Antiviral agents 2. Synthesis of trimeric naphthoquinone analogues of conocurvone and their antiviral evaluation against HIV

Article abstract of DOI:10.1016/j.bmc.2010.06.105

The synthesis of a new series of conocurvone analogues is presented that explores the importance of the pyran rings of conocurvone, their degree of unsaturation as well as the role of alkoxy functionalities as pyran ring replacements, for the inhibition of the HIV-1 integrase (IN) enzyme. Difficulties in synthesising a trimeric naphthoquinone where the central quinone bears a peri-dihydropyran ring was attributed to distortion of the electrophilic dihaloquinone successfully utilised in the past. Increased electron density could also be a factor in reducing reactivity. The desired central dihydropyran bearing trimeric naphthoquinone was successfully synthesised by using a more reactive bromo-tosyloxyquinone intermediate. A maleimide derivative, where the central quinone between the pendant hydroxyquinones was replaced, was successfully synthesised and although it exhibited comparable enzyme inhibitory activity it had negligible HIV inhibitory cellular activity. Compounds were assessed for activity in both in vitro assays using purified recombinant HIV-1 IN and demonstrated superior or comparable activity to conocurvone derivatives previously reported.

Full text of DOI:10.1016/j.bmc.2010.06.105

Bioorganic & Medicinal Chemistry 18 (2010) 6442–6450
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antiviral agents 2. Synthesis of trimeric naphthoquinone analogues
of conocurvone and their antiviral evaluation against HIV
a
a,
Ian T. Crosby a, , David G. Bourke , Eric D. Jones , Paula J. de Bruyn a,à, David Rhodes b,
,
*
Nick Vandegraaff b, , Susan Cox b, , Jonathan A. V. Coates b, , Alan D. Robertson b,§
a Medicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), 381 Royal Parade, Parkville, Victoria 3052, Australia
b AMRAD Operations Pty Ltd, 576 Swan Street, Richmond, Victoria 3121, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of a new series of conocurvone analogues is presented that explores the importance of the
pyran rings of conocurvone, their degree of unsaturation as well as the role of alkoxy functionalities as
pyran ring replacements, for the inhibition of the HIV-1 integrase (IN) enzyme. Difficulties in synthesising
a trimeric naphthoquinone where the central quinone bears a peri-dihydropyran ring was attributed to
distortion of the electrophilic dihaloquinone successfully utilised in the past. Increased electron density
could also be a factor in reducing reactivity. The desired central dihydropyran bearing trimeric naphtho-
quinone was successfully synthesised by using a more reactive bromo-tosyloxyquinone intermediate. A
maleimide derivative, where the central quinone between the pendant hydroxyquinones was replaced,
was successfully synthesised and although it exhibited comparable enzyme inhibitory activity it had neg-
ligible HIV inhibitory cellular activity. Compounds were assessed for activity in both in vitro assays using
purified recombinant HIV-1 IN and demonstrated superior or comparable activity to conocurvone deriv-
atives previously reported.
Received 4 May 2010
Revised 25 June 2010
Accepted 30 June 2010
Available online 3 August 2010
Keywords:
Conocurvone
Naphthoquinone trimers
Anti-HIV activity
HIV integrase inhibition
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
able to reduce viral loads with unprecedented kinetics, this drug
appears to have a low genetic barrier to resistance that results in
Human immunodeficiency virus (HIV) is the causative agent of
acquired immunodeficiency syndrome (AIDS) infection, a pan-
demic disease that continues to exact a serious toll worldwide.1
Currently, there are 25 anti-HIV-1 agents approved for clinical
use, which target a total of four clinically validated viral proteins
(Envelope, Reverse transcriptase, Integrase and Protease) and one
cellular target (CCR5).2 One of two recently FDA-approved drugs,
Raltegravir,3 targets the HIV integrase enzyme, the protein respon-
sible for the integration of newly synthesized HIV-1 DNA into the
host cellular chromosome. This process is absolutely required for
viral replication and can be recapitulated in vitro using recombi-
nant integrase (IN) enzyme. Such assays were pivotal for the iden-
tification and development of integrase strand transfer inhibitors
(INSTIs)4 that culminated in the successful approval of MK-518
(Raltegravir) for clinical use in October 2007. Since the introduc-
tion of Raltegravir into the clinical setting, data suggests that while
relatively rapid selection of drug-resistant viral species.1 Thus,
there is a clear need to investigate novel scaffolds displaying lim-
ited cross-resistance that interfere with HIV-1 IN in a mechanisti-
cally different manner to that of Raltegravir and the other INSTIs
currently in clinical trials.
Inhibitors of integrase have been identified by HTS campaigns
of propriety compound collections5 and by pharmacophore build-
ing and database mining.6,7 The solved crystal structures of 2
known INSTIs in the prototype foamy virus integrase enzyme8 is
anticipated to increase efforts in structure based design ap-
proaches. Screening of natural product libraries against integrase
has produced
a
number of novel inhibitors of integrase9–11
although none have yet progressed into a clinical development
phase. Thus ongoing exploration of the chemical space afforded
by natural products12 continues to be attractive means as identify-
ing new inhibitors. A case in point is conocurvone 1, which was iso-
lated by the NCI using bioassay guided fraction of extracts from the
West Australian Smoke Bush. Conocurvone13 1 has been shown to
an inhibitor of both HIV integrase and HIV mediated cell fu-
sion.14,15 We recently reported16 the synthesis of a series of cono-
curvone 1 analogues based on the core structure 2, where the
stereochemical complications associated with the chiral quater-
nary pyran ring carbons had been removed by gem-dimethyl sub-
stitution. This structural simplification also had the advantage of
* Corresponding author. Tel.: +61 3 99039536; fax: +61 3 99039582.
E-mail address: ian.crosby@pharm.monash.edu.au (I.T. Crosby).
Present address: Avexa Ltd, 576 Swan Street, Richmond, Victoria 3121, Australia.
Present address: Davies Collison Cave, 1 Nicholson Street, Melbourne, Victoria
à
3000, Australia.
§
Present address: Pharmaxis Ltd, 2/10 Rodborough Rd, Frenchs Forest, NSW 2086,
Australia.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.105

I. T. Crosby et al. / Bioorg. Med. Chem. 18 (2010) 6442–6450
6443
making the compounds less lipophilic. Compound 2 was also found
to be well tolerated in rats and fairly well tolerated in beagles but
with some signs of minor heptatoxicity being observed which led
to the decision that no further animal studies would be undertaken
on compound 2.
1,4-naphthoquinone, made by alkylation of 6-hydroxy-1,4-naph-
thoquinone, and 9-hydroxy-3H-naphtho[2,1-b]pyran-7,10-dione 6
using similar chemistry as for the synthesis of compounds 2–4.16
Compounds 7–9 were synthesised from 2,3-dichloro-1,4-naphtho-
quinone and the corresponding methoxy substituted 2-hydroxy-
1,4-naphthoquinones,17,18 again using similar based promoted
condensation.16
O
O
HO
O
O
HO
O
O
O
O
O
O
HO
O
O
O
O
O
O
O
O
RO
HO
O
HO
O
1
6
3 R = Me
4 R = (CH2)2Me
5 R = CH(Me)2
O
O
HO
O
O
O
O
R"
R'
HO
O
O
O
O
O
R
R
O
R'
HO
O
O
2
HO
R"
7
8
9
R = OMe; R', R" = H
R" = OMe; R, R' = H
R', R" = OMe; R = H
This novel class of HIV-inhibitory compounds remains of inter-
est to us due to their unique structure and potential for inhibition
of the recently validated clinical target, HIV integrase. Here we
present the synthesis of a range of naphthoquinone ‘trimers’ that
explore the importance of the pyran rings, their degree of unsatu-
ration, as well as the role of alkoxy functionalities on anti-integrase
and anti-HIV activity.
‘Symmetrical’ trimeric naphthoquinones 10–13 could be syn-
thesised by ‘self-trimerisation’ of the corresponding monomeric
hydroxyquinones by prolonged heating in 1,2-dimethoxyethane
in the presence of Hünig’s base (iPr2NEt).
O
O
HO
O
HO
O
O
O
O
O
O
O
O
O
O
O
O
O
HO
O
HO
O
11
10
O
O
O
HO
OMe
HO
O
O
O
O
MeO
MeO
OMe
OMe
O
O
O
O
HO
OMe
HO
13
12
O
Compound 13 was initially isolated as a self-trimerisation
by-product from the synthesis of 9 in a reaction of 2,3-dichloro-
1,4-naphthoquinone with 2-hydroxy-6,7-dimethoxy-1,4-naphtho-
quinone. For characterisation purposes, 13 was synthesised using
the more reactive 2-bromo-3-(40-toluenesulfonyloxy)-6,7-dime-
thoxy-1,4-naphthoquinone 14, as the corresponding dichloronaph-
2. Results and discussion
2.1. Chemistry
Compounds 2 –4 were synthesised as previously reported.16
Compound 5 was synthesised from 2,3-dichloro-6-isopropoxy-

6444
I. T. Crosby et al. / Bioorg. Med. Chem. 18 (2010) 6442–6450
O
O
O
O
O
MeO
MeO
OH
MeO
MeO
OH
Br
MeO
MeO
OTs
Br
Br2
TsCl / THF
CH2Cl2
O
N Me
O
16
O
14
15
MeO
MeO
OH
iPr2NEt
DME
15
O
O
O
HO
OMe
OMe
O
O
MeO
O
O
OMe
OMe
MeO
HO
13
Scheme 1.
such as 17, also proved to be more difficult to synthesise and a
slightly modified strategy was adopted.
O
O
O
O
O
OH
O
OH
(1) H2, PtO2
(2) Air
O
HO
O
O
6
19
O
O
O
O
SOBr2 / CH2Cl2
O
HO
O
17
TsCl,
py, iPr2NEt
CH2Cl2
O
O
O
O
OH
O
OTs
Br
It was thought that the peri-dihydropyran ring was distorting
the adjacent carbonyl, thereby reducing the reactivity of the qui-
none as well as increasing the electron density of the naphthoqui-
none. In order to increase the reactivity, and to allow for
chemoselection of the central quinone, 8-bromo-9-tosyloxy-3H-
naphtho[2,1-b]pyran-7,10-dione 18 was utilised. This was synthes-
Br
O
20
18
Scheme 2.
ised from 9-hydroxy-3H-naphtho[2,1-b]pyran-7,10-dione
6 by
hydrogenation to 19,16 brominating to give 20 and finally tosyla-
tion to afford 18 (Scheme 2).
thoquinone gave a complex mixtures of products possibly due to
its lower reactivity resulting from the increased electron density
due to the two methoxy groups. Compound 13 was made by reac-
tion of 2-hydroxy-6,7-dimethoxy-1,4-naphthoquinone 1519 with
the reactive 2-bromo-3-(40-toluenesulfonyloxy)-6,7-dimethoxy-
1,4-naphthoquinone 14 (Scheme 1). 2-Bromo-3-(40-toluenesulfo-
nyloxy)-6,7-dimethoxy-1,4-naphthoquinone 14 was synthesised
from 2-hydroxy-6,7-dimethoxy-1,4-naphthoquinone 1519 by bro-
mination to 16 followed by tosylation to afford 14 (Scheme 1).
A compound where the central quinone was substituted with a
pyran ring but had a different type of pendant naphthoquinone,
The bromo-tosyloxy compound 18 proved to be more reactive
with the tosyloxy group preferentially undergoing substitution in
the first base catalysed condensation reaction with a 9-hydroxy-
3H-naphtho[2,1-b]pyran-7,10-dione 6 to afford the bromodimer
21, respectively (Scheme 3). Condensation with a second molecule
of the hydroxyquinone 6 gave the trimeric compound 17 (Scheme
3), although self-trimerisation of the hydroxyquinone 6 gave a
complex reaction mixture and the reaction often ceased before
all of the bromodimer 21 had been consumed. The purification of
O
O
O
O
O
HO
HO
O
O
O
O
O
O
O
OTs
Br
O
O
O
O
6
6
O
Br
iPr2NEt
DME
iPr2NEt
DME
21
18
17
HO
O
Scheme 3.

I. T. Crosby et al. / Bioorg. Med. Chem. 18 (2010) 6442–6450
6445
O
O
HO
O
O
O
Cl
Cl
O
O
O
i
Pr2NEt
N
N
HO
O
O
O
DME
HO
O
O
6
22
23
Scheme 4.
Table 1
Anti-HIV and integrase data for newly synthesized trimeric naphthoquinones
Table 2
Pharmacokinetics for 17
Rat pharmacokineticsa
a
b
c
d
Compound
EC50
(l
M)
CC50
(l
M)
IC50 (30)
IC50 (30-ST)
2
3
4
0.029
0.027
0.009
0.24
0.39
ND
10
10
1
>20
>20
ND
>20
ND
2.5
2.3
2.8
3.0
1.7
ND
1.8
4.5
2.0
2.5
5.0
Clp (mL/h)
Vdss (mL)
0.44
27.7
50.2
45
3.2
7.5
Cmax
t1/2 (h)-IV
Cmax g/mL)-PO
(l
g/mL)-IV
5
>100
3.0
10
11
17
23
(l
9.0
6.0
4
Tmax (h)
t1/2 (h)-PO
%F
0.47
>20
21.7
2.6
a
a
Antiviral activity was calculated following measurement of P24 levels in culture
Average data generated after 5 mg/kg po
supernatants on day 5 of a spreading replication assay performed in the presence of
serial dilutions of compound using a commercially available kit (Vironostika HIV-1
Antigen kit, Biomerieux). Spreading replication assays were performed at an MOI of
0.001 in CEM cells using HIV-1Rf strain.
(n = 2 animals/dose) and 2 mg/kg IV (n = 3
animals/dose).
b
Cytotoxicity assays were performed in CEM cells using the commercially
Table 3
available ATPlite kit according to the manufacturers protocol (Perkin Elmer).
Anti-HIV and integrase data for newly synthesized trimeric naphthoquinones
c
30-Processing assays (30) were performed as previously described11 and assess
Compound EC50 CC50 MW
IC50 (30) IC50 (30-ST) PSAa
the ability of recombinant HIV-1 integrase to catalyse the strand transfer reaction
only.
(l
M) M) M) M)
(
l
(
l
(l
d
30-Strand transfer (30ST) assays were performed as previously described11 and
17
7
8
9
12
13
0.47
>20
>20
>20
16
>20
ND
ND
ND
48
1.8
6.0
13.0
10
12
13
170.57 750.74
161.34 562.48
161.34 562.48
assess the ability of recombinant HIV-1 integrase to catalyse both the 30processing
and strand transfer reactions.
ND
22.5
62
56
>150
179.8
622.53
142.88 502.43
198.26 682.58
ND
ND
10
these reaction mixtures was effected by a combination of flash
chromatography followed by preparative HPLC.
a
PSA calculated using Symyx Draw 3.3.
To explore the nature of the central linker, the central quinone
between the pendant hydroxyquinones was replaced with a
maleimide derivative. Accordingly, N-benzyl-3,4-dichloro-1H-pyr-
role-2,5-dione 22 was reacted with the 9-hydroxy-3H-naph-
Armed with a favourable activity profile in 17 the in vivo phar-
macokinetic properties of this compound was assessed in rats and
the results are summarised in Table 2.
tho[2,1-b]pyran-7,10-dione
analogue 23 (Scheme 4).
6 to afford the maleimide linked
After IV dosing, 17 was slowly absorbed through the stomach
with Tmax of 7.5 h. This could be attributable to the poor aqueous
solubility and dissolution of the compound. Given the high molec-
ular weight (750.8) and PSA (173.2) of 17 intestinal permeability
was expected to be limiting and comparison of the Cmax for IV
and PO confirms this, ultimately resulting in a low bioavailability
(2.6%).
Given these PK results for 17 we turned our attention to modu-
lating the molecular weight and PSA of the class by replacing the
pyran containing naphthoquinone arms with smaller substitutions
and these results are summarised in Table 3.
2.2. Results and discussion
Previously reported compounds 2–4 showed encouraging low
nM potency against HIV infected CEM cells, however we were con-
cerned with emergence of toxicity of this class both in cell culture
(see Table 1) and in vivo (data not shown) which could limit their
therapeutic utility. In order to explore this series further we em-
barked on further chemistry examining the nature of the central
naphthoquinone resulting in compounds 5, 10, 17 and 23. Replac-
ing the n-propyl group of 4 with iso-propyl in 5 shows a loss of
antiviral activity, however, this was balanced by less cellular toxic-
ity, interestingly both compounds showed the same level of activ-
ity in the 30 assay but 5 was inactive in the 30-ST assay.
We firstly removed all functionality of the trimeric system to
give the known compound 12 which showed encouraging antiviral
activity with an EC50 of 16 lM. For compounds 7–9, inclusion of
methoxy substituents had minimal effect on enzymatic activity
Reincorporation of the pyran unit back into the central quinone
in 10 and a reduced pyran unit in compound 17 afforded com-
pounds of similar activities against HIV as 5, and once again with
diminished cellular toxicity. Of note is compound 23 where the
central napthoquinone has been replaced with a maleimide linker.
Although this compound retains comparable enzyme activity to
2–5 and 10, anti-HIV activity was abolished.
compared to 12 but resulted in diminished antiviral activity (all
EC50 >20 lM), suggesting the pyran rings are required for activity.
A comparison of note is the trend of compounds 4–7 which both
feature the same oxygenation pattern around the trimeric core
but vastly different activities against HIV.
In this paper we report a further series of trimeric naphthoqui-
nones and their ability to inhibit HIV and the integrase enzyme.

6446
I. T. Crosby et al. / Bioorg. Med. Chem. 18 (2010) 6442–6450
Although promising anti-HIV data was obtained, the therapeutic
use of these compounds could be limited by their low stomach per-
meability. Although promising anti-HIV data was obtained and
that cell based assays indicated that toxicity was lower for some
compounds, it was deemed that the poor bioavailability in rats
(2.6%) of 17 did not warrant a toxicity study in dogs. Attempts to
improve the chemical properties of the series by reducing molecu-
lar weight diminished activity. Worth noting is the discordance be-
tween enzymatic data and cell based data which suggest inhibition
of HIV may not be related to inhibition of integrase. Future publi-
cations will explore this further.
retention time of 7.9 min by analytical HPLC (gradient) were
combined and triturated with ethyl acetate and hexanes to afford
9-hydroxy-8-(30-(900-hydroxy-300,300-dimethyl-700,1000-dioxo-700,1000-dihy-
dro-300H-naphtho[2,1-b]pyran-800-yl)-1,40-dioxo-60-isopropyloxy-10,40-
dihydronaphthalen-20-yl)-3,3-dimethyl-3H-naphtho[2,1-b]pyran-7,10-
dione 5 (23 mg, 8%) as a orange powder, mp 167–170 °C.
m
.
max 3440
(br w), 2995 (w), 1650 (m), 1645 (m), 1594 (m) cmꢀ1
1H NMR
(300 MHz, CDCl3) 1.40 (6H, d, J 5, OCH(CH3)2), 1.48 (12H, s,
C–CH3), 4.77 (1H, m, OCH(CH3)2), 5.97 (2H, d, J 10, H2, H200), 7.10
(2H, m, H5, H500), 7.18 (1H, d, J 8, H70), 7.56 (1H, br s, H50), 7.69
(2H, d, J 10, H1, H100), 7.92 (2H, m, H6, H600), 8.09 (1H, br d, J 8,
H80). ESI-MS (positive, 30 V) 725 (M+H, 32%), 338 (100), 214 (90).
þ
HR-ESI-MS (positive) Calcd for
725.2045.
C
43H33O11 725.2023; found
3. Experimental
3.1. Chemistry
3.2. Synthesis of methoxy substituted 2-hydroxy-1,4-naphtho-
quinones
3.1.1. General experimental
Most of the general experimental has previously described.16
HPLC was carried out on a C18 column using mixtures of Sol-
vent A (90% acetonitrile in water) and Solvent B (0.05% aqueous
phosphoric acid) detecting at 275 nm. Unless otherwise stated ana-
lytical Gradient HPLC used a flow rate of 1.5 mL/min on a C18 col-
umn beginning elution with 70% A, 30% B, going to 79% A, 21% B
over 15 min, then to 100% A over 3 min and finally 100% A for
8 min. Preparative HPLC was carried out on of solutions of crude
The various methoxy substituted were synthesized by reaction
of the corresponding methoxy substituted 1-tetralones with oxy-
gen in the presence of potassium t-butoxide in t-butanol.17,18
3.2.1. 2-Hydroxy-5-methoxy-1,4-naphthoquinone
3,4-Dihydro-5-methoxy-1(2H)-naphthalenone (2.00 g, 11.48
mmol) afforded 2-hydroxy-5-methoxy-1,4-naphthoquinone (1.10
g, 47%) as fine yellow crystals, mp 170–173 °C (lit.21 176–177 °C).
1H NMR (300 MHz, CDCl3) 3.88 (3H, s, OCH3), 6.00 (1H, s, H3),
7.53 (1H, d, J 8.5, H6), 7.63 (1H, d, J 6.5, H8), 7.74 (1H, distorted
t, H7), 11.19 (1H, br s, OH). ESI-MS (positive, 30 V) 227 (M+Na,
20%), 205 (M+H, 100%).
compounds filtered through HPLC grade filters (0.22 lm) and then
isocratically chromatographed in the specified solvent system at
flow rate 5 mL/min, unless otherwise stated.
3.1.2. 2,3-Dichloro-6-isopropyloxy-1,4-naphthoquinone
To a solution of 2,3-dichloro-6-hydroxynaphthoquinone20 (510
mg, 2.10 mmol) in dichloromethane (30 mL) was added silver(I)
3.2.2. 2-Hydroxy-7-methoxy-1,4-naphthoquinone
3,4-Dihydro-7-methoxy-1(2H)-naphthalenone (520 mg, 2.8 mmol)
gave 2-hydroxy-6-methoxy-1,4-naphthoquinone (214 mg, 36%) as
fluffy orange needles, mp 210–213 °C (lit.18 214–215 °C). 1H NMR
(300 MHz, (CD3)2SO) 3.91 (3H, s, OCH3), 6.07 (1H, s, H3), 7.35
(1H, br d, J 9, H6), 4.08 (1H, br s, H8), 7.88 (1H, br d, J 9, H5).
FAB-MS (3NBA) 205 (M+H).
oxide (973 mg, 4.20 mmol) and 2-bromopropane (394 lL, 4.20
mmol). The mixture was stirred at room temperature for 19 h,
filtered through CeliteÒ and the residue washed with dichloro-
methane until no further colour was eluted. Removal of the vola-
tiles in vacuo gave a brown residue (497 mg) which was purified
by flash chromatography (10% ethyl acetate/hexanes with 1% ace-
tic acid) to afford 2,3-dichloro-6-isopropyloxy-1,4-naphthoquinone
(221 mg, 37%) as yellow microprisms, mp 111–112 °C. mmax 1678
3.2.3. 2-Hydroxy-6,7-dimethoxy-1,4-naphthoquinone 15
3,4-Dihydro-6,7-dimethoxy-1(2H)-naphthalenone (2.00 g, 9.7
mmol) gave 2-hydroxy-6,7-dimethoxy-1,4-naphthoquinone 15
(508 mg, 22%) as fine yellow crystals, mp 210 °C (decomp.) (lit.19
212 °C (decomp.)). 1H NMR (300 MHz, CDCl3) 4.02 (3H, s, OCH3),
4.04 (3H, s, OCH3), 6.24 (1H, s, H3), 7.34 (1H, br s, OH), 7.51, 7.55
(1H, 1H, s, s, H5, H8). ESI-MS (positive, 30 V) 227 (M+Na, 20%),
205 (M+H, 100%). Unreacted starting material (1.31 g, 65.5%) was
also recovered.
(s), 1578 (m) cmꢀ1 1H NMR (300 MHz, CDCl3) 1.40 (6H, d, J 6,
.
OCH(CH3)2), 4.77 (1H, septet, J 6, OCH(CH3)2), 7.17 (1H, dd, J 8, 2,
H7), 7.56 (1H, d, J 2, H5), 8.12 (1H, d, J 8, H8). Calcd for C13H10Cl2O3:
C, 54.8, H, 3.5; found: C, 54.8; H, 3.6.
3.1.3. 9-Hydroxy-8-(30-(900-hydroxy-300,300-dimethyl-700,1000-dioxo-
700,1000-dihydro-300H-naphtho[2,1-b]pyran-800-yl)-1,40-dioxo-60-iso-
propyloxy-10,40-dihydronaphthalen-20-yl)-3,3-dimethyl-3H-naph-
tho[2,1-b]pyran-7,10-dione 5
A mixture of 2,3-dichloro-6-isopropyloxy-1,4-naphthoquinone
(112 mg, 0.393 mmol), 9-hydroxy-3,3-dimethyl-3H-naphtho[2,1-
b]pyran-7,10-dione16 6 (221 mg, 0.864 mmol) and diisopropyleth-
3.2.4. 2-Hydroxy-3-(30-(200-hydroxy-500-methoxy-100,400-dioxo-100,
400-dihydronaphthalene-300-yl)-10,40-dioxo-100,400-dihydronaphtha-
lene-20-yl)-5-methoxy-1,4-naphthoquinone 7
ylamine (684
l
L, 3.92 mmol) in 1,2-dimethoxyethane (10 mL) was
A mixture of 2-hydroxy-5-methoxy-1,4-naphthoquinone (300
mg, 1.46 mmol), 2,3-dichloro-1,4-naphthoquinone (150 mg, 0.66
mmol) and diisopropylethylamine (1.15 mL, 6.60 mmol) in
1,2-dimethoxyethane (15 mL) was heated at reflux for 10 h under
nitrogen. The reaction mixture was cooled, poured into hydrochlo-
ric acid (1 M) and extracted with ethyl acetate and dichlorometh-
ane. The extracts were then combined and washed with aqueous
sodium bicarbonate solution (5%, 3 ꢁ 15 mL). The combined alka-
line extracts were acidified with concentrated hydrochloric acid
and extracted with ethyl acetate and dichloromethane. The com-
bined organic extracts were evaporated in vacuo and the residue
(440 mg) subjected to flash chromatography using 25% ethyl
acetate/hexanes (containing 1% acetic acid) to give a mixture of
heated at reflux for 70 h under nitrogen. The mixture was cooled,
poured into aqueous hydrochloric acid (1 M) and extracted with
ethyl acetate and dichloromethane. The combined extracts were
evaporated and the residue subjected to flash chromatography in
24% ethyl acetate in hexanes containing 1% acetic acid. The result-
ing residue (321 mg) gave a mixture of the hydroxyquinone 6 and
other unidentified compounds. Further elution of the flash column
with 9:1 dichloromethane/methanol containing 1% acetic acid
afforded a red/brown solid (70 mg) which was dissolved in 9:1
ethyl acetate/90% aqueous formic acid and filtered through a plug
of CeliteÒ to give a dark orange solid (33 mg). This was subjected to
preparative HPLC (isocratic 70% A, 30% B) and fractions with a

I. T. Crosby et al. / Bioorg. Med. Chem. 18 (2010) 6442–6450
6447
2-hydroxy-5-methoxy-1,4-naphthoquinone and other unidentified
compounds. Further elution with 9:1 dichloromethane/methanol
(containing 1% acetic acid) afforded a red solid (92 mg). This was
dissolved in ethyl acetate/90% aqueous formic acid 9:1 and filtered
through a plug of CeliteÒ to give a red solid (73 mg) which was
subjected to preparative HPLC (70% A–50% A over 60 min, flow rate
5 mL/min). Fractions that had a retention time of 15.1 min by ana-
lytical HPLC (30% A, 70% B isocratic) were combined and triturated
with ethyl acetate and hexanes to provide 2-hydroxy-3-(30-(200-hy-
droxy-500-methoxy-100,400-dioxo-100,400-dihydronaphthalene-300-yl)-10,40-
dioxo-100,400-dihydronaphthalene-20-yl)-5-methoxy-1,4-naphthoquinone
7 (30 mg, 8%) as a yellow solid, mp 211–213 °C. mmax 3215 (br w),
3.2.6. 2-Hydroxy-3-(30-(200-hydroxy-600,700-dimethoxy-100,400-dioxo-
100,400-dihydronaphthalene-300-yl)-10,40-dioxo-10,40-dihydronaphtha-
lene-20-yl)-6,7-dimethoxy-1,4-naphthoquinone 9
A
mixture of 2-hydroxy-6,7-dimethoxy-1,4-naphthoquinone
(750 mg, 3.20 mmol), 2,3-dichloro-1,4-naphthoquinone (727 mg,
3.20 mmol) and diisopropylethylamine (5.6 mL, 32 mmol) in 1,2-
dimethoxyethane (75 mL) was stirred at room temperature for
23 h under nitrogen. A further portion of 2-hydroxy-6,7-dime-
thoxy-1,4-naphthoquinone (750 mg, 3.20 mmol) was added and
the mixture heated under reflux for an additional 24 h. The reac-
tion was then cooled, poured into hydrochloric acid (1 M) and ex-
tracted with dichloromethane (2 ꢁ 200 mL). The combined
extracts were washed with aqueous sodium bicarbonate (5%,
9 ꢁ 250 mL) during which water was added to break up the emul-
sions that formed. The alkaline extracts were combined, carefully
acidified with concentrated hydrochloric acid and extracted with
chloroform (2 ꢁ 500 mL). The combined extracts were dried and
concentrated in vacuo to provide a brown solid (1.84 g) which
was 80% 9 by analytical HPLC. This residue was dissolved in dichlo-
romethane containing 1% acetic acid (25 mL) and the solution sub-
jected to flash chromatography. Elution with dichloromethane
containing 1% acetic acid (7.0 L) removed unreacted 2-hydroxy-
6,7-dimethoxy-1,4-naphthoquinone, the dimer intermediate and
other unidentified compounds. Elution with chloroform containing
1% acetic acid (5.0 L) afforded the crude product, as a red solid
(1.4 g, 70%). A small sample (126 mg) was subjected to preparative
HPLC (isocratic 70% A, 30% B). Fractions that had a retention time of
10.5 min by analytical HPLC (gradient) were combined to give
2-hydroxy-3-(30-(200-hydroxy-600,700-dimethoxy-100,400-dioxo-100,400-dihy-
dronaphthalene-300-yl)-10,40-dioxo-10,40-dihydronaphthalene-20-yl)-6,7-
dimethoxy-1,4-naphthoquinone 9, as a red solid, mp 215 °C. mmax
1668 (s), 1582 (s), 1276 (br s), 1026 (m) cmꢀ1 1H NMR (300 MHz,
.
CD3OD) 3.90 (6H, s, 2 ꢁ OCH3), 7.39–7.53 (2H, m, H7, H700), 7.61–
7.74 (4H, m, H6, H8, H600, H800), 7.82–7.92 (2H, m, H60, H70), 8.10–
8.20 (2H, m, H50, H80). ESI-MS (positive, 30 V) 601 (M+K, 17%),
585 (M+Na, 30%), 563 (M+H, 100%). HR-ESI-MS (positive) Calcd
þ
for C30H23O8 563.0973; found 563.0960.
3.2.5. 2-Hydroxy-3-(30-(200-hydroxy-700-methoxy-100,400-dioxo-100,400-
dihydronaphthalene-300-yl)-10,40-dioxo-10,40-naphthalene-20-yl)-
1,4-dioxo-1,4-dihydronaphthalene 8
Diisopropylethylamine (0.45 mL) was added to a stirred solu-
tion of 2-hydroxy-7-methoxy-1,4-naphthoquinone (130 mg) and
2,3-dichloro-1,4-naphthoquinone (60 mg) in 1,2-dimethoxyethane
(5 mL). The resulting red solution was heated at reflux for 6 h, al-
lowed to cool, diluted with dichloromethane and washed with
aqueous hydrochloric acid (1 M). The organic phase was dried
and concentrated, and the resulting residue dissolved in a mixture
of dichloromethane (10 mL), acetic anhydride (20 drops) and pyr-
idine (9 drops). After standing for 30 min, water was added and
the mixture was vigorously stirred for 15 min. The organic phase
was washed with aqueous hydrochloric acid (1 M), dried and con-
centrated to give a brown residue which was subjected to flash
chromatography (30% ethyl acetate in hexanes). The major mobile
yellow band afforded 2-acetoxy-3-(30-(200-acetoxy-700-methoxy-
100,400-dioxo-100,400-dihydronaphthalene-300-yl)-10,40-dioxo-10,40-naph-
thalene-20-yl)-7-methoxy-1,4-naphthoquinone (trimeric diacetate
intermediate) as a yellow solid (115 mg, 68%), mp 167–171 °C
3325 (br m), 1668 (br s), 1578 (s), 1510 (s) cmꢀ1 1H NMR
.
(300 MHz, CDCl3) 3.97 (3H, s, OCH3), 4.00 (6H, s, 2 ꢁ OCH3), 4.04
(3H, s, OCH3), 7.45 (2H, br s, H5, H500 or H8, H800 (isomers a and
b)), 7.55, 7.60 (2H, s, H5, H500 or H8, H800, isomer b, isomer a),
7.75–7.84 (2H, m, H60, H70), 8.15–8.24 (2H, m, H50, H80) [isomers
a:b; 5.2:1]. ESI-MS (positive, 30 V) 623 (M+H, 10%), 400 (100), 215
(55). Calcd for C34H22O12ꢃH2O: C, 63.8; H, 3.8; found: C, 63.5; H,
þ
3.7. HR-ESI-MS (positive) Calcd for C34H23O12 623.1184; found
(decomp.). mmax 1784 (m), 1678 (br s), 1660 (s), 1594 (s) cmꢀ1
.
1H
623.1202.
NMR (300 MHz, CDCl3) 2.12 (6H, s, 2 ꢁ OAc), 3.94 (6H, s, 2 ꢁ OMe),
7.24 (2H, dd, J 9, 2, H6, H600), 7.50 (2H, d, J 2, H8, H800), 7.81–7.84 (2H,
m, H60, H70), 8.04 (2H, d, J 9, H5, H500), 8.17–8.20 (2H, m, H50, H80).
m/z (FAB, 3NBA) 647 (M+H, 7%), 646 (MꢀH+1, 3), 604 (13), 563
(22), 562 (40), 358 (16).
3.2.7. 9-Hydroxy-8-(80-(900-hydroxy-300,300-dimethyl-700,1000-dioxo-
700,1000-dihydro-300H-naphtho[2,1-b]pyran-800-yl)-30,30-dimethyl-
70,100-dioxo-,70,100-dihydro-30H-naphtho[2,1-b]pyran-90-yl)-3,3-
dimethyl-3H-naphtho[2,1-b]pyran-7,10-dione 10
Ground aluminium chloride (130 mg) was added to a stirred
solution of the trimeric diacetate intermediate (42 mg) in dichlo-
romethane (3 mL) and the resulting green mixture stirred at
room temperature for 1.5 h. Additional aluminium chloride
(50 mg) was added, stirring maintained for a further 45 min,
the mixture was diluted with dichloromethane (20 mL) and then
washed with aqueous oxalic acid (5%, 2 ꢁ 70 mL). The yellow
organic phase was washed with water, dried and partially
concentrated to a volume of ꢂ5 mL. The solution was heated to
boiling and hexanes (1 mL) carefully added before being allowed
to stand overnight. The solid which precipitated was collected by
filtration and washed with cold dichloromethane to afford the
A solution of 9-hydroxy-3,3-dimethyl-3H-naphtho[2,1-b]pyr-
an-7,10-dione16 6 (500 mg, 1.95 mmol) and diisopropylethylamine
(342 lL, 1.96 mmol) in 1,2-dimethoxyethane (10 mL) was heated
under reflux under nitrogen for 2.5 days whilst monitoring the
reaction by HPLC indicated the significant formation of trimeric
products. The reaction was then cooled, aqueous hydrochloric acid
(1 M) added and the mixture extracted with ethyl acetate until the
extracts were colourless. The combined extracts were washed with
aqueous sodium hydrogen carbonate (5%) until the washings were
colourless. The organic phase was then evaporated to dryness to
leave a residue (593 mg) which was purified by flash chromatogra-
phy (eluting sequentially with 500 mL of 40% ethyl acetate in
hexanes, ethyl acetate, 10% methanol in dichloromethane and
finally methanol; all containing 1% acetic acid). The fractions that
contained trimeric material (rt = 10.0 min, gradient) (280 mg) were
subjected to isocratic preparative HPLC (70% A, 30% B, rt =
15.0 min) to give 9-hydroxy-8-(80-(900-hydroxy-300,300-dimethyl-700,1000-
dioxo-700,1000-dihydro-300H-naphtho[2,1-b]pyran-800-yl)-30,30-dimethyl-
70,100-dioxo-70,100-dihydro-30H-naphtho[2,1-b]pyran-90-yl)-3,3-di-
methyl-3H-naphtho[2,1-b]pyran-7,10-dione 10 (14 mg, 3%), mp
deacetylated trimer
8 as a yellow amorphous solid (27 mg,
75%), mp 238–239 °C (decomp.). 1H NMR (300 MHz, d6-DMSO)
3.89 (6H, s, 2 ꢁ OMe), 7.29, 7.36 (2H, br d, br d, J 8.5, H6, H600
(isomer a), (isomer b)), 7.38 (2H, br s, H8, H800), 7.77, 7.89 (2H,
d, d, J 8.5, H5, H500 (isomer a), (isomer b)), 7.94–7.97 (2H, m,
H60, H70), 8.08–8.11 (2H, m, H50, H80) [isomer a:b; 1:1.2]. m/z
FAB-MS (3NBA) 564 (M+H+1, 15%), 563 (M+H, 40), 662
(M+Hꢀ1, 61), 561 (Mꢀ1, 22), 358 (26). Calcd for C32H18O10.H2O:
C, 66.2; H, 3.5; found: C, 66.2; H, 3.7.
205–211 °C (decomp.). kmax (log e) 223, 275, 293 sh, 401 nm (4.64,

6448
I. T. Crosby et al. / Bioorg. Med. Chem. 18 (2010) 6442–6450
4.40, 4.38, 3.97). mmax 3420 (br m), 1655 (m), 1650 (m), 1564 (m)
cmꢀ1 1H NMR (300 MHz, CDCl3) 1.48 (18H, s, C–CH3), 5.88 (1H, d,
HPLC (gradient) were combined, triturated with ethyl acetate and
hexanes to provide 2-hydroxy-3-(30-(200-hydroxy-100,400-dioxo-
100,400-dihydronaphthalene-300-yl)-10,40-dioxo-10,40-dihydronaphtha-
lene-20-yl)-1,4-naphthoquinone22 12 (14 mg, 4%) as a brown/or-
ange solid, mp 245 °C. mmax 3300 (br w), 1674 (s), 1640 (s), 1592
.
J 10, H20), 5.96 (2H, d, J 10, H2, H200), 7.02–7.15 (3H, m, H5, H50,
H500), 7.64–7.72 (3H, m, H1, H10, H100), 7.91–8.06 (3H, m, H6, H60,
H600). ESI-MS (positive, 100 V) 750 (M+H+1, 22%), 749 (M+H, 46),
559 (48), 388 (100), 145 (63), 129 (49), 117 (68), 112 (54). HR-ESI-
(m) cmꢀ1 1H NMR (300 MHz, CD3OD) 7.65–7.83 (m, 7 ArH),
.
þ
MS (positive) Calcd for C45H33O11 749.2017; found 749.2009.
7.83–7.94 (m, 3 ArH), 7.96–8.09 (m, 3 ArH), 8.11–8.20 (m, 1
ArH). ESI-MS (positive, 30 V) 601 (M+K, 17%), 585 (M+Na, 30%),
503 (M+H, 100%). These data is in good agreement with that for
12 in the NCI patent for 12.22
3.2.8. 9-Hydroxy-8-(80-(900-hydroxy-300,300-dimethyl-700,1000-dioxo-
100,200,700,1000-tetrahydro-300H-naphtho[2,1-b]pyran-800-yl)-30,30-
dimethyl-70,100-dioxo-10,20,70,100-tetrahydro-30H-naphtho[2,1-b]-
pyran-90-yl)-1,2-dihydro-3,3-dimethyl-3H-naphtho[2,1-b]pyran-
7,10-dione 11
3.2.10. 2-Bromo-3-hydroxy-6,7-dimethoxy-1,4-naphthoquinone
16
A
mixture of 9-hydroxy-3,3-dimethyl-1,2-dihydro-3H-naph-
A solution of bromine (77 mg, 0.48 mmol) in dry dichlorometh-
ane (1 mL) was added dropwise to a cooled (ꢀ5 °C), stirred solution
of 2-hydroxy-6,7-dimethoxy-1,4-naphthoquinone19 15 (100 mg,
0.43 mmol) in dry dichloromethane (14 mL) and glacial acetic acid
(1 drop) under nitrogen. After 15 min stirring at room temperature
the volatiles were removed in vacuo to afford, as red micro crystals,
2-bromo-3-hydroxy-6,7-dimethoxy-1,4-naphthoquinone 16 (133
mg, 100%), mp 275–277 °C. mmax 3348 (br m), 1658 (m), 1648
tho[2,1-b]pyran-7,10-dione 1916 (500 mg, 1.94 mmol) and diiso-
propylethylamine (0.337 mL, 1.94 mmol) in 1,2-dimethoxyethane
(10 mL) was heated at reflux for 70 h under nitrogen. The reaction
mixture was cooled, poured into aqueous hydrochloric acid (1 M)
and extracted with ethyl acetate and dichloromethane. The ex-
tracts were combined and the volatiles removed in vacuo. The
resulting residue was dissolved in ethyl acetate (20 mL), washed
with aqueous sodium bicarbonate (5%, 3 ꢁ 20 mL). The organic
phase was separated, dried and volatiles removed in vacuo. The
resulting residue (452 mg) was subjected to flash chromatography
in 25% ethyl acetate in hexanes (containing 1% acetic acid) to give
starting material 19 (44 mg) and other unidentified compounds
(191 mg). Further elution with 9:1 dichloromethane/methanol
(containing 1% acetic acid) followed by 1% acetic acid in methanol
afforded a red solid (420 mg). This solid was dissolved in ethyl ace-
tate/90% aqueous formic acid (9:1) and filtered through CeliteÒ, to
give a dark orange solid (190 mg) which was subjected to prepara-
tive HPLC (isocratic 70% A, 30% B). Fractions that had a retention
time of 11.3 min by analytical HPLC (gradient) were combined
and triturated with ethyl acetate and hexanes to afford 9-hydro-
xy-8-(80-(900-hydroxy-300,300-dimethyl-700,1000-dioxo-100,200,700,1000-tetra-
hydro-300H-naphtho[2,1-b]pyran-800-yl)-30,30-dimethyl-70,100-dioxo-10,
20,70,100-tetrahydro-30H-naphtho[2,1-b]pyran-90-yl)-1,2-dihydro-3,3-
dimethyl-3H-naphtho[2,1-b]pyran-7,10-dione 11 (22 mg, 1.5%) as a
yellow powder, mp 210–215 °C. mmax 3375 (br w), 1658 (br m),
(m), 1632 (m) cmꢀ1 1H NMR (300 MHz, (CD3)2SO) 3.92, 3.93 (3H,
.
3H, s, s, 2 ꢁ OCH3), 7.43, 7.45 (1H, 1H, s, s, H5, H8). ESI-MS (posi-
tive, 30 V) 315 (M[81Br]+H, 95%), 313 (M[79Br]+H, 100), 301 (44),
149 (50). This compound was used without further purification.
3.2.11. 2-Bromo-3-(40-toluenesulfonyloxy)-6,7-dimethoxy-1,4-
naphthoquinone 14
N-Methylmorpholine (67 lL, 0.78 mmol) was added to a stirred
solution of 2-hydroxy-3-bromo-6,7-dimethoxy-1,4-naphthoqui-
none 16 (122 mg, 0.39 mmol) in dry THF (15 mL). After 10 min a
solution of 4-toluenesulfonyl chloride (89 mg, 0.47 mmol) in dry
THF (2 mL) was added and the reaction was stirred at room tem-
perature for 2 h.
(335 L, 3.90 mmol) was added then after 4 drop more N-methyl-
morpholine (200 L, 1.43 mmol) and 4-toluenesulfonyl chloride
A further portion of N-methylmorpholine
l
l
(20 mg, 0.11 mmol) were added. After 20 h hydrochloric acid
(1 M, 10 mL) was added and the products were extracted with
ethyl acetate (10 mL) and dichloromethane (10 mL). The combined
organic extracts were dried and concentrated in vacuo. The result-
ing red solid (204 mg) was purified by flash chromatography, elut-
ing with ethyl acetate:hexane (3:7) to afford 2-bromo-3-(40-
toluenesulfonyloxy)-6,7-dimethoxy-1,4-naphthoquinone 14 (175 mg,
1470 (br m) cmꢀ1 1H NMR (300 MHz, CDCl3) 1.27, 1.29, 1.39
.
(6H, 6H, 6H, s, s, s, 3 ꢁ (CH3)2), 1.43–1.70 (2H, m, 2 ꢁ OH), 1.73–
1.90 (6H, m, H2, H20, H200), 3.08–3.20 (6H, m, H1, H10, H100), 7.00–
7.21 (3H, m, H5, H50, H500), 7.88–7.98, 8.03 (3H, m, d, J 8.5, H6,
H60, H600, isomers b and c, isomer a [isomers a:b:c; 4:1:1]. ESI-MS
(positive, 30 V) 755 (M+H, 7%), 491 (7), 361 (26), 360 (100). HR-
96%), mp 220–221 °C. mmax 1672 (s), 1572 (s) cmꢀ1 1H NMR
.
(300 MHz, CDCl3) 2.51 (3H, s, PhCH3), 4.02, 4.04 (3H, 3H, s, s,
2 ꢁ OCH3), 7.42 (2H, d, J 8, H20, H60), 7.53, 7.57 (2H, s, s, H5, H8),
7.98 (2H, d, J 8, H30, H50). ESI-MS (positive, 30 V) 491 (M[81Br]+Na,
96%), 489 (M[79Br]+Na, 100), 469 (M[81Br]+H, 35), 467 (M[79Br]+H,
33), 143 (42), 135 (65). Calcd for C19H1679BrO7S+ 466.9795; found
466.9814.
þ
ESI-MS (positive) Calcd for C45H39O11 755.2587; found 755.2461.
3.2.9. 2-Hydroxy-3-(30-(200-hydroxy-100,400-dioxo-100,400-dihydrona-
phthalene-300-yl)-10,40-dioxo-10,40-dihydronaphthalene-20-yl)-1,4-
naphthoquinone 1222
A mixture of 2-hydroxy-1,4-naphthoquinone (253 mg, 1.45
mmol), 2,3-dichloro-1,4-naphthoquinone (150 mg, 0.66 mmol)
and diisopropylethylamine (1.15 mL, 6.60 mmol) in 1,2-dime-
thoxyethane (15 mL) was heated at reflux under nitrogen for
14 h. The reaction mixture was cooled, poured into hydrochloric
acid (1 M) and extracted with ethyl acetate and dichloromethane.
The extracts were combined and the volatiles removed in vacuo.
Flash chromatography (25% ethyl acetate/hexanes containing 1%
acetic acid) of the resulting residue (406 mg) gave a mixture of
2-hydroxy-1,4-naphthoquinone and other unidentified com-
pounds. Further elution of the flash column with 10% methanol
in dichloromethane (containing 1% acetic acid) afforded a red solid
(145 mg) which was dissolved in ethyl acetate/90% aqueous formic
acid (9:1) and filtered through a plug of CeliteÒ to give a red solid
(84 mg). This was subjected to preparative HPLC (isocratic 60% A,
40% B). Fractions that had a retention time of 9.9 min by analytical
3.2.12. 2-Hydroxy-3-(30-(200-hydroxy-600,700-dimethoxy-100,400-dioxo-
dihydronaphthalene-300-yl)-10,40-dioxo-60,70-dimethoxy-naphtha-
lene-20-yl)-6,7-dimethoxy-1,4-naphthoquinone 13
A stirred mixture of 2-hydroxy-6,7-dimethoxy-1,4-naphthoqui-
none19 15 (80 mg, 0.35 mmol), 2-bromo-3-(40-toluenesulfonyl-
oxy)-6,7-dimethoxy-1,4-naphthoquinone 14 (75 mg, 0.16 mmol)
and diisopropylethylamine (280 lL, 1.6 mmol) in dry 1,2-dime-
thoxyethane (10 mL) was heated under reflux for 19 h. A second
portion of 2-hydroxy-6,7-dimethoxy-1,4-naphthoquinone 15
(80 mg, 0.35 mmol) and diisopropylethylamine (1.0 mL, 5.7 mmol)
were then added. After a further 77 h heating under reflux, the
mixture was cooled to room temperature and acidified using
hydrochloric acid (1 M). The mixture was extracted with ethyl ace-
tate (15 mL) and dichloromethane (15 mL), the extracts combined,
dried and evaporated. The resulting brown residue (350 mg) was

I. T. Crosby et al. / Bioorg. Med. Chem. 18 (2010) 6442–6450
6449
dissolved in ethyl acetate (100 mL) and extracted with aqueous so-
dium bicarbonate. The combined extracts were acidified with con-
centrated hydrochloric acid and extracted with ethyl acetate
(10 mL). The organic phase was dried and concentrated in vacuo
and the resulting brown solid (100 mg) was subjected to flash
chromatography, eluting with ethyl acetate:acetic acid:hexane
(50:1:49) followed by methanol:acetic acid:dichloromethane
(10:1:89). Evaporation of the fractions from the final elution affor-
ded a brown solid (16 mg) which was dissolved in ethyl ace-
tate:aqueous formic acid (90%) (9:1) and filtered through CeliteÒ.
Removal of the volatiles in vacuo afforded a brown solid (7.6 mg)
which was subjected to preparative HPLC (gradient 50% A, 50% B
to 100% A over 90 min, 5 mL/min). Fractions that had a retention
time of 10.2 min by analytical HPLC were combined to give a yel-
low-orange solid of 2-hydroxy-3-(30-(200-hydroxy-600,700-dimethoxy-
100,400-dioxo-dihydronaphthalene-300-yl)-10,40-dioxo-60,70-dimethoxy-
naphthalene-20-yl)-6,7-dimethoxy-1,4-naphthoquinone 13 (4.3 mg,
3.2.15. 8-(80-Bromo-30,30-dimethyl-70,100-dioxo-10,20,70,100-tetra-
hydro-30H-naphtho[2,1-b]pyran-90-yl)-9-hydroxy-3,3-dimethyl-
3H-naphtho[2,1-b]pyran-7,10-dione 21
Diisopropylethylamine (2.0 mL, 11.5 mmol) to a solution of 8-
bromo-3,3-dimethyl-9-p-toluenesulfonyloxy-1,2-dihydro-3H-naph-
tho[2,1-b]pyran-7,10-dione 20 (1.10 g, 2.23 mmol) and 9-hydroxy-
3,3-dimethyl-3H-naphtho[2,1-b]pyran-7,10-dione16 6 (0.575 g, 2.24
mmol) in 1,2-dimethoxyethane (25 mL) under nitrogen and the
resulting mixture was heated under reflux overnight. The reaction
mixture was cooled, poured into hydrochloric acid (1 M) then ethyl
acetate was added and the organic phase was separated. This was
washed with aqueous sodium hydrogen carbonate solution (5%),
water then dried and the volatiles were removed in vacuo. Flash
chromatography (15–50% ethyl acetate in hexanes with 1% acetic
acid) of the resulting residue (1.27 g) gave a brown solid which
was triturated with ethyl acetate/hexanes to give 8-(80-bromo-
30,30-dimethyl-70,100-dioxo-10,20,70,100-tetrahydro-30H-naphtho[2,1-
b]pyran-90-yl)-9-hydroxy-3,3-dimethyl-3H-naphtho[2,1-b]pyran-
7,10-dione 21 (0.64 g, 50%) as an orange solid, mp 285–295 °C
(decomp.). mmax 3300 (w), 1664 (s), 1632 (m), 1580 (m), 1562
4%), mp 175 °C. kmax (log e) 273, 320 nm (4.57, 4.13). mmax 1664
(m), 1578 (s) cmꢀ1. d (1H) (300 MHz, CDCl3) 3.97, 3.99, 4.00, 4.03
(all s, 6 ꢁ OCH3 (isomers a and b)), 7.42 (2H, br s, 2 ꢁ OH), 7.47
(2H, s, H50, H80), 7.55 (4H, s, H5, H500, H8, H800 (isomer b)), 7.59,
7.61 (s, s, H5, H500, H8 and H800 (isomer a) [isomers a:b; ꢂ2:1].
(m) cmꢀ1 1H NMR (300 MHz, CDCl3) 1.35, 1.37 (3H, 3H, s, s, C0-
.
CH3), 1.50 (6H, s, C–CH3), 1.83 (2H, t, J 7, H20), 3.21 (2H, t, J 7,
H10), 6.03 (1H, d, J 10.5, H2), 7.10, 7.15 (1H, 1H, d, d, J 8.5, H5,
H50), 7.80 (1H, d, J 10.5, H1), 8.03, 8.07 (1H, 1H, d, d, J 8.5, H6,
H60). FAB-MS (3NBA) 578 (M[81Br]+H+1, 12%), 577 (M[81Br]+H,
17), 576 (M[79Br]+H+1, 13), 575 (M[79Br]+H, 14), 497 (23), 496
(46), 495 (39), 481 (26), 213 (38), 187 (35), 165 (82). Calcd for
ESI-MS (positive, 30 V) 683 (M+H, 10%), 391 (40), 279 (55).
þ
HR-ESI-MS (positive) Calcd for
683.1395.
C
36H27O14 683.1395; found
3.2.13. 8-Bromo-9-hydroxy-3,3-dimethyl-1,2-dihydro-3H-
naphtho[2,1-b]pyran-7,10-dione 20
C30H23BrO7: C, 62.6; H, 4.0; found: C, 62.6; H, 4.0.
Bromine (2.1 g, 13.1 mmol) in dry dichloromethane (20 mL).
was added dropwise to a cooled (0 °C) solution of 9-hydroxy-3,3-
dimethyl-1,2-dihydro-3H-naphtho[2,1-b]pyran-7,10-dione16 19
(3.00 g, 11.6 mmol) in dichloromethane (50 mL) containing 10
drops of glacial acetic acid. The cooling bath was removed and
the mixture was stirred at room temperature for 20 min after which
the solvent was evaporated in vacuo to afford, as a bright orange
solid, 8-bromo-9-hydroxy-3,3-dimethyl-1,2-dihydro-3H-naphtho[2,1-
b]pyran-7,10-dione 20 (3.86 g, 99%), mp 201–204 °C. mmax 3328 (br
Evaporation of the trituration filtrate gave more of the dimer 21
(0.145 g, 10%) which was of comparable purity to that of the pre-
cipitated material. Acidification of the alkaline wash and subse-
quent extraction with ethyl acetate afforded 8-bromo-9-hydroxy-
3,3-dimethyl-1,2-dihydro-3H-naphtho[2,1-b]pyran-7,10-dione 19
(0.12 g, 16%).
3.2.16. 9-Hydroxy-8-(80-(900-hydroxy-300,300-dimethyl-700,1000-dioxo-
700,1000-dihydro-300H-naphtho[2,1-b]pyran-800-yl)-30,30-dimethyl-
70,100-dioxo-10,20,70,100-tetrahydro-30H-naphtho[2,1-b]pyran-90-
yl)-3,3-dimethyl-3H-naphtho[2,1-b]pyran-7,10-dione 17
m), 1657 (s), 1642 (s), 1568 (m) cmꢀ1 1H NMR (300 MHz, CDCl3)
.
1.38 (6H, s, C–CH3), 1.89 (2H, t, J 7, H2), 3.27 (2H, t, J 7, H1), 7.12
(1H, d, J 8.5, H5), 7.88 (1H, br s, OH), 8.05 (1H, d, J 8.5, H6). ESI-MS
(positive, 30 V) 377 (M[81Br]+K, 13%), 375 (M[79Br]+K, 13), 356
(M[81Br]+NH4, 23), 354 (M[79Br]+NH4, 25), 339 (M[81Br]+H, 100),
337 (M[79Br]+H, 97), 219 (27), 215 (27), 159 (25), 64 (30). Calcd
for C15H13BrO4: C, 53.5; H, 3.9; found: C, 53.4; H, 3.9.
Diisopropylethylamine (464 lL, 2.66 mmol) was added to a
solution of 8-(80-bromo-30,30-dimethyl-70,100-dioxo-10,20,70,100-tet-
rahydro-30H-naphtho[2,1-b]pyran-90-yl)-9-hydroxy-3,3-dimethyl-
3H-naphtho[2,1-b]pyran-7,10-dione 21 (620 mg, 1.08 mmol) and
9-hydroxy-3,3-dimethyl-3H-naphtho[2,1-b]pyran-7,10-dione16
6
(1.12 g, 4.37 mmol) in 1,2-dimethoxyethane (35 mL) and the
resulting mixture was refluxed under nitrogen. The reaction was
monitored by HPLC until the ratio of the trimers (rt = 10.0 min, gra-
dient) to dimer (rt = 11.5 min, gradient) was ꢂ5 (6–12 h). When
the reaction had reached this stage the mixture was cooled, and
then aqueous hydrochloric acid (1 M) and ethyl acetate were
added. The organic phase was separated, dried and the volatiles
were removed in vacuo. Flash chromatography (25% ethyl acetate
in hexanes containing 1% acetic acid) of the resulting residue
(1.80 g) gave a mixture of starting materials (0.99 g), bromodimer
21 and monomer 6 in the ratio of 9:2 from which monomer 6
can be isolated by extraction with 5% NaHCO3. Further elution of
theflash column using9:1 dichloromethane:methanol(with1%ace-
tic acid) afforded a red solid which was dissolved in ethyl acetate
containing 10% of a 90% aqueous formic acid solution and filtered
through a plug (or short column) of CeliteÒ to give a dark orange so-
lid (644 mg) which was 81% trimers by HPLC. Further elution of the
flash column with 1% acetic acid in methanol gave a semi-solid
which was dissolved in ethyl acetate containing 10% of a 90% formic
acid solution and filtered through a plug of CeliteÒ to give a pale or-
ange solid (194 mg) which was 73% trimers by HPLC.
3.2.14. 8-Bromo-3,3-dimethyl-9-(4-toluenesulfonyloxy)-1,2-
dihydro-3H-naphtho[2,1-b]pyran-7,10-dione 18
A solution of 8-bromo-9-hydroxy-3,3-dimethyl-1,2-dihydro-
3H-naphtho[2,1-b]pyran-7,10-dione 20 (583 mg, 1.71 mmol) in
pyridine (4 mL), diisopropylethylamine (4 mL) and dichlorometh-
ane (4 mL) was cooled to 0 °C and 4-toluenesulfonyl chloride
(350 mg, 1.84 mmol) added. After stirring for 10 min the solution
was added to aqueous hydrochloric acid (2 M) and extracted with
ethyl acetate. The extract was evaporated and the resulting residue
subjected to flash chromatography (10% ethyl acetate in hexanes)
to afford 8-bromo-3,3-dimethyl-9-(4-toluenesulfonyloxy)-1,2-dihy-
dro-3H-naphtho[2,1-b]pyran-7,10-dione 18 (709 mg, 84%), mp
168–169.5 °C. mmax 1680 (s), 1668 (m), 1620 (m), 1578 (w), 1566
(w) cmꢀ1 1H NMR (300 MHz, CDCl3) 1.37 (6H, s, C–CH3), 1.86
.
(2H, t, J 7, H2), 2.49 (3H, s, ArCH3), 3.27 (2H, t, J 7, H1), 7.10 (1H,
d, J 8.5, H5), 7.40 (2H, d, J 8.5, H30), 7.97 (2H, d, J 8.5, 2 ꢁ H20),
8.02 (1H, d, J 8.5, H6). ESI-MS (positive, 70 V) 531 (M[81Br]+K,
16%), 529 (M[79Br]+K, 13), 515 (M[81Br]+Na, 17%), 513
(M[79Br]+Na, 17), 493 (M[81Br]+H, 36%), 491 (M[79Br]+H, 35), 337
(25), 336 (100), 155 (42), 149 (23), 91 (39). Calcd for C21H21BrO6S:
C, 53.8; H, 3.9; found C, 53.6; H, 3.8.

6450
I. T. Crosby et al. / Bioorg. Med. Chem. 18 (2010) 6442–6450
Combining the material obtained as described above from
700,1000-dioxo-700,1000-dihydro-300H-naphtho[2,1-b]pyran-800-yl)1-ben-
zyl-1H-pyrrole-2,5-dione)-3,3-dimethyl-3H-naphtho[2,1-b]pyran-7,
10-dione 23 (66 mg, 19%), mp 165–170 °C. mmax 3346 (br m), 1713
(s), 1655 (s), 1560 (m) cmꢀ1. d (1H) (300 MHz, CDCl3) 1.47 (6H, s,
2 ꢁ (CH3)2), 4.79 (2H, s, NCH2), 5.98 (4H, d, J 10.5, H2 and H200),
7.08 (2H, d, J 8.5, H5 and H500), 7.28–7.47 (5H, m, C6H5), 7.71 (2H,
d, J 10.5, H1 and H100), 7.91 (2H, br s, 2 ꢁ OH), 7.95 (2H, d, J 8.5,
H6 and H600). ESI-MS (positive, 70 V) 734 (M+K, 22%), 719
(M+Na+1, 46), 718 (M+Na, 100), 696 (M+H, 45), 175 (45), 131
(49), 120 (36), 91 (27). Calcd for C41H29NO10: C, 70.8; H, 4.2; N,
five different reactions that used a total of (2.76 g, 4.80 mmol) of
8-(80-bromo-30,30-dimethyl-70,100-dioxo-10,20,70,100-tetrahydro-30H-
naphtho[2,1-b]pyran-90-yl)-9-hydroxy-3,3-dimethyl-3H-naphtho-
[2,1-b]pyran-7,10-dione 17 afforded, after preparative HPLC
purification [70% A, 30% B, (analytical retention time 17.8 min)],
and a final precipitation by dissolving in a small amount of ethyl
acetate and added dropwise to a large volume of hexanes, 9-hydro-
xy-8-(80-(900-hydroxy-300,300-dimethyl-700,1000-dioxo-700,1000-dihydro-300H-
naphtho[2,1-b]pyran-800-yl)-30,30-dimethyl-70,100-dioxo-10,20,70,100-tet-
rahydro-30H-naphtho[2,1-b]pyran-90-yl)-3,3-dimethyl-3H-naphtho[2,1-b]
pyran-7,10-dione 17 (1.008 g, 28%) as a pale orange solid, mp 184–
2.0; found C, 70.7; H, 4.1; N, 1.8. HR-ESI-MS (positive) Calcd for
þ
C
41H30O10 696.1869; found 696.1894.
187 °C (decomp.). kmax (log
e) 218, 275, 295 sh, 389 nm (5.17, 4.95,
4.85, 4.52).
m
max 3388 (br m), 1658 (s), 1648 (s), 1566 (m) cmꢀ1. 1H
Supplementary data
NMR (300 MHz, CDCl3) 1.37, 1.39, 1.46, 1.48, 1.52 (18H, all s, C–
CH3), 1.60 (2H, br s, OH), 1.78–1.90 (2H, m, H20), 3.28 (2H, t, J 7,
H10), 5.97, 5.98 (1H, 1H, d, d, J 10.5, H2, H200), 7.08–7.10, 7.11 and
7.13 (3H, m, d, d, J 6.5, H5, H50, H500 (isomers b and c) and H5,
H50, H500 (isomer a)), 7.43–7.68, 7.69 and 7.71 (2H, m, d, d, J 10.5,
H1, H100 (isomers b and c) and H1, H100 (isomer a)), 7.91–8.00,
8.03 (3H, m, app d, H6, H60, H600 (isomers b and c) and H6, H60,
H600 (isomer a)). ESI-MS (positive, 30 V) 753 (M+H+2, 22%), 752
(M+H+1, 53), 751 (M+H, 100), 397 (19), 381 (24). Calcd for
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.06.105.
References and notes
1. Dickinson, L.; Khoo, S.; Back, D. Antiviral Res. 2010, 85, 176.
2. De Clerk, E. Rev. Med. Virol. 2009, 19, 287.
3. Emery, S.; Winston, A. Lancet 2009, 374, 764.
4. McColl, D. J.; Chen, X. Antiviral Res. 2010, 85, 101.
5. Neamati, N.; Turpin, J. A.; Winslow, H. E.; Christensen, J. L.; Williamson, K.; Orr,
A.; Rice, W. G.; Pommier, Y.; Garofalo, A.; Brizzi, A.; Campiani, G.; Fiorini, I.;
Nacci, V. J. Med. Chem. 1999, 42, 3334.
6. Deng, J.; Dayam, R.; Al-Mawsawi, L. Q.; Neamati, N. Curr. Pharm. Des. 2007, 13, 129.
7. Chen, I.-J.; Nouri Neamati, N.; Nicklaus, M. C.; Ann Orrb, A.; Andersonb, L.;
Barchi, J. J.; Kelleyb, J. A.; Pommier, Y.; MacKerell, A. D. Bioorg. Med. Chem. 2000,
8, 2385.
C
45H34O11: C, 72.0; H, 4.6; found C, 71.7; H, 4.9%.
3.2.17. 9-Hydroxy-8-(40-(900-hydroxy-300,300-dimethyl-700,1000-dioxo-
700,1000-dihydro-300H-naphtho[2,1-b]pyran-800-yl)1-benzyl-1H-pyr-
role-2,5-dione)-3,3-dimethyl-3H-naphtho[2,1-b]pyran-7,10-
dione 23
8. Hare, S.; Gupta, S. S.; Valkov, E.; Engelman, A.; Cherepanov, P. Nature 2010, 464,
232.
A
mixture of 1-benzyl-1H-pyrrole-2,5-dione23 22 (125 mg,
9. Singh, S. B.; Zink, D. L.; Heimbach, B.; Genilloud, O.; Teran, A.; Silverman, K. C.;
Lingham, R. B.; Peter Felock, P.; Hazuda, D. J. Org. Lett. 2002, 4, 1123.
10. Yang, S. S.; Cragg, G. M.; Newman, D. J.; Bader, J. P. J. Nat. Prod. 2001, 64, 265.
11. Ovenden, S. P. B.; Yu, J.; Wan, S. S.; Sberna, G.; Tait, R. M.; Rhodes, D.; Cox, S.;
Coates, J.; Walsh, N. G.; Meurer-Grimes, B. M. Phytochemistry 2004, 65, 3255.
12. Ganesan, A. Curr. Opin. Chem. Biol. 2008, 12, 1.
13. Decosterd, L. A.; Parsons, I. C.; Gustafson, K. R.; Cardellina, J. H., II; McMahon, J.
B.; Cragg, G. M.; Murata, Y.; Pannell, L. K.; Steiner, J. R.; Clardy, J.; Boyd, M. R. J.
Am. Chem. Soc. 1993, 115, 6673.
14. Kearney, M.; Pallansch, L.; Cox, S.; Coates, J.; Buckheit, R. W. J. Antivir. Ther.
2000, 6, 11.
15. Stagliano, K. W.; Emadi, A.; Lu, Z.; Malinakova, H. C.; Twenter, B.; Yu, M.;
Holland, L. E.; Rom, A. M.; Harwood, J. S.; Amin, R.; Johnson, A. A.; Pommier, Y.
Bioorg. Med. Chem. 2006, 14, 5651.
0.49 mmol), 9-hydroxy-3,3-dimethyl-3H-naphtho[2,1-b]pyran-7,10-
dione16
(250 mg, 0.98 mmol) and diisopropylethylamine
(850 L, 4.9 mmol) in 1,2-dimethoxyethane (10 mL) was heated
under reflux for 7.5 h. The mixture was then cooled to room tem-
perature, acidified with hydrochloric acid (1 M) and extracted with
ethyl acetate. Evaporation of the extracts in vacuo to gave a red
solid (376 mg). Analytical HPLC indicated one product in equal
proportions with monomer 6, suggesting incomplete reaction. A
sample of this solid (308 mg), 9-hydroxy-3,3-dimethyl-3H-naph-
tho[2,1-b]pyran-7,10-dione 6 (323 mg, 1.68 mmol) and diisopro-
6
l
pylethylamine (185 lL, 1.06 mmol) in 1,2-dimethoxyethane
16. Crosby, I. T.; Rose, M. L.; Collis, M. P.; de Bruyn, P. J.; Keep, P. L. C.; Robertson, A.
D. Aust. J. Chem. 2008, 61, 768.
(10 mL) was heated under reflux for 56 h. The mixture was then
worked up as before to give a red solid (620 mg) which was sub-
jected to flash chromatography in 30–99% ethyl acetate in hexanes
(containing 1% acetic acid) followed by 10% methanol in dichloro-
methane (containing 1% acetic acid). Evaporation of the fractions
from the final elution afforded a semi-solid which was subjected
to preparative HPLC (isocratic 65% A, 35% B, 4.5 mL/min). Fractions
that had a retention time of 7.3 min by analytical HPLC (gradient)
were combined to give 9-hydroxy-8-(40-(900-hydroxy-300,300-dimethyl-
17. Baillie, A. C.; Thomson, R. H. J. Chem. Soc. (C) 1966, 2184.
18. Kasturi, T. R.; Arunachalam, T. Can. J. Chem. 1966, 44, 1086.
19. Bently, H. R.; Dawson, W.; Pring, F. S. J. Chem. Soc. 1952, 1763.
20. Cameron, D. W.; Feutrill, G. I.; Keep, P. L. C. Tetrahedron Lett. 1989, 30, 5173.
21. Cooke, R. G.; Segal, R. G. Aust. J. Sci. Res. Ser. A 1950, 3, 628.
22. Boyd, M. R.; Cardelina, J. H.; Gustafson, K. R.; Decosterd, L. A.; Parsons, I.;
Pannell, L.; McMahon, J. B.; Cragg, G. M. WO9417055, 1994.
23. Reddy, P. Y.; Kondo, S.; Toru, T.; Ueno, Y. J. Org. Chem. 1997, 62, 2652.