Synthesis and structure-activity relationships of lapacho analogues. 1. Suppression of human keratinocyte hyperproliferation by 2-substituted naphtho[2,3-b]furan-4,9-diones, activation by enzymatic one- and two-electron reduction, and intracellular generation of superoxide

Article abstract of DOI:10.1021/jm3009597

A series of linearly anellated lapacho quinone analogues substituted at the 2-position of the tricyclic naphtho-[2,3-b]furan-4,9-dione system were synthesized and evaluated for their ability to suppress keratinocyte hyperproliferation using HaCaT cells as the primary test system. While very good in vitro potency with IC₅₀ values in the submicromolar range was attained with electron-withdrawing substituents, some compounds were found to induce plasma membrane damage, as evidenced by the release of LDH activity from cytoplasm of the keratinocytes. The most potent analogue against keratinocyte hyperproliferation was the 1,2,4-oxadiazole 18, the potency of which was combined with comparably low cytotoxic membrane damaging effects. Structure-activity relationship studies with either metabolically stable or labile analogues revealed that the quinone moiety was required for activity. Selected compounds were studied in detail for their capability to generate superoxide radicals both in isolated enzymatic one- and two-electron reduction assays as well as in a HaCaT cell-based assay.

Full text of DOI:10.1021/jm3009597

Article
pubs.acs.org/jmc
Synthesis and Structure−Activity Relationships of Lapacho
Analogues. 1. Suppression of Human Keratinocyte
Hyperproliferation by 2‑Substituted Naphtho[2,3‑b]furan-4,9-diones,
Activation by Enzymatic One- and Two-Electron Reduction, and
Intracellular Generation of Superoxide
Alexandra Reichstein, Silke Vortherms, Sven Bannwitz, Jan Tentrop, Helge Prinz, and Klaus Muller*
̈
Institute of Pharmaceutical and Medicinal Chemistry, Westphalian Wilhelms-University, Hittorfstraße 58−62, D-48149 Munster,
̈
Germany
S
* Supporting Information
ABSTRACT: A series of linearly anellated lapacho quinone
analogues substituted at the 2-position of the tricyclic naphtho-
[2,3-b]furan-4,9-dione system were synthesized and evaluated for
their ability to suppress keratinocyte hyperproliferation using
HaCaT cells as the primary test system. While very good in vitro
potency with IC50 values in the submicromolar range was attained
with electron-withdrawing substituents, some compounds were
found to induce plasma membrane damage, as evidenced by the release of LDH activity from cytoplasm of the keratinocytes. The
most potent analogue against keratinocyte hyperproliferation was the 1,2,4-oxadiazole 18, the potency of which was combined
with comparably low cytotoxic membrane damaging effects. Structure−activity relationship studies with either metabolically
stable or labile analogues revealed that the quinone moiety was required for activity. Selected compounds were studied in detail
for their capability to generate superoxide radicals both in isolated enzymatic one- and two-electron reduction assays as well as in
a HaCaT cell-based assay.
Chart 1. Quinones Derived from Lapacho (1, 2, 4a, 5),
Related Structures (7, 7a, 7b), and Antipsoriatic Drugs (3, 6)
INTRODUCTION
■
Lapacho is a commercial natural product obtained from the
inner bark and heartwood of Tabebuia trees indigenous to the
Amazonian rainforest.1,2 It is commonly known as “pau d’arco”,
̂
“ipe-roxo” or “taheebo” and has been used as a longtime folk
medicine in South America against malignant, inflammatory,
infectious, stomach, and skin diseases.1,2 The stem bark of the
tree shows a wide range of biological properties such as
antibacterial, antifungal, and antiinflammatory and, in particular,
antitumor activity.3−6 Today, lapacho is acclaimed to be a
wonder drug for curing cancer2 and also used against disorders
of the immune system, for example psoriasis.1
Lapacho contains a number of naturally occurring quinones,
of which lapachol (1) and the angularly anellated β-lapachone
(2) are the research molecules of interest, as these
naphthoquinones have been related to the anticancer molecular
pharmacology of the plant extract.2 Among the most significant
quinones of the lapacho extract, β-lapachone also displayed
activity comparable to that of the antipsoriatic drug anthralin
(3) against the growth of human keratinocytes, which we have
reported in an earlier paper.7 While lapachol was inactive,
however, the linearly anellated naphtho[2,3-b]furan-4,9-diones
4a and 5 (Chart 1) were among the most potent lapacho
constituents. Moreover, activity against tumor cell lines of
naphtho[2,3-b]furan-4,9-diones8 has stimulated the attention of
medicinal chemists, and as a consequence, several synthetic
analogues have been described.9−14
While most of these studies have focused on antitumor
activities, the effects of lapacho analogues, their mechanism of
action, and structure−activity relationships (SAR) in human
Received: July 5, 2012
Published: July 30, 2012
© 2012 American Chemical Society
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a
Scheme 1
a
Reagents: (a) SeO2, SiO2, microwave, 120 W; (b) H2O2 30%, HOAc, 75 °C; (c) DCC, DMAP, MeOH, appropriate alcohol, 0 °C, RT, 3 h; (d)
quinoline, Cu2Cr2O5, reflux; (e) Na2S2O4, Bu4NBr, THF, KOH, Me2SO4, N2; (f) n-BuLi, THF, −15 °C, appropriate N,N-dimethylcarboxamide, N2;
(g) (NH4)2[Ce(NO3)6], MeCN, H2O, 0 °C.
keratinocytes are still poorly understood. As part of our
ongoing interest in tricyclic structures with antiproliferative
activity such as anthracenones,15 naphtho[2,3-b]thiophen-
4(9H)-ones,16 acridones,17 phenoxazines, and phenothia-
zines,18 we extended our studies to linearly anellated lapacho
analogues. Examples for tricyclic antiproliferative agents with
proven clinical utility against psoriasis are the anthracenone
derivative anthralin (3) and the furobenzopyranone derivative
8-methoxypsoralen (6).19,20 Results from our preliminary
experiments revealed that from the linearly anellated lapacho
analogues, the 2-acetyl substituted 4a showed enhanced activity
in comparison to its unsubstituted analogue 7 and its side chain
reduced forms, the corresponding alkyl and alcohol analogues
7b and 7c, respectively. It is for this reason that we decided to
carry out an extensive study to explore the effect of introducing
other acyl and bioisosteric functionalities at the 2-position of
the tricyclic furan ring system.
With respect to psoriasis, a common, immune-mediated
inflammatory and scaling skin disease, mainly characterized by
excessive growth of keratinocytes,21 we have tested the novel 2-
substituted naphtho[2,3-b]furan-4,9-diones as inhibitors of
keratinocyte hyperproliferation, which is one of the hallmarks
of the disease. We also have measured the generation of
superoxide radicals after incubation of representative analogues
via both one- and two-electron transfer by flavoenzymes and
subsequent autoxidation reactions, as well as their contribution
to intracellular superoxide generation in keratinocytes in order
to determine their ability to redox cycle. The biological
implications of these results with respect to oxidative stress22 in
human keratinocytes and to the potential therapeutic action of
lapacho quinone analogues for the treatment of psoriasis or
other hyperproliferative skin disorders are discussed.
CHEMISTRY
■
The starting 2-methyl-naphtho[2,3-b]furan-4,9-dione (7a) was
synthesized following a literature method.23 Selective oxidation
of the methyl group with SeO2 over a solid support under
microwave irradiation24 gave the carbaldehyde 4, and this in
turn was oxidized to the carboxylic acid 8 using H2O2 and
glacial acetic acid. 2-Carboxylic ester analogues 8a−8k were
obtained from 8 and appropriate alcohols by Steglich
esterification (Scheme 1).25
As also outlined in Scheme 1, the 2-acyl analogues 4b−4g as
well as the carboxamide 12 were prepared from the basic
structure 7. The latter was obtained from acid 8, which was
decarboxylated by means of copper chromite in quinoline in
analogy to a literature procedure.23 Unfortunately, quinone 7 is
completely resistant to electrophilic substitution because of the
deactivation of the furan ring by the quinone carbonyl groups.
Accordingly, to obtain an electron-rich nucleophilic system, 7
was subjected to reductive methylation with dimethyl sulfate
and Na2S2O4 in the presence of tetrabutylammonium bromide
following the procedure of Tanaka.26
Next, 4,9-dimethoxynaphtho[2,3-b]furan (9) was treated
with n-butyllithium and acylated with the appropriate N,N-
dimethylcarboxamides to afford the 2-acylated naphthohydro-
quinone dimethyl ethers 10b−10g. With N,N-diethyl-2,2,2-
trifluoroacetamide, the desired trifluoroacetyl analogue could
not be isolated. However, the reaction proceeded in an
unexpected manner. Thus, carboxamide 11 was formally
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derived from the organolithium intermediate, evidently by
elimination of the trifluoromethyl group instead of diethyl-
amine. A similar observation had been described in the
literature.27 Final oxidation of the naphthohydroquinone ethers
with diammonium cerium(IV) nitrate proceeded with con-
comitant ether cleavage to give the desired 2-acylated target
compounds 4b−4g. In a similar manner, carboxamide 12 was
prepared from 11.
As a key intermediate for the preparation of 2-substituted
naphtho[2,3-b]furan-4,9-diones, carbaldehyde 4 was also used
for the synthesis of analogues bearing α,β-unsaturated side
chains. These were obtained by one of the following synthetic
routes shown in Scheme 2. In the first approach, Knoevenagel
hyde 4 through a Wittig-type30 olefination reaction. The
stereochemical assignment of stilbenes 14a and 14b as E-
isomers was performed through H NMR.
1
Carbaldehyde 4 was also transformed into the corresponding
oxime 15 by treatment with hydroxylamine hydrochloride,
which furnished nitrile 16 upon dehydration with acetic
anhydride. Introduction of a tetrazole moiety at the 2-position
as a bioisostere for the carboxylate group31 was accomplished
according to the procedure described by Lofquist.32 Thus,
nucleophilic attack of NaN3 on nitrile 16, followed by ring
closure, gave the 5-tetrazole analogue 17 (Scheme 2).
Furthermore, 1,2,4-oxadiazole analogue 18 as a bioisosteric
replacement of an ester group33,34 was prepared in a one-pot
reaction35 from the activated carboxylic acid 8 and N-
hydroxypropionamidine.
a
Scheme 2
Finally, analogues 4b and 4d were converted to the
corresponding diacetates 19b and 19d, respectively, by
reductive acetylation36 using zinc in acetic anhydride (Scheme
3).
a
Scheme 3
a
Reagents: (a) Zn, Ac2O, pyridine.
BIOLOGICAL EVALUATION AND DISCUSSION
■
Inhibition of Keratinocyte Hyperproliferation. Com-
pounds prepared in this study were evaluated in a keratinocyte
culture model. Drugs that suppress keratinocyte hyper-
proliferation are potentially useful in the treatment of psoriasis
because a rebalanced homeostatic control of keratinocyte
growth and differentiation is crucial for recovery from psoriatic
to normal epidermis.37 As antihyperproliferative action in cell
cultures may be critical in the management of the hyper-
proliferative component of psoriasis, the sensitivity of HaCaT
keratinocytes to each lapacho analogue was determined directly
by counting the dispersed cells under a phase-contrast
microscope after 48 h of treatment. HaCaT is a rapidly
dividing immortalized human keratinocyte line,38 which mimics
the hyperproliferative epidermis found in psoriasis, as it exhibits
a similar keratinization pattern to psoriatic skin, and these cells
have been widely used as a suitable model for the preclinical
evaluation of novel antipsoriatic agents.39 The biological data
for the novel compounds are summarized in Table 1.
a
Reagents: (a) malonic acid, pyridine, piperidine 110 °C; (b) DCC,
DMAP, EtOH, 0 °C, RT, 3 h; (c) diethyl malonate, benzene,
piperidine, HOAc, reflux; (d) PhCH2PO(OEt)2, NaH, DMSO, N2, 60
°C; (e) (4-cyanobenzyl)triphenylphosphonium chloride, NaH,
DMSO, N2, 60 °C; (f) NH2OH·HCl, K2CO3, EtOH 70%; (g)
Ac2O, reflux; (h) NaN3, NH4Cl, DMF, 90 °C; (i) DCC, CH2Cl2, 0 °C,
N-hydroxypropionamidine, pyridine, reflux.
condensation28 with malonic acid in pyridine provided the
acrylic acid analogue 13a. The 1H NMR showed the
characteristic coupling pattern of an ethenyl spacer, and
coupling constants of the olefinic protons (J = 16 Hz)
indicated compound 13a was in the E-configuration. Ester-
ification gave 13b, and 13c was obtained from the reaction of
carbaldehyde 4 with diethyl malonate. In the second approach,
stilbene 14a was obtained from the Horner−Emmons
reaction29 of carbaldehyde 4 and diethyl benzylphosphonate.
In the third approach, nitrile analogue 14b was prepared from
(4-cyanobenzyl)triphenylphosphonium chloride and carbalde-
In this model, a total of 38 naphtho[2,3-b]furan-4,9-diones
and related structures were tested, of which 15 showed activity
in the low micromolar range (IC50 < 1 μmol/L). In an earlier
report, we established the importance of having a 2-acetyl
functionality at the furan moiety of the tricyclic system, in
enhancement of the antihyperproliferative activity.7 We have
now undertaken a study directed toward understanding the
SAR at the 2-position of the tricyclic ring system. Introduction
of alkyl substituents such as methyl (7a) or ethyl (7b) was
disadvantageous to suppression of keratinocyte hyperprolifera-
tion as compared to unsubstituted 7. However, potency clearly
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increased in the series 7b (alkyl, IC50 12.18 μmol/L) < 7c
(alcohol, IC50 3.70 μmol/L) < 4a (ketone, IC50 0.50 μmol/L),
i.e., with the degree of oxidation of the functional group-bearing
carbon of the 2-substituent. Once we had determined that a
carbonyl group at the 2-position was optimal for substitution,
we decided to investigate the breadth of substituents that would
not only maintain or enhance the potency but also ideally show
low cytotoxicity in the LDH assay as stated below. Thus,
compounds 4b−4g explore the consequences of varying the
substituent attached to the 2-carbonyl group. There appears to
be no special requirement for the nature of this group, as all
compounds were active in the low micromolar range. Small and
branched alkyl groups (4b, 4c), and similarly, phenyl (4d) or
heterocyclic rings (4e−4g), were well tolerated. The nicotinyl
(3-pyridyl) analogue 4g (IC50 0.37 μmol/L) was the strongest
suppressor of keratinocyte hyperproliferation in the 2-acyl
series. However, exchanging the methyl group of ketone 4a for
a hydroxyl group to give the acid 8 resulted in lack of cellular
activity. This may be attributed to the inability of the free acid
to cross the cellular membrane. Indeed, there was a clear
distinction in activity between the free acids and their
corresponding esters. Thus, comparing acid 8 with its esters
8a−8k, activity was restored when the carboxyl group was
esterified, with the ethyl ester 8b (IC50 0.61 μmol/L) being the
most potent suppressor of keratinocyte hyperproliferation of all
esters studied. Lengthening, branching, or cyclizing the alkyl
chain of 8b, as in 8c,d and 8e or 8f, respectively, decreased
potency up to 5-fold, while a terminal phenyl ring (8g and 8h)
was detrimental to activity. Also, replacement of the phenyl ring
of 8h with a thiophene (8i) or a furan ring (8k) gave only
moderately active compounds.
Compounds 13a−13c explore the effect of inserting a double
bond between the furan moiety of the tricyclic system and the
carboxyl group. While the free acrylic acid 13a had no
significant effect on keratinocyte hyperproliferation, its
corresponding ethyl ester 13b as well as ester 13c
demonstrated activity in this assay, supporting the hypothesis
that cellular ineffectiveness of the free acids 8 and 13a was due
to insufficient cell penetration. Furthermore, the stilbenes 14a
and 14b were inactive or only moderately potent in the HaCaT
hyperproliferation assay. Moreover, when replacing the
carboxylic ester of 8b by an amide group, potency was
enhanced. Thus, diethyl amide 12 (IC50 0.24 μmol/L) was
among the most potent compounds of this series.
We next investigated the influence of electron-withdrawing
groups other than a carbonyl at the 2-position. While the nitro
derivative 20 and the oxime 15 were about 4-fold less potent
than the genuine lapacho constituent 4a, nitrile 16 displayed
activity in the low micromolar range. Encouraged by this result,
we also looked for bioisosteric replacement for the carboxylic
acid moiety in 8. For example, replacement with a tetrazole is
well-known and can improve permeability, as this group is
almost 10 times more lipophilic while having similar acidity to
that observed for carboxylic acids.31 However, a tetrazole
substituent (17) did not result in any appreciable activity.
Finally, oxadiazoles have been used as bioisosteric replacements
for carbonyl containing compounds such as esters or
amides.33,34 Therefore, we decided to incorporate a 1,2,4-
oxadiazole moiety to mimic the ester or amide substituent of 8b
and 12, respectively. This resulted in oxadiazole 18, which was
the most potent suppressor of keratinocyte hyperproliferation
in this series. With an IC50 of 0.17 μmol/L, it was slightly more
potent than amide 12 and 4- and 5-fold more potent than the
Table 1. Suppression of Human Keratinocyte
Hyperproliferation by 2-Substituted Naphtho[2,3-b]furan-
4,9-dione Analogues of Lapacho and Related Structures
a
b
AA IC50
(μmol/L)
LDH
compd
X
R
(mU/mL)
c
4
CO
CO
CO
CO
CO
CO
CO
CO
H
H
2.76
0.50
0.63
0.44
0.72
0.67
0.55
0.37
3.85
7.54
12.18
3.70
>30
0.92
0.61
1.32
0.99
2.86
2.93
>30
>30
14.34
8.23
>30
>30
0.24
>30
2.51
1.11
>30
7.73
2.34
0.89
>30
186
c
4a
4b
4c
4d
4e
4f
Me
Et
255
c
270
CH2Me2
Ph
d
c
318
c
2-thienyl
2-furyl
3-pyridyl
169
c
167
c
4g
7
202
d
d
d
d
d
7a
7b
Me
Et
e
7c
8
CHOH
CO
Me
OH
c
8a
CO
OMe
190
c
8b
CO
OEt
187
8c
CO
O-n-Pr
d
d
d
d
d
d
d
d
d
d
8d
CO
OCH2Me2
OCH2CH2Me2
O-cyclohexyl
OPh
8e
CO
8f
CO
8g
CO
8h
CO
OCH2Ph
OCH2-2-thienyl
OCH2-2-furyl
Et, R′ = Me
Ph, R′ = Me
NEt2
8i
CO
8k
CO
10b
10d
12
CO
CO
c
CO
226
13a
13b
13c
14a
14b
15
CHCH
CHCH
CHC
CHCH
CHCH
CH
CN
CO2H
d
d
CO2Et
c
(CO2Et)2
Ph
186
d
d
d
Ph-4-CN
N−OH
c
16
181
c
17
5-(1H-
tetrazole)
380
c
18
5-(1,2,4-
oxadiazole)
3-Et
0.17
157
c
19b
CO
CO
NO2
Et, R′ = Ac
Ph, R′ = Ac
0.80
0.92
2.00
0.70
0.90
15.80
185
c
19d
193
20
d
c
anthralin
β-lapachone
menadione
239
c
268
d
a
Antihyperproliferative activity against keratinocytes. IC50, concen-
tration of test compound required for 50% inhibition of cell growth
(HaCaT). Inhibition of cell growth was significantly different with
b
respect to that of the control, N = 3, P < 0.05. Activity of LDH (mU)
release in HaCaT cells after treatment with 2 μmol/L test compound
c
(N = 3, SD < 10%); Values are significantly different with respect to
vehicle control (0.2% DMSO in the culture medium, 110 mU/mL), P
< 0.05. Brij 35 (polyoxyethyleneglycol dodecyl ether)/ultrasound was
d
e
the positive control (409c mU/mL). Not determined. Reference 7.
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standard antipsoriatic agent anthralin and the naturally
occurring lapacho compound β-lapachone (Table 1), respec-
tively.
effects such as skin toxicity of quinones are linked to ROS
generation.43−47 A characteristic feature of the quinone moiety
of anthraquinones and naphthoquinones in general is its ability
to undergo reversible oxidation−reduction processes. Enzymes
such as NADPH-cytochrome P-450 oxidoreductase (CPR)
catalyze one-electron reduction of the quinone, yielding the
corresponding semiquinone radical as outlined in pathway a of
Scheme 4, whereas NAD(P)H:quinone oxidoreductase 1
Compounds 10b and 10d as well as 19b and 19d (Table 1)
were designed to elucidate the role of the quinone moiety in
the biological action of the lapacho analogues. Both pairs of
compounds are closely related congeners of their parent 2-acyl-
naphtho[2,3-b]furan-4,9-diones 4b and 4d, the biological
activity of which was supposed to be masked in the
naphthohydroquinone dimethyl ether (10b, 10d) or diacetate
(19b, 19d) form, respectively. In the case of the ethers 10b and
10d, the quinone system was masked in such a way to prevent
formation of the parent quinones, whereas acetates 19b and
19d were designed as prodrugs, being cleaved and oxidized
efficiently to parent 4b and 4d in the keratinocyte cultures by
esterases. As expected, both 10b and 10d, which are stable and
cannot be cleaved to the hydroquinone forms, were inactive
against keratinocyte hyperproliferation, suggesting that the
presence of a quinone moiety is a prerequisite for activity in our
novel compounds. In contrast, the effect of reductive
acetylation of the quinone moiety was quite different, with
the metabolically labile esters 19b and 19d suppressing
keratinocyte hyperproliferation in the low micromolar range.
Both precursors were only slightly less active than their
corresponding parent molecules, with IC50 values of 0.80 for
19b vs 0.63 μmol/L for 4b, and 0.92 for 19d vs 0.72 μmol/L
for 4d, respectively.
Scheme 4. Redox cycling Process of a 2-Substituted
a
Naphtho[2,3-b]furan-4,9-dione
Lactate Dehydrogenase Release. HaCaT keratinocytes
were also tested for their susceptibility for the action of the
compounds on plasma membrane integrity. This was assessed
by the activity of LDH (lactate dehydrogenase) released into
the culture supernatant, which is commonly used as an
indicator of plasma membrane damage.40,41 As shown in
Table 1, the most potent lapacho quinone analogues in the
keratinocyte hyperproliferation assay were additionally tested
for LDH release, which was apparent for all compounds and
exceeded that of the vehicle control. Surprisingly, the inactive
tetrazole 17 was the most potent inducer of membrane damage.
The antipsoriatic agent anthralin, the clinical efficacy of which is
limited by irritation of the nonaffected skin surrounding the
psoriatic lesion,19 and both lapacho constituents, the 2-acetyl
analogue 4a and β-lapachone, released significantly large
amounts of LDH. In addition, the 2-acyl analogues 4b, 4d
and the carboxamide 12 were essentially equivalent or even
stronger inducers of cell lysis. In all other cases, cytotoxic
membrane damaging effects as compared to anthralin were
substantially reduced for the lapacho compound analogues, in
particular, when the 2-substituent was terminated with a
heterocycle such as thiophene, furan, and oxadiazole as in 4e,
4f, and 18, respectively.
Superoxide Generation and Redox Cycling. Two major
mechanisms for the biological action of quinoid compounds
have been identified. First, some compounds unsubstituted at
one or both positions of the quinone ring are potent
electrophiles, capable of reacting with, e.g., sulfur nucleophiles
in a 1,4-reductive addition of the Michael type, and thus can
arylate tissue components and causing cell death.42 In the case
of the lapacho naphtho[2,3-b]furan-4,9-dione analogues,
however, both electrophilic positions of the quinone ring are
fused to a furan ring, rendering them inappropriate for such a
reaction.
a
(a) one-electron reduction by CPR/NADPH to its semiquinone
radical and (b) reoxidation back to the quinone resulting in superoxide
generation; (c) two-electron reduction by NQO-1/NADPH to its
hydroquinone and (d) subsequent autoxidation via one-electron
transfer to molecular oxygen with formation of the semiquinone
radical and superoxide.
(NQO-1) catalyzes formally the two-electron reduction,
resulting in the formation of a hydroquinone (pathway c,
Scheme 4).43,48 On the one hand, the semiquinone radical in
turn can react with molecular oxygen and generate superoxide
radical (pathway b, Scheme 4), while on the other, the
hydroquinone autoxidizes with transfer of an electron to
molecular oxygen resulting in semiquinone radical and
concomitant superoxide generation (pathway d, Scheme 4).
In such a fashion, both one- and two-electron reduction
products of the quinone then participate in redox cycling to
generate superoxide as the primary oxygen radical, and the
cycle results in an apparent nonending shunting of electrons
from cellular reducing equivalents to molecular oxygen
generating potentially damaging reactive oxygen species
(ROS).45 Superoxide can react directly with some biomolecules
or with H2O2, the latter formed by enzymatic or spontaneous
dismutation of superoxide, which may generate even more
deleterious oxygen species such as hydroxyl radicals (•OH).22
The formation of various oxygen intermediates and the redox
cycling of the quinone may ultimately lead to a cellular
condition known as oxidative stress,22 which can affect cell
behavior in many ways.49
To provide information on the fundamental capability of
naphtho[2,3-b]furan-4,9-dione derivatives to redox cycle, we
have measured the generation of superoxide after incubation of
selected quinones of this series both in isolated enzyme assays
and in a HaCaT cell-based assay.
Second, there is circumstantial evidence to support the idea
that both the desired biological activity and also some other
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Superoxide generation is commonly determined by the rate
of reduction of ferricytochrome c to ferrocytochrome c.50 To
enhance the specificity of the assay,50 cytochrome c was
succinoylated before use. The specificity was further validated
by addition of superoxide dismutase (SOD), as only the SOD-
inhibitable extent of cytochrome c reduction would indicate a
superoxide-dependent reaction. Catalase was added to scavenge
H2O2, and the high affinity iron chelator DTPA was added to
prevent the generation of hydroxyl radicals.51
Superoxide Generation Through One-Electron Reduction
by CPR/NADPH. To examine one-electron reduction of
naphtho[2,3-b]furan-4,9-diones, we incubated human recombi-
nant CPR with NADPH and the test compounds and measured
the rate of superoxide generation. As shown in Table 2, all of
contrast, incubation with both the 2-acetyl (4a) and oxadiazole
(18) analogues resulted in significantly increased superoxide
generation, which was also observed for menadione as a
positive control. There is considerable data indicating a role for
NQO-1 in the detoxification of redox cycling quinones53,54
because two-electron reduction bypasses semiquinone radical
generation and leads to the formation of a stable hydroquinone
that can be readily conjugated and excreted. However, this is
true only in certain cases because not all hydroquinones are
redox-stable. Originally, the two-electron reduction product of
menadione was regarded as a stable hydroquinone, but later it
was found that oxygen radicals were produced in the presence
of NQO-1.54 This is also confirmed by our study, which shows
a significant increase in superoxide production during the
reduction of menadione by human recombinant NQO-1 (Table
2). Likewise, a significant increase in superoxide generation was
observed after incubation with lapacho constituent 4a and our
novel analogue 18, indicating autoxidation of their hydro-
quinones and redox cycling upon reduction by NQO-1. The
susceptibility to autoxidation of the hydroquinone is important
for it can determine whether or not activation takes place
within the cell.54 Thus, depending upon the properties of the
hydroquinone that is formed and its ability to redox cycle,
appreciable amounts of superoxide may be produced (pathway
d, Scheme 4). While an electron-releasing group such as the 2-
methyl of analogue 7a may impair redox cycling of the quinone,
an electron-withdrawing substituent as in 4a or 18 rather
enables autoxidation of the corresponding hydroquinone
generated in the presence of NQO-1.
Taken together, the results obtained from the isolated
enzyme assays indicate that superoxide radicals are, indeed,
generated following incubation of naphtho[2,3-b]furan-4,9-
diones in the presence of flavoenzymes that act as electron
donors. Under normal conditions, the antioxidant defense
systems such as superoxide dismutase present in the cell are
able to control or prevent adverse effects of oxygen radicals.22
Therefore, it was necessary to show that excessive quantities of
superoxide are also generated within keratinocytes by redox
cycling of lapacho analogues resulting from the normal enzyme
activities of the cell.
Superoxide Generation in Keratinocytes. To directly
assess the level of superoxide generated in quinone-treated
cells, we employed flow cytometry in HaCaT keratinocytes and
a fluorescent probe, dihydroethidium (DHE). This is widely
used for detecting intracellular superoxide.55 The red
fluorescence arising from DHE is usually equated to intra-
cellular superoxide generation, and recent studies proved that
the product of the DHE/superoxide reaction is 2-hydroxyethi-
dium (2-OH-E+) and that 2-OH-E+ is a specific marker for
superoxide.56,57 In these cell-based experiments, we have also
employed menadione as a positive control, as intracellular
generation of superoxide after treatment of cells with this agent
by the use of DHE has been described.56,58
Table 2. Rates of Superoxide Generation by Naphtho[2,3-
b]furan-4,9-diones through One-Electron Reduction by
Human Recombinant CPR and Two-Electron Reduction by
Human Recombinant NQO-1
a
b
•−
•−
compd
4a
O2 (CPR)
O2 (NQO-1)
c
c
3.0 0.2
1.4 0.6
c
7
2.2 0.4
0.4 0.3
0.2 0.2
c
7a
1.2 0.2
c
c
18
1.8 0.2
1.9 0.6
c
c
menadione
2.1 0.4
1.0 0.4
d
control
0.2 0.1
0.2 0.1
a
Superoxide generation in the presence of CPR is expressed as SOD-
inhibitable reduction of succinoylated cytochrome c (μmol/L/min/2
b
mU enzyme) by the test compound (100 μmol/mL). Superoxide
generation in the presence of NQO-1 is expressed as SOD-inhibitable
reduction of cytochrome c (μmol/L/min/unit enzyme) by the test
compound (25 μmol/mL). Each value represents the mean SD, N ≥
c
3. Values are significantly different from vehicle control, P < 0.0001.
d
Rate of reduction with no test compound present (DMSO).
the four lapacho quinone analogues tested were excellent
substrates for CPR. The naturally occurring lapacho constituent
4a produced the highest level of superoxide as determined by
the rate of reduction of SOD-inhibitable succinoylated
cytochrome c. The unsubstituted basic structure 7 showed a
reduction rate comparable to that of menadione48,52 (2-
methylnaphthalene-1,4-dione), which is a well-characterized
redox cycler through one- and two-electron reduction and was
used as a positive control. Also, introduction of a 2-methyl
group or an oxadiazole as in 7a and 18, respectively,
significantly increased the rate of cytochrome c reduction as
compared to controls, but the generation of superoxide was
substantially less than that generated by the 2-acetyl substituted
4a. These results clearly indicate that one-electron reducing
enzymes (e.g., CPR) activate naphtho[2,3-b]furan-4,9-diones
into metabolic products that are superoxide generators, and
furthermore, the rate of superoxide generation depends upon
the nature of the 2-substituent.
Superoxide Generation through Two-Electron Reduction
by NQO-1/NADPH. As a further step toward investigating the
role of redox cycling in the biological action of the novel
compounds, we evaluated the impact of two-electron reduction
by human recombinant NQO-1/NADPH on superoxide
generation. As depicted in Table 2, superoxide generation
after enzymatic two-electron reduction of the basic structure 7
and its 2-methyl analogue 7a followed by hydroquinone
autoxidation did not significantly exceed the control value. By
Acute and Chronic Treatment of HaCaT Keratinocytes
with Lapacho Analogues. In the acute and chronic treatment
experiments, neither the basic structure 7 nor its 2-methyl
analogue 7a had any significant impact on the 2-OH-E+ signals
generated in HaCaT keratinocytes (Table 3). In contrast,
quantitative measurements of the mean fluorescence intensities
from the samples after exposure to 4a and, in particular, 18,
demonstrated markedly enhanced superoxide generation in
keratinocytes in both sets of experiments. Table 3 also presents
the levels of 2-OH-E+ fluorescence in keratinocytes treated with
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function, they elicit different responses to NQO-1 reduction
and superoxide levels in keratinocytes. Surprisingly, inhibition
of NQO-1 and consequently of the two-electron reduction
pathway by dicoumarol increased the levels of superoxide
produced by 7 and 7a, which were not significantly increased
when keratinocytes were treated without the NQO-1 inhibitor
dicoumarol. This strongly suggests that under normal
conditions these quinones were reduced by the two-electron
pathway involving NQO-1 activity, which can be considered
relatively nontoxic. The resulting hydroquinones are stable and
can be readily inactivated by conjugation. This is in good
agreement with the results obtained for 7 and 7a in the isolated
enzyme assay with NQO-1 (Table 2), which did not produce
appreciable amounts of superoxide. However, inhibition of
NQO-1-mediated metabolism of 7 and 7a in keratinocytes by
dicoumarol increased the availability of these quinones for one-
electron reduction and redox cycling, possibly reflecting
competition between NQO-1 and CPR, i.e., pathways c and
a, respectively, of Scheme 4. As a consequence of the inhibited
competition, enhanced 2-OH-E+ fluorescence was observed,
indicative of the predominating CPR pathway with the
consequent generation of superoxide, which is also consistent
with the results of 7 and 7a in the isolated enzyme assay (CPR,
Table 2).
Furthermore, only the 2-acetyl and oxadiazole analogues 4a
and 18, respectively, were potent superoxide generators in the
chronic treatment experiments under conditions comparable to
that of the hyperproliferation model. Interestingly, the presence
of dicoumarol did not potentiate this increase significantly
(Table 3), suggesting that in contrast to 7 and 7a NQO-1
cannot protect keratinocytes against quinone-induced oxidative
stress by these compounds. In support of this, our experiments
using recombinant NQO-1 revealed that unlike 7 and 7a, the
autoxidation of both 4a and 18 resulted in significantly
enhanced superoxide generation (Table 2).
How lapacho compound analogues act at the molecular level
to suppress keratinocyte hyperproliferation is not clear at
present. However, the observation that suppression of
keratinocyte hyperproliferation was accompanied by increased
superoxide generation in lapacho compound analogues treated
cells suggests that ROS mediate, or at least contribute to, the
mechanism of their growth-suppressing effects. Of interest in
this regard, tricyclic antipsoriatic drugs such as anthralin60,61
(3) or 8-methoxypsoralen62,63 (6, Chart 1) have been reported
to exert their action through a ROS-dependent mechanism, and
also, keratinocyte growth suppression by a novel antipsoriatic
benzodiazepine seems to be ROS-mediated.64 Of further
interest is the finding that keratinocytes have drastically lower
mitochondrial superoxide dismutase activity than other skin
cells, inducing a superoxide-driven impairment of mitochon-
drial function, which might be a prerequisite for keratinocyte
differentiation.65 This points to a higher susceptibility of these
cells to oxidative stress, which would be enhanced in the
presence of redox cycling agents. Moreover, the relative
deficiency of the protective enzymes superoxide dismutase,
catalase, and glutathione peroxidase in psoriatic tissue66−68
renders the psoriatic epidermis particularly sensitive to the
action of ROS, and this may as well increase the selective
cytotoxicity of ROS-generating antipsoriatic drugs to psoriatic
involved versus normal epidermis.
Table 3. Superoxide Generation in Human Keratinocytes
Following Acute and Chronic Treatment with Naphtho[2,3-
b]furan-4,9-diones
chronic
dicoumarol
b,c
a
b
treatment O2
d
•−
•−
acute treatment
pretreatment O2
d
d
•−
compd
4a
O2 (MFI)
(MFI)
(MFI)
e
e
e
20.5 7.2
725.5 97.2
452.5 39.2
741.4 127.9
e
7
13.0 0.6
6.4 0.2
601.1 82.1
e
7a
420.2 290.0
1287.0 218.2
431.9 83.5
367.5 80.1
830.9 317.3
e
e
e
18
17.0 3.7
1279.0 320.1
e
menadione
44.0 11.6
457.2 103.4
407.59 68.7
f
control
7.1 5.2
a
HaCaT keratinocytes were incubated with test compound (50 μmol/
b
L) for 30 min. HaCaT keratinocytes were incubated with test
compound (5 μmol/L) for 18 h. HaCaT keratinocytes were
c
preincubated with the NQO-1 inhibitor dicoumarol (5 μmol/L) for
d
25 min before treatment with test compounds. Superoxide generation
is expressed in terms of changes in mean fluorescence intensity (MFI)
e
of 2-OH-E+. Each value represents the mean SD, N = 3. Values are
f
significantly different from vehicle control, P < 0.05. Mean
fluorescence intensity with no test compound present (DMSO).
the known redox cycler menadione. Clearly, menadione
stimulated 2-OH-E+ fluorescence, but a significant increase
was observed only after acute treatment at a relatively high
concentration of 50 μmol/L. This concentration of menadione
has been reported to generate superoxide in human
spermatozoa.58
Effects of Pretreatment with the NQO-1 Inhibitor
Dicoumarol. To determine whether two-electron reduction
of the quinones by NQO-1 is involved in intracellular
superoxide generation, we measured 2-OH-E+ fluorescence in
keratinocytes after preincubation with dicoumarol, a potent
inhibitor of NQO-1.48,59 The concentration of dicoumarol
required for specific inhibition of NQO-1, without significant
impact on the activity of other quinone reducing enzymes, has
been reported to be around 5 μmol/L.59 The results of the
NQO-1 inhibition experiments are summarized in Table 3.
After chronic treatment with lapacho quinone analogues, when
keratinocytes were pretreated with dicoumarol, 2-OH-E+
fluorescence was significantly increased in each case, whereas
it was not appreciably changed in the case of menadione. The
latter finding is somewhat unexpected because it has been
demonstrated that dicoumarol increases the availability of
menadione for one-electron reduction, indicating that NQO-1
plays a protective role in preventing its redox cycling by
competing with the one-electron reduction pathway.48
However, chronic treatment of keratinocytes with menadione
did not significantly increase superoxide generation, whether or
not dicoumarol was added, suggesting that redox activation and
ensuing potential suppression of keratinocyte proliferation of
menadione were neglectable at concentration of 5 μmol/L.
Indeed, as compared to the lapacho quinone analogues (Table
1), relatively high concentrations of menadione were required
for suppression of HaCaT cell hyperproliferation (IC50 = 15.8
μmol/L) under identical conditions. Even though menadione
has proven to be a useful model compound to study the
consequences of oxidative stress both in vitro and in vivo,48,54
the results from our studies do not lend support to an
important role of ROS generated by this quinone, at least in
this model of keratinocyte hyperproliferation.
Next, it is clear from these results that although menadione
and the lapacho compound analogues both possess a quinone
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vitro studies, suppression of keratinocyte hyperproliferation
seems to be superoxide-mediated, leading to subsequent
regulation of keratinocyte growth. These results support further
development of the lapacho analogue class of compounds for
psoriasis and other hyperproliferative skin diseases.
CONCLUSIONS
■
We have designed and prepared novel compounds to answer a
number of questions concerning SAR for suppression of
keratinocyte hyperproliferation among a series of lapacho
naphtho[2,3-b]furan-4,9-dione analogues. In particular, the
consequences of introducing a variety of substituents at the
2-position of the tricyclic ring system have been investigated.
The need for electron-withdrawing substituents such as acyl,
carboxylic ester, carboxamide, or cyano groups is evident. While
very good in vitro potency was attained with these substituents,
a number of compounds evaluated was found to induce plasma
membrane damage, as evidenced by the release of LDH activity
from cytoplasm of the keratinocytes. The three most active
analogues in this series were the nicotinyl derivative 4g,
carboxamide 12, and 1,2,4-oxadiazole 18, which showed potent
suppression of keratinocyte hyperproliferation with IC50 values
in the submicromolar range. It is noteworthy that potency of
oxadiazole 18 was combined with comparably low cytotoxic
keratinocyte membrane-damaging effects. As an important
aspect with respect to SAR, comparison of the metabolically
stable 10b and the labile analogue 19b, which were employed
as masked quinones, indicates that the quinone moiety is a
requirement for activity, as only 19b was active.
EXPERIMENTAL SECTION
■
Melting points were determined with a Kofler melting point apparatus
1
and are uncorrected. H NMR spectra were recorded on a Varian
Mercury 400 plus spectrometer (400 MHz), using tetramethylsilane as
an internal standard. Fourier transform IR spectra were recorded on a
Jasco FT/IR-4100 (ATR) spectrometer by applying ATR correction.
Mass spectra (EI, unless otherwise stated) were obtained on a
Finnigan MAT GCQ instrument (70 eV). Thin layer chromatography
(TLC) was conducted on Merck 60 F254 precoated silica gel plates.
Chromatography refers to column chromatography, which was
performed on Acros Organics silica gel (0.060−0.200 mm, 6 nm)
with CH2Cl2 as eluant unless otherwise stated. Yields have not been
optimized. Elemental analyses were performed by the Microanalysis
Laboratory, University of Munster, using a Vario EL III elemental
̈
analyzer. Analytical data confirmed the purity of the test compounds
was ≥95%.
Compounds 4a,69 7a,23 7b,69 7c,7 9,26 and 2070 were prepared as
described.
4,9-Dioxo-4,9-dihydronaphtho[2,3-b]furan-2-carbaldehyde
(4). To a solution of 7a23 (850 mg, 4.00 mmol) in CH2Cl2 (20 mL)
was added SeO2 (650 mg, 6.00 mmol). SiO2 (4 g) was then added,
excess solvent was evaporated, and the material was exposed to
microwave irradiation (Discover Labmate, CEM) at 120 W (3 × 5
min, TLC-control). The product was purified by chromatography to
afford yellow crystals; 50% yield; mp 194 °C (ref 26 190−101 °C).
General Procedure for the Oxidative Demethylation of
Compounds 10b−10g. 2-Propionylnaphtho[2,3-b]furan-4,9-
dione (4b). To a suspension of 10b (250 mg, 0.88 mmol) in
MeCN (8 mL) and H2O (1.5 mL) at 0 °C was added dropwise within
20 min (NH4)2[Ce(NO3)6] (1.23 g, 2.25 mmol) in MeCN (2.5 mL)
and H2O (2.5 mL) under vigorous stirring. The mixture was allowed
to react for 20 min under the same conditions. Then it was poured
onto ice−water and extracted with CH2Cl2 (3 × 50 mL). The
combined organic phase was washed with a saturated solution of NaCl
(2 × 30 mL), dried over Na2SO4, and then concentrated and purified
by chromatography to afford yellow needles; 75% yield; mp 227 °C;
FTIR 1669 cm−1. 1H NMR (CDCl3) δ 8.28−8.23 (m, 2H), 7.83−7.80
(m, 2H), 7.61 (s, 1H), 3.07 (q, J = 7.44 Hz, 2H), 1.27 (t, J = 7.44 Hz,
3H). MS m/z = 254 (76, M+), 225 (100). Anal. C15H10O4 (C, H).
Analogously, compounds 4c−4g and 12 were prepared from 10c−
10g and 11, respectively. See Supporting Information for details.
Naphtho[2,3-b]furan-4,9-dione (7). A mixture of 8 (1.00 g, 4.13
mmol), quinoline (20 mL), and chromium copper oxide (200 mg) was
refluxed for 2 h and then cooled, diluted with CH2Cl2 (50 mL), and
filtered. The solution was thoroughly washed with HCl (2 mol/L, 5 ×
50 mL), and the combined organic phase was dried over Na2SO4.
Then the solvent was evaporated and the product purified by
chromatography to afford light-yellow, felted needles; 56% yield; mp
225 °C (ref 71 220−221 °C).
4,9-Dioxo-4,9-dihydronaphtho[2,3-b]furan-2-carboxylic
Acid (8). To a suspension of 4 (400 mg, 1.76 mmol) in glacial HOAc
(40 mL) was added H2O2 (30%, 15 mL) at 75 °C, and the mixture was
stirred for 1 h. Then it was concentrated in vacuo, and the product was
filtered by suction, washed with H2O (30 mL), and dried in a drying
apparatus to provide a bright-yellow powder; 91% yield; mp 236 °C.
FTIR 3105, 1709, 1667 cm−1. 1H NMR (DMSO-d6) δ 8.21−8.16 (m,
2H), 7.76−7.74 (m, 2H), 7.65 (s, 1H). MS m/z = 242 (100, M+).
Anal. C13H6O5 (C, H).
General Procedure for the Preparation of Esters 8a−8k.
Methyl 4,9-Dioxo-4,9-dihydronaphtho[2,3-b]furan-2-carboxy-
late (8a). To the carboxylic acid 8 (200 mg, 0.83 mmol) in dry
MeOH (80 mg, 2.48 mmol) was added DMAP (10 mg, 0.08 mmol)
under stirring. Then DCC (200 mg, 0.91 mmol) was added at 0 °C,
In the redox cycling experiments, we studied compounds 4a,
7, 7a, and 18 in detail. Several conclusions can be drawn from
the superoxide generation data available (Tables 2 and 3).
Generally, this class of compounds is susceptible to one-
electron reduction, with all analogues tested generating
superoxide to some degree in the presence of CPR. However,
cell-based assays reveal that two-electron reduction of our
lapacho quinone analogues in keratinocytes may lead either to
activation or deactivation of redox cycling, and the balance
between these processes will presumably depend upon the
relative rates of the one- and two-electron steps and the stability
of the particular hydroquinones that are formed according to
Scheme 4. If the quinones are preferentially and rapidly reduced
to stable hydroquinones (pathway c), and if autoxidation of the
latter (pathway d) is comparatively slow under physiological
conditions, conjugation may occur before reoxidation to the
semiquinone (pathway d). Consequently, both one-electron
transfer (pathway a) and autoxidation of the hydroquinone
(pathway d) and ensuing superoxide generation will be
bypassed in keratinocytes. This was observed for the basic
structure 7 and 2-methyl analogue 7a, which are only moderate
inhibitors of keratinocyte hyperproliferation. In contrast,
quinones that slowly form reactive hydroquinones will not be
prevented from redox cycling in this manner because their
persistence in the keratinocyte will permit one-electron transfer
to molecular oxygen to continue. Moreover, superoxide will
additionally be generated through autoxidation of the hydro-
quinone. Such a scenario may be envisaged for 2-acetyl
analogue 4a or oxadiazole 18, which both suppress hyper-
proliferation of keratinocytes in the low micromolar range. Of
interest, both 4a and 18 are substituted in the 2-position of the
naphtho[2,3-b]furan-4,9-dione with an electron-withdrawing
acetyl or oxadiazole group, respectively. The potential impact of
the functional chemistry of the quinone is supported by
experimental observation that electron-withdrawing groups
make the hydroquinone more susceptible for autoxidation.45
In summary, the most favorable benefit/toxicity profile
compared with the standard antipsoriatic drug anthralin was
determined for oxadiazole analogue 18. On the basis of our in
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and the mixture was stirred for 5 min and then for an additional 3 h at
20 °C. The mixture was filtered, the residue washed with CH2Cl2 (20
mL), and the filtrate evaporated and dissolved in CH2Cl2 (100 mL).
The solution was washed with HCl (0.5 mol/L, 2 × 50 mL), then with
a saturated solution of NaHCO3 (2 × 50 mL), and the organic phase
was dried over Na2SO4. Then the solvent was evaporated and the
product purified by chromatography (CH2Cl2/EtOAc, 97/3) to give
light-yellow crystals; 52% yield; mp 197 °C. FTIR 1738, 1681 cm−1.
1H NMR (CDCl3) δ 8.27−8.22 (m, 2H), 7.81−7.79 (m, 2H), 7.64 (s,
chromatography to afford red, felted needles; 12% yield; mp 239 °C.
FTIR 1656 cm−1. 1H NMR (CDCl3) δ 8.25−8.17 (m, 2H), 7.78−7.74
(m, 2H), 7.57−7.54 (m, 2H), 7.49 (d, J = 16.04 Hz, 1H), 7.42−7.35
(m, 3H), 6.98 (d, J = 16.04 Hz, 1H), 6.95 (s, 1H). MS m/z = 300
(100, M+). Anal. C20H12O3 (C, H).
(E)-4-(2-(4,9-Dioxo-4,9-dihydronaphtho[2,3-b]furan-2-yl)-
vinyl)benzonitrile (14b). 14b was prepared from 4 and (4-
cyanobenzyl)triphenylphosphonium chloride in a similar manner as
described for 14a. See Supporting Information for details.
4,9-Dioxo-4,9-dihydronaphtho[2,3-b]furan-2-carbaldehyde
Oxime (15). A mixture of carbaldehyde 4 (300 mg, 1.33 mmol),
hydroxylamine hydrochloride (300 mg, 4.32 mmol), and K2CO3 (300
mg, 2.16 mmol) in 70% EtOH (15 mL) was stirred at rt for 2 h. Then
H2O (30 mL) was added and the mixture was cooled overnight at 6
°C. The precipitate was sucked off by filtration, washed with H2O, and
dried in a drying apparatus to provide an orange−yellow powder; 94%
yield; mp 275 °C. FTIR 1669 cm−1. 1H NMR (CDCl3) δ 9.91 (s, 1H),
8.21−8.18 (m, 2H), 7.78−7.74 (m, 2H), 7.62 (s, 1H). MS m/z = 241
(100, M+). Anal. C13H7NO4 (C, H, N).
1H), 4.00 (s, 3H). MS m/z = 256 (3, M+), 241 (100). Anal. C14H8O5
(C, H).
Analogously, compounds 8b−8k were prepared from 8. See
Supporting Information for details.
General Procedure for Acylation of 4,9-Dimethoxynaphtho-
[2,3-b]furan (9). 1-(4,9-Dimethoxynaphtho[2,3-b]furan-2-yl)-
propan-1-one (10b). To a mixture of n-butyllithium (2.5 M, 1.33
mL, 3.32 mmol) in THF (3 mL) at −15 °C under N2 was added
dropwise a solution of 926 (380 mg, 1.69 mmol) in THF (15 mL). The
mixture was stirred for 4 h at −15 °C, and then N,N-
dimethylpropionamide (360 mg, 3.54 mmol) in THF (2.5 mL) was
added. The mixture was stirred at rt for 3 h, then poured onto ice−
water, acidified with 10% HCl, and extracted with ether (3 × 50 mL).
The combined organic phase was washed with a saturated solution of
NaCl (50 mL), dried over Na2SO4, and then concentrated and purified
by chromatography to afford a yellow solid; 46% yield; mp 125 °C.
4,9-Dioxo-4,9-dihydronaphtho[2,3-b]furan-2-carbonitrile
(16). A solution of oxime 15 (200 mg, 0.83 mmol) in acetic anhydride
(20 mL) was refluxed for 2 h. It was allowed to cool to rt, then poured
onto ice−water (100 mL), and the precipitate was filtered and washed
with H2O (30 mL). The product was dried in vacuo at 50 °C and
purified by chromatography to afford yellow needles; 65% yield; mp
1
FTIR 1552, 1076 cm−1. H NMR (CDCl3) δ 8.27−8.22 (m, 2H),
1
207 °C. FTIR 2241, 1680 cm−1. H NMR (CDCl3) δ 8.28−8.23 (m,
2H), 7.85−7.83 (m, 2H), 7.60 (s, 1H). MS m/z = 223 (100, M+).
Anal. C13H5NO3 (C, H, N).
7.82−7.79 (m, 2H), 7.60 (s, 1H), 4.31 (s, 3H), 4.24 (s, 3H), 3.06 (q, J
= 7.45 Hz, 2H), 1.26 (t, J = 7.45 Hz, 3H). MS m/z = 284 (60, M•+),
269 (100).
2-(1H-Tetrazol-5-yl)naphtho[2,3-b]furan-4,9-dione (17). To a
solution of 16 (300 mg, 1.35 mmol) in DMF (20 mL) was added
NaN3 (100 mg, 1.50 mmol) and NH4Cl (10 mg, 0.14 mmol), and the
mixture was stirred at 90 °C for 2 h. Then it was cooled to rt, treated
with a saturated solution of NaHCO3 (20 mL), extracted with EtOAc
(2 × 20 mL), and acidified with 36% HCl. It was then extracted with
CH2Cl2 and dried over Na2SO4. The solution was evaporated, and the
product was treated with a few drops of H2O to induce precipitation of
Analogously, compounds 10c−10g were prepared from 9 and the
appropriate N,N-dimethylcarboxamides, and 11 was prepared from 9
and N,N-diethyl-2,2,2-trifluoroacetamide. See Supporting Information
for details.
(E)-3-(4,9-Dioxo-4,9-dihydronaphtho[2,3-b]furan-2-yl)acrylic
Acid (13a). To a solution of malonic acid (130 mg, 1.20 mmol) in
pyridine (20 mL) was added carbaldehyde 4 (230 mg, 1.00 mmol) and
refluxed at 110 °C until the formation of CO2 bubbles had ceased. The
solution was allowed to cool to rt, then poured onto ice−water (50
mL) and acidified with 36% HCl. The precipitate was filtered off,
washed with H2O (20 mL), and then dried in vacuo and recrystallized
from MeOH to give yellow crystals; 41% yield; mp 288 °C. FTIR 1667
1
a yellow powder; 56% yield; mp 287 °C dec. FTIR 1672 cm−1. H
NMR (DMSO-d6) δ 8.16−8.11 (m, 2H), 7.93−7.89 (m, 2H), 7.69 (s,
1H). MS m/z = 266 (9, M+), 238 (100). Anal. C13H6N4O3 (C, H, N).
2-(3-Ethyl-1,2,4-oxadiazol-5-yl)naphtho[2,3-b]furan-4,9-
dione (18). To a solution of 8 (650 mg, 2.70 mmol) in dry CH2Cl2
(15 mL) was added DCC (0.28 g, 1.35 mmol) under N2, and the
mixture was stirred at 0 °C for 1 h. Then it was filtered, and the
solvent was evaporated. The residue was treated with pyridine (15
mL), and a solution of N-hydroxypropionamidine72 (110 mg, 1.30
mmol) in pyridine (2.5 mL) was added dropwise at rt. Then the
mixture was refluxed for 3 h, cooled to rt, poured onto ice−water (100
mL), acidified with HCl (2 mol/L), and extracted with CH2Cl2 (3 ×
50 mL). The combined organic phase was washed with H2O (2 × 50
mL) and dried over Na2SO4. The solution was evaporated, and the
product was purified by chromatography (CH2Cl2/EtOAc, 95/5) to
afford yellow crystals; 18% yield; mp 202 °C. FTIR 1669, 1634 cm−1.
1H NMR (CDCl3) δ 8.27−8.22 (m, 2H), 7.83−7.81 (m, 2H), 7.76 (s,
1
cm−1. H NMR (CDCl3) δ 8.12−8.04 (m, 2H), 7.91−7.83 (m, 2H),
7.56 (s, 1H), 7.54 (d, J = 16.04 Hz, 1H), 6.59 (d, J = 16.04 Hz, 1H).
MS m/z = 268 (100, M+). Anal. C15H8O5 (C, H).
(E)-Ethyl 3-(4,9-Dioxo-4,9-dihydronaphtho[2,3-b]furan-2-yl)-
acrylate (13b). was prepared from 13a as described for 8a. See
Supporting Information for details.
Diethyl 2-[(4,9-Dioxo-4,9-dihydronaphtho[2,3-b]furan-2-yl)-
methylene]malonate (13c). Carbaldehyde 4 (200 mg, 0.88 mmol),
diethyl malonate (0.14 g, 0.88 mmol), piperidine (0.15 mL), and
glacial HOAc (0.5 mL) in benzene (15 mL) were added to a molecular
sieve type 4A (200 mg). The mixture was refluxed for 2 h, then cooled
to rt, and the benzene phase was washed with a solution of 17% NaCl
(2 × 10 mL). The organic phase was dried over Na2SO4 and then
concentrated and purified by chromatography to afford yellow crystals;
43% yield; mp 178 °C. FTIR 1739, 1719, 1667 cm−1. 1H NMR
(CDCl3) δ 8.15−8.13 (m, 2H), 7.73−7.70 (m, 2H), 7.48 (s, 1H), 7.15
(s, 1H), 4.48 (q, J = 7.04 Hz, 2H), 4.28 (q, J = 7.04 Hz, 2H), 1.37 (t, J
= 7.04 Hz, 3H), 1.29 (t, J = 7.04 Hz, 3H). MS m/z = 368 (18, M•+),
284 (100). Anal. C20H16O7 (C, H).
(E)-2-Styrylnaphtho[2,3-b]furan-4,9-dione (14a). To a suspen-
sion of NaH (60% in mineral oil, 0.15 g, 3.87 mmol) in DMSO (7.75
mL) in a dry, 50 mL three-necked flask, purged with N2 at 45 °C, was
added diethyl benzylphosphonate (0.35 g, 1.55 mmol). The mixture
was stirred for 10 min, then carbaldehyde 4 (0.35 g, 1.55 mmol) in
DMSO (7.0 mL) was added, and the mixture was heated at 60 °C and
stirred for 30 min (TLC-control). Then it was poured into H2O (30
mL), acidified with HCl (2 mol/L), and extracted with CH2Cl2 (3 ×
50 mL). The combined organic phase was washed with H2O (3 × 30
mL), dried over Na2SO4, and then concentrated and purified by
1H), 2.88 (q, J = 7.44 Hz, 2H), 1.41 (t, J = 7.44 Hz, 3H). MS m/z =
294 (100, M+). Anal. C16H10N2O4 (C, H, N).
2-Propionylnaphtho[2,3-b]furan-4,9-diyl Diacetate (19b). To
a suspension of 4b (150 mg, 0.59 mmol) and zinc dust (77 mg, 1.18
mmol) in acetic anhydride (5 mL) with stirring was added slowly
pyridine (1.2 mL). An exothermic reaction ensued, and the mixture
was stirred at rt for 1 h. Then it was filtered and treated with H2O (10
mL). The product was dried in vacuo at 50 °C and purified by
chromatography (CH2Cl2/EtOAc, 97/3) to afford white−yellow
1
crystals; 20% yield; mp 145 °C. FTIR 1756, 1719, 1688 cm−1. H
NMR (CDCl3) δ 8.00−7.98 (m, 2H), 7.58−7.51 (m, 2H), 7.47 (s,
1H), 3.04 (q, J = 7.44 Hz, 2H), 2.59 (s, 3H), 2.57 (s, 3H), 1.26 (t, J =
7.44 Hz, 3H). MS m/z = 340 (3, M•+), 256 (100). Anal. C19H16O6 (C,
H).
Analogously, compound 19d was prepared from 4d. See Supporting
Information for details.
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Journal of Medicinal Chemistry
Article
Keratinocyte Culture and Determination of Cell Prolifer-
ation. HaCaT keratinocytes38 were cultured, and the cell proliferation
assay was performed as described previously.73 After 48 h of
incubation, cell growth was determined by enumerating the dispersed
cells by phase contrast microscopy. Inhibition was calculated by the
comparison of the mean values of the test compound (N = 3) with the
control (N = 6−8) activity: (1 − test compound/control) × 100.
Inhibition was statistically significant compared to that of the control
(Student’s t test; P < 0.05). IC50 values were obtained by nonlinear
regression.
Lactate Dehydrogenase Release. The assay was performed as
described.40,41 HaCaT cells were incubated with the test compounds
(2 μM) for 4 h at 37 °C. Extracellular LDH activity was measured
using the UV method with pyruvate, and NADH and is expressed in
mU/mL. Appropriate controls with the vehicle were performed (P <
0.01; N = 3, SD < 10%). Brij 35 (polyoxyethyleneglycol dodecyl
ether)/ultrasound was the positive control.
(1000g), and resuspended in FACS buffer (0.5 mL). Experiments with
DHE were performed in the dark. Controls were performed with no
DHE and with no test compound (DMSO alone). DHE-treated
keratinocytes were immediately evaluated by flow cytometric measure-
ments, which were performed at different time points (0, 10, 20, 30,
60, 90 min). Keratinocytes were kept in CO2 incubator at 37 °C
between measurements. All experiments were run in triplicate.
Flow Cytometry. The fluorescence of the oxidized product 2-OH-
E+ was monitored on a FACSCalibur (Becton Dickinson) flow
cytometer equipped with an argon laser (λ = 488 nm) as a light source,
and the data was collected in the FL2 channel (λ = 550−600 nm, 42
nm bandpass). For each sample, 10000 live cells were examined, and
dead cells were gated out for analysis. The recorded histograms were
analyzed using the software CellQuest Pro and were compared with
histograms of untreated control cells. Data were expressed as mean
fluorescence intensity (MFI).
Superoxide Generation Assays. Enzymatic One-Electron
Reduction. Cytochrome c was succinoylated according to the
procedure of Kuthan et al.74 The extent of succinoylation was
determined by the trinitrobenzenesulfonic acid method according to
the method Finkelstein et al.75 The assay was performed as described
with modification.76 The reaction mixtures, totaling 1 mL in volume,
contained CPR (EC 1.6.2.4; human recombinant, Calbiochem, 2 mU),
catalase (EC 1.11.1.6, bovine liver, Sigma, 70 U), NADPH (0.2 mmol/
L), succinoylated cytochrome c (30 μmol/L), and sufficient buffer
(K2HPO4/KH2PO4, 150 mmol/L; DTPA, 1 mmol/L, bidistilled H2O,
pH 7.4). After 2 min of incubation at 37 °C, succinoylated cytochrome
c (30 μmol/L) was added and superoxide generation was initiated by
addition of NADPH (0.2 mmol/L) and test compound (100 μmol/
mL, DMSO).
Enzymatic Two-Electron Reduction. The same reaction
mixtures were used as described above, except that NQO-1 (DT-
diaphorase, EC 1.6.5.5, human recombinant, Sigma, 0.5 U) was used
for enzymatic reduction of the test compound (25 μmol/mL, DMSO),
native cytochrome c (30 μmol/L) was used for the detection of
superoxide, and the assays were run at 20 °C.
ASSOCIATED CONTENT
* Supporting Information
Supplementary chemical data of compounds 4c−4g, 8c−8k,
10c−10g, 11, 12, 13b, 14b, and 19d and table of analytical data
of all test compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Phone: +49-251-8333324. Fax: 49-251-8332144. E-mail:
kmuller@uni-muenster.de.
■
Notes
The authors declare no competing financial interest.
DEDICATION
■
Dedicated to Prof. Dr. Dr. Wolfgang Wiegrebe, Regensburg, on
the occasion of his 80th birthday.
For both assays, a parallel set of experiments was run to determine
the SOD-inhibitable portion of the total rate of superoxide generation.
Addition of SOD (EC 1.15.1.1, bovine erythrocytes, Sigma, 75 U)
enabled the background reduction rate to be subtracted. The rate of
superoxide generation was measured by the rate of increase in
absorbance at 550 nm on a Merck Spektroquant Pharo 100
spectrophotometer. Rates of reaction were calculated from the initial
2 min of the reaction, and results were expressed in terms of μmol of
SOD-inhibitable (succinoylated) cytochrome c reduced per liter per
minute per unit enzyme using a molar extinction coefficient of 21.1
mM−1 cm−1 for reduced cytochrome c.50 Controls were performed
with no test compound present (DMSO, final concentration of 0.1%).
All reactions were carried out in triplicate.
Intracellular Superoxide Generation. To detect intracellular
superoxide levels,77 HaCaT keratinocytes were cultivated as
described.73 Cells were grown in Dulbecco’s Modified Eagle’s Medium
(DMEM, no. E15−810, PAA) in 6-well plates (2.5 × 105 cells/mL)
supplemented with 10% fetal bovine serum, penicillin (100 units/mL),
and streptomycin (100 μg/mL) in a CO2 incubator for 24 h at 37 °C.
Then the medium was replaced, and cells were treated for 18 h
(chronic treatment) with the test compound (5 μM, DMSO, final
concentration of DMSO in the culture medium was 0.1%) in CO2
incubator for 24 h at 37 °C. Also, acute treatment experiments (30
min, 50 μM test compound) were carried out. In a separate set of
experiments, keratinocytes were preincubated with the NQO-1
inhibitor dicoumarol48,59 (5 μmol/L) for 25 min before treatment
test compounds were added. After incubation with test compound,
DHE (10 μmol/mL) was added, cell culture plates were treated on a
shaker (200 rpm) for 5 min, and cells were allowed to load DHE for
an additional 25 min under incubation conditions. Then the medium
was removed, cells were washed with PBS (0.5 mL/well) and then
trypsinized (0.3 mL/well) for 7 min, treated with FACS buffer
(FACSFlow, BD Biosciences, No. 342003, 0,7 mL), centrifuged
ABBREVIATIONS USED
■
CPR, NADPH-cytochrome P450 oxidoreductase; DCC, N,N′-
dicyclohexylcarbodiimide; DHE, dihydroethidium; DMAP, 4-
dimethylaminopyridine; DTPA, diethylene triamine pentaacetic
acid; NQO-1, NAD(P)H:quinone oxidoreductase 1; ROS,
reactive oxygen species; rt, room temperature; SAR, structure−
activity relationships; SOD, superoxide dismutase
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