Basic quinolinonyl diketo acid derivatives as inhibitors of HIV integrase and their activity against RNase H function of reverse transcriptase

Article abstract of DOI:10.1021/jm5001503

A series of antiviral basic quinolinonyl diketo acid derivatives were developed as inhibitors of HIV-1 IN. Compounds 12d,f,i inhibited HIV-1 IN with IC₅₀ values below 100 nM for strand transfer and showed a 2 order of magnitude selectivity over 3′-processing. These strand transfer selective inhibitors also inhibited HIV-1 RNase H with low micromolar potencies. Molecular modeling studies based on both the HIV-1 IN and RNase H catalytic core domains provided new structural insights for the future development of these compounds as dual HIV-1 IN and RNase H inhibitors.

Full text of DOI:10.1021/jm5001503

Article
pubs.acs.org/jmc
Basic Quinolinonyl Diketo Acid Derivatives as Inhibitors of HIV
Integrase and their Activity against RNase H Function of Reverse
Transcriptase
Roberta Costi,*,† Mathieu Metifiot,‡ Suhman Chung,§ Giuliana Cuzzucoli Crucitti,†
́
Kasthuraiah Maddali,‡ Luca Pescatori,† Antonella Messore,† Valentina Noemi Madia,† Giovanni Pupo,†
Luigi Scipione,† Silvano Tortorella,† Francesco Saverio Di Leva,∥ Sandro Cosconati,⊥ Luciana Marinelli,∥
Ettore Novellino,∥ Stuart F. J. Le Grice,§ Angela Corona,# Yves Pommier,‡ Christophe Marchand,*,‡
and Roberto Di Santo†
†Dipartimento di Chimica e Tecnologie del Farmaco, Istituto Pasteur-Fondazione Cenci Bolognetti, “Sapienza” Universita
̀
di Roma,
P.le Aldo Moro 5, I-00185 Roma, Italy
‡Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 37
Convent Drive, Bethesda, Maryland 20892, United States
§RT Biochemistry Section, HIV Drug Resistance Program, National Cancer Institute, Frederick, Maryland 21702, United States
∥Dipartimento di Farmacia, Universita
⊥DiSTABiF, Seconda Universita
̀
di Napoli “Federico II” Via D. Montesano 49, 80131 Napoli, Italy
di Napoli, Via Vivaldi 43, 81100 Caserta, Italy
#Department of Life and Environmental Sciences, University of Cagliari, 09124 Cagliari, Italy
̀
S
* Supporting Information
ABSTRACT: A series of antiviral basic quinolinonyl diketo
acid derivatives were developed as inhibitors of HIV-1 IN.
Compounds 12d,f,i inhibited HIV-1 IN with IC50 values below
100 nM for strand transfer and showed a 2 order of magnitude
selectivity over 3′-processing. These strand transfer selective
inhibitors also inhibited HIV-1 RNase H with low micromolar
potencies. Molecular modeling studies based on both the HIV-
1 IN and RNase H catalytic core domains provided new
structural insights for the future development of these
compounds as dual HIV-1 IN and RNase H inhibitors.
RT. IN contains three catalytic carboxylate residues, D64,
INTRODUCTION
■
D116, and E152, forming the DDE motif that coordinates two
magnesium atoms of the IN catalytic site. Many HIV-1 IN
inhibitors with metal-complexing properties have been
reported.7 These inhibitors are referred to as strand transfer
IN inhibitors (INSTIs). Three INSTIs, elvitegravir (EVG, 1),
raltegravir (RAL, 2), and dolutegravir (DTG, 3) have already
been approved by the Food and Drug Administration (Figure
1).8,9
RT is another important HIV-1 enzyme and the target of
many anti-HIV drugs. This enzyme has RNA- and DNA-
dependent DNA polymerase, strand displacement, strand
transfer, and RNase H activities.10 RNase H activity, which
degrades RNA from RNA−DNA hybrid molecules, is required
at several steps during reverse transcription and essential for
HIV/AIDS remains one of the most important global health
challenges, especially in sub-Saharan Africa.1,2 Highly active
antiretroviral therapy (HAART),3 the standard of care for HIV/
AIDS, comprises a multitarget regimen combining antiviral
drugs with orthogonal mechanisms of action, thus increasing
the genetic barrier against resistance selection when compared
to monotherapy. Nevertheless, treatment adherence resides
primarily on treatment tolerance and simplicity of admin-
istration, which remains a challenge with multipill HAART
cocktails.4 A single compound capable of inhibiting simulta-
neously two viral targets could represent a therapeutic
alternative. Multitarget inhibitors may alleviate dosing complex-
ity, drug−drug interactions, and toxicities.5 In the field of
medicinal chemistry, the design of active dual inhibitors against
HIV reverse transcriptase (RT) and integrase (IN) is subject of
great interest.6 These inhibitors act on the catalytic sites of the
IN enzyme and the ribonuclease H (RNase H) domain of HIV
Received: October 7, 2013
Published: March 31, 2014
© 2014 American Chemical Society
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Figure 2. 1-Pyrrolidinyl substituted quinolinonyl DKA (7) and general
structure of “base” substituted quinolinonyl DKAs.
of the quinolinonyl diketo acid inhibitors, including the p-fluoro
benzyl group linked to the quinolone nitrogen.
Because of the similarities of the IN and RNase H catalytic
sites and reports on dual activities of a number of compounds
against IN/RNase H, we decide to test our newly designed
quinolonyl DKAs against both enzymes.19b
Herein we describe the synthesis and biological studies of a
series of DKA derivatives that present a p-fluoro benzyl group
on quinolone nitrogen and a variable “base-like” functional
group in position 7 of the quinolone ring as inhibitors of HIV-1
IN. A comparative evaluation of the activity against RNase H
function of the HIV RT of the newly synthesized compounds
was also performed (Figure 2).
RESULTS AND DISCUSSION
■
Figure 1. Anti HIV-1 agents targeting IN (1−3) and RNase H (4−6).
Chemistry. Synthesis of derivatives 11a−g,i and 12a−g,i is
outlined in Scheme 1. 3-Acetyl-4(1H)-quinolinone 8 were
prepared by reaction of 3-fluoroaniline with ethyl orthoformate
and ethyl acetoacetate, which were thermally condensed in the
presence of an inert heating medium (dowtherm A) under
argon atmosphere according to the Yoshizawa procedure.20
Then 8 was alkylated with 4-fluorobenzyl bromide in alkaline
medium (K2CO3) to give the N-1 substituted quinolinone 9.
Derivatives 10a−f and 10h were obtained in few minutes
with good yields by substituting the fluorine atom of 9 with the
appropriate base in the presence of NEt3 under microwave
irradiation. Using the same microwave assisted technique, we
obtained 10g from 9 using N,N-dimethylamine (aq soln 40%)
as base. 10h was converted to the desired chlorinated derivative
10i by reaction with PPh3 in CCl4 and CH2Cl2 using a modified
procedure of a patent of Otsuka Pharmaceutical.21 Derivatives
10a−f and 10i were then condensed with ethyl oxalate, and the
resulting esters 11a−g,i were hydrolyzed to afford the required
acids 12a−g,i (Scheme 1). Chemical, physical, and analytical
data of intermediates 8, 9, and 10a−i, together with the
spectroscopic data, are reported in Supporting Information.
Data for final products 11a−g,i and 12a−g,i are listed in the
Experimental Section.
virus replication. The crystal and NMR structures of isolated
HIV RNase H domain are similar to that of the RNase H
domain in the context of the full-length HIV-RT protein.11
These structures also showed that the folding of the HIV-1
RNase H catalytic core domain (CCD) is similar to that of
HIV-1 IN and, consequently, the catalytic sites of the two
enzymes share a similar geometry. Indeed, also RNase H
features the DDE catalytic motif (comprising D443, E478, and
D498 residues) chelating two magnesium ions, although a
fourth carboxylate residue (D549) is required for catalysis.12
Similar structural characteristics including three aspartate
residues and two magnesium ions at a distance of 3.57 Å
from each other were shown in the DNA polymerase active site
of RT in complex with DNA primer template and an incoming
nucleotide.12
Some diketo acid inhibitors of HIV-1 IN have shown activity
on RNase H,13,14 whereas DNA aptamers used as inhibitors of
RNase H have also been employed to inhibit HIV-1 IN.15
Tropolone (5),16 madurahydroxylactone (6),17 and 2-hydrox-
yquinoline-1,3(2H,4H)-dione (4)18 were recently described to
be able to inhibit both enzymes (Figure 1).
Therefore, concurrent inhibition of HIV-1 RNase H and IN
by compounds with metal-chelating activity could represent an
interesting approach to alleviate some of the problems linked to
multipill combination therapies.
Evaluation of Biological Activities. All newly synthesized
compounds 11a−g,i and 12a−g,i were tested in vitro for their
ability to inhibit RNase H activity of recombinant RT, and on
recombinant IN to evaluate their potencies against its 3′-
processing and strand transfer functions. IN activities were
determined using gel-based assays and the corresponding IC50
values were generated from the dose−response curve and are
summarized in Table 1.
Anti-IN Activities. The newly synthesized DKAs inhibited
IN with nano- to submicromolar potencies for strand transfer
and IC50 values ranging from 2.5 to of 333 μM for 3′-
processing. All compounds exhibited some selectivity for strand
transfer versus 3′-processing, with half of the compounds
showing a selectivity of approximately 2 orders of magnitude or
greater (Table 1). Carboxylic acid-containing compounds were
The work of our research group commenced in the context
of diketo acids (DKAs) compounds as HIV-1 IN inhibitors.19
The design of quinolinone derivatives characterized by one
DKA moiety on position 3 of the quinolinone ring led to HIV-1
IN inhibitors with selectivity for strand transfer versus 3′-
processing. Moreover, inserting a functional group with basic
properties on position 7 of the quinolinone ring generated
derivative 7 (Figure 2), which inhibited ST with nanomolar
IC50 value and submicromolar antiviral activity.19b These results
prompted us to investigate the position 7 of the quinolinone
ring of newly designed DKAs, keeping unchanged the skeleton
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Inhibition of HIV-1 RNase H Activity. The newly
synthesized quinolinone DKA also inhibited the RNase H
function of HIV-1 RT. RNase H inhibition was first calculated
as percentage of inhibition at a single concentration of 10 μM,
then for the most potent compounds the IC50 values were
determined (Table 1). Interestingly, 4-methylpiperazine (11b)
and 4-ethylpiperazine (11c) ester derivatives were more active
than the corresponding acids (12b and 12c, respectively) when
tested against RNase H (41% and 63% inhibition (not shown)
and IC50 18.5 and 26.2 μM, respectively, versus 34% and 22%
of the corresponding acids, Table 1). Five derivatives, including
the lead compound 7b, inhibited RNase H at micromolar
concentration showing IC50s in the range 3.3−6.8 μM: the 4-
acetyl-1-piperazine (12d), the morpholine (12f), the dimethyl-
amine (12g), and the 4-(3-chloropropyl)-1-piperazine (12i)
acid derivatives. As representative examples, compounds 7b,
12f, and 12i were also tested against the polymerase function of
RT and were found inactive up to 40 μM, suggesting that the
observed inhibition is RNase H-mediated and not an indirect
inhibition of the polymerase function of RT. RAL (2), EVG
(1), and DTG (3, data not shown) were also tested for their
ability to inhibit RNase H and found to be inactive,
demonstrating that not all potent INSTIs can efficiently inhibit
this enzyme in vitro (Table 1). These results demonstrate that
in general the best INSTIs (7b and 12d,f,i) were also the most
potent compounds to inhibit RNase H (Table 1).
Scheme 1. Synthetic Route to Quinolinonyl DKAs 11a−g,i
a
and 12a−g,i
Cell-Based Assays. All compounds including the pre-
viously reported 7a, 7b, RAL, and EVG were tested for antiviral
activity and cytotoxicity using HeLa-CD4-LTR-β-gal cells
infected by HIV-1(IIIB). EC50 and CC50 values for compounds
11a−g,i and 12a−g,i are reported in Table 1. In this assay, 7b
exhibited an EC50 value of 14 μM, which is sensibly higher than
the originally reported EC50 value of 0.17 μM in an HTLV-IIIB-
based infection assay.19b Among the tested compounds,
derivatives 11a−c,f,g,i and 12d,f,g show EC50 < 50 μM. In
particular, 11c and 11g are active in the micromolar range
(EC50 = 3.6 and 2.5 μM, respectively), with selectivity indices
(SI) of 14 and 20, respectively. All active compounds were
characterized by low cytotoxicity against the same HeLa-CD4-
LTR-β-gal cells, showing CC50 values >50 μM.
Molecular Modeling. To better rationalize the structure−
activity relationship (SAR) data obtained for this new series of
DKAs, molecular modeling studies were performed. Given the
inhibitory activity of these compounds, docking studies were
undertaken to detect the structural features responsible for the
binding at the active sites of both the aforementioned enzymes.
Several X-ray structures of HIV-1 IN CCD have been
reported,23−38 but unfortunately none of them were cocrystal-
lized with nucleic acids. On the other hand, a number of crystal
structures of the intasome (comprising an IN tetramer
assembled on a pair of viral DNA ends) from the prototype
foamy virus (PFV) have recently been resolved.39−42 These
pioneering studies have demonstrated that two Mg2+ ions are
chelated by residues of the DDE motif (D64, D116, and E152
in HIV-1) inside the active site. Furthermore, both cations
seem to be required for both IN catalytic activity and INSTI
inhibition. Additionally, it has been proven that an induced-fit
mechanism of inhibition for INSTIs occurs through displace-
ment of the terminal 3′-adenosine of the viral DNA from the
active site. Given these recent advances, we decided to revise
our previous theoretical model37 by constructing a new
homology model of the HIV IN CCD/viral DNA complex,
in its INSTI-inhibited form, starting from the structure of full-
a
Reagents and conditions: (i) triethyl orthoformate, ethyl acetoacetate,
Dowtherm A, 95−254 °C, 8 h, 41% yield; (ii) 4-fluorobenzyl bromide,
K2CO3, DMF, 100 °C, 1 h, 29% yield; (iii) proper base, Et3N, DMF,
microwave, 100 W, 153 °C, 10 min, 14−78% yield; (iv) N,N-
dimethylamine aq soln 40%, 100 W, 175 °C, 10 min, 52% yield; (v)
CCl4, CH2Cl2, PPh3, reflux, 24 h, 48% yield; (vi) diethyl oxalate,
C2H5ONa, THF, room temp, 2 h, 76−99% yield; (vii) 1 N NaOH,
THF/CH3OH, room temp, 40 min, 30−100% yield.
always more potent than their corresponding ester counter-
parts.
Three compounds (12d,f,i) inhibited strand transfer with
IC50 values below 100 nM with a 100-fold selectivity over the
3′-processing step. Compounds 12d,f showed an antiviral EC50
of 17 and 26.3 μM, respectively. Compound 12i was unable to
block replication, possibly indicating reduced uptake. The least
active acid-containing compound was 12a (IC50 2.0 μM), in
which the pyrrolidine of the lead was replaced by a piperazine
moiety. This compound was the only one containing an
unsubstituted heteroatom on the 7-substituent. In fact, when
the quinolinone ring was substituted with piperazine
bioisosters, like morpholine (12f) and thiomorpholine (12e),
inhibition of strand transfer activity increased of 1−2 orders of
magnitude (IC50 0.08 and 0.36 μM, respectively). The same
trend was observed when the piperazine hydrogen was replaced
by alkyl or acyl chain. Among the 4-substituted piperazine
derivatives, increased potency was achieved when the N-4 of
the piperazine ring was substituted with acetyl (12d) or 3-
chloropropyl (12i) chains (IC50 0.08 and 0.05 μM,
respectively). Substitution with methyl (12b) or ethyl (12c)
chains lead to submicromolar activity (IC50 0.10 and 0.21 μM,
respectively), indicating that these modifications were not
optimal for strand transfer inhibition. Cumulatively, these
results demonstrate that quinolinone derivatives with piper-
azine bioisoster substitutions are potent INSTIs with antiviral
activity.
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Table 1. Cytotoxicity, Antiviral, Anti-IN, and Anti-RNase H Activities of Compounds 11a−g,i and 12a−g,i
a
Inhibitory concentration 50% (μM) determined against rIN from gel-based assays and expressed as mean SD from at least three independent
b
c
d
experiments. ST: strand transfer. 3′-P: 3′-processing. Percentage of inhibition of RT-associated RNase H activity at fixed concentration of
e
inhibitor 10 μM (%). Inhibitory concentration 50% against HIV-1 RT-associated RNase H activity determined from dose response curves (μM).
f
g
h
Effective concentration 50% in HIV-1 infected HeLa cells (μM). Cytotoxic concentration 50% in HeLa cells (μM). Selectivity index (CC50/EC50).
i
j
k
nd: not determined. Reference 19b. Reference 22.
length IN from PFV in complex with raltegravir (PDB code
3OYA).41 This model was constructed using Prime v3.0
(Schrodinger)43 based on the sequence alignment suggested by
Tang and co-workers.44 This alignment identified conserved
regions and gave the best overlap between the catalytic DDE
motives of the PFV and HIV enzymes. Moreover, original
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Figure 3. (a) Binding mode of compound 7b (yellow) within the HIV-1 IN/DNA model. The CCD is depicted as transparent light-gray surface and
ribbons. Amino acid side chains involved in ligand binding are represented as sticks. The noncleaved (cyan) and processed (orange) viral DNA
strands are shown as ribbon and sticks. Mg2+ metal ions are represented as green spheres. (b) Overlay of compound 7b and raltegravir (magenta) in
the IN binding pocket.
nucleotides were mutated in order to obtain the HIV-1
retroviral DNA sequence. The model was then subjected to the
Prime refinement protocol and finally used for docking studies.
This new theoretical model was first used to perform docking
the precatalytic target capture complex (TCC) from PFV IN
(PDB code 3OS1, Figure 4).40
simulations of raltegravir using Glide v. 5.7 (Schrodinger).45
̈
Interestingly, this inhibitor adopts a binding mode similar to
that shown in the parent crystal structure with its chelating
oxygens oriented toward both active-site Mg2+ cations and its
halobenzyl group fitting into the narrow pocket created by
displacement of the terminal adenosine on the 3′-end of the
viral DNA. These results prompted the docking calculations on
our reference compound 7b, which is an efficient dual INSTI
and RNase H inhibitor (Table 1). In the lowest energy
conformation (GlideScore = −9.24), this compound occupies
the DNA/IN interface with the DKA moiety chelating both the
Mg+ ions (Figure 3a), similarly to the binding mode adopted by
raltegravir (Figure 3b).
Interestingly, the predicted conformation of the DKA moiety
strongly resembles that of a close analogue crystallized in its
unbound conformation,46 suggesting that for these set of
compounds, the unbound conformation would also account for
the bioactive one. Moreover, chelation by the carboxylate group
should explain the higher inhibitory activity displayed by acidic
compounds compared to the corresponding esters despite
sharing the same predicted binding position in the HIV-1 IN
binding site (compare docking pose of 7b in Figure 3a with that
of 7a in Figure S1a in the Supporting Information).
In addition, the quinolinone ring is involved in a parallel-
displaced π−π interaction with the terminal adenosine of the
reactive DNA strand, further stabilizing the ligand in the IN
active site. Moreover, the p-F-benzyl group is embedded in a
hydrophobic cleft created by residues P145 and Q146, forming
a well-oriented π-stacking interaction with the penultimate
cytidine residue of the processed DNA strand. This interaction
pattern (chelation of the Mg2+ cations and insertion in the
DNA/enzyme cleft) is also established by raltegravir in the PFV
IN catalytic site39,41 and in our HIV-1 IN model. Nevertheless,
it is worth mentioning that raltegravir also places its terminal
oxadiazole ring in a cleft, which cannot be explored by our
current set of derivatives. In contrast, our INSTIs feature a basic
substituent in position 7 of the quinolinone ring (−R
substituent, Table 1), which points outward the catalytic site,
as further demonstrated by docking of compound 12i (Figure
S1b in the Supporting Information), which is the most potent
ST inhibitor among the newly synthesized derivatives.
Figure 4. Superimposition between our model of the INSTI/HIV-1
IN CCD/viral DNA ternary complex and the precatalytic TCC from
PFV (PDB code 3OS1). IN enzymes are represented as light-gray
surfaces. The host DNA (green) and the nonprocessed (cyan) and
processed (orange) viral DNA strands are shown as ribbon and sticks.
Compound 7b is depicted as yellow transparent surface and sticks.
This superimposition suggests that our ligands, similarly to
raltegravir, would sterically hamper host DNA binding by
complexing the Mg2+ cofactors and displacing the terminal 3′-
adenosine of the viral DNA, thus accounting for the selective
strand transfer integration step inhibition. On the other hand,
the presence of the viral DNA in the inhibited complex might
also explain why the 3′-processing step is not efficiently
inhibited by the presented ligands.
To explain at molecular level the reasons for the RNase H
inhibition, this enzyme was also considered in our theoretical
studies. Several crystal structures of the full-length wild-type RT
or of its isolated RNase H CCD from HIV-1 in complex with
RNase H inhibitors have been reported.47−50 A superposition
of all of these structures on Cα atoms shows that folding, loops
shape, and the side chain conformations of the majority of
residues within the catalytic site are highly conserved (Figure
S2 in the Supporting Information). On the basis of such an
investigation, we selected the high resolution (2.09 Å) crystal
structure of the full-length RT structure in complex with a
RNase H pyrimidinol carboxylic acid inhibitor and nevirapine
(PDB code 3QIP)49 to carry out docking studies with Glide.
The latter structure is made up by the RNase H domain in
complex with an inhibitor, which is structurally related to our
new series of DKAs. Because this structure is cocrystallized with
Mn2+ ions, we replaced them with the most physiologically
relevant Mg2+ cations, also used in the biochemical assays.
Docking of compound 7b within the RNase H active site shows
To explain the strand transfer-selective inhibiting properties
of our compounds, the predicted INSTI/HIV-1 IN CCD/viral
DNA complex was superimposed with the crystal structure of
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Figure 5. (a) Binding mode of compound 7b (yellow) in the HIV-1 RNase H active site. The active site is shown as transparent white surface and
ribbons. Amino acid side chains important for ligand binding are represented as sticks. Mg2+ metal ions are depicted as green spheres. (b)
Superimposition between our model of the INSTI/RNase H complex and the crystal structure of the full-length HIV-1 RT (p66 subunit) in complex
with an RNA:DNA hybrid (PDB code 4B3O). RNase H residues are represented as white surfaces. The DNA (red) and RNA (light blue) strands
are shown as ribbon and sticks. Compound 7b is depicted as yellow transparent surface and sticks.
that, as happened for IN, also in this case our reference
compound places its DKA branch so as to chelate the two
catalytic Mg2+ cations (Figure 5a). Remarkably, the DKA
moiety again adopts an orientation similar to the unbound
conformation of a closed analogue.46 In addition to the
chelation of the metal ions, the DKA branch H-bonds with the
side chain of N474. Furthermore, compound 7b establishes
favorable lipophilic interactions with the Cβ and Cγ carbons of
the Q475 through its quinolonyl nucleus. Additionally, the p-F-
benzyl group forms favorable contacts with the lipophilic
residues Q500 and W535.
Finally, the pyrrolidine ring is able to take additional contacts
with the Q475 and Y501 residues side chains. Docking of
compound 7a (Figure S3a in the Supporting Information)
shows that, as observed for IN, ester and acidic derivatives
feature analogous predicted binding modes. Thus, also in the
case of RNase H, the generally higher potency displayed by the
acidic compounds might be ascribed to a more tight
coordination of the metal ions by the DKA branch. Finally,
also the newly synthesized derivative 12i, which displays
interesting RNase H inhibitory properties, is predicted to bind
similarly to 7b (Figure S3b in the Supporting Information),
thus suggesting that our INSTIs would all share the same
binding position to the RNase H catalytic site.
derivative occupies through its 2-phenyl indole substituent a
lipophilic pocket shaped by W535, P537, and A538 residues.
This additional cleft might be thus explored by our next-
generation DKAs to achieve an enhanced RNase H inhibitory
profile to obtain dual IN/RNase H inhibitors.
CONCLUSIONS
■
We have designed, synthesized, and evaluated a series of novel
quinolinonyl diketo acid derivatives endowed with a “base-like”
moiety for their inhibition of HIV-1 IN. The compounds were
also tested against the RNase H function of the RT. We
identified nanomolar INSTIs that also exhibited low micro-
molar RNase H inhibition and antiviral properties. Our
molecular modeling study also provided new insights for the
future development of dual inhibitors of HIV-1 IN and RNase
H.
EXPERIMENTAL SECTION
Chemistry. General. Melting points were determined with a Buchi
■
̈
530 capillary apparatus and are uncorrected. Compounds purity were
always >95% determined by high pressure liquid chromatography
(HPLC). HPLC analysis were carried out with a Shimadzu LC-10AD
VP CTO-10AC VP. Column used was generally Discovery Bio Wide
Pore C18 (10 cm × 4.6 mm, 3 μm). Infrared (IR) spectra were
recorded on a PerkinElmer Spectrum-One spectrophotometer. 1H
NMR spectra were recorded on a Bruker AC 400 spectrometer. Merck
silica gel 60 F254 plates were used for analytical TLC. Developed plates
were visualized by UV light. Column chromatography was performed
on silica gel (Merck; 70−230 mesh). Concentration of solution after
reactions and extractions involved the use of a rotary evaporator
operating at reduced pressure of approximately 20 Torr. Analytical
results agreed to within 0.40% of the theoretical values. Dimethyl
sulfoxide-d6 99.9% (code 44,139-2) and deuterochloroform 98.8%
(code 41,675-4) of isotopic purity (Aldrich) were used. Solvents were
reagent grade and, when necessary, were purified and dried by
standard methods. Organic solutions were dried over anhydrous
sodium sulfate (Merck).
To better explain the RNase H inhibitory activity of our
compounds, we superimposed the predicted 7b/RNase H
complex with the recently solved crystal structure of the full
length HIV-1 RT in complex with a RNA:DNA hybrid51 (PDB
code 4B3O) (Figure 5b). This superimposition suggests that
binding of compound 7b would sterically hamper the hosting of
the RNA:DNA hybrid at the RNase H catalytic site, thus
preventing its hydrolysis.
Finally, to provide new hints for future lead optimization
studies, we compared the binding pose of our reference
compound 7b with that of two more potent inhibitors, namely
a naphthyridinone ligand (IC50 ≈ 0.11 μM)48 and a pyrimidinol
Microwave Irradiation Experiments. Microwave reactions were
conducted using a CEM Discover system unit (CEM. Corp.,
Matthews, NC). The machine consists of a continuous focused
microwave-power delivery system with operator selectable power
output from 0 to 300 W. The temperature of the contents of the vessel
was monitored using a calibrated infrared temperature control
mounted under the reaction vessel. All experiments were performed
using a stirring option whereby the contents of the vessel are stirred by
means of a rotating magnetic plate located below the floor of the
microwave cavity and a Teflon-coated magnetic stir bar in the vessel.
3-Acetyl-7-fluoroquinolin-4(1H)-one (8). Triethyl orthoformate
(4.0 g, 27 mmol), ethyl acetoacetate (3.5 g, 27 mmol), 3-fluoroaniline
carboxylic acid (IC50 = 0.23
0.01 μM)49 that were
cocrystallized with the HIV-1 RT enzyme at the RNase H
active site (PDB codes 3LP048 and 3QIP,49 respectively). From
this comparison, it turned out that, similarly to 7b, these
inhibitors chelate both the two Mg2+ cations, although adopting
a slightly different coordination geometry (Figure S4 in the
Supporting Information). On the other hand, due to size and
structural differences, the considered compounds and our
inhibitors establish different interaction patterns at the RNase
H active site. In particular, the pyrimidinol carboxylic acid
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(3.0 g, 27 mmol), and Dowtherm A (5.6 mL) were charged in a three-
necked flask equipped with a condenser and a Dean-Stark apparatus.
This mixture was stirred under argon atmosphere while the
temperature was increased to 95 °C in 1 h then gradually 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 three-necked flask
equipped with thermometer and a Dean-Stark apparatus and heated at
253−254 °C. After addition, the mixture was heated at the same
temperature for 2 h. Then the mixture was cooled at 90 °C, treated
with 2-propanol (10 mL), cooled at 30 °C, filtered, and washed with 2-
propanol and light petroleum ether to give pure derivatives 8 (41%
yield). Chemical and physical data of derivative 8 are reported in the
Supporting Information. For spectroscopic data see ref 19b.
3-Acetyl-7-fluoro-1-[(4-fluorophenyl)methyl]quinolin-4(1H)-
one (9). A mixture of derivative 8 (0.23 g, 1.1 mmol), 4-
fluorophenylmethyl bromide (610 mg, 3.3 mmol), and anhydrous
K2CO3 (210 mg, 1.5 mmol) in dry DMF (10 mL) was stirred at 100
°C for 1 h. After cooling, water was added (40 mL) and the precipitate
that formed was filtered, washed with water and light petroleum ether
in turn, and then dried under IR lamp to provide pure product 9 (29%
yield). Chemical and physical data of derivatives 9 are reported in the
Supporting Information. For spectroscopic data, see ref 19b.
General Procedure for the Synthesis of 7-(Base)-4(1H)-
quinolinones (10a−f,h). A mixture of 9 (2.7 g, 8.5 mmol), proper
amine (25.6 mmol), and TEA (0.8 g, 7.7 mmol) in dry DMF (40 mL)
was irradiated with microwave at 153 °C for 10 min (applied potency
100 W) in an open vessel equipped with a condenser. After cooling,
the reaction mixture was diluted with water (100 mL) and treated with
1 N HCl until pH 7. The solid that formed was collected by filtration.
Purification of crude product was performed by column chromatog-
raphy on silica gel (10:1 chloroform/ethanol as eluent) to give of pure
10a−f,h (14−78% yield). For spectroscopic, chemical, and physical
data of derivatives 10a−f,h, see the Supporting Information.
3-Acetyl-7-(dimethylamino)-1-[(4-fluorophenyl)methyl]-
quinolin-4(1H)-one (10g). A mixture of dimethylamine in H2O
(40%, 1.3 mmol) and quinolinone 9 (0.45 mmol) was irradiated with
microwave at 175 °C, 100 W, for 10 min. After cooling, the reaction
mixture was diluted with water. The formed precipitate was collected
by filtration thorugh a sintered glass septum and washed with water
and diethyl ether. Purification of crude product was performed by
column chromatography on silica gel (chloroform/ethanol 5:1 as
eluent) to give pure product 10g (52% yield). For spectroscopic,
chemical, and physical data of derivatives 10g, see the Supporting
Information.
3-Acetyl-7-[4-(3-chloropropyl)piperazin-1-yl]-1-[(4-
fluorophenyl)methyl]quinolin-4(1H)-one (10i). A suspension of
10h (500 mg, 1.14 mmol) in 3 mL of CCl4 and 3 mL of CH2Cl2 was
added triphenylphosphine (300 mg, 1.14 mmol). The mixture was
heated at reflux for 24 h. The reaction was monitored with TLC (10:1
chloroform/methanol as eluent). After this time, the reaction was
cooled at 25 °C, and the precipitate was filtered. The recovered
organic solution was evaporated to afford 400 mg of crude product.
Purification of crude product was performed by column chromatog-
raphy on silica gel (10:1 chloroform/methanol as eluent) to give of
pure 10i (48% yield). For spectroscopic, chemical, and physical data of
derivatives 10i, see Supporting Information.
General Procedure for the Synthesis of Diketo Esters 11a−
g,i. Freshly prepared sodium ethoxide (390 mg, 5.5 mmol) was added
into a well stirred mixture of the appropriate acetyl derivative 10a−g,i
(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, 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 IR lamp to afford the pure diketo esters 11a−
g,i. Yield (%), melting point (°C), recrystallization solvent, IR, and 1H
NMR are reported for each of the following compounds.
98%; 150−152 °C; washed with 2-propanol; IR ν 3347 (OH enol),
1
1711 (CO ester), 1666 (CO ketone) cm−1. H NMR (DMSO-
d6) δ 1.31 (t, 3H, CH3CH2), 3.45−3.55 (m, 4H, piperazine H), 3.25−
3.35 (m, 4H, piperazine H), 4.31 (q, 4H, CH2CH3), 5.71 (s, 2H, CH2
benzyl), 6.86 (s, 1H, quinolinone C8-H), 7.22−7.31 (m, 3H, benzene
H and quinolinone C6-H), 7.43−7.52 (m, 2H, benzene H), 8.00 (s,
1H, CH enol), 8.10 (d, 1H, quinolinone C5-H), 9.00 (s, 1H,
quinolinone C2-H). Anal. (C26H26FN3O5) C, H, F, N.
4-[1-[(4-Fluorophenyl)methyl]-7-(N-methylpiperazin-1-yl)-4-(1H)-
quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid Ethyl Ester
(11b). Yield 99%; 96−99 °C; washed with 2-propanol; IR ν 3381
(OH enol), 1713 (CO ester), 1605 (ketone) cm−1. 1H NMR
(DMSO-d6) δ 1.02 (s, 3H, CH3 piperazine), 1.22 (t, 3H, CH3CH2),
2.25−2.53 (m, 4H, piperazine H), 3.23−3.31 (m, 4H, piperazine H),
4.10 (q, 4H, CH2CH3), 5.54 (s, 2H, CH2 benzyl), 6.72 (s, 1H,
quinolinone C8-H), 7.05 (d, 1H, quinolinone C6-H), 7.11−7.20 (m,
2H, benzene H), 7.23−7.31 (m, 2H, benzene H), 8.07 (d, 1H,
quinolinone C5-H), 8.55 (s, 1H, CH enol), 8.61 (s, 1H, quinolinone
C2-H). Anal. (C27H28FN3O5) C, H, F, N.
4-[1-[(4-Fluorophenyl)methyl]-7-(N-ethylpiperazin-1-yl)-4-(1H)-
quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid Ethyl Ester (11c).
Yield 99%; 200 °C (dec); washed with 2-propanol; IR ν 3388 (OH
enol), 1711 (CO ester), 1606 (ketone) cm−1. 1H NMR (DMSO-d6)
δ 1.17 (t, 3H, J = 5.5 Hz, CH3CH2 piperazine), 1.21 (t, 3H, CH3CH2),
2.19 (q, 3H, J = 5.5 Hz, CH2CH3 piperazine), 2.30−2.40 (m, 4H,
piperazine H), 3.15−3.30 (m, 4H, piperazine H), 4.11 (q, 4H,
CH2CH3), 5.55 (s, 2H, CH2 benzyl), 6.72 (s, 1H, quinolinone C8-H),
7.01−7.07 (m, 2H, CH enol and quinolinone C6-H), 7.18−7.20 (m,
2H, benzene H), 7.22−7.30 (m, 2H, benzene H), 8.07 (d, 1H,
quinolinone C5-H), 8.54 (s, 1H, quinolinone C2-H). Anal.
(C28H30FN3O5) C, H, F, N.
4-[1-[(4-Fluorophenyl)methyl]-7-(N-acetylpiperazin-1-yl)-4-(1H)-
quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid Ethyl Ester
(11d). Yield 91%; 178−180 °C; washed with 2-propanol; IR ν 3409
(OH enol), 1716 (CO ester), 1604 (ketone) cm−1. 1H NMR
(DMSO-d6) δ 1.24 (t, 3H, CH3CH2), 2.03 (s, 3H, CH3
acetylpiperazine), 3.16−3.35 (m, 4H, piperazine H), 3.45−3.60 (m,
4H, piperazine H), 4.11 (q, 4H, CH2CH3), 5.56 (s, 2H, CH2 benzyl),
6.75 (s, 1H, quinolinone C8-H), 7.01 (s, 1H, CH enol), 7.05 (d, 1H,
quinolinone C6-H), 7.18−7.22 (m, 2H, benzene H), 7.28−7.32 (m,
2H, benzene H), 8.09 (d, 1H, quinolinone C5-H), 8.53 (s, 1H,
quinolinone C2-H). Anal. (C28H28FN3O6) C, H, F, N.
4-[1-[(4-Fluorophenyl)methyl]-7-(thiomorpholin-1-yl)-4-(1H)-qui-
nolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid Ethyl Ester (11e).
Yield 99%; 170 °C; washed with 2-propanol; IR ν 3388 (OH enol),
1
1711 (CO ester), 1606 (ketone) cm−1. H NMR (DMSO-d6) δ
1.21 (t, 3H, CH3CH2), 3.16−3.30 (m, 4H, thiomorpholine H), 3.65−
3.82 (m, 4H, thiomorpholine H), 4.11 (q, 4H, CH2CH3), 5.56 (s, 2H,
CH2 benzyl), 6.77 (s, 1H, quinolinone C8-H), 7.08−7.37 (m, 6H, CH
enol, quinolinone C6-H and benzene H), 8.10 (d, 1H, quinolinone
C5-H), 8.60 (s, 1H, quinolinone C2-H). Anal. (C26H25FN2O5S) C, H,
F, N, S.
4-[1-[(4-Fluorophenyl)methyl]-7-(morpholin-4-yl)-4-(1H)-quinoli-
non-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid Ethyl Ester (11f). Yield
76%; 175−177 °C; trituration on diethyl ether; IR ν 3388 (OH enol),
1
1711 (CO ester), 1606 (ketone) cm−1. H NMR (DMSO-d6) δ
1.21 (t, 3H, CH3CH2), 3.14−3.32 (m, 4H, morpholine H), 3.62−3.80
(m, 4H, morpholine H), 4.11 (q, 4H, CH2CH3), 5.56 (s, 2H, CH2
benzyl), 6.77 (s, 1H, quinolinone C8-H), 7.08−7.37 (m, 6H, CH enol,
quinolinone C6-H and benzene H), 8.10 (d, 1H, quinolinone, C5-H),
8.60 (s, 1H, quinolinone C2-H). Anal. (C26H25FN2O6) C, H, F, N.
4-[1-[(4-Fluorophenyl)methyl]-7-(dimethylamino)-4-(1H)-quinoli-
non-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid Ethyl Ester (11g). Yield
98%; 150 °C (dec); washed with 2-propanol; IR. ν 3345 (OH enol),
1
1710 (CO ester), 1636 (ketone) cm−1. H NMR (DMF-d7) δ 1.30
(t, 3H, CH3CH2), 3.00 (s, 6H, CH3 dimethylamine), 4.19 (q, 4H,
CH2CH3), 5.65 (s, 2H, CH2 benzyl), 6.52 (s, 1H, quinolinone C8-H),
6.88 (s, 1H, quinolinone C6-H), 7.18 (s, 1H, CH enol), 7.21−7.25 (m,
2H, benzene H), 7.45−7.48 (m, 2H, benzene H), 8.19 (d, 1H,
quinolinone C5-H), 8.70 (s, 1H, quinolinone C2-H). Anal.
(C24H23FN2O5) C, H, F, N.
4-[1-[(4-Fluorophenyl)methyl]-7-(piperazin-1-yl)-4-(1H)-quinoli-
non-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid Ethyl Ester (11a). Yield
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Article
4-[1-(4-[(4-Fluorophenyl)methyl]-7-[N-(3-chloroprop-1-yl)-piper-
azin-1-yl]-4-(1H)-quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid
Ethyl Ester (11i). 77%; 175−177 °C; washed with acetone; IR ν 3488
(OH enol), 1723 (CO ester), 1617 (ketone) cm−1. 1H NMR
(DMF-d7) δ 1.37 (t, 3H, CH3CH2), 1.98 (q, 2H, J = 5.5 Hz,
ClCH2CH2CH2N piperazine), 2.48−2.54 (m, 6H, piperazine H and
ClCH2CH2CH2N piperazine), 3.38−3.39 (m, 4H, piperazine H),
3.73−3.76 (q, 4H, CH2CH3), 4.35−4.39 (q, 2H, ClCH2CH2CH2N
piperazine), 5.87 (s, 2H, CH2 benzyl), 6.98 (s, 1H, quinolinone C8-
H), 7.22−7.29 (m, 3H, quinolinone C6-H and benzene H), 7.58−7.62
(m, 2H, benzene H), 8.10 (m, 2H, CH enol and quinolinone C5-H),
9.14 (s, 1H, quinolinone C2-H). Anal. (C29H31ClFN3O5) C, H, Cl, F,
N.
(m, 2H, benzene H), 7.22 (d, 1H, quinolinone C5-H), 8.78 (s, 1H,
quinolinone C2-H), 15.00 (br s, 2H, OH). Anal. (C24H21FN2O5S) C,
H, F, N, S.
4-[1-[(4-Fluorophenyl)methyl]-7-(morpholin-4-yl)-4-(1H)-quinoli-
non-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid (12f). Yield 99%; 187−
189 °C; washed with 2-propanol; IR ν 3500−2000 (OH), 1726 (C
1
O acid), 1600 (CO ketone) cm−1. H NMR (DMSO-d6) δ 3.17−
3.34 (m, 4H, morpholine H), 3.61−3.80 (m, 4H, morpholine H), 5.70
(s, 2H, CH2 benzyl), 6.83 (s, 1H, quinolinone C8-H), 7.10−7.29 (m,
3H, quinolinone C6-H and benzene H), 7.32−7.48 (m, 2H, benzene
H), 7.99 (br s, 1H, CH enol), 8.10 (d, 1H, quinolinone C5-H), 9.02
(s, 1H, quinolinone C2-H), 15.00 (br s, 2H, OH). Anal.
(C24H21FN2O6) C, H, F, N.
General Procedure for the Synthesis of Diketo Acids 12a−
g,i. A mixture of 1 N NaOH (6.5 mL) and the appropriate ester 11a−
g,i (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 yellow solid that formed was collected by filtration,
then washed with water, hot dry ethanol, and light petroleum ether to
afford pure acids 12a−g,i. Yield (%), melting point (°C),
4-[1-[(4-Fluorophenyl)methyl]-7-(dimethylamino)-4-(1H)-quinoli-
non-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid (12g). Yield 98%; 203−
205 °C; washed with 2-propanol; IR ν 3500−2000 (OH), 1735 (C
1
O acid), 1620 (CO ketone) cm−1. H NMR (DMF-d7) δ 3.22 (s,
6H, CH3 dimethylamine), 6.00 (s, 2H, CH2 benzyl), 6.82 (s, 1H,
quinolinone C8-H), 7.14 (s, 1H, quinolinone C6-H), 7.42−7.46 (m,
2H, benzene H), 7.70−7.85 (m, 2H, benzene H), 8.36 (d, 1H,
quinolinone C5-H), 8.41 (s, 1H, CH enol), 9.27 (s, 1H, quinolinone
C2-H), 15.00 (br s, 2H, OH). Anal. (C22H19FN2O5) C, H, F, N.
4-[1-[(4-Fluorophenyl)methyl]-7-[N-(3-chloroprop-1-yl)piperazin-
1-yl]-4-(1H)-quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid
(12i). Yield 100%; 175−177 °C; washed with THF; IR ν 3410−
2000 (OH), 1717 (CO ester), 1655 (ketone) cm−1. 1H NMR
(DMF-d7) δ 2.18 (q, 2H, J = 6.0 Hz, ClCH2CH2CH2N piperazine),
2.73−2.79 (m, 6H, piperazine H and ClCH2CH2CH2N piperazine),
3.50−3.65 (m, 4H, piperazine H), 3.92 (q, 2H, J = 6.0 Hz,
ClCH2CH2CH2N piperazine), 6.03 (s, 2H, CH2 benzyl), 7.16 (s,
1H, quinolinone C8-H), 7.38−7.45 (m, 3H, quinolinone C6-H and
benzene H), 7.74−7.78 (m, 2H, benzene H), 8.37−8.39 (m, 2H, CH
enol and quinolinone C5-H), 9.27 (s, 1H, quinolinone C2-H), 15.00
(br s, 2H, OH). Anal. (C27H27ClFN3O5) C, H, Cl, F, N.
1
recrystallization solvent, IR, H NMR are reported for each of the
following compounds.
4-[1-[(4-Fluorophenyl)methyl]-7-(piperazin-1-yl)-4-(1H)-quinoli-
non-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid (12a). Yield 98%; 224
°C; washed with 2-propanol; IR ν 3500−2000 (OH), 1700 (CO
1
acid), 1649 (CO keton) cm−1. H NMR (DMSO-d6) δ 3.30−3.37
(m, 4H, piperazine H), 3.41−3.50 (m, 4H, piperazine H), 5.64 (s, 2H,
CH2 benzyl), 6.82 (s, 1H, quinolinone C8-H), 7.12−7.20 (m, 3H,
benzene H and quinolinone C6-H), 7.37−7.41 (m, 2H, benzene H),
8.08 (d, 1H, quinolinone C5-H), 8.42 (s, 1H, CH enol), 8.75 (s, 1H,
C2-H quinolinone), 15.00 (br s, 2H, OH). Anal. (C24H22FN3O5) C,
H, F, N.
4-[1-[(4-Fluorophenyl)methyl]-7-(N-methylpiperazin-1-yl)-4-(1H)-
quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid (12b). Yield 99%;
224 °C; washed with 2-propanol; IR ν 3500−2000 (OH), 1700 (C
O acid), 1599 (CO ketone) cm−1. 1H NMR (DMSO-d6) δ 2.38 (s,
3H, CH3 piperazine), 2.61−2.75 (m, 4H, piperazine H), 3.21−3.35
(m, 4H, piperazine H), 5.67 (s, 2H, CH2 benzyl), 6.81 (s, 1H,
quinolinone C8-H), 7.29−7.08 (m, 3H, benzene H, quinolinone C6-
H), 7.31−7.48 (m, 2H, benzene H); 7.69 (br s, 1H, CH enol), 8.07 (d,
1H, quinolinone C5-H), 8.93 (s, 1H, C2-H quinolinone), 15.00 (br s,
2H, OH). Anal. (C25H24FN3O5) C, H, F, N.
Biological Methods. HIV-1 IN Inhibition. HIV-1 IN gel-based
assays were carried out as previously published.52
HIV-1 RT RNase H Inhibition. IC50 values were determined as
previously reported53 using an 18-nt 3′-fluorescein-labeled RNA
annealed to a complementary 18-nt 5′-dabsyl-labeled DNA. To a
96-well plate was added 1 μL of each inhibitor (in DMSO), followed
by 10 μL of the appropriate RT (15−80 ng/mL) in reaction buffer.
Hydrolysis was initiated by adding 10 μL of RNA/DNA hybrid (2.5
μM). Final assay conditions were 50 mM Tris-HCl, pH 8.0, 60 mM
KCl, 10 mM MgCl2, 1% DMSO, 150−800 ng of RT, 250 nM
substrate, and increasing concentrations of inhibitor. Wells containing
only DMSO were used as negative control. Plates were incubated at 37
°C in a Spectramax Gemini EM fluorescence spectrometer for 10 min,
and fluorescence (λex = 485 nm; λem = 520 nm) was measured at 1 min
intervals such that linear initial rates could be measured in the
presence (vi) and absence (v0) of inhibitor. Percent inhibition was
calculated as 100(v0 − vi)/v0, and plotted against log[I]. IC50 values
were determined using Prism5 (GraphPad Software). All assays were
performed in triplicate.
4-[1-[(4-Fluorophenyl)methyl]-7-(N-ethylpiperazin-1-yl)-4-(1H)-
quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid (12c). Yield 99%;
> 240 °C; washed with 2-propanol; IR ν 3500−2000 (OH), 1700
1
(CO acid), 1578 (CO ketone) cm−1. H NMR (DMSO-d6) δ
1.06 (t, 3H, J = 5.5 Hz, CH3CH2 piperazine), 2.22 (q, 3H, J = 5.5 Hz,
CH2CH3 piperazine), 2.28−2.50 (m, 4H, piperazine H), 2.70−3.85
(m, 4H, piperazine H), 5.65 (s, 2H, CH2 benzyl), 6.78 (s, 1H,
quinolinone C8-H), 7.12 (s, 1H, quinolinone C6-H), 7.10−7.20 (m,
2H, benzene H), 7.37 (m, 2H, benzene H), 7.69 (br s, 1H, CH enol),
8.06 (d, 1H, quinolinone C5-H), 8.90 (s, 1H, quinolinone C2-H),
15.00 (br s, 2H, OH). Anal. (C26H26FN3O5) C, H, F, N.
HIV-1 RT Polymerase Inhibition. HIV-1 RT polymerase assays were
4-[1-[(4-Fluorophenyl)methyl]-7-(N-acetylpiperazin-1-yl)-4-(1H)-
quinolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid (12d). Yield 90%;
195−197 °C; washed with acetonitrile; IR ν 3392−2000 (OH), 1716
carried out as previously reported.13b
HIV-1 Replication Inhibition. Compounds antiviral activity was
determined in a cell-based assay according to the procedure described
previously54 and modified as follows. HeLa-CD4-LTR-β-gal cells were
maintained in DMEM with 10% serum and 0.5 mg/mL G418. The day
prior experimentation, 96-well plates were prepared to contain 10000
cells per well in 100 μL of DMEM medium complemented with 10%
serum. On day one, each drug was serial diluted directly on cells
following a 3-fold dilution over 6 points and each well was then filled
to 200 μL with either fresh medium or concentrated viral supernatant
(HIV-1(IIIB), Advanced Biotechnologies Inc.). The highest com-
pound concentration tested was 50 μM. On day two, cells were
washed three times with PBS before adding 200 μL of a solution
containing 50 mM Tris-HCl pH 7.5, 100 mM β-mercaptoethanol,
0.05% Triton X100, and 5 mM 4-methyl-umbelliferyl-β-D-galactopyr-
anoside (4-MUG, Sigma). On day three, sealed plates were read in a
1
(CO acid), 1599 (CO ketone) cm−1. H NMR (DMSO-d6) δ
2.05 (s, 3H, CH3 acetylpiperazine), 3.25−3.42 (m, 4H, piperazine H),
3.50−3.69 (m, 4H, piperazine H), 5.71 (s, 2H, CH2 benzyl), 6.81 (s,
1H, quinolinone C8-H), 7.17−7.28 (m, 3H, benzene H and
quinolinone C6-H), 7.30−7.50 (m, 2H, benzene H), 7.97 (br s, 1H,
CH enol), 8.10 (d, 1H, quinolinone C5-H), 9.01 (s, 1H, quinolinone
C2-H), 15.00 (br s, 2H, OH). Anal. (C26H24FN3O6) C, H, F, N.
4-[1-[(4-Fluorophenyl)methyl]-7-(thiomorpholin-1-yl)-4-(1H)-qui-
nolinon-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid (12e). Yield 30%;
190 °C; washed with 2-propanol; IR ν 3500−2000 (OH), 1711 (C
1
O acid), 1606 (CO ketone) cm−1. H NMR (DMSO-d6) δ 2.43−
2.60 (m, 4H, thiomorpholine H), 3.56−3.75 (m, 4H, thiomorpholine
H), 5.58 (s, 2H, CH2 benzyl), 6.66 (s, 1H, quinolinone C8-H), 7.10−
7.16 (m, 4H, CH enol, quinolinone C6-H and benzene H), 7.32−7.35
3230
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Article
SpectraMax GEMINI-XS (Molecular Devices) with λex/em = 360/460
nm.
All of the pictures were rendered with the UCSF Chimera package
from the Resource for Biocomputing, Visualization, and Informatics at
the University of California, San Francisco.60
Cellular Toxicity. Similarly to the antiviral assays, plates were
prepared with 10,000 HeLa-CD4-LTR-β-gal cells per well and a serial
dilution of compounds in 100 μL. After 24 h of culture, 100 μL of
ATPlite reagent (PerkinElmer) was added to each well. After 5 min at
room temperature, plates’ luminescence was quantified using an
EnVision multilabel reader (PerkinElmer) according to manufacturer’s
instructions.
Homology Modeling. A novel homology model of HIV-1 IN
CCD/viral DNA complex was built using Schrodinger Prime (version
3.0)43 software accessible through the Maestro interface.55 To
construct the model, the crystal structure of full-length IN from
PFV in complex with raltegravir41 (PDB code 3OYA) was used as
template. The sequence alignment used for the homology modeling
was based on the secondary structure alignment of the template and
the HIV-1 IN CCD (PDB code 1QS4)26 on the Cα positions of the
active-site residues Asp64, Asp116, and Glu152 (DDE motif) to
identify the structure conserved regions according to the scheme
suggested by Tang and co-workers.44 Prior to model building, bases of
the 19-mer PFV DNA were mutated in order to obtain the HIV-1
retroviral DNA sequence (GenBank: AAC37875.1). The newly built
model was the subjected to Prime refinement protocol in order to
optimize the active site loop and amino acidic side chains
conformation.
ASSOCIATED CONTENT
* Supporting Information
■
S
Analyses of compounds 8, 9, 10a−i, 11a−g,i, 12a−g,i and
molecular modeling. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*For R.C.: phone, +39-06-49693247; fax, +39-06-49913133; E-
mail, roberta.costi@uniroma1.it.
■
*For C.M.: phone, +1-301-435-2463; fax, +1-301-402-0752. E-
mail, marchand@nih.gov.
Author Contributions
The manuscript was written through contribution of all authors.
All authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
Ligands Setup. The 3D structures of all of the compounds were
generated with the Maestro Build Panel55 and were then submitted to
Polak−Ribiere conjugate gradient minimization (0.0005 kJ/(Å mol)
convergence) using MacroModel (version 9.9).56 DKAs were modeled
in their enolic tautomeric form because it has been clearly established
that such compounds mainly exist in this form in solution and also
given the influence of the two metal cations in the binding site.57
Proteins Setup. The HIV-1 CCD/DNA model and the RNase H
crystal structure (PDB code 3QIP) were prepared for docking
calculations using the “Protein Preparation Wizard” panel of
ACKNOWLEDGMENTS
■
We thank the Italian MIUR for financial support, ISS 40H4,
PRIN 2010-2011 (2010W2KM5L_002). R. Di Santo and R.
Costi thank the FP7 CHAARM project for support. This work
was also supported by the NIH Intramural Research Program,
Center for Cancer Research, National Cancer Institute, and by
NIH grants from the AIDS Intramural Targeted Program
(IATAP).
Schrodinger 2010 molecular modeling package.55 In particular, using
̈
the “preprocess and analyze structure” tool, the bond orders and
disulfide bonds were assigned, all the hydrogen atoms were added, and
all the water molecules in a distance greater than 5 Å from any
heterogroup were deleted; in addition, both Mn2+ ions in the RNase H
crystal structure were replaced with Mg2+ cations. Using Epik 2.0, a
prediction of the side chains heterogroups ionization and tautomeric
states was performed.58 An optimization of the hydrogen-bonding
network was performed using the “H-bond assignment” tool. Finally,
using the “impref utility”, the positions of the hydrogen atoms were
optimized by keeping all the heavy atoms in place.
ABBREVIATIONS USED
■
HAART, highly active antiretroviral therapy; IN, integrase; RT,
reverse transcriptase; RNase H, ribonuclease H; INSTI, strand
transfer IN inhibitor; DKA, diketo acid; SI, selectivity index;
SAR, structure−activity relationship; CCD, catalytic core
domain; PFV, prototype foamy virus; TCC, target capture
complex; IR, infrared
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