Regiocontrolled synthesis and HIV inhibitory activity of unsymmetrical binaphthoquinone and trimeric naphthoquinone derivatives of conocurvone

Article abstract of DOI:10.1016/j.bmc.2006.04.034

Unsymmetrical biquinone and trimeric quinone derivatives were synthesized using halotriflate-biselectrophilic naphthoquinones through stepwise regioselective quinone substitution chemistry and evaluated for their ability to inhibit the cytopathogenic effects of HIV-1 using an MTT colorimetric assay. Compounds were also screened for their ability to inhibit the activity of HIV-1 integrase in vitro. Pyranylated trimeric quinones and biquinones exhibited both antiviral activity and integrase inhibitory activity. Conocurvone 1 and trimeric quinone 21 were the most potent HIV integrase inhibitors in the series. All of the biquinones showed HIV inhibitory activity. Simple methoxy substituted biquinones did not inhibit HIV-1 integrase.

Full text of DOI:10.1016/j.bmc.2006.04.034

Bioorganic & Medicinal Chemistry 14 (2006) 5651–5665
Regiocontrolled synthesis and HIV inhibitory activity
of unsymmetrical binaphthoquinone and trimeric naphthoquinone
derivatives of conocurvone
Kenneth W. Stagliano,a Ashkan Emadi,a Zhenhai Lu,a Helena C. Malinakova,a
Barry Twenter,a Min Yu,a Louis E. Holland,b Amanda M. Rom,b
John S. Harwood,c Ronak Amin,d Allison A. Johnsond and Yves Pommierd,*
aDepartment of Biological, Chemical and Physical Sciences, Illinois Institute of Technology, Chicago, IL 60616, USA
bLife Sciences Operation, IIT Research Institute, Chicago, IL 60616, USA
cDepartment of Chemistry, University of Illinois at Chicago, Chicago, IL 60607, USA
dLaboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute,
National Institutes of Health, Bethesda, MD 20892-4255, USA
Received 24 February 2006; revised 10 April 2006; accepted 13 April 2006
Available online 5 June 2006
Abstract—Unsymmetrical biquinone and trimeric quinone derivatives were synthesized using halotriflate-biselectrophilic naphtho-
quinones through stepwise regioselective quinone substitution chemistry and evaluated for their ability to inhibit the cytopathogenic
effects of HIV-1 using an MTT colorimetric assay. Compounds were also screened for their ability to inhibit the activity of HIV-1
integrase in vitro. Pyranylated trimeric quinones and biquinones exhibited both antiviral activity and integrase inhibitory activity.
Conocurvone 1 and trimeric quinone 21 were the most potent HIV integrase inhibitors in the series. All of the biquinones showed
HIV inhibitory activity. Simple methoxy substituted biquinones did not inhibit HIV-1 integrase.
Published by Elsevier Ltd.
1. Introduction
was suggested that conocurvone 1 may be a dual inhibitor
of both HIV integrase and HIV mediated cell fusion.5,6
Biquinones and higher quinone oligomers are a unique
group of natural products, which possess a diverse array
of biological activities.1 Their structures are based on
two or more quinone units linked together at the quinone
double bond. In almost all cases they possess an element
of symmetry due to their biosynthetic mechanism of
origin, which probably involves oxidative coupling of a
common naphthol intermediate in the key step of the
oligomerization process.2 One intriguing member of
this class is conocurvone 1 isolated from the Western
Australian smoke bush.3 Conocurvone was shown to
inhibit the cytopathogenic effects of HIV-1 in human
T-lymphoblastic cells over a broad concentration range
(ID50 = 0.02 lM; TD50 = 50 lM).3,4 More recently, it
Over the past decade, extensive efforts have been made
resulting in the discovery of a large number of molecules
that can inhibit replication of HIV.7 An essential step in
the HIV life cycle is integration of the viral DNA into
Keywords: Biquinones and trimeric quinones; HIV-1 integrase inhib-
itor; Regiocontrolled synthesis; Conocurvone.
*
Corresponding author. Tel.: +1 301 496 5944; fax: +1 301 402
0752; e-mail: pommier@nih.gov
0968-0896/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmc.2006.04.034

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K. W. Stagliano et al. / Bioorg. Med. Chem. 14 (2006) 5651–5665
the host cell genome. The step is catalyzed by the viral
enzyme, HIV integrase, which is absolutely required
for productive infection8 and therefore, inhibition of
integrase can halt the viral life cycle (for a recent review,
see Ref. 9b).9,10 Integrase catalyzes two separate steps
known as 30-processing (30-P) and DNA strand transfer
(ST). In 30-processing, integrase removes a dinucleotide
next to a conserved cytosine-adenine sequence from
each 30-end of the viral DNA.9,10 Integrase then attaches
the processed 30-end of the viral DNA to the host cell
DNA in the strand transfer reaction.9–12
of the haloquinone unit was achieved by regioselective
substitution of the triflate group in chlorohydroxyqui-
none triflates 4e and 4h (Scheme 1).
The quinone triflates were prepared starting from read-
ily available quinone 1,4-dipoles, Scheme 1.18 Reaction
with either trimethyl silyl halides or the corresponding
hydrohalic acids yielded halohydroxynaphthoquinones
3 in good yields. The chlorohydroxy- and bromohydr-
oxyquinones are most efficiently prepared by reaction
of the dipoles 2 with excess HCl or HBr as previously
reported.18 However, reaction of the dipole 2 with excess
HI yielded a mixture of 2-hydroxy-3-iodonaphthoqui-
none 3d and the corresponding 2-hydroxynaphthoqui-
none 3a, which was difficult to separate.18 Mechanistic
studies revealed that HI was functioning as a quinone
dehalogenating agent.19 For example, reaction of the
dipole 2a with equimolar amounts of HI yielded 3d as
a virtually pure product. Furthermore, when the
iodohydroxyquinone 3d was stirred with excess HI in
methylene chloride at room temperature a quantitative
yield of 2-hydroxynaphthoquinone was obtained. Simi-
lar dehalogenations were observed during reaction of
3b or 3c with excess HI.
An important result of the structural and biochemical
studies on integrase has been the development of practi-
cal assays used to identify novel HIV integrase inhibi-
tors.9,13 These HIV inhibitors not only represent
potential chemotherapeutic lead compounds,7 but as a
collection, they are also useful in databases for pharma-
cophore searching.14 The most promising inhibitors are
proposed to bind to the active site of the integrase
enzyme and chelate important metal co-factors such as
9,10
Mn2+or Mg2+
.
The ability of hydroxyquinones to bind metal cations15
and the inhibition of HIV replication by conocurvone3,4
prompted us to explore whether conocurvone 1 inhibits
HIV-1 integrase in vitro. A series of synthetic trimeric
quinone and biquinone analogs were also tested for
integrase inhibition. In addition, the compounds were
evaluated for their ability to inhibit the cytopathogenic
effects of HIV-1 using an MTT colorimetric assay.16
Initial screening of reaction conditions for the triflation
of hydroxyquinones was first carried out using lawsone
3a as a model. Reaction of 3a with triflic anhydride pro-
vided the known20 para-hydroxynaphthoquinone triflate
4a in 44% yield along with a brilliant red side product
which readily decomposed in solution but whose spec-
tral characteristics were consistent with that of the
ortho-quinone isomer. Triflate 4a also proved to be
unstable requiring storage at ꢀ20 ꢀC under nitrogen.
In contrast, reaction of halohydroxyquinones 3b–i with
1.2 equiv of triflic anhydride provided only a single reg-
ioisomer. Apparently the presence of the halogen atom
on the quinone core exerted a strong regiochemical
directing effect on the triflation reaction.21 The robust
triflates crystallized as brilliant yellow needles and were
air stable at room temperature.
2. Results and discussion
2.1. Chemistry
Trimeric quinones 21–23 (Table 1) were prepared from
the corresponding methoxychlorobiquinones as previ-
ously described.17 Pyranylated biquinones 11 and 19
(Table 2) and halohydroxybiquinones (15a–d and 6b,
Table 3) which lack substituents on the haloquinone
unit were prepared in one step by substitution of a hal-
ogen in 2,3-dichloro- or 2,3-dibromonaphthoquinone by
a hydroxyquinone anion.17 Synthesis of biquinones
(6e,6h, Table 3) with substituents on the aromatic ring
With the triflates in hand, our next objective was to
determine if the triflate group would substitute preferen-
tially in the presence of different types of halogens. In
addition, the regiochemical effect of electron donating
Series 3
Series 1
Series 2
O
O
O
OTf (85)
Cl (15)
OTf (90)
MeO
MeO
OTf (70)
Cl (30)
MeO
MeO
Cl (10)
O
O
4e
4g
O
O
4b
4h
4i
O
O
OTf (100)
I (0)
OTf (95)
I (5)
OTf (90)
I (10)
O
O
O
4d
Figure 1. Regioisomeric ratios of triflate versus halogen substitution during biquinone formation as determined by HPLC.

Table 1. Anti-HIV activity of trimeric naphthoquinone derivatives
O
O
X
O
O
O
O
R3
R2
Y
R1
Compound
X
Y
R1
R2
R3
Integrase inhibitionb MnCl2
Integrase inhibitionb MgCl2
30-Processing (lM)
Strand transfer (lM)
MTT assaya
30-Processing (lM)
Strand transfer (lM)
ID50 (lM)
TD50 (lM)
214,17
22d
23d
OH
OH
OH
OMe
OH
H
H
H
H
H
H
H
22.9 0.2
63.9 10.9
59.1 3.5
8.9 4.3
55.9 12.2
58.3
35.5 9.2
49.5 4.9
45.0 7.1
29.5 3.5
38.0 7.1
41.0 5.7
11 2c
43 3b
NAb
28 1c
>100b
19 1b
H
OMe
OMe
14
OH
OMe
H
72.2 7.4
>1000e
72.2 7.4
>1000e
37.7 4.1
1.3 0.4
24.3 3.1
1.4 0.4
6.7 1.2c
14 2c
22 1c
1
Conocurvone
0.06 0.00c
a MTT assays were conducted as described in Ref. 16.
b Values are expressed as means SEM of three independent assays.
c Values are expressed as means SEM of two independent assays.
d See Ref. 17.
e Conocurvone failed to inhibit integrase in the presence of Mn2+ for three out of four integrase preparations. The inhibition observed for the fourth preparation was 10-fold weaker than inhibition in the
presence of Mg2+. NA, not active.

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K. W. Stagliano et al. / Bioorg. Med. Chem. 14 (2006) 5651–5665
Table 2. Anti-HIV activity of pyranylated binaphthoquinone derivatives
R
O
CH3
X
O
O
O
O
Y
R1 = -CH2CH2CH=C(CH3)2
R2 = -CH3
Compound
X
Y
R
Integrase inhibitionb MnCl2
Integrase inhibitionb MgCl2
MTT assaya
30-Processing (lM) Strand transfer (lM) 30-Processing (lM) Strand transfer (lM) ID50 (lM) TD50 (lM)
19d
8b
OH Cl R1 39.1 8.2
20.7 1.3
44.3 3.3
23 8.5
29.5 3.5
43.5 7.8
30.9 3.5
49.5 8.0
20.5 0.7
29.5 0.7
20.9 3.5
36.3 9.6
0.4 0.1b 2.7 0.1b
1.7 0.3c 4.3 0.8c
2.0 0.3c 6.8 0.6c
2.8 0.4c 5.5 0.1c
OH
OH
I
I
R1 60.2 0.2
R2 22.6 5.1
8a
11
OH Cl R2 75.7 26.8
41.9 6.7
20d
9
OMe Cl R1 >1000
>1000
>1000
>1000
>1000
>1000
>1000
2.3 0.5b 5.4 0.2c
2.7 0.3c 6.3 0.3c
1.8 0.9c 3.5 1.9c
OMe
I
OMe Cl R2
R2 >1000
e
e
e
e
12
a MTT assays were conducted as described in Ref. 16.
b Values are expressed as means SEM of three independent assays.
c Values are expressed as means SEM of three two independent assays.
d See Ref. 17.
e Not tested.
methoxy groups present on the aromatic ring of the
naphthoquinone was also probed. Reaction of the
unsubstituted chlorotriflate 4b with the hydroxyquinone
anion 5 yielded biquinone 6b as the major product
(Scheme 1). Similar results were obtained with the iodo-
triflate 4d to yield 6d. In both cases the triflate group
underwent regioselective substitution by the hydroxy-
quinone anion. The approximate regioisomeric ratios
(shown in parentheses, Fig. 1) revealed that the nature
of the halogen influenced the extent of triflate substitu-
tion. For example, when X = I slightly higher regioselec-
tivity was observed compared to when X = Cl.
gel.22 Repeated attempts to isolate and characterize the
minor regioisomer failed due to facile decomposition.
Pyranylated iodohydroxybiquinones 8 were prepared
using the procedure described above with regioisomeric
ratios (shown in brackets, Scheme 2). The hydroxybiqui-
nones were methylated with trimethyl oxonium tetra-
fluoroborate providing biquinone methyl ethers 16a–e
(Table 3)17 and 9, 12, and 20 (Table 2). The hydroxybiq-
uinones were also converted to dichlorobiquinones
(17a–e) on treatment with oxalyl chloride.23 Regioselec-
tive amination of dichlorobiquinones yielded amino-
chlorobiquinones 18a–c and diaminobiquinone 18d.23
The unreported pyranylated trimeric quinone 14 (Table
3) was prepared as depicted in Scheme 3 by stepwise
substitution of the halogens in dichlorobiquinone 10.
The effect of methoxy group position on regioselectivity
was then studied (series 2 and 3, Fig. 1). Reaction of the
methoxy substituted chlorotriflate 4h and iodotriflate 4i
with hydroxyquinone 3a yielded biquinones 6h and 6i as
major products. In both cases the reactions proceeded
by selective substitution of the triflate group albeit with
decreased regioselectivity compared to the correspond-
ing unsubstituted derivatives 4b and 4d. Interestingly,
the extent of triflate substitution increased in reactions
involving 4e and 4g demonstrating that the position of
the methoxy group does play an important role in con-
trolling regiochemistry. The highest regioselectivity was
achieved with iodotriflate 4g substituted with electron
donating methoxy groups at positions where their
unpaired electrons could not participate in p-donation
to the carbonyl group involved in the conjugate addition
step during triflate substitution. All of the biquinones
6b–i were easily purified from the minor regioisomer
by flash chromatography on oxalic acid coated silica
2.2. Inhibition of HIV-1 integrase catalytic activities
The concentration dependent inhibition of HIV-1
integrase by the compounds presented was tested in an
in vitro assay, as illustrated in Figure 2. IC50 values were
determined for both 30-processing and strand transfer in
the presence of manganese or magnesium cations. All of
the trimeric naphthoquinone derivatives shown in Table
1 inhibited integrase 30-P and ST in the presence of
either manganese or magnesium cations. Conocurvone
1, the most potent derivative, was inhibitory against in-
tegrase only in the presence of magnesium (Figs. 2B–D).
The best synthetic inhibitor of this study was compound
21,4 with an IC50 against integrase ST of 8.9 lM (Figs.
2E and F). Inhibition was retained with hydroxy and

K. W. Stagliano et al. / Bioorg. Med. Chem. 14 (2006) 5651–5665
Table 3. Anti-HIV activity of simple binaphthoquinone derivatives
5655
O
R1
X
R2
R3
O
R8
R7
R6
O
R4
Y
R5
O
Compound
X
Y
R1
R2
R3
R4
R5
R6
R7
R8
MTT Assaya
ID50 (lM) TD50 (lM)
15ae
15be
6a
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OMe
OMe
OMe
OMe
OMe
Cl
Cl
Br
I
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
2.6 0.3
2.3 0.2
1.8 0.5
2.4 0.4
2.7 0.2
NAc
5.3 0.2
5.2 0.4
5.0 0.6
5.3 0.2
5.5 0.1
H
H
H
H
15ce
6b
Cl
Cl
Cl
Cl
Cl
Br
I
OMe
H
H
H
OMe
H
H
15df
6eg
6hg
15ed,e
6d
H
OMe
H
H
>100c
H
H
OMe
H
H
5.3c
12.0c
10.0c
H
H
H
OMe
H
6.2c
H
H
H
OMe
H
2.0 0.1
3.7c
3.6c
4.2c
1.7 0.1b
1.8 0.2b
1.2 0.1b
1.6 0.2b
1.1c
5.7 0.2
6.0c
7.5c
9.8c
H
OMe
H
H
H
6g
I
H
H
OMe
H
H
6i
I
H
H
H
OMe
H
16ae
16be
16ce
16de
16ed,e
17ae
17bf
17cf
17df
17ef
18af
18bf,g
18cf
18df,g
Cl
Br
Cl
Cl
Br
Cl
Cl
Cl
Cl
Cl
NH2
NH2
NH2
NH2
H
H
H
H
2.8 0.0
5.4 0.3b
2.9 0.1
2.5 0.2
2.8c
H
H
H
H
H
OMe
H
H
H
H
H
OMe
H
H
H
H
H
H
OMe
H
H
H
H
H
H
3.3 0.0
7.1 0.3b
3.4 0.2
6.6c
5.4 0.2b
12.0 0.6b
5.7 0.0
11.0c
Cl
Cl
OMe
H
H
H
H
H
OMe
H
H
H
H
Cl
Cl
H
OMe
OH
H
H
H
H
H
H
H
NAc
5.7c
Cl
Cl
H
H
H
H
3.5 0.3b
2.2c
5.7 0.0b
3.6c
OMe
H
H
H
H
H
Cl
NH2
H
OMe
H
H
H
2.4c
0.7 0.0
5.7c
7.9 1.7
H
H
H
H
a MTT assays were conducted as described in Ref. 16. Values are expressed as the mean SEM of three independent assays.
b Two assays performed.
c One assay performed.
d 95:5 mixture of regioisomers.
e See Ref. 17.
f See Ref. 23. All of these compounds failed to inhibit HIV-1 integrase in vitro.
g Four compounds were not tested for integrase inhibition. NA, not active.
methoxy-substitutions at X and Y (compounds 22, 23,
and 14, Table 1). The effect on integrase inhibition
when both X and Y are methoxy groups is unknown.
Pyranylated biquinones inhibited HIV integrase (Table
2), in contrast to the lack of inhibition observed with
simple biquinone derivatives (Table 3). The biquinones
with X = OH (Table 2, compounds 19, 8b,8a, and 11)
moderately inhibited integrase 30-P and ST, with IC50
values between 20 and 75 lM. Replacement of X =
OH with a methoxy group resulted in a loss of inhibition
(compounds 20 and 9). The halogen (Y) and the size of
the R substituents had little effect on integrase inhibition.
over a broad concentration range in our assay. How-
ever, the simplified trimeric quinone 21,4 which lacked
the pyran rings was less active and displayed increased
cytotoxicity compared to conocurvone 1. The monom-
ethyl ether 22 was less active than the dihydroxy deriv-
ative 21.4 Incorporation of a methoxy group on the
aromatic ring in 23 resulted in complete loss of in vitro
anti-HIV activity as determined through repeated anti-
viral assays. Comparison of the activity of 21 and 22
to conocurvone 1 revealed significantly decreased
activity. Fusion of a pyran ring to 22 yielded trimeric
quinone 14 with only a slight increase in anti-HIV
activity.
2.3. HIV inhibitory assay
Comparison of the ID50 and TD50 values of hydroxy-
biquinones (Table 3) revealed no significant difference
between the methoxy group substituted quinones
6b,d,e,g–i and 15c,e, unsubstituted derivatives 15a,b
All of the compounds listed in Tables 1–3 were evalu-
ated for in vitro anti-HIV activity in the CEM-T4 cyto-
protection assay.16 Conocurvone 1 displayed antiviral

5656
K. W. Stagliano et al. / Bioorg. Med. Chem. 14 (2006) 5651–5665
O
O
TMSX (or HX)
CH2Cl2, rt
R2
R1
O
I
R2
R1
OH
X
O
O
3a
3b
2a
2b
2c
R1 = R2 = X = H
R1 = R2 = H (88%)
R1 = R2 = H; X = Cl (76%)
R1 = R2 = H; X = Br (75%)
R1 = R2 = H; X = I (76%)
R1 = OMe; R2 = H (95%)
3c
R1 = H; R2 = OMe (72%)
3d
3e
3f
3g
3h
3i
R1 = OMe; R2 = H; X = Cl (73%)
R1 = OMe; R2 = H; X = Br (78%)
R1 = OMe; R2 = H; X = I (79%)
R1 = H; R2 = OMe; X = Cl (93%)
R1 = H; R2 = OMe; X = I (73%)
O
O
R2
R1
O
X
S
CF3
(CF3SO2)2O / Et3N
CH2Cl2 / 0˚C / 30 min
O
O
4a R1 = R2 = X = H (44%)
4b R1 = R2 = H; X = Cl (85%)
4c R1 = R2 = H; X = Br (85%)
4d R1 = R2 = H; X = I (77%)
4e R1 = OMe; R2 = H; X = Cl (77%)
4f R1 = OMe; R2 = H; X = Br (71%)
4g R1 = OMe; R2 = H; X = I (76%)
4h R1 = H; R2 = OMe; X = Cl (84%)
O
O
HO
4i R1 = H; R2 = OMe; X = I (78%)
O
R3
5
HO
O
Cs2CO3, CH3CN
rt, 4-6 days
R2
R1
R3
O
X
+
regioisomer
R1 = R2 = R3 = H; X = I (72%)
6b R1 = R2 = H; R3 = OMe; X = Cl (56%)
O
6a
R = R = H; R = OMe; X = I (67%)
R2 = R3 = H; R1 = OMe; X = Cl (67%)
R2 = R3 = H; R1 = OMe; X = I (83%)
6d
6e
6g
1
2
3
6h R1 = R3 = H; R2 = OMe; X = Cl (47%)
6i
R1 = R3 = H; R2 = OMe; X = I (60%)
Scheme 1. Regiocontrolled synthesis of unsymmetrical binaphthoquinones.
and 6a, biquinone methyl ethers 16a–e, or dichlorobiq-
uinones 17a–d. Biquinones 15d and 17e which possessed
a peri-methoxy or peri-hydroxy group on one of the qui-
none units lacked activity but possessed similar cytotox-
icity to the other compounds. The diaminobiquinone
18d was one of the most potent derivatives in the simple
biquinone series. The presence of a fused pyran ring on
the aromatic ring of biquinone 19 produced slightly
improved anti-HIV activity (ID50 = 0.4 0.1 lM) by
comparison to the unsubstituted derivative 15a (ID50
=
2.6 0.3 lM, Tables 2 and 3). However, the incorpora-
tion of the pyran ring had little effect on the toxicity. The
length of the aliphatic chain on the pyran ring in 19 (vs
11), the nature of the halogen atom in 8a and 11 and
formation of the biquinone methyl ethers 9, 12, and 20
(Table 2) had little influence.

K. W. Stagliano et al. / Bioorg. Med. Chem. 14 (2006) 5651–5665
5657
R
O
R
CH3
HO
O
O
O
O
CH3
HO
O
Cs2CO3, CH3CN
4d, rt, 7 days
O
I
O
8a
7a
7b
R = -CH3 (60%) [~90:10]
R = -CH3
R = -CH2CH2CH=C(CH3)2
8b
R = -CH CH CH=C(CH ) (51%) [~90:10]
2
2
3 2
CH3
CH3
O
CH3O
O
O
(CH3)3OBF4 / CH2Cl2
[(CH3)2CH]2NC2H5 / rt
93%
O
I
9
O
Scheme 2. Regiocontrolled synthesis of pyranylated iodobinaphthoquinones.
3. Conclusions
integrase even when a hydroxy (X = OH) group was
present. Addition of the pyranyl group resulted in
integrase inhibition. Furthermore, comparison of
compounds 19 and 8a–20 and 9 (Table 2) shows that
the hydroxy (Y = OH) group is required for integrase
inhibition. Therefore, our current hypothesis is that
the pyranyl group is required to correctly position the
hydroxy group for integrase inhibition. It is not known
which portion(s) of the pyranyl structure is responsible
for positioning, but the nature of the aliphatic chain
appears to have no effect. Our studies with the biquinon-
es and trimeric quinones indicate that the natural
derivative conocurvone 1 is the most effective anti-inte-
grase and anti-HIV drug, and that a complex substruc-
ture is required for potent anti-HIV activity. It is
possible that conocurvone 1 and related derivatives
modulate more than one physiologic pathway in HIV,
as suggested earlier.5 Further studies are in progress to
elucidate the roles of the hydroxy and pyranyl groups
of conocurvone derivatives as well as determine their
in vivo mechanism of action.
In conclusion, a series of unsymmetrical biquinone and
trimeric quinone derivatives has been synthesized and
evaluated for their ability to inhibit HIV-1 integrase
and the cytopathogenic effects of HIV in cells. We have
demonstrated that the triflate group undergoes regiose-
lective substitution in halohydroxyquinone triflates to
form unsymmetrical halohydroxybiquinones. The posi-
tion of the methoxy group and the nature of the halogen
atom influenced the regioselectivity. Conocurvone 1
displays potent HIV-inhibitory activity, while exhibiting
low cytotoxicity in non-infected cells. Conocurvone
also inhibits the HIV-1 integrase 30-P and ST reactions
in vitro with greater selectivity for the ST reactions.
Another interesting characteristic of conocurvone 1 is
its selectivity for integrase inhibition in the presence of
magnesium. Prior studies suggested that the ability of
a compound to inhibit integrase in the presence of mag-
nesium correlates with antiviral activity (e.g., the diketo
acids).9,24 Hence integrase inhibition in the presence of
magnesium, which is the case for conocurvone 1, is a po-
sitive attribute of drugs with potential for therapeutic
development.9,24
4. Experimental
4.1. Materials
All of the synthetic trimeric quinones (Table 1) pos-
sessed HIV and integrase inhibitory activity but were
less potent than conocurvone 1. The simplest triquinone,
compound 21, retained anti-HIV and anti-integrase
activities despite the lack of the pyran ring and pyranyl
aliphatic chain present in conocurvone. While the full
conocurvone structure is required for potent inhibition
of HIV, the simple structure of compound 21 is sufficient
for integrase inhibition.
Anhydrous solvents and reagents were purchased from
Aldrich and used as received. All NMR spectra were
recorded on
a
Bruker AvanceTM 500 MHz (or
300 MHz) NMR spectrometer in CDCl3 with CHCl3
as the internal reference. IR spectra were recorded on
a Nicolet Nexus 470 FT-IR in KBr pellets or neat as thin
films on NaCl plates. MS were measured under fast
atom bombardment (FAB) or electron impact (EI) con-
ditions at the University of Notre Dame Mass Spec-
trometry Facility. Melting points were taken in open
capillary tubes and are uncorrected. Analytic thin-layer
chromatography (TLC) was performed on commercial
In contrast to the trimeric quinones, the biquinones (Ta-
bles 2 and 3) require both a hydroxy (=X) and pyranyl
group for anti-integrase and anti-HIV activity. The anti-
viral biquinone derivatives in Table 3 failed to inhibit

5658
K. W. Stagliano et al. / Bioorg. Med. Chem. 14 (2006) 5651–5665
CH3
O
O
CH3
Cs2CO3 / CH3CN
rt / 7 days (61%)
Cl
Cl
HO
+
O
O
O
10
7a
CH3
CH3
CH3
CH3
O
O
O
CH3O
O
O
O
HO
O
(CH3)3OBF4 / CH2Cl2
[(CH3)2CH]2NC2H5 / rt
(99%)
O
Cl
Cl
O
O
CH3
CH3
12
11
O
CH3O
O
O
18-crown-6
NMP / 65˚C / 48 h
O
O
O
(14%)
O
O-K+
O
HO
14
13
O
Scheme 3. Regiocontrolled synthesis of a pyranylated trimeric quinone.
Uniplate silica gel plates (Analtech), 250 lm thickness,
with fluorescent indicator (F-254). Preparative thin-
layer chromatography was performed on commercial
Uniplate silica gel plates (Analtech), 1000 lm thickness,
with fluorescent indicator (F-254). Flash chromatogra-
phy was carried out using 35–70 lm silica gel purchased
from Acros. Oxalic acid coated silica gel was prepared as
described previously.22
as described above, yielded 0.147 g (76%) of 3d as an
orange-brown solid.
Method B. To a stirred solution of the dipole 2a (2.68 g,
7.14 mmol) in 100 mL CH2Cl2 was added 50% HI
(1.28 mL, 0.959 g, 7.5 mmol). The dark orange suspen-
sion was stirred at rt for 3 h during which time a dark
red solution formed. The solution was poured into
100 mL CH2Cl2 and the organic layer washed with
water (pH ꢁ4–5) and 1% Na2S2O3 to remove traces of
I2. The resulting orange solution was dried (MgSO4),
filtered and evaporated to yield a yellow-orange residue.
Precipitation with 50% Et2O/hexanes, as described
above, yielded 1.66 g (77%) of the quinone 3d as an
orange-brown solid: mp = 179–180 ꢀC (CHCl3), lit.
mp = 177–179 ꢀC,17 Rf = 0.19 (50% EtOAc in hexanes);
1H NMR (300 MHz, CDCl3): d 7.72–7.80 (m, 2H),
8.03 (s, 1H), 8.13–8.16 (m, 1H), 8.19–8.22 (m, 1H); IR
4.2. Synthetic procedures
4.2.1.
2-Hydroxy-3-iodo-1,4-naphthoquinone
(3d).
Method A. General procedure. Into an oven dried
50 mL flask, equipped with a magnetic stirrer and under
three cycles of vacuum/nitrogen was added the dipole
(1 mmol) followed by addition of 20 mL of dry CH2Cl2
and 1–2 mmol of TMSX. The suspension was stirred at
room temperature for 16–20 h during which time a solu-
tion formed. The solvent was removed on a rotatory
evaporator and the residue dried under high vacuum
for 3 h. The addition of 2–6 mL of cold (0 ꢀC) 50%
Et2O in hexanes to the residue followed by vigorous mix-
ing with a spatula yielded a precipitate, which was filtered
and dried to yield the desired halohydroxyquinone 3.
(film) 3231 (br s), 1665, 1621, 1574, 1454, 1256 cmꢀ1
.
4.2.2. 3-Iodo-2-hydroxy-6-methoxy-1,4-naphthoquinone
(3g). Reaction of the dipole 2b (0.4031 g, 0.990 mmol)
with TMSI (0.260 mL, 0.365 g, 1.826 mmol), as described
above for the preparation of 3d, yielded 0.257 g (79%) of
the quinone 3g: mp = 189–191 ꢀC (CHCl3); Rf = 0.23
1
Reaction of the dipole 2a (0.243 g, 0.646 mmol) with
TMSI (0.130 mL, 0.182 g, 0.913 mmol), as described
above, yielded a red colored solution, which was poured
into ice water. The organic layer washed with cold water
to remove traces of HI. The organic layer was dried
(MgSO4), filtered, and evaporated to yield a yellow-
orange residue. Precipitation from 50% Et2O/hexanes,
(EtOAc); H NMR (300 MHz, CDCl3): d 3.98 (s, H),
7.18 (dd, J = 8.7, 2.7 Hz, 1H), 7.65 (d, J= 2.7 Hz, 1H),
8.08 (d, J = 8.7 Hz, 1H), 8.16 (s, 1H); 13C NMR
(75 MHz, CDCl3): d 56.4, 90.1, 112.7, 119.7, 121.9,
129.8, 134.4, 160.0, 165.4, 176.1, 179.1; IR (film): 3259
(br s), 1652, 1616, 1251, 1124 cmꢀ1; Anal. Calcd for
C11H7IO4: C, 40.03; H, 2.14. Found: C, 39.80; H, 2.18.

K. W. Stagliano et al. / Bioorg. Med. Chem. 14 (2006) 5651–5665
Conocurvone (µM) + IN, MgCl
5659
2
A
B
CAGT
*
*
0
0
333 111 37 12.3 4.1 1.4 0.5 0.15 0.5
3'-P
CA-OH
P-GT
*
ST
and
21
19
CA
CAGT
CAGT
*
*
C A
*
Conocurvone (µM) + IN, MnCl
E
C
2
Cpd 21 (µM) + IN, MnCl
2
0
0
333 111 37 12.3 4.1 1.4
0
0
333
37
111
12.3 4.1
21
21
19
19
F
D
Figure 2. Inhibition of HIV-1 integrase by trimeric naphthoquinone 21 and conocurvone. (A) Schematic diagram of in vitro integrase assay.
Integrase is reacted with a [50-32P] labeled (*) duplex oligonucleotide corresponding to the final 21 nucleotides of the HIV-1 U5 long terminal repeat.
First, the 50-GT-30 terminal dinucleotide is released by endonucleolytic cleavage, resulting in a 19mer oligonucleotide product (30-processing, 30-P).
Second, the 30-ends are covalently inserted into another oligonucleotide duplex at multiple positions (strand transfer, ST), resulting in the ladder of
bands migrating slower than the 21mer. (B) and (C) Concentration-dependent inhibition of integrase by conocurvone in the presence of magnesium
and manganese. (E) Concentration-dependent inhibition of integrase by compound 21 in the presence of manganese. Results with magnesium were
similar (not shown). The micromolar concentrations of inhibitors are above each gel lane. STP indicates strand transfer products. (D) and (F)
Graphical representation of integrase 30-P (squares) and ST (triangles) inhibition by conocurvone and compound 21. IC50 values were determined by
fit of the data with a sigmoidal dose–response curve.
4.2.3.
3-Chloro-2-hydroxy-7-methoxy-1,4-naphthoqui-
J = 2.7 Hz, 1H), 8.13–8.15 (d, J = 8.7 Hz, 1H); 13C
NMR (125 MHz, CDCl3): d 56.0, 110.9, 119.0, 121.1,
125.3, 129.9, 130.6, 152.6, 163.9, 177.0, 179.6; IR (KBr)
3177 (br s), 2846, 1681, 1635, 1591, 1485, 1435, 1354,
1203, 1145, 1104, 1035, 1013 cmꢀ1; Anal. Calcd for
C11H7ClO4: C, 55.37; H, 2.96. Found: C, 55.11; H, 3.01.
none (3h). To a vigorously stirred suspension of the
dipole 2c (1.00 g, 2.46 mmol) in 25 mL of CHCl3
(25 mL) under N2 at rt, was injected 2.05 mL of concd
HCl (24.6 mmol). The brilliant yellow suspension was
stirred for 48 h at rt during which time a yellow solution
formed. The solution was poured into water and the
organic layer washed with water and then extracted with
10% aqueous NaHCO3. The dark red aqueous layer was
washed with ether and then acidified in a spacious Erlen-
meyer flask with concentrated HCl to pH 2 (litmus). The
yellow suspension was extracted with chloroform and
the organic layer, dried (MgSO4), filtered, and evaporat-
ed to yield 0.55 g (93%) of the quinone 3h as a yellow
4.2.4. 3-Iodo-2-hydroxy-7-methoxy-1,4-naphthoquinone
(3i). Reaction of the dipole 2c (1.40 g, 3.45 mmol) with
TMSI (0.840 mL, 1.18 g, 5.87 mmol), as described above
for the preparation of 3d, yielded 0.837 g (73%) of the
quinone 3i: mp = 181–185 ꢀC; Rf = 0.41 (EtOAc); 1H
NMR (500 MHz, CDCl3): d 3.83 (s, 3H), 7.04–7.07
(dd, J = 10.8, 3.2 Hz, 1H), 7.41 (d, J = 3.3 Hz, 1H),
7.95–7.97 (d, J = 10.7 Hz, 1H); 13C NMR (125 MHz,
CDCl3): d 55.8, 110.7, 120.3, 124.5, 128.3, 129.9,
131.2, 161.2, 163.5, 177.6, 178.9; IR (KBr): 3255 (br s),
1
solid: mp = 221–227 ꢀC, Rf = 0.29 (EtOAc); H NMR
(500 MHz, CDCl3) d 3.96 (s, 3H), 7.24–7.26 (dd,
J = 8.7 Hz, 2.7 Hz, 1H), 7.52 (s, 1H), 7.57 (d,

5660
K. W. Stagliano et al. / Bioorg. Med. Chem. 14 (2006) 5651–5665
2950, 1677, 1629, 1596, 1487, 1433, 1351, 1262, 1133,
1093, 1039, 1002, 906, 892, 853, 789, 745, 682, 662,
567, 483, 455 cmꢀ1; Anal. Calcd for C11H7IO4: C,
40.03; H, 2.14. Found: C, 42.72; H, 2.52.
1150 cmꢀ1; MS (EI) m/z (relative intensity) 432 (M+,
54); 340 (9); 241(62); 215(28); 195 (3); 127 (15); 76
(100); 50 (43); Anal. Calcd for C11H4IF3O5S: C, 30.57;
H, 0.93. Found: C, 30.60; H, 0.90.
4.2.5. 2-Chloro-3-[(trifluoromethanesulfonyl)oxy]-1,4-naph-
thoquinone (4b). The hydroxyquinone 3b (0.60 g,
2.88 mmol) was placed in a 50 mL round-bottomed flask
fitted with a T-bore stopcock. After three vacuum and
nitrogen cycles 25 mL of anhydrous CH2Cl2 was added
and the solution cooled to 0 ꢀC. Freshly distilled Et3N
(0.48 mL, 3.46 mmol) was added followed by 0.58 mL
of triflic anhydride (0.975 g, 3.46 mmol). The light red
reaction mixture was stirred for 10 min at 0 ꢀC and then
for 20 min at rt. The mixture was concentrated to 2 mL
at reduced pressure and the residue loaded onto a short
column of silica and eluted with 20% EtOAc in hexanes.
The eluants were concentrated and the crystals which
formed were filtered to yield 0.836 g (85%) of the triflate
4b as green needles: mp = 120–123 ꢀC; Rf = 0.34 (33%
ether in hexanes); 1H NMR (300 MHz, CDCl3): d
7.85–7.88 (m, 2H), 8.21–8.23 (m, 2H); 13C NMR
(75 MHz, CDCl3): d 116.4, 120.6, 127.8, 128.1, 129.7,
130.8, 135.4 (2C), 148.8, 175.6, 176.7; IR (KBr) 1677,
1594, 1424, 1203, 1172, 1122 cmꢀ1; MS (EI) m/z (relative
intensity) 342 [(M+2)+, 3], 340 (M+, 11), 248 (42), 207
(33), 153 (12), 123 (100), 104 (12), 76 (58); Anal. Calcd
for C11H4ClF3O5S: C, 38.78; H, 1.18. Found: C, 39.18;
H, 1.35.
4.2.8. 2-Chloro-7-methoxy-3-[(trifluoromethanesulfonyl)-
oxy]-1,4-naphthoquinone (4e). Reaction of the hydroxy-
quinone 3e (0.081 g, 0.340 mmol) with Et3N
(0.074 mL, 0.054 g, 0.532 mmol) and triflic anhydride
(0.058 mL, 0.097 g, 0.345 mmol) as described above for
the preparation of 4b, provided a crude solid which
was purified by flash chromatography on silica eluting
with 20% EtOAc in hexanes, as described above, to yield
0.096 g (77%) of the triflate 4e as orange needles:
mp = 167–169 ꢀC, Rf = 0.32 (20% EtOAc in hexanes);
1H NMR (300 MHz, CDCl3): d 3.99 (s, 3H), 7.30 (dd,
J = 8.4, 2.7 Hz, 1H), 7.64 (d, J = 2.7 Hz, 1H), 8.15 (d,
J = 8.7 Hz, 1H); 13C NMR (75 MHz, CDCl3): d 56.5,
112.0, 116.4, 120.6, 121.4, 122.8, 130.3, 133.0, 149.0,
165.3, 174.4, 177.0; IR (film) 1685, 1667, 1421, 1216,
1129, 1021 cmꢀ1; Anal. Calcd for C12H6ClF3O6S: C,
38.88; H, 1.63. Found: C, 38.95; H, 1.72.
4.2.9. 2-Bromo-7-methoxy-3-[(trifluoromethanesulfonyl)-
oxy]-1,4-naphthoquinone (4f). Reaction of the hydroxy-
quinone 3f (0.125 g, 0.444 mmol) with Et3N (0.075 mL,
0.054 g, 0.539 mmol) and triflic anhydride (0.088 mL,
0.147 g, 0.523 mmol) as described above for the prepara-
tion of 4b, provided a crude solid which was purified by
flash chromatography on silica eluting with 33% EtOAc
in hexanes, to yield 0.131 g (71%) of the triflate 4f as or-
ange needles: mp = 169–171 ꢀC, Rf = 0.46 (33% EtOAc
in hexanes); 1H NMR (400 MHz, CDCl3): d 3.99 (s,
3H), 7.30 (dd, J = 6.6, 1.8 Hz, 1H), 7.64 (d, J = 1.8 Hz,
1H), 8.15 (d, J = 6.6 Hz, 1H); 13C NMR (100 MHz,
CDCl3): d 56.4, 112.4, 116.9, 120.1, 121.4, 122.9,
130.5, 133.0, 152.1, 165.4, 174.2, 177.3; IR (film) 1684,
1670, 1423, 1217, 1170, 1135 cmꢀ1; Anal. Calcd for
C12H6BrF3O6S: C, 34.71; H, 1.45. Found: C, 34.63; H,
1.56.
4.2.6. 2-Bromo-3-[(trifluoromethanesulfonyl)oxy]-1,4-naph-
thoquinone (4c). Reaction of the hydroxyquinone 3c
(0.252 g, 0.99 mmol) with Et3N (0.164 mL, 0.120 g,
1.19 mmol) and triflic anhydride (0.199 mL, 0.333 g,
1.18 mmol) as described above for the preparation of
4b, provided a crude solid which was purified by flash
chromatography on silica eluting with 50% EtOAc in
hexane to yield 0.329 g (85%) of the triflate 4c as bright
yellow needles: mp = 114–115 ꢀC; Rf = 0.48 (50% ether
1
in hexanes); H NMR (300 MHz, CDCl3): d 7.85–7.88
(m, 2H), 8.21–8.25 (m, 2H); 13C NMR (100 MHz,
CDCl3): d 116.9, 128.0, 128.5, 129.7, 130.8, 131.1,
135.4, 135.5, 151.8, 175.4, 177.0; IR (KBr) 1676, 1592,
1428, 1215, 1168, 1141 cmꢀ1; MS (EI) m/z (relative
intensity) 386 [(M+2)+, 12], 384 (M+,12), 292 (10), 241
(100), 169 (55), 167 (55), 104 (37), 76 (82); Anal. Calcd
for C11H4BrF3O5S: C, 34.31; H, 1.05. Found: C,
34.14; H, 1.01.
4.2.10. 2-Iodo-7-methoxy-3-[(trifluoromethanesulfonyl)-
oxy]-1,4-naphthoquinone (4g). The hydroxyquinone 3g
(0.118 g, 0.357 mmol) was treated with Et3N
(0.060 mL, 0.043 g, 0.431 mmol) and triflic anhydride
(0.070 mL, 0.117 g, 0.416 mmol) as described above for
the preparation of 4b, providing a crude solid which
was purified by flash chromatography on silica eluting
with 20% EtOAc in hexanes to yield 0.121 g (76%) of
the triflate 4g as orange needles: mp = 144–146 ꢀC,
Rf = 0.40 (20% EtOAc in hexanes); 1H NMR
(300 MHz, CDCl3): d 3.98 (s, 3H), 7.28 (dd, J = 8.4,
2.7 Hz, 1H), 7.62 (d, J = 2.7 Hz, 1H), 8.14 (d,
J = 8.1 Hz, 1H); 13C NMR (75 MHz, CDCl3) d 56.5,
112.7, 113.6, 116.3, 121.3, 123.0, 130.5, 132.1, 157.4,
165.0, 173.1, 178.4; IR (film) 1678, 1668, 1423, 1218,
1205 cmꢀ1; Anal. Calcd for C12H6IF3O6S: C, 31.18; H,
1.30. Found: C, 31.09; H, 1.39.
4.2.7. 2-Iodo-3-[(trifluoromethanesulfonyl)oxy]-1,4-naph-
thoquinone (4d). Reaction of the hydroxyquinone 3d
(0.250 g, 0.883 mmol) with Et3N (0.140 mL, 0.101 g,
1.00 mmol) and triflic anhydride (0.160 mL, 0.268 g,
0.951 mmol) as described above for the preparation of
4b, provided a crude solid which was purified by flash
chromatography on silica eluting with 5% EtOAc in
hexane to yield 0.276 g (77%) of the triflate 4d as yellow
needles: mp = 129–131 ꢀC; Rf = 0.24 (5% EtOAc in hex-
anes); 1H NMR (400 MHz, CDCl3): d 7.85 (pentd,
J = 7.6, 7.2, 2.0, 1.6 Hz, 2H), 8.22–8.23 (m, 1H), 8.24–
8.25 (m, 1H); 13C NMR (100 MHz, CDCl3): d 115.5,
120.1, 128.1, 128.9, 129.9, 130.1, 135.3 (2C), 157.2,
174.5, 178.5; IR (KBr) 1676, 1587, 1420, 1209,
4.2.11. 2-Chloro-6-methoxy-3-[(trifluoromethanesulfonyl)-
oxy]-1,4-naphthoquinone (4h). Reaction of the hydroxy-
quinone 3h (0.540 g, 2.26 mmol) with Et3N (0.375 mL,
0.274 g, 2.71 mmol) and triflic anhydride (0.456 mL,

K. W. Stagliano et al. / Bioorg. Med. Chem. 14 (2006) 5651–5665
5661
0.765 g, 2.71 mmol) as described above for the prepara-
tion of 4b, provided a crude solid which was purified by
flash chromatography on silica eluting with 5% EtOAc
in hexanes to yield 0.705 g (84%) of the triflate 4h as
orange needles: mp = 124–127 ꢀC, Rf = 0.28 (5% EtOAc
in hexanes); 1H NMR (500 MHz, CDCl3): d 3.98 (s,
3H), 7.24–7.26 (dd, J = 8.7 Hz, 2.7 Hz, 1H), 7.62 (d,
J = 2.6 Hz, 1H), 8.14–8.15 (d, J = 8.7 Hz, 1H); 13C
NMR (125 MHz, CDCl3): d 56.2, 111.2, 116.5, 119.6,
121.5, 123.0, 131.1, 131.8, 156.7, 165.0, 174.4, 176.9; IR
(KBr) 3108, 3027, 2998, 2953, 2851, 1783, 1683, 1619,
1585, 1499, 1463, 1436, 1353, 1309, 1212, 1135, 1090,
1012 cmꢀ1; Anal. Calcd for C12H6ClF3O6S: C, 38.88;
H, 1.63. Found: C, 39.28; H, 1.68.
NMR (125 MHz, DMSO-d6) d 122.1, 126.5, 126.8,
127.3, 128.2, 129.3, 130.1, 130.5, 131.5, 132.0, 134.4,
135.0, 135.3, 135.7, 150.6, 155.6, 179.28, 179.33, 181.2,
181.7; FT-IR (KBr) 3334 (br s), 3114, 1675, 1660,
1636, 1589, 1480, 1457, 1371, 1333, 1267, 1214, 1160,
1125, 1080, 1040, 1011 cmꢀ1; MS (FAB) m/z (relative
intensity) 458 [(M+2H)+, 5], 457 [(M+H)+, 3], 391
(40), 330 [(MꢀI)+, 17], 279 (9), 167 (23), 149 (100),
136 (29); HRMS (FAB) Calcd for C20H10127IO5
(M+H)+ was not found but for C20H10O5 (MꢀI)+ calcd:
330.0528. Found: 330.0527.
4.2.14. 30-Chloro-3-hydroxy-7-methoxy-2,20-binaphthale-
nyl-1,4,10,40-tetraone (6b). Reaction of the hydroxyqui-
none
triflate
4b
(0.325 g,
0.96 mmol)
with
4.2.12. 2-Iodo-6-methoxy-3-[(trifluoromethanesulfonyl)-
oxy]-1,4-naphthoquinone (4i). Reaction of the hydroxy-
quinone 3i (0.720 g, 2.18 mmol) with Et3N (0.363 mL,
0.265 g, 2.62 mmol) and triflic anhydride (0.441 mL,
0.739 g, 2.62 mmol) as described above for the prepara-
tion of 4b, provided a crude solid which was purified by
flash chromatography on silica eluting with 5% EtOAc
in hexanes, to yield 0.783 g (78%) of the triflate 4i as a
yellow powder: mp = 135–137 ꢀC, Rf = 0.27 (5% EtOAc
in hexanes); 1H NMR (500 MHz, CDCl3): d 3.99 (s,
3H), 7.27–7.30 (dd, J = 10.9 Hz, 3.4 Hz, 1H), 7.62 (d,
J = 3.3 Hz, 1H), 8.14–8.16 (d, J = 10.9 Hz, 1H); 13C
NMR (125 MHz, CDCl3) d 56.2, 111.3, 120.0, 121.5,
123.9, 130.5, 131.7, 137.0, 148.5, 165.2, 175.4, 175.8;
IR (KBr) 3076, 3006, 2957, 2852, 1768, 1685, 1663,
1588, 1496, 1468, 1421, 1347, 1302, 1235, 1149, 1132,
1019, 1000 cmꢀ1; Anal. Calcd for C12H6IF3O6S: C,
31.18; H, 1.30. Found: C, 31.48; H, 1.45.
hydroxyquinone 5 (0.196 g, 0.96 mmol) and Cs2CO3
(0.626 g, 1.92 mmol) for 6 days, according to the general
procedure described above for the preparation of 6a,
resulted in an 85:15 mixture of 30-chloro-3-hydroxy-7-
methoxy-2,20-binaphthalenyl-1,4,10,40-tetraone 6b and
3-hydroxy-7-methoxy-30-[(trifluoromethanesulfonyl)oxy]-
2,20-binaphthalenyl-1,4,10,40-tetraone. Purification by
flash chromatography on oxalic acid coated silica gel22
with gradual elution from 10% to 50% EtOAc in hexanes
yielded 0.212 g (56%) of the chlorohydroxybiquinone 6b
as a yellow solid: mp = 212–216 ꢀC, Rf = 0.29 (EtOAc),
1H NMR (500 MHz, CDCl3): d 3.88 (s, 3H), 7.10–7.12
(dd, J = 10.7 Hz, 3.1 Hz, 1H), 7.50 (d, J = 3.0 Hz, 1H),
7.70–7.73 (m, 2H), 8.01–8.03 (d, J = 10.9 Hz, 1H), 8.04–
8.05 (m, 1H), 8.12–8.14 (m, 1H); 13C NMR (125 MHz,
CDCl3) d 56.0, 110.7, 115.2, 119.5, 123.4, 127.1, 127.2,
129.1, 131.3, 131.9, 134.0, 134.3, 134.9, 139.8, 145.5,
155.8, 165.1, 177.5, 179.5, 180.4, 181.9; FT-IR (KBr)
3334 (br s), 3085, 2925, 2847, 1675, 1642, 1591, 1499,
1449, 1373, 1274, 1168, 1133, 1102, 1076, 1040,
1013 cmꢀ1; MS (FAB) m/z (relative intensity) 395
[(M+H)+, 6], 360 (5), 307 (18), 289 (12), 255 (6), 220 (7),
173 (13), 154 (100), 136 (89), 107(44); HRMS (FAB) calcd
for C21H1235ClO6 (M+H)+: 395.0322. Found: 395.0333.
4.2.13. 30-Hydroxy-3-iodo-2,20-binaphthalenyl-1,4,10,40-
tetraone (6a). The hydroxyquinone triflate 4d (0.300 g,
0.69 mmol),
2-hydroxy-1,4-naphthoquinone
3a
(0.120 g, 0.69 mmol) and Cs2CO3 (0.450 g, 1.38 mmol)
were placed in a 25 mL round bottom flask fitted with
a T-bore stopcock. After three vacuum and nitrogen
cycles, 10 mL of anhydrous CH3CN was added, and
the suspension stirred at room temperature for 6 days
during which time the color changed to a very dark
red. The mixture was acidified with concentrated HCl
to pH 2 (litmus) forming a yellow-green precipitate.
The suspension was poured into 100 mL of water and
stirred for 1 hour to dissolve the remaining traces of
cesium carbonate. The precipitate was filtered and the
product mixture air-dried for 3 days at rt. The product
consisted of a 90:10 mixture 30-hydroxy-3-iodo-2,20-
binaphthalenyl-1,4,10,40-tetraone 6a and 30-hydroxy-3-
[(trifluoromethanesulfonyl)oxy]-2,20-binaphthalenyl-1,
4,10,40-tetraone as determined by HPLC analyses of the
crude reaction mixture. The molecular weight of the
minor regioisomer was determined by GC–MS analysis
of the crude reaction mixture. Purification by flash chro-
matography on oxalic acid coated silica gel22 with grad-
ual elution from 10% to 50% EtOAc in hexanes yielded
0.226 g (72%) of the iodohydroxybiquinone 6a as a
yellow-orange solid: mp = 213–218 ꢀC, Rf = 0.28
4.2.15. 3-Hydroxy-30-iodo-7-methoxy-2,20-binaphthale-
nyl-1,4,10,40-tetraone (6d). Reaction of the hydroxyqui-
none
triflate
4d
(0.250 g,
0.58 mmol)
with
hydroxyquinone 5 (0.119 g, 0.58 mmol) and Cs2CO3
(0.378 g, 1.16 mmol) for 6 days, according to the general
procedure described above for the preparation of 6a,
resulted in a 95:5 mixture of 3-hydroxy-30-iodo-7-meth-
oxy-2,20-binaphthalenyl-1,4,10,40-tetraone 6d and 3-hy-
droxy-7-methoxy-30-[(trifluoromethanesulfonyl)oxy]-2,20-
binaphthalenyl-1,4,10,40-tetraone. Purification by flash
chromatography on oxalic acid coated silica gel22 with
gradual elution from 10% to 50% EtOAc in hexanes
yielded 0.189 g (67%) of the iodohydroxybiquinone 6d
as an orange-yellow solid: mp = 204–207 ꢀC, Rf = 0.49
1
(EtOAc), H NMR (500 MHz, CDCl3): d 3.84 (s, 3H),
7.06–7.09 (dd, J = 10.8 Hz, 3.1 Hz, 1H), 7.45–7.46 (d,
J = 3.2 Hz, 1H), 7.61–7.69 (m, 2H), 7.97–7.99 (m, 2H),
8.06–8.08 (m, 1H); 13C NMR (125 MHz, CDCl3) d
56.0, 110.6, 119.4, 120.9, 123.5, 127.2, 127.9, 128.4,
129.1, 130.0, 131.7, 133.8, 134.2, 134.8, 150.5, 154.9,
165.0, 178.6, 178.8, 179.7, 181.5; FT-IR (KBr) 3267 (br
s), 2924, 2850, 1730, 1672, 1656, 1642, 1590, 1498, 1437,
1371, 1339, 1299, 1268, 1131, 1073, 1011 cmꢀ1; MS
1
(EtOAc), H NMR (500 MHz, CDCl3): d 7.70 (s, 1H,
br), 7.75–7.81 (m, 3H), 7.84–7.87 (m, 1H), 8.13–8.15
(m, 1H), 8.19–8.20 (m, 2H), 8.22–8.24 (m, 1H); 13C

5662
K. W. Stagliano et al. / Bioorg. Med. Chem. 14 (2006) 5651–5665
(FAB) m/z (relative intensity) 488 [(M+2+H)+, 3], 391
(10), 360 (6), 338 (6), 307 (31), 289 (19), 255 (6), 218
(9), 154 (100), 136 (91), 107 (45); HRMS (FAB) calcd
for C21H13127IO6 (M+2H)+: 487.9757. Found: 487.9759.
oxy-2,20-binaphthalenyl-1,4,10,40-tetraone 6h and 30-hy-
droxy-6-methoxy-3-[(trifluoromethanesulfonyl)oxy]-2,20-
binaphthalenyl-1,4,10,40-tetraone. Purification by flash
chromatography on oxalic acid coated silica gel22 with
gradual elution from 10% to 50% EtOAc in hexanes
yielded 0.200 g (47%) of the chlorohydroxybiquinone
6h as yellow needles: mp = 262–266 ꢀC, Rf = 0.50
4.2.16. 3-Chloro-30-hydroxy-6-methoxy-2,20-binaphthale-
nyl-1,4,10,40-tetraone (6e). Reaction of the hydroxyqui-
1
none
triflate
4e
(0.130 g,
0.35 mmol)
with
(EtOAc), H NMR (500 MHz, CDCl3): d 3.96 (s, 3H),
hydroxyquinone 3a (0.061 g, 0.35 mmol) and Cs2CO3
(0.228 g, 0.70 mmol) for 6 days, according to the general
procedure described above for the preparation of 6a
yielded a 90:10 mixture of 3-chloro-30-hydroxy-6-meth-
oxy-2,20-binaphthalenyl-1,4,10,40-tetraone 6e and 30-hy-
droxy-7-methoxy-3-[(trifluoromethanesulfonyl)oxy]-2,20-
binaphthalenyl-1,4,10,40-tetraone. Purification by flash
chromatography on oxalic acid coated silica gel22 with
gradual elution from 10% to 50% EtOAc in hexanes
yielded 0.092 g (67%) of the chlorohydroxybiquinone
6e as yellow needles: mp = 297–300 ꢀC, Rf = 0.31
7.24–7.27 (dd, J = 8.7 Hz, 2.6 Hz, 1H), 7.57–7.58 (d,
J = 2.6 Hz, 1H), 7.77–7.87 (m, 2H), 8.17–8.19 (d,
J = 8.7 Hz, 1H), 8.17–8.21 (m, 2H); 13C NMR
(125 MHz, CDCl3) d 56.0, 110.7, 115.4, 120.7, 124.8,
126.7, 127.3, 129.3, 130.0, 132.7, 133.5, 133.9, 135.6,
138.1, 146.5, 153.1, 164.6, 176.2, 180.4, 180.7, 181.5;
FT-IR (KBr) 3337 (br s), 3078, 2978, 2944, 2842, 1673,
1651, 1593, 1495, 1460, 1377, 1339, 1284, 1235, 1167,
1141, 1091, 1041, 1011 cmꢀ1; MS (FAB) m/z (relative
intensity) 395 [(M+H)+, 11], 391 (20), 360 (9), 307 (62),
289 (48), 273 (9), 154 (100), 136 (89), 107 (70); HRMS
(FAB) calcd for C21H1235ClO6 (M+H)+: 395.0322.
Found: 395.0319.
1
(EtOAc), H NMR (500 MHz, CDCl3): d 3.99 (s, 3H),
7.24–7.27 (dd, J = 8.7 Hz, 2.6 Hz, 1H), 7.65–7.66 (d,
J = 2.6 Hz, 1H), 7.76–7.86 (m, 2H), 8.08–8.10 (d,
J = 8.6 Hz, 1H), 8.18–8.20 (m, 2H); 13C NMR
(125 MHz, CDCl3) d 56.0, 110.8, 115.5, 121.0, 125.2,
126.7, 127.3, 129.3, 129.8, 132.7, 133.3, 133.5, 135.6,
138.8, 145.2, 153.1, 164.3, 177.5, 179.4, 180.7, 181.5;
FT-IR 3319 (br s), 3076, 2989, 2950, 2844, 1678, 1653,
1582, 1499, 1457, 1378, 1298, 1244, 1216, 1196, 1135,
1076, 1012 cmꢀ1; MS (FAB) m/z (relative intensity) 395
[(M+H)+, 2], 360 (1), 307 (26), 289 (14), 273 (3), 154
(100), 136 (77), 107 (25); HRMS (FAB) calcd for
C21H1235ClO6 (M+H)+: 395.0322. Found: 395.0331.
4.2.19. 30-Hydroxy-3-iodo-7-methoxy-2,20-binaphthale-
nyl-1,4,10,40-tetraone (6i). Reaction of the hydroxyqui-
none triflate 4i (0.400 g, 0.87 mmol) with 2-hydroxy-
naphthoquinone 3a (0.152 g, 0.87 mmol) and Cs2CO3
(0.567 g, 1.74 mmol) for 7 days, according to the general
procedure described above for the preparation of 6a,
resulted in a 90:10 mixture of 30-hydroxy-3-iodo-7-meth-
oxy-2,20-binaphthalenyl-1,4,10,40-tetraone 6i and 30-hy-
droxy-6-methoxy-3-[(trifluoromethanesulfonyl)oxy]-2,20-
binaphthalenyl-1,4,10,40-tetraone. Purification by flash
chromatography on oxalic acid coated silica gel22 with
gradual elution from 10% to 50% EtOAc in hexanes
yielded 0.254 g (60%) of the iodohydroxybiquinone 6i
as a yellow-orange solid: mp = 199–204 ꢀC, Rf = 0.45
4.2.17. 30-Hydroxy-3-iodo-6-methoxy-2,20-binaphthale-
nyl-1,4,10,40-tetraone (6g). Reaction of the hydroxyqui-
none triflate 4g (0.200 g, 0.43 mmol) with 2-hydroxy-
naphthoquinone 3a (0.075 g, 0.43 mmol) and Cs2CO3
(0.280 g, 0.86 mmol) for 6 days, according to the general
procedure described above for the preparation of 6a
yielded 0.173 g (83%) of the iodohydroxybiquinone 6g
as a yellow orange solid: mp = 174–176 ꢀC, Rf = 0.47
1
(EtOAc), H NMR (500 MHz, CDCl3): d 3.96 (s, 3H),
7.20–7.23 (dd, J = 8.7 Hz, 2.7 Hz, 1H), 7.55 (d, J =
2.6 Hz, 1H), 7.77–7.87 (m, 2H), 8.16–8.18 (d, J =
8.7 Hz, 1H), 8.19–8.21 (m, 2H); 13C NMR (125 MHz,
CDCl3) d 56.0, 110.8, 120.6, 120.8, 123.5, 126.7, 127.3,
129.4, 129.9, 130.8, 132.7, 133.5, 133.7, 135.6, 148.9,
152.0, 164.5, 177.2, 178.9, 180.9, 181.2; FT-IR (KBr)
3327 (br s), 2936, 2840, 1729, 1670, 1643, 1592, 1495,
1458, 1444, 1373, 1337, 1281, 1171, 1135, 1087, 1039,
1013 cmꢀ1; MS (FAB) m/z (relative intensity) 488
[(M+2+H)+, 3], 391 (5), 360 (7), 307 (25), 289 (14), 273
(3), 219 (4), 154 (100), 136 (85), 107 (31); HRMS
(FAB) calcd for C21H12127IO6 (M+H)+: 486.9679.
Found: 486.9663.
1
(EtOAc), H NMR (500 MHz, CDCl3): d 3.98 (s, 3H),
7.23–7.26 (dd, J = 8.6 Hz, 2.6 Hz, 1H), 7.65–7.66 (d,
J = 2.6 Hz, 1H), 7.77–7.87 (m, 2H), 8.07–8.08 (d,
J = 8.6 Hz, 1H), 8.19–8.21 (m, 2H); 13C NMR
(125 MHz, CDCl3) d 56.0, 111.6, 120.9 (2C), 125.1,
126.7, 127.3, 129.4, 129.9, 132.1, 132.7, 133.5, 135.6,
149.6, 151.9, 161.9, 164.1, 177.9, 178.4, 180.9, 181.2;
FT-IR (KBr) 3324 (s and br), 2924, 2849, 1734, 1669,
1653, 1587, 1539, 1472, 1374, 1340, 1280, 1230, 1189,
1135, 1068, 1012 cmꢀ1; MS (FAB) m/z (relative intensi-
ty) 488 [(M+2+H)+, 2], 391 (5), 360 (4), 338 (5), 307 (25),
289 (16), 255 (4), 218 (8), 154 (100), 136 (100), 107 (39);
HRMS (FAB) calcd for C21H13127IO6 (M+2H)+:
487.9757. Found: 487.9743.
4.2.20. 9-Hydroxy-8-(3-iodo-1,4-dioxo-1,4-dihydronaph-
thalen-2-yl)-3,3-dimethyl-3H-benzo[f]chromene-7,10-dione
(8a). Reaction of hydroxyquinone triflate 4d (0.700 g,
1.62 mmol) with 9-hydroxy-3,3-dimethyl-3H-benzo[f]-
chromene-7,10-dione 7a25 (0.415 g, 1.62 mmol) and
Cs2CO3 (1.056 g, 3.24 mmol) for 7 days, according to
the general procedure described above for the prepara-
tion of 6a, yielded a 90:10 mixture of 9-hydroxy-8-(3-
iodo-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-3,3-dimeth-
yl-3H-benzo[f]chromene-7,10-dione 8a and 9-hydroxy-
8-(3-[(trifluoromethanesulfonyl)oxy]-1,4-dioxo-1,4-di-
hydronaphthalen-2-yl)-3,3-dimethyl-3H-benzo[f]chro-
4.2.18. 3-Chloro-30-hydroxy-7-methoxy-2,20-binaphthale-
nyl-1,4,10,40-tetraone (6h). Reaction of the hydroxyqui-
none triflate 4h (0.400 g, 1.08 mmol) with 2-hydroxy-
naphthoquinone 3a (0.188 g, 1.08 mmol) and Cs2CO3
(0.704 g, 2.16 mmol) for 7 days, according to the general
procedure described above for the preparation of 6a,
yielded a 70:30 mixture of 3-chloro-30-hydroxy-7-meth-

K. W. Stagliano et al. / Bioorg. Med. Chem. 14 (2006) 5651–5665
5663
mene-7,10-dione. Purification by flash chromatography
on oxalic acid coated silica gel22 eluting with 10%
EtOAc in hexanes, yielded 0.524 g (60%) of the
iodohydroxybiquinone 8a as a red solid: mp = 213–
aration of 6a, yielded 1.537 g (61%) of naphthopyranyl biq-
uinone as a brown solid: mp = 204–208 ꢀC; Rf = 0.21 (50%
EtOAc in hexanes); 1H NMR (500 MHz, CDCl3) d 1.50 (s,
6H), 6.02–6.04 (d, J = 10.3 Hz, 1H), 7.13–7.15 (d,
J = 8.44 Hz, 1H), 7.73–7.81 (m, 3H), 8.01–8.03 (d,
J = 8.44 Hz, 1H), 8.12–8.14 (m, 1H), 8.19–8.22 (m, 1H);
13C NMR (125 MHz, CDCl3) d 28.0 (2C), 77.3, 113.6,
119.5, 121.9, 122.8, 123.3, 126.7, 127.3, 127.4, 129.3,
131.4, 131.8, 134.1, 134.4, 136.7, 138.9, 145.8, 153.2,
158.2, 177.4, 180.4, 180.9, 182.6; FT-IR 3298, 2977, 2926,
1679, 1659, 1631, 1590, 1557, 1466, 1436, 1378, 1355,
1277, 1219, 1188, 1163, 1137, 1120, 1006 cmꢀ1; MS
(FAB) m/z (relative intensity) 449 [(M+2+H)+, 6], 447
[(M+H)+, 10], 411 (6), 397 (5), 307 (11), 213 (10), 154
(100), 136 (72); HRMS (FAB) calcd for C25H1635ClO6
(M+H)+: 447.0635. Found: 447.0623.
1
217 ꢀC, Rf = 0.21 (50% EtOAc in hexanes), H NMR
(500 MHz, CDCl3): d 1.52 (s, 6H), 6.04–6.06 (d,
J = 10.3 Hz, 1H), 7.15–7.17 (d, J = 8.4 Hz, 1H), 7.75–
7.81 (m, 4H), 8.04–8.05 (d, J = 8.4 Hz, 1H), 8.12–8.14
(dd, J = 7.2 Hz, 1.67 Hz, 1H), 8.21–8.23 (dd,
J = 7.2 Hz, 1.65 Hz, 1H); 13C NMR (125 MHz, CDCl3)
d 28.0, 28.0, 119.0, 119.5, 121.9, 122.8, 123.3, 126.8,
127.5, 128.2, 128.5, 128.7, 129.3, 130.1, 131.7, 133.9,
134.3, 136.6, 149.7, 151.9, 158.2, 178.4, 178.8, 180.6,
182.8; FT-IR (KBr) 3318, 2980, 1733, 1656, 1626,
1590, 1562, 1456, 1440, 1380, 1350, 1270, 1162, 1129,
1098, 1075, 1005 cmꢀ1; MS (FAB) m/z (relative intensi-
ty) 539 [(M+H)+, 7], 422 (12), 397 (12), 307 (11), 289 (9),
252 (10), 213 (5), 154 (100), 136 (76), 107 (28); HRMS
(FAB) calcd for C25H16127IO6 (M+H)+: 538.9992.
Found: 539.0004.
4.2.23. 8-(3-Iodo-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-
9-methoxy-3,3-dimethyl-3H-benzo[f]chromene-7,10-dione
(9). The iodohydroxybiquinone 8a (0.094 g, 0.175 mmol)
and trimethyloxonium tetrafluoroborate (0.052 g,
0.35 mmol) were placed in a 50 mL round bottom flask
fitted with a T-bore stopcock. After three vacuum and
nitrogen cycles, 25 mL of anhydrous CH2Cl2 was added
and the suspension stirred at rt for 30 min. N,N-Diiso-
propylethylamine (0.046 mL, 0.034 g, 0.263 mmol) was
added and the dark red solution stirred for 3 h during
which time the color changed to dark yellow. The solu-
tion was diluted with 60 mL of CH2Cl2 and the organic
layer washed successively with water (containing three
drops of concd HCl) then 10% NaHCO3. The organic
layer was dried (MgSO4), filtered, and evaporated to
yield 0.090 g (93%) of the iodomethoxybiquinone 9 as
a brown solid: mp = 90–95 ꢀC; Rf = 0.19 (10% EtOAc
in hexanes); 1H NMR (500 MHz, CDCl3) d 1.50 (s,
6H), 4.09 (s, 3H), 5.97–6.00 (d, J = 13.0 Hz, 1H), 7.09–
7.11 (d, J = 10.4 Hz, 1H), 7.66–7.68 (d, J = 12.9 Hz,
1H), 7.74–7.80 (m, 2H), 7.95–7.97 (d, J = 10.6 Hz,
1H), 8.12–8.14 (m, 1H), 8.21–8.23 (m, 1H); 13C NMR
(125 MHz, CDCl3) d 28.0 (2C), 61.1, 77.3 (CDCl3 over-
laps), 119.7, 121.2, 121.9, 125.9, 126.2, 127.2, 127.5,
128.1, 128.2, 128.6, 130.2, 131.6, 134.0, 134.3, 135.4,
151.1, 157.0, 158.7, 178.3, 179.0, 181.0, 183.2;
FTIR (KBr) 3466, 3398, 2946, 2872, 1670, 1659, 1642,
1592, 1563, 1328, 1320, 1288, 1275, 1266, 1218, 1162,
1119, 1022 cmꢀ1; MS (FAB) m/z (relative intensity)
553 [(M+H)+, 14], 537 (10), 425 (16), 411 (20), 391
(29), 307 (10), 289 (9), 279 (7), 220 (11), 149 (100), 136
(65); HRMS (FAB) calcd for C26H18127IO6 (M+H)+:
553.0148. Found 553.0136.
4.2.21. 9-Hydroxy-8-(3-iodo-1,4-dioxo-1,4-dihydronaphtha-
len-2-yl)-3-methyl-3-(4-methyl-pent-3-enyl)-3H-benzo[f]chro-
mene-7,10-dione (8b). Reaction of the hydroxyquinone
triflate 4d (0.821 g, 1.90 mmol) with teretifolione B 7b25
(0.616 g, 1.90 mmol) and Cs2CO3(1.238 g, 3.80 mmol) for
7 days, according to the general procedure described above
for the preparation of 6a, yielded a 90:10 mixture of 9-hy-
droxy-8-(3-iodo-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-3-
methyl-3-(4-methyl-pent-3-enyl)-3H-benzo[f]chromene-7,
10-dione 8b and 9-hydroxy-8-[3-[(trifluoromethanesulfo-
nyl)oxy]-1,4-dioxo-1,4-dihydronaphthalen-2-yl]-3-meth-
yl-3-(4-methyl-pent-3-enyl)-3H-benzo[f]chromene-7,10-
dione. Purification by flash chromatography on oxalic
acid coated silica gel22 eluting with 10% EtOAc in hex-
anes, yielded 0.587 g (51%) of the iodohydroxybiqui-
none 8b as a dark red solid: mp = 93–97 ꢀC, Rf = 0.30
(50% EtOAc in hexanes), 1H NMR (500 MHz, CDCl3):
d 1.48 (s, 3H), 1.58 (s, 3H), 1.66 (d, J = 3.2 Hz, 3H),
1.71–1.76 (m, 2H), 1.80–1.83 (m, 2H), 2.12 (s, broad,
1H), 5.08–5.11 (m, 1H), 6.00 (d, J = 10.5 Hz, 1H),
7.13–7.15 (d, J = 8.5 Hz, 1H), 7.73–7.80 (m, 2H),
7.85–7.87 (d, J = 10.5 Hz, 1H), 8.03–8.04 (d,
J = 8.5 Hz, 1H), 8.12–8.14 (m, 1H), 8.21–8.23 (m,
1H); 13C NMR (125 MHz, CDCl3) d 17.7, 22.7, 25.6,
26.7, 41.3, 79.6, 119.0, 120.0, 121.8, 122.5, 123.3,
123.4, 126.6, 127.5, 128.2, 128.5, 129.4, 130.1, 131.7,
132.3, 133.9, 134.3, 135.9, 149.7, 151.9, 158.6, 178.4,
178.8, 180.6, 182.8; FT-IR (KBr) 3321, 2966, 2922,
1672, 1645, 1592, 1562, 1460, 1436, 1354, 1264, 1198,
1128, 1075, 1002, 951, 915, 848, 781, 748, 700, 617,
538, 464, 432 cmꢀ1; MS (FAB) m/z (relative intensity)
607 [(M+H)+, 20], 523 (26), 480 (16), 397 (76), 307
(24), 289 (16), 220 (9), 165 (11), 154 (100), 136 (97),
107 (45); HRMS (FAB) calcd for C30H24127IO6
(M+H)+: 607.0618. Found: 607.0629.
4.2.24. 8-(3-Chloro-1,4-dioxo-1,4-dihydronaphthalen-2-
yl)-9-methoxy-3,3-dimethyl-3H-benzo[f]chromene-7,10-
dione (12). Reaction of the chlorohydroxybiquinone
11 (0.500 g, 1.12 mmol), trimethyloxonium tetrafluo-
roborate (0.414 g, 2.8 mmol) and N,N-diisopropyleth-
ylamine (0.217 g, 0.292 mL, 1.68 mmol) for 5 h
according to the general procedure described above
for the preparation of 9 yielded 0.510 g (99%) of the
chloromethoxybiquinone 12 as a dark red solid:
mp = 87–93 ꢀC; Rf = 0.38 (50% EtOAc in hexanes); 1H
NMR (500 MHz, CDCl3) d 1.48–1.52 (s, 6H), 4.09 (s,
3H), 5.97–5.99 (d, J = 12.8 Hz, 1H), 7.09–7.11 (d,
4.2.22. 8-(3-Chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-9-
hydroxy-3,3-dimethyl-3H-benzo[f]chromene-7,10-dione (11).
Reaction of the hydroxyquinone 7a (1.00 g, 3.9 mmol),
2,3-dichloro-1,4-naphthoquinone 10 (0.886 g, 3.9 mmol)
and cesium carbonate (2.542 g, 7.8 mmol) for 7 days,
according to the general procedure described for the prep-

5664
K. W. Stagliano et al. / Bioorg. Med. Chem. 14 (2006) 5651–5665
J = 10.7 Hz, 1H), 7.65–7.68 (d, J = 12.8 Hz, 1H), 7.74–
7.82 (m, 2H), 7.94–7.96 (d, J = 10.7 Hz, 1H), 8.12–8.15
(m, 1H), 8.21–8.24 (m, 1H); 13C NMR (125 MHz,
CDCl3) d 28.0 (2C), 61.1, 77.3 (CDCl3 overlaps),
119.7, 121.2, 121.9, 122.0, 125.9, 126.2, 127.3, 127.4,
128.6, 131.4, 131.8, 134.2, 134.4, 135.4, 140.3, 145.2,
158.2, 158.7, 177.3, 180.6, 181.4, 183.0; FT-IR (KBr)
3071, 2980, 2850, 1677, 1643, 1597, 1562, 1458, 1311,
1278, 1216, 1162, 1119, 1084, 1022 cmꢀ1; MS (FAB)
m/z (relative intensity) 463 [(M+2+H)+, 31], 461
[(M+H)+, 57], 445 (13), 425 (21), 245 (14), 213 (32),
167 (26), 149 (100); HRMS (FAB) calcd for
C26H1835ClO6 (M+H)+: 461.0792. Found: 461.0787.
4.4. Antiviral assay (MTT method)
The antiviral activity of test compounds against HIV
was measured as the inhibition of viral cytopathogenic
effect. CEM-T4 cells used for the assay were maintained
in RPMI-1640 culture medium containing 2 mM L-glu-
tamine and 25 mM HEPES, and supplemented with
10% fetal bovine serum, 50 U/mL of penicillin G, and
50 lg/mL streptomycin sulfate. The antiviral assays
were performed in 96-well tissue culture plates. Cells
were treated with polybrene at a concentration of 2 lg/
mL, and 1 · 104 cells were dispensed into each well.
Appropriate concentrations of test compounds were
prepared in sterile DMSO and diluted with culture
medium to the desired concentrations. Each dilution
of test compound was added to multiple wells of cells,
and the cells were incubated at 37 ꢀC for 1 h. Cells were
then infected at a multiplicity of infection of 0.025
TCID50/cell by the addition of a diluted stock of the
HTLV-IIIB strain of HIV-1. Compounds were tested
in triplicate wells per concentration for infected cells
and in duplicate wells per concentration for uninfected
cells.
4.2.25. 3-Hydroxy-30-(9-methoxy-3,3-dimethyl-7,10-dioxo-
7,10-dihydro-3H-benzo[f]chromen-8-yl)-2,20-binaphthale-
nyl-1,4,10,40-tetraone (14). The chloromethoxybiquinone
12 (0.220 g, 0.48 mmol), hydroxyquinone anion 13
(0.102 g, 0.48 mmol) and 18-crown-6 (0.127 g,
0.48 mmol) were placed in a 25 mL round bottom flask
fitted with a T-bore stopcock. After three vacuum and
nitrogen cycles, 15 mL of anhydrous NMP was added.
The suspension was stirred at 65 ꢀC for 2 days. The dark
red suspension was acidified with concentrated HCl to
pH 2 (litmus) and diluted with 100 mL CHCl3. The mix-
ture was washed with 10% K2CO3 and the organic layer
dried (MgSO4), filtered and evaporated to yield a red
solid. Recrystallization from methanol yielded 0.04 g
(14%) of an orange solid: mp = 169–173 ꢀC; Rf = 0.26
(EtOAc); 1H NMR (500 MHz, DMSO-d6) d 1.36 (s,
6H), 3.82 (s, 3H), 5.88–5.91 (d, J = 13.0 Hz, 1H), 7.05–
7.08 (d, J = 10.6 Hz, 1H), 7.47–7.55 (m, 3H), 7.61–7.72
(m, 4H), 7.81–7.83 (d, J = 9.4 Hz, 1H), 7.97–7.99 (d,
J = 10.5 Hz, 1H), 8.07–8.09 (d, J = 9.4 Hz, 1H); 13C
NMR (125 MHz, DMSO-d6) d 27.8 (2C), 53.0, 76.4,
120.4, 120.5, 124.1, 124.2, 125.7, 126.8, 128.1, 128.3,
128.5, 128.9, 129.4, 131.3, 131.3, 131.4, 131.8, 132.4,
133.1, 133.2, 134.0, 134.3, 137.0, 137.2, 139.6, 156.8
(2C), 160.4, 177.8, 178.4, 180.3, 183.6, 189.8, 197.9;
FT-IR 3439, 2975, 2924, 1669, 1647, 1640, 1586, 1560,
1529, 1457, 1436, 1399, 1363, 1276, 1218, 1146, 1119,
1092, 1019 cmꢀ1; MS (FAB) m/z (relative intensity)
599 [(M+H)+, 26], 539 (20), 338 (19), 255 (78), 154
(60), 109 (100); HRMS (FAB) calcd for C36H23O9
(M+H)+: 599.1342. Found 599.1359.
Assay plates were incubated at 37 ꢀC in a humidi-
fied, 5% CO2 atmosphere and examined microscopi-
cally for toxicity and/or cytopathogenic effect. Once
the viral cytopathogenic effect was maximal (the
eighth day post-infection), the surviving cells in each test
well were quantified using the MTT assay.16 Cytoprotec-
tion and toxicity are reported as the concentration of
drug required to inhibit viral-mediated cytopathicity by
50% of infected controls (ID50) and to cause reduction
of cell growth by 50% of uninfected controls (TD50),
respectively. When a dose-dependent effect for either
anti-HIV activity or cytotoxicity was observed, values
for the 50% effective dose were calculated using the
dose–effect analysis software of Chou and Chou (Else-
vier-Biosoft).
Acknowledgments
We are grateful to the National Institutes of Health
(Grant No. AI43687) for their generous support of this
research. We thank Dr. Jonathan Coates of AMRAD
Operations Pty Ltd (Victoria, Australia) for the gift of
an authentic sample of conocurvone. CEM-T4 cells were
obtained from Dr. J. P. Jacobs through the AIDS Re-
search and Reference Reagent Program, Division of
AIDS, NIAID, NIH. We also thank University of Notre
Dame Mass Spectrometry Facility for assistance with
low- and high-resolution mass spectra. This research
was supported [in part] by the Intramural Research Pro-
gram of the NIH, National Cancer Institute, Center for
Cancer Research.
4.3. HIV-1 integrase assay
The concentration dependent inhibition of test com-
pounds against HIV-1 integrase was measured using an
oligonucleotide based in vitro assay (schematically shown
in Fig. 2A).13 The [50-32P] labeled oligonucleotide duplex
(20 nM) derived from the last 21 base pairs of the HIV-1
U5 LTR was mixed with 400 nM HIV-1 integrase in the
presence of 7.5 mM MnCl2 or MgCl2, 25 mM MOPS,
pH 7.2, and 14.3 mM 2-mercaptoethanol. Inhibitors were
then added and the reactions were incubated at 37 ꢀC for
1 h. Reactions were quenched by the addition of formam-
ide-containing gel loading dye and loaded onto 20%
(19:1) denaturing polyacrylamide gels. The gels were ana-
lyzed using a Molecular Dynamics Phosphorimager
(Sunnyvale, CA). IC50 values were determined from
two or three separate experiments.
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