Structural and theoretical studies of [6-bromo-1-(4-fluorophenylmethyl)-4(1H)-quinolinon-3-yl)]-4-hydroxy-2-oxo-3-butenoic acid as HIV-1 integrase inhibitor

Article abstract of DOI:10.1016/j.bmcl.2009.06.044

Ethyl [6-bromo-1-(4-fluorophenylmethyl)-4(1H)-quinolinon-3-yl]-4-hydroxy-2-oxo-3-butenoate 1 and [6-bromo-1-(4-fluorophenylmethyl)-4(1H)-quinolinon-3-yl)]-4-hydroxy-2-oxo-3-butenoic acid 2 were synthesized as potential HIV-1 integrase inhibitors and evalu

Full text of DOI:10.1016/j.bmcl.2009.06.044

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4806–4809
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structural and theoretical studies of [6-bromo-1-(4-fluorophenylmethyl)-
4(1H)-quinolinon-3-yl)]-4-hydroxy-2-oxo-3-butenoïc acid as HIV-1 integrase
inhibitor
Pierre Vandurm a, , , Christine Cauvin a, , Allan Guiguen b, , Benoît Georges c, Kiet Le Van c,
*
Valérie Martinelli b, Christelle Cardona b, Gladys Mbemba d, Jean-François Mouscadet d, László Hevesi c,à
,
Carine Van Lint b,à, Johan Wouters a,à
a Laboratoire de Chimie Biologique Structurale, Facultés Universitaires Notre-Dame de la Paix (FUNDP), B-5000 Namur, Belgium
b Laboratoire de Virologie Moléculaire, Institut de Biologie et de Médecine Moléculaires, Université Libre de Bruxelles (ULB), B-6041 Gosselies, Belgium
c Laboratoire de Chimie des Matériaux Organiques, Facultés Universitaires Notre-Dame de la Paix (FUNDP), B-5000 Namur, Belgium
d Laboratoire de Biotechnologies et de Pharmacologie Génétique Appliquée, CNRS, Ecole Normale Supérieure de Cachan (ENS), F-94235 Cachan, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Ethyl [6-bromo-1-(4-fluorophenylmethyl)-4(1H)-quinolinon-3-yl]-4-hydroxy-2-oxo-3-butenoate 1 and
[6-bromo-1-(4-fluorophenylmethyl)-4(1H)-quinolinon-3-yl)]-4-hydroxy-2-oxo-3-butenoïc acid 2 were
synthesized as potential HIV-1 integrase inhibitors and evaluated for their enzymatic and antiviral activ-
ity, acidic compound 2 being more potent than ester compound 1. X-ray diffraction analyses and theoret-
ical calculations show that the diketoacid chain of compound 2 is preferentially coplanar with the
quinolinone ring (dihedral angle of 0–30°). Docking studies suggest binding modes in agreement with
structure–activity relationships.
Received 6 May 2009
Revised 8 June 2009
Accepted 11 June 2009
Available online 14 June 2009
Keywords:
HIV-1
Integrase inhibitors
Quinolinone
b-Diketo
Ó 2009 Elsevier Ltd. All rights reserved.
HIV integrase (IN) is currently recognized as an attractive target
against AIDS. It catalyzes the integration of reverse transcribed vir-
al DNA into host cell DNA through a two-step, metal-dependent
process.1 Step one, known as 30 processing (30-P), effects cleavage
of two nucleotides from the two 30 ends of double stranded viral
DNA. The second step, strand transfer (ST), integrates the viral
DNA into the host cell DNA through phosphodiester transesterifi-
cation reactions. Integrase is an especially attractive target for
HIV therapy because the enzyme is needed for viral infectivity
and there are no known host cell counterparts.2
In the past several years, compounds with diverse structural
features have been reported as IN inhibitors.3 Among those IN
inhibitors, compounds containing the b-diketoacid (DKA) moiety
were extensively studied. S13604 (Fig. 1) has entered clinical trials
but its development was stopped due to pharmacokinetic prob-
lems. More recently, other promising DKA-based derivatives have
reached advanced clinical development: raltegravir (Fig. 1) is the
O
O
Cl
N
O
O
NH
N
O
N
N
N
F
HN
NH
S1360
5-CITEP
N
F
O
O
N
O
Cl
O
OH
N
OH
H
N
NH
O
N
H
O
F
N
O
OH
raltegravir
elvitegravir
Figure 1. Structures of DKA-based integrase inhibitors.
* Corresponding author. Tel.: +32 81724569; fax: +32 81725440.
E-mail address: pierre.vandurm@fundp.ac.be (P. Vandurm).
These authors contributed equally to this work.
These authors co-directed the present study (e-mail address: laszlo.hevesi@
fundp.ac.be, cvlint@ulb.ac.be, johan.wouters@fundp.ac.be). CVL is Directeur de
Recherches of the Belgian ‘Fonds de la Recherche Scientifique-FNRS’ (FRS-FNRS).
first FDA approved IN inhibitor (since october 2007) whereas elvi-
tegravir (Fig. 1) is presently in phase III clinical trials.5 All these IN
inhibitors contain a chelating moiety involved in sequestration of
divalent metal cofactors at the enzyme catalytic site.6 A DKA
à
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.06.044

P. Vandurm et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4806–4809
4807
O
N
O
O
F
O
O
O
N
O
O
HO
OH
Br
OR
O
O
O
F
Compound A
1
2
R = Et
R = H
Figure 2. Structures of quinolinone reference compound A8 and 6-bromine
analogues 1 and 2.
compound, the 5-CITEP (Fig. 1), was cocrystallized with the cata-
lytic core domain of the enzyme.7 X-ray crystal structure of the
complex showed that 5-CITEP binds in the middle of the active site
of the enzyme, lying between the three catalytic acidic residues,
D64, D116 and E152, in the vicinity of the active site metal ion.
Recently, some DKA derivatives containing the quinolinone
scaffold have been reported.8,9 Among these, compound A (Fig. 2)
displays a good anti-integrase activity for both 30-P and ST (IC50
Figure 3. Crystal structure of compound 2 (cocrystallized with DMSO) showing the
atom-numbering and hydrogen-bonding scheme. Displacement ellipsoids are
drawn at the 30% level.19 Selected distances (Å) and angles (°) are also reported
for the keto–enol moiety.
(30-P) = 0.44
lM; IC50 (ST) = 0.017 lM). Based on this quinolinone
series, we have synthesized 6-bromide analogues with an ester
(compound 1, Fig. 2) or an acidic group (compound 2, Fig. 2) on
the diketo moiety. In this Letter, we disclose the synthesis, anti-
integrase and antiviral data of these compounds. We discuss also
on conformation and potential binding modes of compound 2 from
crystallographic analyses and modelling studies.
uncomplexed derivative using X-ray diffraction (XRD) and theoret-
ical calculations.
Crystal structure of 2 is shown in Figure 3. This compound
cocrystallized with DMSO, both molecules being linked by hydro-
gen bonds between carboxylic acid group and DMSO oxygen
O33a. The DKA chain and the quinolinone ring of compound 2 are
nearly coplanar with dihedral angle C4–C3–C18–O25 of 177°. The
orientation of the diketo moiety with respect to the quinolinone
ring is governed by intramolecular C2–H2ꢀꢀꢀO25 and C19–H19ꢀꢀꢀO26
hydrogen bonds. In this solid-state structure, the equilibrium be-
tween b-diketo and keto enol forms is displaced in favour of the
keto–enol isomer, the hydrogens on C19 and O25 being clearly iden-
tified by Fourier difference. However, it is difficult to discriminate
between the two potential keto–enol tautomers. Indeed, H25 is in-
volved in a strong hydrogen bond with O24 and the O25–H25 dis-
tance (1.06 Å) is greater than commonly observed for O–H bonds
(0.82 Å). Furthermore, both carbonyl (C18–O25 and C20–O24) and
carbon–carbon (C18–C19 and C19–C20) bond distances are similar
and intermediate between standard single and double bonds,
indicative of strong electronic delocalization within this keto–enol
moiety.
Synthesis of compounds 1 and 2 was achieved by adapting a
method previously reported by Di Santo et al.8 (see Supplementary
data). The requisite intermediate 3-acetyl-6-bromo-4(1H)-quinoli-
none 5 was obtained by reaction of commercially available
4-bromoaniline with ethylethoxymethyleneacetoacetate and sub-
sequent thermic cyclization in diphenyl ether. 5 was then N-alkyl-
ated with 4-fluorobenzyl bromide in alkaline medium to give
compound 6 which then underwent Claisen condensation with
diethyl oxalate in the presence of sodium ethoxide to afford diketo
ester 1. Alkaline hydrolysis of 1 led to 2. Compounds 1 and 2 ob-
tained, respectively, in 74% and 93%, were fully characterized by
IR, 1H NMR, 13C NMR and MS.
Both compounds were evaluated for their anti-integrase,10 anti-
viral activity11 and cytotoxicity (Table 1). Compound 2 exhibited
better inhibitory activity against IN than compound 1.
The antiviral activities are correlated with the anti-integrase
activities, compound
2 displaying an EC50 of 4.0 lM versus
17 M for compound 1 (TZM-bl cells). These data are in agreement
l
with previous results showing higher potency for DKA com-
pounds.12 Concerning anti-integrase activity, both compounds
showed a much better selectivity for the strand transfer versus
30-processing reaction. Interestingly, both derivatives have low
cytotoxicity (CC50 > 1 mM) against HIV-1 infected TZM-bl cells.
In order to predict the potential binding mode of compound 2 in
the enzyme, we first performed conformational analyses on the
In order to further explore the preferential relative orientation
of the DKA chain and quinolinone ring, we performed a conforma-
tional scan on the C19–C18–C3–C2 torsion angle (T1) using a trun-
cated molecule (without bromine atom and p-F-phenyl moiety)
(Fig. 4). The scan was realized considering both potential keto–enol
tautomers (a and b, Fig. 4).
The quantum chemistry calculations were carried out at the
**
pbe1pbe level of theory with 6-31G basis set using GAUSSIAN 03
software.13 At each step of the scan (from 0° to 180° with an incre-
ment of 15°), the conformation was optimized and the energy eval-
Table 1
IN inhibition data (IC50), antiviral activity (EC50, TZM-b cells) and cytotoxicity (CC50
)
of compounds 1 and 2
uated. Figure 4 depicts
conformation and global energy minimum) as a function of torsion
angle T1. Both tautomers present a similar energetic profile (
DE (energy difference between considered
Compound IC50 (30-P)a
(l
M) IC50 (ST)b
(
l
M) EC50 (lM) CC50 (l
M) SIe
>59
>250
c
d
D
E
1
2
78
1.6
0.12
0.03
17
4.0
>1000
>1000
between tautomers less than 1 kcal/mol), the most stable confor-
mation being observed for a torsion angle of 180°. Interestingly,
this conformer corresponds to the crystal structure of compound
2, showing correlations between theoretical and experimental re-
sults. Another local minimum is observed for a T1 value of 30°
a
b
c
Inhibitory concentration 50% for 30-processing.10
Inhibitory concentration 50% for strand transfer.10
Effective concentration 50% (TZM-bl cells).11
Cytotoxic concentration 50% (TZM-bl cells).
d
e
Selectivity index CC50/EC50
.
(DE of 7–8 kcal/mol). The conformer with T1 of 0° is more ener-

4808
P. Vandurm et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4806–4809
30° and 180° were considered for further modelling studies of
compound 2.
tautomer a
tautomer b
O
OH O
O
O
OH
In an attempt to predict the binding mode of compound 2 into
the integrase catalytic core domain, docking studies14 were per-
formed using the X-ray structure of the protein (PDB code: 1BL3,
subunit C).15 Figure 5a depicts the catalytic site located at the sur-
face of the enzyme and composed of three main pockets and Mg2+
metal ion. In the first hydrophilic pocket, the two conserved resi-
dues D64 and D116 chelate the Mg2+ cofactor essential for the cat-
alytic activity of the enzyme. The second pocket is mainly
constituted by hydrophilic residues known to be critical for viral
DNA ends binding (K156, K159)16 or to be associated with resis-
tance to strand transfer inhibitors (N155).17 The third hydrophobic
pocket including P142, Y143, N144, P145, Q146, S147, Q148, G149
and Q62 residues, forms a catalytic loop involved in the binding of
the host DNA and viral DNA ends.18
Docking studies of compound 2 were performed on both tau-
tomers using the most stable conformations identified by theoret-
ical calculations (T1 of 30° and 180°).
Best binding modes were obtained for compound with b keto–
enol tautomeric form. Two predominant poses with DKA moiety
close to Mg2+ metal ion were identified (Fig. 5b). In the first one
(Fig. 5b left, T1 equal to 30°), the Mg2+ is chelated in a bidentate
manner by the two oxygens of the diketo moiety (with D64 and
D116). The p-F-benzyl group points toward the catalytic loop and
OH
OH
O
O
N
Me
N
Me
12
10
8
6
4
tautomer a
tautomer b
2
0
0
30
60
90
120
150
180
T1 torsion angle (°)
**
Figure 4. Conformational results for torsion angle T1 (pbe1pbe/6-31G ). Relative
energies (
reference.
DE) are calculated using the global minimum (T1 = 180°, tautomer b) as
getic due to steric hindrance. As expected, the conformation with
T1 of 90° presents the highest energy due to full lack of conjuga-
tion. Based on these theoretical results, conformers with T1 of
Figure 5. (a) Catalytic domain of integrase15 (pdb code:1BL3) with main residues and Mg2+ metal ion (purple). Pockets are coloured according hydrophobicity of residues
(from blue to brown). This figure was prepared using the MOLCAD module of SYBYL8.0 program.14b (b) Docking results of compound 2 into HIV-1 integrase active site (left,
T1 = 30°; right, T1 = 180°).

P. Vandurm et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4806–4809
4809
the bromine atom inserts between K156 and K159 residues. The
quinolinone ring is close to the N155 side chain. In the second
binding mode depicted in Figure 5b right, the Mg2+ metal ion is
chelated in a bidentate manner by the quinolinone carbonyl oxy-
gen and by the carboxylate group. The bromine atom is oriented
towards the catalytic loop and the p-F-benzyl moiety is located be-
tween K156 and K159. Both binding modes highlight the impor-
tance of the DKA moiety for the chelation of the Mg2+ and
particularly the carboxylate group which could explain the stron-
ger inhibitory potency of acidic compound 2 compared to ester
compound 1. At this stage, it is difficult to discriminate between
the two proposed binding modes and one can not exclude other
potential ones. Indeed, docking studies show some limitations such
as the use of a rigid protein (and metal ion cofactor) and a scoring
function which does not take correctly into account the energetic
contributions issued from metal ion chelation and other weak
interactions such as hydrogen bonds and van der Waals forces. Fur-
thermore, the presence of viral DNA and the existence of a second
Mg2+ in the catalytic pocket can be also considered as recently sug-
gested.20 The obtention of crystal structure of enzyme/DNA/inhib-
itor complexes should help to clarify the inhibition mechanism.
Nevertheless, these modelling studies show the relevance of
synthesizing compounds able to simultaneously chelate metal
ion (pocket 1) and fill the two other depicted cavities (pockets
2 and 3). Within this context, bromine atom could be replaced
by various substituents depending on the binding mode con-
sidered. In the first binding mode (Fig. 5b left), bromine atom
could be substituted by anionic groups able to form electro-
static interactions with K156 and K159 residues. In the second
binding mode (Fig. 5b right), the bromine could be replaced
by big hydrophobic moieties filling pocket 3. These pharmaco-
modulations should increase the inhibitory potency of our ser-
ies of derivatives. Furthermore, resulting structure–activity
relationships should help to discriminate between binding
modes.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.06.044.
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Acknowledgments
This research was supported by grants from the DGTRE (Direc-
tion générale des Technologies, de la Recherche et de l’Energie,
Région Wallonne) (Programs WALEO). P. Vandurm thanks F.R.I.A
for financial support. We also thank the mass spectrometry labora-
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Chemistry: synthetic pathway and compounds spectroscopic
data; biology: protocols for in vitro experiments, antiviral activities
and cytotoxicity measurements; structural and modelling studies:
crystal data and docking parameters. CCDC 730039 contains the
supplementary crystallographic data for this paper. These data
can be obtained free of charge from The Cambridge Crystallo-
graphic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.