Synthesis and DNA binding properties of novel benzo[b]isoquino|2,3-h- naphthyridines

Article abstract of DOI:10.1039/b503281d

Several benzo[b]isoquino[2,3-h]-naphthyridines have been prepared via formal hetero-Diels Alder reaction of N-aryl imines as a key step. These compounds have different side chains at C-11, and a cis or trans configuration at the C-8a,C-14a ring junction.

Full text of DOI:10.1039/b503281d

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A R T I C L E
Synthesis and DNA binding properties of novel
benzo[b]isoquino[2,3-h]-naphthyridines†‡
Oliver Koepler,a Stefania Mazzini,*b Maria Cristina Bellucci,b Rosanna Mondelli,b
Angelika Baro,c Sabine Laschat,*c Marc Hotfilder,d Christophle Viseurd and Wolfgang Freye
a Institut fu¨r Organische Chemie, Technische Universita¨t Braunschweig, Hagenring 30, D-38106,
Braunschweig, Germany
b Dipartimento di Scienze Molecolari Agroalimentari, Universita` di Milano, Via Celoria 2,
20133, Milano, Italy. E-mail: rosanna.mondelli@unimi.it.; Fax: +02-503-16801;
Tel: +02-503-16810
c Institut fu¨r Organische Chemie, Universita¨t Stuttgart, Pfaffenwaldring 55, D-70569, Stuttgart,
Germany. E-mail: sabine.laschat@oc.uni-stuttgart.de; Fax: +49(0)711 685 4285;
Tel: +49 (0)711 685 4565
d Klinik und Poliklinik fu¨r Kinderheilkunde, Pa¨diatrische Ha¨matologie/Onkologie, Domaghstr.
9a, D-48149, Mu¨nster, Germany
e Institut fu¨r Anorganische und Analytische Chemie, Technische Universita¨t Braunschweig,
Hagenring 30, D-38106, Braunschweig, Germany
Received 4th March 2005, Accepted 20th May 2005
First published as an Advance Article on the web 30th June 2005
Several benzo[b]isoquino[2,3-h]-naphthyridines have been prepared via formal hetero-Diels Alder reaction of N-aryl
imines as a key step. These compounds have different side chains at C-11, and a cis or trans configuration at the
C-8a,C-14a ring junction. Binding constants for the interaction with oligonucleotides and polynucleotides were
determined by UV absorption and melting experiments. NMR experiments (NOE) revealed that the cis isomers,
showing a slightly folded structure, preferentially bind to the minor groove of AT-rich oligomers. In contrast, the
trans isomers prefer the CG-rich sequences, leading to cap-complexes with the isoquinoline moiety stacked at the top
of the double helix, in agreement with the flatter shape, and with a preference for the 3ꢀ-terminals, as found for
camptothecins. Models of the complexes were built up by molecular dynamics (MD) calculations, by using the
inter-proton distances derived from the NOE values. Cytotoxicity assays against human Ewing sarcoma cell lines
RD-ES and CAD-ES1 were performed.
benzo[b]isoquino[2,3-h]naphthyridines (e.g. 2), which have less
flexible conformations and an increased cytotoxicity against the
Introduction
Recent studies from our laboratory have shown that iso-
same cell lines.2
quinoline derivatives of type 1 (Scheme 1), exhibit in vitro
The aim of this work is to find out whether or not these
cytotoxic activity against three different human brain tumour
molecules can bind to DNA, as well as the mode of binding
cell lines.1 In order to improve the activity, we synthesized some
to DNA and their specificity for particular DNA sequences.
As DNA remains the main target of antitumour drugs,3–5
knowledge of the mode of binding at a molecular level is
the first step in rational drug design. For these studies, the
tral data and crystal structure data. See http://dx.doi.org/10.1039/
† Electronic supplementary information (ESI) available: NMR spec-
interaction of small molecules with oligonucleotides is a good
model of DNA interaction, because a relatively short sequence
is generally involved in the recognition process. We selected
some of these compounds for a spectroscopic study, comparing
them with camptothecins6 and berberine 3 (Scheme 1), an
isoquinoline plant alkaloid belonging to the structural class of
protoberberines, known to be DNA binders5,7,8 and to show
antitumour activity.9
b503281d
‡ Dedicated to Professor Horst Kunz on occasion of his 65th birthday.
We synthesized analogues of 2 with different side chains
on the benzene ring E (in order to modulate the solubility in
water) and with cis and trans configurations at the C-8a,C-
14a ring junction (Scheme 2). In order to allow convenient
synthesis of analogues, the pentacyclic benzo[b]isoquino[2,3-h]-
naphthyridine system was prepared via a convergent one-pot
imine condensation/hetero-Diels–Alder reaction10 using func-
tionalised aniline derivatives, which could be further modified.
We studied the in vitro cytotoxicity of these compounds and
their interaction with DNA sequences by using UV and NMR
spectroscopy, specifically nuclear Overhauser experiments.11
Poly(dA–dT)2, poly(dG–dC)2 and salmon sperm DNA were
used for UV spectra. The self-complementary duplexes
Scheme 1
2 8 4 8
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 8 4 8 – 2 8 5 8
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
©

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Scheme 2 Preparation of water-soluble benzo[b]isoquino[2,3-h]-1,7-naphthyridine derivatives 9 and 10a, as well as the corresponding
trans-configured derivative 10b starting from tetrahydroisoquinoline-3-carbaldehyde 4.
Table 1 Coupling constant values for the B and C rings of two
selected benzo[b]isoquino[2,3-h]-naphthyridines with all-cis (9) and all-
trans (10b) configurationa
d(AAGAATTCTT)2 (AATT), d(CGACGTCG)2 (ACGT),
d(GCGATCGC)2 (GATC), d(GCATCGCGATGC)2 (CGCG)
and d(CGTATACG)2 (TATA) were used for the NMR study.
J(H,H)
9
10b
Results and discussion
5ax,5eq
7ax,7eq
8ax,8eq
7ax,8ax
7ax,8eq
7eq,8ax
7eq,8eq
8a,8ax
15.3
11.6
13.3
12.8
15.1
11.5
13.1
12.2
Syntheses
Tetrahydroisoquinoline-3-carbaldehyde 4 (S enantiomer) was
obtained in four steps starting from L-phenylalanine in 54%
overall yield.12 As outlined in Scheme 2, condensation of
3.3
4.1
2.7
2.5b,c
3.8
3.0
˚
aldehyde 4 with ethyl 4-aminobenzoate (5) in the presence of 4 A
12.3 (ax,ax)
3.4 (ax,eq)
3.1 (ax,eq)
2.3 (eq,ax)
∼1.5
12.3 (ax,ax)
3.3 (ax,eq)
10.7 (ax,ax)
8.5 (ax,ax)
0.7
10.5 (ax,ax)
4.3 (ax,eq)
15.9
molecular sieves yielded the corresponding intermediate imine
6, which was directly treated with EtAlCl2 to give the pentacyclic
product 7a in 61% yield with high cis diastereoselectivity
(cis/trans = 90 : 10).
8a,8eq
8a,14a
14a,14b
14a,NHb,c,d
14b,15ax
14b,15eq
15ax,15eq
5J(5ax,15ax)b,c
4J(8eq,14a)b,c
4J(8a,NH)b,c
The cyclization can be considered to be a formal hetero-
Diels–Alder reaction, although previous mechanistic studies
with related systems clearly indicated a stepwise mechanism.13,14
Hydrolysis of ethyl ester 7a with KOH gave the carboxylic acid 8
in 89% yield. Conversion of 8 with N,N-dimethylaminoethanol
and DCC afforded ester 9 in 81% yield. Then amide 10a was
prepared in 62% yield from ethyl ester 7a by reaction with
aminoethanol.
11.3 (ax,ax)
4.8 (ax,eq)
16.7
1.1
1.3
1.5
1.3
0
0
a Measured in Hz by 1D experiments, solvent CDCl3, estimated accuracy
within 0.05 Hz, unless otherwise specified. 7b shows the same values
as 10b (see the Supplementary material). b Estimated accuracy 0.1 Hz.
c Proved by decoupling experiments. d These values may be affected by
exchange due to the small amount of acid, present in the unstabilised
CDCl3.
The structure of benzo[b]isoquino[2,3-h]naphthyridines 7a, 8,
9, and 10a was confirmed by H and 13C NMR spectroscopy.
1
The all-cis configuration follows from the values of the coupling
constants involving the protons at C-8a, C-14a, C-14b, and C-15
(see Table 1, and the discussion in the next paragraph).
Compounds with an all-trans configuration would have a
flatter shape, which might induce a different mode of interaction
to the double helix. In order to get access to the trans series,
imine 6 was treated with SnCl4 to give the pentacyclic trans
diastereomer 7b in 52% yield. The diastereomeric ratio of the
crude mixture was 7a : 7b = 0.5 : 99.5 (Scheme 2). Treatment of
ester 7b with aminoethanol and sodium in THF at 80 ◦C gave the
amide 10b in 75% yield. An X-ray crystal structure analysis of
7b and the coupling constants measured for 7b and 10b (Table 1)
proved the relative configuration and the overall flat structure
for the compounds of the trans series, as shown in Fig. 1.15 As
the stereocentre C-14b is S, the absolute configuration must be
8aR,14aR,14bS.
Next we investigated the cyclization of the N-arylimine
11, which was formed in situ from aldehyde 4 and 3,4-
methylenedioxyaniline. As shown in Scheme 3, treatment of
imine 11 with SnCl4 followed by aqueous workup gave a
diastereomeric mixture of the all-cis product 12a (22%) and the
all-trans product 12b (37%), which could be separated by column
chromatography. The decrease in the cis diastereoselectivity is
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 8 4 8 – 2 8 5 8
2 8 4 9

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Fig. 1 ORTEP view of 7b, confirming the relative trans configuration.
Please note that the X-ray numbering is different from the IUPAC
numbering used in Scheme 2 and in the remaining text. Only one of
the two unique molecules in the asymmetric unit it shown.
Scheme 4 Preparation of benzo[b]isoquino[2,3-h]naphthyridine 17
with enhanced flatness in the C-ring.
Scheme 3 Preparation of diastereomers 12a and 12b.
probably due to the presence of two additional electron-donating
ether moieties, which are capable of binding the Lewis acid
and thus of interfering with a chelation-controlled cyclization
mechanism.13,14
In order to improve the flatness of the C-ring in the
benzo[b]isoquino[2,3-h]naphthyridines, we attempted to cyclize
a tethered alkyne moiety instead of an alkene. Tetrahydroiso-
quinoline alcohol 13 was therefore alkylated with 1-bromo-
3-pentyne in the presence of K2CO3 in refluxing toluene to
afford the alcohol 14 in 75% yield (Scheme 4). Compound 14
was submitted to Swern oxidation giving aldehyde 15,16 which
was treated with ethyl 4-aminobenzoate (5) under the usual
conditions without further purification. Subsequent addition
of BF3·OEt2 and aqueous workup after 2 days reaction time
resulted in the formation of the quinoline ester 17 in 38% yield.
When BF3·OEt2 was replaced by EtAlCl2, the chloro compound
18 was isolated in 7% yield as a minor by-product.
The isolation of by-product 18 further supports the cationic
cyclization mechanism depicted in Scheme 5. Thus, in presence
of EtAlCl2, the cyclization of imine 16 should give carbenium
ion 19, which either can be trapped by external chloride to yield
compound 18 or can undergo Friedel–Crafts-type electrophilic
aromatic substitution, followed by tautomerization to give 20.
Oxidation of 20 during workup finally results in aromatization
of the C-ring.
Scheme 5 Proposed cationic cyclization mechanism.
Conformational analysis and relative stereochemistry
The conformation and the relative configuration of 7a, 7b, 9,
10a, and 10b were derived from the H,H coupling constants,
with the help of 2D NOESY and COSY experiments. The values
for two compounds, 9 and 10b as examples of cis and trans
configurations, are reported in Table 1; the data for all com-
pounds are provided in the Supplementary material†. The axial
orientation of 14b-H and 8a-H is proved for both compounds by
the high J values for the coupling with the axial protons of the
vicinal methylene groups, 15-CH2 and 8-CH2, respectively, i.e.
12.3–12.5 Hz for J(8a,8ax) and 11.3–11.8 Hz for J(14b,15ax).
2 8 5 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 8 4 8 – 2 8 5 8

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The small coupling constants with the vicinal protons shown
by H-14a prove the equatorial orientation of this proton and
thus, the all-cis configuration of 7a, 9 and 10a. In contrast, the
values of 10.6 and 8.5 Hz found for J(8a,14a) and J(14a,14b)
in the case of 7b and 10b prove the axial orientation of these
protons, and consequently the all-trans configuration. The lower
value of J(14a,14b) with respect to J(8a,14a), is due to the
electronegativity effect of the nitrogen atom bound to C-14b.
To the same effect are due the low values of J(14a,14b) in the
all-cis stereoisomers, i.e. 1.5–2.0 Hz, compared with the coupling
between equatorial and axial protons in cyclohexanes.17
The conformation of ring C is a perfect chair, while rings B
and D are half-chairs, for all compounds. In the case of the half-
chair conformation, the axial and equatorial orientations stand
for pseudo-axial and pseudo-equatorial; the dihedral angles for
axial–axial protons are 160–180◦, and those for axial–equatorial
are 50–60◦. The molecules are quite rigid and have a folded
structure in the case of the all-cis series, whereas the all-trans
compounds 7b and 10b are rather flat (Fig. 2).
The UV melting experiments were performed with the pair
of stereoisomers 10a and 10b (see Table 2), just to confirm the
results described above. The increase of the melting temperature
of the duplex is 2 ◦C, which means that stabilization (albeit poor)
of the double helix occurs, as found for berberine8 and for other
minor-groove binders.18,19
The aggregation process is of the same order of magnitude
observed for campthotecins;6 the dimerization constants KD
(103 M−1) were included in the calculation of the binding
constants.
The preference for the AT-rich sequence by the cis compounds
and for the CG-rich sequences by the trans isomers was also
1
suggested by the variations of H chemical shift, found in the
titration experiments performed with the same oligonucleotides.
The data are reported in the Supplementary material.† Up-
field shifts of 0.1–0.2 ppm were found for 9 and 10a with
d(AAGAATTCTT)2, but of less than 0.1 ppm with the CG-
rich oligomer d(GCGATCGC)2. The inverse occurs for 10b:
an upfield shift of 0.1–0.2 ppm with d(GCGATCGC)2 and
the other CG-rich oligomers, but of less than 0.1 ppm with
d(AAGAATTCTT)2. A small shift variation was observed for
the protons of the duplexes (upfield Dd in the range 0.05–
0.1). In the case of the complexes 10b/d(GCGATCGC)2 and
10b/d(CGTATACG)2, the imino NH protons of the terminal
CG base-pairs appear shielded by ca. 0.3 ppm, as found for
campthotecines;6 because their signals become very broad, a
more accurate value could not be obtained.
The binding constants, as well as the proton shift variation of
the ligand, are not diagnostic for the mode of binding to DNA,
because their values are the sum of contributions derived from
specific and non-specific interactions. In addition, the external
non-specific ionic interactions of a positively charged drug with
the negatively charged ionic surface of the oligonucleotide are
the main factors responsible for such effects. However, they can
suggest, as in this case, a preference for the AT or CG sequence.
The variation of 31P chemical shifts is negligible, i.e. less than
0.1 ppm, for all the complexes. This is the first evidence for exclu-
dingtheintercalationofthesemoleculesintothedoublehelix.20,21
One example is reported in the Supplementary material.†
Fig. 2 Energy minimized structures of: (a) the all-cis 10a, and (b) the
all-trans 10b stereoisomers.
DNA binding properties
NOE Experiments and structure derivation of the complexes
with d(AAGAATTCTT)2 and d(CGACGTCG)2
We used UV spectroscopy as a first approach for studying
the DNA binding properties of these compounds. The binding
constant, obtained by both UV wavelength variations and UV
melting experiments (Table 2), show that in the case of the cis
isomers 7a, 9 and 10a the interaction with poly(dA–dT)2 is
preferred (K of the order of 104 M−1) over poly(dG–dC)2 (K
of the order of 103 M−1). In contrast, the molecules with a trans
configuration, 7b and 10b, appear to prefer the CG sequence (K
values ca. 104 M−1), whereas the constants for the AT-rich se-
quences are of the order of 102 M−1. The K values obtained with
oligonucleotides of different sequences confirm these results and
allowed us to select the oligomers for the NOE experiments.
1H NOE experiments, which allow the detection of specific con-
tacts between protons of the ligand and protons of the duplex,
were performed in order to recognize the preferred interaction
sites. The resonances of the oligomers were assigned, for both
free and bound species, following well-established procedures11
for the analysis of double-stranded oligonucleotides. The se-
quential assignment of the nucleotide units was performed by
detecting the NOE cross-peaks between the aromatic protons of
the bases and the 1ꢀ,2ꢀ,2ꢀꢀ-ribose protons of the 5ꢀ-neighbour unit,
thus allowing the recognition of the B-DNA structure of the
Table 2 Binding constants (K) for the interaction with oligonucleotides and self-association constants (KD) for benzo[b]isoquino[2,3-
h]naphthyridinesa
K/M−1
7a (all-cis)
7b (all-trans)
9 (all-cis)
10a (all-cis)
10b (all-trans)
Topotecan6
Salmon sperm DNA
Poly(dA–dT)2
Poly(dG–dC)2
(3 0.2) × 103
(1 0.2) × 104
(1 0.1) × 103
(9 0.1) × 102
(1 0.1) × 102
—
(3 0.1) × 104
(8 0.2) × 102
(2 0.3) × 104
(3 0.3) × 102
(5 0.2) × 103
(6 0.1) × 103
(1 0.1) × 104
(7 0.2) × 102
(9 0.2) × 102
(5 0.2) × 104
(5 0.1) × 104
(7 0.2) × 103
(1 0.1) × 104
(1 0.1) × 103
—
—
(6 0.3) × 104
(7 0.3) × 102
(8 0.1) × 104
(9 0.2) × 102
(9 0.1) × 103c
(2 0.1) × 104
(3 0.1) × 104
—
—
(8 0.3) × 104
(4 0.1) × 103
(1 0.2) × 104b
(6 0.1) × 103
—
(2 0.3) × 103
(7 0.1) × 104
(9 0.5) × 102
(1 0.1) × 104
—
d(AAGAATTCTT)2
d(GCGATCGC)2
d(CGACGTCG)2
d(GCATCGCGATGC)2
d(CGTATACG)2
(7 0.1) × 102
—
(2 0.2) × 103
—
(3 0.2) × 103
—
—
—
KD (M−1
)
(7 0.2) × 103
(1 0.2) × 103
(2 0.1) × 103
(1 0.1) × 103
(2 0.1) × 103
a Measured by UV absorption at 20 ◦C, pH 5.8 and with 0.02 M NaCl, unless specified. The values were derived from the experimental data by
solving a system of a non-linear equations and using MATLAB software (v. 5.1), reported in ref. 8. The dimerization costant was included in the
calculations. b K = (4 0.2) × 104 M−1 by UV melting experiments, DT = 2.0 ◦C. c Measured by UV melting experiments, DT = 1.5 ◦C.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 8 4 8 – 2 8 5 8
2 8 5 1

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Table 3 Inter-molecular NOE interactions and inter-proton dis-
The very low solubility in water of 7a and 7b precluded any
NMR experiment in this solvent, but the analogues with amino
or hydroxyalkyl side-chains are enough soluble at pH 5.8–6.0 to
perform NOE experiments. The interaction of 9 and 10a with
d(AAGAATTCTT)2 led to NOE signals involving the aromatic
protons of ring A and protons of the nucleotide located in the
minor groove of the double helix, specifically of the adenines
A4, A5 and of the thymines T6 and T7. The aromatic protons
1-H and 2-H on ring A of the ligand have very close chemical
shifts and cannot be distinguished in the NOE experiments, but
this does not invalidate the results, because they are vicinal and
their partner on the same ring, 3-H, shows interactions with the
same units, i.e. 4ꢀ-H of A4 and 1ꢀ-H of T7 and C8. In addition,
4-H has interactions with 2-H of adenine A4 and with the ribose
protons 1ꢀ-H and 4ꢀ-H of A5. The aromatic proton 13-H located
on ring E shows an interaction with 4ꢀ-H of T6, which indicates
that the 14-NH group is oriented towards the helix.
From the structural model of d(AAGAATTCTT)2 we devel-
oped models for the complexes with 9 and 10a, by docking
the molecules into the DNA in an orientation suggested by
the experimental NOEs. The MD study of the complexes was
performed by using the simulations with CVFF force field on
the picosecond time scale, in presence of a sphere of water
molecules. After a first minimization, a 10 ps simulation was
performed, with no restraints except for the NOE contacts and
the hydrogen bonds between the base pairs. The models of the
complexes with 9 and 10a are very similar, as appears from the
inter-proton distance values; therefore, only the data for 10a
are reported in Table 3, those for the other compounds are in
the Supplementary material†. The result of the interaction of 9
and 10a with d(AAGAATTCTT)2 shows that these molecules
are partial minor-groove binders, as the planar moiety of the
molecule (ring A) is deeply inserted in the groove, ring B
is partially inserted, while the other rings C, D and E are
more external, but with 13-H on ring E relatively close to the
phosphate oxygen atoms of T6 and with the 14-NH group facing
the helix (Fig. 4a). This is a consequence of the folded shape
(Fig. 2), due to the all-cis configuration.
tances in the complexes
9
or 10a/d(AAGAATTCTT)2 and
a
10b/d(CGACGTCG)2
b
˚
Distances/A
AATT
9 or 10a
NOEexp
Strand I
Strand IIc
H-2d
H-1
H-2
H-2
H-1
H-2
H-1
H-2
H-3
H-3
H-3
H-3
H-4
H-4
H-4
H-4
H-4
H-10
H-13
H-2 A4
H-2 A5
H-2 A5
H-1ꢀ A5
H-1ꢀ T6
H-4ꢀ T6
H-1ꢀ T7
H-1ꢀ T7
H-4ꢀ A4
H-1ꢀ T6
H-1ꢀ T7
H-1ꢀ C8
H-2 A5
H-1ꢀ A5
H-4ꢀ A5
H-1ꢀ T6
H-2 A4
H-4ꢀ T6
H-4ꢀ T6
we
m
m
w
m
m
w
s
4.77
4.66
3.19
4.75
3.10
3.95
7.20
8.44
4.95
5.47
10.71
10.03
6.75
4.37
4.37
6.23
5.00
8.92
4.49
13.81
6.78
11.68
10.88
6.21
5.84
4.12
2.50
15.45
8.09
3.00
4.34
10.81
15.11
14.02
10.07
11.58
16.38
12.51
w
zero NOE
m
w
zero NOE
w
w
zero NOE
w
zero NOE
w
b
˚
ACGT
H-1f
H-2
H-3
H-1
H-2
H-3
H-1
H-2
10b
NOEexp
Distances/A
H-8 G8
H-8 G8
H-8 G8
H-2ꢀꢀ G8
H-2ꢀꢀ G8
H-2ꢀ G8
H-1ꢀ G8
H-1ꢀ C7
m
m
w
m
m
w
w
w
3.54
3.35
4.68
3.18
4.14
4.95
4.51
5.00
a Acquired at 20 ◦C, in D2O, at pH 5.8,and with 0.02 M NaCl. b Obtained
from the final structure of the complex after MD calculations. c See
Fig. 4. d 1-H and 2-H have close chemical shifts. e Intensity of the NOE
signals, estimated as follows: w = 4.5–5.5, m = 3.5–4.5, s = 2.5–3.5, by
using as a reference the cross-peak of 5-H/6-H of cytidines (2.5). f 1-H
and 2-H are coincident.
No NOE contacts were detected for 9 and 10a with the
CG-rich oligonucleotides examined. The wider minor groove
of these duplexes might not allow contacts between the ligand
and the walls of the groove, but on the other hand, the AT-rich
minor groove has a relatively negative electrostatic potential,22
which significantly enhances its ability to bind positively charged
molecules. Actually, the binding constant values show that the
interaction of cis stereoisomers with poly(dG–dC)2 and with
CG-rich sequences is poor.
double helix. The formation of the complex was monitored by
titration experiments of the drug against the nucleotide solution,
and the NOESY spectra were acquired at R = [drug]/[DNA] =
1, 1.5 and 2. An example is shown in Fig. 3 and the results are
reported in Table 3.
In contrast, the all-trans compounds 7b and 10b with a
flatter shape, not suitable for minor-groove binding, did not
show any NOE signals with d(AAGAATTCTT)2, but preferred
interactions with the CG-rich sequences. Compound 7b is not
soluble enough for NMR experiments, but when 10b is added to
d(CGACGTCG)2, NOE signals involving the aromatic protons
of ring A appear. In fact, 1-H, 2-H and 3-H interact with protons
of the CG base pairs at the terminal ends of the oligomers,
specifically with 8-H of G8 and with ribose protons of G8 and
C7, whereas zero NOEs were found for 4-H. The bases located
in the inner part of the duplex did not show any contact. The
structural model obtained for this complex shows the interacting
molecule located at the top of the helix, with the A and B
rings stacked with the guanine of the 3ꢀ-terminal ends (Fig. 4b).
Similar cap-complexes were found for topotecan and other
camptothecins,6 with the same preference for the 3ꢀ-terminal
end sites. In order to confirm this preference, we examined
the interaction with d(GCGATCGC)2: in this case the NOE
contacts were found between the above-mentioned protons of
ring A and 6-H of C8 and 2ꢀꢀ-H of G7, but not with protons of C1
and G2, thus confirming the preference for the 3ꢀ-terminal ends.
The formation of cap-complexes might stabilize the double helix
of oligonucleotides, as occurs for the dangling ends, which can
Fig. 3 2D NOESY spectrum of the complex 10a/d(AAGAATTCTT)2,
R = 1 (aromatic and H-1ꢀ region, 20 ◦C, tmix 300 ms, in D2O, pH 5.8,
0.02 M NaCl).
2 8 5 2
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 8 4 8 – 2 8 5 8

View Article Online
Fig. 4 Energy minimized molecular models of the complexes: (a) 10a/d(AAGAATTCTT)2: the planar moiety of the molecule (ring A) is deeply
inserted in the groove (contacts with A4, A5, T6 and T7), ring B is partially inserted, while the other rings C, D and E are more external, but 13-H on
ring E is relatively close to the phosphate oxygen atoms of T6; (b) 10b/d(CGACGTCG)2: the A and B rings are stacked with the guanine G8 of one
of the two 3ꢀ-terminal ends (only one ligand molecule is drawn).
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 8 4 8 – 2 8 5 8
2 8 5 3

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Table 4 LC50 values [lmol L−1
]
of benzo[b]isoquino-[2,3-h]-
Ethyl (8aR,14aS,14bS)-9,9-dimethyl-7,8,8a,9,14,14a,14b,15-
octahydro-5H-benzo[b]isoquino[2,3-h]-1,7-naphthyridine-11-
carboxylate (7a)
naphthyridines 7a,b, 9, 10a,b, 12a,b and 17 against two human
Ewing sarcoma cell linesa
A solution of (S)-N-(4-methyl-3-pentenyl)-1,2,3,4-tetrahydro-
isoquinoline-3-carbaldehyde 4 (3 g, 14 mmol) in CH2Cl2 (15 mL)
and ethyl 4-aminobenzoate 5 (2.31 g, 14 mmol) in CH2Cl2
Cell line
7a
7b
9
10a
10b 12a 12b
17
RD-ES
CAD-ES1
190 197 128
—
353 318 183
427
831
b
21 138 1029 287 145 1550 2216
˚
(10 mL) was added to a suspension of 4 A molecular sieves (20 g)
a LC50 values refer to concentrations at which 50% of the tumour cells
in dry CH2Cl2 under an inert gas atmosphere. After stirring at
room temperature for 12 h, the reaction mixture was filtered
under an inert gas atmosphere through Celite with dry CH2Cl2
as eluent. The filtrate was concentrated to 25 mL, and at −78 ◦C
a 1 M solution of EtAlCl2 in hexane (18 mL, 18 mmol) was
added. The reaction mixture was allowed to warm up to room
temperature, stirred for a further 2 days, and hydrolyzed with
2 M NH4F solution (300 mL). The aqueous layer was extracted
with CH2Cl2 (4 × 100 mL), and the combined organic layers
were dried (MgSO4) and concentrated. The residue was purified
by flash chromatography (Rf 0.41, SiO2, pentane–CH2Cl2–NEt3,
15 : 3 : 1) to give7a (3.37 g, 8.60 mmol, 61%) as colourless needles,
[a]2D2 +218.76 (c 1.13, CH2Cl2). m˜max (KBr)/cm−1 3363 (s), 3070,
3045, 3024, 3001 (m), 2976, 2966, 2949, 2935, 2908, 2895, 2855,
2749 (m), 1682 (s), 1606, 1513, 1501 (s), 1296, 1251 (s), 772, 739
(m). dH (600 MHz, CDCl3) 7.86 (d, J 2.0, 10-H), 7.65 (dd, J 8.4
and 2.0, 12-H), 7.17–7.12 (m, 1,2,3-H), 7.03 (dd, 4-H), 6.46 (d,
J 8.4, 13-H), 4.51 (br dd, NH), 4.30 (q, J 7.1, O–CH2), 3.95 (d,
J 15.2, 5-Heq), 3.71 (br m, 14a-H), 3.38 (dd, J 15.2 and 5J 1.0,
5-Hax), 3.16 (ddd, J 16.7, 11.3 and 5J 1.0, 15-Hax), 3.13 (ddd,
J 11.6, 4.1 and 3.0, 7-Heq), 2.84 (dd, J 16.7 and 4.7, 15-Heq),
2.56 (ddd, J 11.3, 4.7 and 2.1, 14b-H), 2.17 (ddd, J 11.6, 12.6,
3.2, 7-Hax), 1.61 (dqd, J 13.2, 3.2, 3.2, 3.0, 4J8eq,14a 1.2, 8-Heq),
survived. For details see ref. 22. b The observed LC50 value was >1000
lmol L−1
.
stabilize duplexes as much as an additional base-pair,23 but this
should not be relevant in the case of polynucleotides. However,
the capping binding mode of small molecules to nucleotides
has received recent attention for many reasons, for instance
because many biochemical processes involve steps occurring at
the interface between a terminus and a ligand protein.24
Cytotoxicity studies
Compounds 7a,b, 9, 10a,b, 12a,b, and 17b were used for in
vitro cytotoxicity tests against two different human tumour cell
lines, RD-ES and CAD-ES1 (Ewing’s sarcoma) (Table 4).25 The
LC50 values for both cell lines are high, except for compound
7b against CAD-ES1 cells (LC50 = 21 lM), however its activity
appears to be modest. As the binding properties of 7b and 10b are
similar, the lower LC50 of 7b with respectto 9 must be due to other
factors, probably to the easier transport through the membranes
due to its lipophilic characteristics. The same reasons could
explain the lower activity of the present compounds with
respect to the preceding series2 of more lipophilic analogues.
Consequently, further studies are needed in order to improve
and explain the biological activity of these molecules.
4
1.51 (dtd, J 12.5, 3.2, 3.2, J8a,NH 1.3, 8a-H), 1.40 (qd, J 13.2,
12.5, 12.5, 4.1, 8-Hax), 1.40 (s, 9-Me), 1.36 (t, J 7.1, Me), 1.27 (s,
=
9-Me). dC (100 MHz, CDCl3) 167.1 (C O), 146.6 (C-13a), 133.5
(C-4a), 132.6 (C-15a), 128.7 (C-12), 128.4 (C-10), 128.1, 126.3,
125.9 (C-1,2,3), 127.6 (C-9a), 126.0 (C-4), 125.9 (C-1), 118.6 (C-
11), 113.8 (C-13), 60.6 (C-14b), 60.1 (OCH2), 58.0 (C-5), 56.2
(C-7), 49.6 (C-14a), 44.6 (C-8a), 35.3 (C-9), 33.1 (9-Me), 30.9
(C-15), 25.6 (9-Me), 22.1 (C-8), 14.5 (Me). HRMS C25H30N2O2:
calcd. 390.2307, found 390.229.
Conclusions
Lewis acid catalyzed hetero-Diels–Alder reaction and subse-
quent functionalization provides a convenient access to novel
benzo[b]isoquino[2,3-h]naphthyridines. DNA binding experi-
ments with polynucleotides and model oligonucleotides revealed
a preference of the cis diastereomers for AT-rich sequences and
a preference of the trans diastereomers for CG-rich sequences.
NOE experiments showed that the cis isomers bind to the minor
groove of d(AAGAATTCTT)2, whereas the trans isomers lead to
cap-complexes with CG-rich oligonucleotides, with a preference
for the 3ꢀ-terminals, as found for camptothecins. The compounds
showed modest cytotoxicity against two different human tumour
cell lines, RD-ES and CAD-ES1 (Ewing’s sarcoma).
Ethyl (8aR,14aR,14bS)-9,9-dimethyl-7,8,8a,9,14,14a,14b,15-
octahydro-5H-benzo[b]isoquino[2,3-h]-1,7-naphthyridine-11-
carboxylate (7b)
As described above for 7a, using 4 (300 mg, 1.23 mmol), 5
˚
(224 mg, 1.39 mmol), 4 A molecular sieves (2 g), and a 1 M
solution of SnCl4 (1.6 mL, 1.6 mmol), with purification by flash
chromatography (Rf 0.30, SiO2, pentane–CH2Cl2–NEt3, 15 : 3 :
1) was obtained 7b (180 mg, 0.46 mmol, 37%) as a colourless
amorphous solid, [a]2D2 −271.5 (c 1.20, CH2Cl2). Found: C, 76.84;
H, 8.01; N, 7.18. C25H30N2O2 (390.5) requires C, 76.89; H, 7.74;
N, 7.1. m˜max (KBr)/cm−1 3387 (s), 3066, 3028 (m), 2970, 2968,
2930, 2904 (m), 1704 (s), 1607, 1515, 1458 (s), 1296, 1251 (s),
771, 742 (m). dH (600 MHz, CDCl3) 7.96 (d, J 2.0, 10-H), 7.68
(dd, J 8.4 and 2.0, 12-H), 7.17–7.12 (m, 1,2,3-H), 7.05 (dd, 4-H),
6.49 (d, J 8.4, 13-H), 4.45 (br s, NH), 4.31 (q, J 7.1, O–CH2),
Experimental
General
5
3.92 (d, J 15.2, 5-Heq), 3.45 (br d, J 15.2 and J 1.2, 5-Hax),
The self-complementary oligonucleotides d(AAGAATTCTT)2,
d(CGACGTCG)2, d(GCGATCGC)2 and d(GCATCGCG-
ATGC)2, as free acids, were purchased from Roche Diagnostics:
they were synthesized on a 10 lmol scale by solid-phase tech-
niques and purified by gel filtration. Poly(dA–dT)2, poly(dG–
dC)2 and salmon sperm DNA were purchased from Sigma. The
oligonucleotides were dissolved in D2O with low paramagnetic
impurities (99.99% isotope, Aldrich) or in H2O–D2O (90 : 10)
HPLC grade (Aldrich), in the presence of a minimum amount
of NaCl (0.02 M) in order to stabilize the double helix. The
pH was adjusted to 5.8–6.0. Assays for cytotoxicity experiments
were carried out according to the literature.1,2,25 Compound 13
was prepared following a literature method.12
3.22 (dt, J 11.4, 3.8 and 3.0, 7-Heq), 3.19 (dd, J 15.9 and 4.3,
15-Heq), 3.10 (dd, J 10.6 and 8.5, 14a-H), 2.88 (br dd, J 15.9,
10.5 and J 1.2, 15-Hax), 2.29 (ddd, J 11.4, 12.4 and 2.6, 7-
5
Hax), 2.28 (ddd, J 10.5, 4.3 and 8.5, 14b-H), 1.92 (dq, J 13.0,
3.0 and 2.6, 8-Heq), 1.65 (qd, J 13.0, 12.4, 12.3 and 3.8, 8-Hax),
1.53 (ddd, J 10.6, 12.3 and 3.3, H-8a), 1.41 (s, 9-Me), 1.21 (s,
=
9-Me), 1.36 (t, J 7.1, Me). dC (100 MHz, CDCl3) 167.1 (C O),
146.6 (C-13a), 133.7 (C-4a), 132.1 (C-15a), 129.8 (C-9a), 128.9
(C-12), 128.4 (C-10), 128.2, 126.5, 125.9 (C-1,2,3), 126.0 (C-
4), 118.9 (C-11), 113.4 (C-13), 62.9 (C-14b), 60.2 (OCH2), 57.7
(C-5), 56.0 (C-14a), 55.8 (C-7), 44.8 (C-8a), 34.7 (C-9), 33.3 (C-
15), 26.4 (9-Me), 25.7 (9-Me), 23.6 (C-8), 14.5 (Me). m/z: (EI)
390 (M+, 7%), 345 (M+ − OC2H5, 3%), 260 (M+ − 130, 3%),
2 8 5 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 8 4 8 – 2 8 5 8

View Article Online
158 (C11H12N+, 2%), 146 (C10H12N+, 100%), 130 (C9H8N+, 4%),
104(C8H8 , 9%).
4J(8eq,14a) 1.3, 8-Heq), 1.50 (dtd, J 12.3, 3.4, 3.1 and 4J(8a,NH)
1.5, 8a-H), 1.40 (dddd, J 13.3, 12.8, 12.3 and 4.1, 8-Hax), 1.39
(s, 9-Me), 1.27 (s, 9-Me). dC (100 MHz, CDCl3) 167.0 (C O),
+
=
Crystal structure data for 7b. C25H30N2O2, M = 390.51,
crystal size 0.7 × 0.4 × 0.3 mm, monoclinic, space group C2,
146.8 (C-13a), 133.7 (C-15a), 132.6 (C-4a), 128.8 (C-12), 128.6
(C-10), 128.2, 126.4, 125.9, 125.9 (C-1,2,3,4), 127.6 (C-9a), 118.3
(C-11), 113.8 (C-13), 62.3 (OCH2), 60.6 (C-14b), 58.1 (C-5), 57.9
(NCH2), 56.3 (C-7), 49.6 (C-14a), 45.8 (NMe2), 44.7 (C-8a), 35.4
(C-9), 33.1 (9-Me), 30.9 (C-15), 25.5 (9-Me), 22.1 (C-8). m/z (EI)
433 (M+, 1%), 362 (M+ − O(CH2)2N(CH3)2, 7%), 158 (C11H12N+,
◦
˚
a = 24.525(5), b = 8.188(2), c = 26.609(4) A, a = 90 , b =
◦
◦
3
−1
˚
116.853(14) , c = 90 , V = 4767.1(18) A , Dc = 1.088 Mg m ,
−1
˚
T = 293(2) K, Z = 8, l = 0.069 mm , k = 0.71073 A, reflections
collected/unique: 5982/5841 [R(int) = 0.0383], final R indices
[I > 2r(I)]: R1 = 0.0806, wR2 = 0.2046, R indices (all data): R1 =
0.1373, wR2 = 0.2376. CCDC reference number 243553. See
http://dx.doi.org/10.1039/b503281d for crystallographic data
in CIF or other electronic format.
12%), 146 (C10H12N+, 100%), 104 (C8H8 , 10%).
+
(8aR,14aS,14bS)-N-(2-Hydroxyethyl)-9,9-dimethyl-
7,8,8a,9,14,14a,14b,15-octahydro-5H-benzo[b]isoquino-[2,3-h]-
1,7-naphthyridine-11-carboxamide (10a)
(8aR,14aS,14bS)-9,9-Dimethyl-7,8,8a,9,14,14a,14b,15
octahydro-5H-benzo[b]isoquino[2,3-h]-1,7-naphthyridine-11-
carboxylic acid (8)
Under an inert gas atmosphere, aminoethanol (0.31 mL,
5.12 mmol) was added to Na (59 mg, 2.56 mmol) in dry toluene
(10 mL). After the Na had dissolved, a solution of 7a (100 mg,
0.256 mmol) in dry toluene (2 mL) was added dropwise. The
reaction mixture was heated at 80 ◦C for 16 h, cooled to
room temperature and was then hydrolyzed with satd. NH4Cl
solution. The aqueous layer was washed with CH2Cl2 (3 ×
5 mL). The combined organic layers were dried (MgSO4), and
the solvent was removed. The residue was purified by flash
chromatography (SiO2, CH2Cl2–MeOH, 10 : 1 with 5% NEt3) to
give 10a (65 mg, 0.16 mmol, 62%) as yellow crystals, [a]2D2 +127.8
(c 1.05, CHCl3). m˜max (ATR (attenuated total reflection))/cm−1
3315 (s), 3066, 3028 (m), 2965, 2927, 2904, 2871, 2854, 2769 (m),
1603, 1542 (s), 767, 738 (m). dH (600 MHz, CDCl3) 7.70 (d, J 2.2,
10-H), 7.32 (dd, J 8.4 and 2.2, 12-H), 7.18–7.12 (m, 1,2,3-H),
7.03 (dd, 4-H), 6.43 (br t, CONH), 6.48 (d, J 8.4, 13-H), 4.45 (br
m, NH), 3.95 (d, J 15.3, 5-Heq), 3.74 (m, OCH2), 3.69 (br m,
14a-H), 3.58 (m, NCH2), 3.40 (d, J 15.3 and 5J 1.1, 5-Hax), 3.24
(ddd, J 16.7, 11.3 and 5J 1.1, 15-Hax), 3.14 (ddd, J 11.6, 4.1 and
2.9, 7-Heq), 2.83 (dd, J 16.7 and 4.8, 15-Heq), 2.57 (ddd, J 11.3,
4.8 and 2.0, 14b-H), 2.17 (ddd, J 12.8, 11.6 and 2.4, 7-Hax), 1.60
KOH (141 mg, 2.51 mmol) was added to a solution of 7a
(280 mg, 0.71 mmol) in ethanol (10 mL) and water (2 mL), and
the reaction mixture was heated at reflux for 4 h. After being
concentrated, the residual layer was adjusted to pH 8–9 with
6 N HCl (ice-cooling) and extracted four times with CH2Cl2–
methanol (10 : 1). The combined extracts were dried (MgSO4),
and the solvent was removed to give 8 (230 mg, 0.64 mmol,
89%) as an amorphous solid, [a]2D2 +183.15 (c 1.65, MeOH). m˜max
(KBr)/cm−1 3416 (br), 3067, 3028, 2964 (m), 2906, 2873, 2806,
2771 (m), 1703 (s), 1606, 1512, 1497 (s), 1303, 1251 (s), 776, 744
(m). dH (400 MHz, DMSO-d6) 11.9 (s, br, COOH), 7.68 (d, J 1.8,
10-H), 7.48 (dd, J 8.5, 1.8, 12-H), 7.20–7.11 (m, 1,2,3-H), 7.09–
7.03 (m, br, 4-H), 6.76 (d, J 8.5, 13-H), 6.30 (s, br, NH), 3.89 (d,
J 15.5, 5-Heq), 3.66 (s, br, 14a-H), 3.45 (dd, J 16.5 and 11.7, 15-
Hax), 3.26 (d, J 15.5, 5-Hax), 3.03 (m, 7-Heq), 2.70 (dd, J 16.5
and 4.1, 15-Heq), 2.47 (ddd, J 11.7, 4.1 and 1.0, 14b-H), 2.08 (m,
7-Hax), 1.57–1.46 (m, 8-Heq, 8a-H), 1.32 (s, 9-Me), 1.19 (s, 9-
=
Me), 1.14–1.10 (m, 8-Hax). dC (100 MHz, CDCl3) 167.7 (C O),
4
147.7 (C-13a), 133.6 (C-15a), 133.58 (C-4a), 128.3 (C-9a), 128.0
(C-12), 127.6, 126.2, 125.6 (C-10, C-1,2,3), 126.0 (C-4), 116.5
(C-11), 112.8 (C-13), 60.5(C-14b), 57.5 (C-5), 55.6 (C-7), 49.1
(C-14a), 43.4 (C-8a), 34.8 (C-9), 32.9 (9-Me), 30.0 (C-15), 25.2
(9-Me), 21.8 (C-8). m/z (EI): 362 (M+, 3%), 318 (M+ − COOH,
2%), 158 (C11H12N+, 16%), 146 (C10H12N+, 100%), 130 (C9H8N+,
(dqd, J 13.3, 3.3, 3.2, 2.9 and J(8,14a) 1.3, 8-Heq), 1.50 (dtd,
4
J 12.4, 3.2, 3.2 and J(8a,NH) 1.2, 8a-H), 1.38 (s, 9-Me), 1.40
(dddd, J 13.3, 12.8, 12.4 and 4.1, 8-Hax) 1.38 (s, 9-Me), 1.26
=
(s, 9-Me). dC (100 MHz, CDCl3) 169.1 (C O), 145.7 (C-13a),
132.6 (C-15a, C-4a), 128.3, 126.6, 126.3 (C-1,2,3), 128.1 (C-9a),
126.1 (C-10), 126.0 (C-4), 125.5 (C-12), 122.1 (C-11), 114.0
(C-13), 62.8 (OCH2), 60.8 (C-14b), 57.9 (C-5), 56.2 (C-7), 49.5
(C-14a), 44.6 (C-8a), 43.1 (NCH2), 35.5 (C-9), 33.2 (C-16), 30.8
(9-Me), 25.6 (9-Me), 22.0 (C-8). m/z (EI): 387 (M+ − H2O, 1%),
229 (C16H23N+, 5%), 213 (C15H19N+, 5%), 158 (C11H12N+, 15%),
+
31%), 104 (C8H8 , 18%). HRMS C23H26N2O: calcd.362.1994,
found 362.1992.
2-(Dimethylamino)ethyl(8aR,14aS,14bS)-9,9-dimethyl-
7,8,8a,9,14,14a,14b,15-octahydro-5H-benzo[b]isoquino[2,3-h]-
1,7-naphthyridine-11-carboxylate (9)
146 (C10H12N+, 100%), 104 (C8H8 , 12%).
+
(8aR,14aR,14bS)-N-(2-Hydroxyethyl)-9,9-dimethyl-
7,8,8a,9,14,14a,14b,15-octahydro-5H-benzo[b]isoquino-[2,3-h]-
1,7-naphthyridine-11-carboxamide (10b)
DCC (69.8 mg, 0.336 mmol) was added to a suspension of 8
(110 mg, 0.28 mmol) in dry CH2Cl2 (15 mL) at 0 ◦C. After
stirring for 10 min, DMAP (7 mg, 0.06 mmol) and N,N-
dimethylaminoethanol (0.034 mL, 0.336 mmol) in CH2Cl2
(2 mL) were added. The reaction mixture was stirred at room
temperature for 16 h and then filtered through Celite. The filtrate
was washed with aqueous NH4Cl solution (2 × 10 mL), dried
(MgSO4) and concentrated. The residue was purified by flash
chromatography (Rf 0.22, SiO2, pentane–CH2Cl2–NEt3, 10 : 5 :
1) to give 9 (98 mg, 0.23 mmol, 81%) as colourless needles,
[a]2D2 +139.9 (c 0.67, CHCl3). Found: C, 74.49; H, 8.23; N, 9.50.
C27H35N3O2 (433.6) requires C, 74.79; H, 8.14; N, 9.69. m˜max
(KBr)/cm−1 3390 (s), 3067, 3028 (m), 2946, 2904, 2872, 2818,
2768 (m), 1695 (s), 1604, 1512, 1456 (s), 1242 (s), 769, 741 (m).
dH (600 MHz, CDCl3) 7.87 (d, J 2.0, 10-H), 7.64 (dd, J 8.4 and J
2.0, 12-H), 7.17–7.12 (m, 1,2,3-H), 7.03 (dd, 4-H), 6.47 (d, J 8.4,
13-H), 4.49 (br dd, NH), 4.37 (m, OCH2), 3.95 (d, J 15.3, 5-Heq),
3.70 (br m, 14a-H), 3.38 (dd, J 15.3 and 5J 1.1, 5-Hax), 3.16 (br
dd, J 16.7, 11.3 and 5J 1.1, 15-Hax), 3.13 (ddd, J 11.5, 4.1 and
2.7, 7-Heq), 2.84 (dd, J 16.7 and 4.8, 15-Heq), 2.70 (m, NCH2),
2.56 (ddd, J 11.3, 4.8 and 2.3, 14b-H), 2.34 (s, NMe2), 2.17 (ddd,
J 12.8, 11.6 and 3.3, 7-Hax), 1.60 (dqd, J 13.3, 3.3, 3.4, 2.7 and
A solution of 7b (100 mg, 0.256 mmol) in abs. THF (2 mL)
was added to a solution of Na (59 mg, 2.56 mmol) in abs. THF
(10 mL), and the reaction mixture heated at 80 ◦C for 16 h.
The reaction mixture was then cooled to room temperature and
hydrolyzed with satd. NH4Cl solution (10 mL). The aqueous
layer was extracted with CH2Cl2 (3 × 5 mL), and the combined
organic layers were dried (MgSO4). After removal of the solvent,
the residue was purified by flash chromatography (Rf 0.11, SiO2,
CH2Cl2–MeOH, 20 : 1) to give 10b (73 mg, 0.18 mmol, 70%)
as a yellowish amorphous solid, [a]2D2 −222.1 (c 1.21, CHCl3).
Found: C, 73.81; H, 7.81; N, 10.25. C25H31N3O2 (405.5) requires
C, 74.04; H, 7.70; N, 10.36. dH (600 MHz, CDCl3) 7.70 (d, J 2.2,
10-H), 7.32 (dd, J 8.4 and 2.2, 12-H), 7.17–7.12 (m, 1,2,3-H),
7.04 (br m, 4-H), 6.50 (d, J 8.4, 13-H), 6.48 (t, CONH), 4.35
(br s, NH), 3.92 (d, J 15.1, 5-Heq), 3.81 (br t, OCH2), 3.60 (br
5
q, NCH2), 3.45 (br dd, J 15.1 and J 1.3, 5-Hax), 3.22 (dt, J
11.5, 3.8 and 3.0, 7-Heq), 3.18 (dd, J 15.9 and 4.3, 15-Heq),
3.09 (dd, J 10.7 and 8.5, 14a-H), 2.88 (ddd, J 15.9, 10.5 and
5J 1.1, 15-Hax), 2.29 (ddd, J 11.5, 12.2 and 2.5, 7-Hax), 2.28
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 8 4 8 – 2 8 5 8
2 8 5 5

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(ddd, J 10.5, 4.3 and 8.5, 14b-H), 1.92 (dq, J 13.1, 3.0 and 2.5,
8-Heq), 1.65 (qd, J 13.1, 12.3, 12.2 and 3.8, 8-Hax), 1.54 (ddd,
J 10.7, 12.3 and 3.3, 8a-H), 1.48 (s, 9-Me), 1.22 (s, 9-Me). dC
(4 × 20 mL). The combined extracts were dried (MgSO4) and
concentrated. The residue was purified by flash chromatography
(Rf 0.17, SiO2, pentane–EtOAc, 1 : 1), to give 14 (1.97 g,
8.59 mmol, 75%) as a yellowish oil, [a]2D2 −8.9◦ (c 1.02, CHCl3).
Found: C, 78.56; H, 8.30; N, 6.14. C15H19NO (227.32) requires
C, 78.56; H, 8.35; N, 6.11. m˜max (ATR)/cm−1 3393 (br), 3064, 3022
(m), 2916, 2851 (m), 1586, 1495, 1452 (s), 741 (m). dH (400 MHz,
CDCl3) 7.18–7.12 (m, 6,7-H), 7.10–7.05 (m, 5-H), 7.04–7.02 (m,
8-H), 3.94 (d, J 16.4, 1-Ha), 3.77 (d, J 16.4, 1-Hb), 3.58 (dd, J
11.0 and 5.6, 1ꢀꢀ-Ha), 3.54 (dd, J 11.0 and 8.7, 1ꢀꢀ-Hb), 3.19 (dddd,
J 8.7, 7.3, 5.8 and 5.6, 3-H), 2.79 (dd, J 16.7 and 5.8, 4-Ha), 2.73
(ddd, J 12.7, 7.3 and 7.3, 1ꢀ-Ha), 2.58 (dt, J 12.7, 6.4 and 6.4,
1ꢀ-Hb), 2.50 (dd, J 16.7 and 7.3, 4-Hb), 2.37–2.31 (m, 2ꢀ-CH2),
1.78 (t, J 2.5, 4ꢀ-Me). dC (100 MHz, CDCl3) 134.2 (C-4a), 133.9
(C-8a), 128.8 (C-5), 126.9 (C-8), 126.5, 126.0 (C-6,7), 77.6 (C-4ꢀ),
77.4 (C-3ꢀ), 62.3 (C-1ꢀꢀ), 58.8 (C-3), 50.6 (C-1), 50.1 (C-1ꢀ), 26.5
(C-4), 18.8 (C-2ꢀ), 3.4 (4ꢀ-Me). m/z (EI): 198 (M+ − CH2OH,
100%), 196 (M+ −CH2OH − 2, 21%), 176 (C11H14NO+, 80%),
130 (C9H8N+, 17%), 117 (25%), 115 (29%).
=
(100 MHz, CDCl3) 168.9 (C O), 145.7 (C-13a), 133.5 (C-15a),
132.07 (C-4a), 130.1 (C-9a), 128.1, 126.4, 125.9 (C-1,2,3), 126.0
(C-10), 125.8 (C-4), 125.7 (C-12), 122.1 (C-11), 113.6 (C-13),
62.8 (C-19), 62.4 (C-14b), 57.6 (C-5), 55.8 (C-14a), 55.7 (C-7),
44.7 (C-8a), 42.9 (C-18), 34.6 (C-9), 33.2 (C-15), 26.4 (C-16),
25.7 (C-17), 23.5 (C-8). m/z (EI): 387 (M+ − H2O, 12%) 229
(C16H23N+, 5%), 213 (C15H19N+, 5%), 158 (C11H12N+, 12%), 146
(C10H12N+, 100%), 104 (C8H8 , 100%).
+
Preparation of (8aR,15aS,15bS) (12a) and (8aR,15aR,15bS)-
9,9-dimethyl-7,8,8a,9,15,15a,15b,15-octahydro-
5H[1,3]benzodioxolo[5,6-b]isoquino[2,3-h]-1,7-naphthyridine (12b)
As described above for 7a, using 4 (1.5 g, 6.20 mmol), 3,4-
˚
(methylenedioxy)aniline (850 mg, 6.20 mmol), 4 A molecular
sieves (10 g), and a 1 M solution of SnCl4 (8 mL, 8 mmol), with
purification by flash chromatography (SiO2, pentane–CH2Cl2–
NEt3, 15 : 3 : 1), to give 12b in the first fraction as a brown
amorphous solid (Rf 0.41, 800 mg, 2.21 mmol, 37%), and 12a
in the second fraction as a brown amorphous solid (Rf 0.29,
500 mg, 1.38 mmol, 22%). 12a: Found: C, 75.82; H, 7.14, N,
7.78. C23H26N2O2 (362.5) requires C, 76.21; H, 7.23; N, 7.73.
m˜max (ATR)/ cm−1 3389 (s), 2963, 2932 (m), 1620, 1510, 1479 (s),
1030 (s), 746 (m). dH (400 MHz, CDCl3) 7.18–7.13 (m, 2,3-H),
7.11–7.08 (m, 1-H), 7.06–7.01 (m, 4-H), 6.68 (s, 10-H), 6.20 (s,
13-H), 5.81 and 5.79 (d, J 1.4, OCH2O), 4.19 (d, J 16.8, 5-Heq),
3.67 (br s, NH), 3.65 (d, J 16.8, 5-Hax), 3.47 (br s, 14a-H), 3.25
(ddd, J 12.3, 5.0 and 4.9, 14b-H), 3.04 (dd, J 16.9 and 12.3, 15-
Hax), 2.63 (dt, J 11.8, 11.8 and 3.2, 7-Hax), 2.52 (ddd, J 11.8,
4.9 and 2.8, 7-Heq), 2.45 (dd, J 16.9 and 4.9, 15-Heq), 1.62–1.52
(m, 8a-H, 8-Heq), 1.44–1.37 (m, 8-Hax), 1.27 (s, 9-Me), 1.25 (s,
9-Me). dC(100 MHz, CDCl3) 146.0 (C-13a), 139.9 (C-12), 137.4
(C-11), 133.9 (C-4a), 133.3 (C-15a), 128.9 (C-1), 126.7 (C-4),
126.2, 126.1 (C-2,3), 121.0 (C-9a), 105.9 (C-10), 100.2 (OCH2O),
97.3 (C-13), 57.5 (C-14b), 56.6 (C-5), 50.3 (C-14a), 46.3 (C-7),
38.1 (C-8a), 35.1 (C-9), 33.9 (9-Me), 26.5 (9-Me), 24.6 (C-15),
23.3 (C-8). m/z (EI): 362 (M+, 7%), 347 (M+ − OC2H5, 3%), 216
(M+ − 130, 3%), 158 (C11H12N+, 2%), 146 (C10H12N+, 100%),
(3S)-N-(3-Pentynyl)-1,2,3,4-tetrahydroisoquinoline-3-
carbaldehyde (15)
DMSO (0.75 mL, 10.95 mmol) in abs. CH2Cl2 (4 mL) was
added dropwise over 30 min to a solution of (COCl)2 (0.47 mL,
5.60 mmol) in abs. CH2Cl2 (15 mL) at −50 ◦C. After stirring for
15 min, a solution of 14 (500 mg, 2.18 mmol) in abs. CH2Cl2
(10 mL) was added dropwise over 45 min, and the reaction
mixture was stirred at −50 ◦C for 16 h. NEt3 (1.8 mL) was added
dropwise over 30 min, the reaction mixture stirred for a further
15 min and then allowed to warm up to room temperature. The
organic layer was washed with water (3 × 50 mL) and dried
(MgSO4). The solvent was removed under vacuum to give 15
(495 mg, quant.) as a yellow oil. Found: C, 79.38; H, 7.55; N,
6.11. C15H17NO (227.30) requires C, 79.26; H, 7.54; N, 6.16. mmax
(neat)/cm−1 3064 (m), 3047 (m), 3023 (m), 2980 (s), 2925 (s),
2914 (s), 1730 (s), 1454 (s), 745 (s). dH (200 MHz, CDCl3) 9.77
(d, J 1.1, CHO), 7.18–7.12 (m, 5,6,7-H), 7.05–7.03 (m, 8-H),
4.05 and 3.94 (d, J 16.0, 1-CH2), 3.54 (td, J 6.4 and 1.3, 3-H),
3.07–2.73 (m, 4-CH2, 1ꢀ-CH2), 2.45–2.38 (m, 2ꢀ-CH2), 1.79 (t, J
2.5, 3ꢀ-Me). dC (50 MHz, CDCl3) 203.3 (C O), 133.7 (C-4a),
=
130 (C9H8N+, 4%), 104 (C8H8 , 9%). 12b: Found: C, 76.40; H,
+
132.0 (C-8a), 128.7 (C-5), 126.7 (C-8), 126.6 (C-6), 126.3 (C-7),
77.0 (C-3ꢀ), 76.8 (C-4ꢀ), 66.4 (C-3), 53.6 (C-1), 52.1 (C-1ꢀ), 26.0
(C-4), 18.6 (C-2ꢀ), 3.4 (4ꢀ-Me).
7.19; N, 7.80. C23H26N2O2 (362.5) requires C, 76.21; H, 7.23;
N, 7.73. m˜max (ATR)/cm−1 3394(s), 2961, 2928, 2872, 2816 (m),
1631, 1500, 1479 (s), 1033 (s), 740 (m). dH (400 MHz, CDCl3)
7.16–7.10 (m, 1,2,3-H), 7.04–7.03 (m, 4-H), 6.75 (s, 10-H), 6.14
(s, 13-H), 5.81 (s, OCH2O), 3.92 (d, J 15.1, 5-Heq), 3.69 (br s,
NH), 3.43 (d, J 15.1, 5-Hax), 3.21–3.16 (m, 15-Heq, 7-Heq), 2.98
(dd, J 10.4 and 8.4, 14a-H), 2.84 (dd, J 15.7 and 10.4, 15-Hax),
2.30–2.22 (m, 7-Hax, 14b-H), 1.89 (dd, J 12.4, 3.4, 2.9 and 2.9,
8-Heq), 1.64 (dq, J 12.4, 12.4, 12.4 and 3.6, 8-Hax), 1.55 (dt,
J 12.4, 10.4 and 3.4, 8a-H), 1.30 (s, 9-Me), 1.12 (s, 9-Me). dC
(100 MHz, CDCl3) 146.1 (C-13a), 140.2 (C-12), 137.1 (C-11),
133.7 (C-4a), 132.5 (C-15a), 128.2, 126.4, 125.9 (C-1,2,3), 125.8
(C-4), 123.2 (C-9a), 106.3 (C-10), 100.4 (OCH2O), 96.5 (C-13),
63.2 (C-14b), 57.8 (C-5), 56.0 (C-14a), 55.8 (C-7), 45.4 (C-8a),
34.8 (C-9), 33.5 (C-15), 27.2 (9-Me), 27.0 (9-Me), 23.8 (C-8). m/z
(EI): 362 (M+, 7%), 347 (M+ − OC2H5, 3%), 216 (M+ − 130, 3%)
158 (C11H12N+, 2%), 146 (C10H12N+, 100%), 130 (C9H8N+, 4%),
Ethyl (14bS)-9-methyl-7,8,14b,15-tetrahydro-5H-
benzo[b]isoquino[2,3-h]-1,7-naphthyridine-11-carboxylate (17)
As described above for 7a, using 15 (488 mg, 2.15 mmol),
˚
5 (355 mg, 2.15 mmol), 4 A molecular sieves (5 g), and
BF3·OEt2 (0.56 mL, 4.30 mmol), with purification by flash
chromatography (Rf 0.30, SiO2, pentane–CH2Cl2–NEt3, 20 : 2 :
1, to give 17 (300 mg, 0.81 mmol, 38%) as a yellow amorphous
solid, [a]2D2 −267.9 (c 1.06, CHCl3). Found: C, 77.40; H, 6.45; N,
7.54. C24H24N2O2 (372.46) requires C, 77.39; H, 6.49; N, 7.52.
m˜max (ATR)/cm−1 3064, 3027 (m), 2976, 2958, 2925, 2903, 2837,
2754 (m), 1709 (s), 1615, 1581, 1496 (s), 1239 (s), 789, 733 (m). dH
(400 MHz, CDCl3) 8.77 (d, J 1.6, 10-H), 8.16 (dd, J 8.8 and 1.6,
12-H), 7.97 (d, J 8.8, 13-H), 7.26–7.15 (m, 1,2,3-H), 7.12–7.10
(m, 4-H), 4.37 (q, J 7.1, OCH2), 4.04 (d, J 14.9, 5-Heq), 3.83 (dd,
J 16.4 and 4.2, 15-Heq), 3.78 (dd, J 10.8 and 4.2, 14b-H), 3.73 (d,
J 14.9, 5-Hax), 3.25 (ddd, J 11.6, 5.7 and 2.1, 7-Heq), 3.13 (ddd,
J 11.6, 11.6 and 5.7, 8-Hax), 3.05–3.01 (m, 8-Heq), 2.83 (dd, J
16.4 and 10.8, 15-Hax), 2.61 (ddd, J 11.6, 11.6 and 3.9, 7-Hax),
2.68 (s, 9-Me), 1.47 (t, J 7.1, CH2CH3). dC (100 MHz, CDCl3)
+
104 (C8H8 , 9%).
(3S)-3-(Hydroxymethyl)-N-(3-pentynyl)-1,2,3,4-
tetrahydroisoquinoline (14)
Bromopentyne (2 g, 13.60 mmol) was added with ice-cooling
to a suspension of K2CO3 (1.44 g, 11.43 mmol) and 13 (1.85 g,
11.31 mmol) in abs. toluene (20 mL), and the reaction mixture
was heated at reflux for 72 h. After cooling to room temperature,
the reaction mixture was extracted with 2 N HCl (4 × 25 mL).
The aqueous layer was washed with Et2O (3 × 20 mL), adjusted
to pH 8–9 with conc. NH3 solution and extracted with CH2Cl2
=
166.6 (C O), 160.4 (C-14a), 148.2 (C-13a), 142.9 (C-9), 134.6
(C-15a), 133.8 (C-4a), 127.5 (C-11), 126.8 (C-9a), 129.7 (C-10),
128.9 (C-1), 127.8 (C-12), 126.7 (C-13), 126.4, 126.0, 125.7 (C-
2,3,4), 63.6 (C-14b), 61.2 (OCH2), 58.6 (C-5), 50.9 (C-7), 35.1
(C-15), 26.9 (C-8), 14.4 (CH2CH3), 13.7 (9-Me). m/z (EI): 372
2 8 5 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 8 4 8 – 2 8 5 8

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(M+, 100%), 357 (M+ − CH3, 7%), 343 (M+ − C2H5, 8%), 327
(M+ − OC2H5, 8%), 104 (60%).
residual water signal, set at d = 4.80 ppm, for aqueous solutions,
respectively. Estimated accuracy is 0.005 ppm for 1H spectra,
and
0.1 ppm for 13C spectra. The coupling constants were
obtained from one-dimensional spectra and measured in Hz,
with an accuracy of 0.05 Hz, unless specified in Table 1.
The titration experiments were performed by adding increasing
amounts of the drug to the solution (1–3 mM) of the oligonu-
cleotide (DNA), until R = [drug]/[DNA] = 2. As the ligands
are not completely soluble in water, the NMR experiments were
performed by adding a 12 mM solution in DMSO-d6 to the
oligonucleotide.
(11aS)-1-N-(4-Carboxyethylphenyl)-2-(1-chloroethylidene)-
1,2,3,4,6,11-hexahydrobenzo[g]quinolidine (18)
As described above for 7a, using 15 (100 mg, 0.44 mmol), 5
˚
(72 mg, 0.44 mmol), 4 A molecular sieves (1 g), and a 1 M
solution of EtAlCl2 (0.88 mL, 0.88 mmol), with purification by
chromatography (Rf 0.11, SiO2, pentane–CH2Cl2–NEt3, 20 : 2 :
1), to give 18 (67 mg, 0.16 mmol, 37%) as a yellow amorphous
solid, [a]2D2 −76.8 (c 1.07, CHCl3). Found: C, 70.15; H, 6.60;
Cl, 8.67; N, 6.83. C24H27ClN2O2 (410.94) requires C, 70.15; H,
6.6.62; Cl, 8.63; N, 6.82. m˜max (ATR)/cm−1 3365 (br), 3062, 3020
(m), 2926, 2917, 2841 (m), 1697 (s), 1601, 1520, 1476 (s), 1269,
1170, 1100 (s), 768, 747 (m). dH (400 MHz, CDCl3) 7.89–7.86
(m, 3ꢀ,5ꢀ-H), 7.19–7.13 (m, 8,9-H), 7.10–7.06 (m, 10-H), 7.05–
7.01 (m, 7-H), 6.57–6.54 (m, 2ꢀ,6ꢀ-H), 5.48 (d, J 8.5, NH), 4.31
(q, J 7.1, OCH2), 4.31–4.28 (m, 1-H), 4.26 (d, J 16.8, 6-Ha), 3.71
(d, J 16.8, 6-Hb), 3.49 (ddd, J 12.4, 4.9 and 2.5, 11a-H), 2.91 (dd,
J 16.8 and 12.4, 11-Hax), 2.82–2.77 (m, 3-Ha), 2.74–2.61 (m, 4-
CH2), 2.48 (dd, J 16.8 and 4.9, 11-Heq), 2.39–2.31 (m, 3-Hb),
NOESY and ROESY spectra were acquired in the phase-
sensitive TPPI mode, with 2K × 512 complex fids, a spectral
width of 6666.667 Hz, recycling delay of 1.3 s, 72 scans,
at temperature of +20 ◦C, with a mixing time of 300 ms.
TOCSY and ROESY spectra were acquired with the use of
MLEV-17 spin-lock pulse28 (field strength 7576 Hz, 60 ms total
duration). All spectra were transformed and weighted with a 90◦
shifted sine-bell squared function to 1K × 1K real data points.
Water suppression was achieved by the pre-saturation technique,
1
placing the carrier frequency on the H2O resonance. The H
assignments for free and bound molecules were performed by
using the coupling constant values, and NOESY, ROESY, COSY
and TOCSY experiments. In particular, the assignment of 4-H
vs. 1-H in water was established by ROESY, using the NOE
interactions with 5-H and 15-H, respectively, then 2-H vs. 3-H by
the interaction with 1-H and 4-H, respectively. The assignment
of 5-Heq vs. 5-Hax was made by the five-bond coupling between
15-Hax and 5-Hax: the homoallylic coupling is maximum when
the orientation of the interacting protons is parallel to the plane
of the p-bonds. The assignments for the free oligonucleotides
d(AAGAATTCTT)2, d(CGACGTCG)2 and d(GCGATCGC)2
have been reported previously.6,8 The sequential assignment of
nucleotide units in the free nucleotide and in the complexes
was performed by applying well established procedures11 for
the analysis of double-stranded oligomers in the B-DNA form.
The 13C assignments were performed by DEPT and HSQC
experiments.
2.37 (d, J 2.5, 1ꢀꢀ-Me), 1.35 (t, J 7.1, CH2CH3). dC (100 MHz,
ꢀ
=
CDCl3) 166.7 (C O), 150.4 (C-1 ), 133.4 (C-10a), 132.5 (C-6a),
131.5 (C-3ꢀ,5ꢀ), 129.1 (C-7), 129.0 (C-2), 126.9 (C-1ꢀꢀ), 126.8 (C-
10), 126.4 (C-8,9), 119.0 (C-4ꢀ), 112.3 (C-2ꢀ,6ꢀ), 60.2 (OCH2),
58.3 (C-11a), 56.1 (C-6), 53.6 (C-1), 46.2 (C-4), 26.9 (C-3), 23.9
(C-11), 22.3 (1ꢀꢀ-Me), 14.4 (CH2CH3). m/z (EI): 410 (M+, 1%),
374 (M+ − HCl, 40%), 244 (C15H15ClN+, 39%), 146 (C10H12N+,
100%), 130 (C9H8N+, 29%).
UV experiments
The UV spectra were recorded at 25 ◦C on a Perkin–Elmer
Lambda 40 UV-VIS spectrophotometer, and for the melting
experiments on a JASCO W-550 spectrophotometer equipped
with a thermostated cell. Melting of the oligonucleotides
was followed by UV absorbance. Dilution experiments were
performed starting from a concentrated solution (10−4 M) of
the drug in water, which was diluted to 10−7 M, in the presence
of NaCl (0.02 M). For the aggregation process a dimerization
model was considered to be a sufficient approximation,26 because
we have just used the solutions (in the range 10−5–10−7 M)
which gave spectra with the isosbestic point (385, 380, 395,
398 and 402 nm for 7a, 7b, 9, 10a and 10b respectively). The
dimerization constants KD were calculated by using standard
equations. The binding constants K for the nucleotide–drug
interaction were obtained by titration experiments, performed
by adding increasing amounts of a 5.0 × 10−6 M solution in water
of the drug to a 1.0 × 10−5 M solution of the oligonucleotide,
until R = [drug]/[DNA] = 5. Then the binding constants K
were calculated by including the dimerization equilibrium. The
system of non-linear equations8 was solved by using MATLAB
software (v. 5.1). The calculation of thermodynamic data and K
values from equilibrium melting curves was performed following
well-established procedures.27
Molecular modeling
Molecular models were built using a Silicon Graphics 4D35GT
workstation running the INSIGHT II & DISCOVER soft-
ware. MM and MD simulations of oligomers were carried out
starting from the B-DNA conformation generated by using
standard bond lengths and angles contained in the INSIGHT
library. For the models generated by DISCOVER we used the
CVFF force field supported by the Biosym program. The system
was surrounded by a sphere of water molecules with a radius of
˚
25 A. The models of the complexes were developed by docking
the interacting molecules into the DNA in an orientation
suggested by the experimental NOEs. In the first step, we
performed a minimization by DISCOVER, applying 100 steps
of steepest-descendent algorithm followed by conjugate gradient
minimization, until the energy difference between successive
minimization steps was less than 0.1 kcal mol−1. Then, a 10 ps
simulation was run at a constant temperature of 300 K. No
restraints were applied, except for the NOE contacts and the
NMR experiments
1H and 31P NMR spectra of the complexes were recorded
on a Bruker AMX-600 NMR spectrometer, operating at a
frequency of 600.13 MHz for the 1H nucleus and at 242.94 MHz
for the 31P nucleus, equipped with a z-gradient 5 mm reverse
hydrogen bonds between the base pairs (1.7–1.9 A), including
˚
those of the terminal base pairs. The models of AATT/9,
AATT/10a and ACGT/10b yielded structures with total energy
equal to −19588, −19888 and −21698 kcal mol−1, respectively.
An average structure for each complex was created in order
to calculate the RMS deviation over all heavy atoms, relative
to each single frame of the MD calculation; the RMS value
1
probe. H and 13C spectra of the free molecules were recorded
on Bruker AMX-600 and AMX-400 spectrometers. D2O and
H2O–D2O (90 : 10) were used as solvents for the complexes
with the oligonucleotides; CDCl3 and DMSO-d6 were used
for proton and 13C assignments in the free molecules, and for
the measurements of the H,H coupling constants. 1H chemical
shifts (d) were measured in ppm, using as internal references
tetramethylsilane (TMS) for CDCl3 and DMSO-d6, and the
˚
˚
converged to 0.9 0.3 A for AATT models, and 1.0 0.3 A for
ACGT model. The convergence of the energy was ensured and
the stability of the system was checked: a plot of the total energy
of the drug/nucleotide complexes vs. the time of MD simulation
showed that after only 2 ps the systems reached equilibrium.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 8 4 8 – 2 8 5 8
2 8 5 7

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Perkin Trans. 2, 1997, 2083; (c) S. Laschat and J. Lauterwein, J. Org.
Chem., 1993, 58, 2856.
Acknowledgements
General financial support by the Deutsche Forschungsgemein-
schaft, the Fonds der Chemischen Industrie, the DAAD and
CRUI (Vigoni Program) are gratefully acknowledged. We are
also indebted to the University of Milan (Funds FIRST) and
MIUR (Funds COFIN-2003 and FIRB-2003).
11 D. Neuhaus, M. Williamson, The Nuclear Overhauser Effect in
Structural and Conformational Analysis, VCH, New York, 1998.
12 A. Monsees, S. Laschat, S. Kotila, T. Fox and E.-U. Wu¨rthwein,
Liebigs Ann./Recl., 1997, 533.
13 F. Linkert, S. Laschat, S. Kotila and T. Fox, Tetrahedron, 1996, 52,
955.
14 F. Linkert, S. Laschat and M. Knickmeier, Liebigs Ann. Chem., 1995,
985.
15 CCDC-243553 contains the crystallographic data for this paper.
See http://dx.doi.org/10.1039/b503281d for crystallographic data
in CIF or other electronic format.
16 (a) U. Omura and D. Swern, Tetrahedron, 1978, 34, 1651; (b) A. J.
Mancuso, S. L. Huang and D. Swern, J. Org. Chem., 1978, 43, 2480.
17 H. Gu¨nther, Modern NMR Spectroscopy, John Wiley & Sons, New
York, 1995.
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