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

Article abstract of DOI:10.1016/S0968-0896(03)00310-9

Novel antitumoral agents with quinonic structure were synthesized and evaluated for their in vitro cytotoxic activities. This study examines the cytotoxic activities of several aryl benzo[g]isoquinoline-5,10-dione derivatives and a number of aminoacyl dih

Full text of DOI:10.1016/S0968-0896(03)00310-9

Bioorganic & Medicinal Chemistry 11 (2003) 3769–3775
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
Isabel Gomez-Monterrey,a Pietro Campiglia,a Paolo Grieco,a
Maria Vittoria Diurno,a Adele Bolognese,b Paolo La Collac
and Ettore Novellinoa,*
a
`
Dipartimento di Chimica Farmaceutica e Tossicologica, Universita di Napoli ‘Federico II’, Naples, Italy
`
`
Dipartimento di Chimica Organica e Biologica, Universita di Napoli ‘Federico II’, Naples, Italy
b
Dipartimento di Biologia Sperimentale, Sezione di Microbiologia, Universita di Cagliari, 09124 Cagliari, Italy
c
Received 3 January 2003; accepted 8 May 2003
Abstract—Novel antitumoral agents with quinonic structure were synthesized and evaluated for their in vitro cytotoxic activities.
This study examines the cytotoxic activities of several aryl benzo[g]isoquinoline-5,10-dione derivatives and a number of aminoacyl
dihydrothieno[2,3-b]naphtho-4,9-dione (DTNQ) derivatives containing amino acids in position 3 of the ring system. Compound 6
showed remarkable cytotoxic activity at submicromolar concentration not only against several human leukaemia and solid tumour
cell lines, but also toward sensitive and resistant human cell lines.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
In searching for agents with an improved pharmaco-
kinetic properties, potency or spectrum and lower side
effects, a large number of quinone derivatives and rela-
ted compounds have been prepared and several of these
have shown promise in clinical studies.8 Regarding
structural and chemical modifications of this system,
one of the most interesting modifications has been the
introduction of heteroatoms (N, S) into different posi-
tions of the chromophore through incorporation of one
or more of five or six-members heterocyclic ring to the
basic quinone system.9ꢀ11 These bioisosteres would
clearly differ from their carbocyclic counterparts in their
interaction with DNA and enzymes, as well as in their
reduction potential.
Quinone-containing antitumoral drugs such as doxo-
rubicin, and mitoxantrone (Fig. 1) have been estab-
lished as one of the most effective classes of anticancer
agents in clinical use today with broad application in
the treatment of several leukaemia and lymphomas as
well as in combination chemotherapy of solid tumours.1
However, toxic dose-related side effects such as myelo-
suppresion and cardiotoxicity, limited their clinical
application.2,3 The potent anticancer activity as well as
toxic effects described for these compounds are nor-
mally ascribed, at least, to two main mechanisms: one,
which is associated with protein, involves trapping of a
protein enzyme–DNA cleavable intermediate, whereas
the other, a non-protein-associated mechanism, is rela-
ted to redox cycling of the quinone moiety, which pro-
duces damaging free-radical species.4ꢀ7
We have recently reported12 an easy and versatile
method, in a one step reaction, directed towards the
synthesis of two new cyclic quinone derivatives, that is,
the benzo[g]isoquinoline-5,10-dione (2) (structurally
related to aza-anthracene quinones) and the dihy-
drothieno[2,3-b]naphtho-4,9-dione (DTNQ, 3) moiety
(a novel a,a0-disubstituted amino ester).
*Corresponding author. Fax: +39-081-678644; e-mail: novellin@
unina.it
0968-0896/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00310-9

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I. Gomez-Monterrey et al. / Bioorg. Med. Chem. 11 (2003) 3769–3775
properties and purity of the final compounds were
1
assessed by LC–MS, analytical RP-HPLC, H NMR,
and TLC.
Results and Discussion
Figure 1.
The compounds were tested in vitro for the growth
inhibition of MT-4 cell and the results are reported in
Table 1. As shown in table, the aryl-benzoisoquino-
lindione derivatives 2b, 2d, 2e and 2f, inhibited the pro-
liferation of MT-4 cell line at mM concentration. In
particular, the most potent compounds of this series
were 2d and 2f, supporting a chlorine atom at position
30 and/or 40 in aryl ring.
These compounds, prepared by cycloaddition reactions
between naphthoquinone and aryl thiazolidine deriva-
tives (Scheme 1), would clearly retain the planarity,
spatial, and electronic characteristics required for mole-
cular recognition at the cellular level, which seem deter-
mine the antitumoral activity described for structurally
related analogues.9,11
Interesting results were obtained by DTNQ derivatives
containing one amino acid in position 3 of the ring sys-
tem. We first tested the DTNQ moiety in racemic (3)
and in enantiomeric forms (3a and 3b). The enantio-
meric forms were separated and resolved as previously
described.12
This study examines the cytotoxic activities of several
aryl benzo[g]isoquinoline-5,10-dione derivatives and a
number of aminoacyl DTNQ derivatives containing
amino acids in position 3 of the ring system. The incor-
poration of the planar amide group would overall lead
to more effective target-binding affinity.13 In our case,
the amino acids Glycine, b-Alanine and Gly-Gly were
placed as they are nonchiral and conformationally
rather flexible, then l-Phe, d-Phe, and l-Ala were
incorporated, as suitable representatives for apolar,
chiral and restricted residues, which could affect the
affinity with potential target.
The racemic form 3 and the corresponding isomers 3a
and 3b showed similar activity against MT-4 cell line
(CC50=1.2, 1.3, and 3.2 mM, respectively). Subse-
quently, we prepared DTNQ analogues containing
l-Phe (4) and d-Phe (5) amino acids starting from
compound 3, the respective diastereisomeric forms of 4
(4a and 4b) and 5 (5a and 5b) were also resolved and
tested. The biological results demonstrated that both
Chemistry
The 1-aryl-3-ethoxycarbonylbenzo[g]isoquinoline-5,10-
dione derivatives, 2, and the compound 3-amino-3-
ethoxycarbonyl-2,3-dihydrothieno[2,3-b]naphtho -4,9-
dione (DTNQ), (ꢁ)3, were obtained by cycloaddition
reactions between the corresponding thiazolidine deri-
vatives 1a–h and napthoquinone as we previously
described (Scheme 1).12 Subsequently, the DTNQ deri-
vatives containing l- and d-Phe, Gly, l-Ala, b-Ala and
Gly-Gly (4–9), were prepared by coupling of DTNQ
moiety with the corresponding Boc protected amino
acid derivatives using HBTU/HOBt as coupling agents.
After Boc-deprotection with HCl or TFA, the resulting
products were obtained as racemic or diastereisomeric
mixture in high yield (70–75%). The physicochemical
Table 1. Antitumor activity of 1-aryl-3-ethoxycarbonylbenzo[g]-
isoquinoline-5,10-dione derivatives 2a–2h, and 3-substituted-amino-3-
ethoxycarbonyl-2,3-dihydrothiopheno[2,3-b]naptho-4,9-dione deriva-
tives 3–9
Compd
CC50 (mM)a
X
Y
2a
2b
2c
2d
2e
2f
100
13.0
>200
8.2
C6H5
4-CH3C6H4
4-(CH3)NC6H4
4-ClC6H4
3-ClC6H4
3,4-diClC6H3
—
—
—
—
—
—
11.0
8.4
2g
2h
3
(+)3a
(ꢀ)3b
4
4a
4b
5
5a
5b
6
>200
43.0
1.2
3-NO2C6H4
4-NO2C6H4
—
—
H
H
H
—
—
—
—
—
—
—
—
—
—
—
—
—
1.3
3.2
3.2
3.0
3.0
5.6
4.0
6.7
0.06
0.08
0.07
0.40
l-Phe
l-Phe
l-Phe
d-Phe
d-Phe
d-Phe
Gly
l-Ala
b-Ala
Gly-Gly
7
8
9
aCompound dose required to reduce the viability of mock-infected
cells (MT-4) by 50%, as determined by the MTT method.
Scheme 1. Reagents and conditions: (a) Ag2CO3, DBU, Acetonitrile,
rt, 12 h. (b) 1 NHCl, H O/CHCl3, 1 h.
2

I. Gomez-Monterrey et al. / Bioorg. Med. Chem. 11 (2003) 3769–3775
3771
diastereoisomeric mixtures (4, 5) and diastereisomeric
forms (4a, 4b, 5a, 5b) had similar activity and compar-
able to DTNQ (Table 1). These results suggest that the
stereochemistry may not have influence on interaction
of these structures with the target. Thus, we decided to
prepare and test all other compounds in respective
racemic and diastereoisomeric mixtures.
tumour cell lines, SK-MEl-28, MCF7, SK-MES-1,
DU145 (IC50 range=0.1–0.5 mM). Finally, since the
tumour cells possess different mechanisms which con-
fer resistance to a variety of anticancer agents,14 we
evaluated the cytotoxic activity of the compound 6
against the reference agents doxorubicine (Doxo), vin-
cristine (VCR), and etoposide (VP16) in a panel of
sensitive and resistant human cell lines of different
In particular we prepared analogues in which Gly (6),
l-Ala (7), b-Ala (8) and the dipeptide Gly-Gly (9) were
incorporated into DTNQ moiety. Surprisingly, the
compounds 6, 7 and 8 showed a significant increase of
activity of almost 20 times compared to lead compound
3 (CC50 0.06, 0.08, 0.07 and 1.2 mM, respectively), while
compound 9 showed to be active at 0.4 mM.
origin (KBwt, KBMDR, KB7D and KBV20C 15
) (Table 3).
It is worth to noting that compound 6 resulted to have
a fully inhibitory activity to all these resistant cell lines
(IC50 range=0.6–0.9 mM). Further experiments aimed
at defining the target and the mechanisms of the inhi-
bitory effect showed by these molecules are in
progress.
In an effort to understand better the biological profile
of this series of compounds, we choose one of the
most active compounds, that is compound 6, to test in
the panel of human tumour cell lines, using CEM,
L1210, SK-MEl-28, MCF7, SK-MES-1 and DU145
cells (Table 2). In this further study we used as refer-
ence several antineoplastic drugs. In accordance with
the results, this compound showed significant cytotoxic
activity not only against several leukaemia cell lines,
like CEM and L1210 (IC50=0.2 and 0.4 mM respec-
tively), but also toward a number of other solid
In conclusion, we report the synthesis and in vitro bio-
logical evaluation of new quinone derivatives as poten-
tial antitumoral agents. The first results showed that
DTNQ-Gly derivative, 6, was a potent compound
characterized by remarkable in vitro antitumoral activ-
ity toward several leukaemia, solid tumour, and also
resistant human cell lines. These structures represent
potential scaffolds in discovery of new agents with anti-
tumoral activity and the simple synthetic strategy
developed could be used to perform libraries of new
derivatives by combinatorial chemistry.
Table 2. Antitumor activity of 3-(glycyl)amino-3-etoxicarbonyl-2,3-dihydrothieno[2,3-b]naphtho-4,9-dione (6)
Compd
IC50(mM)a
SK-MEl-28e
MT-4b
CEMc
L1210d
MCF7f
SK-MES-1g
DU145h
6
0.06
0.01
0.0004
0.08
ND
0.2
0.4
0.2
0.003
0.2
ND
ND
0.5
0.5
0.05
1.1
0.1
0.2
0.1
0.003
0.4
0.04
0.1
0.07
0.005
0.1
0.01
0.3
Doxo
VCR
VP16
CAMPTO
CisPt
0.01
0.001
0.09
ND
ND
0.06
0.01
0.1
0.01
>100
ND
>100
>100
>100
aCompound concentration required to reduce cell proliferation by 50% as determined by the MTT method, under condition allowing untreated
controls to undergo at least three consecutive rounds of multiplication. Data represent mean values (ꢁSD) for three independent determinations.
bCD4+ human T-cells containing an integrated HTLV-1 genome.
cCD4+ human acute T-lymphoblastic leukaemia.
dMurine T-leukaemic cells.
eHuman skin melanoma.
fHuman breast adenocarcinome.
gHuman lung squamous carcinoma.
hHuman prostate carcinome. Doxorubicine (Doxo), vincristine (VCR), etoposide (VP16), camptothecin (CAMPTO), cisplatinun (CisPt).
Table 3. Effect of 6 on the proliferation of wild-type and drug-resistant KB cells
Compd
IC50(mM)a
b
KBwt
KBMDRc
KB7Dd
KBV20Ce
6
0.4ꢁ0.1
0.03ꢁ0.02
0.006ꢁ0.001
0.6ꢁ0.1
0.6ꢁ0.3
1.8ꢁ0.01
0.7ꢁ0.2
20ꢁ0.01
0.9ꢁ0.4
2.8ꢁ0.2
0.8ꢁ0.1
0.4ꢁ0.1
0.2ꢁ0.09
6.0ꢁ1.7
Doxo
VCR
VP16
0.05ꢁ0.01
20ꢁ0.05
aCompound concentration required to reduce cell proliferation by 50% as determined by the MTT method, under condition allowing untreated
controls to undergo at least three consecutive rounds of multiplication. Data represent mean values (ꢁSD) for three independent determinations.
bKB human nasopharyngeal carcinoma.
cKB subclones passaged in the presence of doxorubicin (Doxo) 0.09 mM.
dKB subclones passaged in the presence of etoposide (VP16) 7 mM.
eKB subclones passaged in the presence of vincristine (VCR) 0.02 mM.

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Experimental
8.32–8.34 (dd, 2H, 6-H and 9-H), 8.85 (s, 1H, 4-H). MS
[M+] calcd for C22H15O4N: 357.9, found: 358.3.
General. Reagents, starting material and solvents were
purchased from commercial suppliers and used as
received. Analytical TLC was performed on a 0.25 mm
layer of silica gel 60 F254 Merck and preparative TLC
on 20ꢂ20 cm glass plates coated with a 2 mm layer of
silica gel PF254 Merck. Silica gel 60 (300–400 mesh),
Merck, was used for flash chromatography. Melting
points were taken on a Kofler apparatus and are
uncorrected. Optical rotations were determined with a
Perkin–Elmer-241 MC polarimeter. 1H NMR spectra
were recorded with a Bruker-500 spectrometer, operat-
ing at 500 MHz. Chemical shifts are reported in d values
(ppm) relative to internal Me4Si and J values are
reported in Hertz (Hz). The ethyl 2(S,R)-(aryl)-1,3-
thiazolidine-4(S)-carboxylate derivatives 1a–h were pre-
pared following the procedure reported16 and were used
directly in the next step without further purification.
1-(40 -Methyl)phenyl-3-ethoxycarbonylbenzo[g]isoquino-
line-5,10-dione (2b). Yellow solid (37%), mp 263–
265 ꢃC. 1H NMR (500 MHz, CDCl3): d 1.40–1.42 (t, 3H,
CH3 ester), 2.45 (s, 3H, p-CH3), 4.49–4.52 (q, 2H, CH2
ester), 7.28–7.30 (m, 2H, aryl), 7.45–7.48 (m, 2H, aryl),
7.83–7.85 (m, 2H, 7-H and 8-H), 8.18–8.20 and 8.31–
8.33 (dd, 2H, 6-H and 9-H), 8.81 (s, 1H, 4-H). MS [M+]
calcd for C23H17O4N: 371.3, found: 371.7.
1-[40-(N-Dimethyl)amino]phenyl-3ethoxycarbonylbenzo[g]-
isoquinoline-5,10-dione (2c). Yellow crystalline solid
(10%), mp 243–244 ꢃC; H NMR (500 MHz, CDCl3): d
1
1.38–1.41 (t, 3H, CH3 ester), 2.98 (s, 6H, N-(CH3)2),
4.42–4.56 (q, 2H, CH2 ester), 6.69–6.71 (d, 2H, aryl),
7.50–7.52 (d, 2H, aryl) 7.76–7.80 (m, 2H, 7-H and 8-H),
8.14–8.16 and 8.22–8.24 (dd, 2H, 6-H and 9-H), 8.60 (s,
1H, 4-H). MS [M+] calcd for C24H20O4N2: 400.1,
found: 400.8.
General procedure for the synthesis of the 1-aryl-3
ethoxycarbonylbenzo[g]isoquinoline-5,10-dione deriva-
tives (2a–h) and 3-amino-3-ethoxycarbonyl-2,3-dihy-
1-(40-Chloro)phenyl-3-ethoxycarbonylbenzo[g]isoquino-
line-5,10-dione (2d). Yellow solid (15%), mp 217–
219 ꢃC; 1H NMR (500 MHz, CDCl3): d 1.45–1.48 (t, 3H,
CH3 ester), 4.51–4.55 (q, 2H, CH2 ester), 7.46–7.51 (dd,
4H, aryl), 7.85–7.88 (m, 2H, 7-H and 8-H), 8.18–8.20
and 8.33–8.35 (dd, 2H, 6-H and 9-H), 8.86 (s, 1H, 4-H).
MS [M+] calcd for C22H14O4NCl: 391.5, found: 392.1.
drothiopheno[2,3-b]naphtho-4,9-dione (3)
According to the procedure previously described,2 the
corresponding thiazolidine 1a–h (1–3 mmol) was dis-
solved in dry acetonitrile (20–50 mL) and naphthoqui-
none (2 equiv), silver carbonate (1 equiv respect to
thiazolidine) and a solution of DBU (0.2 equiv respect
to thiazolidine) in acetonitrile were added. After 12 h at
room temperature, diethyl ether was added, the mixture
was filtered and the solvent was evaporated. The residue
of the reaction was dissolved in chloroform and treated
with 1 NHCl solution (20–30 mL) for 1 h. Then,
chloroform and water were added, the organic phase
was washed with 1 NHCl (2 ꢂ25 mL), water (2ꢂ25 mL)
and dried with Na2SO4 anhydrous. Removal of the sol-
vent and flash chromatography of the residues, using
CHCl3 as eluent, yielded, in each case, the correspond-
ing Diels–Alder adducts 2a–h. In the other hand, the
collected acid aqueous phases were neutralized with
10% NaHCO3 solution and the free amine 3 (DTNQ)
was extracted with chloroform. Flash chromatography
using a gradient of 0–30% ethyl ether in CHCl3 gave
compound 3 as a orange oil: 11% from 1a, 20% from
1b, 50% from 1c, 45% from 1d, 38% from 1e, 45%
1 - (30 - Chloro)phenyl - 3 - ethoxycarbonylbenzo[g]isoquino-
line-5,10-dione (2e). Yellow solid (25%), mp 214–
215 ꢃC; 1H NMR (500 MHz, CDCl3): d 1.46–1.49 (t, 3H,
CH3 ester), 4.52–4.56 (q, 2H, CH2 ester), 7.42–7.44 (d,
1H, aryl), 7.48–7.51 (m, 2H, aryl), 7.54 (s, 1H, aryl),
7.84–7.87 (m, 2H, 7-H and 8-H), 8.17–8.19 and 8.33–
8.35 (dd, 2H, 6-H and 9-H), 8.88 (s, 1H, 4-H). MS [M+]
calcd for C22H14O4NCl: 391.5, found: 392.3.
1-(30,40-Dichloro)phenyl-3-ethoxycarbonylbenzo[g]isoquino-
line-5,10-dione (2f). Yellow solid (20%), mp 271–273 ꢃC;
1H NMR (500 MHz, CDCl3): d 1.46–1.49 (t, 3H, CH3
ester), 4.52–4.56 (q, 2H, CH2 ester), 7.34–7.36 (d, 1H,
aryl), 7.54–7.56 (d, 1H, aryl), 7.66 (s, 1H, aryl), 7.84–
7.87 (m, 2H, 7-H and 8-H), 8.23–8.25 and 8.33–8.35
(dd, 2H, 6-H and 9-H), 8.89 (s, 1H, 4-H). MS [M+]
calcd for C22H13O4NCl2: 425.8, found: 426.3.
1
from 1f, 47% from 1g and 45% from 1h. H N MR
(500 MHz, CDCl3) d, 1.25–1.28 (m, 3H, CH3 ester)
1-(30-Nitro)phenyl-3-ethoxycarbonylbenzo[g]isoquinoline-
5,10-dione (2g). Yellow solid (18%), mp 230–231 ꢃC;
1H NMR (500 MHz, CDCl3): d 1.46–1.49 (t, 3H, CH3
ester), 4.51–4.56 (q, 2H, CH2 ester), 7.65–7.68 (m, 2H,
aryl), 7.80–7.82 (d, 1H, aryl), 7.83 (s, 1H, aryl), 7.85–
7.88 (m, 2H, 7-H and 8-H), 8.16–8.18 and 8.38–8.40
(dd, 2H, 6-H and 9-H), 8.94 (s, 1H, 4-H). MS [M+]
calcd for C22H14O4N2: 402.9, found: 403.6.
0
3.26–3.29 (d, 1H, 2-H, J2,2 =12.3 Hz), 3.82–3.85 (d, 1H,
20-H), 4.27–4.31 (m, 2H, CH2 ester), 7.69–7.76 (m, 2H,
6-H and 7-H) 8.04–8.06 (d, 1H, 8-H or 5-H), 8.09–8.11
(d, 1H, 5-H or 8-H). MS [M+] calcd for C15H13O4NS:
303.3, found: 304.5.
The following substituted benzo[g]isoquinoline-5,10-
dione were prepared by the general method above.
1-(40-Nitro)phenyl-3-ethoxycarbonylbenzo[g]isoquinoline-
5,10-dione (2h). Yellow solid (15%), mp 235–236 ꢃC;
1H NMR (500 MHz, CDCl3): d 1.46–1.49 (t, 3H, CH3
ester), 4.51–4.56 (q, 2H, CH2 ester), 7.66–7.69 (m, 2H,
aryl), 7.80–7.82 (d, 2H, aryl), 7.86–7.89 (m, 2H, 7-H and
8-H), 8.18–8.20 and 8.38–8.40 (dd, 2H, 6-H and 9-H),
1 - Phenyl - 3 - ethoxycarbonylbenzo[g]isoquinoline - 5,10-
dione (2a). Yellow solid (49%), mp 218–219 ꢃC. 1H
NMR (500 MHz, CDCl3): d 1.45–1.48 (t, 3H, CH3
ester), 4.45–4.52 (q, 2H, CH2 ester), 7.05–7.55 (m, 5H,
aryl), 7.83–7.86 (m, 2H, 7-H and 8-H), 8.17–8.19 and

I. Gomez-Monterrey et al. / Bioorg. Med. Chem. 11 (2003) 3769–3775
3773
8.91 (s, 1H, 4-H). MS [M+] calcd for C22H14O4N2:
402.9, found: 403.7.
2H, 6-H and 7-H), 7.78 (s, 1H, NH), 7.97–7.99 (d, 1H,
8-H or 5-H), 8.06–8.08 (d, 1H, 5-H or 8-H).
3-[N-(tert-Butoxycarbonyl)-ꢀ-alanyl]amino-3-ethoxycar-
bonyl-2,3-dihydrothiopheno[2,3-b]-naphtho-4,9-dione (80).
Yellow oil (64% yield). H NMR (500 MHz, CDCl3) d
1.21–1.24 (m, 3H, CH3 ester), 1.37 (s, 9H, Boc), 2.40–
General procedure for the synthesis of the 3-(tert-butoxy-
carbonylaminoacyl)amino-3-ethoxycarbonyl-2,3-dihydro-
thiopheno[2,3-b]naphtho-4,9-dione derivatives (40-90)
1
The compound 3 (1–2 mmol) was dissolved in (3:1)
THF/DMF (30 mL) and the corresponding amino acid
(Boc-l-Phe, Boc-d-Phe, Boc-Gly, Boc-l-Ala, Boc-b-Ala
or Boc-Gly-Gly) (1 equiv), O-Benzotriazole-1-yl-
N,N,N0,N0-tetramethylisouronium hexafluoro phos-
phate (HBTU, 1.2 equiv), hydroxybenzotriazol (HOBt,
1.2 equiv) and diisopropylethylamine (DIEA, 2.4 equiv)
were added successively to that solution; stirring was
continued at room temperature for 48–72 h. After-
wards, the solvents were evaporated, the residue was
dissolved in CHCl3, washed successively with 10% citric
acid (2ꢂ25 mL), 10% NaHCO3 (2ꢂ25 mL), H2O (25
mL), dried over Na2SO4 and evaporated. Flash chro-
matography of the residues with 20–50% gradients of
EtOAc in n-hexane yielded in each case, the (1:1) dia-
steroisomeric mixtures 40–90.
2.43 (m, 2H, b-H), 3.31–3.35 (m, 2H, a-H), 3.70–3.73 (d,
0
0
1H, 2-H J2,2 =12.5 Hz), 3.77–3.80 (d, 1H, 2 -H), 4.25–
4.28 (m, 2H, CH2 ester), 5.15 (s, 1H, NH), 7.30 (s, 1H,
NH), 7.64–7.71 (m, 2H, 6-H and 7-H), 7.96–7.98 (d, 1H,
8-H or 5-H), 8.04–8.06 (d, 1H, 5-H or 8-H).
3-[N-(tert-Butoxycarbonyl)-glycyl-glycyl]amino-3-ethoxy-
carbonyl-2,3-dihydrothiopheno[2,3-b]naphtho-4,9-dione
(90). Yellow foam (61% yield). 1H NMR (500 MHz,
CDCl3) d 1.19–1.12 (m, 3H, CH3 ester), 1.38 (s, 9H,
0
Boc), 3.68–3.71 (d, 1H, 2-H J2,2 =12.5 Hz), 3.76–3.79
(d, 1H, 20-H), 3.83–3.84 and 3.99–4.00 (m, 4H, a, a0-H),
4.24–4.30 (m, 2H, CH2 ester), 3.83–3.84 and 3.99–4.00
(m, 4H, a, a0-H), 5.13 (s,1H, NH), 6.66 (s, 1H, NH),
7.60 (s, 1H, NH), 7.66–7.73 (m, 2H, 6-H and 7-H), 7.98–
8.00 (d, 1H, 8-H or 5-H), 8.06–8.08 (d, 1H, 5-H or 8-H).
3 - [N - (tert - Butoxycarbonyl) - L - phenylalanyl]amino - 3-
ethoxycarbonyl-2,3-dihydrothiopheno[2,3-b]naphtha-4,9-
General procedure for the deprotection of the N-Boc
group
dione (40). Yellow solid (55% yield) mp. 204–206. H
1
NMR (500 MHz, CDCl3) d 1.25–1.28 (m, 3H, CH3
ester), 1.39 (s, 9H, Boc), 2.95–3.04 (m, 2H, b-H), 3.56–
3.59, 3.66–3.68 and 3.78–3.80 (ddd, 2H, 2,20-H), 4.24-
4.30 (m, 2H, CH2 ester), 4.39–4.40 and 5.00–5.01 (m,
1H, a-H), 6.78 (s, 1H, NH), 7.13–7.23 (m, 5H, aryl),
7.71 (s, 1H, NH), 7.76–7.79 (m, 2H, 6-H and 7-H), 7.99–
8.01 (d, 1H, 8-H or 5H), 8.09–8.11 (d, 1H, 5-H or 8-H).
Synthesis of the compounds 4a, 4b, 5a, 5b, 7 and 8.
Method A. 3-[N-(tert-Butoxycarbonyl)-l or d-phenyl-
alanyl or l-alanyl or b-alanyl]amino-3-ethoxycarbonyl-
2,3-dihydrothiopheno[2,3-b]naphtho-4,9-dione (40, 50, 70
or 80) (0.2–0.3 mmol) were dissolved in saturated EtOAc
(HCl) solution (15 mL). After 4–6 h under stirring at
room temperature, the solution was neutralizated with
triethylamine and the solvents were evaporated to dry-
ness. Flash chromatography of 4, 5 with (90:10:1:1)
CH2Cl2–MeOH–HAcO–H2O yielded isolated in each
case, in decreasing order of Rf, the diasteroisomers 4a
and 4b or 5a and 5b. All compounds, including the
compounds 7 and 8, were collected as the chloride salts
after treatment with HCl(g)/ether saturated solution.
3 - [N - (tert - Butoxycarbonyl) - D - phenylalanyl]amino - 3-
ethoxycarbonyl-2,3-dihydrothiopheno[2,3-b]naphtho-4,9-
dione (50). Yellow solid (51% yield), mp 205–207. H
1
NMR (500 MHz, CDCl3) d 1.14–1.19 (m, 3H, CH3
ester), 1.39 (s, 9H, Boc), 2.98–3.05 (m, 2H, b-H), 3.57–
3.60, 3.67–3.70 and 3.78–3.81 (ddd, 2H, 2,20-H), 4.23–
4.33 (m, 2H, CH2 ester), 4.44–4.45 and 4.95–4.96 (m,
1H, a-H), 6.58 (s, 1H, NH), 7.12–7.23 (m, 5H, aryl),
7.61 (s, 1H, NH), 7.69–7.76 (m, 2H, 6-H and 7-H), 7.98–
8.00 (d, 1H, 8-H or 5H), 8.08–8.10 (d, 1H, 5-H or 8-H).
3-(L-Phenylalanyl]amino-3-ethoxycarbonyl-2,3-dihydro-
thiopheno[2,3-b]naphtho-4,9-dione hydrochloride (4a).
Yellow solid (98%), mp. 155–157 ꢃC. 1H
N MR
(500 MHz, CDCl3) d 1.25–1.28 (m, 3H, CH3 ester),
2.66–2.71 and 3.10–3.13 (m, 2H, b-H), 3.60–3.64 (m,
3-[N-(tert-Butoxycarbonyl)-glycyl]amino-3-ethoxycarbo-
nyl-2,3-dihydrothiopheno[2,3-b]naphtho-4,9-dione
(60).
Yellow foam (60% yield). H NMR (500 MHz, CDCl3)
1H, a-H), 3.74–3.77 (d, 1H, 2-H, J2,2 =12.5 Hz), 3.80–
0
3.83 (d, 1H, 20-H), 4.25–4.32 (m, 2H, CH2 ester), 7.34–
7.26 (m, 5H, aryl), 7.68–7.74 (m, 2H, 6-H and 7-H),
7.99–8.01 (d, 1H, 8-H or 5H), 8.09–8.11 (d, 1H, 5-H or
8-H), 8.85 (s, 1H, NH). [a]2D0=+18.1ꢃ (c 3.0, MeOH).
MS [M+] calcd for C24H23O5N2SCl: 486.9. Found:
487.3.
1
d 1.13–1.27 (m, 3H, CH3 ester), 1.43 (s, 9H, Boc), 3.71–
0
0
3.74 (d, 1H, 2-H, J2,2 =12.5 Hz), 3.78–3.81 (d, 1H, 2 -
H), 3.93–3.85 (m, 2H, a-H), 4.23–4.33 (m, 2H, CH2
ester), 5.23 (s, 1H, NH), 7.73–7.75 (m, 2H, 6-H and 7-
H), 7.78 (s, 1H, NH), 7.95–7.97 (d, 1H, 8-H or 5-H),
8.04–8.06 (d, 1H, 5-H or 8-H).
3-(L-Phenylalanyl]amino-3-ethoxycarbonyl-2,3-dihydro-
thiopheno[2,3-b]naphtho-4,9-dione hydrochloride (4b).
Yellow solid (96%), mp. 160–162. H NMR (500 MHz,
CDCl3) d 1.22–1.25 (m, 3H, CH3 ester), 2.68–2.73 and
3-[N-(tert-Butoxycarbonyl)-L-alanyl]amino-3-ethoxycar-
bonyl-2,3-dihydrothiopheno[2,3-b]naphtho-4,9-dione (70).
Yellow oil (59% yield). H NMR (500 MHz, CDCl3) d
1
1
1.20–1.24 (m, 3H, CH3 ester), 1.38 (s, 9H, Boc), 3.40–
3.43 (m, 3H, CH3), 3.69–3.72 and 3.77–3.80 (m, 2H,
2,20-H), 4.07–4.08 and 4.03–4.04 (m, 2H, a-H), 4.21–
4.29 (m, 2H, CH2 ester), 5.23 (s, 1H, NH), 7.67–7.72 (m,
3.24–3.27 (m, 2H, b-H), 3.60–3.64 (m, 1H, a-H), 3.74–
0
0
3.77 (d, 1H, 2-H, J2,2 =12.5 Hz), 3.81–3.84 (d, 1H, 2 -
H), 4.27–4.29 (m, 2H, CH2 ester), 7.36–7.28 (m, 5H,
aryl), 7.63–7.67 (m, 2H, 6-H and 7-H), 7.99–8.01 (d, 1H,

3774
I. Gomez-Monterrey et al. / Bioorg. Med. Chem. 11 (2003) 3769–3775
0
00
J2,2 =12.4 Hz), 3.61–3.64 (d, 1H, 2 -H), 3.90–3.92 (m,
8-H or 5H), 8.08–8.10 (d, 1H, 5-H or 8-H), 8.89 (s, 1H,
NH). [a]2D0=+38.1ꢃ (c 2.5, MeOH). MS [M+] calcd for
C24H23O5N2SCl: 486.9. Found: 487.5.
2H, a-H), 4.15–4.20 (m, 2H, CH2 ester), 7.57–7.67 (m,
2H, 6-H and 7-H), 7.88–7.90 (d, 1H, 8-H or 5-H), 7.97–
7.99 (d, 1H, 5-H or 8-H), 8.23 (s, 1H, NH). MS [M+]
calcd for C19H17O7N2SF3: 475.2. Found: 476.7.
3-(D-Phenylalanyl)amino-3-ethoxycarbonyl-2,3-dihydro-
thiopheno[2,3-b]naphtho-4,9-dione hydrochloride (5a).
1
Yellow solid (96%), mp. 157–158. H NMR (500 MHz,
CDCl3) d 1.22–1.25 (m, 3H, CH3 ester), 2.68–2.73 and
3-(Glycyl-glycyl)amino-3-ethoxycarbonyl-2,3-dihydro-
thiopheno[2,3-b]naphtho-4,9-dione trifluoroacetate (9).
1
3.24–3.27 (m, 2H, b-H), 3.60–3.64 (m, 1H, a-H), 3.74–
Yellow solid (89%) mp. 231–233. H NMR (500 MHz,
CDCl3) d 1.12–1.20 (m, 3H, CH3 ester), 2.72–2.74 (m,
0
0
3.77 (d, 1H, 2-H, J2,2 =12.5 Hz), 3.81–3.84 (d, 1H, 2 -
H), 4.27–4.29 (m, 2H, CH2 ester), 7.36–7.28 (m, 5H,
aryl), 7.63–7.67 (m, 2H, 6-H and 7-H), 7.99–8.01 (d, 1H,
8-H or 5H), 8.08–8.10 (d, 1H, 5-H or 8-H), 8.89 (s, 1H,
NH). [a]2D0=ꢀ17.8ꢃ (c 1.2, MeOH). MS [M+] calcd for
C24H23O5N2SCl: 486.9. Found: 487.8.
2H, a-H), 3.46–3.48 (m, 2H, a0-H), 3.67–3.70 (d, 1H, 2-
0
00
H, J2,2 =12.5 Hz), 3.76–3.79 (d, 1H, 2 -H), 4.15–4.28
(m, 2H, CH2 ester), 7.12 (s, 1H, NH), 7.57–7.63 (m, 2H,
6-H and 7-H), 7.89–7.91 (d, 1H, 8-H or 5H), 7.98–8.00
(d, 1H, 5-H or 8-H), 8.62 (s, 1H, NH). MS [M+] calcd
for C21H20O8N3SF3: 532.3. Found: 532.9.
3-(D-Phenylalanyl)amino-3-ethoxycarbonyl-2,3-dihydro-
thiopheno[2,3-b]naphtho-4,9-dione hydrochloride (5b).
Yellow solid (96%), mp 161–162. H NMR (500 MHz,
CDCl3) d 1.22–1.25 (m, 3H, CH3 ester), 2.68–2.73 and
Biological methods
1
Compounds were dissolved in DMSO at an initial con-
centration of 200 mM and then were serially diluted in
culture medium.
3.24–3.27 (m, 2H, b-H), 3.60–3.64 (m, 1H, a-H), 3.74–
0
0
3.77 (d, 1H, 2-H, J2,2 =12.5 Hz), 3.81–3.84 (d, 1H, 2 -
H), 4.27–4.29 (m, 2H, CH2 ester), 7.36–7.28 (m, 5H,
aryl), 7.63–7.67 (m, 2H, 6-H and 7-H), 7.99–8.01 (d, 1H,
8-H or 5H), 8.08–8.10 (d, 1H, 5-H or 8-H), 8.89 (s, 1H,
Cells
20
NH). [a] D =ꢀ39.2ꢃ (c 3.0, MeOH). MS [M+] calcd for
Cell lines were from American Type Culture Collection
(ATCC). The human nasopharyngeal carcinoma KB
cell line and the drug-resistant subclones KBMDR
C24H23O5N2SCl: 486.9. Found: 487.7.
,
3-(L -Alanyl)amino-3-ethoxycarbonyl -2,3-dihydrothio-
pheno[2,3-b]naphtho-4,9-dione hydrochloride (7). Yellow
foam (86%). H NMR (500 MHz, CDCl3) d 1.18–1.20
(m, 3H, CH3 ester), 2.61–2.62 and 2.65–2.66 (m, 3H,
CH3), 3.30–3.32 and 3.34–3.36 (m, 1H, a-H), 3.67–3.70,
3.77–3.80, and 3.79–3.81 (ddd, 2H, 2,20-H), 4.26–4.29
(m, 2H, CH2 ester), 7.54–7.61 (m, 2H, 6-H and 7-H),
7.97–7.99 (d, 1H, 8-H or 5H), 8.07–8.09 (d, 1H, 5-H or
8-H), 8.24 (s, 1H, NH). MS [M+] calcd for
C18H19O5N2SCl: 411.1. Found: 411.7.
KB7D, and KBV20C were a generous gift of Prof. Y. C.
Cheng, Yale University. H9/IIIb, MT-4, and C8166
(grown in RPMI 1640 containing 10% fetal calf serum
(FCS), 100 U/mL penicillin G, and 100 mg/mL strepto-
mycin) cells were used for anti-HIV assays. Cell cultures
were checked periodically for the absence of myco-
plasma contamination with a MycoTect kit (Gibco).
1
Cytotoxic and antiviral assays
Cytotoxicity of compounds, based on the viability of
mock-infected cells, as monitored by the MTT
method17,18 was evaluated in parallel with their antiviral
activity. Activity of compounds against the HIV-1 mul-
tiplication in acutely infected cells was based on inhibi-
tion of virus-induced cytopathogenicity in MT-4 cells.
3-(ꢀ-Alanyl)amino-3-ethoxycarbonyl-2,3-dihydrothio-
pheno[2,3-b]naphtho-4,9-dione hydrochloride (8). Yellow
foam (95%). H NMR (500 MHz, CDCl3) d 1.23–1.26
1
(m, 3H, CH3 ester), 2.99–3.19 (m, 2H, a-H), 3.38–3.39
00
(m, 2H, b-H), 3.82–3.85 (d, 1H, 2-H, J2,2 =12.5 Hz),
3.94–3.97 (d, 1H, 20-H), 4.26–4.32 (m, 2H, CH2 ester),
7.60–7.74 (m, 2H, 6-H and 7-H), 7.94–7.96 (d, 1H, 8-H
or 5H), 8.07–8.09 (d, 1H, 5-H or 8-H), 8.51 (s, 1H, NH).
MS [M+] calcd for C18H19O5N2SCl: 411.1. Found:
411.8.
Antitumor assays
Exponentially growing leukaemia and lymphoma cells
were resuspended at a density of 1ꢂ105 cells/mL in RPMI
containing serial dilutions of the test drugs. Cell viability
was determined after 4 days at 37 ꢃC by the MTT method.
Activity against cell lines derived from solid tumours was
evaluated in exponentially growing cultures seeded at
5ꢂ104 cells/mL which were allowed to adhere for 16 h to
culture plates before addition of the drugs. Cell viability
was determined by the MTT method for 4 days later.
Synthesis of 3-(glycyl and glycyl-glycyl)amino-3-ethoxy-
carbonyl-2,3-dihydrothiopheno-[2,3-b]naphtho - 4,9 - dione
(6 and 9). Method B. TFA (0.5 mL) was added to a solu-
tion of Boc-protected compounds 60 or 90 (0.1 mmol) in
CH2Cl2 ( 5 mL), stirring was continued for 3–4 h at room
temperature, the reaction mixture was concentrated to
half volume and ether was added. The title compounds as
the trifluoroacetate salt, were racolted by filtration.
Linear regression analysis
3-(Glycyl)amino-3-ethoxycarbonyl-2,3-dihydrothiopheno-
[2,3-b]naphtho-4,9-dione trifluoroacetate (6). Yellow
solid (97%), mp 214–215. H NMR (500 MHz, CDCl3)
d 1.14–1.21 (m, 3H, CH3 ester), 3.57–3.54 (d, 1H, 2-H,
Tumour cell growth at each drug concentration was
expressed as percentage of untreated controls, and the
concentration resulting in 50% (CC50, IC50) growth
inhibition was determined by linear regression analysis.
1

I. Gomez-Monterrey et al. / Bioorg. Med. Chem. 11 (2003) 3769–3775
3775
Acknowledgements
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This work was supported by grant from Regione Cam-
pania ai sensi della L.R.31 Dicembre 1994, No. 41, art.
3, 1ꢃ comma. The NMR spectral data and FAB-Massa
were provided by Centro di Ricerca Interdipartimentale
di Analisi Strumentale, Universita degli Studi di Napoli
‘Federico II’. The assistance of the staff is gratefully
appreciated.
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