Structure-activity relationships and binding mode of styrylquinolines as potent inhibitors of HIV-1 integrase and replication of HIV-1 in cell culture

Article abstract of DOI:10.1021/jm990467o

Our prior studies showed that polyhydroxylated styrylquinolines are potent HIV-1 integrase (IN) inhibitors that block the replication of HIV-1 in cell culture at nontoxic concentrations. To explore the mechanism of action of these inhibitors, various novel styrylquinoline derivatives were synthesized and tested against HIV-1 IN and in cell-based assays. Regarding the in vitro experiments, the structural requirements for biological activity are a carboxyl group at C-7, a hydroxyl group at C-8 in the quinoline subunit, and an ancillary phenyl ring. However the in vitro inhibitory profile tolerates deep alterations of this ring, e.g. by the introduction of various substituents or its replacement by heteroatomic nuclei. Regarding the ex vivo assays, the structural requirements for activity are more stringent than for in vitro inhibition. Thus, in addition to an o-hydroxy acid group in the quinoline, the presence of one ortho pair of substituents at C-3' and C- 4', particularly two hydroxyl groups, in the ancillary phenyl ring is imperatively required for inhibitory potency. Starting from literature data and the SARs developed in this work, a putative binding mode of styrylquinoline inhibitors to HIV-1 IN was derived.

Full text of DOI:10.1021/jm990467o

J . Med. Chem. 2000, 43, 1533-1540
1533
Str u ctu r e-Activity Rela tion sh ip s a n d Bin d in g Mod e of Styr ylqu in olin es a s
P oten t In h ibitor s of HIV-1 In tegr a se a n d Rep lica tion of HIV-1 in Cell Cu ltu r e
†
§
†
†
§
§
Fatima Zouhiri, J ean-Fran c¸ ois Mouscadet, Khalid Mekouar, Didier Desma e¨ le, Delphine Savour e´ , Herv e´ Leh,
§
‡
§
,†
Fr e´ d e´ ric Subra, Marc Le Bret, Christian Auclair, and J ean d'Angelo*
Unit e´ de Chimie Organique, UPRES-A du CNRS 8076, Centre d'Etudes Pharmaceutiques, Universit e´ Paris-Sud, 5 rue J .-B.
Cl e´ ment, 92296 Ch aˆ tenay-Malabry, France; Laboratoire de Physicochimie et de Pharmacologie des Macromol e´ cules
Biologiques, UMR du CNRS 8532, Institut Gustave Roussy, PRII, 39 rue Camille Desmoulins, 94805 Villejuif, France; and
Laboratoire de Physicochimie et de Pharmacologie des Macromol e´ cules Biologiques, UMR du CNRS 8532, LBPA, Ecole
Normale Sup e´ rieure de Cachan, 94235 Cachan, France
Received September 15, 1999
Our prior studies showed that polyhydroxylated styrylquinolines are potent HIV-1 integrase
(
IN) inhibitors that block the replication of HIV-1 in cell culture at nontoxic concentrations.
To explore the mechanism of action of these inhibitors, various novel styrylquinoline derivatives
were synthesized and tested against HIV-1 IN and in cell-based assays. Regarding the in vitro
experiments, the structural requirements for biological activity are a carboxyl group at C-7, a
hydroxyl group at C-8 in the quinoline subunit, and an ancillary phenyl ring. However the in
vitro inhibitory profile tolerates deep alterations of this ring, e.g. by the introduction of various
substituents or its replacement by heteroatomic nuclei. Regarding the ex vivo assays, the
structural requirements for activity are more stringent than for in vitro inhibition. Thus, in
addition to an o-hydroxy acid group in the quinoline, the presence of one ortho pair of
substituents at C-3′ and C-4′, particularly two hydroxyl groups, in the ancillary phenyl ring is
imperatively required for inhibitory potency. Starting from literature data and the SARs
developed in this work, a putative binding mode of styrylquinoline inhibitors to HIV-1 IN was
derived.
In tr od u ction
processing, IN removes two nucleotides from both 3′-
viral DNA ends and forms free 3′-OH groups at a
conserved CA sequence. In the second step, or strand
transfer, free 3′-OH’s are used as nucleophilic agents
and attack phosphoester bonds on opposite strands of
AIDS is essentially a viral disease and should be
treated by antiretroviral agents. Although a combina-
1
tion of antiretroviral therapy has made it possible to
suppress the replication of HIV-1 in infected individuals
to such an extent that it becomes undetectable in the
plasma, the virus persists in reservoirs such as periph-
eral blood mononuclear cells or resting T-lymphocytes
which went back to a latent state after an early
replicative stage.2 Thus, after more than 3 years of
multidrug therapy, it turned out that the HIV-1 infec-
tion can be controlled but not eradicated with current
treatments. Besides, the emergence of resistance against
both reverse transcriptase and protease inhibitors,
which was a frequent issue of the single-agent regimen,
was not completely suppressed by the use of combina-
tion therapy. It is therefore important to identify new
agents that could block the virus at a step of its
replicative cycle which is not yet affected by current
treatments. For these reasons, the integrase (IN), the
third viral enzyme which is strictly required for the
establishment of a stable infection,5 is an attractive
target for new antiviral agents and potential synergy
with currently available HIV reverse transcriptase and
8
target DNA. Systematic screening of potential IN
inhibitors yielded several families of active compounds,
including nucleotides and oligonucleotides, peptides, and
9
-18
small organic molecules.
The most populated family
consists of polyhydroxylated aromatic compounds. Mem-
bers of this group display recurrently the o-diphenol
-4
(catechol) function. Among the numerous classes of HIV
IN inhibitors which have emerged, only a very few of
them exhibit a substantial level of antiviral activity at
nontoxic concentrations. We have recently reported that
polyhydroxylated styrylquinolines are potent HIV IN
inhibitors, block the replication of HIV in cell culture,
1
9
and are devoid of cytotoxicity. However, despite these
remarkable properties, the binding mode of these in-
hibitors remains to be elucidated. In the present paper,
with the aim to explore their mechanism of action,
various novel styrylquinoline derivatives were synthe-
sized and the effects of these structural modifications
were assayed against HIV-1 IN in both 3′-processing
and strand-transfer reactions, as well as against HIV-1
replication in CEM cell culture.
6
protease inhibitors. IN catalyzes two reactions consist-
ing both in the nucleophilic attack of a phosphoester
bond by a hydroxyl group. In the first step, called 3′-
7
Resu lts a n d Discu ssion
*
To whom correspondence should be addressed. Tel: 01 46 83 56
Ch em istr y. Reference compounds 9, 19, 20, and 23
Table 1) have been previously described. Apart from
compounds 15, 28, and 30, all new styrylquinolines were
basically elaborated through condensation between a
9
9. Fax: 01 46 83 57 53. E-mail: jean.dangelo@cep.u-psud.fr.
19
(
†
Universit e´ Paris-Sud.
Institut Gustave Roussy.
Ecole Normale Sup e´ rieure de Cachan.
§
‡
1
0.1021/jm990467o CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/22/2000

1
534 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8
Zouhiri et al.
Sch em e 1
π-electron density of the phenyl subunit, since com-
pounds 14-16, in which this ring bears a p-nitro,
p-amino, and p-acetamido group, respectively, exhibited
similar IC50 values, of approximately 1-4 µM. Likewise,
hydroxylated analogues (4′-hydroxy 17, 3′,5′-dihydroxy
1
4
8, 2′,4′-dihydroxy 19, 3′,4′-dihydroxy 20, 3′-methoxy-
′-hydroxy 21, 3′-hydroxy-4′-methoxy 22, 3′,4′,5′-trihy-
droxy 23, 3′,4′-dihydroxy-5′-methoxy 24, 3′,5′-dimethoxy-
′-hydroxy 25, 3′,5′-dibromo-4′-hydroxy 26, and 3′,4′-
4
dihydroxy-5′-iodo 27) were all active in the range
between 0.3 and 5 µM. In sharp contrast, compounds
2
8 and 29, in which the 7-carbomethoxy-8-hydrox-
Sch em e 2
yquinoline and 5,7-dichloro-8-hydroxyquinoline moieties
are respectively linked to a 3′,4′-catechol half by means
of an ethylenic bridge, were completely devoid of bio-
logical activity. Finally, a full IN inhibitory potency (IC50
of approximate 2.0 µM) was restored in styrylquinoline
3
0, analogue to parent compound 20 where the central
ethylenic linker is replaced by an ethano bridge.
All these results clearly indicate that the presence of
a 7-carboxyl-8-hydroxyl pattern in the quinoline half
and an aromatic or heteroaromatic ancillary subunit are
required for the biological activity of the drug. Replace-
ments of the 7-carboxyl by a carbomethoxy group
(compound 28) or a chlorine atom (compound 29) are
indeed accompanied by a complete loss of inhibitory
potency. However somewhat surprisingly, the in vitro
inhibitory profile not only tolerates deep alterations of
the ancillary phenyl subunit by the introduction of
various substituents or its replacement by heteroaro-
matic nuclei but also is unaffected by the replacement
of the ethylenic linker by a saturated counterpart
2
-methylquinoline moiety 1 and an aromatic or het-
eroaromatic aldehyde 2, followed by neutral or acidic
hydrolysis. 8-Hydroxy-7-quinaldic acid (9) was used as
starting subunit in the syntheses of derivatives 10-27
and 30. Commercially available 5,7-dichloro-2-methyl-
8
-quinolinol (4) was utilized in the preparation of 29
(compound 30). That the present inhibitors more likely
(Scheme 1).
act specifically against the central IN core domain
rather than the end parts of IN was established by
measuring the effect of “nonhydroxylated” derivatives
All aromatic or heteromatic aldehydes used are com-
mercially available, with the exception of 6 and 8
required for the synthesis of 24 and 27), which were
(
1
0-16 upon disintegration reaction catalyzed by the
2
0
prepared through AlCl3-induced demethylation of sy-
ringaldehyde (5) and 5-iodovanillin (7), respectively
(50-212) deletion mutant. All these molecules indeed
displayed good inhibitory potencies in this assay (IC50
range 0.8-12 µM) (Table 2). Incidentally, this experi-
ment also confirms that the presence of hydroxyl group-
(Scheme 2).
Styrylquinoline 15 resulted from the acidic hydrolysis
of 16, and 28 resulted from the condensation between
quinaldine 9 and 3,4-dihydroxybenzaldehyde, followed
by methanolysis of the crude product, using a MeOH-
pyridine mixture. Compound 30 was prepared by cata-
lytic hydrogenation of 20.
(s) in the aromatic (heteroaromatic) half is not an
absolute requirement for in vitro activity.
An ti-HIV Activities a n d Cytotoxicity. Compounds
9
-30 were evaluated for their antiviral activities against
HIV-1 replication in CEM cells. They were tested for
their ability to lower the viral charge in culture super-
natants. CEM cells were infected with HIV-1 and
subsequently treated with increasing drug concentra-
tions. Viral load was estimated 72 h after infection. The
amount of virus was tested by â-galactosidase assay,
with HeLa CD4-â gal cells as reporting cells. Toxicity
was estimated by MTT transformation assay. Results
are listed in Table 3.
Activities a ga in st HIV-1 IN. Each new compound
was screened for inhibitory activity against HIV-1 IN
in both 3′-end-processing and strand-transfer reactions.
Results are illustrated in Table 1. These compounds can
be roughly classified as active (10-27 and 30) and
inactive (9, 28, and 29). Though a complete lack of
inhibitory potency was observed with the parent 8-hy-
droxy-7-quinaldic acid (9), micromolar or submicromolar
activities were obtained with styrylquinolines 10-27,
in which this quinoline half is linked to an aromatic (or
heteromatic) moiety by means of an ethylenic spacer.
One striking feature of the above findings is that the
replacement of the ancillary phenyl ring of prototype
compound 10 by a variety of heteroatomic nuclei (2′-
furyl, 3′-thienyl, and 3′-pyridyl analogues 11-13, re-
spectively) has only a limited effect on anti-HIV-1 IN
activity, with IC50 values ranging from 1.9 to 11 µM.
Also remarkable is the observation that the in vitro
inhibitory potency is relatively independent of the
At first glance, the present molecules can be catego-
rized as inactive, cytotoxic, and active. Inactive com-
pounds comprise quinaldine 9, styrylquinolines 11 and
13 containing a 2′-furyl and 3′-pyridyl ring, respectively,
and 17 and 18, where the phenyl subunit is substituted
by hydroxyl groups at C-4′ and at C-3′ and C-5′,
respectively. Among the cytotoxic compounds, 3′-thienyl
derivative 12 and 30, in which the two subunits are
linked by means of an ethano bridge, exhibited a
relatively weak toxicity (TC50 > 60 µM). Styrylquino-

Styrylquinolines as Inhibitors of HIV IN
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8 1535
Ta ble 1. HIV-1 IN Inhibitory Potencies Determined as Described in the Experimental Section
lines in which the ancillary phenyl nucleus is either
unsubstituted (10) or substituted at C-4′ by a nitro
group (14) or an acetamido function (16) proved to be
moderately cytotoxic (TC50 ranging from 30 to 50 µM).
Finally analogue 23, which possesses three vicinal
hydroxyl functions, and 28 and 29, in which the carboxyl
group at C-7 in the quinoline half is respectively
replaced by a carbomethoxy group or a chlorine atom,
showed a relatively high cytotoxicity (TC50 between 10
and 17 µM). Within the active compounds series,
styrylquinolines where the phenyl half is substituted
at C-4′ by an amino group (15), at C-2′ and C-4′ by two
hydroxyls (19), and at C-3′ by a hydroxyl and at C-4′ by
a methoxy group (22) displayed low anti-HIV-1 activities
(IC50 ranging from 50 to 80 µM). A moderate antiviral
activity (IC50 between 7 and 12 µM) was gained with
styrylquinolines in which the phenyl moiety exhibits the
3
′,4′-dihydroxy-5′-methoxy, 3′,5′-dimethoxy-4′-hydroxy,
and 3′,4′-dihydroxy-5′-iodo trisubstitution patterns (24,
2
3
5, and 27, respectively). Finally, 3′,4′-dihydroxy and
′-methoxy-4′-hydroxy derivatives (20 and 21, respec-

1
536 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8
Zouhiri et al.
Ta ble 2. Effect of “Nonhydroxylated” Styrylquinolines upon
Disintegration Catalyzed by the (50-212) Deletion Mutant
catechol group plays a favorable role in the anti-HIV
profile, its presence is not absolutely required, since the
replacement of the hydroxyl at C-3′ by a methoxy group
does not notably reduce the biological activity. However
it is worthy of note that the presence of three vicinal
hydroxyl groups (compound 23) resulted in high cyto-
toxicity. Finally, replacement of the central ethylenic
linker by an ethano bridge was the cause of significant
collateral cytotoxicity (Table 3).
Bin d in g Mod e of Styr ylqu in olin es. Although the
precise target of styrylquinolines in the ex vivo experi-
ments is not yet firmly identified, current data support
the interpretation that these drugs block HIV-1 replica-
tion directly through inhibition of HIV-1 IN. Thus, the
good correlation between activities in isolated enzyme
models and the ex vivo cellular data reported here is
consistent with this hypothesis. Furthermore, we believe
that the inhibition of virus penetration may be ruled
out for the following reasons: (i) in our conditions of
infection, we observed that the entry of viruses into cells
peaked after 2 h following addition of viruses to the
culture medium; thus, the de novo infection assay was
designed in such a manner that drugs were added to
the cell culture medium only 2 h following infection and
after an extensive wash of cells designed to eliminate
the remaining free viruses; (ii) upon de novo infection
of CEM cells, progeny viruses became detectable in
culture medium after 40 h; thus the viremia estimated
at 72 h was mostly due to viruses produced after only
one round of infection; and (iii) we previously reported
that in a “time of addition” experiment, styrylquinoline
derivatives displayed a maximum activity when added
between 4 and 16 h following infection, a time course
compatible with the inhibition of a post-penetration step
of replication. Finally, since styrylquinolines inhibited
HIV IN but not reverse transcriptase in vitro, we
consider that IN inhibition in infected cells is a plausible
explanation for the ex vivo antiviral activity of these
Ta ble 3. Antiviral Activity and Cytotoxicitya
1
9
inhibitors.
Another striking argument was provided by the
elegant work of Robinson et al. who demonstrated that
a single amino acid change in HIV-1 IN was sufficient
to confer resistance to L-chicoric acid, an inhibitor which
offers some structural resemblance to styrylquinolines.21
Crystal structures of the core domain of HIV-1 IN
a
NR, not reached.
2
+
22
mutants complexed with Mg have been reported.
The magnesium is coordinated in the active site by Asp-
4 and Asp-116, whereas the third residue of the
tively) showed good anti-HIV potencies (IC50 between
and 3 µM).
All these results demonstrate that the structural
6
1
catalytic amino acid triad, Glu-152, does not directly
participate in metal binding. Such a binding site was
unequivocally identified by X-ray structure determina-
tion of a complex between a naphthalenedisulfonic
inhibitor (Y-3) and ASV IN, a protein which exhibits
requirements for ex vivo activity of the drug are more
stringent than for in vitro IN inhibition. Regarding the
quinoline subunit, the effect of its alteration more or
less parallels the in vitro observations. Thus, the
presence of a free carboxyl group at C-7 is critical: its
replacement by a carbomethoxy group or a chlorine
atom resulted in a moderate to high cytotoxicity. In fact,
the other crucial structural requirement concerns the
ancillary aromatic moiety linked to the quinoline moi-
ety. While the IN inhibitory profile tolerates a great
variety of substitution of this ring or its replacement
by heteroaromatic nuclei, the anti-HIV activity impera-
tively requires the presence of one ortho pair of sub-
stituents: a free hydroxyl group at C-4′ and a hydroxyl
or a methoxy group at C-3′. Evidently, though the 3′,4′-
2
3
great similarity with HIV-1 IN. The Y-3 molecule in
the crystal is located in close proximity to the enzyme
active site. Recently, the X-ray structure of the HIV-1
IN catalytic domain complexed with an inhibitor (5CIT-
EP) has been determined. The inhibitor binds centrally
in the active site of the IN and makes a number of close
contacts with the protein. Only minor changes in the
2
4
protein accompany inhibitor binding.
Since it was recognized that HIV-1 IN requires a
divalent cation as cofactor for its enzymatic activity
2
+
(presumably Mg ), and assuming that an o-hydroxy

Styrylquinolines as Inhibitors of HIV IN
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8 1537
particularly two hydroxyls, at this end of the molecule.
A final interesting aspect of the present model is that
the geometrical pattern defined by the functional group
triad at C-7/C-8, C-3′, and C-4′ satisfactorily matches
one of the putative three-point pharmacophores previ-
ously identified by Pommier and co-workers through a
2
6,27
3
D database searching.
Con clu sion s
Styrylquinolines have been recognized as potent
HIV-1 IN inhibitors that block HIV-1 replication in cell-
1
9
based assays. In the present paper, with the aim to
explore their mechanism of action, various novel
styrylquinoline derivatives were synthesized and their
biological properties were evaluated. Since styrylquino-
lines fall within the general category of inhibitors
characterized by two aryl groups linked through a
central spacer, we have examined the molecular deter-
minants of each of these subunits, through a progressive
series of analogues. Regarding the in vitro inhibition,
micromolar or submicromolar activities were obtained
with compounds 10-27 and 30, though 9, 28, and 29
proved to be inactive. These results clearly demonstrate
that the presence of a free carboxyl group at C-7 and a
hydroxyl at C-8 in the quinoline half is required for the
biological activity of the drug. The other prerequisite
for in vitro potency was the presence of an ancillary
aromatic ring. However the in vitro inhibitory profile
tolerates the introduction of various substituents in this
ring or its replacement by heteroatomic nuclei. The
structural requirements for ex vivo activity are more
stringent than for in vitro IN inhibition. In addition to
the presence of an o-hydroxy acid group in the quinoline,
the anti-HIV activity indeed requires the presence of
one ortho pair of substituents in the ancillary phenyl
ring: a free hydroxyl group at C-4′ and a hydroxyl or
methoxy group at C-3′. Examination of SARs developed
for our compounds, interpreted in the light of literature
data, led to a putative binding mode of styrylquinolines
to HIV-1 IN. Since the Mg ion is a cofactor required
for the enzymatic activity of HIV-1 IN, we hypothesized
that the o-hydroxy acid group lies in a possible coordi-
nation to this metal cation. The additional binding force
which plays a significant role in the recognition of the
drug by the protein apparently involves the ancillary
aromatic ring. However, since the exact mechanism by
which styrylquinolines inhibited productive infection is
unknown, the definitive mapping of the binding sites
of these inhibitors awaits further investigation. In
summarizing the above findings, it can be concluded
that the insights so gained are likely to aid the design
and development of new useful HIV-1 IN inhibitors.
HIV-1 IN has indeed become an attractive target for
intervention by chemotherapeutics, because of the rapid
emergence of resistance against both reverse tran-
scriptase and protease inhibitors, currently used in
combination regimens for the treatment of AIDS.28
F igu r e 1. Presumed in vitro binding interactions between
styrylquinolines and the HIV-1 IN catalytic site.
acid group in the quinoline subunit is crucially involved
in both in vitro and ex vivo inhibitory activities, we
hypothesized that this group lies in a possible coordina-
tion to the metal cation. Such an assumption has been
recently reinforced by computational studies. Indeed,
minimization of the energy of interaction between
styrylquinoline drugs on the whole surface of the
catalytic core domain of RSV IN revealed that the best
2
+
fits were found for binding of the drugs on Mg in the
vicinity of the active site.25
The other prerequisite for ex vivo potency was the
presence at the ancillary phenyl ring of a hydroxyl group
at C-4′ and a hydroxyl or a methoxy group at C-3′. These
data suggest that, in addition to an o-hydroxy acid, free
hydroxyl group(s) may be necessary for uptake of the
drugs by the cell or into the virion. Finally, besides the
presence of a carboxylic acid and hydroxyl group(s), the
requirement of a correct size and a geometrical fit seems
also essential for anti-HIV inhibitory potency, since the
replacement of the central ethylenic linker by a satu-
rated counterpart resulted in significant cytotoxicity.
Starting from the above literature data and the SARs
developed in this work, a putative binding mode of
styrylquinolines to HIV-1 IN was derived (Figure 1). The
platform used in designing this model was the binding
2
+
2
+
site of HIV-1 IN mutants, in which a Mg
ion is
coordinated by two catalytic aspartates (Asp-64 and
2
2
Asp-116). Binding interactions between the drug and
the enzyme catalytic site were next tentatively identi-
fied. As mentioned above, since the presence at C-7 and
C-8 of an o-hydroxy acid is required for in vitro activi-
ties, one of the sites of recognition might involve the
2
+
coordination by this group of the Mg
cation. The
second complementary feature for recognition is the
presence of an ancillary phenyl ring. However, since the
in vitro inhibitory profile tolerates deep alterations of
this subunit, e.g. by the introduction of various substit-
uents or it replacement by heteroatomic nuclei, this
second site of recognition clearly involves a nonspecific
binding mode, possibly through a π-stacking interaction.
Regarding the ex vivo activity, the structural require-
ments are more stringent than for in vitro IN inhibition.
As a matter of fact, in that case two additional binding
forces play a significant role in the recognition of the
drug by the protein. These apparently involve hydrogen
bonds at C-3′ and/or C-4′, a situation that is a specific
consequence of the convergence of functional groups,
Exp er im en ta l Section
Melting points were recorded on a capillary tube melting
point apparatus and are uncorrected or by microthermal
analysis on a Mettler FP5 apparatus. Infrared (IR) spectra
were obtained as neat films between NaCl plates or KBr
1
13
pellets. The H and C NMR spectra were recorded in CDCl
3
,
unless otherwise stated; when diffuse, easily exchangeable

1
538 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8
Zouhiri et al.
protons were not listed. Recognition of methyl, methylene,
methine, and quaternary carbon nuclei in 13C NMR spectra
rests on the J -modulated spin-echo sequence. Analytical thin-
layer chromatography was performed on Merck silica gel
(d, J ) 8.6 Hz, 1H); 13C NMR (DMSO-d
6
, 50 MHz) δ 171.4,
160.1, 153.8, 139.1, 138.0, 137.4, 130.7, 130.3, 127.5, 126.6,
1
3
126.4, 126.2, 125.2, 121.3, 115.2, 111.5. Anal. (C16H11NO S‚ /
2
H
2
O) C, H, N.
6
0F254 glass precoated plates (0.25 mm layer). All liquid
(
E)-8-Hyd r oxy-2-[2-(3-p yr id in yl)eth en yl]-7-qu in olin e-
chromatography separations were performed using Merck
silica gel 60 (230-400 mesh ASTM). Ether and tetrahydrofu-
ran (THF) were distilled from Na-benzophenone ketyl. Metha-
nol was dried over magnesium and distilled. Benzene and
ca r boxylic Acid (13). Method C using 3-pyridinecarboxal-
dehyde gave acid 13 in 57% overall yield as yellow crystals:
mp > 260 °C; IR (KBr, cm ) 3600-3000, 2800-1800, 1633,
592; H NMR (CF
-1
1
1
)
1
3
CO
2
D, 400 MHz) δ 9.15 (s, 1H), 8.93 (d, J
2 2
CH Cl were distilled from calcium hydride, under a nitrogen
8.9 Hz, 1H), 8.89 (d, J ) 8.4 Hz, 1H), 7.79 (d, J ) 5.8 Hz,
H), 8.50 (d, J ) 8.9 Hz, 1H), 8.31 (d, J ) 8.7 Hz, 1H), 8.19 (d,
J ) 16.4 Hz, 1H), 8.13 (dd, J ) 8.4, 5.8 Hz, 1H), 8.07 d, J )
atmosphere. All reactions involving air- or water-sensitive
compounds were routinely conducted in glassware which was
flame-dried under a positive pressure of nitrogen. Organic
13
1
1
1
1
3 2
6.4 Hz, 1H), 7.68 (d, J ) 8.7 Hz, 1H); C NMR (CF CO D,
4
layers were dried over anhydrous MgSO . Chemicals obtained
00 MHz) δ 175.6, 155.2, 153.2, 149.5, 147.4, 144.5, 143.6,
from commercial suppliers were used without further purifica-
tion. Elemental analyses were obtained from the Service de
microanalyse, Centre d'Etudes Pharmaceutiques, Ch aˆ tenay-
Malabry, France.
Con d en sa tion of 8-Hyd r oxy-7-qu in a ld ic Acid (9) w ith
Ar om a t ic a n d H et er oa r om a t ic Ald eh yd es. Gen er a l
Meth od A: To a solution of 1.0 equiv of acid 9 in acetic
anhydride (3 mL/mmol) was added 4 equiv of aldehyde. The
mixture was heated under reflux for 16 h and concentrated in
vacuo. The residue was dissolved in 6 N sulfuric acid (4 mL/
mmol) and the resulting solution was heated under reflux for
39.8, 137.3, 134.8, 133.0, 130.7, 130.4, 127.3, 123.8, 121.2,
18.5. Anal. (C17
(
n eca r boxylic Acid (14). Method B using 4-nitrobenzaldehyde
afforded acid 14 in 57% overall yield as brick red crystals: mp
260 °C; IR (KBr, cm ) 3700-2400, 1683, 1594, 1510; H
NMR (DMSO-d , 200 MHz) δ 8.41 (d, J ) 8.6 Hz, 1H), 8.26 (d,
J ) 8.6 Hz, 2H), 8.09-7.96 (m, 4H), 7.83 (d, J ) 8.8 Hz, 1H),
H
12
N
2
O
3
‚2H
2
O) C, H, N.
E)-8-Hyd r oxy-2-[2-(4-n itr op h en yl)eth en yl]-7-qu in oli-
-1
1
>
6
1
3
7
(
1
1
.76 (d, J ) 16.5 Hz, 1H), 7.36 (d, J ) 8.8 Hz, 1H); C NMR
DMSO-d , 50 MHz) δ 171.7, 159.0, 153.4, 147.0, 142.6, 138.0,
37.3, 133.5, 131.1 (2C), 128.3 (2C), 126.7, 124.0 (2C), 122.3,
16.8, 111.0. Anal. (C18 ‚2 H O) C, H, N.
E)-8-Hyd r oxy-2-[2-(4-a m in op h en yl)eth en yl]-7-qu in o-
lin eca r boxylic Acid (15). A suspension of styrylquinoline 16
350 mg, 1.0 mmol) in 6 N sulfuric acid (10 mL) was heated
6
3
h. After beeing cooled 0 °C, 6 N potassium hydroxide was
H
12
N
2
O
5
2
added until neutrality. The mixture was filtered and washed
with water, 2-propanol and finally ether. The solid was
collected and taken up into water, and the suspension was
refluxed for 1 h. The mixture was then filtered; the solid was
recovered and dried under vacuum. The process was repeated
(
(
under reflux for 2 h. After being cooled at 0 °C, 6 N potassium
hydroxide was added until neutrality. The mixture was filtered
and washed with water, 2-propanol and finally ether. The solid
was collected and taken up into water, and the suspension was
refluxed for 1 h. The mixture was then filtered; the solid was
recrystallized from 2-propanol and dried in vacuo to provide
acid 15 (160 mg, 52% yield) as dark brown crystals: mp >
2 2
with CH Cl and ether. The crude product was finally recrys-
tallized from 2-propanol and dried in vacuo.
Gen er a l Meth od B: Method B was identical to method A,
except that recrystallization from 2-propanol was replaced by
washing with hot 2-propanol.
Gen er a l Meth od C: To a solution of 1.0 equiv of acid 9 in
acetic anhydride (3 mL/mmol) was added 4 equiv of aldehyde.
The mixture was heated under reflux for 16 h and concentrated
in vacuo. The residue was dissolved in pyridine (4 mL/mmol),
water (1 mL/mmol) was then added, and the reaction mixture
was refluxed for 3 h. After cooling, the mixture was concen-
trated under reduced pressure. The solid residue was taken
-1
1
2
(
8
1
7
60 °C; IR (KBr, cm ) 3700-2800, 1615, 1586, 1514; H NMR
DMSO-d , 200 MHz) δ 8.47 (d, J ) 8.6 Hz, 1H), 8.16 (d, J )
6
.6 Hz, 1H), 7.87 (d, J ) 15.7 Hz, 1H), 7.83 (d, J ) 8.2 Hz,
H), 7.56-7.39 (m, 3H), 7.08 (d, J ) 8.6 Hz, 1H), 6.63 (d, J )
1
.6 Hz, 2H). Anal. (C18
(
H
14
N
2
O
3
‚ /
4
H
2
O) C, H, N.
E)-8-Hydr oxy-2-[2-(4-acetam idoph en yl)eth en yl]-7-qu in -
olin eca r boxylic Acid (16). Method C using 4-acetamidoben-
zaldehyde gave acid 16 in 30% overall yield as red crystals:
mp > 260 °C; IR (KBr, cm ) 3700-2000, 1679, 1593, 1515;
H NMR (DMSO-d , 200 MHz) δ 10.10 (broad s, 1H), 8.43 (d,
J ) 8.7 Hz, 1H), 8.09 (d, J ) 8.7 Hz, 1H), 7.86 (d, J ) 16.0 Hz,
1H), 7.82 (d, J ) 8.4 Hz, 1H), 7.66 (s, 4 H), 7.55 (d, J ) 16.0
Hz, 1H), 7.25 (d, J ) 8.4 Hz, 1H), 2.05 (s, 3H). Anal.
up into CH
-propanol and ether and dried in vacuo.
E)-8-Hyd r oxy-2-(2-p h en yleth en yl)-7-qu in olin eca r box-
ylic Acid (10). Method A using benzaldehyde afforded acid
0 in 46% overall yield as brick red crystals: mp 178-180 °C;
2 2
Cl and filtered. The solid was washed with
2
-
1
(
1
6
1
-1
1
IR (KBr, cm ) 3600-2400, 1680, 1614, 1590; H NMR (DMSO-
d
6
, 200 MHz) δ 8.41 (d, J ) 8.7 Hz, 1H), 8.07 (d, J ) 8.7 Hz,
H), 7.92 (d, J ) 16.4 Hz, 1H), 7.81 (d, J ) 8.6 Hz, 1H), 7.71
1
(C
20
H
16
N
2
O
4
‚ /
2
H
2
O) C, H, N.
1
(
d, J ) 6.6 Hz, 2H), 7.60 (d, J ) 16.4 Hz, 1H), 7.50-7.35 (m,
(E)-8-Hyd r oxy-2-[2-(4-h yd r oxyp h en yl)eth en yl]-7-qu in -
olin eca r boxylic Acid (17). Method A using 4-hydroxyben-
zaldehyde afforded acid 17 in 54% overall yield as red
3
1
1
H), 7.29 (d, J ) 8.6 Hz, 1H); 13C NMR (DMSO-d
71.5, 160.1, 153.7, 138.2, 137.4, 136.2 (2C), 131.0, 129.1 (3C),
27.5 (2C), 126.6 (2C), 121.7, 115.5, 111.5. Anal. (C18
O) C, H, N.
E)-8-H yd r oxy-2-[2-(2-fu r yl)et h en yl]-7-q u in olin eca r -
boxylic Acid (11). Method C using furfural gave acid 11 in
6% overall yield as brown crystals: mp 215-218 °C; IR (KBr,
6
, 50 MHz) δ
-
1
H
13NO
3
‚
crystals: mp 256-259 °C; IR (KBr, cm ) 3700-2300, 1669,
1
H
2
1578; H NMR (DMSO-d
d, J ) 8.6 Hz, 1H), 8.13 (d, J ) 8.6 Hz, 1H), 7.90 (d, J ) 16.4
Hz, 1H), 7.81 (d, J ) 8.6 Hz, 1H), 7.55 (d, J ) 8.6 Hz, 2H),
.51 (d, J ) 16.4 Hz, 1H), 7.20 (d, J ) 8.6 Hz, 1H), 6.84 (d, J
6
, 200 MHz) δ 9.96 (broad s, 1H), 8.47
(
(
7
5
1
3
-
1
1
) 8.6 Hz, 2 H); C NMR (DMSO-d , 50 MHz) δ 170.8, 160.5,
cm ) 3700-2400, 1630, 1606, 1511; H NMR (DMSO-d
MHz) δ 8.36 (d, J ) 8.7 Hz, 1H), 7.98 (d, J ) 8.7 Hz, 1H), 7.82
s, 1H), 7.80 (d, J ) 8.7 Hz, 1H), 7.78 (d, J ) 16.1 Hz, 1H),
.33 (d, J ) 16.1 Hz, 1H), 7.24 (d, J ) 8.7 Hz, 1H), 6.81 (d, J
3.4 Hz, 1H), 6.63 (dd, J ) 3.4, 1.9 Hz, 1H); 13C NMR
acetone-d , 50 MHz) δ 171.8, 160.7, 153.5, 152.4, 145.0, 138.2,
31.0, 127.0, 124.0, 123.5 (2C), 122.0, 115.3, 113.2, 113.0,
12.4. Anal. (C16 ‚H O) C, H, N.
E)-8-Hyd r oxy-2-[2-(3-th ien yl)eth en yl]-7-qu in olin eca r -
6
, 200
6
1
1
59.2, 153.2, 139.4 (2C), 138.0, 130.5, 129.5 (2C), 127.0, 126.8,
21.2, 120.9, 115.9 (2C), 113.8, 112.3. Anal. (C18 ‚H O)
H
13NO
4
2
(
7
)
(
1
1
C, H, N.
(E)-8-H yd r oxy-2-[2-(3,5-d ih yd r oxyp h en yl)et h en yl]-7-
qu in olin eca r boxylic Acid (18). Method C using 3,5-dihy-
droxybenzaldehyde afforded acid 18 in 15% overall yield as
6
-
1
H
11NO
4
2
red crystals: mp > 260 °C; IR (KBr, cm ) 3700-2200, 1598,
1
(
6
1570; H NMR (DMSO-d , 200 MHz) δ 9.40 (broad s, 1H), 8.42
boxylic Acid (12). Method C using 3-thiophenecarboxalde-
hyde gave acid 12 in 33% overall yield as orange crystals: mp
1
(d, J ) 8.3 Hz, 1H), 8.11 (d, J ) 8.6 Hz, 1H), 7.82 (d, J ) 8.3
Hz, 1H), 7.76 (d, J ) 16.4 Hz, 1H), 7.45 (d, J ) 16.4 Hz, 1H),
75-178 °C; IR (KBr, cm-1) 3400-2300, 1690, 1608, 1514; H
1
13
7.24 (d, J ) 8.3 Hz, 1H), 6.57 (s, 2H), 6.28 (s, 1H); C NMR
NMR (DMSO-d
6
, 200 MHz) δ 8.39 (d, J ) 8.7 Hz, 1H), 8.01(d,
6
(DMSO-d , 50 MHz) δ 171.3, 160.6 159.0 (2C), 153.3, 138.9,
J ) 8.7 Hz, 1H), 7.93 (d, J ) 16.4 Hz, 1H), 7.81 (d, J ) 8.6 Hz,
1
7
137.7, 137.6, 137.0, 131.0, 127.0, 125.5, 121.8, 115.0, 112.1,
.
+
H), 7.83 (d, J ) 0.5 Hz, 1H), 7.65 (dd, J ) 5.0, 2.8 Hz, 1H),
.57 (dd, J ) 5.0, 0.8 Hz, 1H), 7.45 (d, J ) 16.2 Hz, 1H), 7.26
105.7 (2C), 104.4; MS m/e (rel intensity) 323 (M , 0.3), 278
((M - 45) , 19), 159 (60), 44 (100).
.
+

Styrylquinolines as Inhibitors of HIV IN
E)-8-Hyd r oxy-2-[2-(4-h yd r oxy-3-m eth oxyp h en yl)eth e-
n yl]-7-qu in olin eca r boxylic Acid (21). Method A using
vanillin gave acid 21 in 67% overall yield as orange crystals:
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8 1539
(
1H), 8.11 (d, J ) 8.4 Hz, 1H), 7.90-7.70 (m, 2 H), 7.53 (s, 1H),
7.42 (d, J ) 16.4 Hz, 1H), 7.20 (d, J ) 8.6 Hz, 1H), 7.14 (s,
1H); 13C NMR (DMSO-d
, 50 MHz) δ 170.9, 160.4, 153.0, 147.7,
6
-
1
1
mp > 260 °C; IR (KBr, cm ) 3500-2400, 1621, 1581; H NMR
145.2, 137.0, 136.9, 135.9, 130.7, 129.3 (2C), 127.4, 122.4,
3
(
DMSO-d , 200 MHz) δ 9.60 (broad s, 1H), 8.48 (d, J ) 8.8
6
121.1, 113.9, 113.8, 112.5, 85.4. Anal. (C18
H, N.
H
12INO
6
2 2
‚ / H O) C,
Hz, 1H), 8.13 (d, J ) 8.8 Hz, 1H), 7.90 (d, J ) 16.2 Hz, 1H),
7
1
.82 (d, J ) 8.6 Hz, 1H), 7.56 (d, J ) 16.2 Hz, 1H), 7.30 (s,
H), 7.21 (d, J ) 8.6 Hz, 1H), 7.13 (d, J ) 8.1 Hz, 1H), 6.85 (d,
(E)-5,7-Dich lor o-8-h ydr oxy-2-[2-(3,4-dih yd r oxyph en yl)-
et h en yl]-7-q u in olin e (29). To a solution of 5,7-dichloro-8-
hydroxy-2-methyl-7-quinoline (4) (2.28 g, 10 mmol) in acetic
anhydride (30 mL) was added 3,4-dihydroxybenzaldehyde (5.52
g, 40 mmol). The mixture was heated under reflux for 16 h
and concentrated in vacuo. The residue was dissolved in
pyridine (40 mL), water (10 mL) was then added and the
reaction mixture was refluxed for 3 h. After cooling, the
mixture was concentrated under reduced pressure. The solid
1
3
J ) 8.1 Hz, 1H), 3.87 (s, 3H); C NMR (DMSO-d , 50 MHz) δ
6
1
1
5
70.9, 160.6, 153.2, 148.8, 148.1, 139.5, 138.3, 135.8, 130.5,
27.4, 127.2, 122.4, 121.4, 120.8, 115.8, 113.8, 112.4, 110.3,
5.6. Anal. (C19 ‚H O) C, H, N.
E)-8-Hyd r oxy-2-[2-(3-h yd r oxy-4-m eth oxyp h en yl)eth e-
H
15NO
5
2
(
n yl]-7-qu in olin eca r boxylic Acid (22). Method A using
isovanillin gave acid 22 in 54% overall yield as orange
-
1
1
crystals: mp > 260 °C; IR (KBr, cm ) 3700-2400, 1603; H
NMR (DMSO-d , 200 MHz) δ 8.52 (d, J ) 8.6 Hz, 1H), 8.20 (d,
J ) 8.6 Hz, 1H), 7.90 (d, J ) 16.8 Hz, 1H), 7.84 (d, J ) 8.1 Hz,
2 2
residue was taken up into CH Cl and filtered. The crude
product was finally recrystallized from 2-propanol and dried
6
in vacuo to give quinoline 29 (5.2 g, 44% yield) as ocher
-
1
1
H), 7.51 (d, J ) 16.8 Hz, 1H), 7.26 (d, J ) 8.1 Hz, 1H), 7.15
crystals: mp 250-252 °C; IR (KBr, cm ) 3700-2500, 1614,
1
1
3
(
m, 2H), 7.01 (d, J ) 8.6 Hz, 1H), 3.87 (s, 3H); C NMR
6
1599, 1554; H NMR (DMSO-d , 200 MHz) δ 9.40 (broad s,
(DMSO-d
6
, 50 MHz) δ 171.0, 159.3, 153.0, 149.9, 146.9, 140.4,
1H), 8.36 (d, J ) 8.4 Hz, 1H), 8.08 (d, J ) 16.5 Hz, 1H), 7.85
(d, J ) 8.8 Hz, 1H), 7.66 (s, 1H), 7.16 (d, J ) 16.5 Hz, 1H),
7.11(s, 1H), 7.02 (d, J ) 8.1 Hz, 1H), 6.80 (d, J ) 8.1 Hz, 1H);
1
1
39.5, 134.3, 130.5, 128.5, 127.5, 120.9 (3C), 114.4, 113.8,
1
12.8, 112.2, 55.7. Anal. (C19
H
15NO
5
2 2
‚ / H O) C, H, N.
1
3
3
,4-Dih yd r oxy-5-m eth oxyben za ld eh yd e (6). To a solu-
tion of 3,5-dimethoxy-4-hydroxybenzaldehyde (5) (510 mg, 2.8
mmol) in CH Cl (5 mL) was added portionwise aluminum
C NMR (DMSO-d
139.7, 138.0, 133.9, 128.7, 127.3, 124.4, 124.2, 123.0, 121.1,
120.0, 116.8, 116.2, 115.1. Anal. (C17 NO ) C, H, N.
6
, 50 MHz) δ 156.7, 149.4, 148.0, 146.5,
H
11Cl
2
3
2
2
chloride (450 mg, 3.3 mmol). Anhydrous pyridine (1.0 mL, 12.4
mmol) was then added dropwise to the resulting suspension.
After being stirred 72 h at room temperature, the mixture was
8-Hyd r oxy-2-[2-(3,4-d ih yd r oxyp h en yl)et h yl]-7-qu in o-
lin eca r boxylic Acid (30). To a solution of styrylquinoline 20
(200 mg, 0.62 mmol) in DMF (14 mL) was added 50 mg of 10%
Pd/C. The mixture was vigorously stirred for 24 h at 20 °C
under hydrogen atmosphere (4 bar). The catalyst was removed
by filtration through Celite and the filter pack was washed
repeatedly with CH Cl . The filtrate was concentrated in
2 2
vacuo. The solid residue was recrystallized in acetic acid to
afford acid 30 as orange crystals (60 mg, 30% yield): mp 264-
poured into 1 N HCl and was extracted with CH
organic extracts were washed with brine, dried over MgSO
and concentrated under reduced pressure. The residue was
recrystallized from CHCl to give aldehyde 6 (300 mg, 63%)
2 2
Cl . The
4
3
2
0
as colorless crystals: mp 180-181 °C (lit. mp 181-182 °C);
1
H NMR (DMSO-d
s, 3H).
6
, 200 MHz) δ 9.68 (s, 1H), 6.98 (s, 2H), 3.80
-
1
1
(
266 °C; IR (KBr, cm ) 3700-2400, 1656, 1599; H NMR
(
E)-8-Hyd r oxy-2-[2-(4,5-d ih yd r oxy-3-m eth oxyp h en yl)-
6
(DMSO-d , 200 MHz) δ 8.69 (broad s, 2H), 8.43 (d, J ) 8.4
eth en yl]-7-qu in olin eca r boxylic Acid (24). Method A using
Hz, 1H), 7.83 (d, J ) 8.6 Hz, 1H), 7.64 (d, J ) 8.4 Hz, 1H),
7.18 (d, J ) 8.6 Hz, 1H), 6.61 (s, 1H), 6.59 (d, J ) 8.2 Hz, 1H),
4
,5-dihydroxy-3-methoxybenzaldehyde (6) gave acid 24 in 53%
-
1
yield as brick red crystals: mp 215-217 °C; IR (KBr, cm
)
6.47 (dd, J ) 7.8, 1.8 Hz, 1H), 3.26 (t, J ) 6.8 Hz, 2H), 2.88 (t,
1
13
3
700-2200, 1588, 1512; H NMR (DMSO-d
6
, 200 MHz) δ 8.42
J ) 6.8 Hz, 2H); C NMR (DMSO-d , 50 MHz) δ 171.1, 160.8,
6
(d, J ) 8.6 Hz, 1H), 8.14 (d, J ) 8.6 Hz, 1H), 7.84 (m, 2H),
159.5, 145.1, 143.6, 139.9, 136.2, 131.6, 130.7, 127.2, 124.3,
5
119.1, 115.9, 115.6, 113.6, 112.2, 34.5, 33.2. Anal. (C18H15NO ‚
7
1
.51 (d, J ) 16.2 Hz, 1H), 7.18 (d, J ) 8.6 Hz, 1H), 6.88 (s,
H), 6.81 (s, 1H), 3.85 (s, 3H); 13C NMR (DMSO-d
, 50 MHz)
1
2 2
/ H
O) C, H, N.
6
δ 170.9, 160.8, 153.0, 148.7, 146.0, 139.6, 138.9, 136.8, 135.8,
P r ep a r a tion of DNA Su bstr a tes. Oligonucleotides U5B
1
5
30.6, 127.4, 126.4, 121.5, 120.8, 113.5, 112.8, 109.4, 103.2,
5′-GTGTGGAAAATCTCTAGCA, U5AACTGCTAGAGATTTT-
CCACAC, and D38U3 TGCTAGTTCTAGCAGGCCCTTGGGCC-
GGCGCTTGCGCC29 were purchased from Eurogentec and
further purified by denaturing 18% acrylamide gel. For
processing and disintegration assays, 100 pmol of U5A and
D30U5 oligonucleotides were radiolabeled respectively using
3
5.9. Anal. (C19
E)-8-Hyd r oxy-2-[2-(4-h yd r oxy-3,5-d im eth oxyp h en yl)-
eth en yl]-7-qu in olin eca r boxylic Acid (25). Method A using
H
15NO
6
‚ /
2 2
H O) C, H, N.
(
3
,5-dimethoxy-4-hydroxybenzaldehyde (5) gave acid 25 in 38%
-1
overall yield as brick red crystals: mp > 260 °C; IR (KBr, cm
)
1
32
3
700-2500, 1612, 1515; H NMR (DMSO-d
6
, 200 MHz) δ 8.90
T4 polynucleotide kinase (Biolabs) and 50 µCi of [γ- P]ATP
(
broad s, 1H), 8.45 (d, J ) 8.6 Hz, 1H), 8.10 (d, J ) 8.6 Hz,
H), 7.89 (d, J ) 16.3 Hz, 1H), 7.81 (d, J ) 8.6 Hz, 1H), 7.59
d, J ) 16.3 Hz, 1H), 7.21 (d, J ) 8.6 Hz, 1H), 7.02 (s, 2 H),
(s.a. 3000 Ci/mmol). Kinase was heat inactivated and unin-
corporated nucleotide were removed by passage through
Sephadex G-10 (Pharmacia). NaCl was added to the final
concentration of 0.1 M and complementary unlabeled strand
U5B was added to U5A. The mixture was heated to 90 °C for
2 min and the DNA was annealed by slow cooling.
1
(
1
3
3
1
1
6
.84 (s, 6 H); C NMR (DMSO-d , 50 MHz) δ 171.8, 161.4,
54.0, 149.0 (2C), 140.1, 139.1, 138.5, 137.0, 131.3, 127.7,
27.0, 123.0, 121.6, 115.1, 113.0, 105.9 (2C), 56.8 (2C). Anal.
1
(C
20
H
17NO
6
‚ /
2
H
2
O) C, H, N.
HIV-1 IN Assa y. Purified recombinant full-size HIV-1 IN
was a generous gift of Rh oˆ ne-Poulenc-Rorer. Plasmid encoding
the His-tagged soluble deletion mutant F(185)K corresponding
to the (50-212) core domain of HIV-1 IN was generously
provided by Dr. R. Craigie. The protein was expressed and
(
E)-8-Hydr oxy-2-[2-(3,5-dibr om o-4-h ydr oxyph en yl)eth e-
n yl]-7-qu in olin eca r boxylic Acid (26). Method B using 3,5-
dibromo-4-hydroxybenzaldehyde gave acid 26 in 55% yield, as
-
1
brick red crystals: mp > 260 °C; IR (KBr, cm ) 3650-2400,
1
30
1
675, 1611, 1481; H NMR (DMSO-d
8.2 Hz, 1H), 8.05-7.70 (m, 3H), 7.90 (s, 2H), 7.50 (d, J )
6.0 Hz, 1H), 7.25 (d, J ) 8.6 Hz, 1H); 13C NMR (DMSO-d
0 MHz) δ 171.4, 160.0, 153.3, 151.3, 137.8, 137.6, 133.0, 131.0
6
, 200 MHz) δ 8.35 (d, J
purified as described. Processing assay was performed using
)
1
5
0.5 pmol of U5A/U5B substrate in the presence of 1 pmol of
IN in buffer containing 20 mM Tris (pH 7.2), 30 mM NaCl,
10% (w/v) glycerol, 10 mM DTT, 0.01% NP40, supplemented
6
,
(
2C), 130.7, 128.2, 126.6 (2C), 121.5, 115.3, 112.3 (2C), 111.8.
Anal. (C18 ‚H O) C, H, N.
E)-8-Hyd r oxy-2-[2-(3-iod o-4,5-d ih yd r oxyp h en yl)eth e-
n yl]-7-qu in olin eca r boxylic Acid (27). Method B using
2
with 10 mM MnCl . Disintegration assays were performed in
H
11NBr
2
O
4
2
the presence of 0.5 pmol of D38U3 substrate and 2 pmol of IN
core domain. Gels were analyzed using a STORM Molecular
Dynamics phosphorimager. Inhibition in the presence of drugs
was expressed as percent of fractional product compared with
the control.
An tivir a l Assa ys. The lymphocyte cell line CEM was
maintained in a RPMI-1640 (GIBCO Laboratories) supple-
(
3
-iodo-4,5-dihydroxybenzaldehyde (8) gave acid 27 in 49%
-
1
yield, as brick red crystals: mp > 260 °C; IR (KBr, cm
3
δ 10.20 (broad s, 1H), 9.70 (broad s, 1H), 8.46 (d, J ) 8.4 Hz,
)
1
700-2500, 1619, 1587, 1513; H NMR (DMSO-d
6
, 200 MHz)

1
540 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8
Zouhiri et al.
+
mented with 10% fetal calf serum. Hela-CD4 -âGal (P4) cells,
kindly provided by P. Charneau (Institut Pasteur, Paris), were
grown in DMEM with 10% fetal calf serum and 0.5 mg/mL
geneticin. Cell-free viral supernatants were obtained by trans-
fection of P4 cells with HIV-1 PLN4-3 genomic clone. Cells
were plated in triplicate on a 96-well plates (100 µL) and
infected with cell-free virus. Viral supernatants were removed
(12) Artico, M.; Di Santo, R.; Costi, R.; Novellino, E.; Greco, G.; Massa,
S.; Tramontano, E.; Marongiu, M. E.; De Montis, A.; La Colla,
P. Geometrically and conformationally restrained cinnamoyl
compounds as inhibitors of HIV-1 integrase: Synthesis, biologi-
cal evaluation, and molecular modeling. J . Med. Chem. 1998,
4
1, 3948-3960.
(13) Weinberg, B.; Shugars, D. C.; Sherman, P. A.; Sauls, D. L.; Fyfe,
J . A. Cobalamine inhibition of HIV-1 integrase and integration
of HIV-1 DNA into cellular DNA. Biochem. Biophys. Res.
Commun. 1998, 246, 393-397.
2
h after infection and drugs dissolved in DMSO were added
in fresh medium. 72 h later, supernatants were used to infect
Hela-CD4 -âGal cells. The P4 cultures were subsequently
incubated for 24 h and subsequently lysed in a phosphate
(
14) J ing, N.; Hogan, M. E. Stucture-activity of tetrad-forming
oligonucleotides as potent anti-HIV therapeutic drug. J . Biol.
Chem. 1998, 273, 34992-34999.
+
buffer containing 50 mM 2-mercaptoethanol, 10 mM MgSO
4
,
(15) King, P. J .; Ma, G.; Miao, W.; J ia, Q.; McDougall, B. R.; Reinecke,
M. G.; Cornell, C.; Kuan, J .; Kim, T. R.; Robinson, J r., W. E.
Stucture-activity relationships: Analogues of the dicaffeoylqui-
nic and dicaffeoyltartaric acids as potent inhibitors of human
immunodeficiency virus type 1 integrase and replication. J . Med.
Chem. 1999, 42, 497-509.
(16) Klopman, G.; Tu, M. Diversity analysis of 14 156 molecules
tested by the National Cancer Institute for anti-HIV activity
using the quantitative structure-activity relational expert
system MCASE. J . Med Chem. 1999, 42, 992-998.
2
5 mM EDTA, 0.125% NP40. 20 µL of lysate was incubated
3
1
with 100 µL of CPRG-containing buffer. The red staining
intensity was quantified on a multiscan photometer at 570 nm.
Cell viability was estimated by the MTT (Sigma) assay. 20 µL
of a solution of MTT (7.5 mg/mL) in phosphate buffer was
added to each well of the microtiter trays. The plates were
further incubated at 37 °C in a CO
Solubilization of the formazan crystals was achieved by adding
00 mL of 10% SDS, 10 mM HCl. Absorbances were read in a
2
incubator for 4 h.
(
17) Lin, Z.; Neamati, N.; Zhao, H.; Kiryu, Y.; Turpin, J . A.; Aberham,
C.; Strebel, K.; Kohn, K.; Witvrouw, M.; Pannecouque, C.;
Debyser, Z.; De Clercq, E.; Rice, W. G.; Pommier, Y.; Burke, J r.,
T. R. Chicoric acid analogues as of HIV-1 integrase inhibitors.
J . Med. Chem. 1997, 42, 1401-1414.
1
multiscan photometer at 570 nm. In some cases, antiviral
activity was measured by p24 antigen ELISA.
(
18) Singh, S. B.; J ayasuriya, H.; Hazuda, D. L.; Felock, P.; Homnick,
C. F.; Sardana, M.; Patane, M. A. Selective and controlled
hydrolysis of chloropeptin I. HIV-1 integrase activity of frag-
ments. Tetrahedron Lett. 1998, 39, 8769-8770.
Ack n ow led gm en t. This work was supported by
funds from the Agence Nationale de Recherche sur le
Sida (ANRS), the Centre National de la Recherche
Scientifique (CNRS), and the Minist e` re de la Recherche
et de la Technologie. H.L. gratefully acknowledges
fellowship support from the ANRS. Fellowships of the
Fondation pour la Recherche M e´ dicale (Sidaction) to
K.M. and F.Z. are acknowledged. We also thank Prof.
B. Legendre (Facult e´ de Pharmacie de Ch aˆ tenay-Ma-
labry) for microthermal analyses, Dr. J . Mahuteau for
NMR spectral analyses, and Mrs. S. Mairesse-Lebrun
for performing elemental analyses.
(
19) Mekouar, K.; Mouscadet, J .-F.; Desma e¨ le, D.; Subra, F.; Savour e´ ,
D.; Auclair, C.; d'Angelo, J . Styryl-quinoline derivatives: A new
class of potent HIV-1-integrase inhibitors that block HIV-1
replication in CEM cells. J . Med. Chem. 1998, 41, 2846-2857.
20) Anhoury, M.-L.; Crooy, P.; De Neys, R.; Eliaers, J . A simple and
mild method for reducing cyanohydrins to amino-alcohols. J .
Chem. Soc., Perkin Trans. I 1974, 1015-1017.
(
(
21) King, P. J . Robinson, J r., W. E. Resistance to the anti-human
immunodeficiency virus type 1 compound L-chicoric acid results
from a single mutation at amino acid 140 of integrase. J . Virol.
1
998, 72, 8420-8424.
(
22) Goldgur, Y.; Dyda, F.; Hickman, A. B.; J enkins, T. M.; Craigie,
R.; Davies, D. R. Three new structures of the core domain of
HIV-1 integrase: An active site that binds magnesium. Proc.
Natl. Acad. Sci. U.S.A. 1998, 95, 9150-9154.
23) Lubrowski, J .; Yang, F.; Alexandratos, J .; Wlodawer, A.; Zhao,
H.; Burke, J r., T. R.; Neamati, N.; Pommier, Y.; Merkel, G.;
Skalka, A. M. Stucture of the catalytic domain of avian sarcoma
virus integrase with a bound HIV-1 integrase-targeted inhibitor.
Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 4831-4836.
24) Goldgur, Y.; Craigie, R.; Cohen, G. H.; Fujiwara, T.; Yoshinaga,
T.; Fujishita, T.; Sugimoto, H.; Endo, T.; Murai, H.; Davies, D.
R. Structure of the HIV-1 integrase catalytic domain complexed
with an inhibitor: A platform for antiviral drug design. Proc.
Natl. Acad. Sci. U.S.A. 1999, 96, 13040-13043.
25) Ouali, M.; Laboulais, C.; Leh, H.; Gill, D.; Desma e¨ le, D.;
Mekouar, K.; Zouhiri, F.; d'Angelo, J .; Auclair, C.; Mouscadet,
J .-F.; Le Bret, M. Modeling of the inhibition of retroviral
integrases by styrylquinoline derivatives. J . Med. Chem. 2000,
Refer en ces
(
(
1) Ho, D. D.; Neumann, A. U.; Perelson, A. S.; Chen, W.; Leonard,
J . M.; Markowitz, M. Rapid turnover of plasma virions and CD4
lymphocytes in HIV-1 infection. Nature 1995, 373, 123-126.
2) Zhang, L.; Ramratnam, B.; Tenner-Racz, K.; He Y.; Vesanen,
M.; Lewin, S.; Talal, A.; Racz, P.; Perelson, A.; Korber, B.;
Markowitw, M.; Ho, D. Quantifying residual HIV-1 replication
in patients receiving combination antiretroviral therapy. N.
Engl. J . Med. 1999, 340, 1605-1613.
3) Furtado, M.; Callaway, D.; Phair, J .; Kunstman, K.; Stanton,
J .; Macken, C.; Perelson, A.; Wolinsky, S. Persistence of HIV-1
transcription in peripheral blood mononuclear cells in patients
receiving potent antiretroviral therapy. N. Engl. J . Med. 1999,
(
(
(
(
3
40, 1614-1622.
4) Finzi, D.; Silliciano, R. F. Viral dynamics in HIV-1 infection. Cell
998, 93, 665-671.
4
3, in press.
(
(
(
26) Hong, H.; Neamati, N.; Wang, S.; Nicklaus, M. C.; Mazumder,
A.; Zhao, H.; Burke, J r., T. R.; Pommier, Y.; Milne, G. W. A.
Discovery of HIV-1 integrase inhibitors by pharmacophore
searching. J . Med. Chem. 1997, 40, 930-936.
27) Neamati, N.; Hong, H.; Mazumder, A.; Wang, S.; Sunder, S.;
Nicklaus, M. C.; Milne, G. W. A.; Proksa, B.; Pommier, Y.
Depsides and depsidones as inhibitors of HIV-1 integrase:
Discovery of novel inhibitors through 3D database searching.
J . Med. Chem. 1997, 40, 942-951.
28) Robinson, J r., W. E. L-Chicoric acid, an inhibitor of human
immunodeficiency virus type 1 (HIV-1) integrase, improves on
the in vitro anti-HIV-1 effect of Zidovudine plus a protease
inhibitor (AG 1350). Antiviral Res. 1998, 39, 101-111.
29) Chow, S. A.; Vincent, K. A.; Ellison, V.; Brown, P. O. Reversal
of integration and DNA splicing mediated by integrase of human
immunodeficiency virus. Science 1992, 255, 723-726.
1
5) Lafemina, R. L.; Schneider, C. L.; Robbins, H. L.; Callahan, P.
L.; Legrow, K.; Roth, E.; Schleif, W. A.; Emini, E. A. Requirement
of active human immunodeficiency virus type 1 integrase enzyme
for productive infection of human T-lymphoid cells. J . Virol.
(
1
992, 66, 7414-7419.
(
6) De Clerq, E. Toward improved anti-HIV chemotherapy: thera-
peutic strategies for intervention with HIV infections. J . Med.
Chem. 1995, 38, 2491-2517.
(
(
7) Engelman, A.; Mizuuchi, K.; Craigie, R. HIV-1 DNA integra-
tion: mechanism of viral DNA cleavage and DNA strand
transfer. Cell 1991, 67, 1211-1221
(
8) Brown, P. O. Integration of retroviral DNA. Curr. Top. Microbiol.
Immunol. 1990, 157, 18-48.
(
(
9) Neamati, N.; Sunder, S.; Pommier, Y. Design and discovery of
HIV-1 integrase inhibitors. Drug Discov. Today 1997, 2, 487-
(
30) Carteau, S.; Mouscadet, J . F.; Goulaouic, H.; Subra, F.; Auclair,
C. Quantitative in vitro assay for human immunodeficiency virus
deoxyribonucleic acid integration. Arch. Biochem. Biophys. 1993,
4
98.
(
10) Pommier, Y.; Neamati, N. Inhibitors of human immunodeficiency
virus integrase. Adv. Virus Res. 1999, 52, 427-458.
11) Singh, S. B.; Zink, D. L.; Goetz, M. A.; Dombrowski, A. W.;
Polishook, J . D.; Hazuda, D. J . Equisetin and a novel opposite
stereochemical homologue phomasetin, two fungal metabolites
as inhibitors of HIV-1 integrase. Tetrahedron Lett. 1998, 39,
3
00, 756-610.
(
(
31) Hansen, M. S.; Bushman, F. D. Human immunodeficiency virus
type 2 preintegration complexes: Activities in vitro and response
to inhibitors. J . Virol. 1997, 71, 3351-3356.
2
243-2246.
J M990467O