Novel quinolinonyl diketo acid derivatives as HIV-1 integrase inhibitors: Design, synthesis, and biological activities

Article abstract of DOI:10.1021/jm8001422

Novel quinolinonyl diketo acids were designed to obtain integrase (IN) inhibitors selectively active against the strand transfer (ST) step of the HIV integration process. Those new compounds are characterized by a single aryl diketo acid (DKA) chain in co

Full text of DOI:10.1021/jm8001422

4744
J. Med. Chem. 2008, 51, 4744–4750
Novel Quinolinonyl Diketo Acid Derivatives as HIV-1 Integrase Inhibitors: Design, Synthesis,
and Biological Activities
Roberto Di Santo,*,§ Roberta Costi,§ Alessandra Roux,§ Gaetano Miele,§ Giuliana Cuzzucoli Crucitti,§ Alberto Iacovo,§
Federica Rosi,§ Antonio Lavecchia,‡ Luciana Marinelli,‡ Carmen Di Giovanni,‡ Ettore Novellino,‡ Lucia Palmisano,#
Mauro Andreotti,# Roberta Amici,# Clementina Maria Galluzzo,# Lucia Nencioni,| Anna Teresa Palamara,| Yves Pommier,† and
Christophe Marchand*,†
Istituto PasteursFondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, UniVersita` di Roma “La Sapienza”, P.le
A. Moro 5, I-00185 Roma, Italy, Dipartimento di Chimica Farmaceutica e Tossicologica, UniVersita` di Napoli “Federico II”, Via D.
Montesano 49, I-80131 Napoli, Italy, Dipartimento del Farmaco, Istituto Superiore di Sanita`, V.le Regina Elena 299, I-00161 Roma, Italy,
Dipartimento di Scienze di Sanita` Pubblica, UniVersita` di Roma “La Sapienza”, P.le A. Moro 5, I-00185 Roma, Italy, and Laboratory of
Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Building 37, Room 5068,
Bethesda, Maryland 20892-4255
ReceiVed February 10, 2008
Novel quinolinonyl diketo acids were designed to obtain integrase (IN) inhibitors selectively active against
the strand transfer (ST) step of the HIV integration process. Those new compounds are characterized by a
single aryl diketo acid (DKA) chain in comparison to 4, a bifunctional diketo acid reported by our group as
an anti-IN agent highly potent against both the 3′-processing and ST steps. Compound 6d was the most
potent derivative in IN enzyme assays, while 6i showed the highest potency against HIV-1 in acutely infected
cells. The selective inhibition of ST suggested the newly designed monofunctional DKAs bind the IN-DNA
acceptor site without affecting the DNA donor site.
Introduction
IN catalyzes the insertion of the viral cDNA (generated by
reverse transcription of the viral RNA) into the host cell genome.
Integration occurs via a sequence of reactions, which start with
the IN-mediated cleavage of terminal dinucleotide from the 3′-
end of the viral cDNA (termed “3′-processing”, 3′-P) shortly
after reverse transicription in the cytoplasm. Following transfer
of the resulting processed viral cDNA into the nucleus, IN
catalyzes the insertion of both ends into target cellular host
DNA. That second reaction is referred as “strand transfer” (ST).4
In the past 15 years, a range of natural and synthetic compounds
have been identified as inhibitors of recombinant IN enzyme in
biochemical assays. Interestingly, polyhydroxylated aromatics
anddiketocompoundswereamongthefirstinhibitorsidentified.3,7–9
However, those early polyhydroxylated derivatives were later
demonstrated to inhibit viral entry or to be too toxic to be
pursued as therapeutic IN inhibitors.10 More recently, the Merck
and Shionogi companies discovered aryl diketo acid (DKA)
derivatives as selective anti-HIV agents that block the viral
replication cycle via IN inhibition in vivo. Those compounds
are typified by 1-(5-chloroindol-3-yl)-3-hydroxy-3-(2H-tetrazol-
5-yl)propenone (5CITEP, 1) synthesized by Shionogi & Co.
Ltd.11 and by the pyrrole derivative L-731,988 (2) developed
by Merck Research Laboratories12 (Figure 1). They are char-
acterized by their ability to preferentially inhibit ST versus 3′-
P. Chemically, DKA is characterized by a diketo acid moiety
(R and γ ketone and a carboxylic acid), which is believed to be
essential for the inhibitory activity, although the carboxylic
group can be effectively replaced by a bioisoster azole ring
(triazole, tetrazole) (i.e., 1)11 and the 1,3-diketo acid moiety can
be mimicked by a 8-hydroxy-[1,6]naphthyridine ring13 (i.e.,
compound 3, Figure 1).
Three different classes of chemotherapeutic agents are gener-
ally combined to block the replication of human immunodefi-
ciency virus type 1 (HIV-1a) responsible for AIDS and to
prevent the occurrence of resistance: reverse transcriptase
inhibitors (RTI), protease inhibitors (PRI), and fusion inhibitors.
This widespread triple combination therapy is referred to as
HAART (highly active antiretroviral therapy).1 HAART ef-
fectively inhibits HIV replication to such an extent that the virus
becomes undetectable in the blood. However, it fails to eradicate
viruses that are integrated in the host genome or that persist in
cellular and anatomical “reservoirs”. In addition, prolonged drug
exposure led to HIV drug resistance, thus reducing patients’
therapeutically available options.2 The above considerations and
the toxicity of a number of antiretroviral agents have fueled
the discovery of drugs against additional targets. Among them,
HIV integrase (IN), which has no cellular counterpart, has been
intensely studied over the past 15 years.3–5 IN has recently been
fully validated as a therapeutic target with the first FDA
approved IN inhibitor raltegravir.6
* To whom correspondence should be addressed. For R.D.S.: phone and
fax, +39-06-49913150; e-mail, roberto.disanto@uniroma1.it. For C.M.:
phone, +1-301-435-2463; fax, +1-301-402-0752; e-mail, marchand@
nih.gov.
§
Dipartimento di Chimica e Tecnologie del Farmaco, Universita` di Roma
“La Sapienza”.
‡
Universita` di Napoli “Federico II”.
Istituto Superiore di Sanita`.
Dipartimento di Scienze di Sanita` Pubblica, Universita` di Roma “La
#
|
Sapienza”.
†
National Cancer Institute, National Institutes of Health.
a Abbreviations: HIV-1, human immunodeficiency virus type 1; RTI,
reverse transcriptase inhibitors; PRI, protease inhibitors; HAART, highly
active antiretroviral therapy; IN, integrase; 3′-P, 3′-processing; ST, strand
transfer; DKA, aryl diketo acid; 5CITEP, 1-(5-chloroindol-3-yl)-3-hydroxy-
3-(2H-tetrazol-5-yl)propenone; HTECL, high throughput electrochemilu-
minescent; BDKA, bifunctional diketo acid; SAR, structure-activity
relationship.
Recent studies on quinolinonyl diketo acid derivatives led
us to discover the bifunctional compound 4 as a potent IN
inhibitor for both 3′-P and ST.14 Moreover, 4 inhibits HIV-1
replication in acutely infected cells.14 Docking studies on the
binding mode of 4 to the IN catalytic site also suggested a
10.1021/jm8001422 CCC: $40.75
2008 American Chemical Society
Published on Web 07/23/2008

Quinolinonyl Diketo Acid DeriVatiVes as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4745
Scheme 1a
Figure 1. Structures of HIV-1 IN inhibitors belonging to the mono-
and bifunctional DKA class and related 8-hydroxy[1,6]naphthyridine
bioisoster.
a Reagents and conditions: (a) ethyl orthoformate, ethyl acetoacetate,
Dowtherm A, 95-254 °C, 8 h; (b) 4-fluorobenzyl bromide, K2CO3, DMF,
100 °C, 1 h; (c) diethyl oxalate, C2H5ONa, THF, room temp, 2 h; (d) 1 N
NaOH, THF/CH3OH, room temp, 40 min.
Scheme 2a
Figure 2. Structures of newly designed monofunctional quinolinonyl
diketo acid derivatives as HIV-1 IN inhibitors.
peculiar interaction of the drug involving both the acceptor and
the donor DNA binding sites of the enzyme. This hypothesis
was confirmed by our recent cross-linking experiment studies
that pointed out a specific interaction of 4 with K156 and K159
amino acid residues of the IN catalytic core domain.15 This
binding mode could account for the high potency of 4 against
both 3′-P and ST.
The aim of the present project was the design of new
quinolinone derivatives endowed with a selective activity against
ST. This project could provide new information regarding the
interactions of quinolinonyl diketo acids with the IN active site
and consequently increase our knowledge about the catalytic
mechanisms of IN, which in the absence of structural informa-
tion on IN-DNA and drug molecular structures are still far
from being totally elucidated. In the present manuscript, we
describe novel quinolinonyl diketo acid derivatives 5a-i, 6a-i,
7a,b, and 8a,b designed by replacement of the 6-diketo acid
chain of 4, responsible for binding to the donor DNA binding
site, with smaller substituents in the 6-, 7-, or 8-position of the
quinolinone ring (Figure 2). These substituents should exhibit
reduced binding to the donor viral cDNA site due to their small
size/length or to their limited ability to form hydrogen bonds.
Thus, these structural modifications should provide novel
quinolinonyl diketo acid derivatives with increased ST selectivity.
a Reagents and conditions: (a) diethyl oxalate, C2H5ONa, THF, room
temp, 2 h; (b) 1 N NaOH, THF/CH3OH, room temp, 40 min.
Scheme 3a
a Reagents and conditions: (a) pyrrolidine, Et3N, DMF, microwave, 100
W, 153 °C, 10 min; (b) diethyl oxalate, C2H5ONa, THF, room temp, 2 h;
(c) 1 N NaOH, THF/CH3OH, room temp, 40 min.
Results and Discussion
Chemistry. Synthesis of derivatives 5a-i, 6a-i, 7a,b, and
8a,b is outlined in Schemes 1–3. 3-Acetyl-4(1H)-quinolinones
9a-h were prepared by reaction of the proper aniline 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.16 Then, 9a-h were alkylated with 4-fluorobenzyl-
bromide in alkaline medium (K2CO3) to give the N-1 substituted
quinolones 10a-h. These compounds were condensed with
diethyl oxalate in the presence of sodium ethoxide to provide
ethyl esters 5a-h, which were in turn hydrolyzed with 6 N
NaOH to afford the corresponding acids 6a-h (Scheme 1).
Synthesis of 1-unsubstituted derivatives 7a,b and 8a,b was
achieved in a similar fashion by coupling of 4-quinolinones 9e,f
with diethyl oxalate in the presence of the sodium ethoxide,
followed by alkaline hydrolysis of the resulting ethyl esters 7a,b
to provide acids 8a,b (Scheme 2).

4746 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Di Santo et al.
Table 1. Chemical, Physical, and Analytical Data of Derivatives 9a-h and 10a-i
compd
R6
R7
R8
mp (°C)
recryst solventa
yield (%)
analysis
9ab
9b
9c
H
F
H
H
F
H
H
H
F
H
H
Cl
H
H
H
H
F
H
H
Cl
H
H
242-244
>300
270-271
268-270
>300
>300
>300
114-115
213-214
234-235
186-187
193-194
214-216
185-187
215-217
235-237
175-177
a
b
c
b
b
b
b
d
d
e
e
f
e
e
g
d
e
56
29
41
40
29
96
55
19
65
94
100
78
92
73
92
49
56
C, H, N (C11H9NO2)
C, H, N, F (C11H8FNO2)
C, H, N, F (C11H8FNO2)
C, H, N, F (C11H8FNO2)
H
H
Cl
H
H
Cl
H
F
H
H
Cl
H
H
Cl
H
9d
H
H
Cl
H
Cl
H
H
F
H
H
Cl
H
Cl
9eb
9fb
9gb
9h
C, H, N, Cl (C11H8ClNO2)
C, H, N, Cl (C11H8ClNO2)
C, H, N, Cl (C11H8ClNO2)
C, H, N, Cl (C11H7Cl2NO2)
C, H, N, F (C18H14FNO2)
C, H, N, F (C18H13F2NO2)
C, H, N, F (C18H13F2NO2)
C, H, N, F (C18H13F2NO2)
C, H, N, Cl, F (C18H13ClFNO2)
C, H, N, Cl, F (C18H13ClFNO2)
C, H, N, Cl, F (C18H13ClFNO2)
C, H, N, Cl, F (C18H12Cl2FNO2)
C, H, N, F (C22H21FN2O2)
10a
10b
10c
10d
10e
10f
10g
10h
10i
1-pyrrolidinyl
a Recrystallization solvents: (a) aceton, (b) ethanol, (c) isopropanol, (d) toluene, (e) toluene/cyclohexane, (f) benzene/cyclohexane, (g) benzene. b Reference
17.
Derivatives 5i and 6i were synthesized as described in Scheme
2, HTECL assay) as their unsubstituted counterpart 6a. Interest-
ingly, the anti-IN activities appear to be influenced by the
position of the halogen. Indeed, inhibitory activities decrease
in the following order: 8-F(Cl) ∼ 7-F(Cl) > 6-F(Cl) and 7-Cl
> 8-Cl > 6-Cl. The introduction of a second chlorine atom in
the structure of the potent inhibitor 6f led to 6h, which is
approximately 1.5 times less potent than the parent counterpart.
Similarly, replacement of the chlorine atom of 6f with a
pyrrolidine ring led to 6i, which is approximately 1.5 times less
potent than the parent compound. In conclusion, the activities
of compounds with a substituent in the 7-position decrease in
the following order: Cl > F > 1-pyrrolidinyl > H (Table 2,
HTECL assay).
Compounds 5a-i and 6a-i also inhibited integrase with
similar potency in the presence of Mg2+ or Mn2+ (data not
shown). The activity in the presence of Mg2+ in addition to the
selectivity for ST represents a trademark of the most potent
DKAs active against HIV replication in cells.19
Cell-Based Assays. Cytotoxicity and antiviral activities of
compounds 5a-i, 6a-i, 7a,b, and 8a,b are presented in Table
2. Among the tested compounds, derivatives 5a,i, and 6a,e,h,i
show EC50 < 50 µM when tested against HIV-1-infected H9/
HTLVIIIB cells. In particular, 5i and 6a,h,i are active in the
micromolar range, with SI ranging from 48.8 to >1176. The
most potent derivative of this series is 6i (EC50 ) 0.17 µM and
SI > 1176). Compound 6i is in fact more potent than reference
derivatives 2-4. In general, all the active compounds were
characterized by low cytotoxicity against human histiocytic
lymphoma (U937) cell line, showing CC50 values in the range
of 99 to >200 µM.
3. Pyrrolidinyl derivative 10i was obtained in few minutes with
good yields by substitution of fluorine atom of 10c with
pyrrolidine in the presence of NEt3, under microwave irradiation.
10i was then condensed with ethyl oxalate, and the ester 5i that
formed was hydrolyzed to afford the required acid 6i. Chemical,
physical, and analytical data of intermediates 9a-h and 10a-i
are reported in Table 1, while spectroscopic data are shown in
Supporting Information. Data for final products 5a-i, 6a-i,
7a,b, and 8a,b are listed in Experimental Section.
Evaluation of Biological Activities. In Vitro Assays.
Derivatives 5a-i, 6a-i, 7a,b, and 8a,b were tested in vitro for
ST inhibition in the presence of magnesium (Mg2+) using a
recently described high throughput electrochemiluminescent
(HTECL) assay.14 IC50 values were generated from duplicate
experiments (Table 2). Compounds 5a-i, 6a-i, 7a,b, and 8a,b
were also tested for ST and 3′-P using gel-based assays carried
out in the presence of Mg2+ (Figure 3A). IC50 values were
calculated using dose response curves (Figure 3B) and are
summarized in Table 2. The newly synthesized DKAs exhibited
high potency against IN with high selectivity against ST, thus
confirming the hypothesis that the removal of the diketo acid
branch in the 6-position of the quinolinone ring of 4 can lead
to ST-selective inhibitors (6a-i showed 3′-P/ST ratios ranging
from 45 to 667). The acid derivatives 6a-i were more potent
than the corresponding esters 5a-i (Figure 3A and Figure 3B,
compare compounds 6h and 5h), and the 1-p-F-benzyl substi-
tuted quinolinones (5e,f and 6e,f) were markedly more active
than their unsubstituted counterparts (7a,b and 8a,b). This trend
is consistent with the results obtained previously on the
bifunctional DKA (BDKA) series.14 In particular, the relevant
role played by the p-F-benzyl moiety at the 1-position of the
quinolinoneringwaspreviouslynotedfortheDKAderivatives.12,18
The newly synthesized acid derivatives 6a-i were potent
inhibitors, showing IC50 values in the range of 18-40 nM for
the ST step (Table 2, HTECL assay). The most active compound
of this series is 6d with a ST IC50 value of 18 nM, which is
comparable to reference drugs 2 and 3. Removal of the diketo
acid chain from the 6-position of our BDKA lead molecule
(compound 4)14 led to compound 6a that retains the potent anti-
IN activity of the parent derivative with an IC50 value of 40
nM for ST (Table 2, HTECL assay). However, 6a is a more
ST selective inhibitor than 4.14 Introduction of a halogen (F or
Cl) in the 6-, 7-, or 8-position of 6a led to compounds 6b-g,
which show similar anti-IN activities (IC50 ) 18-30 nM, Table
Conclusions
We designed, synthesized, and tested a series of novel
quinolinonyl diketo acid derivatives in both enzyme- and cell-
based assays as anti-HIV-1 agents to selectively target the ST
step of integration. We were able to generate such new
compounds, which are potent ST-selective IN inhibitors and
active against HIV-1 replication in acutely infected cells. These
compounds could be useful tools to study the mechanism of
action of HIV-1 IN.
Experimental Section
Chemistry. General. Melting points were determined on a Buchi
530 melting point apparatus and are uncorrected. Infrared (IR)
spectra were recorded on a Perkin-Elmer Spectrum-one spectro-

Quinolinonyl Diketo Acid DeriVatiVes as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4747
Table 2. Cytotoxicity, Antiviral, and Anti-Integrase Activities of Derivatives 5a-i, 6a-i, 7a,b, and 8a,b
a
anti-IN activity, IC50
antiviral activity
d
e
compd
R6
R7
R8
X
Mg2+ b
Mg2+ c
Mg2+ c
CC50
99
EC50
SIf
2.8
5a
5b
5c
5d
5e
5f
5g
5h
5i
6a
6b
6c
6d
6e
6f
6g
6h
6i
H
F
H
H
F
H
H
Cl
H
Cl
Pyh
H
H
F
H
H
Cl
H
Cl
Pyh
H
Cl
H
Cl
H
H
H
F
H
H
Cl
H
H
H
H
H
F
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
H
H
H
H
H
H
H
H
H
0.67
0.58
1.3
26
21
14
32
3.9
2.3
0.040
0.030
0.020
0.018
0.028
0.019
0.023
0.033
0.028
3.3
0.79, 0.44
0.23, 0.44
1.2, 2.9
5.2
3.0
3.6
5.2
1.8
2.7
0.06, 0.16
0.14
0.050
0.16
0.40
0.11
0.51
0.075
0.18
2.1
224
56
135
34.8
>50
>50
>50
ntg
>333
>333
>333
>333
>333
>333
135
110
14
2.3
4.4
16
6.4
4.0
44
11, 34
14, 9.0
120
>333
166
>200
>200
>200
ntg
H
H
Cl
H
H
Cl
H
H
F
H
H
Cl
H
H
Cl
H
Cl
H
Cl
H
ntg
ntg
>200
>50
>50
4.1
72
>200
>200
>200
>200
>200
>200
>200
>200
156
48.8
>171
1.17
>50
>50
>50
46.1
>50
>50
3.2
H
H
Cl
H
H
4.3
62.5
>1176
>200
0.17
7a
7b
8a
8b
1i
C2H5
C2H5
H
ntg
ntg
>111
>111
37
ntg
ntg
ntg
ntg
H
17
>333
ntg
ntg
2.1
2i
0.05
0.01
0.016
1.0
3i
0.39j
4.29
4
0.017
0.44
>200
>47
a Inhibitory concentration 50% (µM) determined from dose response curves. b Experiments performed in duplicate using the high throughput
electrochemiluminescent (HTECL) assay (ST assay in the presence of Mg2+). c Experiments performed in a gel-based assay in the presence of Mg2+ (see
Figure 3). d Cytotoxic concentration 50% (µM). e Effective concentration 50% (µM). f Selectivity index ) CC50/EC50. g nt: not tested. h Py ) 1-pyrrolidinyl.
i Literature data; see refs 9–11. j This data is referred to EC95.
photometer. 1H NMR spectra were recorded at 400 MHz on a
Bruker AC 400 Ultrashield spectrometer. Merck silica gel 60 F254
plates were used for analytical TLC. Developed plates were
visualized by UV light. Column chromatographies were performed
on silica gel Merck 70-230 mesh. Solvents were reagent grade
and, when necessary, were purified and dried by standard methods.
Concentration of solutions after reactions and extractions involved
the use of a rotatory evaporator (Bu¨chi) operating at a reduced
pressure of approximately 20 Torr. Organic solutions were dried
over anhydrous sodium sulfate. Analytical results agreed to within
(0.40% of the theoretical values.
Microwave Irradiation Experiments. Microwave reactions
were conducted using a CEM Discover synthesis 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.
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 water separator 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 isopropanol
(10 mL), cooled at 30 °C, filtered, and washed with isopropanol
and light petroleum ether in turn to give pure derivatives 9a-h.
Chemical, physical, and analytical data of derivatives 9a-h are
reported in Table 1. For spectroscopic data see Supporting
Information.
General Procedure for the Synthesis of 3-Acetyl-1-(4-fluorophe-
nyl)methyl-4(1H)-quinolinones 10a-h. A mixture of the proper
9a-h derivative (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 the mixture was
cooled, 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 derivatives 10a-h.
Chemical, physical, and analytical data of derivatives 10a-h are
reported in Table 1. For spectroscopic data see Supporting
Information.
3-Acetyl-1-(4-fluorophenyl)methyl-7-(pyrrolidin-1-yl)-4(1H)-
quinolinone (10i). A mixture of 10c (2.7 g, 8.5 mmol), pyrrolidine
(1.8 g, 25.6 mmol), and NEt3 (0.7 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
chromatography on silica gel column (chloroform/ethyl acetate 1:1
General Procedure for the Synthesis of 3-Acetyl-4(1H)-quino-
linones 9a-h. Ethyl orthoformate (4.0 g, 27 mmol), ethyl acetoac-
etate (3.5 g, 27 mmol), the appropriate substituted aniline (27
mmol), and Dowtherm A (5.6 mL) were charged in a three-necked
flask equipped with a water separator. This mixture was stirred
under argon atmosphere, while the temperature was increased to
95 °C in 1 h, then gradually up to 162 °C in a further hour. Then
the mixture was stirred at this temperature for 6 h. After this time,

4748 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Di Santo et al.
Figure 3. Comparison of HIV-1 integrase inhibition by compound 6h and 5h in a gel-based assay and in the presence of Mg2+. (A) Phosphorimager
image showing a representative experiment. 21, 19, and STP correspond to the DNA substrate, the 3′-P product, and the ST products, respectively.
(B) HIV-1 integrase inhibition curves for 3′-P and ST (derived from densitometric analysis of the gels presented in part A).
as eluent) to give 1.7 g (56%) of pure 10i. Chemical, physical, and
analytical data of derivatives 10i are reported in Table 1. For
spectroscopic data see Supporting Information.
4-[7-Fluoro-1-(4-fluorophenyl)methyl-4(1H)-quinolinon-3-yl]-2-
hydroxy-4-oxo-2-butenoic Acid Ethyl Ester (5c). 84%; 151-152
°C; benzene/cyclohexane; IR ν 1726 (CdO ester), 1644 (CdO
1
ketone) cm-1. H NMR (DMSO-d6) δ 1.30 (t, 3H, CH3), 4.30 (q,
General Procedure for the Synthesis of Diketo Esters 5a-i
and 7a,b. Sodium ethoxide (390 mg, 5.5 mmol) was added into a
well stirred mixture of the appropriate acetyl derivative 9a-h or
10a-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, 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 5a-i and 7a,b. Yield (%), melting point (°C),
recrystallization solvent, IR, 1H NMR, and analytical data are
reported for each of the following compounds.
2H, CH2CH3), 5.72 (s, 2H, CH2), 7.17-7.21 (m, 2H, benzene H),
7.31-7.40 (m, 3H, benzene H and quinolinone C6-H), 7.62 (m,
1H, quinolinone C8-H), 7.97 (s, 1H, butenoate C3-H), 8.36 (m,
1H, quinolinone C5-H), 9.10 (s, 1H, quinolinone C2-H), 14.00
(bs, 1H, OH). Anal. (C22H17F2NO5) C, H, N, F.
4-[8-Fluoro-1-(4-fluorophenyl)methyl-4(1H)-quinolinon-3-yl]-2-
hydroxy-4-oxo-2-butenoic Acid Ethyl Ester (5d). 83%; 179-180
°C; benzene/cyclohexane; IR ν 3300 (OH), 1744 (CdO ester), 1648
1
and 1604 (CdO ketone) cm-1. H NMR (DMSO-d6) δ 1.27 (t,
3H, CH3), 4.30 (q, 2H, CH2CH3), 5.78 (s, 2H, CH2), 7.14-7.24
(m, 4H, benzene H), 7.48-7.53 (m, 1H, quinolinone C6-H),
7.59-7.69 (m, 1H, quinolinone C7-H), 7.97 (s, 1H, butenoate
C3-H), 8.17-8.19 (m, 1H, quinolinone C5-H), 9.01 (s, 1H,
quinolinone C2-H), 15.50 (bs, 1H, OH). Anal. (C22H17F2NO5) C,
H, N, F.
4-[1-(4-Fluorophenyl)methyl-4(1H)-quinolinon-3-yl]-2-hydroxy-
4-oxo-2-butenoic Acid Ethyl Ester (5a). 82%; 175-177 °C; ethanol;
IR ν 3400 (OH), 1721 (CdO ester), 1661, 1635, and 1607 (CdO
ketone) cm-1. 1H NMR (CDCl3) δ 1.44 (t, 3H, CH3), 4.42 (q, 2H,
CH2CH3), 5.48 (s, 2H, CH2), 7.06-7.26 (m, 4H, benzene H),
7.37-7.65 (m, 3H, quinolinone C6-H, C7-H, and C8-H), 8.16
(s, 1H, butenoate C3-H), 8.56 (m, 1H, quinolinone C5-H), 8.77
(s, 1H, quinolinone C2-H), 15.50 (bs, 1H, OH). Anal.
(C22H18FNO5) C, H, N, F.
4-[6-Chloro-1-(4-fluorophenyl)methyl-4(1H)-quinolinon-3-yl]-2-
hydroxy-4-oxo-2-butenoic Acid Ethyl Ester (5e). 34%; 131-133
°C; washed with cyclohexane; IR ν 3400 (OH), 1722 (CdO ester),
-1
1
1700 and 1628 (CdO ketone) cm . H NMR (DMSO-d6) δ 1.33
(t, 3H, CH3), 4.33 (q, 2H, CH2CH3), 5.71 (s, 2H, CH2), 7.14-7.34
(m, 4H, benzene H), 7.71-7.77 (m, 2H, quinolinone C7-H and
C8-H), 7.91 (s, 1H, butenoate C3-H), 8.25 (m, 1H, quinolinone
C5-H), 9.09 (s, 1H, quinolinone C2-H), 12.70 (bs, 1H, OH). Anal.
(C22H17ClFNO5) C, H, N, Cl, F.
4-[6-Fluoro-1-(4-fluorophenyl)methyl-4(1H)-quinolinon-3-yl]-2-
hydroxy-4-oxo-2-butenoic Acid Ethyl Ester (5b). 92%; 152-153
°C; toluene/cyclohexane; IR ν 1740 (CdO ester), 1650 and 1624
1
(CdO ketone) cm-1; H NMR (DMSO-d6) δ 1.29 (t, 3H, CH3),
4-[7-Chloro-1-(4-fluorophenyl)methyl-4(1H)-quinolinon-3-yl]-2-
hydroxy-4-oxo-2-butenoic Acid Ethyl Ester (5f). 76%; 173-175
°C; washed with isopropanol; IR ν 3300 (OH), 1727 (CdO ester),
1647 (CdO ketone) cm-1. 1H NMR (DMSO-d6) δ 1.30 (t, 3H, CH3),
4.32 (q, 2H, CH2CH3), 5.76 (s, 2H, CH2), 7.16-7.25 (m, 2H,
benzene H), 7.36-7.38 (m, 2H, benzene H), 7.54 (m, 1H,
quinolinone C6-H), 7.88 (m, 1H, quinolinone C8-H), 7.96 (s,
4.30 (q, 2H, CH2CH3), 5.78 (s, 2H, CH2), 7.16-7.20 (m, 2H,
benzene H), 7.29-7.37 (m, 2H, benzene H), 7.66 (m, 1H,
quinolinone C7-H), 7.81 (m, 1H, quinolinone C8-H), 7.97 (m,
1H, quinolinone C5-H), 8.00 (s, 1H, butenoate C3-H), 9.13 (s,
1H, quinolinone C2-H), 15.33 (bs, 1H, OH). Anal. (C22H17F2NO5)
C, H, N, F.

Quinolinonyl Diketo Acid DeriVatiVes as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4749
1H, butenoate C3-H), 8.29 (m, 1H, quinolinone C5-H), 9.08 (s,
1H, quinolinone C2-H), 12.70 (bs, 1H, OH). Anal. (C22H17-
ClFNO5) C, H, N, Cl, F.
(s, 1H, quinolinone C2-H), 14.67 (bs, 2H, OH). Anal. (C20H13-
F2NO5) C, H, N, F.
4-[7-Fluoro-1-(4-fluorophenyl)methyl-4(1H)-quinolinon-3-yl]-2-
hydroxy-4-oxo-2-butenoic Acid (6c). 95%; 162-163 °C; washed
with anhydrous ethanol; IR ν 3300 (OH), 1719 (CdO acid), 1635
4-[8-Chloro-1-(4-fluorophenyl)methyl-4(1H)-quinolinon-3-yl]-2-
hydroxy-4-oxo-2-butenoic Acid Ethyl Ester (5g). 98%; 164-165
°C; toluene; IR ν 3400 (OH), 1749 (CdO ester), 1644 (CdO
1
(CdO ketone) cm-1. H NMR (DMSO-d6) δ 5.70 (s, 2H, CH2),
1
ketone) cm-1. H NMR (DMSO-d6) δ 1.29 (t, 3H, CH3), 4.30 (q,
7.14-7.21 (m, 2H, benzene H), 7.31-7.37 (m, 3H, benzene H and
quinolinone C6-H), 7.60 (m, 1H, quinolinone C8-H), 7.93 (s,
1H, butenoate C3-H), 8.34 (m, 1H, quinolinone C5-H), 9.05 (s,
1H, quinolinone C2-H), 14.60 (bs, 2H, OH). Anal. (C20H13F2NO5)
C, H, N, F.
2H, CH2CH3), 6.09 (s, 2H, CH2), 7.14-7.16 (m, 4H, benzene H),
7.15 (m, 1H, quinolinone C6-H), 7.82 (m, 1H, quinolinone C7-H),
7.91 (s, 1H, butenoate C3-H), 8.34 (m, 1H, quinolinone C5-H),
8.81 (s, 1H, quinolinone C2-H), 15.25 (bs, 1H, OH). Anal.
(C22H17ClFNO5) C, H, N, Cl, F.
4-[8-Fluoro-1-(4-fluorophenyl)methyl-4(1H)-quinolinon-3-yl]-2-
hydroxy-4-oxo-2-butenoic Acid (6d). 78%; 180-181 °C; washed
with hot anhydrous ethanol; IR ν 3300 (OH), 1732 (CdO acid),
1634 and 1602 (CdO ketone) cm-1. 1H NMR (DMSO-d6) δ 5.86
(s, 2H, CH2), 7.12-7.23 (m, 4H, benzene H), 7.45-7.50 (m, 1H,
quinolinone C6-H), 7.57-7.62 (m, 1H, quinolinone C7-H), 7.70
(s, 1H, butenoate C3-H), 8.16-8.18 (m, 1H, quinolinone C5-H),
8.96 (s, 1H, quinolinone C2-H), 14.30 (bs, 2H, OH). Anal.
(C20H13F2NO5) C, H, N, F.
4-[6-Chloro-1-(4-fluorophenyl)methyl-4(1H)-quinolinon-3-yl]-2-
hydroxy-4-oxo-2-butenoic Acid (6e). 71%; 194-197 °C; DMF/
water; IR ν 3385 (OH), 1710 (CdO acid), 1663 and 1622 (CdO
ketone) cm-1. 1H NMR (DMSO-d6) δ 5.72 (s, 2H, CH2), 7.18-7.32
(m, 4H, benzene H), 7.75-7.85 (m, 2H, quinolinone C7-H and
C8-H), 8.22 (m, 1H, C5-H quinolinone), 7.90 (s, 1H, butenoate
C3-H), 9.08 (s, 1H, quinolinone C2-H), 13.00 (bs, 2H, OH). Anal.
(C20H12ClFNO5) C, H, N, Cl, F.
4-[7-Chloro-1-(4-fluorophenyl)methyl-4(1H)-quinolinon-3-yl]-2-
hydroxy-4-oxo-2-butenoic Acid (6f). 64%; 188-190 °C; washed
with isopropanol; IR ν 3400 (OH), 1734 (CdO acid), 1646 (CdO
ketone) cm-1. 1H NMR (DMSO-d6) δ 5.75 (s, 2H, CH2), 7.21-7.36
(m, 4H, benzene H), 7.55 (m, 1H, quinolinone C6-H), 7.83-7.85
(m, 2H, quinolinone C8-H and butenoate C3-H), 8.30 (m, 1H,
quinolinone C5-H), 9.07 (s, 1H, quinolinone C2-H), 13.00 (bs,
2H, OH). Anal. (C20H12ClFNO5) C, H, N, Cl, F.
4-[6,7-Dichloro-1-(4-fluorophenyl)methyl-4(1H)-quinolinon-3-
yl]-2-hydroxy-4-oxo-2-butenoic Acid Ethyl Ester (5h). 98%; 144-145
°C; toluene/cyclohexane; IR ν 1727 (CdO ester), 1638 (CdO
1
ketone) cm-1. H NMR (DMSO-d6) δ 1.35 (t, 3H, CH3), 4.38 (q,
2H, CH2CH3), 5.82 (s, 2H, CH2), 7.18-7.30 (m, 2H, benzene H),
7.43-7.46 (m, 2H, benzene H), 7.98 (s, 1H, butenoate C3-H),
8.14 (s, 1H, quinolinone C8-H), 8.41 (s, 1H, quinolinone C5-H),
9.14 (s, 1H, quinolinone C2-H), 16.00 (bs, 1H, OH). Anal.
(C22H16Cl2FNO5) C, H, N, Cl, F.
4-[1-(4-Fluorophenyl)methyl-7-(pyrrolidin-1-yl)-4(1H)-quinoli-
non-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid Ethyl Ester (5i). 82%;
170 °C (dec); methanol; IR ν 3300 (OH), 1735 (CdO ester), 1621
1
(CdO ketone) cm-1. H NMR (DMSO-d6) δ 1.31 (t, 3H, CH3),
1.98-2.10 (m, 4H, pyrrolidine H), 3.32-3.36 (m, 1H, pyrrolidine
H), 4.30 (q, 2H, CH2CH3), 5.64 (s, 2H, CH2), 6.37-6.39 (m, 1H,
quinolinone C8-H), 6.77-6.79 (m, 1H, quinolinone C6-H),
7.29-7.32 (m, 2H, benzene H), 7.45-7.48 (m, 2H, benzene H),
8.12-8.16 (m, 2H, quinolinone C5-H and butenoate C3-H), 8.79
(s, 1H, quinolinone C2-H), 13.50 (bs, 1H, OH). Anal.
(C26H25FN2O5) C, H, N, Cl, F.
4-(6-Chloro-4(1H)-quinolinon-3-yl)-2-hydroxy-4-oxo-2-butenoic
Acid Ethyl Ester (7a). 57%; 209-211 °C; ethanol; IR ν 3300 (NH
and OH), 1734 (CdO ester), 1684 (CdO ketone) cm-1. 1H NMR
(DMSO-d6) δ 1.31 (t, 3H, CH3), 4.25 (q, 2H, CH2), 7.40-8.00 (m,
3H, quinolinone C7-H, C8-H and butenoate C3-H), 8.20 (m,
1H, quinolinone C5-H), 8.50 (s, 1H, quinolinone C2-H), 12.75
(bs, 2H, NH and OH). Anal. (C15H12ClNO5) C, H, N, Cl.
4-(7-Chloro-4(1H)-quinolinon-3-yl)-2-hydroxy-4-oxo-2-butenoic
Acid Ethyl Ester (7b). 77%; 187-189 °C; washed with isopropanol;
IR ν 3200, 3064 (NH and OH), 1726 (CdO ester), 1700 and 1624
4-[8-Chloro-1-(4-fluorophenyl)methyl-4(1H)-quinolinon-3-yl]-2-
hydroxy-4-oxo-2-butenoic Acid (6g). 82%; 175-177 °C; washed
with anhydrous ethanol; IR ν 3300 (OH), 1742 (CdO acid), 1637
1
and 1603 (CdO ketone) cm-1. H NMR (DMSO-d6) δ 6.08 (s,
2H, CH2), 7.00-7.30 (m, 4H, benzene H), 7.44-7.46 (m, 1H,
quinolinone C6-H), 7.81-7.83 (m, 2H, butenoate C3-H and
quinolinone C7-H), 8.33-8.35 (m, 1H, quinolinone C5-H), 8.89
(s, 1H, quinolinone C2-H), 14.70 (bs, 2H, OH). Anal.
(C20H13ClFNO5) C, H, N, Cl, F.
-1
1
(CdO ketone) cm . H NMR (DMSO-d6) δ 1.30 (t, 3H, CH3),
4.30 (q, 2H, CH2), 7.47 (m, 1H, quinolinone C6-H), 7.68 (s, 1H,
quinolinone C8-H), 7.94 (s 1H, butenoate C3-H), 8.18 (m, 1H,
quinolinone C5-H), 8.77 (s, 1H, quinolinone C2-H), 13.00 (bs,
1H, NH and OH). Anal. (C15H12ClNO5) C, H, N, Cl.
4-[6,7-Dichloro-1-(4-fluorophenyl)methyl-4(1H)-quinolinon-3-
yl]-2-hydroxy-4-oxo-2-butenoic Acid (6h). 86%; 175-177 °C;
washed with isopropanol; IR ν 3300 (OH), 1722 (CdO acid), 1630
General Procedure for the Synthesis of Diketo Acids 6a-i
and 8a,b. A mixture of 1 N NaOH (6.5 mL) and the appropriate
ester 5a-i or 7a,b (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 6a-i and 8a,b. Yield
(%), melting point (°C), recrystallization solvent, IR, 1H NMR, and
analytical data are reported for each of the following compounds.
4-[1-(4-fluorophenyl)methyl-4(1H)-quinolinon-3-yl]-2-hydroxy-
4-oxo-2-butenoic Acid (6a). 50%; 207-209 °C; washed with
anhydrous ethanol; IR ν 3400 (OH), 1732 (CdO acid), 1619 (CdO
1
(CdO ketone) cm-1. H NMR (DMSO-d6) δ 5.75 (s, 2H, CH2),
7.17-7.22 (m, 2H, benzene H), 7.32-7.39 (m, 2H, benzene H),
7.88 (s, 1H, quinolinone C8-H), 8.06 (s, 1H, butenoate C3-H),
8.32 (s, 1H, quinolinone C5-H), 9.05 (s, 1H, quinolinone C2-H),
16.00 (bs, 2H, OH). Anal. (C20H12Cl2FNO5) C, H, N, Cl, F.
4-[1-(4-Fluorophenyl)methyl-7-(pyrrolidin-1-yl)-4(1H)-quinoli-
non-3-yl]-2-hydroxy-4-oxo-2-butenoic Acid (6i). 87%; 197-199 °C;
washed with isopropanol; IR ν 3300 (OH), 1718 (CdO acid), 1614
1
(CdO ketone) cm-1. H NMR (DMSO-d6) δ 2.06-2.13 (m, 4H,
pyrrolidine H), 3.26-3.30 (m, 4H, pyrrolidine H), 5.77 (s, 2H, CH2),
6.42-6.45 (m, 1H, quinolinone C8-H), 6.81-6.85 (m, 1H,
quinolinone C6-H), 7.18-7.25 (m, 2H, benzene H), 7.49-7.52
(m, 2H, benzene H), 8.02 (s, 1H, butenoate C3-H), 8.15-8.17
(m, 1H, quinolinone C5-H), 9.09 (s, 1H, quinolinone C2-H),
14.00 (bs, 2H, OH). Anal. (C24H21FN2O5) C, H, N, F.
1
ketone) cm-1. H NMR (CDCl3) δ 5.79 (s, 2H, CH2), 7.22-7.38
(m, 4H, benzene H), 7.53-8.34 (m, 5H, quinolinone C6-H, C7-H,
C8-H and butenoate C3-H), 9.16 (s, 1H, quinolinone C2-H),
15.50 (bs, 2H, OH). Anal. (C20H15FNO5) C, H, N, F.
4-[6-Fluoro-1-(4-fluorophenyl)methyl-4(1H)-quinolinon-3-yl]-2-
hydroxy-4-oxo-2-butenoic Acid (6b). 95%; 183-184 °C; washed
with anhydrous ethanol; IR ν 3100 (OH), 1730 (CdO acid), 1613
4-(6-Chloro-4(1H)-quinolinon-3-yl)-2-hydroxy-4-oxo-2-butenoic
Acid (8a). 50%; 220 °C (dec); toluene/cyclohexane; IR ν 3400,
3200 (NH and OH), 1718 (CdO acid), 1640 and 1611 (CdO
1
1
(CdO ketone) cm-1. H NMR (DMSO-d6) δ 5.76 (s, 2H, CH2),
ketone) cm-1. H NMR (DMSO-d6) δ 7.72-7.75 (m, 2H, quino-
7.16-7.20 (m, 2H, benzene H), 7.33-7.36 (m, 2H, benzene H),
7.65 (m, 1H, quinolinone C7-H), 7.80 (m, 1H, quinolinone C8-H),
7.94-7.97 (m, 2H, quinolinone C5-H and butenoate C3-H), 9.10
linone C7-H and C8-H), 8.13 (s, 1H, butenoate C3-H), 8.53
(m, 1H, quinolinone C5-H), 8.80 (s, 1H, quinolinone C2-H),
13.00 (bs, 3H, NH and OH). Anal. (C13H8ClNO5) C, H, N, Cl.

4750 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Di Santo et al.
4-(7-Chloro-4(1H)-quinolinon-3-yl)-2-hydroxy-4-oxo-2-butenoic
Acid (8b). 52%; 200 °C (dec); washed with isopropanol; IR ν 3400,
3200 (NH and OH), 1740 (CdO acid), 1640 and 1616 (CdO ketone)
cm-1. 1H NMR (DMSO-d6) δ 7.47 (m, 1H, quinolinone C6-H) 7.69
(m, 1H, quinolinone C8-H), 7.92 (s, 1H, butenoate C3-H), 8.21 (m,
1H, quinolinone C5-H), 8.88 (s, 1H, quinolinone C2-H), 12.75 (bs,
3H, NH and OH). Anal. (C13H8ClNO5) C, H, N, Cl.
Biological Assays. Integrase Assays. Compounds 5a-i, 6a-i,
7a,b, and 8a,b were tested for their ability to inhibit HIV-1 integrase
in vitro using a gel-based assay in addition to a 96-well plate-based
high throughput electrochemiluminescent assay.14
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 3′-P and ST (for review see ref 4). Briefly, a
DNA-enzyme complex is preformed by mixing the drug at the desired
concentration with 400 nM recombinant HIV-1 integrase in a buffer
containing 50 mM MOPS, pH 7.2, 7.5 mM MnCl2, and 14.3 mM
ꢀ-mercaptoethanol. The integration reaction is then initiated by the
addition of 20 nM 5′-labeled double-stranded DNA template 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 Dynam-
ics phosphorimager (Sunnyvale, CA).
Italian MIUR (PRIN 2006, Grant No. 2006030809), and by the
Intramural Research Program of the Center for Cancer Research,
National Cancer Institute, National Institutes of Health.
Supporting Information Available: Spectroscopic data of
compounds 9a-h and 10a-i; elemental analysis data for derivatives
5a-i, 6a-i, 7a,b, and 8a,b. This material is available free of charge
via the Internet at http://pubs.acs.org.
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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 cells/well. After the infectivity of a virus stock
(HTLV-IIIB) is 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. Culture
were incubated at 37 °C in a 5% CO2 atmosphere for 4 days.
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of a number of drug concentrations) is harvested. HIV p24 antigen is
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RPMI-1640 without phenol red, supplemented with 20% fetal calf
serum and antibiotics. Two hours after plating, compounds were added
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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 isopropanol containing 0.1 N HCl. Absorbance of converted
dye was measured in an ELISA plate reader at a 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% has been called IC50.
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Acknowledgment. This project was supported by the
Ministero della Sanita`, Istituto Superiore di Sanita`, “Programma
Nazionale di ricerca sull’AIDS” (Grant No. 30G.9), by the
JM8001422