Novel bifunctional quinolonyl diketo acid derivatives as HIV-1 integrase inhibitors: Design, synthesis, biological activities, and mechanism of action

Article abstract of DOI:10.1021/jm0511583

The virally encoded integrase protein is an essential enzyme in the life cycle of the HIV-1 virus and represents an attractive and validated target in the development of therapeutics against HIV infection. Drugs that selectively inhibit this enzyme, when

Full text of DOI:10.1021/jm0511583

J. Med. Chem. 2006, 49, 1939-1945
1939
Novel Bifunctional Quinolonyl Diketo Acid Derivatives as HIV-1 Integrase Inhibitors: Design,
Synthesis, Biological Activities, and Mechanism of Action
Roberto Di Santo,*,† Roberta Costi,† Alessandra Roux,† Marino Artico,† Antonio Lavecchia,*,‡ Luciana Marinelli,‡
Ettore Novellino,‡ Lucia Palmisano,§ Mauro Andreotti,§ Roberta Amici,§ Clementina Maria Galluzzo,§ Lucia Nencioni,|
Anna Teresa Palamara,| Yves Pommier, and Christophe Marchand
Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Studi Farmaceutici, and Istituto di Microbiologia, UniVersita` di Roma “La
Sapienza”, P. le A. Moro 5, I-00185 Roma, Italy, Dipartimento del Farmaco, Istituto Superiore di Sanita`, Viale Regina Elena 299, I-00161
Roma, Italy, Dipartimento di Chimica Farmaceutica e Tossicologica, UniVersita` di Napoli “Federico II”, Via D. Montesano 49, I-80131
Napoli, Italy, and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute Building 37, Room 5068,
National Institutes of Health, Bethesda, Maryland 20892-4255
ReceiVed NoVember 18, 2005
The virally encoded integrase protein is an essential enzyme in the life cycle of the HIV-1 virus and represents
an attractive and validated target in the development of therapeutics against HIV infection. Drugs that
selectively inhibit this enzyme, when used in combination with inhibitors of reverse transcriptase and protease,
are believed to be highly effective in suppressing the viral replication. Among the HIV-1 integrase inhibitors,
the â-diketo acids (DKAs) represent a major lead for anti-HIV-1 drug development. In this study, novel
bifunctional quinolonyl diketo acid derivatives were designed, synthesized, and tested for their inhibitory
ability against HIV-1 integrase. The compounds are potent inhibitors of integrase activity. Particularly,
derivative 8 is a potent IN inhibitor for both steps of the reaction (3′-processing and strand transfer) and
exhibits both high antiviral activity against HIV-1 infected cells and low cytotoxicity. Molecular modeling
studies provide a plausible mechanism of action, which is consistent with ligand SARs and enzyme photo-
cross-linking experiments.
Introduction
IN catalyzes the insertion of viral DNA into the host genome
derived from reverse transcription of HIV RNA. Integration
occurs via a multistep sequence of reactions, including (i)
cleavage of a dinucleotide pair from the 3′-end of the viral DNA
(termed “3′-processing”, 3′-P), (ii) insertion of the resulting
shortened strands into the host-cell chromosome (termed “strand
transfer”, ST), and (iii) removal of the two unpaired nucleotides
at the 5′-end of the viral DNA and gap-filling process (for
review, see ref 6).
Three different classes of chemotherapeutic agents are actually
used to inhibit the replication of HIV-1, the etiological agent
of acquired immunodeficiency syndrome (AIDS): the nucleo-
side (NRTI) and non-nucleoside (NNRTI) reverse transcriptase
inhibitors, the protease inhibitors (PRI), and the inhibitors of
the fusion of the virus with the host cell (for review, see ref 1).
The highly active anti-retroviral therapy (HAART), which is
based on the use of a combination of the cited drugs, effectively
inhibits the replication cycle of HIV-1. The advent of HAART
has made possible the suppression of the HIV-1 replication to
such an extent that the virus becomes undetectable in the blood
of infected persons. As a consequence, a decline of both
mortality and morbidity due to the HIV-1 was reported in the
recent years. However, HAART fails to eradicate viral replica-
tion, which persists at a low level in cellular reservoirs, despite
the chemotherapy.2 The ability of HIV-1 to evolve drug
resistance and the toxicity of HAART regimens make integrase
(IN), which is the third virally encoded enzyme required for
HIV-1 replication, a legitimate target for the development of
new drugs. Moreover, IN has no cellular counterpart, and thus
came into sight 10 years ago as a new therapeutic opportunity.3-6
Hopefully, integrase inhibitors will become a potential additive
to HAART or a salvage therapy for patients resistant to currently
available anti-HIV drugs.
Numerous compounds with different structural features have
been reported as IN inhibitors6,7 of which the most important
class is typified by an aryl â-diketo acid motif (DKAs)8
(compound 1,9 Figure 1). DKAs selectively inhibit the ST
reaction of IN and exhibit antiviral activity against HIV-1-
infected cells in a manner consistent with inhibition of integra-
tion.8,10 These compounds are also useful tools to explore the
molecular mechanism of IN.8,11,12
More recently, some bifunctional DKAs (BDKAs) were
reported, which are characterized by the presence of two diketo
acid chains in the skeleton of the IN inhibitor (compounds 2-4,
Figure 1).13-15 However, the BDKAs reported so far were less
potent than mono functional counterparts and endowed with low
antiviral activities. These properties could be ascribed to the
lack of some important structural features in the reported
BDKAs. In fact, structure-activity relationships on monofunc-
tional aryl diketo acids led to the conclusion that the highest
activity was obtained when the central aromatic ring is 1,3-
disubstituted with the diketo acid chain and a benzyl moiety
(see compound 1, Figure 1). In such a way, the angle between
the two lines extended from the above groups is around 120°.9
* To whom correspondence should be addressed. R.D.S.: phone and
fax, +39-6-49913150; e-mail, roberto.disanto@uniroma1.it. A.L.: phone
and fax, +39-81-678613; e-mail, lavecchi@unina.it.
† Dipartimento di Studi Farmaceutici, Istituto Superiore di Sanita`.
‡ Dipartimento di Chimica Farmaceutica e Tossicologica, Universita` di
Napoli “Federico II”.
The aim of the present work was to obtain new BDKAs with
improved activity against IN and HIV-1-infected cells. Thus,
on the basis of our prior experience,16 we designed new
§ Dipartimento del Farmaco, Instituto Superiore di Sanita`.
| Istituto di Microbiologia, Universita` di Roma “La Sapienza”.
Laboratory of Molecular Pharmacology, National Cancer Institute.
10.1021/jm0511583 CCC: $33.50 © 2006 American Chemical Society
Published on Web 02/17/2006

1940 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Di Santo et al.
Scheme 1a
Figure 1. Structures of selected HIV-1 IN inhibitors belonging to the
mono and bifunctional DKAs.
a Reagents and conditions: (a) ethyl ethoxymethyleneacetoacetate, neat,
120 °C 5 min, 73%; (b) Ph2O, reflux 50 min, 56%; (c) 4-flurobenzyl
bromide, K2CO3, DMF, reflux 2 h, 88%; (d) diethyl oxalate, C2H5ONa,
THF‚C2H5OH, rt 2 h, 5 (100%), 45 min, 7 (88%); (e) 1 N NaOH, rt 1.5 h,
6 (52%), 8 (77%).
Figure 2. Structural features of the newly designed bifunctional
quinolonyl diketo acid derivatives.
F-benzyl-substituted quinolinones (7, 8) are more active than
the unsubstituted counterparts (5, 6). Derivative 8 is the most
potent derivative with IC50 values for strand transfer around 15
nM (Figure 3 and Table 1). Compound 8 is selective for ST
inhibiton with IC50 values approximately 20-fold lower for ST
than for 3′-P (Figure 3, compare panels B and C; Table 1).
Compound 8 also inhibits integrase with similar potency in the
presence of Mg2+ or Mn2+ (Table 1). The activity in the
presence of Mg2+ in addition to the selectivity for ST represents
a trademark of the most potent DKAs.11 Compound 8 is to our
knowledge, the most potent bifunctional diketo acid derivative
reported to date.
inhibitors 5-8 based on the 4-(4(1H)-quinolinon-3-yl)-2,4-
dioxobutanoic acid skeleton (Figure 2). This scaffold was chosen
because (i) it is easy to alkylate the 4(1H)-quinolinone at
1-position with a benzyl group to obtain a 1,3-disubstituted
compound that well fits the geometric requirements for an
optimal IN inhibitory activity and (ii) a second diketo acid
function can readily be introduced at position 6 of the aromatic
ring via an acetyl intermediate. Moreover, with the aim to
characterize the mode of binding of the novel compounds within
the IN, docking studies were performed and a molecular basis
of enzyme inhibition is proposed.
Cell-Based Assay. Antiviral activities are presented in Table
1. Compound 8 showed good antiviral efficacy in HIV-1-
infected H9/HTLVIIIB cells (EC50 ) 4.29 µM, EC90 ) 40 µM)
and low cytotoxicity (CC50 > 200 µM, SI > 46.6). 6 and 7
were 8 and 5 times, respectively, less potent than the parent
compound 8, while 5 was inactive below 50 µM concentration.
Note that the quinolonyl derivative 8 showed higher potency
in inhibiting the replication cycle of HIV-1 in cell-based assays
(EC50) and better SI than the reference derivatives 3 and 4.
These structure-activity relationships emphasize the rel-
evance of the free carboxylic acid group and the p-F-benzyl
moiety for both tight binding with IN and high antiviral potency.
Docking Studies. To elucidate the binding mode of the most
active compound (8) within the IN catalytic core domain (CCD),
docking calculations were carried out using the X-ray structure
1BIS (subunit B).17 A Mg2+ ion, was placed in the active site
between the carboxylate oxygen atoms of residues D64 and
D116, considering the geometry of the Mg2+ ion present in the
PDB structure 1QS4 (subunit A).18
To take into account protein flexibility, 8 was docked to an
ensemble of protein snapshots taken from a 1-ns molecular
dynamics (MD) simulation, according to the relaxed-complex
method.19 The program AutoDock 3.0.5 was chosen because it
utilizes a fully flexible ligand in its docking algorithm and
because it has been shown to successfully reproduce many
crystal structure complexes.20 Details of the docking setup and
the MD simulation protocol are provided as Supporting Infor-
mation. The protein turned out to be very stable throughout the
Results and Discussion
Chemistry. The synthesis of derivatives 5-8 is outlined in
Scheme 1. The key intermediate, 3,6-diacetyl-4(1H)-quinolinone
(9), was obtained by reaction of 4-acetylaniline with ethyl
ethoxymethyleneacetoacetate, followed by thermic cyclization
in diphenyl ether. 9 was then alkylated with 4-fluorobenzyl
bromide in alkaline medium (K2CO3) to afford N-1 substituted
quinolinone 10. Acetyl derivatives 9 and 10 underwent Claisen
condensation with diethyl oxalate in the presence of sodium
ethoxide as a catalyst to afford the diketo esters 5 and 7, which
were in turn hydrolyzed with 6 N NaOH to the corresponding
acid derivatives 6 and 8. The chemical and physical data of
compounds 5-10 are reported in the Experimental Section; the
spectroscopic data of derivatives 5-8 are given in the Support-
ing Information.
Evaluation of Biological Activities. In Vitro Assays.
Derivatives 5-8 were tested in vitro for ST inhibition in the
presence of magnesium (Mg2+) using a novel electrochemilu-
minescent assay to generate IC50 values from triplicate experi-
ments (Table 1). Compounds 5-8 were also tested for ST and
3′-P using gel-based assays in the presence of Mg2+ or Mn2+
(Figure 3). IC50 values were calculated using dose-response
curves (Figure 3B,C) and are summarized in Table 1. The newly
synthesized BDKAs exhibit potent inhibitory activity against
IN for both ST and 3′-P steps. The acid derivatives (6, 8) are
more potent than the corresponding esters (5, 7), and the 1-p-

HIV-1 Integrase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 1941
Table 1. Cytotoxicity and Antiviral and Anti-Integrase Activities of Derivatives 5-8
a
anti-IN activity: IC50
ST
3′-P
antiviral activity
d
e
compd
R
X
Mg2+ b
Mg2+ c
Mn2+ c
Mg2+ c
Mn2+ c
CC50
EC50
SIf
5
6
7
H
C2H5
H
C2H5
H
77 ( 13
9.1 ( 1.2
0.38 ( 0.01
0.016 ( 0.004
39
2.9
0.29
0.017
80
28
22
21
0.44
>333
>4.1
40
0.20
7.8
>200
>200
191
>50
36.3
20
-
H
0.43
0.25
0.012
1.83
6.5
5.5
9.6
>47
-
Bzg
Bzg
8
>200
4.29h
nr
2i
3i
4i
nrj
82
1.8
>25
81
61k
17
-
4.8
0.2
a Inhibitory concentration 50% (µM) determined from dose-response curves. b Experiments performed in triplicate using the BioVeris assay (ST assay
in the presence of MgCl2). c Experiments performed on gels in the presence of MnCl2 or MgCl2 (see Figure 3). d Cytotoxic concentration 50% (µM). e Effective
concentration 50% (µM). f Selectivity index ) CC50/EC50. g Bz ) CH2-4-F-Ph. h For this compound, EC90 ) 40 µM was determined. i Literature data; see
refs 11-13. j nr: not reported. k Percentage of inhibition obtained at a concentration of 25 µM.
Figure 3. Comparison of HIV-1 integrase inhibition by compound 7 and 8 in the presence of Mg2+. (A) Phosphorimager image showing a
representatitve experiment. 21, 19, and STP correspond to DNA substrate, 3′-processing product, and strand transfer products, respectively. (B)
HIV-1 integrase inhibition curve (derived from densitometric analysis of typical experiments) for 3′-processing. (C) HIV-1 integrase inhibition
curve for strand transfer.

1942 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Di Santo et al.
Figure 4. The two predominant binding modes of 8 (yellow) to MD snapshots of the IN core domain. Residues lining the ligand position are
highlighted. The metal ion is represented as a magenta sphere.
MD simulation, with the exception of the three loop regions
139-147, 165-172, and 185-196. The former, which we refer
to as “catalytic loop” is located close to the active site. Its
importance for the catalytic activity together with the flexible
nature are well-known.21 According to previous MD studies,
the catalytic loop moves back and widens the active site region.22
The Mg2+ coordination by D64 and D116 residues was always
conserved during the production run.
Docking of 8 to the MD snapshots, collected every 100 ps,
revealed two predominant binding modes, which are shown in
Figure 4. In the binding orientation shown in Figure 4a, the
carboxylate group of one diketo acid chain of the ligand chelates
the Mg2+ metal ion, whereas the other one inserts between
residues K156 and K159, forming H-bonds with both side chains
and with N155 CO backbone. The p-F-benzyl group points
toward a hydrophobic pocket formed by the catalytic loop
residues Y143, P142, I141, and G140 and by residues I60, Q62,
V77, V79, H114, G149, V150, I151, E152, S153, and M154.
Particularly, a favorable electrostatic interaction occurs between
the fluorine atom on the benzyl ring and the amide group of
Figure 5. Model of IN CCD-DNA complex with 8 (rendered as CPK)
bound to the active site. Donor DNA is in red and acceptor DNA is in
green.
Q62 residue. The quinoline ring of the molecule forms a stacked
amide-aromatic interaction with the N155 side chain, while
the quinoline carbonyl oxygen enables a hydrogen bond to the
T66 side chain. In our model, T66, S153, and M154, whose
mutations were found to confer resistance to DKAs, are all in
close proximity of the ligand.8
Thus, the free carboxylic functions are necessary for a strong
interaction with the IN enzyme, particularly the metal ion. On
the other hand, the drop in activity of 6 compared to 8 in both
ST and 3′-P (see Table 1) can be clearly attributed to the absence
of the p-F-benzyl moiety, which in our model inserts into the
hydrophobic pocket, leading to a more efficient binding.
According to previous findings on BDKAs activity,13 8 is
effective on both ST and 3′-P processes catalyzed by HIV-1
IN and in this feature differs from the reported monofunctional
DKAs, which are generally found to be selective toward the
ST. In this respect, it was hypothesized that BDKAs can bind
both DNA acceptor and donor site of HIV-1 IN, being inhibitors
of both ST and 3′-P processes, whereas the monofunctional
DKAs selectively bind the acceptor site, being effective on ST.13
Here, in an effort to gain more insight into the mechanism of
action of the reported ligands, a re-evaluation of the above-
described docking results was performed with the aid of the
theoretical model of HIV-1 IN tetramer complexed with both
A significant alternative to the described binding mode is
given by a docking position in which the metal ion is chelated
in a bidentate manner by the quinoline carbonyl oxygen and
by one oxygen of the diketo acid function, while the hydroxyl
and the carboxylate groups of the same branch contact the D116
and the N117 side chains, respectively (Figure 4b). On the
opposite side of the molecule, the other diketo acid arm
elongates toward the K156 and K159 residues contacting
exclusively the latter amino acid. The p-F-benzyl group entirely
inserts in the hydrophobic pocket, where it is stabilized by
hydrophobic interactions with I151 and P142 side chains.
Although the two binding modes differ in some details, it is
worth noting that, in both docking solutions, the ligand interacts
with the same enzyme attachment points: (i) the metal ion, (ii)
the K156 and/or K159 residues, and (iii) the hydrophobic pocket.
The reduced inhibitory potencies of diketo esters 5 and 7
compared to their respective acids 6 and 8 is due to the ability
of both carboxylic functionalities to chelate the metal ion and
to interact with the above-mentioned lysine residues, whereas
the diketo esters weakly bind to these portions of the enzyme.
donor and acceptor DNA developed by McCammon et al.23
A
visual inspection of 8 docked into the CCD-DNA complex
(Figure 5) led us to the following considerations: (i) the diketo
acid arm, which coordinates the Mg2+ metal ion, is clearly
important in anchoring the ligand to the catalytic site. It is worth

HIV-1 Integrase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 1943
suspension was stirred for 2.5 h at 100 °C. After cooling, cold water
(0-4 °C) was added (40 mL) and the precipitate was filtered,
washed with water and light petroleum ether in turn and then dried
under an IR lamp to provide 10 (330 mg, 89%); mp 213-214 °C
(toluene).
General Procedure for the Synthesis of Diketo Esters 5 and
7. Sodium ethoxide (390 mg, 5.5 mmol) was added into a well-
stirred mixture of 9 or 10 (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 (2 h for 5, 45 min for
7) and then was poured into n-hexane (50 mL). The collected
precipitate was vigorously stirred for 30 min in 1 N HCl (50 mL).
The yellow solid that formed was filtered, washed with water, and
dried under an IR lamp. 5: 100% yield; mp >300 °C (DMF/water).
Anal. (C21H19NO9) C, H, N. 7: 88% yield; mp 189-190 °C
(toluene). Anal. (C28H24FNO9) C, H, N, F.
General Procedure for the Synthesis of Diketo Acids 6 and
8. A mixture of 1 N NaOH (6.5 mL) and compound 5 or 7 (1.3
mmol) in THF/methanol 1:1 (12 mL) was stirred at room temper-
ature for 18 h and then poured onto crushed ice. The aqueous layer
was separated and treated with 1 N HCl until reaching pH 3, and
the yellow solid that formed was collected by filtration and then
washed with water, hot dry ethanol, and light petroleum ether. 6:
52% yield; mp >300 °C (washed with hot acetone). Anal. (C17H11-
NO9) C, H, N. 8: 77% yield; mp 178-180 °C (washed with hot
dry ethanol). Anal. (C24H16FNO9) C, H, N, F.
noting that while this arm seems not to interfere with the donor
DNA binding, it may physically hamper the acceptor DNA
binding. In our model, either the acceptor DNA or the ligand
contact the N117 residue (see Figure 4b). This amino acid plays
a crucial role in the enzyme function as its mutation leads to
an IN preferentially defective in the ST step of integration.24
(ii) The other diketo acid function, inserting between K156 and
K159 residues, would interfere with the donor DNA recognition
and binding. This result is in accordance with photo-cross-
linking data, which reveal that the above-mentioned lysines are
critical for donor DNA binding.25 This could explain the ability
of 8 to inhibit the IN catalyzed 3′-P reaction. (iii) The p-F-
benzyl group, which inserts in the hydrophobic pocket, would
reduce the catalytic loop mobility and physically hamper the
binding of the acceptor DNA. In the proposed model, both DNA
molecules are in proximity of the loop; particularly, the acceptor
DNA is in close contact with I151 and P142, which contribute
to the ligand binding (Figure 4b). This finding also makes sense
with respect to the experimental observation that the benzyl
group in DKAs ligands is the primary determinant of the ST
inhibition.13
Conclusions
We have designed, synthesized, and tested in both enzyme-
and cell-based assays novel BDKAs as anti-HIV-1 agents
targeted to IN. These compounds are potent inhibitors of both
the ST and 3′-P steps and are endowed with antiviral activity.
Docking experiments have helped to provide a framework for
interpreting the inhibitory activity of our BDKAs. As more
information becomes available to better characterize the details
of DNA-IN interaction, this issue will be resolved more
definitively. Moreover, studies on monofunctional derivatives
related to 5-8 are in progress, with the aim to elucidate
differences in the mechanisms of action.
Biological Assays. Integrase Assays. Compounds 5-8 were
tested for their ability to inhibit HIV-1 integrase in vitro using a
gel-based assay in addition to a plate-based electrochemiluminescent
assay.
In the gel assay, a 5′-end-labeled 21-mer double-stranded DNA
oligonucleotide, corresponding to the last 21 bases of the U5 viral
LTR, is used to follow both the 3′-processing and strand transfer
steps of the integration reaction (for review, see ref 6). Briefly, a
DNA-enzyme complex is preformed by mixing 500 nM of
recombinant HIV-1 integrase and 20 nM of 5′-labeled double-
stranded DNA template in a buffer containing 50 mM MOPS, pH
7.2, 7.5 mM MnCl2 or MgCl2, and 14.3 mM â-mercaptoethanol
for 15 min on ice. The integration reaction is then initiated by
addition of the drug and continued in a total volume of 10 µL for
60 min at 37 °C. The reaction samples are stopped by adding the
same volume of electrophoresis denaturing dye and loaded on 20%
19/1 acrylamide denaturing gel. Gels were exposed overnight and
analyzed using a Molecular Dynamics Phosphorimager (Sunnyvale,
CA).
The plate-based electrochemiluminescent assay was performed
using a BioVeris M-SERIES Analyzer (Gaithersburg, MD). DNA
substrates were obtained from BioVeris and used according to the
manufacturer’s recommendations. Briefly, donor DNA is incubated
for 30 min at 37 °C in the presence of 250 nM of recombinant
HIV-1 integrase. After addition of the drug, the integration reaction
is initiated by addition of target DNA. Reaction is carried out for
60 min at 37 °C and then read on the BioVeris M-SERIES
Analyzer.
Experimental Section
Chemistry. General. Melting points were determined with a
Buchi 530 capillary apparatus and are uncorrected. Infrared (IR)
1
spectra were recorded on a Spectrum-one spectrophotometer. H
NMR spectra were recorded on a Bruker AC 400 spectrometer.
Merck silica gel 60 F254 plates were used for analytical TLC.
Column chromatographies were performed on silica gel Merck 70-
230 mesh. Concentration of solutions after reactions and extractions
involved the use of a rotatory evaporator operating at a reduced
pressure of approximately 20 Torr. Analytical results agreed to
within (0.40% of the theoretical values.
2-[[(4-Acetylphenyl)amino]methylene]-3-oxobutanoic Acid Eth-
yl Ester. A mixture of 4-aminoacetophenone (2.0 g, 0.015 mol)
and 2-(ethoxymethylene)-3-oxobutanoic acid ethyl ester26 (2.8 g,
0.015 mol) was heated at 120 °C for 5 min. After cooling, the solid
that formed was washed with n-hexane and with a small amount
of cold CHCl3 to obtain pure 2-[[(4-acetylphenyl)amino]methylene]-
3-oxobutanoic acid ethyl ester (3.0 g, 73%), mp 156-158 °C
(benzene), which was used in the next reaction without further
purification.
3,6-Diacetyl-4(1H)-quinolinone (9). 2-[[(4-Acetylphenyl)amino]-
methylene]-3-oxobutanoic acid ethyl ester (5.0 g, 0.018 mol) was
dissolved in boiling diphenyl ether (50 mL) and refluxed for 50
min. After cooling, the mixture was treated with n-hexane (100
mL) and the precipitate that formed was collected and filtered on
a short silica gel column (ethyl acetate and then chloroform/
methanol 10:1 as eluent) to afford 9 (2.3 g, 56%); mp >300 °C
(DMF/water).
3,6-Diacetyl-1-(4-fluorophenylmethyl)-4(1H)-quinolinone (10).
A solution of 9 (250 mg, 1.1 mmol) in dry DMF (10 mL) was
treated with anhydrous K2CO3 (210 mg, 1.5 mmol) and 4-fluo-
rophenylmethyl bromide (610 mg, 3.3 mmol) and the resulting
Anti-HIV Assays in Cultured Cell Lines. The anti-HIV drug
testing was performed in 96-well plates with a defined, previously
titered inoculum of a laboratory strain (HTLV-IIIB) to minimize
the inoculum effect.
In brief, all compounds were dissolved in dimethyl sulfoxide
and diluted in cell culture medium at concentrations ranging from
0.1 to 50 µM. Exponentially growing human T lymphocytes (H9
cell line) were added at 5000 cell/well. After the infectivity of a
virus stock (HTLV-IIIB) was quantified, an aliquot containing 1
× 105 50% tissue culture infectious dose (TCID50) per 5000 H9
cells per well is used as inoculum in each set of in vitro infections
of H9 cells. Uninfected cells with the compound served as a toxicity
control, and infected and uninfected cells without the compound
served as basic controls. Cultures were incubated at 37 °C in a 5%
CO2 atmosphere for 4 days. Supernatant fluid of infected wells (in
the absence of drug and at each of a number of drug concentrations)
was harvested. HIV p24 antigen was quantified and the 50%

1944 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Di Santo et al.
inhibitory concentration (IC50) of drug was determined using the
median effect equation.
geometry optimization was carried out employing the DISCOVER
program with the CVFF force field.
Cytotoxicity Assays. The cytotoxicity of test compounds was
evaluated on human histiocytic lymphoma (U937) cell line obtained
from the American Type Culture Collection (ATCC, Rockville,
MD). Cells were plated in a 96 well-plate at a concentration of 5
× 103/mL in RPMI-1640 without phenol red, supplemented with
20% fetal calf serum and antibiotics. Two hours after plating,
compounds were added at different concentrations ranging from 1
µg/mL to 200 mg/mL and cells were incubated at 37 °C for 24 h.
Then, cells were incubated at 37 °C for 3 h with 1 mg/mL of MTT
(Sigma-Aldrich, Milan, Italy). After incubation, the remaining
water-insoluble formazan was solubilized in absolute 2-propanol
containing 0.1 N HCl. Absorbance of converted dye was measured
in an ELISA plate reader at the wavelength of 570 nm. The
cytotoxicity of the compounds was calculated as the percentage
reduction of the viable cells compared with the drug-free control
culture. The drug concentration required to reduce the cell viability
by 50% is called IC50.
Molecular Modeling. Molecular Dynamics (MD) Simulation.
The catalytic core domain (CCD) structure of HIV-1 integrase (IN)
was taken from PDB structure 1BIS.17 The chain B of the X-ray
structure was chosen for our studies, and a Mg2+ ion was placed
in the active site between the carboxylate oxygen atoms of amino
acid residues D64 and D116 considering the geometry of the Mg2+
ion that was present in the subunit A of the PDB structure 1QS4.18
To sample the conformational space of IN enzyme, a 1-ns MD
simulation was carried out in explicit solvent. All the water
molecules present in the X-ray structure of the enzyme were
removed and all hydrogen atoms were added to the residues,
considering residues Arg, Lys, Glu, and Asp in their charged form,
while all His residues were considered neutral by default. To make
the system electroneutral, a total of two neutralizing counterions
(Cl-) were added with the aid of the program VEGA (v 1.5.0).27
The enzyme was then soaked with a 10 Å water layer. The
calculations were carried out with the DISCOVER module of the
INSIGHT II program using the CVFF force field.28 A multiple-
step procedure was used. The complex was energetically minimized
with 5000 steps of a steepest descent minimization, followed by
3000 steps of conjugate gradient minimization to adjust the water
molecules and the counterions locally and to eliminate any residual
geometrical strain, while the heavy atoms of the enzyme were kept
fixed. The minimized solvated system was used as the initial struc-
ture for an equilibration stage (100 ps), followed by a production
run (1 ns). In the equilibration stage, energy minimization of the
protein side chains was achieved by employing 5000 steps of
steepest descent and 3000 steps of conjugate gradient algorithm.
The system was then heated gradually starting from 10 to 300 K
in 5-ps steps and was equilibrated with temperature bath coupling
(300 K). During the equilibration and the production run, the
backbone of the enzyme secondary structures was kept fixed. A
cutoff of 13 Å was used for nonbonded interactions. Coordinates
and energies were saved every 10 ps, yielding 100 structures. For
the purpose of automatic docking, a snapshot was taken each
100 ps.
(1) Ligand Setup. The structure of 8 was generated from the
standard fragment library of the SYBYL software version 7.0.29
Geometry optimizations were achieved with the SYBYL/MAXI-
MIN2 minimizer by applying the BFGS algorithm30 with a
convergence criterion of 0.001 kcal/mol and by employing the
TRIPOS force field. Partial atomic charges were assigned using
the Gasteiger and Marsili formalism31 as implemented in the
SYBYL package. 8 was modeled in its keto-enolic form with the
negatively charged carboxylate groups. Six flexible torsions were
specified: two around the quinoline ring, two about the benzyl
group, and two allowing the hydroxyl groups to rotate.
(2) Protein Setup. The protein structure was set up for docking
as follows: the unpolar hydrogens were removed and Kollman
united-atom partial charges were assigned. Solvation parameters
were added to the protein-DNA complex using the ADDSOL
utility of the AutoDock program. The grid maps were calculated
with AutoGrid. The grids were chosen to be large enough to include
a significant part of the protein around the catalytic site. In all cases,
we used grid maps with 61× 61× 61 points with a grid-point
spacing of 0.375 Å. The center of the grid was set to be coincident
with the Mg2+ ion in the active site of catalytic domain.
Acknowledgment. The authors thank Prof. J. Andrew
McCammon for the donation of the IN-DNA complex coor-
dinates. We also thank Dr. M. Zancato (University of Padova,
Italy) for performing elemental analyses. This project was
supported by Ministero della Sanita`, Istituto Superiore di Sanita`,
“Programma Nazionale di ricerca sull’ AIDS” (Grant No.
30F.19), Italian MIUR (PRIN 2004), and in part by the Center
for Cancer Research, National Cancer Institute, National
Institutes of Health.
Supporting Information Available: Spectroscopic data for
compounds 5-10, elemental analyses for derivatives 5-8, and
details of the docking setup and the MD simulation protocol. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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