Design, synthesis, and cytotoxic evaluation of a new series of 3-substituted spiro[(dihydropyrazine-2,5-dione)-6,3′-(2′,3′- dihydrothieno[2,3-b]naphtho-4′,9′-dione)] derivatives

Article abstract of DOI:10.1021/jm0612158

A series of 3-substituted spiro[(dihydropyrazine-2,5-dione)-6,3′- (2′,3′-dihydrothieno[2,3-b]naphtho-4′,9′-dione)] derivatives were prepared using an easy synthetic route via condensation of the 3-amino-3-(ethoxycarbonyl)-2,3-dihydrothieno[2,3-b]naphtho-4

Full text of DOI:10.1021/jm0612158

J. Med. Chem. 2007, 50, 1787-1798
1787
Design, Synthesis, and Cytotoxic Evaluation of a New Series of 3-Substituted
Spiro[(dihydropyrazine-2,5-dione)-6,3′-(2′,3′-dihydrothieno[2,3-b]naphtho-4′,9′-dione)]
Derivatives
Isabel Gomez-Monterrey,† Pietro Campiglia,‡ Alfonso Carotenuto,† Daniela Califano,§ Claudio Pisano,| Loredana Vesci,|
Teresa Lama,† Alessia Bertamino,† Marina Sala,† Antonio Mazzella di Bosco,† Paolo Grieco,† and Ettore Novellino*,†
Dipartimento di Chimica Farmaceutica e Tossicologica, UniVersity of Naples “Federico II”, Napoli, Dipartimento di Scienze Farmaceutica,
UniVersita` di Salerno, Salerno I-84084, Fisciano, Oncologia Sperimentale E, Istituto dei Tumori Fondazione “G. Pascale”, Napoli, and Area
di Ricerca OncologicasR&S, Sigma-Tau S.p.A., Pomezia, Roma, Italy
ReceiVed October 18, 2006
A series of 3-substituted spiro[(dihydropyrazine-2,5-dione)-6,3′-(2′,3′-dihydrothieno[2,3-b]naphtho-4′,9′-
dione)] derivatives were prepared using an easy synthetic route via condensation of the 3-amino-3-
(ethoxycarbonyl)-2,3-dihydrothieno[2,3-b]naphtho-4,9-dione system and amino acids followed by intramo-
lecular lactamization. Amino acids containing alkyl and aryl, linear and cyclic, polar and apolar, and basic
and acid residues were incorporated. Evaluation of these analogues against the MCF-7 human breast carcinoma
and SW 620 human colon carcinoma cell lines revealed, for the 3S,3′R isomers derived from Pro (7a), Cys
(11a), and Met (12a) and the 3R,3′S isomer derived from D-Pro (7c), a cytotoxic potency comparable to or
greater than that of doxorubicin. Some of these selected analogues were potent cytotoxic agents in several
other sensible and resistant human solid tumor cell lines and may be able to circumvent the multiple-drug-
resistance mechanism. In particular, only a partial cross-resistance to the compounds 7, 11, and 12 was
observed in selected tumor cell sublines known to be resistant to doxorubicin (MCF-7/Dx and A2780/Dx),
whereas a very low level of cross-resistance to compounds 7 and 11 was found in a tumor cell subline
selected for resistance to cisplatin (A2780/DDP). In addition, the topoisomerase II inhibition activity and
DNA-binding properties were investigated.
Introduction
The antitumoral anthracyclines daunorubicin and doxorubicin
and the synthetic anthracene-9,10-dione mitoxantrone are potent
agents in clinical use today with broad application in the
treatment of several leukemias and lymphomas as well as in
combination chemotherapy of solid tumors.1,2 The therapeutic
relevance for these quinone derivatives has prompted intensive
research into new structures with the goal of bypassing
significant problems that limit the utility of these compounds,
notably toxic dose-related side effects and development of drug
resistance.3-5 A large number of quinonic derivatives have been
prepared, and several of these have shown promise in clinical
studies.6,7 Prominent examples are the azaanthracene derivative
6,9-bis[(2-aminomethyl)amino]benz[g]isoquinoline-5,10-di-
one dimaleate salt (BBR 2778),8 the anthrapyrazole derivative
losoxantrone (DuP-941)9,10 (Figure 1), and some anthracenyl
peptide derivatives which have been shown to be non-cross-
resistant in MDR and AT-MDR cell lines and exhibited in vivo
activity against tumor xenografts.11,12 The mechanisms of cell-
killing induction by these compounds are multimodal in nature.
Their planarity allows an intercalation between specific base
pairs of DNA, while their redox properties are linked to the
production of radical species in biological systems.13 The
chemical and biological activity exhibited by these quinonic
compounds is greatly affected by the different substituents of
the planar ring system. The side chain usually bears one or two
positive charges to establish an electrostatic interaction with the
Figure 1. Structure of BBR 2778, losoxantrone, and the DTNQ
template and its derivatives.
phosphate backbone of the polynucleotide. In addition, they meet
the requirements for effective poisoning of topoisomerase II.14-16
In fact, the mechanism of action appears to be mainly related
to drug binding to the so-called “cleavable complex” formed
by DNA and topoisomerase II.17
* To whom correspondence should be addressed. Phone: +39-081-
678643. Fax: +39-081-678644. E-mail: novellin@unina.it.
† University of Naples “Federico II”.
‡ Universita` di Salerno.
§ Istituto dei Tumori Fondazione “G. Pascale”.
| Sigma-Tau S.p.A.
10.1021/jm0612158 CCC: $37.00 © 2007 American Chemical Society
Published on Web 03/22/2007

1788 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Gomez-Monterrey et al.
Scheme 1a
a Reagents and conditions: (i) Fmoc-Aa-OH, HBTU, HOBt, DMF, rt, 24 h; (ii) 20% piperidine/CH2Cl2, rt, 30 min, then MeOH, TEA, reflux, 1-3 h; (iii)
20% TFA in dichloromethane.
Therefore, the development of innovative agents and/or novel
combinations is needed to improve the therapeutic effectiveness
of this important class of compounds. In this regard, we have
recently highlighted the potential value of a new planar ring
system, the 3-amino-3-(ethoxycarbonyl)-2,3-dihydrothieno[2,3-
b]naphtho-4,9-dione system (I, DTNQ), as a template in the
development of effective cytotoxic agents (Figure 1).18-20
Previously, we explored the biological properties of two series
related to the lead compound I. In the first series, we introduced
amino acid and dipeptide residues in position 3 of the planar
structure (general formula II).19 The compound 3-(glycylamino)-
3-(ethoxycarbonyl)-2,3-dihydrothieno[2,3-b]naphtho-4,9-di-
one (2a, R ) H) retains cytotoxic activity at submicromolar
concentration against several human leukemia and solid tumor
cell lines. In the second series, we designed a set of spirohy-
dantoin derivatives in which the planar chromophore (DTNQ)
was combined, through the hydantoin ring, to different alkyl-,
hydroxyalkyl-, and aminoalkyl-substituted side chains (general
formula III). The 3-[2-(N,N-dimethylamino)ethyl or propyl]-
spiro[(dihydroimidazo-2,4-dione)-5,3′-(2′,3′-dihydrothieno[2,3-
b]naphtho-4′,9′-dione)] hydrochloride derivatives (3a, 3b) showed
remarkable biological activity, not only against several human
solid tumor cell lines (comparable to that of doxorubicin), but
also toward doxorubicin- and cis-platinum-resistant human cell
lines.20 The increment of cytotoxic activity observed for these
DTNQ-based derivatives seems to indicate that the conforma-
tional restriction introduced by the hydantoin ring allows an
optimal location of the planar ring relative to the side chain.
On the basis of this finding, we decided to modify both the
dimension and side-chain nature of the spiro nucleus attached
to the DTNQ chromophore. Thus, a series of 3-substituted spiro-
[(dihydropyrazine-2,5-dione)-6,3′-(2′,3′-dihydrothieno[2,3-b]-
naphtho-4′,9′-dione)] derivatives as well as the pentacyclic
analogues spiro[(hexahydropyrrolo[1,2-a]pyrazine-1,4-dione)-
6,3′-(2′,3′-dihydrothieno[2,3-b]naphtho-4′,9′-dione)] were de-
signed and synthesized (general formula IV). Our synthetic
approach involves the condensation of the 3-amino-3-(ethoxy-
carbonyl)-2,3-dihydrothieno[2,3-b]naphtho-4,9-dione system with
conveniently protected amino acids and subsequent intramo-
lecular lactamization. This modification has been performed to
define which functional side chain is critical for cytotoxic
activity and to explore the structural parameters associated with
the biological activity. Thus, Gly, Ala, Leu, L- and D-Pro, Phe,
Tyr, Ser, Cys, Met, Lys, His, Asp, and Asna were incorporated
as suitable representatives for alkyl and aryl, linear and cyclic,
polar and apolar, and basic and acid residues. The present paper
deals with the synthesis, structural determination, and cytotoxic
activity of this new series of compounds. In addition, some of
the most active compounds were evaluated for their topoi-
somerase II inhibition activity and DNA-binding properties.
Results and Discussion
Chemistry. The preparation of the spirodiketopiperazine
derivatives was performed following the synthetic route de-
scribed in Scheme 1. The starting DTNQ was obtained by
cycloaddition reaction between the 2-(4′-chlorophenyl)thiazo-
lidine and naphthoquinone as we previously described.18 The
condensation of the DTNQ system with the corresponding
N-Fmoc- and side-chain-protected amino acids, using HBTU
and HOBt as condensating agents, afforded pseudodipeptide 2
or 2′ (from proline) as a mixture of diastereoisomers. Mildly
acid-sensitive side-chain protecting groups (X ) Boc, tBu, Trt)
were used for the synthesis of the derivatives containing Ser,
a Abbreviations: AAA, amino acid analysis; Boc, tert-butyloxycarbonyl;
t
Trt, trityl; Bu, tert-butyl; CH3CN, acetonitrile; DCM, dichoromethane;
DIPEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; DQF-
COSY, double-quantum-filtered correlated spectroscopy; Et3SiH, triethyl-
silane; FAB-MS, fast-atom bombardment mass spectrometry; Fmoc,
9-fluorenylmethoxycarbonyl; HOBt, N-hydroxybenzotriazole; HBTU, 2-(1H-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; MD,
molecular dynamic; NOE, nuclear Overhauser effect; NOESY, nuclear
Overhauser enhancement spectroscopy; Pbf, 2,2,4,6,7-pentamethyldihy-
drobenzofuran-5-sulfonyl; FC, flash chromatography; RP-HPLC, reversed-
phase high-performance liquid chromatography; SDS, sodium dodecyl
sulfate; TFA, trifluoroacetic acid; TLC, thin-layer chromatography; TOCSY,
total correlated spectroscopy; Trt, triphenylmethyl (trityl). Abbreviations
used for amino acids and designation of peptides follow the rules of the
IUPAC-IUB Commission of Biochemical Nomenclature in J. Biol. Chem.
1972, 247, 977-983. Amino acid symbols denote the L-configuration unless
indicated otherwise.

Synthesis of a New Series of DTNQ-Based DeriVatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1789
Table 1. Cytotoxic Activity of 3-Substituted Spiro[(dihydropirazine-2,5-dione)-6,3′-(2′,3′-dihydrothieno[2,3-b]naphtho-4′,9′-dione)] Derivatives
4a,b-6a,b and 8a,b-16a,b and Spiro[(hexahydropyrrolo[1,2-a]pyrazine-1,4-dione)-6,3′-(2′,3′-dihydrothieno[2,3-b]naphtho-4′,9′-dione)] Derivatives
7a-c
IC50a (µM ( SD)
stereochemistry
compd
3,3′
R
MCF-7b
SW620c
2a
4a,b
4a
4b
5a
5b
6a
6b
7a
7b
7c
7d
8a
8b
9a
9b
10a
10b
11a
11b
12a
12b
13ad
13bd
14a
14b
15a
15b
16a
16b
doxorubicin
3SR
3′SR
3′R
3′S
H
H
H
H
CH3
CH3
0.196 ( 0.005
0.047 ( 0.001
0.046 ( 0.001
0.049 ( 0.001
0.053 ( 0.013
0.098 ( 0.010
0.090 ( 0.013
0.160 ( 0.007
0.020 ( 0.004
0.263 ( 0.047
0.017 ( 0.008
0.213 ( 0.060
0.060 ( 0.010
0.140 ( 0.040
0.085 ( 0.009
0.154 ( 0.010
0.243 ( 0.030
0.456 ( 0.050
0.029 ( 0.009
0.069 ( 0.009
0.036 ( 0.009
0.108 ( 0.019
0.083 ( 0.011
0.179 ( 0.007
0.660 ( 0.111
1.850 ( 0.102
1.700 ( 0.110
1.930 ( 0.050
0.351 ( 0.030
0.835 ( 0.013
0.022 ( 0.008
1.100 ( 0.001
0.245 ( 0.036
0.267 ( 0.008
0.326 ( 0.010
0.287 ( 0.014
0.776 ( 0.014
0.414 ( 0.030
0.476 ( 0.011
0.120 ( 0.030
0.840 ( 0.030
0.010 ( 0.020
1.200 ( 0.300
0.445 ( 0.170
0.459 ( 0.180
0.700 ( 0.050
1.000 ( 0.300
0.894 ( 0.040
1.530 ( 0.250
0.210 ( 0.008
0.224 ( 0.050
0.229 ( 0.031
0.765 ( 0.100
0.825 ( 0.079
1.715 ( 0.160
1.929 ( 0.163
>4000
3S,3′R
3S,3′S
3S,3′R
3S,3′S
3S,3′R
3S,3′S
3R,3′S
3R,3′R
3S,3′R
3S,3′S
3S,3′R
3S,3′S
3S,3′R
3S,3′S
3R,3′R
3R,3′S
3S,3′R
3S,3′S
3S,3′R
3S,3′S
3S,3′R
3S,3′S
3S,3′R
3S,3′S
3S,3′R
3S,3′S
CH2CH(CH3)2
CH2CH(CH3)2
cyclo(CH2CH2CH2)-
cyclo(CH2CH2CH2)-
cyclo(CH2CH2CH2)-
cyclo(CH2CH2CH2)-
CH2C6H5
CH2C6H5
CH2C6H4(4-OH)
CH2C6H4(4-OH)
CH2OH
CH2OH
CH2SH
CH2SH
CH2CH2SCH3
CH2CH2SCH3
(CH2)4NH2
(CH2)4NH2
CH2-imidazole
CH2-imidazole
CH2COOH
CH2COOH
CH2CONH2
0.285 ( 0.059
>4000
1.528 ( 0.032
3.353 ( 0.079
0.178 ( 0.003
CH2CONH2
a Data represent mean values ((SD) for three independent determinations. b Human breast carcinoma cell line. c Human colon carcinoma cell line. d Evaluated
as TFA salts.
Cys, Lys, Hys, Asp, and Asn. Then, Fmoc-amino deprotection
in 20% piperidine/CH2Cl2 and successive treatment with TEA
in MeOH at reflux afforded the corresponding 3-substituted
spiro[(dihydropyrazine-2,5-dione)-6,3′-(2′,3′-dihydrothieno[2,3-
b]naphtho-4′,9′-dione)] derivatives 4-6, 8, and 12 and the spiro-
[(hexahydropyrrolo[1,2-a]pyrazine-1,4-dione)-6,3′-(2′,3′-dihy-
drothieno[2,3-b]naphtho-4′,9′-dione)] derivatives 7 in 46-60%
overall yields.
Finally, after removal of the side-chain protecting group using
20% TFA in dichloromethane, the final compounds 9-11 and
13-16 were obtained in 36-53% overall yields. Since the
synthetic pathway used for the preparation of the starting DTNQ
generates an asymmetric carbon (C-3) in this system, the final
compound 4 (derived from Gly) was obtained as a racemic
mixture, while the other compounds were obtained as diaste-
reoisomer mixtures. The synthesis of the enantiomerically pure
compounds 4a and 4b was performed starting from Fmoc-Gly
and pure enantiomers of DTNQ, which were resolved according
to the procedure previously described.18,21 For the other
compounds (5-16), HPLC analysis of the resulting crude
products showed the presence of both 3S,3′R (a) and 3S,3′S (b)
diastereoisomers (a/b ) 3/2 to 1/1 ratio), which in all cases
were separated after flash chromatography. The same procedure
allowed the separation of 7c (3R,3′S) and 7d (3R,3′R) obtained
from D-Pro. The physicochemical properties and purity of the
final compounds were assessed by TLC, LC-MS, analytical
RP-HPLC, and NMR analysis (Experimental Section).
Stereochemistry Determination. The determination of the
absolute configuration at the C-3′ asymmetric center was
performed by an NMR study of representative derivatives 7a,
7b, 8a, 8b, 9a, 9b, 14a, and 14b, which showed a major
descrimination by NMR. The only difference observed in the
2D NOESY22 spectra of “type a” derivatives compared to “type
b” (Supporting Information) was the presence in the last of NOE
contacts of both H-2′a (H-2′ low-field signal) and H-2′b (H-2′
high-field signal) with H-3 (corresponding to HR of the proline
moiety). Inspections of molecular models obtained by molecular
dynamic simulation (Experimental Section) showed that the
distances of both H-2′(s) and H-3 were about 3.9 Å in the 3S,3′S
isomer and about 5.2 Å in the 3S,3′R isomer. Hence, the
stereochemistry 3S,3′R could be assigned to compounds of type
a and 3S,3′S to compounds of type b. The above assignment is
in accordance with the downfield shift observed for the H-2′a
signal in compounds a compared to the same signal in

1790 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Gomez-Monterrey et al.
Table 2. Inhibition of Multiple Human Tumor Cell Lines by Selected Compounds
IC50a (µM ( SD)
NCI-H460
lung
HCT-116
ilocecal
PC-3
prostate
LoVo
colon
MeVo
melanoma
compd
A2780ovarian
7a
7b
7c
7d
11a
11b
12a
12b
doxorubicin
0.105 ( 0.001
0.530 ( 0.03
0.036 ( 0.002
0.190 ( 0.02
0.190 ( 0.02
0.460 ( 0.15
0.180 ( 0.03
0.624 ( 0.05
0.007 ( 0.001
0.114 ( 0.01
0.994 ( 0.10
0.052 ( 0.01
0.170 ( 0.01
0.470 ( 0.04
0.730 ( 0.06
0.256 ( 0.03
0.917 ( 0.03
0.010 ( 0.008
0.090 ( 0.010
0.838 ( 0.030
0.240 ( 0.002
3.300 ( 0.100
0.350 ( 0.030
0.660 ( 0.020
0.256 ( 0.010
0.766 ( 0.050
0.153 ( 0.01
0.610 ( 0.09
0.110 ( 0.01
1.900 ( 0.30
0.307 ( 0.05
0.367 ( 0.058
0.293 ( 0.017
0.582 ( 0.05
0.420 ( 0.03
1.410 ( 0.10
0.320 ( 0.06
1.700 ( 0.02
0.870 ( 0.10
1.300 ( 0.10
0.540 ( 0.07
0.620 ( 0.05
0.033 ( 0.004
0.075 ( 0.01
0.570 ( 0.004
0.150 ( 0.01
7.200 ( 1.60
0.075 ( 0.02
0.567 ( 0.10
0.057 ( 0.01
1.130 ( 0.05
0.820 ( 0.10
a Data represent mean values ((SD) for three independent determinations.
compounds b, which, actually, represents the most significant
resonance difference between a and b 1H NMR spectra.
Molecular models showed that the distance between H-2′a and
carbonyl oxygen O-5 is smaller in compounds a compared to
compounds b (2.5-2.7 Å vs 2.8-3.0 Å). It can be hypothesized
that the resulting deshielding of H-2′a explains the observed
resonance shift. Once the stereochemistry of C-3′ in stereoisomer
couples 7, 8, 9, and 14 was assigned, we acquired the CD spectra
of these compounds. We expected a significant difference in
their CD spectra due to the inversion of the chiral center C-3′.
In fact, the inversion of a chiral center in the immediate vicinity
of a chromophore group is also expected to invert the sign of
the Cotton effects of the relevant absorption band(s).23 Actually,
the CD spectrum of compounds 7a, 8a, 9a, and 14a (3S,3′R)
showed a negative Cotton effect centered around 320 nm and
a positive Cotton effect around 290 nm, while compounds 7b,
8b, 9b, and 14b (3S,3′S) showed inverted Cotton effects at the
same wavelengths (Supporting Information). Starting from these
results, we were able to assign the absolute stereochemistry of
the other a/b diastereisomer couples on the basis of their CD
spectra (Supporting Information). Interestingly, in all diasterei-
somer couples, a downfield shift of the H-2′a signal confirmed
the assignment of the relevant compound to the 3S,3′R series.
Finally, the stereochemistries of compounds 7c and 7d were
easily discriminated by NMR and CD analysis considering their
enantiomeric relationship with compounds 7a and 7b, respec-
tively.
diastereoisomers were, in general, 2- or 3-fold more potent than
the corresponding 3S,3′S and 3R,3′R diastereoisomers. The
incorporation at position 3 of a small alkyl substituent (CH3,
5a) was well tolerated, but a marginal loss in activity was
obtained with compound 6 (2-fold) by increasing the steric bulk
of the alkyl side chain. A further conformational restriction
introduced by the L- and D-Pro amino acids gave the most
interesting and potent derivatives of this series. In fact, 7a and
its enantiomer 7c (IC50 ) 20 and 17 nM, respectively) were
10-fold more potent than the corresponding diasteroisomers 7b
and 7d and showed an activity similar to that of doxorubicin
on the MCF-7 cell line. In addition, compound 7c was 17-fold
more active than doxorubicin on the SW 620 cell line (IC50
10 vs 178 nM).
)
The presence of a benzyl group (8a,b), relatively rigid and
more electron rich when compared to the alkycyclic proline side
chain, led to significant loss of activity (3-fold). Replacement
of a hydroxybenzyl group (9a,b) instead of a benzyl group
further decreased the cytotoxic activity. The incorporation of
amino acids containing sulfur gave the potent derivatives 11a
and 12a, which retained a cytotoxic level similar to that of
compounds 7a and 7c and doxorubicin (IC50 ) 29 and 36 nM,
respectively). The compounds containing hydroxyl (10a), amino
(13a), or imidazole (14a) groups in the side chain were
respectively 12-, 4-, and 33-fold less potent than compound 7a.
Overall, these data suggest that a hydrophobic interaction might
exist between the target and this molecular area of the drugs.
Finally, the presence of a free carboxyl function in 15 led to a
dramatic reduction in cell growth inhibition of these cell lines.
This observation would indicate that the diketopiperazine
molecular area might interact with the DNA minor groove,
and thereby, a negative charge in this area would be disfavored.
In fact, the amide analogue 16a was 5-fold more potent than
15a.
In Vitro Cytotoxicity. Preliminarily, the cytotoxic activity
of all diketopiperazine derivatives was evaluated toward the
MCF-7 human breast carcinoma and SW 620 human colon
carcinoma cell lines (Table 1). For comparative purposes,
doxorubicin and the linear pseudodipeptide analogue 2a were
also included in the assay. As shown in Table 1, compound 4
in racemically and enantiomerically pure forms showed a potent
cytotoxic activity on the MCF-7 and SW 620 cell lines, being
about 4-fold more active with respect to compound 2a and only
2-fold less potent than doxorubicin.
To further determine their antitumor spectra and drug-
resistance profile, compounds 7, 11, and 12 were selected and
screened against a panel of human tumor cell lines (Table 2),
including selected breast and ovarian cellular sublines resistant
to doxorubicin (MCF-7/Dx and A2780/Dx) and resistant to
cisplatin (A2780/DDP) (Table 3). As observed in the case of
the MCF-7 and SW620 cell lines, the diastereoisomers with
configuration 3S,3′R (7a, 11a, 12a) or 3R,3′S (7c) were the most
potent derivatives. In the tested A2780 (ovarian), NCI-H460
(lung), HCT-116 (ilocecal), and PC-3 (prostate) cell systems,
compounds 7a and 7c showed marked cytotoxic potency (Table
2) and were less active against LoVo (colon) tumor cells (IC50
) 420 and 320 nM, respectively). Interestingly, compounds 7a,
11a, and 12a were 11-, 11-, and 14-fold more potent than
doxorubicin in MeVo (melanoma) tumor cells (IC50 ) 75, 75,
and 57 nM).
The improved antitumor activity and spectra of these newly
synthesized compounds, compared to the linear 2a, demonstrated
that C3′ chemical modification was indeed an effective approach
to optimize the activity profiles of DTNQ derivatives.19
The results in Table 1 reveal some important structure-
activity relationships. First, the wide activity range observed
for compounds 5-16 (IC50 ) 17-1930 nM, on the MCF-7 cell
line) indicated that the nature of the substituents on C-3 of the
diketopiperazine moiety markedly affects the activity profile
of these compounds. Second, the relative configurations at the
3 and 3′ carbons have an important influence on the cytotoxic
activity of these compounds, defining a preferential positioning
of the side-chain substituents. Thus, the 3S,3′R and 3R,3′S

Synthesis of a New Series of DTNQ-Based DeriVatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1791
Table 3. Inhibition of Drug-Resistant Tumor Cell Lines by Selected Compounds
IC50a (µM ( SD)
MCF-7
breast
MCF-7/Dxb
breast
A2780
ovarian
A2780/Dxb
ovarian
A2780/DDPd
ovarian
compd
RIc
RIc
RIc
7a
7b
7c
7d
11a
11b
12a
12b
doxorubicin
0.020 ( 0.004
0.263 ( 0.047
0.017 ( 0.008
0.213 ( 0.060
0.029 ( 0.009
0.069 ( 0.009
0.036 ( 0.009
0.108 ( 0.019
0.022 ( 0.008
0.074 ( 0.03
0.340 ( 0.03
0.041 ( 0.020
0.234 ( 0.10
0.032 ( 0.01
0.193 ( 0.02
0.158 ( 0.03
0.302 ( 0.02
5.300 ( 0.40
3.7
1.3
2.4
1.1
1.1
2.8
4.4
2.8
286
0.105 ( 0.001
0.530 ( 0.03
0.036 ( 0.002
0.190 ( 0.02
0.190 ( 0.02
0.460 ( 0.15
0.180 ( 0.03
0.624 ( 0.05
0.007 ( 0.001
0.160 ( 0.02
0.760 ( 0.07
0.150 ( 0.01
3.200 ( 0.10
1.200 ( 0.30
0.980 ( 0.20
0.450 ( 0.05
1.560 ( 0.20
0.290 ( 0.01
1.5
1.4
5.8
4.2
6.3
2.1
2.5
2.5
41
0.256 ( 0.03
0.793 ( 0.01
0.420 ( 0.01
5.500 ( 0.09
1.890 ( 0.30
1.130 ( 0.10
0.789 ( 0.02
3.359 ( 0.02
0.007 ( 0.001
2.4
1.5
10
11.7
10
2.4
4.4
4.4
1e
a Data represent mean values ((SD) for three independent determinations. b Subline resistant to doxorubicin. c Resistance index: ratio between the IC50
of resistant and sensitive tumor cell lines. d Subline resistant to cisplatin. e The RI for cisplatin on A2780/ DDP was 28.
Figure 2. Effects of compounds 5a, 6a, and 7b on the DNA-relaxing
activity of human top2: lane 1, pBR322 DNA; lane 2, pBR322 + top2;
lane 3, pBR322 + top2 + VP-16, 25 µM; lanes 4, 5, 6, and 7, pBR322
+ top2 + 5a (1, 5, 10, and 25 µM, respectively); lanes 8, 9, 10, and
11, pBR322 + top2 + 6a (1, 5, 10, and 25 µM, respectively); lanes
12, 13, 14, and 15, pBR322 + top2 + 7b (1, 5, 10, and 25 µM,
respectively).
Figure 3. (a) STD NMR spectrum of the 7a (1 mM)/DNA (50 µM)
complex recorded upon saturation at -1 ppm. (b) Corresponding 1D
When tested against multiple resistance tumor cells (Table
3), these compounds retained good inhibition against selected
proton spectrum.
breast and ovarian cellular subclones resistant to both doxoru-
bicin (MCF-7/Dx and A2780/Dx) and cisplatin (A2780/DDP).
In particular, the MCF-7/Dx cell line exhibited a low level of
cross-resistance to all derivatives 7, 11, and 12 (RI (resistance
index) ) 1.1-4.4), whereas a very low cross-resistance to 7a,
7b, 11b, 12a, and 12b was observed in the ovarian doxorubicin-
resistant subline A-2780/Dx (RI ) 1.4-2.5). Moreover, the
cisplatin-resistant ovarian subclone (A2780/DDP) showed low
levels of cross-resistance to compounds 7a, 7b, and 11b (RI )
2.4, 1.5, and 2.4, respectively).
Drug Inhibition of DNA Relaxation Induced by Topoi-
somerase II (top2). The relative drug activity in inhibiting top2-
mediated DNA relaxation was studied using supercoiled pBR322
DNA as a substrate for human top2 and etoposide (VP-16) as
a top2-specific inhibitor.24 The preliminary experiments indi-
cated that the compounds containing linear and cyclic alkyl side
chains (5a, 6a, and 7b) exhibited potent inhibitory activity at a
concentration of 10 µM (Figure 2). The most potent cytotoxic
compounds 7a, 7c, 11a, and 12a inhibited top2 catalytic activity
in the concentration range 25-50 µM (data not shown), whereas
the remaining compounds did not inhibit top2 catalytic activity
at a concentration of 50 µM.
activity less than perfect. A similar finding was reported
previously with 9-substituted acridine derivatives, acridine
polyamine conjugates, and indenoisoquinolines and topoi-
somerase I or II inhibition.25-27
DNA Binding Properties. Saturation Transfer Difference
(STD) NMR. Compound 7a was tested to see whether it
interacts with DNA using the STD-NMR28,29 technique. STD-
NMR is a technique that can be used to characterize and identify
binding. This technique has become increasingly important as
a tool in the investigation of biomolecular recognition phenom-
ena. The STD-NMR technique is a method of epitope mapping
and ligand screening by NMR spectroscopy. Resonances of the
macromolecule are selectively saturated, and in a binding ligand,
enhancements are observed in the difference (STD-NMR)
spectrum arising from subtraction of this spectrum from a
reference spectrum in which the macromolecule is not saturated.
Protons of the ligand which are in close contact with the
macromolecule can easily be identified from the STD-NMR
spectrum, because they are saturated to the highest degree. They
should have a stronger STD, and this allows direct observation
of areas of the ligand that comprises the epitope. We investigated
the interaction of compound 7a with calf thymus DNA. The
1
These results demonstrate that there is not an exact correlation
between cytotoxicity in cancer cell cultures and top2 inhibitory
activity. Differences in cellular uptake, distribution within the
cell, and additional targets within the cell may all play a role,
thus making the correlation of cytotoxicity with top2 inhibitory
1D H NMR and STD-NMR spectra of 7a in the presence of
1
calf thymus DNA are shown in Figure 3. The 1D H NMR
spectrum of 7a in the presence of calf thymus DNA (Figure
3b) displayed broad signals around 3.0-0.8 ppm, normal for
long-chain DNA, and sharp resonance signals at 3.52, 3.11, 2.55,

1792 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Gomez-Monterrey et al.
Figure 4. A side view of the energy-minimized average structure from the 70 ps period of the MD simulation of complexes (a) 7a/[d(ACGTACGT)]2
and (b) 7b/[d(ACGTACGT)]2.
and 1.90 and around 1.3 ppm arising from low molecular weight
molecules of the buffer solution (EDTA, Tris) and impurities.
These latter peaks were not present in the corresponding STD
spectrum because these molecules do not bind to the DNA
(Figure 3a). In contrast, all the proton resonances of 7a were
observed in the STD spectrum, demonstrating that 7a/DNA
interactions did occur.
) 1.38 for the quasi-planar tricyclic portion and BMI ) 0.28
for the proline portion. This result can be explained assuming
two different DNA binding modes for 7a. An intercalative mode
of binding is sustained by its tricyclic planar core, and an
external backbone binding can be attributed to its proline moiety.
Interestingly, the DF-STD finding for 7a is very similar to that
observed for doxorubicin (BMI ) 1.33 for the intercalating
tricyclic chromophoric core and BMI ) 0.20 for the amino sugar
moiety).30 We performed the same analysis on compound 16a,
which bears a more flexible side chain compared to compound
7a and which shows an intermediate cytotoxic activity in this
series. A DF-STD profile similar to that of 7a was observed
for 16a, indicating an analogous binding mode for these
compounds.
Model Building and MD Simulation of the 7a,b/DNA
Complexes. Models of a DNA octamer complexed to com-
pounds 7a and 7b were built by automatic docking (AutoDock)
and extensive energy minimization and equilibration31 on the
basis of the NMR structure of a bisdaunorubicin (WP631) bound
to [d(ACGTACGT)]2 (see the Experimental Section for de-
tails).32
From the AutoDock result, the DTNQ chromophore of 7a
binds at the middle 5′-GC-3′ base pairs of double-stranded DNA
by intercalation, whereas diketopiperazine and proline rings are
placed externally (Figure 4a). Starting from the best scored
conformation of the complex 7a/DNA given by AutoDock, a
free MD trajectory (70 ps) was generated at 300 K using the
Discover module present in InsightII (Accelrys, San Diego, CA).
The trajectory was sampled every 1 ps and examined visually
by using the InsightII program. Inspection of the MD trajectory,
during which the drug remains inserted in the DNA and fully
intercalating at the 5′-GC-3′ steps, led to the following observa-
tions: Watson-Crick base-base hydrogen bond distances are
conserved during the simulation with an average value around
2 Å; an average Watson-Crick base-base distance C1′-C1′
and base-base angle N1-C1′-C1′ for pyrimidines or N9-
C1′-C1′ for purines are 10.9 Å and 64.4° with root-mean-square
deviations (rmsd’s) of 0.3 Å and 9°, respectively, in agreement
with standard values for B-DNA (10.85 Å and 51.5°).33 After
Di Micco et al. have recently devised an elegant STD-based
method, named DF-STD (differential frequency STD) spec-
troscopy,30 that allows the study of small-molecule-DNA
binding modes. In particular, 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
conditions, in which saturation is centered either in the aromatic
or in the low-field aliphatic spectral regions. Due to the
anisotropy in the efficiency of saturation diffusion along the
vertical and horizontal dimensions of helical DNA, 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. Con-
versely, an external ligand presenting the majority of its binding
surface in contact with the DNA phosphodiester backbone
should be more affected by irradiation in the deoxyribose region
rather than by irradiation in the DNA aromatic region. To
facilitate analysis of the DF-STD data, the authors introduced
the “binding mode index” (BMI), a numerical parameter that
expresses the relative sensitivity of ligand protons to the
perturbation arising from each type of saturation (that is, base
versus sugar/backbone saturation). Three BMI ranges could be
extrapolated: 0 < BMI < 0.50 for external (nonspecific)
electrostatic backbone binding, 0.90 < BMI < 1.10 for minor-
groove binding, and 1.20 (0.90) < BMI < 1.50 for base-pair
intercalation. DF-STD analysis of compound 7a was performed
using the poly(dG-dC)‚poly(dG-dC) copolymer as the DNA
target since the absence of the thymine methyl groups allows a
selective irradiation of the aliphatic (0.5 ppm) or aromatic (9.00
ppm) regions. Two different BMI values were obtained: BMI

Synthesis of a New Series of DTNQ-Based DeriVatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1793
60 F254 (Merck) and preparative TLC on 20 × 20 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 uncor-
rected. Optical rotations were determined with a Perkin-Elmer-241
MC polarimeter. Mass spectra were obtained using an FAB-MS
spectrometer. Analytical RPHPLC was performed on a Vydac C-18
(25 × 0.46 cm) columm, using a tunable UV detector set at 215
nm. Elution was performed with a linear gradient from 10% to
60% acetonitrile in 0.1% aqueous TFA over 55 min at a flow rate
of 1 mL/min. The DTNQ system was synthesized according to the
procedure previously described,20 using ethyl 2-(4-chlorophenyl)-
1,3-thiazolidine-4-carboxylate and naphthoquinone as starting mate-
rial. The enantiomerically pure (+)- and (-)-DTNQ were synthe-
sized according to the procedure previously described.20,21
General Procedure for the Synthesis of 3-Substituted Spiro-
[(dihydropyrazine-2,5-dione)-6,3′-(2′,3′-dihydrothieno[2,3-b]-
naphtho-4′,9′-dione)] Derivatives 4-6 and 8-16 and Spiro-
[(hexahydropyrrolo[1,2-a]pyrazine-1,4-dione)-6,3′-(2′,3′-
dihydrothieno[2,3-b]naphtho-4′,9′-dione)] Derivatives 7. To a
solution of DTNQ (1-2 mmol) in dichloromethane/DMF (20 mL/5
mL) were successively added the corresponding N-Fmoc-protected
amino acid Gly, Ala, Leu, Pro, D-Pro, Phe, Tyr(tBu), Ser(tBu), Cys-
(Trt), Met, Lys(Boc), Hys(Trt), Asp(tBu), or Asn(Trt) (1.1 equiv),
HBTU (1.2 equiv), HOBt (1.2 equiv), and DIEA (2.4 equiv), and
stirring was continued at room temperature for 48 h. Afterward,
the reaction mixture was diluted with dichloromethane (20 mL),
and the resulting solution was washed successively with 10% citric
acid (2 × 25 mL), 10% NaHCO3 (2 × 25 mL), and water (2 × 25
mL), dried over Na2SO4, and evaporated to dryness. The residue
was dissolved in CH2Cl2 (5 mL), and a solution of 20% piperidine
in DMF (5-10 mL) was added. Stirring was continued for 30 min
at room temperature and for 2 h at reflux temperature. The reaction
mixture was allowed to warm to room temperature and evaporated
to dryness. Flash chromatography of the residues, using different
eluent systems, yielded, in each case, the correspondent final
spirodiketopiprerazine derivatives 4-8 and 12 and the side-chain-
protected derivatives 9′-11′ and 13′-16′.
the initial heating to 300 K, the rmsd profile, calculated for all
atoms from the initial energy-minimized complex, remains stable
for the rest of the simulation (1.8 ( 0.3 Å), indicating that the
drug/DNA complex settles into a well-defined and stable
configuration during the simulation. As shown in Figure 4a,
the complex 7a/[d(ACGTACGT)]2 is stabilized by hydrogen
bonds and by π-π stacking forces. The amide N1 hydrogen of
7a forms hydrogen bonds with N2 and N3 atoms of the
guanidine G15 at the intercalation site. Furthermore, one of the
amine hydrogens on N2 of guanine G3 forms another hydrogen
bond with O5 of the diketopiperazine ring of 7a. These
hydrogen-bonding interactions were present in the initial model
and were maintained for the whole length of the monitored
simulation period.
The model reported in Figure 4a suggests that the intercalation
of 7a with DNA also involves π-π stacking interactions of
the quinone moiety with the five- and six-membered rings of
the overlying guanine G15 and of the underlying guanine G3.
Finally, favorable van der Waals contacts are observable
between the proline Câ methylene and the C4′ methine group
of the deoxyribose ring of the guanine G15 residue. This model
fully agrees with the experimental STD-NMR results.
For compound 7b, we obtained very similar results in the
simulation study (Figure 4b). The most important difference
observed in the 7b/DNA complex compared to the 7a/DNA
complex was the absence, in the first, of the hydrogen bonds
between the N1 hydrogen of the drug with N2 and N3 atoms
of the guanidine G3. Accordingly, the rmsd profile of the 7b/
DNA complex (calculated for all atoms from the initial energy-
minimized complex) during the MD simulation increased to 4.2
( 0.3 Å, indicating that it was less stable than the 7a/DNA
complex. The different stabilities of these complexes can explain
the reduction in cytotoxic potency (about 10-fold) of 7b
compared to 7a observed for almost all the tested cell lines (see
above).
General Procedure for Removal of the Side-Chain Protecting
Groups. A solution of side-chain-protected derivatives 9′-11′ and
13′-16′ (0.03-0.05 mM) in dichloromethane (10 mL) was treated
with trifluoroacetic acid (2 mL) and stirred at room temperature
for 1-3 h. After evaporation to dryness, the treatment of the residue
with dichloromethane and diethyl ether yielded the corresponding
final compounds 9-11 and 13-16. The purity of all these
compounds was assessed as being >98% using HPLC, microanaly-
ses, and NMR spectroscopy.
Conclusions
We report here the synthesis and biological evaluation of new
spirodiketopiperazine derivatives of the dihydrothieno[2,3-b]-
naphtho-4,9-dione system which exhibit activity against different
tumor cell lines. Most active compounds 7a,c, 11a, and 12a
showed similar or improved activities compared to doxorubicin
on the MCF-7 and SW620 cell lines. The ability of these
compounds to overcome potential multiple-drug-resistance
mechanisms has also been demonstrated by their cytotoxicity
on doxorubicin- and cisplatin-resistant cell lines. STD-NMR
spectroscopy investigation performed on compounds 7a and 16a
demonstrated that they interact with DNA with a dual binding
mode: intercalative for the tricyclic planar core and external
considering the side-chain moiety. A three-dimensional struc-
tural model was developed in which compounds 7a and 7b
intercalate at the middle 5′-GC-3′ base pairs of double-stranded
DNA. The complexes are stabilized by π-π stacking interac-
tions, by a proposed set of hydrogen bonds, and by van der
Waals contacts. This proposed interaction model might explain
the high cytotoxicity of the active compounds. Other factors
such as differences in cellular uptake, distribution within the
cell, and/or an additional target within the cell might also affect
the cytotoxicity of these derivatives.
Data for spiro[(dihydropyrazine-2,5-dione)-6,3′-(2′,3′-dihy-
drothieno[2,3-b]naphtho-4′,9′-dione)] (4): FC in 10% MeOH in
CHCl3; yellow solid (51%); mp 197-201 °C; HPLC tR 17.92 min;
1H NMR (500 MHz, CD3OD) δ 3.55 (d, 1H, J ) 12.9 Hz, H-2′b),
4.06 (d, 1H, J ) 16.2 Hz, H-3) 4.21 (d, 1H, H-2′a), 4.27 (d, 1H,
H-3′), 7.69-7.76 (m, 2H, H-6′ and H-7′), 8.03 (d, 1H, J ) 7.1 Hz,
H-5′ or H-8′), 8.07 (d, 1H, H-8′ or H-5′); FAB-MS m/z calcd for
C15H10N2O4S 314.32, found 314.51. The pure enantiomers (3′R)-
4a and (3′S)-4b were prepared from the corresponding (+)- and
(-)-DTNQ and glycine following the general procedure indicated
20
above. Data for 4a: [R]D ) -24.8° (c 1.0, MeOH); mp 196-
20
198 °C. Data for 4b: [R]D ) +25.5° (c 1.2, MeOH); mp 197-
199 °C.
Data for (3S,3′R)-3-methylspiro[(dihydropyrazine-2,5-dione)-
6,3′-(2′,3′-dihydrothieno[2,3-b]naphtho-4′,9′-dione)] (5a): FC in
20% MeOH in CHCl3; yellow solid (32%); mp 205-206 °C; HPLC
tR 17.22 min; [R]D20 ) -7.5 (c 1.1, MeOH). 1H NMR (500 MHz,
CD3OD): δ, 1.23 (m, 3H, CH3), 3.40 (d, 1H, J ) 12.8 Hz, H-2′b),
3.52-3.56 (m, 1H, H-3), 3.58 (d, 1H, H-2′a), 7.67-7.73 (m, 2H,
H-6′ and H-7′), 8.00 (d, 1H, J ) 7.4 Hz, H-8′); 8.03 (d, 1H, J )
7.5 Hz, H-5′). FAB-MS m/z calc for C16H12N2O4S 328.34, found
328.39.
Experimental Section
General Procedures. 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
3(S),3′S)-3-Methylspiro[(dihydropirazin-2,5-dione)-6,3′-(2′,3′-
dihydrothieno[2,3-b]naphtho-4′,9′-dione)] (5b). FC in 20% MeOH

1794 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Gomez-Monterrey et al.
in CHCl3. Yellow solid (28%), mp 210-211 °C. HPLC tR 16.31
H-6′ and H-7′), 7.98 (d, 1H, J ) 7.3 Hz, H-8′), 8.06 (d, 1H, J )
7.4 Hz, H-5′); FAB-MS m/z calcd for C22H16N2O4S 404.44, found
404.63.
20
1
min. [R]D ) +36.1 (c 1.0, MeOH); H NMR (500 MHz, CD3-
OD) δ 1.22 (m, 3H, CH3), 3.53 (d, 1H, J ) 12.9 Hz, H-2′b), 3.56
(d, 1H, H-2′a), 3.59-3.62 (m, 1H, H-3), 7.83-7.89 (m, 2H, H-6′
and H-7′), 7.90 (d, 1H, J ) 7.4 Hz, H-8′), 8.01 (d, 1H, J ) 7.5 Hz,
H-5′); FAB-MS m/z calcd for C16H12N2O4S 328.34, found 328.51.
Data for (3S,3′S)-3-benzylspiro[(dihydropyrazine-2,5-dione)-
6,3′-(2′,3′-dihydrothieno[2,3-b]naphtho-4′,9′-dione)] (8b): FC in
5% MeOH in CHCl3; yellow solid (26%); mp 263-265 °C; HPLC
tR 23.74 min; [R]D20 ) -8.3 (c 1.1, MeOH); 1H NMR (500 MHz,
DMSO-d6) δ 3.08-3.12 (m, 2H, H-â), 3.62 (d, 1H, J ) 12.6 Hz,
H-2′b), 3.87 (d, 1H, H-2′a), 4.40-4.42 (m, 1H, H-3), 7.22-7.30
(m, 5H, aryl), 7.83-7.85 (m, 2H, H-6′ and H-7′), 8.01 (d, 1H, J )
7.5 Hz, H-8′), 8.03 (d, 1H, J ) 7.4 Hz, H-5′), 8.38 (s, 1H, NH),
8.80 (s, 1H, NH); 1H NMR (500 MHz, CDCl3) δ 3.38 (d, 1H, J )
12.9 Hz, H-2′b), 3.45-3.47 (m, 1H, H-â), 3.51-3.53 (m, 1H, H-â),
4.21-4.23 (m, 1H, H-3), 4.26 (d, 1H, H-2′a), 6.18 (s, 1H, NH),
6.30 (s, 1H, NH), 7.29-7.38 (m, 5H, aryl), 7.68-7.78 (m, 2H,
H-6′ and H-7′), 8.05 (d, 1H, J ) 7.4 Hz, H-8′), 8.11 (d, 1H, J )
7.4 Hz, H-5′); FAB-MS m/z calcd for C22H16N2O4S 404.44, found
404.51.
Data for (3S,3′R)-3-sec-butylspiro[(dihydropyrazine-2,5-di-
one)-6,3′-(2′,3′-dihydrothieno[2,3-b]naphtho-4′,9′-dione)] (6a):
FC in AcOEt; yellow solid (27%); mp 223-224 °C; HPLC tR 22.51
20
1
min; [R]D ) -9.0 (c 1.0, MeOH); H NMR (500 MHz, CDCl3)
δ 1.26 (s, 6H, H-δ), 1.69-1.75 (m, 1H, H-γ), 1.79-1.82 (m, 1H,
H-â), 1.89-1.93 (m, 1H, H-â), 3.34 (d, 1H, J ) 12.9 Hz, H-2′b),
4.37 (d, 1H, H-2′a), 4.46-4.49 (m, 1H, H-3), 6.32 (s, 1H, NH),
6.70 (s, 1H, NH), 7.67-7.73 (m, 2H, H-6′ and H-7′), 8.05 (d, 1H,
J ) 7.5 Hz, H-8′), 8.09 (d, 1H, J ) 7.5 Hz, H-5′); FAB-MS m/z
calcd for C19H18N2O4S 370.42, found 370.63.
Data for (3S,3′S)-3-sec-butylspiro[(dihydropyrazine-2,5-di-
one)-6,3′-(2′,3′-dihydrothieno[2,3-b]naphtho-4′,9′-dione)] (6b):
FC in AcOEt; yellow solid (23%); mp 226-228 °C; HPLC tR 21.20
min; [R]D20 ) +31.7 (c 1.0, MeOH); 1H NMR (500 MHz, CDCl3)
δ 1.26 (s, 6H, H-δ), 1.87-1.94 (m, 1H, H-γ), 1.95-2.05 (m, 1H,
H-â), 2.17-2.21 (m, 1H, H-â), 3.38 (d, 1H, J ) 12.9 Hz, H-2′b),
4.14-4.16 (m, 1H, H-3), 4.25 (d, 1H, H-2′a), 6.26 (s, 1H, NH),
6.58 (s, 1H, NH), 7.68-7.75(m, 2H, H-6′ and H-7′), 8.03 (d, 1H,
J ) 7.4 Hz, H-8′), 8.08 (d, 1H, J ) 7.5 Hz, H-5′); FAB-MS m/z
calcd for C19H18N2O4S 370.42, found 370.61.
Data for (3S,3′R)-3-[4-(tert-butyloxy)benzyl]spiro[(dihydro-
pyrazine-2,5-dione)-6,3′-(2′,3′-dihydrothieno[2,3-b]naphtho-4′,9′-
dione)] (9′a): FC in AcOEt; yield 24%; 1H NMR (500 MHz,
CDCl3) δ 1.32 (s, 9H, CH3), 2.68 (d, 1H, J ) 12.9 Hz, H-2′b),
3.11-3.15 (m, 1H, H-â), 3.28-3.32 (m, 1H, H-â), 4.05 (d, 1H,
H-2′a), 4.66-4.71 (m, 1H, H-3), 6.07 (s, 1H, NH), 6.24 (s, 1H,
NH), 7.00 (d, 2H, J ) 6.8 Hz, aryl), 7.19 (d, 2H, J ) 6.9 Hz,
aryl), 7.70-7.74 (m, 2H, H-6′ and H-7′), 8.00 (d, 1H, J ) 7.4 Hz,
H-8′), 8.06 (d, 1H, J ) 7.5 Hz, H-5′). After removal of the tert-
butyl group the corresponding (3S,3′R)-3-(4-hydroxybenzyl)spiro-
[(dihydropyrazine-2,5-dione)-6,3′-(2′,3′-dihydrothieno[2,3-b]naphtho-
4′,9′-dione)] (9a) was quantitatively obtained as a yellow solid:
Data for (3S,3′R)-3-spiro[(hexahydropyrrolo[1,2-a]pyrazine-
1,4-dione)-6,3′-(2′,3′-dihydrothieno[2,3-b]naphtho-4′,9′-dione)]
20
(7a): FC in AcOEt; yellow solid (30%); mp 199-203 °C; [R]D
20
) -98.3 (c 1.2, MeOH); HPLC tR 20.68 min; 1H NMR (500 MHz,
DMSO-d6) δ 1.81-1.83 (m, 1H, H-γ), 1.87-1.90 (m, 2H, H-γ
and H-â), 2.13-2.15 (m, 1H, H-â), 3.19-3.21 (m, 1H, H-δ), 3.43
(d, 1H, J ) 12.6 Hz, H-2′b), 3.59-3.61 (m, 1H, H-δ), 4.12 (d, 1H,
H-2′a), 4.41-4.43 (m, 1H, H-3), 7.83-7.86 (m, 2H, H-6′ and H-7′),
8.00 (d, 1H, J ) 7.5 Hz, H-8′), 8.03 (d, 1H, J ) 7.4 Hz, H-5′),
8.91(s, 1H, NH); 1H NMR (500 MHz, CDCl3) δ 1.99-2.15(m, 3H,
H-â and H-γ), 2.47-2.51 (m, 1H, H-â), 3.27 (d, 1H, J ) 12.6 Hz,
H-2′b), 3.69-3.73 (m, 2H, H-δ), 4.61 (d, 1H, H-2′a), 4.64-4.66
(m, 1H, H-3), 6.69 (s, 1H, NH), 7.69-7.75 (m, 2H, H-6′ and H-7′),
7.99 (d, 1H, J ) 7.4 Hz, H-8′), 8.06 (d, 1H, J ) 7.4 Hz, H-5′);
FAB-MS m/z calcd for C18H14N2O4S 354.38, found 354.50.
mp 282-283 °C; HPLC tR 28.2 min; [R]D ) -32.0 (c 2.0,
1
MeOH); H NMR (500 MHz, DMSO-d6) δ 2.72-2.74 (m, 1H,
H-â), 3.03-3.05 (m, 1H, H-â), 3.73 (d, 1H, J ) 12.6 Hz, H-2′b),
3.82 (d, 1H, H-2′a), 4.38-4.40 (m, 1H, H-3), 6.95 (d, 2H, J ) 7.4
Hz, aryl), 7.12 (d, 2H, J ) 7.4 Hz, aryl), 7.804-7.87 (m, 2H, H-6′
and H-7′), 7.97 (d, 1H, J ) 7.5 Hz, H-8′), 8.01 (d, 1H, J ) 7.4 Hz,
1
H-5′), 8.72 (s, 1H, NH), 8.79 (s, 1H, NH); H NMR (500 MHz,
CDCl3) δ 2.68 (d, 1H, J ) 12.9 Hz, H-2′b), 3.11-3.15 (m, 1H,
H-â), 3.28-3.32 (m, 1H, H-â), 4.03 (d, 1H, H-2′a), 4.66-4.69 (m,
1H, H-3), 6.07 (s, 1H, NH), 6.24 (s, 1H, NH), 7.00 (d, 2H, J ) 6.9
Hz, aryl), 7.19 (d, 2H, J ) 6.9 Hz, aryl), 7.70-7.74 (m, 2H, H-6′
and H-7′), 8.00 (d, 1H, J ) 7.4 Hz, H-8′), 8.08 (d, 1H, J ) 7.4 Hz,
H-5′); FAB-MS m/z calcd for C22H16N2O5S 420.44, found 420.41.
Data for (3S,3′S)-3-[4-(tert-butyloxy)benzyl]spiro[(dihydro-
pyrazine-2,5-dione)-6,3′-(2′,3′-dihydrothieno[2,3-b]naphtho-4′,9′-
dione)] (9′b): FC in AcOEt; yield 32%; 1H NMR (500 MHz,
CDCl3) δ 1.32 (s, 9H, CH3), 3.40 (d, 1H, J ) 12.8 Hz, H-2′b),
3.45-3.47 (m, 2H, H-â), 4.21-4.23 (m, 1H, H-3), 4.29 (d, 1H,
H-2′a), 5.89 (s, 1H, NH), 6.51 (s, 1H, NH), 7.03 (d, 2H, J ) 7.4
Hz, aryl), 7.33 (d, 2H, J ) 7.4 Hz, aryl), 7.70-7.78 (m, 2H, H-6′
and H-7′), 8.10-8.13 (m, 2H, H-8′ and H-5′). After removal of
the tert-butyl group the corresponding (3S,3′S)-3-(4-hydroxybenzyl)-
spiro[(dihydropyrazine-2,5-dione)-6,3′-(2′,3′-dihydrothieno[2,3-b]-
naphtho-4′,9′-dione)] (9b) was obtained as a yellow solid (91%):
Data for (3S,3′S)-spiro[(hexahydropyrrolo[1,2-a]pyrazine-1,4-
dione)-6,3′-(2′,3′-dihydrothieno[2,3-b]naphtho-4′,9′-dione)] (7b):
FC in AcOEt; yellow solid (23%); mp 210-212 °C; HPLC tR 18.57
min; [R]D20 ) -74.6 (c 1.0, MeOH); 1H NMR (500 MHz, DMSO-
d6) δ 1.97-2.02 (m, 2H, H-γ), 2.05-2.08 (m, 1H, H-â), 2.13-
2.15 (m, 1H, H-â), 3.40-3.43 (m, 1H, H-δ), 3.44-3.47 (m, 1H,
H-δ), 3.90 (d, 1H, J ) 12.6 Hz, H-2′b), 3.95 (d, 1H, H-2′a), 4.47-
4.50 (m, 1H, H-3), 8.06-8.15 (m, 2H, H-6′ and H-7′), 8.18 (d,
1H, J ) 7.4 Hz, H-8′), 8.21 (d, 1H, J ) 7.4 Hz, H-5′), 8.93 (s, 1H,
1
NH); H NMR (500 MHz, CDCl3) δ 1.80-1.84 (m, 1H, H-γ),
1.92-2.06 (m, 2H, H-â, H-γ), 2.33-2.36 (m, 1H, H-â), 3.32 (d,
1H, J ) 12.6 MHz, H-2′b), 3.49-3.51 (m, 1H, H-δ), 3.84-3.86
(m, 1H, H-δ), 4.11 (d, 1H, H-2′a), 4.14-4.16 (m, 1H, H-3′), 6.77
(s, 1H, NH), 7.70-7.73 (m, 2H, H-6′ and H-7′), 8.01 (d, 1H, J )
7.4 Hz, H-8′), 8.08 (d, 1H, J ) 7.4 Hz, H-5′); FAB-MS m/z calcd
for C18H14N2O4S 354.38, found 354.51.
20
mp 293-294 °C; HPLC tR 29.6 min; [R]D ) +11.1 (c 1.5,
1
MeOH); H NMR (500 MHz, DMSO-d6) δ 3.04-3.06 (m, 1H,
H-â), 3.08-3.10 (m, 1H, H-â), 3.69 (d, 1H, J ) 12.6 Hz, H-2′b),
3.82 (d, 1H, H-2′a), 4.38-4.40 (m, 1H, H-3), 6.90 (d, 2H, J ) 7.4
Hz, aryl), 7.23 (d, 2H, J ) 7.4 Hz, aryl), 7.84-7.89 (m, 2H, H-6′
and H-7′), 7.98 (d, 1H, J ) 7.5 Hz, H-8′), 8.03 (d, 1H, J ) 7.4 Hz,
Data for (3S,3′R)-3-benzylspiro[(dihydropyrazine-2,5-dione)-
6,3′-(2′,3′-dihydrothieno[2,3-b]naphtho-4′,9′ -dione)] (8a): FC in
5% MeOH in CHCl3; yellow solid (20%); mp 249-251 °C; HPLC
tR 23.15 min; [R]D20 ) -51.0 (c 1.0, MeOH); 1H NMR (500 MHz,
DMSO-d6) δ 3.10-3.14 (m, 2H, H-â), 3.61 (d, 1H, J ) 12.6 Hz,
H-2′b), 3.83 (d, 1H, H-2′a), 4.38-4.40 (m, 1H, H-3), 7.24-7.31
(m, 5H, aryl), 7.83-7.85 (m, 2H, H-6′ and H-7′), 7.92 (d, 1H, J )
7.5 Hz, H-8′), 7.98 (d, 1H, J ) 7.4 Hz, H-5′), 8.71 (s, 1H, NH),
8.83 (s, 1H, NH); 1H NMR (500 MHz, Cl3CD) δ 2.60 (d, 1H, J )
12.9 Hz, H-2′b), 3.11-3.15 (m, 1H, H-â), 3.33-3.36 (m, 1H, H-â),
3.97 (d, 1H, H-2′a), 4.66-4.71 (m, 1H, H-3), 6.33 (s, 1H, NH),
6.57 (s, 1H, NH), 7.29-7.38 (m, 5H, aryl), 7.68-7.74 (m, 2H,
1
H-5′), 8.41 (s, 1H, NH), 8.79 (s, 1H, NH); H NMR (500 MHz,
CDCl3) δ 3.36 (d, 1H, J ) 12.8 Hz, H-2′b), 3.40-3.44 (m, 2H,
H-â), 4.19-4.21 (m, 1H, H-3), 4.27 (d, 1H, H-2′a), 6.00 (s, 1H,
NH), 6.58 (s, 1H, NH), 7.03 (d, 2H, J ) 7.4 Hz, aryl), 7.33 (d, 2H,
J ) 7.4 Hz, aryl), 7.70-7.78 (m, 2H, H-6′ and H-7′), 8.00 (d, 1H,
J ) 7.4 Hz, H-8′), 8.03 (d, 1H, J ) 7.4 Hz, H-5′); FAB-MS m/z
calcd for C22H16N2O5S 420.44, found 420.41.
Data for (3S,3′R)-3-[(tert-butyloxy)methyl]spiro[dihydropy-
razine-2,5-dione-6,3′-(2′,3′-dihydrothieno[2,3-b]naphtho-4′,9′-di-
one)] (10′a): FC in AcOEt; yield 26%; 1H NMR (500 MHz, CDCl3)
δ 1.40 (s, 9H, CH3), 3.41 (d, 1H, J ) 12.6 Hz, H-2′b), 3.69-3.72

Synthesis of a New Series of DTNQ-Based DeriVatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1795
(m, 1H, H-â), 3.82-3.84 (m, 1H, H-â), 4.35 (d, 1H, H-2′a), 4.58-
4.60 (m, 1H, H-3), 6.57 (s, 1H, NH), 6.64 (s, 1H, NH), 7.69-7.75
(m, 2H, H-6′ and H-7′), 8.00 (d, 1H, J ) 7.3 Hz, H-8′), 8.07 (d,
1H, J ) 7.4 Hz, H-5′). After removal of the tert-butyl group the
corresponding (3S,3′R)-3-(hydroxymethyl)spiro[dihydropyrazine-
2,5-dione-6,3′-(2′,3′-dihydrothieno[2,3-b]naphtho-4′,9′-dione)] (10a)
was obtained as a yellow solid (91%); mp 239-241 °C; HPLC tR
22.97 min; [R]D ) -9.8 (c 0.8, MeOH); H NMR (500 MHz,
CD3OD) δ 3.59 (d, 1H, J ) 12.6 Hz, H-2′b), 3.60-3.63 (m, 1H,
H-â), 3.84-3.86 (m, 1H, H-â), 4.01 (d, 1H, H-2′a), 4.20-4.23 (m,
1H, H-3), 7.77-7.82 (m, 2H, H-6′ and H-7′), 8.04 (d, 1H, J ) 7.4
Hz, H-8′), 8.09 (d, 1H, J ) 7.4 Hz, H-5′); FAB-MS m/z calcd for
C16H12N2O5S 344.34, found 344.51.
(d, 1H, J ) 7.4 Hz, H-5′); FAB-MS m/z calcd for C18H16N2O4S2
388.46, found 388.31.
Data for (3S,3′S)-3-[(methylthio)ethyl]spiro[dihydropyrazine-
2,5-dione-6,3′-(2′,3′-dihydrothieno[2,3-b]naphtho-4′,9′-dione)]
(12b): FC in AcOEt; yellow solid (26%); mp 251-253 °C; HPLC
tR 20.80 min; [R]D20 ) +39.1 (c 1.1, MeOH); 1H NMR (500 MHz,
CDCl3) δ 2.12 (s, 3H, CH3), 2.36-2.40 (m, 2H, H-â), 2.71-2.76
(m, 2H, H-γ), 3.31 (d, 1H, J ) 12.8 Hz, H-2′b), 4.12 (d, 1H, H-2′a),
4.28-4.32 (m, 1H, H-3), 6.45 (s, 1H, NH), 6.83 (s, 1H, NH), 7.70-
7.76 (m, 2H, H-6′ and H-7′), 8.01 (d, 1H, J ) 7.4 Hz, H-8′), 8.11
(d, 1H, J ) 7.4 Hz, H-5′); FAB-MS m/z calcd for C18H16N2O4S2
388.46, found 388.29.
Data for (3S,3′R)-3-[(N-(tert-butyloxycarbonyl)amino)butyl]-
spiro[(dihydropyrazine-2,5-dione)-6,3′-(2′,3′-dihydrothieno[2,3-
b]naphtho-4′,9′-dione)] (13′a): FC in AcOEt; yield 22%; 1H NMR
(500 MHz, CDCl3) δ 1.41 (s, 9H, CH3), 1.52-1.55 (m, 4H, H-γ
and H-δ), 1.94-1.96 (m, 2H, H-â), 3.11-3.14 (m, 2H, H-ꢀ), 3.36
(d, 1H, J ) 12.9 Hz, H-2′b); 4.36 (d, 1H, H-2′a), 4.50-4.52 (m,
1H, H-3), 4.64 (s, 1H, NHBoc), 6.61 (s, 1H, NH), 6.85 (s, 1H,
NH), 7.66-7.72 (m, 2H, H-6′ and H-7′), 7.97 (d, 1H, J ) 7.4 Hz,
H-8′), 8.05 (d, 1H, J ) 7.4 Hz, H-5′). After removal of the N-Boc
protecting group the corresponding (3S,3′R)-3-(aminobutyl)spiro-
[(dihydropyrazine-2,5-dione)-6,3′-(2′,3′-dihydrothieno[2,3-b]naph-
20
1
Data for (3S,3′S)-3-[(tert-butyloxy)methyl]spiro[dihydropy-
razine-2,5-dione-6,3′-(2′,3′-dihydrothieno[2,3-b]naphtho-4′,9′-di-
one)] (10′b): FC in AcOEt; yield 26%; 1H NMR (500 MHz, CDCl3)
δ 1.40 (s, 9H, CH3), 3.41 (d, 1H, J ) 12.6 Hz, H-2′b), 3.91-3.94
(m, 1H, H-â), 4.03-4.07 (m, 1H, H-â), 4.26-4.30 (m, 2H, H-2′a
and H-3), 6.35 (s, 1H, NH), 6.38 (s, 1H, NH), 7.67-7.75 (m, 2H,
H-6′ and H-7′), 8.03 (d, 1H, J ) 7.4 Hz, H-8′), 8.08 (d, 1H, J )
7.4 Hz, H-5′). After removal of the tert-butyl group the corre-
sponding (3S,3′S)-3-(hydroxymethyl)spiro[dihydropyrazine-2,5-di-
one-6,3′-(2′,3′-dihydrothieno[2,3-b]naphtho-4′,9′-dione)] (10b) was
obtained as a yellow solid (90%): mp 248-249 °C; HPLC tR 23.74
tho-4′,9′-dione)] trifluoroacetate (13a) was quantitatively obtained
20
1
20
min; [R]D ) +33.6 (c 1.0, MeOH); H NMR (500 MHz, CD3-
OD) δ 3.60 (d, 1H, J ) 12.6 Hz, H-2′b), 3.74-3.76 (m, 1H, H-â),
4.00-4.03 (m, 1H, H-â), 4.11 (d, 1H, H-2′a), 4.16-4.19 (m, 1H,
H-3), 7.77-7.83 (m, 2H, H-6′ and H-7′), 8.04 (d, 1H, J ) 7.4 Hz,
H-8′), 8.08 (d, 1H, J ) 7.4 Hz, H-5′); FAB-MS m/z calcd for
C16H12N2O5S 344.34, found 344.51.
as a yellow solid: mp 244-246 °C; HPLC tR 14.52 min; [R]D
)
1
-2.2 (c 1.0, water); H NMR (500 MHz, CD3OD) δ 1.02-1.07
(m, 2H, H-γ), 1.44-1.56 (m, 4H, H-δ and H-â), 2.79-2.82 (m,
2H, H-ꢀ), 3.47 (d, 1H, J ) 12.6 Hz, H-2′b), 4.16 (d, 1H, H-2′a),
4.50-4.52 (m, 1H, H-3), 7.76-7.79 (m, 2H, H-6′ and H-7′), 8.01
(d, 1H, J ) 7.4 Hz, H-8′), 8.05 (d, 1H, J ) 7.4 Hz, H-5′); FAB-
MS m/z calcd for C19H19N3O4S‚TFA 499.10, found 499.14.
Data for (3S,3′S)-3-[(N-(tert-butyloxycarbonyl)amino)butyl]-
spiro[(dihydropyrazine-2,5-dione)-6,3′-(2′,3′-dihydrothieno[2,3-
b]naphtho-4′,9′-dione)] (13′b): FC in AcOEt; yield 20%; 1H NMR
(500 MHz, CDCl3) δ 1.40 (s, 9H, CH3), 1.99-2.04 (m, 2H, H-γ),
2.16-2.23 (m, 2H, H-δ), 3.11-3.15 (m, 2H, H-â), 3.19-3.21 (m,
2H, H-ꢀ), 3.39 (d, 1H, J ) 12.9 Hz, H-2′b), 4.14-4.16 (m, 1H,
H-3), 4.20 (d, 1H, H-2′a), 4. 84 (s, 1H, NHBoc), 6.72 (s, 1H, NH),
6.88 (s, 1H, NH), 7.66-7.73 (m, 2H, H-6′ and H-7′), 8.01 (d, 1H,
J ) 7.3 Hz, H-8′), 8.06 (d, 1H, J ) 7.4 Hz, H-5′). After removal
of the N-Boc group the corresponding (3S,3′S)-3-(aminobutyl)spiro-
[(dihydropyrazine-2,5-dione)-6,3′-(2′,3′-dihydrothieno[2,3-b]naph-
Data for (3R,3′R)-3-[(tritylthio)methyl]spiro[dihydropyrazine-
2,5-dione-6,3′-(2′,3′-dihydrothieno[2,3-b] naphtho-4′,9′-dione)]
1
(11′a): FC in AcOEt; yield 26%; H NMR (500 MHz, CDCl3) δ
2.79-2.89 (m, 2H, H-â), 3.26 (d, 1H, J ) 12.6 Hz, H-2′b), 3.98-
4.00 (m, 1H, H-3), 4.28 (d, 1H, H-2′a), 5.99 (s, 1H, NH), 6.54 (s,
1H, NH), 7.26-7.48 and 7.50-7.54 (m, 15H, aryl), 7.67-7.75 (m,
2H, H-6′ and H-7′), 8.00 (d, 1H, J ) 7.4 Hz, H-8′), 8.05 (d, 1H, J
) 7.4 Hz, H-5′). After removal of the trityl group the corresponding
(3R,3′R)-3-(mercaptomethyl)spiro[(dihydropyrazine-2,5-dione)-6,3′-
(2′,3′-dihydrothieno[2,3-b]naphtho-4′,9′-dione)] (11a) was obtained
as a yellow solid (80%): mp 181-183 °C; HPLC tR 21.69 min;
20
1
[R]D ) -16.2 (c 1.5, MeOH); H NMR (500 MHz, CD3OD) δ
3.11-3.15 (m, 2H, H-â), 3.64 (d, 1H, J ) 12.6 Hz, H-2′b), 4.23
(d, 1H, H-2′a), 4.34-4.36 (m, 1H, H-3), 7.77-7.83 (m, 2H, H-6′
and H-7′), 8.05 (d, 1H, J ) 7.4 Hz, H-8′), 8.09 (d, 1H, J ) 7.4 Hz,
H-5′); FAB-MS m/z calcd for C16H12N2O4S2 360.40, found 360.25.
Data for (3R,3′S)-3-[(tritylthio)methyl]spiro[dihydropyrazine-
2,5-dione-6,3′-(2′,3′-dihydrothieno[2,3-b]naphtho-4′,9′-dione)]
tho-4′,9′-dione)] trifluoroacetate (13b) was quantitatively obtained
20
as a yellow solid: mp 251-253 °C; HPLC tR 11.27 min; [R]D
)
1
+42.7 (c 1.0, water); H NMR (500 MHz, CD3OD) δ 1.02-1.07
(m, 2H, H-γ), 1.44-1.56 (m, 4H, H-δ and H-â), 2.79-2.82 (m,
2H, H-ꢀ), 3.47 (d, 1H, J ) 12.6 Hz, H-2′b), 4.16 (d, 1H, H-2′a),
4.50-4.52 (m, 1H, H-3), 7.76-7.79 (m, 2H, H-6′ and H-7′), 8.01
(d, 1H, J ) 7.4 Hz, H-8′), 8.05 (d, 1H, J ) 7.4 Hz, H-5′); FAB-
MS m/z calcd for C19H19N3O4S‚TFA 499.10, found 499.27
Data for (3S,3′R)-3-[(1-trityl-1H-imidazol-4-yl)methyl]spiro-
[dihydropyrazine-2,5-dione-6,3′-(2′,3′-dihydrothieno[2,3-b]naph-
1
(11′b): FC in AcOEt; yield 20%; H NMR (500 MHz, CDCl3) δ
3.07-3.18 (m, 2H, H-â), 3.25 (d, 1H, J ) 12.7 Hz, H-2′b), 3.49-
3.52 (m, 1H, H-3), 4.18 (d, 1H, H-2′a), 6.22 (s, 1H, NH), 6.36 (s,
1H, NH), 7.20-7.538 (m, 15H, aryl), 7.66-7.73 (m, 2H, H-6′ and
H-7′), 8.00 (d, 1H, J ) 7.4 Hz, H-8′), 8.05 (d, 1H, J ) 7.4 Hz,
H-5′). After removal of the trityl group the corresponding (3R,3′S)-
3-(mercaptomethyl)spiro[(dihydropyrazine-2,5-dione)-6,3′-(2′,3′-di-
hydrothieno[2,3-b]naphtho-4′,9′-dione)] (11b) was obtained as a
1
tho-4′,9′-dione)] (14′a): FC in AcOEt; yield 20%; H NMR (500
MHz, CDCl3) δ 2.86-2.93 (m, 1H, H-â), 3.27 (d, 1H, J ) 12.8
Hz, H-2′b), 3.32-3.35 (m, 1H, H-â), 4.41 (d, 1H, H-2′a), 4.69-
4.71 (m, 1H, H-3), 6.63 (s, 1H, NH), 6.70 (s, 1H, NH), 7.02-7.35
(m, 15H, aryl), 7.40 (s, 1H, imidazole), 7.66-7.73 (m, 2H, H-6′,
H-7′), 7.98 (d, 1H, J ) 7.3 Hz, H-8′), 8.06 (d, 1H, J ) 7.3 Hz,
H-5′), 8.23 (s, 1H, imidazol). After removal of the trityl group the
corresponding (3S,3′R)-3-(1H-imidazol-4-ylmethyl)spiro[dihydro-
pyrazine-2,5-dione-6,3′-(2′,3′-dihydrothieno[2,3-b]naphtho-4′,9′-di-
one)] (14a) was quantitatively obtained as a yellow solid: mp 259-
20
yellow solid (82%): mp 198-201 °C; HPLC tR 20.56 min; [R]D
1
) +27.9 (c 1.1, MeOH); H NMR (500 MHz, CD3OD) δ 3.14-
3.16 (m, 1H, H-â), 3.19-3.21 (m, 1H, H-â), 3.58 (d, 1H, J ) 12.6
Hz, H-2′b), 4.09 (d, 1H, H-2′a), 4.15-4.18 (m, 1H, H-3), 7.77-
7.83 (m, 2H, H-6′ and H-7′), 8.05 (d, 1H, J ) 7.4 Hz, H-8′), 8.09
(d, 1H, J ) 7.4 Hz, H-5′); FAB-MS m/z calcd for C16H12N2O4S2
360.40, found 360.26.
Data for (3S,3′R)-3-[(methylthio)ethyl]spiro[dihydropyrazine-
2,5-dione-6,3′-(2′,3′-dihydrothieno[2,3-b]naphtho-4′,9′-dione)]
(12a): FC in AcOEt; yellow solid (26%); mp 242-244 °C; HPLC
tR 21.86 min; [R]D20 ) -3.0 (c 1.0, MeOH); 1H NMR (500 MHz,
CDCl3) δ 2.10 (s, 3H, CH3), 2.26-2.29 (m, 2H, H-â), 2.64-2.74
(m, 2H, H-γ), 3.28 (d, 1H, J ) 12.9 Hz, H-2′b), 4.40 (d, 1H, H-2′a),
4.54-4.60 (m, 1H, H-3), 6.40 (s, 1H, NH), 6.63 (s, 1H, NH), 7.69-
7.73 (m, 2H, H-6′ and H-7′), 8.00 (d, 1H, J ) 7.4 Hz, H-8′), 8.07
20
1
261 °C; HPLC tR 17.77 min; [R]D ) -18.8 (c 1.0, MeOH); H
NMR (500 MHz, DMSO-d6) δ 3.08-3.14 (m, 2H, H-â), 3.57 (d,
1H, J ) 12.6 Hz, H-2′b), 3.921 (d, 1H, H-2′a), 4.40-4.41 (m, 1H,
H-3), 7.20 (s, 1H, imidazole), 7.83-7.86 (m, 2H, H-6′ and H-7′),
8.01 (d, 1H, J ) 7.5 Hz, H-8′), 8.04 (d, 1H, J ) 7.4 Hz, H-5′),
1
8.61 (s, 1H, imidazole), 8.92 (s, 1H, NH), 9.00 (s, 1H, NH); H
NMR (500 MHz, CD3OD) δ 2.96 (d, 1H, J ) 12.8 Hz, H-2′b),
3.14-3.19 (m, 1H, H-â), 3.27-3.30 (m, 1H, H-â), 3.89 (d, 1H,
H-2′a), 4.56-4.60 (m, 1H, H-3), 7.20 (s, 1H, imidazole), 7.68-

1796 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Gomez-Monterrey et al.
7.72 (m, 2H, H-6′, H-7′), 7.92 (d, 1H, J ) 7.3 Hz, H-8′), 7.97 (d,
1H, J ) 7.3 Hz, H-5′), 8.37 (s, 1H, imidazole); FAB-MS m/z calcd
for C19H14N4O4S 394.41, found 394.53.
) 7.4 Hz, H-8′,), 8.06 (d, 1H, J ) 7.4 Hz, H-5′). After removal of
the trityl group the corresponding (3S,3′R)-3-[(aminocarbonyl)-
methyl]spiro[(dihydropyrazine-2,5-dione)-6,3′-(2′,3′-dihydrothieno-
[2,3-b]naphtho-4′,9′-dione )] (16a) was quantitatively obtained as
Data for (3S,3′S)-3-[(1-trityl-1H-imidazol-4-yl)methyl]spiro-
[dihydropyrazine-2,5-dione-6,3′-(2′,3′-dihydrothieno[2,3-b]naph-
tho-4′,9′-dione)] (14′b): FC in AcOEt; yield 20%; H NMR (500
20
a yellow solid: mp 225-228 °C; HPLC tR 18.21 min; [R]D
)
1
1
+7.2 (c 1.5, MeOH); H NMR (500 MHz, CD3OD) δ 2.96-3.00
(m, 2H, H-â), 3.61 (d, 1H, J ) 12.9 Hz, H-2′b), 4.18 (d, 1H, H-2′a),
4.41-4.46 (m, 1H, H-3), 7.76-7.83 (m, 2H, H-6′, H-7′), 8.02 (d,
1H, J ) 7.4 Hz, H-8′), 8.06 (d, 1H, J ) 7.4 Hz, H-5′); FAB-MS
m/z calcd for C17H13N3O5S 371.37, found 371.53.
MHz, CDCl3) δ 3.10-3.13 (m, 1H, H-â), 3.19-3.23 (m, 1H, CH2),
3.38 (d, 1H, J ) 12.8 Hz, H-2′b), 4.24 (d, 1H, H-2′a), 4.38-4.41
(m, 1H, H-3), 6.61 (s, 1H, NH), 6.65 (s, 1H, NH), 7.02-7.35 (m,
15H, aryl), 7.39 (s, 1H, imidazole), 7.61-7.63 (t, 1H, J ) 7.4 Hz,
H-6′), 7.68-7.70 (t, 1H, J ) 7.4 Hz, H-7′), 7.94-7.98 (m, 2H,
H-8′ and H-5′), 8.27 (s, 1H, imidazole). After removal of the trityl
group the corresponding (3S,3′S)-3-(1H-imidazol-4-ylmethyl)spiro-
[dihydropyrazine-2,5-dione-6,3′-(2′,3 ′-dihydrothieno[2,3-b]naphtho-
4′,9′-dione)] (14b) was quantitatively obtained as a yellow solid:
Data for (3S,3′S)-3-[(N-Tritylaminocarbonyl)methyl]spiro-
[(dihydropyrazine-2,5-dione)-6,3′-(2′,3′-dihydrothieno[2,3-b]-
naphtho-4′,9′-dione)] (16b′): FC in AcOEt; yield 25%; 1H NMR
(500 MHz, CDCl3) δ 2.90-2.94 (m, 1H, H-â), 2.99-3.023 (m,
1H, H-â), 3.30 (d, 1H, J ) 12.9 Hz, H-2′b), 4.19 (d, 1H, H-2′a),
4.36-4.38 (m, 1H, H-3), 6.90 (s, 1H, NH), 7.14 (s, 1H, NH), 7.28-
7.35 (m, 15H, aryl), 7.53-7.56 (m, 1H, H-6′), 7.61 (s, 1H, NH),
7.69-7.72 (m, 1H, H-7′), 7.84-7.86 (m, 2H, H-5′ and H-8′). After
removal of the trityl group the corresponding (3S,3′S)-3-[(ami-
nocarbonyl)methyl]spiro[(dihydropyrazine-2,5-dione)-6,3′-(2′,3′-di-
hydrothieno[2,3-b]naphtho-4′,9′-dione)] (16b) was quantitatively
obtained as a yellow solid: mp 218-220 °C; HPLC tR 18.53 min;
20
mp 265-268 °C; HPLC tR 16.54 min; [R]D ) +27.8 (c 1.0,
1
MeOH); H NMR (500 MHz, DMSO-d6) δ 2.94-2.97 (m, 1H,
H-â), 3.10-3.13 (m, 1H, H-â), 3.68 (d, 1H, J ) 12.6 Hz, H-2′b),
3.82 (d, 1H, H-2′a), 4.18-4.20 (m, 1H, H-3), 6.96 (s, 1H,
imidazole), 7.83-7.86 (m, 2H, H-6′ and H-7′), 8.03 (d, 1H, J )
7.5 Hz, H-8′), 8.06 (d, 1H, J ) 7.4 Hz, H-5′), 8.42 (s, 1H,
imidazole), 8.91(s, 1H, NH), 8.94(s, 1H, NH); 1H NMR (500 MHz,
CD3OD) δ 3.10 (d, 1H, J ) 12.8 Hz, H-2′b), 3.19-3.23 (m, 1H,
H-â), 3.29-3.32 (m, 1H, H-â), 3.38 (d, 1H, H-2′a), 4.40-4.43 (m,
1H, H-3), 7.28 (s, 1H, imidazole), 7.69-7.74 (m, 2H, H-6′, H-7′),
7.91 (d, 1H, J ) 7.4 Hz, H-8′), 7.97 (d, 1H, J ) 7.4 Hz, H-5′),
8.35 (s, 1H, imidazole); FAB-MS m/z calcd for C19H14N4O4S
394.41, found 394.53.
20
1
[R]D ) +51.2 (c 1.0, MeOH); H NMR (500 MHz, CD3OD) δ
2.81-2.85 (m, 1H, H-â), 2.98-3.00 (m, 1H, H-â), 3.70 (d, 1H, J
) 12.9 Hz, H-2′b), 4.15 (d, 1H, H-2′a), 4.50-4.52 (m, 1H, H-3),
7.78-7.85 (m, 2H, H-6′, H-7′), 8.04 (d, 1H, J ) 7.4 Hz, H-8′),
8.08 (d, 1H, J ) 7.4 Hz, H-5′); FAB-MS m/z calcd for C17H13N3O5S
371.37, found 371.53.
Cell Lines. Human tumor cell lines were used in this study, NCI-
H460 lung carcinoma, MCF-7 breast carcinoma, SW 620 and LoVo
colon carcinoma, HCT-116 ilocecal carcinoma, PC-3 prostate
carcinoma, MeVo melanoma, and A2780 ovarian carcinoma, as
well as two sublines selected for resistance to doxorubicin (A2780/
Dx and MCF-7/Dx) and a subline selected for resistance to cisplatin
(A2780/DDP). Sensitive tumor cells were obtained from the
American Type Culture Collection, whereas resistant tumor cells
were from the Istituto Tumori of Milan. All cell lines were grown
as monolayers in RPMI-1640 (Life Technologies, Inc., New York)
containing 10% fetal bovine serum (Life Technologies, Inc.).
Drug Treatment. Cells were grown in a volume of 100 µL at
approximately 10% confluency in 96-well multititer plates and were
allowed to attach and recover for another 24 h. Varying concentra-
tions of drugs alone were then added to each well, the plates were
incubated in an atmosphere of 5% CO2 and 95% air at 37 °C for
an additional 24 h, and then the plates were washed to remove
drug and incubated for 48 h. Control cultures included equivalent
amounts of vehicle used to solubilize each molecule. Experimental
agent nos. 3-10 were solubilized in DMSO while nos. 11-17 and
doxorubicin were dissolved in water.
Sulforhodamine B Assay. At the end of the treatment, cell
viability was assessed by the sulforhodamine B (SRB) assay.34 Data
were expressed as T/C (%) ) (OD of treated cells/OD of control
cells) × 100, and the concentration of the test compound causing
a 50% inhibition of cell growth (IC50) was calculated from the dose/
effect curve for each tested compound. Every assay was performed
in triplicate, and the drug IC50 of each cell line was the average of
at least three independent experiments.
Human Topoisomerase IIr Relaxation Assay. Purified top2
was purchased from TopoGEN (Columbus, OH). For the assay 0.2
µg of supercoiled pBR322 DNA (Takara Shuzo Co., Ltd., Otsu,
Japan) was relaxed by 1 unit of top2 in a total volume of 20 µL of
assay buffer in the presence of various concentrations of the drugs
for 1 h at 30 °C. At the end of the incubation period the DNA
samples were subjected to electrophoresis in a 1% agarose gel with
TBE running buffer at a voltage of 1 V/cm for approximately 18
h. After the run the gel was stained with 1 µg/mL ethidium bromide
for 30 min followed by destaining in distilled water for 30 min.
The DNA topoisomers were photographed under UV light.
Circular Dichroism (CD). All CD spectra were recorded using
a JASCO J710 spectropolarimeter, with a cell of 1 mm path length.
The CD analyses were performed using a measurement range from
Data for (3S,3′R)-3-[(tert-butyloxycarbonyl)methyl]spiro-
[(dihydropyrazine-2,5-dione)-6,3′-(2′,3′-dihydrothieno[2,3-b]-
naphtho-4′,9′-dione)] (15′a): FC in AcOEt; yield 20%; H NMR
1
(500 MHz, CDCl3) δ 1.48 (s, 9H, CH3), 2.77-2.81 (m, 1H, H-â),
3.05-3.18 (m, 1H, H-â), 3.47 (d, 1H, J ) 12.8 Hz, H-2′b), 4.39
(d, 1H, H-2′a), 4.75-4.77 (m, 1H, H-3), 6.60 (s, 1H, NH), 6.86 (s,
1H, NH), 7.66-7.73 (m, 2H, H-6′, H-7′), 8.00 (d, 1H, J ) 7.4 Hz,
H-8′), 8.10 (d, 1H, J ) 7.4 Hz, H-5′). After removal of the tert-
butyl group the corresponding (3S,3′R)-3-[(hydroxycarbonyl)-
methyl]spiro[(dihydropyrazine-2,5-dione)-6,3′-(2′,3′-dihydrothieno-
[2,3-b]naphtho-4′,9′-dione)] (15a) was obtained as a yellow solid
20
(90%): mp 280-282 °C; HPLC tR 13.2 min; [R]D ) +1.8 (c
1
1.0, MeOH); H NMR (500 MHz, DMSO-d6) δ 2.36-2.39 (m,
1H, H-â), 2.73-2.75 (m, 1H, H-â), 3.27 (d, 1H, J ) 12.8 Hz,
H-2′b), 3.49 (d, 1H, H-2′a), 4.32-4.35 (m, 1H, H-3), 7.68-7.74
(m, 2H, H-6′, H-7′), 7.86 (s, 1H, NH), 8.00 (d, 1H, J ) 7.4 Hz,
H-8′), 8.03 (s, 1H, NH), 8.10 (d, 1H, J ) 7.4 Hz, H-5′); FAB-MS
m/z calcd for C17H12N2O6S 372.04, found 372.10.
Data for (3S,3′S)-3-[(tert-butyloxycarbonyl)methyl]spiro-
[(dihydropyrazine-2,5-dione)-6,3′-(2′,3′-dihydrothieno[2,3-b]-
naphtho-4′,9′-dione)] (15′b): FC in AcOEt; yield 20%; 1H NMR
(500 MHz, CDCl3) δ 1.36 (s, 9H, CH3), 3.08-3.20 (m, 1H, H-â),
3.45 (d, 1H, J ) 12.8 Hz, H-2′b), 3.46-3.49 (m, 1H, H-â), 4.30
(d, 1H, H-2′a), 4.50-4.53 (m, 1H, H-3), 6.60 (s, 1H, NH), 6.90 (s,
1H, NH), 7.71-7.76 (m, 2H, H-6′, H-7′), 8.01 (d, 1H, J ) 7.4 Hz,
H-8′), 8.10 (d, 1H, J ) 7.4 Hz, H-5′). After removal of the tert-
butyl group the corresponding (3S,3′S)-3-[(hydroxycarbonyl)methyl]-
spiro[(dihydropyrazine-2,5-dione)-6,3′-(2′,3′-dihydrothieno[2,3-b]-
naphtho-4′,9′-dione)] (15b) was obtained as a yellow solid (93%):
mp 291-292 °C; HPLC tR 14.1 min; [R]D20 +46.2 (c 1.00, MeOH);
1H NMR (500 MHz, DMSO-d6) δ 2.58-2.61 (m, 1H, H-â), 2.79-
2.81 (m, 1H, H-â), 3.31 (d, 1H, J ) 12.8 Hz, H-2′b), 3.36 (d, 1H,
H-2′a), 4.54-4.57 (m, 1H, H-3), 7.66-7.73 (m, 2H, H-6′, H-7′),
7.78 (s, 1H, NH), 8.00 (d, 1H, J ) 7.4 Hz, H-8′), 8.01 (s, 1H,
NH), 8.10 (d, 1H, J ) 7.4 Hz, H-5′); FAB-MS m/z calcd for
C17H12N2O6S 372.04, found 372.13.
Data for (3S,3′R)-3-[(N-Tritylaminocarbonyl)methyl]spiro-
[(dihydropyrazine-2,5-dione)-6,3′-(2′,3′- dihydrothieno[2,3-b]-
1
naphtho-4′,9′-dione)] (16a′): FC in AcOEt; yield 22%; H NMR
(500 MHz, CDCl3) δ 2.85-2.90 (m, 1H, H-â), 3.08-3.13 (m, 1H,
H-â), 3.21 (d, 1H, J ) 12.8 Hz, H-2′b), 4.20 (d, 1H, H-2′a), 4.78-
4.80 (m, 1H, H-3), 6.43 (s, 1H, NH), 6.89 (s, 1H, NH), 7.28-7.31
(m, 15H, aryl), 7.67-7.74 (m, 2H, H-6′ and H-7′), 7.99 (d, 1H, J

Synthesis of a New Series of DTNQ-Based DeriVatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1797
500 to 240 nm, a 2 nm bandwidth, four accumulations, and a 100
nm/min scanning speed at 25 °C.
The grid was centered on the central axis of DNA and particularly
between 5′-C2G7-3′ and 5′-G3C6-3′ of double-strain DNA. Of the
three different search algorithms offered by AutoDock 3.0.5, the
Lamarckian genetic algorithm (LGA) was applied to model the
interaction/binding between DNA and compounds 7a and 7b. For
the local search, the so-called pseudo-Solis and Wets algorithm39
was used. The docking process was performed in two steps. In the
first short step, consisting of 100 LGA runs, the initial position of
the ligand was random. The population was 50, the maximum
number of generations was 27000, and the maximum number of
energy evaluations was limited to 250000. The best ligand
orientation in the first step, based on the score criteria, was used
as the input position for the second docking step, where the number
of energy evaluations was 2.5 × 106. The second step provided
the most probable ligand geometries and orientations in the binding
pocket. The resultant ligand orientations and conformations were
scored on the basis of the docking and binding energies (the cutoff
value for the energies was 2 kcal/mol). Multiple docking runs can
increase the performance of docking programs,40 as was shown
specifically in the case of AutoDock.41 To meet aspects of
calculation time and data size on one hand and convergence criteria
and statistical relevance on the other hand, 50 independent docking
runs were performed for each docking case. Cluster analysis was
performed on the docked results using a root-mean-square (rms)
tolerance of 0.5 Å.
b. Molecular Dynamics Simulations. The best scored complex
structure obtained by AutoDock was subjected to molecular
dynamics calculations. First, the complex was energy-minimized
using the steepest descent algorithm until the maximum rms
derivative was less than 0.01 kcal/Å, followed by MD production
runs of 70 ps, initiated after the system was heated from 100 to
300 K over 2 ps. During molecular dynamics, frame structures were
saved every 1 ps. The average conformation was extracted and again
energetically minimized by a combination of steepest descent and
a conjugate gradient minimization algorithm until the maximum
derivative was less than 0.01 kcal/Å. The Homology InsightII
module was used to check some structural properties (bond
distances, bond angles, torsion angles, rmsd, hydrogen bonds). The
hydrogen bond criterion was a maximum donor-acceptor distance
of 3.5 Å and a minimum donor-proton-acceptor angle of 120°.
NMR Spectroscopy. NMR experiments were performed on a
Varian Mercury 500 MHz spectrometer at 298 K. NMR samples
were prepared by dissolving each compound (about 5 mM) in 0.6
mL of CDCl3 (Sigma-Aldrich). For the absolute stereochemistry
determination, compounds 7a and 7b were dissolved in DMSO-d6
(Sigma-Aldrich) since this solvent allowed a better dispersion of
the aliphatic proton signals. 2D NOESY22 spectra were recorded
in the phase-sensitive mode, and data block sizes were 2048
addresses in t2 and 512 equidistant t1 values. Before Fourier
transformation, the time domain data matrices were multiplied by
shifted sin2 functions in both dimensions.
a. STD-NMR. STD-NMR experiments were performed on a
Varian Inova 700 MHz spectrometer at 298 K. NMR samples were
prepared by dissolving the ligand and the calf thymus DNA (Sigma-
Aldrich) or poly(dG-dC)‚poly(dG-dC) copolymer (Pharmacia Bio-
chemicals) in D2O (600 µL, 99.996%, CIL Laboratories) containing
phosphate-buffered saline at pH 7.1. A high ligand-receptor molar
excess (20:1) was used for the best STD effects. In particular, the
concentration of 7a and 16a was 1.0 mM, whereas that of the DNA
was 50 µM, expressed as the molarity of phosphate groups. 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, 64 scans were recorded for
the reference STD spectrum, whereas 1024 scans were recorded
for each DF-STD spectrum (saturation time 2 s). The relative STD
effect was calculated for each signal as the difference between the
intensity (expressed as the 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 9.0 and
0.5 ppm.30
Computational Chemistry. Molecular modeling and graphics
manipulations were performed using the InsightII software package
(Accelrys, San Diego, CA) running on a Silicon Graphics Octane2
workstation and on a Dell Precision 780, a dual-core Pentium D
3800 GHz machine running Linux RedHat Enterprise WS 4.0 as
the operating system.
a. Model Building and Ligand Docking. The NMR structure
of the DNA octamer [d(ACGTACGT)]2 complexed with WP631
(PDB entry code 1AL9)32 was retrieved from the RCSB Protein
Data Bank.35 To obtain the DNA octamer [d(ACGTACGT)]2, used
for our docking, the ligand WP631 was removed from the original
PDB file. Missing hydrogens and CVFF (consistent-valence force
field)36 partial atomic charges were added using the Biopolymer
module of the InsightII program (Accelrys). After assignment of
bond orders, missing hydrogen atoms were added and a short
minimization (100 steepest descent steps using the CVFF with a
gradient convergence value of 0.05 kcal/(mol Å)) was performed
using the Discover module present in the InsightII program to
release any internal strain. Then in the AutoDockTools package,31
the partial atomic charges were calculated using the Gasteiger-
Marsili method,37 and after merging of nonpolar hydrogens, rotable
bonds were assigned. The 3D structures of compounds 7a and 7b
were constructed using the module Builder of the InsightII program
and then optimized using a molecular dynamics simulation calcula-
tion performed by a simulated annealing method,38 in vacuo, using
the CVFF in the InsightII/Discover software packages.
Automated docking studies were carried out using AutoDock
version 3.0.5.31 The grid maps representing the DNA in the actual
docking process were calculated with AutoGrid (part of the
AutoDock package). The grids (one for each atom type in the ligand,
plus one for electrostatic interactions) were chosen to be sufficiently
large to include not only the active site but also significant portions
of the surrounding surface. The points of the grids were thus 50 ×
50 × 50 Å with a grid spacing of 0.375 Å (roughly a quarter of
the length of a carbon-carbon single bond), and because the
location of the original ligand in the complex was known, the cubic
grids were centered on the ligand’s binding site.
Acknowledgment. The LC-MS and NMR spectral data
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.
Supporting Information Available: Microanalytical, 13C NMR,
and CD data for all test compounds and 2D NOESY spectra for
compounds 7a, 7b, 8a, 8b, 9a, 9b, 14a, and 14b. This material is
available free of charge via the Internet at http://pubs.acs.org.
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