New 2-arylnaphthalenediols and triol inhibitors of HIV-1 integrase - Discovery of a new polyhydroxylated antiviral agent

Article abstract of DOI:10.1016/j.bmc.2010.05.059

A series of 13 hydroxylated 2-arylnaphthalenes have been synthesized and evaluated as HIV-1 integrase inhibitors. 7-(3,4,5-Trihydroxyphenyl)naphthalene- 1,2,3-triol 1c revealed chemical instability upon storage, leading to the isolation of a dimer 5c which was also tested. In the 2-arylnaphthalene series, all compounds were active against HIV-1 IN with IC₅₀'s within the 1-10 μM range, except for 1c and 5c which displayed submicromolar activity. Antiviral activity against HIV-1 replication was measured on 1b-c and 5c. Amongst the tested molecules, only 5c was found to present antiviral properties with a low cytotoxicity on two different cell lines.

Full text of DOI:10.1016/j.bmc.2010.05.059

Bioorganic & Medicinal Chemistry 18 (2010) 5194–5201
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New 2-arylnaphthalenediols and triol inhibitors of HIV-1
integrase—Discovery of a new polyhydroxylated antiviral agent
Cédric Maurin a, Cédric Lion a, Fabrice Bailly a, Nadia Touati a, Hervé Vezin a, Gladys Mbemba b,
Jean François Mouscadet b, Zeger Debyser c, Myriam Witvrouw c, Philippe Cotelle a,
*
a Laboratoire de Chimie Organique et Macromoléculaire, UMR CNRS 8009, Université des Sciences et Technologies de Lille, 59655 Villeneuve d’Ascq, France
b Laboratoire de Biotechnologies et Pharmacologie génétique Appliquée, UMR CNRS 8113, ENS Cachan, 61 avenue du président Wilson, 94235 Cachan, France
c Molecular Medicine, K.U.Leuven and IRC KULAK, Kapucijnenvoer 33, B-3000 Leuven, Flanders, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 13 hydroxylated 2-arylnaphthalenes have been synthesized and evaluated as HIV-1 integrase
inhibitors. 7-(3,4,5-Trihydroxyphenyl)naphthalene-1,2,3-triol 1c revealed chemical instability upon stor-
age, leading to the isolation of a dimer 5c which was also tested. In the 2-arylnaphthalene series, all com-
Received 3 April 2010
Revised 20 May 2010
Accepted 21 May 2010
Available online 26 May 2010
pounds were active against HIV-1 IN with IC50’s within the 1–10 lM range, except for 1c and 5c which
displayed submicromolar activity. Antiviral activity against HIV-1 replication was measured on 1b–c
and 5c. Amongst the tested molecules, only 5c was found to present antiviral properties with a low cyto-
toxicity on two different cell lines.
Keywords:
6-Arylnaphthalene-1,2-diols
HIV-1 Integrase inhibitors
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
of clinical success.7 Therefore, it is highly relevant to develop a sec-
ond generation of integrase inhibitors with a new mechanism of
Combination therapy, comprising at least three anti-human
immunodeficiency virus (anti-HIV) drugs, has become the standard
treatment of AIDS. Virtually all drugs that have been licensed for
clinical use in the treatment of HIV infections fall into one of the
following five categories: (i) nucleoside/nucleotide reverse trans-
criptase inhibitors (NRTIs), (ii) non-nucleoside reverse transcrip-
tase inhibitors (NNRTIs), (iii) protease inhibitors (PIs), (iv) entry
inhibitors, and (v) integrase inhibitors.1 Highly active antiretroviral
therapy (HAART) strategy still suffers from issues of patient com-
pliance, cost, deleterious side effects, and emerging drug
resistance.
Although integrase (IN) has been pursued for many years as a
potential target for the development of new anti-HIV compounds,
the first integrase inhibitor licensed for clinical use, raltegravir
(Scheme 1), was only approved in October 2007. Multiple resistant
mutations have since surfaced in both treatment-experienced and
treatment-naïve patients.2 This viral resistance most often results
from the substitution of one of three amino acids—Y143, Q148,
or N155—usually in combination with at least one other mutation.3
Other integrase inhibitor under recent clinical trials (i.e., elvitegra-
vir in phase III, GSK-364735 and BMS-707035 halted in phase II)
present strong cross-resistance with raltegravir4–6 and the devel-
opment of these compounds may yield a relatively low amount
action.
IN catalyzes the two-steps of the integration process, namely
30-processing and strand transfer reactions in the presence of a diva-
lent cation (while Mn2+ has been widely used for in vitro assays,
Mg2+ is thought to be the cation acting as a cofactor of the enzyme
in vivo).8 In the past several years, numerous compounds with var-
ious structural features have been reported as IN inhibitors, the
most developed of which are b-ketoenol derivatives and polyphe-
nols.9 Raltegravir and other integrase inhibitors under clinical trials
are more or less derived from the diketoacid derivatives family and
selectively inhibit the strand transfer reaction. A plethora of IN inhib-
itors has arised from the polyphenol family, however very few of them
actually inhibit viral replication. They are generally non-selective
integrase inhibitors and inhibiting both the 30-processing and the
strand transfer reactions in the same magnitude.
We previously reported the synthesis and IN inhibition evalua-
tion of a first series of catechol and bis-catechol derivatives, two of
which were found to be active against IN and five presented moder-
ate antiviral activity with a relatively high toxicity.10 In the present
work, we prepared a series of new hydroxylated 2-arylnaphthalenes
1a–n.
Regarding the first 2-phenylnaphthalene series, we had previ-
ously demonstrated that (i) the ortho-dihydroxy substitution must
be born by the naphthalenyl moiety, (ii) a 1,3-dimethyl substitu-
tion (on the naphthalene nucleus) is not recommended for HIV-1
IN inhibitory activity, (iii) catechols on the ancillary aromatic units
* Corresponding author. Tel.: +33 320 43 48 58; fax: +33 320 33 63 09.
E-mail address: philippe.cotelle@univ-lille1.fr (P. Cotelle).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.05.059

C. Maurin et al. / Bioorg. Med. Chem. 18 (2010) 5194–5201
5195
OH
O
F
O
N
O
O
Cl
OH
N
H
N
N
F
H3CO
HO
HN
Elvitegravir
Raltegravir
O
N
O
OH
N
O
O
N
N
F
OH
N
H
GSK-364735
Scheme 1.
may be replaced by a trifluoromethyl group. We therefore de-
signed a second set of 13 hydroxylated 2-arylnaphthalenes where
the substitution on the ancillary phenyl ring was chosen on the ba-
sis of our own experience (incorporation of a CF3 group) and on the
facts that lead HIV-1 IN inhibitors comprise a fluorophenyl unit
and that the presence of at least one hydroxy group is favorable.
Finally, we serendipitously found that 7-(3,4,5-trihydroxyphe-
nyl)naphthalene-1,2,3-triol 1c is sensitive to oxidation thus lead-
ing to isolation of dimer 5c. Whereas 1c was found to be toxic,
5c revealed good antiviral properties.
ated o-naphthoquinone ring. Both in 1H and 13C NMR spectroscopy,
the FID’s were found to be very short, denoting a rapid relaxation
phenomenon.
Synthetic polymers obtained from the oxidation of caffeic acid
with hydrogen peroxide were found to be active against HIV-1 and
2.13 As in the case of 5c, at the solid state, they gave ESR signals
attributed to semiquinonic radical. Our previous experience14 of
radical organic material led us to record the ESR spectrum of o-naph-
thaquinone dimer 5c. This spectrum shown in Figure 1 is a result of
two paramagnetic signals composed by three lines and centered at
g = 2.006. The signal intensities suggest two types of radical species
in the sample. The central signal is assigned to a majority of mono-
radical (S = 1/2) typical of a semiquinonic radical anion. The doublet
with a splitting of 46.8 mT corresponds to a minority of biradical
triplet spin state (S = 1). The dipole spin–spin interaction of the
biradical allows us to calculate the distance between radical centers.
This distance rAB which separates the radical pairs can be attributed
by the dipole splitting term, D.
2. Results and discussion
2.1. Chemistry
2-Phenylnaphthalenes 1a–c were prepared by an acid-pro-
moted dimerization of di (tri)methoxyphenylethanals 2a–c,11 fol-
lowed by an exhaustive demethylation of the methyl ethers11
using boron tribromide (Scheme 2) (yields = 54–78%). 2-Phenyl-
naphthalenes 1d–n were synthesized in a two-steps procedure
using the Suzuki coupling reaction of 2-bromo-5,6-dimethoxy-
naphthalene12 6 with conveniently substituted phenylboronic
acids 3d–n (yields = 30–85%) leading to the methoxylated deriva-
tives 4d–n followed by demethylation of the methoxy groups
using boron tribromide (Scheme 3) (yields = 47–95%). In our hands,
solutions of 1c (in acetone, ethyl acetate, DMSO) were found to
present an unexpected instability with time. Prolonged storage of
a solid, dry sample of 1c also resulted in darkness leading ulti-
mately to a black solid 5c which was isolated, characterized and
tested as IN inhibitor. Its structure (Scheme 4) was established
on the basis of spectroscopic data. The 1H NMR spectra of 5c in
DMSO-d6 and acetone-d6 indicate the presence of two 1,2,4 trisub-
stituted aromatic patterns and three singlets (respectively, inte-
grating for one, two and two protons each). The dimerization is
comforted by the observation of 28 peaks (four of them corre-
sponding to two carbons) on the 13C spectrum. The deshielded
peaks on the 13C NMR spectra can be attributed to a tetraoxygen-
D ¼ ðb2 gAgB=ꢀhr3 Þ ð1 ꢀ 3 cos2 hAB
Þ
AB
hAB is the angle between the pair director and the external field. The
other terms have their usual meanings. For hAB = p/2, rAB (nm) =
(52.04/D (MHz))1/3. The D value extracted from ESR spectra was
around 1310 MHz and the distance rAB deduced was approximately
3.4 0.5 Å.
This very short distance cannot be explained on the basis of
intramolecular interactions due to the absence of folding of the
bulky dimeric molecule 5c. We therefore suppose that this inter-
radical distance may provide from intermolecular interactions.
2.2. Antiintegrase activity
Compounds 1a–n and 5c were screened for inhibitory activities
against purified HIV-1 IN on the overall reaction, that is, both
Mg2+-dependent 30-processing and strand transfer reactions
(Table 1). In the case of 5c, the IC50’s on the two reactions catalyzed
by IN were measured. In our previous paper on a first series of
2-phenylnaphthalene derivatives,10 we found that best integrase
inhibition was obtained when the ortho-dihydroxy moiety was
born by the naphthalene nucleus and the two aromatic rings were
not orthogonal (no substitution on the positions ortho of the phe-
nyl ring). In the present work, a series of 7-arylnaphthalene-1,2-
diols was synthesized displaying good IN inhibitory action with
R1
R2
CHO
1) HCl, dioxane
1a-c
R3
2) BBr3
IC50 ranging between 1 and 10 lM. However there is a slight
R4
54-78%
difference between the bis-catechols 1b and 1d and the naphtha-
lene-1,2-diols 1e–n. Bis-catechols 1b and 1d gave the same result
on IN than 1a showing that the relative position of the catechol
2a-c
Scheme 2.

5196
C. Maurin et al. / Bioorg. Med. Chem. 18 (2010) 5194–5201
OMe
R2
MeO
R1
R3
Pd0(PPh3)4
1) BBr3/CH2Cl2
4d-n
1d-n
+
Benzene/K2CO3 2M
30-85%
2) H2O
Br
(HO)2B
47-95%
3d-n
6
Scheme 3.
HO
O
HO
[ox]
OH
OH
OH
HO
O
OH
OH
1c
OH
OH
OH
OH
OH
HO
HO
HO
O
OH
OH
OH
[ox]
OH
OH
HO
O
HO
OH
OH
OH
OH
O
O
O
5c
OH
OH
Scheme 4.
S=1
S=1/2
S=1
40000
30000
20000
10000
0
x 1000
x 1000
-10000
-20000
-30000
-40000
2800
3000
3200
3400
3600
3800
4000
Magnetic field (G)
Figure 1. ESR_CW spectrum at room temperature of 5c powder with g = 2.006.
moiety is not critical and confirming the importance of the pres-
ence of two catechols. In deep contrast with our previous results,
the substitution of the phenyl ring is practically ineffective to give
SAR information possibly because this part of the inhibitor does
not participate in the interaction with the enzyme. The linker be-
tween the two aromatic moieties (absent in the present case) has
to be modified, that is, by adding one or more sp3 atom in order
to increase to degree of freedom and allow this part of the inhibitor
to interact with the enzyme. Adding a third hydroxyl group did not
transfer reaction (IC50 (30-P) = 450 nM and IC50 (ST) = 170 nM). In
deep contrast with chicoric acid13 which inhibits selectively the
30-processing step, 5c inhibits both catalyzed reactions in the same
magnitude with a slight difference in favor of ST.
2.3. Antiviral activity
Synthetic polyphenols and natural tannins are known to pres-
ent antiviral activity.16 Compounds 1b–d and 5c were evaluated
ex vivo for their antiviral activity against HIV-1 replication in
CEM cells (Table 2). 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 concentra-
significantly increase the inhibition of IN (IC50 of 0.5
1–2 M for 1a–b and 1d).
The dimer 5c strongly inhibited the overall activity of IN
(IC50 = 0.85 M) with no significant selectivity toward the strand
lM for 1c vs
l
l

C. Maurin et al. / Bioorg. Med. Chem. 18 (2010) 5194–5201
5197
Table 1
biradical has also been characterized. It is a new example of the re-
dox instability of some polyphenols. However, in this particular
case, the final dimer was found to be less toxic than the initial pol-
yphenol 1c. The various oxidation processes are probably responsi-
ble for the toxicity of 1c via formation of semiquinonic radicals.
Compound 5c presents remarkable antiviral activity (compared
to other polyphenols) and is amongst the most active antiviral pol-
yphenol reported to date.
HIV-1 IN inhibitory potencies of compounds 1a–n
R1
OH
HO
R2
R3
R1
R1
R2
R3
R2
R3
R4
R4
R4
1d-n
1a-c
4. Experimental
4.1. Chemistry
a
Compound
R1
R2
R3
R4
IC50 in
2.110
lM
1a
1b
1c
5c
H
OH
H
OH
OH
OH
OH
H
OH
H
H
OH
1.00 0.03
0.50 0.1
0.85 0.53 (overall)
0.45 0.07 (30-P)
0.17 0.14 (ST)
2.2 0.2
6.0 2.6
10.0 4.24
4.8 0.14
8.1 3.1
6.0 0.54
10.0 0.24
10.0 0.31
7.3 0.1
All solvents were of commercial quality used from freshly opened
containers and were dried and purified by conventional methods.
Reagents were purchased from Aldrich-chimie (St Quentin-Falla-
vier, France). Mps were determined on a Reichert Thermopan appa-
ratus, equipped with a microscope and are uncorrected. NMR
spectra were obtained on an AC 300 Bruker spectrometer in the
appropriate deuterated solvent with TMS as internal reference (1H
300 MHz, 13C 75 MHz). Mass spectra were recorded on a Thermo-
Finnigan PolarisQ mass spectrometer (70 eV, Electronic Impact) or
on a Voyager DE STR mass spectrometer (Applied Biosystems) (MAL-
DI-TOF). Elemental analyses were performed by CNRS laboratories
(Vernaison).
1d
1e
1f
1g
1h
1i
1j
1k
1l
H
H
OH
H
H
CF3
H
H
F
H
OH
H
H
OH
H
H
CF3
H
H
F
H
OH
H
H
H
OH
H
H
CF3
H
H
F
H
H
H
H
H
H
H
H
H
H
H
1m
1n
9.6 0.13
8.0 0.1
H
a
Concentration giving 50% inhibition of the integrase overall activity.
4.2. Demethylation using boron tribromide—General procedure
tions of drugs. The amount of virus was assayed by b-galactosidase
assay, with HeLaCD4-b-gal cells as reporting cells. Toxicity was
To a solution of the methylated compound in dichloromethane
(1 mmol in 20 mL) boron tribromide (1 M in dichloromethane,
(x +1) mmol, x corresponds to the number of methoxy group) was
added dropwise. The mixture was stirred at room temperature for
1 h. The synthesis of 1c requires 10 equiv of BBr3 and 48 h at reflux.
estimated by MTT transformation assay. No activity (>100
against the HIV-1 replication or cytotoxicity (>100 M) was found
for 1b. Compound 1d was found to be slightly active (EC50 = 40 M,
CC50 >100 M). In deep contrast with 1a–b and 1d, 1c was found to
lM)
l
l
l
be very toxic. This could be related to its unstability. However the
isolated air-oxidized dimeric compound 5c revealed a good antivi-
4.3. Purification—General procedure
ral activity with an EC50 of about 1.15
lM and a low toxicity CC50 of
85 M giving a therapeutic index of 74. So we may assume that
l
Twenty milliliters of distilled water was slowly added and after
stirring for additional 15 min, the precipitate was filtered, washed
with water then with dichloromethane, and dried over P2O5.
Crystallization from acetone gave pure compounds.
compound 1c gives other toxic oxidized (or non-oxidized) com-
pounds during the cellular assays. The antiviral activity of 5c was
also evaluated in the HIV-1 infection of MT-4 cells. The toxicity
of 5c was slightly lower (75.6 lM) than in the HeLaCD4-b-gal cel-
lular test but its antiviral activity slightly decreased (3.9
the therapeutic index dropped to 19.
lM) and
4.3.1. 6-(2,3-Dihydroxyphenyl)naphthalene-1,2-diol 1b
Light brown solid; yield = 54%; mp = 107–109 °C; 1H NMR
3
(acetone-d6): 6.81 (t, 1H, JH5 –H4 = 3JH5 –H6 = 7.2 Hz, H5 ); 6.86
0
0
0
0
0
(dd, 1H, 3JH4 –H5 = 7.2 Hz, 4JH4 –H6 = 2.0 Hz, H4 ); 6.91 (dd, 1H, JH6 –H5
3
0
0
0
0
0
0
0
3. Conclusion
3
3
0
0
0
= 7.2 Hz, JH6 –H4 = 2.0 Hz, H6 ); 7.19 (d, 1H, JH3–H4 = 8.7 Hz, H3);
3
3
7.34 (d, 1H, JH4–H3 = 8.7 Hz, H4); 7.66 (dd, 1H, JH7–H8 = 8.7 Hz,
In this paper we report the anti-integrase activities of 14
2-phenylnaphthalenediols and triols. We demonstrated the insta-
bility of compound 1c which readily undergoes air oxidation over
a few days and the remarkable antiviral properties of the resulting
o-naphthoquinone 5c that exists (partially) as a radical. A minor
4JH7–H5 = 1.7 Hz, H7); 7.92 (d, 1H, JH5–H7 = 1.7 Hz, H5); 8.12 (d, 1H,
4
3JH8–H7 = 8.7 Hz, H8); 13C NMR (acetone-d6): 114.8 (CH); 118.8
(CH); 120.5 (CH); 120.6 (CH); 121.2 (CH); 122.4 (CH); 125.4 (C);
127.2 (C); 127.6 (CH); 128.4 (CH); 129.7 (C); 134.3 (C); 138.8 (C);
140.7 (C); 143.5 (C); 146.1 (C); MS (EI): m/z (%) = 268 ([M+], 10);
238 (100); 221 (38); 95 (34); 84 (47); 83 (70); 59 (52); Elemental
Anal. Calcd C, 71.64; H, 4.51. Found: C, 71.31; H, 4.68.
Table 2
Antiviral activities of compounds 1b–c and 5c compared to L-chicoric acid
Compound
Cell lines
CC50
(l
M)
EC50 (lM)
Tic
a
b
4.3.2. 7-(3,4,5-Trihydroxyphenyl)naphthalene-1,2,3-triol 1c
Gray solid; yield = 75%; mp >250 °C; 1H NMR (DMSO-d6): 6.77
Chicoric acid
MT-4
115 2115
12.7 8.515
9
5
1a
1b
1c
1d
5c
5c
HeLa (P4)
HeLa (P4)
MT-4
HeLa (P4)
HeLa (P4)
MT-4
5110
9.910
3
0
0
(s, 2H, H2 ,6 ); 6.80 (s, 1H, H4); 7.40 (dd, 1H, JH6–H5 = 8.55 Hz,
>100
1
>100
85 15
75.6 5.5
>100
>1
40
1.15 0.85
3.9 0.5
4JH6–H8 = 1.95 Hz, H6); 7.51 (d, 1H, JH5–H6 = 8.55 Hz, H5); 8.10 (d,
3
1H, JH8–H6 = 1.95 Hz, H8); 13C NMR (DMSO-d6): 100.8 (CH); 105.3
4
74
19
(2 CH); 117.2 (CH); 121.0 (C); 122.1 (CH); 126.0 (CH); 126.9 (C);
131.5 (C); 132.2 (C); 132.5 (C); 133.7 (C); 138.8 (C); 146.5 (2 C);
147.3 (C); MS (MALDI-TOF): m/z (%) = 301 ([M++H], 16); 300 ([M+],
100); 299 (10); Elemental Anal. Calcd C, 64.00; H, 4.03. Found: C,
63.69; H 4.24.
a
b
c
Cytotoxic concentration 50%.
Effective concentration 50%.
Therapeutic index = CC50/EC50
.

5198
C. Maurin et al. / Bioorg. Med. Chem. 18 (2010) 5194–5201
3
4
0
0
4.3.3. 4-(2,6-Dihydroxy-4-(6,7,8-trihydroxynaphthalene-2-yl)-
phenoxy)-7-(3,4,5-trihydroxyphenyl) naphthalene-1,2,3-triol 5c
H4); 7.72 (m, 2H, H2 ,6 ); 7.75 (dd, 1H, JH7–H8 = 8.8 Hz, JH7–H5
4
= 1.7 Hz, H7); 8.00 (d, 1H, JH5–H7 = 1.7 Hz, H5); 8.21 (d, 1H,
1H NMR (DMSO-d6): 6.65 (s, 2H, H2 ,6 ); 6.71 (s, 1H, H4); 6.74 (s,
3JH8–H7 = 8.8 Hz, H8); 13C NMR (CDCl3): 56.9 (CH3); 61.2 (CH3);
115.7 (CH); 122.0 (CH); 124.5 (CH); 125.6 (CH); 125.9 (CH);
127.2 (CH); 127.3 (2 ꢁ CH); 128.2 (C); 128.8 (2 ꢁ CH); 130.0 (C);
136.8 (C); 141.1 (C); 142.9 (C); 148.5 (C).
0
0
3
0
0
2H,
H
2 ,6 ); 7.33 (d, 1H, JH5–H6 = 8.2 Hz, H5); 7.69 (dd, 1H,
3JH6–H5 = 8.2 Hz, JH6–H8 = 2.1 Hz, H6); 7.74 (d, 1H, JH5–H6 = 7.9 Hz,
H5); 7.99 (dd, 1H, JH6–H5 = 7.9 Hz, JH6–H8 = 1.8 Hz, H6); 8.09 (d,
4
3
3
4
1H, JH8–H6 = 2.1 Hz, H8); 8.16 (d, 1H, JH6–H8 = 1.8 Hz, H6); 13C
NMR (DMSO-d6): 105.5 (2 CH); 105.6 (2 CH); 122.1; 124.4;
126.3; 128.1; 128.3; 130.1; 130.5; 131.3; 132.1; 132.2; 133.1;
134.3; 134.5; 139.3; 139.8; 140.5; 140.8; 142.2; 143.3; 145.3;
146.6 (2C); 146.8 (2C); 164.9; 166.7; 180.5; 188.7; MS (EI): m/z
(%) = 314 (72); 286 (100); 151 (95); Elemental Anal. Calcd for
4
4
4.4.3. 6-(2-Methoxyphenyl)-1,2-dimethoxynaphthalene 4f
Pale yellow powder; yield = 47%; mp = 121–122 °C; 1H NMR
(CDCl3): 3.86 (s, 3H, OCH3); 4.03 (s, 3H, OCH3); 4.05 (s, 3H,
3
4
0
0
0
0
0
OCH3); 7.05 (dd, 1H, JH3 –H4 = 8.1 Hz, JH3 –H5 = 1.0 Hz, H3 ); 7.10
(td, 1H, JH5 –H6 = 3JH5 –H4 = 7.6 Hz, JH5 –H3 = 1.0 Hz, H5 ); 7.32 (d,
3
4
0
0
0
0
0
0
0
3
3
0
0
C32H20O12: C, 64.44; H, 3.38. Found: C, 64.77; H, 3.02.
1H, JH3–H4 = 9.0 Hz, H3); 7.38 (ddd, 1H, JH4 –H3 = 8.1 Hz,
JH4 –H5 = 7.6 Hz, JH4 –H6 = 1.7 Hz, H4 ); 7.45 (dd, 1H, JH6 –H5
= 7.6 Hz, JH6 –H4 = 1.7 Hz, H6 ); 7.66 (d, 1H, JH4–H3 = 9.0 Hz, H4);
3
4
3
0
0
0
0
0
0
0
4
3
0
0
0
4.3.4. 6-Bromo-1,2-dimethoxynaphthalene 6 was obtained
according to published procedure11 as beige solid, mp = 50–
52 °C (lit.12 51–53 °C)
3
4
7.73 (dd, 1H, JH7–H8 = 8.8 Hz, JH7–H5 = 1.7 Hz, H7); 7.94 (d, 1H,
4JH5–H7 = 1.7 Hz, H5); 8.18 (d, 1H, JH8–H7 = 8.8 Hz, H8); 13C NMR
3
1H NMR (CDCl3): 3.99 (s, 3H, OCH3); 4.00 (s, 3H, OCH3); 7.31 (d,
(CDCl3): 55.6 (CH3); 57.0 (CH3); 61.2 (CH3); 111.3 (CH); 115.3
(CH); 120.8 (CH); 121.0 (CH); 124.5 (CH); 127.9 (CH); 128.1 (C);
128.4 (CH); 128.7 (CH); 129.8 (C); 130.7 (C); 131.1 (CH); 134.5
(C); 143.0 (C); 148.4 (C); 156.6 (C); MS (MALDI-TOF): m/z
(%) = 295 (23); 294 ([M+], 100); Elemental Anal. Calcd C, 77.53; H,
6.16. Found: C, 77.27; H, 6.15.
3
3
1H, JH3–H4 = 9.0 Hz, H3); 7.51 (d, 1H, JH4–H3 = 9.0 Hz, H4); 7.53
3
4
4
(dd, 1H, JH7–H8 = 9.0 Hz, JH7–H5 = 2.0 Hz, H7); 7.94 (d, 1H, JH5–H7
= 2.0 Hz, H5); 8.00 (d, 1H, JH8–H7 = 9.0 Hz, H8); 13C NMR(CDCl3):
3
56.8 (CH3); 61.2 (CH3); 116.2 (CH); 117.9 (C); 123.2 (CH);
123.3 (CH); 127.5 (C); 129.4 (CH); 129.5 (CH); 130.6 (C); 143.0 (C);
148.6 (C).
4.4.4. 6-(3-Methoxyphenyl)-1,2-dimethoxynaphthalene 4g
White powder; yield = 56%; mp = 107–108 °C; 1H NMR (CDCl3):
3.91 (s, 3H, OCH3); 4.03 (s, 3H, OCH3); 4.05 (s, 3H, OCH3); 6.94
4.4. Preparation of 4d–n
3
4
4
0
0
0
0
0
0
0
A mixture of 6-bromo-1,2-dimethoxynaphthalene 6 (2 mmol
534 mg), the convenient arylboronic acid (2 mmol), tetrakistriphe-
nylphosphine palladium(0) (70 mg) in benzene (2 mL) and an 2 M
aqueous solution of potassium carbonate (2 mL) was refluxed for
3 h. The mixture was cooled to room temperature and chloroform
(8 mL) and saturated aqueous solution of sodium hydrogenocar-
bonate (8 mL) were added. The aqueous layer was separated and
extracted twice with chloroform (10 mL). The organic layers were
grouped, washed with saturated aqueous solution of sodium
hydrogenocarbonate (10 mL). The organic layer was dried over so-
dium sulfate and evaporated to give a residue.
Compounds 4d–h were crystallized from acetone. In the cases
of 4i–n, the residue was dissolved in petroleum ether (200 mL
for 500 mg of residue) and filtered. After evaporation to dryness,
crystallization in a minimum of petroleum ether gave pure 4i–n.
In the cases of 4i–k, the residue was chromatographed on silica
column (eluent: dichloromethane) and crystallized from petro-
leum ether.
(ddd, 1H, JH4 –H5 = 8.0 Hz, JH4 –H2 = 2.0 Hz, JH4 –H6 = 0.9 Hz, H4 );
4
3
0
0
0
0
0
7.27 (d, 1H, JH2 –H4 = 2.0 Hz, H2 ); 7.32 (dd, 1H, JH6 –H5 = 7.8 Hz,
4
3
0
0
0
JH6 –H4 = 0.9 Hz, H6 ); 7.34 (d, 1H, JH3–H4 = 8.9 Hz, H3); 7.41 (dd,
1H, JH5 –H4 = 8.0 Hz, JH5 –H6 = 7.8 Hz, H5 ); 7.68 (d, 1H, JH4–H3
= 8.9 Hz, H4); 7.76 (dd, 1H, JH7–H8 = 8.8 Hz, JH7–H5 = 1.8 Hz, H7);
3
3
3
0
0
0
0
0
3
4
4
3
8.00 (d, 1H, JH5–H7 = 1.8 Hz, H5); 8.21 (d, 1H, JH8–H7 = 8.8 Hz, H8);
13C NMR (CDCl3): 55.4 (CH3); 57.0 (CH3). 61.3 (CH3); 112.7 (CH);
113.1 (CH); 115.8 (CH); 119.9 (CH); 122.1 (CH); 124.6 (CH); 125.7
(CH); 126.0 (CH); 128.4 (C); 129.9 (CH); 130.0 (C); 136.8 (C); 142.8
(C); 143.0 (C); 148.6 (C); 160.1 (C); MS (EI): m/z (%) = 295 (21);
294 (100); 279 (42); 251 (36); 236 (36); Elemental Anal. Calcd C,
77.53; H, 6.16. Found: C, 77.65; H, 6.21.
4.4.5. 6-(4-Methoxyphenyl)-1,2-dimethoxynaphthalene 4h
Gray powder; yield = 72%; mp = 148–149 °C; 1H NMR (CDCl3):
3.89 (s, 3H, OCH3); 4.03 (s, 3H, OCH3); 4.05 (s, 3H, OCH3); 7.04 (d,
3
3
0
0
0
0
0
0
2H, JH3 ,5 –H2 ,6 = 8.8 Hz, H3 ,5 ); 7.32 (d, 1H, JH3–H4 = 8.8 Hz, H3);
3
3
0
0
0
0
7.65 (d, 1H, JH4–H3 = 8.8 Hz, H4); 7.67 (d, 2H, JH2 ,6 –H3 ,5 = 8.8 Hz,
3
4
0
0
H
2 ,6 ); 7.74 (dd, 1H, JH7–H8 = 8.8 Hz, JH7–H5 = 1.7 Hz, H7); 7.95 (d,
4.4.1. 6-(3,4-Dimethoxyphenyl)-1,2-dimethoxynaphthalene 4d
1H, 4JH5–H7 = 1.7 Hz, H5); 8.20 (d, 1H, 3JH8–H7 = 8.8 Hz, H8); 13C NMR
(CDCl3): 55.4 (CH3); 56.9 (CH3); 61.2 (CH3); 114.3 (2 ꢁ CH); 115.7
(CH); 121.9 (CH); 124.4 (CH); 124.8 (CH); 125.7 (CH); 127.9 (C);
128.3 (2 ꢁ CH); 130.1 (C); 133.6 (C); 136.4 (C); 142.9 (C); 148.2
(C); 159.1 (C); MS (MALDI-TOF): m/z (%) = 296 (20); 295 (73); 294
([M+], 100); Elemental Anal. Calcd C, 77.53; H, 6.16. Found: C,
77.30; H, 6.11.
White powder; yield = 30%; mp = 129–130 °C; 1H NMR (CDCl3):
3.93 (s, 3H, OCH3); 3.98 (s, 3H, OCH3); 3.99 (s, 3H, OCH3); 4.02 (s,
3
0
0
0
0
3H, OCH3); 6.96 (d, 1H, JH5 –H6 = 8.1 Hz, H5 ); 7.23 (s, 1H, H2 );
3
3
0
0
0
7.24 (d, 1H, JH6 –H5 = 8.1 Hz, H6 ); 7.30 (d, 1H, JH3–H4 = 9.0 Hz,
H3); 7.63 (d, 1H, JH4–H3 = 9.0 Hz, H4); 7.71 (dd, 1H, JH7–H8
3
3
4
4
= 8.8 Hz, JH7–H5 = 1.9 Hz, H7); 7.92 (d, 1H, JH5–H7 = 1.9 Hz, H5);
8.17 (d, 1H, JH8–H7 = 8.8 Hz, H8); 13C NMR (CDCl3): 56.0
3
(2 ꢁ CH3); 56.9 (CH3); 61.2 (CH3); 110.5 (CH); 111.6 (CH); 115.7
(CH); 119.6 (CH); 121.9 (CH); 124.4 (CH); 125.0 (CH); 125.8
(CH); 128.0 (C); 130.0 (C); 134.1 (C); 136.6 (C); 142.9 (C); 148.3
(C); 148.6 (C); 149.2 (C); MS (MALDI-TOF): m/z (%) = 325 (32);
324 ([M+], 100); Elemental Anal. Calcd C, 74.06; H, 6.21. Found:
C, 74.24; H, 6.32.
4.4.6. 1,2-Dimethoxy-6-(2-trifluoromethylphenyl)naphthalene
4i
White powder; yield = 58%; mp = 86–87 °C; 1H NMR (CDCl3):
4.01 (s, 3H, OCH3); 4.03 (s, 3H, OCH3); 7.33 (d, 1H, 3JH3–H4 = 9.0 Hz,
H3); 7.40 (d, 1H, 3J = 7.6 Hz, H3 or H6 ); 7.45 (br d, 1H,
0
0
JH7–H8 = 8.5 Hz, H7); 7.48 (t, 3J = 7.6 Hz, H4 or H5 ); 7.58 (t,
3
0
0
3
3
0
0
J = 7.6 Hz, H4 or H5 ); 7.62 (d, 1H, JH4–H3 = 9.0 Hz, H4); 7.72 (br s,
1H, H5); 7.78 (d, 1H, 3J = 7.6 Hz, H3 or H6 ); 8.14 (d, 1H,
0
0
4.4.2. 6-Phenyl-1,2-dimethoxynaphthalene 4e
Gray powder; yield = 37%; mp = 114–115 °C (lit.12 114–115 °C);
3JH8–H7 = 8.5 Hz, H8); 13C NMR (CDCl3): 56.9 (s, CH3); 61.2 (s, CH3);
1H NMR (CDCl3): 4.02 (s, 3H, OCH3); 4.04 (s, 3H, OCH3); 7.33 (d, 1H,
115.8 (s, CH); 120.9 (s, CH); 124.3 (q, CF3, JC–F = 272.2 Hz); 124.5
1
3
3
4
3
0
0
0
0
0
0
JH3–H4 = 9.0 Hz, H3); 7.38 (tt, 1H, JH4 –H3 ,5 = 7.5 Hz, JH4 –H2 ,6
(s, CH); 126.1 (q, CH, JC–F = 5.4 Hz); 127.4 (s, CH); 127.5 (q, CH,
3
4
= 1.3 Hz, H4 ); 7.49 (m, 2H, H3 ,5 ); 7.67 (d, 1H, JH4–H3 = 9.0 Hz,
4JC–F = 1.7 Hz); 127.7 (q, CH, JC–F = 1.7 Hz); 128.3 (s, C); 128.6
0
0
0

C. Maurin et al. / Bioorg. Med. Chem. 18 (2010) 5194–5201
5199
3
(q, C, 2JC–F = 29.8 Hz); 129.1 (s, C); 131.4 (s, CH); 132.4 (s, CH); 135.7
(s, C); 141.4 (q, C, 3JC–F = 1.7 Hz); 143.0 (s, C); 148.7 (s, C); MS (EI): m/
z (%) = 333 (20); 332 ([M+], 98); 317 (49); 289 (28); 250 (21); 249
(100); Elemental Anal. Calcd C, 68.67; H, 4.55; F, 17.15. Found: C,
68.88; H, 4.49; F, 17.33.
130.2 (d, CH, JC–F = 8.6 Hz); 135.4 (s, C); 142.8 (s, C); 143.4 (d, C,
3JC–F = 7.3 Hz); 148.6 (s, C); 163.2 (d, C, JC–F = 245.4 Hz); MS (EI):
1
m/z (%) = 283 (25); 282 ([M+], 100); 267 (52); 239 (69); 224 (36);
196 (36); Elemental Anal. Calcd C, 76.58; H, 5.36; F, 6.73. Found:
C, 76.35; H, 5.47; F, 6.52.
4.4.7. 1,2-Dimethoxy-6-(3-trifluoromethylphenyl)naphthalene 4j
4.4.11. 6-(4-Fluorophenyl)-1,2-dimethoxynaphthalene 4n
Cream powder; yield = 85%; mp = 65–67 °C; 1H NMR (CDCl3):
White powder; yield = 78%; mp = 118–119 °C; 1H NMR (CDCl3):
3
3
0
0
0
0
4.02 (s, 3H, OCH3); 4.06 (s, 3H, OCH3); 7.34 (d, 1H, JH3–H4 = 9.0
4.02 (s, 3H, OCH3); 4.04 (s, 3H, OCH3); 7.17 (t, 2H, JH3 ,5 –H2 ,6
=
3
3
3
3
0
0
0
0
0
0
0
0
0
Hz, H3); 7.58 (d, 1H, JH4 –H5 = 7.6 Hz, H4 ); 7.62 (t, 1H, JH5 –H4
=
JH3 ,5 –F = 8.6 Hz, H3 ,5 ); 7.33 (d, 1H, JH3–H4 = 8.9 Hz, H3); 7.65 (d,
3
3
3
3
0
0
0
0
0
0
0
0
0
JH5 –H6 = 7.6 Hz, H5 ); 7.67 (d, 1H, JH4–H3 = 9.0 Hz, H4); 7.73 (dd,
1H, JH4–H3 = 8.9 Hz, H4); 7.66 (d, 2H, JH2 ,6 –H3 ,5 = 8.6 Hz, H2 ,6 );
3
4
3
3
4
0
0
1H, JH7–H8 = 8.8 Hz, JH7–H5 = 1.9 Hz, H7); 7.86 (d, 1H, JH6 –H5
=
7.69 (dd, 1H, JH7–H8 = 8.8 Hz, JH7–H5 = 1.7 Hz, H7); 7.93 (d, 1H,
4
3
7.6 Hz, H6 ); 7.99 (br s, 1H); 7.99 (d, 1H, JH5–H7 = 1.9 Hz, H5);
4JH5–H7 = 1.7 Hz, H5); 8.20 (d, 1H, JH8–H7 = 8.8 Hz, H8); 13C NMR
0
8.24 (d, 1H, 3JH8–H7 = 8.8 Hz, H8); 13C NMR (CDCl3): 56.8 (s, CH3);
(CDCl3): 56.9 (s, CH3); 61.2 (s, CH3); 115.7 (d, 2 ꢁ CH, JC–F
2
3
61.2 (s, CH3); 115.8 (s, CH); 122.4 (s, CH); 123.8 (q, CH, JC–F
3.9 Hz); 123.9 (q, CH, JC–F = 3.9 Hz); 124.3 (q, CF3, JC–F
=
=
= 21.4 Hz); 115.8 (s, CH); 122.1 (s, CH); 124.5 (s, CH); 125.4 (s,
3
1
3
CH); 125.7 (s, CH); 128.1 (s, C); 128.8 (d, 2 ꢁ CH, JC–F = 7.9 Hz);
4
272.2 Hz); 124.7 (s, CH); 125.4 (s, CH); 126.0 (s, CH); 128.6 (s, C);
129.9 (s, C); 135.8 (s, C); 137.2 (d, C, JC–F = 3.0 Hz); 142.9 (s, C);
2
1
129.3 (s, CH); 129.8 (s, C); 130.5 (s, CH); 131.2 (q, C, JC–F
=
148.5 (s, C); 162.4 (d, C, JC–F = 246.0 Hz); MS (MALDI-TOF): m/z
32.3 Hz); 135.2 (s, C); 141.9 (s, C); 142.9 (s, C); 148.8 (s, C); MS
(EI): m/z (%) = 332 ([M+], 100); 317 (43); 289 (42); 274 (22); 269
(31); 249 (27); Elemental Anal. Calcd C, 68.67; H, 4.55; F, 17.15.
Found: C, 68.48; H, 4.64; F, 17.27.
(%) = 283 (42); 282 ([M+], 100); Elemental Anal. Calcd C, 76.58; H,
5.36; F, 6.73. Found: C, 76.72; H, 5.39; F, 6.59.
Compounds 1d–n were obtained using the same procedure of
compounds 1b–c. Compounds 1i–n were isolated in the dichloro-
methane layer. After evaporation, they were crystallized in a min-
imum of dichloromethane.
4.4.8. 2-Dimethoxy-6-(4-trifluoromethylphenyl)naphthalene 4k
White powder; yield = 55%; mp = 131–132 °C; 1H NMR (CDCl3):
4.02 (s, 3H, OCH3); 4.04 (s, 3H, OCH3); 7.34 (d, 1H, 3JH3–H4 = 9.0 Hz,
H3); 7.66 (d, 1H, 3JH4–H3 = 9.0 Hz, H4); 7.71 (dd, 1H, 3JH7–H8 = 8.8 Hz,
4.4.12. 6-(3,4-Dihydroxyphenyl)naphthalene-1,2-diol 1d
Brown powder; yield = 95%; mp >250 °C; 1H NMR (acetone-d6):
4
3
3
3
0
0
0
0
0
0
0
0
0
0
0
JH7–H5 = 1.7 Hz, H7); 7.71 (d, 2H, JH2 ,6 –H3 ,5 = 7.9 Hz, H2 ,6 or
6.83 (d, 1H, JH5 –H6 = 8.2 Hz, H5 ); 7.11 (dd, 1H, JH6 –H5 = 8.2 Hz,
3
4
3
0
0
0
0
0
0
0
0
0
0
0
0
0
H
3 ,5 ); 7.78 (d, 2H, JH2 ,6 –H3 ,5 = 7.9 Hz, H2 ,6 or H3 ,5 ); 7.98 (d,
JH6 –H2 = 2.2 Hz, H6 ); 7.20 (d, 1H, JH3–H4 = 8.8 Hz, H3); 7.25 (d,
1H, JH2 –H6 = 2.2 Hz, H2 ); 7.36 (d, 1H, JH4–H3 = 8.8 Hz, H4); 7.64
(dd, 1H, JH7–H8 = 8.7 Hz, JH7–H5 = 1.7 Hz, H7); 7.89 (d, 1H, JH5–H7
1H, JH5–H7 = 1.7 Hz, H5); 8.23 (d, 1H, JH8–H7 = 8.8 Hz, H8); 13C
4
3
4
3
0
0
0
3
4
4
NMR (CDCl3): 56.8 (s, OCH3); 61.2 (s, OCH3); 115.8 (s, CH); 122.3
1
3
(s, CH); 124.4 (q, CF3, JC–F = 271.9 Hz); 124.7 (s, CH); 125.4 (s,
= 1.7 Hz, H5); 8.03 (br s, 4H, 4 ꢁ OH); 8.14 (d, 1H, JH8–H7 = 8.7 Hz,
3
CH); 125.8 (q, 2 ꢁ CH, JC–F = 3.7 Hz); 126.1 (s, CH); 127.5 (s,
H8); 13C NMR (acetone-d6): 114.8 (CH); 116.6 (CH); 119.1 (CH);
119.3 (CH); 120.5 (CH); 122.2 (CH); 125.0 (CH); 125.1 (CH);
125.3 (C); 130.5 (C); 134.1 (C); 136.5 (C); 138.7 (C); 140.5 (C);
145.5 (C); 146.2 (C); MS (EI): m/z (%) = 268 ([M+], 24); 238 (56);
165 (29); 119 (37); 105 (100); 91 (63); 77 (72); 67 (59); Elemental
Anal. Calcd C, 71.64; H, 4.51. Found: C, 71.42; H, 4.63.
2
2 ꢁ CH); 128.6 (s, C); 129.1 (q, C, JC–F = 32.4 Hz); 129.7 (s, C);
135.2 (s, C); 142.9 (s, C); 144.6 (s, C); 148.9 (s, C); MS (MALDI-
TOF): m/z (%) = 333 (21); 332 ([M+], 100); Elemental Anal. Calcd
C, 68.67; H, 4.55; F, 17.15. Found: C, 68.85; H, 4.60; F, 17.02.
4.4.9. 6-(2-Fluorophenyl)-1,2-dimethoxynaphthalene 4l
Beige powder; yield = 54%; mp = 82–84 °C; 1H NMR (CDCl3): 4.01
(s, 3H, OCH3); 4.02 (s, 3H, OCH3); 7.18 (ddd, 1H. JH3 –F = 10.7 Hz,
4.4.13. 6-Phenylnaphthalene-1,2-diol 1e
3
Light brown powder; yield = 65%; mp = 178–180 °C (dec); 1H
0
3
4
3
3
0
0
0
0
0
0
0
JH3 –H4 = 8.1 Hz, JH3 –H5 = 1.5 Hz, H3 ); 7.24 (td, 1H, JH5 –H4
=
NMR (acetone-d6): 7.24 (d, 1H, JH3–H4 = 8.8 Hz, H3); 7.35 (tt, 1H,
3
4
3
4
3
0
0
0
0
0
0
0
0
0
0
0
0
0
JH5 –H6 = 7.5 Hz, JH5 –H3 = 1.5 Hz, H5 ); 7.30–7.37 (m, 1H, H4 );
JH4 –H3 ,5 = 7.3 Hz, JH4 –H2 ,6 = 1.3 Hz, H4 ); 7.42 (d, 1H, JH4–H3 =
7.32 (d, 1H, JH3–H4 = 8.9 Hz, H3); 7.54 (td, 1H, JH6 –H5 = 4JH6 –F
8.8 Hz, H4); 7.48 (m, 2H, H3 ,5 ); 7.73 (dd, 1H, JH7–H8 = 8.8 Hz, 4J
3
3
3
0
0
0
0
0
4
3
4
0
0
0
0
0
= 7.5 Hz, JH6 –H4 = 1.9 Hz, H5 ); 7.65 (d, 1H, JH4–H3 = 8.9 Hz, H4);
H7–H5 = 1.9 Hz, H7); 7.76 (m, 2H, H2 ,6 ); 8.02 (d, 1H, JH5–H7
=
7.68 (dt, 1H, JH7–H8 = 8.8 Hz, JH7–H5 = 5JH7–F = 1.8 Hz, H7); 7.96 (br
1.9 Hz, H5); 8.22 (d, 1H, JH8–H7 = 8.8 Hz, H8); 13C NMR (acetone-
d6): 119.2 (CH); 120.7 (CH); 122.4 (CH); 125.0 (CH); 125.7 (C);
126.1 (CH); 127.7 (2 ꢁ CH); 127.8 (CH); 129.7 (2 ꢁ CH); 130.4
(C); 136.3 (C); 138.7 (C); 140.8 (C); 141.9 (C); MS (MALDI-TOF):
m/z (%) = 238 (25); 237 (58); 236 ([M+], 100); 235 (40); Elemental
Anal. Calcd C, 81.34; H, 5.12. Found: C, 81.18; H, 5.21.
3
4
3
s, 1H, H5); 8.19 (d, 1H, 3JH8–H7 = 8.8 Hz, H8); 13C NMR (CDCl3): 56.9
2
(s, CH3); 61.2 (s, CH3); 115.6 (s, CH); 116.2 (d, CH, JC–F = 22.6 Hz);
121.6 (s, CH); 124.5 (d, CH, JC–F = 3.7 Hz); 124.7 (s, CH); 127.4 (d,
CH, JC–F = 3.0 Hz); 128.0 (d, CH, JC–F = 3.0 Hz); 128.4 (s, C); 129.0
4
4
4
3
2
(d, CH, JC–F = 8.6 Hz); 129.1 (d, C, JC–F = 13.7 Hz); 129.7 (s, C);
131.0 (d, CH, 3JC–F = 3.0 Hz); 131.5 (d, C, 3JC–F = 1.8 Hz); 142.9 (s, C);
1
148.8 (s, C); 160.1 (d, C, JC–F = 247.8 Hz); MS (MALDI-TOF): m/z
4.4.14. 6-(2-Hydroxyphenyl)naphthalene-1,2-diol 1f
(%) = 283 (50); 282 ([M+], 100); Elemental Anal. Calcd C, 76.58; H,
5.36; F, 6.73. Found: C, 76.39; H, 5.43; F, 6.85.
Dark brown powder; yield = 47%; mp = 168 °C (dec); 1H NMR
0
0
(acetone-d6): 6.76–6.88 (m, 2H, H3 and H5 ); 7.04–7.36 (m, 3H,
0
0
H3, H4 and H6 ); 7.57–7.78 (m, 2H, H7 and H4 ); 7.93 (br s, 1H,
OH); 8.11–8.24 (m, 2H, H5 and H8); 8.53 (br s, 2H, 2 ꢁ OH); 13C
NMR (acetone-d6): 116.9 (CH); 119.1 (CH); 120.7 (CH); 122.2
(CH); 122.3 (CH); 124.9 (CH); 125.8 (C); 126.0 (CH); 129.0 (C);
129.1 (CH); 129.2 (CH); 130.4 (C); 136.5 (C); 138.8 (C); 140.7 (C);
154.6 (C); MS (MALDI-TOF): m/z (%) = 253 (38); 252 ([M+], 100);
Elemental Anal. Calcd C, 76.18; H, 4.79. Found: C, 76.49; H, 4.96.
4.4.10. 6-(3-Fluorophenyl)-1,2-dimethoxynaphthalene 4m
Cream prism; yield = 67%; mp = 95–97 °C; 1H NMR (CDCl3):
0
4.01 (s, 3H, OCH3); 4.02 (s, 3H, OCH3); 7.02–7.08 (m, 1H, H4 );
3
0
0
7.33 (d, 1H, JH3–H4 = 9.0 Hz, H3); 7.38–7.50 (m, 3H, H2 , H5 ,
3
0
and H6 ); 7.66 (d, 1H, JH4–H3 = 9.0 Hz, H4); 7.70 (dd, 1H,
3JH7–H8 = 8.8 Hz, JH7–H5 = 1.7 Hz, H7); 7.96 (d, 1H, JH5–H7 = 1.7 Hz,
4
4
H5); 8.19 (d, 1H, JH8–H7 = 8.8 Hz, H8); 13C NMR (CDCl3): 56.8 (s,
3
2
CH3); 61.1 (s, CH3); 113.9 (d, CH, JC–F = 21.4 Hz); 114.0 (d, CH,
4.4.15. 6-(3-Hydroxyphenyl)naphthalene-1,2-diol 1g
2JC–F = 22.0 Hz); 115.7 (s, CH); 122.1 (s, CH); 122.8 (s, CH); 124.6
Light brown powder; yield = 75%; mp = 186–187 °C (dec);
3
0
0
(s, CH); 125.4 (s, CH); 125.7 (s, CH); 128.4 (s, C); 129.7 (s, C);
1H NMR (acetone-d6): 6.85 (ddd, 1H,
JH4 –H5 = 7.9 Hz,

5200
C. Maurin et al. / Bioorg. Med. Chem. 18 (2010) 5194–5201
4
4
0
0
0
0
0
0
JH4 –H2 = 2.4 Hz, JH4 –H6 = 1.2 Hz, H4 ); 7.22–7.33 (m, 4H, H2 and
0
Anal. Calcd C, 67.11; H, 3.64; F, 18.73. Found: C, 67.02; H, 3.75; F,
18.82.
3
0
H6 and H3 and H5 ); 7.41 (d, 1H, JH4–H3 = 8.5 Hz, H4); 7.70 (dd, 1H,
3JH7–H8 = 8.8 Hz, JH7–H5 = 1.7 Hz, H7); 7.84 (br s, 1H, OH); 7.99 (d,
4
4
3
1H, JH5–H7 = 1.7 Hz, H5); 8.20 (d, 1H, JH8–H7 = 8.8 Hz, H8); 8.40 (br
s, 2H, 2 ꢁ OH); 13C NMR (acetone-d6): 114.6 (CH); 114.9 (CH);
119.0 (CH); 119.2 (CH); 120.7 (CH); 122.3 (CH); 125.0 (CH); 125.7
(C); 126.1 (CH); 130.4 (C); 130.7 (CH); 136.4 (C); 138.8 (C); 140.8
(C); 143.5 (C); 158.7 (C); MS (EI): m/z (%) = 253 (42); 252 ([M+],
100); 251 (21); Elemental Anal. Calcd C, 76.18; H, 4.79. Found: C,
75.97; H, 4.87.
4.4.20. 6-(2-Fluorophenyl)naphthalene-1,2-diol 1l
Green powder; yield = 95%; mp = 145–147 °C (dec); 1H NMR
0
0
(acetone-d6): 7.22–7.34 (m, 2H, H3 and H5 ); 7.25 (d, 1H,
3
3
0
JH3–H4 = 8.8 Hz, H3); 7.37–7.45 (m, 1H, H4 ); 7.41 (d, 1H, JH4–H3
=
0
8.8 Hz, H4); 7.60–7.65 (m, 2H, H7 and H6 ); 7.88 (s, 1H, OH); 7.94
3
(br s, 1H, H5); 8.20 (d, 1H, JH8–H7 = 8.8 Hz, H8); 8.46 (s, 1H, OH);
13C NMR (acetone-d6): 116.8 (d, CH, 2JC–F = 22.6 Hz); 119.2 (s, CH);
4
120.7 (s, CH); 121.9 (s, CH); 125.5 (d, CH, JC–F = 3.7 Hz); 125.7 (s,
4
4
4.4.16. 6-(4-Hydroxyphenyl)naphthalene-1,2-diol 1h
C); 126.6 (d, CH, JC–F = 3.0 Hz); 128.6 (d, CH, JC–F = 3.0 Hz); 129.8
Brown powder; yield = 91%; mp >250 °C (dec); 1H NMR (ace-
(d, CH, JC–F = 8.6 Hz); 129.9 (d, C, JC–F = 13.4 Hz); 130.0 (s, C);
3
2
3
tone-d6): 6.96 (d, 2H, JH3 ,5 –H2 ,6 = 8.5 Hz, H3 ,5 ); 7.21 (d, 1H,
131.2 (d, C, 3JC–F = 1.2 Hz); 131.8 (d, CH, 3JC–F = 3.7 Hz); 138.7 (s, C);
0
0
0
0
0
0
3JH3–H4 = 8.5 Hz, H3); 7.37 (d, 1H, JH4–H3 = 8.5 Hz, H4); 7.62 (d,
141.0 (s, C); 160.7 (d, C, JC–F = 246.0 Hz); MS (ei): m/z (%) = 255
3
1
3
3
2H, JH2 ,6 –H3 ,5 = 8.5 Hz, H2 ,6 ); 7.68 (dd, 1H, JH7–H8 = 8.8 Hz,
(24); 254 ([M+], 100); 253 (33); Elemental Anal. Calcd C, 75.58; H,
4.36; F, 7.47. Found: C, 75.25; H, 4.49; F, 7.30.
0
0
0
0
0
0
4JH7–H5 = 1.7 Hz, H7); 7.80 (s, 1H, OH); 7.93 (d, 1H, JH5–H7
4
3
= 1.7 Hz, H5); 8.17 (d, 1H, JH8–H7 = 8.8 Hz, H8); 8.34 (s, 1H, OH);
8.46 (s, 1H, OH); 13C NMR (acetone-d6): 116.5 (2 ꢁ CH); 119.1
(CH); 120.5 (CH); 122.2 (CH); 124.9 (CH); 125.0 (CH); 125.2 (C);
128.8 (2 ꢁ CH); 130.5 (C); 133.2 (C); 136.3 (C); 138.8 (C); 140.4
(C); 157.7 (C); MS (EI): m/z (%) = 252 ([M+]. 5); 250 (7); 223 (19);
222 (100); 194 (18); 165 (20); Elemental Anal. Calcd C, 76.18; H,
4.79. Found: C, 76.04; H, 4.90.
4.4.21. 6-(3-Fluorophenyl)naphthalene-1,2-diol 1m
Green powder; yield = 90%; mp = 146–148 °C; 1H NMR (acetone-
3
3
4
0
0
0
0
0
d6): 7.11 (dddd, 1H, JH4 –F = 9.2 Hz, JH4 –H5 = 7.8 Hz, JH4 –H2
=
4
3
0
0
0
2.7 Hz, JH4 –H6 = 1.0 Hz, H4 ); 7.25 (d, 1H, JH3–H4 = 8.8 Hz, H3);
7.43 (d, 1H, JH4–H3 = 8.8 Hz, H4); 7.47–7.56 (m, 2H, H2 and H5 );
3
0
0
3
4
4
0
0
0
0
0
0
7.61 (ddd, 1H, JH6 –H5 = 7.8 Hz, JH6 –H2 = 1.7 Hz, JH6 –H4 = 1.0 Hz,
H6 ); 7.74 (dd, 1H, JH7–H8 = 8.8 Hz, 4JH7–H5 = 2.0 Hz, H7); 7.90 (s, 1H,
3
0
4
4.4.17. 6-(2-Trifluoromethylphenyl)naphthalene-1,2-diol 1i
OH); 8.07 (d, 1H. JH5–H7 = 2.0 Hz, H5); 8.22 (d, 1H,3JH8–H7
=
Brown powder; yield = 85%; mp = 110 °C (dec); 1H NMR (ace-
8.8 Hz,H8); 8.50 (s,1H,OH); 13C NMR (acetone-d6): 114.2 (d, CH, 2J
3
tone-d6): 7.24 (d, 1H, JH3–H4 = 8.8 Hz, H3); 7.31 (d, 1H,
C–F = 22.0 Hz);114.3 (d, CH, 2JC–F = 21.4 Hz); 119.3 (s, CH); 120.9 (s,
3
4
3JH4–H3 = 8.8 Hz, H4); 7.32 (dm, 1H, JH7–H8 = 8.8 Hz, H7); 7.45 (dm,
CH); 122.5 (s, CH); 123.5 (d, CH, JC–F = 3.1 Hz); 124.7 (s, CH);
3
1H, 3JH6 –H5 = 6.9 Hz, H6 ); 7.59 (ddm, 1H, JH4 –H5 = 8.1 Hz, 3JH4 –H3
125.9 (s, C); 126.5 (s, CH); 130.3 (s, C); 131.4 (d, CH, 3JC–F = 8.6 Hz);
0
0
0
0
0
0
0
3
4
0
0
0
= 7.8 Hz, H4 ); 7.63 (m, 1H, H5); 7.70 (ddm, 1H, JH5 –H4 = 8.1 Hz,
JH5 –H6 = 6.9 Hz, H5 ); 7.80 (dm, JH3 –H4 = 7.8 Hz, H3 ); 7.90 (br s,
134.8 (d, C, JC–F = 2.5 Hz); 138.8 (s, C); 141.1 (s, C); 144.5 (d, C,
3
3
1
3JC–F = 7.9 Hz); 164.1 (d, C, JC–F = 243.5 Hz); MS (MALDI-TOF): m/z
0
0
0
0
0
0
3
1H, OH); 8.11 (d, 1H. JH8–H7 = 8.8 Hz, H8); 8.47 (br s, 1H, OH); MS
(MALDI-TOF): m/z (%) = 305 (42); 304 ([M+]. 100); 303 (33); Elemen-
tal Anal. Calcd C, 67.11; H, 3.64; F, 18.73. Found: C, 67.45; H, 3.78; F,
18.49. Since 1i rapidly degraded in acetone solution the 13C NMR
spectra cannot be recorded.
(%) = 255 (40); 254 ([M+]. 100); 253 (26); Elemental Anal. Calcd C,
75.58; H, 4.36; F, 7.47. Found: C, 75.81; H, 4.28; F, 7.33.
4.4.22. 6-(4-Fluorophenyl)naphthalene-1,2-diol 1n
Green powder; yield = 69%; mp = 161–163 °C (dec); 1H NMR
3
0
0
(acetone-d6): 7.21–7.26 (m, 2H, H3 .5 ); 7.24 (d, 1H, JH3–H4 = 8.6 Hz,
4.4.18. 6-(3-Trifluoromethylphenyl)naphthalene-1,2-diol 1j
H3); 7.40 (d, 1H, 3JH4–H3 = 8.6 Hz, H4); 7.69 (dd, 1H, 3JH7–H8 = 8.8 Hz,
JH7–H5 = 1.7 Hz, H7); 7.76–7.81 (m, 2H, H2 .6 ); 7.87 (s, 1H, OH);
7.99 (d, 1H, JH5–H7 = 1.7 Hz, H5); 8.20 (d, 1H, JH8–H7 = 8.8 Hz, H8);
8.46 (s, 1H, OH); 13C NMR (acetone-d6): 116.3 (d, 2 ꢁ CH,
2JC–F = 21.4 Hz); 119.3 (s, CH); 120.7 (s, CH); 122.5 (s, CH); 124.9 (s,
Gray powder; yield = 90%; mp = 154–157 °C; 1H NMR (acetone-
4
0
0
3
3
4
3
d6): 7.29 (d, 1H, JH3–H4 = 8.7 Hz, H3); 7.46 (d, 1H, JH4–H3 = 8.7 Hz,
3
0
0
H4); 7.67–7.69 (m, 2H, H5 and H6 ); 7.78 (dd, 1H, JH7–H8 = 8.8 Hz,
JH7–H5 = 1.9 Hz, H7); 7.94 (br s, 1H, OH); 8.03 (m, 1H. H4 ); 8.08
4
0
4
3
0
(br s, 1H, H2 ); 8.16 (d, 1H, JH5–H7 = 1.9 Hz, H5); 8.27 (d, 1H,
CH); 125.6 (s, C); 126.1 (s, CH); 129.5 (d, 2 ꢁ CH, JC–F = 8.6 Hz);
3JH8–H7 = 8.8 Hz, H8); 8.53 (br s, 1H, OH); 13C NMR (acetone-d6):
130.3 (s, C); 135.2 (s, C); 138.3 (d, C, JC–F = 3.0 Hz); 138.8 (s, C);
4
1
119.4 (s, CH); 121.0 (s, CH); 122.7 (s, CH); 124.2 (q, CH,
140.9 (s, C); 163.1 (d, C, JC–F = 244.1 Hz); MS (ei): m/z (%) = 254
3
3JC–F = 3.7 Hz); 124.3 (q, CH, JC–F = 3.7 Hz); 124.7 (s, CH); 125.4
([M+]. 32); 224 (100); 196 (36); Elemental Anal. Calcd C, 75.58; H,
4.36; F, 7.47. Found: C, 75.76; H, 4.30; F, 7.58.
1
(q, CF3, JC–F = 271.6 Hz); 126.0 (s, C); 126.8 (s, CH); 130.3 (s, C);
2
130.6 (s, CH); 131.5 (s, CH); 131.5 (q, C, JC–F = 31.7 Hz); 134.6 (s,
C); 138.8 (s, C); 141.3 (s, C); 143.0 (s, C); MS (MALDI-TOF): m/z
(%) = 305 (33); 304 ([M+]. 100); 303 (19); Elemental Anal. Calcd
C, 67.11; H, 3.64; F, 18.73. Found: C, 67.32; H, 3.71; F, 18.54.
4.5. ESR spectroscopy
The CW X-band ESR measurement were carried out on a Bruker
Elexys 580_FT spectrometer at room temperature. The using
parameters are 6.34 microwave power and 10 G amplitude modu-
lation. The sample powder was analyzed in a quartz tube inserted
in a standard cavity.
4.4.19. 6-(4-Trifluoromethylphenyl)naphthalene-1,2-diol 1k
Green powder; yield = 94%; mp = 114–117 °C (dec); 1H NMR
3
(acetone-d6): 7.28 (d, 1H, JH3–H4 = 8.7 Hz, H3); 7.45 (d, 1H,
3
4
3JH4–H3 = 8.7 Hz, H4); 7.73 (dd, 1H, JH7–H8 = 8.8 Hz, JH7–H5
3
0
0
0
0
0
0
0
0
= 1.95 Hz, H7); 7.80 (d, 2H, JH3 .5 –H2 .6 = 8.8 Hz, H3 .5 or H2 .6 );
4.6. Integrase inhibition
3
0
0
0
0
0
0
7.94 (br s, 1H, OH); 7.97 (d, 2H, JH3 .5 –H2 .6 = 8.8 Hz, H3 .5 or
4
0
0
H2 .6 ); 8.12 (d, 1H, JH5–H7 = 1.95 Hz, H5); 8.26 (d, 1H,
3JH8–H7 = 8.8 Hz, H8); 8.54 (br s, 1H, OH); 13C NMR (acetone-d6):
119.3 (s, CH); 121.0 (s, CH); 122.7 (s, CH); 124.7 (s, CH); 125.5
To determine the susceptibility of the HIV-1 integrase enzyme
to different compounds, we used an enzyme-linked immunosor-
bent assay. This assay uses an oligonucleotide substrate in which
one oligo (50-ACTGCTAGAGATTTTCCACACTGACTAAAAGGGTC-30)
is labeled with biotin on the 30 end and in which the other oligo
is labeled with digoxigenin at the 50 end. For the overall
integration assay, the second 50-digoxigenin-labeled oligo is
1
3
(q, CF3, JC–F = 271.0 Hz); 126.0 (s, C); 126.5 (q, CH, JC–F = 3.7 Hz);
2
126.9 (s, CH); 128.2 (s, CH); 129.1 (q, C, JC–F = 32.3 Hz); 130.2 (s,
C); 134.6 (s, C); 138.7 (s, C); 141.2 (s, C); 145.8 (s, C); MS (MAL-
DI-TOF): m/z (%) = 305 (50); 304 ([M+], 100); 303 (51); Elemental

C. Maurin et al. / Bioorg. Med. Chem. 18 (2010) 5194–5201
5201
50-GACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGT-30. The integr-
ase was diluted in 750 mM NaCl, 10 mM Tris (pH 7.6), 10% glyc-
erol, 1 mM b-mercaptoethanol, and 0.1 mg/mL bovine serum
HIV strains, which is defined as the EC50, was determined. In par-
allel, the 50% cytotoxic concentration (CC50) was determined.
albumin. To perform the reaction, 4
sponds to a concentration of WT integrase17 of 1.6
of annealed oligos (7 nM) were added in a final reaction volume
of 40 L containing 10 mM MgCl2, 5 mM DTT, 20 mM HEPES
(pH 7.5), 5% PEG, and 15% DMSO. The reaction was carried out
for 1 h at 37 °C. These reactions were followed by an immunosor-
bent assay on avidin-coated plates.18 30-Processing and strand
transfer assays were performed according to previously reported
methods.19
l
L of diluted integrase (corre-
Acknowledgments
lM) and 4
lL
This work was financially supported by Grants from la Région
Nord Pas-de-Calais, le Centre National de la Recherche Scientifique
(CNRS), l0Agence Nationale de la Recherche contre le Sida (ANRS),
and the European Commission (LSHB-CT-2003-503480). The Euro-
pean TRIoH Consortium is gratefully acknowledged. The Mass
Spectrometry facility used in this study was funded by the Euro-
pean Community (FEDER), the Région Nord-Pas de Calais (France),
the CNRS, and the Université des Sciences et Technologies de Lille.
We are grateful to Martine Michiels, Nam Joo Vanderveken, and
Barbara Van Remoortel for excellent technical assistance.
l
4.7. In vitro anti-HIV and drug susceptibility assays
4.7.1. Hela-CD4+-ßGal (P4) cells
References and notes
The lymphocytes cell line CEM4fx was maintained in RPMI-
1640 medium supplemented with 10% fetal calf serum. Hela-
CD4+-ßGal (P4) cells were grown in DMEM with 10% fetal calf ser-
um and 0.5 mg/mL geneticin. Cell-free viral supernatants were ob-
tained by trans-fection of P4 cells with HIV-1 PLN4-3 genomic
clone.20 CEM4fx cells were plated in triplicate on a 96-well plate
1. De Clercq, E. Int. J. Antimicrob. Agents 2009, 33, 307.
2. Low, A.; Mohri, H.; Markowitz, M. 14th Conference on Retroviruses and
Opportunistic Infections Feb 25–28, 2007. Los Angeles, CA.
3. Cooper, D. A.; Steigbigel, R. T.; Gatell, J. M.; Rockstroh, J. K.; Katlama, C.; Yeni, P.;
Lazzarin, A.; Clotet, B.; Kumar, P. N.; Eron, J. E.; Schechter, M.; Markowitz, M.;
Loutfy, M. R.; Lennox, J. L.; Zhao, J.; Chen, J.; Ryan, D. M.; Rhodes, R. R.; Killar, J.
A.; Gilde, L. R.; Strohmaier, K. M.; Meibohm, A. R.; Miller, M. D.; Hazuda, D. J.;
Nesly, M. L.; DiNubile, M. J.; Isaacs, R. D.; Tippler, H.; Nguyen, B. Y. N. Eng. J. Med.
2008, 359, 355.
4. Malet, I.; Delelis, O.; Valantin, M. A.; Montes, B.; Soulie, C.; Wirden, M.;
Tchertanov, L.; Peytavin, G.; Reynes, J.; Mouscadet, J. F.; Katlama, C.; Calvez, V.;
Marcelin, A. G. Antimicrob. Agents Chemother. 2008, 52, 1351.
5. Goethals, O.; Clayton, R.; Van Ginderen, M.; Vereycken, I.; Wagemans, E.;
Geluykens, P.; Dockx, K.; Strijbos, R.; Smits, V.; Vos, A.; Meersseman, G.;
Jochmans, D.; Vermeire, K.; Schols, D.; Hallenberger, S.; Hertogs, K. J. Virol.
2008, 82, 10366.
6. Kobayashi, M.; Nakahara, K.; Seki, T.; Miki, S.; Kawauchi, S.; Suyuma, A.;
Wakasa-Morimoto, C.; Kodama, M.; Endoh, T.; Oosugi, E.; Matsushita, Y.;
Murai, H.; Fujishita, T.; Yoshinaga, T.; Garvey, E.; Foster, S.; Underwood, M.;
Johns, B.; Sato, A.; Fujiwara, T. Antiviral Res. 2008, 80, 213.
7. Serrao, E.; Odde, S.; Ramkumar, K.; Neamati, N. Retrovirology 2009, 6, 25.
8. Cowan, J. A. The Biological Chemistry of Magnesium; VCH: New York, 1995. pp 1–
23.
9. (a) Maurin, C.; Bailly, F.; Cotelle, P. Curr. Med. Chem. 2003, 10, 1795; (b)
Ramkumar, K.; Serrao, E.; Odde, S.; Neamati, N. Med. Res. Rev. 2010
doi:10.1002/med.20194.
10. Dupont, R.; Jeanson, L.; Mouscadet, J.-F.; Cotelle, P. Bioorg. Med. Chem. Lett.
2001, 11, 3175.
11. Maurin, C.; Bailly, F.; Cotelle, P. Tetrahedron 2005, 61, 7054.
12. Burke, T. R., Jr.; Lim, B.; Marquez, V. E.; Li, Z. H.; Bolen, J. B.; Stefanova, I.; Horak,
I. D. J. Med. Chem. 1993, 36, 425.
13. Nakashima, H.; Murakami, T.; Yamamoto, N.; Naoe, T.; Kawazoe, Y.; Konno, K.;
Sakagami, H. Chem. Pharm. Bull. 1992, 40, 2102.
(100 lL) and infected with cell-free virus. Viral supernatants were
removed 2 h after infection and drugs dissolved in DMSO were
added in fresh medium. Infected cells were grown in the presence
of drugs for 3 days. Supernatants were then collected at t = 72 h
and used to infect P4 cells. P4 cultures were incubated for 24 h
and subsequently lyzed in a phosphate buffer containing 50 mM
2-mercaptoethanol, 10 mM MgSO4, 25 mM EDTA, 0.125% NP40.
20 lL of lyzate was incubated with 100 lL of CPRG-containing buf-
fer. The red staining intensity was quantified on a multiscan pho-
tometer at 570 nm. CEM4fx cell viability was estimated by the
MTT (Sigma) assay after 3 days treatment with drugs (20 lL). A
solution of (7.5 mg/mL) in phosphate buffer was added to each
well of microtiter trays. Plates were further incubated at 37 °C in
a CO2 incubator for 4 h. Solubilization of formazan crystals was
achieved by adding 100 mL of 10% SDS, 10 mM HCI. Absorbance
was read in a multiscan photometer at 570 nm. Experiments were
performed in triplicate and averaged.
4.7.2. Lymphocyte MT-4 cell
The inhibitory effect of antiviral drugs on the HIV-1-induced
cytopathic effect (CPE) in human lymphocyte MT-4 cell culture
was determined by the MT-4/MTT assay.21 This assay is based on
the reduction of the yellow colored 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) by mitochondrial dehy-
drogenase of metabolically active cells to a blue formazan deriva-
tive, which can be measured spectrophotometrically. The 50%
cell culture infective dose (CCID50) of the HIV-1 (IIIB) strain was
determined by titration of the virus stock using MT-4 cells. For
the drug susceptibility assays, MT-4 cells were infected with
100ꢀ300 CCID50 of the virus stock in the presence of fivefold serial
dilutions of the antiviral drugs. The concentration of various com-
pounds achieving 50% protection against the CPE of the different
14. Cotelle, P.; Vezin, H. Res. Chem. Intermed. 2003, 29, 365.
15. Pluymers, W.; Neamati, N.; Pannecouque, C.; Fikkert, V.; Marchand, C.; Burke,
T. R.; Pommier, Y.; Shols, D.; De Clercq, E.; Debyser, Z.; Witvrouw, M. Mol.
Pharmacol. 2000, 58, 641.
16. Cos, P.; Maes, L.; Vlietinck, A.; Pieters, L. Planta Med. 2008, 74, 1323.
17. Debyser, Z.; Cherepanov, P.; Pluymers, W.; De Clercq, E. Methods Mol. Biol.
2001, 160, 139.
18. Hwang, Y.; Rhodes, D.; Bushman, F. Nucleic Acids Res. 2000, 28, 4884.
19. Busschots, K.; Voet, A.; De Mayer, M.; Rain, J. C.; Emiliani, S.; Benarous, R.;
Desender, L.; Debyser, Z.; Christ, F. J. Mol. Biol. 2007, 365, 1480.
20. Jeanson, L.; Subra, F.; Vaganay, S.; Hervy, M.; Marangoni, E.; Bourhis, J.;
Mouscadet, J. F. Virology 2002, 300, 100.
21. Pauwels, R.; Balzarini, J.; Baba, M.; Snoeck, R.; Schols, D.; Herdewijn, P.;
Desmyter, J.; De Clercq, E. J. Virol. Methods 1988, 20, 309.