Antiproliferative activity of synthetic naphthoquinones related to lapachol. First synthesis of 5-hydroxylapachol

Article abstract of DOI:10.1016/j.bmc.2010.02.032

A series of 5-hydroxy-1,4-naphthoquinones analogues was synthesized from juglone (6) and their antiproliferative activity against a representative panel of six human solid tumor cell lines has been investigated. The 2,5-dihydroxy-3-(3-methylbut-2-enyl)naphthalene-1,4-dione (4) and 2,3-dihydro-5-hydroxy-2-(prop-1-en-2-yl)naphtho[2,3-b]furan-4,9-dione (27) were the most potent antiproliferative agents with GI₅₀ values of 0.42-8.1 and 0.80-2.2 μM, respectively. The results provide insight into the correlation between some structural properties of 5-hydroxynaphthoquinones and their antiproliferative activity.

Full text of DOI:10.1016/j.bmc.2010.02.032

Bioorganic & Medicinal Chemistry 18 (2010) 2621–2630
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antiproliferative activity of synthetic naphthoquinones related to lapachol.
First synthesis of 5-hydroxylapachol
Evelyn L. Bonifazi , Carla Ríos-Luci b,c, Leticia G. León b,c, Gerardo Burton , José M. Padrón b,c,*,
Rosana I. Misico
a
a
a
a,
*
Departamento de Química Orgánica and UMYMFOR (CONICET-UBA), Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón 2,
Ciudad Universitaria, C1428EHA Buenos Aires, Argentina
b
Instituto Universitario de Bio-Orgánica Antonio González (IUBO-AG), Universidad de La Laguna, C/Astrofísico Francisco Sánchez 2, 38206 La Laguna, Tenerife, Spain
BioLab, Instituto Canario de Investigación del Cáncer (ICIC), C/Astrofísico Francisco Sánchez 2, 38206 La Laguna, Tenerife, Spain
c
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 5-hydroxy-1,4-naphthoquinones analogues was synthesized from juglone (6) and their anti-
proliferative activity against a representative panel of six human solid tumor cell lines has been investi-
gated. The 2,5-dihydroxy-3-(3-methylbut-2-enyl)naphthalene-1,4-dione (4) and 2,3-dihydro-5-hydroxy-
Received 7 December 2009
Revised 14 February 2010
Accepted 18 February 2010
Available online 23 February 2010
2-(prop-1-en-2-yl)naphtho[2,3-b]furan-4,9-dione (27) were the most potent antiproliferative agents
with GI50 values of 0.42–8.1 and 0.80–2.2 M, respectively. The results provide insight into the correla-
l
tion between some structural properties of 5-hydroxynaphthoquinones and their antiproliferative
activity.
Keywords:
Naphthoquinone
Lapachone
Ó 2010 Elsevier Ltd. All rights reserved.
Lapachol
Antiproliferative
1
. Introduction
product of lapachol (3), has been intensely investigated for clinical
1
1–13
use in cancer chemotherapy.
b-lapachone has been known for more than 20 years, the detailed
mechanism of action remains largely unknown.
Although the cytotoxic action of
1
4
Quinones are widely distributed in nature and have a variety of
roles in organisms. In addition to functional constituents of various
biochemical systems such as ubiquinone and vitamin K1, they also
may be found in dyes, or acting as defensive compounds. There-
fore, quinone derivatives may be toxic to cells by a number of
mechanisms including redox cycling,1 arylation, intercalation,
induction of DNA strand breaks, generation of free radicals and
alkylation via quinone methide formation.2 Several clinically
important anticancer drugs such as daunorubicin (1) and mitomy-
cin C (2) contain the quinone moiety as a relevant part of their
structures (Fig. 1) and there is an increasing number of reports con-
cerning the biological evaluation of synthetic analogues and new
natural products related to this class of compounds.3 Within the
active quinones, lapachol (3) a natural naphthoquinone, and many
heterocyclic derivatives were investigated during the past years,
It is well-known that the presence of mono- or dihydroxy
groups at C-5 or C-5 and C-8 positions of the naphthoquinone
moiety, respectively, induces a higher toxic effect to cells due to
15
an increased efficiency of redox cycling. In this context, we have
directed our efforts to obtain novel synthetic derivatives contain-
ing a hydroxyl group at the C-5 position. Herein, we report the first
synthesis of 5-hydroxylapachol (4) and the preparation of new
derivatives of (3) and (5) with diverse substitution patterns at
the C-2 and C-3 positions of the naphthoquinone scaffold. These
compounds were prepared to establish structure–activity relation-
ships when considering their antiproliferative activities against a
representative panel of human solid tumor cell lines comprising
A2780 (ovarian), SW1573 (non-small cell lung), WiDr (colon),
T-47D (breast), HBL-100 (breast), and HeLa (cervix).
4–6
antifungal,7 trypanocidal
8
mainly due to their antibacterial,
and anticancer activities.9 More recently, 5-hydroxylapachol (4)
was isolated from the root heart wood of Tectona grandis and, like
lapachol (3), was found to be cytotoxic to Artemia salina (brine
shrimp) with an LC50 of 5 ppm.10 b-Lapachone (5), a cyclization
2
2
. Results and discussion
.1. Chemistry
The target naphthoquinones were synthesized from juglone (6)
and lapachol (3). As shown in Scheme 1, the addition of an aqueous
solution of dimethylamine to juglone (6) gave a 2.4:1 mixture of
*
Corresponding authors. Tel./fax: +54 11 45763385.
E-mail addresses: jmpadron@ull.es (J.M. Padrón), misicori@qo.fcen.uba.ar, misi
cori@gmail.com (R.I. Misico).
0
968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.02.032

2
622
E. L. Bonifazi et al. / Bioorg. Med. Chem. 18 (2010) 2621–2630
ence of sodium iodide and triethylamine in DMSO (method A) gave
the mixture of naphthoquinones 4 and 12–14 in low yield.18
Interestingly, when DMF was used as the reaction solvent the
C-alkylated compound 4 was the major product (57%), and no
cyclization products were obtained. Changing the base to potas-
sium or lithium carbonate resulted in low yields of 4 and formation
of significant amounts of, o-alkylation (12), Claisen rearrangement
(15), and dialkylation (16) products. To the best of our knowledge,
this is the first reported procedure for the synthesis of the natural
product 5-hydroxylapachol (4). The formation of the iodinated
product 13 may be explained as a result of the electrophilic addi-
tion of iodine (arising from the oxidation of iodide by DMSO)19
to the side chain double bond of 15, followed by cyclization to give
2
0,21
the dihydrofuran ring.
Similarly, compound 14 could originate
from 4.
Scheme 3 shows the prenylation of 3-hydroxyjuglone (10) with
1
-bromo-3-methylbut-2-ene. As in the previous case, the reaction
of 10 with 1-bromo-3-methylbut-2-ene in the presence of sodium
iodide and triethylamine in DMF gave compounds 17, 18, and 21
while the reaction in DMSO gave a mixture of compounds 17–20.
When the prenylation was carried out in DMF with potassium car-
bonate 17, 18, 21, and 22 were obtained.
Figure 1. Structure of naturally occurring quinones.
With 5-hydroxylapachol (4) in hand, we prepared several deriv-
atives with the purpose of performing SAR studies. The 5-deoxy
analogues derived from lapachol (3) were also prepared for com-
parison purposes. Thus, treatment of compound 4 with dimethyl
sulfate led to compounds 23 and 24 in 8% and 47% yields, respec-
tively (Scheme 4). In addition, we prepared the set of tricyclic
derivatives 25–30 shown in Scheme 5. Treatment of 3 with 3 equiv
of CAN in dry acetonitrile gave dihydrofurans 25 (30%) and 26
2
2
(
51%) via intramolecular cyclization. When the same procedure
was applied to 4 the resulting quinones 27 and 28, were obtained
in 7% and 12%, respectively. Treatment of 3 with dilute H SO led to
-lapachone (29) and b-lapachone (5), respectively. The reaction
2
4
2
3
a
2 4
of 5-hydroxylapachol (4) with concentrated H SO gave only com-
pound 30 in 97% yield.
Scheme 1. Preparation of hydroxyjuglones 9 and 10.
The iodinated quinones 13, 14 and 20 were obtained in low
yield but preliminary testing indicated an interesting biological
2
5
-dimethylaminojuglone (7) and 3-dimethylaminojuglone (8) in
1% yield. Subsequent deamination of 7 and 8 with concentrated
1
6
Table 1
Reaction of hydroxyjuglones 9 and 10 with 1-bromo-3-methylbut-2-ene
HCl afforded quantitatively 2-hydroxyjuglone (9) and 3-hydroxy-
juglone (10), respectively. Due to the poor yield of 8 we carried
out an alternative approach through the intermediate 3-aminojug-
lone (11) obtained in 94% yield from juglone (6) and sodium azide
Reaction conditionsa
Products formed (% yield)
9
9
9
0
0
0
A
B
C
A
B
C
4 (13), 12 (2), 13 (2), 14 (6)
4 (57), 12 (19), 15 (traces)
4 (6), 12 (25), 15 (20), 16 (13)
17 (10), 18 (19), 19 (24), 20 (0.9)
17 (55), 18 (21), 21 (traces)
17 (7), 18 (19), 21 (20), 22 (10)
1
7
in 1 N HCl solution. The acidic hydrolysis of 11 led to compound
1
1
1
1
0 in 91% overall yield.
The prenylation of 2-hydroxyjuglone (9) with 1-bromo-3-meth-
ylbut-2-ene was attempted under different reaction conditions
Scheme 2, Table 1). Treatment of 9 with allyl bromide in the pres-
a
(
3 3 2 3
A) NaI, Et N, DMSO; (B) NaI, Et N, DMF; (C) K CO , DMF.
(
Scheme 2. Reaction of 2-hydroxyjuglone (9) with 1-bromo-3-methylbut-2-ene.

E. L. Bonifazi et al. / Bioorg. Med. Chem. 18 (2010) 2621–2630
2623
Scheme 3. Reaction of 3-hydroxyjuglone (10) with 1-bromo-3-methylbut-2-ene.
2
.2. Biological activity
As a model for the anticancer activity we used the representa-
tive panel of human solid tumor cell lines A2780 (ovarian), HBL-
00 (breast), HeLa (cervix), SW1573 (non-small cell lung), T-47D
breast), and WiDr (colon). The in vitro antiproliferative activity
of the synthesized naphthoquinones was evaluated using the Na-
1
(
2
4
Scheme 4. Reaction of 5-hydroxylapachol with dimethyl sulfate.
tional Cancer Institute (NCI) protocol after 48 h of drug exposure
using the sulforhodamine B (SRB) assay. In this method, for each
drug a dose–response curve was generated. The effect was defined
as percentage of growth (PG), where 50% growth inhibition (GI50),
represented the drug concentration at which PG was +50.25 The re-
sults are summarized in Table 2 together with the lipophilicity
(
C log P) of the compounds, evaluated by in silico calculation based
2
6
on their chemical structures. The data show that lipophilicity dif-
ferences are not relevant to the observed activities.
The comparison between lapachol (3) and the hydroxy deriva-
tives 4 and 17 showed that the presence and position of a hydroxy
group attached to the aromatic ring affects the ability to suppress
the growth of the tumor cell lines. As shown in Table 2 the results
obtained with 5-hydroxylapachol (4) were remarkable. In fact,
compound 4 was one of the most active products evaluated with
GI50 values in the range 0.42–8.1 lM. The antiproliferative effect
2
7
of 8-hydroxylapachol (17) carrying the hydroxyl at C-8, was low-
er than compound 4 although it was comparable to that of lapachol
(3). Thus, the presence of a phenolic hydroxy group at C-5 seems to
play an important role in increasing antiproliferative effect. A sim-
ilar effect was observed when comparing 25 and 27, supporting the
beneficial role of the C-5 hydroxyl described above.
In this particular context, the influence of the isoprenyl side
chain on the antiproliferative activity was evaluated. When the
side chain is an 1,1-dimethylallyl group instead of the naturally
occurring prenyl group as in compounds 15 and 21, the activity ob-
served was lower than that of lapachol (5) and without differences
between the regioisomers. The lack of a side chain as in commer-
cial juglone (6) and compounds 7–11 resulted in poor activity.
However the presence of a nitrogen atom at C-2 in the quinone ring
increased the activity within the group, with compound 11 being
the most active. An improved selectivity was also observed when
the free hydroxyl groups in the quinone ring of 5- (4) and 8-
hydroxylapachol (17) were converted to the methyl ethers (16
and 22). The above results show that the absence of the prenyl side
chain produces weakly active derivatives, and suggests that not
only the size but the relative position of the side chain is crucial
to achieve a better activity profile.
2 4
Scheme 5. Reaction of lapachol (3) and 5-hydroxylapachol (4) with CAN and H SO .
activity (see below), thus we sought for a more efficient synthetic
procedure for their preparation. As shown in Scheme 6, the addi-
tion of 3-methylbut-2-en-1-ol to 9 under Mitsunobu’s conditions
(
3
DIAD, Ph P) led to a 5:1 mixture of compounds 12 and 15. Claisen
rearrangement of 12 in ethanol at 60 °C gave 15 in 92% yield. The
addition of iodine to compound 15 gave the sought iodinated
naphthoquinone 13 (66%) and its isomer 31 (7%). The same reac-
tion sequence applied to 3-hydroxyjuglone (10) gave compounds
2
1
0 and 32 in 7.1% and 12.5% overall yield, respectively. Compounds
4 (41%) and 33 (6%) were obtained by addition of iodine to 5-
Several compounds assayed were furanonaphthoquinones (13,
14, 20 and 25–28) and pyranonaphthoquinones (5, 19, 29 and
30). Some compounds of the series, displayed high antiproliferative
hydroxylapachol (4).

2
624
E. L. Bonifazi et al. / Bioorg. Med. Chem. 18 (2010) 2621–2630
Scheme 6. Preparation of iodinated compounds 13 and 20.
Table 2
Lipophilicity and GI50 values for the in vitro screening of naphthoquinones against human solid tumor cellsa
Compds
Cell line
C log P
A2780 (ovarian)
HBL-100 (breast)
HeLa (cervix)
SW1573 (lung)
T-47D (breast)
WiDr (colon)
3
4
5
6
7
8
9
3.7
4.0
1.7
3.7
2.4
2.4
2.0
2.0
2.3
3.7
4.7
4.6
3.9
5.7
4.0
3.7
3.3
4.7
3.9
5.7
4.1
3.9
3.4
0.7
3.7
1.0
3.2
3.5
1.9 (±0.5)
0.57 (±0.16)
1.1 (±0.8)
64 (±34)
16 (±4.0)
2.4 (±1.2)
>100
7.8 (±4.6)
0.60 (±0.12)
0.69 (±0.39)
66 (±28)
30 (±2.8)
5.7 (±1.3)
>100
2.3 (±0.8)
0.42 (±0.11)
0.81 (±0.45)
89 (±17)
19 (±1.8)
7.1 (±3.6)
>100
34 (±6.5)
0.70 (±0.23)
0.76 (±0.39)
50 (±13)
35 (±5.5)
5.3 (±2.2)
>100
76 (±29)
8.1 (±3.7)
2.3 (±0.4)
77 (±30)
38 (±8.0)
34 (±4.8)
>100
36 (±9.8)
6.3 (±1.9)
2.0 (±0.2)
81 (±26)
32 (±4.5)
17 (±5.5)
>100
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
28 (±8.1)
3.1 (±0.9)
12 (±4.1)
1.7 (±0.5)
2.1 (±1.2)
26 (±5.1)
1.0 (±0.3)
3.9 (±0.6)
2.0 (±0.5)
2.4 (±0.1)
1.6 (±0.3)
21 (±1.9)
5.9 (±0.7)
1.8 (±0.4)
21 (±5.6)
2.6 (±0.2)
2.1 (±0.3)
0.82 (±0.36)
2.2 (±0.2)
4.0 (±0.4)
3.2 (±0.9)
>100
90 (±14)
86 (±13.2)
2.0 (±0.8)
17 (±10.6)
1.3 (±0.3)
2.3 (±0.5)
36 (±6.3)
4.3 (±0.3)
18 (±2.7)
7.7 (±1.6)
4.8 (±1.4)
0.40 (±0.13)
3.8 (±1.3)
5.1 (±2.4)
4.8 (±0.2)
5.8 (±1.7)
1.9 (±0.9)
2.5 (±0.5)
0.83 (±0.35)
2.8 (±1.3)
3.1 (±0.8)
2.7 (±0.7)
>100
>100
2.9 (±0.3)
27 (±2.7)
1.9 (±0.3)
2.0 (±0.5)
31 (±3.0)
1.6 (±0.2)
21 (±4.1)
19 (±6.1)
3.9 (±1.5)
0.41 (±0.05)
16 (±1.8)
20 (±1.4)
20 (±6.9)
17 (±2.3)
2.3 (±0.2)
2.1 (±0.2)
0.94 (±0.36)
2.8 (±0.5)
14 (±4.5)
2.6 (±0.8)
5.7 (±2.4)
18 (±3.0)
1.7 (±0.4)
2.4 (±0.7)
28 (±1.6)
1.9 (±0.2)
2.6 (±0.4)
4.5 (±1.6)
2.3 (±0.3)
1.3 (±0.3)
24 (±5.3)
20 (±0.6)
3.5 (±0.6)
18 (±1.0)
2.1 (±0.9)
1.8 (±0.2)
0.80 (±0.41)
2.2 (±0.34)
15 (±2.6)
12 (±1.2)
4.0 (±0.8)
28 (±5.3)
1.8 (±0.4)
2.3 (±0.4)
27 (±2.3)
21 (±4.5)
30 (±4.7)
23 (±6.1)
19 (±2.6)
2.5 (±0.4)
30 (±4.6)
28 (±6.9)
22 (±4.3)
45 (±8.1)
21 (±4.6)
20 (±8.0)
2.2 (±0.7)
23 (±2.8)
25 (±3.5)
21 (±4.7)
3.5 (±0.3)
18 (±7.4)
2.0 (±0.03)
1.9 (±0.2)
31 (±1.6)
11 (±3.8)
19 (±2.4)
7.3 (±3.2)
19 (±6.0)
2.0 (±0.2)
23 (±1.8)
22 (±6.1)
20 (±2.7)
28 (±5.5)
2.7 (±0.3)
4.8 (±1.6)
2.0 (±0.1)
18 (±2.1)
26 (±0.6)
18 (±1.6)
a
GI50 values are given in
lM and are mean of three to ten experiments, standard deviation is given in parentheses.
activity, with the furanonaphthoquinone 27 exhibiting the highest
activity (GI50 values of 0.80–2.2 M) comparable to b-lapachone
5). When comparing the activity of 27 with those of the analogue
furanonaphthoquinones 14 and 28, the observed decrease in activ-
ity may be ascribed to the replacement of the exocyclic double
bond by an oxygenated function like a nitrate group or by iodine.
l
(

E. L. Bonifazi et al. / Bioorg. Med. Chem. 18 (2010) 2621–2630
2625
3
. Conclusion
uct was purified by column chromatography on silica gel (hexanes/
EtOAc, gradient) to give 7 (450 mg, 36% yield) as a bright red solid;
1
6
1
13
In summary, we described the first synthesis of the antiprolifer-
mp 148–149 °C (from EtOH) (lit. 147 °C). The H and C NMR
30
ative agent, 5-hydroxylapachol (4), through prenylation of 2-
hydroxyjuglone (9). The conventional structure–activity relation-
ships indicate that antiproliferative activity is favored by naphtho-
quinones possessing a 5-hydroxy group in the aromatic ring.
Additionally, this activity is favored in furanonaphthoquinones
with compound 27 being the most active member of this and all
series. This result is in agreement with recent findings by Iida
et al.2 and point toward 5-hydroxynaphthoquinones as a promis-
ing group of lapachone analogues.
data were consistent with that reported in the literature. MS m/
z (%) 217 (M , 100), 202 (27), 188 (12), 174 (8), 160 (8), 121 (6),
89 (8), 68 (9), 63 (11), 44 (24). Anal. Calcd for C12H11NO : C,
3
66.35; H, 5.10; N, 6.45. Found: C, 66.12; H 5.09; N, 6.44. Further
elution gave compound 8 (187 mg, 15%) as violet crystals; mp
157–158 °C (from EtOH) (lit. 156 °C). The H NMR data were con-
sistent with that reported in the literature.
(125.77 MHz, CDCl ): 188.3 (C-4), 182.0 (C-1), 161.7 (C-8), 151.6
(C-2), 136.8 (C-6), 132.9 (C-10), 122.5 (C-7), 117.9 (C-5), 115.4
+
1
6
1
3
0
13
C
NMR
8
3
+
(
C-9), 108.2 (C-3), 43.0 (CH
3
). MS m/z (%) 217 (M , 100), 202 (36),
1
88 (8), 174 (10), 160 (7), 121 (5), 89 (8), 68 (7), 63 (11), 44 (32).
4
4
. Experimental
4
.2.2. 2,5-Dihydroxy-1,4-naphthalenedione (2-hydroxyjuglone, 9)
A suspension of 7 (1.52 g, 7.0 mmol) in conc. HCl (75 mL) was
.1. General procedure
heated under reflux for 5 h. After cooling, the reaction mixture
was diluted with water, and extracted with CH Cl . The organic
layer was dried over anhydrous Na SO and evaporated to dryness.
Melting points were taken on a Fisher–Johns apparatus and are
2
2
uncorrected. IR spectra were recorded in thin films using KBr disks
on a Nicolet Magna 550 FT-IR spectrophotometer. NMR spectra were
2
4
The residue was purified by column chromatography (hexane/
1
13
acquired at 500.13 ( H NMR) and 125.77 MHz ( C NMR) on a Bruker
EtOAc, 2:8) to give 9 (1.30 g, 98%) as red crystals; mp 219–220 °C
1
13
Avance II 500 or at 200.13 ( H NMR) and 50.32 MHz ( C NMR) on a
BrukerAC 200 spectrometer. Chemicalshifts aregiven inppmdown-
field from TMS as internal standard, J values are given in Hz. Multi-
plicity determinations and 2D spectra were obtained using
standard Bruker software. Low-resolution EI mass spectra (LR EIMS)
were collected on a Shimadzu QP-5000 mass spectrometer at 70 eV
by direct inlet. High-resolution EI mass spectra (HR EIMS) were
determinedonanAgilentLCTOFmassspectrometer. Elementalanal-
yses were performed on an Exeter CE 440 analyzer. All solvents were
of AR grade except dichloromethane (DCM) which was of LR grade
and distilled before use. When necessary, the purification of solvents
and starting materials was carried out using standard procedures.
31
1
13
(
dec.) (lit. 218–220 °C (dec.)). The H and C NMR data were con-
32
+
sistent with that reported in the literature. MS m/z (%) 190 (M ,
00), 162 (25), 134 (35), 121 (91), 105 (14), 92 (26), 69 (23), 63
39), 51 (30).
1
(
4
.2.3. 2,8-Dihydroxy-1,4-naphthalenedione (3-hydroxyjuglone, 10)
A suspension of compound 8 (or 11) (2.4 mmol) in conc. HCl
(
165 mL) was heated under reflux for 24 h. The mixture was
poured into cold water and extracted with CH Cl . The organic
layer was dried over anhydrous Na SO , filtered, and the solvent
2
2
2
4
evaporated. The residue was purified by column chromatography
on silica gel (hexane/EtOAc, 2:8) and crystallized from dilute acetic
acid to afford 10 (442 mg, 97%) as orange crystals; mp 219–221 °C
Lapachol
(2-hydroxy-3-(3-methylbut-2-enyl)naphthalene-1,4-
dione, 3) and juglone (5-hydroxynaphthalene-1,4-dione, 6) were
used as starting materials to synthesize the training set of naphtho-
quinones. Lapachol (3) was isolated by extraction of the powdered
wood of Tabebuia impetiginosa (Bignoniaceae) with a cold solution
31
1
13
(
from acetic acid) (lit. 218–222 °C). The H and C NMR data
32
were consistent with that reported in the literature. MS m/z (%)
90 (M , 100), 162 (24), 134 (17), 121 (62), 105 (11), 92 (26), 69
+
1
(
22), 63 (30), 51 (19).
1
8,29
of sodium carbonate.
drich Co. The -lapachone (3,4-dihydro-2,2-dimethyl-2H-benzo-
g]chromene-5,10-dione, 29) and b-lapachone (3,4-dihydro-2,2-di-
methyl-2H-benzo[h]chromene-5,6-dione, 5) were obtained by
Juglone (6) was purchased from Sigma–Al-
a
4
.2.4. 2-Amino-8-hydroxynaphthalene-1,4-dione (3-
[
aminojuglone, 11)
To a stirred solution of juglone (6) (100 mg, 0.57 mmol) in
2
3
intramolecular cyclization of lapachol (3) using sulfuric acid,
4
.8 mL of methanol under an argon atmosphere was added a solu-
whereas compounds 25 and 26 were obtained by intramolecular
cyclization of 3 using CAN following known procedures.22 Reactions
were monitored using thin-layer chromatography (TLC) on alumi-
num-backedprecoatedSilicaGel60F254plates(EMerck). Ingeneral
naphthoquinones are highly colored and were visible on a TLC plate;
colorless compounds were detected using UV light. Flash chroma-
tography was carried out using Silica Gel 60 (230–400 mesh) with
the solvent system indicated in the individual procedures. All sol-
vent ratios are quoted as vol/vol. Full characterization data are in-
cluded for known compounds where these data are incompletely
reported in the literature. All new compounds were characterized
tion of sodium azide (220 mg, 3.38 mmol) in 1.6 mL of water and
acidified to pH 4 (with 1 N HCl). The reaction was heated at 30–
3
5 °C for 22 h, and then the mixture was cooled, and extracted
with EtOAc. The organic layer was washed successively with water
and brine, dried over anhydrous Na SO , and concentrated. The
2
4
residue was purified by column chromatography on silica gel (hex-
ane/EtOAc, gradient) to give 11 (101 mg, 94%) as red crystals; mp
17
1
2
54–255 °C (lit. 253–254 °C). The H NMR data were consistent
17 13
with that reported in the literature.
C NMR (125.77 MHz,
CD OD): 188.4 (C-4), 185.6 (C-1), 163.7 (C-8), 153.4 (C-2), 139.4
3
(
(
(
C-6), 135.9 (C-10), 124.2 (C-7), 116.2 (C-9), 112.1 (C-5), 104.5
C-3). MS m/z (%) 189 (M , 100), 173 (2.3), 162 (61), 145 (6), 133
38), 121 (56), 104 (15), 92 (51), 68 (24), 63 (36), 41 (27).
1
13
by HRMS, IR and NMR. Assignment of H and C spectra was made
using COSY, HSQC and HMBC spectra for all new compounds.
+
4
4
.2. Chemistry
4.2.5. Reaction of 2-hydroxyjuglone (9) with 1-bromo-3-
methylbut-2-ene
4.2.5.1. Method a. The reaction of 9 (108 mg, 0.57 mmol) with 1-
.2.1. 2-(Dimethylamino)-5-hydroxy-1,4-naphthalenedione (2-
N,N-dimethylaminojuglone, 7) and 2-(dimethylamino)-8-hydr-
oxy-1,4-naphthalenedione (3-N,N-dimethylaminojuglone, 8)
The reaction of juglone (6) (1 g, 5.75 mmol) with an aqueous
solution of dimethylamine (1.7 mL, 37% w/v) was carried out fol-
bromo-3-methylbut-2-ene (76.5 lL, 0.66 mmol) in DMSO was car-
18
ried following the procedure described by Jiang et al. The reac-
tion mixture was fractionated by column chromatography on
silica gel (hexane/EtOAc, gradient) to give quinones 4 (19 mg,
13%), 12 (2.9 mg, 2%), 13 (4.4 mg, 2%), and 14 (13.1 mg, 6%).
16
lowing the procedure described by Thomson. The reaction prod-

2
626
E. L. Bonifazi et al. / Bioorg. Med. Chem. 18 (2010) 2621–2630
4
.2.5.1.1. 2,5-Dihydroxy-3-(3-methylbut-2-enyl)naphthalene-1,4-
J = 8.2, 7.6 Hz, H-7), 7.28 (1H, dd, J = 8.4, 1.2 Hz, H-6), 6.28 (1H,
0
0
dione (5-hydroxylapachol, 4). Orange crystals; mp 143–144 °C
dd, J = 17.5, 10.6 Hz, H-2 ), 5.00 (1H, dd, J = 17.5, 0.8 Hz, H-3 ),
1
0
1
13
0
0
0
13
(
lit. 144–145 °C). The spectroscopic data ( H NMR, C NMR,
4.97 (1H, dd, J = 10.6, 0.9 Hz, H-3 ), 1.57 (6H, s, CH
3
-4 and 5 ).
C
1
0
and MS) were consistent with that reported in the literature.
NMR (125.77 MHz, CDCl
153.5 (C-2), 148.1 (C-2 ), 134.7 (C-7), 128.5 (C-9), 127.5 (C-3),
3
): 191.3 (C-4), 181.5 (C-1), 161.5 (C-5),
0
4.2.5.1.2. 2-(3-Methylbut-2-enyloxy)-5-hydroxynaphthalene-1,4-
0
0
dione (12). Dark yellow crystals; mp 135–136 °C (from isopropa-
nol). IR (KBr) 3404, 2936, 2910, 1689, 1633, 1590, 1452, 1376,
241, 852, 740 cm
OH-5), 7.66 (1H, dd, J = 7.5, 1.1 Hz, H-8), 7.56 (1H, t, J = 8.0 Hz, H-
), 7.26 (1H, dd, J = 8.4, 1.1 Hz, H-6), 6.08 (1H, s, H-3), 5.48 (1H,
tsept, J = 6.7, 1.3 Hz, H-2 ), 4.60 (2H, d, J = 6.9 Hz, H-1 ), 1.81 (3H,
s, CH
90.9 (C-4), 179.53 (C-1), 161.0 (C-5), 160.2 (C-2), 140.9 (C-3 ),
35.4 (C-7), 131.1 (C-9), 125.0 (C-6), 119.5 (C-8), 116.9 (C-2 ),
14.1 (C-10), 110.0 (C-3), 66.7 (C-1 ), 25.8 (C-4 ), 18.4 (C-5 ). MS
m/z (%) 258 (M , 10), 243 (10), 225 (2), 215 (3), 197 (1), 190 (99),
73 (3), 162 (33), 145 (6), 134 (11), 121 (33), 105 (56), 89 (23),
9 (91), 63 (36), 41 (100). HRMS found: 258.0894 (M) calcd for
126.7 (C-6), 118.9 (C-8), 115.2 (C-10), 109.8 (C-3 ), 41.0 (C-1 ),
0
0
+
28.5 (C-4 and C-5 ). MS m/z (%) 258 (M , 63), 243 (100), 229
ꢀ
1 1
1
.
3
H NMR (500.13 MHz, CDCl ): 12.26 (1H, s,
(12), 215 (22), 197 (13), 187 (11), 169 (10), 159 (8), 141 (11),
131 (9), 121 (18), 103 (7), 92 (17), 77 (16), 63 (13). HRMS found:
+
7
259.0970 (M+H) calcd for C15
15
H O
4
: 259.0970.
0
0
0
0
13
3
-4 ), 1.76 (3H, s, CH
3
-5 ). C NMR (50.32 MHz, CDCl
3
0
):
4.2.5.3. Method c. A solution of 9 (50.0 mg, 0.26 mmol) in DMF
(1.15 mL) was added to K CO (36.5 mg, 0.26 mmol) or Li CO
1
1
1
2
3
2
3
0
(19.2 mg, 0.26 mmol) in DMF (0.350 mL), and stirred for 15 min
at room temperature under an argon atmosphere. 1-Bromo-3-
methylbut-2-ene (0.0761 mL, 0.66 mmol) in DMF (0.115 mL) was
added dropwise over 15 min, stirring was continued for 15 min
at the same temperature, and then the reaction mixture was
heated to 40 °C. After 2 h the mixture was cooled and water was
added to stop the reaction. The reaction mixture was extracted
with ether and the organic layer was washed successively with
0
0
0
+
1
6
C
+
15
H
14
O
4
: 258.0892.
.2.5.1.3. 2,3-Dihydro-5-hydroxy-2-(iodomethyl)-3,3-dimethyl-
naphtho[2,3-b]furan-4,9-dione (13). Yellow crystals; mp 114–
4
1
7
7
15 °C. IR (KBr) 2967, 2927, 2872, 1730, 1682, 1633, 1454, 765,
2 4
NaCl (ss), water, and dried over anhydrous Na SO . The solvent
ꢀ1
1
05 cm
.
H NMR (500.13 MHz, CDCl
3
): 12.29 (1H, s, OH-5),
was evaporated under reduced pressure and the residue was frac-
tionated by column chromatography on silica gel (cyclohexane/tol-
uene, gradient), to give quinones 4 (3.7 mg, 6%), 12 (16.6 mg, 25%),
15 (13.7 mg, 20%) and 16 (11 mg, 13%).
4.2.5.3.1. 2-(3-Methylbut-2-enyloxy)-5-hydroxy-3-(3-methylbut-
2-enyl)naphthalene-1,4-dione (16). Yellow oil. IR (KBr) 3386,
.61 (1H, dd, J = 7.4, 1.1 Hz, H-8), 7.53 (1H, t, J = 7.9 Hz, H-7), 7.24
(
1H, dd, J = 8.4, 1.0 Hz, H-6), 4.76 (1H, t, J = 7.0 Hz, H-2), 3.47 (1H,
dd, J = 11.0, 7.1 Hz, H-10a), 3.39 (1H, dd, J = 11.0, 7.0 Hz, H-10b),
.64 (3H, s, CH -11), 1.44 (3H, s, CH
-12). 13C NMR (125.77 MHz,
CDCl ): 188.4 (C-4), 177.1 (C-9), 161.4 (C-5), 158.0 (C-9a), 135.1
C-7), 131.5 (C-8a), 130.5 (C-3a), 125.9 (C-6), 119.4 (C-8), 115.0
C-4a), 94.0 (C-2), 46.0 (C-3), 27.6 (C-11), 19.7 (C-12), -2.1 (C-10).
1
3
3
3
ꢀ
1
1
(
(
2962, 2927, 2857, 1730, 1627, 1462, 1265 cm
(500.13 MHz, CDCl ): 12.31 (1H, s, OH-5), 7.56 (1H, dd, J = 7.5,
1.7 Hz, H-8), 7.53 (1H, t, J = 7.7 Hz, H-7), 7.21 (1H, dd, J = 7.8,
.
H NMR
3
+
MS m/z (%) 384 (M , 100), 369 (M–CH
3
, 15), 257 (M–I, 22), 242
0
0
(
(
MꢀI–CH
3
, 39), 215 (16), 199 (8), 187 (15), 128 (11), 115 (17), 92
1.7 Hz, H-6), 5.45 (1H, tsept, J = 7.7, 1.9 Hz, H-2 ), 5.10 (1H, tsept,
J = 7.0, 1.7 Hz H-2 ), 4.93 (2H, d, J = 7.3 Hz, H-1 ), 3.27 (2H, d,
+
0
00
15), 69 (12), 63 (18), 41 (34). HRMS found: 384.9932 (M+H) calcd
0
0
00
for C15
H
14
O
4
I: 384.9937.
J = 7.1 Hz, H-1 ), 1.77 (3H, s, CH
3
-5 ), 1.76 (3H, s, CH
3
-4 ), 1.72
0
0
0
13
4.2.5.1.4.
2,3-Dihydro-5-hydroxy-2-(2-iodopropano-2-yl)naph-
(3H, s, CH
3
-5 ), 1.68 (3H, d, J = 0.7 Hz, CH
3
-4 ).
C NMR
tho[2,3-b]furan-4,9-dione (14). Orange crystals; mp 113–114 °C.
(125.77 MHz, CDCl
(C-2), 139.7 (C-3 ), 135.2 (C-7), 134.7 (C-3), 133.7 (C-3 ), 131.7
3
): 190.9 (C-4), 181.3 (C-1), 160.9 (C-5), 157.6
0
0
0
IR (KBr) 2975, 2920, 2846, 1677, 1627, 1612, 1457, 1260, 770,
ꢀ1
1
0
00
6
86 cm
.
H NMR (500.13 MHz, CDCl
3
): 12.19 (1H, s, OH-5),
(C-9), 124.4 (C-6), 119.9 (C-2 ), 119.8 (C-2 ), 118.8 (C-8), 114.5
0
0
00
0
0
7
.64 (1H, dd, J = 7.5, 1.1 Hz, H-8), 7.54 (1H, t, J = 8.0 Hz, H-7), 7.24
(C-10), 70.1 (C-1 ), 25.8 (C-4 ), 25.8 (C-4 ), 22.5 (C-1 ), 18.1 (C-
0
0
0
+
(
1H, dd, J = 8.4, 1.1 Hz, H-6), 4.30 (1H, dd, J = 8.2, 5.5 Hz, H-2),
5 ), 17.9 (C-5 ). MS m/z (%) 326 (M ,1), 279 (12), 258 (61), 243
3
.36 (1H, dd, J = 19.0, 5.5 Hz, H-3a), 3.18 (1H, dd, J = 19.1, 8.3 Hz,
(100), 225 (10), 215 (21), 197 (8), 187 (7), 131 (5), 121 (17), 104
13
H-3b), 1.66 (3H, s, CH
3
-11), 1.60 (3H, s, CH
3
-12).
C NMR
(7), 83(11), 71(21), 58 (47), 41(32). HRMS found: 327.1595
+
(
(
(
125.77 MHz, CDCl
C-9a), 135.4 (C-7), 131.0 (C-8a), 125.0 (C-6), 119.4 (C-8), 118.7
C-3a), 113.9 (C-4a), 80.8 (C-2), 30.3 (C-3), 27.2 (C-10), 26.5 (C-
3
): 188.9 (C-4), 178.7 (C-9), 161.0 (C-5), 154.3
(M+H) calcd for C20
H
23
O
4
: 327.1596.
4.2.6. Reaction of 3-hydroxyjuglone (10) with 1-bromo-3-
methylbut-2-ene
+
1
1), 24.9 (C-12). MS m/z (%) 384 (M , 61), 257 (M-I, 76), 215
(
100), 187 (16), 121 (23), 92 (19), 69 (98), 63 (30), 41 (48). HRMS
4.2.6.1. Method a. The reaction of 3-hydroxyjuglone (10)
+
found: 384.9937 (M+H) calcd for C15
H
14
O
4
I: 384.9937.
(162 mg,
0.855 mmol)
with
1-bromo-3-methylbut-2-ene
(
0.115 mL, 0.99 mmol) was carried out following the procedure
4
.2.5.2. Method b. To 2-hydroxyjuglone (9) (50 mg, 0.26 mmol),
in a dried round bottom flask, was added 1-bromo-3-methylbut-
-ene (37 L, 0.316 mmol), sodium iodide (47.4 mg, 0.316 mmol),
described for 2-hydroxyjuglone (method a). The resulting mixture
was fractionated by column chromatography on silica gel (hex-
ane/EtOAc, gradient), to give quinones 17 (22.1 mg, 10%), 18
(41.9 mg, 19%), 19 (5.4 mg, 2.4%) and 20 (3.0 mg, 0.9%).
2
l
triethylamine (0.045 mL, 0.316 mmol) and DMF (0.350 mL). The
mixture was stirred at room temperature under a nitrogen atmo-
sphere for 1 h, and then heated to 40 °C. After 4 h, the reaction mix-
ture was cooled diluted with water and extracted with CH
organic layer was washed successively with NaHCO (5% w/v) and
water, and dried over anhydrous Na SO . The solvent was evapo-
rated under reduced pressure and the residue was fractionated
by column chromatography on silica gel (hexane/EtOAc, gradient),
to give quinones 4 (39 mg, 57%), 12 (12.6 mg, 19%) and traces of 15.
4.2.6.1.1. 3,5-Dihydroxy-2-(3-methylbut-2-enyl)naphthalene-1,4-
2
7
33
dione (17)
.
Orange needles; mp 122–123 °C (from EtOH) (lit.
1
2
Cl
2
. The
121.5–123.0 °C). H NMR (500.13 MHz, CDCl
3
): 11.11 (1H, s, OH-
3
8), 7.65 (1H, dd, J = 7.5, 1.5 Hz, H-5), 7.62 (1H, t, J = 7.7 Hz, H-6),
0
2
4
7.19 (1H, dd, J = 8.1, 1.5 Hz, H-7), 5.18 (1H, m, H-2 ), 3.29 (2H, d,
0
0
0
13
J = 7.4 Hz, H-1 ), 1.78 (3H, s, CH
3
-5 ), 1.68 (3H, s, CH
3
-4 ).
C
NMR (125.77 MHz, CDCl
8), 152.3 (C-2), 137.5 (C-6), 134.1 (C-3 ), 132.7 (C-10), 124.7
3
): 185.0 (C-1), 183.7 (C-4), 161.1 (C-
0
0
4
.2.5.2.1. 2,5-Dihydroxy-3-(2-methylbut-3-en-2-yl)naphthalene-
,4-dione (15). Orange crystals; mp 123–124 °C. IR (KBr) 3246,
989, 2951, 2926, 1725, 1661, 1631, 1485, 1463, 1267, 765,
(C-3), 123.1 (C-7), 119.7 (C-5), 119.3 (C-2 ), 113.0 (C-9), 25.8
0
0
0
+
1
(C-5 ), 22.7 (C-1 ), 17.9 (C-4 ). MS m/z (%) 258 (M , 100), 244
(69), 225 (31), 215 (54), 195 (25), 187 (21), 175 (24), 165 (15),
149 (30), 131 (23), 121 (2), 103 (23), 92 (46), 77 (50), 65 (59),
41 (72).
2
7
7
ꢀ1
1
02 cm
.
3
H NMR (500.13 MHz, CDCl ): 12.55 (1H, s, OH-5),
.97 (1H, s, OH-2), 7.62 (1H, dd, J = 7.4, 1.2 Hz, H-8), 7.52 (1H, dd,

E. L. Bonifazi et al. / Bioorg. Med. Chem. 18 (2010) 2621–2630
2627
4
.2.6.1.2. 2-(3-Methylbut-2-enyloxy)-8-hydroxynaphthalene-1,4-
c). The resulting mixture was fractionated by column chromatogra-
phy on silica gel (hexane/EtOAc, gradient), to give quinones 17
(10.3 mg, 7%), 18 (27.9 mg, 19%), 21 (29.5 mg, 20%) and 22
(18.6 mg, 10%).
dione (18). Orange needles; mp 133–135 °C (from EtOH). IR
KBr) 3564, 3068, 2969, 2915, 1648, 1597, 1580, 1457, 1300,
205, 826, 730 cm . H NMR (500.13 MHz, CDCl ): 11.79 (1H, s,
3
(
1
ꢀ1 1
OH-8), 7.62 (2H, m, H-5 and H-6), 7.22 (1H, dd, J = 6.9, 2.8, H-7),
4.2.6.3.1. 3-(3-Methylbut-2-enyloxy)-5-hydroxy-2-(3-methylbut-
0
27
6
.13 (1H, s, H-3), 5.49 (1H, tsept, J = 6.6, 1.8 Hz H-2 ), 4.58 (2H, d,
2-enyl)naphthalene-1,4-dione (22)
.
Yellow crystals; mp 72–
0
0
0
13
J = 6.9 Hz, H-1 ), 1.81 (3H, s, CH
3
-4 ), 1.76 (3H, s, CH
3
-5 ). C NMR
73 °C (from EtOH). IR (KBr) 3418, 2967, 2920, 2872, 1635, 1609,
ꢀ1 1
(
50.32 MHz, CDCl
3
): 185.2 (C-1), 184.1 (C-4), 161.9 (C-8), 159.1
3
1457, 1273, 752, 715 cm . H NMR (500.13 MHz, CDCl ): 11.88
0
(
C-2), 141.0 (C-3 ), 137.0 (C-6), 132.0 (C-10), 123.7 (C-7), 118.8
(1H, s, OH-8), 7.60 (1H, dd, J = 7.5, 1.4 Hz, H-5), 7.57 (1H, t,
J = 7.9 Hz, H-6), 7.20 (1H, dd, J = 8.1, 1.4 Hz, H-7), 5.48 (1H, tsept,
0
0
(
C-5), 116.9 (C-2 ), 114.3 (C-9), 111.0 (C-3), 66.5 (C-1 ), 25.8 (C-
0
0
+
00
0
4
), 18.4 (C-5 ). MS m/z (%) 258 (M , 90), 240 (21), 231 (47), 213
J = 7.2, 1.4 Hz H-2 ), 5.08 (1H, tsept, J = 7.2, 1.4 Hz, H-2 ), 4.86
0
0
0
(
13), 203 (12), 192 (100), 173 (12), 161 (16), 145 (12), 133 (11),
(2H, d, J = 7.3 Hz, H-1 ), 3.29 (2H, d, J = 7.3 Hz, H-1 ), 1.77 (6H, s,
-5 and 4 ), 1.73 (3H, s, CH
4 ); C NMR (125.77 MHz, CDCl
(C-8), 156.3 (C-2), 139.8 (C-3 ), 137.2 (C-3 ), 136.3 (C-6), 133.7
(C-3), 132.2 (C-10), 123.7 (C-7), 119.9 (C-2 ), 119.7 (C-2 ), 118.9
(C-5), 114.4 (C-9), 70.4 (C-1 ), 25.8 (C-4 ), 25.8 (C-4 ), 23.4 (C-1 ),
18.1 (C-5 ), 17.9 (C-5 ). MS m/z (%) 326 (M , 0.6), 258 (48), 243
0
00
00
1
2
21 (23), 105 (74), 89 (30), 68 (82), 63 (53), 53 (62); HRMS found:
58.0899, calcd for C15 : 258.0892.
.2.6.1.3. 9-Hydroxy-2,2-dimethyl-2H-benzo[g]chromene-5,10-
dione -caryopterone, 19). Orange needles; mp 144–145 °C
CH
3
3
-5 ), 1.67 (3H, d, J = 1.1 Hz CH
): 187.0 (C-1), 184.6 (C-4), 161.4
3
-
0
13
H
14
O
4
3
0
0
0
4
0
00
(a
3
3
1
00
00
0
0
(
(
1
1
dec.) (from EtOH) (lit.
500.13 MHz, CDCl ): 11.88 (1H, s, OH-9), 7.64 (1H, dd, J = 7.5,
.1 Hz, H-6), 7.59 (1H, t, J = 7.8 Hz, H-7), 7.21 (1H, dd, J = 8.3,
.0 Hz, H-8), 6.64 (1H, d, J = 10.0 Hz, H-4), 5.74 (1H, d, J = 10.0 Hz,
-11 and 12); 13C NMR (125.77 MHz, CDCl
):
84.7 (C-10), 181.0 (C-5), 161.5 (C-9), 152.1 (C-10a), 136.7 (C-7),
31.6 (C-5a), 131.3 (C-3), 124.0 (C-8), 119.0 (C-6), 118.6 (C-4a),
15.4 (C-4), 114.5 (C-9a), 80.7 (C-2), 28.4 (CH -11 and 12). MS
144–145.5 °C (dec)).
H
NMR
0
0
0
+
3
(67), 225 (5), 215 (12), 204 (7), 187 (5), 175 (14), 165 (13), 149
(19), 128 (5), 121 (14), 103 (5), 92 (13), 69 (100), 55 (19), 41
+
H-3), 1.57 (6H, s, CH
3
3
(35); HRMS (M+H) 326.1532, calcd for C20
H
22
4
O : 326.1518.
1
1
1
4.2.7. 5-Hydroxy-2-methoxy-3-(3-methylbut-2-
enyl)naphthalene-1,4-dione (23) and 2,5-dimethoxy-3-(3-
methylbut-2-enyl)naphthalene-1,4-dione (24)
3
+
m/z (%) 256 (M , 21), 241 (100), 228 (3), 213 (25), 182 (5), 173
5), 149 (7), 121 (16), 92 (13), 77 (8), 63 (21), 43 (18).
.2.6.1.4. 2,3-Dihydro-8-hydroxy-2-(iodomethyl)-3,3-dimethyl-
naphtho[2,3-b]furan-4,9-dione (20). Orange oil. IR (KBr) 2962,
(
5-Hydroxylapachol (4) (17.8 mg, 0.069 mmol) was added to a
stirred mixture of potassium carbonate (52 mg, 0.38 mmol) and
acetone (1.3 mL) at room temperature followed by dimethyl sul-
fate (0.0103 mL, 0.11 mmol). After 20 h the starting material had
disappeared (tlc), the solvent was removed under vacuum and
the solid was extracted with EtOAc, washed with brine and
water, dried over anhydrous sodium sulfate, and the solvent
evaporated under reduced pressure. The residue was fraction-
ated by column chromatography on silica gel (hexane/EtOAc,
gradient), to give quinones 23 (1.5 mg, 8%) and 24 (4.0 mg,
47%).
4
ꢀ1
1
2
(
922, 1640, 1609, 1457, 1370, 1276, 752, 707 cm
500.13 MHz, CDCl ): 11.62 (1H, s, OH-8), 7.60 (2H, m, H-5 and
H-6), 7.20 (1H, dd, J = 7.4, 2.2 Hz, H-7), 4.74 (1H, t, J = 6.7 Hz, H-
.
H NMR
3
2
6
), 3.68 (1H, dd, J = 11.0, 6.8 Hz, H-10a), 3.60 (1H, dd, J = 11.1,
1
3
.7 Hz, H-10b), 1.62 (3H, s, CH
): 182.6 (C-9), 181.1 (C-4), 161.9 (C-8),
57.3 (C-9a), 137.0 (C-6), 133.45 (C-4a), 130.6 (C-3a), 124.1 (C-7),
19.1 (C-5), 114.5 (C-8a), 93.1 (C-2), 46.1 (C-3), 27.5 (C-10), 27.2
3
-11), 1.43 (3H, s, CH
3
-12).
C
NMR (125.77 MHz, CDCl
1
1
3
+
(
(
(
C-11), 19.8 (C-12). MS m/z (%) 384 (M , 0.6), 323 (36), 257
+
M ꢀI, 100), 242 (66), 213 (40), 199 (25), 185 (25), 128 (30), 115
4.2.7.1. 5-Hydroxy-2-methoxy-3-(3-methylbut-2-enyl)naphtha-
lene-1,4-dione (5-hydroxy-2-methoxylapachol, 23). Yellow
crystals; mp 60–61 °C. IR (KBr) 2954, 2930, 2854, 1674, 1633,
52), 103 (27), 92 (71), 77 (37), 63 (43), 41 (48); HRMS found:
3
13 4
83.9849, calcd for C15H O I: 383.9859.
ꢀ1
1
1
601, 1457, 1310, 1266, 762, 697 cm
.
H NMR (500.13 MHz,
4
0
0
.2.6.2. Method b. The reaction of 3-hydroxyjuglone (10) (81 mg,
.428 mmol) with 1-bromo-3-methylbut-2-ene (57.4 L,
.50 mmol) was carried out as described for compound 9 (method
CDCl
3
): 12.29 (1H, s, OH-5), 7.57 (1H, dd, J = 7.5, 1.7 Hz, H-8),
l
7.54 (1H, t, J = 7.7 Hz, H-7), 7.22 (1H, dd, J = 7.8, 1.7 Hz, H-6), 5.11
(1H, tsept, J = 7.2, 1.4 Hz, H-2 ), 4.14 (3H, s, OCH
0
3
), 3.27 (2H, d,
0
0
b). The resulting residue was fractionated by column chromatogra-
phy on silica gel (hexane/EtOAc, gradient), to give quinones 17
J = 7.3 Hz, H-1 ), 1.78 (3H, s, CH
3
-5 ), 1.69 (3H, d, J = 1.1 Hz, CH
3
-
0
13
4 ). C NMR (125.77 MHz, CDCl
3
): 190.9 (C-4), 181.2 (C-1), 160.9
0
(
60.7 mg, 55%), 18 (23.2 mg, 21%) and traces of 21.
(C-5), 158.0 (C-2), 135.3 (C-7), 133.9 (C-3 ), 131.6 (C-9), 124.6 (C-
3), 124.5 (C-6), 119.8 (C-2 ), 118.9 (C-8), 114.4 (C-10), 61.3
(OCH ), 25.8 (C-4 ), 22.4 (C-1 ), 17.9 (C-5 ). MS m/z (%) 272 (M ,
3
0
4
.2.6.2.1. 3,5-Dihydroxy-2-(2-methylbut-3-en-2-yl)naphthalene-
27
0
0
0
+
1,4-dione (21)
.
Orange needles; mp 118–119 °C (dec.) (from
EtOH). IR (KBr) 3313, 2956, 2927, 2867, 1734, 1635, 1459, 1286,
81), 257 (51), 239 (42), 229 (71), 211 (46), 197 (20), 187 (16),
173 (26), 165 (24), 149 (19), 128 (13), 115 (39), 103 (15), 92
(29), 77 (25), 63 (39); HRMS found: 272.1055, calcd for
ꢀ1 1
7
52, 705 cm
3
. H NMR (500.13 MHz, CDCl ): 10.92 (1H, s, OH-
8
), 7.72 (1H, s, OH-2), 7.63 (1H, t, J = 7.9 Hz, H-6), 7.58 (1H, dd,
J = 7.5, 1.2 Hz, H-5), 7.17 (1H, dd, J = 8.4, 1.2 Hz, H-7), 6.27 (1H,
C
16
H
16
O
4
: 272.1049.
0
0
dd, J = 17.5, 10.6 Hz, H-2 ), 5.01 (1H, dd, J = 17.5, 0.9 Hz, H-3 ),
0
0
0
13
4
.97 (1H, dd, J = 10.6, 0.9 Hz, H-3 ), 1.55 (6H, s, CH
3
-4 and 5 ).
C
4.2.7.2.
2,5-Dimethoxy-3-(3-methylbut-2-enyl)naphthalene-
NMR (125.77 MHz, CDCl ): 185.2 (C-1), 184.0 (C-4), 160.7 (C-8),
3
1,4-dione (2,5-dimethoxylapachol, 24). Orange oil. IR (KBr)
0
ꢀ1
1
1
1
2
2
1
2
52.5 (C-2), 147.9 (C-2 ), 137.9 (C-6), 133.9 (C-10), 129.6 (C-3),
2930, 2841, 1667, 1614, 1580, 1470, 1444, 1239, 762 cm
. H
0
0
22.3 (C-7), 119.8 (C-5), 112.5 (C-9), 109.8 (C-3 ), 41.1 (C-1 ),
NMR (500.13 MHz, CDCl ): 7.70 (1H, dd, J = 7.6, 1.1 Hz, H-8),
3
0
0
+
8.1 (C-4 and 5 ). MS m/z (%) 258 (M , 60), 243 (100), 229 (16),
7.60 (1H, dd, J = 8.4, 7.7 Hz, H-7), 7.26 (1H, dd, J = 8.4, 0.9 Hz, H-
0
15 (40), 197 (14), 187 (10), 175 (9), 159 (6), 141 (12), 128 (14),
6), 5.13 (1H, tsept, J = 7.1, 1.3, Hz, H-2 ), 4.05 (3H, s, OCH
3
0
at C-
15 (36), 103 (13), 92 (30), 77 (30), 63 (44), 41 (54); HRMS found:
2), 3.99 (3H, s, OCH
3
at C-5), 3.27 (2H, d, J = 7.3 Hz, H-1 ), 1.77
-5 ), 1.66 (3H, d, J = 1.0 Hz, CH
+
0
0
13
59.0968 (M+H) , calcd for C15
15
H O
4
: 259.0970.
(3H, s, CH
3
3
-4 ).
C
NMR
(
125.77 MHz, CDCl
3
): 184.7 (C-4), 181.8 (C-1), 159.3 (C-5), 155.7
0
4
.2.6.3. Method c. The reaction of 3-hydroxyjuglone (10)
L,
.66 mmol) was carried out as described for compound 9 (method
(C-2), 136.4 (C-3), 134.1 (C-7), 133.9 (C-9), 133.4 (C-3 ), 120.3
(C-2 ), 119.6 (C-10), 118.9 (C-8), 117.9 (C-6), 60.8 (OCH
56.5 (OCH
0
(
108 mg, 0.57 mmol) with 1-bromo-3-methylbut-2-ene (76.5
l
3
at C-2),
0
0
0
0
3
at C-5), 25.8 (C-4 ), 23.2 (C-1 ), 17.9 (C-5 ). MS m/z

2
628
E. L. Bonifazi et al. / Bioorg. Med. Chem. 18 (2010) 2621–2630
+
(
1
(
2
%) 286 (M , 20), 271 (16), 255 (13), 243 (14), 228 (11), 179 (13),
49 (9), 135 (11), 115 (12), 105 (11), 91 (11), 76 (33), 63 (18), 43
4.2.11. Synthesis of 2-(3-methylbut-2-enyloxy)-8-
hydroxynaphthalene-1,4-dione (18) and 3,5-dihydroxy-2-(2-
methylbut-3-en-2-yl)naphthalene-1,4-dione (21) using
+
17 19 4
100); HRMS found: 287.1281 (M+H) , calcd for C H O :
0
87.1283.
Mitsunobu s conditions
3
-Hydroxyjuglone (10) (100.0 mg, 0.54 mmol), 3-methylbut-2-
4
.2.8. Reaction of 5-hydroxylapachol (4) with CAN
The reaction of 5-hydroxylapachol (4) (30.0 mg, 0.12 mmol)
en-1-ol (0.162 mL, 1.6 mmol) and triphenylphosphine (419 mg,
1.6 mmol) were dissolved in dry THF (5 mL), and then DIAD
(0.12 mL, 0.6 mmol) in THF (2.5 mL) was added. The reaction was
stirred 1 h at room temperature and then concentrated in vacuo.
The residue was fractionated by column chromatography on silica
gel (hexane/EtOAc, gradient), to give quinones 18 (55%) and 21
(10%).
with CAN (191 mg, 0.35 mmol) was carried out following the pro-
cedure described by Eyong et al.22 The reaction residue was puri-
fied by flash chromatography (silica gel, eluting with mixtures of
hexane/EtOAc, gradient), to give 27 (2.1 mg, 7%) and 28 (4.3 mg,
1
2%).
4
.2.12. Synthesis of 2,5-dihydroxy-3-(2-methylbut-3-en-2-
4
.2.8.1. 2,3-Dihydro-5-hydroxy-2-(prop-1-en-2-yl)naphtho[2,3-b]-
yl)naphthalene-1,4-dione (15) by Claisen rearrangement
Compound 12 (77.0 mg, 0.3 mmol) in EtOH (4 mL) was heated
at 60 °C overnight. The reaction mixture was cooled to room tem-
perature, and the solvent evaporated under reduced pressure. The
residue was fractionated by column chromatography on silica gel
furan-4,9-dione (5-hydroxydehydroiso-a-lapachone, 27). Orange
34
crystals; mp 118–119 °C (from EtOH) (lit. 118–121 °C). IR (KBr) 3595,
ꢀ1
2
961, 2926, 1676, 1636, 1455, 1282, 1226, 832, 765 cm . The spectro-
scopic data (NMR, MS) were consistent with that reported in the
34
literature.
(
hexane/EtOAc, gradient), to give quinone 15 (70.8 mg, 92%).
4
.2.8.2. 2,3-Dihydro-5-hydroxy-2-(2-nitratepropan-2-yl)naph-
4
.2.13. Synthesis of 3,5-dihydroxy-2-(2-methylbut-3-en-2-
tho[2,3-b]furan-4,9-dione (28). Orange crystal; mp 157–
yl)naphthalene-1,4-dione (21) by Claisen rearrangement
Compound 21 (90% yield) was obtained from compound 18 fol-
lowing the procedure described previously for compound 15.
1
1
CDCl
58 °C (from EtOH). IR (KBr) 3412, 2946, 2920, 2846, 1677,
ꢀ1 1
638, 1627, 1606, 1456, 1292 cm . H NMR (500.13 MHz,
): 12.13 (1H, s, OH-5), 7.65 (1H, dd, J = 7.4, 1.1 Hz, H-
), 7.56 (1H, t, J = 7.9 Hz, H-7), 7.27 (1H, dd, J = 7.9, 0.8 Hz,
3
8
4
.2.14. Synthesis of 2,3-dihydro-5-hydroxy-2-(iodomethyl)-3,3-
H-6), 5.19 (1H, dd, J = 11.0, 8.8 Hz, H-2), 3.27 (1H, dd,
J = 17.4, 11.0 Hz, H-3a), 3.19 (1H, dd, J = 17.4, 8.9 Hz, H-3b),
dimethylnaphtho[2,3-b]furan-4,9-dione (13) and 2,3-dihydro-
9-hydroxy-2-(iodomethyl)-3,3-dimethylnaphtho[1,2-b]furan-
4
13
1
.71 (3H, s, CH
3
-11), 1.68 (3H, s, CH
): 187.8 (C-4), 176.4 (C-9), 161.3 (C-5),
60.4 (C-9a), 135.3 (C-7), 131.7 (C-8a), 125.9 (C-6), 123.7 (C-
a), 119.7 (C-8), 114.7 (C-4a), 89.7 (C-10), 88.4 (C-2), 28.1
3
-12);
C
NMR
,5-dione (31)
(
125.77 MHz, CDCl
3
Compound 15 (18.2 mg, 0.070 mmol) in dichloromethane
1
3
(
2 mL) was treated at room temperature with a solution of iodine
+
(57.6 mg, 0.46 mmol) in a mixture of dichloromethane (1.9 mL)
and pyridine (0.25 mL). The reaction mixture was stirred for
40 min at room temperature, followed by addition of cold water.
The organic phase was washed with 10% sodium carbonate fol-
(
C-3), 21.4 (C-11), 20.3 (C-12). MS m/z (%) 319 (M , 16), 257
MꢀNO , 3.5), 215 (71), 159 (16), 131 (15), 103 (12), 92 (8),
7 (22), 59 (20), 43 (100); HRMS found: 320.0774 (M+H)
calcd for C15 : 320.0770.
(
7
3
+
H14NO
7
lowed by cold water and dried with anhydrous Na
2 4
SO . The solvent
was evaporated under vacuum and the residue fractionated by col-
umn chromatography on silica gel (hexane/EtOAc, gradient), to
give quinones 13 (17.7 mg, 66%) and 31 (1.9. mg, 7%).
4
.2.9. 3,4-Dihydro-6-hydroxy-2,2-dimethyl-2H-benzo[g]chro-
mene-5,10-dione (6-hydroxy- -lapachone, 30)
4
The reaction of 5-hydroxylapachol (4) with H SO was carried
a
2
out following the procedure described in Ref. 23. The reaction
residue was purified by column chromatography on silica gel
4
.2.14.1. 12,3-Dihydro-9-hydroxy-2-(iodomethyl)-3,3-dimeth-
ylnaphtho[1,2-b]furan-4,5-dione (31). Orange crystals; mp
1
1
(
hexane/EtOAc, gradient) to give 30 (97%) as yellow crystals;
54–155 °C. IR (KBr) 3450, 2925, 2854, 1727, 1608, 1461,
3
0
1
mp 179–180 °C (lit. 178–180 °C). The H NMR spectrum was
ꢀ1 1
3
259, 1039, 945 cm . H NMR (500.13 MHz, CDCl ): 7.89 (1H,
consistent with that reported in the literature.33
125.77 MHz, CDCl ): 190.1 (C-5), 179.3 (C-10), 160.9 (C-6),
55.3 (C-10a), 135.0 (C-8), 131.2 (C-9a), 124.7 (C-7), 119.5 (C-
a), 119.1 (C-9), 114.1 (C-5a), 78.6 (C-2), 31.3 (C-3), 26.5 (CH
13
C
NMR
s, OH-9), 7.71 (1H, dd, J = 7.5, 1.0 Hz, H-6), 7.48 (1H, dd, J = 8.4,
.5 Hz, H-7), 7.22 (1H, dd, J = 8.5, 1.0 Hz, H-8), 4.88 (1H, dd,
J = 9.4, 4.8, H-2), 3.49 (1H, dd, J = 11.1, 4.8 Hz, H-10a), 3.46 (1H,
dd, J = 11.1, 9.4 Hz, H-10b), 1.55 (3H, s, CH -11), 1.37 (3H, s,
): 180.5 (C-5), 175.3 (C-
), 166.4 (C-9b), 155.1 (C-9), 133.7 (C-7), 131.3 (C-5a), 125.0
(
3
7
1
4
1
2
3
-
+
3
1), 16.1 (C-4). MS m/z (%) 258 (M , 74), 243 (M-CH
3
, 100),
13
3 3
CH -12). C NMR (125.77 MHz, CDCl
15 (15), 173 (29), 149 (14), 121 (15), 89 (18), 74 (14), 63
4
(
39), 41 (85).
(
(
(
(
C-8), 123.6 (C-6), 122.2 (C-3a), 110.7 (C-9a), 96.8 (C-2), 44.2
C-3), 26.6 (C-11), 19.8 (C-12), 0.0 (C-10). MS m/z (%) 384
M ), 369 (M-CH
57), 214 (31), 199 (8), 187 (23), 127 (19), 115 (18), 92 (22),
9 (100), 63 (61), 41 (82). HRMS found: 384.9933 (M+H)+ calcd
I: 384.9937.
+
4
.2.10. Synthesis of 2-(3-methylbut-2-enyloxy)-5-
3 3
, 7), 257 (M–I, 29), 242 (MꢀI–CH , 17), 229
hydroxynaphthalene-1,4-dione (12) and 2,5-dihydroxy-3-(2-
methylbut-3-en-2-yl)naphthalene-1,4-dione (15) using
6
0
Mitsunobu s conditions
14 4
for C15H O
2
-Hydroxyjuglone (9) (92.8 mg, 0.5 mmol), 3-methylbut-2-en-
1
0
-ol (0.0609 mL, 0.6 mmol) and triphenylphosphine (157 mg,
.6 mmol) were dissolved in dry THF (5 mL), and then DIAD
4
.2.15. Synthesis of 2,3-dihydro-8-hydroxy-2-(iodomethyl)-3,3-
dimethylnaphtho[2,3-b]furan-4,9-dione (20) and 2,3-dihydro-
6
4
(
0.12 mL, 0.6 mmol) in THF (2.5 mL) was added. The reaction was
-hydroxy-2-(iodomethyl)-3,3-dimethylnaphtho[1,2-b]furan-
,5-dione (32)
Compounds 20 (12%) and 32 (21%) were obtained from com-
stirred overnight at room temperature and then concentrated in
vacuo. The residue was fractionated by column chromatography
on silica gel (hexane/EtOAc, gradient), to give quinones 12
pound 21 following the procedure described previously for com-
pound 13.
(
77.4 mg, 60%) and 15 (15.5 mg, 12%), respectively.

E. L. Bonifazi et al. / Bioorg. Med. Chem. 18 (2010) 2621–2630
2629
4
.2.15.1. 2,3-Dihydro-6-hydroxy-2-(iodomethyl)-3,3-dimethyl-
4.3.1. Chemosensitivity testing
naphtho[1,2-b]furan-4,5-dione (32). Orange crystal; mp 215–
Chemosensitivity tests were performed using the SRB assay of
the NCI with slight modifications. Briefly, pure compounds were
initially dissolved in DMSO at 400 times the desired final maxi-
mum test concentration. Control cells were exposed to an equiva-
lent concentration of DMSO (0.25% v/v, negative control). Each
agent was tested in triplicates at different dilutions in the range
2
9
7
9
17 °C. IR (KBr) 3384, 2981, 2914, 1643, 1440, 1210, 1041,
ꢀ1
1
52 cm
.
3
H NMR (500.13 MHz, CDCl ): 11.89 (1H, s, OH-6),
.57 (1H, dd, J = 8.7, 7.3 Hz, H-8), 7.26 (1H, dd, J = 7.3, 1.0 Hz, H-
), 7.13 (1H, dd, J = 8.7, 1.0 Hz, H-7), 4.77 (1H, dd, J = 7.2, 6.7 Hz,
H-2), 3.43 (2H, m, H-10a and H-10b), 1.56 (3H, s, CH
3
-11), 1.36
1
3
(
(
(
3H, s, CH
3
-12). C NMR (125.77 MHz, CDCl
3
): 185.0 (C-5), 174.9
1–100
Drug incubation times were 48 h, after which time cells were pre-
cipitated with 25 L ice-cold 50% (w/v) trichloroacetic acid and
lM. The drug treatment was started on day 1 after plating.
C-4), 166.4 (C-9b), 164.5 (C-6), 137.7 (C-8), 127.1 (C-9a), 123.2
C-7), 123.1 (C-3a), 117.7 (C-9), 113.4 (C-5a), 95.1 (C-2), 45.3 (C-
l
+
3
3
2
6
3
), 27.0 (C-11), 19.6 (C-12), ꢀ0.6 (C-10). MS m/z (%) 384 (M , 33),
fixed for 60 min at 4 °C. Then the SRB assay was performed. The
+
69 (18), 356 (5), 257 (M ꢀI, 24), 242 (27), 229 (60), 214 (47),
optical density (OD) of each well was measured at 492 nm, using
0
13 (38), 199 (19), 187 (33), 127 (15), 115 (19), 92 (24), 69 (70),
3 (43), 41 (100); HRMS found: 383.9863, calcd for C15
83.9859.
BioTek s PowerWave XS Absorbance Microplate Reader. Values
H
13
O
4
I:
were corrected for background OD from wells only containing
medium. The percentage growth (PG) was calculated with respect
to untreated control cells (C) at each of the drug concentration lev-
els based on the difference in OD at the start (T0) and end of drug
exposure (T), according to NCI formulas. Therefore, if T is greater
than or equal to T0 the calculation is 100 ꢁ [(T ꢀ T0)/(C ꢀ T0)]. If
4
2
.2.16. Synthesis of 2,3-dihydro-5-hydroxy-2-(2-iodopropano-
-yl)naphtho[2,3-b]furan-4,9-dione (14) and 3,4-dihydro-10-
hydroxy-3-iodo-2,2-dimethyl-2H-benzo[h]chromene-5,6-dione
(
33)
T is less than T0 denoting cell killing the calculation is
5
-Hydroxylapachol (4) (40 mg, 0.16 mmol) in dichloromethane
100 ꢁ [(T ꢀ T0)/(T0)]. The effect is defined as percentage of growth,
where 50% growth inhibition (GI50) represents the concentration at
which PG is +50. With these calculations a PG value of 0 corre-
sponds to the amount of cells present at the start of drug exposure,
while negative PG values denote net cell kill.
(
(
(
4.5 mL) was treated at room temperature with a solution of iodine
130 mg, 0.51 mmol) dissolved in a mixture of dichloromethane
4.2 mL) and pyridine (0.5 mL). The reaction mixture was stirred
for 30 min at room temperature, followed by addition of cold
water. The organic phase was washed with 10% sodium carbonate
(
3 ꢁ 12 mL), followed by cold water (3 ꢁ 12 mL). After drying over
Acknowledgements
sodium sulfate, the solvent was evaporated under vacuum. The
residue was submitted to column chromatography on silica gel
This work was financed by the Consejo Nacional de Investigac-
iones Científicas y Técnicas and Universidad de Buenos Aires
(
hexane/EtOAc, gradient) and the quinones 14 (41%) and 33 (6%)
were isolated
(
X-470). Financial support co-financed by the EU-FEDER: the Span-
ish MICIIN (CTQ2008-06806-C02-01/BQU), MSC (RTICC RD06/
020/1046); Canary Islands’ ACIISI (PI 2007/021) and FUNCIS
REDESFAC PI 01/06 and 35/06). L.G.L. thanks the Spanish
4
.2.16.1. 3,4-Dihydro-10-hydroxy-3-iodo-2,2-dimethyl-2H-
0
(
benzo[h]chromene-5,6-dione (33). Red crystals; mp 125–126 °C.
IR (KBr) 3419, 2984, 1695, 1645, 1606, 1589, 1456, 1385, 1292,
1
MSC-FIS for a postdoctoral contract. J.M.P. thanks the Spanish
MEC-FSE for a Ramón y Cajal contract. E.L.B. thanks CONICET for
a fellowship.
ꢀ1
1
267, 833, 781, 662 cm
3
. H NMR (500.13 MHz, CDCl ): 8.79
(
1H, s, OH-10), 7.74 (1H, dd, J = 7.5, 1.3 Hz, H-7), 7.42 (1H, dd,
J = 8.3, 7.8 Hz, H-8), 7.20 (1H, dd, J = 8.4, 1.3 Hz, H-9), 4.39 (1H,
dd, J = 8.7, 5.5 Hz, H-3), 3.34 (1H, dd, J = 18.2, 5.5 Hz, H-4a), 3.14
Supplementary data
(
1H, dd, J = 18.2, 8.7 Hz, H-4b), 1.78 (3H, s, CH
3
-11), 1.74 (3H, s,
): 178.6 (C-6), 177.5 (C-5),
1
3
CH
3
-12). C NMR (125.77 MHz, CDCl
3
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.02.032.
1
9
2
63.3 (C-10b), 155.9 (C-10), 132.6 (C-8), 130.9 (C-6a), 126.3 (C-
), 123.4 (C-7), 113.9 (C-10a), 111.7 (C-4a), 83.8 (C-2), 30.3 (C-4),
+
7.1 (C-11), 26.1 (C-3), 25.1 (C-12). MS m/z (%) 384 (M , 20), 257
References and notes
+
(
3
M ꢀI, 21), 215 (38), 175 (9), 121 (12), 69 (54); HRMS found:
13 4
83.9862, calcd for C15H O I: 383.9859.
1.
2.
3.
4.
Miller, M. G.; Rodgers, A.; Cohen, G. M. Biochem. Pharmacol. 1986, 35, 1177.
Moore, H. W. Science 1977, 197, 527.
MartinOwton, W. J. Chem. Soc., Perkin Trans. 1 1999, 2409.
Eyong, K. O.; Folefoc, G. N.; Kuete, V.; Beng, V. P.; Krohn, K.; Hussain, H.;
Nkengfack, A. E.; Saeftel, M.; Sarite, S. R.; Hoerauf, A. Phytochemistry 2006, 67,
605.
4
.3. Cells, culture and plating
The human solid tumor cell lines A2780 (ovarian), SW1573
5
6
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