Synthesis of new pyrrolo-, and pyrido-anellated quinazolinones as potential antiproliferative agents

Article abstract of DOI:10.1016/j.tet.2016.10.055

Some pyrrolo[3,2-f]quinazolinones (compounds 4–12), two pyrido[3,2-f]- and two pyrido[2,3-g]quinazolinones (compounds 15, 16 and 22, 23, respectively) were prepared via multistep syntheses. The tricyclic pyrroloquinazolinone nucleus was built starting fro

Full text of DOI:10.1016/j.tet.2016.10.055

Accepted Manuscript
Synthesis of new pyrrolo-, and pyrido-anellated quinazolinones as potential
antiproliferative agents
Davide Carta, Lisa Dalla Via, Aída Nelly García-Argáez, Maria Grazia Ferlin
PII:
S0040-4020(16)31114-0
10.1016/j.tet.2016.10.055
TET 28196
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 22 July 2016
Revised Date: 4 October 2016
Accepted Date: 20 October 2016
Please cite this article as: Carta D, Via LD, García-Argáez AN, Ferlin MG, Synthesis of new pyrrolo-, and
pyrido-anellated quinazolinones as potential antiproliferative agents, Tetrahedron (2016), doi: 10.1016/
j.tet.2016.10.055.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Synthesis of new pyrrolo-, and pyrido-anellated quinazolinones as antiproliferative agents

ACCEPTED MANUSCRIPT
Synthesis of new pyrrolo-, and pyrido-anellated quinazolinones as potential
antiproliferative agents
Davide Carta,a Lisa Dalla Viaa,*, Aída Nelly García-Argáeza,b, Maria Grazia Ferlina
aDepartment of Pharmaceutical and Pharmacological Science, University of Padova, Via F.
Marzolo 5, 35131 Padova, Italy.
bFondazione per la Biologia e la Medicina della Rigenerazione T.E.S.-Tissue Engineering and
Signalling Onlus, Via F. Marzolo,13, 35131 Padova, Italy
Abstract
Some pyrrolo[3,2-f]quinazolinones (compounds 4-12), two pyrido[3,2-f]- and two pyrido[2,3-
g]quinazolinones (compounds 15, 16 and 22, 23, respectively) were prepared via multistep
syntheses. The tricyclic pyrroloquinazolinone nucleus was built starting from 5-aminoindole via a
modified reported pathway to form the pyrimidine ring. To synthesize the tricyclic
pyridoquinazolinones, new pathways were designed: the angular moiety was obtained starting from
6-aminoquinazolinone to form the pyridine ring and the linear one starting from the 6-
aminoquinolinone to form the pyrimidine ring. An interesting antiproliferative activity was found
for compound 23, which was able to form a molecular intercalative complex with DNA and to
interfere with the relaxation activity of both topoisomerase I and II. The activation of the apoptotic
pathway was also demonstrated.
Keywords: pyrroloquinazolinone; pyridoquinazolinone; antiproliferative activity; DNA
intercalation; DNA topoisomerases.
* corresponding author. Phone: +39 0498275712; fax: +39 0498275366; e-mail address:
lisa.dallavia@unipd.it
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1. Introduction
The quinazoline skeleton is found in a number of biologically active molecules and in particular,
4(3H)-quinazolinone derivatives display a wide range of biological properties, well documented in
the literature.1 Quinazolinone derivatives have also been developed as antiproliferative agents
acting as inhibitors of thymidylate synthase2 or of tubuline polymerization.3 Heterocycle-fused
quinazolinones are less described and pyrimidoquinazolinones, due to their polycyclic planar
structure bearing more or less complex side groups, were proposed as intercalators and
topoisomerase II inhibitors.4,5
Although many papers describe various polyheterocycle alkylamino-substituted derivatives acting
as intercalators and topoisomerase II inhibitors, those focusing on the synthesis and biological
profiles of pyrrolo- and pyridoquinazolinones are very few.6
Considering the richness of the chemical, physicochemical and pharmacological properties of
quinazoline derivatives, the preparation of new fused quinazoline structures remains a worthy area
of research.
In the past few years we have focused our interest on the synthesis and biological evaluation of
some pyrroloquinolines and pyridocarbazoles as analogues of ellipticine, a well-known intercalating
anti-topoisomerase II agent.7,8 The ability of ellipticine to intercalate into DNA and to alter
topoisomerase II activity appears to play an important role in its antineoplastic activity.9,10
Recently, we have moved to the synthesis and characterization of some new poly-heterocycles
containing the quinazolinone scaffold fused to a pyrrolo or pyridine ring to build both angular and
linear tricycles. Figure 1 shows the structures of clinically used prototypic topoisomerase inhibitors
(top), inhibitors in clinical development (middle), our previous pyrroloquinoline derivatives,7 and
the new structures proposed in this paper (bottom). The structural similarity among all these
compounds is evident: the presence of a planar platform linking side polar groups, two crucial
elements for topoisomerase inhibition.11-14
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H
N
O
O
O
OH
OH
N
OH
O
OH
HN
OH
HO
OH
O
N
CH3
N
OMe
OH
OH
NH2
HN
O
O
OH
N
H
O
OH
Topotecan
Doxorubicin
Mitoxantrone
O
N
O
O
O
OH
N
HN
O
N
O
HO
H3C
HN
N
H3CO
H3CO
N
N
N
S
(CH2)3
N
N
O
N
O
CH3
NSC 725776
C-1311
Vosaroxin
COOCH3
O
N
R
R3
O
O
R
O
N
N
O
N
R
N
N
N
N
N
R1
HN
R2
R
HN
H3C
OCH3
O
R
pyrido[2,3-g]quinazolinone
pyrido[3,2-f]quinazolinone
pyrrolo[3,2-f]quinoline
pyrrolo[3,2-f]quinazolinone
R = diethylaminoethyl
R1 = ethyl
R2 = dimethylamino, N-methyl-piperazino
R3 = H, CN, CHO, CH2OH
Figure 1. Structures of clinically used prototypic topoisomerase inhibitors (top), inhibitors in
clinical development (middle), previous pyrroloquinolines,7 new pyrrolo- and pyrido-quinazolinone
structures proposed in this paper (bottom).
2. Results and discussion
2.1. Chemistry
The synthesis of pyrrolo[3,2-f]quinazolinone derivatives 4-8 described in Scheme 1 was
accomplished adopting a methodology previously reported to form the pyrimidine ring starting from
an aromatic amine.15 Thus, 5-aminoindoles 1-3 obtained by us as previously reported16 were
submitted to
a
one pot reaction by successive addition of the reagents:
ethoxycarbonylisothiocyanate, diethylamine or methylpiperazine, and HgCl2 to the reaction mixture
with the indicated stoichiometry. Finally, when the reaction mixture was subjected to thermal
cyclization, the 2-alkyl amino-substituted pyrrolo[3,2-f]quinazolinones 4-8 were formed by means
3

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of a regio-selective reaction.17 Indeed, it is worthy to note that the products obtained possess
exclusively angular structures, and any product with linear geometry was not observed by proton
1
NMR spectroscopy. The H NMR spectra of compounds 4-8 showed two doublets at about δ 7.64
and 7.92 for an ortho coupling (J = 8.7 Hz) assigned to H-5 and H-6, respectively, and not two
singlets as would be expected for a linear structure. In Scheme 1, the other four
pyrroloquinazolinone derivatives described are obtained by modifying compounds 6 and 7 at
position 8. Firstly, aldehydic compounds 9 and 10 were prepared by reacting 6 and 7 with POCl3
and DMF, then 9 and 10 were reduced by NaBH4 in methanol to alcoholic derivatives 11 and 12.
In Scheme 2A and 2B, the total synthesis of two pyridoquinazolinone derivatives, one angular (16)
and one linear (23) is described. The only successful approach starting from 13, was to react it with
the Meldrum’s acid in trimethylorthoformate to obtain compound 14 and then thermal cyclization in
diphenyl ether (15), as described in Scheme 2A. Due to the poor solubility of tricycle 15 in every
solvent, the tricycle, without any purification was submitted to alkylation with
chloroethyldiethylamine in DMF, and the sole obtained 3-alkylated derivative was treated with HCl
gas in absolute ethanol to furnish its mono-hydrocloride 16.
Figure 2. HMBC (A, blue arrows) and NOE (B, red arrows) correlations of compound 16.
The exact structure of compound 16 was confirmed by HRMS (m/z [M+H+] calculated for
+
C17H21N4O2 , 313.1759) and 1D and 2D NMR experiments (see the Supplementary Material). The
1H NMR (400 MHz, DMSO-d6) spectrum of compound 16 showed a triplet at 0.81 ppm (6H, J=
7.08 Hz) and a quartet at 2.49 ppm (4H, J= 7.24 Hz) both generated by the ethyldiethylamino chain.
Additionally the triplets at 2.75 and 4.34 ppm (J= 5.87 Hz and J= 5.68 Hz, respectively), correspond
to the ethylene bridge of the side chain. The presence of two doublets at 7.02 (H-8) and 8.66 (H-9)
characterized by a coupling constant of 4.50 Hz is distinctive for the two ortho protons belonging to
the pyridine ring. The singlet at 8.67 ppm (H-3) and two doublets at 7.92 (H-5) and 8.30 (H-6) ppm
with a coupling constant of 9.10 Hz belong to the quinazolinone nucleus. The sharp singlet at 15.11
ppm is assigned to the hydroxyl proton at position 10 that is de-shielded by the proximity of the
carbonyl group. This signal and the presence of two doublets at 7.92 and 8.30 ppm of the ortho
coupling H-5 and H-6 are in agreement with a [3,2-f] angular geometry of compound 16.
4

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Both confirmation of specific geometry and complete structural elucidation were realized by means
of 2D NMR experiments like NOESY, HSQC and HMBC. By HSQC and HMBC experiments it
was possible to determine the site of alkylation, that is the 2-N position as well as the HMBC
experiments (Figure 2A, blue arrows), that showed a significant correlation between the protons at
4.23 ppm (t, 2H, NCH2CH2N), and carbon signals at 149.22 (C-3) and 164.15 (C-1). The hydroxyl
proton at 15.11 ppm showed clear correlations with the carbon signals at 111.37 (C-9), 117.77 (C-
10a) and 163.39 (C-10) ppm stating an undoubtable presence of a hydroxyl group at position 10.
The NOESY spectrum of 16 (see Supplementary Material) displays correlations between the
methylene bridge and the quinazolinone ring system (Figure 2B, red arrows): for example, the
proton at 15.11 ppm is next to the proton at 7.02 ppm (H-9) on the same ring, while H-3, H-5 and
H-6 showed no NOE effect with other aromatic proton, further evidence of the [3,2-f] geometry, an
occurrence that cannot be fully understood from the 1D or COSY spectra.
In Scheme 2B, the synthetic route to a linear pyridoquinazolinone starts with the commercial
available 5-amino-2-nitrobenzoic acid which was transformed into its methyl ester before reaction
with Meldrum’s acid and trimethylorthoformate to provide intermediate 18. This was thermally
cyclized at 250 °C (diphenyl ether) to give a mixture of the two isomeric quinolinones 19a and 19b
in which the desired compound 19a was present in a higher percentage (63%). Unfortunately, at this
stage the two isomers couldn’t be separated by any method, thus the mixture was submitted to
alkaline hydrolysis (NaOH 20% in methanol) to give the corresponding carboxylic acids. However,
only the acid 20 was obtained, while due to its intrinsic chemical stability the isomer 19b didn’t
hydrolyze in those conditions, and so was easily removed from the reaction mixture. Successively,
the quinolinone 20 was catalytically reduced (H2, C/Pd 10%) to amino-compound 21 and this was
reacted with trimethylorthoformate and CH3COONH4 in a MW oven (250W, 5 min)18 to give the
linear pyridoquinazolinone 22. This was the method giving the best yields of 13%. The linearity of
compound 22 was confirmed by 1H NMR spectroscopy, which showed that two singlets at 8.30 and
8.40 δ assigned to H-10 and H-5 respectively were among the other expected signals. Alkylation
with chloroethyldiethylamine in anhydrous DMF furnished the bis-substituted derivative that finally
was transformed into its dihydrochloride 23 by treatment with HCl dry gas in absolute ethanol. The
exact structure of compound 23 was identified by HRMS (m/2z [M+2H+] calculated for
2+
C23H35N5O2 , 206.6390; found, 206.6384) that proves the presence of two diethylaminoethyl side
1
1
chains, and H and 13C NMR spectrometry. In the H NMR spectrum of 23, the lack of broad
signals of the H-3 and H-6 protons of compound 22 was diagnostic. Additionally, the chemical
shifts and the coupling constants of the two doublets due to the H-7 and H-8 (8.03 δ, J=7.44 and
6.07, J= 7.38 Hz) of the pyridone ring in 22 changed into 8.72 δ, J=5.50 Hz and 6.96 δ, J=5.55 Hz
of the pyridine ring in compound 23.
5

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Scheme 1: synthesis of pyrrolo[3,2-f]quinazolinone derivatives 4-12
R3
N
R2
R3
R2
R2
S
N C
NH
COOC2H5
R2
H
O
∆
N C
NH
COOC2H5
b
H2N
R1
N
a
NH
R3
N
R1
N
R1
N
N
R1
1-3
4- 8
1: R1 = H; R2 = H
2: R1 = C2H5; R2 = H
3: R1 = C2H5; R2 = CN
4: R1 = H; R2 = H; R3 = N(CH3)2; 30.0%
5: R1 = H; R2 = H; R3 = N(CH2)2(CH2)2NCH3; 25.0%
6: R1 = C2H5; R2 = H; R3 = N(CH3)2; 25.0%
7: R1 = C2H5; R2 = H; R3 = N(CH2)2(CH2)2NCH3; 57.0%
8: R1 = C2H5; R2 = CN; R3 = N(CH3)2; 35.0%
CH2OH
O
CHO
O
N
N
c
d
N
NH
R3
6, 7
NH
R3
N
11, 12
9, 10
9, 11: R3 = N(CH3)2; 87.0%, 52.0%
10, 12: R3 = N(CH2)2(CH2)2NCH3; 92.2%, 86.5%
Reagents and conditions:
a) DMF, SCNCO2C2H5, r.t., 1 h; b) dimethylamine or methylpiperazine, HgCl2, r.t.,
12 h, refluxing 2 h; c) POCl3, DMF, 30-40°C, 4 h; d) methanol, NaBH4, r.t., 3-4 h.
Scheme 2: synthetic routes to pyridoquinazolinone derivatives 16 and 23.
6

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A
N
O
O
O
O
O
O
OH
O
N
O
H
NH
N
N
N
H2N
O
N
c, d
a
b
NH
NH
HCl
N
HN
45.0%
85.8%
N
93.0%
13
14
15
16
Reagents and conditions:
a) Meldrum's acid, trimethylorthoformate, refluxing, 3+2 h; b) diphenylether, 250° C, 15 min;
c) chloroethylendiethylamine, NaH, anhydrous DMF, TEA, rt,12 h; d) EtOAc, HCl dry gas;
B
O
O
O
NO2
O
O
O
O
NO2
O
NO2
OH
NO2
O
NO2
a
b
c
O
N
H
N
H
H2N
N
H
H2N
O
85.8%
93.0%
77.0%
O
O
18
O
O
17
37%
19a 63%
19b
O
d
N
N
O
O
O
O
O
N
H
N
O
NH2
OH
NO2
OH
f
NO2
e
g, h
46.8%
NH
N
+
2HCl
23
O
N
N
N
N
H
13.2%
94.0%
H
H
O
O
O
19b
22
21
20
N
73.0%
Reagents and conditions:
a) HCl dry gas, EtOH abs, refluxing, 18 h, 89%; b) Meldrum's acid, trimethylorthoformate, refluxing, 2 + 18 h;
c) diphenylether, 250° C, 15 min; d) methanol, NaOH 20%, rt, 24 h; e) C/Pd 10%, H2, rt, 1 h; f) trimetilorthoformate,
CH3COONH4, MW, 250W, 5 min; g) choroethylendiethylamine, NaH 60%, anhydrous DMF, r.t., 12 h; h) ethyl acetate, HCl dry gas.
2.2. Biological activity
2.2.1 Antiproliferative effect against human tumor cell lines. The antiproliferative activity of the
new pyrrolo- and pyrido-quinazolinone derivatives was evaluated by means of an in vitro assay on
HeLa (human cervix adenocarcinoma), H460 (large cell lung carcinoma) and MSTO-211H (human
biphasic mesothelioma) cell lines. The results were expressed as GI50 values, which is concentration
(µM) of derivative able to induce 50% of growth inhibition with respect to the control culture. The
ellipticine was taken as reference compound.
As regards the pyrroloquinazolinones 4-12, the most active derivatives appear to be 11 and 12, both
characterized by a hydroxymethyl side chain in position 3 of the pyrrole ring. In detail, while 4-10
do not affect cell growth up to 50 µM concentration, for 11 and 12 detectable GI50 values, ranging
from 16.0 to 49.0 µM, are found.
Inside the pyridoquinazolinone series (compounds 15, 16, 22 and 23), the derivative 23 shows a
notable antiproliferative activity, with low- (2.9 ± 0.9 µM on HeLa) or sub-micromolar (0.38 ± 0.04
µM and 0.42 ± 0.06 µM on H460 and MSTO-211H, respectively) GI50 values. Interestingly, 23
shows cytotoxicity to MSTO-211H cells, higher with respect to that exerted by the reference drug,
ellipticine. For this latter drug indeed, a GI50 value of 0.77 ± 0.14 µM is obtained. Otherwise, all
other synthesised pyrido-quinazolinones do not exert any significant antiproliferative effect, having
7

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GI50 higher than 50 µM in all cell lines taken into account. In particular, by comparing the
unsubstituted pyridoquinazolinone 22 with 23, this latter characterised by two diethylaminoalkyl
side chains, it could be affirmed that they are essential for the occurrence of an antiproliferative
effect, as already demonstrated for some benzothiopyranoindole derivatives.19
2.2.2 Determination of apoptosis. To evaluate cell death mechanism, flow cytometry analysis was
performed with the most sensitive H460 cells, by using Annexin V-FITC and DNA-specific dye
propidium iodide labelling. The obtained results are shown in Figure 3A as dot plots, and in Figure
3B the corresponding percentage are depicted as histograms. By increasing the concentration of 23
from 0 to 40 µM, a decrease in the percentage of viable cells from about 90% to about 75% takes
place. This decrease is paralleled by the enhancement of the apoptotic cells, in both early and late
apoptosis, that becomes about 20% at the highest concentration. Otherwise, at all considered
concentrations, the percentage of cells undergoing a necrosis process is practically negligible and
constant (less than 6%).
A
B
100
80
viable
60
40
20
0
apoptotic
necrotic
0
10
40
compound 23 (µM)
Figure 3. Flow cytometry analysis of cell death induced by 23 (A) Dot plots of the flow cytometry
analysis performed on H460 cells labelled with Annexin V-FITC and propidium iodide after 18 h of
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treatment with different concentrations of 23. (B) The histograms show the percentage of viable
(Q3), early and late apoptotic (Q4+Q2) and necrotic (Q1) cells. Values are the mean ± SD of four
independent experiments.
2.2.3 Interaction with DNA. The presence of the planar pyridoquinazolinone chromophore
suggested that the antiproliferative effect exerted by 23 could result from the ability to form an
intercalative complex with DNA. Moreover, according to previous results,19 the presence of
diethylaminoalkyl side chains, thanks to the basic nitrogen, protonated at physiological pH, could
allow an easier interaction between the nucleic acid and the compound.
To pursue this question, flow linear dichroism (LD) experiments were performed in the presence of
salmon testes DNA. Figure 4 shows the UV-vis spectrum of derivative 23 (A) and LD spectra of a
DNA solution in the presence of 23 at different [drug]/[DNA] ratios (B).
The LD spectrum of the macromolecule shows the typical negative signal at 260 nm, due to the
absorption of nucleotide base pairs, in accordance with previous data.8 The addition of the test
compound induces the occurrence of a dose-dependent negative signal at higher wavelengths (about
320-420 nm) at which only the pyridoquinazolinone chromophore can absorb. Because the small
pyridoquinazolinone molecule cannot itself become oriented in the flow field, the presence of a
signal at 320-420 nm suggest that 23 is able to form a complex with DNA. The negative sign of this
signal indicates an orientation of the chromophore parallel to the plane of DNA base pairs, thus it
can be concluded that the complexation occurs through an intercalative mode of binding.
9

1.0
0.8
0.6
0.4
0.2
0.0
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250
300
350
400
450
500
550
0.0
-0.1
-0.2
-0.3
-0.4
-0.5
-0.6
R = 0.00
R = 0.04
R = 0.08
0.00
-0.01
-0.02
-0.03
-0.04
-0.05
-0.06
-0.07
250
300
350
400
450
500
550
wavelength (nm)
250
300
350
400
450
500
550
wavelength (nm)
Figure 4. Absorbance (A) and linear dichroism (LD) spectra of compound 23 at different
[drug]/[DNA] ratios (R) = 0, 0.04 and 0.08. [DNA]=1.9x10-3 M.
2.2.4 Effect on topoisomerases. A number of studies highlighted topoisomerases as the biological
targets of many intercalative anticancer agents.14,20,21 In particular, several clinically used anticancer
drugs, designed as topoisomerase poisons, promote the stabilization of the enzyme-mediated DNA
cleavage, while some agents, generally referred as catalytic inhibitors, interfere with a step of the
catalytic cycle.
In this connection, first of all we evaluated the effect of 23 on the catalytic activity of both
topoisomerase I (Fig. 5A) and topoisomerase II (Figure 5B). The incubation of supercoiled DNA
(lane DNA) with topoisomerase I (lane topo I) induces the relaxation of supercoiled DNA as
demonstrated by the appearance of a series of topoisomers. In the presence of increasing
concentrations of the tested pyridoquinazolinone, from 0.5 to 2.5 µM, a concentration-dependent
disappearance of the bands corresponding to the relaxed forms occurs, indicating an inhibition of
the relaxation activity of the enzyme. A similar behaviour can be observed when supercoiled DNA
is incubated in the presence of topoisomerase II (Figure 5B). Indeed, in these experimental
conditions, the enzyme promotes the relaxation of supercoiled DNA (lane topo II), and the addition
of 23 causes a concentration-dependent decrease of topoisomers. It is noteworthy that in both cases,
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by increasing the concentration of 23, a decrease in the intensity of DNA bands can be observed,
and, moreover, at very high concentrations (10-20 µM) a decrease in electrophoretic mobility of the
band corresponding to supercoiled DNA occurs. These results suggest that the inhibition of
topoisomerase I and II relaxation activity could be due to DNA intercalation rather than through a
poisoning effect.
Figure 5. Effect of 23 on relaxation of supercoiled pBR322 DNA by human recombinant
topoisomerase I (A) and II (B).
Actually, derivative 23 does not promote DNA cleavage by topoisomerase I or topoisomerase II in
contrast to camptothecin and m-AMSA, respectively, taken as reference (Figure 6A and B). Indeed,
the intensity of the band corresponding to nicked DNA in the presence of topoisomerase I (Figure
6A) and linear DNA for topoisomerase II, (Figure 6B) is not amplified by the addition of 23.
Otherwise, pyridoquinazolinone derivative is able to induce a marked decrease in the
electrophoretic mobility of relaxed DNA that can be related to an effective intercalative ability.
11

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Figure 6. Effect of 23 on the stabilization of covalent DNA-topoisomerase I (A) and DNA-
topoisomerase II (B) complex.
3. Conclusions
Some novel pyrrolo[3,2-f]quinazolinones (compounds 4-12) and pyridoquinazolinones (compounds
15, 16, 22 and 23) were synthesized following different multi-step pathways. Intermediates and
final compounds were fully characterized by instrumental techniques, in particular the exact
structure of the angular pyridoquinazolinone 16 was achieved by HRMS and 1D and 2D NMR
experiments. The latter allowed observation of the HMBC and NOE correlations for compound 16
and by this way to confirm the unexpected structure shown in figure 2. Worthy to note is that
compound 23 was effortlessly obtained thanks to the selective hydrolytic reaction of the
intermediate quinazolinone 19a in mixture with 19b, giving the corresponding easy isolable acid
20.
The investigation on the antiproliferative effect highlighted the ability of the linear pyrido[2,3-
g]quinazolinone 23 to induce a significant growth inhibition on all tested cell lines, with GI50 values
in the low- or sub-micromolar range. Linear flow dichroism experiments demonstrated the capacity
12

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of the planar chromophore to insert between DNA base pairs. The investigation of the effect of 23
on topoisomerase I and II catalytic activity showed a notable inhibition of the relaxation ability of
supercoiled DNA mediated by the two nuclear enzymes. These results suggest that the effective
intercalative complexation between 23 and DNA could perturb the macromolecule conformation,
thus interfering with topoisomerase catalytic activity.
4. Material and methods
4.1 General experimental conditions
Melting points were determined on a Buchi M-560 capillary melting point apparatus and are
uncorrected. Infrared spectra were recorded on a PerkinElmer 1760 FTIR spectrometer with
potassium bromide pressed disks and on a Varian ATR FTIR; all values are expressed in cm−1.
1
UV−Vis spectra were recorded on a Thermo Helyos α spectrometer. H NMR spectra were
determined on Bruker 300 and 400 MHz spectrometers, with the solvents indicated; chemical shifts
are reported in δ (ppm) downfield from tetramethylsilane as internal reference. Coupling constants
are given in Hertz. Integrals were satisfactorily in line with those expected on the basis of the
compound structure. Elemental analyses were performed in the Microanalytical Laboratory,
Department of Pharmaceutical Sciences, University of Padova, on a PerkinElmer C, H, N elemental
analyzer model 240B, and analyses indicated by the symbols of the elements were within ±0.4% of
the theoretical values. Analytical data are presented in detail for each final compound in the
Supplementary Data. Mass spectra were obtained on a Mat 112 Varian Mat Bremen (70 eV) mass
spectrometer and on an Applied Biosystems Mariner System 5220 LC/MS (nozzle potential 140
eV). Column flash chromatography was performed on Merck silica gel (250−400 mesh ASTM);
chemical reactions were monitored by analytical thin-layer chromatography (TLC) on Merck silica
gel 60 F-254 glass plates. Solutions were concentrated on a rotary evaporator under reduced
pressure. Starting materials were purchased from Sigma-Aldrich and Alfa Aesar, and solvents were
purchased from Carlo Erba, Fluka and Lab-Scan. DMSO was obtained anhydrous by distillation
under vacuum and stored on molecular sieves.
The purity of new tested compounds was checked by HPLC on a VARIAN ProStar model 210, with
detector DAD VARIAN ProStar 335. The analysis was performed with a flow of 1 mL/min, using a
C-18 column (250 mm × 4.6 mm), a particle size of 5 ꢀm, and a loop of 10 ꢀL. The detector was set
at 300 nm. The mobile phase consisted of phase A (Milli-Q H2O, 18.0 Mꢁ, TFA 0.05%) and phase
B (95% MeCN, 5% phase A). The gradient elution was performed as reported: 0 min, % B = 10;
0−20 min, % B = 90; 25 min, % B = 90; 26 min, % B = 10; 31 min, % B = 10.
13

ACCEPTED MANUSCRIPT
4.1.1 General procedure for the synthesis of pyrroloquinazolinones (4−8).
In a 100 mL flask, the appropriate amino-indole derivative 1-316 (0.30 g, 1.87 mmol) dissolved in
DMF (30 mL) was added with ethoxycarbonylisothiocyanate (0.21 mL, 1.87 mmol) and the mixture
was stirred at RT for 1 h. The progress of the reaction was checked by TLC (EtOAc/n-Hex, 8:2)
until disappearance of the starting material and formation of the first intermediate. At the end, the
mixture was cooled at 0 °C and added to dimethylamine (0.28 mL, 5.61 mmol) and HgCl2 (0.5 g,
1.87 mmol) and was left to stir at RT overnight. At the end, after that the formation of the second
intermediate was completed (TLC, EtOAc/n-Hex, 8:2), the mixture was heated under reflux (160
°C) for about 2 hours until the previously mentioned intermediate disappeared with the concomitant
formation of a new fluorescent spot corresponding to the final compound (TLC, CHCl3/MeOH, 9:1)
was evident. The reaction took about 40 hours. Then the mixture was cooled, filtered through a
celite pad. The filtrate was evaporated under vacuum to dryness and the obtained solid was
recrystallized from acetonitrile.
4.1.1.1 3-(Dimethylamino)-2H-pyrrolo[3,2-f]quinazolin-1(7H)-one (4)
Grey powder. Yield: 30%; mp: 290 °C (decomposition); [Found: C, 62.9; H, 5.28; N, 24.45.
C12H12N4O requires C, 63.2; H, 5.30; N, 24.6%]; Rf 0.29 (CHCl3/MeOH, 9:1); νmax (ATR, ZnSe)
3400-2800 (br), 1691, 1470, 1390, 1255, 1105 cm-1; δH (400 MHz, DMSO-d6) 3.29 (s, 6H,
N(CH3)2), 7.16 (dd, 1H, Ј 2.7 and 2.1 Hz, H-9), 7.62 (d, 1H, Ј 2.7 Hz, H-8), 7.64 (d, 1H, Ј 8.7 Hz,
H-5), 7.92 (d, 1H, Ј 8.7 Hz, H-6), 11.86 (s, 1H, NH), 11.90 ppm (bs, 1H, CONH); δC (101 MHz,
DMSO-d6) 39.72 (N(CH3)2), 102.69 (C-9), 105.84 (C-9b), 111.21 (C-9a), 120.58 (C-5), 123.86 (C-
6), 129.23 (C-6a), 133.05 (C-8), 149.78 (C-4a), 150.15 (C-3), 161.50 ppm (C-1); HRMS (ESI-MS,
140 eV): m/z [M+H+] C12H13N4O+ requires 229.1084; found, 229.1078; RP-C18 HPLC: tR 11.7 min,
97.5%.
4.1.1.2 3-(4-Methylpiperazin-1-yl)-2H-pyrrolo[3,2-f]quinazolin-1(7H)-one (5)
Grey solid. Yield: 25%; mp: ≥290 °C (decomposition); [Found: C, 63.6; H, 6.05; N, 24.70.
C15H17N5O requires C, 63.40; H, 6.03; N, 24.65%]; Rf 0.22 (CHCl3/MeOH, 9:1); νmax (ATR, ZnSe)
3410-2850 (br), 1690, 1475, 1392, 1260, 1110 cm-1; δH NMR (400 MHz, DMSO-d6) 2.78 (s, 3H,
NCH3), 3.10-2.98 (m, 4H, N(CH2)2(CH2)2NCH3), 4.30-4.17 (m, 4H, N(CH2)2(CH2)2NCH3), 7.11 (d,
1H, Ј 8.6 Hz, H-5), 7.15 (dd, 1H, J 3.07 and 2.01 Hz, H-9), 7.51 (dd, 1H, Ј 8.6 and 2.1 Hz, H-6),
7.81 (dd, 1H, Ј 3.02 and 0.7 Hz, H-8), 11.47 (s, 1H, NH), 11.53 ppm (s, 1H, CONH); δC NMR (101
MHz, DMSO-d6) 42.73 (N(CH2)2(CH2)2NCH3), 42.89 (N(CH2)2(CH2)2NCH3), 52.09 (NCH3),
103.14 (C-9), 109.01 (C-9b), 119.56 (C-9a), 123.83 (C-5), 126.45 (C-6), 127.82 (C-8), 132.52 (C-
14

ACCEPTED MANUSCRIPT
6a), 146.07 (C-3), 149.10 (C-4a), 163.43 ppm (C-1); HRMS (ESI-MS, 140 eV): m/z [M+H+]
C15H18N5O+ requires 284.1506; found, 284.1510; RP-C18 HPLC: tR 14.7 min, 99.7%.
4.1.1.3 3-(Dimethylamino)-7-ethyl-2H-pyrrolo[3,2-f]quinazolin-1(7H)-one (6)
Beige solid. Yield: 25%; mp: 250 °C (decomposition); [Found: C, 65.40; H, 6.27; N, 21.80.
C14H16N4O requires C, 65.6; H, 6.29; N, 21.9%]; Rf 0.62 (CHCl3/MeOH, 9:1); νmax (ATR, ZnSe)
3450-3330 (br), 1690, 1475, 1392, 1260, 1110 cm-1; δH NMR (400 MHz, DMSO-d6) 1.36 (t, Ј 7.3
Hz, 3H, CH2CH3), 2.87 (s, 6H, N(CH3)2), 4.26 (q, Ј 7.3 Hz, 2H, CH2CH3), 7.12 (d, Ј 2.9 Hz, 1H, H-
9), 7.20 (d, Ј 8.8 Hz, 1H, H-5), 7.50 (d, Ј 2.9 Hz, 1H, H-8), 7.80 (d, Ј 8.8 Hz, 1H, H-6), 11.33 ppm
(s, 1H, CONH); δC NMR (101 MHz, DMSO-d6) 16.30 (CH2CH3), 38.71 (N(CH3)2), 40.90
(CH2CH3), 102.30 (C-9), 106.99 (C-9b), 114.71 (C-9a), 118.16 (C-6), 120.08 (C-5), 124.39 (C-8),
130.45 (C-6a), 145.24 (C-3), 148.88 (C-4a), 162.91 ppm (C-1); HRMS (ESI-MS, 140 eV): m/z
[M+H+] C14H17N4O+ requires 257.1397; found, 257.1395; RP-C18 HPLC: tR 16.45 min, 98.5%.
4.1.1.4 7-Ethyl-3-(4-methylpiperazin-1-yl)-2H-pyrrolo[3,2-f]quinazolin-1(7H)-one (7)
Brown solid. Yield: 57%; mp: 280 °C (decomposition); [Found: C, 65.40; H, 6.78; N, 22.43.
C17H21N5O requires C, 65.6; H, 6.80; N, 22.50%]; Rf 0.31 (CHCl3/MeOH, 9:1); νmax (ATR, ZnSe)
3450-3330 (br), 1688, 1474, 1387, 1254, 1109 cm-1; δH NMR (400 MHz, DMSO-d6) 1.37 (t, Ј 7.3
Hz, 3H, CH2CH3), 2.78 (s, 3H, NCH3), 3.10-2.86 (m, 4H, N(CH2)2(CH2)2NCH3), 4.42-4.30 (m, 4H,
N(CH2)2(CH2)2NCH3), 4.27 (q, Ј 7.3 Hz, 2H, CH2CH3), 7.11 (d, Ј 8.6 Hz, 1H, H-5), 7.14 (dd, Ј 3.0
Hz and J 0.74 Hz, 1H, H-9), 7.51 (d, Ј 2.9 Hz, 1H, H-8), 7.85 (d, Ј 9.2 Hz, 1H, H-6), 11.53 ppm (s,
1H, CONH); δC (101 MHz, DMSO-d6) 16.19 (CH2CH3), 40.82 (CH2CH3), 42.84
(N(CH2)2(CH2)2NCH3), 43.01 (N(CH2)2(CH2)2NCH3), 52.18 (NCH3), 102.33 (C-9), 108.47 (C-9b),
119.12 (C-9a), 124.49 (C-5), 125.83 (C-6), 129.43 (C-8), 131.91 (C-6a), 146.22 (3-C), 149.72 (4a-
C), 163.43 ppm (C-1); HRMS (ESI-MS, 140 eV): m/z [M+H+] C17H22N5O+ requires 312.1819;
found, 312.1802 and 156.5916; RP-C18 HPLC: tR 16.47 min, 97.4%.
4.1.1.5
3-(Dimethylamino)-7-ethyl-2,7-dihydro-1-oxo-1H-pyrrolo[3,2-f]quinazoline-9-
carbonitrile (8)
Slight pink solid. Yield 35%; mp: 260-264 °C; [Found: C, 63.9; H, 5.36; N, 24.85. C15H15N5O
requires C, 64; H, 5.37; N, 24.90%]; Rf 0.46 (CHCl3/MeOH, 9:1); νmax (ATR, ZnSe) 3460-3320
(br), 2240-2215, 1692, 1463, 1374, 1250, 1110 cm-1; δH NMR (400 MHz, DMSO-d6) 1.40 (t, Ј 7.3
Hz, 3H, CH2CH3), 3.33 (s, 6H, N(CH3)2), 4.31 (q, Ј 7.3 Hz, 2H, CH2CH3), 7.21 (d, Ј 8.9 Hz, 1H, H-
5), 7.90 (d, Ј 8.9 Hz, 1H, H-6), 8.32 (s, 1H, H-8), 11.44 ppm (s, 1H, CONH); δC NMR (101 MHz,
DMSO-d6) 15.66 (CH2CH3), 37.71 (N(CH3)2), 41.63 (CH2CH3), 86.31 (C-9), 110.01 (CN), 117.62
15

ACCEPTED MANUSCRIPT
(C-9b), 118.59 (C-9a), 120.35 (C-6), 121.90 (C-5), 131.10 (C-6a), 138.61 (C-8), 149.18 (C-4a),
151.28 (C-3), 162.82 ppm (C-1); HRMS (ESI-MS, 140 eV): m/z [M+H+] C15H16N5O+ requires
282.1349; found, 282.1342; RP-C18 HPLC: tR 13.95 min, 96.5%.
4.1.1.6
3-(Dimethylamino)-7-ethyl-2,7-dihydro-1-oxo-1H-pyrrolo[3,2-f]quinazoline-9-
carbaldehyde (9)
As a common procedure, in a 2-neck 100 mL flask, POCl3 (1.5 mL) was introduced slowly to DMF
(3 mL), keeping the temperature around 10˚C by means of a water/ice bath. After about 15 min, a
mixture of pyrroloquinazolinone 6 (0.30 g, 1.17 mmol) and DMF (2 mL) was added portion wise,
maintaining the temperature below 10 °C. The reaction mixture was then heated at 50 °C. for about
3 h while the progress was checked by TLC (CHCl3/MeOH, 9:1). At the end of the reaction the
mixture was cooled to RT and then added with water and ice before making it alkaline with 10%
NaOH. The formed final suspension was filtered and the filtrate was extracted with EtOAc. The
combined extracts were evaporated to dryness under vacuum giving a sandy coloured solid. Yield
87%; mp: 260 °C (decomposition); [Found: C, 63.20; H, 5.66; N, 19.67. C15H16N4O2 requires C,
63.4; H, 5.67; N, 19.7%]; Rf 0.53 (CHCl3/MeOH, 9:1); νmax (ATR, ZnSe) 3470-3330 (br), 2830-
2810, 2750-2720, 1765-1645, 1470, 1390, 1235, 1115 cm-1; δH NMR (400 MHz, DMSO-d6) 1.41 (t,
Ј 7.3 Hz, 3H, CH2CH3), 3.12 (s, 6H, N(CH3)2), 4.34 (q, Ј 7.3 Hz, 2H, CH2CH3), 7.24 (d, Ј 8.8 Hz,
1H, H-6), 7.92 (d, Ј 8.9 Hz, 1H, H-5), 8.19 (s, 1H, H-8), 11.05 (s, 1H, CHO), 11.15 ppm (s, 1H,
CONH); δC NMR (101 MHz, DMSO-d6) 15.74 (CH2CH3), 37.58 (N(CH3)2), 41.71 (CH2CH3),
102.06 (C-9), 109.44 (C-9b), 118.90 (C-6), 120.04 (C-9a), 121.48 (C-5), 132.49 (C-6a), 133.89 (C-
8), 147.23 (C-3), 149.01 (C-4a), 162.99 (C-1), 191.79 ppm (CHO); HRMS (ESI-MS, 140 eV): m/z
+
[M+H+] C15H17N4O2 requires 285.1346; found, 285.1341; RP-C18 HPLC: tR 12.38 min, 97.4%.
4.1.1.7
7-Ethyl-3-(4-methylpiperazin-1-yl)-2H-pyrrolo[3,2-f]quinazolin-1(7H)-one-9-
carbaldehyde (10)
Compound 10 was obtained as reported for compound 9, by reacting 1 g of compound 7 and
obtaining 0.563 g of a pale yellow powdery solid. Yield: 52%; mp: 290 °C (decomposition);
[Found: C, 63.6; H, 6.23; N, 20.6. C18H21N5O2 requires C, 63.7; H, 6.24; N, 20.6%]; Rf 0.24
(CHCl3/MeOH, 9:1; νmax (ATR, ZnSe) 3465-3320 (br), 2828, 2735, 1724, 1481, 1388, 1223, 1112
cm-1; δH NMR (400 MHz, DMSO-d6) 1.40 (t, J 7.3 Hz, 3H, CH2CH3), 2.20 (s, 3H, NCH3), 2.55-
2.39 (m, 4H, N(CH2)2(CH2)2NCH3), 3.62-3.46 (m, 4H, N(CH2)2(CH2)2NCH3), 4.35 (q, Ј 7.3 Hz,
2H, CH2CH3), 7.23 (d, Ј 9.2 Hz, 1H, H-5), 7.96 (d, Ј 8.9 Hz, 1H, H-6), 8.23 (s, 1H, H-8), 11.15 (s,
1H, CHO), 11.49 ppm (s, 1H, CONH); δC NMR (101 MHz, DMSO-d6) 15.75 (CH2CH3), 41.68
(CH2CH3), 42.92 (N(CH2)2(CH2)2NCH3), 44.12 (N(CH2)2(CH2)2NCH3), 53.34 (NCH3), 110.40 (C-
16

ACCEPTED MANUSCRIPT
9), 111.01 (C-9b), 115.93 (C-9a), 118.89 (C-6), 121.51 (C-5), 133.03 (C-6a), 134.38 (C-8), 145.84
(C-4a), 149.65 (C-3), 161.19 (C-1), 191.51 ppm (CHO); HRMS (ESI-MS, 140 eV): m/z [M+H+]
+
C18H22N5O2 requires 340.1768; found, 340.1721 and 170.5959; RP-C18 HPLC: tR 15.50 min,
98.0%.
4.1.2 General Procedure for the Synthesis of 9-Methyl-Alcohol-Substituted Pyrroloquinazoline
Derivatives 11, 12. In a 250 mL flask, on the basis of the starting material, a double molar amount
of NaBH4 was suspended in MeOH (5 mL). The ethylpyrroloquinazoline 9-carbaldehyde derivative
9, 10 (3−5 mmol) dissolved in of MeOH (10 mL) was slowly added under inert atmosphere (N2)
and at room temperature. The course of the reaction was monitored by TLC, in all cases noting the
appearance of a spot with a Rf lower than that of the starting 9-carbaldehyde compound. At the end
of the reaction lasting about 3 h, the excess of NaBH4 was quenched with a saturated aqueous
solution of NH4Cl and the solid was separated by filtration and washed with EtOAc some times.
The total filtrate was dried with anhydrous Na2SO4 and evaporated under vacuum, yielding the
crude reaction product which was purified by liquid chromatography.
4.1.2.1 3-(Dimethylamino)-7-ethyl-9-(hydroxymethyl)-2H-pyrrolo[3,2-f]quinazolin-1(7H)-one
(11)
The general procedure was followed by adding dropwise a solution of the 9-carbaldehyde derivative
9 (2.445 g, 3.77 mmol) in MeOH (10 mL) to a suspension of NaBH4 (0.539 g,7.54 mmol) in MeOH
(5 mL). At the end of the reaction (2 h), by workup of the reaction mixture, a crude yellow-orange
solid was obtained (1.024 g). After purification in a chromatographic column (eluent CHCl3/MeOH,
9:1) a yellow solid corresponding to compound 11 was obtained (0.953 g). Yield: 92.2%; mp: 244
°C (decomposition); [Found: C, 62.8; H, 6.33; N, 19.53. C15H18N4O2 requires C, 62.9; H, 6.34; N,
19.6%]; Rf 0.84 (CHCl3/MeOH, 9:1); νmax (ATR, ZnSe) 3660-3250 (br), 1645, 1430, 1388, 1075,
1050 cm-1; δH NMR (400 MHz, DMSO-d6) 1.43 (t, Ј 7.25 Hz, 3H, CH2CH3), 3.24 (s, 6H, N(CH3)2),
4.38 (q, Ј 7.26 Hz, 2H, CH2CH3), 5.10 ppm (d, J 5.31 Hz, 2H, -CH2OH); 5.59 (t, J 5.31 Hz, 1H, -
OH), 7.25 (d, Ј 9.16 Hz, 1H, H-6), 7.88 (d, Ј 9.04 Hz, 1H, H-5), 7.99 (s, 1H, H-8), 11.39 ppm (s,
1H, CONH); δC NMR (101 MHz, DMSO-d6) 16.08 (CH2CH3), 38.34 (N(CH3)2), 42.04 (CH2CH3),
60.98 (CH2OH), 104.44 (C-9), 110.12 (C-9b), 119.45 (C-6), 121.73 (C-9a), 122.83 (C-5), 133.94
(C-6a), 140.12 (C-8), 148.01 (C-3), 149.23 (C-4a), 163.44 ppm (C-1); HRMS (ESI-MS, 140 eV):
+
m/z [M+H+] C15H19N4O2 requires 287.1503; found, 287.1513; RP-C18 HPLC: tR 10.52 min,
98.5%.
17

ACCEPTED MANUSCRIPT
4.1.2.2
3-(4-methylpiperazin-1-yl)-7-ethyl-9-(hydroxymethyl)-2H-pyrrolo[3,2-f]quinazolin-
1(7H)-one (12)
Following the general procedure, to a suspension of NaBH4 (0.275 g, 7.23 mmol) in MeOH (5 mL)
was slowly added the 9-carbaldehyde derivative 10 (1.253 g, 3.62 mmol) in MeOH (10 mL). At the
end of the reaction (2 h), on TLC a yellow spot having a Rf lower than that of the starting
compound. By workup of the reaction mixture a crude yellow-beige solid residue was obtained
(1.050 g), which was purified on chromatographic column (CHCl3/MeOH, 9:1), yielding compound
12 as a beige solid residue (0.950 g). Yield: 86.5%; mp: 275 °C (decomposition); [Found: C, 63.2;
H, 6.78; N, 20.47. C18H23N5O2 requires C, 63.3; H, 6.79; N, 20.5%]; Rf 0.89 (CHCl3/MeOH, 9:1);
νmax (ATR, ZnSe) 3650-3200, 1680, 1415, 1400, 1112, 987 cm-1; δH NMR (400 MHz, DMSO-d6)
1.45 (t, J 7.47 Hz, 3H, CH2CH3), 2.24 (s, 3H, NCH3), 2.65-2.44 (m, 4H, N(CH2)2(CH2)2NCH3),
3.42-3.28 (m, 4H, N(CH2)2(CH2)2NCH3), 4.42 (q, Ј 7.42 Hz, 2H, CH2CH3), 5.09 (d, J 5.51 Hz, 2H,
-CH2OH), 5.57 (t, J 5.51 Hz, 1H, -OH), 7.34 (d, Ј 9.07 Hz, 1H, H-5), 7.92 (d, Ј 9.03 Hz 1H, H-6),
8.04 (s, 1H, H-8), 11.54 ppm (s, 1H, CONH); δC NMR (101 MHz, DMSO-d6) 16.01 (CH2CH3),
42.04 (CH2CH3), 42.88 (N(CH2)2(CH2)2NCH3), 45.02 (N(CH2)2(CH2)2NCH3), 54.11 (NCH3), 60.45
(CH2OH) 111.43 (C-9), 115.23 (C-9b), 120.04 (C-9a), 122.23 (C-6), 125.43 (C-5), 132.98 (C-6a),
134.90 (C-8), 145.23 (C-4a), 150.03 (C-3), 163.87 ppm (C-1); HRMS (ESI-MS, 140 eV): m/z
+
[M+H+] C18H24N5O2 requires 342.1925; found, 342.1921 and 171.5994; RP-C18 HPLC: tR 10.99
min, 96.8%.
4.1.3 6,6-Dimethyl-3-{[(4-oxo-3,4-dihydroquinazolin-6-yl)amino]methylidene}oxane-2,4-dione
(14)
A solution of Meldrum’s acid (1.427 g, 9.91 mmol) in an excess of methyl orthoformate (15 mL)
was heated to reflux for two hours under nitrogen atmosphere. The solution was then cooled to
room temperature and the starting 6-aminoquinazolinone (13, 1.228 g, 7.62 mmol) was added
portion wise. The mixture thus obtained was maintained at reflux for 6 hours, checking the progress
of the reaction by TLC (eluent CHCl3/MeOH, 8:2). The reaction mixture was partly evaporated in a
rotavapor, then taken up with water and subsequently extracted with EtOAc. The combined extracts
were dried with anhydrous sodium sulfate, filtered and then evaporated to dryness. The crude
reaction residue was purified by silica gel column chromatography (eluent CHCl3/MeOH, 8:2) and
additionally re-crystallized from MeOH yielding 2.063 g (6.54 mmol) of a yellow powdery solid.
Yield: 85.8%; mp: 247 °C; Rf 0.52 (CHCl3/MeOH, 8:2); νmax (ATR, ZnSe) 3421-3108, 1750, 1740,
1675, 1415, 1400, 1112, 987 cm-1;δH NMR (400 MHz, DMSO-d6) 1.69 (s, 6H, C(CH3)2), 7.72 (d, J
8.83, 1H, H-8), 8.04 (dd, J 8.83 Hz and J 2.70 Hz, 1H, H-7), 8.09 (d, J 3.26 Hz, 1H, H-2), 8.19 (d, J
2.63 Hz, 1H, H-5), 8.62 (d, J 14.74 Hz, 1H, =CHNH), 11.40 (d, J 14.46 Hz, 1H, =CHNH), 12.34
18

ACCEPTED MANUSCRIPT
ppm (bs, 1H, CONH); δC NMR (101 MHz, DMSO-d6) 26.86 (C(CH3)2), 87.72 (=CHNH), 104.54
(C(CH3)2), 115.54 (C-5), 123.63 (C-4a), 126.39 (C-7), 129.05 (C-8), 145.83 (C-2), 147.10 (C-8a),
159.92 (C-4), 163.15 (C=O), 163.96 (C=O), 167.65 (C-4), 197.58 ppm (C=CH); HRMS (ESI-MS,
+
140 eV): m/z [M+H+] C15H14N3O5 requires 316.0933; found, 316.0930.
4.1.4 10-Hydroxy-1H,2H,7H,10H-pyrido[3,2-f]quinazolin-1-one (15)
Boiling Ph2O (~10 mL) was quickly added to a flask containing the quinazolinone derivative 14
(2.063 g, 6.54 mmol). The reaction mixture was maintained at reflux for 15min and then cooled to
room temperature. Subsequently, Et2O (20mL) was added to the mixture and the formation of a
precipitate was observed which was allowed to deposit at room temperature overnight. It was then
collected, washed several times with Et2O and dried in a vacuum oven, resulting in 1.302 g of a
brownish powdery solid. Yield: 93.0%; mp > 330 °C (decomposition); [Found: C, 61.8; H, 3.30; N,
19.67. C11H7N3O2 requires C, 62; H, 3.31; N, 19.7%]; Rf 0.47 (blue fluorescent spot, CHCl3/MeOH,
8:2); νmax (ATR, ZnSe) 3455-3100, 1702, 1680, 1447, 1394, 1117, 999 cm-1; HRMS (ESI-MS, 140
eV): m/z [M+H+] C11H8N3O2 requires 214.0617; found, 214.0625.
+
4.1.5
2-[2-(Diethylamino)ethyl]-10-hydroxy-1H,2H,7H,10H-pyrido[3,2-f]quinazolin-1-one
hydrochloride (16)
In a dried 100 mL flask, excess NaH (60 % dispersion in mineral oil, 0.680 g, 2.82 mmol) was
added and washed three times with toluene. Via a funnel, compound 15 (0.200 g, 0.94 mmol)
suspended in anhydrous DMF (5 mL) was added dropwise, and the mixture changed from light
brown to deep brown with concomitant development of hydrogen fumes. The reaction mixture was
left stirring for 1 h at room temperature. Then, the halo-compound 2-chloro-N,N-
diethylethanamine(threefold molar excess over starting compound, 0.485 g, 2.82 mmol) and
triethylamine (0.39 mL, 2.82 mmol, 0.728 g/mL) both dissolved in anhydrous DMF (1 mL) were
added, and the solution was left stirring at room temperature for 5 h, monitoring reaction progress
by TLC (CHCl3/MeOH, 8:2). At the end, H2O (25 mL) was added to the reaction mixture, which
was then extracted with EtOAc. The combined extracts were washed several times with H2O, brine,
dried over anhydrous MgSO4, filtered and finally evaporated to dryness by rotary evaporation,
obtaining a yellow semisolid residue which was then triturated with cyclohexane. Finally, the
powdery solid was dissolved in an EtOAc/absolute EtOH, 2:1 mixture, and dry HCl gas was
bubbled into the solution until a yellowish precipitate formed. The mixture was left at 0−4 °C
overnight, and the resulting precipitate was collected and dried under vacuum to yield 0.125 g of a
powdery gold solid. Yield: 43%; mp: 268 °C; [Found: C, 58.4; H, 6.06; N, 16.02. C17H20N4O2
requires C, 58.5; H, 6.07; N, 16.1%]; Rf 0.54 (blue fluorescent spot, CHCl3/MeOH, 8:2); νmax (ATR,
19

ACCEPTED MANUSCRIPT
ZnSe) 3455-3100, 2700-2250 (br), 1704, 1688, 1450, 1390, 1116, 998 cm-1; δH NMR (400 MHz,
DMSO-d6) 0.81 (t, J 7.08 Hz, 6H, CH2CH3), 2.49 (q, J 7.24 Hz, 4H, CH2CH3), 2.75 (t, J 5.87 Hz,
2H, NCH2CH2N); 4.23 (t, J 5.68 Hz, 2H, NCH2CH2N), 7.02 (d, J 4.50 Hz, 1H, H-9), 7.92 (d, J 9.20
Hz, 1H, H-5), 8.16 (bs, 1H, NHCl), 8.30 (d, J 9.01 Hz, 1H, H-6), 8.66 (d, J 4.50 Hz, 1H, H-8), 8.67
(s, 1H, H-3), 15.11 ppm (C(10)-OH); δC NMR (101 MHz, DMSO-d6) 12.44 (CH2CH3), 46.18
(NCH2CH2N), 47.10 (CH2CH3), 50.38 (NCH2CH2N), 111.37 (C-9), 116.15 (C-10b), 117.77 (C-
10a), 130.37 (C-5), 139.41 (C-6), 149.22 (C-3), 150.50 (C-4a), 151.57 (C-6a), 152.01 (C-8), 163.39
+
(C-10), 164.15 ppm (C-1); HRMS (ESI-MS, 140 eV): m/z [M+H+] C17H21N4O2 requires 313.1759;
found, 313.1710 and 157.0925; RP-C18 HPLC: tR 4.89 min, 96.2%.
4.1.6 Ethyl 5-amino-2-nitrobenzoate (17)
In a 250 mL round-bottomed flask, commercial 5-amino-2-nitrobenzoic acid (1.088 g, 5.97 mmol)
was dissolved in absolute EtOH (100 mL). The mixture was then saturated with dry HCl gas,
refluxed and stirred for 6 h. After 6 h all the starting reagent had disappeared, so EtOH was
removed under reduced pressure. The resulting residue was treated with sat’d NaHCO3 and
extracted with EtOAc (2×20 mL). The organic extracts were washed with water, brine, dried over
Na2SO4 and finally concentrated to give 1.129 g (5.37 mmol) of a pure brown powdery solid. Yield:
89%; Rf 0.75 (CHCl3/MeOH, 8:2); mp: 105 °C; δH NMR (400 MHz, DMSO-d6) 1.25 (t, J 7.06 Hz,
3H, CH2CH3), 4.28 (q, J 7.04 Hz, 2H, CH2CH3), 6.60 (d, J 2.46 Hz, 1H, H-5), 6.67 (dd, J 9.10 Hz
and J 2.48 Hz, 1H, H-4), 6.90 (bs, 2H, -NH2), 7.91 ppm (d, J 9.08, 1H, H-3); δC NMR (101 MHz,
DMSO-d6) 13.64 (CH2CH3), 61.44 (CH2CH3), 111.37 (C-4), 113.11 (C-5), 127.41 (C-3), 132.44
(C-2), 154.81 (C-5), 166.65 ppm (COO).
4.1.7 Ethyl 5-{[(2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-ylidene)methyl]amino}-2-nitrobenzoate
(18)
A solution of Meldrum’s acid (1.006 g, 6.98 mmol) in an excess of methyl orthoformate (15 mL)
was heated to reflux for two hours under nitrogen atmosphere. The solution was then cooled to
room temperature and the starting ethyl 5-amino-2-nitrobenzoate (17, 1.129 g, 5.37 mmol) was
added portion wise. The mixture thus obtained was maintained at reflux for 6 hours, checking the
progress of the reaction by TLC (eluent CHCl3/MeOH, 8:2). The reaction mixture was partly
evaporated in a rotavapor, then taken up with water and subsequently extracted with EtOAc. The
combined extracts were dried with anhydrous sodium sulfate, filtered and then evaporated to
dryness. The crude reaction residue was purified by silica gel column chromatography (eluent
CHCl3/MeOH, 9:1) and additionally re-crystallized from MeOH yielding 1.011 g (2.77 mmol) of a
crystalline light brown powdery solid. Yield: 52.0%; mp: 221 °C Rf 0.79 (CHCl3/MeOH, 9:1); νmax
20

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(ATR, ZnSe) 3108, 2987, 1748, 1731, 1648, 1490, 1315, 870, 706 cm-1; δH NMR (400 MHz,
DMSO-d6) 1.31 (t, J 7.09 Hz, 3H, CH2CH3), 1.70 (s, 6H, C(CH3)2, 4.35 (q, J 7.10, 2H, CH2CH3),
7.96 (dd, J 8.94 Hz and J 2.51 Hz, 1H, H-4), 8.09 (d, J 2.53 Hz, 1H, H-6), 8.17 (d, J 8.92 Hz, 1H,
H-3), 8.69 (d, J 14.34 Hz, 1H, C=CH), 11.39 ppm (d, J 14.36 Hz, 1H, NH); δC NMR (101 MHz,
DMSO-d6) 14.16 (CH2CH3), 27.03 (C(CH3)2), 62.74 (CH2CH3), 89.75 (C(CH3)2), 104.94 (C=CH),
120.02 (C-6), 122.12 (C-4), 126.56 (C-3), 129.70 (C-1), 143.75 (C-5), 143.85 (C-2), 153.94
(C=CH), 162.88 (CO), 163.57 (CO), 165.03 ppm (COO); HRMS (ESI-MS, 140 eV): m/z [M+H+]
+
C16H17N2O8 requires 365.0985; found, 365.0982.
4.1.8 Ethyl 6-nitro-4-oxo-1,4-dihydroquinoline-7-carboxylate (19a) and ethyl 6-nitro-4-oxo-
1,4-dihydroquinoline-5-carboxylate (19b)
Boiling Ph2O (~10 mL) was quickly added to a flask containing derivative 18 (0.505 g, 1.39 mmol).
The reaction mixture was maintained at reflux for 15 min and then cooled to room temperature.
Subsequently, Et2O (20mL) was added to the mixture and the formation of a precipitate was
observed which was allowed to deposit at room temperature overnight. It was then collected,
washed several times with Et2O and dried in a vacuum oven, resulting in 0.283 g of a brownish
powdery solid constituted by an irresolvable mixture of 19a and 19b. Yield: 77.0%; Rf 0.63 and
0.61 (CHCl3/MeOH, 8:2); mp: 134 °C; δH NMR (400 MHz, DMSO-d6) 1.30-1.16 (m, 6H,
CH2CH3), 4.36 (q, J 7.08 Hz, 4H, CH2CH3), 6.14 (d, J 7.55 Hz, 1H, H-3, 19b), 6.23 (d, J 7.57 Hz,
1H, H-3, 19a), 7.77 (d, 1H, J 9.27 Hz, H-7, 19b), 7.88 (s, 1H, H-8, 19a), 8.03 (d, J 7.46 Hz, 1H, H-
2, 19b), 8.10 (d, J 7.63 Hz, 1H, H-2, 19a), 8.40 (d, J 9.32 Hz, 1H, H-8, 19b), 8.65 (s, 1H, H-5, 19a),
12.34 ppm (bs, 2H, NH). Yield determined by NMR molar ratio evaluation: 63%, 19a; 37%, 19b;
+
HRMS (ESI-MS, 140 eV): m/z [M+H+] C12H11N2O5 requires 263.0668; found, 263.0660.
4.1.9 6-Nitro-4-oxo-1,4-dihydroquinoline-7-carboxylic acid (20)
In a 250 mL round-bottomed flask, the unresolvable mixture of isomers 19a and 19b (0.645 g, 2.46
mmol) was dissolved in MeOH and the resulting solution was cooled by mean of an ice bath.
Approximately 10 mL of a NaOH 20% solution were consequently added and the mixture was
allowed to stand at room temperature overnight. After 24 h only one of the TLC spot was
completely disappeared, MeOH was removed by mean of a rotary evaporator and the resulting
water remnants were extracted with EtOAc (3×20 mL). The combined organic fractions were dried
with anhydrous sodium sulfate, filtered and then evaporated to dryness, leading to a yellow
powdery product, corresponding to 19b (0.270 g, 1.02 mmol). Yield: 41.4%; Rf 0.61
(CHCl3/MeOH, 8:2); mp: 132.8; νmax (ATR, ZnSe) 3420-3075, 2988, 1698, 1650, 1488, 1112, 980
cm-1; °C; δH NMR (400 MHz, DMSO-d6) 1.32 (t, J 7.27 Hz, 3H, CH2CH3), 4.34 (q, J 7.24 Hz, 2H,
21

ACCEPTED MANUSCRIPT
CH2CH3), 6.14 (d, J 7.44 Hz, 1H, H-3), 7.75 (d, 1H, J 9.04 Hz, H-7), 8.06 (d, J 7.45 Hz, 1H, H-2),
8.42 (d, J 9.10 Hz, 1H, H-8), 12.21 ppm (bs, 1H, NH); δC NMR (101 MHz, DMSO-d6) 14.13
(CH2CH3), 61.18 (CH2CH3), 109.05 (C-3), 113.14 (C-5), 117.29 (C-7), 120.08 (C-5), 120.09 (C-8),
140.00 (C-2), 141.10 (C-8a), 148.37 (C-6), 164.50 (COOEt), 173.76 (C-4); HRMS (ESI-MS, 140
+
eV): m/z [M+H+] C12H11N2O5 requires 263.0668; found, 263.0664. As the alkaline water fraction
was made acidic (HCl 37%), a brown precipitate immediately formed. Collected by filtration on a
G4 gooch filter, the resulting gummy solid was washed several times with water and finally dried in
a vaccum oven overnight. 0.420 g (1.79 mmol) of a powdery deep brown solid compound 20 were
obtained. Yield: 73%; mp: 311 °C; Rf 0.10 (CHCl3/MeOH, 8:2); νmax (ATR, ZnSe) 3550-2650,
1780, 1698, 1650, 1437, 1340, 1250 cm-1; δH NMR (400 MHz, DMSO-d6) 6.23 (d, J 7.53 Hz, 1H,
H-3), 7.95 (s, 1H, H-8), 8.09 (d, 1H, J 7.50 Hz, H-2), 8.59 (s, 1H, H-5), 12.74 ppm (bs, 1H, NH); δC
NMR (101 MHz, DMSO-d6) 110.67 (C-3), 120.21 (C-8), 122.31 (C-5), 125.08 (C-4a), 131.17 (C-
8a), 141.18 (C-2), 141.92 (C-7), 142.16 (C-6), 174.62 (C-4), 175.57 ppm (COOH); HRMS (ESI-
+
MS, 140 eV): m/z [M+H+] C10H7N2O5 requires 235.0355; found, 235.0349.
4.1.10 6-Amino-4-oxo-1,4-dihydroquinoline-7-carboxylic acid (21)
Into a three-necked flask of 500 mL, previously dried in an oven, about 0.300 g of C/Pd 10% and
approximately 60 mL of EtOAcwere placed. After connecting the flask to an elastomer balloon
containing hydrogen gas, the mixture was stirred at room temperature for 1 h in order to saturate the
suspension of C/Pd with hydrogen. Next, derivative 20 (0.832 g, 3.17 mmol) in MeOH (250 mL)
was added dropwise to the suspension, and the mixture was stirred under hydrogen at atmospheric
pressure and heated by means of an oil bath at 50−60 °C for 15 h, monitoring the progress of the
reaction by TLC analysis (n-Butanole/H2O/AcOH, 3:1:1). At the end of the reaction, the mixture
was filtered by mean of a celite pad, and the solution was concentrated to dryness on a rotavapor to
give 0.609 g (2.98 mmol) of semisolid dark purple product. Yield: 94%; mp: 311 °C; Rf 0.58 (green
fluorescent spot, n-Butanole/H2O/AcOH, 3:1:1); νmax (ATR, ZnSe) 3550-2650, 1777, 1699, 1635,
1605, 1440, 1102, 987 cm-1; δH NMR (400 MHz, DMSO-d6) 5.37 (bs, 2H, NH2), 6.05 (d, J 6.99 Hz,
1H, H-3), 7.39 (s, 1H, H-8), 7.89 (d, J 6.93 Hz, 1H, H-2), 8.17 (s, 1H, H-8), 12.32 ppm (bs, 1H,
NH); δC NMR (101 MHz, DMSO-d6) 105.61 (C-3), 107.96 (C-4a), 116.25 (C-5), 122.18 (C-8),
130.01 (C-7), 139.07 (C-8a), 142.61 (C-2), 146.65 (C-6), 168.48 (COOH), 173.72 ppm (C-4);
+
HRMS (ESI-MS, 140 eV): m/z [M+H+] C10H9N2O3 requires 205.0613; found, 205.0609.
4.1.11 3H,4H,6H,9H-Pyrido[2,3-g]quinazoline-4,9-dione (22)
A mixture of 21 (0.200g, 0.98 mmol), trimethylorthoformate (2 mL) and ammonium acetate (0.075
g, 0.98 mmol) was placed in a tall beaker. The beaker was covered with a stemless funnel and then
22

ACCEPTED MANUSCRIPT
irradiated in a domestic microwave oven for 2 min with a power of 210 W. After a cooling time of
about 5 min, the beaker and contents were irradiated at 250 W for an addition 5 min. The resultant
residue was triturated with water and filtered off and finally purified by liquid column
chromatography (CHCl3/MeOH, 8:2). 0.028 g (0.13 mmol) of a pale brown solid were obtained.
Yield: 13.2%; mp: 177 °C; [Found: C, 61.8; H, 3.30; N, 19.66. C11H7N3O2 requires C, 62; H, 3.31;
N, 19.7%]; Rf 0.32 (blue fluorescent spot, CHCl3/MeOH, 8:2); νmax (ATR, ZnSe) 3400-2800 (br),
1745, 1691, 1488, 1382, 1263, 1112 cm-1; δH NMR (400 MHz, DMSO-d6) 6.07 (d, J 7.38 Hz, 1H,
H-8), 8.03 (d, J 7.44 Hz, 1H, H-7), 8.05 (s, 1H, H-2), 8.30 (s, 1H, H-10), 8.40 (s, 1H, H-5), 11.54
(bs, 1H, N(6)H), 12.40 ppm (bs, 1H, N(3)H); δC NMR (101 MHz, DMSO-d6) 108.55 (C-8), 116.87
(C-5), 124.18 (C-10), 126.31 (C-4a), 130.87 (C-9a), 138.76 (C-5a), 141.89 (C-7), 143.72 (C-10a),
+
145.18 (C-2), 161.45 (C-4), 177.56 ppm (C-9); HRMS (ESI-MS, 140 eV): m/z [M+H+] C11H8N3O2
requires 214.0616; found, 214.0617.
4.1.12 9-[2-(Diethylamino)ethoxy]-3-[2-(diethylamino)ethyl]-3H,4H-pyrido[2,3-g]quinazolin-4-
one dihydrochloride (23)
In a dried 100 mL flask, excess NaH (60 % dispersion in mineral oil, 0.040 g, 1.40 mmol) was
placed and washed three times with toluene. Via a funnel, compound 22 (0.100 g, 0.46 mmol)
suspended in anhydrous DMF (1 mL) was added dropwise, and the mixture changed from light
brown to deep brown with concomitant development of hydrogen fumes. The reaction mixture was
left stirring for 1 h at room temperature. Then, the halo-compound 2-chloro-N,N-
diethylethanamine(sixfold molar excess over starting compound, 0.484 g, 2.80 mmol) and
triethylamine (0.39 mL, 2.80 mmol, 0.728 g/mL) both dissolved in anhydrous DMF (0.5 mL) were
added, and the solution was left stirring at room temperature for 5 h, monitoring reaction progress
by TLC (CHCl3/MeOH, 8:2). At the end, H2O (10 mL) was added to the reaction mixture, which
was then extracted with EtOAc. The combined extracts were washed several times with H2O, brine,
dried over anhydrous MgSO4, filtered and finally evaporated to dryness by rotary evaporation,
obtaining a yellow semisolid residue which was then triturated with cyclohexane. Finally, the
powdery solid was dissolved in an EtOAc/absolute EtOH, 2:1 mixture, and dry HCl gas was
bubbled into the solution until a yellowish precipitate formed. The mixture was left at 0−4 °C
overnight, and the resulting precipitate was collected and dried under vacuum to yield 0.105 g (0.21
mmol) of a powdery yellow solid. Yield: 46.8%; mp: 243 °C; [Found: C, 56.9; H, 7.26; N, 14.43.
C23H34N5O2 requires C, 57; H, 7.28; N, 14.5%]; Rf 0.75 (blue fluorescent spot, CHCl3/MeOH, 8:2);
νmax (ATR, ZnSe) 3425-2867 (br), 2750-2534 (br), 1685, 1478, 1334, 1276, 1124 cm-1; δH NMR
(400 MHz, DMSO-d6) δ 0.85-0.98 (m, 12H, CH2CH3), 2.51-2.71 (m, 8H, CH2CH3), 2.86 (t, J 7.25
23

ACCEPTED MANUSCRIPT
Hz, 2H, OCH2CH2N), 2.91 (t, J 7.24 Hz, 2H, NCH2CH2N), 4.11 (t, J 7.25 Hz, 2H, NCH2CH2N),
4.30 (t, J 7.26 Hz, 2H, OCH2CH2N), 6.96 (d, J 5.55 Hz, 1H, H-8), 8.10 (s, 1H, H-10), 8.37 (s, 1H,
H-2), 8.42 (s, 1H, H-5), 8.72 ppm (d, J 5.50 Hz, 1H, H-7); δC NMR (101 MHz, DMSO-d6) δ 11.62-
11.74 (CH2CH3), 47.12 (NCH2CH2N(CH2CH3)2), 47.69 (OCH2CH2N(CH2CH3)2), 50.89
(NCH2CH2N(CH2CH3)2), 52.94 (NCH2CH2N(CH2CH3)2), 53.83 (OCH2CH2N(CH2CH3)2), 68.16
(OCH2CH2N(CH2CH3)2), 110.72 (C-8), 118.72 (C-5), 125.24 (C-10), 126.37 (C-4a), 128.87 (C-9a),
140.82 (C-5a), 144.82 (C-7), 146.98 (C-10a), 147.72 (C-2), 163.56 (C-9), 165.18 ppm (C-4);
HRMS (ESI-MS, 140 eV): m/2z [M+2H2+] C23H35N5O22+ requires, 206.6390; found, 206.6384; RP-
C18 HPLC: tR 8.96 min, 99.5%.
4.2 Inhibition Growth Assay
HeLa (human cervix adenocarcinoma cells) were grown in Nutrient Mixture F-12 [HAM] (Sigma
Chemical Co.); H460 (large cell lung carcinoma) and MSTO-211H (human biphasic mesothelioma)
were grown in RPMI 1640 (Sigma Chemical Co.) supplemented with 2.38 g/L Hepes, 0.11 g/L
pyruvate sodium and 2.5 g/L glucose. 1.5 g/L NaHCO3, 10% heat-inactivated fetal calf serum
(Invitrogen), 100 U/mL penicillin, 100 ꢀg/mL streptomycin, and 0.25 ꢀg/mL amphotericin B
(Sigma Chemical Co.) were added to both media. The cells were cultured at 37°C in a moist
atmosphere of 5% carbon dioxide in air. Cells (2.5-3 x 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 incubated for a further 72 h. A Trypan blue assay was performed to
determine cell viability. Cytotoxicity data were expressed as GI50 values, i.e., the concentration of
the test agent inducing 50% reduction in cell number compared with control cultures.
4.3. Nucleic acids
Salmon testes DNA was purchased from Sigma Chemical Company. The DNA concentration was
determined using extinction coefficient 6600 M-1 cm-1 at 260 nm. pBR322 DNA was purchased
from Fermentas Life Sciences.
4.4. Linear flow dichroism
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 was defined as:
LD(λ) = A//(λ) - A
(λ)
24

ACCEPTED MANUSCRIPT
where A// and A correspond to the absorbances of the sample when polarized light was oriented
parallel or perpendicular to the flow direction, respectively. The orientation was produced by a
device designed by Wada and Kozawa22 at a shear gradient of 500-700 rpm, and each spectrum was
accumulated twice.
Aqueous solutions of salmon testes DNA (1.9 x 10-3M) in 10 mM ETN buffer (containing 0.01 M
TRIS, 1 mM EDTA (pH 7.0) and 0.01 M NaCl) were used. Spectra were recorded at 25°C at
[drug]/[DNA] = 0, 0.04 and 0.08.
4.5. DNA topoisomerase relaxation assay
Supercoiled pBR322 plasmid DNA (0.25 µg) was incubated with 1U topoisomerase II (human
recombinant topoisomerase II α, USB Corporation) or 2U topoisomerase I (calf thymus
topoisomerase I, Topogen) and the test compound as indicated for 60 min at 37°C in 20 µL reaction
buffer.
Reactions were stopped by adding 4 µL stop buffer (5% SDS, 0.125% bromophenol blue and 25%
glycerol), 50 µg/mL proteinase K (Sigma) and incubating for a further 30 min at 37°C. The samples
were separated by electrophoresis on 1% agarose gel at room temperature. The gels were stained
with ethidium bromide 1 µg/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.
4.6. Topoisomerase I-mediated DNA cleavage
Reaction mixtures (20 µL) containing 35 mM Tris-HCl (pH = 8.0), 72 mM KCl, 5 mM MgCl2, 5
mM DTT, 5 mM spermidine, 0.01% bovine serum albumin, 20 ng pBR322 plasmid DNA (Fer-
mentas Life Sciences), 5 U topoisomerase I (human recombinant topoisomerase I, TopoGen) and
test compounds were incubated for 60 min at 37 oC. Reactions were stopped by adding 4 µL of stop
buffer (5% SDS, 0.125% bromophenol blue and 25% glycerol), 50 µg/ mL proteinase K (Sigma)
o
and incubating for a further 30 min at 37 C. The samples were separated by electrophoresis on a
1% agarose gel containing ethidium bromide 0.5 µg/mL (Sigma) at room temperature in TBE buffer
(0.09 M Tris-borate and 0.002 M EDTA), transilluminated by UV light, and fluorescence emission
was visualized by a CCD camera coupled to a Bio-Rad Gel Doc XR apparatus.
4.7. Topoisomerase II-mediated DNA cleavage
Supercoiled pBR322 plasmid DNA (0.25 µg) was incubated with 10 U topoisomerase II (human
recombinant topoisomerase II α, USB) and the test compounds, as indicated, for 60 min at 37 °C in
20 µL reaction buffer.
25

ACCEPTED MANUSCRIPT
Reactions were stopped by adding 4 µL stop buffer (5% SDS, 0.125% bromophenol blue and 25%
glycerol), 50 µg/mL proteinase K (Sigma) and incubating for a further 30 min at 37 °C. The
samples were separated by electrophoresis on a 1% agarose gel containing ethidium bromide 0.5
µg/mL in TBE buffer (0.09 M Tris-borate and 0.002 M EDTA), transilluminated by UV light, and
fluorescence emission was visualized by a CCD camera coupled to a Bio-Rad Gel Doc XR
apparatus.
4.8. Evaluation of apoptotic cell death by Annexin V-FITC and propidium iodide staining
To detect phosphatidylserine translocation from the inner face to the outer surface of plasma
membrane, a FITC Annexin V Apoptosis Detection Kit I (BD Pharmigen) was used.
H460 cells (2.0 x 105) were seeded into each cell culture plate in complete growth medium. After
incubation for 24 h the test agents were added at the indicated concentrations and cells were
incubated for a further 18 h. After treatment, cells were centrifuged and resuspended at 106 cells/mL
in binding buffer. Cell suspensions (100 µL) were added with Annexin V-FITC and propidium
iodide (PI) as indicated by the supplier’s instructions, and incubated for 15 min at room temperature
in the dark. The populations of Annexin V-negative/PI-negative (viable), Annexin V-positive/PI-
negative (early apoptosis), Annexin V-positive/PI-positive (late apoptosis) and Annexin V-
negative/PI-positive (necrosis) cells were evaluated by FACSCanto II flow cytometer (Becton–
Dickinson, Mountain View, CA).23
Acknowledgments
The present work has been carried out with financial support of the Italian Ministry of Education,
University and Research (MIUR).
The Supplementary Material is available free of charge on the Elsevier Publications Website.
Associated content: 1H NMR spectrum, 13C NMR spectrum, 1H-13C HSQC correlation table, 1H-13C
1
1
HMBC and H-1H NOESY correlation table of compound 16; H NMR spectrum detail of
compound 22; HPLC trace of compounds 16 and 23; Elemental Analyses of compounds 4-12, 15,
16, 22 and 23.
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