DNA intercalators based on (1,10-phenanthrolin-2-yl)isoxazolidin-5-yl core with better growth inhibition and selectivity than cisplatin upon head and neck squamous cells carcinoma

Article abstract of DOI:10.1016/j.ejmech.2017.11.067

((3RS,5SR)- and ((3RS,5RS)-2-(2-methoxybenzyl)-3-(1,10-phenanthrolin-2-yl)isoxazolidin-5-yl)methanol have been synthesized, according to 1,3-dipolar cycloaddition methodology, as DNA intercalating agents and evaluated for their anticancer activity against

Full text of DOI:10.1016/j.ejmech.2017.11.067

Accepted Manuscript
DNA intercalators based on (1,10-phenanthrolin-2-yl)isoxazolidin-5-yl core with better
growth inhibition and selectivity than cisplatin upon head and neck squamous cells
carcinoma
Maria G. Varrica, Chiara Zagni, Placido G. Mineo, Giuseppe Floresta, Giulia
Monciino, Venerando Pistarà, Antonio Abbadessa, Angelo Nicosia, Rogerio M.
Castilho, Emanuele Amata, Antonio Rescifina
PII:
S0223-5234(17)30970-4
10.1016/j.ejmech.2017.11.067
EJMECH 9942
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 October 2017
Revised Date: 22 November 2017
Accepted Date: 24 November 2017
Please cite this article as: M.G. Varrica, C. Zagni, P.G. Mineo, G. Floresta, G. Monciino, V. Pistarà,
A. Abbadessa, A. Nicosia, R.M. Castilho, E. Amata, A. Rescifina, DNA intercalators based on (1,10-
phenanthrolin-2-yl)isoxazolidin-5-yl core with better growth inhibition and selectivity than cisplatin upon
head and neck squamous cells carcinoma, European Journal of Medicinal Chemistry (2017), doi:
10.1016/j.ejmech.2017.11.067.
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ACCEPTED MANUSCRIPT
DNA intercalators based on (1,10-phenanthrolin-2-yl)isoxazolidin-5-yl core with better
growth inhibition and selectivity than cisplatin upon head and neck squamous cells carcinoma
a
a
b–e
a,b
a
Maria G. Varrica , Chiara Zagni , Placido G. Mineo , Giuseppe Floresta , Giulia Monciino ,
a
b
b
f
Venerando Pistarà , Antonio Abbadessa , Angelo Nicosia , Rogerio M. Castilho , Emanuele
a
a,*
Amata , Antonio Rescifina
a
Dipartimento di Scienze del Farmaco, Università degli Studi di Catania, V.le A. Doria, 95125
Catania, Italy
b
Dipartimento di Scienze Chimiche, Università di Catania; Viale A. Doria, 6; 95125- Catania, Italy.
c
CNR-IPCB Istituto per i Polimeri, Compositi e Biomateriali; Via P. Gaifami 18, I-95126 Catania,
Italy
d
CNR-IPCF Istituto per i Processi Chimico-Fisici; Viale F. Stagno d’Alcontres 37, I-98158,
Messina, Italy
e
INSTM Consorzio Interuniversitario Nazionale per la Scienza e Tecnologia dei Materiali (UdR of
Catania); Viale A. Doria, 6; I-95125, Catania, Italy
f
Laboratory of Epithelial Biology, Department of Periodontics and Oral Medicine, University of
Michigan School of Dentistry, Ann Arbor, MI 48109-1078, USA
Corresponding author:
Antonio Rescifina arescifina@unict.it
Graphical Abstract
Highlights
•
•
•
New 1,10-phenanthrolin-2-yl-substituted isoxazolidines have been synthetized
The molecules act as DNA intercalators with anticancer activity
The anticancer activity against the cisplatin-resistant HN13 cell lines was of 4.5 µM
Keywords
DNA-intercalators; 1,3-Dipolar cycloaddition; Isoxazolidines; HeLa; Head and neck squamous cells
carcinoma

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Abstract
(3RS,5SR)-
(
and
((3RS,5RS)-2-(2-methoxybenzyl)-3-(1,10-phenanthrolin-2-yl)isoxazolidin-5-
yl)methanol have been synthesized, according to 1,3-dipolar cycloaddition methodology, as DNA
intercalating agents and evaluated for their anticancer activity against human cervical carcinoma
HeLa and head and neck squamous cells carcinoma cell lines. The synthesized compounds
exhibited good cytotoxic activity with IC50 better than cisplatin, used as the main and effective
treatment for HNSCC, and a 24.3–72.0-fold selectivity respect to the 184B5 non-cancerous
immortalized breast epithelial cell lines. Unwinding assay, circular dichroism data, and Uv-vis
melting experiments confirmed that these compounds act as DNA intercalators with a binding
–
4
–1
constant in the order of 10 M . Docking studies showed that both compounds can interact as
intercalating agent with both poly-d(AT) and poly-d(GC) , preferring an entrance by the minor
2
2
groove of the poly-d(AT)2.

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1. Introduction
The discovery of new compounds with antitumor activity is one of the most important goals in
medicinal chemistry [1-4]. An important class of chemotherapeutic agents used in cancer therapy is
represented by small molecules that interact with DNA. Two broad classes of noncovalent DNA-
binding agents have been identified, the intercalators [5, 6] and the groove binders [7]. Intercalators
are small molecules that can reversibly bind in between adjacent base pairs of double-stranded
DNA (dsDNA) by inserting a planar aromatic chromophore. This process causes lengthening,
stiffening and unwinding of the DNA helix. Groove binders fit into the DNA minor groove causing
little perturbation of the DNA structure [8].
Representative examples of classical intercalators are acridine derivatives such as DACA [9], or
acridine orange [10], dyes such as methylene blue [11], phenanthridine derivatives such ethidium
bromide [12], doxorubicin [13], quinacrine [14], and actinomycin [15] (Fig. 1).
Br
N
N
N
N
N
N
O
N
H2N
NH2
H
DACA
Acridine
Ethidium bromide
O
O
OH
OH
N
S
OH
N
HN
O
N
N
O
O
OH
O
O
Cl
NH2
OH
Quinacrine
Methylene blue
Doxorubicin
SO2CH3
NH
H
O
N
H3CO
OH
OH
O
O
HN
OH
OH
N
N
O
HO
O
HN
N
H
N
Amsacrine
Camptothecin
Mitoxantrone
Fig. 1. Selected DNA intercalating agents and Topoisomerase inhibitors.
In 1970 a group of enzymes, called topoisomerases [16], involved in the control of the shape of
DNA, was discovered. These enzymes control the topology of the supercoiled DNA double helix

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during the transcription of replication of cellular genetic materials. They allow the relaxation of
supercoiled DNA through a mechanism implicating the breakage of a phosphodiester bond of either
one strand (Topo I) or both strands (Topo II) of the duplex DNA [17]. Among topoisomerase I
poisons camptothecin and its derivatives are the best representatives [18], while topoisomerase II
inhibitors examples are amsacrine and mitoxantrone [19] (Fig. 1).
Following our ongoing interest in the synthesis and biological evaluation of new DNA intercalators
[
20-25], here we report the synthesis and the biological activity of novel functionalized
isoxazolidines where the aromatic moiety has been replaced by 1,10-phenanthroline ring [26, 27].
We reasoned that phenanthrolin-isoxazolidine conjugates might provide new compounds for the
development of bioactive mimics. Thus, the main focus of this paper is to design a new
phenanthrolin-isoxazolidines and verify whether these new hybrid agents induce antiproliferative
effect, leading to cell growth perturbation.
Compounds 6 have been synthesized in good yields according to the 1,3-dipolar cycloaddition
methodology [28-32], which is a chemical reaction between a 1,3-dipole and a dipolarophile to
produce five-membered rings, just reported by us in an eco-friendly procedure [33]. The ability of
the new compounds to intercalate into DNA and inhibit Topo I enzyme was tested in vitro.
Moreover, the compounds were screened over three carcinoma cell lines, human cervical carcinoma
(HeLa) and head and neck squamous cells carcinoma (HNSCCs), in order to test their ability to
inhibit cancer cells growth. The compounds showed anticancer activity in the low micromolar range
with the cis stereoisomer slightly more active than the trans one.
Finally, circular dichroism, Uv-vis melting experiments, and docking studies demonstrate that
compounds 6 effectively intercalate between DNA base-pairs approaching the duplex from the
minor groove and quantitatively justify the observed biological results.
2. Results and discussion
2.1. Chemistry
The strategy of the synthetic approach is based on the construction of new nitrone 5, which has been

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prepared as a mixture of Z/E isomers (9:1), in 47% yield, by reaction of 1,10-phenanthroline-2-
carbaldehyde 4 [34] and N-(2-methoxybenzyl)hydroxylamine 3 [35].
1
The H NMR spectrum of crude reaction shows the presence of two diagnostic methine protons at
9.18 and 9.19 ppm indicative of the presence of E/Z isomers. NOE measurements allow assigning
the configuration of the major compound. In particular, irradiation of the benzylic proton at 5.24
ppm induced a positive NOE effect on the downfield resonance of methine proton at 9.18
representative of a cis relationship between these protons, thus suggesting a Z configuration for this
nitrone. On the contrary, no NOE effect was observed for the minor compounds when the benzylic
proton at 5.29 was irradiated.
Subsequent reaction of nitrone 5 with allyl alcohol in excess at 100 °C in a sealed tube for 3 h
afforded a mixture of two isoxazolidines 6 (Scheme 1). The cycloaddition reaction proceeds with
high regioselectivity but low stereoselectivity giving rise only 5-substituted regioisomers in a
cis/trans configuration and in 1:1 ratio.
Scheme 1. Synthesis of target isoxazolidine derivatives. Reagents and conditions: a) sodium acetate, CH Cl , 30 min at
2
2
0
°C and then at room temperature overnight; b) allyl alcohol, sealed tube 100 °C for 3 h.

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1
13
The structure of adducts has been elucidated by H NMR and C NMR spectroscopies and MS
1
spectrometry. The H NMR spectrum of 6 as C regioisomers exhibit the diagnostic resonance of
5
the H protons at 4.34 and 4.55 ppm, while the methylene protons at C resonate at 2.62–2.66 and
5
4
2.91–3.10 ppm. As regards the 4-substituted regioisomers it is known that these compounds do not
show resonance at these chemical shift values.
The cis/trans configuration of the obtained adducts has been determined by NOE experiments. In
particular, irradiation of H (4.55 ppm) in compound 6a induces a positive NOE effect on the
5
downfield resonance of methylene proton at C (H ), centered at 3.10 ppm, and on H , centered at
4
4b
3
4
.75 ppm. Conversely, the irradiation of H not induced any positive NOE effect on H and H
4a 3 5
protons, but produces a positive effect on H and the hydroxymethylenic protons linked to C of
4
b
5
isoxazolidine ring. These results revealed that H , H , and H are in a cis relationship. Irradiation of
3
4b
5
H in compound 6b induces a NOE enhancement for the downfield resonance of methylene protons
5
at C (H ) centered at 2.91 ppm; irradiation of H centered at 4.81 ppm produced a positive NOE
4
4b
3
on H4a, while the irradiation of H induced a positive NOE on H , H and the hydroxymethylenic
4a
3
4b
protons at C . Finally, the irradiation of H generated a positive NOE on H . These data
5
4b
5
unequivocally indicate that compound 6b is in a trans configuration. (Scheme 1).
2
2
.2. Biological evaluation
.2.1. Cytotoxicity assays
The cytotoxicity of compounds 6 was evaluated in vitro against HeLa and HNSCCs cell lines HN6
and HN13. As a screening assay, the cytotoxicity was tested using an MTT tetrazolium reduction
assay and expressed as IC values where IC is the drug concentration causing 50% inhibition of
50
50
the cell growth. Results shown in Table 1 indicates that the isoxazolidines 6 displayed good
cytotoxicity against the tested cell lines. In particular, the new derivatives 6 showed a slightly
improved cytotoxic activity toward HeLa cells respect to the compounds previously reported by us
exhibiting IC50 values in the low micromolar range [22, 23]. The stereochemical configuration
disclosed a low impact on the activity. In fact, the cis isoxazolidine 6a was slightly more active than

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the trans one 6b over all the cell lines tested.
Table 1
a
In vitro efficacy assay of compounds 6 on selected HNSCC cell lines and on non-cancerous cells expressed as IC50
(
µM) at 24 h
Compound
HeLa
HN6
6.3 ±0.7
24.8 ±1.1
21.6 ±1.5
HN13
184B5
6
6
a
b
13.3 ±0.2
40.2 ±1.4
43.3 ±2.3
4.5 ±0.6
25.4 ±1.0
41.6 ±2.4
324 ±12
381 ±17
45.4 ±2.9
Cisplatin
a
Mean of three independent triplicate experiments ± standard error.
Interesting, compound 6a displayed better growth inhibition than cisplatin (3.4–9.2-fold), here
chosen as reference compound because it is an established and effective treatment for HNSCC [36];
moreover, and more importantly, these cell lines are particularly resistant to cisplatin. Finally, to
determine if the newly synthesized compounds have differential cytotoxic effects on cancer and
non-cancer cells, their cytotoxicity was also evaluated using 184B5 non-cancerous immortalized
breast epithelial cell lines. We found that the antiproliferative activity of compound 6a was not
pronounced against the non-cancerous cell lines (Table 1) with 24.3, 51.4, and 72.0-fold selectivity
respect to the more resistant Hela, HN6, and HN13 cancer cell lines, respectively.
2.2.2. Unwinding assay
DNA topoisomerases are essential enzymes that exert their important cellular roles by relaxing the
superhelical tension [37]. Topoisomerase I (Topo I) topoisomerases catalyze the relaxation of
supercoiled DNA by introducing transient single-stranded DNA breaks in one of the phosphodiester
backbones of the duplex DNA. This results in a reversible Topo-I/DNA covalent complex followed
by the reorganization and the reconnection of the damaged DNA strand.
In order to investigate the ability of compounds 6 to inhibit Topo I or effectively intercalate into
DNA base pairs, we performed an agarose-gel electrophoresis experiment ad-hoc designed, based
on the different electrophoretic mobility of supercoiled and relaxed conformations of DNA. The
relaxation assay using topoisomerase I is one of the most robust approaches to evidence
intercalation of small molecules into DNA [38].

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Relaxed plasmid DNA (pGEM-T Easy) was incubated with Topo I enzyme alone or in the presence
of the examined compound 6. Topo I inhibitors will prevent the enzyme from changing the state of
the relaxed DNA; whereas, in the presence of an intercalator, Topo I will convert the relaxed DNA
into a supercoiled state. As seen in the right side of Fig. 2, relaxed pGEM-T Easy plasmid substrate
was converted to negatively supercoiled molecules by treatment with Topo I in the presence of
molecules 6 at a concentration of 10 nM, indicating that it acts as DNA intercalator.
Fig. 2. Intercalation into DNA of compounds 6. Intercalation was monitored by conversion of relaxed (R) pGEM-T
Easy plasmid to negatively supercoiled molecules (SC). An agarose gel stained with ethidium bromide is shown.
Control reactions were carried out in the absence of enzyme [from left to right: SC, R, compound 6a + plasmid + Topo
I, compound 6b + plasmid + Topo I].
2.3. Circular dichroism
To gain a deeper insight into the changes of polynucleotide properties induced by 6 binding, with
the aim to prove, or not, the postulated intercalation between base pairs, we studied, by circular
dichroism (CD) technique, the behavior of this compound on interaction with calf-thymus DNA (ct-
DNA).
The CD spectrum of ct-DNA, in B form, displays two conserved peaks at 246 and 276 nm; the first
one, negative, is due to right-handed helicity whereas the second one, positive, is due to base
stacking [39].
The CD titration spectra of ct-DNA, monitored in the presence of increasing amounts of 6a were
shown in Fig. 3. Increasing concentration of 6a decreases the intensity of 276 nm signals, without
any shift in its positions, and a concomitant appearance of a negative induced CD (ICD) signal at

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271 nm (as a proof of the intercalation phenomenon). These results are consistent with an
intercalative binding of the 1,10-phenanthroline moiety. In particular, the changes in the intrinsic
CD spectrum of the ct-DNA reflect the diminished helicity (helix unwinding) and the extent of base
stacking accompanied by stabilization of the right-handed B conformation of ct-DNA, as frequently
observed for intercalators [40].
Fig. 3. CD spectra of 6a/ct-DNA system titration at 25 °C (up) and UV-Vis spectrum of 6a (down). Increasing
concentration of 6a decreases the intensity of the CD signal.
The CD results of compound 6b (Fig. 4) are similar to that of compound 6a but without the ICD.
Finally, the small negative ICD signal at 271 nm further prove the intercalation phenomenon
contemporarily establishing the geometry of ligand: the ICD sign is in accord with the pyrene
moiety perpendicular to the DNA axis with its long direction almost parallel to the base-pair long
axis [41, 42]. It can be noticed that, although compounds 6a and 6b are chiral species, they are a

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racemic mixture. In fact, these systems show a silent CD spectrum, indicating that the observed ICD
phenomenon is genuine.
These CD experimental data are qualitatively well in accord to those obtained in silico (Table 2).
Fig. 4. CD spectra of 6b/ct-DNA system titration at 25 °C (up) and UV-Vis spectrum of 6b (down). Increasing
concentration of 6b decreases the intensity of the CD signal.
2.4. Evaluation of binding constants by means of ct-DNA melting experiments
To investigate the quantitative interaction between ct-DNA and compounds 6, the thermal melting
of the DNA/6 complexes were evaluated by means of thermally controlled UV-vis experiments. In
this way, because of the interactions of 6 with DNA, a variation of the melting temperature is
recorded, revealing an enhancement of the ct-DNA melting temperature.
From UV-vis measurements, in absence of 6, the melting of ct-DNA in PBS solution results 354 K,
that increased at 358 K and 355 K when it is in mixture with compounds 6a and 6b, respectively.
From the melting temperature differences between free ct-DNA and the ct-DNA/6 complexes (Fig.
S1), using the equation 1, it is possible to calculate the binding constants of the drug molecule with
ct-DNA [43].

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ꢀ
ꢀ
ꢄ
− ꢁ
= ꢃ
ꢉ ln ꢊ( 1 + ꢋꢌ ꢎ) eq. 1
ꢍ
ꢁ
°ꢂ
ꢅ∆ꢆꢇꢈ
ꢂ
In this equation, T° is the optical melting temperature of the free ct-DNA, T is the optical melting
m
m
temperature of ct-DNA in the presence of the intercalating molecule, ∆Hwc is the enthalpy of ct-
DNA melting, R is the gas constant, KT , is the drug binding constant at T , is the free drug
activity, and n is the site size of the drug binding. The binding constants (KT ), calculated from the
α
m
m
m
4
–1
3
–1
melting curves, result 4.53•10 M and 9.26•10 M for 6a and 6b, respectively.
These experimental data quantitatively parallel those obtained both in vitro and in silico (Tables 1
and 2) and, taken together with the Topoisomerase 1 and CD results, suggest that the biological
activity observed for compounds 6 is effectively due to their intercalation between DNA base-pairs.
2.5. Docking studies
In order to confirm and rationalize the observed biological results and to get more insight into the
intercalation modality, the supramolecular complexes of synthesized compounds with DNA have
been investigated by molecular modeling methodology. Although the obtained compounds are
racemates, we have already demonstrated that compounds with a 3R configuration possess the best
intercalating properties [22]. Thus, all molecular docking calculations were performed on 3R
stereoisomers.
For in silico studies, we adopted an already validated molecular modeling template consisting of
three steps [23], employing, for the minimization, the AMBER14 force field including the OL15
DNA fine-tuning, that is one of the most accurate force fields widely used for proteins and DNA
[
44].
The possibility to achieve complexes by binding along the groove was a priori excluded according
to previously reported results [21].
The obtained results showed that compound 6a show the best intercalative properties. In particular,
the inspection of Table 2 indicates that compound 6a exhibits the highest activity, followed by 6b,
according to the biological results. Moreover, compounds 6, in general, show a preference for the
poly-AT fragment intercalating from the minor groove.
Fig. 5 reports a plot of compound 6a intercalated into poly-d(AT) from minor groove. The
2
interaction of this compound with nucleobases involve one hydrogen bond between the hydroxylic

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group of compounds 6a and an adenine of the DNA backbone. However, other important
hydrophobic and van der Waals interactions are established between the DNA and the intercalating
portion of the molecule (1,10-phenanthroline ring). Particularly, this hydrogen bond is not present
neither in the complex of the same molecule with poly-d(AT) from the major groove nor in the
2
complexes of 6a with poly-d(GC) . Thus, we speculate that the selectivity and the potency of the
2
molecule 6a, respect to 6b, is probably due to this hydrogen bond, which can be classified as a
moderate, mostly electrostatic interaction.
Table 2
a
Calculated binding energies for compounds 6 intercalated in d(AT) and d(GC) dodecamers
2
2
Compound
Poly-AT
From major groove
–7.61
From minor groove
–10.01
(
(
3R,5S)-6a (cis)
3R,5R)-6b (trans)
–7.21
–9.62
Poly-GC
From major groove
From minor groove
(
3R,5S)-6a (cis)
3R,5R)-6b (trans)
All values are in kcal/mol.
–7.61
–7.14
–8.88
–7.96
(
a

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Fig. 5. Interaction of 6a with poly-d(AT) from minor groove. a) Front view, b) side view, c) 2D-interactions.
2
3. Conclusions
In this study, we report the synthesis and the biological properties of two novel 1,10-
phenanthroline-bearing isoxazolidinyl derivatives. The obtained compounds show an interesting
antitumor activity, due to DNA intercalation, over three carcinoma cell lines. All of the obtained
compounds were tested for their in vitro cytotoxic activity toward three different human tumor cell
lines, and the most-potent one, 6a, showed an IC of 4.5 µM toward the HNSCCs cell lines HN13.
5
0
Moreover, compound 6a showed a 72-fold selectivity upon the 184B5 non-cancer immortalized
breast epithelial cell lines. The HNSCCs is the sixth leading cancer by incidence worldwide.
Patients with HNSCCs are treated with multiple-modality therapies concerning surgery, radiation,

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and chemotherapy. However, long-term survival rates in patients with advanced-stage HNSCCs
have not increased significantly in the past 30 years [45, 46]. Although cisplatin is currently the
most commonly used chemotherapeutic agent for HNSCCs, its administration is associated with
systemic toxicities that often reduce compliance and prevent timely completion of therapy. To the
best of our knowledge compound 6a is the first intercalating agent that possesses a better growth
inhibition (3.4–9.2-fold), upon cancerous HN6 and HN13 cell lines, and selectivity (24.4–65.9-
fold), upon non-cancerous 184B5 cell lines, than cisplatin.
The intercalating properties, determined by electrophoresis (unwinding assay) and circular
dichroism, and the estimated binding constants, calculated by Uv-vis melting point measurements,
are consistent with molecular docking studies.
4. Experimental Section
4.1. Chemistry
Solvents and reagents were used as received from commercial sources. Melting points were
determined with a Kofler apparatus and are reported uncorrected. Elemental analyses were
1
performed with a Perkin–Elmer elemental analyzer. NMR spectra ( H NMR recorded at 500 MHz,
1
3
C NMR recorded at 125 MHz) were obtained on Varian Instruments and are referenced in ppm
relative to TMS or the solvent signal. Thin-layer chromatographic separations were performed on
Merck silica gel 60-F254 precoated aluminum plates. Flash chromatography was accomplished on
Merck silica gel (200–400 mesh). The circular dichroism spectra were recorded by means of
JASCO J-815 spectropolarimeter equipped with a 150W Xenon lamp. The ellipticity was obtained
calibrating the instruments with a 0.06% (w/v) aqueous solution of ammonium d-10-
camphorsulfonate and with a 0.08% (w/v) aqueous solution of tris(ethylenediamine)cobalt(III)
chloride complex 2{(–)-∆-[Co(en) ]Cl }•NaCl•6 H O. The measurements, corrected for the
3
3
2
contribution from cell and solvent, were performed at a constant temperature of 25 °C in quartz
cells. The temperature of J-815 was controlled by means of a Jasco PTC-423S/15 Peltier-type

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temperature control system cooled with an external water circulator. The spectra have been
corrected to take into account the dilution effect after each addition. UV-visible spectra were
recorded both at room and variable temperature using a Shimadzu Model 1601 spectrophotometer
equipped with a Peltier unit, in quartz cells, using PBS solution as a solvent. Compounds 2 and 4,
although commercially available, were synthesized as reported in the literature [47, 48].
4.1.1. Synthesis of N-(2-methoxybenzyl)hydroxylammonium chloride 3
To a solution of 2-methoxybenzaldehyde oxime 2 (2.15 g, 0.014 mol) in dry CH OH (30 mL),
3
containing a trace of methyl orange, NaBH CN (1.37 g, 21.9 mmol) was added. Subsequently, 2.5
3
M HCl–MeOH was added dropwise with stirring to maintain the red color of the solution for 30
min. The reaction mixture was further stirred for 4 h, and methanol was removed under vacuum to
1
give a white solid. Yield 98%, m.p. 136–139 °C. H NMR (DMSO, 500 MHz): 3.81 (s, 3H, CH ),
3
1
3
4
5
.27 (s, 2H, CH ), 6.96–7.43 (m, 4H), 10.98 (bs, 2H, NH ), 11.46 (bs, 1H, OH). C NMR (DMSO,
2 2
00 MHz): 48.96, 55.52, 110.95, 117.61, 120.08, 130.80, 132.07, 157.66. Anal. Calcd for
C H NO •HCl: C, 50.67; H, 6.38; N, 7.39%. Found: C, 50.78; H, 6.37; N, 7.37%.
8
11
2
4.1.2. Synthesis of N-(2-methoxybenzyl)-1-(1,10-phenanthrolin-2-yl)methanimine oxides 5
To a solution of sodium acetate (0.79 g, 0.0096 mol) in dichloromethane (30 mL), cooled at 0 °C,
was added the N-(2-methoxybenzyl)hydroxylammonium chloride 3 (1.33 g, 0.0096 mol) and
successively, dropwise, the corresponding 1,10-phenantroline-2-carbaldehyde 4 (0.67 g, 0.0032
mol). The reaction mixture was then stirred for 30 min at 0 °C and then at room temperature
overnight. After this time, organic solvent was removed under reduced pressure and the obtained
solid was purified by silica gel flash-chromatography (CHCl3 saturated with ammonium
hydroxide/cyclohexane 40:60) to give the pure Z nitrone (Z)-5. Z. Yield 55%; yellow solid m.p.
1
1
98–204 °C. H NMR (CDCl , 500 MHz): 3.87 (s, 3H,), 5.24 (d, 2H, J=4.2 Hz), 6.94–8.32 (m,
3
13
1
0H), 9.18 (d, 1H, J=4.2 Hz), 9.51 (d, 1H, J=8.5 Hz). C NMR (CDCl , 500 MHz): 55.54, 66.50,
3

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1
1
4
10.82, 120.85, 123.16, 123.25, 126.44, 127.10, 128.31, 129.42, 130.84, 132.13, 135.10, 136.28,
36.35, 136.76, 141.32, 143.45, 148.37, 150.33, 158.34. Anal. Calcd for C H N O : C, 73.45; H,
21
17
3
2
.99; N, 12.24%. Found: C, 73.32; H, 4.97; N, 12.28%.
1
Further elution gave 5.5% of E nitrone (E)-5. Yellow solid m.p. 175–180 °C. H NMR (CDCl , 500
3
MHz): 3.94 (s, 3H, OCH ), 5.29 (s, 2H, CH ), 6.94–8.33 (m, 10H), 8.29 (s, 1H), 9.19 (s, 1H), 9.48
3
2
(d, 1H, J=8.5 Hz).
4.2. General procedure for the synthesis of isoxazolines 6
A solution of nitrone 5 (0.50 g, 0.012 mol) and allyl alcohol (7 mL) in a sealed tube equipped with a
stir bar, was allowed to react at 100 °C for 3 h. The mixture was evaporated and the residue was
purified by flash chromatography on a silica gel (CHCl /CH OH/Et N 98:1:1).
3
3
3
4.2.1. ((3RS,5SR)-2-(2-methoxybenzyl)-3-(1,10-phenanthrolin-2-yl)isoxazolidin-5-yl)methanol 6a
1
Yield 44%, yellow solid. H NMR (CDCl , 500 MHz): 2.66 (ddd, 1H, J=6.8, 7.7 and 14.3 Hz, H ),
3
4a
3
.10 (ddd, 1H, J=7.1, 7.9 and 14.3 Hz, H ), 3.31 (bs, 1H, OH), 3.67 (dd, 1H, J = 3.1 and 12.5 Hz,
4b
H ), 3.74 (s, 3H, OMe), 3.82 (dd, 1H, J=4.7 and 12.5 Hz, H ), 4.06 (d, 1H, J=13.5 Hz, H ), 4.18
5
’a
5’b
2’a
(
d, 1H, J=13.5 Hz, H ), 4.55 (dddd, 1H, J=3.1, 4.7, 6.8 and 7.1 Hz, H ), 4.75 (dd, 1H, J=7.7 and
2’b 5
13
7
.9 Hz, H ), 6.79–7.19 (m, 4H), 748–8.25 (m, 6H), 9.22 (dd, 1H, J = 1.2 and 4.1 Hz, H ). C NMR
3
9
(
CDCl , 125 MHz): 38.30, 54.92, 55.23, 64.86, 71.95, 77.70, 110.21, 120.40, 121.46, 122.89,
3
1
1
5
25.64, 126.25, 126.42, 127.90, 128.37, 128.83, 130.29, 136.15, 136.82, 145.30, 150.37, 155.23,
57.33, 161.27. Anal. Calcd for C H N O : C, 71.80; H, 5.77; N, 10.47%. Found: C, 71.99; H,
2
4
23
3
3
.76; N, 10.49%.
4.2.2. ((3RS,5RS)-2-(2-methoxybenzyl)-3-(1,10-phenanthrolin-2-yl)isoxazolidin-5-yl)methanol 6b
1
Yield 35%, yellow solid. H NMR (CDCl , 500 MHz): 2.62 (ddd, 1H, J=6.8, 7.7 and 14.3 Hz, H ),
3
4a
2
.91 (ddd, 1H, J = 7.1, 7.9 and 14.3 Hz, H ), 3.27 (bs, 1H, OH), 3.63 (dd, 1H, J = 3.1 and 12.5 Hz,
4b

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H ), 3.71 (s, 3H, OMe), 3.88 (dd, 1H, J = 4.7 and 12.5 Hz, H ), 4.12 (s, 2H, H ), 4.34 (dddd, 1H,
5
’a
5’b
2’
J = 3.1, 4.7, 6.8 and 7.1 Hz, H ), 4.81 (dd, 1H, J = 7.7 and 7.9 Hz, H ), 6.79–7.19 (m, 4H), 748–
5
3
1
3
8
5
1
.25 (m, 6H), 9.24 (dd, 1H, J = 1.2 and 4.1 Hz, H ). C NMR (CDCl , 125 MHz): 38.94, 55.28,
9 3
5.69, 63.55, 72.05, 78.51, 110.28, 120.53, 121.37, 122.89, 125.84, 126.23, 126.50, 128.33, 128.88,
30.03, 130.63, 136.16, 136.81, 145.87, 148.02, 150.88, 157.12, 161.79. Anal. Calcd for
C H N O : C, 71.80; H, 5.77; N, 10.47%. Found: C, 71.66; H, 5.78; N, 10.44%.
24
23
3
3
4
4
.3. Biology
.3.1. Cell viability assay
HN6, HN13, HeLa, and 184B5 cell viability was measured using a commercial MTT assay
CellTiter 96 Aqueous One Solution Assay, Promega Co., USA), according to the Manufacturer's
(
instructions. The assay is based on the ability of viable cells to metabolize yellow 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to violet formazan that can be detected
4
spectrophotometrically. Cells were seeded in 96-well plates at a density of 1×10 cells per well in
complete medium containing 10% fetal bovine serum and antibiotics and incubated in 5% CO and
2
maintained in a 5% CO , 95% humidity atmosphere at 37 °C. Subsequent, cells were washed with
2
serum-free medium and subsequently incubated for 24 h after treatment with test compounds.
Successively, 20 ꢀL of fresh MTT solution (5 mg/mL) was added to each well and incubated with
cells at 37 °C for additional 4 h. The supernatant was removed and 100 ꢀL of DMSO was added to
each well to dissolve the formazan crystals formed by the cellular reduction of MTT. The
absorbance values of each well were measured by a plate reader at a test wavelength of 490 nm.
The assays were performed in triplicate and a non-linear regression analysis was used to get dose-
response curves.
4.3.2. DNA unwinding assay
The assay was carried out using negatively supercoiled or relaxed pGEM-T Easy plasmid. Relaxed

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plasmid was generated by treating negatively supercoiled pGEM-T Easy with Topo I. Reaction
mixtures (10 mL final volume) containing 0.3 mg of supercoiled or relaxed pGEM-T Easy, Topo I
and the studied compounds 6 at concentration of 10 nM were incubated in reaction buffer (50 mM
Tris–HCl pH 7.5, 20 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, and BSA at 0.3 mg/mL) for 30
min at 37 °C. The reactions were terminated by the addition of 0.5% SDS and 0.5 mg/mL
proteinase K. Samples were incubated for 30 min at 50°C. Next, 1.2 mL of 10X loading buffer
(20% Ficol 400; 0.1 M EDTA, pH 8.0, 1.0% SDS, and 0.25% bromphenol blue) were added and the
reactions mixtures loaded onto a 1% agarose gel made in 1X TBE buffer. The gel was run in 1X
TBE containing 0.1% SDS. After electrophoresis, DNA bands were stained with ethidium bromide
(10 mg/mL) and visualized by transillumination with ultraviolet light (300 nm).
4.3.3. CD Titration
CD spectra were acquired in a standard quartz cell of 1 cm path length in the 240–400 nm range.
For each spectrum, 5 runs were averaged with a 5 min equilibration interval before each scan. All
the spectra were recorded using fixed concentration of ct-DNA (12.6 µM in base pair) in the
absence or in presence of different concentrations of 6a or 6b (4.26 mM solution in DMSO) ranging
from 0 to about 35 µM.
4.3.4. ct-DNA melting experiments
The ct-DNA and DNA/6 complexes melting curves were obtained in the range 308–388 K. The ct-
DNA sample (31 µM in base pair) was mixed with 31 µM (saturating drug concentrations) of 6a or
6b in PBS solution. The temperature of the Peltier was raised from 308 to 388 K with a heating rate
of 1 K/min and monitoring the absorbance change at 260 nm [49, 50]. Melting point was obtained
from the mid-point of the melting curve.
4.3.5. Molecular modeling studies

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All poly-d(AT) and poly-d(GC) fragments were 3′- and 5′-end-capped with a phosphate group and
2
2
the system configured as a fully anionic oligonucleotide. Subsequently, the geometry was fully
minimized, using Yasara software (ver. 17.4.17) [51] with a convergence criterion of 0.005
kcal/mol per Å, assigning a distance-dependent dielectric of 1.0, 1–4 scale factors of 0.833 for the
electrostatic part and of 0.5 for the van der Waals one, and the nonbonded cutoff on 8 Å.
Docking was performed using AutoDock 4.2.5.1 [52] using the default docking parameters, the
point charges were initially assigned according to the AMBER14 force field and then damped to
mimic the less polar Gasteiger charges used to optimize the AutoDock scoring function. The setup
was done with the YASARA molecular modeling program.
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