Studies on quinones. Part 42: Synthesis of furylquinone and hydroquinones with antiproliferative activity against human tumor cell lines

Article abstract of DOI:10.1016/j.bmc.2007.10.028

The preparation of furyl-1,4-quinone and hydroquinones by reaction of 2-furaldehyde N,N-dimethylhydrazone with benzo- and naphthoquinones is reported. Access to furylnaphthoquinones from unactivated quinones requires acid-induced conditions, however oxidative coupling reactions of activated quinones proceed under neutral conditions. The in vitro cytotoxic activity of the prepared compounds against a panel of three human cancer cell lines has been studied. Most of the furyl-1,4-quinones exhibited good antiproliferative activity (GI₅₀ = 6.5-33.5 μm) against the MCF-7, NCI-H460, and SF-268 (CNS cancer) cell lines chosen for testing.

Full text of DOI:10.1016/j.bmc.2007.10.028

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 862–868
Studies on quinones. Part 42: Synthesis of furylquinone
and hydroquinones with antiproliferative activity
against human tumor cell linesq
Julio Benites,a Jaime A. Valderrama,b,* Felipe Rivera,b Leonel Rojo,a Nair Campos,c
c
d
´
´
Madalena Pedro and Marıa Sa¨o Jose Nascimento
aDepartamento de Ciencias Qu´ımicas y Farmace´uticas, Universidad Arturo Prat, Casilla 121, Iquique, Chile
bFacultad de Qu´ımica, Pontificia Universidad Cato´lica de Chile, Casilla 306, Santiago, Chile
c
ˆ
´
Centro de Estudos de Qu´ımica Organica, Fitoquımica e Farmacologia de Universidade do Porto,
Faculdade de Farma´cia da Universidade do Porto, Porto Portugal
dLaborato´rio de Microbiologia da Faculdade de Farma´cia da Universidade do Porto, Porto Portugal
Received 6 June 2007; revised 28 September 2007; accepted 10 October 2007
Available online 13 October 2007
Abstract—The preparation of furyl-1,4-quinone and hydroquinones by reaction of 2-furaldehyde N,N-dimethylhydrazone with ben-
zo- and naphthoquinones is reported. Access to furylnaphthoquinones from unactivated quinones requires acid-induced conditions,
however oxidative coupling reactions of activated quinones proceed under neutral conditions. The in vitro cytotoxic activity of the
prepared compounds against a panel of three human cancer cell lines has been studied. Most of the furyl-1,4-quinones exhibited
good antiproliferative activity (GI50 = 6.5–33.5 lm) against the MCF-7, NCI-H460, and SF-268 (CNS cancer) cell lines chosen
for testing.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
essential for the cytotoxic effects. The evaluation also
showed that the lipophilic fragment, derived from the
antitumoral sesquiterpene euryfuran, seems to attenuate
the cytotoxicity.10,11
Natural and synthetic quinonoid compounds are well-
known substances which possess a variety of biological
properties such as antibacterial, antifungal, antiproto-
zoal, inhibition of HIV-1 reverse transcriptase, and anti-
tumor activity.2–7 Some of these pharmacological effects
have been attributed to the formation of DNA-damag-
ing anion-radical intermediates formed by bioreduction
of the quinone nucleus.8 Biological redox activity is rec-
ognized as playing a key role in a number of processes,
including the triggering of cellular events that can be
exploited for therapeutic uses.9
Our reported procedures for the synthesis of euryfuryl-
quinone and hydroquinones were developed by furyla-
tion of quinones through oxidative coupling and
Michael addition reactions. The arylation reaction pro-
ceeds under mild conditions for benzoquinones contain-
ing an electron-withdrawing substituent (activated
quinones) to give the corresponding euryfurylhydroqui-
none (i.e., compound 1, Fig. 1). Depending on the qui-
none substituents, the Michael adducts undergo in situ
redox reactions to give the respective euryfurylquinones.
Previous work on the synthesis and antiprotozoan activ-
ity of euryfuryl-1,4-quinones and hydroquinones indi-
cates that quinone and hydroquinone groups are
OH
OH
O
O
O
O
Keywords: Quinones; Michael addition; Oxidative coupling;
Cytotoxicity.
O
q
For Part 41, see Ref. 1.
Corresponding author. Tel.: +56 2 6864432; fax: +56 2 6864744;
e-mail: jvalderr@uc.cl
2
1
*
Figure 1.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.10.028

J. Benites et al. / Bioorg. Med. Chem. 16 (2008) 862–868
863
In the case of the preparation of euryfurylnaphthoqui-
nones (compound 2, Fig. 1), the oxidative coupling reac-
tion with quinones not bearing electron-withdrawing
groups requires acetic acid to induce the Michael addi-
tion of the euryfuran to the quinone double bond.
quinone 6, which were heated in boiling toluene for
24 h (Scheme 1). The presence of the starting com-
pounds 3 and 6 together with trace amounts of a red po-
lar substance was detected (TLC) in the resulting deep
red reaction mixture.
In this communication, we disclose the design and chem-
ical synthesis of furylquinone and hydroquinones, and
their activity to inhibit the in vitro growth of three hu-
man tumor cell lines. The choice of a furyl substituent
like 2-dialkylfur-5-yl was justified by the following rea-
sons: (a) its occurrence in antitumor12 and antitrypanos-
omal drugs13 of the nifurtimox series, (b) a probable
synergistic effect in the biological activity by combina-
tion with a quinone system in one molecule, and (c)
precedents on the preparation of dimethylhydrazofu-
ryl-1,4-quinones by oxidative coupling of furfural N,N-
dimethylhydrazone with 1,4-quinones.14
We assumed that the lack of formation of furyl-1,4-qui-
nones 5 and 7 is due to a non favorable equilibrium dis-
placement from the substrates toward the Michael
adduct intermediates. Based on this assumption, the
reactions of nucleophile 3 with quinones 4 and 6 were
studied in the presence of silver(I) oxide. We expected
that this oxidant would induce an irreversible conver-
sion of the addition products to the corresponding furyl-
quinones 5 and 7, thus preventing a retro-Michael
reaction. However, TLC analysis of the reaction mixture
after 24 h showed the presence of the substrates together
with trace amounts of polar products.
Based on our previous results on the reaction of euryfu-
ran with unactivated quinones,10 we attempted to pre-
pare furylquinones by using acetic acid to induce the
oxidative coupling reaction. Treatment of nucleophile
3 with quinones 4 and 6 was carried out in acetic acid
solutions at room temperature. This yielded the ex-
pected furylquinones 5 and 7 in 63 and 42% yield,
respectively (Scheme 2). Under similar conditions furyl-
quinones 9 and 11 (Scheme 2) were prepared in 77 and
69% yield by reaction of hydrazone 3 with 2-chloro-
1,4-naphthoquinone 8 and 2-methoxy-1,4-benzoquinone
10 (Scheme 2).
2. Results and discussion
2.1. Chemistry
We initiated the synthesis of furylquinone and hydro-
quinones by attempting the preparation of furylquinone
5 from commercially available furfural dimethylhydraz-
one 3 and 1,4-naphthoquinone 4 by the procedure re-
ported by Potts and col.14 The treatment was
conducted by refluxing compounds 3 and 4 in toluene
(Scheme 1). Analysis (TLC) of the reaction mixture after
2 h showed the presence of 3 and 4 and no reaction was
detected even after 10 h of reflux. Similar results were
found in the treatment of hydrazone 3 with 1,4-benzo-
The results indicate that oxidative coupling reactions of
hydrazone 3 with quinones 4, 6, 8, and 10 are induced by
O
O
N
NMe2
N NMe2
O
O
O
O
O
NMe2
6
N
4
O
O
O
Ph-Me
Ph-Me
O
3
5
7
Scheme 1.
O
O
O
N
NMe2
N
NMe2
OMe
O
O
O
O
O
NMe2
N
O
O
4
10
MeO
3
11
O
O
O
O
5
Cl
6
O
8
N
NMe2
N
NMe2
O
O
O
O
O
O
Cl
7
9
Scheme 2. Formation of furyl-1,4-quinones 5, 7, 9, and 11 in acetic acid at room temperature.

864
J. Benites et al. / Bioorg. Med. Chem. 16 (2008) 862–868
the acetic acid, which probably acts by decreasing the
LUMO energies of electrophiles 4, 6, 8, and 10, thus
improving the HOMO–LUMO interactions with nucleo-
phile 3.
Furylquinone 16 was prepared by oxidative coupling of
formylbenzoquinone 12b with hydrazone 3 using the
procedure for preparing quinone 14. The treatment
yielded 16 (94%) as a deep green solid unstable upon
exposure to air. Furylhydroquinone 17, a quite stable
yellow solid compound, was prepared in 74% yield by
reduction of quinone 16 with Zn–AcOH.
Next we studied the reaction of compound 3 with acti-
vated benzoquinones. A preliminary trial with 3 and
acetylbenzoquinone 12a (1.1 equiv) in dichloromethane
at room temperature was studied. The reaction pro-
ceeds rapidly (TLC) to give a mixture of compounds
The different stabilities of acylhydroquinones 13 and 17
under aerobic conditions are probably related to the ste-
ric hindrance between the substituents on the aromatic
ring. These interactions are clearly seen in molecular
models of compounds 13 and 17 (Fig. 2).
1
13–15 as evidenced by H NMR analysis of the crude.
This result indicates that 12a acts as a Michael accep-
tor and as an oxidant in the oxidative coupling reac-
tion. In the first step, a conjugate addition of 3 to
quinone 12a proceeds to give the Michael adduct 13
which, by a further redox reaction with 12a, yielded
furyl-1,4-benzoquinone 14 and 2,5-dihydroxyacetophe-
none 15 (Scheme 3).
It is interesting to point out that the optimized confor-
mation of 13 and 17 performed with the CSChem3D
software, reveals differences for the distances between
the oxygen of the acyl group and the hydrogen atom
of the o-hydroxyl group, suggesting a more favorable
intramolecular hydrogen bonding stabilization for com-
pound 17 than 13.
In order to control the reaction between 3 and 12a to-
ward the formation of furylquinone 14, the oxidative
coupling reaction of 3 with quinone 12a was attempted
in the presence of silver(I) oxide as a co-oxidant. Qui-
none 12a, generated by oxidation from 2,5-dihydroxy-
acetophenone 15 with Ag2O, was reacted in situ with
3. The treatment, performed with 4 equivalents of sil-
ver(I) oxide, provides furylquinone 14 as a blue syrupy
product in nearly quantitative yield (Scheme 4).
1
We have found (TLC and H NMR) that furylquinone
2.062Å
2.071Å
14 undergoes partial conversion to hydroquinone 13
on exposure to air. Moreover, attempts to obtain 13
by catalytic hydrogenation of quinone 14 showed that
13 undergoes partial aerobic oxidation to 14.
13
17
Figure 2. C@Oꢀ ꢀ ꢀHO distances in the minimized molecular models of
compounds 13 and 17.
N
NMe2
N
NMe2
OH
O
O
Me
NMe2
OH
OH
O
O
O
O
N
O
12a
O
Me
+
+
O
redox
O
OH Me
O
Me
13
12a
3
14
15
Scheme 3. Reaction of hydrazone 3 with acetylbenzoquinone 12a.
N
NMe2
N
NMe2
O
R
OH
OH
O
O
O
O
Zn
+3
O
AcOH
O
Ag2O, CH2Cl2
O
O
R
12a. R = Me
12b. R = H
17
14. R = Me
16. R= H
H2, Pd-C
[O]
O
N
NMe2
OH
OH
Me
O
13
Scheme 4. Oxidative coupling reaction of quinones 14 and 16 with 3.

J. Benites et al. / Bioorg. Med. Chem. 16 (2008) 862–868
865
N
NMe2
toxic activities and LUMO energies for quinones 5, 9,
and 11, the above observations can be explained by
the argument that the quinones inhibit the cell prolifer-
ation through an oxidative stress mechanism which
arises from the capacity of quinone compounds to enter
redox cycles.9,18
O
OH
O
OH
OH
O
HCl
THF-H2O
OH
OH
O
O
17
18
O
O
OH
OH
Cl
O
SC4H9
HS-
nC4H9
HCl
Comparison of the inhibitory activity of compounds 17–
20 (entries 5–8) indicates moderate inhibition (GI50
638.3 lM) against the MCF-7 and SF-268 cell lines.
Since no significant differences were found by changing
the substituents located in the 6-position of the acylhy-
droquinone systems, except for compound 20, appar-
ently the acylhydroquinone core seems to be essential
for the cytotoxic activity.
O
Me
O
R = H
R = Me
OH
R
19
12. R = Me
15. R = H
20
Scheme 5. Preparation of 6-substituted dihydroxybenzaldehydes 18–
20.
In order to acquire information on the influence of the
dimethylhydrazonofuryl substituent on the biological
activity of furylhydroquinone 17, compounds 18, 19,
and 20 were prepared to be included in the biological
study.
It is noteworthy that while compounds 5, 9, and 11
showed to be equally potent for the three cell lines, com-
pounds 17–20 displayed slight inhibition on the NCI-
H460 cell line. This different cell line response may re-
flect tumor type-specific sensitivity to these
compounds.
a
Dialdehyde 18 was obtained in 68% yield by acid-in-
duced hydrolysis of hydrazone 17. Acylhydroquinones
19 and 20 were prepared by conjugate addition of
1-butanethiol and hydrogen chloride to the respective
quinones 12 and 15 (Scheme 5), according to previously
reported procedures.15,16
3. Conclusions
In summary, a number of furyl-1,4-quinone and hydro-
quinones were synthesized through oxidative coupling
reactions from 1,4-quinones and furfural dimethylhyd-
razone. These compounds were tested in an in vitro
cytotoxic assay against three cancer cell lines: MCF-7
(breast adenocarcinoma), NCI-H460 (non-small cell
lung cancer), and SF-268 (CNS cancer). Some com-
pounds showed good inhibition of the growth of the
tested cancer cells, with GI50 in the low-middle micro-
molar range. The biological evaluation indicates that
furyl-1,4-quinones 5, 7, and 11 were more active than
hydroquinones 17–20. These results, together with the
values of the LUMO energies of 5, 7, and 11, suggest
that cell growth inhibition by quinones is related to
the generation of active oxygen species (ROS) after re-
dox cycling. The described oxidative coupling chemistry
and biological evaluation provided significant informa-
tion about the molecular design of new cytotoxic agents
based on quinonoid compounds.
2.2. Biology
Compounds 5, 7, 9, 11, and 17–20 were evaluated for
their capacities to inhibit the in vitro growth of MCF-
7 (breast adenocarcinoma), NCI-H460 (non-small cell
lung cancer), and SF-268 (CNS cancer) cell lines, after
48-h continuous exposure. The results, expressed as
the concentration causing 50% of cell growth inhibition
(GI50), are summarized in Table 1.
All the compounds exhibited a growth inhibiting effect
(GI50 633.5 lM) against the MCF-7 breast adenocarci-
noma cell line. Furyl-1,4-quinones 5, 9, and 11 exhibited
growth inhibitory effect levels (GI50 627.2 lM) lower
than furylhydroquinones 17–20 against non-small cell
lung cancer (NCI-H460) and CNS cancer cell (SF-268).
Although, in most cases the growth inhibiting effects
were moderate, more potent effects were found with qui-
nones 5, 7, 9, and 11 (entries 1–4) than with hydroqui-
nones 17–20 (entries 5–8). This could be explained
assuming that the cytotoxic activity of these compounds
depends on their abilities to accept electrons. In the case
of quinone compounds the inductive effects of the fused
aromatic ring, Cl and OMe substituents on the 1,4-
benzoquinone moiety of 5, 9, and 11 probably determine
their greater cytotoxicities with respect to that of 7. The
electron affinities of quinones 5, 7, 9, and 11, evaluated
through the LUMO energies,17 support this assumption.
In fact, calculation of the LUMO energies of quinones 5
(ꢁ0.7220 eV), 7 (ꢁ0.6883 eV), 9 (ꢁ0.9992 eV), and 11
(ꢁ0.7629)17 indicates that the less cytotoxic quinone 7
possesses the lowest affinity with the highest value of
the LUMO energy. In spite of no correlation of cyto-
4. Experimental
4.1. Chemical synthesis
All reagents were of commercial quality, reagent grade,
and were used without further purification. Melting
points (mp) were determined on a Ko¨fler hot-stage
apparatus and are uncorrected. IR spectra were re-
corded on a Bruker vector 22-FT spectrophotometer
using KBr discs, and wavenumbers are given in cmꢁ1
.
Proton nuclear magnetic resonance (1H NMR) spectra
were measured at 200 and 400 MHz on Bruker AM-
200 and AM-400 spectrometers. Chemical shifts are ex-
pressed in ppm downfield relative to TMS (d scale), and
coupling constants (J) are reported in Hz. Carbon
nuclear magnetic resonance (13C NMR) spectra were

866
J. Benites et al. / Bioorg. Med. Chem. 16 (2008) 862–868
Table 1. Effect of furylquinones and hydroquinones on the growth of three human tumor cell lines
Entry Compound
GI50 (lM)
MCF-7
NCI-H460
SF-268
N
NMe2
N
NMe2
O
O
1
5
7
9
7.7 1.2
6.8 1.9
148.2 13.3
10.9 1.6
6.5 0.9
O
N
O
O
O
2
3
33.5 0.8
100.3 5.1
NMe2
O
O
O
8.1 1.4
9.3 1.4
Cl
N
NMe2
O
O
4
5
11
17
18
8.2 0.7
27.2 1.5
9.1 0.7
MeO
OH
O
N
NMe2
O
29.4 0.9
93.0 3.9
38.3 3.4
OH
OH
O
O
O
6
7
20.3 0.2
80.2 4.4
35.8 1.5
OH
OH
O
SC4H9
Me
19
20
29.2 1.5
63.3 1.7
27.5 2.4
OH
OH
O
Cl
8
9
15.6 0.6
42.8 8.2
121.5 2.0
94.0 8.7
18.8 0.5
93.0 7.0
OH
O
Doxorubicina
Results are means SEM of 6–9 independent experiments performed in duplicate.
a Data from the positive control doxorubicin are expressed as nM.
measured at 50 and 100 MHz on Bruker AM-200 and
AM-400 spectrometers. High resolution mass spectra
were run at the Universidad de Granada, Spain. Silica
gel 60 (70–230 mesh) and TLC aluminum foil 60 F254
(Merck, Darmstadt) were used for preparative column
and analytical TLC, respectively.
purification by column chromatography (1:1 hexane/
ethyl acetate) to yield pure 5 as a blue solid (175 mg,
63%); mp 172–173 ꢁC (lit.,14 175–176 ꢁC); IR (KBr): v
1674 and 1642 (C@O), 1541 (C@N); 1H NMR
(200 MHz, CDCl3): d 8.11–8.03 (m, 2H, 5- and 8-H),
7.76–7.68 (m, 2H, 6- and 7-H), 7.64 (d, 1H, J = 3.7 Hz,
30- or 40-H), 7.29 (s, 1H, 3-H), 6.99 (s, 1H, CH@N), 6.57
(d, 1H, J = 3.7 Hz, 40- or 30-H), 3.06 (s, 6H, 2· Me); 13C
NMR (50 MHz, CDCl3): d 184.6, 183.5, 156.1, 145.6,
134.6, 133.8, 133.3, 132.4, 132.3, 126.6, 126.1, 125.8,
121.9, 119.7, 109.7, 42.5 (2C). HRFABMS calcd for
C17H14N2O3Na [M+Na]+ 317.0902, found 317.0917.
4.1.1. 2-(5-N,N-Dimethylhydrazonofur-2-yl)-1,4-naphtho-
quinone (5). A suspension of hydrazone 3 (66 mg,
0.5 mmol) and 4 (150 mg, 0.9 mmol) in glacial acetic acid
(15 mL) was vigorously stirred for 12 h at room tempera-
ture. The reaction mixture was poured into water
(100 mL), neutralized with sodium hydrogencarbonate,
and extracted with dichloromethane (2· 30 mL). The
organic extract was washed with water and dried over
MgSO4. The solvent was evaporated under reduced
pressure and the residue was submitted to successive
4.1.2. 2-(5-N,N-Dimethylhydrazonofur-2-yl)-1,4-benzo-
quinone (7). A suspension of 6 (312 mg, 2.9 mmol) and
hydrazone 3 (200 mg, 1.4 mmol) in glacial acetic acid
(15 mL) was vigorously stirred for 24 h at room

J. Benites et al. / Bioorg. Med. Chem. 16 (2008) 862–868
867
temperature. The reaction mixture was poured into
water (100 mL), neutralized with sodium hydrogencar-
bonate, and extracted with dichloromethane (2 ·
30 mL). The organic extract was washed with water
and dried over MgSO4. The solvent was evaporated
under reduced pressure and the residue was column
chromatographed (1:1 hexane/ethyl acetate) to give pure
7 as a blue solid (297 mg, 42%); mp 126–127 ꢁC (lit.,14
mp 126–127 ꢁC); IR (KBr): v 1660 and 1640 (C@O);
4.1.5. 3-Acetyl-2-(5-N,N-dimethylhydrazonofur-2-yl)-1,4-
benzoquinone (14). A suspension of hydrazone 3 (152 mg,
1.1 mmol), 12a (150 mg, 1.0 mmol), silver(I) oxide
(928 mg, 4 mmol), and magnesium sulfate (500 mg) in
benzene (25 mL) was vigorously stirred for 1 h at room
temperature. The deep blue solution was filtered and
the solids were washed with benzene. The filtrate was
evaporated under vacuum and the residue was purified
by column chromatography (dichloromethane) to give
quinone 14 (280 mg, 98%) as a blue viscous oil: IR
(KBr) v 1700 (C@O acetyl), 1660 and 1630 (C@O
quinone); 1H NMR (200 MHz): d 7.62 (d, 1H, J = 4 Hz,
40-H), 6.93 (s, 1H, N@CH), 6.77 (s, 2H, 5- and 6-H),
6.60 (d, 1H, J = 4 Hz, 30-H), 3.10 (s, 6H, NMe2), 2.60
(s, 3H, COMe). Anal. calcd for C15H14 N2 O4: C, 62.93;
H, 4.93; N, 9.79. Found: C, 62.52; H, 5.05; N, 9.85.
1
1596 (C@N); H NMR (200 MHz, CDCl3): d 7.48 (d,
1H, J = 3.7 Hz, 30- or 40-H), 7.0 (s, 1H, 3-H), 6.77 (s,
2H, 5- and 6-H), 6.68 (s, 1H, CH@N), 6.59 (d, 1H,
J = 3.7 Hz, 40- or 30-H), 3.60 (s, 6H, 2· Me); 13C
NMR (50 MHz, CDCl3): d 189.2, 188.4, 151.9, 151.3,
151.1, 142.0, 124.1, 123.9, 110.4, 109.5, 108.6, 42.8,
42.7. HRFABMS calcd for C13H12N2O3Na [M+Na]+
367.0745, found 367.0756.
4.1.6. 2,5-Dihydroxy-6-(5-N,N-dimethylhydrazonofur-2-
yl)-benzaldehyde (17). A suspension of hydrazone 3
(284 mg, 2.05 mmol), 2,5-dihydroxybenzaldehyde (276 mg,
2 mmol), and silver(I) oxide (1.86 g, 8 mmol) in benzene
(25 mL) was vigorously stirred for 1 h at room tempera-
ture. The resulting deep blue mixture was filtered, the
solids were washed with benzene, and the filtrate was
evaporated under reduced pressure to give crude quinone
16 (511 mg, 94%) as an unstable green solid. Crude
quinone 16 (300 mg, 1.1 mmol), zinc dust (1 g,
15.4 mmol), and glacial acetic acid (1 mL) in dichloro-
methane (10 mL) were stirred for 5 min at room tempera-
ture. The mixture was filtered and the solid washed with
dichloromethane. The filtrate was washed with water
and the organic phase was dried over magnesium sulfate
and evaporated under reduced pressure. The residue
was purified by column chromatography (chloroform)
to afford furylhydroquinone 17 as orange crystals
(233 mg, 78%), mp 127–128 ꢁC. IR (KBr): v 3180 (OH),
1649 (C@O), 1556 (C@N); 1H NMR (200 MHz, CDCl3):
d 11.51 (s, 1H, OH), 9.89 (s, 1H, CHO), 7.18 (d, 1H,
J = 9.1 Hz 6-H), 7.03 (s, 1H, N@CH), 6.89 (d, 1H,
J = 9.1 Hz, 3- or 4-H), 6.58 (d, 1H, J = 3.4 Hz, 30- or
40-H), 6.49 (d, 1H, J = 3.4 Hz, 40- or 30-H), 3.01 (s, 6H,
2· Me); 13C NMR (50 MHz, CDCl3): d196.2, 156.9,
153.9, 146.8, 144.4, 126.6, 120.8, 119.3, 117.9, 117.1,
115.7, 108.1, 42.6 (2C). HRFABMS calcd for
C14H14N2O4Na [M+Na]+ 297.0841, found 297.0854.
4.1.3. 3-Chloro-2-(5-N,N-dimethylhydrazonofur-2-yl)-1,4-
naphthoquinone (9). A suspension of hydrazone 3
(71.8 mg, 0.52 mmol) and 8 (192.0 mg, 1.0 mmol) in
glacial acetic acid (15 mL) was vigorously stirred for
8 h at room temperature. The reaction mixture was
poured into water (100 mL), neutralized with sodium
hydrogencarbonate, and extracted with dichlorometh-
ane (2 · 30 mL). The organic extract was washed with
water and dried over MgSO4. The solvent was evapo-
rated under reduced pressure and the residue was puri-
fied by column chromatography (1:1 hexane/ethyl
acetate) to yield pure 9 as a blue solid (254 mg, 77%);
mp 139–140 ꢁC; IR (KBr): v 1673 and 1652 (C@O);
1532 (C@N); 1H -NMR (200 MHz, CDCl3): d 8.18–
8.07 (m, 2H, 5- and 8-H), 7.75–7.71 (m, 2H, 6- and 7-
H), 7.65 (d, 1H, J = 3.8 Hz, 30- or 40-H), 7.08 (s, 1H,
CH@N), 6.69 (d, 1H, J = 3.8 Hz, 40- or 30-H), 3.08 (s,
6H, 2· Me); 13C NMR (50 MHz, CDCl3): d 181.9,
178.0, 157.0, 144.3, 134.9, 134.0, 133.8, 132.3, 132.2,
131.4, 127.1, 126.8, 124.8, 119.9, 108.3, 42.5 (2C)
HRFABMS calcd for C17H13ClN2O3 [M+H]+
329.0692, found 329.0686.
4.1.4. 2-(5-N,N-Dimethylhydrazonofur-2-yl)-5-methoxy-
1,4-benzoquinone (11). A suspension of hydrazone 3
(72 mg, 052 mmol) and 10 (152 mg, 1.1 mmol) in gla-
cial acetic acid (15 mL) was vigorously stirred for 12
h at room temperature. The reaction mixture was
poured into water (100 mL), neutralized with sodium
hydrogencarbonate, and extracted with dichlorometh-
ane (2· 30 mL). The organic extract was washed with
water and dried over MgSO4. The solvent was
evaporated under reduced pressure and the residue
was purified by column chromatography (1:1 hexane/
ethyl acetate) to yield pure 11 as a blue solid
(208 mg, 69%); mp 108–110 ꢁC; IR (KBr): v 1659 and
1616 (C@O); 1561 (C@N); 1H NMR (200 MHz,
CDCl3): d 7.56 (d, 1H, J = 3.8 Hz, 30- or 40-H), 7.00
(s br, 2H, 3- and 6-H), 6.58 (d, 1H, J = 3.8 Hz, 40- or
30-H), 5.88 (s, 1H, CH@N), 3.84 (s, 3H, OMe), 3.08
(s, 6H, 2· Me); 13C NMR (50 MHz, CDCl3): d
186.1, 181.5, 159.2, 156.6, 145.6, 133.3, 122.7, 121.5,
119.4, 109.9, 107.4, 56.2, 42.5 (2C); HRFABMS calcd
for C14H14N2O4Na [M+Na]+ 297.0851, found
297.0856.
4.1.7. 2,5-Dihydroxy-6-(5-formylfur-2-yl)-benzaldehyde
(18). A solution of 17 (64 mg, 0.24 mmol) and hydro-
chloric acid (5 mL, 1.3 N) in aqueous methanol
(20 mL, 50%) was left for 3d at room temperature.
The mixture was diluted with water and extracted with
dichloromethane (2· 20 mL). The organic extract was
dried over magnesium sulfate and evaporated under re-
duced pressure. The residue was purified by column
chromatography to give 18 (38 mg, 68%) as a yellow so-
lid, mp 168–169 ꢁC. IR (KBr): v 3354 (OH), 1643
(C@O). 1H NMR (400 MHz, CDCl3): d 11.01 (br s,
1H, OH), 10.01 (s, 1H, CHO), 9.96 (s br, 1H, OH),
9.61 (s, 1H, CHO), 7.64 (d, 1H, J = 3.5 Hz, 30- or 40-
H), 7.25 (d, 1H, J = 8.8 Hz, 3- or 4-H), 7.03 (d, 1H,
J = 8.8 Hz, 4- or 3-H), 6.99 (d, 1H, J = 3.5 Hz, 40- or
30-H). 13C NMR (100 MHz, COMe2-d6) 116.1, 117.7,
120.2, 126.2, 148.5, 152.3 (2C), 153.1, 153.2, 156.0,

868
J. Benites et al. / Bioorg. Med. Chem. 16 (2008) 862–868
177.5, 196.8; HRFABMS calcd for C12H8O5Na
[M+Na]+ 255.0269, found 255.0263.
was calculated as described.20 Doxorubicin (Sigma
Chemical Co.), used as a positive control, was tested
in the same way.
4.1.8. 2,5-Dihydroxy-6-chlorobenzaldehyde (20). A sus-
pension of 2,5-dihydroxybenzaldehyde (170 mg,
1.23 mmol), silver (II) oxide (510 mg, 2.2 mmol), magne-
sium sulfate (500 mg), and dichloromethane (35 mL)
was vigorously stirred for 2 h. The mixture was filtered
and the solids were washed with dichloromethane
(15 mL). Dry hydrogen chloride was bubbled through
the filtrate for 5 min and the resulting solution was left
overnight at room temperature. The solvent was re-
moved and the residue was purified by column chroma-
tography (dichloromethane) to give pure 20 as a gold
yellow solid (146 mg, 69%); mp 155–157 ꢁC; IR(KBr) v
3271 (OH), 2773 and 2719 (C@O); 1H NMR
(200 MHz, CDCl3): d 11.50 (s, 1H, C2-OH), 10.35 (s,
1H, CHO), 7.26 (d, 1H, J = 9 Hz, 3-H or 4-H), 6.88
(d, 1H, J = 9 Hz, 4-H or 3-H), 5.41 (s, 1H, C5-OH);
13C NMR (50 MHz, CDCl3): d 194.7, 157.9, 144.5,
125.9, 120.6, 117.8, 115.8. HRFABMS calcd for
C7H5O3Cl [M]+ 171.9927, found 171.9921.
Acknowledgments
Financial support by FONDECYT (Grant 1020885),
FCT (I& D Nꢁ 226/94), POCTI (QCA III), and FEDER
is gratefully acknowledged. Madalena Pedro is a recipient
of a PhD grant from FCT (SFRH/BD/1456/2000). The
authors thank the National Cancer Institute, Bethesda,
MD (USA), for kindly providing the tumor cell lines.
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