Synthesis, structural, and biological evaluation of bis- heteroarylmaleimides and bis-heterofused imides

Article abstract of DOI:10.1016/j.bmc.2011.08.016

Bis-2,3-heteroarylmaleimides and polyheterocondensed imides joined through nitrogen atoms of the N,N′-bis(ethyl)-1,3-propanediamine linker were prepared from substituted maleic anhydrides and symmetrical diamines in good to satisfactory yields and short r

Full text of DOI:10.1016/j.bmc.2011.08.016

Bioorganic & Medicinal Chemistry 19 (2011) 5291–5299
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, structural, and biological evaluation of bis-heteroarylmaleimides
and bis-heterofused imides
Nicola Ferri b, Tiziano Radice a, Manuela Antonino b, Egle Maria Beccalli a, Stella Tinelli c, Franco Zunino c,
Alberto Corsini b, Graziella Pratesi c, Enzio M. Ragg d, Maria Luisa Gelmi a, Alessandro Contini a,
⇑
a Dipartimento di Scienze Molecolari Applicate ai Biosistemi – Sezione di Chimica Organica ‘‘A. Marchesini’’, Università degli Studi di Milano, Via Venezian 21, 20133 Milano, Italy
b Dipartimento di Scienze Farmacologiche, Università degli Studi di Milano, Via Balzaretti 9, 20133 Milano, Italy
c Fondazione IRCCS Istituto Nazionale Tumori, Via Venezian 1, 20133 Milano, Italy
d Dipartimento di Scienze Molecolari Agroalimentari – Sezione di Chimica Organica, Università degli Studi di Milano, Via Celoria 2, 20133 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Bis-2,3-heteroarylmaleimides and polyheterocondensed imides joined through nitrogen atoms of the
N,N0-bis(ethyl)-1,3-propanediamine linker were prepared from substituted maleic anhydrides and sym-
metrical diamines in good to satisfactory yields and short reaction times using microwave heating. The
novel molecules were shown to inhibit proliferation of human tumor cells (NCI-H460 lung carcinoma)
and rat aortic smooth muscle cells (SMCs) with variable potencies. Compound 11a, the most potent
one of the series, showed IC50 values comparable to those observed for the leading molecule elinafide
in both cell lines, but with a higher selectivity toward human tumor cells. Compound 11a affected
G1/S phase transition of the cell cycle, showed in vitro DNA intercalating activity and in vivo antitumor
activity. A thorough structural analysis of the 11a-DNA complex was also made by mean of NMR and
computational techniques.
Received 27 June 2011
Revised 28 July 2011
Accepted 9 August 2011
Available online 16 August 2011
Keywords:
DNA intercalator
Microwave chemistry
NMR spectroscopy
Molecular dynamics
Biological activity
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
The antitumor activity of 3 seems to be related to its ability to
stabilize the DNA-intercalator-topoisomerase II ternary complex
Several examples of bis-intercalator agents, whose typical
features are the presence of two identical (i.e., naphthalimide,1–7
benzonaphthalimide,8 imidazoacridanone,9,10 antracyclinone,11,12
acridinecarboxamide,13 phenazinecarboxamide,14,15 rebeccamy-
cin,16 and anthrapyrazole,17 rings) or dissimilar chromofores18,19
connected by a flexible chain containing nitrogen atoms, are re-
ported in the literature. Studies related to the features of the
chain, both concerning length and structural constraints, are also
reported.8,14,20 Furthermore, SAR studies were reported by
Braña et al. on elinafide analogs containing aromatic fused hetero-
cycles,21–25 aryl or heteroaryl substituted elinafides as well as their
aza-analogs.26 More recently, we reported the biological activity of
a series of molecules belonging to the classes of 2,3-heteroaryl
substituted maleimides of kind 1 and heterofused imides of kind
1. By contrast, its parent elinafide (4, Fig. 2) and its derivatives
are potent bis-intercalator of DNA1–5 and do not stabilize the topo
II-DNA complex.25,26 DNA intercalation assays on selected com-
pounds of series 2 showed that, in contrast to 4, they do not di-
rectly interact with DNA.27
As documented by the literature, the preparation of dimeric
compounds represents an attractive strategy to increase both
O
Ar2
Ar1
Et
Ar = 2-(N-methyl) pyrrolyl,
N
2-furyl, 2-thienyl,
3-indolyl
N
Et
O
1
2 (Fig. 1), characterized by heteroatom and
p-system extension
diversity, against human tumor cells (NCI-H460 lung carcinoma)
Y
and rat aortic smooth muscle cells (SMCs).27
X = NMe, S, CH=CH
Y = O, S, CH=CH
The antiproliferative profile of some of the above compounds is
comparable to the one of amonafide (3, Fig. 2) which is classified as
a mono-intercalator of DNA.
O
X
N
Et
N
O
Et
2
⇑
Corresponding author. Tel.: +39 02 503 14480; fax: +39 02 503 14476.
E-mail address: alessandro.contini@unimi.it (A. Contini).
Figure 1. 2,3-Heteroaryl maleimides 1 and heterofused imides 2.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.08.016

5292
N. Ferri et al. / Bioorg. Med. Chem. 19 (2011) 5291–5299
6 required DMF as the solvent and microwave heating at 450 W
NMe2
O
and 130 °C (5 min). The reaction between the anhydride 5c and
the amine 8 gave compound 9 in quantitative yield (CH2Cl2,
100 °C, 450 W, 15 min).
O
N
As preliminary biological assays revealed that 7a, d, and h were
the most promising moieties (see Table 1), the preparation of cor-
responding bis-heterofused imides 11a–c was planned. Starting
from 10a and b and using traditional heating the reaction was
unsuccessful and a mixture of compounds, not isolable in pure
form, was formed. On the other hand, satisfactory results were
achieved by making 10a to react with 6 in DMF using microwave
(450 W) at 100 °C. Compound 11a was obtained after 15 min and
was isolated in pure form in 60% yield (Scheme 2).
NH2
amonafide 3
O
O
N
O
H
H
N
N
N
The microwave assisted reaction (15 min) was also performed
starting from 10b (600 W, 120 °C) and 10c (450 W, 130 °C) provid-
ing compounds 11b (24%) and 11c (65%), respectively.
O
elinafide 4
Figure 2. DNA-interacting compounds.
3. Biological evaluation
The antiproliferative effect of compounds of the series 7, 9, and
11 was evaluated, after three days of exposure, in cultured rat aor-
tic SMCs and in human NCI-H460 lung tumor cell lines (Table 1).
The IC50 values for the novel molecules ranged from 0.09 to
DNA binding affinities and selectivity. In this study, we report the
synthesis, the antiproliferative activity and the DNA intercalation
of new bis-intercalator agents belonging to the classes of
2,3-heteroaryl substituted maleimides and heterofused imides
using, as the linker, the N,N0-bis-(2-aminoethyl)-1,3-propanedi-
amine which is generally considered the most suited one, as dem-
onstrated by several studies.8,14 In analogy with our previous
study,27 the human NCI-H460 lung carcinoma cell line and a rat
aortic SMC cell line were used due to the interest of DNA-interca-
lating agents in the therapy of tumor as well as in cardiovascular
diseases. Compound 11a was selected as the most potent one, elic-
iting antiproliferative effects comparable to those of 4 on both cell
types. This compound was shown to directly interact with DNA
in vitro and to inhibit cellular proliferation by interfering with
the progression of G1/S phase of the cell cycle. Moreover, in human
NCI-H460 tumor xenografts it was shown to reduce tumor growth
in a dose-dependent manner.
more than 30 lM in SMCs, and from 0.003 to more than 30 lM
in the tumor cell line. Within compounds 7, characterized by the
presence of two heteroaryl substituents on the maleimide ring,
compound 7a was found moderately potent in both cell lines,
while 7d and h were the most effective on NCIH-460 and SMCs,
respectively. Instead, within the polyheterocondensed imides 11,
compound 11a had potency levels comparable to those of 4, while
the others proved to be less potent. Importantly, compound 11a
decreased cell proliferation of the tumor cell line with an IC50 value
30-fold lower than that calculated for rat SMCs, that is, 0.003
lM
versus 0.09 M, respectively, while compound 11b, although less
l
potent than 11a, showed an even more selective antiproliferative
effect with an about 500-fold preference toward the human tumor
cell line (Table 1). It should be noted that, in the same experimen-
tal conditions, the reference compound 4 showed a really modest
selectivity (1.5-fold) toward the NCIH-460 line.
2. Chemistry
It should also be noted that, for compounds 11a and b, dimer-
ization using an amine-type linker of the proper length and phys-
icochemical properties has a dramatic influence on the biological
activity. Indeed, the corresponding N-diethylamminoethyl substi-
tuted monomers 2, previously reported as compound 12a
The preparation of compounds 7 took advantage of a very effi-
cient procedure consisting in the use of microwave heating
(Scheme 1).
Compounds 7a–d, f, and g were prepared in good yields
(40–98%) from the anhydrides 5a–d, f, and g and N,N0-bis-(2-ami-
noethyl)-1,3-propanediamine 6 operating in CH2Cl2 at 100 °C and
450 W in 15 min. Instead, the preparation of 7e (30%) and 7h
(82%) from the corresponding anhydrides 5e and 5f and diamine
(X = NMe, Y = O, IC50 = 5.3 and 8.6
respectively) and 12d (X = S, Y = O, IC50 >30
lines),27 in the same experimental conditions showed a potency
lM on SMC and NCI-H460,
lM on both cell
Ar2
Ar2
O
Ar2
O
O
NH2(CH2)2NH(CH2)2NH(CH2)2NH2
6
Ar1
Ar1
O
N
N
i
Ar1
N
N
H
H
O
O
O
5
7
a: Ar1 = 2-(N-methyl)pyrrolyl, Ar2 = 2-furyl
b: Ar1 = 2-(N-methyl)pyrrolyl, Ar2 = 2-thienyl
c: Ar1 = Ar2 = 2-thienyl
i
NH2(CH2)3
N
N (CH2)3NH2
8
d: Ar1 = 2-thienyl, Ar2 = 2-furyl
e: Ar1 = 3-indolyl, Ar2 = 2-(N-methyl)pyrrolyl
f: Ar1 = 3-indolyl, Ar2 = 2-thienyl
g: Ar1 = 3-indolyl, Ar2 = phenyl
h: Ar1 = Ar2 = phenyl
S
S
O
O
S
S
N
N
N
N
O
O
9
Scheme 1. Synthesis of bis-2,3-heteroaryl maleimides 7 and 9. Reagents and conditions: (i) 7a–d,f,g, 9: CH2Cl2, Mw (450 W, 100 °C); (ii) 7e,h: DMF, Mw (450 W, 130 °C).

N. Ferri et al. / Bioorg. Med. Chem. 19 (2011) 5291–5299
5293
Table 1
Table 1 (continued)
Antiproliferative effects of compounds 7, 9 and 11 in rat aortic SMCs and human
tumor cell line NCI-H460
Compd. Imide moiety
IC50 (lM) SMCs IC50 (lM) NCIH-460
Compd. Imide moiety
IC50 (lM) SMCs IC50 (lM) NCIH-460
O
11c
0.23
0.4
N
4
0.012
0.73
0.008
0.65
O
O
N
O
NMe
O
7a
Y
O
O
O
N
NMe
S
O
X
a: X = N-Me, Y = O
b: X = S, Y = O
7b
7c
7d
1.8
1.3
3.5
3.4
4.3
0.5
O
10a-c
c: X = Y = CH=CH
O
O
O
N
S
S
Y
Y
O
O
O
O
N
N
N
S
X
X
N
H
N
H
O
O
O
11a-c
Scheme 2. Synthesis of bis-heterofused imides 11. Reagents and conditions: (i)
DMF, Mw (10a: 450 W, 100 °C, 10b: 600 W, 120 °C; 10c: 450 W, 130 °C).
O
N
H
N
Table 2
MeN
Effect of compound 11a on cell cycle of human NCI-H460 cells
7e
7f
16.5
3.9
4.2
Incubation
G0/G1 (%)
S (%)
G2/M (%)
O
O
0.4% FCS
10% FCS
10% FCS + 11a
10% FCS + 4
64.25 0.68
54.39 2.0
76.63 1.62
68.40 1.582.0
11.17 1.16
20.51 0.14
10.56 1.79
13.44 1.56
24.59 1.84
25.10 1.85
12.81 0.16
18.15 0.02
N
H
N
S
4.7
Results are representative of three replicate experiments.
O
O
N
H
N
from about 100 to about 3000-folds lower than the dimers. Curi-
ously, the same ratio was not observed for compound 11c, where
the corresponding monomer (12i, in Ref. 27) resulted almost
7g
1.0
3.9
1.2
equally potent (IC50 = 0.68 and 0.9
respectively).
lM on SMC and NCI-H460,
O
O
N
To further investigate the antiproliferative mechanism of action
of compound 11a, we analyzed the cell cycle progression of human
NCI-H460 cells by flow cytometry analysis. Incubation of NCI-H460
cells with 0.4% FCS led to accumulation of cells at G0/G1 phase
(64.25 0.68%) with only a small percentage at S phase (11.17
1.16%). After incubation for 20 h with 10% FCS we observed a sig-
nificant increase in the proportion of NCI-H460 cells in S phase
(20.51 0.14%), which was decreased to 13.44 1.56% by
7h
0.62
O
O
O
N
S
S
9
>30
>30
0.08
lM of 4 and to 10.56 1.791% by 0.003 lM of compound
11a (Table 2).
O
N
Importantly, we did not observe a significant increase in the
percentage of cells at sub-G0/G1 phase, demonstrating a specific
antiproliferative activity of both 4 and 11a, without induction of
apoptosis (Fig. 3).
With the aim to evaluate if the antiproliferative effect of com-
pound 11a was a consequence of DNA intercalation or an inhibi-
tory action on the DNA topoisomerase II activity, we performed
an in vitro DNA topoisomerase II activity assay (Fig. 4). Incubation
O
MeN
11a
11b
0.09
0.003
0.07
O
O
N
O
O
S
>30
of compounds 11a and 4, at concentrations of 0.1 lM, with plasmid
DNA in the presence of DNA topoisomerase II altered the DNA elec-
O
N
trophoretic mobility on agarose gel, indicating a possible inhibitory

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N. Ferri et al. / Bioorg. Med. Chem. 19 (2011) 5291–5299
Figure 3. Effect of compounds 4 and 11a on cell cycle progression of human NCI-H460 cells. Cells were seeded at a density of 2 ꢀ 105 per 35-mm dish and incubated with
RPMI supplemented with 10% FCS. Twenty-four hours later, the medium was changed with medium containing 0.4% FCS to stop cell growth, and the cultures were incubated
for 2 days. At this time, the medium was replaced with medium containing 10% FCS, in the presence or absence of compound 4 (0.08
lM) or compound 11a (0.003 lM). After
18 h, at 37 °C, cell cycle analysis was performed by FACS. The results are representative of three independent experiments conducted in duplicate.
effect on topoisomerase II (Fig. 4A). However, a similar shift of the
DNA electrophoretic mobility was also observed in the absence of
the topoisomerase II, suggesting that both compounds efficiently
interacted with plasmid DNA (Fig. 4B). These results indicated that
the bis-intercalating agent 4 as well as compound 11a did not af-
fect the activity of the DNA topoisomerase II, but elicited their anti-
proliferative action through a direct interaction to DNA.
16.9 mg/kg, which resulted active (P <0.01 vs controls) and well
tolerated. A clear dose–response was observed in the antitumor ef-
fect of the compound, since the lower dose tested was unable to
significantly inhibit tumor growth (Fig. 5).
Compound 11a was selected for in vivo testing and antitumor
activity evaluation, due to its high potency against the NCI-H460
tumor cell line. Tumor fragments were implanted by sc injection
in both flanks of nude athymic mice. The compound was adminis-
tered iv, from day 2, every 4th day for four times (q4dx4). Control
mice received vehicle alone (DMSO 10% in water). Due to the
low solubility of the molecule, the higher dose tested was
Figure 5. Antitumor activity for compound 11a on human NCI-H460 lung tumor
xenograft. Tumors were generated by s.c. implant of fragment in both flanks of
athymic nude mice. Mice were treated i.v. at days 2, 6, 10, and 14 (arrows) with:
solvent (X); compound, 10 mg/Kg (N) or 16.9 mg/Kg (j). Mean tumor weight of 8
mice/group are shown. Bars: Standard errors. ⁄P <0.01 by Student’s t-test.
Figure 4. Effect of 4 and 11a on DNA topoisomerase II activity. (1) Linear pRYG
DNA; (2) Supercoiled pRYG DNA; (3) pRYG DNA + Topo II; (4) pRYG DNA + Topo
II + 4; (5) pRYG DNA + Topo II + 11a; (6) pRYG DNA + 4; (7) pRYG DNA + 11a.

N. Ferri et al. / Bioorg. Med. Chem. 19 (2011) 5291–5299
5295
for the complete assignment of the ligand aromatic protons, as well
as their intermolecular NOE interactions are also reported in Fig-
ure 7 (Chemical shift assignments are listed in Table S1, Supple-
mentary data). Particularly meaningful are the two well
separated resonances at 3.17 and 3.58 ppm, attributed to the
methyl groups (11a-6Ha and 11a-6Hb) residing on the ligand pyrr-
olyl moieties. Each methyl group displays intra-residue NOE inter-
actions with the protons at adjacent positions (11a-5Ha and 5Hb at
d 7.0 and 6.35 ppm, respectively), as well as strong intermolecular
NOEs, respectively, with G2,G3-10H and C10,C11-5H. These base-
pairs define an intercalation site constituted by the G2G3/C10C11
DNA region. Due to the self-complementarity of the hexanucleo-
tide, a second intercalation site is defined by the symmetry related
segment C4C5/G8G9.
4. Structural evaluation of the DNA-11a complex
In order to further characterize the binding properties of 11a to
DNA, the self-complementary oligodeoxynucleotide d(CGGCCG)2
was chosen as model, on the basis of previous DNA foot-printing
studies pointing out the preference for CG-rich sequences of
bis-naphthalimides.24,25
The 1H NMR spectrum of 11a-trifluoroacetate, dissolved in D2O,
is characterized by a general line-broadening and a high-field shift
by approximately 1.5 ppm, with respect to the free base, of the
aromatic proton resonances, a clear indication of extensive self-
aggregation. When the hexanucleotide d(CGGCCG)2 is added to
the 11a-trifluoroacetate solution an even more pronounced loss
of resolution is observed, at low DNA/drug ratios, due to the forma-
tion of high molecular weight aggregates. This is mainly caused by
the polyelectrolyte nature of DNA, which initially favors nonspe-
cific external binding of the ligand. However, at DNA/drug values
greater than 0.25, new sharp and well resolved resonances appear
with increasing intensity until a unitary molar ratio is reached,
thus suggesting the formation of one or more well-defined com-
plexes when DNA and ligand are present at comparable concentra-
tions (see Fig. S1, Supplementary data).
The splitting observed for both ligand and DNA 1H resonances
clearly indicates that two main complexes exist in solution, differ-
ing only by the orientation of the ligand aromatic moiety, which al-
ways intercalates between the G2:C11 and G3:C10 base pairs. The
first orientation (orientation A) is defined by the pyrrolyl and
furanyl moieties interacting respectively with G2G3 and C10C11 res-
idues of DNA, whereas the second one (orientation B) involves the
pyrrolyl moiety interacting with C10C11 residues (see Fig. 6). These
two orientations are present in solution in equal amounts, as indi-
cated by the respective resonance intensities measured in the
1D-NMR spectra (see Fig. 7 on top).
In principle, four different orientations of 11a could be devised
for its intercalation into the hexanucleotide, of which only two
maintain the overall C2-symmetry of the complex (i.e., those with
the two aromatic moieties in antiparallel orientation, see Figure 6A
and C) while the other two (those with the aromatic systems in
parallel orientation, Figure 6B), destroy the inherent molecular
symmetry and give rise to intrinsically different chemical environ-
ments for each proton residing on DNA strands and ligand.
As a result, a resonance splitting would be expected for hydro-
gen atoms residing in close proximity of the binding site. Indeed,
such splitting was observed especially when cooling the solution
below room temperature, thus proving the existence of several ex-
change equilibria involving more than one complex with a rate
comparable to the NMR time-scale. An example of such splitting
is shown in Figure 7, which depicts sections of a 2D-NOESY exper-
iment performed at 15 °C on a 1:1 complex of 11a-trifluoroacetate
with 50-d(CGGCCG)2-30.
For the sake of clarity, oligonucleotide residues have separately
been numbered as follows: 50-d(C1G2G3C4C5G6)-30/50-d(C7G8G9C10
C11G12)-30, while ligand protons have been numbered following IU-
PAC rules (labels ‘a’ and ‘b’ refer to the two aromatic moieties,
becoming magnetically nonequivalent in the case of a nonsymmet-
ric complex). The 2D-NOESY spectrum (see also Fig. S2,Supplemen-
tary data, for details) actually displays three kinds of correlations:
(a) intra-residue NOE interactions involving protons that reside
either on DNA or on ligand moieties (continuous lines); (b) inter-
molecular NOEs involving proton pairs residing on both ligand
and DNA (continuous lines); and (c) cross-peaks due to chemical
exchange of the various orientations of ligand within DNA (dotted
lines). Examples of some significant intra-molecular interactions
Figure 7. Expansion of the 2D-NOESY spectrum (low-field and methyl regions)
performed on the 11a-trifluoroacetate:d(CGGCCG)2 complex in D2O (3 ꢀ 10ꢁ4 M,
NaCl 25 ꢀ 10ꢁ3 M, pH 5.5) at 15 °C, tmix = 0.24 s. Continuous lines: inter- and intra-
molecular NOEs; dotted lines: cross-peaks due to chemical exchange. The corre-
sponding 1D-spectrum is shown on top, together with the most significant
resonance assignments.
Figure 6. Possible binding orientations of compound 11a intercalated into
d(CGGCCG)2.

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N. Ferri et al. / Bioorg. Med. Chem. 19 (2011) 5291–5299
Table 3
Experimentala (dexp), calculatedb NOE distances (<dcalc>) and errorsc (err).derived for 11a intercalated into d(CGGCCG)2 major and minor grooves (orientation B). All values are
given in Å
Proton pair
i
dexp
<dcalc> (major)
err (major)
<dcalc> (minor)
err (minor)
j
G2–H10
G3–H8
H6a
H6a
H6b
H5b
H5b
H6b
H5b
H6b
H3a
H3a
H2a
H2a
H3a
H2a
H2a
H3a
H2a
H3a
H2a
H3a
H4a
C5–H6
2.5
3.6
4.1
3.7
3.0
2.9
3.9
3.5
3.0
2.9
2.6
3.1
3.2
2.8
4.0
2.9
3.0
3.2
2.8
2.7
3.1
2.4
2.7
4.1
5.0
2.9
3.0
3.6
4.6
3.8
4.0
3.2
2.8
3.2
2.9
3.6
3.9
3.7
3.4
4.4
3.7
2.8
2.8
2.4
ꢁ0.2
ꢁ0.5
ꢁ0.9
0.8
3.0
5.7
4.2
6.3
5.7
4.1
4.6
3.9
8.7
8.2
5.9
6.5
8.5
5.3
3.9
10.6
5.8
8.1
7.9
2.7
2.8
2.4
ꢁ0.5
ꢁ2.1
ꢁ0.1
ꢁ2.6
ꢁ2.7
ꢁ1.2
ꢁ0.7
ꢁ0.5
ꢁ5.7
ꢁ5.3
ꢁ3.2
ꢁ3.4
ꢁ5.3
ꢁ2.4
0.1
C4–H10
C4–2H20
C4–1H20
C4–H5
C5–H5
C5–H5
C10–H10
C10–1H20
C10–1H20
C10–2H20
C10–2H20
C10–H6
C10–H5
C11–H10
C11–H5
C11–H5
C11–H6
H2a
0.0
ꢁ0.7
ꢁ0.7
ꢁ0.3
ꢁ1.0
ꢁ0.3
ꢁ0.1
ꢁ0.1
0.3
ꢁ0.8
0.0
ꢁ0.8
ꢁ0.3
ꢁ1.2
ꢁ0.8
ꢁ0.1
0.3
ꢁ7.6
ꢁ2.8
ꢁ4.9
ꢁ5.0
ꢁ0.1
0.3
H3a
C5–H5
0.0
0.0
St. deviation
0.5
2.2
a
Interproton distances derived from experimental 2D-NOESY cross-peak volumes by applying the following equation dij = dref ꢀ (Vref/Vij)1/6, where dref and Vref refer to
proton pairs at fixed distance chosen as reference.
P
b
-6
theoretical distances calculated as averages from the 8 ns production MD run with the following equation: dcalc = (
d )
ꢁ1/6/N.
i=1,N
i
c
err = (dexp ꢁ dcalc).
This corresponds to a 180° flip of the ligand aromatic system
within the intercalation site, occurring on a relatively long time-
scale, with rates estimated about 0.7 sꢁ1 on the basis of the ex-
change cross-peak volumes measured at 15 °C. This value is in line
with that one reported for the bulkiest derivatives of compound
4.28,29 Thus, compound 11a is likely to behave as other known
bis-imide intercalators that were shown to be major groove
binders.21,28
In order to fully ascertain whether 11a is a major- or a minor-
groove binder, a quantitative analysis of NOE-based inter-proton
distances was necessary, supported by molecular modeling and
independent molecular dynamics (MD) calculations. Structures of
compound 11a in the three orientations A, B, and C intercalated
in either the major and minor groove were built and subjected to
an unrestrained molecular dynamics run with the explicit inclu-
sion of water molecules as solvent. After proper equilibration, an
8 ns simulation run was produced and the trajectories were pro-
cessed using the MM-PBSA approach, which in previous studies
was shown to be able to discriminate binding conformations on
an energetic basis with an error between 1.5 and 2.5 kcal/mol, on
the average.30,31 The computed binding free energy differences
showed a moderate preference for the major groove conformations
(SD: 0.5 and 2.2 Å, respectively). It should be noted that MD simu-
lations were carried out without any experimentally derived re-
straint, thus giving support to the validity of the adopted protocol.
In conclusion, those results, obtained from NMR experiments
and theoretical calculations, indicate that compound 11a is able
to bis-intercalate at the level of the major groove in two possible
and energetically equivalent binding modes, which only differ in
the orientation of the heterocyclic moiety within the intercalation
site and equilibrate in solution, probably through the (slow) forma-
tion of a mono-intercalated intermediate (see Scheme 3).
Such molecular intermediate escaped NMR detection, but its
concentration must necessarily be very low. In the case of elina-
fide:d(ATGCAT)2 complex, an alternative ring-flip mechanism has
also been proposed, involving internal rotation within the interca-
lation site.29 As this alternative motion should be linked with the
opening and closing of external base-pairs, a motion that certainly
occurs in the less stable A:T base-pairs at terminal positions, we
feel that in our case the ‘slide-out and ring-flip’ mechanism is more
likely to occur, given the ‘CG-rich’ character of our oligonucleotide
and the increased diameter of the aromatic moiety in 11a, with re-
spect to elinafide and the tested derivatives.
Hydrogen bond analysis revealed that the bis-intercalated com-
plexes are well stabilized by two hydrogen bonds between the pro-
tonated amino groups of the linker chain and the carbonyl groups
of G3 and G9 facing the major groove, as shown in Figure 8.
(D
G: ꢁ49.5 vs ꢁ48.5 kcal/mol, as the average among A, B, and C
conformations), but energy differences resulted well below the
accuracy limits of the method (Table S2, Supplementary data).
Aiming to discriminate between major and minor groove bind-
ing preferences, theoretical NOE distances were calculated from
MD trajectories and compared to experimental NOE distances. Cal-
culated distances, averaged from orientation B over the whole 8 ns
trajectory, are reported in Table 3.
Orientation B was chosen as the most representative of all the
possible local orientations, but comparable results were obtained
also by performing the same distance analysis on orientations A
and C. Theoretical values obtained for the major groove complex
fit particularly well with experimental NOE distances, while
significant deviations were obtained for the minor groove complex
Scheme 3. The ‘‘slide-out and ring-flip’’ mechanism.

N. Ferri et al. / Bioorg. Med. Chem. 19 (2011) 5291–5299
5297
6.1.1. General procedure for the preparation of bis-maleimides
7a–d,f,g and 9
Amine 6 (0.025 mL, 0.15 mmol) or 8 (0.031 mL, 0.15 mmol) was
added to a solution of anhydride 5 (0.3 mmol) in CH2Cl2 (3 mL).
The mixture was stirred under microwave heating (100 °C,
450 W) in the presence of a Weflon stirrer (7a–d,f,g, 9: 15 min).
The mixture was cooled at room temperature and water (5 mL)
was added. The organic layer was separated, dried over anhydrous
Na2SO4 and filtered. The solvent was evaporated and the residue
was purified by crystallization. In case of 5g, a solid was formed
during the reaction which was filtered from the hot solution,
washed with CH2Cl2 to give pure compound 7g. Pure compound
9 was directly obtained from the reaction mixture by addition of
Et2O and washing the solid with the same solvent.
6.1.2. General procedure for the preparation of 7e,h
N,N0-Bis-(2-Aminoethyl)-1,3-propanediamine (6) (0.03 mL,
0.17 mmol) and anhydride 5e or h (0.34 mmol) were dissolved in
DMF (8 mL). The mixture was stirred under microwave heating
(130 °C, 450 W) in the presence of a Weflon stirrer for 5 min. The
mixture was cooled at room temperature and diluted with a sol-
vent (5e: AcOEt, 15 mL; 5h: Et2O, 15 mL) and brine (10 mL). The
organic layer was separated, washed with water, dried over anhy-
drous Na2SO4 and filtered. Pure compound 7e was obtained after
crystallization. Instead, when the ethereal solution was
concenterd, a yellow solid was separated corresponding to pure 7h.
Figure 8. Geometry of 11a bis-intercalated into DNA (orientation B) obtained by
averaging 8000 frames extracted from the 8 ns MD simulation (rotation and
translations were removed before averaging). H-bonds are reported as dotted lines,
together with their average population (%).
6.1.3. General procedure for the preparation of compounds
11a–c
Although in complex with DNA, the ligand undergoes significant
internal motions, as both the aromatic moiety and the linker chain
independently reorient themselves within a two-state jump model
(see Fig. S4, Supplementary data).
To a solution of anhydride 10 (0.39 mmol) in DMF (6 mL), was
added N,N0-bis(2-aminoethyl)-1,3-propanediamine 6 (0.033 mL,
0.19 mmol). The mixture was stirred under microwave heating
(10a: 450 W, 100 °C; 10b: 600 W, 120 °C; 10c: 450 W, 130 °C) for
15 min. Starting from 10a,b, the mixture was then diluted with
Et2O (15 mL) and brine (10 mL). A solid was formed, filtered and
washed with Et2O to give pure compounds 11a,b. Starting from
10c, a solid was directly formed in the reaction mixture which
was collected and washed with Et2O affording pure 11c.
5. Conclusion
In conclusion, new bis-2,3-heteroarylmaleimides
7
and
polyheterocondensed imides 11 jointed through nitrogen atoms
of the N,N0-bis(ethyl)-1,3-propanediamine linker were prepared
and their ability to inhibit in vitro the proliferation of human tu-
mor cells (NCI-H460 lung carcinoma) and rat aortic smooth muscle
cells (SMCs) was evaluated. 11a resulted the most potent com-
pound of the series and showed to be selective for the NCIH-460
line, with an IC50 value 30-fold lower than that calculated for rat
6.2. Biological methods
6.2.1. Cell culture
The human NCI-H460 lung tumor cells and primary rat aortic
SMCs were used in this study. SMCs cultured from the
intimal-medial layers of aorta of male Sprague–Dawley rats
(200–250 g) were grown in monolayers at 37 °C in a humidified
atmosphere of 5% CO2 in DMEM supplemented with 10% (v/v) fetal
calf serum (FCS), 100 U/ml penicillin, 0.1 mg/mL streptomycin,
20 mM tricine buffer, and 1% (v/v) non-essential amino acid solu-
tion. Cells were used between the fourth and tenth passages. The
human NCI-H460 lung carcinoma cells (ATCC HTB 177) were
cultured in RPMI-1640 supplemented with 10% FCS. DMEM and
RPMI-1640 were purchased from SIGMA (Milan, Italy), trypsin
ethylenediamine tetraacetate, penicillin (10.000 U/mL), streptomy-
cin (10 mg/mL), tricine buffer (1 M, pH 7.4), non-essential amino
acid solution (100ꢀ), and fetal calf serum (FCS) were purchased
from Invitrogen (Carlsbad, CA, USA). Disposable culture flasks
and Petri dishes are from Corning Glassworks (Corning, NY).
SMCs, (i.e., 0.003 lM versus 0.09 lM, respectively). In terms of
potency, 11a is comparable to the lead compound 4 in both cell
lines, but for this latter a really modest selectivity (1.5-fold) toward
the tumor cell line was observed. The parent compound 11b,
although less potent than 11a, showed an even more selective
antiproliferative effect with an about 500-fold preference toward
the human tumor cell line. The antitumor activity of compound
11a was also demonstrated by in vivo studies in mice on human
NCI-H460 lung tumor xenograft. Finally, a thorough structural
analysis of the 11a-DNA complex was also made by means of
NMR and computational techniques. The DNA intercalating ability
of bis-2,3-heteroarylmaleimides, initially observed through in vitro
assays, was thus confirmed also providing useful insights into the
dynamic behavior of the complex which might lead to further
structural optimization of this class of bis-intercalators.
6. Experimental
6.1. Chemistry
6.2.2. Cell proliferation assay
Rat SMCs were seeded at a density of 2 ꢀ 105 cells/Petri dish
(35 mm) and incubated with DMEM supplemented with 10% FCS.
Twenty-four hours later, the medium was changed to one contain-
ing 0.4% FCS to stop cell growth, and the cultures were incubated
for 72 h. At this time (time 0), the medium was replaced with
one containing 10% FCS in the presence or absence of known
Compounds 5a,b,d,e,f,g,32 5c,27 10a,b27 and 10c33 were pre-
pared according to known procedures. Compound 5h is a commer-
cial available compound.

5298
N. Ferri et al. / Bioorg. Med. Chem. 19 (2011) 5291–5299
concentrations of the tested compounds, and the incubation was
continued for a further 72 h at 37 °C. NCIH460 cells were seeded
at a density of 8 ꢀ 104 cells/Petri dish (35 mm), and incubated with
RPMI-1640 supplemented with 10% FCS. Twenty-four hours after
seeding, cells were exposed to test compound, then harvested
72 h later. Cell proliferation was evaluated by cell counting after
trypsinization of the monolayers with use of a Coulter Counter
model ZM.34 All the compounds were dissolved in DMSO prior to
dilution, being the final concentration of DMSO at a maximum of
1%. The concentration of compounds required to inhibit 50% of cell
proliferation (IC50) was calculated by linear regression analysis of
the logarithm of the concentration (in micromoles per liter)
versus logit.
for four times. For statistical analysis, TW was compared in treated
versus control mice by unpaired Student’s t-test (two-tailed).
6.3. NMR experiments
6.3.1. Sample preparation
The oligonucleotide 50-d(CGGCCG)-30 was purchased as sodium
salt from Roche Diagnostics (Monza, Italy) and utilized without
further purification. It was dissolved in D2O (99.9% isotopic purity,
Isotec, USA) at 2 mM concentration, in the presence of 0.025 M
NaCl. pH values were corrected to 5.5 by adding small amounts
of 1 mM NaOD or 1 mM DCl solutions. The trifluoroacetate salt of
11a was prepared by dissolving 1 mg of the free base in trifluoro-
ethanol (TFE, Sigma–Aldrich, Milano, Italy) and adding two equiv-
alents of trifluoroacetic acid dissolved in TFE. The mixture was
taken to dryness under vacuum. The obtained trifluoroacetate salt
was dissolved in D2O 99.9%, in the presence of NaCl 25 mM. The pH
value of the solution was adjusted to 5.5 as described above. The
final concentration was 0.3 mM, as checked with a calibrated
external reference.
6.2.3. DNA topoisomerase II activity assay
For the assay the supercoiled pRYG DNA was utilized as sub-
strate according to protocol provided by TopoGen Inc. (Columbus,
USA). Supercoiled pRYG DNA was incubated in a reaction buffer
containing 10 mM Tris–HCl, pH 7.9, 1 mM EDTA, 150 mM NaCl,
0.1% BSA, 0.1 mM spermidine, and 5% glycerol in the presence of
two units of DNA toposiomerase II. The tested compounds were
added at the indicated concentrations and after 10 min at 37 °C
the reaction was stopped by addition of the stop buffer containing
the loading dye (15 sarkosyl, 0.025% bromophenol blue, 5% glyc-
erol). Then the reaction mixture was analyzed on a 1% agarose
gel by running at 80 V in TAE buffer (400 mM Tris–base, 10 mM
EDTA, 200 mM sodium acetate, pH 8.3). Gels were stained with
ethidium bromide and the image acquired with Gel Doc acquisition
system and Quantity One software (Bio-Rad). The analysis of the
DNA intercalating activity of the tested compounds were carried
out under the same experimental condition in the absence of the
DNA toposiomerase II.
6.3.2. NMR experiments
The NMR spectra were recorded on a Bruker AV600 spectrome-
ter operating at 600.10 MHz (proton frequency). Chemical shifts (d)
are measured in ppm, with 1H spectra referenced to external DSS
(2,2-dimethyl-2-silapentane-5-sulfonate sodium salt) set at
0.00 ppm. Estimated accuracy is within 0.03 ppm. Titration exper-
iments were performed by adding increasing amounts of oligonu-
cleotide stock solutions to 11a–trifluoroacetate dissolved in D2O.
Phase sensitive NOESY spectra were acquired at temperatures
ranging from 5 °C to 25 °C in TPPI (Time Proportional Phase Incre-
ments) mode, with 640 ꢀ 4 K complex FIDs, spectral width of
8400 Hz, recycling delay of 1.3 s, 80 scans. Mixing times ranged from
50 ms to 300 ms. TOCSY spectra were acquired with the use of
MLEV-17 spin-lock pulse (field strength 10.000 Hz, 20–80 ms total
duration). For samples dissolved in D2O, water suppression was
achieved by pre-saturation, placing the carrier frequency on the
HDO resonance. All spectra were transformed and weighted with a
90° shifted sine-bell squared function to 2 K ꢀ 2 K real data points.
Proton assignments for 11a, both free and in the complex with
d(CGGCCG)2, were accomplished by means of standard two-
dimensional homonuclear correlation techniques. Protons residing
on the N-methyl pyrrole moiety were assigned by means of the
observed NOE interactions with the methyl groups, resonating
at 3.17 and 3.58 ppm, and by the vicinal coupling constant
values, measured on resolution-enhanced spectra (3J4,5 = 2.6 Hz;
3J2,3 = 1.9 Hz). The 1H NMR sequential assignments of d(CGGCCG)2
with and without ligand were performed by applying well estab-
lished procedures for the analysis of double-stranded type
B-DNA.38 Spectral manipulation and analysis were performed
respectively with TOPSPIN (v. 1.3, Bruker Biospin, Rheinstetten,
Germany) and SPARKY.39
6.2.4. Cell cycle analysis
Flow cytometry was utilized to analyze cell cycle distribution.
Cells were trypsinized and centrifuged for 5 min at 1.000 rpm. Pel-
lets were fixed with 62% ethanol, then resuspended in 0.4 mL of
staining buffer of propidium iodide (10
lg/mL RNAse, 1% NP-40,
5
lg/ml propidium iodide, in H20). Samples were placed in the
dark for 30 min and the fluorescence of individual nuclei was
measured. Nuclear propidium Iodide fluorescence signal was re-
corded on the FL3 channel of a FACS scan flow cytometer (Becton
Dickinson). The number of cells in G0/G1, S and G2/M phases
was expressed as percentages of total events (10.000 cells).35
6.2.5. In vivo antitumoral evaluation
The study was carried out using female athymic Swiss nude
mice, 8–10 weeks old (Charles River, Calco, Italy). Mice were kept
in aminar flow rooms at constant temperature and humidity with
free access to food and water. Experimental protocols were ap-
proved by the Ethics Committee for Animal Experimentation of
the Istituto Nazionale Tumori, Milan, according to the United King-
dom Coordinating Committee on Cancer Research Guidelines.36
The human lung carcinoma NCI-H460 was maintained in vivo
by serial s.c. passages of tumor fragments (about 2 ꢀ 2 ꢀ 6 mm)
in healthy mice, as described previously.37 The experimental
groups included 4–5 mice bearing bilateral s.c. tumors. Tumor frag-
ments were implanted on day 0, and tumor growth was followed
by biweekly measurements of tumor diameters with a Vernier cal-
iper. Tumor weight (TW) was calculated according to the formula:
TW (mg) = tumor volume (mm3) = d2 ꢀ D/2 where d and D are the
shortest and the longest diameter, respectively. Treatment started
at day 2, when TW was around 50 mg. Compound 11a was dis-
solved in DMSO to a final concentration of 10% in sterile water,
and was delivered iv according to a every 4th day schedule (q4d)
6.4. Molecular modeling
Using the PDB structure 1CX3 as a template, molecular models
were built with the MOE software package40 and compound 11a
was manually docked in the DNA double strand. A total of six mod-
els were prepared according to all the possible relative orientations
of the heterocyclic moieties within either the major and the minor
groove. All models were subjected to a preliminary geometry opti-
mization (rmsd = 0.1) using the default MMFF94x force field, the
default distance dependent solvation model for water and by keep-
ing a positional restraints on DNA heavy atoms of 10 kcal/mol.
AM1-BCC partial atomic charges compatible with the General
Amber Force Field (gaff)41 were calculated for compound 11a using

N. Ferri et al. / Bioorg. Med. Chem. 19 (2011) 5291–5299
5299
the antechamber module implemented in the Amber9 suite.42,43
A
14. Gamage, S. A.; Spicer, J. A.; Finlay, G. J.; Stewart, A.; Charlton, P.; Baguley, B. C.;
Denny, W. A. J. Med. Chem. 2001, 44, 1407.
15. Spicer, J. A.; Gamage, S. A.; Rewcastle, G. W.; Finlay, G. J.; Baguley, B. C.; Denny,
W. A. J. Med. Chem. 2000, 43, 1350.
topological file and a coordinate set was prepared for the complex
using the leap module of the Amber9 package accordingly to the
ff03 and gaff force fields.44,45 The complex was solvated by placing
a periodic box of TIP3P water up to a distance of 10 Å from the DNA
complex (total vdW box size: 53.0 Å ꢀ 50.9 Å ꢀ 50.1 Å), and the
system was neutralized by adding Na+ ions. After a careful equili-
bration of the water box, the full system was gently heated through
six 5 ps molecular dynamic (MD) runs where phosphate backbone
restraints were gradually reduced from 10 to 5 kcal/mol, while the
temperature was raised from 0 to 300 K. The heating was followed
by a 100 ps run in NVT ensemble and four 100 ps run in NPT
ensemble where backbone restraints were gradually reduced from
5 to 0 kcal/mol. Finally, 8 ns NPT production runs were performed
at 300 K without any restraints, with the exception of bonds
involving hydrogen atoms which were constrained through the
SHAKE algorithm.46 A time step of 0.002 ps was used and frames
were saved every 500 steps (1 frame/ps). Resulting trajectories
were post-processed using the MM-PBSA approach in order to esti-
mate relative energies of binding.47 Trajectory analysis were per-
formed with the ptraj module of the Amber9 package. Molecular
graphics images were produced with either MOE or the UCSF
Chimera package.48
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Supplementary data
Supplementary data associated (NMR details and extended ta-
bles, further computational details, spectroscopic and analytical
data for compounds 7a–h, 9, 11a–c) with this article can be found,
in the online version, at doi:10.1016/j.bmc.2011.08.016.
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