Styrylquinoline derivatives: A new class of potent HIV-1 integrase inhibitors that block HIV-1 replication in CEM cells

Article abstract of DOI:10.1021/jm980043e

On the basis of the fact that several polynucleotidyl transferases, related to HIV integrase, contain in their active site two divalent metal cations, separated by ca. 4 ?, new potential HIV integrase inhibitors were designed, in which a quinoline substructure is linked to an aryl nucleus possessing various hydroxy substitution patterns, by means of an ethylenic spacer. Although the most active compounds contain the catechol structure, this group is not essential for the activity, since compound 21 that lacks such a moiety is a potent drug, implicating the presence of a different pharmacophore. The most promising styrylquinolines thus synthesized inhibit HIV-1 integrase in vitro at micromolar or submicromolar concentrations and block HIV replication in CEM cells, with no significant cellular toxicity in a 5-day period assay. These inhibitors are active against integrase core domain-mediated disintegration, suggesting that fragment 50-212 is their actual target. These new styrylquinolines may provide lead compounds for the development of novel antiretroviral agents for AIDS therapeutics, based upon inhibition of HIV integrase. They might also be used in the elucidation of the mechanism of inhibition of this enzyme; e.g., they could serve as candidates for cocrystallization studies with HIV integrase.

Full text of DOI:10.1021/jm980043e

2846
J . Med. Chem. 1998, 41, 2846-2857
Styr ylqu in olin e Der iva tives: A New Cla ss of P oten t HIV-1 In tegr a se In h ibitor s
Th a t Block HIV-1 Rep lica tion in CEM Cells
Khalid Mekouar,† J ean-Franc¸ois Mouscadet,§ Didier Desmae¨le,† Fre´de´ric Subra,§ Herve´ Leh,§ Delphine Savoure´,§
Christian Auclair,§ and J ean d’Angelo*,†
Unite´ de Chimie Organique, URA du CNRS 1843, Centre d’Etudes Pharmaceutiques, Universite´ Paris-Sud,
5 rue J .-B. Cle´ment, 92296 Chaˆtenay-Malabry, France, and Laboratoire de Physicochimie et de Pharmacologie des
Macromole´cules Biologiques, URA du CNRS 147, Institut Gustave Roussy, PRII, 39 rue Camille Desmoulins,
94805 Villejuif, France
Received J anuary 21, 1998
On the basis of the fact that several polynucleotidyl transferases, related to HIV integrase,
contain in their active site two divalent metal cations, separated by ca. 4 Å, new potential HIV
integrase inhibitors were designed, in which a quinoline substructure is linked to an aryl nucleus
possessing various hydroxy substitution patterns, by means of an ethylenic spacer. Although
the most active compounds contain the catechol structure, this group is not essential for the
activity, since compound 21 that lacks such a moiety is a potent drug, implicating the presence
of a different pharmacophore. The most promising styrylquinolines thus synthesized inhibit
HIV-1 integrase in vitro at micromolar or submicromolar concentrations and block HIV
replication in CEM cells, with no significant cellular toxicity in a 5-day period assay. These
inhibitors are active against integrase core domain-mediated disintegration, suggesting that
fragment 50-212 is their actual target. These new styrylquinolines may provide lead
compounds for the development of novel antiretroviral agents for AIDS therapeutics, based
upon inhibition of HIV integrase. They might also be used in the elucidation of the mechanism
of inhibition of this enzyme; e.g., they could serve as candidates for cocrystallization studies
with HIV integrase.
In tr od u ction
groups: DNA ligands, C-terminal domain ligands, and
compounds that interfere with the catalytic domain of
the protein. The first family contains nonspecific in-
tercalating agents,8,9 as well as more specific oligonucle-
otides targeting integrase binding sites on both LTRs.10,11
The second class includes polyanionic drugs, such as
suramin which interacts with the highly basic C-ter-
minal domain.12 Finally, the third group embodies hy-
droxylated aromatic compounds including aurintricar-
boxylic acids,13 bis-catechols,14 CAPE, flavones and flav-
onoids,15 curcumin,16 tyrphostins,17 lignanolides,18 cou-
marin derivatives,19 cosalanes,20 hydrazide derivatives,21
and depsides and depsidones.22 Systematic screening
or more rational studies23-27 of these families allowed
to identify possible pharmacophores,23 such as the cate-
chol structure.16 Finally, peptides obtained by rational28
or combinatorial research strategies29 were also de-
scribed. Overall, while many integrase inhibitors have
now been identified, only a handfull displayed antiviral
activity in cell culture. This poorly populated group
comprises lignanolides,18 curcumin,16 aurintricarboxylic
acids,13 dicaffeoylquinic acids and analogues,30,31 diaryl
sulfones,23 and finally G-rich oligonucleotides.32 To
date, only one serious candidate has entered clinical
assay. Surprisingly, it is a G-rich oligonucleotide that
was shown to inhibit integrase activity both in vitro and
in vivo.32-34 However, despite its remarkable antiviral
properties, the clinical use of such an oligonucleotide
still rests on the capacity to produce amounts of the
compound compatible with therapeutic exigencies.35
AIDS is essentially a viral disease and should be
treated by antiretroviral agents.1 From this standpoint,
HIV DNA integration into genomic DNA of the host cell,
a crucial step in the replicative life cycle of the virus,
constitutes a particularly attractive target for AIDS
chemotherapeutics, including potential synergy with
currently available HIV reverse transcriptase and pro-
tease inhibitors.2 The integration step, which is medi-
ated by a viral protein, integrase (IN), is required for
the production of progeny viruses.3 IN directs two dis-
tinct reactions: terminal cleavage at each 3′ end of the
proviral DNA removing a pair of bases and strand trans-
fer which results in the joining of each processed 3′ end
to 5′-phosphates in the target DNA.4 The terminal
cleavage and strand-transfer reactions can be modeled
in vitro, by using purified recombinant IN protein.5
Under simple reaction conditions, purified IN requires
a double-stranded DNA substrate mimicking the viral
LTRs extremity. In the absence of any other nucleic
species, homologous integration is observed, whereas
addition of a nonspecific target leads to heterologous
integration. Moreover, integrase is capable of catalyzing
the reverse reaction termed “disintegration”.6 The
disintegration assay involves a short oligonucleotide, the
structure of which resembles the reaction intermediate.7
Systematic screening of potential inhibitors has been
undertaken using those three activity assays.
Several families of IN inhibitors have now been
identified. Most of them can be classified into three
Consequently, we set out to synthesize a new class of
compound based upon the quinoline structure, with the
† Universite´ Paris-Sud.
§ Institut Gustave Roussy.
S0022-2623(98)00043-0 CCC: $15.00 © 1998 American Chemical Society
Published on Web 06/25/1998

Styrylquinoline Derivatives
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2847
Sch em e 1
view to identify new structurally simple inhibitors of
HIV integrase. In this report, we describe new
styrylquinoline derivatives that inhibit HIV integrase
in vitro at micromolar or submicromolar concentrations,
block HIV replication in CEM cells, and are devoid of
cellular toxicity.
Resu lts a n d Discu ssion
Ch em istr y. It is manifest that the presence of
divalent cations is essential for the catalytic activity of
HIV integrase.36-38 Today it is not clear whether
cations are directly involved in catalysis, in integrase
activation through a metal-induced conformational
change,39,40 or in both. However, several lines of
evidence support the proposal that a metal cofactor is
coordinated by the acidic residues of the D,D(35)E
motive that constitutes the active site of the enzyme.
In view of the fact that several polynucleotidyl trans-
ferases, related to HIV integrase, contain in their active
site two divalent metal cations, separated by ca. 4
Å,41-46 we planed to design new potential HIV integrase
inhibitors, in which two metal-chelating subunits are
linked by means of an appropriate central spacer. We
reasoned that 8-hydroxyquinoline (“oxine”), a well-
known reagent used in analytical chemistry for the
determination of numerous divalent metals, including
manganese, zinc, and magnesium, might constitute the
basic structure of one of the chelating systems. An aryl
nucleus possessing various hydroxy substitution pat-
terns was chosen for the second chelating subunit, on
the basis of the observation that the most potent HIV
integrase inhibitors known to date generally contain an
ortho-dihydroxylated (catechol-type) aromatic moiety. In
support of this endeavor, a series of quinoline-containing
compounds were synthesized and tested in vitro against
HIV integrase and ex vivo against CEM cells infected
with HIV-1.
Sch em e 2
Etherification of commercially available 8-hydroxy-
quinaldine (1) with (chloromethyl)safrole (22)47 gave 2.
Condensation of 1 with acid chlorides 23 and 24 led to
the corresponding esters 3 and 4, respectively. Car-
boxylation of 1 under the Kolbe-Schmitt conditions
furnished the known acid 548 (Scheme 1).
An ti-In tegr a se Activities. First, styrylquinoline
derivatives were tested against 3′-processing and strand-
transfer activities. Strand-transfer experiments were
performed using a plasmid as a target in order to sim-
plify the quantitative calculation52 (see the Experi-
mental Section). The in vitro anti-HIV integrase ac-
tivity of the molecules synthesized is summarized in
Table 1.
These compounds can be roughly classified as inac-
tive, 1-12, and active, 13-21. In turn, the latter class
of molecules can be subdivided into three families:
moderately potent drugs 13 and 14 fail to show complete
inhibition at 100 µM; potent compound 15 has very little
effect at 1 µM, although it shows IC50 below 10 µM; and
highly potent drugs 16-21 gave rise to a marked effect
at 1 µM. A complete lack of inhibitory potency was
observed with the parent 8-hydroxyquinaldine (1) and
with its O-alkylated and O-acylated derivatives 2 (data
not shown), 3, and 4. However, a promising observation
was made in this series: quinaldine 5 bearing a hy-
droxyl group at C-8 and a carboxyl function at C-7
exhibited a discrete but significant activity. Styrylquin-
olines 6-12, in which a quinoline moiety is linked to
an aryl nucleus by means of an ethylenic spacer, proved
to be completely devoid of activity, despite the presence
of a catechol center in some of these compounds (9-
Basically, the elaboration of the central ethylenic
linker, of pure E geometry, in styrylquinolines 6-21 was
secured through the Perkin condensation between
quinaldines 25 and aromatic aldehydes 26 (Scheme 2).49
Syntheses of substrates 8, 11, 14, and 16 employed
the following peculiar protocols. The aldehyde partner
in the preparation of the symmetrical substrate 8 was
the known aldehyde 27,50 prepared from 1. The starting
material in the preparation of nitrile 16 was the known
aldehyde 28,51 which was converted into oxime 29.
Concomitant dehydration of the oxime function of 29
took place during the next Perkin condensation, which
thus delivered nitrile 16. Addition of aldehyde 30,
obtained from 28, to Wittig reagent prepared from
phosphonium salt 31 gave after removal of the protect-
ing groups triol 32, the precursor through Perkin
condensation of the bis-ethylenic derivative 14. Amino
derivative 11 was prepared from commercially available
8-nitroquinaldine (33) by a three-step sequence involv-
ing Perkin condensation into 34, tin(II) chloride reduc-
tion, and finally hydrolysis of the acetoxy groups
(Scheme 3).

2848 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Mekouar et al.
Sch em e 3
In t er a ct ion w it h t h e Ca t a lyt ic Cor e Dom a in .
Integrase can catalyze a reverse reaction called “disin-
tegration” reaction.6 Unlike processing and strand
transfer, this reaction may be carried out by the core
domain of integrase lacking both N- and C-terminal
domains.54 Therefore, inhibition of integrase-mediated
disintegration activity is good evidence for a mechanism
involving direct interaction with the catalytic domain.
To determine whether styrylquinoline derivatives act
at the core domain of integrase, disintegration using the
soluble 50-212 core domain of HIV integrase was
performed.55 Table 2 shows the activity of active
styrylquinoline derivatives against the reaction cata-
lyzed by this domain.
All compounds 13-21 that were active against inte-
gration inhibited disintegration as well. Compounds
18-21 which displayed IC50 values below 1 µM are
highly active, the 3,4,5-trihydroxylated 19 being the
most potent, in agreement with results of strand-
transfer assay. Again, salts 18 and 20 were as active
as parent compounds 17 and 19. This result strongly
supports the hypothesis that the target site of styrylquin-
oline derivatives is located in the catalytic core of IN.
To reinforce this conclusion, another set of experiments
was undertaken. As a matter of fact, integrase belongs
to the superfamily of polynucleotidyl transferases.45
Structural resemblances between polynucleotidyl trans-
ferases and type II restriction enzyme catalytic do-
mains have been reported,56 suggesting similar chem-
ical mechanisms for reaction pathways in both fam-
ilies. Consequently, a common effect against integrase
and a member of the type II restriction enzyme family
should provide another evidence for an interaction with
their structurally related catalytic domain. To support
this hypothesis, we tested the lead compound 17 against
the EcoRI-mediated cleavage of a linearized plasmid
(see the Experimental Section). Results are shown in
Figure 1.
11). When an additional hydroxyl group was introduced
at the C-8 position in the quinoline half of inactive
styrylquinoline 9, a moderate inhibitory potency was
gained (compare 13 or 14 with 9). However, the
replacement of the three hydroxyl groups of 13 by
acetoxy substituents completely abolished the activity
(compare 12 with 13); thus the inhibitory potency is
clearly associated with the presence of “ free” phenolic
hydroxyl groups. When a carboxyl or nitrile function
and a hydroxyl group were both introduced in the
quinoline subunit, at the summits C-7 and C-8, respec-
tively, potent to highly potent HIV integrase inhibitors
were designed (compounds 15-21). Styrylquinoline 15,
in which the phenyl half possesses the 2,4-m-dihydroxy
substitution pattern, showed good potency. High in-
hibitory activities were finally obtained with molecules
16-20, where the phenyl nucleus is now 3,4-ortho-
dihydroxylated or 3,4,5-trihydroxylated. Note in this
respect that in the lower-energy conformer of 13 (com-
pound having the minimun requirement for activity),
the pattern of the oxygen atom triad (interatomic
distances of 9.46, 7.67, and 2.72 Å) satisfactorily matches
one of the putative three-point pharmacophores previ-
ously identified by Pommier and co-workers22,25 (for a
recent indentification of a four-point pharmacophore, see
ref 53). It is noteworthy that, compared with the
catechol-containing compound 17, the same level of
activity was gained with substrate 21, in which the
phenyl subunit now carries a carboxyl function at C-3
and a hydroxyl group at C-4. Thus, the latter example
clearly demonstrates that the design of potent HIV
integrase inhibitors does not necessarily require the
presence of an ortho-dihydroxylated (catechol-type) aro-
matic moiety.
It can be observed that 17 is actually an inhibitor of
EcoRI. However, the IC50 for EcoRI inhibition, roughly
equal to 50 µM, was markedly higher than the one
calculated for integrase inhibition (ca.. 1 µM, see Table
1). Since concentrations of EcoRI and integrase in
either assays were roughly equal, the IC50 discrepancy
likely results from specificity of the drug toward inte-
grase. From this standpoint, it is important to note that
neither 17 nor 19 was active against HIV reverse
transcriptase in vitro (data not shown) for concentra-
tions ranking up to 100 µM. In conclusion, styrylquino-
line derivatives most likely interact with the IN core
domain. It is in agreement with previous reports which
showed that hydroxylated compounds interfere with the
HIV IN core domain. However, despite the fact that
catechol-containing styrylquinolines were the most ef-
fective derivatives, it is important to note that deriva-
tives 15 and 21, lacking the catechol structure, were
active at concentrations lower than 10 µM, suggesting
the presence of a different pharmacophore.
Although IN displays more activity in vitro with Mn2+
as the cationic cofactor, it is generally admitted that
Mg2+ is the actual cofactor in vivo.56 Since styrylquino-
line derivatives were designed to interfere with the
active site of integrase, it was of interest to investigate
their properties against Mg2+-dependent activity. How-

Styrylquinoline Derivatives
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2849
Ta ble 1. HIV-1 Integrase Inhibitory Potencies Determined as Described in the Experimental Section
ever, under our standard conditions, the strand-transfer
activity of IN was virtually undetectable. To adress that
issue, we designed a different assay in which the full-
length IN was first incubated on ice in the presence of
10 mM of Mg2+
subsequently added with or without the potential
inhibitor, and the reaction mixture was incubated for
10 min at 4 °C. The target DNA was eventually added,
.
The 500-bp viral substrate was

2850 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Mekouar et al.
Ta ble 2. Effect of Active Styrylquinolines Derivatives upon
Disintegration Catalyzed by the Deletion Mutant (50-212)
compd
IC50 (µM)
13
15
16
17
18
19
21
2.5
2.5
3.6
0.9
0.7
0.24
0.6
F igu r e 2. Phosphorimager picture of Mg2+-dependent activity
of HIV IN in the presence of compound 19; 10 ng of 500-bp
viral substrate and 40 ng of pSP70 were incubated in the
presence of 2 pmol of HIV-1 IN and increasing concentrations
of compound 19, in a buffer containing 10 mM MgCl2. Lanes
1 and 12: DNA size marker. Lanes 2-10: concentration-
dependent inhibition of IN activity by compound 19 in the
presence of Mg2+. Drug concentrations are indicated above
each lane. Lane 11: incubation of viral substrate and 2 pmol
of HIV-1 IN without drug. Lane 12: viral substrate without
IN. Reaction products are shown on the right; (A) 500-bp viral
DNA, (B) homologous integration products, (C) heterologous
integration products.
activities against HIV-1 replication in CEM cells. They
were tested for their ability to lower the viral charge in
culture supernatants. CEM cells were infected with
HIV-1 and subsequently treated with increasing con-
centrations of styrylquinoline derivatives. Viral load
was estimated 72 h after infection. The amount of virus
was assayed by â-galactosidase assay, with HeLaCD4-
â-gal cells as reporting cells. Antiviral assays were
confirmed by virion-associated p24 antigen assay in
supernatants. More interestingly, several compounds
were active against viral replication as shown in Table
4. Toxicity was estimated by MTT transformation
assay. As shown in Table 3, most compounds were
devoid of cellular toxicity up to 100 µM, which was the
highest concentration tested. Under our standard
conditions, antiviral activity was tested after 72 h
postinfection. To determine wether some cytoxicity
could build up over a longer period of time, toxicity of
compounds 17, 15, and 21 was also tested 120 h after
infection. Again, very moderate or no toxicity at all was
detected with active compounds 17, 15, and 21 (see
Table 4).
F igu r e 1. Phosphorimager picture of EcoRI activity assay.
Plasmid pSP65 was linearized using SspI restriction enzyme
and radiolabeled with T4 kinase. Incubation of the resulting
30005-bp DNA fragment with 0.1 U of EcoRI gave rise to 2101-
and 904-bp DNA fragments. Lanes 1 and 9: linear DNA size
marker. Lanes 2-8: concentration-dependent inhibition of
EcoRI activity by compound 17. Drug concentrations are
indicated above each lane.
and the reaction was allowed to proceed for 60 mn at
37 °C. Preincubation with Mg2+ gave rise to a signifi-
cant increase in strand-transfer activity allowing us to
test the compounds with these new conditions. As
shown in Figure 2 for compound 19, inhibition of strand-
transfer reaction (IC50 ) 1.9 mM) was readily observed
with Mg2+ as cofactor.
Ex Vivo Activity of Styr ylqu in olin e Der iva tives.
Compounds 13-21 were evaluated for their antiviral
In vitro active compound 15 exhibited a moderate
activity ex vivo with IC50 between 50 and 100 µM and

Styrylquinoline Derivatives
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2851
Ta ble 3. Cytotoxicity of Quinoline Derivatives
compd
TC50 (µM)
TC90 (µM)
5
7
9
13
15
16
17
18
19
21
>100
50.1
12.3
8.1
>100
>100
100
71
>100
>100
6.2
56.3
>100
>100
10.7
>100
>100
>100
93.3
>100
Ta ble 4. Inhibition of HIV-1 Replication in CEM Cells by
Quinoline Derivatives
antiviral activity
toxicity at
(µM) on CEM cells
100 µM (%)
compd
IC25
IC50
IC90
72 h
120 h
5
13
15
17
>100
>100
3.6
>100
0
99
0
1.2
26.3
0.46
F igu r e 4. Effect of time of drug addition on HIV replication
inhibition by compound 17. CEM cells infected with HIV-
79.8
1.3a
20b
20b
1.2
>100
<10
<10
12.3a
0
1
PLN4-3 were treated at 2 h (oblique bars), 4 h (horizontal bars),
>100b
10.7
>100
0
100
0
8 h (dotted bars), and 24 h (filled bars) after virus infection
with compound 17 at 1 and 10 mM. Viral load in supernatants
was monitored after 72 h by HeLaâ-gal cell infection assay.
SEM of triplicate samples are shown as error bars.
18
19
21
0.4
20.9
62
0
a
b
As measured by P4 infection. As measured by P24 ELISA
test.
terestingly, viral replication could be followed visually,
since it gave rise to syncytia. As a matter of fact, the
antiviral effect led to a marked decrease of the amount
of these syncytia, which became virtually undetectable
above 50 µM. This anti-HIV activity of compound 17
was confirmed by measuring p24. Sodium salt 18 led
to the same antiviral activity. It has been previously
suggested that polyhydroxycarboxylates such as dicaf-
feoylquinic acids could interfere with virus penetra-
tion.22 Consequently, to investigate further the anti-
integrase activity of 17, ex vivo, kinetic experiments
were performed. Compound 17 was added respectively
2, 4, 8, and 24 h after infection. Cells were washed after
virus treatment in order to eliminate virus that had not
penetrated into cells. Again, a marked antiviral effect
was observed (Figure 4).
However, percent of inhibition was dependent upon
the time of addition. Inhibition was maximized when
the drug was added after 4 and 8 h. Clearance and/or
degradation of the drug can account for the lower effect
when the drug was added only 2 h after infection.
Adding the drug after 24 h gave rise to a markedly
decrease of the drug activity. One can infer from this
experiment that drug was effective during the first
round of viral replication. Since interference with virus
penetration was not possible, drugs must be active
against an early postpenetration step. Reverse tran-
scription is generally believed to begin in HIV virions
and to continue during the first hour of infection.
Reverse transcription leads to the synthesis of viral
DNA, which enters a nucleoprotein complex containing
at least integrase and possibly other viral and cellular
proteins. This preintegration complex, often called PIC,
migrates into the cell nucleus where it is covalently
inserted into host genome between 8 and 16 h postin-
fection. Recent reports57 suggested that polyhydroxy-
lated inhibitors of HIV IN are most likely active against
the assembly of preintegration complexes. Accordingly,
some compounds that are potent inhibitors of recombi-
F igu r e 3. Antiviral activity (b) and cytotoxicity (9) of
compound 17. CEM cells were infected with cell-free virus; 2
h after virus addition, cells were washed to remove unbound
virus, and fresh medium containing increasing concentration
of compound 17 dissolved in DMSO was added. At 72 h
postinfection, supernatants were collected and used to infect
HeLaCD4+-â-gal cells. Cultures were then incubated for 24 h
before lysis and subsequent incubation with CPRG for 1 h.
The red-staining intensity was quantified on a multiscan
photometer at 570 nm. After 72 h, cell viability was estimated
by the MTT assay. Results are the average of three experi-
ments.
was devoid of cytotoxicity. Styrylquinolines 13 and 19
were also active but proved to be dramatically cytotoxic.
However, it is worthy to note that the most effective
drug 19 (IC50 ) 0.4 µM) ex vivo was the best lead in
vitro. Finally, compound 17 yielded the most promising
results as shown in Figure 3.
This compound gave rise to a strong antiviral effect
as measured by â-gal assay, whereas no toxicity on
either infected or noninfected cells was observed. In-

2852 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Mekouar et al.
potassium carbonate (2.10 g, 15 mmol) in dry acetone (15 mL)
was added dropwise a solution of dimethoxycaffeoyl chloride
(1.60 g, 7.41 mmol) in acetone (10 mL). After stirring for 4 h
at 20 °C, the reaction mixture was filtered and the filtrate
concentrated in vacuo. The oily residue was chromatographed
on silica gel (AcOEt:cyclohexane ) 1:2) to provide a solid which
was recrystallized from ethyl acetate to afford 3 (1.4 g, 54%)
as colorless crystals: mp 119-121 °C; IR (neat) 1723, 1633,
nant IN were found to be poorly active against integra-
tion mediated by purified preintegration complexes.58
These results indicate that such inhibitors must be
present in cells at the time of PIC formation to be fully
active. Our data are consistent with such a model. It
can be argued that kinetic results are compatible with
inhibition of reverse transcription as well. To rule out
such a possibility, compounds 17 and 19 were tested
against HIV reverse transcriptase in vitro. No effect
against the enzyme was observed in the range of
concentrations tested (1 nM-100 µM).
1
1600; H NMR (CDCl3, 200 MHz) δ 8.04 (d, J ) 8.6 Hz, 1H),
7.91 (d, J ) 15.8 Hz, 1H), 7.70-7.60 (m, 1H), 7.51-7.44 (m,
2H), 7.28 (d, J ) 8.6 Hz, 1H), 7.25-7.16 (m, 2H), 6.89 (d, J )
8.8 Hz, 1H), 6.78 (d, J ) 16.0 Hz, 1H), 3.93 (s, 3H), 3.92 (s,
3H), 2.70 (s, 3H); 13C NMR (CDCl3, 50 MHz) δ 165.9, 159.4,
151.3, 149.2, 147.0, 146.4, 135.9, 127.7, 127.3, 125.4, 125.1,
123.0, 122.5, 121.4, 115.0, 110.9, 109.6, 55.9, 55.8, 25.7. Anal.
(C21H19NO4) C, H, N.
Con clu sion
In summary, the present styrylquinoline derivatives
constitute a new class of HIV integrase inhibitors that
display a marked effect against HIV replication ex vivo.
Since most of these compounds are devoid of cytotoxicity
at concentrations up to 100 µM, they represent a new
lead for the development of anti-HIV drugs. Further-
more, seeing that several of the these inhibitors exhibit
a strong activity, they might be used in the elucidation
of the mechanism of inhibition of HIV integrase; e.g.,
they could serve as candidates for cocrystallization
studies with this enzyme.
3,4-Bis[[[(1,1-d im et h ylet h yl)d im et h yl]silyl]oxy]cin -
n a m yl Ch lor id e (24). To a solution of caffeic acid (3.5 g, 19.4
mmol) and imidazole (3.94 g, 58 mmol) in anhydrous DMF (20
mL) was added tert-butyldimethylchlorosilane (8.7 g, 58 mmol)
at 0 °C. The resulting mixture was stirred at 20 °C for 16 h.
Water (20 mL) was then added, and the reaction mixture
stirred for 10 min. The mixture was extracted with ether. The
combined organic extracts were dried and concentrated in
vacuo. CH2Cl2 (100 mL) and DMF (0.5 mL) were added to
the oily residue. The solution was cooled to 0 °C, and oxalyl
chloride (3.43 g, 27 mmol) was added dropwise. After stirring
for 1 h at 20 °C, the reaction mixture was concentrated under
vaccum to leave crude acid chloride 24, which was used directly
in the next step without further purification.
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
(E)-Qu in a ld in -8-yl 3,4-Dih yd r oxycin n a m a te (4). To a
solution of 8-hydroxyquinaldine (1) (3.34 g, 21 mmol) and
potassium carbonate (5.80 g, 42 mmol) in dry acetone (50 mL)
was added dropwise a solution of crude acid chloride 24 (9.0
g, 21 mmol) in acetone (30 mL). After stirring for 16 h at 20
°C, the reaction mixture was filtered and the filtrate concen-
trated in vacuo. The oily residue was taken into dry THF (10
mL), and a 1.1 M solution of tetrabutylammonium fluoride in
THF (11.6 mL, 126 mmol) was added. After the mixture
stirred at 20 °C for 1 h, acetic acid (5 mL) and water (20 mL)
were added. The mixture was diluted with ethyl acetate, the
organic layer separated, and the aqueous phase extracted with
two 20-mL portions of ethyl acetate. The combined organic
extracts were dried and concentrated. The solid residue was
recrystallized from 2-propanol to give colorless crystals of 4
(2.28 g, 33%): mp 201-203 °C; IR (KBr) 3600-2400, 1694,
1
pellets. The H and 13C NMR spectra were recorded in CDCl3,
unless otherwise stated; when diffuse, easily exchangeable
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
60F254 glass precoated plates (0.25-mm layer). All liquid
chromatography separations were performed using Merck
silica gel 60 (230-400 mesh ASTM). Ether and tetrahydro-
furan (THF) were distilled from sodium benzophenone ketyl.
Methanol was dried over magnesium and distilled. Benzene
and CH2Cl2 were distilled from calcium hydride, under a
nitrogen 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 layers were dried over anhydrous MgSO4. Chemicals
obtained from commercial suppliers were used without further
purification. Elemental analyses were obtained from the
Service de microanalyse, Centre d’Etudes Pharmaceutiques,
Chaˆtenay-Malabry, France.
1
1634, 1605; H NMR (DMSO-d6, 200 MHz) δ 8.28 (d, J ) 8.4
Hz, 1H), 7.82 (dd, J ) 6.4, 3.0 Hz, 1H), 7.72 (d, J ) 16.0 Hz,
1H), 7.60-7.46 (m, 2H), 7.42 (d, J ) 8.4 Hz, 1H), 7.20-7.10
(m, 2H), 6.81 (d, J ) 8.0 Hz, 1H), 6.65 (d, J ) 16.0 Hz, 1H),
2.50 (s, 3H); 13C NMR (DMSO-d6, 50 MHz) δ 165.4, 159.0,
148.9, 147.0, 146.8, 145.7, 140.4, 136.3, 127.5, 125.7, 125.6,
125.5, 122.8, 121.9, 121.7, 115.9, 115.2, 113.2, 25.3. Anal.
(C19H15NO4) C, H, N.
8-[[(2-Allyl-4,5-(m eth ylen ed ioxy)p h en yl)m eth yl]oxy]-
qu in a ld in e (2). To a mixture of 8-hydroxyquinaldine (1) (2.21
g, 13.9 mmol), potassium carbonate (3.80 g, 27.8 mmol) and
potassium iodide (4.60 g, 27.8 mmol), in dry acetonitrile (50
mL) was added dropwise a solution of (chloromethyl)safrole47
(3.60 g, 18.0 mmol) in acetonitrile (20 mL). After being stirred
for 18 h at 20 °C, the reaction mixture was poured into water
and extracted with three 50-mL portions of CH2Cl2. The
organic layer was dried and concentrated in vacuo to leave a
solid which was recrystallized from isopropyl ether to provide
colorless crystals of 2 (3.0 g, 65%): mp 118-119 °C; IR (KBr)
3061, 1638, 1617, 1603; 1H NMR (CDCl3, 200 MHz) δ 7.96 (d,
J ) 8.4 Hz, 1H), 7.40-7.20 (m, 3H), 7.03 (s, 1H), 6.94 (dd, J )
6.4, 2.4 Hz, 1H), 6.68 (s, 1H), 6.10-5.80 (m, 1H), 5.86 (s, 2H),
5.27 (s, 2H), 5.10-4.90 (m, 3H), 3.41 (d, J ) 6.4 Hz, 1H), 2.75
(s, 3H); 13C NMR (CDCl3, 50 MHz) δ 158.2, 154.0, 147.3, 146.3,
136.9, 136.1, 131.3, 128.4, 127.8, 125.6, 122.6, 120.1, 116.0,
110.9, 110.1, 108.9, 103.3, 101.0, 69.0, 37.0, 25.8. Anal.
(C21H19NO3) C, H, N.
8-Hyd r oxy-7-qu in a ld ic Acid (5).48 To a suspension of
8-hydroxyquinaldine (1) (29 g, 0.18 mol) in toluene (130 mL)
was added potassium hydoxide (11.3 g, 0.20 mol). The stirred
mixture was heated under reflux for 24 h, collecting the water
of the reaction in a Dean-Stark trap. After the mixture cooled
at 20 °C, DMF (100 mL) was added, and the Dean-Stark trap
was replaced with a distillation column. The reaction mixture
was progressively heated until most of the toluene had been
distilled. When the temperature reached 140 °C, a stream of
CO2 was passed into the solution and continued throughout
the reaction. The distillation of the solvents was continued
while the temperature was gradualy raised to 160 °C. The
reaction mixture was heated for 2 h at this temperature and
then cooled to 20 °C. The stream of CO2 was stopped, and
water (250 mL) was added. The solution was acidified to pH
7 with concentrated HCl, and the mixture was extracted with
ethyl acetate. The aqueous phase was acidified to pH 4.2, and
the precipitate was filtered, washed with water, and dried in
vacuo. The crude acid was recrystallized in 2-propanol to give
acid 5 (8.0 g, 22%) as yellow crystals: mp 206-208 °C (lit.48
(E)-Qu in a ld in -8-yl 3,4-Dim eth oxycin n a m a te (3). To a
mixture of 8-hydroxyquinaldine (1) (1.17 g, 7.41 mmol) and

Styrylquinoline Derivatives
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2853
1
mp 207-215 °C); IR (neat) 3433, 3200-2400, 1700, 1633; H
NMR (DMSO-d6, 400 MHz) δ 8.54 (d, J ) 8.5 Hz, 1H), 7.86 (d,
J ) 8.6 Hz, 1H), 7.71 (d, J ) 8.5 Hz, 1H), 7.20 (d, J ) 8.6 Hz,
1H), 2.80 (s, 3H); 13C NMR (DMSO-d6, 50 MHz) δ 170.7 (C),
160.8 (C), 156.2 (C), 140.9 (CH), 135.0 (C), 130.4 (C), 127.5
(CH), 125.0 (CH), 112.8 (CH), 112.6 (C), 22.4 (CH3). Anal.
(C11H9NO3‚1/4H2O) C, H, N.
(E)-2-(2-P h en yleth en yl)-8-a cetoxyqu in olin e (6). To a
solution of 8-hydroxyquinaldine (1) (0.60 g, 3.7 mmol) in acetic
anhydride (6 mL) was added benzaldehyde (0.79 g, 7.5 mmol).
The mixture was heated under reflux for 16 h and concentrated
in vacuo. The oily residue was chromatographed on silica gel
(AcOEt:cyclohexane ) 1:4) to provide a solid which was
recrystallized from di-n-butyl ether to afford 6 (0.6 g, 56%) as
colorless crystals: mp 113-114 °C; IR (neat) 1769, 1619, 1598;
1H NMR (CDCl3, 200 MHz) δ 8.12 (d, J ) 8.6 Hz, 1H), 7.80-
7.58 (m, 5H), 7.55-7.30 (m, 5H), 7.28 (d, J ) 8.0 Hz, 1H), 2.50
(s, 3H); 13C NMR (CDCl3, 50 MHz) δ 169.9, 155.9, 147.5, 141.1,
136.5, 136.4, 134.8, 129.0, 128.9 (2C), 128.8, 128.6, 127.4 (2C),
125.7, 125.6, 121.7, 120.3, 21.1. Anal. (C19H15NO2) C, H, N.
(E)-2-[2-(3,4-Dih yd r oxyp h en yl)eth en yl]-8-n itr oqu in o-
lin e (10). To a solution of 8-nitroquinaldine (3.0 g, 16 mmol)
in acetic anhydride (30 mL) was added 3,4-diacetoxybenzal-
dehyde (3.86 g, 18.3 mmol). The mixture was heated under
reflux for 36 h and concentrated in vacuo. The residue was
dissolved in pyridine (36 mL), and the resulting solution was
heated under reflux. Water (12 mL) was then added and the
reaction mixture refluxed for 3 h. After the mixture cooled,
water (100 mL) and CH2Cl2 (100 mL) were added to the
mixture. The organic phase was separated and the aqueous
phase extracted with CH2Cl2. The combined organic extracts
were dried and concentrated. The solid residue residue was
chromatographed on silica gel (AcOEt:cyclohexane ) 3:1) to
provide a solid which was recrystallized from 2-propanol to
afford 10 (2.7 g, 55%) as brown crystals: mp 198-199 °C; IR
(KBr) 3526, 3200-2400, 1638, 1600; 1H NMR (DMSO-d6, 200
MHz) δ 8.47 (d, J ) 8.7 Hz, 1H), 8.19 (d, J ) 8.2 Hz, 2H), 7,99
(d, J ) 8.7 Hz, 1H), 7.75-7.60 (m, 2H), 7.16-7.05 (m, 2H),
7.01 (dd, J ) 8.4, 2.0 Hz, 1H), 6.79 (d, J ) 8.4 Hz, 1H); 13C
NMR (DMSO-d6, 50 MHz) δ 158.2, 147.5, 147.3, 145.7, 138.5,
137.0, 136.7, 131.7, 127.6, 127.4, 124.7, 124.4, 123.5, 121.5,
120.3, 115.9, 114.3. Anal. (C17H12N2O4) C, H, N.
(E)-2-(2-P h en yleth en yl)-8-h yd r oxyqu in olin e (7). A so-
lution of acetoxyquinoline 6 (1.2 g, 4.1 mmol) in pyridine (20
mL) was heated under reflux, water (8 mL) was then added,
and the reaction mixture refluxed for 3 h. After the mixture
cooled, water (20 mL) and CH2Cl2 were added to the mixture.
The organic phase was separated and the aqueous phase
extracted with CH2Cl2. The combined organic extracts were
dried and concentrated. The solid residue was recrystallized
from di-n-butyl ether to afford yellow crystals of 7 (0.95 g,
92%): mp 96-100 °C; IR (KBr) 3402, 3060, 1641, 1602; 1H
NMR (CDCl3, 200 MHz) δ 7.97 (d, J ) 8.6 Hz, 1H), 7.59 (d, J
) 16.2 Hz, 1H), 7.50 (d, J ) 8.4 Hz, 1H), 7.50 (m, 1H), 7.35-
7.10 (m, 6H), 7.03 (dd, J ) 8.8, 1.4 Hz, 1H); 13C NMR (CDCl3,
50 MHz) δ 153.5, 152.0, 137.9, 136.3 (2C), 134.2, 128.7 (2C),
128.6, 128.0, 127.6, 127.2 (3C), 120.2, 117.6, 110.0. Anal.
(C17H13NO) C, H, N.
(E)-1,2-Bis[2-(8-h yd r oxyqu in olyl)]eth ylen e (8). A solu-
tion of aldehyde 2750 (9.40 g, 43 mmol) and 8-acetoxyquinaldine
(8.78 g, 43 mmol) in acetic anhydride (100 mL) and acetic acid
(50 mL) was heated under reflux for 8 h and concentrated in
vacuo. Aqueous sodium bicarbonate was added, and the
mixture was extracted with CH2Cl2. The organic extracts were
dried and concentrated to leave a yellow solid, which was
dissolved in refluxing pyridine (40 mL). Water (10 mL) was
then added, and the reaction mixture refluxed for 6 h. After
standing at 0 °C for 8 h, the reaction mixture was filtered.
The solid washed with ether and dried in vacuo to give
compound 8 (2.50 g, 18%) as yellow crystals: mp >220 °C; IR
(KBr) 3362, 1633, 1596; 1H NMR (DMSO-d6, 200 MHz) δ 9.30
(broad s, 2H), 8.28 (d, J ) 8.6 Hz, 2H), 8.20 (s, 2H), 7.85 (d, J
) 8.6 Hz, 2H), 7.35-7.25 (m, 4H), 7.06 (dd, J ) 7.0, 2.0 Hz,
2H); 13C NMR (DMSO-d6, 50 MHz) δ 153.2, 153.0, 138.5, 136.8,
133.7, 128.1, 127.6, 121.6, 117.7, 111.5. Anal. (C20H14N2O2)
C, H, N.
(E)-2-[2-(3,4-Dih yd r oxyp h en yl)et h en yl]qu in olin e (9).
To a solution of quinaldine (0.9 g, 6.3 mmol) in acetic anhydride
(10 mL) was added 3,4-diacetoxybenzaldehyde (1.48 g, 7.0
mmol). The mixture was heated under reflux for 12 h and
concentrated in vacuo. The residue was dissolved in pyridine
(20 mL), and the resulting solution was heated under reflux.
Water (8 mL) was then added and the reaction mixture
refluxed for 3 h. After the mixture cooled, water (20 mL) and
CH2Cl2 (20 mL) were added to the mixture. The organic phase
was separated and the aqueous phase extracted with CH2Cl2.
The combined organic extracts were dried and concentrated.
The solid residue was recrystallized from 2-propanol to give
yellow crystals of 9 (1.51 g, 91%): mp 246-251 °C; IR (KBr)
3536, 3050-2200, 1602; 1H NMR (DMSO-d6, 200 MHz) δ 9.30
(broad s, 2H), 8.35 (d, J ) 8.8 Hz, 1H), 8.10-7.70 (m, 5H),
7.60 (t, J ) 6.8 Hz, 1H), 7.20 (m, 2H), 7.10 (dd, J ) 8.0, 1.0
Hz, 1H), 6.85 (d, J ) 8.8 Hz, 1H); 13C NMR (DMSO-d6, 50
MHz) δ 156.3, 147.8, 146.9, 145.7, 136.4, 134.8, 129.8, 128.6,
127.9 (2C), 126.9, 125.9, 125.4, 119.9 (2C), 116.0, 114.1. Anal.
(C17H13NO2‚1/4H2O) C, H, N.
(E)-2-[2-(3,4-Dih yd r oxyp h en yl)eth en yl]-8-a m in oqu in o-
lin e (11). To a solution of 8-nitroquinaldine (3.0 g, 16 mmol)
in acetic anhydride (30 mL) was added 3,4-diacetoxybenzal-
dehyde (3.86 g, 18.3 mmol). The mixture was heated under
reflux for 36 h and concentrated in vacuo. The residue was
dissolved in ethanol (30 mL); tin(II) chloride (7.2 g, 38 mmol)
was then added, and the reaction mixture stirred for 1 h. The
solution was cooled to 0 °C; water (10 mL) was added. The
mixture was basified to pH 8 with aqueous sodium carbonate
and extracted with CH2Cl2. The combined organic extracts
were dried and concentrated to leave crude 2-[2-(3,4-diacetoxy-
phenyl)ethenyl]-8-aminoquinoline which was directly hydro-
lyzed in the next step. The obtained product (2.0 g, 5.5 mmol)
was dissolved in refluxing pyridine (36 mL), water (12 mL)
was added, and the reaction mixture refluxed for 3 h. After
the mixture cooled, water (40 mL) and CH2Cl2 (50 mL) were
added to the mixture. The organic phase was separated and
the aqueous phase extracted with CH2Cl2. The combined
organic extracts were dried and concentrated. The solid
residue was chromatographed on silica gel (CH2Cl2:MeOH )
13:1) to provide amine 11 (0,18 g, 5%) as a brown amorphous
1
solid: IR (KBr) 3600-2600, 1633, 1597; H NMR (DMSO-d6,
200 MHz) δ 9.20 (broad s, 2H), 8.07 (d, J ) 8.7 Hz, 1H), 7.70
(d, J ) 16.4 Hz, 1H), 7.66 (d, J ) 8.2 Hz, 1H), 7.25-6.90 (m,
5H), 6.90-6.70 (m, 2H), 5.93 (s, 2H); 13C NMR (DMSO-d6, 50
MHz) δ 152.6, 146.5, 145.6, 145.0, 137.1, 136.1, 133.6, 128.2,
127.3, 127.0, 125.4, 119.9, 119.6, 115.9, 113.8, 113.6, 108.9.
Anal. (C17H14N2O2‚1/4H2O) C, H, N.
(E)-2-[2-(3,4-Dia cetoxyp h en yl)eth en yl]-8-a cetoxyqu in -
olin e (12). To a solution of 8-acetoxyquinaldine (2.8 g, 14
mmol) in acetic anhydride (30 mL) was added 3,4-diacetoxy-
benzaldehyde (3.86 g, 18.3 mmol). The mixture was heated
under reflux for 24 h and concentrated in vacuo. The solid
residue was taken into ether and filtered on a sintered glass
funnel. The solid was thoroughly washed with ether to leave
compound 12 (3.0 g, 53%) as a red solid: mp 163-165 °C; IR
1
(KBr) 1777, 1596; H NMR (CDCl3, 200 MHz) δ 8.13 (d, J )
8.8 Hz, 1H), 7.80-7.56 (m, 3H), 7.54-7.40 (m, 4H), 7.26-
7.19 (m, 2H), 2.55 (s, 3H), 2.33 (s, 3H), 2.31 (s, 3H); 13C
NMR (CDCl3, 50 MHz) δ 168.1 (3C), 155.3, 147.4, 142.1, 140.1,
136.4, 135.5, 132.8, 130.1, 128.6, 125.8, 125.5 (2C), 125.3,
123.7, 121.8, 121.7, 120.2, 21.0, 20.7 (2C). Anal. (C23H19NO6)
C, H, N.
(E)-2-[2-(3,4-Hyd r oxyp h en yl)eth en yl]-8-h yd r oxyqu in o-
lin e (13). A solution of compound 12 (1.37 g, 3.4 mmol) in
pyridine (10 mL) was heated under reflux. Water (5 mL) was
then added, and the reaction mixture refluxed for 3 h. After
the mixture cooled, water (30 mL) was added and the reaction
mixture was stored at 0 °C overnight. The crystals were
collected by filtration and washed with water, ethanol, and
finally ether to give quinoline 13 (0.53 g, 56%) as bright-red
crystals: mp 232-235 °C; IR (KBr) 3600-2400, 1589; 1H NMR

2854 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Mekouar et al.
(acetone-d6, 200 MHz) δ 8.23 (d, J ) 8.7 Hz, 1H), 7.91 (d, J )
16.2 Hz, 1H), 7.72 (d, J ) 8.7 Hz, 1H), 7.40-7.30 (m, 2H), 7.22
(s large, 1H), 7.21 (d, J ) 16.2 Hz, 1H), 7.10-7.05 (m, 2H),
6.87 (d, J ) 8.1 Hz, 1H); 13C NMR (DMSO-d6, 50 MHz) δ 154.1,
152.9, 146.8, 145.7, 138.2, 136.4, 135.1, 128.2, 127.5, 126.7,
(E)-8-H yd r oxy-2-[2-(3,4-d ih yd r oxyp h en yl)et h en yl]-7-
qu in olin eca r boxylic Acid (17). Method A using 3,4-diac-
etoxybenzaldehyde afforded acid 17 in 50% overall yield as
red crystals: mp g230 °C; IR (KBr) 3400-2600, 1667, 1590;
1H NMR (DMSO-d6, 200 MHz) δ 8.48 (d, J ) 8.8 Hz, 1H), 8.15
(d, J ) 8.8 Hz, 1H), 7.84 (d, J ) 16.2 Hz, 1H), 7.82 (d, J ) 8.4
Hz, 1H), 7.45 (d, J ) 16.2 Hz, 1H), 7.16 (d, J ) 8.6 Hz, 1H),
7.09 (d, J ) 1.5 Hz, 1H), 7.04 (dd, J ) 8.2; 1.5 Hz, 1H), 6.80
(d, J ) 8.2 Hz, 1H); 13C NMR (DMSO-d6, 50 MHz) δ 170.6,
160.8, 152.8, 148.0, 145.7, 140.0, 139.2, 135.2, 130.5, 127.5,
127.3, 120.9, 120.7, 120.2, 116.0, 114.2, 113.2, 112.8. Anal.
(C18H13NO5‚1/4H2O) C, H, N.
(E)-8-Hyd r oxy-2-[2-(3,4,5-t r ih yd r oxyp h en yl)et h en yl]-
7-qu in olin eca r boxylic Acid (19). Method A using 3,4,5-
triacetoxybenzaldehyde afforded acid 19 in 40% overall yield
as red crystals: mp g230 °C; IR (KBr) 3600-2400, 1630, 1585;
1H NMR (DMSO-d6, 200 MHz) δ 8.51 (d, J ) 8,8 Hz, 1H), 8.21
(d, J ) 8.8 Hz, 1H), 7.84 (d, J ) 8.4 Hz, 1H), 7.80 (d, J ) 16.1
Hz, 1H), 7.44 (d, J ) 16.1 Hz, 1H), 7.16 (d, J ) 8.4 Hz, 1H),
6.68 (s, 2H); 13C NMR (DMSO-d6, 50 MHz) δ 170.6, 161.0,
152.7, 146.4 (2C), 140.4, 140.1, 136.3, 134.9, 130.5, 127.8,
124.7, 120.8, 119.8, 117.7, 116.0, 114.0, 111.2. Anal. (C17H13
-
NO3‚1/4H2O) C, H, N.
7-[2-(3,4-Dih yd r oxyp h en yl)eth en yl]-8-h yd r oxyqu in a l-
d in e (32). To a suspension of phosphonium salt 31 (6.9 g, 10
mmol) in anhydrous THF (35 mL) was added at - 78 °C a 1.5
M hexane solution of n-BuLi (6.9 mL, 10.3 mmol). The
temperature was gradually raised to room temperature, and
a solution of 8-acetoxyquinaldine-7-carbaldehyde (31) (1.7 g,
7.4 mmol) in THF (15 mL) was added dropwise. After the
mixture stirred for 2 h at room temperature, water (10 mL)
was added and the mixture was filtered. The filtrate was
extracted with three 50-mL portions of CH2Cl2. The combined
organic extracts were dried and concentrated in vacuo. The
residue was taken up in THF (10 mL) and treated with a 1 M
THF solution of n-Bu4NF (16 mL, 16 mmol). After the mixture
stirred for 1 h at 20 °C, 10% aqueous acetic acid (30 mL) was
added and the mixture was extracted with AcOEt. The
combined organic extracts were dried and concentrated to give
0.60 g of 32, as a yellow solid which was directly used in the
next step without further purification.
(E ,E )-2,7-B is [2-(3,4-h y d r o x y p h e n y l)e t h e n y l]-8-h y -
d r oxyqu in olin e (14). To a solution of crude compound 32
(0,6 g, 2 mmol) in dry acetone (10 mL) were added at 0 °C
potassium carbonate (1.7 g, 12 mmol) and acetyl chloride
(0,94 g, 12 mmol). After being stirred for 2 h at 20 °C, the re-
action mixture was filtered and concentrated in vacuo. Water
was added, and the mixture was extracted with CH2Cl2. The
combined organic extracts were dried and concentrated. The
residue was taken up into acetic anhydride (8 mL), and 3,4-
diacetoxybenzaldehyde (0.28 g, 1.3 mmol) was added. The
mixture was heated under reflux for 4 days, and concentrated
in vacuo. The obtained product was dissolved in refluxing
pyridine (10 mL), water (4 mL) was added, and the reaction
mixture refluxed for 3 h. After the mixture cooled, water (40
mL) and CH2Cl2 (50 mL) were added to the mixture. The
organic phase was separated and the aqueous phase extracted
with CH2Cl2. The combined organic extracts were dried and
concentrated. The solid residue was chromatographed on silica
gel (CH2Cl2:MeOH ) 13:1) to give compound 14 as a red solid
(0.07 g, 2% overall yield from 31): mp >220 °C; IR (KBr) 3600-
2600, 1597; 1H NMR (acetone-d6, 200 MHz) δ 8.20 (d, J ) 8.7
Hz, 1H), 7.91 (d, J ) 16.4 Hz, 1H), 7.76 (d, J ) 9.1 Hz, 1H),
7.67 (d, J ) 8.1 Hz, 1H), 7.40 (d, J ) 16.4 Hz, 1H), 7.32-7.15
(m, 5H), 7.08 (d, J ) 8.9 Hz, 1H), 6.98 (d, J ) 7.8 Hz, 1H),
6.85 (m, 2H). Anal. (C25H19NO5‚H2O) C, H, N.
Gen er a l P r oced u r e A. Con d en sa tion of 8-Hyd r oxy-7-
qu in a ld ic Acid (5) w ith Ar om a tic Ald eh yd es. To a solu-
tion of 1.0 equiv of acid 5 in acetic anhydride (3 mL/mmol)
was added 1.3 equiv of aldehyde. The mixture was heated
under reflux for 24 h and concentrated in vacuo. The residue
was dissolved in pyridine (4 mL/mmol), the resulting solution
was heated under reflux, water (1 mL/mmol) was then added,
and the reaction mixture refluxed for 3 h. After the mixture
cooled, water and CH2Cl2 were added with stirring. After
standing at 0 °C for 8 h the reaction mixture was filtered. The
solid was washed with methanol, CH2Cl2, and finally ether
and dried in vacuo to provide acids 15, 17, 19, and 21.
(E)-8-H yd r oxy-2-[2-(2,4-d ih yd r oxyp h en yl)et h en yl]-7-
qu in olin eca r boxylic Acid (15). Method A using 2,4-diac-
etoxybenzaldehyde afforded acid 15 in 39% overall yield as
red crystals: mp >220 °C; IR (KBr) 3600-2400, 1587; 1H
NMR (DMSO-d6, 200 MHz) δ 8.46 (d, J ) 8.9 Hz, 1H), 8.13
(d, J ) 8.9 Hz, 1H), 8.03 (d, J ) 16.4 Hz, 1H), 7.81 (d, J ) 8.6
Hz, 1H), 7.65 (d, J ) 16.4 Hz, 1H), 7.44 (d, J ) 8.5 Hz, 1H),
7.13 (d, J ) 8.5 Hz, 1H), 6.39 (s, 1H), 6.35 (d, J ) 8.5 Hz, 1H);
13C NMR (DMSO-d6, 50 MHz) δ 170.7, 161.1, 160.9, 158.5,
153.3, 140.7, 135.6, 134.5, 130.3, 129.9, 127.7, 120.3, 118.8,
114.3, 113.5, 112.6, 108.2, 102.7. Anal. (C18H13NO5‚H2O) C,
H, N.
126.2, 120.8, 120.0, 113.2, 112.9, 107.4 (2C). Anal. (C18H13
-
NO6‚1/2H2O) C, H, N.
(E )-8-H yd r oxy-2-[2-(3-ca r b oxyl-4-h yd r oxyp h e n yl)-
eth en yl]-7-qu in olin e ca r boxylic Acid (21). Method A
using 5-formylsalicylic acid afforded diacid 21 in 38% overall
yield as red crystals: mp >220 °C; IR (KBr) 3600-2200, 1665,
1
1610, 1590; H NMR (DMSO-d6, 200 MHz) δ 8.47 (d, J ) 8.8
Hz, 1H), 8.17-8.07 (m, 2H), 7.98 (d, J ) 16.4 Hz, 1H), 7.90
(dd, J ) 8.8, 2.0 Hz, 1H), 7.82 (d, J ) 8.4 Hz, 1H), 7.58 (d, J
) 16.4 Hz, 1H), 7.23 (d, J ) 8.6 Hz, 1H), 7.04 (d, J ) 8.6 Hz,
1H). Anal. (C19H13NO6‚H2O) C, H, N.
8-Hyd r oxy-7-qu in a ld in eca r ba ld eh yd e Oxim e (29). To
a solution of 8-hydroxy-7-quinaldinecarbaldehyde (28)51 (1.0
g, 5.3 mmol) in acetic acid (20 mL) were added hydroxylamine
hydrochloride (0,69 g, 10 mmol) and sodium acetate (0.82 g,
10 mmol). The resulting mixture was heated at 100 °C for 1
h. After cooling the reaction mixture was concentrated under
reduced pressure. The residue was poured into water and
extracted with three 50-mL portions of CH2Cl2. The combined
organic extracts were dried and concentrated to give oxime
29 (0.70 g, 65%) as a yellow solid: mp 171-175 °C; IR (KBr)
3400-2400, 1643, 1614, 1563; 1H NMR (DMSO-d6, 200 MHz)
(only the major anti isomer is described) δ 8.50 (s, 1H), 8.16
(d, J ) 8.4 Hz, 1H), 7.70 (d, J ) 8.6 Hz, 1H), 7.42 (d, J ) 8.3
Hz, 1H), 7.32 (d, J ) 8.6 Hz, 1H), 2.68 (s, 3H); 13C NMR
(DMSO-d6, 50 MHz) δ 157.4, 150.8, 144.7, 138.1, 136.2, 127.2,
123.2, 122.9, 117.9, 115.2, 24.7.
(E )-7-Cya n o-8-h yd r oxy-2-[2-(2,4-d ih yd r oxyp h e n yl)
eth en yl]-7-qu in olin e (16). A mixture of oxime 29 (0.60 g,
2.9 mmol) and 3,4-diacetoxybenzaldehyde (0.72 g, 3.4 mmol)
in acetic anhydride (10 mL) was heated under reflux for 24 h
and concentrated in vacuo. After cooling the reaction mix-
ture was concentrated under reduced pressure. The resi-
due was dissolved in pyridine (10 mL), and the solution was
heated under reflux. Water (8 mL) was then added, and the
reaction mixture refluxed for 3 h. After the mixture cooled,
water (20 mL) and CH2Cl2 were added to the mixture. The
organic phase was separated and the aqueous phase extracted
with CH2Cl2. The combined organic extracts were dried and
concentrated. The solid residue was recrystallized from 2-pro-
panol to afford dark-red crystals of 16 (0.63 g, 71%): mp
239-244 °C; IR (KBr) 3600-2600, 2194, 1630, 1603; 1H
NMR (DMSO-d6, 200 MHz) δ 8.32 (d, J ) 8.6 Hz, 1H), 8.04 (d,
J ) 16.2 Hz, 1H), 7.87 (d, J ) 8.6 Hz, 1H), 7.54 (d, J ) 8.5
Hz, 1H), 7.41 (d, J ) 8.5 Hz, 1H), 7.16 (d, J ) 16.2 Hz, 1H),
7.10 (s, 1H), 7.00 (d, J ) 8.4 Hz, 1H), 6.79 (d, J ) 8.2 Hz, 1H);
13C NMR (DMSO-d6, 50 MHz) δ 158.2, 155.7, 149.6, 147.0,
145.6, 137.6, 136.7, 129.6, 127.8, 126.5, 123.9, 123.7, 123.3,
119.9, 118.5, 117.1, 115.9, 114.0. Anal. (C18H12N2O3‚1/4H2O)
C, H, N.
Gen er a l P r oced u r e B. Syn th esis of Sod iu m Sa lts 18
a n d 20. To a suspension of the acid (0.3 mmol) in degassed
water was added, a 0.1 M solution of sodium hydroxide

Styrylquinoline Derivatives
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2855
dropwise (3 mL, 0.3 mmol) under nitrogen atmosphere. The
obtained solution was lyophilized (Christ Lyophilizer, D) over
24 h (-40 °C/+40 °C) to give the expected salt as a brown solid
(quantitative).
An tivir a l Assa ys. The lymphocyte cell line CEM was
maintained in RPMI-1640 medium (GIBCO Laboratories)
supplemented with 10% fetal calf serum. HeLaCD4+-â-gal
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
transfection of CEM cells with HIV-1 PLN4-3 genomic clone.
Cells were plated in triplicate on 96-well plates (100 µL) and
infected with cell-free virus. Viral supernatants were re-
moved 2 h after infection, and drugs dissolved in DMSO
were added in fresh medium; 72 h later, supernatants were
used to infect HeLaCD4+-â-gal cells. The P4 cultures were
subsequently incubated for 24 h and subsequently lysed in a
phosphate buffer containing 50 mM 2-mercaptoethanol, 10
mM MgSO4, 25 mM EDTA, and 0.125% NP40; 20 µL of lysate
was incubated with 100 µL of CPRG containing buffer.58 The
red staining intensity was quantified on a multiscan photom-
eter 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 CO2
incubator for 4 h. Solubilization of the formazan crystals was
achieved by adding 100 mL of 10% SDS, 10 mM HCl.
Absorbances were read in a multiscan photometer at 570 nm.
In some cases, antiviral activity was measured by p24 antigen
ELISA.
(E)-8-H yd r oxy-2-[2-(3,4-d ih yd r oxyp h en yl)et h en yl]-7-
qu in olin eca r boxylic Acid Sod iu m Sa lt (18). Method B
starting from acid 17 gave salt 18 as a brown amorphous
1
solid: IR (KBr) 3600-2200, 1630, 1598; H NMR (DMSO-d6,
200 MHz) δ 8.08 (d, J ) 8.6 Hz, 1H), 7.84 (d, J ) 8.6 Hz, 1H),
7.75 (d, J ) 8.4 Hz, 1H), 7.49 (d, J ) 16.4 Hz, 1H), 7.24 (d, J
) 16.4 Hz, 1H), 7.13 (s, 1H), 6.95 (m, 2H), 6.74 (d, J ) 7.8 Hz,
1H); 13C NMR (DMSO-d6, 50 MHz) δ 171.6, 163.7, 154.0, 147.7,
146.2, 141.1, 136.0, 133.6, 129.9, 127.5, 127.0, 126.0, 119.5,
119.2, 115.8, 114.9, 113.7, 111.9.
(E)-8-Hyd r oxy-2-[2-(3,4,5-t r ih yd r oxyp h en yl)et h en yl]-
7-qu in olin eca r boxylic Acid Sod iu m Sa lt (20). Method B
starting from acid 18 gave salt 20 as a brown amorphous solid:
mp g300 °C; IR (KBr) 3700-2400, 1596; 1H NMR (DMSO-d6,
200 MHz) δ 8.06 (d, J ) 8.6 Hz, 1H), 7.84 (d, J ) 8.6 Hz, 1H),
7.75 (d, J ) 8.2 Hz, 1H), 7.41 (d, J ) 16.4 Hz, 1H), 7.16 (d, J
) 16.4 Hz, 1H), 6.94 (d, J ) 8.4 Hz, 1H), 6.65 (s, 2H); 13C NMR
(DMSO-d6, 50 MHz) δ 171.6, 163.5, 154.0, 146.5, 146.2 (2C),
141.0, 136.0, 134.2, 129.7, 126.8, 125.7, 125.6, 119.2, 115.0,
112.0, 106.2 (2C).
P r ep a r a tion of DNA Su bstr a tes. Oligonucleotides U5B
5′-GTGTGGAAAATCTCTAGCA, U5A ACTGCTAGAGATTTTC-
CACAC, and D38U3 TGCTAGTTCTAGCAGGCCCTTGGGC-
CGGCG-CTTGCGCC6 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
T4 polynucleotide kinase (Biolabs) and 50 µCi of [γ-32P]ATP
(sp. act. 3000 Ci/mmol). Kinase was heat-inactivated, and
unincorporated nucleotides 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. For the
integration experiment (strand transfer), a 492-bp DNA
substrate which contains LTR termini at each end to mimic
the unintegrated proviral DNA genome was used as donor
substrate. Plasmid pSP70 was used as acceptor substrate. The
492-bp DNA donor substrate was prepared from plasmid
pU3U5.59
HIV-1 In tegr a se Assa y. Purified recombinant full-size
HIV-1 integrase was a generous gift of Rhoˆne-Poulenc-Rorer.
Plasmid encoding the His-tagged soluble deletion mutant
F(185)K corresponding to the (50-212) core domain of HIV-1
integrase was generously provided by Dr. R. Craigie. The
protein was expressed and purified as described in ref 52.
Processing assay was performed using 0.5 pmol of U5A/U5B
substrate in the presence of 1 pmol of integrase in buffer
containing 20 mM Tris (pH 7.2), 30 mM NaCl, 10% (w/v)
glycerol, 10 mM DTT, 0.01% NP40, supplemented with 10 mM
MnCl2. Disintegration assays were performed in the presence
of 0.5 pmol of D38U3 substrate and 2 pmol of integrase core
domain. Finally, integration reactions were performed using
10 ng of donor and 40 ng of target DNA, in the presence of 1
pmol of IN. Gels were analyzed using a STORM Molecular
Dynamics phosphorimager. Inhibition in the presence of drugs
is expressed as percent of fractional product compared with
the control.
EcoRI Clea va ge Assa y. EcoRI enzyme and pSP70 were
from Biolabs; 1-kb DNA size marker (Pharmacia) was labeled
using T4 polynucleotide kinase-exchange reaction. pSP70 was
linearized with restriction enzyme SspI and labeled using T4
polynucleotide-exchange reaction in the presence of 50 µCi of
[γ-32P]ATP (sp. act. 3000 Ci/mmol); 2 nM of the labeled
fragment was incubated at 37 °C with 0.1 unit of EcoRI in
the presence of increasing concentrations of drugs dissolved
in DMSO, in a final volume of 10 µL. Final concentration of
DMSO was 10%. An aliquot of the reaction was loaded on a
0.8% agarose gel. Results were analyzed using a STORM 840
Molecular Dynamics phosphorimager.
Ack n ow led gm en t. A fellowship from the Fondation
pour la Recherche Me´dicale (Sidaction) to K.M. is
acknowledged. This work was in part supported by
funds from the Agence Nationale de Recherche sur le
Sida (ANRS). We thank also Professor B. Legendre
(Faculte´ de Pharmacie de Chaˆtenay-Malabry) for mi-
crothermal analyses, Mrs. S. Mairesse-Lebrun for per-
forming elemental analyses, and M. E. Franque for
skillfull help.
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