Benzoquinazoline derivatives as new agents affecting DNA processing

Article abstract of DOI:10.1016/j.bmc.2010.12.037

A new series of benzo[h]quinazoline and benzo[f]quinazoline derivatives was prepared and studied for the biological activity. The compounds carrying a dimethylaminoethyl side chain (6c, 8c and 12) inhibit cell growth. The ability to form a molecular compl

Full text of DOI:10.1016/j.bmc.2010.12.037

Bioorganic & Medicinal Chemistry 19 (2011) 1197–1204
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Benzoquinazoline derivatives as new agents affecting DNA processing
Giovanni Marzaro a, Lisa Dalla Via a, Antonio Toninello b, Adriano Guiotto a, Adriana Chilin a,
⇑
a Department of Pharmaceutical Sciences, University of Padova, Via Marzolo 5, 35131 Padova, Italy
b Department of Biological Chemistry, University of Padova, Via G. Colombo 3, 35121 Padova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of benzo[h]quinazoline and benzo[f]quinazoline derivatives was prepared and studied for
the biological activity. The compounds carrying a dimethylaminoethyl side chain (6c, 8c and 12) inhibit
cell growth. The ability to form a molecular complex with DNA and to interfere with topoII and topoI
relaxation activity was evidenced for the most active 6c and 8c, along with the capacity to induce apop-
tosis on HeLa cells.
Received 13 October 2010
Revised 29 November 2010
Accepted 15 December 2010
Available online 19 December 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Antitumor agents
Benzoquinazoline
DNA
Topoisomerase
1. Introduction
effective dual antitopo activity, delaying the onset of resistance,
such as phenazine-1-carboxamide derivatives8 and 8,9-benzo[a]
Cancer is still one of the leading cause of death worldwide and
any effort to find new and efficient antitumoral agents is helpful
and required.
Among the possible approaches to achieve antitumor activity,
compounds interacting with DNA play a major role.1 Many of these
drugs exert their antitumor activity by binding to DNA or to en-
zymes critical for DNA regular functions, thus triggering a cellular
response, which eventually leads to cell death.
The class of topoisomerase inhibitors is widely investigated
because topoisomerases (both topoI and topoII) represent an
important target for successful cancer therapy,2 and these enzymes
are often overexpressed in many tumors.3
phenazine-11-carboxamide derivatives,9 among which XR 11576
(3, Fig. 1) has entered clinical trials.10 Finally, similar profile was
found for TAS-103 (4, Fig. 1), initially identified as dual inhibitor,11
although it predominantly acts as a topoIIa
inhibitor.12
All these compounds share some common features, such as a
nitrogen heterocyclic ring system and an amino substituent like
the methanesulfonamidoaniline or a dimethylaminoethyl group
(typical of dual inhibitors). By assembling different but similar nitro-
gen scaffolds to obtain structurally novel dual topo inhibitors, we
designed a new series of benzo[h]quinazoline (A) and benzo[f]qui-
The antitopo agents show a great variability in their chemical
structures. Many potent inhibitors share an acridine skeleton:
among them, a lead compound is m-amsacrine (1) (m-AMSA,
Fig. 1), a 9-aminoacridine derivative, whose strong antiprolifera-
tive activity is due to its interaction with topoII.4
Despite its good antitumor activity, especially against leukemia,
m-AMSA presented problems of drug resistance.5 A valid alternative
to single enzyme inhibitors was found with dual topoI/topoII inhib-
itors, which target two enzymes working at different points of cell
cycle, thus circumventing resistance phenomena. XR5000 (2)
(DACA, Fig. 1), an acridine-4-carboxamide derivative structurally
related to m-AMSA, was the first dual inhibitor to enter clinical
trial,6 and it proves to be able to overcome drug resistance.7 Other
structurally different classes of compounds also demonstrated
MeO
HN
NHSO2Me
N
NMe2
O
N
N
1
H
2
OH
N
N
MeO
Me
N
NMe2
O
N
H
O
HN
NMe2
3
4
⇑
Corresponding author. Tel.: +39 0498275349; fax: +39 0498275366.
E-mail address: adriana.chilin@unipd.it (A. Chilin).
Figure 1. Structures of m-AMSA (1), DACA (2), XR11576 (3) and TAS-103 (4).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.12.037

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G. Marzaro et al. / Bioorg. Med. Chem. 19 (2011) 1197–1204
N
N
N
H, OMe
HN
N
NHSO2Me
R =
HN
R
R
NMe2
A
B
Figure 2. Structures of benzo[h]quinazoline (A) and benzo[f]quinazoline (B).
nazoline (B) derivatives bearing the AMSA substituent or the
dimethylaminoethyl substituent (Fig. 2).
First of all, molecular modeling studies were performed to
evaluate the effect of acridine nucleus replacement with the two iso-
meric benzoquinazoline scaffolds. Then the synthesized compounds
were tested for their antiproliferative effect on human tumor cell
lines. Linear flow dichroism experiments (LD) were performed to
establish the ability to form a complex with DNA and the ability to
interfere with topoI and topoII activities was investigated.
Figure 3. Distance of the acridine nitrogen from the nitrogen atoms of the DNA
bases.
2. Results and discussion
2.1. Molecular modeling studies
Molecular modeling studies were performed on m-AMSA and
on all the designed compounds, to investigate the binding mode
of m-AMSA with the DNA and to explore the opportunities to re-
place acridine nucleus with the two isomeric benzoquinazoline
scaffolds. Since no structure of m-AMSA intercalated in the DNA
was available in the PDB, the nucleic acid template was extracted
from a RX complex of another acridine derivative intercalated in
a double strand DNA (PDB code: 1G3X). Lamarkian Genetic Algo-
rithm was used to generate 10 independent searches and to assess
the binding energy for all the compounds.13 Intercalation energies
are reported in Table 1. In any case the intercalation into the DNA
was calculated to be energetically favorable.
Figure 4. Superimposition of minimized structure for isolated (green) and inter-
calated m-AMSA (colored by atoms).
Several features of binding mode of m-AMSA in the DNA were
extracted from the docking studies.
Analysis of the intercalated structure revealed that the nitrogen
of the acridine moiety was placed in the middle of the DNA axis, so
that it was equidistant (about 4 Å) from all the nitrogen atoms of
the bases (Fig. 3).
Moreover, superimposition of the intercalated structure and the
isolated m-AMSA (Fig. 4), revealed that a large variation on dihe-
dral angle between acridine nucleus and aniline side chain was re-
quired to allow interaction with DNA, according to other
experimental observations.14
Figure 5. Superimposition of m-AMSA (green colored), 6b and 7b analogs (red and
cyan colored, respectively) into the intercalation site of DNA.
On the contrary, for the benzoquinazoline derivatives bearing
the same aniline side chain of m-AMSA, a lower variation of the
dihedral angle between intercalated and isolated structures was
observed (data not shown). Thus, in the case of benzoquinazoline
derivatives, a lower penalty for dihedral free energy variation
was observed with respect to m-AMSA.
calculated interaction energies suggested that the replacement of
acridine with benzoquinazoline could lead to intercalative
compounds.
2.2. Chemistry
The good spatial correlation among docked benzoquinazoline
structures and m-AMSA (see an example inFig. 5) and the favorable
The starting 6-bromobenzo[h]quinazoline (5) and 6-bromo-
benzo[f]quinazoline (7) were synthesized from the corresponding
4-bromonaphthyl-1-amine and 4-bromonaphthyl-2-amine, as
previously described.15 The bromobenzoquinazolines 5 and 7 were
coupled with the 40-aminomethane-sulfonanilides and with
N,N-dimethylethylendiamine under modified Buchwald–Hartwig
conditions using microwave (MW) irradiation to provide
compounds 6a–c and 8a–c (Scheme 1). The reaction was carried
out with palladium acetate, BINAP and cesium carbonate in water
instead of classical toluene, which is not only a toxic compound but
also a bad solvent for MAOS (microwave assisted organic synthe-
sis) because of its unfavorable dipole moment.
Table 1
Intercalation energy from docking studies
Compound
Average interaction energy (kcal/mol)
m-AMSA
ꢀ8.28
ꢀ8.14
ꢀ8.24
ꢀ7.47
ꢀ8.17
ꢀ8.15
ꢀ7.47
6a
6b
6c
8a
8b
8c

G. Marzaro et al. / Bioorg. Med. Chem. 19 (2011) 1197–1204
1199
H
N
A)
B)
N
N
N
N
O
N
HN
N
N
a: R =
a
a
NHSO2Me
Br
R
13
14
5
6a-c
HN
c
b
b: R =
c: R =
N
N
NMe2
MeO
HN
NHSO2Me
H
Cl
N
N
N
N
N
N
N
c
NMe2
a
Br
R
15
16
7
8a-c
Scheme 3. Reagents and conditions: (a) CAN, AcOH/H2O, rt, 5 min; (d) CCl4, Ph3P,
CH2Cl2, reflux, 3 h; (c) N,N-dimethylethylenediamine, iPrOH, 80 °C, MW, 15 min.
Scheme 1. Reagents and conditions: (a) 40-aminomethanesulfonanilides or
N,N-dimethylethylenediamine, Pd(OAc)2, BINAP, Cs2CO3, H2O, 150 °C, MW, 20 min.
Table 2
Besides the 6-substituted derivatives, we synthesized
compounds with the side chain in the pirimidine ring of the tricy-
clic nucleus, in order to verify the importance of other substitution
position, considering that DACA, XR11576 and TAS-103 bear the
aminoalkyl substituent close to the heterocyclic nitrogen. In this
case, the functionalization was carried out only with the dimeth-
ylaminoethylamino chain.
The benzo[h]quinazoline (9), achievable only by debromination
of 5 with sodium ethoxide in abs EtOH under MW irradiation,15
was selectively oxidized with cerium ammonium nitrate (CAN) in
aqueous acetic acid at the 4 position, then submitted to chlorina-
tion with POCl3 and Et3N and finally coupled with N,N-dimethyl-
ethylendiamine in iPrOH under MW irradiation, obtaining
compound 12 (Scheme 2).
Cell growth inhibition in the presence of the test compounds
a
Compound
IC50
(lM)
HeLa
HL-60
A431
6a
6b
6c
8a
8b
8c
12
Ellipticine
m-AMSA
>100
>100
15.7 1.7
>100
>100
23.2 1.8
>100
0.31 0.01
0.012 0.002
26.5 2.9
26.9 3.3
10.8 1.3
57.7 4.1
>100
3.6 0.8
>100
77.7 4.5
4.3 0.2
50.5 2.3
0.47 0.03
0.17 0.02
>100
30.7 4.5
4.3 0.4
61.6 4.9
0.64 0.02
0.0060 0.0011
a
The results have been expressed as IC50 values, that is, the concentration of
compound able to induce a 50% reduction in viable cell number relative to control.
An analogous synthetic route was followed for the benzo[f]qui-
nazoline derivative 16. The starting benzo[f]quinazoline (13), ob-
tained from naphthyl-2-amine,15 was selectively oxidized with
cerium ammonium nitrate (CAN) in aqueous acetic acid at the 1
position, then submitted to chlorination with CCl4 and triphenil-
phosphine in dichloroethane, obtaining the chloro derivative 15
(Scheme 3).
leukemia) and A431 (human squamous carcinoma) cell lines and
expressed as IC50 values (Table 2). Ellipticine and m-AMSA were
used as reference compounds.
The ability of the AMSA-like derivatives 6a–b and 8a–b to inhi-
bit cell growth was very weak, nevertheless it must be pointed out
that these compounds showed a rather low solubility in aqueous
media.
Owing to the known instability of 4-chloro derivatives,16 com-
pound 15 was immediately submitted to substitution step, but
any attempt to carry out the reaction gave only the starting quinaz-
olin-1-one (14), due to the ease of hydrolysis of chlorine group
which was competitive with the substitution reaction.
The derivatization of the benzoquinazoline scaffold with a dim-
ethylaminoethylamino chain allowed a significant increase in sol-
ubility, and an enhancement in cytotoxicity, but with different
extent, depending upon the substitution position. Compounds 6c
and 8c, bearing the side chain in the central benzene ring of the
benzoquinazoline chromophore, showed appreciable antiprolifera-
tive activity with IC50 values in the micromolar range for all the
tested cell lines. On the contrary, compound 12, bearing the side
chain in the pyrimidine ring of the benzoquinazoline nucleus
showed a weak cytotoxicity and only in HL-60 and A431 cell lines.
Thus, the introduction of a dimethylaminoethylamino side
chain in the benzoquinazoline chromophore gives rise to an anti-
proliferative effect, which results significantly increased when
the side chain is inserted in the central benzene ring (compounds
6c and 8c). This could be explained by the better binding mode
shown in the modeling studies for both the derivatives with re-
spect to compound 12 (Average Interaction Energy = ꢀ7.15 kcal/
mol) (Fig. 6).
2.3. Antiproliferative activity
The ability of the new derivatives to inhibit cell growth was
evaluated on HeLa (cervix adenocarcinoma), HL-60 (promyelocytic
N
N
N
N
N
NH
O
a
b
5
Br
9
10
c
d
N
N
N
N
As previously described for m-AMSA, an efficient interaction
between ligands and DNA required that the nitrogen atoms of
the central aromatic nucleus must be placed in the middle of
DNA axis. As a consequence, the scaffold placement drives also
the side chain positioning: compound 6c (Fig. 6A) could place the
side chain into the minor groove, thus favouring the intercalation,
while compound 12 did not (Fig. 6B).
NH Me
N
Cl
Me
12
11
Scheme 2. Reagents and conditions: (a) EtONa, abs EtOH, 110 °C, MW, 15 min; (b)
CAN, AcOH/H2O, rt, 5 min; (c) POCl3, Et3N, reflux, 3 h; (d) N,N-dimethylethylene-
diamine, iPrOH, 80 °C, MW, 15 min.

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G. Marzaro et al. / Bioorg. Med. Chem. 19 (2011) 1197–1204
Figure 6. Binding poses predicted for 6c (A) and 12 (B) into DNA.
2.4. Interaction with DNA
To investigate a possible mechanism of action involved in the
cytotoxicity of the most active compounds, linear flow dichroism
(LD) experiments were performed in the presence of salmon testes
DNA at different [drug]/[DNA] ratios. Indeed, the planarity of the
benzoquinazoline chromophore suggests for the new derivatives
the ability to form an intercalative complex with DNA.
Figure 7 shows the spectra obtained in the presence of 6c and 8c
at the same [drug]/[DNA] ratio (Fig. 7A) and the spectra of 12 at dif-
ferent [drug]/[DNA] ratios (Fig. 7B).
Figure 7. Linear flow dichroism spectra: (A) DNA alone (a) and in the presence of 6c
(b) and 8c (c) at [drug]/[DNA] = 0.08; (B) DNA alone (a) and in the presence of 12 at
[drug]/[DNA] = 0.08 (b) and 0.16 (c).
The DNA spectrum (traces a) show at 260 nm the typical nega-
tive dichroic signal, while in the presence of new benzoquinazo-
a
b
c
d
e
f
g
h
i
j
k
l
m
lines (traces
b and c) a further negative signal at higher
relaxed DNA
wavelengths (300–450 nm) occurs. The occurrence of this latter
signal in the presence of test compound (spectra b and c) indicates
that the planar benzoquinazoline nucleus forms a molecular com-
plex with DNA and its negative sign means an intercalative geom-
etry of binding.
These results indicate for the tricyclic benzoquinazoline chro-
mophore the ability to insert between base pairs. Nevertheless, this
capacity is shared by all the compounds, both the most active 6c
and 8c and also by 12. As a consequence, the capacity to form a
molecular complex with DNA cannot explain the significant differ-
ence in cytotoxicity exerted by 12 with respect to the others two
derivatives (Table 2).
supercoiled DNA
Figure 8. Effect of derivatives 6c, 8c and 12 on the relaxation of supercoiled
plasmid DNA by human recombinant topoII. Lane a: 0.25 g pBR322 DNA; lane b: as
lane a in the presence of 1 U topoII; lanes c–f: as lane b in the presence of 1, 10, 50,
100 M 6c, respectively; lanes g–j: as lane b in the presence of 1, 10, 50, 100 M 8c,
respectively; lane k: as lane b in the presence of 100 M 12; lane l: as lane b in the
l
l
l
l
presence of 8
l
M m-AMSA; lane m: as lane b in the presence of solvent alone.
A
B
a
a
b
c
d
e
f
b
c
d
e
f
linear DNA
2.5. Effect on topos activity
The ability of the new benzoquinazoline derivatives to form an
intercalative complex with DNA suggested that their antiprolifera-
tive effect could be related to the interference with nuclear
enzymes involved in DNA processing, like topos.17 Figures 8 and
9 show the effect of 6c, 8c and 12 on the relaxation of supercoiled
plasmid pBR322 DNA catalyzed by topoII.
In detail, both 6c and 8c inhibit the relaxation ability of the
enzyme in a concentration-dependent manner (lanes c–f and lanes
g–j,Fig. 8), and the inhibition becomes evident at 50 lM concentra-
supercoiled DNA
relaxed DNA
Figure 9. Determination of cleavable complex by human recombinant topoII
induced by 6c (A) and 8c (B). Lanes a: 0.25 g pBR322 DNA; lanes b: as lanes a in the
presence of 10 U topoII; lanes c–e: as lanes b in the presence of 10, 50, 100 M test
compound, respectively; lanes f: as lanes b in the presence of 8 M m-AMSA.
l
l
l

G. Marzaro et al. / Bioorg. Med. Chem. 19 (2011) 1197–1204
1201
a
b
c
d
tions (lanes e and i) as demonstrated by the disappearance of re-
laxed DNA and the appearance of the supercoiled form. Neverthe-
less, this latter DNA shows a clear shift in the electrophoretic
mobility with respect to the control (lanes a). At higher concentra-
tion (100 lM, lanes f and j) the supercoiled DNA and the mobility
shift of the band with respect to the control are even more evident
and this effect should be attributable to an efficient intercalative
capacity of both 6c and 8c.
No effect on topoII catalyzed relaxation is exerted by 12, even at
the higher concentration (100 lM, lane k) and this result, in agree-
ment with the data shown in Table 2, suggests a correlation be-
tween the topoII inhibitory activity and the cytotoxicity.
Taken together these results suggest that the different position
of the aminoalkylamino side chain inside the benzoquinazoline nu-
cleus plays a crucial role for the interaction of the derivatives with
DNA that, in the case of 6c and 8c, makes the macromolecule less
available for the enzymatic processing.
Figure 11. Agarose gel electrophoresis of nuclear DNA from HeLa cells. Lane a:
untreated control; lane b, 50 M cisplatin; lane c: 200 M 6c; lane d: 200 M 8c.
l
l
l
The agents interfering with the catalytic cycle of topoII are
divided into two classes: poisons, which stabilize the covalent
DNA–topoII complex (also known as the cleavable complex) and
catalytic inhibitors, which act on any of the other steps in the
catalytic cycle. Many intercalative agents, such as adriamycin,
m-AMSA and mitoxantrone, are able to stimulate the formation
of the cleavable complex, thus acting as poisons.18–20 The occur-
rence of the covalent intermediate can be demonstrated experi-
mentally by the enzyme-dependent formation of linear from
supercoiled DNA. Figure 9 shows a cleavable complex assay
performed in the presence of increasing concentrations of benzo-
tic process in HeLa cells. A biochemical hallmark of apoptosis is the
cleavage of nuclear DNA following the activation of endonucleases
that degrades the high order chromatine into mono and oligonu-
cleosomal DNA fragments. This process gives rise to a ‘ladder’ of
nucleosomal-size multimers.21 Figure 11 shows the gel electropho-
resis of DNA extracted from HeLa cells incubated in the presence of
6c (lane c), 8c (lane d) and cisplatin (lane b), taken as reference
compound. The results show for both benzoquinazoline derivatives
the occurrence of the typical DNA fragmentation, thus suggesting
that the cell death provoked by the test compounds occurs through
the induction of the apoptotic process.
quinazolines 6c and 8c and 8 lM m-AMSA, taken as reference
compound.
As expected, m-AMSA, a well-known topoII poison, induces the
formation of linear DNA (lanes f). Nevertheless, both benzoquinaz-
oline derivatives are unable to stabilise the formation of double
strand breaks (linear DNA) also at higher concentration (100 lM,
lanes e). Thus, 6c and 8c have to be defined as topoII catalytic
3. Conclusion
The design, synthesis and biological evaluation of a series of
benzo[h]quinazoline (6a–c and 12) and benzo[f]quinazoline
(8a–c) derivatives are reported. The compounds carrying a dimeth-
ylaminoethyl side chain (6c, 8c and 12) inhibit cell growth: in
particular, 6c and 8c are more cytotoxic than 12. Both benzoqui-
nazoline nuclei intercalate between DNA base pairs, and the ability
to interfere with topoII and topoI relaxation was evidenced for the
most active 6c and 8c, carrying the dimethylaminoethyl side chain
on the benzene of the quinazoline chromophore.
These results suggest that the mechanism of action responsible
for their cytotoxicity lies in the ability to interact with the macro-
molecule in such a way that the DNA processing enzymes, topoI
and topoII, cannot use the macromolecule as substrate for their
relaxation activity. This hypothesis is further supported by the fact
that both active compounds (6c and 8c) are unable to act as topoII
poisons.
inhibitors.
The lack of topoII poisoning effect along with the capacity to
form an effective intercalative complex with DNA prompt us to
investigate also on the ability to interfere with another DNA-re-
lated enzyme such topoI. Figure 10 A and B show the relaxation
of supercoiled DNA catalyzed by the enzyme in the absence (lane
b) and in the presence of increasing amounts of 6c and 8c, respec-
tively (lanes c–g).
The addition of the benzoquinazoline derivatives induces a con-
centration-dependent inhibition on the relaxation capacity, which
appears comparable to that evidenced on topoII (Fig. 8).
2.6. DNA fragmentation
The occurrence of nuclear DNA fragmentation after incubation
of HeLa cells with 6c and 8c suggests that the cytotoxic effect in-
duced by the benzoquinazoline derivatives is the result of the acti-
vation of the apoptotic process.
To get further details into the cytotoxic effect of 6c and 8c, we
investigate on the ability of these derivatives to induce the apopto-
In conclusion, the reported results allow to draw some interest-
ing structure–activity relationships. In particular, the comparison
between 6a–c and 8a–c evidenced that both the nitrogen nuclei,
benzo[h]quinazoline or benzo[f]quinazoline, induce a similar bio-
logical effect. On the contrary, inside each series, the insertion of
a protonatable dimethylaminoethylamino side chain seems to be
essential for the occurrence of a significant antiproliferative activ-
ity. Also the position of this side chain is an important structural
requirement, as demonstrated by the different behavior of 6c and
12. On the basis of these considerations, the new 6-aminoalkyl
substituted benzo[h]quinazolines and benzo[f]quinazolines can
be considered as an interesting model worthy to be further devel-
oped and studied with the aim to design more effective drugs.
A
a
B
b
c
d
e
f
g
a
b
c
d
e
f g
relaxed DNA
supercoiled DNA
Figure 10. Effect of derivatives 6c (A) and 8c (B) on the relaxation of supercoiled
plasmid DNA by human recombinant topoI. Lanes a: 0.25 g pBR322 DNA; lanes b:
as lanes a in the presence of 2 U topoI; lanes c–g: as lanes b in the presence of 1, 10,
25, 50, 100 M test compound, respectively.
l
l

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G. Marzaro et al. / Bioorg. Med. Chem. 19 (2011) 1197–1204
4.2.1.2. N-[4-(Benzo[h]quinazolin-60-ylamino)-3-methoxy-
phenyl]methanesulfonamide (6b). Yield 62%; mp: 260 °C; IR
4. Experimental section
4.1. Computational methodologies
(KBr) 3360, 2935, 2865, 1655, 1575, 1520, 1435, 1330, 1200,
1150, 980, 770 cmꢀ1 1H NMR (DMSO-d6): d = 9.22–9.17 (m, 3H,
;
20-H, 40-H and 70-H or 100-H), 8.53 (d, J = 8.0, 70-H or 100-H), 7.97–
7.85 (m, 3H, 80-H, 90-H and NHSO2CH3), 7.25 (d, J = 8.5, 1H, 5-H),
7.03 (d, J = 2.1, 1H, 2-H), 6.89 (dd, J = 8.5, J = 2.1, 1H, 6-H), 6.74 (s,
1H, 50-H), 3.29 (s, 3H, OCH3), 3.05 (s, 3H, NHSO2CH3); 13C NMR
(DMSO-d6) d = 156.2, 153.4, 152.0, 144.6, 141.6, 135.5, 129.9,
128.8, 127.6, 126.4, 125.6, 124.5, 124.4, 122.6, 112.5, 104.9,
100.0, 55.4, 39.0; HRMS-ESI: m/z [M+H]+ calcd for C20H19N4O3S:
395.1172, found 395.1331; Anal. calcd for C20H18N4O3S: C, 60.90;
H, 4.60; N, 14.20; S, 8.13; found C, 60.88; H, 4.62; N, 14.24; S, 8.12.
All modeling studies were carried out on a 4 CPU (Intel Core™2
Quad CPU Q9550 2.83 GHz) ACPI ꢁ64 based PC. DNA structure was
downloaded from Protein Data Bank (PDB ID: 1G3X) and modified
employing Autodock 4.1 software.22 Minimum energy conforma-
tion for each isolated compound was derived using MarvinSketch
software23 and was used as the initial tridimensional structure
for the docking. All docking studies were performed with Autodock
4.1 software employing the Lemarkian Genetic Algorithm, generat-
ing 10 independent docking pose for each compound. In all the
cases, the population size was set to 150 and the maximum num-
ber of evaluation was set to 250,000. The dimension and the posi-
tion of the docking grid were set in a way that the compounds
could place their side chains in the minor groove. The DNA was
considered as a rigid molecule, while the ligands were considered
as flexible molecules. The intercalative energies were calculated as
the mean of the docked structures obtained for each compound.
4.2.1.3. 6-[N-(Dimethylamino)ethyl]aminobenzo[h]quinazoline
(6c). Yield 49%; mp: >300 °C; IR (KBr) 3425, 2940, 2865, 1650,
1575, 1510, 1460, 1200, 1055, 830, 770 cmꢀ1 1H NMR (CDCl3):
;
d = 9.20 (s, 1H, 4-H), 9.13 (s, 1H, 2-H), 9.29 (d, J = 7.9, 1H, 7-H or
10-H), 8.00 (d, J = 7.9, 1H, 7-H or 10-H), 7.81 (m, 2H, 8-H and 9-
H), 6.57 (s, 1H, 5-H), 5.61 (s, 1H, NH), 3.39 (t, J = 6.6, 2H,
NHCH2CH2N(CH3)2), 2.77 (t, J = 6.6, 2H, NHCH2CH2N(CH3)2), 2.33
(s, 6H, N(CH3)2); 13C NMR (CDCl3): d = 155.5, 151.8, 145.5, 143.3,
130.4, 129.9, 128.4, 127.5, 125.4, 125.4, 120.5, 95.4, 57.2, 45.0,
40.7; HRMS-ESI: m/z [M+H]+ calcd for C16H19N4: 267.1610, found
267.1593; Anal. calcd for C16H18N4: C, 72.15; H, 6.81; N, 21.04;
found C, 72.16; H, 6.75; N, 21.10.
4.2. Chemistry
Melting points were determined on a Gallenkamp MFB-595-
010M melting point apparatus and are uncorrected. Analytical
TLC was performed on pre-coated 60 F254 silica gel plates
(0.25 mm; Merck) developing with a CHCl3/MeOH mixture (9:1).
Preparative column chromatography was performed using silica
gel 60 (0.063–0.100 mm; Merck). The microwave irradiation was
performed in a CEM DiscoverÒ monomode reactor with the tem-
perature monitored by a built-in infrared sensor. IR spectra were
recorded on a Perkin–Elmer 1760 FT-IR spectrometer. 1H NMR
spectra were recorded on a Bruker AMX300 spectrometer with
TMS as internal standard. Chemical shift values are reported in
ppm and coupling constants are reported in Hz. HRMS spectra
were obtained using an ESI-TOF Mariner 5220 (Applied Biosystem)
mass spectrometer with direct injection of the sample and collect-
ing data in the positive ion mode. Elemental analyses were per-
formed on a Perkin–Elmer 2400 Analyzer and are within 0.4% of
theoretical values. Starting benzoquinazolines 5, 7 and 13 were
prepared according to literature methods.15
4.2.1.4. N-[4-(Benzo[f]quinazolin-60-ylamino)phenyl]methane-
sulfonamide (8a). Yield 28%; mp: 295 °C; IR (KBr) 3350, 2930,
2865, 1650, 1580, 1510, 1460, 1325, 1225, 1150, 1055, 980, 830,
770 cmꢀ1 1H NMR (DMSO-d6): d = 9.86 (s, 1H, 10-H), 8.95 (s, 1H,
;
30-H), 8.93 (d, J = 7.8, 1H, 70-H or 100-H), 8.59 (d, J = 7.8, 1H, 70-H
or 100-H), 7.85–7.76 (m, 2H, 80-H and 90-H), 7.03–6.91 (m, 4H, 2-
H, 3-H, 5-H and 6-H), 6.74 (s, 1H, 50-H), 2.59 (s, 3H, NHSO2CH3);
13C NMR (DMSO-d6): d = 156.1, 152.6, 152.5, 147.6, 137.0, 134.4,
129.4, 128.7, 127.8, 124.9, 124.3, 123.1, 122.7, 121.6, 116.9,
101.5, 39.0; HRMS-ESI: m/z [M+H]+ calcd for C19H17N4O2S:
365.1067, found 365.1205; Anal. calcd for C19H16N4O2S: C, 62.62;
H, 4.43; N, 15.37; S, 8.80; found C, 62.62; H, 4.46; N, 15.39; S, 8.85.
4.2.1.5. N-[4-(Benzo[f]quinazolin-60-ylamino)-3-methoxy-
phenyl]methanesulfonamide (8b). Yield 12%; mp: 266 °C; IR
(KBr) 3440, 2935, 2865, 1655, 1580, 1515, 1460, 1330, 1150, 975,
4.2.1. General procedure for 6-substituted benzo[h]
830 cmꢀ1 1H NMR (DMSO-d6): d = 9.93 (s, 1H, 10-H), 8.99–8.95
;
quinazolines 6a–c and 6-substituted benzo[f]quinazolines 8a–c
A mixture of 5 or 7 (1.0 mmol), N-(4-aminophenyl)-methanesul-
fonamide or N-(4-amino-3-methoxyphenyl)methane-sulfonamide24
or N,N-dimethylethylenediamine (1.2 mmol), Pd(OAc)2 (0.03 mmol),
BINAP (0.04 mmol), Cs2CO3 (1.5 mmol) in H2O (10 mL) was
microwave irradiated at 150 °C (power set point 150 W, ramp time
1 min, hold time 20 min). After cooling, the mixture was extracted
with EtOAc (3 ꢁ 20 mL), and the organic phase was evaporated under
reduced pressure. The residue was purified by column chromatogra-
phy (eluent: CHCl3/MeOH, 9/1) to give 6 or 8.
(m, 2H, 30-H e 70-H or 100-H), 8.60 (d, J = 8.4, 1H, 70-H or 100-H),
8.53 (s all, 1H, NHSO2CH3), 7.88–7.79 (m, 2H, 80-H and 90-H), 7.30
(d, J = 8.4, 1H, 5-H), 7.04 (d, J = 1.9, 1H, 2.H), 6.93 (dd, J = 8.4,
J = 1.9, 1H, 6-H), 6.41 (s, 1H, 50-H), 3.73 (s, 3H, OCH3), 3.06 (s, 3H,
NHSO2CH3); 13C NMR (DMSO-d6): d = 156.1, 154.8, 152.7, 152.3,
148.5, 137.3, 129.2, 128.5, 128.4, 127.7, 124.4, 124.1, 123.0,
122.7, 116.4, 111.8, 104.3, 100.5, 55.4; HRMS-ESI: m/z [M+H]+ calcd
for
C20H19N4O3S: 395.1172, found 395.1261; Anal. calcd for
C20H18N4O3S: C, 60.90; H, 4.60; N, 14.20; S, 8.13; found C, 60.91;
H, 4.65; N, 14.18; S, 8.10.
4.2.1.1. N-[4-(Benzo[h]quinazolin-60-ylamino)phenyl]methane-
sulfonamide (6a). Yield 45%; mp: 265 °C; IR (KBr) 3395, 2935,
4.2.1.6. 6-[N-(Dimethylamino)ethyl]aminobenzo[f]quinazoline
(8c). Yield 50%; mp: >300 °C; IR (KBr) 3425, 2940, 2865, 1650,
2865, 1620, 1590, 1510, 1430, 1320, 1220, 1155, 975, 830 cmꢀ1
;
1H NMR (DMSO-d6): d = 9.31 (s, 1H, 40-H), 9.24–9.21 (m, 2H, 20-
He 70-H or 100-H), 8.54 (broad s, 1H, NHSO2CH3), 8.50 (d, J = 8.0,
1H, 70-H or 100-H), 7.98–7.87 (m, 2H, 80-H and 90-H), 7.38 (s, 1H,
50-H), 7.32–7.23 (m, 4H, 2-H, 3-H, 5-H and 6-H), 2.98 (s, 3H,
NHSO2CH3); 13C NMR (DMSO-d6): d = 156.6, 152.5, 145.1, 140.5,
139.3, 132.1, 130.1, 130.0, 129.5, 27.8, 124.4, 124.2, 122.7, 122.4,
121.3, 101.9, 38.8; HRMS-ESI: m/z [M+H]+ calcd for C19H17N4O2S:
365.1067, found 365.0962; Anal. calcd for C19H16N4O2S: C, 62.62;
H, 4.43; N, 15.37; S, 8.80; found C, 62.68; H, 4.41; N, 15.35; S, 8.79.
1585, 1545, 1460, 1360, 1250, 1125, 1055, 995, 830, 770 cmꢀ1
;
1H NMR (CDCl3): d = 10.01 (s, 1H, 1-H), 9.21 (s, 1H, 3-H), 9.49 (s,
1H, NH), 8.96 (d, J = 7.5, 1H, 10-H), 8.54 (d, J = 7.5, 1H, 7-H), 7.98
(m, 2H, 9-H and 8-H), 7.05 (s, 1H, 5-H), 3.94 (m, 2H,
NHCH2CH2N(CH3)2), 3.50 (t, J = 6.6, 2H, NHCH2CH2N(CH3)2), 2.91
(s, 6H, N(CH3)2), 2.35 (s, 9H, 3ꢂCH3SO3H); 13C NMR (CDCl3):
d = 156.5, 154.0, 151.7, 149.1, 128.5, 128.4, 128.1, 127.8, 122.5,
122.3, 121.2, 97.5, 56.9, 44.9, 40.2; HRMS-ESI: m/z [M+H]+ calcd
for
C16H19N4: 267.1610, found 267.1599; Anal. calcd for

G. Marzaro et al. / Bioorg. Med. Chem. 19 (2011) 1197–1204
1203
C16H18N4: C, 72.15; H, 6.81; N, 21.04; found C, 72.18; H, 6.85; N,
21.06.
4.2.7. 1-Chlorobenzo[f]quinazoline (15)
A mixture of 14 (0.40 g, 2.0 mmoli), triphenylphosphine (2.1 g,
8.0 mmoli) and CCl4 (1.9 ml, 20.0 mmoli) in CH2Cl2 (5 ml) was stir-
red at reflux for 3 h. After cooling, the reaction mixture was con-
centrated under reduced pressure and the residue was purified
by column chromatography (eluent: CHCl3) to give 15; (0.21 g,
yield 48%); mp: dec on heating; 1H NMR (CDCl3): d = 1H NMR
(CDCl3): d = 9.75 (d, J = 8.4, 1H, 7-H or 10-H), 9.11 (s, 1H, 3-H),
8.23 (d, J = 9.0, 1H, 5-H or 6-H), 8.02 (d, J = 8.4, 1H, 7-H or 10-H),
7.93 (d, J = 9.0, 1H, 5-H or 6-H), 7.87-7.75 (m, 2H, 8-H and 9-H).
4.2.2. Benzo[h]quinazoline (9)
A mixture of 5 (1.3 g, 5.0 mmol) and ethanolic EtONa (21% wt,
20 mL) was microwave irradiated at 110 °C (power set point
100 W, ramp time 30 s, hold time 15 min). After cooling, the mix-
ture was diluted with sat. NH4Cl (200 mL), extracted with CHCl3
(3 ꢁ 80 mL), and the organic phase was evaporated under reduced
pressure to give 9 (0.88 g, yield 98%); mp: 120 °C; 1H NMR (DMSO-
d6): d = 9.42 (s, 1H, 4-H), 9.28 (s, 1H, 2-H), 9.21–9.18 (m, 1H, 7-H or
10-H), 7.90–7.87 (m, 1H, 7-H or 10-H), 7.82 (d, J = 8.9, 1H, 5-H or
6-H), 7.77–7.73 (m, 2H, 8-H and 9-H), 7.64 (d, J = 8.9, 1H, 5-H or
6-H).
4.2.8. Substitution reaction on 15
A mixture of 15 (0.25 g, 1.1 mmoli), N,N-dimethyl-ethylenedia-
mine (0.36 ml, 3.3 mmoli) in iPrOH (5 ml) was microwave irradi-
ated at 80 °C (three cycles as follow: power set point 100 W,
ramp time 30 s, hold time 15 min). After cooling, the reaction mix-
ture was concentrated under reduced pressure and the residue was
purified by column chromatography (eluent: CHCl3/MeOH, 9/1) to
give only 14.
4.2.3. Benzo[h]quinazolin-4(3H)-one (10)
A solution of CAN (4.4 g, 8.0 mmoli) in H2O (12 ml) was added
under stirring at room temperature to a solution of 9 (0.40 g,
2.0 mmol) in AcOH (1 ml). The resulting precipitated was collected
by filtration, washed with AcOH, to give 10 (0.4 g, yield 98%); mp:
>300 °C; 1H NMR (DMSO-d6): d = 8.92 (dd, J = 7.2, J = 1.6, 1H, 7-H or
10-H), 8.37 (s, 1H, 2-H), 8.09–8.04 (m, 2H, 5-H or 6-H, and 7-H or
10-H), 7.96 (t, J = 8.8, 1H, 5-H o 6-H), 7.82–7.71 (m, 2H, 8-H and
9-H).
4.3. Biology
4.3.1. Cell cultures
HL-60 (human myeloid leukemic cells) were grown in RPMI
1640 (Sigma Chemical Co.) supplemented with 15% heat-inacti-
vated fetal calf serum (Biological Industries), HeLa (human cervix
adenocarcinoma cells) in Nutrient Mixture F-12 [HAM] (Sigma
Chemical Co.) supplemented with 10% heat-inactivated fetal calf
serum (Biological Industries) and A431 (human squamous carci-
noma cells) in D-MEM (Sigma Chemical Co.) supplemented with
10% heat-inactivated fetal calf serum (Biological Industries).
4.2.4. 4-Chlorobenzo[h]quinazoline (11)
A mixture of 10 (0.40 g, 1.9 mmoli), POCl3 (5 ml) and TEA (2 ml)
was stirred at reflux for 3 h. After cooling, the reaction mixture was
concentrated under reduced pressure and the residue was sus-
pended in EtOAc (100 mL). The organic phase was washed with
sat. NaHCO3 (100 mL), then with H2O (100 mL) and evaporated un-
der reduced pressure to give 11 (0.25 g, yield 63%); mp: 115 °C; 1H
NMR (CDCl3): d = 9.22 (d, J = 7.6, 1H, 7-H or 10-H), 9.16 (s, 1H, 2-H),
8.04 (d, J = 9.1, 1H, 5-H or 6-H), 7.97–7.90 (m, 2H, 5-H or 6-H and
7-H or 10-H), 7.87–7.75 (m, 2H, 8-H and 9-H).
100 U/mL penicillin, 100 lg/mL streptomycin and 0.25 lg/mL
amphotericin B (Sigma Chemical Co.) were added to the media.
The cells were cultured at 37 °C in a moist atmosphere of 5%
carbon dioxide in air.
4.2.5. 4-[N-(Dimethylamino)ethyl]aminobenzo[h]quinazoline
(12)
A mixture of 11 (80 mg, 0.33 mmoli), N,N-dimethylethylenedi-
amine (36 lL, 0.33 mmoli) in iPrOH (2 ml) was microwave irradi-
4.3.2. Inhibition growth assay
HL-60 cells (4 ꢁ 104) were seeded into each well of a 24-well
cell culture plate. After incubation for 24 h, various concentrations
of the test agents were added to the complete medium and incu-
bated for a further 72 h. HeLa and A431 (4 ꢁ 104) cells were seeded
into each well of a 24-well cell culture plate. After incubation for
24 h, the medium was replaced with an equal volume of fresh
medium, and various concentrations of the test agents were added.
The cells were then incubated in standard conditions for a further
72 h. A trypan blue assay was performed to determine cell viabil-
ity. Cytotoxicity data were expressed as IC50 values, that is, the
concentration of the test agent inducing 50% reduction in cell num-
ber compared with control cultures.
ated at 80 °C (three cycles as follow: power set point 100 W,
ramp time 30 s, hold time 15 min). After cooling, the reaction mix-
ture was concentrated under reduced pressure and the residue was
purified by column chromatography (eluent: CHCl3/MeOH, 9/1) to
give 12 (70 mg, yield 80%; mp: >300 °C; IR (KBr) 3435, 2940, 2865,
1650, 1620, 1525, 1360, 1220, 1110, 1055, 830, 770 cmꢀ1; 1H NMR
(CDCl3): d = 9.17 (m, 1H, 7-H or 10-H), 8.85 (s, 1H, 2-H), 7.91–7.88
(m, 1H, 7-H or 10-H), 7.79 (d, J = 8.8, 1H, 5-H or 6-H), 7.73–7.70 (m,
3H, 5-H or 6-H and 8-H and 9-H), 6.91 (s, 1H, NH), 3.79 (t, J = 6.3,
2H, NHCH2CH2N(CH3)2), 2.78 (t, J = 6.3, 2H, NHCH2CH2N(CH3)2),
2.41 (s, 6H, N(CH3)2); 13C NMR (CDCl3): d = 155.2, 150.3, 146.8,
146.0, 135.5, 129.2, 128.4, 127.0, 126.7, 124.5, 121.1, 96.4, 57.1,
44.9, 40.4; HRMS-ESI: m/z [M+H]+ calcd for C16H19N4: 267.1610,
found 267.1588; Anal. calcd for C16H18N4: C, 72.15; H, 6.81; N,
21.04; found C, 72.13; H, 6.80; N, 21.10.
4.3.3. Nucleic acids
Salmon testes DNA was purchased from Sigma Chemical Com-
pany. Its hypochromicity, determined according to Marmur and
Doty,25 was over 35%. pBR322 DNA was purchased from Fermentas
Life Sciences.
4.2.6. Benzo[f]quinazolin-1(2H)-one (14)
4.3.4. Linear flow dichroism
A solution of CAN (4.4 g, 8.0 mmoli) in H2O (12 ml) was added
under stirring at room temperature to a solution of 13 (0.40 g,
2.0 mmol) in AcOH (1 ml). The resulting precipitated was collected
by filtration, washed with AcOH, to give 14 (0.40 g, yield 98%); mp:
260 °C; 1H NMR (DMSO-d6): d = 12.63 (s, 1H, NH), 9.86 (d, J = 8.2,
1H, 7-H or 10-H), 8.30 (d, J = 8.2, 1H, 7-H or 10-H), 8.28 (s, 1H,
2-H), 8.07 (d, J = 8.4, 1H, 5-H or 6-H), 8.05 (d, J = 8.4, 1H, 5-H or
6-H), 7.85 (t, J = 8.2, 1H, 8-H o 10-H), 7.60 (t, J = 8.2, 1H, 8-H o
10-H).
Linear dichroism (LD) measurements were performed on a Jasco
J500A circular dichroism spectropolarimeter converted for LD and
equipped with an IBM PC and a Jasco J interface. Linear dichroism is
defined as:
LDðkÞ ¼ A==ðkÞ ꢀ A?ðkÞ
where A// and A\ correspond to the absorbances of the sample
when polarized light is oriented parallel or perpendicular to the
flow direction, respectively. The orientation is produced by a device

1204
G. Marzaro et al. / Bioorg. Med. Chem. 19 (2011) 1197–1204
designed by Wada and Kozawa26 at a shear gradient of 500–
700 rpm and each spectrum was accumulated four times. A solution
of salmon testes DNA (1.9 ꢁ 10ꢀ3 M) in ETN buffer (containing
10 mM TRIS, 10 mM NaCl, and 1 mM EDTA, pH 7) was used. Spectra
were recorded at 25 °C at different [drug]/[DNA] ratios.
Acknowledgments
The present work has been carried out with financial supports
of the Italian Ministry for University and Research (MIUR), Rome
(Italy).
4.3.5. DNA topoisomerases relaxation assay
References and notes
Supercoiled pBR322 plasmid DNA (0.25 lg, Fermentas Life Sci-
ences) was incubated with 1 U topoisomerase II (USB) or 2 U topo-
isomerase I (USB) and the test compounds as indicated, for 60 min
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at 37 °C in 20
L stop buffer (5% SDS, 0.125% bromophenol blue and 25% glyc-
erol), 50 g/mL proteinase K (Sigma) and incubating for a further
lL reaction buffer. Reactions were stopped by adding
4
l
l
30 min at 37 °C. The samples were separated by electrophoresis
on a 1% agarose gel at room temperature. The gels were stained
with ethidium bromide 1 lg/mL in TAE buffer, transilluminated
by UV light, and fluorescence emission was visualized by a CCD
camera coupled to a Bio-Rad Gel Doc XR apparatus.
4.3.6. Topoisomerase II cleavage assay
Reaction mixtures (20
50 mM NaCl, 50 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 15
BSA, 1 mM ATP, 0.25 g pBR322 plasmid DNA, 10 U topoisomerase
II and test compounds were incubated for 60 min at 37 °C. Reac-
tions were stopped by adding 4 L stop buffer (5% SDS, 0.125% bro-
mophenol blue and 25% glycerol), 50 g/mL proteinase K (Sigma)
lL) containing 10 mM Tris–HCl (pH 7.9),
l
g/mL
l
l
l
and incubating for a further 30 min at 37 °C. The samples were sep-
arated by electrophoresis on a 1% agarose gel containing ethidium
bromide 0.5 lg/mL at room temperature.
18. Chen, A. Y.; Liu, L. F. Annu. Rev. Pharmacol. Toxicol. 1994, 34, 191.
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4.3.7. DNA isolation and electrophoresis
HeLa (5 ꢁ 105) cells were incubated in standard conditions for
24 h at 37 °C, then the medium was replaced with an equal volume
of fresh medium and the test agent was added at the indicated con-
centration. Control cells were grown under the same conditions
with the addition of the solvent alone. After 24 h of incubation,
DNA from control and treated cells was extracted according to
the procedure described by Sambrook et al.27 The isolated DNA
was dissolved in TE buffer (Tris 10 mM, pH 8, EDTA 1 mM) and
analyzed by agarose (1%) gel electrophoresis. The gel was stained
24. Brennan, S. T.; Colbry, N. L.; Leeds, R. L.; Leja, B.; Priebe, S. R.; Reily, M. D.;
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with ethidium bromide solution (1 lg/mL), transilluminated by
UV light and fluorescence emission visualized using a CCD camera
coupled to a Bio-Rad Gel Doc 1000 apparatus.