Synthesis, DNA and protein interactions and human topoisomerase inhibition of novel Spiroacridine derivatives

Article abstract of DOI:10.1016/j.bmc.2018.10.038

Nine new spiroacridine derivatives were synthetized by introducing cyano-N-acylhydrazone group between the acridine and phenyl-substituted rings followed by spontaneous cyclization. The new compounds were assayed for their DNA binding properties, human to

Full text of DOI:10.1016/j.bmc.2018.10.038

Accepted Manuscript
Synthesis, DNA and protein interactions and human topoisomerase inhibition
of novel Spiro-acridine derivatives
Rawny Galdino Gouveia, Amélia Galdino Ribeiro, Miguel Ângelo Santos
Pinheiro Segundo, Jamerson Ferreira de Oliveira, Maria do Carmo Alves de
Lima, Túlio Ricardo Couto de Lima Souza, Sinara Mônica Vitalino de Almeida,
Ricardo Olímpio de Moura
PII:
S0968-0896(18)31591-8
https://doi.org/10.1016/j.bmc.2018.10.038
BMC 14597
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 September 2018
24 October 2018
29 October 2018
Please cite this article as: Gouveia, R.G., Ribeiro, A.G., Segundo, M.A.S., de Oliveira, J.F., de Lima, M.d.C., de
Lima Souza, T.R.C., de Almeida, S.M.V., de Moura, R.O., Synthesis, DNA and protein interactions and human
topoisomerase inhibition of novel Spiro-acridine derivatives, Bioorganic & Medicinal Chemistry (2018), doi:https://
doi.org/10.1016/j.bmc.2018.10.038
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Synthesis, DNA and protein interactions and human topoisomerase inhibition of
novel Spiro-acridine derivatives
Rawny Galdino Gouveiaa, Amélia Galdino Ribeirob, Miguel Ângelo Santos Pinheiro
Segundoa, Jamerson Ferreira de Oliveirab, Maria do Carmo Alves de Limab, Túlio
Ricardo Couto de Lima Souzac, Sinara Mônica Vitalino de Almeidad,e *, Ricardo
Olímpio de Mouraa,f.
a Laboratório de Síntese e Vetorização de Moléculas, Universidade Estadual da Paraíba,
Departamento de Ciências Biológicas, João Pessoa, PB, Brazil.
b Laboratório de Química e Inovação Terapêutica (LQIT) - Departamento de
Antibióticos, Universidade Federal de Pernambuco, Recife, PE, Brazil.
c Unidade Acadêmica de Serra Talhada, Universidade Federal Rural de Pernambuco,
Serra Talhada-PE, Brazil.
d Laboratório de Imunopatologia Keizo Asami (LIKA), Universidade Federal de
Pernambuco, Recife, PE, Brazil.
e Universidade de Pernambuco (UPE), Faculdade de Ciências, Educação e Tecnologia
de Garanhuns (FACETEG), Garanhuns, PE, Brazil.
f Departamento de Ciências Farmacêuticas, Centro de Ciências Biológicas e da Saúde,
Universidade Estadual da Paraíba - Bodocongó, Campina Grande, PB, Brazil.
*Corresponding author
Dr. Sinara Monica Vitalino de Almeida
E-mail: sinara.monica@upe.br
Rua Capitão Pedro Rodrigues, 105, São José, Garanhuns, Pernambuco, Brazil.
55294-902. Telephone: + 55 87 37618212.

Abstract
Nine new spiro-acridine derivatives were synthetized by introducing cyano-N-
acylhydrazone group between the acridine and phenyl-substituted rings followed by
spontaneous cyclization. The new compounds were assayed for their DNA binding
properties, human topoisomerase II inhibition and bovine serum albumin (BSA)
interaction. Besides, docking analysis were performed in order to better understanding
the biomolecule-compounds interactions. All compounds interacted with BSA which
was demonstrated by the fluorescence suppression constant of 104 M-1. Compounds
with chloro and NO2 substituents at that para-position on phenyl ring demonstrated the
best results for BSA interaction. DNA binding constant determined by UV-vis data
demonstrated high values for AMTAC-11 and AMTAC-14, 1.1 x 108 M−1 and 4.8 x 106
M−1, respectively, and all others presented constant values of 105 M−1. AMTAC-06 with
chloro at para-position on phenyl ring presented a topoisomerase II inhibition of
84.34% in comparison to the positive controls used. Docking studies indicated that
AMTAC-06 is able to intercalate the DNA base pairs at topoisomerase II active site,
preventing DNA religation, in a process known as poisoning. Topoisomerase enzyme
inhibition result was correlated to BSA interaction profile, since AMTAC-06 showed
the best results in both analysis. The findings obtained here proved that methoxy or
chloro substitution on phenyl ring at para-position is fundamental for in vitro activity of
new spiro-acridine derivatives, and indicates that AMTAC-06 is a promising entity and
should serve as a lead compound in the development of new DNA and protein binder,
as well as human topoisomerase II inhibitors.
Keywords: Spiro-acridine; protein interaction; DNA-binding; Topoisomerase II.

1. Introduction
Acridine derivatives are compounds presenting a planar ring that can suffer
different chemical modifications yielding new structures with diverse biological
activities, such as antibacterial, antiparasitic, antioxidant and antitumor due the
interaction with several molecular targets.1-6 The extent of the biological activities of
acridine derivatives has been attributed to the planarity of the ring and consequent
insertion between the DNA pair of bases besides the interaction and inhibition of cell
essential enzymes such as topoisomerases type I and II, telomerase and protein
kinases.5, 7-9
DNA and topoisomerase are well established target for many organic
compounds and the simultaneous interaction in the interface between the biomolecules
are widely explored for synthesis of promising compounds with antitumor activity,10
since these compounds can induce the cell cycle arrest and trigger cell apoptosis.8, 11,12
Recent researches have been proposed that the broad extent of biological activities of
acridine derivatives cannot be explained just by the interaction with nucleic acids and
that these different biological activities can be significantly caused by direct interactions
with proteins including receptor, kinases and enzymes. Acridines interact with proteins
both directly or as a component of nucleic acid-acridine complex.13
The interaction studies between new chemical compounds and proteins have
been made with bovine serum albumin (BSA) because its homology with human serum
albumin (HSA), abundance in plasma and action as transporter and delivery
biomolecule increasing the efficacy of treatment simultaneous to the side effects
decreasing.14 The intrinsic fluorescence of BSA due to tryptophan residues can indicate
the compound-protein interaction. Depending on substituents, acridine ring is
hydrophobic and is mostly protonated under physiological pH. Positive charge can be
recognized by carboxylate of Asp and Glu residues.13 This interaction can be detected
by changes in fluorescence of BSA.15,16 Furthermore, the information obtained by BSA
interaction assays can be used to predict the pharmacokinetic profile of new compounds
and their biological potential.14
A new and promising class of acridine derivatives is the spiro-acridines. The
acridine ring reactivity at carbon C-9 allows a spontaneous cyclization by an
intramolecular nucleophilic attack and subsequent ready formation of the five-
membered spiro ring attached to acridine.17 Salem et al.18 synthetized new spiro

tria(thia)zolidine-acridines and evaluated their interaction with DNA by different
techniques. The compounds interacted with DNA with binding constant between 0.34 -
0.93 × 104 M-1 indicating that the cyclization process did not impair the interaction with
DNA. The compounds were also able to inhibit the cell HL-60 proliferation, one
derivative inhibited both topoisomerase I and II enzyme activities with significant
action on mitochondrial physiology.
Almeida et al.19 have also produced new spiro-acridines by introducing cyano-
N-acylhydrazone group between the acridine and (non- and methoxy-substituted)
phenyl rings followed by spontaneous cyclization. The authors evaluated the DNA
binding properties, human topoisomerase I and II inhibition assay and antiproliferative
of the new compounds. The results demonstrated that both compounds are specific for
human topoisomerase II inhibition and retained the DNA interaction ability. Based on
this previous data, our group proposed the synthesis of new spiro-acridine derivatives
with different substituents on phenyl ring and the evaluation of the chemical
modifications influence on the DNA and protein binding, as well as the human
topoisomerase IIinhibition activity.
2. Results and discussion
2.1. Chemistry
In order to find new molecules for cancer treatment, new spiro-acridines with
different substituents were synthetized from the basic structure of AMTAC-01
according Almeida et al.19 Scheme 1 demonstrated the reaction sequence used in this
work and the structure of each respective spiro-acridine derivatives. As well established
in previous works,20,21 diphenylamine with zinc dichloride in acetic acid were used for
the synthesis of 9-methylacridine22 followed by the oxidation of 9-methylacridine with
pyridinium chlorochromate (PCC)23 resulting 9-acridinaldehyde compound. In the
following step, 9-acridinaldehyde reacted in absolute ethanol and triethylamine with the
respective substituted N-benzylidene-2-cyanoacetohydrazide intermediary through a
Knoevenagel condensation to yield the compounds that suffered a spontaneous
cyclization between N1 of the hydrazine and acridine C-9 carbon.19 For the final
compounds the yield was higher than 50%, with the best result for AMTAC-09

(86.7%). The structures of all new derivatives were determined by 1H and 13C NMR, IR
and high-resolution mass spectrometry.
Scheme 1. Synthesis of spiro-acridine derivatives. Reagents and conditions: (a)
CH3CO2H, EtOH, 45 °C; (b) CH3CO2H, ZnCl2, 230 °C; (c) PCC, CH2Cl2,25 °C; (d)
EtOH, triethylamine, 60 °C.
Through 1H NMR analysis, it was possible to identify characteristic singlets of
the spiro-acridine proton (NH), with chemical shifts ranging from δ 8.59 to 9.93 ppm.
1
Other feature observed in the H NMR of new compounds was the presence of protons
from the chemical bond of C=C (vinyl) and C=N (imine) with displacement at δ 8.54-
1
8.55 ppm and 7.38-8.42 ppm, respectively. Besides, H NMR spectra of all derivatives
demonstrated characteristic singlet, (s), doublet (d), doublet of doublet (dd), triplet (t)
and multiplet (m) for aromatic groups of acridine ring similar to the literature.19 In its
turn, the 13C NMR spectra also confirmed the structures by the presence of signals
corresponding to spiro-acridine carbon atoms. It was observed the displacement
between 69.33-71.59 that points out the quaternary carbon formed after the spontaneous
cyclization process.17 It was also possible to visualize the displacements between 161.38
and 163.70 ppm that are characteristic of the carbon in the carbonyl groups (C=O).

IR results also helped for structural characterization of the new spiro-acridine
derivatives. The following bands were observed: 3296-3331 cm-1, relative to the NH of
acridine ring; 1678-1724 cm-1, related to carbonyl group; 2237-2239 cm-1 corresponding
to nitrile group for all compounds. Finally, mass spectrometry (MS) was useful to
confirm the identity of the new compounds.
2.2. BSA interaction study
Albumin is the most abundant human plasma protein with functions related to
blood osmotic pressure and transport, metabolism, delivery and removal of exogenous
and endogenous substances.16 For playing most of these roles, albumin needs to bind to
organic compounds, as drugs for example, and influences its bioavailability, stability
and toxicity.24,25 Therefore, determining if and understanding how albumin binds to
chemical compounds, such as spiro-acridines, can help to predict the biological
behaviour of the derivatives, since studies demonstrated that this protein serves
efficiently as compound transporter to the target site resulting in higher efficacy of
treatment and reduced side effects.14,26
In this study, BSA was used for establishing the protein binding profile of
spiro-acridines. BSA is employed in several studies due to its high similarity to the
HSA, besides it is easy to evaluate the compound-BSA binding process by fluorescence
emission of its tryptophan residues (Trp) that change the emissive properties after
interaction.26,27 For BSA-spiro-acridine interaction evaluation, a solution of BSA (10
µM) in absence and presence of different concentrations (2-20 µM) of each derivative
was analysed by excitation at 285 nm and emission scan from 310 to 400 nm. Table 1
shows the results of BSA fluorescence in the presence and absence of each compound.
It is worthwhile to highlight that the compounds were no fluorescent by excitation at
285 nm.
Table 1. Fluorescence data of Bovin Serum Albumin (BSA) in the absence and
presence of spiro-acridines
Red shift in
λmax free
λmax bound
c
KSV b (M−1)
Compound
emission
Δλ (nm)
15
I/I0 (%)
(nm)a
(nm)
AMTAC-06
AMTAC-07
AMTAC-08
AMTAC-09
AMTAC-10
AMTAC-11
AMTAC-13
325
320
322
321
321
324
322
340
342
342
342
342
342
343
3.4 x 104
2.3 x 104
2.0 x 104
1.9 x 104
2.1 x 104
2.7 x 104
2.3 x 104
60.30
55.11
48.01
42.53
45.77
54.95
51.46
22
20
21
21
22
21

AMTAC-14
AMTAC-16
322
325
343
343
21
22
3.1 x 104
2.3 x 104
71.74
58.57
a Tryptophan BSA fluorescence obtained by excitation at 285 nm and scanning emission
from 310-400 nm.
b Stern–Volmer quenching constant, KSV, obtained from quenching of BSA fluorescence
by AMTAC’s.
c Percentage of protein fluorescence decreasing, where I0 and I are fluorescence
intensities in the absence and presence of AMTAC’s compounds obtained from
fluorimetric titrations.
As shown in Table 1, the addition of all compounds to BSA solution resulted in
fluorescence quenching and red shift (Δλ) of the emission maxima wavelength, with the
higher alteration promoted by AMTAC-14 (71.74%). These results indicate that
interaction between BSA-compound occurred for two reasons: (a) Trp residues are
located in hydrophobic pockets that can serve as sites for compound binding, which can
lead to loss of excitation energy by transference to the compound and decreasing of
fluorescence intensity; (b) the formation of complex BSA-compound can change the
protein conformation with disruption of the Trp hydrophobic environment that in turn
promotes the fluorescence quenching. The first mechanism is the most likely since
conformation changes could impair the drug carrying and delivery after compound
binding, besides it was still observed fluorescence at the high compound concentration
and chemical substituent influenced the emission decrease profile. This can be
supported by the AMTAC-14 result that bears the -NO2 moiety with high electron-
withdrawing effect causing an aromatic electron catching with consequent fluorescent
quenching. The BSA fluorescence spectra in both the absence and presence of
AMTAC-14 (0–20 µM) are given in Figure 1 (for the other derivatives see
Supplementary Information).

Figure 1. Emission spectrum of BSA (10 µM) in the absence and presence of the
AMTAC-14. [AMTAC-14] = 0, 2, 4, 6, 8, 10 and 20 µM. The arrow (↓) shows a
reduction of intensity on the concentration of the complex at room.
To better understand quantitatively the behavior of BSA fluorescence
suppression promoted by spiro-acridine derivatives, the magnitude of quenching was
analyzed by the Stern-Volmer equation (see experimental section), where Ksv is the
fluorescence quenching constant determined by linear regression plot graph of relative
emission intensity (F0/F) versus derivative concentrations (Q).28 All compounds caused
quenching of BSA fluorescence emission (Table 1). The KSV values decreased as
follows: 06>14>11>13,16,07>10>08>09. The high values presented by AMTAC-06,
AMTAC-14 and AMTAC-11 suggest a strong binding towards BSA. These results can
be explained by the hydrophobicity (π) and electronic effects (σ) of the aromatic para-
substituents, as stated by Craig.29 Derivative AMTAC-14 bears a NO2 as substituent
that present a high electronic (+σ) and lower lipophilic (+π) effects. AMTAC-06 and
AMTAC-11 are para- and mono-substituted, similar to AMTAC-14, with -Chloro
(positive electronic and lipophilic effects) and -N(CH3)2 (negative electronic and
positive hydrophobic effects), respectively.29 This last chemical group is ionizable
depending on the pH favouring the hydrogen bond formation at the para-position of the
new compounds. On the other hand, derivatives bearing two substituents presented the
lower KSV values presumably due to a steric hindrance that impaired the formation of

BSA-derivative complex. To our knowledge, this is the first work that evaluated the
interaction between BSA and spiro-acridine derivatives. These findings can help to
better understand the biological activity of these new compounds in anticancer activity
tests, as already demonstrated for novel substituted triazine-benzimidazole analogues.14
2.3. DNA binding assay
Acridine derivatives are well established DNA binders and this mechanism
contributes to its anticancer activity.10 Different spiro-acridine derivatives maintain their
DNA-binding ability that can be correlated with cytostatic and antiproliferative actions
against cancer cells, as well as topoisomerase inhibition.18,19 In order to determine the
interaction between DNA and the new spiro-acridine derivatives, electronic absorption
spectroscopy was performed to identify compound spectrum changes in the presence of
increasing amounts of DNA. Electronic absorptions of the derivatives were recorded in
Tris-HCl buffer (pH 7.6). As expected, all derivatives showed absorption in the region
around 250-400 nm.19 The maxima absorption wavelengths of compounds in the
absence and presence of ctDNA can be seen in Table 2.
Table 2. UV–vis absorption data of spiro-acridines in the absence and presence of
ctDNA
Δλ
(nm)
λmax
free
(nm)
λmax
bound
(nm)
Extinction
coefficient
(ε) M−1
Hyperchr
(%)a
Hypochr
(%)b
Compound
Kb M−1
AMTAC-06
AMTAC-07
AMTAC-08
AMTAC-09
AMTAC-10
AMTAC-11
AMTAC-13
AMTAC-14
AMTAC-16
325
320
322
300
270
341
335
335
272
321
323
273
379
262
343
333
334
268
4
3
51
79
8
2
2
1
4
-
44.27
18.900
12.100
12.400
19.100
34.400
17.700
14.100
31.600
34.500
7.6 x 105
1.5 x 105
8.2 x105
6.7 x 105
1.6 x 105
1.1 x 108
3.7 x 105
4.8 x 106
2.6 x 105
45.78
65.29
-
-
-
-
-
-
-
100
30
7.14
8.06
13.46
14.43
a
Hyperchromicity for complexes formed by compounds and 100 µM of ctDNA in
comparison to free ligands.
b
Hypochromicity for complexes formed by compounds and 100 µM of ctDNA in
comparison to free ligands.
All derivatives exhibited a shift of the maxima absorption wavelength (Δλ) in the
presence of ctDNA which suggests a complex formation between DNA-compound. The

model of McGhee and von Hippel30 was used to estimate the binding constants (Kb)
from the spectrophotometric data. The highest Kb values were demonstrated by
AMTAC-11 and AMTAC-14, 1.1 x 108 M−1 and 4.8 x 106 M−1, respectively, and all
others presented Kb values > 105 M−1. These binding constant values correspond to the
typical range for complexes formed between acridine derivatives and DNA,10 and the
high ctDNA binding constants indicate greater affinity of the newly synthesized spiro-
acridine ligands to ctDNA. The Kb values of AMTAC-11 and AMTAC-14 can be
attributed to the mono-substitution at para-position on phenyl ring which could
decrease the steric hindrance caused by others groups as demonstrated by the diphenyl
moiety in the derivatives produced by Salem et al.18 Contrary to protein interaction, Kb
values suggest that di-substitution on phenyl ring with ionizable and electron-
withdrawing groups prompt the in vitro interaction between spiro-acridine and DNA.
Considering that DNA is the site of action of essential enzymes for cancer cell
proliferation, such as topoisomerase, high DNA binding values can indicate better
enzyme inhibition and consequent cell death.
2.4. Antitopoisomerase II activity
DNA topoisomerases are essential enzymes for cell proliferation and survival
because they are involved in DNA metabolism during transcription and replication
processes, both in eukaryotic as well in prokaryotic cells. According to the action
mechanism, topoisomerase enzymes are classified in type I or type II, which differ in
their abilities to cut either one or two strands of DNA, respectively.31 Compounds that
inhibit the topoisomerase function are used as antitumor agents, as for example the
amsacrine (mAMSA), an acridine derivative that is a specific inhibitor of human
topoisomerase II with DNA intercalation contributing for the mechanism and
specificity against cancer cells.32 Almeida et al.19 demonstrated that two new spiro-
acridine derivatives (AMTAC-01 and AMTAC-02) were specific for inhibition of
human topoisomerase IItopo II) that is expressed preferentially in proliferating
cells. Both compounds were no active against human topoisomerase type I. Based on
these previous data, plasmid pUC19 relaxation was studied with agarose gel
electrophoresis in order to investigate the effect of the new spiro-acridine derivatives on

topo II activity, using the mAMSA as positive control, besides to compare to the
results of AMTAC-01 and AMTAC-02. The results are demonstrated in Figure 2.
Figure 2. Effects of spiro-acridine derivatives and positive control on the inhibition of
human topoisomerase II Native supercoiled pUC19 was incubated for 30 min at 37◦C
with 2 units of human topo II in the absence (lane 2) or presence of ligands at
concentration 100 µM. Inhibition of topo II-induced DNA relaxation by mAMSA
(lane 3) was used as positive control. DNA samples were run on agarose gel followed
by ethidium bromide staining. The supercoiled band of each lane was analyzed by optic
densitometry and the density is presented in the respective upper graphic.
The topo II enzyme was able to relax the plasmid DNA (lane 2) in the absence
of the compounds. In contrast, the positive control (lane 3) and active compounds
caused the enzyme inhibition which affected the unwinding of the supercoiled DNA,
leading to a band pattern similar to the plasmid only (lane 1). In other words, if the
compound is active against topo IIactivity, the supercoiled DNA band is present on
the electrophoresis gel. As demonstrated in Figure 2, most of the new spiro-acridine
derivatives inhibited in some proportion the enzyme. The average values of intensity
obtained by densitometry results were used to calculate the enzyme inhibition
percentage to better analyze the effect. Table 3 presents the values of enzyme inhibition
for each derivative and positive control.

Table 3. Topo II inhibition activity of spiro-acridine derivatives.
AMTAC
Compounds
m-AMSA
01
02
06
07
08
09
10
11
13
14
16
% of enzyme
inhibition
100%
9.78 88.11 84.34 70.72 61.70 1.64 15.91 1.64 1.64 43.74 20.62
AMTAC-02 was better than other derivatives, presenting similar results to m-
AMSA with enzyme inhibition of 88.11% followed by AMTAC-06, AMTAC-07,
AMTAC-08 and AMTAC-14 with values of inhibition of 84.34, 70.72, 61.70 and
43.74%, respectively. These results can be explained by the electronic distribution,
hydrophobic features and stereochemistry of the substituents of the new derivatives. As
demonstrated previously by Almeida et al.,19 AMTAC-02 was better than AMTAC-01
both in antiproliferative assay as well as in topo enzyme inhibition test, probably due to
the stabilization of ternary complex by the methoxy group on the phenyl ring. From the
new series, AMTAC-06 and AMTAC-07 showed the best results which suggests that
para-substitution on phenyl ring favours the biological activity. Therefore, methoxy,
chloro and fluorine moieties at para-position stabilize the ternary complex formed by
DNA-enzyme-derivative. These chemical substituents present positive electronic and
hydrophobic effects (+σ +π) which contributed for enzyme inhibition. This explanation
is corroborated by the poor inhibition presented by AMTAC-11 that bears a para-
substitution with -N(CH3)2, a group with negative electronic effect and positive
hydrophobicity.29
Di-substitution with chloro also impaired the topo II inhibition in a position-
dependent manner, as visualized by the results of AMTAC-08 and AMTAC-10. The
former was more active (61.70%) than the last (15.91%). It is worthwhile to highlight
that the mono-substitution with chloro promoted the best inhibition pattern (AMTAC-
06), suggesting that two groups at different positions probably caused a steric hindrance
that impairs the enzyme inhibition. It was also verified that the position of the group
NO2 influenced the activity of the derivatives, with the best inhibition result observed
for the para-substituted compound AMTAC-14 (43.74%) against the ortho-substituted
AMTAC-13 (1.64%). Therefore, the mono and para-substitution on phenyl ring of
spiro-acridine derivatives is fundamental for topoisomerase enzyme inhibition, which is
confirmed by the inactivity of AMTAC-09, a derivative without para-substitution, even
presenting a methoxy moiety that was the most favourable for inhibition activity in

AMTAC-02 structure.19 These findings allowed the identification of chemical groups
and the best positions for phenyl substitution of spiro-acridine derivatives that favours
the topoisomerase inhibition, confirming the importance of methoxy moiety on phenyl
ring as demonstrated by Ketron et al.32 and at the para-position for better interaction
with amino acids of enzyme active site, as confirmed by the activity of AMTAC-06.
4. In Silico studies
4.1. Molecular docking: Bovine Serum Albumin
Molecular docking was used to investigate the preferred binding site and
ligand/receptor interactions of the test compounds to BSA. Results are presented in
Table 4 and show that AMTAC-06, 07, 08, 13 and 16 bind to the BSA in a
conformation that allows direct interaction to Trp-213, which is supported by our
experimental findings. The lead compound in this study, AMTAC-06, docked to BSA
with a free binding energy of -9.22 kcal mol-1. The conformation of the AMTAC-06-
BSA complex with the lowest energy, as well as ligand/protein interactions, are shown
in Figure 3.
Table 4. Docking score and interacting residues of test compounds with BSA
Compound
Binding Hydrogen
Hydrophobic Interactions
Energy
kcal
Bond
Pi-alkyl
Alkyl
Pi-sigma
Pi-pi
stacked
mol-1
AMTAC-06
-9.22
Ser-453
Arg-194, Leu-197,
Arg-198, Ala-209,
Val-342, Leu-346,
Leu-454, Val-481
Arg-198, Leu-197,
Ala-209, Val-342,
Leu-346, Val-481
Pro-110, Lys-114,
Leu-115, Arg-144,
Arg185, Leu189
Phe-205, Ala-209,
Ala-212, Leu-346,
Ala-349, Lys-350,
Leu-480, Val-481
Lys-114, Leu-115,
Pro-117, Ile-141,
Arg-144, Leu-189,
Arg185
Leu-480
Trp-213
AMTAC-07
AMTAC-08
AMTAC-09
-9.97
-8.39
-8.40
Ser-343
Arg-144
Leu-197,
Leu-480
Trp-213
Trp-213
-
Leu-197,
Leu-346,
Leu-480
Leu-346
Lys-350,
Asn-482
AMTAC-10
AMTAC-11
-9.18
-8.81
Leu-112,
Leu-115
-
-
-
-
-
Lys-114, Leu-115,
Pro-117, Arg-144,
Arg-185

AMTAC-13
-8.82
-
Arg-194, Leu-197,
Arg-198, Tpr-213,
Leu-456, Arg-483,
Arg-484
Leu-197,
Arg-483
-
AMTAC-14
AMTAC-16
-9.05
-9.50
Lys-431,
Arg-435
Ser-453
Leu-189, Ala-193,
Arg-196, Arg-458
Leu-197, Arg-198,
Ala-209, Val-342,
Leu-346, Val-481
-
-
Leu-197,
Leu-480
Trp-213
Figure 3. (a) Docked pose of AMTAC-06 (in yellow) against BSA ribbon structure.
Trp213 is indicated in blue. (b) Binding interactions of AMTAC-06 with the surrounding
amino acid residues.
The molecular docking results suggest AMTAC-06 binds to the hydrophobic
pocket between subdomains IIA and IIIA in BSA. The carbonyl oxygen makes a
hydrogen bond to Ser-453, and the chlorine containing ring interacts with Leu-454, Val-
342 and Leu-197 via hydrophobic interactions. The spiro-acridine rings are surrounded
by Leu-346, Val-481, Ala-209, Leu-480, Leu-197, Arg-198 and Trp-213, also via
hydrophobic interactions. The close proximity to Trp-213 must allow for a strong
interaction with this residue, and may be the reason for the pronounced quenching
observed at the fluorescence assay.
4.2. Molecular docking: Topoisomerase II / DNA

In order to better understand the mechanism involving topoisomerase inhibition,
a docking study was performed between AMTAC-02, AMTAC-06 and mAMSA, which
showed the best inhibition results, and a topo II / DNA model from PDB (5GWK)
containing the co-crystalized inhibitor etoposide. In this study, the inhibitors were
docked to the active site occupied by etoposide. These results are presented in Table 5
and Figure 4.
Table 5. Docking score and interacting residues of test compounds with topoisomerase
II in complex with DNA
Binding
Carbon
Hydrogen
Bond
Hydrophobic Interactions
Hydrogen
Bond
Compound
Energy
kcal mol-1
Pi-alkyl
Pi-pi stacked
A-12
C-8
T-9
A-12
T-9
G-13
C-8
-8.51
-8.65
-
-
Glu-461
-
Arg-487
AMTAC-02
AMTAC-06
Arg-487
Arg-487
Asp-463
Ser-464
Arg-487
-10.34
-
mAMSA

Figure 4. Binding modes of the topo II active site with (a) Etoposide; (b) mAMSA; (c)
AMTAC-02; (d) AMTAC-06. Docked molecules are in yellow, the co-crystalized
ligand etoposide is in blue.
The docking results show that AMTAC-02, AMTAC-06 and mAMSA were well
accommodated in the active site of the topo II / DNA complex. As expected, mAMSA,
an acridine derivative used as positive control in this study, was able to intercalate
between the DNA base pairs and make hydrogen bonds with three amino acid residues
(Asp-463, Ser-464 and Arg-487). The spiro-acridines AMTAC-02 and AMTAC-06 also
docked to the active site by intercalating the DNA base pairs, and presented pi-alkyl
interactions with Arg-487 via one of the spiro-acridine rings. Additionally, AMTAC-02
presented one carbon-hydrogen bond with Glu-461. The presence of multiple aromatic
rings in these compounds allows for pi-stacking interactions with the nucleotides, which
is known as the driving force in the DNA intercalation process.33 As previously
discussed, one can notice that the interaction with the amino acid residues surrounding
the active site seems to improve the inhibitory potential of these compounds, by
stabilization of the non-intercalated side chain of the molecules. These results, together

with our experimental data, present relevant evidence that these compounds inhibit
topoisomerase II activity by preventing DNA religation after the double strand breaks
promoted by the enzyme, a process known as poisoning.34
4.3. ADMET prediction
In silico ADMET predictions were performed to evaluate the pharmacokinetic
and toxicity profiles of the synthetized compounds. Two common ways to evaluate the
oral bioavailability potential of a compound are the Lipinski’s and Veber’s rules.35,36
Lipinski’s rules states that the potential drug is orally active when meets the following
criteria: a) molecular weight ≤ 500 g mol-1; b) LogP ≤ 5 (or MLogP ≤ 4.15); c) number
of hydrogen bond acceptors ≤ 10; d) number of hydrogen bond donors ≤5. In order to
attend to Veber’s rules, a compound must present a TPSA ≤ 140 Å2 and have a
maximum of 10 rotatable bonds. Our results show that all compounds are in agreement
with both Lipinski’s and Veber’s rules (Table 6).
Table 6. In-Silico Pharmacokinetic data estimated at SwissADME or pkCSM
webservices.
Compound
LogPb LogSc
Mol.
Weight
(g mol-1)
410.86
394.40
445.30
485.33
445.30
419.48
421.41
436.46
H bond
H bond
Rot.
TPSA
(Å2)d
Int.
Abs.
(%)e
96.3
92.6
94.1
93.4
96.8
100
donors acceptors Bondsa
AMTAC-06
AMTAC-07
AMTAC-08
AMTAC-09
AMTAC-10
AMTAC-11
AMTAC-14
AMTAC-16
1
1
1
1
1
1
1
2
3
4
3
4
3
3
5
5
2
2
2
3
2
3
3
4
3.48
3.38
3.96
3.24
3.96
2.87
2.09
2.34
-5.98
-5.44
-6.63
-6.22
-6.63
-5.53
68.49
68.49
68.49
77.72
68.49
71.73
100
84.3
-6.12 114.31
-5.94 97.95
aNumber of rotatable bonds in the molecule.
bSwissADME Moriguchi log of octanol-water partition coefficient.
cSwissADME Ali log of aqueous solubility.
dSwissADME calculation of Topological Polar Surface Area (TPSA).
epkCSM prediction of the proportion of compound absorption though the human small
intestine.
Additionally, the calculated LogS values show that AMTAC-06, 07, 11 and 16
must be moderately soluble in water (LogS between -6 and -4), while AMTAC-08, 09,
10 and 14 are expected to be poorly soluble (LogS between -10 and -6). The intestinal
absorption prediction for these compounds show that all compounds (except AMTAC-

16) must be readily absorbed by the human small intestine, with values >90%. These
results show that the evaluated compounds must present an adequate profile regarding
oral bioavailability, in special AMTAC-06, 07 and 11. This information will be useful
in future in vivo biological activity evaluation of these new spiro-acridine derivatives.
5. Conclusion
New spiro-acridine derivatives were synthetized in good yields and their
chemical structures were confirmed. All compounds were assayed for their BSA and
DNA in vitro interaction besides the topoisomerase inhibition activity. BSA
fluorescence quenching indicated that the compounds interacted with the protein (104
M-1), with AMTAC-06 and AMTAC-14 presenting the best results, which suggests that
para-position with chloro and NO2 favoured protein-compound interaction. ctDNA
binding assay by UV-vis spectroscopy proved that the derivatives conserved their DNA
interaction potential with high constant binding values for AMTAC-11 and AMTAC-
14, 1.1 x 108 M−1 and 4.8 x 106 M−1, respectively, and all others presented Kb values =
105 M−1. In human topo II inhibition test, AMTAC-06 with chloro at para-position on
phenyl ring presented a high inhibition value (84.34%), similar to the positive controls
used. Docking studies showed that AMTAC-06 is able to intercalate the DNA base
pairs at topo II active site, preventing DNA religation, in a process known as
poisoning. Topo enzyme inhibition result was correlated to BSA interaction profile,
since AMTAC-06 showed the best results in both analysis. This finding suggests that
BSA is useful both to predict the serum behavior of new compounds as well as to
explore the potential of enzyme inhibition such as topoisomerase. This work proved that
methoxy or chloro substitution on phenyl ring at para-position is fundamental for in
vitro activity of new spiro-acridine derivatives, and indicates that AMTAC-06 is a
promising entity and should serve as a lead compound in the development of new anti-
cancer agents.
6. Experimental
6.1. Material and methods
The starting materials for intermediate reagent synthesis 2-cyanoacetohydrazide
benzaldehyde were purchased from Sigma Aldrich (Saint Louis, MO, USA) and used as
received, as well as the calf thymus DNA (ctDNA) and Bovine Serum Albumin (BSA).

All common solvents used were chemically pure and AR grade and supplied from
Aldrich, Merck and Fluka and used without purification. Melting points were measured
in capillary tubes on a Quimis Model 340 apparatus (Quimis, Diadema, Brasil). Thin
Layer Cromatography (TLC) was performed on Fluka Analytical silica gel thickness 0.2
mm plates with fluorescent indicator 254 nm and the disclosures were made by UV
light (254 or 365 nm). The Nuclear Magnetic Resonance spectra of Hydrogen and
Carbon were obtained on Agilent-NMR spectrometer, mod. Mercury Plus multinuclear
300MHz, magneto-NMR OXFORD 300 (operating at 400 or 300 MHz for 1H and 100
or 75 MHz for 13C) using DMSO-d6 as solvent and tetramethylsilane (TMS) as internal
standard. The processing of the spectra was done in MestReC 4.8.6.0 software.
Chemical shifts are reported in parts per million. The multiplicities are reported as
follows: singlet (s), doublet (d), doublet of doublet (dd), triplet (t) and multiplet (m).
Infrared spectra were obtained with KBr discs on IRPrestige-21 spectrometer,
Shimadzu®. The data were processed with Origin 8.0 software. The measures the exact
masses of the molecular ions were obtained on a Shimadzu electrospray LC / MS-IT-
TOF in a positive way, Shimadzu®. UV–Vis spectra were measured on an Ultraspec
3000 PRO UV–Visible spectrophotometer (Biochrom Ltd., Cambridge, UK) and
fluorescence spectra on a JASCO FP-6300 (Jasco Corporation, Tokyo, Japan)
spectrofluorometer.
6.2. Preparation of spiro-acridines
The intermediate syntheses were accomplished as described previously:19 to a solution
of equimolar quantities of 2-cyanoacetohydrazide and aromatic aldehyde in absolute
ethanol, 20 drops of acetic acid were added. The mixture was stirred at room
temperature until TLC indicated the end of reaction. The resulting precipitate was
filtered, washed with ethanol and the following intermediate products were obtained:
(Z)-2-cyano-N-(4-chlorobenzylidene)acetohydrazide;
dichlorobenzylidene)acetohydrazide;
nitrobenzylidene)acetohydrazide;
(Z)-2-cyano-N-(2,6-
(Z)-2-cyano-N-(2-
(Z)-2-cyano-N-(2,4-
dichlorobenzylidene)acetohydrazide;
methylbenzylidene)acetohydrazide;
(Z)-2-cyano-N-(4-
(Z)-2-cyano-N-(3-ethoxy-4-
hydroxybenzylidene)acetohydrazide;
(Z)-N-(5-bromo-2-methoxybenzylidene)-2-
cyanoacetohydrazide; (Z)-2-cyano-N-(4-fluorobenzylidene)acetohydrazide; (Z)-2-

cyano-N-(4-(dimethylamino)benzylidene)acetohydrazide;
(Z)-2-cyano-N-(4-
nitrobenzylidene)acetohydrazide. For the final AMTAC synthesis, each of one these
intermediates reagents and 9-acrinaldehyde (1.0 equiv),22,23 15 drops of trielhylamine
were refluxed in ethanol (10 mL) at 60◦C until TLC indicated the end of reaction (4 h).
Afterwards, the mixture was filtered and washed with water and ethanol. The products
were then analyzed by 1H NMR, 13C NMR, mass spectroscopy and infrared.
6.2.1.
1-(4-chlorobenzylideneamino)-5-oxo-1,5-dihydro-10H-spiro[acridine-9,2-
pyrrole]-4-carbonitrile (AMTAC-06)
Yellow powder. Formula: C24H15ClN4O; M.W.: 410.8551 g/mol; Yield: 24%; Melting
1
Point: 240-242º C; Rf: 0.54 (n-hexane/EtOAc 7:3). H-NMR (DMSO-d6): δ 9.82 (s,
1H, NH), 8.55 (s, 1H, CH), 8.42 (s, 1H, N=CH), 7.38 (m, 4H, aromatic), 7.26 (m, 2H,
13
spiro-acridine), 7.00 (m, 4H, spiro-acridine), 6.82 (m, 2H, spiro-acridine). C NMR: δ
69.33, 108.58, 111.76, 112.78, 115.58, 120.90, 127.50, 128.93, 129.47, 130.65, 133.03,
135.56, 139.05, 146.84, 161.18, 161.38. IR (KBr, cm-1): 3315.63 (NH), 3089.96 (CH),
2239.35 (CN), 1676.14 (C=O), 1485.18, 1334.74 (aromatic CN), 790.81 (Ar-Cl).
HRMS m/z [M + H]+: calculated: 410.8551; found: 411.0870.
6.2.2.
1-(4-fluorobenzylideneamino)-5'-oxo-1,5-dihydro-10H-spiro[acridine-9,2-
pyrrole]-4-carbonitrile (AMTAC-07)
Yellow powder. Formula: C24H15FN4O; M.W.: 394.4005 g/mol; Yield: 55.7%; Melting
Point: 206-209º C; Rf: 0.58 (n-hexane/EtOAc 7:3). 1H-NMR (DMSO-d6): δ 9.79 (s, 1H,
NH), 8.53 (s,1H, CH), 8.48 (s, 1H, N=CH), 7.40 (m, 2H, aromatic), 7.26 (m, 2H,
acridine), 7.14 (m, 2H, aromatic), 7.00 (m, 4H, acridine), 6.82 (m, 2H, acridine). 13C
NMR: δ 69.37, 108.68, 111.89, 112.78, 115.57, 116.32, 116.54, 120.87, 127.46, 129.52,
129.61, 130.58, 130.74, 130.77, 139.08, 147.40, 161.07, 161.07, 161.33, 162.54,
165.01. IR (KBr, cm-1): 3317.56 (NH), 2239.35 (CN), 1701 and 2157 (C=O), 1485.18,
1234.44 (C-F).
HRMS m/z [M + H]+: calculated: 394.4005; found: 395.1100.
6.2.3.
1-(2,4-dichlorobenzylideneamino)-5-oxo-1,5-dihydro-10H-spiro[acridine-9,2-
pyrrole]-4-carbonitrile (AMTAC-08)
Brown powder. Formula: C24H14Cl2N4O; M.W.: 445.3002 g/mol; Yield: 58.1%;
1
Melting Point: 178-180º C; Rf: 0.51 (n-hexane/EtOAc 7:3). H-NMR (DMSO-d6): δ

9.91 (s, 1H, NH), 8.58 (s, 1H, CH), 8.18 (s,1H, N=CH), 7.65 (d, J=10Hz, 1H, aromatic),
7.53 (d, 1H, aromatic), 7.35 (dd, J=10 and 5Hz, 1H, aromatic), 7.26 (m, 2H, spiro-
acridine), 6.99 (m, 4H, spiro-acridine), 6.81 (m, 2H, spiro-acridine). 13C NMR: δ 70.95,
110.50, 112.89, 114.94, 117.78, 123.16, 129.85, 130.01, 130.69, 131.96, 132.50,
133.12, 136.74, 138.09, 141.16, 141.62, 163.40, 163.68. IR (KBr, cm-1): 3323.34 (NH),
3091.89 (CH), 2239.35 (CN), 1678.06 (C=O).
HRMS m/z [M + H]+: calculated: 445.3002; found: 445.0760.
6.2.4. 1-(5-bromo-2-methoxybenzylideneamino)-5-oxo-1,5-dihydro-10H-spiro[acridine-
9,2-pyrrole]-4-carbonitrile (AMTAC-09)
Yellow powder. Formula: C25H17BrN4O2; M.W.: 485.3321 g/mol; Yield: 86.7%;
1
Melting Point: 208-210º C; Rf: 0.49 (n-hexane/EtOAc 7:3). H-NMR (DMSO-d6): δ
9.93 (s, 1H NH), 8.57 (s, 1H, CH), 8.36 (s, 1H, N=CH), 7.53 (d, J=3,5 Hz, 1H,
aromatic), 7.48 (dd, J= 3 e 11Hz, 1H, aromatic), 7.29 (dt, J=2 and 10.5Hz, 2H, spiro-
acridine), 7.01 (m, 4H, spiro-acridine), 6.93 (d, J=11Hz, 1H, aromatic), 6.83 (dt, J= 1.5
and 10Hz 2H, spiro-acridine). 13C NMR: δ 56.53, 56.58, 68.98, 108.61, 111.25, 112.85,
112.90, 115.05, 115.54, 120.86, 124.41, 127.19, 127.66, 130.83, 134.61, 139.09,
139.11, 140.19, 157.51, 161.27. IR (KBr, cm-1): 3300.20 (NH), 3078.39 (CH), 2239.35
(CN), 1489.04, 1267.23 and 1055.06 (ethers C-O), 752.2382 (Ar-Br).
HRMS m/z [M + H]+: calculated: 485.3321; found: 484.9840.
6.2.5.
1-(2,6-dichlorobenzylideneamino)-5-oxo-1,5-dihydro-10H-spiro[acridine-9,2-
pyrrole]-4-carbonitrile (AMTAC-10)
Yellow powder. Formula: C24H14Cl2N4O; M.W.: 445.3002 g/mol; Yield: 77.1%;
1
Melting Point: 217-219º C; Rf: 0.58 (n-hexane/EtOAc 7:3). H-NMR (DMSO-d6): δ
9.76 (s, 1H, NH), 8.57 (s, 1H,CH), 8.31 ppm (s, 1H, N=CH), 7.36 (m, 2H, aromatic),
7.30 (m, 1H, aromatic), 7.25 (m, 2H, spiro-acridine), 6.99 (m, 4H, spiro-acridine), 6.84
(m, 2H, spiro-acridine). 13C NMR: δ 68.92, 108.33, 111.19, 112.68, 115.64, 120.88,
127.61, 129.40, 130.40, 130.71, 131.84, 134.10, 138.93, 141.68, 161.42, 161.55. IR
(KBr, cm-1): 3331.06 (NH), 3089.96 (CH), 2237.42 (CN), 1537.26 (C=N), 783.09 (Ar-
Cl).
HRMS m/z [M + H]+: calculated: 445.3002; found: 445.0370.

6.2.6.
1-(4-dimethylaminobenzylideneamino)-5-oxo-1,5-dihydro-10H-spiro[acridine-
9,2-pyrrole]-4-carbonitrile (AMTAC-11)
Orange powder. Formula: C26H21N5O; M.W.: 419.4778 g/mol; Yield: 80.6%; Melting
Point: 214-216º C; Rf: 0.38 (n-hexane/EtOAc 7:3). 1H-NMR (DMSO-d6): δ 9.81 (s, 1H,
NH), 8.50 (s, 1H, CH), 8.35 (s, 1H,N=CH), 7.28 (m, 2H, spiro-acridine), 7.21 (d, J=
8Hz, 2H, aromatic), 7.01 (m, 4H, spiro-acridine), 6.85 (m, 2H, spiro-acridine), 6.61 (m,
2H, aromatic), 2.89 (s, 6H, N(CH3)2). 13C NMR: δ 40.09, 69.29, 108.64, 112.03, 112.26,
113.07, 115.51, 120.83, 121.21, 127.49, 128.93, 130.48, 139.11, 150.38, 152.29,
160.62, 160.98. IR (KBr, cm-1): 3304.06 (NH), 3084.17 (CH), 2239.35 (CN), 1481.33
(C=N).
HRMS m/z [M + H]+: calculated: 419.4778; found: 420.1600.
6.2.7. 1-(2-nitrobenzylideneamino)-5-oxo-1,5-dihydro-10H-spiro[acridine-9,2-pyrrole]-
4-carbonitrile (AMTAC-13)
Yellow powder. Formula: C24H15N5O3; M.W.: 421.4076 g/mol; Yield: 61%;
1
Melting Point: 212-215º C; Rf: 0.48 (n-hexane/EtOAc 7:3). H-NMR (DMSO-d6): δ
9.83 (s, 1H, NH), 8.57 (s, 1H, CH), 8.56 (s, 1H, N=CH), 7.90 (m,1H, aromatic), 7.71
(m, 1H, aromatic), 7.64 (m, 1H, aromatic), 7.54 (m, 1H, aromatic), 7.26 (m, 2H, spiro-
acridine), 6.99 (m, 4H, spiro-acridine), 6.81 (m, 2H, spiro-acridine). 13C NMR: δ 69.16,
107.97, 111.00, 112.70, 115.77, 120.88, 125.11, 127.36, 127.81, 128.34, 130.75,
131.54, 134.20, 139.02, 141.08, 148.40, 161.42, 161.74. IR (KBr, cm-1): 3325.27 (NH),
3095.74 (CH), 2237.42 (CN), 1556.55 and 1342.45 (Ar-NO2).
HRMS m/z [M + H]+: calculated: 421.4076; found: 422.0970.
6.2.8. 1-(4-nitrobenzylideneamino)-5-oxo-1,5-dihydro-10H-spiro[acridine-9,2-pyrrole]-
4-carbonitrile (AMTAC-14)
Brown powder. Formula: C24H15N5O3; M.W.: 421.4076 g/mol; Yield: 86.4%; Melting
1
Point: 281-283º C; Rf: 0.5 (n-hexane/EtOAc 7:3). H-NMR (DMSO-d6): δ 9.87 (s, 1H,
NH), 8.59 (s,1H, CH), 8.45 (s, 1H, N=CH), 8.15 (d, J=10Hz, 2H, aromatic), 7.59 (m,
2H, aromatic), 7.27 (m, 2H, spiro-acridine), 7.02 (m, 4H, spiro-acridine), 6.83 (m, 2H,
spiro-acridine). 13C NMR: δ 71.59, 110.69, 113.69, 114.86, 117.84, 123.16, 126.78,
129.71, 130.41, 132.96, 141.24, 142.35, 147.01, 150.72, 163.70. IR (KBr, cm-1):
3325.27 (NH), 3048.17 (CH), 2239.35 (CN), 1724.36 (C=O), 1517.97 and 1435.03 (Ar-
NO2).

HRMS m/z [M + H]+: calculated: 421.4076; found: 422.1040.
6.2.9. 1-(3-ethoxy-4-hydroxybenzylideneamino)-5-oxo-1,5-dihydro-10H-spiro[acridine-
9,2-pyrrole]-4-carbonitrile (AMTAC-16)
Yellow powder. Formula: C26H20N4O3; M.W.: 436.4620 g/mol; Yield: 70.3%; Melting
Point: 210-213º C; Rf: 0.38 (n-hexane/EtOAc 7:3). 1H-NMR (DMSO-d6): δ 9.83 (s, 1H,
NH), 9.52 (s, 1H, OH), 8.54 (s, 1H, CH), 8.25 (s, 1H, N=CH), 7.27 (m, 2H, aromatic),
7.00 (m, 1H aromatic and 4H spiro-acridine), 6.83 (m, 2H, spiro-acridine), 6.70 (s, 2H,
aromatic), 3.93 (q, J= 5 and 15 Hz ,2H, OCH2), 1.29 (t, J= 5 and 15 Hz, 3H, CH3). 13C
NMR: δ 17.26, 66.37, 71.40, 111.10, 112. 89, 114.32, 115.11, 117.65, 118.10, 123.00,
124.52, 127.71, 129.63, 132.72, 141.32, 149.69, 151.00, 152.33, 162.78. 163.28. IR
(KBr, cm-1): 3516.23 (Ar-OH), 3296.34 (NH), 2241.28 (CN), 1487.11, 1269.16 (Ar-
OCH2CH3), 1203.58 (phenol C-O).
HRMS m/z [M + H]+: calculated: 436.4620; found: 437.4620.
6.3. ctDNA interaction evaluation by UV-vis absorption
ctDNA was solubilized in Tris-HCl (0.01 M, pH 7.6) buffer and the DNA concentration
was determined using the molar extinction coefficient of 6600 M-1 cm-1 at 260 nm.37
DNA concentration was expressed as micromolar equivalents of the base pairs. Spiro-
acridine derivatives were dissolved in DMSO at a concentration of 1 mM (stock
solution) from which the working solutions were prepared by dilution using Tris-HCl
(0.01 M, pH 7.6) buffer at concentrations ranging from 1 to 10 µM. The interaction
studies were performed by maintaining the ctDNA concentration (10 µM pb) and
varying the derivatives concentrations (1-10 µM) up to relation of 1:1 molar. Before the
measurements, the system was stirred and incubated at room temperature for 10
minutes. The measurements were performed in a rectangular quartz cuvette with 1 cm
path length, at 24 ºC. The intrinsic binding constant (Kb) was obtained by fitting the
data to equation 1:30
[DNA]/(εa – εf) = [DNA]/(εb – εf) + 1 / Kb (εb – εf)
(1)
εa, εb and εf were the apparent, bound, and free extinction coefficients, respectively.
Plot fitting of [DNA]/(εa – εf) vs. [DNA] used Kb obtained from the ratio of the slope
to the Y intercept. The binding data were fitted using a SigmaPlot 10.0 software.

6.4. BSA interaction assay
The BSA tryptophan fluorescence measurements were performed with solution
concentration of 10 µM of BSA in Tris-HCl (0.01 M, pH 7.6) buffer, at 24 ºC in a
rectangular quartz cuvette with 1 cm path length. Emission spectra were recorded in the
region 310–400 nm after excitation at 285 nm both in the absence and the presence of
spiro-acridine derivatives. Fluorescence titrations were conducted by the addition of
increasing amounts of AMTAC’s (0-10 µM) directly into the cell containing solutions
of BSA. Fluorescence emission changes were used to calculate the Stern-Volmer
constant according the equation 2:28
F0 / F = 1 + KSV [Q]
(2)
where F0 and F are the fluorescence intensities in the absence and presence of spiro-
acridine derivatives. KSV is the constant linear suppression. [Q] is the concentration of
derivatives. The plot of relative emission intensity (F0/F) versus [Q] was used to obtain
the constant from the linear regression. The binding data were fitted using a SigmaPlot
10.0 software.
6.5. Human topoisomerase II inhibition assay
The human topoisomerase IIα inhibition activity was determined according Almeida et
al.19 Briefly, the relaxation of 100 ng of pUC19 DNA plasmid (from Sigma) by 4.0 unit
of recombinant human (p170) topoisomerase IIα (Sigma-Aldrich) in buffer (50 mM
Tris-HCl, pH 8.0, 120 mM KCl, 10 mM MgCl2, 0.5 mM ATP and 0.5 mM
dithiothreitol) was evaluated after incubation for 30 min at 37 °C without or with spiro-
acridines. All AMTAC’s compounds were tested at the concentration of 100 μM and
m-AMSA was used as positive control in the same concentration. Besides, AMTAC-01
and AMTAC-02 were also tested to compare the topo inhibition in relation to the
substitution of the new derivatives. Reactions were terminated with 1% SDS and
digested with proteinase K. Agarose gel (0.8%) electrophoresis was performed in SB
buffer (NaOH + boric acid) at 7 V/cm for 2 h. The gel was stained with ethidium
bromide (1 mg/ml) and photographed under UV light. Quantitative evaluation of the gel
bands corresponding to supercoiled DNA was performed by densitometry using an

image-processing software (Scion Image, version Beta 4.0.2.). The optic densities of the
bands for each AMTAC derivative, as well as all controls (only plasmid DNA, DNA +
topo and mAMSA) were obtained from which the values of density average were
obtained and used for calculating the enzyme inhibition percentage of the compounds in
relation to the positive control mAMSA.
6.6. In silico studies
6.6.1. In silico ADME and Molecular Docking Analysis
In silico molecular interaction analysis studies on the test compounds with BSA and
topoisomerase 2 were performed using AutoDock4.2.6 in combination with the
Lamarckian genetic algorithm.38 The ADME profiles of the synthetized compounds
were investigated with the use of SwissADME and pkCSM webplatforms.39,40
6.6.2 Molecular docking
6.6.2.1. Ligand Structure Preparation
The 3D structures were built with Avogadro 1.2.0 software and fully optimized with
PM6 semiempirical method implemented in the MOPAC2016.41-43 The optimized
ligands were then saved as pdb files. With the use of AutoDockTools-1.5.6, the non-
polar hydrogens were merged with the corresponding carbons, then partial charge of
atoms were calculated using the Gasteiger procedure implemented in the
AutoDockTools package. The rotatable bonds of the ligands were defined, the structures
were saved as pdbqt and used for docking studies.
6.6.2.2. Protein Structure Preparation
The crystal structures of BSA (PDB ID: 4OR0) and topo 2 (PDB ID: 5GWK) were
retrieved from RCSB Protein Data Bank. With the use of Dassault Systèmes BIOVIA
Discovery Studio Visualizer (v16.1.0.15350),44 water molecules and other heteroatoms
were removed. Magnesium ions from topo II were kept. Then, using AutoDockTools,
non-polar hydrogens were merged, and polar hydrogens added to the structure of the
proteins. Kollman charges were added and the structure was saved as pdbqt for the
docking studies. A partial charge of +0.8 was manually attributed to magnesium ions.

6.6.2.3. Docking procedure
The 3D grid was created by the Autogrid algorithm to generate the grid parameter file.
The grid spacing was 0.0375 nm in each dimension. For Topoisomerase, each grid map
consisted of a 60 × 60 × 60 grid point, and the center was set to the position of the co-
crystalized ligand located at the Chain A. For albumin, in order to cover all binding sites
in the protein, two grid maps were generated, each one consisted of a 126 x 126 x 126
grid point, centered in binding sites I and II.
The Lamarckian genetic algorithm in AutoDock4.2.6 was applied to search the
best conformation and orientation of the ligands. The global optimization was started
with a population of 150 randomly positioned individuals with a maximum of 2500000
energy evaluations and a maximum of 27000 generations. During each docking
experiment, 100 runs were carried out, generating 100 conformations for
Topoisomerase and 200 for BSA. The resulting docking poses were analyzed using
AutoDockTools and Discovery Studio Visualizer.
Acknowledgments
Authors are thankful to CNPq (Conselho Nacional de Desenvolvimento Científico e
Tecnológico, Brazil, grant number 407355/2016-2), CAPES (Fundação Coordenação de
Aperfeiçoamento de Pessoal de Ensino Superior, Brazil), FACEPE (Fundo de Amparo à
Ciência e Tecnologia de Pernambuco, Brazil) for financial support.
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