N-Substituted Quinolinonyl Diketo Acid Derivatives as HIV Integrase Strand Transfer Inhibitors and Their Activity against RNase H Function of Reverse Transcriptase

Article abstract of DOI:10.1021/acs.jmedchem.5b00159

Bifunctional quinolinonyl DKA derivatives were first described as nonselective inhibitors of 3′-processing (3′-P) and strand transfer (ST) functions of HIV-1 integrase (IN), while 7-aminosubstituted quinolinonyl derivatives were proven IN strand transfer inhibitors (INSTIs) that also displayed activity against ribonuclease H (RNase H). In this study, we describe the design, synthesis, and biological evaluation of new quinolinonyl diketo acid (DKA) derivatives characterized by variously substituted alkylating groups on the nitrogen atom of the quinolinone ring. Removal of the second DKA branch of bifunctional DKAs, and the amino group in position 7 of quinolinone ring combined with a fine-tuning of the substituents on the benzyl group in position 1 of the quinolinone, increased selectivity for IN ST activity. In vitro, the most potent compound was 11j (IC₅₀ = 10 nM), while the most active compounds against HIV infected cells were ester derivatives 10j and 10l. In general, the activity against RNase H was negligible, with only a few compounds active at concentrations higher than 10 μM. The binding mode of the most potent IN inhibitor 11j within the IN catalytic core domain (CCD) is described as well as its binding mode within the RNase H catalytic site to rationalize its selectivity. (Chemical Presented).

Full text of DOI:10.1021/acs.jmedchem.5b00159

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Article
N-Substitued Quinolinonyl Diketo Acid Derivatives as
HIV Integrase Strand Transfer Inhibitors and their Activity
against RNase H Function of Reverse Transcriptase
Luca Pescatori, Mathieu Métifiot, Suhman Chung, Takashi Masaoka, Giuliana Cuzzucoli Crucitti,
Antonella Messore, Giovanni Pupo, Valentina Noemi Madia, Francesco Saccoliti, Luigi Scipione, Silvano
Tortorella, Francesco Saverio Di Leva, Sandro Cosconati, Luciana Marinelli, Ettore Novellino, Stuart
Francis John Le Grice, Yves Pommier, Christophe Marchand, Roberta Costi, and Roberto Di Santo
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00159 • Publication Date (Web): 11 May 2015
Downloaded from http://pubs.acs.org on May 12, 2015
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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NꢀSubstitued Quinolinonyl Diketo Acid Derivatives as
HIV Integrase Strand Transfer Inhibitors and their
Activity against RNase H Function of Reverse
Transcriptase
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†
ƒ
⊥
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Luca Pescatori, Mathieu Métifiot, Suhman Chung, Takashi Masoaka, Giuliana Cuzzucoli Crucitti,
†
†
†
†
†
Antonella Messore, Giovanni Pupo, Valentina Noemi Madia, Francesco Saccoliti, Luigi Scipione,
†
‡
∫
‡
Silvano Tortorella, Francesco Saverio Di Leva, Sandro Cosconati, Luciana Marinelli, Ettore
‡
⊥
ƒ
*,ƒ
*,†
Novellino, Stuart F. J. Le Grice, Yves Pommier, Christophe Marchand, Roberta Costi, and
†
Roberto Di Santo
†
Dipartimento di Chimica e Tecnologie del Farmaco, Istituto PasteurꢀFondazione Cenci Bolognetti,
Sapienza” Università di Roma, Pꢀle Aldo Moro 5, Iꢀ00185, Roma, Italy
“
ƒLaboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute,
National Institutes of Health, Building 37, Room 5068, Bethesda, Maryland 20892ꢀ4255, United States
⊥
Resistance Mechanisms Laboratory, HIV Drug Resistance Program, Center for Cancer Research,
National Cancer Institute, National Institutes of Health, Frederick, Maryland, United States
‡ Dipartimento di Farmacia, Università di Napoli “Federico II” Via D. Montesano 49, 80131 Napoli,
Italy
∫
DiSTABiF, Seconda Università di Napoli, Via Vivaldi 43, 81100 Caserta, Italy
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RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required
according to the journal that you are submitting your paper to)
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Abstract. Bifunctional quinolinonyl DKA derivatives were firstly described as nonꢀselective inhibitors
of 3’ꢀprocessing (3’ꢀP) and strand transfer (ST) functions of HIVꢀ1 integrase (IN), while 7ꢀ
aminosubstituted quinolinonyl derivatives were proven IN strand transfer inhibitors (INSTIs) that also
displayed activity against ribonuclease H (RNase H). In this study we describe the design, synthesis and
biological evaluation of new quinolinonyl diketo acid (DKA) derivatives caracterized by variously
substituted alkylating groups on nitrogen atom of the quinolinone ring. Removal of the second DKA
branch of bifunctional DKAs, and the amino group in position 7 of quinolinone ring combined with a
fine tuning of the substituents on the benzyl group in position 1 of the quinolinone, increased selectivity
for IN ST activity. In vitro, the most potent compound was 11j (IC = 10 nM), while the most active
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compounds against HIV infected cells were ester derivatives 10j and 10l. In general, the activity against
RNase H was negligible, with only few compounds active at concentrations higher than 10 ꢀM. The
binding mode of the most potent IN inhibitor 11j within the IN catalytic core domain (CCD) is
described, as well as its binding mode within the RNase H catalytic site to rationalize its selectivity.
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Introduction
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Human immunodeficiency virus type 1 (HIVꢀ1) integrase (IN) and reverse trascriptase (RT) are two
enzymes encoded by pol gene and are essential for virus replication. IN mediates insertion of the
doubleꢀstranded DNA provirus into the host genome, while RT synthesizes this DNA using viral RNA
as a template, and degrading RNA of the RNAꢀDNA replication intermediate in the process. As such,
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both events have been desirable terapeutical targets.
The role of IN during HIVꢀ1 replication begins in the cytoplasm after completion of reverse
trascription. The resulting DNA double stranded DNA is subjected to two reactions. The first of these
involves removing of the 3’ꢀterminal GT dinucleotides adjacent to the highly conserved CA at 3’ꢀend
creating reactive 3’ꢀOH ends in both strands of the viral DNA, and is termed 3’ꢀprocessing (3’ꢀP).
During this step, IN, viral DNA and both host (barrier to autointegration factor (BAF), heatꢀshock
protein 60 (HSP60), high mobility group protein (HMG), lens epitheliumꢀderived growth factor,
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3
(
LEDGF)) and viral (matrix, viral protein R (Vpr), p7/nucleocapsid and RT) proteins form a
preintegration complex (PIC), which is transported into the nucleus. This is followed by a strand
transfer (ST) step, involving a concerted nucleophilic attack by the two reactive 3’ꢀOH recessed ends of
the viral DNA (DNA donor), formed during 3’ꢀP, on the host DNA (DNA acceptor). The gap formed
after the nucleophilic attack between the 5’ꢀend of DNA donor and 3’ꢀend of DNA acceptor is filled
probably by the cellular DNA repair enzymes, finishing the integration process.
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Integration is the final step before irreversibile and productive HIVꢀ1 infection of the target cell.
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Although identified as an attractive target more then 12 years ago, the first drug targeting IN
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raltegravir, 1) was approved by the Food and Drug Administration (FDA) (Figure 1) only in late 2007
as part of combination antiretroviral therapy.
RT is the most actively and longest studied among the enzymes encoded by the pol gene, displaying
both DNA polymearase and ribonuclease H (RNase H) activities, the latter catalyzing degradation of
RNA of the RNAꢀDNA hybrid intermediate to permit the synthesis of (+) strand DNA. Although RT is
a multifunctional enzyme, all approved nucleoside and nonꢀnucleoside RT inhibitors target only its
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DNA polymerase function. IN and the RNase H domain of RT belong to a broader class of
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polynucleotidyl trasferases and share structural similarities. Both enzymes require Mg for catalysis,
and the metal ion is chelated by a highly conserved motif consisting in three acidic aminoacid residues,
also referred to as DDE motif. Within the IN catalytic core, this motif is represented by the three
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conserved acidic residues D64, D116 and E152 involved in the coordination of the two catalytic Mg
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ions. In the RNase H domain, this motif comprises D443, E478, D498 and D549 aminoacids which
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also chelate two magnesium ions.
Given the structural similarities between the catalytic domains of these HIVꢀ1 enzymes, several
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compounds initially developed as IN inhibitors including diketo acids (DKAs) 2 and 3 (Figure 1), and
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DNA aptamers have also been screened against RNase H. Recently, three structurally different
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compounds have been described as dual inhibitors: tropolone 4, 2ꢀhydroxyisoquinolinꢀ1,3(2H,4H)ꢀ
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dione 5 and madurahydroxylactone 6 (Figure 1).
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OH
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OH
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OH
O
HO
H C
N
OH
3
O
OH O
O
6
5
Figure 1. Structures of dual IN and RNase H inhibitors.
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Each one of these classes of inhibitors characteristically presents three aligned heteroatoms capable of
binding divalent metal ions.
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Recently, quinolinonyl diketo acidꢀderived HIVꢀ1 IN inhibitors were described. First bifunctional
DKA 7 (Fig. 2) was reported as nonꢀselective 3’ꢀP and ST inhibitor, interacting with both the acceptor
and donor DNA bindig sites of the enzyme. Indeed, 3’ꢀP was inhibited with an IC of 0.20 ꢀM, in
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comparison with ST reaction that was inhibited with IC of 0.012 ꢀM. This compound was modified to
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obtain a series of quinolinonyl diketo acid derivatives, in which the 6ꢀdiketo acid chain responsable of
’ꢀP inhibition was removed, providing selective ST inhibitors. In particular, compound 8 showed good
inhibitory activity with an IC value of 33 nM with no activity against 3’ꢀP step up to a 50 ꢀM
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concentration (Figure 2). The proven capability of the DKA chain to chelate the catalyticallyꢀcritical
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metal ions made these potential inhibitors of both enzymes.
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OH
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OH
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OH
COOH
Cl
Cl
HOOC
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7
Selective ST inhibitor
Activity against 3'-P
F
F
IC50 3'-P = 200 nM
IC50 ST = 12 nM
IC50 3'-P > 50 ꢀM
IC50 ST = 33 nM
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N
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OH
COOH
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N
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F
IC50 RNase H = 3.3 ꢀM
IC50 IN ST = 80 nM
Activity against IN and RNase H
Figure 2. HIVꢀ1 IN inhibitors of the mono and bifunctional quinolinonyl DKA class.
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In recent years, a number of IN inhibitors have been tested against RNase H function, and recently we
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reported derivatives of 9 as IN inhibitors endowed with a moderate activity against RNase H.
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However, the amino function that was introduced in that series does not increase neither the potency
against the IN enzyme, nor the activity against RNaseH. Thus we decided to design new quinolinone
derivatives to better understand the structureꢀactivity relationships within this series of inhibitors. In
particular, we describe herein the design, synthesis and the biological evaluation of a series of
quinolinonyl DKA derivatives 10a-aa and 11a-aa that are caracterized by a variously substituted benzyl
group or by an aryl or heteroaryl alkyl moiety on nitrogen atom of the quinolinone ring (Figure 3). In
fact, the benzyl portion of IN strand transfer inhibitors (INSTIs) is known to be relevant for the binding
of these inhibitors to the IN/DNA complex. Thus, a study of its influence on the activity of this series of
quinolnyl DKAs against IN and its role on the inhibition of RNase H fuction is vital. Moreover, the role
of the arylmethyl group linked on quinolonyl nitrogen was not previously studied within this series of
inhibitors.
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Role of N substituent
Role of N substituent
n
Ar
R6
R4
R5
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0a-w X = C H
10x-aa X = C H5
11x-aa X = H
n= 1 or 4
Ar = Ph, Py, Naph, Qui
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11a-w X = H
Figure 3. Stuctures of the newly designed quinolinonyl diketo acid derivatives 10a-aa and 11a-aa (Ph =
phenyl, Py = 2ꢀpyridinyl, Naph = 2ꢀnaphtyl Qui = 2ꢀquinolinyl). For specific structures and molecules
see Tables 1 and 2.
Result and Discussion
Chemistry. Compounds 10aꢀaa and 11aꢀaa were synthesized according to the pathway reported in
Scheme 1. 3ꢀAcetylꢀ4(1H)ꢀquinolinones 12a-c were prepared by reaction of aniline with ethyl
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orthoformate and ethyl acetoacetate, which were thermally condensed in the presence of an inert heating
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medium (Dowtherm A) under argon atmosphere, according to the Yoshizawa procedure. Nꢀ1
substituted quinolones 13a-aa were obtained via alkylation of compounds 12a-aa with the proper aryl
alkyl bromide in alkaline medium (K
condensation reaction with diethyl oxalate and sodium ethoxide as the catalyst to provide ethyl esters
0aꢀaa, which were in turn hydrolyzed with 1 N NaOH to afford the corresponding acids 11aꢀaa
Scheme 1). Chemical, physical, and spectroscopic data of final products 10aꢀaa and 11aꢀaa are
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CO
3
). Compounds 12a-aa were then subjected to a Claisen
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(
described in the Experimental Section, while data of intermediates 12a-c, 13a-aa and analyses of 10aꢀ
aa and 11aꢀaa are reported in Supporting Information section.
a
Scheme 1. Synthetic Route to Quinolinonyl DKAs 10a-aa and 11a-aa
O
N
O
O
O
a
b
c
R
R
R
NH2
N
H
12a-c
R1 13a-aa
O
N
O
OH
O
O
OH
OCH CH
2
3
OH
d
R
R
O
O
N
R1
10a-aa
R1
11a-aa
a
Reagents and conditions: (a) ethyl orthoformate, ethyl acetoacetate, Dowtherm A, 95ꢀ254 °C, 8 h,
2ꢀ58% yield; (b) arylalkyl bromide, K CO , DMF, 100 °C, 50ꢀ75% yield; (c) diethyl oxalate,
5
2
3
C H ONa, THF, room temp, 10 min, 62ꢀ100% yield; (d) 1 N NaOH, THF/CH OH, room temp, 40 min,
2
5
3
3
8ꢀ100% yield.
Evaluation of Biological Activities. In Vitro Assays. Newly synthesized compounds 10a-aa and
11a-aa were tested in vitro with respect to RTꢀassociated RNase H function, as well as INꢀassociated
3
’ꢀP and ST activities. ST and 3’ꢀP were evaluated using gelꢀbased assay carried out in presence of
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Mg and the corresponding IC values were calculated using a doseꢀresponse curves. The results of
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these assays are summarized in Table 1 and Table 2.
Several of the newly synthesized compounds were potent IN inhibitors showing high activity against
the ST reaction and low activity against 3’ꢀP and RNase H function. As expected from previous
findings, acid derivatives were always more active then the corresponding esters when tested against IN.
Moreover, the Nꢀbenzyl substituted derivatives 11a-w were more active than the Nꢀarylalkyl substituted
counterparts 11x-aa on both IN and RNase H (Table 1 and Table 2).
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Anti-IN activities. Acid derivatives 11a-w were very active ST inhibitors, showing IC values in the
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nanomolar range (640ꢀ10 nM), with the exception of 11k,o,q that exibited IC values in the micromolar
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range (3.2−7.0 ꢀM).
Monoꢀ (11a-i) and diꢀsubstituted (11j-w) benzyl derivatives showed comparable activities against IN.
In fact, in the first case the IC values were in the range of 0.64ꢀ0.015 ꢀM, while in the second one
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were in the range of 0.22ꢀ0.010 ꢀM.
The most active compound of the series was the 2,4ꢀF acid derivative 11j, with an IC value of 10
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nM. Four additional compounds, the 2ꢀF, 3ꢀF, 2,6ꢀF and 2,3ꢀF,Cl derivatives (11b,d,l,s), demonstrated
2
comparable potencies (IC values of 16 nM, 15 nM, 19 nM and 18 nM respectively). Interestingly, the
50
fluorine substituent was best tolerated, particularly when bound in position 2 of the benzyl ring, both in
mono and disubstitutedꢀbenzyl derivatives. This trend was highlighted in the series of the diꢀsubstituted
benzyl derivatives, comparing the most active compound 11j with the other compounds 11l-u. In fact,
when the fluorine atom was bound in position 2 of the benzyl ring, inhibitory activity increased even if
there was a second substituent on the phenyl ring. For example, compounds 11l (2,6ꢀF ) and 11s (2,3ꢀ
2
F,Cl) showed inhibitory activities comparable to 11j. When the fluorine atom in position 2 was replaced
by a chlorine atom (11o-p) or a methyl group (11q), activity decreased of 1ꢀ2 order of magnitude (IC50
values of 2.3 ꢀM, 0.27 ꢀM and 7.0 ꢀM, respectively). An exception was the derivative 11v, 2ꢀ
fluorobenzyl substituted, characterized by a chlorine atom in 8ꢀposition of the aromatic ring of the
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Journal of Medicinal Chemistry
Page 10 of 44
quinolinonic nucleus. This derivative was 10 times less active than its parent 2ꢀfluoro benzyl substituted
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
counterpart 11b, (IC values 0.13 ꢀM and 0.016 ꢀM, respectively).
50
Among monosubstituted benzyl derivatives 11a-i, replacing the fluorine atom in the 2ꢀposition with a
methoxy group led to a decrease in the activity (11c, IC value of 38 nM), as well as its substitution
5
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
with an hydrogen atom (11a, IC value of 34 nM). The shift of the fluorine atom from postition 2 to 3,
50
gave a compound that had the same inhibitory activity (11d, IC value of 15 nM), while its shift to
5
0
position 4 slightly decreased activity (8, IC value of 40 nM). When the fluorine atom in position 4 was
5
0
replaced by other atoms (11f) or group (11g,h-i), activity decreased by an order of magnitude (IC50
values 0.45ꢀ0.64 ꢀM).
Anti-RH acitivities. The quinolinonyl diketo acids derivatives 10a-aa and 11a-aa were tested for
RNase H inhibition, calculated as a percentage of inhibition at a concentration of 10 ꢀM. IC values
5
0
have been determined for derivatives showing % of inhibition >30%.
In general, the ester derivatives 10a-aa poorly inhibited the RNase H function at 10 ꢀM, and in all
cases the mesaured IC were always >50 ꢀM. Although the acid counterparts were more effective, their
50
IC were poor compared to their antiꢀIN potency. The most active compounds were the acid derivatives
5
0
1
1g, 11l and 11p showing IC values of ~10 ꢀM. Among the monoꢀsubstituted benzyl derivatives (10a-
5
0
i and 11a-i), substitution in position 4 was best tolerated. In particular, in the ester series, a nitro group
10i) increased the inhibitory activity of the molecule (31.9% inhibition); among this series, inhibitory
activity decreased in the following order: NO > OH > Cl > OCH > F. In the acid series, increased
(
2
3
activity was highlighted by the presence of a chlorine atom (11f, 56.2% inhibition); among this series,
inhibitory activity decreased in the order: Cl > F > OCH ≈ OH ≈ NO .
3
2
Interestingly, compound 8 was tested as a representative example of the nonꢀbasic quinolonyl DKA
16b
series previously reported
recombinant RNase H.
and was confirmed weak inhibitor, showing IC50 = 42 ꢀM against
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Journal of Medicinal Chemistry
The disubstituted benzyl derivatives 10j-w and 11j-w, showed a good inhibitory activity when two
chlorine atoms were the substituents of the benzyl moiety (10o-p, 51 and 55% inhibition, respectively;
11o-p, 68 and 86.3% inhibition, respectively). In general, the fluorine atom lead to decreased activity,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
even then in the F ꢀbenzyl and Cl,Fꢀbenzyl disubstituted derivatives (10j-n and 10s-u, 3ꢀ24% inhibition;
2
1
1j-n and 11s-u, 30ꢀ57.9% inhibition), with respect to the most active acid compound 2,6ꢀCl2ꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
disubstituted derivative 11p (86.3% inhibition).
Cell-Based Assays. Newly synthesized compounds were tested for antiviral activity and cytotoxicity
using HeLaꢀCD4ꢀLTRꢀβꢀgal cells infected by HIVꢀ1(IIIB). Elvitegravir (EVG) and 1 were used as
reference compounds. EC and CC values for compounds 10a-aa and 11a-aa are reported in Table 1
50
50
and Table 2. Among these, derivatives 10a-e,g,j,l-n,p,s,t,w and 11a-d,f,j,l,m,s,w,y show EC < 50 ꢁM.
5
0
It is noteworthy that although less active in biochemical assays, esters 10a-e,g,j,l-n,p,s,t,w and acids
1a-d,f,j,l,m,s,w,y are almost equipotent in cell based assays. In particular, the most active compounds
are the ester derivatives 10j and 10l and acid 11f that are active in the submicromolar range (EC =
1
5
0
0
.58, < 0.2 and 0.87 ꢁM, respectively), with selectivity indices (SI) higher than 50 (>86.2, >250, >57.5,
respectively). Notably, compound 10l showed an incresased antiviral activity if compared with
1
6,18
quinolinonyl DKA reported in our previous papers.
All active compounds were characterized by low
cytotoxicity against the same HeLaꢀCD4ꢀLTRꢀβꢀgal cells, showing CC values >50 ꢁM, with the sole
5
0
exception of 10e showing a CC of 44 ꢀM.
5
0
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Journal of Medicinal Chemistry
Page 12 of 44
Table 1. Cytotoxicity, Antiviral, Anti-IN, and Anti-RNase H Activities of Compounds 10a-w and
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
1a-w
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Activity in enzyme assays
IN
Antiviral activity and
cytotoxicity
RNaseH
a
IC (ꢀM)
50
CC50
d(ꢀM) SI
R
% in. at
10 ꢀM
EC50
(ꢀM)
e
Cpd
R
R
2
R
3
R
4
R
5
X
3’ꢀP
ST
IC (ꢀM)
50
c
b
6
f
10a
10b
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
F
Et >1000
Et 125
Et >333
Et >12.3
Et >333
Et >37
Et >333
Et >37
Et >37
Et
0.70
0.28
1.4
ꢀ3.3
2.8
11.4
ꢀ9.4
4.9
16.1
26.0
10.0
31.9
3
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
1
>50
>50
>50
>50
44
>50
F
H
1.5
>33.4
>19.2
>29.4
5.2
1
1
1
1
1
0c
0d
0e
0f
OCH
H
3
H
2.6
H
4.9
1.7
H
OCH
H
H
H
H
H
H
H
F
H
16
8.5
3
H
Cl
OH
OCH
>37
1.4
>50
30.9
>50
>50
0.58
>50
<0.2
9.8
nd
0g
H
>50
nd
>1.6
10h
H
3
>37
>37
2.2
1
1
1
1
1
0i
H
NO
F
2
nd
0j
F
>50
nd
>86.2
0k
0l
F
H
Et
3.7
30
F
H
H
H
F
Et >12.3
Et
0.25
5.5
4.1
24
>50
>50
>50
nd
>250
>5.1
>1.5
0m
H
F
H
H
H
10n
10o
H
F
H
Et
11
24
32.4
>50
30.2
>50
Cl
Cl
H
H
H
Cl
H
H
H
H
Et
>111
32
51
>50
>50
>50
1
1
0p
0q
Cl Et
Et
55.0
33
>50
nd
>1.7
CH
CH
H
>111
3
3
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Page 13 of 44
Journal of Medicinal Chemistry
1
0r
ꢀ
H
CH
3
H
CH
H
Et
1.3
21
nd
>50
nd
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
10s
10t
ꢀ
ꢀ
F
Cl
Cl
Cl
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
Et 1.08
Et 266
Et >333
Et
1.08
3.1
21
nd
10
>50
>50
nd
>5
H
H
F
F
18
nd
17.4
>50
11
>2.9
1
1
1
1
1
0u 8ꢀCl
0w 7ꢀCl
F
25.08
24
nd
H
2.8
9.6
45.1
45.2
2.4
1.9
56.2
44.4
45.8
42.4
46
nd
>50
>50
>50
>50
>50
nd
>4.5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1a
1b
1c
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
4.0
1.2
>4.1
4.6
>4.1
22
0.034
0.016
0.038
0.015
0.14
0.54
0.45
0.51
0.64
0.010
3.2
nd
15.5
2.6
H
>50
>19.2
>5.4
>1.8
OCH
H
H
H
H
H
H
F
H
16.3 ± 0.5
nd
9.3
3
11d
H
28.2
>50
0.87
>50
>50
>50
13.8
>50
8.1
1
1
1
1
1
1e
1f
OCH
H
3
H
nd
Cl
OH
OCH
>50
>50
nd
>57.5
1g
1h
1i
H
>37
28
9.5 ± 0.4
>50
H
nd
3
H
NO
F
27
47.0 ± 1.5
35.9 ± 0.8
>50
nd
2
11j
H
>50
nd
>3.6
1
1
1
1
1
1k
1l
F
H
H
37
F
H
H
H
H
F
0.7
0.019
0.11
0.22
2.3
57.9
44
10.8 ± 0.4
>50
>50
>50
nd
>6.2
>3.1
1m
1n
1o
H
H
Cl
Cl
F
F
H
H
H
16.2
>50
>50
>50
>50
>50
F
H
30
>50
H
Cl
H
H
H
H
68
44.7 ± 2.0
10.0 ± 0.3
19.6 ± 0.6
37.8 ± 0.9
nd
11p
11q
H
Cl H
0.27
7.0
86.3
52
nd
CH
H
H
CH
H
H
H
H
H
nd
3
3
1
1r
CH
3
CH
0.05
47
nd
3
1
1
1
1s
1t
ꢀ
ꢀ
F
Cl
Cl
Cl
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
1.75
21
0.018
0.19
12
nd
23.4
>50
50
>50
nd
>2.1
H
H
F
25
nd
1u 8ꢀCl
F
9.8
0.096
0.13
16
nd
nd
11v 8ꢀCl
11w 7ꢀCl
1
H
H
20.5
45
>50
>50
17
nd
F
H
36.5
22.6 ± 0.5
>50
>2.9
g
0.087±
0.0236
±0.0046
1
2.8± 6
h
nd
nd
>100
>50
>50
>2118
0
.008
EVG
8.1±
0.028±
0.006
0.0142
±0.0052
g
g
91 ± 8
>3521
4
.2
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Journal of Medicinal Chemistry
Page 14 of 44
a
b
Inhibitory concentration 50% (ꢀM) determined from dose response curves. Percentage of inhibition
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
c
d
determined at compound concentration of 10 ꢀM. Effective concentration 50% (ꢀM). Cytotoxic
concentration 50% (ꢀM). SI = CC /EC . nd: not determined. Reference 20. Reference 18.
e
f
g
h
50
50
Table 2. Cytotoxicity, Antiviral, Anti-IN, and Anti-RNase H Activities of Compounds 10x-aa and
1
1x-aa
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Activity in enzyme assays
IN
Antiviral activity and cytotoxicity
RNaseH
a
IC (ꢀM)
50
%
ibn. at 10
x
c
d
e
R
X
3’ꢀP
ST
120
32
IC (ꢀM)
EC (ꢀM)
CC (ꢀM) SI
50
50
50
ꢀM
f
1
1
0x
0y
Et
Et
>333
>333
19.4
29.7
nd
nd
>50
>50
nd
nd
nd
1
0z
Et
>333
14
ꢀ5.6
nd
>50
1
1
0aa
1x
Et
H
H
>1000
>333
18
3.5
4.4
nd
nd
nd
>50
>50
4.8
nd
nd
<0.45
<0.45
33.0
40.7
11y
>50
nd
>10.4
1
1
1z
H
H
>1000
70
16
ꢀ2.6
nd
nd
>50
1aa
0.40
16.4
>50
nd
a
b
Inhibitory concentration 50% (ꢀM) determined from dose response curves. Percentage of inhibition
c
d
determined at compound concentration of 10 ꢀM. Effective concentration 50% (ꢀM). Cytotoxic
concentration 50% (ꢀM). SI = CC /EC . nd: not determined.
e
f
5
0
50
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Page 15 of 44
Journal of Medicinal Chemistry
Molecular Modeling. To better rationalize structureꢀactivity relationships (SARs) for the newly
identified quinolinonyl DKAs, molecular docking studies were performed on compound 11j which is
the most potent INSTI (10 nM) within this series while displaying insignificant activity against RNase
H. Computations were aimed at elucidating the structural requisites responsible for binding at the IN
active sites, but with residual activity against RNase H.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Our recently built homology model of the IN catalytic core domain (CCD)/viral DNA was used to
18
carry out docking studies. This model was constructed starting from the coordinates of the fullꢀlength
structure of prototype foamy virus (PFV) IN in complex with 1 (PDB code 3OYA) and two active site
2
+
21
Mg ions required for both catalytic activity and chelation by INSTIs. According to docking results,
compound 11j lies at the IN/DNA interface with the O, O, O donor triad of the DKA branch chelating
2
+
the Mg ions, and the central hydroxyl anionic oxygen atom bridging between the two cations (Figure
4
a). This results in formation of 5ꢀ and 6ꢀmembered chelate rings with the two metal centers.
Interestingly, the 5ꢀ,6ꢀmembered chelate ring binding motif appears to be one of the most critical
features of highly effective INSTIs, as underscored by recent theoretical studies on the role of the metal
22
binding groups within this class of anti HIVꢀ1 drugs. Additionally, chelation by the carboxylate group
might explain the higher inhibitory potency of acidic compounds with respect to the corresponding
esters (see 10a-aa vs 11a-aa). Besides coordination of the metal centers, additional key interactions are
established by our reference compound within the active site. In particular, the quinolinonyl ring
establishes hydrophobic contacts with the Cβ and Cγ carbons of Q148 and is involved in a parallelꢀ
displaced πꢀπ interaction with the terminal adenosine at the 3’ end of the nucleic acid substrate, which is
2
1
known to be displaced through an inducedꢀfit mechanism. Furthermore, the pꢀfluorobenzyl group fits
into a narrow pocket created by rearrangement of the aforementioned terminal adenosine and the
enzyme surface. Herein, the pꢀFꢀbenzyl branch establishes a πꢀstacking interaction with the penultimate
cytidine of the DNA reactive strand and favorable hydrophobic contacts with Q146 and P145.
Interestingly, these two amino acids are directly involved in separation of the viral DNA strands upon
2
3
the ST reaction. Indeed, these interactions appear to influence the inhibitory potency of these INSTIs
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Journal of Medicinal Chemistry
Page 16 of 44
as demonstrated by the potencies of compounds bearing the fluorine atoms in different positions of the
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
aromatic ring (compounds 11b, 11d, 11j-n, 11s, 11t, 11u, 11v). This is in line with SARs data achieved
2
4
for different INSTIs by us and other groups. Nevertheless, it should be pointed out that the role of the
23
fluorobenzyl group in the IN recognition is rather “enigmatic” as outlined by Hare and coꢀworkers. On
the other hand, the importance of the fluorine substitution on the benzyl group is also proved by
compounds bearing different halogens (11f, 11o and 11p) and different groups (11e, 11g-i, 11o-q)
which are generally less active than the fluorinated analogues. Additionally, limited extension of the
pocket at the IN/DNA interface would also explain why replacement of the benzyl substituent with
bulkier groups (compounds 11x-aa) results in a loss of the inhibitory activity against IN.
0
1
2
3
4
5
6
7
8
9
0
1
2
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9
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6
7
8
9
0
Overall, these results support the theory according to which INSTIs should inhibit the ST reaction by
2
+
’
contacting the Mg cofactors and displacing the terminal 3 ꢀadenosine of the viral DNA, thus
interfering with the recognition between IN/viral DNA binary complex and the host cell DNA (see SI
2
3
Figure S1). Indeed, the binding mode provided by our docking calculations is shared by the majority
2
5,26,27,28
21
of INSTIs including other quinolinonyl DKAs
and also 1 (Figure 4b).
Figure 4. a) Binding mode of compound 11j (yellow) within the HIVꢀ1 IN/DNA model. Catalytic core
domain is depicted as transparent light gray surface and ribbons. Amino acid side chains important for
the ligand binding are represented as sticks. The nonꢀtransferred (cyan) and reactive (orange) viral DNA
2
+
strands are shown as ribbon and sticks. Mg metal ions are represented as green spheres. b) Overlay of
compound 11j and 1 (magenta) in the IN binding pocket.
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Journal of Medicinal Chemistry
A rationalization for residual activity against RNase H shown by some compounds of this series was
also performed. The 2.09 Å resolution crystal structure of fullꢀlength HIVꢀ1 RT in complex with a
RNase H pyrimidinol carboxylic acid inhibitor and the nonnucleoside inhibitor nevirapine (PDB code
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
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4
4
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4
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5
5
5
5
5
5
5
5
6
3
QIP) was selected for docking calculations. This structure shows the RNase H domain in complex with
an inhibitor which is more structurally related to our DKA compounds as compared to the other ligands
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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0
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8
9
0
2
9,30,31
coꢀcrystallized with the fullꢀlength enzyme or the isolated RNase H domain.
Before running
2+
docking calculations, Mn ions in complex with this structure were replaced with physiologically
2
+
relevant Mg cations, which were also present in the biochemical and cellꢀbased assays. In the
2
+
predicted binding conformation, 11j chelates both catalytic Mg cations with its DKA branch (Figure
) in a 5ꢀ,6ꢀmembered chelate ring binding arrangement, whereas the carboxylate terminal group Hꢀ
5
bonds with the amide of N474. As already observed for IN, such a chelate geometry would explain the
general higher inhibitory activity displayed by acidic ligands compared to the corresponding esters. In
addition chelating the metal cations, 11j establishes favorable lipophilic interactions with the Cβ and Cγ
carbons of Q475 through its quinolinonyl nucleus while the benzyl substituent, although being partially
solvent exposed, is able to contact the lipophilic residues Q500, and W535. Indeed, the generally lower
1
8
RNase H activity of this series with respect our previous study can be ascribed to the lack of additional
interactions established with residues such as Y501 which is highly conserved and is a critical
component of the RNase H primer grip. Moreover, recent experimental data demonstrate that mutating
this residue is also negatively affecting the inhibithory potency of other chemotypes bearing the DKA
32
moiety.
1
8
In conclusion, and in support of previously reported series, we suggest that the quinolinonyl DKAs
presented herein could exert their RNase H inhibitory activity by sterically hindering fitting of the viral
DNA:RNA hybrid at the catalytitic site, thus hampering the degradation of the RNA strand (see SI
1
8
Figure S2).
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1
2
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7
8
9
1
1
1
1
1
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1
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0
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9
0
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4
5
6
7
8
9
0
Figure 5. Binding mode of compound 11j (yellow) within the HIVꢀ1 RNase H active site. CCD is
shown as transparent white surface and ribbons. Amino acid side chains important for the ligand binding
2
+
are represented as sticks. Mg metal ions are depicted as green spheres.
A final consideration sould be done on the higher activity of 10j in cellꢀbased assays compared to that
of 11j. Although ester 10j showed a similar binding pose of 11j at both the IN and RNase H active sites
(see Figure S3 and compare with Figures 4 a) and 5), its higher activity against HIV infected cells
would be rationalized hypothesizing that 10j is likely to better penetrate cell membrane than its
corresponding acid and, once in the cell, it would be hydrolyzed to 11j that may represent the effective
inhibiting form of both the IN and RNase H enzymes.
Conclusion
In this paper we describe the design, synthesis, and biological assays of a series of new quinolinonyl
diketo acid derivatives that are caracterized by variously substituted alkylating groups on the nitrogen
atom of the quinolinone ring. These derivatives were tested in both enzymeꢀ and cellꢀbased assays as
antiꢀHIVꢀ1 agents to selectively target the ST step of integration, testing in parallel the RNase H
function of RT. We generated new compounds, which are able to selectively inhibit IN and only
marginally the RNase H. Five acid compounds resulted to be active against IN in the nanomolar range
and, among them, compound 11j (IC = 10 nM against IN) inhibited RNase H activity with an IC =
5
0
50
3
5.9 ꢀM. These compounds were also active against HIVꢀ1 replication in acutely infected cells, as well.
However, the potency in cellꢀbased assays was lower if compared to the activity against ST, suggesting
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Journal of Medicinal Chemistry
that the acid derivatives are less prone to cell membrane penetration. Thus, the most active compounds
against HIV infected cells turned out to be ester derivatives 10j and 10l.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
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2
2
2
2
2
2
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4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
In conclusion, the design of this new quinolinonyl DKAs was based on i) removal of the second DKA
branch of bifunctional DKAs, that directed the activity against 3’ꢀP, ii) removal of the amino group in
position 7 of quinolinone ring that addressed the activity on both IN and RNase H, and iii) fine tuning of
the substituents on the benzyl group in position 1 of the quinolinone, that preferentially addressed
activity against IN ST. Further studies will be necessary to improve the activity of these compounds
against HIV infected cells.
0
1
2
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5
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7
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9
0
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9
0
Experimental Section
Chemistry. General. Melting points were determined on a Bobby Stuart Scientific SMP1 melting
point apparatus and are uncorrected. Compounds purity were always >95% determined by high pressure
liquid chromatography (HPLC). HPLC analysis were carried out with a Shimadzu LCꢀ10AD VP CTOꢀ
10AC VP. Column used was generally Discovery Bio Wide Pore C18 (10 cm X 4.6 mm, 3 ꢁm). IR
spectra were recorded on a PerkinꢀElmer Spectrumꢀone spectrophotometer. 1H NMR spectra were
recorded at 400 MHz on a Broker AC 400 Ultrashield 10 spectrophotometer (400 MHz).
Dimethylsulfoxideꢀd6 99.9% (code 44,139ꢀ2) and deuterochloroform 98.8% (code 41,675ꢀ4) of isotopic
purity (Aldrich) were used. Column chromatographies were performed on silica gel (Merck; 70ꢀ230
mesh) column or alluminium oxide (Sigmaꢀaldrich; 150 mesh) column. All compounds were routinely
checked on TLC by using aluminiumꢀbaked silica gel plates (Fluka DCꢀAlufolien Kieselgel 60 F254) or
alluminium oxide (Fluka DCꢀAlufolien Aluminium oxide). Developed plates were visualized by UV
light. Solvents were reagent grade and, when necessary, were purified and dried by standard methods.
Concentration of solutions after reactions and extractions involved the use of rotary evaporator (Büchi)
operating at a reduced pressure (ca. 20 Torr). Organic solutions were dried over anhydrous sodium
sulfate (Merck). All reactions were carried out under nitrogen; all solvents were freshly distilled under
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Page 20 of 44
nitrogen and stored over molecular sieves for at least 3 h prior to use. Analytical results agreed to within
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
±
0.40% of the theoretical values.
3-Acetyl-4(1H)-quinolinones 12a-c. Ethyl orthoformate (4.0 g, 27 mmol), ethyl acetoacetate (3.5 g,
2
7 mmol), the proper aniline (27 mmol), and Dowtherm A (5.6 mL) were charged in a round bottom
threeꢀnecked flask equipped with a water separator. This mixture was stirred under argon atmosphere,
while the temperature was increased to 95 °C in 1 h, then gradually up to 162 °C in a further hour. Then
the mixture was stirred at this temperature for 6 h. After this time, the resulting solution was added in
portions during 3 h into 42 mL of Dowtherm A stirred in a round bottom threeꢀnecked flask equipped
with thermometer and water separator and heated at 253ꢀ254 °C. After the addition the mixture was
heated at the same temperature for 2 h. Then the mixture was cooled at 90 °C, treated with isopropanol
(10 mL), cooled at 30 °C, filtered, and washed with isopropanol and light petroleum ether in turn to give
pure derivatives 12a-c. For chemical, physical, analytical and spectroscopic data of compounds 12a-c
see Supporting Information.
0
1
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2
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0
1
2
3
4
5
6
7
8
9
0
General Procedure for the Synthesis of 3-Acetyl-1-(aryl) methyl-4(1H)-quinolinones (13a-aa). A
mixture of 12a-c (1.1 mmol), the proper arylmethyl bromide (3.3 mmol), and anhydrous K CO (210
2
3
mg, 1.5 mmol) in dry DMF (10 mL) was stirred at 100 °C for 1 h. After the mixture was cooled, water
was added (40 mL) and the formed precipitate was filtered, washed with water and light petroleum ether
in turn, and then dried under IR lamp to provide pure derivatives 13a-aa. For chemical, physical,
analytical and spectroscopic data of compounds 13a-aa see Supporting Information.
General Procedure for the Synthesis of Diketo Esters 10a-aa. Sodium ethoxide (390 mg, 5.5
mmol) was added into a well stirred mixture of the appropriate acetyl derivative 13a-aa (2.7 mmol) and
diethyl oxalate (790 mg, 5.4 mmol) in anhydrous THF (2.7 mL) under nitrogen atmosphere. The
mixture was stirred at room temperature for 2 h, then was poured into nꢀhexane (50 mL). The collected
precipitate was vigorously stirred for 30 min in 1 N HCl (50 mL). The formed yellow solid was filtered,
washed with water, and dried under IR lamp to afford the pure diketo esters 10a-aa. Yield (%), melting
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Journal of Medicinal Chemistry
1
point (°C), recrystallization solvent, IR, H NMR, and analytical data for each of the following
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
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3
3
3
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4
4
4
4
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5
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5
5
5
5
5
6
compounds are reported.
4-[1-Phenylmethyl-4(1H)-quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid Ethyl Ester (10a).
8
9 %; 141ꢀ142 °C; benzene; IR ν 3400 (OH), 1740 (C=O ester), 1648, 1629, and 1609 (C=O ketone)
ꢀ1 1
cm . H NMR (DMSOꢀd ) δ 1.29 (t, 3H, CH CH ), 4.29 (q, 2H, CH CH ), 5.76 (s, 2H, CH ), 7.22ꢀ7.29
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
2
3
2
3
2
(
m, 5H, benzene H), 7.48 (m, 1H, quinolinone C6ꢀH), 7.70ꢀ7.71 (m, 2H, quinolinone C7ꢀH and C8ꢀH ),
.01 (s, 1H, butenoate C3ꢀH), 8.31 (m, 1H, quinolinone C5ꢀH), 9.12 (s, 1H, quinolinone C2ꢀH), 15.50
(bs, 1H, OH). Anal. (C22H19NO5) C, H, N.
-[1-(2-Fluorophenyl)methyl-4(1H)-quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid Ethyl
Ester (10b). 92 %; 152ꢀ153 °C; toluene; IR ν 3200 (OH), 1744 (C=O ester), 1651, and 1612 (C=O
8
4
ꢀ1 1
ketone) cm . H NMR (DMSOꢀd ) δ 1.28 (t, 3H, CH CH ), 4.29 (q, 2H, CH CH ), 5.81 (s, 2H, CH ),
6
2
3
2
3
2
7
.12ꢀ7.28 (m, 4H, benzene H), 7.47 (m, 1H, quinolinone C6ꢀH), 7.68ꢀ7.76 (m, 2H, quinolinone C7ꢀH
and C8ꢀH ), 7.99 (s, 1H, butenoate C3ꢀH), 8.31 (m, 1H, quinolinone C5ꢀH), 9.09 (s, 1H, quinolinone
C2ꢀH), 15.00 (bs, 1H, OH). Anal. (C22H18FNO5) C, H, F, N.
4
-[1-(2-Methoxyphenyl)methyl-4(1H)-quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid Ethyl
Ester (10c). 98 %; 154ꢀ156 °C; toluene; IR ν 3200 (OH), 1726 (C=O ester), 1631, and 1601 (C=O
ꢀ1 1
ketone) cm . H NMR (DMSOꢀd ) δ 1.29 (t, 3H, CH CH ), 3.82 (s, 3H, CH ), 4.29 (q, 2H, CH CH ),
6
2
3
3
2
3
5
.63 (s, 2H, CH ), 6.89 (t, 1H, benzene H), 7.06 (d, 1H, benzene H), 7.13 (d, 1H, benzene H), 7.30 (t,
2
1H, benzene H), 7.49 (m, 1H, quinolinone C6ꢀH), 7.71ꢀ7.76 (m, 2H, quinolinone C7ꢀH and C8ꢀH ), 8.00
s, 1H, butenoate C3ꢀH), 8.31 (m, 1H, quinolinone C5ꢀH), 9.05 (s, 1H, quinolinone C2ꢀH), 14.20 (bs,
(
1
H, OH). Anal. (C22H21NO6) C, H, N.
4-[1-(3-Fluorophenyl)methyl-4(1H)-quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid Ethyl
Ester (10d). 94 %; 174ꢀ175 °C; methanol; IR ν 3138 (OH), 1740 (C=O ester), 1650 and 1613 (C=O
ꢀ1 1
ketone) cm . H NMR (DMSOꢀd ) δ 1.27 (t, 3H, CH CH ), 4.30 (q, 2H, CH CH ), 5.78 (s, 2H, CH ),
6
2
3
2
3
2
7
.01ꢀ7.20 (m, 3H, benzene H), 7.38 (m, 1H, benzene H), 7.49 (m, 1H, quinolinone C6ꢀH), 7.65ꢀ7.74 (m,
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2
H, quinolinone C7ꢀH and C8ꢀH ), 8.01 (s, 1H, butenoate C3ꢀH), 8.31 (m, 1H, quinolinone C5ꢀH), 9.12
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(s, 1H, quinolinone C2ꢀH), 15.00 (bs, 1H, OH). Anal. (C22H18FNO5) C, H, F, N.
4-[1-(3-Methoxyphenyl)methyl-4(1H)-quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid Ethyl
Ester (10e). 93 %; 177ꢀ179 °C; toluene; IR ν 3146 (OH), 1729 (C=O ester), 1628 and 1603 (C=O
ꢀ1 1
ketone) cm . H NMR (DMSOꢀd ) δ 1.29 (t, 3H, CH CH ), 3.69 (s, 3H, CH ), 4.27 (q, 2H, CH CH ),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
2
3
3
2
3
5
.72 (s, 2H, CH ), 6.74 (d, 1H, benzene H), 6.84ꢀ6.89 (m, 2H, benzene H), 7.24 (t, 1H, benzene H), 7.49
2
(
m, 1H, quinolinone C6ꢀH), 7.69ꢀ7.75 (m, 2H, quinolinone C7ꢀH and C8ꢀH ), 8.01 (s, 1H, butenoate
C3ꢀH), 8.31 (m, 1H, quinolinone C5ꢀH), 9.10 (s, 1H, quinolinone C2ꢀH), 15.40 (bs, 1H, OH). Anal.
C22H21NO6) C, H, N.
-[1-(4-Chlorophenyl)methyl-4(1H)-quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid Ethyl
Ester (10f). 91 %; 174ꢀ176 °C; toluene; IR ν 3130 (OH), 1745 (C=O ester), 1643 and 1611 (C=O
(
4
ꢀ1
1
ketone) cm . H NMR (DMSOꢀd ) δ 1.30 (t, 3H, CH CH ), 4.29 (q, 2H, CH CH ), 5.77 (s, 2H, CH ),
6
2
3
2
3
2
7
.30ꢀ7.32 (m, 2H, benzene H), 7.40ꢀ7.42 (m, 2H, benzene H), 7.50 (m, 1H, quinolinone C6ꢀH), 7.67 (m,
1H, quinolinone C8ꢀH ), 7.73 (m, 1H, quinolinone C7ꢀH), 8.01 (s, 1H, butenoate C3ꢀH), 8.30 (m, 1H,
quinolinone C5ꢀH), 9.14 (s, 1H, quinolinone C2ꢀH), 15.60 (bs, 1H, OH). Anal. (C22H18ClNO5) C, H,
Cl, N.
4
-[1-(4-Hydroxyphenyl)methyl-4(1H)-quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid Ethyl
ꢀ1
Ester (10g). 64 %; 179ꢀ181 °C; methanol; IR ν 3141 (OH), 1733 (C=O ester), 1600 (C=O ketone) cm .
1
H NMR (DMSOꢀd ) δ 1.30 (t, 3H, CH CH ), 4.31 (q, 2H, CH CH ), 5.61 (s, 2H, CH ), 6.71ꢀ6.73 (m,
6
2
3
2
3
2
2
H, benzene H), 7.13ꢀ7.15 (m, 2H, benzene H), 7.50 (m, 1H, quinolinone C6ꢀH), 7.74 (m, 1H,
quinolinone C7ꢀH ), 7.79 (m, 1H, quinolinone C8ꢀH), 8.00 (s, 1H, butenoate C3ꢀH), 8.31 (m, 1H,
quinolinone C5ꢀH), 9.06 (s, 1H, quinolinone C2ꢀH), 9.51 (bs, 1H, OH phenole), 14.00 (bs, 1H, OH).
Anal. (C22H19NO6) C, H, N.
4
-[1-(4-Methoxyphenyl)methyl-4(1H)-quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid Ethyl
ꢀ1
Ester (10h). 90 %; 154ꢀ156 °C; methanol; IR ν 1734 (C=O ester), 1638 and 1602 (C=O ketone) cm .
1
H NMR (DMSOꢀd ) δ 1.28 (t, 3H, CH CH ), 3.69 (s, 3H, CH ), 4.29 (q, 2H, CH CH ), 5.67 (s, 2H,
6
2
3
3
2
3
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Journal of Medicinal Chemistry
CH ), 6.89ꢀ6.91 (m, 2H, benzene H), 7.24ꢀ7.26 (m, 2H, benzene H), 7.50 (m, 1H, quinolinone C6ꢀH),
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7
.71ꢀ7.79 (m, 2H, quinolinone C7ꢀH and C8ꢀH ), 8.00 (s, 1H, butenoate C3ꢀH), 8.31 (m, 1H,
quinolinone C5ꢀH), 9.10 (s, 1H, quinolinone C2ꢀH), 15.60 (bs, 1H, OH). Anal. (C22H21NO6) C, H, N.
-[1-(4-Nitrophenyl)methyl-4(1H)-quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid Ethyl
4
ꢀ1 1
Ester (10i). 88 %; 175ꢀ176 °C; toluene; IR ν 1718 (C=O ester), 1638 and 1607 (C=O ketone) cm . H
NMR (DMSOꢀd ) δ 1.30 (t, 3H, CH CH ), 4.32 (q, 2H, CH CH ), 5.94 (s, 2H, CH ), 7.46ꢀ7.54 (m, 3H,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
2
3
2
3
2
benzene H and quinolinone C6ꢀH), 7.60 (m, 1H, quinolinone C8ꢀH), 7.68 (m, 1H, quinolinone C7ꢀH),
8.02 (s, 1H, butenoate C3ꢀH), 8.18ꢀ8.20 (m, 2H, benzene H), 8.33 (m, 1H, quinolinone C5ꢀH), 9.18 (s,
1
H, quinolinone C2ꢀH), 15.60 (bs, 1H, OH). Anal. (C22H18N2O7) C, H, N.
-[1-(2,4-Difluorophenyl)methyl-4(1H)-quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid Ethyl
4
ꢀ1 1
Ester (10j). 96 %; 180 °C; methanol; IR ν (C=O ester), and (C=O ketone) cm . H NMR (DMSOꢀd ) δ
6
1
7
.35 (t, 3H, CH CH ), 4.36 (q, 2H, CH CH ), 5.84 (s, 2H, CH ), 7.10 (t, 1H, quinolinone C6ꢀH), 7.41ꢀ
2 3 2 3 2
.57 (m, 3H, benzene H), 7.76ꢀ7.83 (m, 2H, quinolinone C8ꢀH and quinolinone C7ꢀH), 8.05 (s, 1H,
butenoate C3ꢀH), 8.38 (d, 1H, quinolinone C5ꢀH), 9.14 (s, 1H, quinolinone C2ꢀH), 14.60 (bs, 1H, OH).
Anal. (C22H17F2NO5) C, H, F, N.
4-[1-(2,5-Difluorophenyl)methyl-4(1H)-quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid Ethyl
Ester (10k). 62 %; 160ꢀ163 °C; toluene; IR ν 3432 (OH), 1736 (C=O ester), 1637 and 1602 (C=O
ꢀ1
1
ketone) cm . H NMR (DMSOꢀd ) δ 1.35 (t, 3H, CH CH ), 4.35 (q, 2H, CH CH ), 5.86 (s, 2H, CH ),
6
2
3
2
3
2
7.18ꢀ7.41 (m, 3H, benzene H), 7.58 (t, 1H, quinolinone C6ꢀH), 7.75 (m, 1H, quinolinone C8ꢀH), 7.82
m, 1H, quinolinone C7ꢀH), 8.06 (s, 1H, butenoate C3ꢀH), 8.38 (m, 1H, quinolinone C5ꢀH), 9.16 (s, 1H,
(
quinolinone C2ꢀH), 14.60 (bs, 1H, OH). Anal. (C22H17F2NO5) C, H, F, N.
4-[1-(2,6-Difluorophenyl)methyl-4(1H)-quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid Ethyl
Ester (10l). 85 %; 172ꢀ173 °C; benzene/toluene; IR ν 3432 (OH), 1736 (C=O ester), 1637 and 1602
ꢀ1 1
(
C=O ketone) cm . H NMR (DMSOꢀd ) δ 1.28 (t, 3H, CH CH ), 4.30 (q, 2H, CH CH ), 5.84 (s, 2H,
6 2 3 2 3
CH ), 7.14ꢀ7.19 (t, 2H, benzene H), 7.43ꢀ7.53 (m, 2H, benzene H and quinolinone C6ꢀH), 7.68 (t, 1H,
2
quinolinone C7ꢀH), 7.80 (d, 1H, quinolinone C8ꢀH), 8.00 (s, 1H, butenoate C3ꢀH), 8.30 (m, 1H,
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Journal of Medicinal Chemistry
Page 24 of 44
quinolinone C5ꢀH), 9.11 (s, 1H, quinolinone C2ꢀH), 15.00 (bs, 1H, OH). Anal. (C22H17F2NO5) C, H,
F, N.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4-[1-(3,4-Difluorophenyl)methyl-4(1H)-quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid Ethyl
Ester (10m). 100%; 175ꢀ176 °C; benzene; IR ν 3432 (OH), 1719 (C=O ester), 1648 and 1612 (C=O
ꢀ1 1
ketone) cm . H NMR (DMSOꢀd ) δ 1.35 (t, 3H, CH CH ), 4.38 (q, 2H, CH CH ), 5.80 (s, 2H, CH ),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
2
3
2
3
2
7
.19 (t, 1H, quinolinone C6ꢀH), 7.41ꢀ7.57 (m, 3H, benzene H), 7.72ꢀ7.80 (m, 2H, quinolinone C8ꢀH and
quinolinone C7ꢀH), 8.06 (s, 1H, butenoate C3ꢀH), 8.38 (d, 1H, quinolinone C5ꢀH), 9.17 (s, 1H,
quinolinone C2ꢀH), 14.60 (bs, 1H, OH). Anal. (C22H17F2NO5) C, H, F, N.
4
-[1-(3,5-Difluorophenyl)methyl-4(1H)-quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid Ethyl
Ester (10n). 90 %; 174ꢀ175 °C; methanol; IR ν 3432 (OH), 1736 (C=O ester), 1642 and 1626 (C=O
ꢀ1 1
ketone) cm . H NMR (DMSOꢀd ) δ 1.34 (t, 3H, CH CH ), 4.36 (q, 2H, CH CH ), 5.84 (s, 2H, CH ),
6
2
3
2
3
2
7
1
.05ꢀ7.23 (m, 3H, benzene H), 7.58 (t, 1H, quinolinone C6ꢀH), 7.69 (d, 1H, quinolinone C8ꢀH), 7.79 (t,
H, quinolinone C7ꢀH), 8.04 (s, 1H, butenoate C3ꢀH), 8.38 (m, 1H, quinolinone C5ꢀH), 9.16 (s, 1H,
quinolinone C2ꢀH), 14.60 (bs, 1H, OH). Anal. (C22H17F2NO5) C, H, F, N.
-[1-(2,4-Dichlorophenyl)methyl-4(1H)-quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid Ethyl
4
ꢀ1 1
Ester (10o). 94 %; 200ꢀ201 °C; toluene; IR ν 1735 (C=O ester), 1641 and 1632 (C=O ketone) cm . H
NMR (DMSOꢀd ) δ 1.34 (t, 3H, CH CH ), 4.35 (q, 2H, CH CH ), 5.84 (s, 2H, CH ), 6.99 (d, 1H,
6
2
3
2
3
2
benzene H), 7.37 (dd, 1H, benzene H), 7.50ꢀ7.61 (m, 2H, quinolinone C6ꢀH and C8ꢀH), 7.78ꢀ7.83 (m,
2H, benzene H and quinolinone C7ꢀH), 8.06 (s, 1H, butenoate C3ꢀH), 8.41 (dd, 1H, quinolinone C5ꢀH),
9
.13 (s, 1H, quinolinone C2ꢀH), 15.00 (bs, 1H, OH). Anal. (C22H17Cl2NO5) C, H, Cl, N.
-[1-(2,6-Dichlorophenyl)methyl-4(1H)-quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid Ethyl
4
ꢀ1 1
Ester (10p). 82 %; 184ꢀ186 °C; toluene; IR ν (C=O ester), and (C=O ketone) cm . H NMR (DMSOꢀ
d ) δ 1.32 (t, 3H, CH CH ), 4.35 (q, 2H, CH CH ), 5.90 (s, 2H, CH ), 7.61ꢀ7.67 (m, 2H, benzene H),
6
2
3
2
3
2
7
.73ꢀ7.75 (m, 2H, quinolinone C6ꢀH and C8ꢀH), 7.94 (t, 1H, benzene H), 8.03ꢀ8.04 (m, 2H, quinolinone
C7ꢀH and butenoate C3ꢀH), 8.42 (d, 1H, quinolinone C5ꢀH), 8.46 (s, 1H, quinolinone C2ꢀH), 15.00 (bs,
H, OH). Anal. (C22H17Cl2NO5) C, H, Cl, N.
1
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Journal of Medicinal Chemistry
4
-[1-(2,4-Dimethylphenyl)methyl-4(1H)-quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic
Acid
1
2
ꢀ1
Ethyl Ester (10q). 95 %; 181 °C; toluene; IR ν 1727 (C=O ester), 1638 and 1600 (C=O ketone) cm .
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
H NMR (DMSOꢀd ) δ 1.32 (t, 3H, CH CH ), 2.24 (s, 3H, CH ), 2.36 (s, 3H, CH ), 4.32 (q, 2H,
6
2
3
3
3
CH CH ), 5.69 (s, 2H, CH ), 6.58 (d, 1H, benzene H), 6.90 (d, 1H, benzene H), 7.12 (s, 1H, benzene H),
2
3
2
7
.53ꢀ7.61 (m, 2H, quinolinone C6ꢀH and C8ꢀH), 7.76 (m, 1H, quinolinone C7ꢀH), 8.05 (s, 1H,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
butenoate C3ꢀH), 8.37 (d, 1H, quinolinone C5ꢀH), 8.94 (s, 1H, quinolinone C2ꢀH), 15.00 (bs, 1H, OH).
Anal. (C24H23NO5) C, H, N.
4-[1-(3,5-Dimethylphenyl)methyl-4(1H)-quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic
Acid
ꢀ1
Ethyl Ester (10r). 89 %; 181 °C; methanol; IR ν 1716 (C=O ester), 1625 and 1601 (C=O ketone) cm .
1
H NMR (DMSOꢀd ) δ 1.35 (t, 3H, CH CH ), 2.24 (s, 6H, CH ), 4.35 (q, 2H, CH CH ), 5.72 (s, 2H,
6
2
3
3
2
3
CH ), 6.89ꢀ6.96 (m, 3H, benzene H), 7.54 (t, 1H, quinolinone C6ꢀH), 7.71ꢀ7.77 (m, 2H, quinolinone
2
C7ꢀH and C8ꢀH), 8.05 (s, 1H, butenoate C3ꢀH), 8.37 (d, 1H, quinolinone C5ꢀH), 9.13 (s, 1H,
quinolinone C2ꢀH), 15.00 (bs, 1H, OH). Anal. (C24H23NO5) C, H, N.
4
-[1-(3-Chloro-2-fluorophenyl)methyl-4(1H)-quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid
Ethyl Ester (10s). 79 %; 167ꢀ169 °C; benzene; IR ν 1746 (C=O ester), 1648 and 1601 (C=O ketone)
ꢀ1 1
cm . H NMR (DMSOꢀd ) δ 1.32 (t, 3H, CH CH ), 4.32 (q, 2H, CH CH ), 5.89 (s, 2H, CH ), 7.13ꢀ7.18
6
2
3
2
3
2
(
m, 2H, benzene H and quinolinone C6ꢀH), 7.55ꢀ7.77 (m, 4H, quinolinone C7ꢀH, C8ꢀH and benzene H),
.03 (s, 1H, butenoate C3ꢀH), 8.35 (d, 1H, quinolinone C5ꢀH), 9.13 (s, 1H, quinolinone C2ꢀH), 15.00
(bs, 1H, OH). Anal. (C22H17ClFNO5) C, H, Cl, F, N.
-[1-(3-Chloro-4-fluorophenyl)methyl-4(1H)-quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid
Ethyl Ester (10t). 84 %; 160ꢀ161 °C; methanol; IR ν 1746 (C=O ester), 1648 and 1601 (C=O ketone)
8
4
ꢀ1 1
cm . H NMR (DMSOꢀd ) δ 1.32 (t, 3H, CH CH ), 4.32 (q, 2H, CH CH ), 5.77 (s, 2H, CH ), 7.30ꢀ7.72
6
2
3
2
3
2
(m, 6H, quinolinone C6ꢀH, C7ꢀH, C8ꢀH and benzene H), 8.03 (s, 1H, butenoate C3ꢀH), 8.33 (d, 1H,
quinolinone C5ꢀH), 9.15 (s, 1H, quinolinone C2ꢀH), 15.00 (bs, 1H, OH). Anal. (C22H17ClFNO5) C, H,
Cl, F, N.
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8
-Chloro-4-[1-(3-chloro-4-fluorophenyl)methyl-4(1H)-quinolinon-3-yl]-2-hydroxy-4-oxo-2-
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
butenoic Acid Ethyl Ester (10u). 70 %; 166ꢀ167 °C; ethanol; IR ν 1746 (C=O ester), 1648 and 1601
ꢀ1 1
(C=O ketone) cm . H NMR (DMSOꢀd ) δ 1.32 (t, 3H, CH CH ), 4.33 (q, 2H, CH CH ), 6.10 (s, 2H,
6
2
3
2
3
CH ), 7.11 (m, 1H, quinolinone C6ꢀH), 7.34ꢀ7.50 (m, 3H, benzene H), 7.80ꢀ7.85 (m, 2H, quinolinone
2
C7ꢀH and C8ꢀH) 7.95 (s, 1H, butenoate C3ꢀH), 8.38 (d, 1H, quinolinone C5ꢀH), 8.91 (s, 1H,
quinolinone C2ꢀH), 15.00 (bs, 1H, OH). Anal. (C22H16Cl2FNO5) C, H, Cl, F, N.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
-[7-Chloro-1-(2-fluorophenyl)methyl-4(1H)-quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid
ꢀ
1
Ethyl Ester (10w). 85 %; 175 °C; methanol; IR ν 1721 (C=O ester), 1627 and 1585 (C=O ketone) cm .
1
H NMR (DMSOꢀd ) δ 1.35 (t, 3H, CH CH ), 4.36 (q, 2H, CH CH ), 5.88 (s, 2H, CH ), 7.22ꢀ7.48 (m,
6
2
3
2
3
2
4
H, benzene H), 7.60 (t, 1H, quinolinone C6ꢀH), 7.90 (m, 2H, quinolinone C7ꢀH and C8ꢀH), 8.00 (s,
1H, butenoate C3ꢀH), 8.35 (d, 1H, quinolinone C5ꢀH), 9.11 (s, 1H, quinolinone C2ꢀH), 15.00 (bs, 1H,
OH). Anal. (C22H17FClNO5) C, H, N, F, Cl.
2
-Hydroxy-4-oxo-4-(4-oxo-1-(4-phenylbutyl)-1,4-dihydroquinolin-3-yl)but-2-enoic Acid Ethyl
ꢀ
1 1
Ester (10x). 95 %; 88ꢀ90 °C; cyclohxane; IR ν 1725 (C=O ester), 1630 and 1604 (C=O ketone) cm . H
NMR (DMSOꢀd ) δ 1.24 (t, 3H, CH CH ), 1.60 (m, 2H, buthyl H), 1.75 (m, 2H, buthyl H), 2.58 (m, 2H,
6
2
3
buthyl H), 4.25 (m, 2H, buthyl H), 4.45 (q, 2H, CH CH ), 7.11ꢀ7.25 (m, 5H, benzene H and quinolinone
2
3
C6ꢀH), 7.49 (m, 1H, quinolinone C8ꢀH), 7.74ꢀ7.78 (m, 2H, butenoate C3ꢀH and quinolinone C7ꢀH),
.30 (d, 1H, quinolinone C5ꢀH), 8.76 (s, 1H, quinolinone C2ꢀH), 15.00 (bs, 1H, OH). Anal.
(C25H25NO5) C, H, N.
-Hydroxy-4-(1-(naphthalen-2-ylmethyl)-4-oxo-1,4-dihydroquinolin-3-yl)-4-oxobut-2-enoic Acid
Ethyl Ester (10y). 87 %; 153ꢀ154 °C; DMF/H O; IR ν 1734 (C=O ester), 1629 and 1603 (C=O ketone)
8
2
2
ꢀ
1 1
cm . H NMR (DMSOꢀd ) δ 1.28 (t, 3H, CH CH ), 4.32 (q, 2H, CH CH ), 5.94 (s, 2H, CH ), 7.49ꢀ7.56
6
2
3
2
3
2
(
m, 4H, naphthyl H), 7.66ꢀ7.75 (m, 2H, naphthyl H), 7.79 (s, 1H, naphthyl H), 7.84ꢀ7.92 (m, 3H,
quinolinone C6ꢀH, C7ꢀH and C8ꢀH), 8.04 (s, 1H, butenoate C3ꢀH), 8.32 (d, 1H, quinolinone C5ꢀH),
9
.20 (s, 1H, quinolinone C2ꢀH), 15.00 (bs, 1H, OH). Anal. (C26H21NO5) C, H, N.
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Journal of Medicinal Chemistry
2
-hydroxy-4-oxo-4-(4-oxo-1-(pyridin-4-ylmethyl)-1,4-dihydroquinolin-3-yl)but-2-enoic
Acid
1
2
ꢀ1 1
Ethyl Ester (10z). 66 %; 168ꢀ169 °C; DMF/H O; IR ν 1717 (C=O ester), 1645 (C=O ketone) cm . H
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
NMR (DMSOꢀd ) δ 1.28 (t, 3H, CH CH ), 4.28 (q, 2H, CH CH ), 5.82 (s, 2H, CH ), 7.08 (s, 2H,
6
2
3
2
3
2
picoline H), 7.49 (t, 1H, quinolinone C6ꢀH), 7.55 (d, 1H, quinolinone C8ꢀH), 7.79 (t, 1H, quinolinone
C7ꢀH), 7.95 (s, 1H, butenoate C3ꢀH), 8.32 (m, 1H, quinolinone C5ꢀH), 8.50 (s, 2H, picoline H), 9.10 (s,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
H, quinolinone C2ꢀH), 15.50 (bs, 1H, OH). Anal. (C21H18N2O5) C, H, N.
-hydroxy-4-oxo-4-(4-oxo-1-(quinolin-2-ylmethyl)-1,4-dihydroquinolin-3-yl)but-2-enoic
2
Acid
ꢀ1
Ethyl Ester (10aa). 100 %; 237ꢀ238 °C; DMF/H O; IR ν 1712 (C=O ester), 1634 (C=O ketone) cm .
2
1
H NMR (DMSOꢀd ) δ 1.16 (t, 3H, CH CH ), 4.30 (q, 2H, CH CH ), 5.98 (s, 2H, CH ), 7.11ꢀ7.94 (m,
6
2
3
2
3
2
9
H, quinolinone C6ꢀH, C7ꢀH, C8ꢀH, quinoline H), 8.25ꢀ8.37 (m, 2H, quinolinone C5ꢀH and quinoline
H), 9.04 (s, 1H, quinolinone C2ꢀH), 15.00 (bs, 1H, OH). Anal. (C25H20N2O5) C, H, N.
General Procedure for the Synthesis of Diketo Acids 11a-aa. A mixture of 1 N NaOH (6.5 mL)
and the appropriate ester 10a-aa (1.3 mmol) in 1:1 THF/methanol (12 mL) was stirred at room
temperature for 40 min and then poured onto crushed ice. The aqueous layer was separated and treated
with 1 N HCl until pH 3 was reached, and the formed yellow solid was collected by filtration, then
washed with water, hot dry ethanol, and light petroleum ether to afford pure acids 11a-aa. Yield (%),
1
melting point (°C), recrystallization solvent, IR, H NMR, and analytical data for each of the following
compounds are reported.
4-[1-Phenylmethyl-4(1H)-quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid (11a). 92 %; 141ꢀ
ꢀ1 1
1
42 °C; DMF/H O; IR ν 3424 (OH), 1726 (C=O acid), 1625 and 1603 (C=O ketone) cm . H NMR
2
(
DMSOꢀd ) δ 5.75 (s, 2H, CH ), 7.22ꢀ7.35 (m, 5H, benzene H), 7.44 (m, 1H, quinolinone C6ꢀH), 7.65ꢀ
6 2
7.69 (m, 2H, quinolinone C7ꢀH and C8ꢀH ), 7.90 (s, 1H, butenoate C3ꢀH), 8.30 (m, 1H, quinolinone C5ꢀ
H), 9.08 (s, 1H, quinolinone C2ꢀH), 14.00ꢀ16.00 (bs, 2H, OH). Anal. (C19H15NO5) C, H, N.
4
-[1-(2-Fluorophenyl)methyl-4(1H)-quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid (11b). 83
ꢀ1 1
%
; 172ꢀ174 °C; toluene; IR ν 3200 (OH), 1748 (C=O acid), 1621 (C=O ketone) cm . H NMR (DMSOꢀ
d ) δ 5.81 (s, 2H, CH ), 7.14ꢀ7.27 (m, 3H, benzene H), 7.35 (m, 1H, benzene H), 7.50 (m, 1H,
6
2
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Journal of Medicinal Chemistry
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quinolinone C6ꢀH), 7.70ꢀ7.73 (m, 2H, quinolinone C7ꢀH and C8ꢀH ), 7.95 (s, 1H, butenoate C3ꢀH),
.31 (d, 1H, quinolinone C5ꢀH), 9.08 (s, 1H, quinolinone C2ꢀH), 14.00ꢀ16.00 (bs, 2H, OH). Anal.
(C20H14FNO5) C, H, F, N.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
8
4
-[1-(2-Methoxyphenyl)methyl-4(1H)-quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid (11c).
ꢀ
9
4 %; 172ꢀ175 °C; washed with dry ethanol; IR ν 3149 (OH), 1742 (C=O acid), 1620 (C=O ketone) cm
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
.
H NMR (DMSOꢀd ) δ 3.82 (s, 3H, CH ), 5.61 (s, 2H, CH ), 6.89 (t, 1H, benzene H), 7.01ꢀ7.12 (m,
6 3 2
2
H, benzene H), 7.0 (t, 1H, benzene H), 7.42ꢀ7.49 (m, 1H, quinolinone C6ꢀH), 7.71ꢀ7.74 (m, 2H,
quinolinone C7ꢀH and C8ꢀH ), 7.94 (s, 1H, butenoate C3ꢀH), 8.30 (d, 1H, quinolinone C5ꢀH), 9.02 (s,
H, quinolinone C2ꢀH), 14.00ꢀ16.00 (bs, 2H, OH). Anal. (C21H17NO6) C, H, N.
-[1-(3-Fluorophenyl)methyl-4(1H)-quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid (11d). 60
%; 158ꢀ159 °C; washed with hot dry ethanol; IR ν 3392 (OH), 1719 (C=O acid), 1667 and 1620 (C=O
1
4
ꢀ1
1
ketone) cm . H NMR (DMSOꢀd ) δ 5.76 (s, 2H, CH ), 7.05ꢀ7.18 (m, 3H, benzene H), 7.36 (m, 1H,
6
2
benzene H), 7.47 (m, 1H, quinolinone C6ꢀH), 7.66ꢀ7.68 (m, 2H, quinolinone C7ꢀH and C8ꢀH ), 7.88 (s,
1H, butenoate C3ꢀH), 8.30 (d, 1H, quinolinone C5ꢀH), 9.07 (s, 1H, quinolinone C2ꢀH), 14.00ꢀ16.00 (bs,
2
H, OH). Anal. (C20H14FNO5) C, H, F, N.
4-[1-(3-Methoxyphenyl)methyl-4(1H)-quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid (11e).
ꢀ
8
0 %; 179ꢀ181 °C; washed with dry ethanol; IR ν 3002 (OH), 1748 (C=O acid), 1602 (C=O ketone) cm
1
1
.
H NMR (DMSOꢀd ) δ 3.70 (s, 3H, CH ), 5.72 (s, 2H, CH ), 6.75 (d, 1H, benzene H), 6.84ꢀ6.89 (m,
6 3 2
2H, benzene H), 7.24 (t, 1H, benzene H), 7.49 (m, 1H, quinolinone C6ꢀH), 7.69ꢀ7.74 (m, 2H,
quinolinone C7ꢀH and C8ꢀH ), 7.98 (s, 1H, butenoate C3ꢀH), 8.30 (d, 1H, quinolinone C5ꢀH), 9.08 (s,
1
H, quinolinone C2ꢀH), 13.90 (bs, 1H, OH), 15.60 (bs, 1H, OH). Anal. (C21H17NO6) C, H, N.
4-[1-(4-Chlorophenyl)methyl-4(1H)-quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid (11f). 96
%; 177ꢀ179 °C; washed with hot dry ethanol; IR ν 3250 (OH), 1741 (C=O acid), 1643 and 1611 (C=O
ꢀ1 1
ketone) cm . H NMR (DMSOꢀd ) δ 5.76 (s, 2H, CH ), 7.25ꢀ7.31 (m, 2H, benzene H), 7.39ꢀ7.41 (m,
6
2
2
H, benzene H), 7.49 (m, 1H, quinolinone C6ꢀH), 7.65ꢀ7.73 (m, 2H, quinolinone C8ꢀH and C7ꢀH), 7.96
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Journal of Medicinal Chemistry
(
s, 1H, butenoate C3ꢀH), 8.30 (d, 1H, quinolinone C5ꢀH), 9.11 (s, 1H, quinolinone C2ꢀH), 14.00ꢀ16.00
bs, 2H, OH). Anal. (C20H14ClNO5) C, H, Cl, N.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
4-[1-(4-Hydroxyphenyl)methyl-4(1H)-quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid (11g).
ꢀ
7
7 %; 207ꢀ208 °C; washed with dry ethanol; IR ν 3135 (OH), 1718 (C=O acid), 1600 (C=O ketone) cm
1
1
.
H NMR (DMSOꢀd ) δ 5.59 (s, 2H, CH ), 6.71ꢀ6.73 (m, 2H, benzene H), 7.11ꢀ7.13 (m, 2H, benzene
6 2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H), 7.47 (m, 1H, quinolinone C6ꢀH), 7.67ꢀ7.78 (m, 2H, quinolinone C8ꢀH and C7ꢀH), 7.92 (s, 1H,
butenoate C3ꢀH), 8.29 (d, 1H, quinolinone C5ꢀH), 9.00 (s, 1H, quinolinone C2ꢀH), 9.50 (bs, 1H, OH
phenole), 14.00ꢀ16.00 (bs, 2H, OH). Anal. (C20H15NO5) C, H, N.
4
-[1-(4-Methoxyphenyl)methyl-4(1H)-quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid (11h).
1
00 %; 152 °C; washed with dry ethanol; IR ν 3400 (OH), 1726 (C=O acid), 1620 and 1611 (C=O
ꢀ1 1
ketone) cm . H NMR (DMSOꢀd ) δ 3.69 (s, 3H, CH ), 5.66 (s, 2H, CH ), 6.88ꢀ6.90 (m, 2H, benzene
6
3
2
H), 7.24ꢀ7.26 (m, 2H, benzene H), 7.50 (m, 1H, quinolinone C6ꢀH), 7.70ꢀ7.78 (m, 2H, quinolinone C7ꢀ
H and C8ꢀH ), 7.93 (s, 1H, butenoate C3ꢀH), 8.30 (d, 1H, quinolinone C5ꢀH), 9.07 (s, 1H, quinolinone
C2ꢀH), 14.00ꢀ16.00 (bs, 2H, OH). Anal. (C21H17NO6) C, H, N.
4
-[1-(4-Nitrophenyl)methyl-4(1H)-quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid (11i). 90
%
; 230ꢀ232 °C; washed with dry ethanol; IR ν 3100 (OH), 1731 (C=O acid), 1634 and 1602 (C=O
ꢀ1 1
ketone) cm . H NMR (DMSOꢀd ) δ 5.87 (s, 2H, CH ), 7.45ꢀ7.52 (m, 3H, benzene H and quinolinone
6
2
C6ꢀH), 7.58 (m, 1H, quinolinone C8ꢀH), 7.68 (m, 2H, quinolinone C7ꢀH), 7.92 (s, 1H, butenoate C3ꢀH),
8.18ꢀ8.20 (m, 2H, benzene H), 8.32 (d, 1H, quinolinone C5ꢀH), 9.12 (s, 1H, quinolinone C2ꢀH), 14.00ꢀ
1
6.00 (bs, 2H, OH). Anal. (C20H14N2O7) C, H, N.
-[1-(2,4-Difluorophenyl)methyl-4(1H)-quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid (11j).
4
ꢀ1 1
84 %; 182 °C; ethanol; IR ν 3400 (OH), 1729 (C=O acid), and 1620 and 1608 (C=O ketone) cm . H
NMR (DMSOꢀd ) δ 5.84 (s, 2H, CH ), 7.11 (t, 1H, quinolinone C6ꢀH), 7.37ꢀ7.40 (m, 2H, benzene H),
6
2
7
.57 (d, 1H, quinolinone C8ꢀH), 7.77ꢀ7.81 (m, 2H, benzene H and quinolinone C7ꢀH), 8.01 (s, 1H,
butenoate C3ꢀH), 8.37 (d, 1H, quinolinone C5ꢀH), 9.13 (s, 1H, quinolinone C2ꢀH), 14.60 (bs, 1H, OH),
5.90 (bs, 1H, OH). Anal. (C20H13F2NO5) C, H, F, N.
1
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