Synthesis of structurally simplified analogues of aplidinone A, a pro-apoptotic marine thiazinoquinone

Article abstract of DOI:10.1016/j.bmc.2009.11.063

The synthesis of analogues of aplidinone A (7), a prenylated quinone isolated from the Mediterranean ascidian Aplidium conicum, has been performed. This work not only allowed confirming the structural assignment of aplidinone A, previously made with the s

Full text of DOI:10.1016/j.bmc.2009.11.063

Bioorganic & Medicinal Chemistry 18 (2010) 719–727
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of structurally simplified analogues of aplidinone A,
a pro-apoptotic marine thiazinoquinone
c
Anna Aiello a, Ernesto Fattorusso a, Paolo Luciano b, Marialuisa Menna a, , Marco A. Calzado ,
Eduardo Muñoz c, Francesco Bonadies d, Marcella Guiso d, Maria Filomena Sanasi d,
Gianfranco Cocco d, Rosario Nicoletti d
*
a Dipartimento di Chimica delle Sostanze Naturali, Università degli Studi di Napoli ‘Federico II’, Via D. Montesano 49, 80131 Napoli, Italy
b Centro Servizi Interdipartimentale di Analisi Strumentale (C.S.I.A.S.), Università degli Studi di Napoli ‘Federico II’, Via D. Montesano 49, Napoli, Italy
c Departamento de Biología Celular, Fisiología e Inmunología, Universidad de Córdoba, Avda Menéndez Pidal s/n, 14004 Córdoba, Spain
d Dipartimento di Chimica, Università La Sapienza di Roma, P. le Aldo Moro 5, 00185 Roma, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 July 2009
Revised 20 November 2009
Accepted 27 November 2009
Available online 6 December 2009
The synthesis of analogues of aplidinone A (7), a prenylated quinone isolated from the Mediterranean
ascidian Aplidium conicum, has been performed. This work not only allowed confirming the structural
assignment of aplidinone A, previously made with the support of GIAO shielding calculations, but, above
all, made a series of structurally related quinone derivatives (compounds 8–13 and the natural metabo-
lite) available for a screening in vitro for cytotoxic and pro-apoptotic activity and for SAR studies. The
study evidenced one of the synthetic analogues (11) as a potent cytotoxic and pro-apoptotic agent against
Dedicated to the memory of Professor
Rodolfo A. Nicolaus
several tumor cell lines which also inhibits the TNFa-induced NF-jB activation in a human leukemia T
cell line. This exemplifies the potential of a natural product to qualify as lead structure for medicinal
chemistry campaigns, affording simplified analogues with better bioactivity and easier to synthesize.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Ascidians
Marine natural products
Quinones
Pro-apoptotic compounds
ROS production
1. Introduction
presence of an unusual 1,1-dioxo-1,4-thiazine ring fused to a qui-
none moiety. As for the aplidinones, the whole of the NMR data
Naturally occurring prenylated 1,4-benzoquinones and hydro-
quinones are commonly found in a variety of organisms and play
important roles in several metabolic processes, such as photosyn-
thesis and electron transport.1,2 Examples of these compounds have
been isolated from marine sources, mostly from tunicates belonging
to the order of Aplousobranchiata (family Polyclinidae), such as asci-
dians of the genus Aplidium. They show a wide array of different
structures, originated by intra- and inter-molecular cyclizations
and/or rearrangements of the original terpene hydroquinone/qui-
none skeleton, thus giving macrocyclic or polycyclic structures; they
are often linked to amino acids or taurine residues.3–14
Recently, in the frame of our search for bioactive new molecules
from Mediterranean tunicates, we have investigated the ascidian
Aplidium conicum and this study yielded a large group of new
prenylated quinones we named conicaquinones (1 and 2),15 thiap-
lidiaquinones (3 and 4),16 and aplidinones (5–7).17 These com-
pounds are structurally quite different, but they share the
were not enough to unambiguously define the structure of the het-
erocyclic ring; an uncertainty still remained about the relative po-
sition of the sulfur and the nitrogen position in the ring. The
reported regiochemistry was assigned as suggested for aplidinone
A (7) by comparison of its experimental 13C NMR chemical shifts
with those predicted by GIAO18 shielding calculations for regioi-
somers models A1 (8) and A2 (9). In the present paper we report
about a synthetic study performed in order to validate the struc-
tural assignment of aplidinone A made by theoretical means. Syn-
thesis of model compounds A1 and A2 has been carried out and
allowed us to confirm the proposed regiochemistry. In addition,
the designed synthetic procedure was adapted to prepare further
quinone analogues, 10–13. Both aplidinone A and its synthetic ana-
logues were shown to possess interesting cytotoxic effects; SAR
studies revealed that 11 is the most potent cytotoxic and pro-apop-
totic agent against several tumor cell lines and also inhibits TNF
a-
induced NF- B activation in a human leukemia T cell line.
j
This exemplifies the potential of a natural product to qualify as
lead structure for medicinal chemistry campaigns, affording sim-
plified analogues with better bioactivity and easier to synthesize.
* Corresponding author. Tel.: +39 081 678518; fax: +39 081 678552.
E-mail address: mlmenna@unina.it (M. Menna).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.11.063

720
A. Aiello et al. / Bioorg. Med. Chem. 18 (2010) 719–727
8 and 9. The isolation of the two isomers 8 and 9 was achieved by
2. Results and discussion
HPLC separation of this mixture; their structures were univocally
assigned by spectral studies as described below.
2.1. Synthesis of model compounds A1 (8) and A2 (9): validation
of the structure assigned to aplidinone A (7)
ESI-MS data (see Section 4) of 8 and 9 indicated that the two com-
pounds were isomers with the molecular formula C11H13NO5S. Anal-
ysis of 1D and 2D NMR spectral data of 8 (CDCl3) allowed the
assignment of all 1H and 13C NMR signals, which are reported in
Table 1. A firstset of signals, namelythose relevant to the bicyclicnu-
cleus [dH: 3.28 (m, 2H-2), 4.07 (m, 2H-3), 4.24 (s, OCH3), 6.81 (br s,
NH); dC: 48.7 (C-2), 39.9 (C-3), 62.2 (OCH3), 144.2 (C-4a), 179.0 (C-
5), 128.6 (C-6), 157.3 (C-7), 173.9 (C-8), 108.7 (C-8a)], appeared al-
most identical to those of aplidinone A.17 The remaining signals
[dH: 2.40 (q, J = 7.5 Hz, 2H-10), 0.99 (t, J = 7.5 Hz, 3H-20); dC: 16.2 (C-
10), 12.4 (C-20)] were obviously due to the ethyl group which replaces
in structure 8 the geranyl moiety linked to the benzoquinone ring of
the natural compound 7. The position of the ethyl group, with
respect to nitrogen and sulfur atoms of the heterocyclic ring, was as-
signed through assessment of proton–carbon long-range couplings,
reported in Table 1. Particularly, diagnostic cross peaks were ob-
served in the HMBC spectrum of 8 between the methylene protons
at d 2.40 (2H-10) and the carbonyl resonance at d 179 (C-5), as well
as between the sulfur-linked methylene protons at d 3.28 (2H-2)
and the other carbonyl signal at d 173.9 (C-8). These key correlations
indicated that the ethyl chain was linked at C-6, thus defining the
structure of 8 as 6-ethyl-7-methoxy-3,4-dihydro-1,1-dioxo-2H-
Heterocyclic systems like aplidinone A, having a 1,1-dioxo-1,4-
thiazinic ring condensed with a benzoquinone ring, are not com-
mon in literature. This kind of compounds has been synthesized
for the first time in 198820 by condensation of hypotaurine with
naphthoquinone using the known conjugate addition reaction of
amines and sulfinic acids with naphthoquinones21 to give substi-
tuted naphthoquinones. Later, Harada et al. worked out the synthe-
sis of adociaquinones A and B using the same protocol.22
Although this route has the disadvantage of being not regioselec-
tive (see Scheme 1), we recognized it as a simple and versatile way to
synthesize in relatively few steps aplidinone A derivatives, starting
from an appropriate benzoquinone derivative. Thus, compounds 8
and 9 have been prepared according to the synthetic path reported
in Scheme 2. The alkylation of methoxy-hydroquinone (14) was
tested with diethyl sulfate in the presence of potassium carbonate,
butthereactionyieldeda mixtureofmonoand bisether. Satisfactory
results, instead, were obtained by using cesium carbonate as base
under argon atmosphere. It has been shown that the introduction
of an alkyl residue on 1,2,4-trimethoxy benzene may be selective
in the 3-position.23 So, ethylation of compound 15 in the correct po-
sition was obtained allowing to react the Li derivative with diethyl
sulfate for 24 h at room temperature under argon. The product
(16) showed the expected 1H NMR spectrum which exhibits the
ortho coupling between the two aromatic protons (see Section 4).
Oxidationof 16to quinonewas carriedoutwitha largeexcess (molar
ratio 8:1) of cerium ammonium nitrate (CAN).
4
1,4-benzothiazine-5,8-dione. It is to be noted that the JC8–H2 cou-
pling gives rise to a weak correlation peak, which could not be
detected in the HMBC spectrum of aplidinone A due to the very small
amounts of natural compound isolated.
1H and 13C NMR features of compound 9 were nearly identical
to those of 8, with regard to the number and shape of the signals.
However, differences were observed in the chemical shift values of
a few NMR signals (see Table 1); significantly different were the
Quinone 17 was obtained with 62% of yield; its condensation
with hypotaurine22 afforded, as expected, a mixture of compounds
O
O
O
O
O
H
N
O
X
Y
X
Y
X
S
S
+
+
H2N
OH
S
Y
N
H
O
O
O
O
O
Scheme 1.
OEt
OH
OEt
n-BuLi, Et2SO4
Cs2CO3, Et2SO4
H3CO
H3CO
H3CO
OEt
16
OH
14
OEt
15
(NH4)Ce(NO3)6
0oC, 1 min.
O
O
O
O
O
O
O
H
N
S
NH2CH2CH2SO2H
100oC, 1min.
+
S
N
H
H3CO
H3CO
H3CO
O
O
O
9
17
8
Scheme 2.

A. Aiello et al. / Bioorg. Med. Chem. 18 (2010) 719–727
721
Table 1
NMR data (CDCl3) of compounds 8 and 9
Pos.
8
9
dC
dH, mult. (J in Hz)
HMBC
dC
dH, mult. (J in Hz)
HMBC
1
—
—
—
—
—
—
2
3
4
48.7
39.9
—
3.28 m
4.07 m
—
3, 8, 8a
2, 4a
—
48.7
39.7
—
3.28 m
4.04 m
—
3, 8, 8a
2, 4a
—
4a
5
6
7
8
144.2
179.0
128.6
157.3
173.9
108.7
16.2
12.4
62.2
—
—
—
—
—
—
—
—
—
—
—
—
—
142.7
176.4
152.8
139.7
178.0
110.1
17.0
13.4
60.8
—
—
—
—
—
—
—
—
—
—
—
—
—
8a
10
2.40 q (7.5)
0.99 t (7.5)
4.24 s
5, 6, 7, 20
6, 10
7
2.50 q (7.5)
1.08 t (7.5)
3.91 s
6, 7, 8, 20
7, 10
6
20
OCH3
NH
6.81 br s
—
6.50 br s
—
chemical shift values of the carbonyl carbons [d 176.4 vs d 179.0
(C-5) and d 178.0 vs d 173.9 (C-8)] and of the methoxyl protons
(d 3.91 vs d 4.24). Thus, it was evident that the two compounds
were isomerically differing in the location of the substituents on
the quinone ring. The alternative 7-ethyl-6-methoxy-3,4-dihy-
dro-1,1-dioxo-2H-1,4-benzothiazine-5,8-dione structure was univ-
ocally assigned to 9 by analysis of HMBC spectrum, as well. Both
2- and 10-methylene protons were indeed correlated to the same
carbonyl carbon resonating at d 178.0 (C-8), thus indicating that
in 9 the ethyl chain was linked at C-7 rather than at C-6. The whole
series of HMBC correlations were consistent with the proposed
assignment (see Table 1).
Compounds 8 and 9 were obtained in a ratio of 8:2 in favor of 9.
The regioselectivity observed in our case merits a comment. Data
in the literature are related to reactions of hypotaurine with qui-
nones lacking strong directing groups (i.e., electron releasing
groups). In these examples mixtures roughly 1:1 were formed and,
therefore, we suggest that the effect of the methoxyl group strongly
orients the attack of nucleophilic sulfur on the quinone ring.
The close agreement between the signals of bicyclic skeleton of
compound 8 and aplidinone A strongly sustained the structure
assignment previously made for the natural compound by theoret-
ical means.17 In addition, we compared the experimental 13C
chemical shifts of 8 and 9 to the corresponding theoretical ones ob-
tained from GIAO calculation24 (see Section 4). For both structures,
the correlation coefficient (R) value was around 0.998, indicative of
a very good fit between the two set of parameters and, thus, repre-
sented a further validation of the theoretical applied method.
2.2. Synthesis of compounds 10–13
Quinones10–13 were synthesizedthrough a route slightlydiffer-
ent from that used for the preparation of 8 and 9, since trial experi-
ments have shown a drop in the yields of alkylation with long
chain alkyl bromides. Therefore, the following route, starting from
1,3-dimethoxybenzene (18), in analogy with a similar path re-
ported,25 was followed (Scheme 3). The lithium derivative of resor-
cinol dimethyl ether was reacted with two different alkyl
bromides; yields were 58% with octyl bromide (1,3-dimethoxy-2-
octylbenzene, 19) and 35% with tetradecyl bromide (1,3-dime-
thoxy-2-tetradecylbenzene, 19a). The oxidation of these substrates
withperaceticacid(preparedin situ) affordedin bothcases mixtures
(20 and 21; 20a and 21a, respectively), which were further oxidized
with nitric acid, giving quinones 21 (32% yield) and 21a (30% yield),
respectively, as sole products. Both 21 and 21a were reacted with
hypotaurine and afforded mixtures 10/11 and 12/13, respectively.
Products10–13wereisolatedasindividualcompoundsbyHPLCsep-
arations; as expected, for each couple of regioisomers, one was
formed in large excess in respect to the other. Their chemical charac-
terization was easily performed through spectral analysis (Table 2),
referring to the NMR spectral assignments made for 8 and 9. Actu-
ally, carbon and proton resonances relative to the bicyclic nucleus
OCH3
O
OCH3
OCH3
-
H2O2
n-BuLi, Br(CH2)nCH3
n
n
n
+
CH3COOH
THF,dry 24h
H3CO
H3CO
H3CO
H3CO
OH
O
n = 7 19
18
O
n = 7 20
n = 13 19a
HNO3
n = 13 20a
n = 7 21
n = 13 21a
O
O
O
O
H
N
S
n
n
n
NH2CH2CH2SO2H
+
S
N
H
H3CO
H3CO
H3CO
O
O
O
O
O
n = 7 10
n = 13 12
n = 7 11
n = 13 13
n = 7 21
n = 13 21a
Scheme 3.

722
A. Aiello et al. / Bioorg. Med. Chem. 18 (2010) 719–727
Table 2
NMR data of compounds 10–13
Pos.
10
11
12
13
dC
dH, mult. (J in Hz)
dC
dH, mult. (J in Hz)
dC
dH, mult. (J in Hz)
dC
dH, mult. (J in Hz)
1
—
—
—
—
—
—
—
—
2
3
4
48.7
39.7
—
3.29 m
4.06 m
—
48.7
39.5
—
3.28 m
4.06 m
—
48.8
39.7
—
3.29 m
4.06 m
—
48.7
39.5
—
3.28 m
4.06 m
—
4a
6
7
144.3
127.7
157.6
174.1
22.9
28.1
29.6
29.1
31.8
22.6
13.9
—
—
—
—
62.2
—
—
—
—
—
142.8
152.9
138.8
178.1
23.5
28.8
29.5
29.1
31.7
22.3
13.6
—
—
—
—
60.6
—
—
—
—
—
144.3
127.7
157.6
174.1
22.9
28.1
29.6
29.1
29.1
29.1
29.1
29.1
31.8
22.6
13.9
62.1
—
—
—
—
—
142.8
152.9
138.8
178.1
23.5
28.8
29.5
29.1
29.1
29.1
29.1
29.1
31.7
22.3
13.6
60.7
—
—
—
—
—
8
10
2.38 t (7.5)
1.36
1.25
1.27
1.25
1.28
0.88 t (7.5)
—
—
—
2.47 t (7.5)
1.43
1.26
1.26
1.24
1.27
0.87 t (7.5)
—
—
—
2.38 t (7.5)
1.36
1.25
1.27
1.27
1.27
1.27
1.27
1.25
2.47 t (7.5)
1.43
1.26
1.26
1.26
1.26
1.26
1.26
1.24
20
30
40–50
60
70
80
90–110
120
130
140
OCH3
NH
1.28
1.27
—
—
0.88 t (7.5)
4.26 s
6.81 br s
0.87 t (7.5)
3.92 s
6.50 br s
4.25 s
6.81 br s
3.91 s
6.50 br s
of the couples of compounds 10/12 and 11/13 were virtually identi-
cal to those of compounds 8 and 9, respectively, thus defining the
structures 10–13 as those reported in Figure 1.
esterases cleave the ester bonds, producing the hydrophilic and
fluorescent dye calcein, which cannot leave the cell via the plasma
membrane unless the cells are damaged by cytotoxic compounds.
The test compounds were dissolved in DMSO and a dilution series
in the medium was prepared. Cells were incubated with the pre-
pared solutions, the final content of DMSO in the cell cultures
being never higher than 1%; the IC50 values for the test compounds
are shown in Table 4. Compound 11 showed potent cytotoxic activ-
ities toward all the cell lines tested, indicating that it targets a com-
mon pathway in cancer cells. Compounds 9 and 13 were both less
active than 11, demonstrating a definite structure activity relation-
ship for the size of the alkyl chain.
2.3. Pro-apoptotic properties of compounds 7–13
A large number of quinones, both synthetic and naturally occur-
ring, have been screened for their antitumor activity; particularly,
it has been shown that they lead to oxidative stress by means of an
increase of oxygen reactive species (ROS) and the depletion of re-
duced glutathione (GSH) amount26 and this action has been related
to the beginning of apoptosis.27,28
We have previously shown that thiaplidiaquinones 3 and 4 in-
duce generation of ROS and apoptosis in the Jurkat cell line.16 On
this cell line we have now investigated the cytotoxic activity of
aplidinone A (7), as well as that of its synthetic analogues 8–13.
2.4. Inhibition of TNFa-induced NF-jB activation
Another important factor in cancer development and progres-
sion is the regulation of many anti-apoptotic genes by the NF-
jB
Aplidinone A induced cytotoxicity with an IC50 about 45
cytotoxic activity was enhanced with the modifications introduced
in 11 (IC50 ꢀ 20 M) and decreased in 8–10, 12, and 13 (Table 3).
lM; the
family of transcription factors.29–31 Also in this process, ROS play
an important role; in fact, ROS are continuously produced during
normal aerobic metabolism by the mitochondria electron chain
and their level is related to many different physiological stim-
uli.32,33 Low-medium levels of ROS can protect cell from apoptosis
by activating antioxidant mechanisms, including activation of the
l
To further discriminate whether aplidinone A analogues in-
duced cell death by either necrosis or apoptosis we treated the
cells with compounds 8–11 for 24 h and cell cycle analyses were
carried out. Treated cells showed a significant loss of nuclear
DNA (chromatinolysis) measured by an increase in the frequency
of subdiploid (apoptotic) cells (Fig. 2). As expected, 11 was the
most potent apoptotic inducer in this group of analogues.
NF-
j
B pathway.34 Conversely, high levels of intracellular ROS re-
B pathway and in the induction
sult in the inhibition of the NF-
j
of cell death by either apoptosis or necrosis.
Thus, we investigated the effects of synthetic compounds 8–13 on
the NF-kB pathway (aplidinone A could not be tested due to the very
limited amounts of this compound in our hands) through the use of
Comparisonof thebiological activity of aplidinone A with that of 8,
10, and 12, all having the 6-alkyl-7-methoxy-3,4-dihydro-1,1-dioxo-
2H-1,4-benzothiazine-5,8-dione structure and differing each other
only in the nature of the side chain, indicates a critical role of this part
structure for the induction of apoptosis. Further interesting conclu-
sions can be drawn by comparing the cytotoxic effects of compound
10 to those of 11, having the same side chain and the alternative 7-al-
kyl-6-methoxy-3,4-dihydro-1,1-dioxo-2H-1,4-benzothiazine-5,8-
dione structure. This ‘unnatural’ structure appears to positively affect
the cytotoxic activity, since compound 11 is substantially more cyto-
toxic than the relevant isomer 10 with the 6-alkyl-7-methoxy-3,4-
dihydro-1,1-dioxo-2H-1,4-benzothiazine-5,8-dione structure.
The cytotoxic effect of compounds 9, 11, and 13 was then stud-
ied against a series of tumor cell lines by the calcein-AM method.
Calcein-AM is a fluorogenic, highly lipid-soluble dye that rapidly
penetrates the plasma membrane. Inside the cell, endogenous
the NF-
compound 11 was the only compound that showed a strong anti-
NF- B activity. It is to be noted that compound 8, lacking cytotoxic
activity, at high concentrations also showed some inhibitory activity
on the NF- B pathway indicating that ROS inducing activity is not
jB-dependent luciferase assay. As depicted in Figure 3,
j
j
always coupled with NF-jB inhibition.
3. Conclusions
Our results revealed compound 11 as a potent pro-apoptotic
agent, active against several tumor cell lines, and also capable to
inhibit the TNFa-induced NF-jB activation in a human leukemia
T cell line. Actually, using aplidinone A as a lead structure, we have

A. Aiello et al. / Bioorg. Med. Chem. 18 (2010) 719–727
723
O
O
O
O
O
H
N
S
S
N
H
2
O
O
1
O
O
OH
O
OH
O
O
H
N
H
N
O
S
S
O
O
O
O
O
3
4
O
O
H
N
H
N
H2N
S
HN
S
O
6
5
O
O
O
O
O
O
SO3
O
O
H
N
H
H
N
1'
6
7
N
H3CO
4a
5
8
3
2
2'
H3CO
8a
H3CO
S
S
S
O
O
O
O
O
O
O
O
O
O
7
9
8
O
O
O
O
H
N
H
N
H
N
H
N
H3CO
H3CO
7
13
S
H3CO
S
S
H3CO
S
7
13
O
O
O
O
O
O
O
O
O
O
O
13
12
11
10
Figure 1. Aplidinone A (7) and its synthetic analogues.
on the structural analogy between quinone derivatives and coen-
zyme Q is that 11 may interfere with the CoQ binding site of the
CoQ-dependent cellular redox systems at the mitochondria and
at the plasma membrane. This could lead to a redirection of the
normal electron flow, generating an excess of ROS, inhibiting NF-
kB activation and inducing apoptosis in tumor cells.
Table 3
Cytotoxity of aplidinone A (7) and its synthetic derivatives 8–13 on Jurkat leukemia
cell line
Compound
7
8
9
10
11
12
13
a
IC50
44.5
6
>50
>50
>50
20.7
2
>50
>50
a
IC50 values of test compound (lM) using PI staining as a cell viability test.
4. Experimental section
obtained a simplified analogue with better bioactivity and easier to
synthesize and this is an illustrative case of the potential of a nat-
ural product to act as lead structure for drug discovery. At the mo-
ment, there is no evidence about the mechanism by which 11
causes cytotoxic effects. However, a plausible hypothesis based
4.1. General experimental procedures
ESI mass spectra were performed on a hybrid quadrupole-TOF
massspectrometer, dissolving thesampleinMeOH. Thespectrawere

724
A. Aiello et al. / Bioorg. Med. Chem. 18 (2010) 719–727
Figure 2. Apoptotic effects of compounds 8–9 and 10–11 on the human T leukemia Jurkat cell line.
at 64 K points (digital resolution: 0.09 Hz). Homonuclear (1H–1H)
and heteronuclear (1H–13C) connectivitieswere determined by COSY
Table 4
Cytotoxity of quinone analogues toward five tumor cellsa
and HSQC experiments, respectively. Two and three bond 1H–13
C
Compound
Cell line
AGS
connectivities were determined by gradient 2D HMBC experiments
optimized for a 2,3J of 8 Hz. Routine 1H and 13C NMR spectra were re-
corded with a Varian Mercury 300 MHz or a Varian Gemini 200 MHz.
Medium-pressure liquid chromatographies (MPLC) were performed
ona Buchi861apparatuswith SiO2 (230–400mesh)packedcolumns.
High performance liquid chromatography (HPLC) separations were
achieved on a Knauer 501 apparatus equipped with an RI detector.
293T
A549
HCT116
HeLa
9
11
13
32.6
8.7
47.9
>50
13.0
50.8
>50
7.2
45.0
>50
14.1
44.8
>50
24.8
47.9
a
IC50 values of test compound (lM) using calcein-AM as cell viability test.
recordedbyinfusion intotheESIsourceusing MeOH asthesolvent. EI
mass spectra were obtained on GC–MS HP 5890, HP 5971A Mass
selective detector. 1H (700 MHz) and 13C (175 MHz) NMR spectra
were recorded on a Varian INOVA spectrometer, respectively, chem-
ical shifts were referenced to the residual solvent signal (CDCl3:
dH = 7.26, dC = 77.0). For an accurate measurement of the coupling
constants, the one-dimensional 1H NMR spectra were transformed
4.2. Reagents
Commercial reagents: Sigma–Aldrich. Solvents: Carlo Erba. TLC:
Silica Gel 60 F254 (plates 5 ꢁ 20, 0.25 mm) Merck. Preparative TLC:
Silica Gel 60 F254 plates (20 ꢁ 20, 2 mm). Spots revealed by UV
lamp then by spraying with 2 N sulfuric acid and heating at
120,00
100,00
80,00
60,00
40,00
20,00
0,00
10 25 50 10 25 50 10 25 50 10 25 50 10 25 50 10 25 50 µM
8
9
10
11
12
13
TNF
α
Figure 3. Effect of compounds 8–13 in TNFa-mediated NF-jB activation. Results show the percentage of NF-jB-dependent luciferase activity inhibition in 5.1 cells pretreated
with the compounds. The luciferase activity induced by TNF
a
in the absence of the compounds was considered 100% of activation.

A. Aiello et al. / Bioorg. Med. Chem. 18 (2010) 719–727
725
120 °C. ‘Acidic’ silica gel was prepared by treating Silica Gel 60
Merck with 1 N HCl for 24 h, washing with water until the chlorine
test was negative, activating for 48 h at 120 °C, then equilibrating
with 10% of water. Anhydrous solvents: Sigma–Aldrich (DMF) or
prepared by distillation according to standard procedures.
at rt. The mixture was diluted with water and extracted with ethyl
acetate four times. The organic phase was washed with brine, dried
over sodium sulfate, and filtered. Solvent removal gave 360 mg of
17 (62%), subsequently purified by filtration on ‘acidic’ silica gel
(eluent: CHCl3). ESI-MS: MH+ = 167 m/z. 1H NMR (CDCl3): d 6.62
(1H, d, J = 10.0 Hz, H-6 or H-5); 6.53 (1H, d, J = 10.0 Hz, H-5 or H-
6); 3.96 (3H, s, –OCH3); 2.36 (2H, q, J = 7.4 Hz, –CH2CH3); 0.98
(3H, t, J = 7.4 Hz, –CH2CH3).
4.3. Cell cultures
The adherent tumoral cell lines 293T (kidney cancer), AGS (gas-
tric cancer), A549 (lung cancer), HeLa (cervix cancer) and HCT116
(colon cancer) were maintained in DMEM medium (Lonza, Basel,
Switzerland) supplemented with 10% heat-inactivated FBS, 2 mM
4.7. Condensation of quinone 17 with hypotaurine
325 mg (1.96 mmol) of quinone 17 were dissolved in 26 mL of a
mixture of EtOH/CH3CN 1:1 and heated in a water bath. Then, a solu-
tion of hypotaurine (235 mg, 2.15 mmol) in 13 mL of water was
added in portions. The yellow solution became orange. Most of the
ethanol was removed in vacuo and the residue was poured into
water. The mixture was extracted with ethyl acetate (three times)
andtheorganicphasewaswashedwithbrine, driedoversodiumsul-
fate, and filtered. Solvent removal afforded a mixture of the isomers
8 and 9 (148 mg, 28%) which was separated as reported below.
glutamine, penicillin (50 U/mL) and streptomycin (50 lg/mL)
(complete medium). Jurkat cells (Leukemia) were maintained in
exponential growth in RPMI 1640 (Lonza), containing 10% FBS,
2 mM glutamine, and antibiotics. The cells were grown at 37 °C
in a 5% CO2 humidified atmosphere. The 5.1 cell line is a Jurkat de-
rived clone stably transfected with a plasmid containing the lucif-
erase gene driven by the HIV-1-LTR promoter and was maintained
in exponential growth in complete RPMI 1640 medium.
4.4. 1,4-Diethoxy-2-methoxybenzene (15)
4.8. 1,3-Dimethoxy-2-octylbenzene (19) and 1,3-dimethoxy-2-
tetradecylbenzene (19a)
To 2 g (14.2 mmol) of methoxy-hydroquinone 14, dissolved in
100 mL of dry DMF, 9.26 g (28.2 mmol) of Cs2CO3 were added under
argon atmosphere. 3.72 mL (28.2 mmol) of diethyl sulfate were then
added drop wise; the mixture was stirred for 30 min and heated for
22 h at 120 °C (bath temp). The dark liquid was cooled and 30 mL of
aqueous concd NH3/methanol 1:1 were added and the mixture was
left for 60 min under stirring. Methanol was removed under vac-
uum;thesolutionwas thenpoured into200 mLof coldwater andex-
tracted three times with ethyl acetate (50 mL each). The collected
organic extracts were washed with brine, dried with sodium sulfate
and filtered; the solvent removal afforded 15 (2.70 g, 97%) suffi-
ciently pure for the following reaction. EIMS: M+Å = m/z 196. 1H
NMR (CDCl3): d 6.78 (1H, d, J = 8.8 Hz, H-6); 6.51 (1H, d, J = 2.8 Hz,
H-3); 6.35 (1H, dd, J = 8.8, 2.8 Hz, H-5); 4.03 (2H, q, J = 14.0, 7.0 Hz,
–OCH2CH3) 3.98 (2H, q, J = 14.0, 7.0 Hz, –OCH2CH3); 3.84 (3H, s, –
OCH3); 1.42 (3H, t, J = 7.0 Hz, –OCH2CH3); 1.39 (3H, t, J = 7.0 Hz, –
OCH2CH3).
Resorcinol dimethyl ether (18, 1 g, 7.25 mmol) was dissolved in
anhydrous THF (15 mL). Then, 7 mL of a n-BuLi 1.6 M solution
(11 mmol) were added, under argon atmosphere at 0 °C. After
1 h, the temperature was allowed to rise to 25 °C and the mixture
was left under stirring for 2 h. Temperature was again lowered at
0 °C, and the bromoalkane (13 mmol; 2.2 mL of Br-octane or
3.9 mL of Br-tetradecane) was added in portions. Stirring was con-
tinued at rt for 18 h. The reaction mixture was poured into water
and the organic products extracted with ethyl acetate. The extract
was washed with 2 N HCl and water, dried over anhydrous sodium
sulfate, and filtered; then, the solvent was evaporated.
Chromatography on silica gel of crude 19 (SiO2/crude 19 50:1;
eluent: hexane/Et2O 9:1) gave pure 19 (1.05 g, 58%). EIMS: M+Å
=
m/z 250. 1H NMR (CDCl3): d 7.12 (1H, t, J = 8.4 Hz, H-5); 6.54 (2H,
d, J = 8.4 Hz, H-4 and H-6); 3.81 (6H, s, –OCH3); 2.63 (2H, t,
J = 7.4 Hz, –CH2(CH2)6CH3); 1.1–1.3 (12H, 6CH2); 0.89 (3H, t,
J = 6.4 Hz, –CH2(CH2)6CH3).
4.5. 1,4-Diethoxy-2-ethyl-3-methoxybenzene (16)
Chromatography on silica gel of crude 19a (SiO2/crude 19a
50:1; eluent: hexane/ethyl acetate 95:5) gave pure 19a (850 mg,
1.97 g (10 mmol) of 1,4 diethoxy-2-methoxybenzene (15) were
dissolved in 25 mL of anhydrous ether and 13.5 mL of a n-BuLi
1.6 M solution (20 mmol) were added, under argon atmosphere at
rt; the mixture was stirred for 1 h. Then, 5 mL of diethyl sulfate
(20 mmol) were added and the mixture was left under stirring for
24 h (the end of the reaction was controlled with TLC, eluent: chlo-
roform/hexane 7:3). After this time, 25 mL of aqueous concd NH3/
methanol 1:1 were added and the mixture was left for 20 min under
stirring. The mixture was poured into cold water (150 mL) and ex-
tracted three times with ether. The ethereal solution was washed
with brine several times, dried over sodium sulfate, and filtered. Sol-
vent removal gave 2.20 g of 16 (98%), sufficiently pure for the follow-
ing reaction. EIMS: M+Å = m/z 224. 1H NMR (CDCl3): d 6.60 (1H, d,
J = 8.8 Hz, H-6); 6.43 (1H, d, J = 8.8 Hz, H-5); 4.01 (2H, q, J = 14.0,
7.0 Hz, –OCH2CH3); 3.69 (2H, q, J = 14.0, 7.0 Hz, –OCH2CH3); 3.77
(3H, s, –OCH3); 2.60 (2H, q, J = 7.6 Hz, –CH2CH3); 1.42 (3H, t,
J = 7.0 Hz, –OCH2CH3); 1.39 (3H, t, J = 7.0 Hz, –OCH2CH3); 1.13 (3H,
t, J = 7.6 Hz, –CH2CH3).
35%). EIMS: M+Å = m/z 334. 1H NMR (CDCl3):
d 7.12 (1H, t,
J = 8.4 Hz, H-5); 6.54 (2H, d, J = 8.4 Hz, H-4 and H-6); 3.81 (6H, s,
–OCH3); 2.63 (2H, t, J = 7.4 Hz, –CH2(CH2)12CH3); 1.1–1.3 (24H,
12CH2); 0.89 (3H, t, J = 6.4 Hz, –CH2(CH2)12CH3).
4.9. 2-Octyl-3-methoxycyclohexa-2,5-diene-1,4-dione (21) and
2-tetradecyl-3-methoxycyclohexa-2,5-diene-1,4-dione (21a)
The 1,3-dimethoxy-2-alkyl-benzene (2.51 mmol; 630 mg of 19
and 840 mg of 19a, respectively) was dissolved in a minimum vol-
ume of glacial acetic acid. p-Toluene sulfonic acid (50 mg) and
0.4 mL of 34% H2O2 solution were added and the mixture was
heated at 50 °C for 1 h. The mixture was poured into water and ex-
tracted with AcOEt (three times). The extract was washed with
water and dried over anhydrous sodium sulfate; after filtration,
the solvent removed in vacuo. Purification on ‘acidic’ silica gel,
(SiO2/reaction mixture 15:1; eluent: hexane/ethyl acetate 9:1)
afforded the mixtures of compounds 20/21 and 20a/21a, respec-
tively, which were submitted to the further oxidation. The mixture
of compounds 20 and 21, as well as that of compounds 20a and
21a, was dissolved in glacial acetic acid (3 mL) and 0.23 mL of con-
cd HNO3 were added in portions at 0 °C under stirring. After 15 h,
the reaction was complete (TLC hexane/ethyl acetate 9:1). The
4.6. 2-Ethyl-3-methoxycyclohexa-2,5-diene-1,4-dione (17)
600 mg (3.5 mmol) of 16 dissolved in 90 mL of acetonitrile were
added to a solution of CAN (14.4 g, 26.4 mmol) in water (110 mL)

726
A. Aiello et al. / Bioorg. Med. Chem. 18 (2010) 719–727
mixture was poured into water and extracted with AcOEt (three
times). The extract was washed with water, dried over anhydrous
sodium sulfate, and filtered. Removal of the solvent gave crude
products 21 (490 mg) and 21a (620 mg), respectively, which were
purified by chromatography on ‘acidic’ silica gel (SiO2/compound
20:1; eluent: hexane/ethyl acetate 8:2). Compounds 21 (201 mg,
32%) and 21a (252 mg, 30%) were thus obtained sufficiently pure
for the following reaction.
6-Tetradecyl-7-methoxy-3,4-dihydro-1,1-dioxo-2H-1,4-benzo-
thiazine-5,8-dione (12)
ESI-MS (positive ion mode): m/z 462 [M+Na]+; HPLC: EtOAc/
hexane 6:4 (v/v), (tR): 11.0 min (single peak). 1H and 13C NMR data
are reported in Table 2.
7-Tetradecyl-6-methoxy-3,4-dihydro-1,1-dioxo-2H-1,4-benzo-
thiazine-5,8-dione (13)
1H NMR of compound 21 (CDCl3): 6.67 (1H, d, J = 9.9 Hz, H-6 or
H-5); 6.58 (1H, d, J = 9.9 Hz, H-5 or H-6); 4.01 (3H, s, –OCH3); 2.42
(2H, t, J = 7.2 Hz, –CH2(CH2)6CH3); 1.1–1.5 (12H, 6CH2); 0.87 (3H, t,
J = 7.2 Hz, –CH2(CH2)6CH3). ESI-MS: MH+ = m/z 251.
ESI-MS (positive ion mode): m/z 462 [M+Na]+; HPLC: EtOAc/
hexane 6:4 (v/v), (tR): 22.0 min. 1H and 13C NMR data are reported
in Table 2.
1H NMR of compound 21a (CDCl3): d 6.67 (1H, d, J = 9.9 Hz, H-6
or H-5); 6.58 (1H, d, J = 9.9 Hz, H-5 or H-6); 4.01 (3H, s, OCH3); 2.42
(2H, t, J = 7.2 Hz, –CH2(CH2)12CH3); 1.1–1.6 (24H, 12CH2); 0.87 (3H,
t, J = 7.2 Hz, –CH2(CH2)12CH3). ESI-MS: MH+ = m/z 335.
4.12. Computational details
DFT calculations were performed on a Pentium-4 processor at
3.0 GHz using the GAUSSIAN03 package (Revision B.05). The hypoth-
esized rotamers for A1 and A2 were optimized at the hybrid DFT
mPW1PW91 level using the 6-31G(d) basis set. GIAO 13C calcula-
tions were performed using the mPW1PW91 functional and 6-
31G(d,p) basis set, using as input the geometry previously opti-
mized at the mPW1PW91/6-31G(d) level. For these calculations,
the IEF-PCM solvent continuum model, as implemented in GAUSSIAN
(methanol solvent), was used.
4.10. Condensation of quinones 21 and 21a with hypotaurine
290 mg of 21 or 390 mg of 21a (1.16 mmol) were dissolved in
EtOH/CH3CN 1:1. The solutions were heated at 70 °C and hypotau-
rine (185 mg, 1.69 mmol), dissolved in the minimum amount of
water, was added in portions. The mixtures turned from yellow
to red orange. The end of reactions was controlled by TLC (eluent:
ethyl acetate/hexane 10:3). After solvent removing in vacuo, each
mixture was poured into water and extracted with EtOAc (three
times). The organic layer was washed with water, dried over anhy-
drous sodium sulfate, and filtered. Solvent removal gave residues
containing mixtures of compounds 10 and 11 (from 21, 163 mg,
39% yield) as well as of compounds 12 and 13 (from 21a,
158 mg, 31% yield); they were separated as reported below.
4.13. Cytotoxicity assays in Jurkat cells
The cells (106) were treated with increasing concentrations of
the compounds for 24 h and cytotoxicity assays were performed
by propidium iodide staining. Treated cells were incubated at room
temperature for 1 min in the presence of 10
lg/mL of propidium
iodide (PI) (in a total volume of 500 L of PBS). PI is a charged
l
hydrophilic compound, so living cells are impermeable to it. When
cell membranes are disrupted and the osmotic equilibrium is im-
paired (i.e., necrosis or late apoptosis) this compound can rapidly
penetrate into the cells and bind nucleic acids by charge affinity.
PI bound to nucleic acids exhibits red fluorescence upon excitation
with the 488 nm spectral line of the argon-ion laser of the flow
cytometer. After incubation, cells were immediately analyzed by
flow cytometry using an EPIC XL analyzer (Beckman-Coulter, Hia-
leah, MI).
4.11. Separation of crude mixtures of 8–9, 10–11, and 12–13
isomers
Separation of isomers 8 and 9 mixture (100 mg) was achieved
by HPLC on a SiO2 column (Luna 5
l
m, 250 ꢁ 4.60 mm) eluting
with EtOAc/hexane 6:4 (v/v) and afforded pure compounds 8
(17 mg) and 9 (62 mg). Mixture of isomers 10 and 11 (70 mg), as
well as that of 12 and 13 (50 mg), were separated in the same con-
ditions and yielded pure compounds 10 (5 mg), 11 (42 mg), 12
(3 mg), and 13 (30 mg).
4.14. Determination of nuclear DNA loss and cell cycle analysis
6-Ethyl-7-methoxy-3,4-dihydro-1,1-dioxo-2H-1,4-benzothiazine-
5,8-dione (8)
The percentage of cells undergoing chromatinolysis (subdiploid
cells) was determined as previously described.19 Briefly, the con-
trol and treated cells were fixed in ethanol (70%) for 24 h at 4 °C.
Then, the cells were washed twice with PBS containing 4% glucose
and subjected to RNA digestion (using RNAse-A, 50 U/mL) and PI
(20 mg/mL) staining in PBS for 1 h at RT. After incubation, cells
were immediately analyzed by flow cytometry using an EPIC XL
analyzer. With this method, low molecular weight DNA leaks from
the ethanol-fixed apoptotic cells and the subsequent staining al-
lows determination of the percentage of subdiploid cells (the
sub-G0/G1 fraction).
ESI-MS (positive ion mode): m/z 294 [M+Na]+; HPLC: EtOAc/
hexane 6:4 (v/v), (tR): 13.5 min (single peak). 1H and 13C NMR data
are reported in Table 1.
7-Ethyl-6-methoxy-3,4-dihydro-1,1-dioxo-2H-1,4-benzothi-
azine-5,8-dione (9)
ESI-MS (positive ion mode): m/z 294 [M+Na]+; HPLC: EtOAc/
hexane 6:4 (v/v), (tR): 23.0 min (single peak). 1H and 13C NMR data
are reported in Table 1.
6-Octyl-7-methoxy-3,4-dihydro-1,1-dioxo-2H-1,4-benzothi-
azine-5,8-dione (10)
4.15. Calcein uptake assay
ESI-MS (positive ion mode): m/z 378 [M+Na]+; HPLC: EtOAc/
hexane 6:4 (v/v), (tR): 12.0 min (single peak). 1H and 13C NMR data
are reported in Table 2.
The tumor adherent cell lines (104/well) were cultured in com-
plete medium in 96-well plates and incubated with increasing con-
centrations of the compounds for another 48 h. After treatment,
calcein-AM was added (final concentration 1 lM) and the cells
7-Octyl-6-methoxy-3,4-dihydro-1,1-dioxo-2H-1,4-benzothi-
azine-5,8-dione (11)
were incubated for 60 min. The uptake was then stopped by trans-
ferring the plates on ice and washing the cells twice with HBSS pre-
cooled to 4 °C. The fluorescence of the calcein generated within the
cells was analyzed in a Tecan Pro plate reader with 485-nm excita-
tion and 535-nm emission filters.
ESI-MS (positive ion mode): m/z 378 [M+Na]+; HPLC: EtOAc/
hexane 6:4 (v/v), (tR): 21.0 min (single peak). 1H and 13C NMR data
are reported in Table 2.

A. Aiello et al. / Bioorg. Med. Chem. 18 (2010) 719–727
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Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.;
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the relative light units (RLU)/
specific transactivation expressed as the percentage of transcrip-
tional activity compared to TNF alone (100%) or by showing the
lg of protein was calculated and the
a
absolute RLU numbers. All the experiments were repeated at least
four times.
Acknowledgments
This work was supported by MIUR (PRIN—Natural compounds
and synthetic analogues with antitumor activity). E.M. and M.A.C.
were supported by MEC Grant SAF2007-60305 and by the Spanish
RIS Network ‘Red de Investigación en SIDA’ (RD06-0006).
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