Synthesis of symmetrically substituted 3,3-dibenzyl-4-hydroxy-3,4-dihydro- 1H-quinolin-2-ones, as novel quinoline derivatives with antibacterial activity

Article abstract of DOI:10.1016/j.ejmech.2014.05.024

A novel series of symmetrically substituted 3,3-dibenzyl-4-hydroxy-3,4- dihydro-1H-quinolin-2-ones was synthesized and tested as antimicrobials. The minimum inhibitory concentration (MIC) values of the most active heterocycles were slightly higher than those exhibited by levofloxacin, employed as comparator. Structural factors affecting the activity were explored along three diversification points, including the substituents of the aromatic rings of the 3-benzyl moieties, as well as the functionalization of both, the homocyclic ring of the heterocycle and the quinolonic nitrogen atom. 6-Chloro and 3,3-bis(4′-chlorobenzyl) derivatives showed the lower MIC values. Optimally substituted heterocycles were synthesized, which exhibited enhanced activity.

Full text of DOI:10.1016/j.ejmech.2014.05.024

Accepted Manuscript
Synthesis of Symmetrically Substituted 3,3-Dibenzyl-4-hydroxy-3,4-dihydro-1H-
quinolin-2-ones, as Novel Quinoline Derivatives with Antibacterial Activity
Matías D. Ferretti, Alexandre T. Neto, Ademir F. Morel, Teodoro S. Kaufman, Enrique
L. Larghi
PII:
S0223-5234(14)00438-3
10.1016/j.ejmech.2014.05.024
EJMECH 6980
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 December 2013
Revised Date: 24 April 2014
Accepted Date: 6 May 2014
Please cite this article as: M.D. Ferretti, A.T. Neto, A.F. Morel, T.S. Kaufman, E.L. Larghi, Synthesis of
Symmetrically Substituted 3,3-Dibenzyl-4-hydroxy-3,4-dihydro-1H-quinolin-2-ones, as Novel Quinoline
Derivatives with Antibacterial Activity, European Journal of Medicinal Chemistry (2014), doi: 10.1016/
j.ejmech.2014.05.024.
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ACCEPTED MANUSCRIPT
Synthesis of Symmetrically Substituted 3,3-Dibenzyl-4-hydroxy-3,4-dihydro-
1
H- quinolin-2-ones, as Novel Quinoline Derivatives with Antibacterial Activity
*
Matías D. Ferretti, Alexandre T. Neto, Ademir F. Morel, Teodoro S. Kaufman and
Enrique L. Larghi
OH
R3
Ar
*
R
3
,3-Dibenzyl-4-hydroxy-3,4-dihydro-1H-quinolin-2-ones were synthesized and tested as antimicrobials.
N
O
MIC and MLC values were slightly higher than those exhibited by levofloxacin, employed as
comparator. SAR were explored along three diversification points (both aromatic rings and the
quinolonic nitrogen). 6-Chloro and 3,3-bis(4’-chlorobenzyl) derivatives showed the lower MIC
values.
R1

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Synthesis of Symmetrically Substituted 3,3-Dibenzyl-4-hydroxy-3,4-dihydro-1H-
quinolin-2-ones, as Novel Quinoline Derivatives with Antibacterial Activity
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b
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Matías D. Ferretti, Alexandre T. Neto, Ademir F. Morel, Teodoro S. Kaufman and
a,*
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Enrique L. Larghi
a
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Instituto de Química Rosario (CONICET-UNR), Suipacha 531, Rosario, S2002LRK,
República Argentina. Tel/fax: +54-341-4370477, ext. 103.
E-mail: larghi@iquir-conicet.gov.ar (Enrique L. Larghi)
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Núcleo de Pesquisas em Produtos Naturais, Prédio 18, Universidade Federal de Santa
María, Santa Maria, RS, Brazil. Tel/fax: +55-55-3220-8869
E-mail: afmorel@base.ufsm.br (Ademir F. Morel)
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Abstract - A novel series of symmetrically substituted 3,3-dibenzyl-4-hydroxy-3,4-
dihydro-1H-quinolin-2-ones was synthesized and tested as antimicrobials. The
minimum inhibitory concentration (MIC) values of the most active heterocycles were
slightly higher than those exhibited by levofloxacin, employed as comparator. Structural
factors affecting the activity were explored along three diversification points, including
the substituents of the aromatic rings of the 3-benzyl moieties, as well as the
functionalization of both, the homocyclic ring of the heterocycle and the quinolonic
nitrogen atom. 6-Chloro and 3,3-bis(4’-chlorobenzyl) derivatives showed the lower
MIC values. Optimally substituted heterocycles were synthesized, which exhibited
enhanced activity.
Keywords: Bioactive compounds; Novel antibacterial heterocycles; Broad spectrum;
3,3-Dibenzyl-4-hydroxy-3,4-dihydro-1H-quinolin-2-ones.
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1. Introduction
Bacterial infection is one of the most complex global health issues of this century.
The rise of resistant microorganisms is perceived as a serious threat, which aggravates
the problem. As a result, increasing efforts have been placed during recent years
towards the search for new antimicrobials [1] in order to neutralize this hazard. If left
unsolved, this can evolve into a potentially dangerous public health crisis of worldwide
dimensions [2].
Quinolines are privileged heterocycles which have been developed into important
therapeutic agents, embodying a wide variety of biological activities [3]. Antimicrobial
activity has been found among 4-oxo-1,4-dihydro-quinoline-3-carboxylic acids
(collectively known as ‘quinolones’), 1H-quinolin-2-ones (carbostyrils) [4] and 1H-
quinoline-2,4-diones (4-hydroxy-2-quinolones) [5].
The quinolones are one of the most useful and versatile families of antibacterial
agents [6]. The first generation of quinolones was developed after observations
regarding nalidixic acid (1, Figure 1), a side product found during the synthesis of the
antimalarial agent chloroquine. Continued research resulted in the discovery of more
suitable analogs, successively leading to second and third generations of these
antibacterials, with representatives such as ciprofloxacin (2) and levofloxacin (3),
respectively, characterized by improvements in their potency, spectrum and
bioavailability. Thus, nalidixic acid and its congeners were active only against Gram-
negative microorganisms exhibiting poor pharmacokinetics, while ciprofloxacin
increased both the spectrum and activity against aerobic Gram-negatives and
levofloxacin broadened the spectrum to include some aerobic Gram-positive bacteria.
Fourth generation quinolones, like moxifloxacin, are also currently available [6a]. These
methoxy-quinolones are even more active against Gram-positives and anaerobes
bacteria.
Functionalization of the related carbostyril skeleton is also a fertile source of
biologically important molecules that exert a plethora of bio- and pharmacological
activities. These include enzyme inhibitors [7] and receptor antagonists [8], as well as
antithrombotic [9], antiviral (against hepatitis B [10] and HIV [11]), antihelmintic [12],
antioxidant [13], antiproliferative [14], anti-tumor [15], and cytotoxic against human
tumor cell lines [16] agents. However, relatively few 1H-quinolin-2-one derivatives
have been reported as antibacterials [17]; among them, the 4-substituted analogs
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rationally designed by the group of Jayashree, based on docking experiments with
bacterial DNA gyrase [18].
With regards to the antibacterial activity of 1H-quinoline-2,4-diones [19], O’Donnell
et al. [20] recently performed a detailed study on the inhibition of bacterial growth by a
series of sixty synthetic and natural products, carrying different substitution patterns on
their homo and heterocyclic rings.
Symmetrically and unsymmetrically 3,3-disubstitued 1H-quinoline-2-one derivatives
are structurally related compounds that have been found in nature. They are represented
by compound 4 [21], the HIV-1RT inhibitor buchapine (5, buchapsine, IC = 8 µM)
5
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[22], the derivatives of which exhibited antitumor and antimycobacterial activity [23]
and the cytotoxic and pro-apoptotic agent severibuxine (6) [24]. Analogous compounds
have also been synthesized for structural analysis and method development purposes
[25], as selective 5-HT6 receptor antagonists [26] and as intermediates towards
antithrombotic agents [27]. In addition, the quinoline-2,4-dione derivative 7 has been
used as a cleft-type receptor in chiral recognition [28].
In a previous study, we have observed through bioautographic techniques that 3,3-
disubstituted-1H-quinoline-2,4-diones, including the 3,3-dimethyl, 3,3-diallyl and 3,3-
dibenzyl analogs (8), exhibited antibacterial activity. In this preliminary work, we have
explored the activity of the related 3,3-dibenzyl-4-hydroxy derivative 9, which
displayed intriguing bacterial growth inhibition [29]. On the other hand, Daruwala et al.
proved that related 3,3-dialkyl-4-hydroxy-3,4-dihydro-1H-quinolin-2-one 10 was
relatively non-toxic (LD > 150 mg/Kg) and a mild sedative, in the method involving
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sodium pentobarbital sleeping time potentiation (ED = 85 mg/Kg) [30].
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Compounds 9 and 10 belong to a class of heterocycles, the antimicrobial activity of
which has not been yet systematically explored. Therefore, we decided to perform a
more detailed study, by executing the synthesis of a series of 3,3-dibenzyl-4-hydroxy-
3,4-dihydro-1H-quinolin-2-ones, carrying out structural modifications at three main
points and evaluating their antimicrobial activity against a panel of microorganisms.
Centres subjected to structural variation were the isocyclic ring, the C-3 pendant benzyl
moieties and position 1 of the quinolinic core.
4

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O
N
O
O
N
CO H
F
CO H
F
CO H
2
2
2
Me
N
N
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N
O
Me
, Nalidixic acid
H
Me
Me
3, Levofloxacin
1
2, Ciprofloxacin
O
Bu
Bu
O
N
H
O
R3'
R4' R4
O
O
O
R3'
H
R3
R6
4
R3
R
EtO
C
N
2
3
N
R1
O
H
S
N
R1
2
O
:
O
1
8
R = H; R= H, Me, Hal;
7
1
3
3'
6
3
3'
4
5
R = Me; R =R = CH =CMe ; R = H
R =R = alkyl, alkenyl, Ph;
2
2
1
6
3
4
4'
R =R = H, R = CH =CMe ;
R =R = =O;
2
2
3
'
1
4
3
3'
4'
R = C(Me )CH=CH
9 R=R =R = H; R =R = Ph= R = OH
2
2
1
3
3'
6
3
3'
1
4'
6
R =R = H; R = geranyl; R = OH
10 R=R =R = H; R = Me; R = OH
9
4
9
9
5
6
Figure 1. Representative examples of the quinolone antimicrobials (1-3) and examples of natural (4-6)
and synthetic (7, 8) 3,3-disubstitued-1H-quinoline-2,4-diones and related compounds (9, 10).
9
9
9
7
8
9
2. Results and Discussion
2.1. Chemistry
1
1
1
1
1
1
00
01
02
03
04
05
A library of symmetrically substituted 3,3-dibenzyl-4-hydroxy-3,4-dihydro-1H-
quinolin-2-one derivatives (11) was prepared, employing a modular approach based on
the retrosynthetic analysis shown in Scheme 1, which hinged upon the intermediacy of
4-hydroxy-1H-quinoline-2-one intermediates (13) and uncovered anilines (15) and
diethyl malonate (16) as suitable starting materials.
4'
R3
'
2
OH
O
Ar
O
Cyclization OH
C-Alkylation
Ar
O
5
4
3
6
7
Ar
EtO
4
a
+
8
a
N
2
O
N
H
Ar
X
N
O
NH2
EtO
O
1
R
8
R
R
R
H
14
R1
Amidation
N-Alkylation
11
12
13
15
16
1
06
1
1
07
08
Scheme 1. Retrosynthetic analysis of the target 3,3-dibenzyl-4-hydroxy-3,4-dihydro-1H-quinolin-2-ones
11.
1
1
09
10
In this way, substituents with different H-bond donor/acceptor characteristics and
steric/electronic properties were introduced during the syntheses through the use of
1
11
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12
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properly substituted aromatic precursors 14 and 15 leading to 12. N-alkylation of the
latter provided a third point of structural diversification.
Accordingly, six substituted anilines were condensed at high temperature with
diethyl malonate (16) under solventless condition, affording the expected bis-
malonamide intermediates (17-22) in 44-91% yield (Scheme 2). In turn, these dianilides
were cyclized under promotion of polyphosphoric acid, as eco-friendly Lewis acid, at
120 °C during a standard time of 6 h [31], to give the desired 4-hydroxy-carbostyryls
(23-28) in 17-98% yield.
Scheme 2. Synthesis of 3,3-dibenzyl-4-hydroxy-3,4-dihydro-1H-quinolin-2-ones 45-60.
OH
3
4
2
O
ii
i
R
R
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NH2
1
N
CH
2
2
6
N
O
H
H
1
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1
1
1
1
5a R= H
17 R= H
23 R= H
5b R= 4-F
5c R= 4-Cl
18 R= 4-F
19 R= 4-Cl
24 R= 6-F
25 R= 6-Cl
iii
5d R= 4-OMe
5e R= 4-Me
5f R= 2-MeO
20 R= 4-OMe
21 R= 4-Me
22 R= 2-MeO
26 R= 6-MeO
27 R= 6-Me
28 R= 8-MeO
v
OH Ar
O
Ar
O
O
Ar
O
5
4
1
3
v
6
iv
Ar
Ar
Ar
R
4a
R
7
R
8
a
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N
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H
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5 R= H, Ar= C
6 R= 6-F, Ar= C
7 R= 6-Cl, Ar= C
8 R= 6-MeO, Ar= C
9 R= 6-Me, Ar= C
0 R= 8-MeO, Ar= C
1 R= H, Ar= 4'-MeOC
6
H
5
, R = H
39 R= H, Ar= C
40 R= H, Ar= C
41 R= H, Ar= C
42 R= H, Ar= C
6
H
6
H
6
H
6
H
5
5
5
5
, R = Me
29 R= H, Ar= C
30 R= 6-F, Ar= C
31 R= 6-Cl, Ar= C
6 5
H
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6
H
5
, R = H
, R = Et
6 5
H
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1
6
H
5
, R = H
, R = Bn
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H
5
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1
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H
5
, R = H
, R = CH
2
CO
2
Et
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3
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3
3
2 R= 6-MeO, Ar= C H5
6
1
1
6
H
5
, R = H
43 R= 6-Cl, Ar= 4'-ClC
44 R= 6-Cl, Ar= 4'-ClC
6
H
H
4
4
, R = Et
3 R= 6-Me, Ar= C
H
5
6
1
1
6
H
5
, R = H
6
, R = 4"-ClC
6
H
4
CH
2
4R= 8-MeO, Ar= C
6
H
5
1
6
H
4
, R = H
5 R= H, Ar= 4'-MeOC H
1
6
2 R= H; Ar= 4'-ClC
3 R= H; Ar= 2'-ClC
6
H
H
4
, R = H
, R = H
6 R= H, Ar = 4'-ClC
6
H
H
4
4
1
6
4
1
37 R= H, Ar = 2'-ClC
6
4 R= H; Ar= C
5 R= H; Ar= C
6 R= H; Ar= C
7 R= H; Ar= C
6
6
6
6
H
H
H
H
5
5
5
5
, R = Me
, R = Et
, R = Bn
, R = CH
1
6
38 R= 6-Cl, Ar = 4'-ClC H4
1
1
2
CO
2
Et
1
8 R= 6-Cl, Ar= 4'-ClC
9 R= 6-Cl, Ar= 4'-ClC
0 R= 6-Cl, Ar= 4'-ClC
6
H
6
H
6
H
4
4
4
, R = H
, R = Et
, R = 4"-ClC
1
1
6
H
4
CH
2
1
22
1
1
1
23
24
25
Reagents and conditions: i) H C(CO Et) (16), neat, 210-220 ºC, 1-2 h. ii) PPA, 130-140 ºC, 6 h. iii)
2 2 2
1
1
ArCH X (X= Br, Cl), K CO , PhMe/H O (1:1, v/v), 110 °C, 24-48 h. iv) (R O) SO or R Br, K CO ,
2
2
3
2
2
2
2
3
MeCN, 80 °C, 24-40 h. v) NaBH , EtOAc-EtOH (1:1, v/v), r.t., 2-5 h.
4
1
1
1
1
26
27
28
29
With these synthetic intermediates in hand, and in order to introduce the second point
of diversification, the regioselective C-3 dibenzylation of heterocycles 23-28 was next
undertaken, employing an adaptation of previous procedures, with benzyl halides as
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alkylating agents and K CO as base [32]. Under these conditions, the desired 3,3-
2
3
dibenzyl-1H-quinoline-2,4-diones 29-38 were obtained in 34-69% yield after 24-36 h.
With the purpose of including the third point of structural variation, the
regioselective N-benzylation of 2,4-dione 29 was initially explored in anhydrous DMF,
employing NaH as base [33]; however, only decomposition products were detected.
Better results were obtained when the K PO /N,N-dimethylacetamide (DMA) reagent
3
4
system was used [34] which afforded exclusively the desired N-benzylated product (41),
albeit in moderate yield (36%).
The selective N-alkylation was carried out employing K CO in refluxing MeCN
2
3
[35]. This procedure accepted a diversity of electrophiles and allowed access to N-
alkylated products 39-44 in good to very good yields (60-89%).
Finally, the ketonic carbonyl moiety of the quinoline-2,4-diones 29-44 was
selectively reduced with NaBH in a EtOAc-EtOH mixture, uneventfully rendering the
4
expected carbinols (45-60) in 37-99% yield.
2.2. Biological activity
Initially, the 3,3-dibenzyl-4-hydroxy-3,4-dihydro-1H-quinolin-2-ones 44-53 were
tested as antimicrobials employing the broth micro dilution technique, with levofloxacin
(3) as comparator. The results (Table 1) revealed that among the 3,3-dibenzyl congeners
45-50, the derivatives on the homocyclic ring exhibited the lowest MIC. The 6-chloro
derivative 47 was the most potent compound, followed by the 6-fluoro analog 46. These
values were 2-4 times lower than those observed for 45 (unsubstituted on the aromatic
ring of the heterocyclic scaffold) and the 6-methyl (49), 6-methoxy (48) and 8-methoxy
substituted derivatives (50).
Functionalization of the 3-benzyl substituents strongly affected the antibacterial
activity. Thus, while the 4’-MeO substituted analog 51 was as active as the non-
substituted precursor 45, the 3,3-bis(4’-chlorobenzyl) derivative 52 showed MIC values
8-60 times lower than 45 indicating a higher level of antibacterial activity. In addition,
the position of the chlorine atom within the aromatic ring of the benzyl group of the
analogs also proved to be relevant, as the MIC value of 4’-chloro derivative 52 was up
to eight times lower than that of its 2’-chloro substituted congener 53. These
observations suggest that steric and also electronic factors may have some impact on the
potency of these compounds.
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Compounds 54-57 served to understand the effect of N-substitution. When compared
to 45, it was observed that the introduction of small alkyl groups such as methyl (54)
and ethyl (55) afforded minor improvements on the bioactivity and only against
Bacillus subtilis and Escherichia coli, while the presence of the bulkier benzyl group
(56) afforded a product which displayed essentially the same profile of the parent
heterocycle 45 and installation of an alkyl acetate moiety (57) resulted in a substance
devoid of activity.
1
1
71
72
Table 1. MIC and MLC (µg/mL) results obtained for the 3,3-dibenzyl-4-hydroxy-3,4-
dihydro-1H-quinolin-2-ones 45-60 against a panel of five microorganisms.
a
Microorganisms
Gram-positive
Gram-negative
B. subtilis
MIC/MLC
100 > 200
S. aureus
S. sonnei
P. aeruginosa
MIC/MLC
E. coli
Compound
MIC/MLC
50 > 200
50 > 200
25 > 200
100 > 200
50 > 200
100 > 200
MIC/MLC
50 > 200
25 > 200
12.5 > 200
100 > 200
50 > 200
100 > 200
100 > 200
MIC/MLC
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
100 > 200 100 > 200
50 > 200 25 > 200
50 > 200 12.5 200
100 > 200 100 > 200
100 > 200 25 > 200
25
200
200
12.5
50 > 200
50 > 200
100 > 200
100 > 200
100 > 200 100 > 200
100 > 200 100 > 200
100
6.25
12.5
200
100
200
1.56
12.5
25
6.25
12.5
100
200
100
100 > 200 12.5
100 > 200
100 > 200 100 > 200
100 1.52
25
200
200
50 > 200
12.5 > 200
100 > 200
100 > 200
100 > 200
100 > 200
-
100 > 200
200 > 200
200 > 200
50 > 200
> 200
-- 100 > 200
-- 100 > 200
> 200
3.12
-- > 200
> 200
--
12.5
100
100
3.12
> 200
50
50
25
3.12
6.25
50
12.5
50
3.12
50
100 3.12
50
3.12
50
50
50
50
100
> 100 > 200
3.12 3.12
100
3.12
50
50
50 > 100
Levofloxacin
3.12
3.12
3.12
3.12 3.12
3.12
1
1
73
74
a. Mean of 4 determinations.
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1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
06
07
08
From these results it may be inferred that functionalization of the nitrogen atom
affects the potency of the heterocycles and that the nature of the substituent has some
influence on it, presumable through steric (54 and 55 vs. 56 and 57) as well as
electronic (55 and 56 vs. 57) effects.
Based on the structures of the heterocycles that showed lower MIC values, a new
compound was designed to embody modifications at two diversification points;
therefore compound 58 carrying a 6-chloro substituent and two 4’-chlorobenzyl groups
on C-3, was synthesized through the intermediacy of 38. The resulting trichloro
derivative retained the activity, exhibiting approximately the same spectrum and about
one half the MIC of the parent compound 52, demonstrating that the effects of the
substituents were non-additive.
In a further modification, and taking into account the improved performance of 55
with regards to 45, compound 59 (the N-ethyl derivative of 58) was synthesized by N-
alkylation of 38, followed by reduction of the resulting quinoline-2,4-dione 43.
Interestingly, this compound displayed a narrower spectrum, remaining a similar MIC
value than 58 against Gram-positive bacteria such as B. subtilis and S. aureus, while
being essentially inactive against the Gram-negative test strains (Table 1). Not
unexpectedly, however, in light of the profile of 56, no improvements were observed
when the bulkier and more lipophilic 4”-chlorobenzyl moiety was attached to the
nitrogen atom (60).
Interestingly, the tested compounds exhibited comparatively higher MIC values
when exposed to P. aeruginosa. This observation reflects some degree of selectivity on
the side of the assayed compounds. P. aeruginosa has demonstrated to exhibit
differential sensitivity against different quinolone antibacterials [36].
Compounds 58 and 59 were also examined against an additional set of five clinically
relevant bacteria. The results (Table 2) revealed only subtle differences between their
profiles.
Therefore, in order to get some insights on the mode of action of these heterocycles,
the behavior responses of 58 and 59 against Enterococcus spp. were studied, at
concentration levels in the range 0.5-8 times the MIC, and the responses were assessed
against those of ampicillin and levofloxacin (3), as comparators. The modes of action of
these antibacterials (interaction with PBPs and type II bacterial topoisomerases,
respectively), trigger a series of events which may lead (on a species specific basis) to
filamentation or lysis, thus modifying the turbidity of the medium. The determination of
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the optical densities at 620 nm (OD ) was employed as a measure of the turbidity of
6
20
the medium [37], and in both cases controls devoid of the antibacterials were used,
which displayed a continuous increase of the OD620 due to normal bacterial growth. The
results are displayed in Figures 2 and 3.
Table 2. MIC and MLC (µg/mL) results for 58 and 59 against selected
a
microorganisms.
Microorganisms
Gram-positive
Gram-negative
Enterococcus Enterobacter
Salmonella
typhimurium
Burkholderia
cepacia
Morganella
morganii
spp.
aerogenes
Compound
MIC MLC MIC MLC
MIC MLC MIC MLC MIC MLC
5
5
8
9
6.25
6.25
0.78
50
25
50
50
>100
>100
0.78
50
50
100
100
6.25
6.25
0.78
6.25
12.5
0.78
1.56 3.12
3.12 12.5
0.78 1.56
Levofloxacin
0.78
0.78
0.78 0.78
2
2
16
17
a. Mean of 4 determinations.
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
The experiments revealed that the turbidity diminished rapidly after treatment with
58 (Figure 2A) at concentrations as low as the MIC (6.25 µg/mL). In the presence of 58
the media exhibited a typical decrease in the OD620 after a short initial growth period,
resembling the response of ampicillin (Figure 2C); however, the effect of 58 took place
at lower concentration and the variation in turbidity levels was more pronounced.
Interestingly, at 3.12 µg/mL, the medium containing 53 displayed an initial decay in the
OD , followed by bacterial regrowth after 3 h.
6
20
A closer inspection of the curves of Enterococcus spp. treated with 58 (Figure 2A)
revealed an apparent paradox. The decrease of OD620 was lower at higher
concentrations of the tested heterocycle. This is reminiscent to the behavior of nalidixic
acid (1) and related quinolones, as well as other antibacterials (Eagle effect) [38].
Nalidixic acid was found to be most bactericidal at 50-200 µg/mL. However,
concentrations of 1 above this range reduced rather than increased its bactericidal effect.
At higher levels, around 400 µg/mL, the drug was relatively bacteriostatic. When the
mode of action of nalidixic acid was investigated, it was found that at the most
bactericidal concentration the drug inhibited production of DNA, but not RNA or
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2
2
34
35
36
protein synthesis. However, at higher concentrations, where the drug was least
bactericidal, RNA and protein synthesis were both found to be inhibited [39].
2
37
2
2
2
2
38
39
40
41
Figure 2. Growth inhibition effect of compounds 58 (A) and 59 (B) against Enterococcus spp. For the
sake of comparison, the responses of ampicillin (C) and levofloxacin (D) are also shown. Cells were
incubated in casein soy broth medium, and each compound was added at time zero. OD620= Optical
density at 620 nm. Average of readings from cuadruplicate experiments; with error < 5%.
2
2
2
2
2
2
2
2
2
2
42
43
44
45
46
47
48
49
50
51
On the other side, compound 59 was also capable of causing growth retardation in a
concentration-dependent manner, exhibiting response curves (Figure 2B) similar to
those of levofloxacin (Figure 2D). These curves were characterized by a relatively rapid
increase of the OD620 during the first 3 h, followed by increments at a much slower pace
after the fourth hour. Additionally, compound 58 was found to be a more effective
antibacterial than ampicillin (Figure 2A and 2C), reaching higher inhibition levels in a
shorter time.
A similar behavior was noted when cultures of Morganella morganii were exposed
to the heterocycles 58 and 59 at concentrations between 3.12 and 50 µg/mL (Figure 3).
Compound 58 caused a decrease in OD620 at the MIC (6.25 µg/mL) and above (Figure
3A); furthermore, the curves exhibited characteristics analogous to those observed when
the heterocycle was made to interact with Enterococcus spp. (Figure 2A). On the
contrary, the N-ethyl derivative 59 displayed a different behavior (Figure 3B); its effects
2
2
2
2
52
53
54
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were not significant up to 12.5 µg/mL (2 × MIC), causing a noticeable concentration-
dependent growth retardation above 25 µg/mL.
2
2
2
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2
2
2
2
57
58
59
60
61
62
63
64
The mechanism of action of the tested 3,3-dibenzyl-4-hydroxy-3,4-dihydro-1H-
quinolin-2-ones is still unclear. Nevertheless, these compounds lack the 3-carboxy-4-
oxo motif, characteristic of the quinolone antibacterials, which is the minimal structural
+
+
requirement for chelation with Mg at the DNA-gyrase site [40]. Thus, 58 and 59
would in principle be operating under a different mechanism from that of the traditional
quinolones, however further experimentation is needed in order to solve this question.
2
65
2
2
2
66
67
68
Figure 3. Growth inhibition effect of compounds 58 (A) and 59 (B) against Morganella morganii. Cells
were incubated in casein soy broth medium, and each compound was added at time zero. OD620= Optical
density at 620 nm. Average of readings from cuadruplicate experiments; error < 5%.
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
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73
74
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3. Conclusions
The synthesis and biological activity of a library of sixteen 3,4-dihydro-1H-quinolin-
2-one derivatives, embodying a 3,3-dibenzyl-4-hydroxy pattern and three main sites of
structural diversification, were reported. The design of the library allowed gaining
preliminary insights into the structural requirements for the activity of the heterocycles
and helped to improve their antibacterial profile.
In some cases, the antibacterial activity of the heterocycles against a panel of five
different microorganisms, including Gram-positive and Gram-negative strains reached
levels comparable to those of levofloxacin. However, unlike the latter, these novel
compounds are of simple preparation, which can be performed from easily available
starting materials and low cost reagents.
Further experimentation is under way in order to acquire knowledge on the influence
of the configuration of C-4 hydroxyl group of the 3,3-dibenzyl-4-hydroxy-3,4-dihydro-
1H-quinolin-2-ones on their activity or potency. This will shed light on the mechanism
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of action of these heterocycles, and help to establish a predictive SAR model. The
results will be published in due time.
2
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4. Experimental section
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14
15
16
17
4.1. General conditions
Melting points were taken on an Ernst Leitz Wetzlar model 350 hot-stage
1
13
19
microscope and are reported uncorrected. The H, C and F NMR spectra were
usually acquired in CDCl (unless otherwise stated) in a Bruker Avance spectrometer
3
1
13
19
(300.13, 75.48 and 282.23 MHz for H, C and F, respectively) with tetramethylsilane
(TMS) as internal standard. The chemical shifts (δ) are reported in ppm downfield from
TMS and the coupling constants (J) are expressed in Hertz. The DEPT 135 experiment
1
3
was used to aid the interpretation of the fully decoupled C NMR spectra. In special
cases, 2D NMR experiments (COSY, HMBC and HSQC) were also employed. Low
resolution mass spectra (EI = 70 eV) were obtained with a Shimadzu QP-2010plus
instrument by direct injection with a probe temperature program (T = 40 °C, T = 350
Init
End
°C, ∆t= 20 °C/min). High resolution mass spectra were obtained with a Bruker
MicroTOF-Q II instrument (Bruker Daltonics, Billerica, MA). Detection of the ions was
performed in electrospray ionization, positive ion mode.
The reactions requiring inert atmospheres were carried out under dry Argon,
employing oven-dried glassware. The reagents were used as received; dry DMF was
prepared by distillation from anhydrous BaO; absolute EtOH was prepared by
distillation after refluxing the reagent for 24 h over I -activated Mg turnings; dry MeCN
2
was prepared by a 4 h reflux over P O followed by distillation; anhydrous solvents
2
5
were stored in dry Young ampoules. In the conventional purification procedure, the
residue was submitted to flash column chromatography with silica gel 60 H (particle
size= 63–200 µm). The elution was carried out with hexane-EtOAc mixtures, under N2
positive pressure and employing gradient of solvent polarity techniques.
All new compounds gave single spots on TLC plates of Kieselgel 60 GF , run in
2
54
different hexane-EtOAc and CH Cl -PhMe solvent systems. The chromatographic spots
2
2
were detected by exposure of the plates to UV light (254 nm), followed by spraying with
ethanolic ninhydrin (amines) or with ethanolic p-anisaldehyde/sulfuric acid reagent and
careful heating for 2 min.
The effect of the compounds on bacterial growth was tested in 96-well microplates
placed in a Spectra Max M2 microplate reader (Molecular Devices Corp.) programmed
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32
33
to obtain measurements at 23 °C in order to prevent the evaporation of samples [33].
The optical densities of the cell cultures were recorded at 620 nm (OD ) at 15 min
6
20
intervals with shaking (30 s) between measurements. Bacterial concentrations of the cell
cultures were adjusted employing a SP-22 spectrophotometer (Biospectro, Brazil).
4.2. General procedure for the synthesis of dianilides 17-22
The aniline (15a-f, 2.0 equiv.) and diethyl malonate (16, 1.0 equiv.) were mixed in a
round bottom flask, the mixture was degassed under Argon and placed in a pre-heated
bath at 210-220 °C, allowing to escape the EtOH formed. Once the evolution of EtOH
had stopped (1-2 h), the reaction was cooled to room temperature and the thus formed
solid was washed several times with EtOAc.
1
3
4.2.1. N ,N -Diphenylmalonamide (17).- Yield: 11.6 g (91%). White solid; m.p.:
230–232 ºC (Lit. 228–230 ºC [41]). IR (KBr, ν): 3273 (N-H), 3149 (C-H), 1668 (C=O),
-1 1
1647, 1597, 1535, 1498, 1444, 1413, 1357, 1249, 752 and 688 cm . H NMR (DMSO-
d ): δ 3.49 (s, 2H, CH ), 7.06 (t, J = 7.8, 2H, H-4), 7.32 (t, J = 7.8, 4H, H-3), 7.62 (d, J
6
2
1
3
3
34
= 7.8, 4H, H-2) and 10.17 (s, 2H, NH). C NMR (DMSO-d ): δ 45.9 (CH ), 119.0 (4 ×
6 2
3
3
3
3
3
3
3
3
3
35
36
37
38
39
40
41
42
43
C-2), 123.4 (2 × C-4), 128.7 (4 × C-3), 138.9 (2 × C-1) and 165.3 (2 × C=O). EI-MS
+
(m/z, %): 254 (M , 41), 135 (55), 120 (8), 93 (100), 91 (13) and 77 (22).
1
3
4.2.2. N ,N -bis(4-Fluorophenyl)malonamide (18).- Yield: 0.28 g (44%). White
solid; m.p.: 211–213 ºC (Lit. 214–216 ºC [42]). IR (KBr, ν): 3286 (N-H), 3088 (C-H),
-1 1
1672, 1645 (C=O), 1618, 1558, 1512, 1409, 1222 and 752 cm . H NMR (DMSO-d ): δ
6
3.42 (s, 2H, CH ), 7.13 (t, J = 9.1, 4H, H-2), 7.58 (dd, J = 5.0 and 9.1, 4H, H-3) and
2
1
3
2
10.20 (s, 2H, NH). C NMR (DMSO-d ): δ 46.1 (CH ), 115.8 (d, J = 22.5, 4 × C-3),
6
2
CF
3
4
1
121.3 (d, J = 8.2, 4 × C-2), 135.7 (d, J = 2.2, 2 × C-1), 158.5 (d, J = 238.7, 2 × C-
CF
CF
CF
1
9
3
3
3
3
3
3
3
44
45
46
47
48
49
50
4) and 165.7 (2 × C=O). F NMR (DMSO-d ): δ -119.1 (m, Ar-F). EI-MS (m/z, %):
6
+
290 (M , 56), 153 (42), 137 (8), 111 (100), 95 (10), 83 (21) and 75 (7).
1
3
4.2.3. N ,N -bis(4-Chlorophenyl)malonamide (19).- Yield: 1.4 g (49%). White
solid; m.p.: 250–252 ºC (Lit. 256–257 ºC [41]). IR (KBr, ν): 3253 (N-H), 3045 (C-H),
-1 1
1643 (C=O), 1533, 1012, 977 and 501 cm . H NMR (DMSO-d ): δ 3.48 (s, 2H, CH ),
6
2
1
3
7.37 (d, J = 8.8, 4H, H-2), 7.63 (d, J = 8.8, 4H, H-3) and 10.33 (s, 2H, NH). C NMR
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51
(DMSO-d ): δ 45.8 (CH ), 120.6 (4 × C-2), 126.9 (2 × C-4), 128.6 (4 × C-3), 137.8 (2 ×
6
2
+
3
3
3
3
3
3
3
52
53
54
55
56
57
58
C-1) and 165.4 (2 × C=O). EI-MS (m/z, %): 322 (M , 51), 169 (39), 153 (12), 127 (100),
99 (14), 91 (34) and 75 (13).
1
3
4.2.4. N ,N -bis(4-Methoxyphenyl)malonamide (20).- White solid. Yield: 0.4 g
(63%). White solid; m.p.: 233–235 ºC (Lit. 232–234 ºC [41]). IR (KBr, ν): 3269 (N-H),
-1 1
2954 (C-H), 1645 (C=O), 1616, 1533, 1508, 1411, 1244, 1035 and 821 cm . H NMR
(DMSO-d ): δ 3.39 (s, 2H, CH ), 3.72 (s, 6H, ArOCH ), 6.88 (d, J = 9.0, 4H, H-3), 7.51
6
2
3
1
3
3
3
59
60
(d, J = 9.0, 4H, H-2) and 10.00 (s, 2H, NH). C NMR (DMSO-d ): δ 45.5 (CH ), 55.7
6 2
(2 × ArOCH ), 113.8 (4 × C-3), 120.6 (4 × C-2), 132.1 (2 × C-1), 155.3 (2 × C-4) and
3
+
3
3
3
3
3
3
3
3
61
62
63
64
65
66
67
68
164.9 (2 × C=O). EI-MS (m/z, %): 314 (M , 100), 165 (41), 149 (7), 121 (73), 108 (37)
and 80 (7).
1
3
4.2.5. N ,N -di(p-Tolyl)malonamide (21).- Yield: 0.47 g (71%). White solid; m.p.:
246–248 ºC (Lit. 251–253 ºC [43]). IR (KBr, ν): 3269 (N-H), 3034 (C-H), 1649 (C=O),
-1 1
1614, 1531, 1514, 1404 and 1357 cm . H NMR (DMSO-d ): δ 2.25 (s, 6H, ArCH ),
6
3
3.42 (s, 2H, CH ), 7.11 (d, J = 8.3, 4H, H-3), 7.48 (d, J = 8.5, 4H, H-2) and 10.05 (s,
2
1
3
2H, NH). C NMR (DMSO-d ): δ 20.3 (2 × ArCH ), 45.7 (CH ), 119.0 (4 × C-3), 129.1
6
3
2
3
3
3
3
3
3
3
69
70
71
72
73
74
75
(4 × C-2), 132.2 (2 × C-1), 136.4 (2 × C-4) and 165.2 (2 × C=O). EI-MS (m/z, %): 282
+
(M , 100), 149 (48), 133 (7), 106 (90), 91 (23) and 77 (18).
1
3
4.2.6. N ,N -bis(2-Methoxyphenyl)malonamide (22).- Yield: 0.63 g (49%). White
solid; m.p.: 160–162 ºC (Lit. 163 ºC [41]). IR (KBr, ν): 3367 (N-H), 3292 (N-H), 3005
-1 1
(C-H), 1674 (C=O), 1597, 1529, 1489, 1463, 1255, 1228, 1118 and 1024 cm . H NMR
(DMSO-d ): δ 3.68 (s, 2H, CH ), 3.82 (s, 6H, ArOCH ), 6.86-7.11 (m, 8H, 2 × ArH),
6
2
3
1
3
3
3
76
77
8.00 (d, J = 7.6, 2H, H-6) and 9.67 (s, 2H, NH). C NMR (DMSO-d ): δ 45.5 (CH ),
6 2
55.7 (2 × ArOCH ), 110.1 (2 × C-3), 120.2 (2 × Ar), 120.7 (2 × Ar), 124.2 (2 × Ar),
3
+
3
3
3
3
3
3
78
79
80
81
82
83
127.1 (2 × C-1), 148.4 (2 × C-2) and 164.7 (2 × C=O). EI-MS (m/z, %): 314 (M , 100),
165 (33), 134 (11), 123 (68), 108 (50) and 80 (9).
4.3. General procedure for the synthesis of 4-hydroxy-1H-quinolin-2-ones 23-28
The dianilide (1.0 equiv.) was placed in a round bottom flask and mixed with 10
times (by weight) of polyphosphoric acid. The syrupy mixture was heated at 130-140 °C
1
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during 6 h, when it was poured onto crushed ice and made alkaline with 4 N NaOH. The
solids were filtered off and the remaining liquids were transferred to a separation funnel
and washed with Et O (3 × 5 mL). The aqueous phase was transferred to a beaker placed
2
in an ice bath and acidified with 6 N HCl to pH= 1, causing precipitation of the 4-
hydroxy-3,4-dihydro-1H-quinolin-2-one product. The solid was filtered in a Büchner
funnel, washed several times with H O and dried in a desiccator over P O , under
2
2
5
vacuum during 24 h. This product was used without further purification.
4.3.1. 4-Hydroxy-1H-quinolin-2-one (23) [14c].- Yield: 1.26 g (67%). Pale pinkish
solid; m.p.: 350–352 ºC (Lit. > 300ºC [44]). IR (KBr, ν): 3400 (O-H, N-H), 3093 (C-H),
2951 (C-H), 2908, 2821, 2762, 1635 (C=O), 1593, 1506, 1419, 1379, 1332, 1234 and
-1 1
866 cm . H NMR (DMSO-d ): δ 5.74 (s, 1H, H-3), 7.13 (t, J = 7.7, 1H, H-6), 7.25 (d,
6
J = 8.3, 1H, H-8), 7.44-7.52 (m, 1H, H-7), 7.78 (d, J = 7.8, 1H, H-5), 11.17 (s, 2H, OH
1
3
and NH). C NMR (DMSO-d ): δ 98.1 (C-3), 115.0 (C-4a and C-8), 120.9 (C-5), 122.6
6
(C-6), 130.7 (C-7), 139.1 (C-8a), 162.5 (C-4)* and 163.5 (C-2)*. EI-MS (m/z, %): 161
+
(M , 100), 133 (16), 119 (83), 105 (19), 92 (68) and 77 (11).
4.3.2. 6-Fluoro-4-hydroxy-1H-quinolin-2-one (24) [14c].- Yield: 0.10 g (52%).
White solid; m.p.: 356 ºC, dec. (Lit. 345 ºC [42]). IR (KBr, ν): 3446 (O-H), 3097 (N-H),
-1 1
2949 (C-H), 2899, 1647 (C=O), 1600, 1517, 1456, 1319, 1251, 1240 and 1197 cm . H
NMR (DMSO-d ): δ 5.94 (s, 1H, H-3), 7.27-7.55 (m, 3H, H-5, H-7 and H-8) and 11.31
6
1
3
2
4
4
4
4
4
4
4
4
4
4
4
4
05
06
07
08
09
10
11
12
13
14
15
16
(bs, 2H, OH and NH). C NMR (DMSO-d ): δ 99.6 (C-3), 108.0 (d, J = 23.7, C-5),
6 CF
3
3
2
116.3 (d, J = 8.1, C-4a), 117.5 (d, J = 8.1, C-8), 119.2 (d, J = 24.2, C-7), 136.3
CF
CF
CF
1
4
19
(C-8a), 157.1 (d, J = 237.5, C-6), 162.1 (d, J = 2.8, C-4) and 163.8 (C-2). F NMR
CF
CF
+
(DMSO-d ): δ -121.5 (m, Ar-F). EI-MS (m/z, %): 179 (M , 100), 151 (10), 137 (83),
6
123 (19), 110 (53), 95 (16), 82 (29) and 75 (19).
4.3.3. 6-Chloro-4-hydroxy-1H-quinolin-2-one (25) [45].- Yield: 0.81 g (98%).
White solid; m.p.: 308 ºC, dec. (Lit. 350 ºC [42]). IR (KBr, ν): 3124 (N-H), 2972 (C-H),
-
1 1
2837, 1670 (C=O), 1610, 1302, 1231 and 998 cm . H NMR (DMSO-d ): δ 5.78 (s, 1H,
6
H-3), 7.28 (d, J = 8.7, 1H, H-8), 7.54 (d, J = 7.6, 1H, H-7), 7.73 (s, 1H, H-5) and 11.39
1
3
(bs, 2H, OH and NH). C NMR (DMSO-d ): δ 99.0 (C-3), 116.2 (C-4a), 117.1 (C-5),
6
121.7 (C-8), 125.1 (C-6), 130.7 (C-7), 137.8 (C-8a), 161.3 (C-4) and 163.3 (C-2). EI-
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MS (m/z, %): 195 (M , 100), 167 (12), 153 (92), 139 (15), 126 (37), 99 (13), 90 (23) and
75 (29).
4.3.4. 6-Methoxy-4-hydroxy-1H-quinolin-2-one (26).- Yield: 0.45 g (19%). White
solid; m.p.: 310–312 ºC (lit. > 300 ºC [44]). IR (KBr, ν): 3344 (O-H), 3143 (N-H), 2993
-1 1
(C-H), 2839, 1670 (C=O), 1614, 1037, 898 and 821 cm . H NMR (DMSO-d ): δ 3.75
6
(s, 3H, Ar-OCH ), 5.71 (s, 1H, H-3), 7.07-7.22 (m, 3H), 11.06 (s, 1H, NH) and 11.26 (s,
3
1
3
1H, OH). C NMR (DMSO-d ): δ 55.5 (Ar-OCH ), 98.6 (C-3), 103.9 (C-5), 115.5 (C-
6
3
4a), 116.7 (CH), 120.1 (CH), 133.7 (C-8a), 153.8 (C-6), 162.1 (C-4) and 163.3 (C-2).
+
EI-MS (m/z, %): 191 (M , 100), 176 (56), 149 (22), 134 (65), 121 (25), 106 (84) and 80
(20).
4.3.5. 6-Methyl-4-hydroxy-1H-quinolin-2-one (27).- Yield: 0.46 g (17%). White
solid; m.p.: 259 ºC, dec. [44]. IR (KBr, ν): 3419 (O-H), 3288 (N-H), 2916 (C-H), 1670
-1 1
(C=O), 1610, 1244, 1091 and 1041 cm . H NMR (DMSO-d ): δ 2.33 (s, 3H, ArCH ),
6
3
5.73 (s, 1H, H-3), 7.16 (d, J = 8.2, 1H, H-8), 7.30 (dd, J = 1.7 and 6.4, 1H, H-7), 7.56
1
3
(s, 1H, H-5) and 11.10 (s, 1H). C NMR (DMSO-d ): δ 21.0 (ArCH ), 97.7 (C-3), 115.6
6
3
(C-4a), 116.0 (C-8), 122.6 (C-5), 131.2 (C-6), 132.9 (C-7), 137.3 (C-8a), 163.7 (C-4)*
+
and 163.8 (C-2).* EI-MS (m/z, %): 175 (M , 100), 147 (8), 133 (95), 119 (20), 106 (42),
104 (58), 91 (15) and 77 (42).
4.3.6. 8-Methoxy-4-hydroxy-1H-quinolin-2-one (28).- Yield: 0.60 g (36%). White
solid; m.p.: 246–248 ºC (Lit. 248 ºC [46]). IR (KBr, ν): 3149 (N-H), 2960 (C-H), 2835,
-1 1
1663 (C=O), 1591, 1504, 1317, 1242 and 1053 cm . H NMR (DMSO-d ): δ 3.88 (s,
6
3H, ArOCH ), 5.75 (s, 1H, H-3), 7.03-7.19 (m, 2H, H-5,8), 7.37 (dd, J = 1.6, 7.6, 1H,
3
1
3
H-7), 10.09 (s, 1H, NH) and 11.31 (s, 1H, OH). C NMR (DMSO-d ): δ 56.4
6
(ArOCH ), 99.1 (C-3), 111.7 (C-7), 114.7 (C-5), 116.0 (C-5a), 121.4 (C-6), 129.7 (C-
3
+
8a), 146.1 (C-8), 163.0 (C-4)* and 163.3 (C-3).* EI-MS (m/z, %): 191 (M , 100), 190
(55), 162 (45), 148 (29), 134 (41), 121 (31), 106 (52), 91 (28) and 77 (18).
4.4. General procedure for the C,C-dibenzylation of the 4-hydroxy-1H-quinolin-2-
ones 29-38
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The 4-hydroxy-3,4-dihydro-1H-quinolin-2-one (1.0 equiv.), K CO (8.0 equiv.) and
2
3
H O (9.0 mL/mmol), were placed in a round bottom flask and the mixture was stirred
2
until complete dissolution of the heterocycle (approximately 15 min.). Then, a solution
of the benzyl halide (6.0 equiv., 9.0 mL/mmol) in toluene was added. The reaction was
refluxed during 48 h, cooled to room temperature, diluted with distilled H O and
2
extracted with EtOAc (3 × 25 mL). The combined organic extracts were washed with
brine (2 × 10 mL), dried over MgSO , concentrated under reduced pressure and the
4
residue was purified by column chromatography.
4.4.1. 3,3-Dibenzyl-1H-quinoline-2,4-dione (29).- Yield: 0.097 g (46%). White
solid; m.p.: 187–189 ºC (Lit. 206–208 ºC [32c]). IR (KBr, ν): 3286 (N-H), 3066 (C-H),
1695 (C=O, ketone), 1684, 1661 (C=O, amide), 1645, 1610, 1598, 1485, 1358, 1247,
-1 1
1211, 763, 705 and 503 cm . H NMR: δ 3.43 (d, J = 12.8, 2H, ArCH ), 3.51 (d, J =
2
12.8, 2H, ArCH ), 6.45 (d, J = 8.2, 1H, H-8), 6.93 (t, J = 7.5, 1H, H-7), 6.99-7.24 (m,
2
1
3
11H, 2 × ArH and H-6), 7.77 (dd, J = 1.0 and 7.9, 1H, H-5) and 8.44 (s, 1H, NH). C
NMR: δ 45.2 (2 × ArCH ), 64.6 (C-3), 115.3 (C-8), 119.8 (C-8a), 123.0 (C-6), 126.8 (2
2
× p-C H CH and C-5), 127.9 (4 × m-C H CH ), 129.7 (4 × o-C H CH ), 135.5 (2 ×
6
5
2
6
5
2
6
5
2
ipso-C H CH ), 135.7 (C-7), 139.8 (C-4a), 173.0 (C-2) and 197.0 (C-4). EI-MS (m/z,
6
5
2
+
%): 340 (M , 32), 250 (60), 153 (8), 141 (9), 127 (14), 111 (25), 99 (33), 91 (46), 85
(68) and 71 (100).
4.4.2. 3,3-Dibenzyl-6-fluoro-1H-quinoline-2,4-dione (30).- Yield: 0.11 g (61%).
White solid; m.p.: 176–178 ºC. IR (KBr, ν): 3230 (N-H), 3034 (C-H), 1685 (C=O,
-1 1
ketone), 1653 (C=O, amide), 1624, 1500, 1411, 1363, 1249 and 1166 cm . H NMR: δ
3.43 (d, J = 12.8, 2H, ArCH ), 3.49 (d, J = 12.8, 2H, ArCH ), 6.49 (dd, J = 4.0 and 8.8,
2
2
1H, H-8), 6.94-7.12 (m, 11H, 2 × ArH and H-7), 7.44 (dd, J = 3.0, 8.2, 1H, H-5) and
1
3
2
8.72 (s, 1H, NH). C NMR: δ 45.3 (2 × ArCH ), 64.4 (C-3), 112.4 (d, J = 23.1, C-7),
2
CF
3
3
2
117.2 (d, J = 7.1, C-8), 120.7 (d, J = 6.6, C-4a), 123.3 (d, J = 24.2, C-5), 126.9 (2
CF
CF
CF
× p-C H CH ), 128.1 (4 × m-C H CH ), 129.7 (4 × o-C H CH ), 135.4 (2 × ipso-
6
5
2
6
5
2
6
5
2
1
19
C H CH ), 136.4 (C-8a), 158.3 (d, J = 244.2, C-6), 173.2 (C-2) and 196.5 (C-4). F
6
5
2
CF
+
NMR: δ -118.5 (m, Ar-F). EI-MS (m/z, %): 359 (M , 2.4), 268 (100), 250 (3), 138 (4),
+
131 (4), 103 (7), 91 (72) and 77 (5). HRMS (ESI–TOF), m/z found: 382.1208 [M+Na] ,
C H FNNaO requires 382.1214.
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4.4.3. 3,3-Dibenzyl-6-chloro-1H-quinoline-2,4-dione (31).- Yield: 0.13 g (69%).
White solid; m.p.: 201–203 ºC. IR (KBr, ν): 3325 (N-H), 2929 (C-H), 1697 (C=O,
-1
1
ketone), 1654 (C=O, amide), 1606, 1489, 1280, 1247 and 1192 cm H NMR: δ 3.42
.
(d, J = 12.8, 2H, ArCH ), 3.50 (d, J = 12.8, 2H, ArCH ), 6.46 (dd, J = 2.2 and 8.6, 1H,
2
2
H-8), 6.96-7.13 (m, 10H, 2 × ArH), 7.21 (dd, J = 2.4 and 8.6, 1H, H-7), 7.74 (d, J = 2.4,
1
3
1H, H-5) and 8.75 (s, 1H, NH). C NMR: δ 45.3 (2 × ArCH ), 64.7 (C-3), 117.1 (C-8),
2
120.7 (C-4a), 126.4 (C-5), 127.0 (2 × p-C H CH ), 128.1 (4 × m-C H CH ), 128.7 (C-
6
5
2
6
5
2
8a), 129.7 (4 × o-C H CH ), 135.3 (2 × ipso-C H CH ), 135.6 (C-7), 138.5 (C-6), 173.3
6
5
2
6
5
2
+
(C-2) and 196.3 (C-4). EI-MS (m/z, %): 375 (M , 2.4), 284 (100), 266 (3), 154 (3), 131
(5), 115 (3), 103 (7), 91 (69) and 77 (4). HRMS (ESI–TOF), m/z found: 398.0919
+
[M+Na] , C H ClNNaO requires 398.0918.
2
3
18
2
4.4.4. 3,3-Dibenzyl-6-methoxy-1H-quinoline-2,4-dione (32).- Yield: 0.023 g
(40%). White solid; m.p.: 180–182 ºC. IR (KBr, ν): 3175 (N-H), 3057 (C-H), 2902 (C-
-1
1
H), 1685 (C=O, ketone), 1651 (C=O, amide), 1508, 1490, 1419 and 1350 cm . H
NMR: δ 3.42 (d, J = 12.8, 2H, ArCH ), 3.49 (d, J = 12.8, 2H, ArCH ), 3.76 (s, 3H, Ar-
2
2
OCH ), 6.51 (d, J = 8.8, 1H, H-8), 6.88 (dd, J = 3.0, 8.8, 1H, H-7), 6.97-7.13 (m, 10H,
3
1
3
2 × ArH), 7.23 (d, J = 3.0, 1H, H-5) and 9.37 (s, 1H, NH). C NMR: δ 45.2 (2 ×
ArCH ), 55.7 (ArOCH ), 64.1 (C-3), 107.9 (C-8), 117.3 (C-5), 120.3 (C-8a), 124.5 (C-
2
3
7), 126.8 (2 × p-C H CH ), 128.0 (4 × m-C H CH ), 129.7 (4 × o-C H CH ), 134.5 (C-
6
5
2
6
5
2
6
5
2
4a), 135.6 (2 × ipso-C H CH ), 155.5 (C-6), 173.6 (C-2) and 197.1 (C-4). EI-MS (m/z,
6
5
2
+
%): 371 (M , 9), 280 (100), 150 (3), 131 (6), 106 (3) and 91 (45). HRMS (ESI–TOF),
+
m/z found: 394.1404 [M+Na] , C H NNaO requires 394.1414.
24
21
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4.4.5. 3,3-Dibenzyl-6-methyl-1H-quinoline-2,4-dione (33).- Yield: 0.035 g (38%).
White solid; m.p.: 200–202 ºC. IR (KBr, ν): 3311 (N-H), 3084 (C-H), 2929 (C-H), 1689
-1 1
(C=O, ketone), 1651 (C=O, amide), 1618, 1508, 1419 and 704 cm . H NMR: δ 2.23 (s,
3H, Ar-CH ), 3.41 (d, J = 12.8, 2H, ArCH ), 3.49 (d, J = 12.8, 2H, ArCH ), 6.34 (d, J =
3
2
2
8.0, 1H, H-8), 7.00-7.13 (m, 11H, 2 × ArH and H-7), 7.55-7.59 (m, 1H, H-5) and 7.92
1
3
(s, 1H, NH). C NMR: δ 20.5 (ArCH ), 45.2 (2 × ArCH ), 64.5 (C-3), 115.5 (C-H),
3
2
119.7 (C-8a), 126.6 (C-H), 126.8 (2 × p-C H CH ), 128.0 (4 × m-C H CH ), 129.8 (4 ×
6
5
2
6
5
2
o-C H CH ), 132.8 (C-6), 135.7 (2 × ipso-C H CH ), 136.8 (C-H), 138.0 (C-4a), 173.4
6
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2
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44
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46
47
(C-2) and 197.2 (C-4). EI-MS (m/z, %): 355 (M , 1.6), 264 (100), 246 (2), 131 (5), 103
+
(6), 91 (35) and 77 (6). HRMS (ESI–TOF), m/z found: 356.1635 [M+H] , C H NO
24
22
2
requires 356.1645.
4.4.6. 3,3-Dibenzyl-8-methoxy-1H-quinoline-2,4-dione (34).- Yield: 0.14 g (62%).
White solid; m.p.: 196–198 ºC. IR (KBr, ν): 3184 (N-H), 3064 (C-H), 2929 (C-H), 1693
-1 1
(C=O, ketone), 1654 (C=O, amide), 1506, 1382 and 1267 cm . H NMR: δ 3.42 (d, J =
12.8, 2H, ArCH ), 3.48 (d, J = 12.8, 2H, ArCH ), 3.70 (s, 3H, ArOCH ), 6.77 (dd, J =
2
2
3
1.4 and 8.0, 1H, H-8), 6.84 (t, J = 7.8, 1H, H-7), 6.99-7.13 (m, 10H, 2 × ArH), 7.34 (dd,
1
3
J = 1.0 and 7.8, 1H, H-5) and 7.83 (s, 1H, NH). C NMR: δ 45.4 (2 × ArCH ), 60.0
2
(ArOCH ), 64.7 (C-3), 115.8 (C-7), 118.0 (C-5), 120.1 (C-4a), 122.2 (C-6), 126.8 (2 ×
3
p-C H CH ), 128.0 (4 × m-C H CH ), 129.7 (4 × o-C H CH ), 130.6 (C-8a), 135.9 (2 ×
6
5
2
6
5
2
6
5
2
+
ipso-C H CH ), 145.2 (C-8), 172.0 (C-2) and 197.3 (C-4). EI-MS (m/z, %): 371 (M ,
6
5
2
10), 370 (26), 280 (100), 264 (6), 131 (6), 91 (67) and 71 (22). HRMS (ESI–TOF), m/z
+
found: 394.1415 [M+Na] , C H NNaO requires 394.1414.
24
21
3
4.4.7. 3,3-bis(4’-Methoxybenzyl)-1H-quinoline-2,4-dione (35).- Yield: 0.064 g
(52%). White solid; m.p.: 179–181 ºC. IR (KBr, ν): 3266 (N-H), 2930 (C-H), 2837,
1682 (C=O, ketone), 1658 (C=O, amide), 1640, 1599, 1510, 1490, 1438, 1379, 1253,
-1 1
1180 and 824 cm . H NMR: δ 3.34 (d, J = 13.1, 2H, ArCH ), 3.42 (d, J = 13.1, 2H,
2
ArCH ), 3.61 (s, 6H, ArOCH ), 6.46-7.32 (m, 11H, 2 × ArH, H-6, H-7 and H-8), 7.79
2
3
1
3
(dd, J = 1.0 and 7.8, 1H, H-5) and 8.52 (s, 1H, NH). C NMR: δ 44.2 (2 × ArCH ),
2
54.9 (2 × ArOCH ), 65.0 (C-3), 113.3 (4 × C-3’), 115.6 (C-8), 119.8 (C-8a), 123.0 (C-
3
6), 126.8 (C-5), 127.7 (4 × C-2’), 130.7 (2 × C-1’), 135.7 (C-7), 140.1 (C-4a), 158.2 (2 ×
+
C-4’), 173.5 (C-2) and 197.3 (C-4). EI-MS (m/z, %): 401 (M , 1.6), 280 (69), 161 (2),
+
121 (100), 91 (6) and 77 (10). HRMS (ESI–TOF), m/z found: 424.1510 [M+Na] ,
C H NNaO requires 424.1519.
25
23
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4.4.8. 3,3-bis(4’-Chlorobenzyl)-1H-quinoline-2,4-dione (36).- Yield: 0.040 g
(40%). White solid; m.p.: 232–234 ºC. IR (KBr, ν): 3178 (N-H), 3055 (C-H), 2993 (C-
-1 1
H), 2931, 1689 (C=O, ketone), 1653 (C=O, amide), 1595, 1487 and 1388 cm . H NMR:
δ 3.36 (d, J = 12.8, 2H, ArCH ), 3.44 (d, J = 12.8, 2H, ArCH ), 6.51 (d, J = 6.0, 1H, H-
2
2
8), 6.95-7.07 (m, 9H, 2 × ArH and H-7), 7.33 (dt, J = 1.4 and 7.6, 1H, H-6), 7.79 (d, J =
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6.9, 1H, H-5) and 8.25 (s, 1H, NH). C NMR: δ 44.5 (2 × ArCH ), 64.7 (C-3), 115.5
2
(C-5), 119.8 (C-8a), 123.6 (C-6), 127.1 (C-5), 128.3 (4 × C-3’), 131.2 (4 × C-2’), 132.9
(2 × C-4’), 133.9 (2 × C-1’), 136.3 (C-7), 139.8 (C-4a), 172.3 (C-2) and 196.8 (C-4). EI-
+
MS (m/z, %): 409 (M , 0.9), 284 (100), 248 (9), 165 (3), 125 (40), 120 (6), 89 (11) and
+
71 (3). HRMS (ESI–TOF), m/z found: 432.0510 [M+Na] , C H Cl NNaO requires
23
17
2
2
432.0529.
4.4.9. 3,3-bis(2’-Chlorobenzyl)-1H-quinoline-2,4-dione (37).- Yield: 0.048 g
(38%). White solid; m.p.: 172–174 ºC. IR (KBr, ν): 3243 (N-H), 1691 (C=O, ketone),
-1 1
1660 (C=O, amide), 1613, 1596, 1485, 1474, 1436, 1373, 1227, 755 and 740 cm . H
NMR: δ 3.61 (d, J = 14.5, 2H, ArCH ), 3.70 (d, J = 14.5, 2H, ArCH ), 6.66 (d, J = 8.1,
2
2
1H, H-8), 6.86-7.24 (m, 9H, o-ClC H CH and H-7), 7.39 (t, J = 7.7, 1H, H-6), 7.88 (d,
6
4
2
1
3
J = 6.9, 1H, H-5) and 9.65 (s, 1H, NH). C NMR: δ 40.3 (2 × ArCH ), 61.2 (C-3),
2
116.2 (C-5), 119.7 (C-8a), 123.5 (C-6), 126.4 (2 × Ar), 127.6 (C-5), 128.0 (2 × Ar),
129.5 (2 × Ar), 130.2 (2 × Ar), 133.7 (2 × Ar), 134.9 (2 × Ar), 136.0 (C-7), 140.2 (C-
+
4a), 173.6 (C-2) and 195.2 (C-4). EI-MS (m/z, %): 410 (M , 0.05), 374 (45), 284 (7),
+
248 (100), 125 (20) and 89 (5). HRMS (ESI–TOF), m/z found: 410.0704 [M+H] ,
C H Cl NO requires 410.0709.
23
18
2
2
4.4.10. 3,3-bis(4’-Chlorobenzyl)-6-chloro-1H-quinoline-2,4-dione (38).- Yield:
0.039 g (34%). White solid; m.p.: 204–206 ºC. IR (KBr, ν): 3259 (N-H), 3069 (C-H),
2958 (C-H), 1685 (C=O, ketone), 1662 (C=O, amide), 1489, 1408, 1363, 1095, 837 and
-1 1
815 cm . H NMR: δ 3.36 (d, J = 12.8, 2H, ArCH ), 3.43 (d, J = 12.8, 2H, ArCH ),
2
2
6.51 (d, J = 8.4, 1H, H-8), 6.97-7.07 (m, 8H, 2 × ArH), 7.39 (dd, J = 2.2 and 8.4, 1H,
1
3
H-7), 7.75 (d, J = 2.2, 1H, H-5) and 8.40 (s, 1H, NH). C NMR: δ 44.6 (2 × ArCH ),
2
64.7 (C-3), 117.1 (C-8), 120.4 (C-4a), 126.5 (C-5), 128.4 (4 × Ar), 129.2 (C-8a), 131.2
(4 × Ar), 133.1 (2 × Ar), 133.7 (2 × Ar), 136.2 (C-7), 138.1 (C-6), 172.3 (C-2) and
+
196.0 (C-4). EI-MS (m/z, %): 443 (M , 1), 318 (65), 282 (9), 165 (3), 125 (100), 99 (3)
+
and 89 (9). HRMS (ESI–TOF), m/z found: 466.0119 [M+Na] , C H Cl NNaO
2
3
16
3
2
requires 466.0139.
4.5. General Procedures for the N-alkylation of the 3,3-dibenzyl-1H-quinoline-2,4-
diones 39-44
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4.5.1. Procedure A. The 1H-quinoline-2,4-dione (1.0 equiv.), K CO (3.0 equiv.)
2
3
and dry acetonitrile (30 mL/mmol) were placed in a round bottom flask equipped with a
Liebig condenser. After stirring for 5 min., the alkylating agent (1.2 equiv.) was added
dropwise and the system was refluxed under Argon atmosphere until complete
consumption of the starting material (24-48 h). Then, the reaction was cooled and the
product was concentrated under reduced pressure. The residue was column
chromatographed, to afford the N-alkylated product.
4.5.2. Procedure B. The 1H-quinoline-2,4-dione (1.0 equiv.) was dissolved in
anhydrous DMA (13 mL/mmol) and anhydrous K PO (6.0 equiv.) was immediately
3
4
added. The system was placed under an Argon atmosphere, and treated dropwise with
the alkylating agent (1.2 equiv.), while the suspension was vigorously stirred at 120 °C.
Once verified the complete consumption of the starting material, the volatiles were
distilled off under vacuum, and the remaining oily material was purified by column
chromatography to afford the N-alkylated product.
4.5.3. 3,3-Dibenzyl-1-methyl-1H-quinoline-2,4-dione (39).- Synthesized after
Procedure A. Yield: 0.012 g (60%). White solid; m.p.: 126–128 ºC (Lit. 126-128 ºC
[29a]). IR (KBr, ν): 2926 (C-H), 1685 (C=O, ketone), 1651 (C=O, amide), 1598, 1490,
-1 1
1473, 1456, 1367, 1082, 804, 761 and 704 cm . H NMR: δ 3.16 (s, 3H, CH N), 3.47 (s,
3
4H, ArCH ), 6.62 (d, J = 8.2, 1H, H-8), 6.90-7.08 (m, 11H, 2 × ArH and H-6), 7.26-
2
13
7.34 (m, 1H, H-7) and 7.84 (dd, J = 1.6 and 7.8, 1H, H-5). C NMR: δ 29.1 (CH N),
3
45.9 (2 × ArCH ), 64.6 (C-3), 114.2 (C-8), 121.3 (C-8a), 122.5 (C-6), 126.6 (2 × Ar),
2
127.1 (C-5), 127.8 (4 × Ar), 129.7 (4 × Ar), 135.8 (2 × Ar), 135.9 (C-7), 142.6 (C-4a),
+
172.0 (C-2) and 197.1 (C-4). EI-MS (m/z, %): 355 (M , 1.1), 264 (100), 134 (15), 103
(8), 91 (64) and 77 (15).
4.5.4. 3,3-Dibenzyl-1-ethyl-1H-quinoline-2,4-dione (40).- Synthesized after
Procedure A. Yield: 0.018 g (77%). White solid; m.p.: 145–147 ºC. IR (KBr, ν): 3064
(C-H), 2933 (C-H), 1685 (C=O, ketone), 1647 (C=O, amide), 1598, 1471, 1377 and
-1 1
1303 cm . H NMR: δ 1.01 (t, J = 7.1, 3H, CH CH N), 3.48 (s, 4H, ArCH ), 3.81 (q, J
3
2
2
= 7.1, 2H, CH CH N), 6.65 (d, J = 8.4, 1H, H-8), 6.92 (t, J = 7.5, 1H, H-6), 6.97-7.15
3
2
1
3
(m, 10H, 2 × ArH), 7.26-7.37 (m, 1H, H-7) and 7.85 (dd, J = 1.6 and 7.8, 1H, H-5). C
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NMR: δ 11.8 (CH CH N), 37.0 (CH CH N), 46.1 (2 × ArCH ), 64.1 (C-3), 114.1 (C-8),
3
2
3
2
2
121.5 (C-8a), 122.4 (C-6), 126.6 (2 × Ar), 127.4 (C-5), 127.8 (4 × Ar), 129.8 (4 × Ar),
+
135.8 (2 × Ar), 141.6 (C-4a), 171.7 (C-2) and 197.5 (C-4). EI-MS (m/z, %): 369 (M ,
1.3), 278 (100), 148 (5), 103 (4), 91 (29) and 77 (6). HRMS (ESI–TOF), m/z found:
+
370.1806 [M+H] , C H NO requires 370.1802.
25
24
2
4.5.5. 1,3,3-Tribenzyl-1H-quinoline-2,4-dione (41).- Synthesized after Procedure
B. Yield: 0.014 g (36%). White solid; m.p.: 149–151 ºC. IR (KBr, ν): 3030 (C-H), 2926
(C-H), 1683 (C=O, ketone), 1651 (C=O, amide), 1597, 1492, 1467, 1454, 1436, 1377
-1 1
and 1298 cm . H NMR: δ 3.48 (d, J = 12.6, 2H, ArCH ), 3.62 (d, J = 12.6, 2H,
2
ArCH ), 4.99 (s, 2H, ArCH N), 6.46 (d, J = 8.2, 1H, H-8), 6.54-6.62 (m, 2H, ArH), 6.91
2
2
(t, J = 7.2, 1H, H-6), 7.01-7.19 (m, 14H, 3 × ArH and H-7) and 7.91 (dd, J = 1.8 and
1
3
7.8, 1H, H-5). C NMR: δ 45.4 (2 × ArCH ), 46.3 (ArCH N), 64.8 (C-3), 115.4 (C-8),
2
2
121.5 (C-4a), 122.7 (C-6), 125.9 (Ar), 126.8 (2 × Ar), 126.9 (2 × Ar), 127.2 (C-5), 128.0
(4 × Ar), 128.6 (2 × Ar), 129.9 (4 × Ar), 135.2 (Ar), 135.8 (2 × Ar), 135.9 (C-7), 141.8
+
(C-8a), 172.3 (C-2) and 197.6 (C-4). EI-MS (m/z, %): 431 (M , 1.2), 340 (82), 234 (4),
+
208 (12), 91 (100) and 77 (4). HRMS (ESI–TOF), m/z found: 454.1799 [M+Na] ,
C H NNaO requires 454.1783.
30
25
2
4.5.6. Ethyl 2-(3,3-dibenzyl-2,4-dioxo-3,4-dihydroquinolin-1H-yl)acetate (42).-
Synthesized after Procedure A. Yield: 0.022 g (89%); colorless oil. IR (NaCl, ν): 3062
(C-H), 2933 (C-H), 1748 (C=O, ester) 1693 (C=O, ketone), 1658 (C=O, amide), 1600,
-1 1
1373, 1197, 437 and 410 cm . H NMR: δ 1.16 (t, J = 7.0, 3H, CH CH O), 3.47 (s, 4H,
3
2
ArCH ), 4.14 (q, J = 7.1, 2H, CH CH O), 4.51 (s, 2H, EtO CCH ), 6.47 (d, J = 8.2, 1H,
2
3
2
2
2
H-8), 6.94-7.10 (m, 11H, Ar-H and H-7), 7.27-7.36 (m, 1H, H-6) and 7.86 (dd, J = 1.8
1
3
and 7.8, 1H, H-5). C NMR: δ 14.0 (CH CH O), 43.9 (EtO CCH ), 45.5 (2 × ArCH ),
3
2
2
2
2
6
40
61.5 (CH CH O), 64.4 (C-3), 113.8 (C-8), 121.2 (C-8a), 123.0 (C-6), 126.7 (2 × Ar),
3 2
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6
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42
43
44
45
127.6 (C-5), 127.9 (4 × Ar), 129.8 (4 × Ar), 135.6 (C-7), 135.9 (2 × Ar), 141.6 (C-4a),
+
167.7 (EtO CCH ), 172.3 (C-2) and 196.4 (C-4). EI-MS (m/z, %): 427 (M , 2), 336
2
2
(100), 290 (34), 262 (50), 234 (3), 132 (23), 91 (41) and 77 (8). HRMS (ESI–TOF), m/z
+
found: 428.1839 [M+Na] , C H NO requires 428.1856.
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4.5.7. 6-Chloro-3,3-bis-(4’-chlorobenzyl)-1-ethyl-1H-quinoline-2,4-dione (43).-
Synthesized after Procedure A. Yield: 0.019 g (80%). White solid; m.p.: 151–153 ºC. IR
(KBr, ν): 2936 (C-H), 1686 (C=O, ketone), 1653 (C=O, amide), 1597, 1489, 1435,
-1 1
1359, 1273, 1016 and 817 cm . H NMR: δ 1.02 (t, J = 7.1, 3H, CH CH N), 3.41 (s,
3
2
4H, ArCH ), 3.81 (q, J = 7.1, 2H, CH CH N), 6.67 (d, J = 8.8, 1H, H-8), 6.95 (d, J =
2
3
2
8.3, 4H, ArH), 7.02 (d, J = 8.3, 4H, ArH), 7.32 (dd, J = 8.8, 2.5, 1H, H-7) and 7.81 (d, J
1
3
= 2.5, 1H, H-5). C NMR: δ 11.9 (CH CH N), 37.3 (CH CH N), 45.3 (2 × ArCH ),
3
2
3
2
2
6
53
64.1 (C-3), 116.1 (C-8), 122.1 (C-4a), 126.8 (C-5), 128.2 (4 × Ar), 128.6 (C-8a), 131.2
(4 × Ar), 132.9 (2 × Ar), 133.9 (2 × Ar), 136.1 (C-7), 140.1 (C-6), 171.2 (C-2) and 196.3
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61
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63
64
+
(C-4). EI-MS (m/z, %): 471 (M , 2.5), 346 (100), 182 (7), 125 (60) and 89 (10). HRMS
+
(ESI–TOF), m/z found: 494.0446 [M+Na] , C H Cl NNaO requires 494.0452.
2
5
20
3
2
4.5.8.
1,3,3-tris(4’-Chlorobenzyl)-6-chloro-1H-quinoline-2,4-dione
(44).-
Synthesized after Procedure B. Yield: 0.062 g (57%). Glassy solid; m.p.: 144–146 ºC.
IR (KBr, ν): 3053 (C-H), 2940 (C-H), 1695 (C=O, ketone), 1660 (C=O, amide), 1595,
-1 1
1485, 1436, 1409, 1360, 1252, 1096, 1015 and 822 cm . H NMR: δ 3.40 (d, J = 12.7,
2H, ArCH ), 3.57 (d, J = 12.7, 2H, ArCH ), 4.95 (bs, 2H, ArCH N), 6.43 (m, 3H, ArH
2
2
2
and H-8), 6.97 (d, J = 8.4, 4H, ArH), 7.07 (d, J = 8.4, 4H, ArH), 7,09-7.19 (m, 3H, ArH
1
3
and H-7) and 7.88 (d, J = 2.6, 1H, H-5). C NMR: δ 44.9 (ArCH N), 45.6 (2 × ArCH ),
2
2
6
65
64.7 (C-3), 117.2 (C-8), 122.1 (C-4a), 126.8 (C-5), 127.2 (2 × Ar), 128.5 (4 × Ar), 129.0
(2 × Ar), 129.2 (C-8a), 131.2 (4 × Ar), 133.0 (Ar), 133.1 (2 × Ar), 133.3 (Ar), 134.0 (2 ×
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77
78
+
Ar), 136.1 (C-7), 140.0 (C-6), 171.7 (C-2) and 196.4 (C-4). EI-MS (m/z, %): 569 (M ,
2), 442 (22), 276 (4), 127 (30), 125 (100) and 89 (12). HRMS (ESI–TOF), m/z found:
+
590.0195 [M+Na] , C H Cl NNaO requires 590.0219.
3
0
21
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4.6. General procedure for the synthesis of the 3,3-dibenzyl-4-hydroxy-3,4-dihydro-
1H-quinolin-2-ones 45-60
The 3,3-dibenzyl-1H-quinoline-2,4-dione (1.0 equiv.) was dissolved in a 1:1 mixture
of EtOAc/EtOH (10 mL/mmol) contained in a round bottom flask. The solution was
cooled in an ice bath, treated with NaBH (1 mol equiv.), and allowed to react at room
4
temperature until complete consumption of the starting material was observed. Then, the
reaction was cooled at 0 °C and saturated NH Cl solution was cautiously added; after
4
stirring for 15 min., the reaction products were extracted with EtOAc (3 × 25 mL). The
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combined organic extracts were washed with brine, dried over MgSO and concentrated
4
under reduced pressure, leaving a residue which upon column chromatography
furnished the desired carbinol.
4.6.1. 3,3-Dibenzyl-4-hydroxy-3,4-dihydro-1H-quinolin-2-one (45).- Yield: 0.025
g (99%). White solid; m.p.: 171–173 ºC. IR (KBr, ν): 3403 (O-H), 3245 (N-H), 1666
-1 1
(C=O, amide), 1599, 1491, 1377, 1256, 1048, 763, 743 and 702 cm . H NMR: δ 2.00
(s, 1H, OH), 2.77 (d, J = 13.7, 1H, ArCH ), 3.01 (d, J = 13.7, 1H, ArCH ), 3.09 (d, J =
2
2
13.7, 1H, ArCH ), 3.38 (d, J = 13.7, 1H, ArCH ), 4.69 (s, 1H, H-4), 6.56 (d, J = 7.6,
2
2
1H, H-8), 7.02 (t, J = 7.4, 1H, H-7), 7.10-7.24 (m, 9H, 2 × ArH and H-6), 7.26-7.38 (m,
1
3
6
6
89
90
3H, ArH and H-5) and 8.16 (s, 1H, NH). C NMR: δ 36.9 (ArCH ), 37.9 (ArCH ), 53.0
2 2
(C-3), 69.7 (C-4), 114.5 (C-8), 123.3 (C-7), 125.9 (C-5), 126.1 (C-8a), 126.6 (2 × Ar),
127.9 (2 × Ar), 128.2 (2 × Ar), 128.6 (C-6), 130.3 (2 × Ar), 130.8 (2 × Ar), 134.3 (C-
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93
94
95
96
97
98
99
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02
03
04
+
4a), 136.5 (Ar), 137.3 (Ar) and 172.5 (C-2). EI-MS (m/z, %): 343 (M , 0.3), 252 (100),
234 (12), 216 (4), 174 (21) and 91 (41). HRMS (ESI–TOF), m/z found: 366.1464
+
[M+Na] , C H NNaO requires 366.1465.
2
3
21
2
4.6.2. 3,3-Dibenzyl-6-fluoro-4-hydroxy-3,4-dihydro-1H-quinolin-2-one (46).-
Yield: 0.046 g (91%). White solid; m.p.: 187–189 ºC. IR (KBr, ν): 3384 (O-H), 3242
(N-H), 3030 (C-H), 2922 (C-H), 1666 (C=O, amide), 1505, 1253, 1237, 1144, 819 and
-1 1
701 cm . H NMR: δ 2.80 (d, J = 13.7, 1H, ArCH ), 2.98 (d, J = 13.9, 1H, ArCH ),
2
2
3.03 (d, J = 13.9, 1H, ArCH ), 3.51 (d, J = 13.7, 1H, ArCH ), 4.69 (d, J = 4.8, 1H, H-
2
2
4), 6.49 (dd, J = 4.6 and 8.6, 1H, H-8), 6.79 (dt, J = 2.8 and 8.6, 1H), 7.07-7.25 (m,
1
3
10H, 2 × ArH), 7.29-7.35 (m, 1H) and 8.77 (s, 1H, NH). C NMR: δ 37.3 (ArCH ),
2
2
2
38.1 (ArCH ), 52.9 (C-3), 69.1 (C-4), 112.8 (d, J = 24.2, Ar), 114.8 (d, J = 23.1,
2
CF
CF
3
Ar), 115.8 (d, J = 7.7, Ar), 126.7 (Ar), 126.8 (Ar), 127.9 (2 × Ar), 128.4 (2 × Ar),
CF
1
7
7
7
7
7
7
7
05
06
07
08
09
10
11
128.5 (C), 130.2 (2 × Ar and C), 130.6 (2 × Ar), 136.2 (Ar), 137.4 (Ar), 159.2 (d, J =
CF
1
9
+
242.0, C-6) and 172.4 (C-2). F NMR: δ -119.2 (m, Ar-F). EI-MS (m/z, %): 361 (M ,
0.9), 270 (100), 252 (12), 234 (7), 192 (19) and 91 (42). HRMS (ESI–TOF), m/z found:
+
384.1367 [M+Na] , C H FNNaO requires 384.1370.
2
3
20
2
4.6.3. 3,3-Dibenzyl-6-chloro-4-hydroxy-3,4-dihydro-1H-quinolin-2-one (47).-
Yield: 0.032 g (65%). White solid; m.p.: 194–196 ºC. IR (KBr, ν): 3419 (O-H), 3240
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(N-H), 3061 (C-H), 2922 (C-H), 1668 (C=O, amide), 1595, 1492, 1375, 1298, 1049 and
-1 1
821 cm . H NMR: δ 2.79 (d, J = 13.7, 1H, ArCH ), 2.99 (d, J = 13.7, 1H, ArCH ),
2
2
3.04 (d, J = 13.7, 1H, ArCH ), 3.48 (d, J = 13.7, 1H, ArCH ), 4.68 (d, J = 6.2, 1H, H-
2
2
4), 6.43 (d, J = 8.2, 1H, H-8), 7.05 (dd, J = 2.4 and 8.4, 1H, H-7), 7.13-7.23 (m, 6H, 2 ×
1
3
ArH), 7.27-7.40 (m, 4H, ArH) and 7.95 (s, 1H, NH). C NMR: δ 37.3 (ArCH ), 38.2
2
(ArCH ), 53.0 (C-3), 69.0 (C-4), 115.5 (C-H), 125.9 (C-H), 126.7 (Ar), 126.8 (Ar),
2
127.9 (2 ×Ar), 128.1 (C), 128.2 (C-H), 128.4 (2 × Ar), 128.5 (C), 130.2 (2 × Ar), 130.7
(2 × Ar), 132.6 (C-6), 136.1 (Ar), 137.3 (Ar) and 172.0 (C-2). EI-MS (m/z, %): 377
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7
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7
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7
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20
21
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23
24
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26
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28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
+
(M , 1.2), 286 (100), 268 (12), 250 (5), 208 (14), 115 (4) and 91 (52). HRMS (ESI–
+
TOF), m/z found: 400.1063 [M+Na] , C H ClNNaO requires 400.1075.
2
3
20
2
4.6.4. 3,3-Dibenzyl-4-hydroxy-6-methoxy-3,4-dihydro-1H-quinolin-2-one (48).-
Yield: 0.018 g (92%). White solid; m.p.: 150–152 ºC. IR (KBr, ν): 3207 (O-H, N-H),
-1
1
3061 (C-H), 2929 (C-H), 1683 (C=O, amide), 1500, 1373, 1249 and 1159 cm . H
NMR: δ 2.22 (s, 1H, OH), 2.98 (d, J = 13.6, 1H, ArCH ), 3.05 (d, J = 13.8, 1H,
2
ArCH ), 3.39 (d, J = 13.8, 1H, ArCH ), 3.39 (d, J = 13.6, 1H, ArCH ), 3.77 (s, 3H,
2
2
2
ArOCH ), 4.66 (s, 1H, H-4), 6.53 (d, J = 8.5, 1H, H-8), 6.67 (dd, J = 2.4 and 8.5, 1H,
3
H-7), 6.94 (d, J = 2.4, 1H, H-5), 7.10-7.24 (m, 8H, 2 × ArH), 7.31 (d, J = 6.6, 2H, ArH)
1
3
and 8.92 (s, 1H, NH). C NMR: δ 36.8 (ArCH ), 37.6 (ArCH ), 52.8 (C-3), 55.6 (2 ×
2
2
ArOCH ), 69.8 (C-4), 111.6 (C-H), 113.6 (C-H), 115.9 (C-H), 126.6 (Ar), 127.6 (C),
3
127.7 (C), 127.9 (2 × Ar), 128.2 (2 × Ar), 130.3 (2 × Ar), 130.8 (2 × Ar), 136.6 (Ar),
+
137.5 (Ar), 156.1 (C-6) and 172.7 (C-2). EI-MS (m/z, %): 373 (M , 26), 282 (100), 264
(19), 204 (6), 151 (6), 123 (5) and 91 (34). HRMS (ESI–TOF), m/z found: 396.1572
+
[M+Na] , C H NNaO requires 396.1570.
2
4
23
3
4.6.5. 3,3-Dibenzyl-4-hydroxy-6-methyl-3,4-dihydro-1H-quinolin-2-one (49).-
Yield: 0.011 g (73%). Colorless oil. IR (NaCl, ν): 3444 (O-H), 3392 (N-H), 3055 (C-H),
-1
2929 (C-H), 2873, 1732, 1672 (C=O, amide), 1373, 1265, 1249, 738, 702 and 437 cm .
1
H NMR: δ 2.29 (s, 3H, ArCH ), 2.02 (d, J = 4.8, 1H, OH), 2.74 (d, J = 13.7, 1H,
3
ArCH ), 2.99 (d, J = 13.7, 1H, ArCH ), 3.08 (d, J = 13.7, 1H, ArCH ), 3.33 (d, J =
2
2
2
13.7, 1H, ArCH ), 4.64 (d, J = 4.8, 1H, H-4), 6.48 (d, J = 7.8, 1H, H-8), 6.94 (d, J =
2
1
3
8.0, 1H, H-7), 7.10-7.31 (m, 11H, 2 × ArH and H-5) and 8.26 (s, 1H, NH). C NMR: δ
20.9 (ArCH ), 36.6 (ArCH ), 37.6 (ArCH ), 52.9 (C-3), 69.9 (C-4), 114.6 (C-H), 125.9
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(C), 126.6 (2 × Ar and C), 127.9 (2 × Ar), 128.2 (2 × Ar), 129.0 (C-H), 130.3 (2 × Ar),
130.8 (2 × Ar), 131.9 (C), 132.9 (C), 136.8 (Ar), 137.4 (Ar), 172.4 (C-2). EI-MS (m/z,
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7
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7
7
7
7
7
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47
48
49
50
51
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58
+
%): 357 (M , 2), 266 (100), 248 (15), 188 (12), 135 (6), 107 (8) and 91 (33). HRMS
+
(ESI–TOF), m/z found: 380.1628 [M+Na] , C H NNaO requires 380.1621.
2
4
23
2
4.6.6. 3,3-Dibenzyl-4-hydroxy-8-methoxy-3,4-dihydro-1H-quinolin-2-one (50).-
Yield: 0.011 g (37%). White solid; m.p.: 182–184 ºC. IR (KBr, ν): 3371 (O-H), 2929
-1
1
(C-H), 1653 (C=O, amide), 1494, 1448, 1417, 1369, 1273, 1247 and 1055 cm . H
NMR: δ 2.08 (d, J = 5.6, 1H, OH), 2.75 (d, J = 13.7, 1H, ArCH ), 3.02 (d, J = 13.7,
2
1H, ArCH ), 3.09 (d, J = 13.7, 1H, ArCH ), 3.36 (d, J = 13.7, 1H, ArCH ), 3.76 (s, 3H,
2
2
2
ArOCH ), 4.67 (d, J = 4.4, 1H, H-4), 6.64-6.72 (m, 1H, H-7), 6.89-6.97 (m, 2H, H-5
3
1
3
and H-6), 7.08-7.30 (m, 10H, 2 × ArH) and 7.78 (s, 1H, NH). C NMR: δ 37.1
(ArCH ), 38.2 (ArCH ), 52.9 (C-3), 55.8 (ArOCH ), 69.7 (C-4), 110.1 (C-H), 117.7 (C-
2
2
3
H), 123.0 (C-H), 123.5 (C), 126.5 (2 × Ar), 126.6 (C), 127.9 (2 × Ar), 128.1 (2 × Ar),
130.3 (2 × Ar), 130.8 (2 × Ar), 136.6 (Ar), 137.4 (Ar), 145.1 (C-8) and 171.4 (C-2). EI-
7
7
7
7
7
7
7
7
7
7
7
7
7
7
59
60
61
62
63
64
65
66
67
68
69
70
71
72
+
MS (m/z, %): 373 (M , 4), 282 (100), 264 (9), 249 (5), 204 (11), 151 (4), 123 (5) and 91
+
(28). HRMS (ESI–TOF) m/z found: 396.1583 [M+Na] , C H NNaO requires
24
23
3
396.1570.
4.6.7. 3,3-bis(4-Methoxybenzyl)-4-hydroxy-3,4-dihydro-1H-quinolin-2-one (51).-
Yield: 0.030 g (99%). White solid; m.p.: 155–157 ºC. IR (KBr, ν): 3397 (O-H), 3237
(N-H), 2914 (C-H), 1667 (C=O, amide), 1612, 1596, 1511, 1378, 1300, 1251, 1176 and
-1 1
1032 cm . H NMR: δ 2.08 (s, 1H, OH), 2.70 (d, J = 13.9, 1H, ArCH ), 2.94 (d, J =
2
13.9, 1H, ArCH ), 3.02 (d, J = 13.9, 1H, ArCH ), 3.28 (d, J = 13.9, 1H, ArCH ), 3.69
2
2
2
(s, 3H, OCH ), 3.73 (s, 3H, OCH ), 4.68 (s, 1H, H-4), 6.59 (dd, J = 0.8 and 7.8, 1H, H-
3
3
8), 6.67-6.80 (m, 4H, p-CH OC H CH ), 7.02 (dt, J = 1.0, 7.4, 1H, H-7), 7.10-7.23 (m,
3
6
4
2
1
3
5H, p-CH OC H CH and H-6), 7.33 (d, J = 7.4, 1H, H-5) and 8.51 (s, 1H, NH). C
3
6
4
2
NMR: δ 35.8 (ArCH ), 36.8 (ArCH ), 52.9 (C-3), 55.1 (2 × ArOCH ), 69.8 (C-4), 113.4
2
2
3
7
7
7
7
7
73
74
75
76
77
(2 × Ar), 113.6 (2 × Ar), 114.7 (C-8), 123.3 (C-7), 126.0 (C-5), 126.3 (C-8a), 128.5 (2 ×
Ar), 128.6 (C-6), 129.3 (2 × Ar), 131.3 (Ar), 131.7 (Ar), 134.4 (C-4a), 158.2 (Ar), 158.3
+
(Ar) and 172.9 (C-2). EI-MS (m/z, %): 403 (M , 2), 282 (100), 264 (25), 174 (22), 121
+
(95), 91 (6) and 77 (10). HRMS (ESI–TOF), m/z found: 426.1683 [M+Na] ,
C H NNaO requires 426.1676.
25
25
4
2
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4.6.8. 3,3-bis(4-Chlorobenzyl)-4-hydroxy-3,4-dihydro-1H-quinolin-2-one (52).-
Yield: 0.060 g (63%). White solid; m.p.: 95–97 ºC. IR (KBr, ν): 3383 (O-H), 3265 (N-
H), 2924 (C-H), 1670 (C=O, amide), 1614, 1597, 1489, 1409, 1382, 1091, 1016 and
-1 1
754 cm . H NMR: δ 2.02 (d, J = 5.8, 1H, OH), 2.71 (d, J = 13.7, 1H, ArCH ), 2.97 (d,
2
J = 13.7, 1H, ArCH ), 3.04 (d, J = 13.7, 1H, ArCH ), 3.28 (d, J = 13.7, 1H, ArCH ),
2
2
2
4.64 (d, J = 5.6, 1H, H-4), 6.54 (dd, J = 0.8, 7.6, 1H, H-8), 7.04 (dt, J = 1.0, 7.6, 1H, H-
7), 7.08-7.23 (m, 9H, p-ClC H CH , H-5), 7.32 (d, J = 6.8, 1H, H-6) and 8.01 (s, 1H,
6
4
2
1
3
NH). C NMR: δ 36.2 (ArCH ), 37.3 (ArCH ), 52.8 (C-3), 69.4 (C-4), 114.6 (C-8),
2
2
7
87
123.6 (C-7), 125.9 (Ar and C-5), 128.0 (2 × Ar), 128.2 (2 × Ar), 128.8 (Ar and C-8a),
131.6 (2 × Ar), 132.0 (2 × Ar), 132.6 (C-6), 134.1 (C-4a), 134.7 (Ar), 135.5 (Ar) and
7
7
7
7
7
7
7
7
7
7
7
88
89
90
91
92
93
94
95
96
97
98
+
171.8 (C-2). EI-MS (m/z, %): 411 (M , 0.4), 286 (100), 268 (19), 250 (6), 233 (8), 174
+
(23), 125 (34) and 93 (9). HRMS (ESI–TOF), m/z found: 434.0676 [M+Na] ,
C H Cl NaNO requires 434.0685.
23
19
2
2
4.6.9. 3,3-bis(2-Chlorobenzyl)-4-hydroxy-3,4-dihydro-1H-quinolin-2-one (53).-
Yield: 0.038 g (99%). Colorless oil. IR (NaCl, ν): 3209 (O-H, N-H), 3063 (C-H), 2925
-
(C-H), 1667 (C=O, amide), 1589, 1475, 1440, 1362, 1264, 1053, 1043, 754 and 735 cm
1
1
. H NMR: δ 2.83 (s, 1H, OH), 3.22 (d, J = 14.2, 1H, ArCH ), 3.37 (d, J = 9.7, 1H,
2
ArCH ), 3.42 (d, J = 9.7, 1H, ArCH ), 3.55 (d, J = 14.2, 1H, ArCH ), 4.80 (s, 1H, H-4),
2
2
2
6.60 (d, J = 7.6, 1H, H-8), 6.95-7.63 (m, 11H, 2 × ArH, H-5, H-6 and H-7) and 9.21 (s,
1
3
7
8
8
8
8
8
8
8
8
8
8
8
99
00
01
02
03
04
05
06
07
08
09
10
1H, NH). C NMR: δ 33.5 (ArCH ), 33.6 (ArCH ), 54.3 (C-3), 69.2 (C-4), 114.4 (C-8),
2 2
123.5 (C-7), 125.1 (C-8a), 126.2 (Ar and C-5), 127.2 (Ar), 128.0 (Ar), 128.2 (Ar), 128.5
(C-6), 129.4 (2 × Ar), 132.2 (Ar), 132.9 (Ar), 134.0 (Ar), 134.4 (Ar), 134.6 (C-4a),
+
135.4 (Ar), 135.5 (Ar) and 172.7 (C-2). EI-MS (m/z, %): 411 (M , 0.07), 376 (100), 286
(20), 250 (15), 234 (15), 174 (9) and 125 (22). HRMS (ESI–TOF), m/z found: 434.0682
+
[M+Na] , C H Cl NNaO requires 434.0685.
2
3
19
2
2
4.6.10. 3,3-Dibenzyl-4-hydroxy-1-methyl-3,4-dihydro-1H-quinolin-2-one (54).-
Yield: 0.060 g (52%). White solid; m.p.: 147–149 ºC. IR (KBr, ν): 3408 (O-H), 3028
-1 1
(C-H), 2933 (C-H), 2879, 1653 (C=O, amide), 1602, 1473, 1367, 1124 and 700 cm . H
NMR: δ 1.96 (s, 1H, OH), 2.70 (d, J = 13.7, 1H, ArCH ), 3.04 (s, 2H, ArCH ), 3.24 (s,
2
2
3H, NCH ), 3.38 (d, J = 13.7, 1H, ArCH ), 4.60 (s, 1H, H-4), 6.68 (d, J = 8.0, 1H, H-8),
3
2
2
8