New pterocarpanquinones: Synthesis, antineoplasic activity on cultured human malignant cell lines and TNF-α modulation in human PBMC cells

Article abstract of DOI:10.1016/j.bmc.2009.12.073

A new pterocarpanquinone (5a) was synthesized through a palladium catalyzed oxyarylation reaction and was transformed, through electrophilic substitution reaction, into derivatives 5b-d. These compounds showed to be active against human leukemic cell lines and human lung cancer cell lines. Even multidrug resistant cells were sensitive to 5a, which presented low toxicity toward peripheral blood mononuclear cells (PBMC) cells and decreased the production of TNF-α by these cells. In the laboratory these pterocarpanquinones were reduced by sodium dithionite in the presence of thiophenol at physiological pH, as NAD(P)H quinone oxidoredutase-1 (NQO1) catalyzed two-electron reduction, and the resulting hydroquinone undergo structural rearrangements, leading to the formation of Michael acceptors, which were intercepted as adducts of thiophenol. These results suggest that these compounds could be activated by bioreduction.

Full text of DOI:10.1016/j.bmc.2009.12.073

Bioorganic & Medicinal Chemistry 18 (2010) 1610–1616
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New pterocarpanquinones: Synthesis, antineoplasic activity on cultured
human malignant cell lines and TNF-a modulation in human PBMC cells
Chaquip D. Netto a, Alcides J. M. da Silva b, Eduardo J. S. Salustiano c, Thiago S. Bacelar c, Ingred G. Riça d,
Moises C. M. Cavalcante d, Vivian M. Rumjanek c, Paulo R. R. Costa b,
*
a Laboratório Integrado Multiusuário II, Instituto Macaé de Metrologia e Tecnologia, Universidade Federal do Rio de Janeiro, Campus Macaé, Brazil
b Laboratório de Química Bioorgânica, Núcleo de Pesquisas de Produtos Naturais, Centro de Ciências da Saúde, Bloco H, Universidade Federal do Rio de Janeiro, RJ 21941-590, Brazil
c Laboratório de Imunologia Tumoral, Instituto de Bioquímica Médica, Centro de Ciências da Saúde, Bloco H, Universidade Federal do Rio de Janeiro, RJ 21941-590, Brazil
d Laboratório Integrado Multiusuário I Prof. Vera Koatz, Instituto Macaé de Metrologia e Tecnologia, Universidade Federal do Rio de Janeiro, Campus Macaé, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
A new pterocarpanquinone (5a) was synthesized through a palladium catalyzed oxyarylation reaction
and was transformed, through electrophilic substitution reaction, into derivatives 5b–d. These com-
pounds showed to be active against human leukemic cell lines and human lung cancer cell lines. Even
multidrug resistant cells were sensitive to 5a, which presented low toxicity toward peripheral blood
Received 2 November 2009
Revised 24 December 2009
Accepted 31 December 2009
Available online 6 January 2010
mononuclear cells (PBMC) cells and decreased the production of TNF-a by these cells. In the laboratory
these pterocarpanquinones were reduced by sodium dithionite in the presence of thiophenol at physio-
logical pH, as NAD(P)H quinone oxidoredutase-1 (NQO1) catalyzed two-electron reduction, and the
resulting hydroquinone undergo structural rearrangements, leading to the formation of Michael accep-
tors, which were intercepted as adducts of thiophenol. These results suggest that these compounds could
be activated by bioreduction.
Keywords:
Naphthoquinone
Pterocarpan
Catalytic oxa-Heck reaction
Cancer
Ó 2010 Elsevier Ltd. All rights reserved.
TNF-a modulation
Bioreduction
1. Introduction
As part of a program directed at the discovery of new anticancer
drugs, we previously synthesized the natural pterocarpan 1 (Fig. 1)
Naturally occurring para-quinones and their synthetic analogs
are an important source of antineoplasic products1–4 and some of
these compounds have been used in clinic as anticancer5–10 and
antiparasitic5 drugs. Anthraquinones such as daunorubicin and
doxorubicin, used in cancer chemotherapy, act through DNA inter-
calation. These and other xenobiotic para-quinones can be reduced
at the mitochondria to the corresponding semiquinone radicals
through action of cytochrome P450 reductases (one electron
reduction)1,2,5,6,10 and the resulting semiquinones can transfer
one electron to molecular oxygen. The resulting superoxide anion
can be transformed into hydroxyl radical which initiates a cascade
of events leading to oxidative stress. Alternatively, enzymes such
as NAD(P)H quinone oxidoredutase-1 (NQO1) catalyze two-elec-
tron reduction of xenobiotic para-quinones to the corresponding
hydroquinones, which are subsequently conjugated (phase II
metabolism) and them excreted.10–12
and derivatives.19,20 We showed that the compound 1 was active
against different leukemic cell lines, including MDR cell lines,
and presented low cytotoxicity against lymphocytes activated by
the mitogen phytohemagglutinin (PHA).20 However, once cathe-
cols can be transformed in vivo into the corresponding ortho-qui-
nones, compound 2 was considered as a possible metabolite of 1
and was prepared by the oxidation of the catechol system at ring
A in 1.20 ortho-Quinone 2 was more potent against leukemic cells
but presented high cytotoxicity against lymphocytes activated by
PHA.20 In order to prepare more active and less toxic compounds,
we designed new derivatives, named pterocarpanqunones, in
which the pro-toxic cathecol group was changed by a naphthoqui-
none group. These compounds can also be seen as analogs of Kal-
afungin, an antimicrobial agent obtained from the culture broth
of a soil isolate of Streptomyces tanashiensis, sharing in their struc-
tures the dihydrofuran moiety.21,22
However, some hydroquinones can undergo structural rear-
rangements leading to the formation of Michael acceptors, that
can alkylate essential nucleophiles in the target cells. This process
is known as bioreductive activation.10,13–18
Pterocarpanquinones 4a–g were synthesized and presented
antineoplasic effect on human leukemic cell lines mentioned
above23 and MCF-7 breast cancer cell line.24 Some of these ptero-
carpanquinones were also active in fresh leukemic cell samples
obtained from patients and overexpressing ATP-binding cassette
transporters (ABC) proteins.23 On the other hand, these pterocar-
panquinones showed low cytotoxicity to peripheral blood
* Corresponding author. Tel.: +55 21 2562 6793; fax: +55 21 2562 6512.
E-mail address: prrcosta@ism.com.br (P.R.R. Costa).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.12.073

C. D. Netto et al. / Bioorg. Med. Chem. 18 (2010) 1610–1616
1611
phenols,19,24,25 oxygenated ortho-iodo phenols are more difficult
to obtain and only few examples describing the iodination of these
very reactive substrates are reported in the literature. In our hands,
phenol 7 could not be transformed into the corresponding ortho-
iodophenol 9 when allowed to react with N-iodosuccinimide in tri-
fluoroacetic acid or acetonitrile.31,32 In both cases a complex mix-
ture of products was formed. However, 9 could be prepared from
the corresponding ortho-mercuryphenol 8d by reaction with I2.
On the other hand, the ortho-bromophenol 10 was prepared from
phenol 7 by reaction with NBS (Scheme 1).33,34
O
A
OH
A
O
OH
A
O
O
B
HO
O
B
O
D
B
C
H
H
O
H
C
C
H
H
H
O
D
O
O
O
O
O
O
D
Kalafungin (3)
( )-2
( )-1
O
O
O
O
O
H
H
Unfortunately, attempts to accomplish the oxyarylation of 6 by
9 or 10 under the conditions favoring the neutral mechanism
(Pd(OAc)2, Et3N, Condition B)26,27 or under conditions favoring
the cationic mechanism (Pd(OAc)2, Ag2CO3, Condition C)28 did
not lead to the desired adduct 4d (Table 1, entries 5 and 6).
Probably, under the conditions employed it was not possible to
accomplish the oxidative addition of Pd(0) in the C–I bond in ortho-
iodophenol 9 and ortho-bromophenol 10. This step is disfavored by
the presence of electron releasing groups in the aromatic ring.
Next, we decided to accomplish the catalytic oxyarylation reac-
tion of 6 using ortho-iodophenol (11). Compound 5a was not
formed in condition B (Scheme 3, Table 2, entry 1), but in condition
C this pterocarpanquinone was obtained in 41% yield (entry 2). A
similar yield was obtained in the absence of PPh3 (entry 3).
Pterocarpanquinones 5b–d were obtained from 5a by electro-
philic substitution, taking advantage of the great reactivity of E-
ring for electrophilic aromatic substitution over the A-ring, which
is deactivated due to the conjugation with the carbonyl groups of
the quinone moiety (Scheme 4).33,35
H
H
R1
OR1
OR2
O
O
E
O
O
E
( )-4
a, R1,R2=CH2
b, R1=Bn, R2=Bn
c, R1=Me, R2=Bn
d R1=Bn, R2=Me
( )-5
a, R1=H
e, R1=Me, R2=H
f, R1=H, R2=Me
g, R1=H, R2=H
b, R1=NO2
c R1=Cl
d, R1=Br
Figure 1. Pterocarpans, lapachol, and pterocarpanquinones.
mononuclear cells (PBMC) activated by PHA.23 While in MCF-7
cells the antineoplasic activity was stronger for compounds bear-
ing a phenol group at the E ring, the antileukemic profile was inde-
pendent on the oxygenation pattern at this ring.24 So, in order to
know more about the structure–activity relationships, we decided
to synthesise a new derivative (5a) without substitution at the E
ring. Related non phenolic derivatives 5b–d were also prepared
and their antineoplasic activity was also evaluated. Antineoplasic
effect of these new pterocarpanquinones on leukemia and lung hu-
man cell lines, and TNF-a modulation in PBMC cells was studied.
2.2. Pharmacology
2. Results
The pterocapanquinones 5a–d were evaluated on two human
leukemic cell lines, K562 and HL-60. K562 cells, from a chronic
2.1. Chemistry
Although very promising, pterocarpanquinones 4a–d were syn-
thesized24 through the oxyarylation of chromenquinone 6 by
ortho-mercuryphenols 8a–d under the conditions developed by
Horino and Cols25 (stoichiometric PdCl2, LiCl in acetone, condition
A, Scheme 2, Table 1, entries 1–4). However, this procedure is
expensive and the use of organomercurial reagents are not recom-
mended to prepare drug candidates.
In contrast to the Heck reaction, only few examples of catalytic
oxyarylation (oxa-Heck reaction) of olefins are described in the liter-
ature, and in these cases ortho-iodophenols26–29 were used instead
ortho-mercuryphenols and we decided to try these more adequate
procedures using 9 as source of the organopalladium species to pre-
pare the pterocarpanquinone 4d. Once arylbromides are also sub-
strates for Heck reactions, compound 10 was also studied.30
In contrast with the easy preparation of oxygenated ortho-mer-
cury phenols by direct mercuration of the corresponding
OBn
HO
OCH3
iii
7
i
Br
OBn
I
OBn
ii
ClHg
HO
OBn
HO
OCH3
HO
OCH3
10
9
OCH3
8d
Scheme 1. Synthesis of o-halophenols. Reagents and conditions: (i) Hg(OAc)2/LiCl/
rt/quantitative yield; (ii) I2, THF, rt, quantitative yield; (iii) NBS, CH3CN, À30 °C, 75%.
O
X
OR1
OR2
O
conditions
A, B or C
+
Table 1
Yields and major conditions for reactions showed in Scheme 1
HO
8, 9 or 10
O
6
Entry
(Ar–X)
Conditions
4 (yield%)
8a
, X=HgCl, R1=R2=CH2
1
2
3
4
5
6
8a
8b
8c
8d
9
A
A
A
A
4a (57)
4b (50)
4c (58)
4d (55)
—
8b, X=HgCl, R1=Bn,R2=Bn
8c, X=HgCl, R1=CH3,R2=Bn
O
O
8d
, X=HgCl, R1=Bn, R2= CH3
9, X=I, R1=Bn,R2=CH3
10
H
C
, X=Br, R1=Bn,R2=CH3
10
B or C
—
H
OR1
OR2
Scheme 2. Synthesis for pterocarpanquinones 4a–d.
O
O
Conditions A = stoichiometric PdCl2, LiCl in acetone at room temperature for 12 h.
Conditions B = 10 mol % Pd(OAc)2, 20 mol % PPh3, 3.0 equiv Et3N, acetone, reflux for
12 h. Conditions C = 10 mol % Pd(OAc)2, 20 mol % PPh3, 3.0 equiv Ag2CO3, acetone,
reflux for 12 h.
( )-4

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C. D. Netto et al. / Bioorg. Med. Chem. 18 (2010) 1610–1616
(excepted for 5b) HL-60 cell lines (Table 3), we decided to investi-
O
O
gate its pharmacological properties in more details in other se-
lected leukemic cells.
I
10 mol% of Pd(OAc)2
+
As multidrug resistant (MDR) is one of the most important
problems in cancer chemotherapy, the antineoplasic effect of 5a
was tested on Lucena-1 (Table 4). This cell line, derived from
K562 and originally selected for resistance to the vinca alkaloid
vincristine, is a MDR cell and overexpresses ATP-binding cassette
sub-family B member 1 protein (ABCB1), a transmembrane protein
of 170 KDa which belongs to the ABC superfamily of transporters
and is codified by MDR-1 gene.38 This protein is responsible for
removing xenobiotics from the cell, being related to the process
of MDR.38
HO
3 eq. base,
acetone, reflux
11
O
6
O
O
H
H
O
O
5a
( )-
Scheme 3. Synthesis of pterocarpanquinone 5a by catalytic oxy-arylation of 6.
Table 4 shows the IC50 obtained for 5a on Lucena-1, Raji, Jurkat
and Daudi human leukemic cell lines. Lucena-1 is also resistant to
oxidative stress39 and was slightly more resistant than K562. Jur-
kat, Raji and Daudi are human lymphocytic cell lines. Jurkat, a T
cell leukemia with high levels of Bcl-2 expression, was more resis-
tant than the other leukemic cell lines. Compound 5a was very bio-
selective and did not show significant cytotoxicity for PBMC
Table 2
Yields and major conditions for reactions showed in Scheme 3
Entry
Condition
PPh3
5a (yield%)
1
2
3
B
C
C
0.2 equiv
0.2 equiv
—
—
41
40
activated by PHA (IC50 > 20
as reference.
lM). Mitomycin C (Mit. C) was used
Conditions B = 10 mol % Pd(OAc)2, 20 mol % PPh3, 3.0 equiv Et3N, acetone, reflux for
12 h. Conditions C = 10 mol % Pd(OAc)2, 20 mol % PPh3, 3.0 equivAg2CO3, acetone,
reflux for 12 h.
Not only leukemic cells were sensitive to quinone 5a. Table 5
shows that this quinone was very active against a small cell lung
cancer cell line (GLC-4), and to a lesser extent to non-small cell
lung cancer cell lines (A549 and H460). It is worth to mention that
GLC-4 and A549 cell lines present high expression of MRP-1 pro-
tein (MDR phenotype).40,41
Currently it is known that inflammation and cancer are often
associated. The neoplastic microenvironment is rich in cytokines,
chemokines and inflammatory enzymes that can become it more
favorable to the tumor development.42 One of the most important
O
O
O
O
i or ii or iii
A
H
H
H
H
O
O
E
R1
O
O
5a
( )-
5b c,d
,
( )-
inflammatory molecule is tumor necrosis factor (TNF-
a), a cyto-
i, HNO3 /CH2Cl2 /0 o
ii, NCS/CH3CN/rt
iii, NBS/CH3CN/rt
C
b, R1=NO2 (50%)
c, R1=Cl (55%)
d, R1=Br (57%)
kine that drives inflammatory reactions. Despite its name, TNF-
a
mediates inflammation-induced tumor growth and can act as an
endogenous tumor promoter.43,44 To determine whether 5a could
modulate the production of TNF-
with lipopolysaccharide (LPS) and this quinone in different concen-
trations for 2 h. The levels of TNF- in PBMC supernatants showed
that 5a significantly reduced TNF- liberation at 25 M concentra-
a, we incubated human PBMC
Scheme 4. Synthesis of pterocarpanquinones 5b–d.
a
a
l
myeloid leukemia, contains high levels of intracellular glutathione
(GSH) and are resistant to oxidative stress.36 Cell viability greater
than 90% was observed, even after treatment of these cells with
tion (Fig. 2). Moreover, the highest concentration tested (100 lM)
H2O2 100 l
M.37 In contrast, HL-60 cells, a pro-myelocytic leuke-
mia, presents a low level of antioxidant defense and is sensible
to oxidative stress.36 In Table 3 are presented the results obtained,
showing that these new pterocarpanquinones are as active as com-
pounds 1 and 4f, used as reference in this study. Mitomycin C was
used as reference too. Since 5a was the more potent in K562 and
Table 4
Antineoplasic effect of pterocarpanquinone 5a in leukemia cancer cell lines and PBMC
(IC50 in
l
M)a
Quinone
Lucena-1
Raji
Jurkat
Daudi
PBMC
5a
Mit. C
2.75
2.75
3.32
ND
6.77
ND
3.10
0.45
>20
4.03
ND = not done.
a
Table 3
Results are reported as IC50 values (concentration required to inhibit cell
growth by 50%) in micromolar. Data represent the means of three independent
experiments, with each concentration tested in triplicate and SD values were lower
than 15%. Assays were performed as described in Section 5.
Antineoplasic effect of pterocarpanquinones 5a,b,d,e in K562 and HL-60 cell lines
(IC50 in l
M)a
Quinone
K562
HL-60
5a
5b
5c
5d
1
3
4f
1.67
3.48
6.77
5.70
2.95
16.04
2.18
0.47
2.00
0.40
4.87
4.87
2.10
ND
Table 5
Antineoplasic effect of pterocarpanquinones 5a on lung cancer cell lines, A549, H460,
and GLC-4 cell lines (IC50
,
l
M)a
A549
11.21
Quinone
5a
H460
12.86
GLC-4
5.17
ND
ND
Mitomycin C
ND = not done.
ND = not done.
a
a
Results are reported as IC50 values (concentration required to inhibit cell
growth by 50%) in micromolar. Data represent the means of three independent
experiments, with each concentration tested in triplicate and SD values were lower
than 15%. Assays were performed as described in Section 5.
Results are reported as IC50 values (concentration required to inhibit cell
growth by 50%) in micromolar. Data represent the means of three independent
experiments, with each concentration tested in triplicate and SD values were lower
than 15%. Assays were performed as described in Section 5.

C. D. Netto et al. / Bioorg. Med. Chem. 18 (2010) 1610–1616
1613
p < 0.05
p < 0.05
O
OH
1600
1400
1200
1000
800
600
400
200
0
O
O
i
H
H
reduction
R1
H
H
R1
O
O
O
O
H
4a 5a 5b
( )-
,
,
B
R2
R2
rearrangment
OH
O
O
O
conjugate addition
H oxidation by air
H
OH
R1
HO
O
O
SPh
13a,13b,13c
R2
Control
R2
Vehicle
10
25
50
100
LPS
R1
5a (μM)
LPS (2μg/mL)
Scheme 5. Reductive activation of pterocarpanquinones 4a, 5a, and 5b. Reagents
and conditions: (i) sodium dithionite, Tris/HCl (pH 7.4), PhSH, 22% for 13a,
quantitative for 13b and 67% for 13c. Compound 13a: R1,R2 = –OCH2O–; 13b:
R1 = R2 = H; 13c, R1 = NO2, R2 = H.
Figure 2. Effect of 5a on LPS-stimulated TNF-
a secretion by PBMC incubated for
2 h. Data are representative of three experiments and are mean SEM.
was able to inhibit almost 100% of TNF-
pared to LPS-stimulated PBMC treated with vehicle.
Its worth to note that under this condition 5a was still less toxic
to PBMC activated by PHA. In PBMC stimulated by LPS, more than
70% of cell viability was observed even in the presence of 100 lM
a liberation when com-
nite at buffered pH 7.4 in the presence of thiophenol led to the ad-
ducts of thiophenol 13a, 13b, and 13c, respectively, strongly
suggesting that these compounds could be activated by bioreduc-
tion, acting as alkylating agents (Scheme 5). This mechanism of ac-
tion could explain the potency of the antineoplasic effect of these
compounds on cell lines resistant to oxidative stress.
of 5a (data not shown).
It is interesting to mention that K562 and probably Lucena-1
expresses a high level of NQO2 (an isoform of NQO1), the enzyme
responsible for two electron reduction of quinones to hydroqui-
nones.45-47
3. Discussion
Similar to what has been observed for other para-naphthoqui-
nones,1,2,5,6,10 it seems reasonable to accept that pterocarpanqui-
nones 4a–d and 5a–d can be transformed through one-electron
reduction into the corresponding semiquinone radicals. These rad-
icals can initiate a cascade of reactions leading to oxidative stress
of the cell. The antineoplasic effect of these compounds on HL-
60, Raji, and Daudi cell lines could be associated with this mecha-
nism of action. However, Jurkat, K562 and Lucena-1 cell lines are
resistant to oxidative stress and, at least in these cases, an alterna-
tive mechanism of action should be operating. Pterocarpanquinon-
es 4 and 5 share with the pyranonaphthoquinone Kalafungin (3) an
important structural feature, the presence of a C–O bond at D-ring
(Fig. 3). It was proposed that Kalafungin acts as antibiotic through a
mechanism13 similar to that suggested for Mitomycin C and ana-
logs,5–7,17 and antitumor drugs model quinomethanes14,15 (biore-
ductive activation), being activated after reduction by NQO1 (two
electron reduction). The resulting hydroquinone undergo a struc-
tural rearrangement, furnishing a Michael acceptor which react
with essential nucleophiles in the cell, as proposed by Moore and
Czeniak.13,16 The first experimental evidence for this metabolic
pathway was supported by Brimble and Nairn through the reduc-
tion of the O-methyl ether of 3 by sodium dithionite in the pres-
ence of sulfur nucleophiles, leading to the incorporation of these
nucleophiles.13
4. Conclusions
Although pterocarpanquinones 4a–d and 5a–d have similar
pharmacological profile, the conditions used to prepare 5a–d are
less expensive, using catalytic amount of Pd(OAc)2 and avoiding
the use of organomercurial reagents.
Pterocarpanquinone 5a showed potent antineoplasic effect
against leukemic cell lines, including those with the MDR pheno-
type, suggesting that this compound is not a substrate to ABCB1,
the transporter that confers resistance to Lucena-1 cells. Although
to a lesser degree, 5a was also effective against non-small lung can-
cer cell lines (A549 and H460) known to express different levels of
the MDR transporters ABCB1 and ABCC1.48,49 The small cell lung
cancer cell line GLC-4 was, however, more sensitive to 5a.
One of the most important pro-inflammatory cytokines, TNF-
has a critical role in carcinogenesis too.50 Interestingly, several
studies have demonstrated endogenous TNF- as a tumor pro-
moter and metastatic factor.51–53 Significant levels of TNF-
was
a
a
a
found in tumor microenvironment of various human cancers,
including those of breast, ovarian, prostate, lymphoma, melanoma
and leukemia.54 We demonstrated that 5a inhibited TNF-
a libera-
tion in human PBMC cells. This effect revealed 5a as potentially
even more effective against cancer, since it can act by two mecha-
nisms, directly by killing tumor cells and indirectly, resolving the
inflammatory environment that supports tumor development.
The key feature to the understanding the cytotoxic effect of
these compounds on cell lines resistant to oxidative stress seems
to be the bioreductive activation mechanism, transforming these
compounds into potent Michael acceptors, especially in cells that
overexpress NQO1. It is possible that this mechanism of action
overcomes the antioxidant defenses present in these cells. The
low toxicity in vitro (PBMC activated by PHA) and the efficient syn-
thesis of 5a, show that this compound is a good candidate to fur-
ther evaluations in vivo.
A similar result was obtained in our laboratory for 4a, 5a and 5b
(Scheme 5). The reduction of these compounds by sodium dithio-
O
OH
A
O
B
O
C
O
D
A
B
H
C
H
O
D
H
H
O
O
E
R1
R2
O
O
( )-4 or 5
12
Figure 3. Pterocarpanquinones 4 and 5 and Kalafungin (12).

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C. D. Netto et al. / Bioorg. Med. Chem. 18 (2010) 1610–1616
was recorded on a Bruker Avance 400 (400.013 MHz) spectrometer
5. Experimental
5.1. Pharmacology
at ambient temperature. All J values are given in Hz. Chemical
shifts are expressed in parts per million downfield shift from tetra-
methylsilane as an internal standard, and reported as position (dH)
(relative integral, multiplicity (s = singlet, d = doublet, dd = double
doublet, dt = double triplet, m = multiplet), coupling constant
(J Hz) and assignment. 13C NMR spectrum was recorded on a Bru-
ker Avance 400 (100.003 MHz) spectrometer at ambient tempera-
ture with complete proton decoupling. Data are expressed in parts
per million downfield shift from tetramethylsilane as an internal
standard and reported as position (dC).
5.1.1. Cell lines
All cell lines used in this work were maintained in RPMI-1640
medium (Sigma–Aldrich Corp. St. Louis, MO, USA), supplemented
with 50 lM b-mercaptoethanol, 25 mM Hepes, pH adjusted to
7.4 with NaOH, 60 mg/L penicillin, 100 mg/L streptomycin and
10% fetal calf serum (FCS) (Gibco, Grand Island, NY, USA), inacti-
vated at 56 °C for 1 h. Daudi cells were supplemented with 20%
FCS. Vincristine sulfate (60 nM) (Sigma–Aldrich) was added to Luc-
ena-1 in order to maintain the MDR phenotype.
5.2.1. Synthesis of pterocarpanquinone 5a
To a stirred solution of 6 (106 mg, 0.5 mmol) in acetone (10 ml),
2-iodophenol 11 (83 mg, 0.75 mmol), silver-carbonate (413 mg,
1.5 mmol), PPh3 (26.2 mg, 0.1 mmol, 20 mol %) and Pd(OAc)2
(11.2 mg, 0.05 mmol, 10 mol %) were added. The reaction mixture
was refluxed for 16 h and filtered in celite with ethyl acetate. The
organic layer was washed with brine, dried over anhyd Na2SO4
and concentrated. The crude product was washed in n-hexane
and purified by flash chromatography on silica. After column chro-
matography using n-hexane/ethyl acetate (95:5) as eluant, this
compound was obtained as a yellow solid in 41% yield, mp
145 °C. A 40% yield was observed in absence of PPh3.
5.1.2. Isolation of PBMC
Peripheral blood mononuclear cells (PBMC) were obtained by
fractionating heparinized blood from healthy volunteers. Blood
was heparinized and fractionated on Ficoll–hypaque (Hystopaque)
(GE Healthcare, Uppsala, Sweden) by density gradient centrifuga-
tion. The PBMC fraction was washed twice and resuspended in
RPMI-1640, supplemented as described above, and the cell density
was adjusted to 106 cells/mL. Cells were incubated with 5
lg/mL of
the mitogen phytohemagglutinin (PHA) (Sigma–Aldrich), in the
presence or absence of the compounds being tested.
1H NMR (CDCl3) d 8.21–8.10 (m, 2H); 7.78–7.68 (m, 2H); 7.30–
7.18 (m, 2H); 6.98–6.91 (m, 2H); 5.66 (d, J = 6.69 Hz; 1H); 4.59 (dd,
J = 11.08, 5.03 Hz; 1H); 3.81 (t, J = 11.08 Hz; 1H); 3.64–3.53 (m,
1H); 13C NMR (CDCl3, 75 MHz) d 183.25 (C); 179.28 (C); 158.73
(C); 157.01 (C); 134.54 (CH); 133.38 (CH); 130.58 (C); 130.57 (C);
129.58 (CH); 126.49 (CH); 126.42 (CH); 125.11 (C); 124.51 (CH);
121.23 (CH); 118.16 (C); 110.80 (CH); 72.32 (CH); 67.09 (CH2);
38.37 (CH); LRMS (EI) m/z 304.
5.1.3. Cell treatment
Leukemic cell lines K562, Lucena-1, Daudi, Raji and Jurkat were
exposed to 5a in culture for 24 h, 48 h, or 72 h. Lung cancer cells
A549, H460 and GLC-4 were let to adhere for 24 h before being ex-
posed to 5a. Briefly, 2 Â 104 cells/mL in 200
lL were seeded in 96-
well microtiter plates in drug-free medium or in medium contain-
ing different concentrations of 5a and maintained for 72 h at 37 °C
in an atmosphere of 5% CO2, and cell viability was then measured.
5.2.2. Eletrophilic substitution reactions on 5a. Synthesis of 5b,
5c and 5d
5.1.4. Cell viability
Cell viability was determined by the 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium (MTT) colorimetric assay. MTT can
be reduced by dehydrogenases present in active mitochondria of
living cells. After incubation in the presence or absence of the com-
pound being tested, 20
added to each well. Plates were then kept at 37 °C in 5% CO2 for 3 h.
After centrifugation, 200 L of DMSO (Sigma–Aldrich) was added
to all wells in order to dissolve the dark blue crystals formed by
the reduction of MTT. Absorbance was measured with a Tecan Sun-
rise ELISA reader at 490 nm. Absorbance was directly proportional
to the amount of formazan (reduction product) present, indicating
the percentage of living cells. The IC50 values were obtained by
nonlinear regression on the GraphPad Prism v4.0 program (Graph-
Pad Software, San Diego, CA, USA).
5.2.2.1. Pterocarpanquinone 5b.
To
a
solution of 5a
(100 mg, 0.33 mmol) in 3.5 mL of CHCl3 was added fuming HNO3
(0.5 ml) at À30 °C. The reaction mixture was stirred for 2 h at same
temperature. After this time, the TLC showed the formation of a
product more polar than start material. The reactional mixture
was dropped into cold water and then extracted with ethyl acetate.
The organic layer was washed with brine, dried over anhyd Na2SO4
and concentrated in vacuum. The crude product was purified by
flash chromatography on silica to furnish a yellow solid (57.6 mg,
0.165 mmol) in 50% yield, mp 250 °C.
lL of MTT (5 mg/mL) (Sigma–Aldrich) was
l
1H NMR (CDCl3) d8.24–8.16 (m, 4H); 7.86–7.77 (m, 2H); 7.02 (d,
1H, J = 8.6 Hz); 5.90 (d, 1H, J = 6.3 Hz); 4.67 (d, 1H, J = 4.1 and
10.9 Hz); 3.90 (t, 1H, 10.8 Hz); 3.78–3.75 (m, 1H); 13C NMR (CDCl3,
75 MHz) d 179.0 (C); 164.2 (C); 157.4 (C); 142.6 (C); 134.9 (CH);
133.9 (CH); 131.8 (C); 130.7 (C); 127.2 (CH); 126.8 (CH); 126.8
(CH); 121.2 (CH); 117.3 (C); 110. 9 (CH); 74.8 (CH); 66.6 (CH2);
38.1 (CH). LRMS (EI) m/z 349.
5.1.5. TNF-
PBMC were cultured in 24-well plates at 106 cells/mL. Stimula-
tion of PBMC was induced by 2 g/mL of Escherichia Coli 055:B5
a release assay
l
LPS (Sigma, Chemical Co., St Louis, MO, USA) for 2 h (37 °C, 5%
CO2). Additionally, groups of cells were incubated at same time
with 0.5% DMSO in RPMI 1640 (vehicle) with or without 5a in dif-
5.2.2.2. Pterocarpanquinone 5c.
To a solution of 5a (50 mg,
0.165 mmol) in 5 mL of CH3CN was added NCS (100 mg,
0.75 mmol) at room temperature. The reaction mixture was stirred
for 24 h. After the reaction was complete, the solvent was evapo-
rated under reduced pressure and ethyl acetate was added. The or-
ganic layer was washed with brine, dried over anhyd Na2SO4 and
concentrated. The crude product was purified by flash chromatog-
raphy on silica to furnish a yellow solid (30.8 mg, 0.091 mmol) in
55% yield, mp 228 °C.
ferent concentrations (10, 25, 50 and 100
supernatants were collected and analyzed by ELISA. TNF-
TNF- levels in PBMC supernatants were determined using sand-
lM). After stimulation,
a
Assay-
a
wich-ELISA kits (R&D Systems, USA), with sensitivities of 4 pg/mL.
5.2. Chemistry
1H NMR (CDCl3) d 8.18 (d, 1H, J = 7.5 Hz); 8.13 (d, 1H, J = 7.5 Hz);
7.81–7.71(m, 2H); 7.25 (d, 1H, J = 2.1 Hz); 7.17 (dd, 1H, J = 8.5,
2.1 Hz) 6.85 (d, 1H, J = 8.5 Hz); 5.69 (d, 1H, J = 6.7 Hz); 4.57 (dd,
1H, J = 11.2, 5.1 Hz); 3.81 (t, 1H, J = 11.1 Hz); 3.61- 3.55 (m, 1H);
Melting points were determined with a Thomas–Hoover appa-
ratus and are uncorrected. Column chromatography was per-
formed on silica gel 230–400 mesh (Aldrich). 1H NMR spectrum

C. D. Netto et al. / Bioorg. Med. Chem. 18 (2010) 1610–1616
1615
13C NMR (CDCl3, 75 MHz) d 183.31 (C); 179.3 (C); 157.63 (C);
5.2.3. Synthesis of ortho-halophenols 9 and 10
5.2.3.1. ortho-Iodophenol 9. To a stirred 0.1 M solution of
157.20 (C); 134.8 (CH); 133.66 (CH); 131.98 (C); 130.76 (C);
129.71 (CH); 127.14 (C); 126.72 (CH); 126.17 (C); 124.87 (CH);
117.96 (C); 111.96 (CH); 73.16 (CH); 66.92 (CH2); 33.67 (CH);
LRMS (EI) m/z 338.
organomercurial 8d in THF at À78 °C was slowly added a 1 M solu-
tion of I2 in THF until the reaction mixture was purple. Immedi-
ately was added a solution of NaHSO3 (20% w/v). The organic
layer was extracted with ethyl acetate, dried over anhyd Na2SO4.
The solvent was removed under reduced pressure to furnish a dark
oil. The product was used in crude form.
5.2.2.3. Pterocarpanquinone 5d.
To a solution of 5a (50 mg,
0.165 mmol) in 5 mL of CH3CN was added NBS (106.5 mg,
0.6 mmol) at room temperature. The reaction mixture was stirred
for 24 h. After the reaction was complete, the solvent was evapo-
rated under reduced pressure and ethyl acetate was added. The or-
ganic layer was washed with brine, dried over anhyd Na2SO4 and
concentrated. The crude product was purified by flash chromatog-
raphy on silica to furnish a yellow solid (36.3 mg, 0.095 mmol) in
57% yield, mp 228 °C.
1H NMR (CDCl3) d 7.48–7.25 (m, 5H), 7.10 (s, 1H), 6.60(s, 1H),
5.00 (s, 2H), 3.82 (s, 3H).
5.2.3.2. ortho-Bromophenol 10.
To a stirred solution of phe-
nol 7 (920 mg, 4 mmol) in 15 mL of CH3CN at 0 °C was added NBS
(784 mg, 4.4 mmol). After the reaction was complete (10 min) the
reactionmixturewasextractedwithethylacetate, washedwithbrine,
dried over anhyd Na2SO4. The solvent was removed under reduced
pressure. The crude product was purified by flash chromatography
(hexane/ethyl acetate 7:3)onsilica tofurnish a darksolid in75% yield.
1H NMR (CDCl3) d 7.50–7.20 (m, 5H), 6.99 (s, 1H), 6.62 (s, 1H),
5.02 (s, 2H), 3.82 (s, 3H).
1H NMR (CDCl3) d 8.2 (m, 2H);7.8 (m, 2H); 7.4 (d, 1H,
J = 1.97 Hz); 7.35 (dd, 1H, J = 8.5 and 2.14 Hz); 6.85 (d, 1H,
J = 8.45 Hz) 5.7 (d, 1H, J = 5.9 Hz); 4.6 (dd, 1H, J = 11.3, 6.0 Hz);
3.8 (t, 1H, J = 11.0 Hz); 3.6 (m, 1H); 13C NMR (CDCl3, 75 MHz) d
183.16 (C); 179.15 (C); 158.0 (C); 157.06 (C); 134.67 (CH);
133.54 (CH); 132.48 (CH); 131.82 (C); 130.6 (C); 127.6 (CH);
126.58 (CH); 117.78 (C); 113.04 (C); 112.43 (C); 112.2 (CH); 73.0
(CH); 66.8 (CH2); 38.44 (CH); LRMS (EI) m/z 382 (100%), 383,
384, 385, 386.
Acknowledgments
Our research was supported by Grants from FINEP, Programa de
Oncobiologia-UFRJ, PRONEX, FAPERJ, CNPq and CAPES. C.D.N. and
P.R.R.C. are supported by CNPq fellowship and E.J.S.S. by FAF/FECD
fellowship. We are grateful to Dr. Ottilia R. Affonso-Mitidieri for
useful suggestions and Central Analítica NPPN-UFRJ for the analyt-
ical data.
5.2.2.4. General procedure for reductive thioalkylation.
A
solution of pterocarpanquinone (0.06 mmol) in 3:1 THF–MeOH
(8.0 mL) and Tris–HCl buffer (pH 7.4, 3.0 mL) was degassed for
15 min with dry nitrogen. To this solution was added sodium
dithionite (254.04 mg 1.46 mmol) to effect hydroquinone forma-
tion, followed by a solution of the thiophenol (24.6 lL, 0.24 mmol)
References and notes
in degassed 3:1 THF–MeOH (2.0 mL). The reaction was stirred at
room temperature under nitrogen atmosphere and monitored
periodically by TLC. The reaction mixture was extracted by ethyl
acetate and the organic layer was washed with brine and dried
over sodium sulfate. The solvent was removed under reduced pres-
sure. The crude product was purified by flash chromatography
using 9:1 hexane/ethyl acetate as eluant.
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