, H-30, H-50), 7.41 (2H, d, J = 8.53 Hz, H-20, H-60), 7.57
(1H, t, J = 7.27 Hz, H-6), 7.74 (1H, d, J = 9.09 Hz, H-5), 7.87 (1H, t,
J = 7.27 Hz, H-7), 8.52 (1H, d, J = 8.00 Hz, H-8). Anal. (C19H19NO2)
C, H, N.
2-(4-Methoxyphenyl)-1-methylquinolin-4(1H)-one (22d).63
To a suspension of 2-(4-hydroxyphenyl)-1-methylquinolin-4(1H)-one
22c62 (0.20 g, 0.80 mmol) and K2CO3 (0.22 g, 1.60 mmol) in dry DMF
(10 mL) was added dropwise of MeI (0.34 g, 0.15 mL, 2.40 mmol) and
warmed to 70 °C for 1.5 h under magnetic stirring. The mixture was
poured in water and the solid obtained, was filtrated, dried and purified by
column chromatography CHCl3:MeOH (99:1) to give 0.10 g of the title
compound 22d63 as a yellow solid (yield 47%, mp 135.5ꢀ138.0 °C). 1H
NMR (CDCl3): δ 3.60 (3H, s, NCH3), 3.85 (3H, s, OCH3), 6.25 (1H, s,
H-3), 7.00 (2H, d, J = 8.85 Hz, H-30, H-50), 7.30ꢀ7.45 (3H, m, H-20, H-60,
H-6), 7.55 (1H, d, J = 8.60 Hz, H-5) 8.00 (1H, dt, J = 8.62 Hz and J =
1.71, H-7), 8.50 (1H, dd, J = 8.00 and 1.70 Hz, H-8). Anal. (C17H15NO2)
C, H, N.
2-(4-Hydroxyphenyl)-1-methylquinolin-4(1H)-one (22c).62
To a suspension of NaH (0.93 g, 38.6 mmol) in dry DMF (25 mL)
was added dropwise a solution of 4-{[(2-acetylphenyl)(methyl)amino]-
carbonyl}phenyl acetate 16b (2.40 g, 7.70 mmol) in dry DMF (30 mL)
and maintained for 1 h under magnetic stirring at rt. After decomposition
of the excess of NaH with EtOAc, the mixture was poured in water and
extracted with EtOAc (3 ꢁ 50 mL). From the evaporation under reduced
pressure of the organic solvent and after purification with flash column
chromatography CHCl3:MeOH (99:1 f80:20), the title compound
22c62 was obtained as a white solid (yield 52%, mp 335.3ꢀ336.5 °C).
1H NMR (DMSO-d6): δ 3.50 (3H, s, NꢀCH3), 5.90 (1 H, s, H-3), 6.90
(2H, d, J = 8.53 Hz, H-30, H-50), 7.30ꢀ7.50 (3H, m, H-20, H-60, H-6),
5732
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Journal of Medicinal Chemistry
ARTICLE
2-(4-Ethoxyphenyl)-1-methylquinolin-4(1H)-one (22e).
With the same procedure described for compound 22d,63 using EtI
instead of MeI, the title compound 22e was obtained as a white solid
(yield 65%, mp 200.0ꢀ201.8 °C). 1H NMR (CDCl3): δ 1.45 (3H, t, J =
7 Hz, OCH2CH3), 3.68 (3H, s, NCH3), 4.10 (2H, q, J = 7 Hz,
OCH2CH3), 6.45 (1H, s, H-3), 7.00 (2H, d, J = 8.50 Hz, H-30, H-50),
7.35 (2H, d, J = 8.50 Hz, H-20, H-60), 7.00 (1H, t, J = 7.27 Hz, H-6), 7.58
(1H, d, J = 8.80 Hz, H-5), 7.74 (1H, d, J = 7.80 Hz, H-7), 8.48 (1H, d, J =
7.80 Hz, H-8). Anal. (C18H17NO2) C, H, N.
1-Methyl-2-(4-propoxyphenyl)quinolin-4(1H)-one (22f).
With the same procedure described for compound 22d,63 using n-PrI
instead of MeI, the title compound 22f was obtained as a white solid
(yield 38%, mp 147.7ꢀ148.5 °C). This compound was also obtained in
mixture with compound 21f following the procedure described above
(see the procedure for spectral data).
2-(4-Isopropoxyphenyl)-1-methylquinolin-4(1H)-one (22g).
With the same procedure described for compound 22d,63 using i-PrI
instead of MeI, the title compound 22g was obtained as a white solid
(yield 55%, mp 173.2ꢀ175.1 °C). 1H NMR (CDCl3): δ 1.40 (6H, d, J =
6.00 Hz, OCH(CH3)2), 3.75 (3H, s, NCH3), 4.52ꢀ4.72 (1H, m,
OCH(CH3)2), 6.58 (1H, s, H-3), 6.98 (2,H, d, J = 8.70 Hz, H-30,
H-50), 7.33 (2H, d, J = 8.70 Hz, H-20, H-60), 7.47 (1H, t, J = 7.10 Hz,
H-6), 7.62 (1H, d, J = 8.50 Hz, H-5), 7.75 (1H, dd, J = 6.90 and 1.60 Hz,
H-7), 8.48 (1H, dd, J = 8.00 and 1.30 Hz, H-8). Anal. (C19H19NO2)
C, H, N.
2-{4-[2-(Dimethylamino)ethoxy]phenyl}-1-methylquino-
lin-4(1H)-one (22h). With the same procedure described for com-
pound 22d,63 using (2-chloroethyl)dimethylamine hydrochloride
instead of MeI, the title compound 22h was obtained, after purification
with flash column chromatography CH2Cl2:MeOH (95:5), as a yellow-
ish solid (yield 16%, mp 122.0ꢀ125.0 °C). 1H NMR (CDCl3): δ 2.44
(6H, s, NCH3), 2.85 (2H, t, J = 5.63 Hz, CH2N), 3.67 (3H, s, CH2N),
4.20 (2H, t J = 5.67 Hz, OCH2), 6.35 (1H, s, H-3), 7.08 (2H, d, J = 8.74
Hz, H-30, H-50), 7.44 (2H, d, J = 11.86 Hz, H-20, H-60), 7.40ꢀ7.53 (1H,
m, H-6), 7.59 (1H, d, J = 8.52 Hz, H-5), 7.69ꢀ7.81 (1H, m, H-7), 8.54
(1-H, d, J = 8.00 Hz, H-8). Anal. (C20H22N2O2) C, H, N.
chromatography CH2Cl2:MeOH (90:10), as a white solid (yield 14%,
mp 132.2ꢀ132.9 °C). 1H NMR (CDCl3): δ 2.56 (4H, t, J = 4.84 Hz,
NCH2), 2.81 (2H, t, J = 5.83 Hz, CH2N), 3.59 (3H, s, NCH3), 3.71
(4H, t, J = 4.75 Hz, CH2O), 4.14 (2H, t, J = 5.55 Hz, OCH2), 6.25 (1H, s,
H-3), 6.97 (2H, d, J = 8.84 Hz, H-30, H-50), 7.30 (2H, d, J = 8.87 Hz,
H-20, H-60), 7.30ꢀ7.48 (1H, m, H-6), 7.58 (1H, d, J = 11.68 Hz, H-5),
7.60ꢀ7.70 (1H, m, H-7), 8.46 (1-H, dd, J = 7.91 and 1.79 Hz, H-8).
Anal. (C22H24N2O3) C, H, N.
6,7-Dimethoxy-1-methyl-2-(4-propoxyphenyl)quinolin-
4(1H)-one (23f). With the same procedure described for compound
22d,63 starting from 2-(4-hydroxyphenyl)-6,7-dimethoxy-1-methylqui-
nolin-4(1H)-one 23c, and using n-PrI instead of MeI, the title com-
pound 23f was obtained, after purification with a flash chromatographic
column CH2Cl2:MeOH (99:1), as a white solid (yield 24%, mp 258.2ꢀ
259.5 °C). 1H NMR (CDCl3): δ 1.05 (3 H, t, J = 7.43, OCH2CH2CH3),
1.54ꢀ1.57 (2 H, m, OCH2CH2CH3), 3.55ꢀ3.65 (2 H, m, OCH2-
CH2CH3), 3.90ꢀ4.05 (9 H, m, two OCH3, NCH3), 6.25 (1 H, s, H-3),
6.85 (1 H, s, H-8), 6.95 (2 H, d, J = 8.67 Hz, H-30, H-50), 7.30 (2 H, d, J =
8.67 Hz, H-20, H-60), 7.85 (1 H, s, H-5). Anal. (C21H23NO4) C, H, N.
6,7-Dihydroxy-1-methyl-2-phenylquinolin-4(1H)-one (26a).
To a solution of 6,7-dimethoxy-1-methyl-2-phenylquinolin-4(1H)-one
23a64 (0.20 g, 0.60 mmol) in dry CH2Cl2 (20 mL) was added dropwise
of a solution 1 M of BBr3 (8.5 mL, 8.50 mmol) in CH2Cl2 and maintained
for 3 h at room temperature under magnetic stirring. The mixture was
poured in water and the obtained precipitate, after crystallization in a
mixture of MeOH/CH2Cl2, gave 0.12 g of the title compound 26a as a
white solid (yield 82%, mp 350.0ꢀ352.2 °C). 1H NMR (MeOD):
δ 3.70 (3H, s, NCH3), 6.20 (1H, s, H-3), 7.20 (1H, s, H-8), 7.40ꢀ7.65
(5H, m, H-20, H-30, H-40, H-50, H-60), 7.80 (1 H, s, H-5). Anal. (C16H13-
NO3) C, H, N.
6,7-Dihydroxy-2-(4-hydroxyphenyl)-1-methylquinolin-
4(1H)-one (26c). With the same procedure described for compound
26a, starting from 2-(4-hydroxyphenyl)-6,7-dimethoxy-1-methylquino-
lin-4(1H)-one 23c, the title compound 26c was obtained as a white solid
(yield 99%, mp 368.5ꢀ369.6 °C). 1H NMR (DMSO-d6): δ 3.50 (3H, s,
NCH3), 5.75 (1H, s, H-3), 6.80 (2H, d, J = 8.52 Hz, H-30, H-50), 7.00
(1H, s, H-8), 7.25 (2H, d, J = 8.52 Hz, H-20, H-60), 7.50 (1H, s, H-5),
9.75ꢀ10.00 (3H, bs, 3 OH). Anal. (C16H13NO4) C, H, N.
2-{4-[2-(Diethylamino)ethoxy]phenyl}-1-methylquino-
lin-4(1H)-one (22i). With the same procedure described for com-
pound 22d,63 using (2-chloroethyl)diethylamine hydrochloride instead
of MeI, the title compound 22i was obtained, after purification with flash
chromatography column CH2Cl2:MeOH (99:1), as brownish solid
7-Hydroxy-6-methoxy-1-methyl-2-(4-propoxyphenyl)-
quinolin-4(1H)-one (27f). To a solution of 6,7-dimethoxy-1-methyl-
2-(4-propoxyphenyl)quinolin-4(1H)-one 23f (0.10 g, 0.30 mmol) in
dry DMF (10 mL) was added of LiCl (0.06 g, 1.40 mmol) and
maintained for 4 days at reflux. The mixture was poured in water,
acidified with HCl 2N at pH = 6, and the obtained precipitate, after
filtration and purification by a chromatographic column CHCl3:MeOH
(95:5), gave 0.012 g of the title compound 27f as a white solid (yield
13%, mp 271.3ꢀ273.0 °C). 1H NMR (DMSO-d6): δ 1.02 (3H, t, J =
7.50 Hz, CH3), 1.70ꢀ1.85 (2H, m, CH2CH3), 3.50 (3H, s, NCH3), 3.88
(3H, s, OCH3), 4.00 (2H, t, J = 6.50 Hz, OCH2), 5.82 (1H, s,
H-3), 7.00ꢀ7.15 (3H, m, H-30, H-50, H-8),7.40 (2H, d, J = 8.85 Hz,
H-20, H-60), 7.55 (1H, s, H-5), 10.20 (1H, bs, OH). Anal. (C20H21NO4)
C, H, N.
N,N-Diethyl-2-{[2-(4-propoxyphenyl)quinolin-4-yl]oxy}-
ethanamine Hydrochloride (28f). A suspension of 2-(4-propox-
yphenyl)quinolin-4-ol 20f (0.15 g, 0.54 mmol) and K2CO3 (0.22 g, 1.62
mmol) in dry DMF (10 mL) was maintained for 30 min under magnetic
stirring at room temperature. Then was added dropwise a solution of
(2-chloroethyl)diethylamine hydrochloride (0.15 g, 1.08 mmol) in dry
DMF (5 mL) and warmed at 110 °C for 5 h. The mixture was poured in
water and extracted with Et2O (5 ꢁ 30 mL). The organic phase was
dried with Na2SO4 and bubbled with HCl g to obtain 0.12 g of a
white solid that resulted in the title compound 28f as hydrochloride
(yield 54%, mp 243.8ꢀ245.0 °C). 1H NMR (CDCl3): δ 1.00ꢀ
1.25 (9H, m, OCH2CH2CH3 and both NCH2CH3), 1.75ꢀ2.00 (2H,
1
(yield 25%, mp 104.0ꢀ106.0 °C). H NMR (CDCl3): δ 1.15 (6H, t,
J = 7.13 Hz, NCH2CH3), 2.71 (4H, q, J = 7.13 Hz, NCH2CH3), 2.96
(2H, t, J = 6.27 Hz, CH2N), 3.67 (3H, s NCH3), 4.16 (2H, t, J = 6.25 Hz,
OCH2), 6.34 (1H, s, H-3), 7.07 (2H, d, J = 9.71 Hz, H-30, H-50), 7.39
(2H, d, J = 6.68 Hz, H-20, H-60), 7.40ꢀ7.50 (1H, m, H-6), 7.59 (1H, d,
J = 8.33 Hz, H-5), 7.72ꢀ7.79 (1H, m, H-7), 8.55 (1H, dd, J = 798 and
1.55 Hz, H-8). Anal. (C22H26N2O2) C, H, N.
1-Methyl-2-[4-(2-piperidin-1-ylethoxy)phenyl]quinolin-
4(1H)-one (22j). With the same procedure described for compound
22d,63 using 1-(2-chloroethyl)piperidine hydrochloride instead of MeI,
the title compound 22j was obtained, after purification with flash column
chromatography CH2Cl2:MeOH (90:10), as a brownish solid (yield
35%, mp 144.0ꢀ146.0 °C). 1H NMR (CDCl3): δ 1.43ꢀ1.51 (2H, m,
NCH2CH2CH2), 1.52ꢀ1.75 (4H, m, NCH2CH2CH2), 2.61 (4H, t, J =
5.03 Hz, NCH2CH2CH2), 2.86 (2H, t, J = 6.00 Hz, CH2N), 3.68 (3H, s,
NCH3), 4.22 (2H, t, J = 6.09 Hz, OCH2), 6.35 (1H, s, H-3), 7.07 (2H, d,
J = 8.74 Hz, H-30, H-50), 7.38 (2H, d, J = 6.74 Hz, H-20, H-60), 7.42ꢀ7.53
(1H, m, H-6), 7.58 (1H, d, J = 10.66 Hz, H-5), 7.70ꢀ7.82 (1H, m, H-7),
8.55 (1-H, dd, J = 7.98 and 1.62 Hz, H-8). Anal. (C23H26N2O2) C, H, N.
1-Methyl-2-[4-(2-morpholin-4-ylethoxy)phenyl]quinolin-
4(1H)-one (22k). With the same procedure described for compound
22d,63 using 4-(2-chloroethyl)morpholine instead of MeI, the title
compound 22k was obtained, after purification with flash column
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Journal of Medicinal Chemistry
ARTICLE
m, OCH2CH2CH3), 2.70 (4H, q, J = 7.16 Hz, NCH2CH3), 3.05 (2H, t,
J = 6.10 Hz, OCH2CH2N), 4.00 (2H, t, J = 6.65 Hz, OCH2CH2CH3),
4.35 (2H, t, J = 6.10 Hz, OCH2CH2N), 7.00 (2H, d, J = 6.80 Hz, H-30,
H-50), 7.15 (1H, s, H-3), 7.45 (1H, t, J = 8.20 Hz, H-6), 7.70 (1H, t, J =
8.22 Hz, H-7), 8.00ꢀ8.10 (3H, m, H-20, H-60, H-8), 8.25 (1H, d, J = 7.34
Hz, H-5). Anal. (C24H31ClN2O2) C, H, N.
N,N-Diethyl-2-({2-[4-(2-piperidin-1-ylethoxy)phenyl]-
quinolin-4-yl}oxy)ethanamine (28j). With the same procedure
described for compound 28f, starting from 2-[4-(2-piperidin-1-ylethoxy)-
phenyl]quinolin-4-ol 20j, without bubbling HCl g, the title compound
28j was obtained as the free base, after purification by a flash chromato-
graphic column CH2Cl2:MeOH (95:5), as a brown oil (yield 47%). 1H
NMR (CDCl3): δ 1.05 (6H, t, J = 7.10 Hz, NCH2CH3), 1.30ꢀ1.45 (2H,
m, piperidinic CH2), 1.50ꢀ1.70 (4H, m, piperidinic CH2), 2.40ꢀ2.55
(4H, m, piperidinic CH2), 2.67 (4H, q, J = 7.16 Hz, NCH2CH3), 2.77
(2H, t, J = 5.98 Hz, CH2N), 3.04 (2H, t, J = 6.06 Hz, CH2N), 4.15 (2H, t,
J = 3.32 Hz, OCH2), 4.32 (2H, t, J = 2.43 Hz, OCH2), 6.99 (2H, d, J =
8.70 Hz, H-30, H-50), 7.11 (1H, s, H-3), 7.36ꢀ7.43 (1H, m, H-7),
7.58ꢀ7.67 (1H, m, H-6), 7.99ꢀ8.14 (4H, m, ArꢀH). Anal. (C28H37-
N3O2) C, H, N.
4-(2-Piperidin-1-ylethoxy)-2-(4-propoxyphenyl)quino-
line (29f). A mixture of 2-(4-propoxyphenyl)quinolin-4-ol 20f (0.15 g,
0.54 mmol), t-BuOK (0.18 g, 1.60 mmol), and 1-(2-chloroethyl)-
piperidine hydrochloride (0.20 g, 1.10 mmol) in dry DMF (5 mL)
was maintained for 8 h at 100 °C under magnetic stirring. After cooling,
the mixture was poured in water and the obtained precipitate was
filtered, dried, and crystallized from EtOH to obtain 0.10 g of the title
compound 29f as a white solid (yield 51%, mp 95.2ꢀ97.6 °C). 1H NMR
(CDCl3): δ 1.10 (3H, t, J = 7.00 Hz, OCH2CH2CH3), 1.50ꢀ1.65 (2H,
m, piperidinic CH2), 1.75ꢀ2.00 (4H, m, OCH2CH2CH3 and piper-
idinic CH2), 2.70ꢀ2.80 (4H, m, piperidinic CH2), 3.20 (2H, t, J = 5.30
Hz, OCH2CH2N), 4.00 (2H, t, J = 6.70 Hz, OCH2CH2CH3), 4.55 (2H,
t, J = 5.60 Hz, OCH2CH2N), 7.05 (2H, d, J = 8.70 Hz, H-30,H-50), 7.20
(1H, s, H-3), 7.45 (1H, t, J = 8.15 Hz, H-6), 7.70 (1H, t, J = 8.15 Hz,
H-7), 8.00ꢀ8.15 (4H, m, H-20, H-60, H-5,H-8). Anal. (C25H30N2O2)
C, H, N.
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’ ASSOCIATED CONTENT
Supporting Information. 1Hꢀ H 2D-NOESY NMR
1
S
b
spectral data of compound 27f and elemental analysis data for
target compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
(20) Doan, T. L.; Fung, H. B.; Mehta, D.; Riska, P. F. Tigecycline: a
glycylcycline antimicrobial agent. Clin. Ther. 2006, 28, 1079–1106.
(21) Hooper, D. C. Fluoroquinolone resistance among Gram-positive
cocci. Lancet Infect. Dis 2002, 2, 530–538.
Corresponding Author
*Phone: +39 75 5855130. Fax: +39 75 5855115. E-mail: stefano.
sabatini@unipg.it.
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’ ABBREVIATIONS USED
MDR, multidrug resistance; MFS, major facilitator superfamily;
EPI, efflux pump inhibitor; EtBr, ethidium bromide
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