Synthesis and structure-activity relationships of lapacho analogues. 2. modification of the basic naphtho[2,3- b ]furan-4,9-dione, redox activation, and suppression of human keratinocyte hyperproliferation by 8-hydroxynaphtho[2,3- b ]thiophene-4,9-diones

Article abstract of DOI:10.1021/jm500754d

The basic structure of linearly anellated lapacho quinones, naphtho[2,3-b]furan-4,9-dione (7), was modified in the search for novel agents against keratinocyte hyperproliferation. The synthesis and structure-activity relationships of several heterocycle-fused naphthoquinones as well as a full range of 2- and 7-substituted derivatives of one of these, 8-hydroxynaphtho[2,3- b]thiophene-4,9-dione (8a), are described. Out of a total of 71 analogues, particularly 2-thenoyl-substituted 26l, 2-nicotinoyl-substituted 26m, and 2-oxadiazole-substituted 35a compared favorably with the antipsoriatic agent anthralin. Their potency for suppression of keratinocyte hyperproliferation, which was evaluated using HaCaT cells as a model, was combined with comparably low membrane-damaging effects toward keratinocytes, as established by the release of lactate dehydrogenase activity from the cytoplasm of the cells. With respect to the mechanism of action, redox activation of lapacho quinones by one- and two-electron reduction in isolated enzymatic assays was studied, and their potential to generate superoxide was confirmed in the keratinocyte-based hyperproliferation assay.

Full text of DOI:10.1021/jm500754d

Article
pubs.acs.org/jmc
Synthesis and Structure−Activity Relationships of Lapacho
Analogues. 2. Modification of the Basic Naphtho[2,3‑b]furan-4,9-
dione, Redox Activation, and Suppression of Human Keratinocyte
Hyperproliferation by 8‑Hydroxynaphtho[2,3‑b]thiophene-4,9-
diones
Sven Bannwitz, Dirk Krane, Silke Vortherms, Tobias Kalin, Cathrin Lindenschmidt,
Nader Zahedi Golpayegani, Jan Tentrop, Helge Prinz, and Klaus Muller*
̈
Institute of Pharmaceutical and Medicinal Chemistry, PharmaCampus, Westphalian Wilhelms University, Corrensstrasse 48, D-48149
Munster, Germany
̈
S
* Supporting Information
ABSTRACT: The basic structure of linearly anellated lapacho quinones,
naphtho[2,3-b]furan-4,9-dione (7), was modified in the search for novel
agents against keratinocyte hyperproliferation. The synthesis and
structure−activity relationships of several heterocycle-fused naphthoqui-
nones as well as a full range of 2- and 7-substituted derivatives of one of
these, 8-hydroxynaphtho[2,3-b]thiophene-4,9-dione (8a), are described.
Out of a total of 71 analogues, particularly 2-thenoyl-substituted 26l, 2-
nicotinoyl-substituted 26m, and 2-oxadiazole-substituted 35a compared
favorably with the antipsoriatic agent anthralin. Their potency for
suppression of keratinocyte hyperproliferation, which was evaluated
using HaCaT cells as a model, was combined with comparably low
membrane-damaging effects toward keratinocytes, as established by the
release of lactate dehydrogenase activity from the cytoplasm of the cells.
With respect to the mechanism of action, redox activation of lapacho quinones by one- and two-electron reduction in isolated
enzymatic assays was studied, and their potential to generate superoxide was confirmed in the keratinocyte-based
hyperproliferation assay.
heartwood of the lapacho (Tabebuia) tree from the
Bignoniaceae family.1−3 Commonly known as “pau-d’arco”,
lapacho is widely used as a folk medicine for the treatment of a
variety of diseases by the local people in South America.4,5 Its
remarkable pharmacological properties, including antibacterial,
antifungal, antiinflammatory, and antitumor activities, are cited
in the literature.6−11 As a consequence of its biological
significance, quinone 3 has become an attractive synthetic
target, and several studies have been carried out toward the
development of antitumor agents.12−17
INTRODUCTION
■
Derivatives of the naturally occurring substance lapachol (1),
such as the angularly anellated β-lapachone (2) or the linearly
anellated naphtho[2,3-b]furan-4,9-diones 3 and 4 (Chart 1),
are among the principal constituents of the inner bark and
Chart 1. Lapacho Quinones 1−4 and Antipsoriatic Drugs 5
and 6
Moreover, lapacho is today used against disorders of the
immune system and skin diseases such as psoriasis,4 which is
supported by an earlier study in which we assessed potent
inhibitory action of a number of lapacho compounds against
the growth of human keratinocytes at sufficiently low
concentration, and in particular, quinones 2−4 displayed
activity comparable to that of the antipsoriatic drug anthralin
(5).18 In our continuing search for potent antiproliferative
agents based on a three-ring structure such as anthracenones,19
Received: May 15, 2014
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jm500754d | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 1. Suppression of Human Keratinocyte Hyperproliferation by Simple Analogues of Basic Naphtho[2,3-b]furan-4,9-dione
a
compd
X
Y
Z
R1
R2
AA, IC50 (μmol/L)
b
3
O
CH
CO
H
COMe
0.50
0.70
3.85
4.80
2.75
13.50
3.77
>30
2.76
2.50
6.12
>30
>30
>30
0.90
1.20
0.44
0.40
3.50
1.28
>30
>30
5, anthralin
b
7
O
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
CO
CO
CO
CO
CO
CO
CO
CH2
H
H
8
S
H
H
8a
S
OH
H
8b
8c
S
OMe
H
S
Cl
H
8d
8e
S
NH2
H
S
NHCOMe
H
9
S
OH
H
H
10
11
12
12a
13
14
18
18a
18b
18c
21
21a
Se
NH
NH
NMe
O
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
H
H
H
H
H
N
H
H
N
H
H
S
N
H
H
S
CH
CH
CH
CH
N
H
COMe
COMe
COMe
COMe
COMe
COMe
S
OH
OMe
Cl
H
S
S
NH
NMe
N
H
a
Antihyperproliferative activity against keratinocytes. IC50 = concentration of test compound required for 50% inhibition of cell growth (HaCaT).
Inhibition of cell growth was significantly different with respect to that of the control, N = 3, P < 0.05. Reference 23.
b
naphtho[2,3-b]thiophen-4(9H)-ones,20 acridones,21 and phe-
noxazines and phenothiazines,22 these findings encouraged the
design and synthesis of a novel series of linearly anellated
lapacho analogues and their evaluation for suppression of
keratinocyte hyperproliferation, which we have reported
recently.23 Anthralin (5) and 8-methoxypsoralen (6) exemplify
established drugs with a tricyclic structure (Chart 1) in the
clinical treatment of psoriasis.24,25
scaling skin disorder, mainly characterized by excessive growth
of keratinocytes.26
With respect to redox activation of the novel analogues,
testing procedures for the generation of superoxide by the
compounds are identical to those described in our recent
paper.23
CHEMISTRY
■
In this paper we describe the initial modifications of the basic
naphtho[2,3-b]furan-4,9-dione (7; Table 1) of lapacho
ingredient 3 in which several closely related ring systems
were compared and also a full range of analogues of one of
these, 8-hydroxynaphtho[2,3-b]thiophene-4,9-dione (8a),
which is bioisosteric to the basic structure of lapacho
constituent 4 (Chart 1) and is found to be among the most
active structural types. Thus, the first goal of this investigation
was the synthesis and biological evaluation of heterocyclic-ring-
fused naphthoquinones and derivatives thereof. These thio-
phene (8, 8a−8e), selenophene (10), pyrrole (11), imidazole
(12, 12a), oxazole (13), and thiazole (14) analogues are
chromophore-modified naphtho[2,3-b]furan-4,9-diones and
should maintain the planarity and spatial and electronic
characteristics of the basic lapacho quinone 7 that may be
necessary for molecular recognition at the cellular level. The
second goal was to further delineate the structure−activity
relationships (SARs) for lapacho quinone analogues. To do
this, many congeners of 8a with side chains occupying the 2- or
7-position on the thiophene-anellated skeleton were synthe-
sized and evaluated for their ability to suppress keratinocyte
hyperproliferation. This may be critical for the management of
psoriasis, a common, immune-mediated inflammatory and
The target molecule for the synthesis of the desired analogues
was 8-hydroxynaphtho[2,3-b]thiophene-4,9-dione (8a). The
methodology for its preparation is outlined in Scheme 1. In
following the procedure of Weinmayr,27,28 thenoylbenzoic acid
15 was prepared from 3-nitrophthalic anhydride by Grignard
reaction and separated from its isomer. The nitro group of 15
was reduced with ferrous sulfate in aqueous ammonia to the
amino intermediate 16, and direct ring closure upon heating
with sulfuric/boric acid yielded aminonaphtho[2,3-b]-
thiophene-4,9-dione (8d). While the conditions used do not
ensure control of regioselectivity and could result in rearrange-
ments to produce the 5-amino isomer, this process gave only
isomerically pure 8d. The latter was converted to the target
molecule 8a by diazotation and subsequent hydrolysis of the
diazonium salt. The X-ray crystal structure of 8a (see the
Supporting Information for details) clearly confirmed the 8-
position of the substituent, whereas the corresponding 5-
hydroxy isomer29 could not be detected. Furthermore,
reduction of 8a with stannous chloride in acetic acid occurred
at the keto group remote from the hydroxyl group, as in the
case of anthraquinone reduction to anthrones,30 and provided
naphthothiophenone 9. Even though this analogue lacks the
quinone moiety, it is closely related to anthralin (5) of the
B
dx.doi.org/10.1021/jm500754d | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
tively. These were treated with n-butyllithium and acetylated
with N,N-dimethylacetamide to afford the 2-acetylated
naphthohydroquinone dimethyl ethers 20b and 20c. Final
oxidation of the naphthohydroquinone ethers with diammo-
nium cerium(IV) nitrate proceeded with concomitant ether
cleavage to give the desired 2-acetylated target compounds 18b
and 18c, and ether cleavage of the methoxy derivative 18b with
aluminum chloride provided the 8-hydroxy derivative 18a.
Likewise, 5,8-dihydroxy-substituted 2-acetyl analogue 40 was
prepared starting from 36. 8-Unsubstituted 2-acetyl analogue
18 (not depicted; see Table 1) was obtained in an alternative
approach from naphtho[2,3-b]thiophene32 by 2-acetylation as
described above, and the resulting 2-acetylnaphthothiophene
(20) was then oxidized with chromium(VI) oxide.
Scheme 1
The 2-acetyl analogues 21 and 21a of the naphtho[2,3-
d]imidazole-4,9-diones 1233 and 12a34 (Table 1), respectively,
were obtained by oxidation of 2-(1-hydroxyethyl)-1H-naphtho-
[2,3-d]imidazole35 with an excess of potassium dichromate to
afford 21, which upon treatment with methyl iodide gave the
N-methyl analogue 21a (Scheme 3).
a
Reagents and conditions: (a) 2-bromothiophene, Mg, Et2O, benzene,
45 °C, or selenophene, AlCl3, CH2Cl2; (b) FeSO4, NH3, reflux; (c)
H2SO4, 120 °C; (d) (1) HOAc, H2SO4, 70 °C; (2) NaNO2, H2SO4, 0
°C; (3) H2O, H2SO4, reflux; (e) SnCl2, HCl, HOAc, reflux.
a
Scheme 3
anthrone class of antipsoriatic agents. For the synthesis of
seleno analogue 10, Friedel−Crafts conditions proved entirely
satisfactory for the condensation of phthalic anhydride with
selenophene to the acid 17.
The 2-acetyl analogues 18a−18c were prepared from the
corresponding basic structures 8a and 8c (Scheme 2), which
were subjected to reductive methylation. The phase-transfer
method of Kraus31 with sodium dithionite and dimethyl sulfate
in the presence of tetrabutylammonium bromide afforded the
electron-rich nucleophilic 8-methoxy- and 8-chloro-substituted
4,9-dimethoxynaphtho[2,3-b]thiophenes 19b and 19c, respec-
a
Reagents and conditions: (a) K2Cr2O7, H2SO4, H2O, reflux; (b)
CH3I, K2CO3, DMF, −60 °C.
The preparation of 7-substituted derivatives (Table 2) of the
selected lapacho skeleton 8a is outlined in Scheme 4. As in our
earlier studies on anthrone chemistry,36 we have used the
Marschalk reaction,37,38 which enables the direct attachment of
alkyl side chains to hydroxyanthraquinones. We have now
successfully adopted this method to provide access to ortho-
substituted 8-hydroxynaphtho[2,3-b]thiophene-4,9-diones.
While 8a itself is an electron-deficient quinone system,
reduction with sodium dithionite results in an electron-rich,
highly nucleophilic hydroxynaphthothiophene intermediate
that reacts with aldehydes and after thermal elimination of
the hydroxy substituent directly yields the alkylated products.
The direct alkylation of 8a proceeded cleanly with aliphatic
aldehydes, benzaldehyde, and benzaldehydes with substituents
deactivating the phenyl ring to provide the 7-substituted
compounds 22a−22d and 22i−22p. Attempts to alkylate 8a
with benzaldehydes bearing electron-donating groups failed.
However, hydroxymethylation of 8a and reoxidation of the
reaction mixture afforded the alcohol 22u, which was converted
into the chloride 22v by thionyl chloride. Friedel−Crafts
alkylation of methoxybenzenes with 22v gave analogues 22s
and 22t. Carboxylate esters 22e−22h and 22q,r were obtained
by esterification of the carboxylic acids 22e and 22o,p,
respectively.
a
Scheme 2
The 2-substituted 8-hydroxynaphtho[2,3-b]thiophene-4,9-
diones of Table 3 were prepared by the general method of
Scheme 5. For functionalization of 8a by a substitution
reaction, protection of the phenolic system was necessary and
a more activated tricyclic structure was required. Therefore, 8a
was exhaustively methylated as described above (Scheme 2) to
a
Reagents and conditions: (a) Na2S2O4, Bu4NBr, THF, KOH,
Me2SO4, N2; (b) n-BuLi, THF, −15 °C, N,N-dimethylacetamide,
N2; (c) (NH4)2[Ce(NO3)6], MeCN, H2O, 0 °C; (d) AlCl3, CH2Cl2,
rt.
C
dx.doi.org/10.1021/jm500754d | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Table 2. Suppression of Human Keratinocyte
Hyperproliferation by 7-Substituted 8-Hydroxynaphtho[2,3-
b]thiophene-4,9-diones
Scheme 4
a
b
compd
22a
R
AA, IC50 (μmol/L) LDH (mU/mL)
H
5.3
d
22b
Me
Pr
11.4
>30
21.5
2.8
d, c
d
22c
22d
CO2H
d
22e
CO2Me
d
22f
CO2Et
2.1
d
22g
CO2Pr
4.5
d
22h
CO2Bn
>30
8.5
d
22i
furan
127
107
d
22k
Ph
10.1
13.8
16.7
14.2
25.7
>30
19.1
>30
23.2
>30
1.2
22l
Ph-4-Me
Ph-4-CF3
Ph-4-CN
Ph-2-CO2H
Ph-4-CO2H
Ph-2-CO2Me
Ph-4-CO2Me
Ph-4-OMe
Ph-3,4-(OMe)2
OH
22m
22n
d
d
22o
d
22p
d
22q
d
22r
d
a
R is defined in Table 2. Reagents and conditions: (a) Na2S2O4,
NaOH, N2, 60 °C, 20 min; (b) RCHO, Na2S2O4, N2, 90 °C, 12 h, O2,
HCl; (c) appropriate alcohol, H2SO4, reflux, 3 h; (d) (1) Na2S2O4,
MeOH, NaOH, HCHO, N2, 0−5 °C, 3 h; (2) H2O2, HCl; (e)
CH2Cl2, SOCl2; (f) anisole or veratrole, AlCl3, CH2Cl2, reflux, 4 h.
22s
d
22t
d
c
c
c
c
22u
214
284
239
268
d
23
0.70
0.70
0.90
15.8
anthralin
β-lapachone
menadione
followed by deprotection of the 8-methoxy group with
aluminum chloride to give the desired 8-hydroxy-substituted
target compounds, i.e., 2-acyl analogues 26a−26m, 2-carboxylic
acid 29a, esters 29b−29e, amide 32, and oxadiazoles 35a and
35b.
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
respect to that of the control, N = 3, P < 0.05. Activity of LDH (mU)
b
release in HaCaT cells after treatment with 2 μmol/L test compound
(N = 3, SD < 10%). Values are significantly different with respect to
those of the vehicle control (0.2% DMSO in the culture medium, 110
c
BIOLOGICAL EVALUATION AND DISCUSSION
■
Initial Evaluation of Simple Analogues as Inhibitors of
Keratinocyte Hyperproliferation and SARs for the
Heterocyclic Ring System. As shown in Table 1 we initially
examined the antihyperproliferative activity against keratino-
cytes of some basic heterocycle-fused naphthoquinones, i.e.,
simple analogues of the basic structure 7 of lapacho-derived 2-
acetylnaphtho[2,3-b]furan-4,9-dione (3) in which the furan
oxygen was replaced by sulfur (8), selenium (10), or nitrogen
(11). Furthermore, an additional heteroatom was introduced
(12−14).
The ability of these compounds to suppress keratinocyte
hyperproliferation is 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.42 As a good model for the
evaluation of our compounds, we used HaCaT cells, a rapidly
dividing immortalized human keratinocyte line.43 These cells
mimic the hyperproliferative epidermis, show a keratinization
pattern similar to that of psoriatic skin, and have been widely
used for the preclinical evaluation of novel antipsoriatic
agents.44 The antipsoriatic agent anthralin and the naturally
occurring β-lapachone were used as standard compounds
(Table 2).
mU/mL), P < 0.05. Brij 35 (polyoxyethylene glycol dodecyl ether)/
d
ultrasound was the positive control (409 mU/mL). Not determined.
provide the electron-rich 4,8,9-trimethoxynaphtho[2,3-b]-
thiophene (19b), and the desired 2-acyl precursors 24a−24m
as well as carboxylic acid 27a were prepared by metalation of
19b with n-butyllithium and quenching the resulting anion with
the appropriate N,N-dimethylcarboxamide or dry ice, respec-
tively, analogously to the procedure of Tanaka.39 In a similar
manner, carboxamide 30 was obtained with N,N-diethyl-2,2,2-
trifluoroacetamide, as in this case the trifluoromethyl group was
eliminated from the organolithium intermediate rather than
diethylamine. 2-Carboxylic ester analogues 27b−27e were
obtained from 27a and appropriate alcohols by Steglich
esterification.40 Furthermore, 1,2,4-oxadiazole analogues 33a
and 33b were also prepared from carboxylic acid 27a and N-
hydroxypropionamidine or N-hydroxy-3-phenylpropionami-
dine, respectively, in a one-pot reaction.41 As also outlined in
Scheme 5, these 2-substituted 4,8,9-trimethoxynaphtho[2,3-
b]thiophenes 24a−24m, 27a−27e, and 30, as well as 33a and
33b, were then oxidized with diammonium cerium(IV) nitrate
to the quinone stage (25a−25m, 28a−28e, 31, 34a, 34b)
D
dx.doi.org/10.1021/jm500754d | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
even abolished activity, unless it was converted into its N-
acetylated form 8e. However, interesting inhibitory activity was
obtained by adding an 8-hydroxy group (8a), and an even more
potent compound was found when the 2-position of the fused
heterocycle was acetylated, as in 18a. Finally, quinone 8a was
also reduced to the naphthothiophenone 9 to explore an
analogue that contains chemical features on which the
minimum structure of antipsoriatic activity is based45 and is
most similar to the potent antipsoriatic agent anthralin (5).
Unfortunately, this analogue was only slightly more potent than
8a and substantially less potent than anthralin.
Table 3. Suppression of Human Keratinocyte
Hyperproliferation by 2-Substituted 8-Hydroxynaphtho[2,3-
b]thiophene-4,9-diones
a
b
compd
R
AA, IC50 (μmol/L)
LDH (mU/mL)
26a
26b
26c
26d
26e
26f
H
1.00
0.38
0.45
4.42
>30
d
Evaluation of 7-Substituted 8-Hydroxynaphtho[2,3-
b]thiophene-4,9-diones. With a rudimentary understanding
of the tricyclic skeleton SARs, we turned our attention to side
chain modifications on naphthoquinone skeleton 8a. Among
our first thoughts was to introduce carbon side chains into
position 7, as this is favored by the presence of the 8-hydroxy
group. The ability of the resulting compounds to suppress
keratinocyte hyperproliferation is shown in Table 2. Com-
pounds 22a−22c and 22i−22t explore the consequences of
adding an aliphatic or arylmethyl side chain to the 7-position of
the naphthoquinone skeleton. Increasing the lipophilic
character of the compounds appears to be disadvantageous
for their potency, which is demonstrated by the loss of potency
for the set of 7-alkyl homologues; the order was methyl > ethyl
> butyl. Furthermore, 7-benzyl-substituted analogues were all
less active than the parent 8a. Compounds 22l−22t were
prepared to study the effects of further substitution on the
benzyl group, but these agents were less potent, which was also
observed for those bearing hydrophilic groups (22o−22r). This
indicated that both electronic and solubility effects might not be
at play in the SARs of 7-substituted analogues. Analogues 22d−
22h explore introduction of an acetic acid side chain and some
corresponding esters. This was done primarily to improve the
hydrophilic character of the molecules. Methyl ester 22e was
equipotent to parent 8a, whereas ethyl ester 22f was somewhat
more potent. Finally, 7-hydroxymethylated 22u was the most
potent compound in this series. Potency was further improved
by an additional 2-acetyl group, with an IC50 of 0.70 μM for
analogue 23, but not as potent as 2-acetylated 8a; cf. lapacho
compound 18a, IC50 = 0.40 μM (Table 1). The overall trend of
lower potency seems to indicate that introduction of this
structural modification in position 7 of the naphtho[2,3-
b]thiophene-4,9-dione is generally unfavorable, however.
Effects of Varying the 2-Acetyl Side Chain. The third
part of this investigation examined the effects of varying the 2-
acetyl group (18a) on the selected 8-hydroxynaphtho[2,3-
b]thiophene-4,9-dione (8a) to define SARs for this position
(Table 3). The choice of the 2-acyl and 2-carboxylic ester
groups was guided by our previous study on the naphthofuran
series of analogues in which a carbonyl group at the 2-position
was found optimal for substitution.23 Comparing the
homologues series of small and branched alkyl groups (18a
of Table 1, 26b, 26c), these compounds were active in the
submicromolar range, whereas a terminal phenyl ring in 26d
and 26f lowered potency or gave an inactive compound (26e).
Furthermore, a phenyl (26g) ring directly attached to the
carbonyl group was tolerated well, with an IC50 of 0.80 μM,
which is of the same order of magnitude as those of anthralin
and β-lapachone. However, aromatic substitution was not
successful, independently of more hydrophobic and electron-
withdrawing (26h) or electron-donating (26i, 26k) effects,
leading to a decrease in potency. Replacing the phenyl ring in
c
Et
244
CHMe2
CH2Ph
d
d
d
d
(CH2)2Ph
CH2OPh
Ph
11.15
0.80
5.64
4.72
7.07
0.29
0.30
>30
c
26g
26h
26i
419
Ph-4-Cl
d
d
d
Ph-4-OMe
Ph-2-OH-4-OMe
2-thienyl
3-pyridine
OH
26k
26l
c
158
c
26m
29a
29b
29c
29d
29e
32
154
d
c
OEt
0.76
0.92
11.90
>30
233
OCHMe2
OCH2Ph-4-OMe
O(CH2)2Ph
NEt2
d
d
d
c
0.36
0.19
0.48
330
c
35a
35b
Et
150
(CH2)2Ph
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
those of the vehicle control (0.2% DMSO in the culture medium, 110
mU/mL), P < 0.05. Brij 35 (polyoxyethylene glycol dodecyl ether)/
d
ultrasound was the positive control (409 mU/mL). Not determined.
The preliminary evaluation of SARs for the basic
naphthoquinone skeletons in this model indicated that most
of them indeed possessed valuable, but not outstanding,
activity. Attempts at changing the nature of the heterocyclic
ring system met with varied success. Bioisosteric replacement of
the furan oxygen of the basic lapacho structure 7 with nitrogen
or introduction of a second nitrogen atom was detrimental to
activity, as the pertinent pyrrole-fused (11) and imidazole-fused
(12 and 12a) agents were totally inactive. This was also
observed for their 2-acetylated derivatives 21 and 21a,
respectively, which are aza analogues of the original lapacho
quinones 3 and 4. On the other hand, oxazole (13) and thiazole
(14) analogues revealed interesting activity; however, attempts
to introduce a 2-acetyl group as in the original lapacho
quinones 3 and 4 failed. Furthermore, replacement of oxygen
for sulfur (8) almost retained activity. This led us to evaluate
some 8-substituted naphthoquinone skeletons (8a−8e) of the
thiophene analogue 8. These structures were then acetylated at
C-2 as in 3. From inspection it became clear that introduction
of an 8-chlorine atom, 8c, did not substantially alter the
inhibitory potency in the keratinocyte hyperproliferation assay
relative to that of 7 and 8, whereas an 8-methoxy group, 8b, led
to a clearly less potent structure, and an 8-amino group, 8d,
E
dx.doi.org/10.1021/jm500754d | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 5
a
R is defined in Table 3. Reagents and conditions: (a) n-BuLi, THF, −15 °C, appropriate N,N-dimethylcarboxamide, N2; (b) (NH4)2[Ce(NO3)6],
MeCN, H2O, 0 °C; (c) AlCl3, CH2Cl2, rt; (d) n-BuLi, THF, −15 °C, CO2, THF, −70 °C, N2; (e) DCC, DMAP, ROH, 0 °C, rt; (f) DCC, CH2Cl2, 0
°C, appropriate N-hydroxyalkylamidine, pyridine, reflux.
Table 4. Suppression of Human Keratinocyte Hyperproliferation by 4,8,9-Trimethoxynaphtho[2,3-b]thiophenes, 8-
Methoxynaphtho[2,3-b]thiophene-4,9-diones, and 5,8-Dihydroxynaphtho[2,3-b]thiophene-4,9-diones
a
a
compd
R
AA, IC50 (μmol/L)
compd
R
AA, IC50 (μmol/L)
8b
H
H
13.50
>30
>30
>30
3.50
5.70
27b
28b
33a
34a
36
CO2Et
CO2Et
>30
2.87
>30
3.52
2.70
0.50
19b
24b
24m
25b
25m
COEt
5-(1,2,4-oxadiazole)
5-(1,2,4-oxadiazole)
H
nicotinoyl
COEt
nicotinoyl
40
COMe
a
Antihyperproliferative activity against keratinocytes. IC50 = concentration of test compound required for 50% inhibition of cell growth (HaCaT).
Inhibition of cell growth was significantly different with respect to that of the control, N = 3, P < 0.05.
26g with thiophene (26l) or pyridine (26m) gave bioisosteric
molecules whose potency was nearly 3-fold improved. An acid
substituent at position 2 renders the compound inactive (29a).
The deleterious effect of the presence of the free carboxylic acid
group on potency could be overcome by esterification. Thus,
ethyl ester 29b demonstrated activity, and with an IC50 of 0.76
μM against keratinocyte hyperproliferation it was equipotent to
anthralin, supporting the hypothesis that cellular inactivity of
29a was due to insufficient cell penetration. Similar to the 2-
acyl series, a terminal phenyl ring of the ester chain diminished
potency (29d) or abolished activity (29e). Potency was further
enhanced when the ester group of 29b was replaced with a
pertinent amide group, with analogue 32 (IC50 = 0.36 μM)
being among the most potent compounds of this series. As the
replacement of the ester with an amide was compatible with
good activity, it was obviously of interest to see what would
happen when the same replacement was made with a
bioisosteric group that would mimic the carbonyl-containing
ester or amide groups of 29b and 32, such as the 1,2,4-
oxadiazole moiety.46,47 Accordingly, we found that the
oxadiazoles 35a and 35b were highly potent, IC50 = 0.19 and
0.48 μM, confirming a similar observation made in our previous
study.23
Other Structural Features. Table 4 shows, for a limited
set of analogues, the effect on activity against keratinocyte
hyperproliferation of other structural features. There are four
pairs of compounds where the effect of O-methylation of the 8-
hydroxy group can be explored by comparing the correspond-
ing 8-methoxy and 8-hydroxy analogues of Table 3. In all cases
the 8-hydroxy analogues are the more potent inhibitors, which
is in good agreement with the observation made in the initial
study with the pairs of basic structures 8a/8b and 18a/18b
(Table 1). The differences vary from 4- to 9-fold (25b/26b,
28b/29b) and up to 19-fold for the nicotinoyl-substituted
25m/26m and oxadiazoles 34a/35a. In addition, there are five
pairs of compounds where the basic structures (8b/19b) and
F
dx.doi.org/10.1021/jm500754d | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 5. Rates of Superoxide Generation by 8-Hydroxynaphtho[2,3-b]thiophene-4,9-diones through One-Electron Reduction
by Human Recombinant CPR and Two-Electron Reduction by Human Recombinant NQO-1
•−
•−
rate of O2 generation
rate of O2 generation
a
b
a
b
compd
CPR
NQO-1
compd
CPR
NQO-1
c
c
c
c
c
8a
1.8 0.4
1.1 0.1
1.0 0.1
0.3 0.2
menadione
2.1 0.4
1.0 0.4
0.2 0.1
c
d
18a
35a
0.6 0.2
0.7 0.1
control
0.2 0.1
c
a
The rate of superoxide generation in the presence of CPR is expressed as the rate of SOD-inhibitable reduction of succinoylated cytochrome c
b
(μmol/L/min/2 mU of enzyme) by the test compound (100 μmol/mL). Each value represents the mean SD, N ≥ 3 The rate of superoxide
generation in the presence of NQO-1 is expressed as the rate of SOD-inhibitable reduction of cytochrome c (μmol/L/min/U of enzyme) by the test
compound (25 μmol/mL). Each value represents the mean SD, N ≥ 3. Values are significantly different from those of the vehicle control, P <
0.0001. Rate of reduction with no test compound present (DMSO).
c
d
those bearing propionyl (24b/25b), nicotinoyl (24m/25m),
ethyl carboxylate (27b/28b), and oxadiazole (33a/34a) side
chains can be directly compared. The results for these sets of
compounds suggest the requirement for a quinone moiety.
When the quinone form is masked by reductive methylation to
afford the 4,8,9-trimethoxynaphtho[2,3-b]thiophenes, in all five
of these cases activity against keratinocyte hyperproliferation is
completely lost. Finally, compounds 36 and 40 explore the
consequences of an additional 5-hydroxy group, which would
allow the presence of a fully internally hydrogen-bonded
quinone function. There appears to be no requirement for
hydrogen bond donor capability at position 5, as the 2-acetyl
derivative 40 and the monohydrogen-bonded analogue 18a as
well as the pair of basic structures 36 and 8a differ little in their
potency
Lactate Dehydrogenase Release. A major concern in the
testing of potential inhibitors of keratinocyte hyperproliferation
is to confirm that the compound does not interfere with the
functioning of the cell membrane by causing leakage of
cytoplasm through it. Therefore, the action on the cell cultures
by the lapacho analogous compounds was assessed by the
activity of lactate dehydrogenase (LDH) activity released into
the culture supernatant. The release of LDH is commonly used
as an indicator of plasma membrane damage.48,49 Tables 2 and
3 show the membrane-damaging properties of some selected
potent inhibitors of keratinocyte hyperproliferation. The most
potent 7-substituted inhibitor of Table 2, hydroxymethyl-
substituted 22n, also significantly released LDH, while LDH
released by weaker inhibitors such as furan 22i and compound
22k was not significant. Whereas all tested compounds of Table
3 released significantly large amounts of LDH, there was clear
distinction in potency of inducing cell lysis within the 2-
substituted series. Thus, comparing the strongest inducers of
membrane damage with the weakest, i.e., compound 26g with
26l/26m, and 32 with 35a, respectively, less than half the
amount of LDH was released when the phenyl ring or the
amide group was replaced with a thiophene/pyridine or an
oxadiazole ring, respectively, suggesting that bioisosteric
replacement is a useful measure for substantially reducing
membrane-damaging effects. In this assay, the heterocycle-
substituted analogues 26l, 26m, and 35a also compared
favorably with anthralin, the clinical efficacy of which is limited
by staining and irritation of the nonaffected skin,24 and β-
lapachone, as LDH release by these agents significantly
exceeded that of the vehicle control (Table 2) and was much
more pronounced than that of the bioisosteric analogues.
Enzymatic Redox Activation. While a number of
biological effects of lapacho-derived quinones have been
described, their mechanism of action and their key cellular
targets remain largely unknown. Due to their electrophilic and
redox-active nature, the major mechanisms of quinones include
alkylation of cellular nucleophiles such as thiols and redox
cycling initiated through one-electron reduction by NADPH−
cytochrome P450 oxidoreductase (CPR) or two-electron
reduction by NAD(P)H:quinone oxidoreductase 1 (NQO-
1).50−52 Both the desired biological action and some other
effects such as skin toxicity of quinones have been attributed to
the generation of reactive oxygen species (ROS) through
enzyme-mediated redox cycling.50−56 The reduction of
quinones typically occurs in two sequential reactions to
generate hydroquinones via semiquinone radicals that can
react back to the parent compound, thus perpetuating a redox
cycle. Concomitantly, electron transfer to molecular oxygen
generates superoxide and other species therefrom, such as
H2O2 and hydroxyl radicals (•OH). Finally, the depletion of the
antioxidant defense systems may cause oxidative stress,57 which
results in the failure of normal cellular functions and even cell
death.58 Our recent study revealed that lapacho quinones were
activated by both CPR- and NQO-1-catalyzed one- and two-
electron reduction, respectively, to generate superoxide, which
we also confirmed in a keratinocyte-based assay.23
Superoxide Generation in Isolated Enzyme Assays. The
rate of superoxide generation was measured using a
spectrophotometric assay that employs ferricytochrome c as
the terminal electron acceptor, which was specified by (1)
succinoylation of cytochrome c, (2) addition of superoxide
dismutase (SOD) to ensure that only the SOD-inhibitable,
superoxide-dependent extent of cytochrome c reduction was
measured,59 (3) addition of catalase to scavenge H2O2, and (4)
DTPA to prevent the generation of hydroxyl radicals.60
The ability of selected 8-hydroxynaphtho[2,3-b]thiophene-
4,9-diones to stimulate superoxide generation was determined,
and the results are presented in Table 5. For comparison, the
known redox cycler menadione61,62 (2-methylnaphthalene-1,4-
dione) was used as a positive control. In the one-electron
reduction catalyzed by human recombinant CPR and NADPH,
the 2-unsubstituted structure 8a generated the highest amount
of superoxide, whereas 2-acetylation (18a) or an oxadiazole
ring in position 2 (35a) substantially decreased activation
through one-electron transfer, which was significant for all three
lapacho analogues. Accordingly, the semiquinone radicals
generated from these compounds in turn can efficiently react
with molecular oxygen and generate superoxide. However,
when the compounds were incubated with human recombinant
NQO-1/NADPH to study the contribution of two-electron
reduction, superoxide generation of the basic structure 8a did
not significantly exceed the control value. By contrast, both the
2-acetyl (18a) and oxadiazole (35a) analogues significantly
G
dx.doi.org/10.1021/jm500754d | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 6. Superoxide Generation in Human Keratinocytes and Percentage of Apoptotic Cells Following Acute and Chronic
Treatment with 8-Hydroxynaphtho[2,3-b]thiophene-4,9-diones
a
•−
O2 generation (MFI)
percentage of apoptotic cells
b
c
c
,
d
c
c,d
compd
8a
acute treatment
chronic treatment
dicoumarol pretreatment
chronic treatment
dicoumarol pretreatment
e
e
e
e
e
e
23.8 3.7
610.6 258.7
717.7 91.1
813.0 85.0
747.0 36.3
4.9 1.4
6.1 3.1
7.3 4.7
20.3 12.1
3.1 1.1
4.3 2.6
e
e
e
e
18a
23.7 3.8
921.4 149.2
23.0 13.2
77.6 23.0
4.8 2.3
e
e
e
35a
20.9 16.6
802.2 400.1
e
menadione
44.0 11.6
431.9 83.5
367.5 80.1
457.2 103.4
407.6 68.7
f
control
7.1 5.2
3.0 1.0
a
Superoxide generation is expressed in terms of changes in the mean fluorescence intensity (MFI) of 2-OH-E+. Each value represents the mean
b
c
SD, N = 3. HaCaT keratinocytes were incubated with test compound (50 μmol/L) for 30 min. HaCaT keratinocytes were incubated with test
compound (5 μmol/L) for 18 h. HaCaT keratinocytes were preincubated with the NQO-1 inhibitor dicoumarol (5 μmol/L) for 25 min before
treatment with test compounds. Values are significantly different from those of the vehicle control, P < 0.05. Mean fluorescence intensity with no
d
e
f
test compound present (DMSO).
generated increased amounts of superoxide, suggesting that
their primary hydroquinones produced by two-electron
reduction were not stable and readily autoxidized. This may
be related to an electron-withdrawing substituent in position 2,
which enables redox cycling and superoxide generation.23
Superoxide Generation in Keratinocytes. To prove that
redox cycling of lapacho quinones also results from the normal
enzyme activities of keratinocytes, superoxide generation was
directly evaluated in a whole-cell assay. For the detection and
quantification of superoxide under the conditions of the
hyperproliferation assay in HaCaT keratinocytes, we employed
flow cytometry using dihydroethidium (DHE) as the
fluorescent probe.63 The fluorescence intensity arising from
the product of the DHE/superoxide reaction, 2-hydroxyethi-
dium (2-OH-E+), is a measure of intracellular superoxide
generation and a specific marker for superoxide.64,65
Menadione served as a positive control, as intracellular
superoxide generation by the use of DHE has been described
for this naphthoquinone.64,66
Acute and Chronic Treatment of HaCaT Keratinocytes
with Lapacho Analogues. Quantitative measurements of the
mean fluorescence intensities from the samples demonstrated
significantly increased superoxide generation following both
acute and chronic treatment with the three lapacho compounds
(Table 6). However, menadione significantly enhanced the
levels of 2-OH-E+ fluorescence only at relatively high
concentrations (50 μmol/L) where superoxide generation in
human spermatozoa has been described.66
Effects of Pretreatment with the NQO-1 Inhibitor
Dicoumarol. To demonstrate the role of NQO-1 through a
two-electron reduction of lapacho quinones, we determined
intracellular superoxide generation in keratinocytes after
preincubation with dicoumarol. This is a commonly used
inhibitor of NQO-1,61,67 which competes with NADH for
binding and prevents reduction of various quinones. As shown
in Table 6, chronic treatment of dicoumarol-pretreated
keratinocytes with lapacho analogues significantly increased 2-
OH-E+ fluorescence in each case. In control experiments, alone
or with dicoumarol, chronic treatment of keratinocytes with
menadione did not significantly increase superoxide generation
at a concentration of 5 μmol/L, which is in line with the
observation that relatively high concentrations of menadione
were required for suppression of HaCaT cell hyperproliferation
(IC50 = 15.8 μmol/L, Table 2).
the early phase of apoptosis stain positive for 7-AAD in flow
cytometry, depending on the concentration of the fluorescent
intercalator.68 All lapacho analogues (5 μM/L) significantly
increased early apoptosis compared to the control (Table 6),
with oxadiazole 35a having the greatest effect. In contrast, the
basic structure 8a only slightly triggered apoptosis, and
induction of cell death by menadione was not significant. In
contrast to its effect on intracellular superoxide generation,
pretreatment of the keratinocytes with dicoumarol substantially
reduced induction of apoptosis by about 3-fold.
The biological implications of oxidative stress57 in human
keratinocytes induced by lapacho analogues with respect to
their potential therapeutic action for the treatment of psoriasis
or other hyperproliferative skin disorders has been fully
discussed in the first part of this study.23 According to the
results of this paper, the selected 8-hydroxynaphtho[2,3-
b]thiophene-4,9-dione (8a) and its 2-substituted analogues
18a and 35a were potent generators of superoxide radicals in
the chronic treatment experiments under conditions compara-
ble to those of the hyperproliferation model. The observation
that pretreatment of keratinocytes with the NQO-1 inhibitor
dicoumarol did not substantially modulate increased superoxide
generation may suggest that NQO-1 cannot protect keratino-
cytes against oxidative stress induced by these compounds. The
large amounts of superoxide produced may indicate an
increased availability of these quinones for one-electron
reduction and redox cycling by CPR. Surprisingly, when we
extended these studies to compare the ability of the three
lapacho quinones to trigger cell death, either alone or in the
presence of dicoumarol, keratinocytes pretreated with the
NQO-1 inhibitor were protected against apoptosis induced by
the 2-substituted analogues 18a and 35a. This clearly shows
that NQO-1 is involved in the activation of these quinones.
However, the nature of the activated species responsible for a
possible reaction with DNA or other cellular macromolecules,
such as lipids and proteins, to cause damage to the cell, remains
unclear, as superoxide formation did not correlate with the
induction of apoptosis. In this context, the possible
contribution of the oxadiazole substituent at the quinone, as
in the highly potent quinone 35a, is currently under
investigation in our laboratory.
CONCLUSIONS
■
In summary, we have described modifications to three
structural features present in the basic naphtho[2,3-b]furan-
4,9-dione (7) of lapacho compounds that could influence
biological activity and may help to further delineate SARs for
To further examine the action of lapacho analogues in
HaCaT keratinocytes after chronic treatment, apoptosis was
measured by 7-aminoactinomycin D (7-AAD) staining. Cells in
H
dx.doi.org/10.1021/jm500754d | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
stirring was added anhydrous K2CO3 (3.00 g, 21.72 mmol), and the
reaction mixture was heated to reflux. Dimethyl sulfate (1.03 mL,
10.86 mmol) was added dropwise, and the mixture was refluxed for an
additional 4 h until the reaction was completed (TLC control). Then
it was cooled to rt, filtered by suction, and washed with acetone (20
mL). The solvent was evaporated and the residue purified by
chromatography to afford light yellow, felted needles: 72% yield; mp
suppression of keratinocyte hyperproliferation. To this end, a
total of 71 analogues of 7 were prepared and evaluated.
Information obtained from our initial SAR studies that pyrrole-
or imidazole-fused systems were inactive, coupled to the fact
that 2-acylation of pertinent thiazole- or oxazole-fused systems
was not efficient, led us to explore the effect of introducing a
variety of substituents at the 2- and 7-positions of the selected
thiophene-fused 8-hydroxy-substitued naphthoquinone 8a.
While 7-substitution was generally less beneficial for improving
potency, potent inhibitors of keratinocyte hyperproliferation
were obtained with electron-withdrawing functionalities such as
acyl, carboxylic acids, and carboxamide or their bioisosteric
replacement with a 1,2,4-oxadiazole ring at position 2 of the
tricyclic system. In particular, analogues 26l, 26m, and 35a with
a thiophene, a pyridine, or an oxadiazole ring, respectively,
compared favorably with the antipsoriatic agent anthralin, as
their potency was combined with comparably low membrane-
damaging effects toward keratinocytes. The biological activity of
the lapacho analogues is thought to be due, in part, to their
activation by enzymatic reductases such as CPR or NQO-1 to
the semiquinones or hydroquinones, respectively. These
intermediates react with oxygen to generate superoxide,
which we have detected in the hyperproliferation assay.
However, the amount of superoxide produced by lapacho
quinones did not correlate with their ability to induce cell
death.
69
1
164 °C (lit. mp 164−166 °C); FTIR 1670 cm−1; H NMR (CDCl3)
δ 7.91 (dd, 3J = 7.62 Hz, 4J = 1.18 Hz, 1H), 7.70 (t, 1H), 7.63 (d, 2H),
7.33 (d, 1H), 4.04 (s, 3H); MS m/z 244 (100, M+). Anal. (C13H8O3S)
C, H.
N-(4,9-Dioxo-4,9-dihydronaphtho[2,3-b]thiophene-8-yl)-
acetamide (8e). A mixture of 8d (0.25 g, 1.09 mmol) in acetic
anhydride (5 mL) and H2SO4 (96%, 0.10 mL) was heated to reflux for
20 min. Then it was poured into ice−water (100 mL) and extracted
with CH2Cl2 (3 × 50 mL). The combined organic phase was washed
with water (3 × 50 mL), dried over Na2SO4, concentrated, and
purified by chromatography to afford orange needles: 54% yield; mp
198−200 °C; FTIR 1702 (CONH), 1662 (CO), 1627 (CO···HN)
1
3
4
cm−1; H NMR (CDCl3) δ 12.04 (s, 1H), 9.02 (dd, J = 8.6 Hz, J =
1.17 Hz, 1H), 7.92 (dd, 1H), 7.67 (d, J = 5.08 Hz, 1H), 7.65 (t, J =
8.21 Hz, 1H), 7.61 (d, J = 5.08 Hz, 1H), 2.25 (s, 3H); MS m/z 271
(23, M•+), 229 (100). Anal. (C14H9NO3S) C, H.
8-Hydroxynaphtho[2,3-b]thiophen-9(4H)-one (9). To a sol-
ution of 8a (0.30 g, 1.30 mmol) in glacial acetic acid (40 mL) heated
to reflux was added, dropwise over 3 h, a solution of SnCl2 (5.55 g,
24.60 mmol) in 37% HCl (9.7 mL). The solution was then cooled to
rt, filtered, diluted with water (40 mL), and extracted with CH2Cl2 (2
× 40 mL). The combined organic phase was washed with water (3 ×
40 mL), dried over Na2SO4, concentrated, and purified by
chromatography to afford yellow crystals: 3% yield; mp 165−168
°C; FTIR 1627 (CO···HO) cm−1; 1H NMR (CDCl3) δ 12.60 (s, 1H),
7.73 (d, J = 5.08 Hz, 1H), 7.39 (t, 1H), 7.10 (d, J = 5.08 Hz, 1H), 6.88
(dd, 3J = 7.62 Hz, 4J = 0.98 Hz, 1H), 6.86 (dd, J = 8.21 Hz, 1H), 4.25
(s, 2H); MS m/z 216 (100, M•+). Anal. (C12H8O2S) C, H.
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 (attenuated total reflection, 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 the eluant unless otherwise
stated. Yields have not been optimized. Elemental analyses were
Naphtho[2,3-b]selenophene-4,9-dione (10). A solution of 17
(1.00 g, 3.58 mmol) in 96% H2SO4 (20 mL) was heated at 100 °C for
1 h. Then the mixture was poured onto ice−water (100 mL) and
extracted with CH2Cl2 (2 × 50 mL). The combined organic phase was
dried over Na2SO4, concentrated, and purified by chromatography to
afford yellow-orange crystals: 41% yield; mp 239 °C; FTIR 1674 cm−1;
1H NMR (CDCl3) δ 8.42 (d, 1H), 8.26−8.20 (m, 2H), 8.01 (d, 1H),
7.79−7.71 (m, 2H); MS m/z 262 (100, M+). Anal. (C12H6O2Se) C, H.
2-(Selenophene-2-ylcarbonyl)benzoic Acid (17). Phthalic
anhydride (14.80 g, 100 mmol) in CH2Cl2 (100 mL) was slowly
added to a suspension of anhydrous AlCl3 (29.43 g, 220 mmol) in
CH2Cl2 (75 mL), and the mixture was stirred at rt for 30 min. Then
selenophene (13.10 g, 100 mmol) was added dropwise within 30 min
under stirring. The mixture was stirred for an additional 2 h, treated
with HCl (0.5 mol/L, 125 mL), and stirred for a further 1 h. Then the
aqueous phase was extracted with CH2Cl2 (3 × 50 mL) and the
combined organic phase extracted with NaOH (2 mol/L, 2 × 100
mL). The product was precipitated with 37% HCl, filtered by suction,
and recrystallized from MeOH/water (1/1) to afford white crystals:
42% yield; mp 147 °C; FTIR 1701, 1652 cm−1; 1H NMR (DMSO-d6)
δ 8.03−8.02 (d, 1H), 7.95−7.93 (d, 1H), 7.87−7.86 (d, 1H), 7.72−
7.62 (m, 2H), 7.27−7.21 (m, 2H); MS m/z 280 (23, M+), 187 (100).
2-Acetylnaphtho[2,3-b]thiophene-4,9-dione (18). To a sol-
ution of 1-(naphtho[2,3-b]thiophene-2-yl)ethan-1-one (20; 0.20 g,
0.88 mmol) in glacial acetic acid (10 mL) was added with stirring at
room temperature, dropwise over 1 h, a solution of CrO3 (0.33 g, 3.29
mmol) in glacial acetic acid (5 mL) and water (5 mL). The solution
was stirred for an additional 60 min at rt, poured into water (100 mL),
and extracted with CH2Cl2 (3 × 50 mL). The combined organic phase
was washed with a saturated solution of NaCl (2 × 50 mL), dried over
Na2SO4, concentrated, and purified by chromatography to afford
yellow crystals: 64% yield; mp 253−254 °C; FTIR 1668 (CO), 1650
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 5,30 7,23 8,69 8c,27 8d,27,28 11,70 12,33 12a,34 13,71 14,72
15,28 16,28 and 3673 were prepared as described.
8-Hydroxynaphtho[2,3-b]thiophene-4,9-dione (8a). To a
solution of 8-aminonaphtho[2,3-b]thiophene-4,9-dione (8d;27 2.50 g,
10.91 mmol) in glacial acetic acid (36 mL) was added 96% H2SO4 (10
mL) at 70 °C. The mixture was stirred for 10 min, and a small amount
of insoluble material was removed by filtration. The filtrate was cooled
in an ice bath (0 °C), a solution of NaNO2 (0.982 g, 14.23 mmol) in
water (2 mL) was added dropwise over 30 min, and the reaction
mixture was kept at 0 °C for 75 min. Urea (0.325 g, 5.41 mmol) in
water (2 mL) was added to quench any unreacted nitrous acid, and the
reaction mixture was stirred for an additional 30 min at 0 °C. The cold
solution was added over 1 h to a refluxing solution of 96% H2SO4 (33
mL) in water (260 mL). The reaction was heated at reflux for 3 h until
N2 evolution ceased and then cooled in an ice bath. The brown-red
crystals were filtered by suction, dried in vacuo, and purified by
chromatography to afford orange crystals: 64% yield; mp 188 °C;
1
FTIR 1665, 1626 (CO···HO) cm−1; H NMR (CDCl3) δ 12.18 (s,
1H), 7.77 (d, 1H), 7.77 (d, J = 5.08 Hz, 1H), 7.71 (d, J = 5.09 Hz,
1H), 7.63 (t, 1H), 7.27 (dd, 1H); MS m/z 230 (100, M+). Anal.
(C12H6O3S) C, H. For the crystallographic data of 8a, see the
Supporting Information.
8-Methoxynaphtho[2,3-b]thiophene-4,9-dione (8b). To a
solution of 8a (0.50 g, 2.17 mmol) in dry acetone (20 mL) with
1
(CO) cm−1; H NMR (CDCl3) δ 8.26−8.14 (m, 2H), 8.10 (s, 1H),
7.80−7.65 (m, 2H), 2.60 (s, 3H); MS m/z 256 (100). Anal.
(C14H8O3S) C, H.
I
dx.doi.org/10.1021/jm500754d | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
General Procedure for the Cleavage of Methyl Ethers. 2-
Acetyl-8-hydroxynaphtho[2,3-b]thiophene-4,9-dione (18a). Anhy-
drous AlCl3 (0.54 g, 4.05 mmol) was added in portions to a solution of
18b (0.100 g, 0.349 mmol) in dry CH2Cl2 (17 mL), and the mixture
was stirred at rt for 30 min. Then it was treated with water (150 mL),
acidified with HCl (2 mol/L), stirred for an additional 1 h, and
extracted with CH2Cl2 (3 × 50 mL). The combined organic phase was
washed with water (3 × 50 mL), dried over Na2SO4, concentrated, and
purified by chromatography to afford orange crystals: 64% yield; mp
at reflux and treated with K2Cr2O7 (8.83 g, 30 mmol). After 1 h the
mixture was cooled and filtered. The product was dried in vacuo over
P2O5 and purified by chromatography to afford yellow crystals: 53%
1
yield; mp > 350 °C; FTIR 1686, 1662 cm−1; H NMR (DMSO-d6) δ
14.96 (br s, 1H), 8.10−8.08 (m, 2H), 7.88−7.84 (m, 2H), 2.65 (s,
3H); MS m/z 240 (62, M+), 212 (100). Anal. (C13H8N2O3) C, H, N.
2-Acetyl-1-methyl-1H-naphtho[2,3-d]imidazole-4,9-dione (21a).
A mixture of 21 (0.50 g, 2.08 mmol) and anhydrous K2CO3 (2.00 g,
15 mmol) in dry DMF (50 mL) was cooled to −60 °C in a dry ice/
acetone bath under stirring while iodomethane (0.31 mL, 5 mmol)
was added dropwise. After 30 min the mixture was allowed to return to
rt and stirred for an additional 18 h. Then it was poured into water
(150 mL) and filtered by suction. The product was dried in vacuo over
P2O5 and purified by chromatography to afford a pale yellow powder:
79% yield; mp 204 °C; FTIR 1679 cm−1; 1H NMR (CDCl3) δ 8.28−
8.15 (m, 2H), 7.78−7.75 (m, 2H), 4.43 (s, 3H), 2.82 (s, 3H); MS m/z
254 (100, M+). Anal. (C14H10N2O3) C, H, N.
1
253−254 °C; FTIR 1664 cm−1; H NMR (CDCl3) δ 12.05 (s, 1H,
OH), 8.15 (s, 1H), 7.80 (dd, 3J = 7.43 Hz, 4J = 0.97 Hz, 1H), 7.68 (t,
1H), 7.31 (d, 1H), 2.68 (s, 3H); MS m/z 272 (79, M+), 257 (100).
Anal. (C14H8O4S) C, H.
General Procedure for the Oxidative Demethylation to
Naphtho[2,3-b]thiophene-4,9-diones. 2-Acetyl-8-
methoxynaphtho[2,3-b]thiophene-4,9-dione (18b). To a suspension
of 20b (0.30 g, 0.95 mmol) in MeCN (8 mL) and water (1.5 mL) at 0
°C was added dropwise within 20 min (NH4)2[Ce(NO3)6] (1.39 g,
2.54 mmol) in MeCN (2.5 mL) and water (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 (50 mL) and extracted
with CH2Cl2 (3 × 50 mL). The combined organic phase was washed
with a saturated solution of NaCl (2 × 50 mL), dried over Na2SO4,
concentrated, and purified by chromatography to afford light yellow,
Methyl 2-(8-Hydroxy-4,9-dioxo-4,9-dihydronaphtho[2,3-b]-
thiophene-7-yl)acetate (22e). To the carboxylic acid 22d (0.15 g,
0.52 mmol) in MeOH (15 mL) was added 96% H2SO4 (0.1 mL), and
the mixture was heated to reflux for 3 h. Then it was allowed to cool to
rt, diluted with ice−water (50 mL), and extracted with CH2Cl2 (3 ×
30 mL). The combined organic phase was washed with water, dried
over Na2SO4, concentrated, and purified by chromatography to
provide an orange product: 51% yield; mp 173 °C; FTIR 1738 (ester),
1
felted needles: 64% yield; mp 260−262 °C; FTIR 1668 cm−1; H
3
4
1
1658 (CO), 1622 (CO···HO) cm−1; H NMR (CDCl3) δ 12.55 (s,
NMR (CDCl3) δ 8.10 (s, 1H), 7.92 (dd, J = 7.62 Hz, J = 1.17 Hz,
1H), 7.74 (t, 1H), 7.35 (d, J = 8.40 Hz, 1H), 4.05 (s, 3H), 2.65 (s,
3H); MS m/z 286 (100, M+). Anal. (C15H10O4S) C, H.
1H), 7.77 (d, 3J = 5.08 Hz, 1H), 7.73 (d, 3J = 7.83 Hz, 1H), 7.70 (d, 3J
= 5.08 Hz, 1H), 7.57 (d, 3J = 7.44 Hz, 1H), 3.78 (s, 2H), 3.74 (s, 3H);
MS m/z 302 (100, M•+). Anal. (C15H10O5S) C, H.
Analogously, compounds 18c and 39 were prepared from 20c and
38, respectively. See the Supporting Information for details.
Analogously, compounds 22f−22h, 22q, and 22r were prepared
from the appropriate alcohols and 22d, 22o, and 22p, respectively. See
the Supporting Information for details.
4,8,9-Trimethoxynaphtho[2,3-b]thiophene (19b). To a solution of
8a (5.00 g, 21.72 mmol) and tetrabutylammonium bromide (0.70 g,
0.22 mmol) in dry THF (220 mL) under N2 was added dropwise
sodium dithionite (22.72 g, 130.43 mmol) in water (110 mL). The
mixture was vigorously stirred for 30 min and then treated with
sodium hydroxide (28.04 g, 500 mmol) in water (110 mL). After 30
min it was cooled to 0 °C in an ice bath, dimethyl sulfate (43.48 mL,
458.48 mmol) was added, and the mixture was then stirred for 2 h at 0
°C. Then it was allowed to warm to rt, stirred for an additional 2 h,
and extracted with CH2Cl2 (3 × 200 mL). The combined organic
phase was washed with a saturated solution of NaCl (200 mL), dried
over Na2SO4, and evaporated, and the residue was purified by
chromatography to afford yellow crystals: 77% yield; mp 82 °C; FTIR
3077, 2836 cm−1; 1H NMR (CDCl3) δ 7.88 (dd, 1H), 7.53 (d, J = 5.47
Hz, 1H), 7.45 (d, J = 5.67 Hz, 1H), 7.37 (t, J = 7.42 Hz, 1H), 6.85 (d, J
= 7.23 Hz, 1H), 4.09 (s, 3H), 4.05 (s, 3H), 4.04 (s, 3H); MS m/z 274
(72, M•+), 259 (100). Anal. (C15H14O3S) C, H.
General Procedure for the Marschalk Reaction of 8-
Hydroxynaphtho[2,3-b]thiophene-4,9-dione (8a) with Various
Benzaldehydes. 7-Benzyl-8-hydroxynaphtho[2,3-b]-thiophene-
4,9-dione (22k). To a solution of 8a (0.80 g, 3.47 mmol) in NaOH
(1 mol/L, 100 mL) was added Na2S2O4 (0.91 g, 5.21 mmol) under N2.
The solution was heated to 60 °C for 20 min. Benzaldehyde (1.84 g,
17.35 mmol) was added, and the temperature was raised to 90 °C.
After 12 h the reaction mixture was cooled, aerated for 30 min, and
allowed to cool to rt. Then it was poured onto water (200 mL),
acidified with HCl (2 mol/L), and extracted with CH2Cl2 (3 × 100
mL). The combined organic phase was washed with water, dried over
Na2SO4, concentrated, and purified by chromatography (CH2Cl2/
hexane, 8/2) to provide red crystals: 7% yield; mp 145 °C; FTIR 1658
1
(CO), 1621 (CO···HO) cm−1; H NMR (CDCl3) δ 12.61 (s, 1H),
3
3
3
7.75 (d, J = 5.08 Hz, 1H), 7.70 (d, J = 7.82 Hz, 1H), 7.69 (d, J =
3
4
Analogously, compounds 19c and 37 were prepared from 8c and
36,73 respectively. See the Supporting Information for details.
General Procedure for 2-Acylation of 19b. 1-(4,8,9-
Trimethoxynaphtho[2,3-b]thiophene-2-yl)ethan-1-one (20b). To a
mixture of n-butyllithium (1.6 M, 5.40 mL, 8.64 mmol) in dry THF
(8.5 mL) at −15 °C under N2 was added dropwise a solution of 19b
(1.20 g, 4.37 mmol) in dry THF (43 mL). The mixture was stirred for
4 h at −15 °C, and then N,N-dimethylacetamide (0.86 mL, 9.17
mmol) in THF (7 mL) was added. The mixture was stirred at rt for 3
h, poured onto ice−water (85 mL), acidified with HCl (2 mol/L), and
extracted with ether (3 × 50 mL). The combined organic phase was
washed with a saturated solution of NaCl (3 × 50 mL), dried over
Na2SO4, concentrated, and purified by chromatography to afford light
4.70 Hz, 1H), 7.41 (dd, J = 7.82 Hz, J = 0.78 Hz, 1H), 7.31−7.25
(m, 5H), 4.09 (s, 2H); MS m/z 320 (100, M•+). Anal. (C19H12O3S) C,
H.
Analogously, compounds 22a−22d, 22i, and 22l−22p were
prepared from the appropriate aldehydes. See the Supporting
Information for details.
8-Hydroxy-7-(4-methoxybenzyl)naphtho[2,3-b]thiophene-4,9-
dione (22s). To a suspension of 22v (0.25 g, 0.90 mmol) and
anhydrous AlCl3 (0.54 g, 4.04 mmol) in CH2Cl2 (30 mL) was added
anisole (0.15 g, 1.35 mmol). The reaction mixture was refluxed for 4 h
and then poured onto ice−HCl (100 mL) with stirring. The product
was extracted with CH2Cl2 (3 × 50 mL), the organic phase dried over
Na2SO4, and the residue purified by chromatography (CH2Cl2/
hexane, 6/4) to give an orange powder: 19% yield; mp 140 °C; FTIR
1
yellow crystals: 23% yield; mp 190−192 °C; FTIR 1665 cm−1; H
1
1654 (CO), 1621 (CO···HO) cm−1; H NMR (CDCl3) δ 12.61 (s,
NMR (CDCl3) δ 8.14 (s, 1H), 7.86 (dd, 1H), 7.39 (t, 1H), 6.90 (d, J =
7.62 Hz, 1H), 4.14 (s, 3H), 4.05 (s, 3H), 4.03 (s, 3H), 2.72 (s, 3H);
MS m/z 316 (93, M•+), 301 (100).
1H), 7.75 (d, 3J = 5.09 Hz, 1H), 7.68 (d, 3J = 5.09 Hz, 1H), 7.67 (d, 3J
= 7.82 Hz, 1H), 7.39 (d, 3J = 8.21 Hz, 1H), 7.18 (d, 3J = 8.61 Hz, 2H),
3
Analogously, 2-acetyl derivatives 20, 20c, and 38 were prepared
from naphtho[2,3-b]thiophene,32 19c, and 37, respectively. See the
Supporting Information for details.
6.85 (d, J = 9.00 Hz, 2H), 4.02 (s, 2H), 3.78 (s, 3H); MS m/z 350
(100, M•+). Anal. (C20H14O4S) C, H.
Analogously, compound 22t was prepared from veratrole. See the
2-Acetyl-1H-naphtho[2,3-d]imidazole-4,9-dione (21). A solution
of ( )-2-(1-hydroxyethyl)-1H-naphtho[2,3-d]imidazole35 (2.00 g,
9.43 mmol) in 96% H2SO4 (8.3 mL) and water (150 mL) was heated
Supporting Information for details.
8-Hydroxy-7-(hydroxymethyl)naphtho[2,3-b]thiophene-4,9-
dione (22u). A suspension of 8a (1.00 g, 4.34 mmol) in MeOH (100
J
dx.doi.org/10.1021/jm500754d | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
mL) was cooled to 0 °C, and NaOH (2 mol/L, 15 mL) was added
under N2. The mixture was stirred for 15 min, treated with Na2S2O4
(3.00 g, 17.21 mmol) in water (15 mL), and stirred for an additional
20 min. A solution of formaldehyde (37%, 9 mL) was added dropwise,
and then the mixture was stirred for 3 h at 0−5 °C under N2 (TLC
control). The reaction mixture was then treated with H2O2 (2 mL),
poured onto ice−water (200 mL), and acidified with HCl (6 mol/L)
until the color changed to orange. The aqueous phase was extracted
with CH2Cl2 (3 × 100 mL). The combined organic phase was washed
with water, dried over Na2SO4, and concentrated. Petroleum ether was
added to induce precipitation of the product at 0 °C. The precipitate
was filtered by suction, washed with petroleum ether/CH2Cl2 (1/1, 20
mL), dried in vacuo, and purified by chromatography (CH2Cl2/ethyl
acetate, 1/1) to provide orange crystals: 37% yield; mp 185 °C; FTIR
mmol) under stirring. Then DCC (1.07 g, 5.18 mmol) was added at 0
°C, and the mixture was stirred for 10 min and then for an additional 3
h at rt. The mixture was filtered and the filtrate evaporated and
dissolved in CH2Cl2 (100 mL). The solution was washed with HCl
(0.5 mol/L, 2 × 50 mL) and 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 to give yellow needles: 53% yield; mp 134−135 °C;
FTIR 1702 (CO) cm−1; 1H NMR (CDCl3) δ 8.27 (s, 1H), 7.87 (d, J =
8.6 Hz, 1H), 7.38 (t, 1H), 6.88 (dd, J = 7.62 Hz, 1H), 4.43 (q, 2H),
4.13 (s, 3H), 4.05 (s, 3H), 4.03 (s, 3H), 1.45 (t, 3H); MS m/z 346
(83, M•+), 331 (100). Anal. (C18H18O5S) C, H.
Analogously, compounds 27c−27e were prepared from 27a. See
the Supporting Information for details.
1
3311 (OH), 1660 (CO), 1612 (CO···HO) cm−1; H NMR (DMSO-
3-Ethyl-5-(4,8,9-trimethoxynaphtho[2,3-b]thiophene-2-yl)-1,2,4-
oxadiazole (33a). To a solution of 27a (1.20 g, 3.77 mmol) in dry
CH2Cl2 (20 mL) was added DCC (0.39 g, 1.89 mmol) under N2, and
the mixture was stirred at 0 °C for 1 h. Then it was filtered and the
solvent evaporated. The residue was treated with pyridine (20 mL),
and a solution of N-hydroxypropionamidine74 (0.16 g, 1.82 mmol) in
pyridine (4 mL) was added dropwise at rt. Then the mixture was
refluxed for 3 h, cooled to rt, poured onto ice−water (200 mL),
acidified with HCl (2 mol/L), and extracted with CH2Cl2 (3 × 50
mL). The combined organic phase was washed with water (2 × 200
mL) and dried over Na2SO4. The solution was evaporated, and the
product was purified by chromatography (CH2Cl2/ethyl acetate, 9/1)
to afford light yellow needles: 25% yield; mp 171−173 °C; FTIR 1599,
1571 cm−1; 1H NMR (CDCl3) δ 8.34 (s, 1H), 7.89 (dd, 3J = 8.61 Hz,
4J = 0.78 Hz, 1H), 7.41 (t, J = 8.60 Hz, 1H), 6.91 (d, J = 7.44 Hz, 1H),
4.15 (s, 3H), 4.06 (s, 3H), 4.05 (s, 3H), 2.86 (q, 2H), 1.42 (t, 3H);
MS m/z 370 (87, M•+), 355 (100. Anal. (C19H18N2O4S) C, H, N.
Analogously, compound 33b was prepared from 27a. See the
Supporting Information for details.
Keratinocyte Culture and Determination of Cell Prolifer-
ation. HaCaT keratinocytes43 were cultured, and the cell proliferation
assay was performed as described previously.36 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.
d6) δ 12.32 (s, 1H), 8.24 (d, 3J = 4.92 Hz, 1H), 7.83 (d, 3J = 7.82 Hz,
3
3
1H), 7.68 (d, J = 5.08 Hz, 1H) 7.67 (d, 1H), 5.45 (t, J = 5.48 Hz,
3
1H), 4.61 (d, J = 5.09 Hz, 2H); MS m/z 260 (95, M•+), 231 (100).
Anal. (C13H8O4S) C, H.
Analogously, compound 23 was prepared from 18a. See the
Supporting Information for details.
7-Chloromethyl-8-hydroxynaphtho[2,3-b]thiophene-4,9-dione
(22v). A solution of 22u (1.65 g, 6.34 mmol), thionyl chloride (2.26 g,
19.02 mmol), and DMF (1 mL) was stirred in dry CH2Cl2 (30 mL)
for 2 h at rt. Then the solvent and excess thionyl chloride were
removed in vacuo. The resulting residue was treated with a mixture of
ether (20 mL) and petroleum ether (10 mL) and filtered by suction to
afford an orange product: 83% yield; mp 130 °C; FTIR 1657 (CO),
1619 (CO···HO) cm−1; 1H NMR (CDCl3) δ 12.50 (s, 1H), 8.27 (d, 3J
= 4.69 Hz, 1H), 7.90 (d, 3J = 7.82 Hz, 1H), 7.69 (d, 3J = 5.09 Hz, 1H),
3
7.65 (d, J = 7.82 Hz, 1H), 4.82 (s, 2H); MS m/z 280 (20, M•+)
(37Cl), 278 (50, M•+) (35Cl), 243 (100).
According to the general procedure for 2-acylation as described for
20b, compounds 24a−24m were prepared from 4,8,9-
trimethoxynaphtho[2,3-b]thiophene (19b) and the appropriate N,N-
dimethylcarboxamides and 30 was prepared from 19b and N,N-
diethyl-2,2,2-trifluoroacetamide. See the Supporting Information for
details.
According to the general procedure for the oxidative demethylation
as described for 18b, compounds 25a−25m were prepared from 24a−
24m, compounds 28a−28e from 27a−27e, and compounds 31, 34a,
and 34b from 30, 33a, and 33b, respectively. See the Supporting
Information for details.
Lactate Dehydrogenase Release. The assay was performed as
described.48,49 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
milliunits per milliliter. Appropriate controls with the vehicle were
performed (P < 0.01, N = 3, SD < 10%). Brij 35 (polyoxyethylene
glycol dodecyl ether)/ultrasound was the positive control.
According to the general procedure for the cleavage of methyl
ethers as described for 18a, compounds 26a−26m were prepared from
25a−25m, compounds 29a−29e were prepared from 28a−28e,
compounds 32, 35a, and 35b were prepared from 31, 34a, and 34b,
and compound 40 was prepared from 39, respectively. See the
Supporting Information for details.
4,8,9-Trimethoxynaphtho[2,3-b]thiophene-2-carboxylic Acid
(27a). To a mixture of n-butyllithium (1.6 M, 5.40 mL, 8.60 mmol)
in dry THF (8.5 mL) at −15 °C under N2 was added dropwise a
solution of 19b (1.50 g, 5.47 mmol) in dry THF (55 mL). The
mixture was stirred and cooled to −78 °C, and then dry ice (27.00 g,
613.62 mmol) was added in portions under N2. The mixture was
stirred at −78 °C for 10 min, allowed to warm to −20 °C within 30
min, and then poured into HCl (2 mol/L, 45 mL). The organic phase
was separated and the aqueous phase extracted with ether (2 × 50
mL). The combined organic phase was extracted with NaOH (1 mol/
L, 3 × 100 mL) and the aqueous phase acidified with HCl (2 mol/L)
to pH 2. The carboxylic acid was extracted with CH2Cl2 (3 × 200
mL), the organic phase dried over Na2SO4, and then the solvent
removed in vacuo to afford a light yellow solid: 82% yield; mp 273−
Superoxide Generation Assays (Enzymatic One-Electron
Reduction, Enzymatic Two-Electron Reduction). Both assays
were performed exactly as previously described in full detail.23
Intracellular Superoxide Generation. To detect intracellular
superoxide levels,75 HaCaT keratinocytes were cultivated as
described.36 The cells were grown in Dulbecco’s modified Eagle’s
medium (DMEM; no. E15-810, PAA) in six-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 the cells were treated
for 18 h (chronic treatment) with the test compound (5 μM, DMSO;
the final concentration of DMSO in the culture medium was 0.1%) in
a 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 dicoumarol61,67 (5 μmol/L) for 25 min before the treatment
test compounds were added. After incubation with the test compound,
DHE (10 μmol/mL) was added, the cell culture plates were treated on
a shaker (200 rpm) for 5 min, and the cells were allowed to load DHE
for an additional 25 min under incubation conditions. Then the
1
275 °C; FTIR 1677 (CO2H) cm−1; H NMR (DMSO-d6) δ 8.15 (s,
1H), 7.78 (d, J = 8.40 Hz, 1H), 7.43 (t, 1H), 7.03 (d, J = 7.63 Hz, 1H),
4.06 (s, 3H), 3.97 (s, 3H), 3.90 (s, 3H); MS m/z 318 (84, M•+), 303
(100).
Ethyl 4,8,9-Trimethoxynaphtho[2,3-b]thiophene-2-carboxylate
(27b). To the carboxylic acid 27a (1.50 g, 4.71 mmol) in absolute
ethanol (0.82 mL, 14.13 mmol) was added DMAP (0.05 g, 0.41
K
dx.doi.org/10.1021/jm500754d | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
medium was removed, and the cells were washed with PBS (0.5 mL/
well), trypsinized (0.3 mL/well) for 7 min, treated with FACS buffer
(FACSFlow, BD Biosciences, no. 342003, 0.7 mL), centrifuged
(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, and 90 min). Keratinocytes were kept in the 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 were collected in the FL2 channel (λ = 550−600 nm, 42
nm bandpass). For each sample, 10 000 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
the histograms of untreated control cells. Data are expressed as the
mean fluorescence intensity (MFI).
(6) Arenas, P. Medicine and magic among the Maka Indians of the
Paraguayan Chaco. J. Ethnopharmacol. 1987, 21, 279−295.
(7) Bastien, J. W. Pharmacopeia of Qollahuaya Andeans. J.
Ethnopharmacol. 1983, 8, 97−111.
(8) Hartwell, J. L. Plants used against cancer. A survey. Lloydia 1968,
31, 71−170.
(9) Queiroz, M. L. S.; Valadares, M. C.; Torello, C. O.; Ramos, A. L.;
Oliveira, A. B.; Rocha, F. D.; Arruda, V. A.; Accorci, W. R.
Comparative studies of the effects of Tabebuia avellanedae bark extract
and β-lapachone on the hematopoietic response of tumour-bearing
mice. J. Ethnopharmacol. 2008, 117, 228−235.
(10) de Castro, S. L.; Emery, F. S.; da Silva, E. N., Jr. Synthesis of
quinoidal molecules: strategies towards bioactive compounds with an
emphasis on lapachones. Eur. J. Med. Chem. 2013, 69, 678−700.
̀
(11) Fiorito, S.; Epifano, F.; Bruyere, C.; Mathieu, V.; Kiss, R.;
Genovese, S. Growth inhibitory activity for cancer cell lines of lapachol
and its natural and semi-synthetic derivatives. Bioorg. Med. Chem. Lett.
2014, 24, 454−457.
(12) Eyong, K. O.; Kumar, P. S.; Kuete, V.; Folefoc, G. N.;
Nkengfack, E. A.; Baskaran, S. Semisynthesis and antitumoral activity
of 2-acetylfuranonaphthoquinone and other naphthoquinone deriva-
tives from lapachol. Bioorg. Med. Chem. Lett. 2008, 18, 5387−5390.
(13) Takano, A.; Hashimoto, K.; Ogawa, M.; Koyanagi, J.; Kurihara,
T.; Wakabayashi, H.; Kikuchi, H.; Nakamura, Y.; Motohashi, N.;
Sakagami, H.; Yamamoto, K.; Tanaka, A. Tumor-specific cytotoxicity
and type of cell death induced by naphtho[2,3-b]furan-4,9-diones and
related compounds in human tumor cell lines: relationship to
electronic structure. Anticancer Res. 2009, 29, 455−464.
Flow Cytometric Detection of 7-Aminoactinomycin D (7-
AAD) Positive Cells. Cells were grown and exposed to test
compounds or preincubated with dicoumarol exactly as described
above. Evaluation of apoptosis68 was performed using 7-AAD (10 μg/
mL) according to the manufacturer’s instructions. At least 10 000 cells
were acquired, and data were collected in the FL3 channel and
analyzed by CellQuest Pro.
ASSOCIATED CONTENT
* Supporting Information
■
S
(14) Wu, C.; Johnson, R. K.; Mattern, M. R.; Wong, J. C.; Kingston,
D. G. I. Synthesis of furanonaphthoquinones with hydroxyamino side
chains. J. Nat. Prod. 1999, 62, 963−968.
Supplementary chemical data of compounds 18c, 19c, 20, 20c,
22a−22r, 22t, 23, 24a−24m, 25a−25m, 26a−26m, 27c−27e,
28a−28e, 29a−29c, 30−32, 33b, 34a, 34b, 35a, 35b, and 37−
40, X-ray structure of 8a, and analytical data of all test
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
(15) Yamashita, M.; Kaneko, M.; Tokuda, H.; Nishimura, K.;
Kumeda, Y.; Iida, A. Synthesis and evaluation of bioactive
naphthoquinones from the Brazilian medicinal plant, Tabebuia
avellanedae. Bioorg. Med. Chem. 2009, 17, 6286−6291.
́ ́
(16) Jimenez-Alonso, S.; Guasch, J.; Estevez-Braun, A.; Ratera, I.;
Veciana, J.; Ravelo, A. G. Electronic and cytotoxic properties of 2-
amino-naphtho[2,3-b]furan-4,9-diones. J. Org. Chem. 2011, 76, 1634−
1643.
AUTHOR INFORMATION
Corresponding Author
*Phone: +49-251-8333324. E-mail: kmuller@uni-muenster.de.
■
(17) Gomez-Monterrey, I.; Campiglia, P.; Aquino, C.; Bertamino, A.;
Granata, I.; Carotenuto, A.; Brancaccio, D.; Stiuso, P.; Scognamiglio,
I.; Rusciano, M. R.; Maione, A. S.; Illario, M.; Grieco, P.; Maresca, B.;
Novellino, E. Design, synthesis, and cytotoxic evaluation of acyl
derivatives of 3-aminonaphtho[2,3-b]thiophene-4,9-dione, a quinone-
based system. J. Med. Chem. 2011, 54, 4077−4091.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS USED
■
(18) Muller, K.; Sellmer, A.; Wiegrebe, W. Potential antipsoriatic
̈
7-AAD, 7-aminoactinomycin D; CPR, NADPH−cytochrome
P450 oxidoreductase; DCC, N,N′-dicyclohexylcarbodiimide;
DHE, dihydroethidium; DMAP, 4-(dimethylamino)pyridine;
DTPA, diethylenetriaminepentaacetic acid; NQO-1, NAD(P)-
H:quinone oxidoreductase 1; ROS, reactive oxygen species; rt,
room temperature; SAR, structure−activity relationship; SOD,
superoxide dismutase
agents: lapacho compounds as potent inhibitors of HaCaT cell growth.
J. Nat. Prod. 1999, 62, 1134−1136.
(19) Muller, K.; Reindl, H.; Breu, K. Antipsoriatic anthrones with
̈
modulated redox properties. 5. Potent inhibition of human
keratinocyte growth, induction of keratinocyte differentiation, and
reduced membrane damage by novel 10-arylacetyl-1,8-dihydroxy-
9(10H)-anthracenones. J. Med. Chem. 2001, 44, 814−821.
(20) Zuse, A.; Schmidt, P.; Baasner, S.; Bohm, K. J.; Muller, K.;
̈
̈
REFERENCES
Gerlach, M.; Gunther, E. G.; Unger, E.; Prinz, H. Sulfonate derivatives
̈
■
of naphtho[2,3-b]thiophen-4(9H)-one and 9(10H)-anthracenone as
highly active antimicrotubule agents. Synthesis, antiproliferative
activity, and inhibition of tubulin polymerization. J. Med. Chem.
2007, 50, 6059−6066.
(1) Burnett, A. R.; Thomson, R. H. Naturally occuring quinones. Part
X. The quinoid constituents of Tabebuia avellanedae (Bignoniaceae). J.
Chem. Soc. C 1967, 2100−2104.
(2) Rao, M. M.; Kingston, D. G. I. Plant anticancer agents. XII.
Isolation and structure elucidation of new cytotoxic quinones from
Tabebuia cassinoides. J. Nat. Prod. 1982, 45, 600−604.
(3) Epifano, F.; Genovese, S.; Fiorito, S.; Mathieu, V.; Kiss, R.
Lapachol and its congeners as anticancer agents: a review. Phytochem.
Rev. 2014, 13, 37−49.
(21) Putic, A.; Stecher, L.; Prinz, H.; Muller, K. Structure−activity
̈
relationship studies of acridones as potential antipsoriatic agents. 1.
Synthesis and antiproliferative activity of simple N-unsubstituted 10H-
acridin-9-ones against human keratinocyte growth. Eur. J. Med. Chem.
2010, 45, 3299−3310.
(22) Prinz, H.; Chamasmani, B.; Vogel, K.; Bohm, K. J.; Aicher, B.;
̈
(4) Jones, K. Pau d’arco: Immune Power from the Rain Forest; Healing
Arts Press: Rochester, VT, 1995.
Gerlach, M.; Gunther, E. G.; Amon, P.; Ivanov, I.; Muller, K. N-
̈
̈
Benzoylated phenoxazines and phenothiazines: synthesis, antiprolifer-
ative activity and inhibition of tubulin polymerization. J. Med. Chem.
2011, 4247−4263.
́
(5) Gomez Castellanos, J. R.; Prieto, J. M.; Heinrich, M. Red lapacho
(Tabebuia impetiginosa)a global ethnopharmacological commodity?
J. Ethnopharmacol. 2009, 121, 1−13.
L
dx.doi.org/10.1021/jm500754d | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(23) Reichstein, A.; Vortherms, S.; Bannwitz, S.; Tentrop, J.; Prinz,
(44) George, S. E.; Anderson, R. J.; Cunningham, A.; Donaldson, M.;
Groundwater, P. W. Evaluation of a range of anti-proliferative assays
for the preclinical screening of anti-psoriatic drugs: a comparison of
colorimetric and fluorimetric assays with the thymidine incorporation
assay. Assay Drug Dev. Technol. 2010, 8, 384−395.
H.; Muller, K. 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. J. Med. Chem. 2012, 55, 7273−7284.
(45) Muller, K.; Prinz, H.; Gawlik, I.; Ziereis, K.; Huang, H.-S. Simple
̈
(24) Harris, D. R. Old wine in new bottles: the revival of anthralin.
Cutis 1998, 62, 201−203.
analogues of anthralin: unusual specificity of structure and
antiproliferative activity. J. Med. Chem. 1997, 40, 3773−3780.
(46) Chen, X.; Wang, W. The use of bioisosteric groups in lead
optimization. Annu. Rep. Med. Chem. 2003, 38, 333−346.
(25) Inzinger, M.; Heschl, B.; Weger, W.; Hofer, A.; Legat, F. J.;
Gruber-Wackernagel, A.; Tilz, H.; Salmhofer, W.; Quehenberger, F.;
Wolf, P. Efficacy of psoralen plus ultraviolet A therapy vs. biologics in
moderate to severe chronic plaque psoriasis: retrospective data analysis
of a patient registry. Br. J. Dermatol. 2011, 165, 640−645.
(26) Lowes, M. A.; Bowcock, A. M.; Krueger, J. G. Pathogenesis and
therapy of psoriasis. Nature 2007, 445, 866−873.
̀
(47) Bostrom, J.; Hogner, A.; Llinas, A.; Wellner, E.; Plowright, A. T.
̈
Oxadiazoles in medicinal chemistry. J. Med. Chem. 2012, 55, 1817−
1830.
(48) Bonnekoh, B.; Farkas, B.; Geisel, J.; Mahrle, G. Lactate
dehydrogenase release as an indicator of dithranol-induced membrane
injury in cultured human keratinocytes. Arch. Dermatol. Res. 1990, 282,
325−329.
(27) Schroeder, H. E.; Weinmayr, V. The synthesis of thiophan-
thraquinones from thenoyl and thenylbenzoic acids. J. Am. Chem. Soc.
1952, 74, 4357−4361.
(49) Muller, K.; Huang, H.-S.; Wiegrebe, W. Antipsoriatic anthrones
̈
with modulated redox properties. 3. 10-Thio-substituted 1,8-
dihydroxy-9(10H)-anthracenones as inhibitors of keratinocyte growth,
5-lipoxygenase, and the formation of 12(S)-HETE in mouse
epidermis. J. Med. Chem. 1996, 39, 3132−3138.
(28) Weinmayr, V. o-(2-Thenoyl)-benzoic acids and thiophanthra-
quinones. J. Am. Chem. Soc. 1952, 74, 4353−4357.
(29) Muller, K.; Sellmer, A.; Salvesen, J. Lapacho compounds, their
̈
preparation, and methods of use for the treatment of cell proliferative
disorders. PCT Int. Appl. WO 2004026253 A2 20040401, 2004.
(50) Bolton, J. L.; Trush, M. A.; Penning, T. M.; Dryhurst, G.;
Monks, T. J. Role of quinones in toxicology. Chem. Res. Toxicol. 2000,
13, 135−160.
(51) Powis, G. Free radical formation by antitumor quinones. Free
Radical Biol. Med. 1989, 6, 63−101.
(52) Brunmark, A.; Cadenas, E. Redox and addition chemistry of
quinoid compounds and its biological implications. Free Radical Biol.
Med. 1989, 7, 435−447.
(53) Song, Y.; Buettner, G. R. Thermodynamic and kinetic
considerations for the reaction of semiquinone radicals to form
superoxide and hydrogen peroxide. Free Radical Biol. Med. 2010, 49,
919−962.
(54) Doering, M.; Ba, L. A.; Lilienthal, N.; Nicco, C.; Scherer, C.;
Abbas, M.; Zada, A. A. P.; Coriat, R.; Burkholz, T.; Wessjohann, L.;
Diederich, M.; Batteux, F.; Herling, M.; Jacob, C. Synthesis and
selective anticancer activity of organochalcogen based redox catalysts.
J. Med. Chem. 2010, 53, 6954−6963.
(55) da Silva, E. N., Jr.; Cavalcanti, B. C.; Guimaraes, T. T.; Pinto, M.
d. F. R.; Cabral, I. O.; Pessoa, C.; Costa-Lotufo, L. V.; de Moraes, M.
O.; de Andrade, C. K. Z.; dos Santos, M. R.; de Simone, C. A.; Goulart,
M. O. F.; Pinto, A. V. Synthesis and evaluation of quinonoid
compounds against tumor cell lines. Eur. J. Med. Chem. 2011, 46, 399−
410.
(30) Prinz, H.; Wiegrebe, W.; Muller, K. Synthesis of anthracenones.
̈
1. Sodium dithionite reduction of peri-substituted anthracenediones. J.
Org. Chem. 1996, 61, 2853−2856.
(31) Kraus, G. A.; Man, T. O. An improved reductive methylation
procedure for quinones. Synth. Commun. 1986, 16, 1037−1042.
(32) Tedjamulia, M. L.; Tominaga, Y.; Castle, R. N.; Lee, M. L. The
synthesis of dinaphthothiophenes. J. Heterocycl. Chem. 1983, 20,
1143−1148.
(33) Hoover, J. R. E.; Day, A. R. Preparation of some imidazole
derivatives of 1,4-naphthoquinone. J. Am. Chem. Soc. 1954, 76, 4148−
4152.
̈
(34) Fries, K.; Walter, R.; Schilling, K. Uber tricyclische
Verbindungen, in denen Naphtalin mit einem Heterocyclus anelliert
ist. Liebigs Ann. Chem. 1935, 516, 248−285.
(35) Zellner, H.; Zellner, G.; Koppl, F.; Dirnberger, J. Reaktion von
̈
Brenztraubensaure mit o-Diaminen, 3. Mitt.: Synthesen von 2-(α-
̈
Keto-alkyl)-benzimidazolen. Monatsh. Chem. 1967, 98, 643−665.
(36) Muller, K.; Leukel, P.; Ziereis, K.; Gawlik, I. Antipsoriatic
anthrones with modulated redox properties. 2. Novel derivatives of
chrysarobin and isochrysarobinantiproliferative activity and 5-
lipoxygenase inhibition. J. Med. Chem. 1994, 37, 1660−1669.
(37) Marschalk, C.; Koenig, F.; Ouroussoff, N. Nouvelle met
d’introduction des chaines laterales dans le noyau anthraquinonique.
Bull. Soc. Chim. Fr. 1936, 3, 1545−1575.
(38) Krohn, K. Synthesis of anthracyclinones by electrophilic and
nucleophilic addition to anthraquinones. Tetrahedron 1990, 46, 291−
318.
̈
́
hode
́
́ ́
(56) Lizzi, F.; Veronesi, G.; Belluti, F.; Bergamini, C.; Lopez-Sanchez,
A.; Kaiser, M.; Brun, R.; Krauth-Siegel, R. L.; Hall, D. G.; Rivas, L.;
Bolognesi, M. L. Conjugation of quinones with natural polyamines:
toward an expanded antitrypanosomatid profile. J. Med. Chem. 2012,
55, 10490−10500.
(39) Koyanagi, J.; Yamamoto, K.; Nakayama, K.; Tanaka, A. A new
synthetic route to 2-substituted naphtho[2,3-b]furan-4,9-dione. J.
Heterocycl. Chem. 1997, 34, 407−412.
(57) Sies, H. Biochemistry of oxidative stress. Angew. Chem., Int. Ed.
Engl. 1986, 25, 1058−1071.
(58) Halliwell, B. Oxidative stress and cancer: have we moved
forward? Biochem. J. 2007, 401, 1−11.
(59) Tarpey, M. M.; Wink, D. A.; Grisham, M. B. Methods for
detection of reactive metabolites of oxygen and nitrogen: in vitro and
in vivo considerations. Am. J. Physiol.: Regul., Integr. Comp. Physiol.
2004, 286, R431−R444.
(60) Halliwell, B. Superoxide-dependent formation of hydroxyl
radicals in the presence of iron chelates. Is it a mechanism for hydroxyl
radical production in biochemical systems? FEBS Lett. 1978, 92, 321−
326.
(40) Neises, B.; Steglich, W. 4-Dialkylaminopyridines as acylation
catalysts. 5. Simple method for esterification of carboxylic acids. Angew.
Chem., Int. Ed. Engl. 1978, 17, 522−524.
(41) Borg, S.; Estenne-Bouhtou, G.; Luthman, K.; Csoregh, I.;
̈
Hesselink, W.; Hacksell, U. Synthesis of 1,2,4-oxadiazole-, 1,3,4-
oxadiazole-, and 1,2,4-triazole-derived dipeptidomimetics. J. Org. Chem.
1995, 60, 3112−3120.
(42) Pol, A.; Bergers, M.; Schalkwijk, J. Comparison of
antiproliferative effects of experimental and established antipsoriatic
drugs on human keratinocytes, using a simple 96-well-plate assay. In
Vitro Cell. Dev. Biol.: Anim. 2003, 39, 36−42.
(43) Boukamp, P.; Petrussevska, R. T.; Breitkreutz, D.; Hornung, J.;
Markham, A.; Fusenig, N. E. Normal keratinization in a spontaneously
immortalized aneuploid human keratinocyte cell line. J. Cell Biol. 1988,
106, 761−771.
(61) Thor, H.; Smith, M. T.; Hartzell, P.; Bellomo, G.; Jewell, S. A.;
Orrenius, S. The metabolism of menadione (2-methyl-1,4-naphtho-
quinone) by isolated hepatocytes. A study of the implications of
oxidative stress in intact cells. J. Biol. Chem. 1982, 257, 12419−12425.
̈
(62) Buffinton, G. D.; Ollinger, K.; Brunmark, A.; Cadenas, E. DT-
diaphorase-catalysed reduction of 1,4-naphthoquinone derivatives and
M
dx.doi.org/10.1021/jm500754d | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
glutathionyl-quinone conjugates. Effect of substituents on autoxidation
rates. Biochem. J. 1989, 257, 561−571.
(63) Zhao, H.; Kalivendi, S.; Joseph, J.; Zhang, H.; Joseph, J.;
́
Nithipatikom, K.; Vasquez-Vivar, J.; Kalyanaraman, B. Superoxide
reacts with hydroethidine but forms a fluorescent product that is
distinctly different from ethidium: potential implications in intra-
cellular fluorescence detection of superoxide. Free Radical Biol. Med.
2003, 34, 1359−1368.
(64) Zhao, H.; Joseph, J.; Fales, H. M.; Sokoloski, E. A.; Levine, R. L.;
Vasquez-Vivar, J.; Kalyanaraman, B. Detection and characterization of
the product of hydroethidine and intracellular superoxide by HPLC
and limitations of fluorescence. Proc. Natl. Acad. Sci. U.S.A. 2005, 102,
5727−5732.
(65) Zielonka, J.; Vasquez-Vivar, J.; Kalyanaraman, B. Detection of 2-
hydroxyethidium in cellular systems: a unique marker product of
superoxide and hydroethidine. Nat. Protoc. 2008, 3, 8−21.
(66) De Iuliis, G. N.; Wingate, J. K.; Koppers, A. J.; McLaughlin, E.
A.; Aitken, R. J. Definitive evidence for the nonmitochondrial
production of superoxide anion by human spermatozoa. J. Clin.
Endocrinol. Metab. 2006, 91, 1968−1975.
(67) Tan, A. S.; Berridge, M. V. Evidence for NAD(P)H:quinone
oxidoreductase 1 (NQO1)-mediated quinone-dependent redox cycling
via plasma membrane electron transport: a sensitive cellular assay for
NQO1. Free Radical Biol. Med. 2010, 48, 421−429.
(68) Zembruski, N. C. L.; Stache, V.; Haefeli, W. E.; Weiss, J. 7-
Aminoactinomycin D for apoptosis staining in flow cytometry. Anal.
Biochem. 2012, 429, 79−81.
(69) Zani, C. L.; Chiari, E.; Krettli, A. U.; Murta, S. M.; Cunningham,
M. L.; Fairlamb, A. H.; Romanha, A. J. Anti-plasmodial and anti-
trypanosomal activity of synthetic naphtho[2,3-b]thiophen-4,9-qui-
nones. Bioorg. Med. Chem. 1997, 5, 2185−2192.
(70) Watanabe, T.; Miyagi, C.; Murakami, Y. Synthetic studies on
indoles and related compounds. XXXI. Chemical confirmation of the
synthetic route for the benz[f ]indole skeleton and its application. J.
Heterocycl. Chem. 1993, 30, 217−224.
(71) de Oliveira, C. G. T.; Ferreira, V. F.; Freitas, C.; Carballido, J. M.
Synthesis and antimicrobial evaluation of oxazole-1,4-naphthquinones.
Heterocycl. Commun. 2002, 8, 199−204.
(72) Boggust, W. A.; Cocker, W.; Schwarz, J. C. P.; Stuart, E. R.
Some naphthothiazoles. J. Chem. Soc. 1950, 680−682.
(73) Krapcho, A. P.; Petry, M. E.; Hacker, M. P. Heterosubstituted
anthracene-9,10-dione analogues. The synthesis and antitumor
evaluation of 5,8-bis[(aminoalkyl)amino]naphtho[2,3-b]thiophene-
4,9-diones. J. Med. Chem. 1990, 33, 2651−2655.
(74) Moloney, G. P.; Martin, G. R.; Mathews, N.; MacLennan, S.;
Dodsworth, S.; Sang, P. Y.; Knight, C.; Maxwell, M.; Glen, R. C.
Synthesis and serotonergic activity of 2-oxadiazolyl-5-substituted-N,N-
dimethyltryptamines: novel antagonists for the vascular 5-HT1B-like
receptor. J. Chem. Soc., Perkin Trans. 1 1999, 2725−2733.
́
(75) Mukhopadhyay, P.; Rajesh, M.; Yoshihiro, K.; Hasko, G.;
Pacher, P. Simple quantitative detection of mitochondrial superoxide
production in live cells. Biochem. Biophys. Res. Commun. 2007, 358,
203−208.
N
dx.doi.org/10.1021/jm500754d | J. Med. Chem. XXXX, XXX, XXX−XXX