Design, synthesis, and cytotoxic evaluation of acyl derivatives of 3-aminonaphtho[2,3-b ]thiophene-4,9-dione, a quinone-based system

Article abstract of DOI:10.1021/jm200094h

A series of 3-acyl derivatives of the dihydronaphtho[2,3-b]thiophen-4,9- dione system were studied with respect to cytotoxicity and topoisomerase II inhibitory activity. These analogues were designed as electron-deficient anthraquinone analogues with pote

Full text of DOI:10.1021/jm200094h

ARTICLE
pubs.acs.org/jmc
Design, Synthesis, and Cytotoxic Evaluation of Acyl
Derivatives of 3-Aminonaphtho[2,3-b]thiophene-4,9-dione,
a Quinone-Based System
Isabel Gomez-Monterrey,† Pietro Campiglia,‡ Claudio Aquino,§ Alessia Bertamino,† Ilaria Granata,‡
Alfonso Carotenuto,† Diego Brancaccio,† Paola Stiuso,|| Ilaria Scognamiglio,|| M. Rosaria Rusciano,^
Angela Serena Maione,^ Maddalena Illario,^ Paolo Grieco,† Bruno Maresca,‡ and Ettore Novellino*,†
†Department of Pharmaceutical and Toxicological Chemistry, University of Naples “Federico II”, Naples, Italy
‡Department of Pharmaceutical Science, Division of BioMedicine, University of Salerno, Salerno, Italy
§Kellogg School of Science and Technology at The Scripps Research Institute, Scripps Florida, Jupiter, Florida, United States
Department of Biochemistry and Biophysics, Second University of Naples, Naples, Italy
^Department of Experimental Pharmacology, University of Naples “Federico II”, Naples, Italy
S Supporting Information
b
ABSTRACT:
A series of 3-acyl derivatives of the
dihydronaphtho[2,3-b]thiophen-4,9-dione system were studied
with respect to cytotoxicity and topoisomerase II inhibitory
activity. These analogues were designed as electron-deficient
anthraquinone analogues with potential intercalation ability.
Derivatives 3-(diethylamino)-N-(4,9-dioxo-4,9-dihydronaphtho-
[2,3-b]thiophen-3-yl)propanamide (11m) and 3-(2-(dimethyl-
amino)ethylamino)-N-(4,9-dioxo-4,9-dihydronaphtho[2,3-b]-
thiophen-3-yl)propanamide (11p) showed a high efficacy in cell
lines that were highly resistant to treatment with doxorubicin,
such as MDA-MB435 (melanoma), IGROV (ovarian), and SF-
295 (glioblastoma) human cell lines. Both compounds inhibit
topoisomerase II mediated relaxation of DNA, while only 11p
incites arrest at the S phase in Caco-2 cells, inducing a delay of cell cycle progression and an increase of cell differentiation. The ability
of these derivatives to modulate small heat shock proteins and cardiotoxicy effects was also explored. In addition, the DNA-binding
properties of these compounds were investigated and discussed.
’ INTRODUCTION
system through a five- or six-membered heterocycle or the
presence of a cycloalkyl as the fifth ring was an effective approach
to identify new compounds endowed with potent cytotoxic
activity and able to overcome multidrug resistance of tumor
cells. Thus, the 3-glycylamino-3-(ethoxycarbonyl)-2,3-dihy-
drothieno[2,3-b]naphtho-4,9-dione (5),13 the spirohydantoin
derivatives 3-[2-(N,N-dimethylamino)ethyl or propyl]-
spiro[(dihydroimidazo-2,4-dione)-5,30-(20,30-dihydrothieno[2,3-b]-
naphtho-40,90-dione)] (6a,b),14 and the spirodiketopiperazine
derivatives 4-[(2-N,N-dimethyl)amino]ethylspiro[(dihydropirazin-
2,5-dione)-6,30-(20,30-dihydrothieno[2,3-b]naphtho-40,90-dione)
(7)15 and spiro[(hexahydropyrrolo[1,2-a]pyrazine-1,4-dione)-
6,30-(20,30-dihydrothieno[2,3-b]naphtho-40,90-dione)] (8)16 showed
remarkable cytotoxic activity against several solid tumors and
doxorubicin- and cis-platinum-resistant human cell lines.
Anthracyclines are among the most effective and useful antic-
ancer agents developed, and they are used to treat more types of
cancer than any other chemotherapy agent.1,2 Their clinical
importance has stimulated wide research3ꢀ6 directed to the
development of new structurally related compounds with the
goal of bypassing significant problems that limit their utility, such
as their failure in resistant tumors expressing the ABCB1
(MDR1) gene7ꢀ9 and the emergence of severe short- and
long-term side effects associated with bone marrow and myo-
cardial cell toxicity.10,11 With this aim, our research group has
developed different series of quinone-based compounds contain-
ing the 3-amino-3-(ethoxycarbonyl)-2,3-dihydrothieno[2,3-b]-
naphtho-4,9-dione system (4, DTNQ) as chromophore
(Figure 1).12 The effected modifications on this template and
the analysis of the structureꢀactivity relationship (SAR) on the
different synthesized series showed that the incorporation of a
distal protonated alkylamine linked to the chromophore DTNQ
Received: January 27, 2011
Published: May 09, 2011
r
2011 American Chemical Society
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In addition, STD NMR spectroscopy investigation performed
on compounds 7 and 8 demonstrated that these derivatives
interact with DNA with a dual binding mode: intercalative for the
dihydrothieno[2,3-b]naphtho-4,9-dionetricyclic core and exter-
nal considering the side chain moiety.15,16 However, even though
these derivatives had many of the structural characteristics of
classical quinone-based DNA intercalating agents, they were not
able to inhibit topoisomerase II (topo II) at equicytotoxic
concentrations, indicating that other factors such as differences
in cellular uptake, distribution within the cell, and additional
targets within the cell might also affect the cytotoxicity of these
derivatives.17
planar chromophore. This quinone-based amine system showed
interesting cytotoxic activity toward the MCF-7 human breast
carcinoma (IC50 = 3.2 μM) and SW 620 human colon carcinoma
cell lines (IC50 = 4.0 μM), indicating its potential as a template in
the development of efficient cytotoxic agents. The new system
presents a more “planar core” compared to initial DTNQ
structure and an amine group able to be functionalized with
appropriate side chain in a defined orientation with respect to the
chromophore, thus guaranteeing two of the main structural
requisites for the antineoplastic activity of intercalating agents.
According to literature data, among heterocyclic quinones
endowed with cytotoxic activity, those containing a thiophene
nucleus fused to a quinone system have received little attention,
despite the antitumoral activity of thiophene analogues of
daunomycin and mitoxantrone described by the work groups
of Kita19 and Krapcho,20 respectively.
Now we have considered the possibility of using a new DTNQ
derivative, the 3-aminonaphtho[2,3-b]thiophene-4,9-dione (9,
TNQ) recently synthesized in our laboratories,18 as a more
Thus, we developed a series of 3-substituted aminonaphtho-
[2,3-b]thiophene-4,9-dione derivatives in which the amine group
of the planar chromophore (TNQ) was linked to several amino
acids (Gly, Ala, Phe, Lys, Pro, β-Ala), substituted alkylcarbonyl
chains (hydroxyacetyl, hydroxypropionyl, (N,N-diethyl)aminoacetyl,
(N,N-diethyl)aminopropionyl, 2-morpholinacetyl, 3-morpholin-
propionyl, (N0,N0-methyl)(N-aminoethyl)aminopropionyl, thioa-
cetyl, thiopropionyl), and carbamoyl chains (propyl, amino-
ethyl), which represent the side chain functionalities of the more
active compounds of the precedent series. The objectives of this
investigation are (a) validation of the TNQ system as template in
the development of new quinone-based antitumoral agents, thus
exploring new chemical spaces; (b) identification of the structural
parameters that are important for the cytotoxic activity, through a
comparative study of the structureꢀactivity relationships
(SARs) of TNQ derivatives; and (c) exploration of the basic
biochemical events correlated to cytotoxic activity of new
derivatives. The present paper deals with the preliminary studies
concerning the synthesis of novel TNQ derivatives, their
Figure 1. Structures of some DTNQ derivatives and the new TNQ
system.
Scheme 1a
a Reagents and conditions: (i) Boc-Aaa-OH, HBTU, HOBt, DIPEA in DMF, room temperature; (ii) DBU in MeOH/H2O, room temperature;
(iii) TFA/DCM, TES.
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Scheme 2a
a Reagents and conditions: (i) chloroacetyl chloride, TEA in THF; (ii) bromopropionyl chloride, TEA in THF; (iii) DBU in MeOH/H2O, room
temperature; (iv) nucleophilic reagents in THF, TEA, reflux temperature; (v) for 11i and 11n 20% TFA in dichloromethane; (vi) for 11j, 11k, 11m, 11o,
and 11p HCl (g)/diethyl ether solution.
cytotoxic activity, interaction with topo II and DNA, influence on
cell cycle progression, modulation of small heat shock proteins,
and cardiotoxicy effects.
11aꢀf were obtained as trifluoroacetate salts in 40ꢀ48% overall
yields.
Two homologue series of compounds containing a linear
substituted alkyl chain were synthesized from 3-(20-chloro)-
acetamide-3-ethoxycarbonyl-2,3-dihydrothieno[2,3-b]naphtho-
4,9-dione (12) and 3-(acrylamido)-3-ethoxycarbonyl-2,3-dihy-
drothieno[2,3-b]naphtho-4,9-dione (13), respectively, following
a similar methodology (Scheme 2).
Condensation of 4 with chloroacethyl chloride in THF, using
triethylamine as base, afforded the (20-chloro)acetamide deriva-
tive 12 with 92% yield. Under these conditions, the reaction of 4
with bromopropionyl chloride gave the 3-bromopropionamide
intermediate (90% yield), which partially evolved to β-elimina-
tion product 3-(acrylamido)-3-ethoxycarbonyl-2,3-dihydrothieno-
[2,3-b]naphtho-4,9-dione (13) during workup of reaction. De-
carboxylation performed on 12 and 13 intermediates gave directly
the 2-hydroxyacetamide (11g) and 3-hydroxypropyonamide
(11l) as final compounds, respectively. Nucleophilic displace-
ment of the chlorine atom (12) or Michael-type addiction to
acrylamido moiety (13) using diethylamine, triphenylmetha-
nethiol, morpholine, or N,N-diethylethylendiamine, in THF
and triethylamine at reflux, readily provided the corresponding
’ RESULT AND DISCUSSION
Chemistry. The synthetic approach to new 3-substituted
aminonaphtho[2,3-b]thiophene-4,9-dione derivatives was based
on the capacity of the DTNQ system and its 3-N-acyl derivatives
to undergo oxidative decarboxylation during the hydrolysis of
3-ethyl ester group under basic conditions, as we recently
described.18 Condensation of 3-amino-3-ethoxycarbonyl-2,3-
dihydrothieno[2,3-b]naphtho-4,9-dione (4, DTNQ) with differ-
ent Boc-amino acids (a = Gly, b= Ala, c = Phe, d = Lys, e = Pro,
f = β-Ala,) using HBTU, HOBt, and DIPEA in DMF afforded, with
high yields (50ꢀ65%), the appropriate pseudodipeptide intermedi-
ates 100aꢀf, as shown in Scheme 1. Treatment with DBU in
MeOH/H2O medium directly gave the corresponding decarboxy-
lated intermediates 110aꢀf in 76ꢀ82% yields. Finally, after removal
of the Boc protecting group using 20% TFA in dichloro-
methane and triethylsilane as scavenger, the final compounds
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Scheme 3a
a Reagents and conditions: (i) (Boc)2O; (ii) DBU in MeOH/H2O, room temperature; (iii) 50% TFA/dichloromethane; (iv) chloroacetyl chloride, TEA
in THF; (v) (N,N-dimethyl)ethylenediamine in THF, TEA, reflux temperature; (vi) triphosgene, TEA, THF, room temperature, 10 min, then R-NH2.
acetamide (120hꢀk) or propionamide (130mꢀp) analogues.
Basic hydrolysis of these derivatives afforded the corresponding
decarboxylated compounds (11hꢀj and 11mꢀp) except in the
case of 120k (R = HNCH2CH2N(CH3)2). In fact, under the cited
conditions, this intermediate gave the cyclic derivative 4-[(2-N,
N-dimethyl)amino]ethylspiro[(dihydropirazin-2,5-dione)-6,30-
(20,30-dihydrothieno[2,3-b]naphtho-40,90-dione)] (7) previously
described.15 Then final compounds presenting an amine func-
tionality 11j, 11k, 11m, 11o, and 11p were treated with a
solution of gaseous hydrochloric acid in diethyl ether to provide
corresponding hydrochloride salts. This was found to both aid
purification and provide an improved solubility profile for the
biological assays. The final thioacetamide 11i and thiopropyo-
namide 11n derivatives were obtained after S-Trt deprotection
using 20% TFA in dichloromethane in quantitative yields.
For the synthesis of compound 11k and the urea-based
derivatives 11q and 11r we chose an alternative route that
implied the use of 3-aminonaptho[2,3-b]thiophene-4.9-dione
(TNQ, 9) as starting material (Scheme 3). The condensation
of 9, obtained after deprotection of the corresponding N-Boc
TNQ (14) using 50% TFA in dichloromethane,18 with chlor-
oacethyl chloride afforded the (20-chloro)acetamide intermedi-
ate 15 (88% yield). Reaction of 15 with diethylamine in THF and
triethylamine at reflux afforded the final derivative 11k. Com-
pounds 11q and 11r were obtained by treatment of 9 with
triphosgene and TEA in THF followed by addiction of propy-
lamine or N,N-dimethylethylendiamine. Also in this case, the use
of 9 as starting material was necessary because the corresponding
N-carbamoyl derivatives of DTNQ (compounds 60) evolved
rapidly to spirohydantoin derivatives 6 under hydrolytic
conditions.14
Results in Table 1 confirmed compound 9 as a potential
scaffold for new antitumoral agents with a cytotoxic activity in the
micromolar range on the three cell lines used in the assay. The
improved antitumor activity and spectra of some of the newly
synthesized compounds, compared to 9, demonstrated that
chemical modification at C-3 was an effective approach to
optimize the activity profiles of TNQ moiety. The wide activity
range observed for compounds 11aꢀr (IC50 from 0.6 to >40
μM) indicated that the nature of substituents on the amine group
at the C-3 position markedly affects the activity profile of these
compounds. Incorporation through the 3-amino group of differ-
ent amino acids was well tolerated in the case of linear amino
acids such as glycine (11a). The presence of amino acids
containing alkyl (Ala, 11b) or benzyl (Phe, 11c) side chain,
relatively more rigid and more electron rich when compared to
unsubstituted side chain, led to significant loss of activity,
especially in the MDA231 cell line. This negative effect was
more noteworthy with the introduction of an alkylamino side
chain (Lys, 11d). The incorporation of Pro gave the derivative
11e, which turned out to be the most active in the leukemic cell
line (IC50 = 0.9 μM).
Other interesting results were obtained with the incorporation
of a primary or tertiary amine to the end of the ethyl side chains.
Compounds 11f, 11m, and 11p retained cytotoxic levels similar
to those of doxorubicin on the SW 620 cell line, with IC50 of 0.8,
1.5, and 0.6 μM, respectively, and maintained the activity on the
MDA231 and U937 cell lines within the micromolar range
(2.0ꢀ3.7 and 1.1ꢀ1.7 μM, respectively). These derivatives were
2- to 5-fold more potent than their methylene homologues (11a,
11h, and 11k, respectively) on all the cell lines. Congeners with a
hydroxyl (compounds 11g and 11l), thiol (compounds 11i and
11n), or morpholin (compounds 11j and 11o) groups were
remarkably less potent compared to their primary and tertiary
amine analogues.
In Vitro Cytotoxicity. TNQ derivatives were first examined
for antiproliferative activity against the MDA231 human breast
carcinoma, SW 620 human colon carcinoma, and U937 human
leukemic monocyte lymphoma cell lines, and the obtained IC50
values are summarized in Table 1. For comparative purposes, the
template 9 and doxorubicin were also included in the assay.
Finally, the incorporation of an alkyl or alkylamino side chain
through a ureide group led to a decrease of the activity in the
resultant analogues 11q and 11r, respectively. These results
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Table 1. Cytotoxic Activities of 3-(Amino)naphtho[2,3-b]thiophene-4,9-dione (9) and 3-[(Acyl)amino]naphtho[2,3-b]-
thiophene-4,9-dione Derivatives (11aꢀf)
IC50 ( SD a (μM)
topo II activitye
10 μM
compd
R
MDA231b
SW620c
U937d
5 μM
9
11.3 ( 0.4
6.2 ( 4.6
>40
4.0 ( 0.3
2.3 ( 0.4
12.4 ( 1.5
30.50 ( 6.4
>40
10.1 ( 0.4
7.0 ( 0.07
9.1 ( 0.2
f
11a
CH2NH2
f
11b
11c
CH(CH3)NH2
f
CH[CH2(C6H5)]NH2
>40
>40
þ
þ
f
11d
11e
CH[(CH2)4NH2]NH2
2-pyrrolidinyl f
>40
20 ( 0.01
0.9 ( 0.06
1.7 ( 0.01
15.1 ( 0.06
5.1 ( 0.07
30.1 ( 0.04
4.3 ( 0.02
4.0 ( 0.03
15 ( 0.06
1.1 ( 0.01
23.9 ( 0.34
7.2 ( 0.07
1.3 ( 0.03
10.1 ( 0.50
9.8 ( 0.20
0.93 ( 0.01
6.7 ( 2.5
3.7 ( 0.9
10.1 ( 0.2
8.5 ( 0.12
13.6 ( 0.15
7.1 ( 0.2
4.9 ( 0.4
9.2 ( 0.6
2.5 ( 0.1
15.2 ( 0.1
10.1 ( 1.3
2.0 ( 0.1
9.5 ( 0.52
8.7 ( 0.30
1.13 ( 0.01
5.4 ( 0.1
0.8 ( 0.27
18.5 ( 0.7
4.0 ( 0.14
20.9 ( 0.16
10.8 ( 0.12
2.1 ( 0.3
20.3 ( 0.8
1.5 ( 0.2
20.7 ( 0.8
20.1 ( 0.3
0.6 ( 0.08
6.5 ( 1.20
5.9 ( 0.20
0.12 ( 0.01
f
11f
CH2CH2NH2
11g
CH2OH
g
11h
11i
CH2N(CH2CH3)2
CH2SH
11j
CH2-morpholine g
g
11k
11l
CH2NH(CH2)2N(CH3)2
(CH2)2OH
g
11m
11n
11o
11p
11q
11r
(CH2)2N(CH2CH3)2
þ
þþþ
þþþ
0
(CH2)2SH
(CH2)2-morpholine g
g
(CH2)2NH(CH2)2N(CH3)2
þþ
g
NH(CH2)2CH3
g
NH(CH2)2N(CH3)2
doxorubicin
0
a Data represent mean values ((SD) for three independent determinations. b Human melanoma cell line. c Human colon carcinoma cell line. d Human
leukemic monocyte lymphoma cell line. e The semiquantitative evaluation of topo II mediated DNA relaxation activity was as follows: þþþ, high; þþ,
intermediate; þ, low; 0, absent. The rest of the compounds were not tested. f Evaluated as TFA salts. g Evaluated as HCl salts.
imply a minor tolerance to structural modifications in this series
compared to precedent series.
micromolar range against undifferentiated Caco-2 tumor colon cell
lines (IC50 = 0.8ꢀ1.0 μM) while being 4-fold less active (IC50
=
To further determine the antitumor spectra, the most potent
compounds 11f, 11m, and 11p were selected and screened against
a panel of human tumor cell lines, including MDA-MB435 and
SK-MEL 28 (melanoma), IGROV (ovarian), SF-295 and SNB-19
(glioblastoma), and Colo205, HT-29, and undifferentiated Caco-2
(colon). Differentiated Caco-2, a well accepted model of normal
cell line because of its ability to acquire the phenotype of mature
small-intestinal cell,21 was utilized to characterize a safety profile of
the compounds at least in terms of “cell selectivity”.22
As observed in Table 2, selected compounds were more potent
than doxorubicin on the melanoma, colon, and CNS human tumor
cell lines, with IC50 in the range 0.1ꢀ1.0 μM. Compounds 11m and
11p turned out to be the most active derivatives against SK-MEL 28
human melanoma cell line (IC50 = 0.6 and 0.3 μM, respectively)
and were equipotent to doxorubicin (IC50 = 0.4 μM). Analogous to
that observed in the previously described series,14ꢀ16 these com-
pounds showed a remarkable activity against tumor cell lines
generally highly resistant to treatment with doxorubicin. Com-
pounds 11m and 11p presented a cytotoxic activity in the
3.8ꢀ4.1 μM) on differentiated Caco-2 cell line. These data
indicated a good profile of cell selectivity for our derivatives
(selectivity index SI ≈ 0.22) especially if they are compared with
the high toxicity data obtained with doxorubicin (SI = 11.1).
Subcellular Distribution of TNQ Derivatives in MCF-7 Cell
Line. Distribution of the labeled forms of our derivatives within
the cell was investigated by confocal microscopy in MCF-7 cell
line, using 50 nM 11m (IC50= 0.5 μM) and 11p (IC50 = 0.6 μM).
As shown in Figure 2, these TNQ derivatives are clearly localized
in the nuclei, indicating a site of cytotoxic action similar to those
of classic quinone-based intercalators.23
Topoisomerase Inhibition. A number of quinone antitumor
drugs are thought to be cytotoxic by virtue of their ability to
stabilize a covalent topo IIꢀDNA intermediate, the cleavable
complex.24 Topo II is an essential enzyme that plays an important
role in DNA replication, repair, transcription, and chromosome
segregation.25 Topo II alters the topological state of nucleic acids
by passing an intact DNA helix through a transient break which
generates a separate DNA helix.26 We analyzed the possibility
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Table 2. Inhibition of Multiple Human Tumor Cell Lines by Selected Compound
IC50 ( SD a (μM)
origin of tumor
melanoma
cell line
11f
11m
11p
doxorubicin
MDA-MB435
SK-MEL 28
IGROV
0.4 ( 0.10
1.5 ( 0.08
1.2 ( 0.30
2.8 ( 0.20
1.6 ( 0.60
0.4 ( 0.04
0.6 ( 0.08
2.6 ( 0.2
0.5 ( 0.08
0.6 ( 0.08
2.5 ( 0.10
0.6 ( 0.06
0.7 ( 0.04
0.9 ( 0.05
0.8 ( 0.05
1.0 ( 0.6
0.5 ( 0.09
0.3 ( 0.07
2.0 ( 0.20
0.6 ( 0.09
0.9 ( 0.10
1.1 ( 0.05
0.5 ( 0.10
0.8 ( 0.03
3.8 ( 0.09
1.3 ( 0.21
0.6 ( 0.09
1.3 ( 0.30
4.4 ( 0.50
0.8 ( 0.05
1.5 ( 0.30
1.1 ( 0.20
6.7 ( 0.80
0.6 ( 0.05
ovarian
glioblastoma
SF-295
SNB-19
colon
Colo205
HT-29
Caco-2b
Caco-2c
6.1 ( 0.32
4.1 ( 0.10
SI d
11f
11m
11p
doxorubicin
11.1
0.43
0.25
0.21
a Data represent mean values (SD) for three independent determinations. b Preconfluent Caco-2 cell line. c Postconfluent Caco-2 cell line.
d SI = selectivity index = (IC50 on undifferentiated Caco-2 cell line)/(IC50 on differentiated Caco-2 cell line).
Figure 2. Distribution of labeled 11m and 11p in MCF-7 cells by confocal microscopy.
that compounds 11m and 11p could inhibit the activity of topo
II. The effect of cytotoxic compounds 11m and 11p and of the
inactive compound 11c on the strand passage activity of topo II
was determined by the enzyme-mediated negatively supercoiled
pBR322 relaxation.27
As indicated in Figure 3, compounds 11m and 11p displayed
significant inhibition of topo II mediated relaxation in a concen-
tration-dependent mode, while 11c does not inhibit this activity
at the concentrations tested. These results, shown also in Table 1
as semiquantitative form, parallel the cytotoxicity data enumer-
ated in the same table, thus suggesting a behavior similar to that
of classical intercalators. Moreover, at the assay concentrations,
the doxorubicin showed a lack of activity (see Supporting
Information) which agrees with the results described in different
studies.28 These studies show that doxorubicin inhibits topo II
only at 0.04ꢀ0.92 μM, while at higher concentration the
inhibition is either diminished or totally abolished.
DNA Binding Properties by NMR. Representative com-
pounds 11c, 11m, and 11p were tested to see if they interact
with DNA, using both saturation transfer difference (STD)29 and
waterꢀligand observed via gradient spectroscopy (WaterLOGSY)
NMR techniques.30 STD NMR and WaterLOGSY are techni-
ques that can be used to characterize and identify binding. These
techniques have become increasingly important as tools in the
investigation of biomolecular recognition phenomena.31 In the
STD NMR, resonances of the macromolecule are selectively
saturated, and in a binding ligand, enhancements are observed in
the difference (STD NMR) spectrum resulting from subtraction
of this spectrum from a reference spectrum in which the
macromolecule is not saturated. All the proton resonances of
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11m and 11p were observed in the STD spectra acquired in the
that 11c does not interact with DNA. The same results were
obtained using the WaterLOGSY experiment. In this experiment,
the large bulk water magnetization is partially transferred via the
macromoleculeꢀligand complex to the free ligand. Because of
the very different tumbling times of the free ligand and of the
macromoleculeꢀligand complex, LOGSY signals are typically
negative for free ligands in solution and relatively less negative or
positive for binders in the presence of the macromolecule.
Figure 5 shows the WaterLOGSY spectra of 11c, 11m, and
presence of poly(dG-dC) poly(dG-dC) copolymer as the DNA
3
target (Figure 4), demonstrating that 11m/DNA and 11p/DNA
interactions did occur. In contrast, the absence of the proton
resonances of 11c in its STD spectra (Figure 4) demonstrates
11p with and without the poly(dG-dC) poly(dG-dC) copoly-
3
mer. As observed, 11m and 11p signals became positive in the
presence of DNA while 11c signals remain negative demonstrat-
ing that 11m and 11p but not 11c interact with the DNA
polymer.
Furthermore, we applied the so-called DF-STD (differential
frequency STD) spectroscopy,32 to study the binding modes of
11m and 11p with the DNA. The method allows the discrimina-
tion of base-pair intercalators and minor-groove and external
binders. The approach is based on the comparison of two parallel
sets of STD experiments performed under the same experimental
Figure 3. Effects of compounds 11p, 11m, and 11c on the topo II
mediated DNA cleavage. Supercoiled plasmid pBR 322 (0.5 pmol) was
incubated with 1 unit of purified human topo II in the presence or
absence of the tested agents: (lane 1), supercoiled DNA; (lane 2),
relaxed DNA enzyme control; (lanes 3 and 4) 5 and 10 μM 11p; (lanes 5
and 6) 5 and 10 μM 11m; (lanes 7 and 8) 5 and 10 μM 11c.
Figure 4. 1D proton spectra (a, d, g) and the corresponding STD NMR spectra recorded upon saturation at 10 ppm (b, e, h) and ꢀ1 ppm (c, f, i) of
11c/DNA, 11m/DNA, and 11p/DNA complexes, respectively. The STD NMR spectra were plotted with the same noise level.
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Figure 5. Water LOGSY spectra of 11c, 11m, and 11p in the absence (b, d, e) or in the presence (a, c, e) of poly(dG-dC) poly(dG-dC) copolymer. The
3
asterisk (/) indicates the DMSO residual signal.
conditions, in which saturation is centered either in the aromatic
or in the low-field aliphatic spectral regions.
A ligand making proximate contacts with aromatic base
protons, such as an intercalator sandwiched by consecutive base
pairs, would receive more saturation upon irradiation of DNA
aromatic protons rather than irradiation of deoxyribose protons.
The converse would be true for an external ligand. The “binding
mode index” (BMI), a numerical parameter that expresses the
relative sensitivity of ligand protons to the perturbation arising
from base versus sugar/backbone saturation was used.32 Three
BMI ranges were defined in the original contribution:32 0 < BMI <
0.50 for external (nonspecific) electrostatic backbone binding;
Figure 6. Effects of 11m, 11p, and 11c on the distribution of Caco-2 cell
0.90 < BMI < 1.10 for minor-groove binding; 1.20 (0.90) < BMI <
1.50 for base-pair intercalation. DF-STD analysis of compound
11m gave different BMI values: BMI = 0.86, for the aliphatic
populations data represent the percentage of cells in each phase of cell
cycle. For 11m: G1, 29%; G2/M, 28%; S, 41%. For 11p: G1, 20%; G2/
M, 26%, S, 53%. For 11c: G1, 37%; G2/M, 18%, S, 44%.
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signals and BMI = 1.35 for the aromatic signals. This result can be
explained assuming two different DNA binding modes for 11m.
An intercalative mode of binding is sustained by its tricyclic
planar core, and an external backbone binding can be attributed
to its side chain. This is similar to that observed for doxorubicin32
and for compounds 7 and 8 in our previous work.15,16 Consider-
ing 11p, BMI = 1.01 was measured for the aromatic protons and
BMI = 0.90 for the aliphatics. These BMI values are compatible
with both intercalative and minor-groove binders.
Cell Cycle Effects. To investigate the cytotoxic effects of these
derivatives in more detail, we examined the effects on cell cycle
progression in CaCo-2 cell line. The percentage of these cells in
G1, S, and G2/M phases was analyzed after 48 h of treatment with
1 μM 11m, 11p, and 11c (Figure 6). Under these conditions, the
control cells were in phase G1 (42%), G2/M (21%), and S (36%).
The treatment with 11p resulted in a significant accumulation of
cells in the S phase, while concomitantly the G1 populations
decreased. About 53% of the CaCo-2 cells treated with this
compound were arrested at the S phase. Under the same condi-
tions, the treatment with 11m induces a weak increase of cell in
both G2 and S phases while the treatment with 11c does not
modify significantly the distribution of Caco-2 cell populations
Accordingly, treatment of Caco-2 cells with 1 μM of our
derivatives for 48 h induced an increase of cyclin A expression33
only in the case of 11p (43%; see Supporting Information),
indicating that the cell cycle progression of cells in the S phase
was prompted. The expression of cyclin A was not upset in
treated Caco-2 cells with 11m and 11c.
Since cell division arrest is one of the prerequisites for cell
differentiation,34 we determined the effect of our molecules on
Caco-2 differentiation. In Figure 7 we report alkaline phospha-
tase (ALP) activity, a marker of enterocytic differentiation
correlated to the postconfluent phase.35
Figure 7. Differentiation of Caco-2 cells assessed by measurement of
alkaline phosphatase activity after 48 h of culture in the presence of 0, 1,
5, and 10 μM 11p, 11m, and 11c.
Treatment of preconfluent Caco-2 with 1 μM 11p increased
ALP activity of 35% (p < 0.005). A more significant increase of
the differentiation, with ALP augment of >180%, was only
obtained by treatment of Caco-2 cells with 5 μM 11p or 10
μM 11m for 48 h. All these preliminary results suggested that for
this series the cell growth inhibition was not related to cell cycle
perturbation.
Modulation of Heat Shock Protein (Hsp) Expression. Small
heat shock proteins are involved in a variety of cellular processes
including cell growth and differentiation.36 We previously re-
ported the ability of a DTNQ analogue, compound 8, to
modulate the heat shock protein expression on Caco-2 cells.17
In order to evaluate the behavior of the new synthesized
derivatives, we carried out a preliminary study on the effect of
11m and 11p at 1 and 5 μM in Hsp27 expression in Caco-2 cells
for 48 h. Hsp27 is weakly expressed in Caco-2 (Figure 8), and
Figure 8. Effects of Caco-2 cells treatment with 1 and 5 μM 11p and
11m on Hsp27 expression.
Figure 9. Results of cell viability assay of 11m, 11p, and doxorubicin on H9C2 cells at 1 μM.
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treatment of this cell line with 11p led to a significant dose-
dependent increase of its expression. 11m produced a weak
enhancement of Hsp27 expression at 1 μM, which was not
observed at 5 μM.
F254, and preparative TLC was performed on 20 cm ꢁ 20 cm glass
plates coated with a 0.5 mm layer of Merck silica gel PF254. Flash and
gravity chromatographic purification was performed using 230ꢀ400
mesh silica gel unless otherwise noted. Melting points were taken on a
Kofler apparatus and are uncorrected. 1H NMR and 13C NMR spectra
were recorded with a Varian 400 spectrometer, operating at 400 and 100
MHz, respectively. Chemical shifts are reported in δ values (ppm)
relative to internal Me4Si, and J values are reported in hertz (Hz). ESI-
MS experiments were performed on an Applied Biosystem API 2000
triple-quadrupole spectrometer. Combustion microanalyses were per-
formed on a Carlo Erba CNH 1106 analyzer, and all reported values are
within 0.4% of calculated values. These elemental analysis results
confirmed g95% purity.
Cardiomyocyte Cell Viability. It is well-known that the
clinical use of anthracyclines, specially doxorubicin, in the treat-
ment of many neoplastic diseases is limited by cumulative
cardiotoxicty.37 One cause of this effect has been attributed to
the redox process involving the quinone system which results in
the formation of reactive oxygen species and ultimately in
myocyte death. In order to evaluate the potential toxicity of
our quinone ring, we examined the cell viability in cardiac derived
H9C2 myocytes exposed to 1 μM 11m, 11p, and doxorubicin for
24, 48, 72, 96, and 120 h. Previous studies reported in the
literature used this cell line as a model system to evaluate the
cardiotoxicity caused by doxorubicin.38
As shown in Figure 9, treatment with doxorubicin induced
cardiotoxicity in a time-dependent manner37 while compounds
11m and 11p maintained good cell viability after 120 h (74% and
76%, respectively). The possible correlation between the reduced
cardiotoxicity and the Hsp27 modulation39 will be the subject of
more in-depth studies.
General Procedure for the Synthesis of 3-[(Acyl)amino]-
naphtho[2,3-b]thiophene-4,9-dione Trifluoroacetate Salts
(11aꢀf). The 3-amino-3-ethoxycarbonyl-2,3-dihydrothieno[2,3-b]-
naphtho-4,9-dione system (DTNQ) (1), the 3-(N-tert-butyloxyamino-
acyl)amino-3-ethoxycarbonyl-2,3-dihydrothieno[2,3-b]naphtho-4,9-
dione (100aꢀf), and the 3-(N-tert-butyloxyaminoacyl)aminonaphtho-
[2,3-b]thiophene-4,9-dione (110aꢀf) derivatives were synthesized
according to the refs 12, 13, and 17, respectively. Then TFA was added
to a solution of decarboxylated Boc-protected derivatives (110aꢀf) (0.1
mmol) in DCM (10 mL), using triethylsilane as scavenger. Stirring was
continued for 3ꢀ4 h at room temperature. The reaction mixture was
concentrated to half volume, and ether was added. The title compounds
as the trifluoroacetate salt were collected by filtration as yellow solids.
3-[(Glycyl)amino]naphtho[2,3-b]thiophene-4,9-dione Tri-
’ CONCLUSIONS
We report the synthesis and biological evaluation of a series of
quinone-based derivatives, designed as conjugated structures
linking a planar naphtho[2,3-b]thiophenedione core with differ-
ent acyl-substituted groups. Among the designed molecules,
compounds containing a 3-(diethylamino)propanamide (11m)
or 3-(2-(dimethylamino)ethylamino)propanamide (11p) proto-
natable side chain showed a greater cytotoxic potency than
doxorubicin against cell lines that were highly resistant to
treatment with this drug, such as the melanoma (MDA-
MB435), glioblastoma (SF-295), and colon (SW 620, Col205,
and HT-29) human tumor cell lines,
Preliminary results about the mechanism of action indicate that
these derivatives had a significant effect on topoisomerase II activity
targeting the nuclear DNA, which is generally considered as an
attractive target for anticancer therapy. The NMR results suggested
that DNA interactions do occur for highly active compounds 11m
and 11p but not for inactive compound 11c. Experimental data
indicate that 11m and 11p intercalate the DNA through their
aromatic portion. Furthermore, a nonintercalative mode of binding
to DNA can also hold for 11p. These data revealed significant
similarities in the cytotoxic behavior and the site of action of these
compounds compared to classical intercalators. However, the
compounds tested showed a minor influence on the regulation of
the cell cycle and only derivative 11p prolonged the S phase of the
Caco-2 cell cycle inducing both delay of cell cycle progression in
responsive cells and moderate cellular differentiation. This latter
compound also showed a high ability to increase Hsp27 expression.
Finally, the compounds under study affect the viability of H9C2
cells after chronic treatment to a lesser extent than does doxor-
ubicin. Further development and more in-depth studies on the
mechanism of action of this series are in progress.
1
fluoroacetate (11a). Yield: 45%. Mp 207ꢀ208 ꢀC. H NMR (400
MHz, CD3OD) δ: 4.10 (s, 2H, CH2); 7.85ꢀ7.87 (m, 2H, H-6 and H-7);
8.20ꢀ8.23 (m, 2H, H-5 and H-8); 8.50 (s, 1H, H-2). 13C NMR (100
MHz, CD3OD) δ: 45.7(CH2), 119.2 (C-2), 127.5 (C-6 and C-7), 129.4
(C-3), 132.9 (C-8a), 134.2 (C-4a), 134.7 (C-5 and C-8), 135.9 (C-3a),
147.1 (C-9a), 171.8, 180.7, and 182.7 (CdO). ESI-MS m/z calcd for
C16H11F3N2O5S, 400.03; found, 400.11.
3-[(L-Alanyl)amino]naphtho[2,3-b]thiophene-4,9-dione
1
Trifluoroacetate (11b). Yield: 45%. Mp 206 ꢀC. H NMR (400
MHz, CD3OD) δ: 1.69 (d, 3H, J = 7.2 Hz, CH3); 4.38ꢀ4.41 (q, 1H,
CH); 7.87ꢀ7.89 (m, 2H, H-6 and H-7); 8.22ꢀ8.28 (m, 2H, H-5 and
H-8); 8.49 (s, 1H, H-2). 13C NMR (100 MHz, CD3OD) δ: 21.7 (CH3),
50.1 (RCH), 118.8 (C-2), 126.6 (C-6 and C-7), 129.8 (C-3), 133.1 (C-
8a), 133.9 (C-4a), 134.6 (C-5 and C-8), 135.1 (C-3a), 147.2 (C-9a),
172.1, 180.9, and 182.5 (CdO). ESI-MS m/z calcd for
C17H13F3N2O5S, 414.05; found, 414.12.
3-[(L-Phenylyl)amino]naphtho[2,3-b]thiophene-4,9-dione
Trifluoroacetate (11c). Yield: 41%. Mp 195ꢀ196 ꢀC. 1H NMR (400
MHz, CD3OD) δ 2.96ꢀ3.07 (2 H, m, βCH2); 4.44ꢀ4.47 (1 H, m,
RCH); 7.12ꢀ7.22 (5 H, m, aryl); 7.87ꢀ7.89 (2 H, m, H-6 and H 7);
8.22ꢀ8.25 (2 H, m, H-5 and H 8); 8.47 (1H, s, H-2). 13C NMR (100
MHz, CD3OD) δ: 37.9 (βCH2), 50.6 (RCH), 118.8 (C-2), 127.9 (C-6
and C-7), 125.9, 127.6, 128.3, 128.9, and 137.9 (aryl), 131.4 (C-3), 133.6
(C-8a), 134.2 (C-4a), 134.9 (C-5 and C-8), 139.0 (C-3a), 142.5 (C-9a),
172.7, 179.8, and 181.9 (CdO). ESI-MS m/z calcd for C23H17-
F3N2O5S, 490.08; found, 490.01.
3-[(L-Lysyl)amino]naphtho[2,3-b]thiophene-4,9-dione
Bistrifluoroacetate (11d). Yield: 42%. Mp 241ꢀ242 ꢀC. 1H NMR
(400 MHz, D2O) δ: 1.13ꢀ1.19 (m, 2H, γCH2); 1.37ꢀ1.40 (m, 2H,
δCH2); 1.59ꢀ1.61 (m, 2H, βCH2); 2.83ꢀ2.88 (m, 2H, εCH2);
3.56ꢀ3.58 (m, 1H, RCH); 7.52ꢀ7.55 (m, 2H, H-6 and H-7);
7.60ꢀ7.70 (m, 2H, H-5 and H-8); 7.94 (s, 1H, H-2). 13C NMR (100
MHz, CD3OD) δ: 22.7 (γCH2), 31.5 (δCH2), 36.4 (βCH2), 45.7
(εCH2), 53.1 (RCH), 119.8 (C-2), 126.9 and 127.3 (C-6 and C-7),
128.9 (C-3), 132.2 (C-8a), 133.5 (C-4a), 134.5 (C-5 and C-8), 135.6
(C-3a), 146.2 (C-9a), 172.6, 181.7, and 182.2 (CdO). ESI-MS m/z
calcd for C22H21F6N3O7S, 585.10; found, 585.21.
’ EXPERIMENTAL SECTION
General. Reagents, starting materials, and solvents were purchased
from commercial suppliers and used as received. Analytical TLC was
performed on plates coated with a 0.25 mm layer of Merck silica gel 60
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3-[(L-Prolyl)amino]naphtho[2,3-b]thiophene-4,9-dione
Trifluoroacetate (11e). Yield: 40%. Mp 197ꢀ198 ꢀC. 1H NMR (400
MHz, CD3OD) δ: 2.17ꢀ2.19 (m, 2H, γCH2); 2.25ꢀ2.29 (m, 1H,
βCH2); 2.63ꢀ2.66 (m, 1H, βCH2); 3.45ꢀ3.50 (m, 2H, δCH2);
4.73ꢀ4.75 (m, 1H, RCH2); 7.87ꢀ7.88 (m, 2H, H-6 and H-7);
8.21ꢀ8.24 (m, 2H, H-5 and H-8); 8.46 (s, 1H, H-2). 13C NMR (100
MHz, CD3OD) δ: 23.9 (γCH2), 29.5 (βCH2), 46.2 (δCH2), 60.7
(RCH), 120.4 (C-2), 126.8 and 127.0 (C-6 and C-7), 129.5 (C-3), 132.9
(C-8a), 133.4 (C-4a), 134.2 and 134.4 (C-5 and C-8), 135.8 (C-3a),
147.6 (C-9a), 171.9, 181.2, and 182.8 (CdO). ESI-MS m/z calcd for
C19H15F3N2O5S, 440.07; found, 440.13.
3-[(β-Alanyl)amino]naphtho[2,3-b]thiophene-4,9-dione
Trifluoroacetate (11f). Yield: 48%. Mp 205ꢀ207 ꢀC. 1H NMR (400
MHz, CD3OD) δ: 2.77ꢀ2.80 (t, 2H, RCH2); 3.06ꢀ3.11 (t, 2H,
βCH2); 7.88ꢀ7.91 (m, 2H, H-6 and H-7); 8.15ꢀ8.19 (m, 2H, H-5
and H-8); 8.42 (s, 1H, H-2). 13C NMR (100 MHz, CD3OD) δ: 37.4
(RCH2), 40.1 (CH2NH2), 119.9 (C-2), 126.9 (C-6 and C-7), 128.9 (C-
3), 131.9 (C-8a), 134.2 (C-4a), 134.5(C-5 and C-8), 139.0 (C-3a), 144.1
(C-9a), 170.9, 178.7, and 181.2 (CdO). ESI-MS m/z calcd for
C17H13F3N2O5S, 414.05; found, 414.06.
N-(4,9-Dioxo-4,9-dihydronaphtho[2,3-b]thiophen-3-yl)-
2-hydroxyacetamide (11g). DBU (0.6 mmol) was added dropwise
to a solution of 3-(20-chloro)acetamide-3-ethoxycarbonyl-2,3-dihydro-
thieno[2,3-b]naphtho-4,9-dione (12)15 (0.2 mmol) in methanolꢀwater
(9:1, 10 mL). After 30 min at room temperature the solvents were
evaporated and the reaction residues were dissolved in chloroform and
washed with water and dried with Na2SO4. The title compound was
purified by flash chromatography (FC) using ethyl acetate as eluent.
Yellow solid (43%). Mp 181ꢀ183 ꢀC. 1H NMR (400 MHz, CDCl3) δ:
4.12 (s, 2H, CH2); 7.79ꢀ7.81 (m, 2H, H-6 and H-7); 8.24ꢀ8.27 (m,
2H, H-5 and H-8); 8.50 (s, 1H, H-2); 11.23 (s, 1H, NH). 13C NMR (100
MHz, CD3OD) δ: 65.2(CH2), 119.9 (C-2), 126.7 (C-6 and C-7), 129.6
(C-3), 132.6 (C-8a), 134.0 (C-4a), 134.3 (C-5 and C-8), 136.8 (C-3a),
146.2 (C-9a), 171.9, 181.5, and 183.0 (CdO). ESI-MS m/z calcd for
C14H9NO4S, 287.03; found, 287.19.
General Procedure for the Synthesis of N-(4,9-Dioxo-4,9-
dihydronaphtho[2,3-b]thiophen-3-yl)-2-(substituted)aceta-
mide (11hꢀj). To a solution of 12 (0.3ꢀ0.4 mmol) in THF (20 mL)
were added the corresponding nucleophilic agents: N,N-diethylamine or
triphenylmethanethiol or morpholine (3.0 equiv) and DIPEA (2 equiv).
After the mixture was stirred at reflux temperature for 1ꢀ3 h, the solvent
was evaporated. Then the residues (120hꢀj) were dissolved in metha-
nolꢀwater (9:1, 20 mL) and DBU (0.9ꢀ1.2 equiv) was added dropwise
to these solutions. The reaction mixtures were stirred for 0.5ꢀ1.5 h.
Then the solvents were evaporated and the reaction residues were
dissolved in chloroform and washed with water and dried with Na2SO4.
The corresponding free bases of compounds 11h and 11i were first
purified by FC using DCM/methanol 9/1 as the eluent system. Then the
treatment with a HCl (g)/diethyl ether solution gave the final com-
pounds as hydrochloride salts. Compound 11j was obtained after Trt
removal using a 50% TFA/DCM solution. All compounds were
obtained as yellow solids.
Mp 213-214 ꢀC. 1H NMR (400 MHz, CD3OD) δ: 3.29ꢀ3.32 (t, 4H,
CH2N); 4.15ꢀ4.18 (t, 4H, CH2O); 4.39 (s, 2H, CH2,); 7.86ꢀ7.89 (m,
2H, H-6 and H-7); 8.22ꢀ8.26 (m, 2H, H-5 and H-8); 8.49 (s, 1H, H-2).
13C NMR (100 MHz, CD3OD) δ: 48.5 (CH2N), 50.9 (RCH2), 63.2
(CH2O), 118.7 (C-2), 127.1 (C-6 and C-7), 129.7 (C-3), 132.9 (C-8a),
133.7 (C-4a), 135.5 (C-5 and C-8), 138.1 (C-3a), 143.6 (C-9a), 172.6,
178.7, and 181.6 (CdO). ESI-MS m/z calcd for C18H17ClN2O4S,
392.06; found, 392.15.
N-(4,9-Dioxo-4,9-dihydronaphtho[2,3-b]thiophen-3-yl)-
2-mercaptoacetamide (11j). FC: n-hexane/ethyl acetate 1/1.
1
Yield: 43%. Mp 183ꢀ184 ꢀC. H NMR (400 MHz, CDCl3) δ: 3.50
(s, 2H, CH2); 7.79ꢀ7.81(m, 2H, H-6 and H-7); 8.25ꢀ8.27 (m, 2H, H-5
and H-8); 8.50 (s, 1H, H-2); 11.21 (s, 1H, NH). 13C NMR (100 MHz,
CDCl3) δ: 29.2 (CH2), 119.3 (C-2), 127.5 (C-6 and C-7), 129.7 (C-3),
133.7 (C-8a), 134.1 (C-4a), 134.3 and 134.3 (C-5 and C-8), 137.9 (C-
3a), 145.3 (C-9a), 170.5, 180.1, and 182.3 (CdO). ESI-MS m/z calcd
for C14H9NO3S2, 303.00; found, 303.26.
2-(2-(Dimethylamino)ethylamino)-N-(4,9-dioxo-4,9-dihydro-
naphtho[2,3-b]thiophen-3-yl)acetamide Dihydrochloride Salt -
(11k). The 3-aminonaphtho[2,3-b]thiophene-4,9-dione (TNQ, 9,1 g,
4.4 mmol)17 was dissolved in THF (30 mL). Chloroacetyl chloride (0.6
g, 5.3 mmol) and TEA (2.2 equiv) were added to the solution. The
mixture was stirred at room temperature for 1 h, after which chloroform
and water were added. The organic layer was washed with water and
dried. Concentration of the chloroform phase gave the crude product
2-chloro-N-(4,9-dioxo-4,9-dihydronaphtho[2,3-b]thiophen-3-yl)aceta-
mide as an oil (14, 1.2 g, 89%). 1H NMR (400 MHz, CDCl3) δ 3.82 (s,
2H, CH2,); 7.81ꢀ7.851 (m, 2H, H-6 and H-7); 8.26ꢀ8.29 (m, 2H, H-5
and H-8); 8.41 (s, 1H, H-2); 11.23 (s, 1H, NH). This was used directly in
the next step without further purification. 14 (150 mg, 0.5 mmol) was
dissolved in THF (10 mL), and the mixture was added to a solution of N,
N-dimethylethylendiamine (mL, mmol) in THF (10 mL). After the
mixture was stirred at reflux temperature for 6 h, the solvent was
evaporated and the residue was purified by flash chromatography with
DCM/MeOH 9/1. Then treatment with a HCl (g)/diethyl ether
solution gave the title compound as a yellow solid (140 mg, 65%).
Mp 212ꢀ213 ꢀC. 1H NMR (400 MHz, CD3OD) δ: 2.76 (s, 6H, CH3);
3.02ꢀ3.05 (m, 2H, CH2); 3.19ꢀ3.21 (m, 2H, CH2); 3.41 (sbr, 2H,
CH2), 7.84ꢀ7.86 (m, 2H, H-6 and H-7); 8.18ꢀ8.23 (m, 2H, H-5 and
H-8); 8.38 (s, 1H, H-2). 13C NMR (100 MHz, CD3OD) δ: 42.0 (CH2),
42.8 (CH3), 45.7 (CH2), 53.1 (CH2), 118.8 (C-2), 126.9 (C-6 and C-7),
128.9 (C-3), 133.0 (C-8a), 133.9 (C-4a), 134.2 (C-5 and C-8), 137.5
(C-3a), 144.0 (C-9a), 171.9, 179.1, and 182.3 (CdO). ESI-MS m/z
calcd for C18H21Cl2N3O3S, 429.07; found, 429.18.
3-(30-Bromo)propanamide-3-ethoxycarbonyl-2,3-dihydro-
thieno[2,3-b]naphtho-4,9-dione and 3-Acrylamido-3-ethoxy-
carbonyl-2,3-dihydrothieno[2,3-b]naphtho-4,9-dione (13).
DTNQ (3.3 mmol) was dissolved in THF. Bromopropanoyl chloride
(4 mmol) and TEA (8.2 equiv) were added to the solution. The mixture
was stirred at room temperature for 2 h, after which chloroform and
water were added. The organic layer was washed with water and dried.
Concentration of the chloroform phase gave the crude product, which
was purified by flash chromatography with DCM/MeOH 9.5/0.5. The
2-(N0,N0-Diethylamino)-N-(4,9-dioxo-4,9-dihydronaphtho-
[2,3-b]thiophen-3-yl)acetamide Hydrochloride (11h). Yield:
43%. Mp 209ꢀ210 ꢀC. 1H NMR (400 MHz, CD3OD) δ: 1.37ꢀ1.40 (t,
6H, CH3); 3.31ꢀ3.34 (q, 4H, CH2); 4.38 (s, 2H, CH2,); 7.85ꢀ7.87 (m,
2H, H-6 and H-7); 8.21ꢀ8.26 (m, 2H, H-5 and H-8); 8.52 (s, 1H, H-2).
13C NMR (100 MHz, CD3OD) δ 15.8 (CH3), 47.9 (CH2), 51.5
(RCH2), 119.2 (C-2), 126.4 (C-6 and C-7), 129.5 (C-3), 132.9 (C-
8a), 133.9 (C-4a), 135.1 (C-5 and C-8), 137.9 (C-3a), 144.9 (C-9a),
172.8, 179.9, and 181.9 (CdO). ESI-MS m/z calcd for
C18H19ClN2O3S, 378.08; found, 377.98.
1
title compounds in 1:1 ratio were obtained as a yellow oil (89%). H
NMR (400 MHz, CDCl3) δ: 1.23ꢀ1.25 (m, 6H, CH3); 2.71 (d, 1H, J=
6.5 Hz, CH2); 2.82ꢀ2.85 (m, 1H, CH2); 3.69ꢀ3.63 (m, 6H, CH2Br and
H-2); 4.21ꢀ4.30 (m, 4H, CH2); 5.91 (d, 1H, J = 16.0 Hz, CHdCH2);
5.68 (d, 1H, J = 9.9 Hz, CHdCH2); 6.23 (dd, 1H, J = 16.2 and 10.0 Hz);
7.40 (s, 2H, NH); 7.67ꢀ7.71 (m, 4H, H-6 and H-7); 7.98 (d, 2H, J = 6.8
Hz, H-5); 8.07 (d, 2H, J = 7.6 Hz, H-8).
N-(4,9-Dioxo-4,9-dihydronaphtho[2,3-b]thiophen-3-yl)-
3-hydroxypropionamide (11l). DBU (0.6 mmol) was added
dropwise to a solution of 13(0.1 mmol) in methanolꢀwater (9:1,
10 mL). After 30 min at room temperature the solvents were evaporated
N-(4,9-Dioxo-4,9-dihydronaphtho[2,3-b]thiophen-3-yl)-2-
(N0-morpholine)acetamide Hydrochloride (11i). Yield: 43%.
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Journal of Medicinal Chemistry
ARTICLE
and the reaction residues were dissolved in chloroform and washed with
water and dried with Na2SO4. The title compound was purified by flash
chromatography (FC) using ethyl acetate as eluent. Yellow solid (56%).
Mp 152ꢀ153 ꢀC. 1H NMR (400 MHz, CDCl3) δ: 2.74ꢀ2.77 (m, 2H,
CH2); 3.77ꢀ3.79 (m, 2H, CH2); 7.76ꢀ7.79 (m, 2H, H-6 and H-7);
8.23ꢀ8.24 (m, 2H, H-5 and H-8); 8.52 (s, 1H, H-2); 10.75 (s, 1H, NH).
13C NMR (100 MHz, CD3OD) δ: 38.1 (R-CH2), 68.3 (CH2OH), 119.4
(C-2), 127.4 (C-6 and C-7), 128.9 (C-3), 132.7 (C-8a), 134.1 (C-4a),
134.2 and 134.3 (C-5 and C-8), 138.5 (C-3a), 143.2 (C-9a), 171.2,
179.1, and 181.3 (CdO). ESI-MS m/z calcd for C15H11NO4S, 301.04;
found, 301.22.
General Procedure for the Synthesis of N-(4,9-Dioxo-
4,9-dihydronaphtho[2,3-b]thiophen-3-yl)-3-(substituted)
propanamide (11mꢀp). To a solution of 13 (0.1ꢀ0.3 mmol) in
THF (20 mL) were added N,N-diethylamine or triphenylmethanethiol
or morpholine or N,N-dimethylethylendiamine (1.1 equiv) and DIPEA
(2 equiv). After the mixture was stirred at reflux temperature for 12ꢀ24
h, the solvent was evaporated. Then the residues (130mꢀp) were
dissolved in methanolꢀwater (9:1, 20 mL) and DBU (5 equiv) was
added dropwise to these solutions. The reaction mixtures were stirred
for 0.5ꢀ1 h. Then the solvents were evaporated and the reaction
residues were dissolved in chloroform and washed with water and dried
with Na2SO4. The corresponding free bases of compounds 11m, 11n,
and 11p were first purified by FC using DCM/methanol 9/1 as the
eluent system. Then treatment with a HCl (g)/diethyl ether solution
gave the final compounds as hydrochloride salts and yellow solids.
Compound protected 11o was purified by FC using n-hexane/ethyl
acetate 3/2 as eluent. Then the final compound was obtained after Trt
removal with a 50% TFA/DCM solution.
(m, 2H, CH2N(Me)2); 3.47ꢀ3.50(m, 2H, βCH2); 3.50ꢀ3.53 (m, 2H,
NHCH2); 7.85ꢀ7.87 (m, 2H, H-6 and H-7); 8.20ꢀ8.25 (m, 2H, H-5
and H-8); 8.49 (s, 1H, H-2). 13C NMR (100 MHz, CD3OD) δ: 32.1
(RCH2), 42.4 (NHCH2), 42.8 (CH3), 43.9 (βCH2), 53.1
(CH2N(Me)2), 119.5 (C-2), 126.8 and 127.0 (C-6 and C-7), 129.0
(C-3), 132.9 (C-8a), 133.5 (C-4a), 134.2 and 134.3 (C-5 and C-8),
137.5 (C-3a), 144.0 (C-9a), 171.9, 178.9, and 182.5 (CdO). ESI-MS
m/z calcd for C19H23Cl2N3O3S, 443.08; found, 443.18.
General Procedure for the Synthesis of 1-(4,9-Dioxo-4,9-
dihydronaphtho[2,3-b]thiophen-3-yl)-3-(substituted)urea
(11q,r). Bis(trichloromethyl)carbonate (118 mg, 0.4 mmol) was added
to a solution of TNQ (9, 230 mg, 1 mmol) in dry THF (25 mL) at room
temperature. Then TEA (0.3 mL, 2 mmol) was added dropwise for 5
min, with stirring continuing for an additional 10 min. Afterward, a
solution of corresponding amines, propylamine, or N,N-dimethylami-
noethylamine (1.1 equiv) in dry THF (5 mL) and TEA (0.3 mL, 2
mmol) was added, and the mixture was stirred for 1 h. Then the reaction
mixture was diluted with chloroform, washed with water, dried over
Na2SO4, and evaporated to dryness. Flash chromatography of the
residues using n-hexane/ethyl acetate 4/1 or DCM/MeOH 9/1 as
eluents yielded, in each case, the correspondent urea derivatives as
yellow solids.
The final compound 11r was obtained as the hydrochloride salt by
treatment of the corresponding free bases with a HCl (g)/diethyl ether
solution.
1-(4,9-Dioxo-4,9-dihydronaphtho[2,3-b]thiophen-3-yl)-
1
3-propylurea (11q). Yield: 75%. Mp 198ꢀ199 ꢀC. H NMR (400
MHz, CDCl3) δ: 0.98ꢀ1.02 (m, 3H, CH3); 1.60ꢀ1.64 (m, 2H, CH2);
3.27ꢀ3.32 (m, 2H, CH2); 4.83 (s, 1H, NH); 7.76ꢀ7.78 (m, 2H, H-6
and H-7); 8.19ꢀ8.21 (m, 2H, H-5 and H-8); 8.23 (s, 1H, H-2); 9.51 (s,
1H, NH). 13C NMR (100 MHz, CDCl3) δ: 11.5 (CH3), 23.4 (CH2),
42.7 (NHCH2), 116.2 (C-2), 127.2 and 127.4 (C-6 and C-7), 124.9 (C-
3), 131.6 (C-8a), 133.6 (C-4a), 134.0 and 134.3 (C-5 and C-8), 136.4
(C-3a), 142.9 (C-9a), 154.9, 177.6, and 182.1 (CdO). ESI-MS m/z
calcd for C16H14N2O3S, 314.07; found, 314.22.
3-(Diethylamino)-N-(4,9-dioxo-4,9-dihydronaphtho[2,3-
b]thiophen-3-yl)propanamide Hydrochloride (11m). Yield:
1
43%. Mp 201ꢀ202 ꢀC. H NMR (400 MHz, CD3OD) δ: 1.36ꢀ1.40
(t, 6H, CH3); 3.10ꢀ3.13 (m, 2H, CH2); 3.30ꢀ3.34 (q, 4H, CH2);
3.56ꢀ3.59 (m, 2H, CH2); 7.85ꢀ7.87 (m, 2H, H-6 and H-7); 8.20ꢀ8.24
(m, 2H, H-5 and H-8); 8.48 (s, 1H, H-2). 13C NMR (100 MHz,
CD3OD) δ: 15.8 (CH3), 35.8 (RCH2), 47.9 (CH2CH3), 51.5 (βCH2),
118.6 (C-2), 126.9 (C-6 and C-7), 129.2 (C-3), 132.7 (C-8a), 133.1 (C-
4a), 134.8 (C-5 and C-8), 138.9 (C-3a), 145.0 (C-9a), 172.5, 178.9, and
182.6 (CdO). ESI-MS m/z calcd for C19H21ClN2O3S, 392.10; found,
390.17.
1-(2-(Dimethylamino)ethyl)-3-(4,9-dioxo-4,9-dihydro-
naphtho[2,3-b]thiophen-3-yl)urea Hydrochloride (11r).
Yield: 69%. Mp 216ꢀ217 ꢀC. 1H NMR (400 MHz, CD3OD) δ:
3.23ꢀ3.25 (m, 2H, CH2); 3.30 (s, 6H, CH3); 3.59ꢀ3.61 (m, 2H,
CH2); 7.85ꢀ7.87 (m, 2H, H-6 and H-7); 8.21 (s, 1H, H-2); 8.22ꢀ8.24
(m, 2H, H-5 and H-8). 13C NMR (100 MHz, CD3OD) δ: 35.4
(NHCH2), 42.9 [N(CH3)2], 58.3 [CH2N(CH3)2], 116.0 (C-2), 126.7
and 127.9 (C-6 and C-7), 125.3 (C-3), 132.6 (C-8a), 134.7 (C-4a), 134.1
(C-5 and C-8), 136.1 (C-3a), 143.5 (C-9a), 154.6, 178.2, and 181.1
(CdO). ESI-MS m/z calcd for C17H18ClN3O3S, 379.08; found, 378.97.
Biology. Dulbecco’s modified Eagle’s medium (DMEM), fetal
bovine serum (FBS), trypsinꢀEDTA solution (1ꢁ), penicillin and
streptomycin, and phosphate-buffered saline (PBS) were from Cambrex
Biosciences. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT), propidium iodide (PI), Triton X-100, sodium citrate,
formamide, and mouse monoclonal anti-tubulin were purchased from
Sigma (Milan, Italy). Rabbit polyclonal anti-cyclin A primary antibody
were from Cell Signaling Technology (Celbio; Milan, Italy). ECL
reagent was obtained from Amersham Pharmacia Biotech, U.K.
Cell Culture. Human breast MDA231, human colon carcinoma
SW620, Colo205, HT-29, and Caco-2, human monocytic leukemia
U937, human melanoma MDA-MB435 and SK-MEL28, human ovarian
cancer IGROV, and human glioblastoma SF-295 and SNB-19 cell lines
were grown at 37 ꢀC in Dulbecco’s modified Eagle’s medium containing
10 mM glucose (DMEM-HG) supplemented with 10% fetal calf serum
and 100 units/mL each of penicillin and streptomycin and 2 mmol/L
glutamine. In each experiment, cells were placed in fresh medium,
cultured in the presence of synthesized compounds (from 0.1 to 25 mM)
and followed for further analyses.
N-(4,9-Dioxo-4,9-dihydronaphtho[2,3-b]thiophen-3-yl)-
3-morpholinopropanamide Hydrochloride (11n). Yield: 48%.
Mp 207ꢀ208 ꢀC. 1H NMR (400 MHz, CD3OD) δ: 3.11ꢀ3.14 (t, 2H,
RCH2); 3.32ꢀ3.37 (m, 4H, CH2N); 3.48ꢀ3.50 (m, 2H, βCH2);
3.98ꢀ4.01 (m, 4H, CH2O); 7.86ꢀ7.88 (m, 2H, H-6 and H-7);
8.19ꢀ8.24 (m, 2H, H-5 and H-8); 8.50 (s, 1H, H-2). 13C NMR (100
MHz, CD3OD) δ: 37.3 (R-CH2), 47.5 (CH2N), 50.9 (βCH2), 67.2
(CH2O), 119.1 (C-2), 126.8 (C-6 and C-7), 130.1 (C-3), 132.6 (C-8a),
133.9 (C-4a), 135.1 (C-5 and C-8), 138.7 (C-3a), 145.5 (C-9a), 172.3,
178.5, and 181.9 (CdO). ESI-MS m/z calcd for C19H19ClN2O4S,
406.08; found 406.17.
N-(4,9-Dioxo-4,9-dihydronaphtho[2,3-b]thiophen-3-yl)-
3-mercaptopropanamide (11o). Yield: 42%. Mp 181ꢀ182 ꢀC. 1H
NMR (400 MHz, CDCl3) δ: 2.82ꢀ2.85 (m, 2H, CH2); 2.91ꢀ2.95 (m,
2H, CH2); 7.79ꢀ7.81 (m, 2H, H-6 and H-7); 8.21ꢀ8.26 (m, 2H, H-5
and H-8); 8.48 (s, 1H, H-2); 10.33 (s, 1H, NH). 13C NMR (100 MHz,
CDCl3) δ: 23.9 (CH2SH), 39.5 (R-CH2), 119.9 (C-2), 127.6 (C-6 and
C-7), 128.0 (C-3), 132.9 (C-8a), 134.0 (C-4a), 134.4 (C-5 and C-8),
138.9 (C-3a), 144.8 (C-9a), 172.1, 179.8, and 181.8 (CdO). ESI-MS
m/z calcd for C15H11NO3S2, 317.02; found, 317.18.
3-(2-(Dimethylamino)ethylamino)-N-(4,9-dioxo-4,9-dihydro-
naphtho[2,3-b]thiophen-3-yl)propanamide Dihydrochlor-
ide (11p). Yield: 45%. Mp 227ꢀ228 ꢀC. 1H NMR (400 MHz,
CD3OD) δ: 2.98 (s, 6H, CH3); 3.09ꢀ3.12 (t, 2H, RCH2); 3.29ꢀ3.31
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Cell Viability Assay. Cell viability for all cell lines was determined
using the 3-[4,5-demethylthiazol-2,5-diphenyl-2H-tetrazolium bromide
(MTT) colorimetric assay. The test is based on the ability of mitochon-
drial dehydrogenase to convert, in viable cells, the yellow MTT reagent
(Sigma Chemical Co., St. Louis, MO) into a soluble blue formazan dye.
Cells were seeded into 96-well plates to a density of 105 cells/100 μL
well. After 24 h of growth to allow attachment to the wells, compounds
were added at various concentrations (from 0.1 to 25 mM). After 24 or
48 h of growth and after removal of the culture medium, 100 μL/well
medium containing 1 mg/mL MTT was added. Cell cultures were
further incubated at 37 ꢀC for 2 h in the dark. The solution was then
gently aspirated from each well, and the formazan crystals within the
cells were dissolved with 100 μL of DMSO. Optical densities were read
at 550 nm using a Multiskan Spectrum Thermo Electron Corporation
reader. Results were expressed as percentage relative to vehicle-treated
control (0.5% DMSO was added to untreated cells). IC50
(concentration eliciting 50% inhibition) values were determined by
linear and polynomial regression. Experiments were performed in
triplicate.
Topo II Mediated Supercoiled pBR322 Relaxation. DNA
relaxation assays were based on the procedure of Osheroffet al.26b
Reaction buffer contained 10 mM Tris-HCl (pH 7.9), 50 mM KCl,
50 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA, and 15 μg/mL bovine
serum albumin (BSA), 0.15 μg of supercoiled pBR322, 4 units of topo II
in a total of 20 μL. Relaxation was employed at 37 ꢀC for 6 min and
stopped by the addition of 3 μL of stop solution (100 mM EDTA, 0.5%
SDS, 50% glycerol, 0.05% bromophenol blue). Electrophoresis was
carried out in a 1% agarose gel in 0.5ꢁ TBE (89 mM Tris base, 89 mM
boric acid, and 2 mM EDTA) at 4 V/cm for 1 h. DNA bands were stained
with 0.5 μg/mL ethidium bromide (EB) solution and photographed
through a gel document system GDS8000 (UVP). The amount of DNA
bands was quantified by Gel 1D intermediate software.
Confocal Microscopy. For immunocytochemistry, cells were fixed
in 0.04 g/L paraformaldehyde for 30 min at 4 ꢀC and permeabilized with
0.01 g/L Triton X-100 for 30 min at 4 C. Cells were then washed and
stained with Hoechst 33342 (Vector, Burlingame, CA). Images were
acquired with a LSM510 inverted confocal microscope (Zeiss, Oberko-
chen, Germany) using a 63ꢁ oil objective and processed using LSM
software (Zeiss).
Flow Cytometry: Analysis of Cell Cycle. CaCo-2 cells were
seeded in six multiwell plates at a density of 25 ꢁ 105 cells/plate. After
48 h of incubation with 11m and 11p derivatives and doxorubicin in
DMEM without serum at 37 ꢀC, cells were washed in PBS, pelleted,
centrifuged, and directly stained in a propidium iodide (PI) solution (50
mg of PI in 0.1% sodium citrate, 0.1% NP40, pH 7.4) for 30 min at 4 ꢀC
in the dark. Flow cytometric analysis was performed using a FACScan
flow cytometer (Becton Dickinson, San Jose, CA). To evaluate the cell
cycle, PI fluorescence was collected as FL2 (linear scale) by the ModFIT
software (Becton Dickinson). For the evaluation of intracellular DNA
content, at least 20 000 events for each point were analyzed in at least
three different experiments giving a SD of less than 5%.
Tris-buffered saline/Tween 20/1% milk powder. This step was followed
by incubation with the corresponding horseradish peroxidase conjugated
antibody (anti-rabbit-IgG 1:6000; Biosource, Germany). Bands were read
by enhanced chemiluminescence (ECL kit, Amersham, Germany).
Alkaline Phosphatase Activity. Alkaline phosphatase (ALP)
activity was used as marker of the degree of cell differentiation. Attached
and floating cells were washed and lysed with 0.25% sodium deoxycho-
late, essentially as described by Herz et al.40 ALP activity was determined
using Sigma Diagnostics ALP reagent (no. 245). Total cellular protein
content of the samples was determined in a microassay procedure as
described by Bradford41 using the coomassie protein assay reagent kit
(Pierce). ALP activity was calculated as units of activity per milligram of
protein.
H9C2 Cell Viability. Cardiomyoblasts H9C2 were cultured in
Dulbecco’s minimal essential medium (DMEM, GIBCO) supplemen-
ted with 0.1 g/L fetal bovine serum (FBS, GIBCO), 200 mg/mL
L-glutamine, 100 units/mL penicillin, and 10 mg/mL streptomycin
(Sigma-Aldrich) at 37 ꢀC in 0.95 g/L airꢀ0.05 g/L CO2. The H9C2
samples were studied between passages 4 and 10. The MTT colorimetric
assay (Invitrogen, San Diego, CA) was used to evaluate cell proliferation
in the presence or absence of inhibitors. Briefly, H9C2 cells were plated
into 96 multiwells at a density of 2000 cells/well in quadruplicate.
Inhibitors (11m, 11p, and doxorubicin) were added to each well at 1 μM
for the indicated time points. Then an amount of 10 μL of MTT reagent
was added to each well. The plate was returned to the cell culture
incubator for 2 h. The absorbance in each well, including the blanks, was
measured at 570 nm in a microplate plate reader.
Statistical Analysis. Data were expressed as the mean ( standard
deviation (SD). Statistical significance was assessed by the Student t test.
P adjustment for multiple comparisons was done by the Holm method
(sequential Bonferonni correction method). P < 0.05 was considered
statistically significant.
STD NMR and WaterLOGSY Spectroscopy. STD NMR29 and
WaterLOGSY30 experiments were performed on a Varian Inova 700
MHz spectrometer at 25 ꢀC. NMR samples were prepared by dissolving
the ligand and the poly(dGdC) poly(dG-dC) copolymer (Pharmacia
3
Biochemicals) in H2O/D2O 9:1 (final volume of 600 μL; D2O 99.996%,
CIL Laboratories) containing phosphate buffered saline (100 mM) at
pH 7.1. A high ligandꢀreceptor molar excess (20:1) was used. In
particular, the concentrations of 11c, 11m, and 11p were 1.0 mM,
whereas that of the DNA was 50 μM, expressed as molarity of phosphate
groups. Water suppression was achieved by using the double-pulsed field
gradient spinꢀecho (DPFGSE) scheme.42 The STD effects of the
individual protons were calculated for each compound relative to a
reference spectrum with off-resonance saturation at δ = ꢀ16 ppm.
Typically, 512 scans were recorded for each DF-STD spectrum
(saturation time of 2 s). The relative STD effect was calculated for each
signal as the difference between the intensity (expressed as S/N ratio) of
one signal in the on-resonance STD spectrum and that of the same signal
in the off-resonance NMR spectrum divided by the intensity of the same
signal in the off-resonance spectrum. BMI values were obtained as the
ratio of the relative STD effects upon irradiation at 10.0 and ꢀ1.0 ppm.31
The absence of STD effects in samples in which the DNA was not added
ensured a selective macromolecule saturation. WaterLOGSY NMR
experiments employed a 20 ms selective Gaussian 180ꢀ pulse at the
water signal frequency and an NOE mixing time of 1.5 s.
Western Blot Assay. The effects of 11m, 11p, and 11c on
expression of cyclin A and of 11m and 11p on Hsp27, were determined
by Western blots. Compound-stimulated and unstimulated (control)
cell lysates were prepared using an ice cold lysis buffer (50 mM Tris,
150 mM NaCl, 10 mM EDTA, 1% Triton) supplemented with a mixture
of protease inhibitors containing antipain, bestatin, chymostatin, leu-
peptin, pepstatin, phosphoramidon, Pefabloc, EDTA, and aprotinin
(Boehringer, Mannheim, Germany). Equivalent protein samples were
resolved on 8ꢀ12% sodium dodecyl sulfate (SDS)ꢀpolyacrylamide gels
and transferred to nitrocellulose membranes (Bio-Rad, Germany). For
immunodetection, membranes were incubated overnight with specific
antibody at the concentrations indicated in the manufacter’s protocol
(Santa Cruz Biotechnology). The two antibodies were diluted in
’ ASSOCIATED CONTENT
S
Supporting Information. Microanalysis data for all com-
b
pounds; data on the expression of cyclin A in control and induced
by 11p, 11m, and 11c; and data of effects of doxorubicin on the
topo II mediated DNA cleavage. This material is available free of
charge via the Internet at http://pubs.acs.org.
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Journal of Medicinal Chemistry
ARTICLE
’ AUTHOR INFORMATION
(11) Minotti, G.; Menna, P.; Salvatorelli, E.; Cairo, G.; Gianni, L.
Anthracyclines: molecular advances and pharmacologic developments
in antitumor activity and cardiotoxicity. Pharmacol. Rev. 2004,
56, 185–229.
(12) Gomez-Monterrey, I.; Campiglia, P; Mazzoni, O.; Novellino,
E.; Diurno, M. V. Cycloaddition reactions of thiazolidine derivatives. An
approach to the synthesis of new functionalized heterocyclic systems.
Tetrahedron Lett. 2001, 42, 5755–5757.
Corresponding Author
*Phone: þ39-081-678643. Fax: þ39-081-678644. E-mail:
novellin@unina.it.
’ ACKNOWLEDGMENT
(13) Gomez-Monterrey, I.; Campiglia, P.; Grieco, P.; Diurno, M. V.;
Bolognese, A.; La Colla, P.; Novellino, E. New Benzo[g]isoquinoline-
5,10-diones and dihydrothieno[2,3-b]naphtho-4,9-dione derivatives:
synthesis and biological evaluation as potential antitumoral agents.
Bioorg. Med. Chem. 2003, 11, 3769–3775.
The ESI-MS and NMR spectra data were provided by Centro
di Servizio Interdipartimentale di Analisi Strumentale (CSIAS),
Universitꢀa degli Studi di Napoli “Federico II”, Italy. The assis-
tance of the staff is gratefully appreciated.
(14) Gomez-Monterrey, I.; Santelli, G.; Campiglia, P.; Califano, D.;
Falasconi, F.; Pisano, C.; Vesci, L.; Lama, T.; Grieco, P.; Novellino, E.
Synthesis and cytotoxic evaluation of novel spirohydantoin derivatives of
the dihydrothieno[2,3-b]naphtho-4,9-dione system. J. Med. Chem. 2005,
48, 1152–1157.
(15) Gomez-Monterrey, I.; Campiglia, P.; Carotenuto, A.; Stiuso, P.;
Bertamino, A.; Sala, M.; Aquino, C.; Grieco, P.; Morello, S.; Pinto, A.;
Ianelli, P.; Novellino, E. Spiro[(dihydropyrazin-2,5-dione)-6,30-(20,30-
dihydrothieno[2,3-b]naphtho-40,90-dione)]-based cytotoxic agents:
structure activity relationship studies on the substituent at N4-position
of the diketopiperazine domain. J. Med. Chem. 2008, 51, 2924–2932.
(16) Gomez-Monterrey, I.; Campiglia, P.; Carotenuto, A.; Califano,
D.; Pisano, C.; Vesci, L.; Lama, T.; Bertamino, A.; Sala, M.; Mazzella di
Bosco, A.; Grieco, P.; Novellino, E. Design, synthesis, and cytotoxic
evaluation of a new series of 3-substituted spiro[(dihydropyrazine-2,5-
dione)-6,30-(20,30-dihydrothieno[2,3-b]naphtho-40,90-dione)] deriva-
tives. J. Med. Chem. 2007, 50, 1787–1798.
’ ABBREVIATIONS USED
Boc, tert-butyloxycarbonyl; HBTU, 2-(1H-benzotriazole-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate; HOBt, N-hy-
droxybenzotriazole; DIPEA, N,N-diisopropylethylamine; DMF,
N,Ndimethylformamide; THF, tetrahydrofuran; DCM, dichloro-
methane; TES, triethylsilane; DBU, 1,8-diazabicyclo(5.4.9)-
undec-7-ene; TLC, thin-layer chromatography; ES-MS, electro-
spray mass spectrometry; SD, standard deviation; STD, satura-
tion transfer difference; DF, differential frequency; NOE, nuclear
Overhauser effect; WaterLOGSY, waterꢀligand observed via
gradient spectroscopy
’ ADDITIONAL NOTE
Abbreviations used for amino acids follow the rules of the
IUPAC-IUB Commission of Biochemical Nomenclature in
J. Biol. Chem. 1972, 247, 977ꢀ983. Amino acid symbols denote
L-configuration.
(17) Gomez-Monterrey, I.; Campiglia, P.; Bertamino, A.; Aquino,
C.; Sala, M.; Grieco, P.; Dicitore, A; Vanacore, D.; Porta, A.; Maresca, B.;
Novellino, E.; Stiuso, P. A novel quinone-based derivative (DTNQ-Pro)
induces apoptotic death via modulation/reduction of heat shock protein
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