New spiroacridine derivatives with DNA-binding and topoisomerase I inhibition activity

Article abstract of DOI:10.1016/j.tetlet.2016.10.108

Eight spiroacridine derivatives containing the isoxazoline ring were synthesized and characterized using elemental analysis, IR, UV–vis, and NMR measurements. Their interactions with calf thymus DNA were extensively studied by various spectroscopic techni

Full text of DOI:10.1016/j.tetlet.2016.10.108

Accepted Manuscript
New spiro-acridine derivatives with DNA-binding and Topoisomerase I inhib-
ition activity
Danica Sabolová, Mária Vilková, Ján Imrich, Ivan Potoč ň ák
PII:
S0040-4039(16)31441-1
DOI:
http://dx.doi.org/10.1016/j.tetlet.2016.10.108
TETL 48278
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
16 August 2016
24 October 2016
28 October 2016
Please cite this article as: Sabolová, D., Vilková, M., Imrich, J., Potoč ň ák, I., New spiro-acridine derivatives with
DNA-binding and Topoisomerase I inhibition activity, Tetrahedron Letters (2016), doi: http://dx.doi.org/10.1016/
j.tetlet.2016.10.108
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Graphical abstract

New spiro-acridine derivatives with DNA-binding and Topoisomerase I inhibition
activity
Danica Sabolováa*, Mária Vilkováb, Ján Imrichb, Ivan Potočňákc
a Department of Biochemistry, Institute of Chemistry, Faculty of Science, P. J. Šafárik University, Moyzesova 11, 041 54
Košice, Slovakia. Corresponding author: danica.sabolova@upjs.sk; phone: +421552341263
b Department of Organic Chemistry, Institute of Chemistry, Faculty of Science, P. J. Šafárik University, Moyzesova 11, 041
54 Košice, Slovakia
c Department of Inorganic Chemistry, Institute of Chemistry, Faculty of Science, P. J. Šafárik University, Moyzesova 11, 041
54 Košice, Slovakia
Abstract
Eight spiro-acridine derivatives containing the isoxazoline ring were synthesized and characterized using elemental analysis,
IR, UV-Vis, and NMR measurements. Their interactions with calf thymus DNA were extensively studied by various
spectroscopic techniques and gel electrophoresis. The UV-visible and CD measurements implied that these derivatives
interact with calf thymus DNA through intercalation. The Stern-Volmer quenching constants were determined and ranged
from 0.126×104 to 1.394×104 M-1. A topoisomerase I inhibition assay was performed with the spiro-acridine derivatives.
Key words
spiro-acridine, isoxazoline, single crystal X-ray analysis, drug-DNA interaction, topoisomerase I inhibition
Acridine and its derivatives are interesting chemical families containing a planar aromatic chromophore which is
able to bind into DNA by intercalation and thereby inhibit crucial classes of enzymes involved in the regulation of
DNA, especially topoisomerases1 and telomerases.2 The strong fluorescence properties exhibited by acridine
pharmacophores have led to their use in a number of fields of biological research, including as chemiluminescent
agents, DNA intercalators, fluorescence reagents for the labeling of biomolecules, and chemical sensors in
fluorescence spectroscopy.3-5 Additionally, it has been reported that natural and non-natural isoxazolines and
spirocyclic isoxazoline derivatives have shown promising antiproliferative effects on a variety of cancer cell lines.6
As part of our efforts to develop novel acridine derivatives possessing bioactive heterocyclic substituents, which
are able to interfere with cellular processes, we have searched for new synthetic approaches for their preparation. The
1,3-dipolar cycloaddition reaction between nitrile oxides (NO) and alkenes is of considerable interest as an efficient
way to prepare isoxazolines. For that reason, we have examined these reactions for the synthesis of new isoxazoline
derivatives, which were screened for their DNA binding and topoisomerase I inhibition activities.
Herein, we report the three step synthesis of 10'-methyl-3-substituted-4H,10'H-spiro[acridine-9',5-[1,2]oxazoles] 5ah
from the reactive acridine dipolarophile intermediate 4 which was synthesized according to Scheme 1. 9-Methylacridine (2)
was converted into 9,10-dimethylacridinium methyl sulfate (3) using dimethyl sulfate according to a literature procedure,7
followed by subsequent elimination to generate the exocyclic double bond of derivative 4. Dipolarophile 4 was trapped with
NO 7ah to afford the corresponding spiro-acridine products via addition to the exomethylenic double bond. NO 7ah were
prepared according to literature procedures.8 The treatment of compound 4 with NO 7ah yielded only a single regioisomer

5. The regioselectivity of these 1,3-dipolar cycloaddition reactions was not surprising as intermediate 4 represents a
phenylogous enamine which is unambiguously polarized and regioselectively trapped by dipoles. Notably, in the reaction of
dipolarophile 4 with NO 7b, 3-brominated spiroisoxazoline analogue 5c was also isolated in 20% yield when the NO reagent
used was contaminated with 3-bromo-2,4,6-trimethoxybenzonitrile oxide (7c) which was partially formed as a by-product
after the synthesis of 7b from its bromooxime precursor.
The 1,3-dipolar cycloaddition reaction yields of compound 4 with NO compounds 7ah were low due to various
reasons, including degradation of nitrile oxides in the reaction mixture, which occurred faster for nitrile oxides 7d, e, gi than
for 7ac, f, as well as in situ release of reactive species from their precursors in the reaction mixture.
CH3
CH3
DMS
toluene
CH3COOH
220 °C
6 h
reflux
2 h
N+
NH
N
-
CH3OSO3
1
2
CH3
(67%)
3
(82%)
R3
R1
N
+
-
R4
R
C
N
O
CH2
TEA
1''
3
7ah
CH2Cl2
CH2Cl2
R2
1
4
O
rt
14 h
rt
1 h
8'
5'
1'
N
8'a
9'a
X
CH3
4
10'a
4'a
N
4'
R3
R4
CH3
R1
5ah
a: R1=R2=R3=R4 = H (47%)
O
N
b: R1=R2=R3= OCH3, R4= H (28%)
c: R1=R2=R3= OCH3, R4= Br (20%)
d: R1=R2= Cl, R3=R4= H (52%)
e: R1=R2=R4= H, R3= OCH3 (39%)
f: R1=R2=R4= H, R3= Br (70%)
g: R1=R2=R3= CH3, R4= H (57%)
h: R1=R2=R4= H, R3= NO2 (35%)
R2
N
CH3
6ah
OCH
OCH
3
Cl
Cl
3
H
CO
H CO
3
3
7a:
7e:
7b:
7f:
7c:
7g:
7d:
OCH
Br
OCH
3
3
CH
3
H
C
H
CO
Br
O
N
3
3
7h:
2
CH
3
Scheme 1. Synthesis of 10'-methyl-3-substituted-4H,10'H-spiro[acridine-9',5-[1,2]oxazoles] 5a–h.
In previous work9,10 we found that the polarization of the dipolarophile CH=CH double bond as expressed by
13C chemical shifts was useful for explaining the reaction regioselectivity. In this case, similar explanations were
impossible due to the instability of dipolarophile 4, which was generated in situ. The structural characterization of
compounds 5ah was achieved using 1D and 2D NMR techniques. The 1H, 13C and 15N NMR chemical shifts obtained
1
1
for 5a, as well as the H,13C-HMBC and H,15N-HMBC correlations, allowed unambiguous assignment of all atoms
(Fig. 1) and was further confirmed by single crystal X-ray data (Fig. 2).

Figure 1. Selected 1H,13C-HMBC () and 1H,15N-HMBC () correlations in derivative 5a.
Figure 2. Single crystal X-ray structure of derivative 5a.
Absorption spectroscopy is one of the most commonly employed instrumental techniques for studying DNA
interactions with small ligands. The spectral titration of spiro-acridines 5ah was followed by monitoring the UV-Vis
absorbance changes in the range of 240–450 nm. Upon addition of calf thymus DNA (CT DNA), the absorption bands
showed significant hypochromism and a slight red shift (Table 1). The representative UV-vis spectrum of compound
5h is shown in Figure 3. The observed hypochromism is caused by the DNA binding activity of the investigated
compounds resulting from partial unwinding of the DNA double helix and consequent conformational changes.11
A
decrease of peak intensities with an absorbance reduction of approximately 14% to 35% from its initial value was
observed. The hypochromism, due to a strong interaction between the electronic states of the intercalating
chromophore and those of the DNA bases, was noted to be similar to previous reports,12 suggesting the close proximity
of the acridine ring and DNA. The presence of an isosbestic point at 400 nm indicated spectroscopically distinct
chromophores, namely, free and bound species. Such behavior is generally associated with intercalation as a dominant
binding mode.13

Figure 3. Spectrophotometric titration of derivative 5h (6 μM) in 0.01 M Tris buffer (pH 7.3, 24 C) with increasing concentration of CT
DNA (from top to bottom, step 1.56μM).
Table 1. UV-Vis absorption characteristics and binding constants of compounds 5a–h.
λmax1
(nm)
λmax2
(nm)
Δλ1
(nm)
Δλ2
(nm)
Compound
Hypochromism (%)
Ksv (M-1)
298
297
300
298
294
298
297
298
-
4
2
2
5
6
3
2
5
-
-
34.78
19.05
21.43
28.75
19.57
27.08
13.95
29.79
-
0.389×104
0.429×104
0.481×104
0.384×104
0.126×104
1.131×104
0.332×104
1.394×104
5a
5b
5c
5d
5e
5f
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
5g
5h
346
12
27.08
The results are similar to those previously reported by our group for comparable intercalators, methyl 2-[2-
(acridin-9-yl)imino-3-R-4-oxo-1,3-thiazolidin-5-ylidene]acetates (R = sec-Bu, tert-Bu, 4-Br-C6H4) which were
prepared from thioureas and dimethyl acetylenedicarboxylate.14
For better information about the DNA binding properties of the investigated spiro-acridines 5ah,
competitive binding experiments based on the displacement of ethidium bromide (EB) from CT DNA were performed.
In this assay, molecules that are able to bind to DNA via the same mode as EB are able to displace EB from the DNA
helix. The amount of fluorescence quenching of the DNA–EB system can be used to determine the extent of
intercalation between the examined molecule and DNA.15,16 The representative emission spectra of EB bound to DNA
in the absence and presence of compound 5h is shown in Figure 4; similar reduction of the fluorescence maxima was
observed for derivatives 5ag. Upon addition of the spiro-acridines, a significant decrease in the fluorescence intensity
was observed, suggesting that the compounds bind to DNA by the intercalative mode. The fluorescence quenching of
DNA-bound EB can be well described by the linear Stern-Volmer equation17 in which the spiro-acridine derivatives
are the quenchers. The Stern-Volmer quenching constants (KSV) obtained from the linear quenching plot was estimated
to range from 0.126×104 to 1.394×104 M-1 (Table 1).
Figure 4. Fluorescence emission spectra of EB bound to CT DNA in the absence and presence of increasing amounts of derivative 5h. Inset:
the corresponding Stern–Volmer plot for the quenching process of EB by 5h (0-25.2 μM), at 2.8 μM intervals. Arrow indicates change upon
increasing concentration.

CD experiments were performed in order to define the orientation of the compounds with respect to the DNA
helix. The B-form conformation of DNA shows two conservative CD bands in the UV region, a positive band at 278
nm due to the base stacking and a negative band at 246 nm due to the polynucleotide helicity.1,15 Upon incubation of
the compounds with CT DNA, the CD spectra displayed minor changes of both the positive and negative bands (Fig.
5). The helical band at 246 nm, corresponding to the extent of DNA unwinding, exhibited a decrease and slight blue
shift for all compounds in the same order as that of bis-3,6-alkylamidoacridines.18 CD spectral measurements
confirmed the intercalating binding mode of the studied compounds.
Figure 5. CD spectra of CT DNA in the absence (black solid line) and presence (red dashed line) of derivative 5h (0.15 mM), in 0.01 M Tris
buffer (pH 7.4, 24 °C).
The planar polycyclic structure of the acridines allows them to easily intercalate into double-stranded DNA,
which can interfere with DNA regulatory enzymes such as topoisomerases.19 Topoisomerases (TOPO I, II) are
enzymes involved in the supercoiling of DNA, and play important roles in many aspects of DNA processing. As a
result, these molecular targets are being studied for the development of a new generation of inhibitory agents.20 There
are two mechanisms by which an intercalator can affect topoisomerase activity and thereby stop cell proliferation.
Firstly through intercalation, in which the binding site of the topoisomerase is occupied and formation of the enzyme–
DNA complex is hindered, and secondly through the formation of a ternary complex between DNA, the intercalator
and topoisomerase, a structure which is significantly more stable than the DNA–topoisomerase complex.1,21
A standard plasmid cleavage assay was used to study the effect of compounds 5ah (10, 20 and 30 µM) on
calf thymus topoisomerase I by DNA gel electrophoresis. This method provides a direct means of determining whether
the compounds affect the unwinding of a supercoiled (SC) duplex DNA substrate; the enzyme converts SC DNA to
relaxed (R) DNA and nicked open circular (NC) DNA. If Topo I retains its normal activity, one would expect to
observe the disappearance of the SC DNA band (fastest band) and the appearance of bands for R DNA and NC
DNA.22 On the contrary, if the activity of the enzyme is inhibited, no R DNA or NC DNA would be detected.
As shown in Figure 6, compounds 5a, fh at 30 µM inhibit TOPO I, as indicated by the negative SC form as
the main band (lanes 5, 20, 23, 26), while the bands of NC and R form disappeared. However, compounds 5be
showed only partial inhibition of topoisomerase I at the same concentration.

Figure 6. The inhibition of human Topo I by compounds 5ah. Lane 1: pUC19 DNA; lane 2: DNA+ Topo I; lanes 3-5: DNA + Topo I + 5a
(10, 20, 30 µM); lanes 6-8: DNA + Topo I + 5b (10, 20, 30 µM); lanes 9-11: DNA + Topo I + 5c (10, 20, 30 µM); lanes 12-14: DNA + Topo
I + 5d (10, 20, 30 µM); lanes 15-17: DNA + Topo I + 5e (10, 20, 30 µM); lanes 18-20: DNA + Topo I + 5f (10, 20, 30 µM); lanes 21-23:
DNA + Topo I + 5g (10, 20, 30 µM); lanes 24-26: DNA + Topo I + 5h (10, 20, 30 µM);. Topo I and pUC19 were incubated for 30 minutes at
37 °C in the presence or absence of compounds 5ah. SC - supercoiled DNA, R - relaxed DNA, NC - nicked open circular DNA.
In summary, we have synthesized and characterized eight novel spiro-acridines via the regioselective 1,3-
dipolar cycloaddition reaction of an acridine dipolarophile containing an exocyclic double bond and nitrile oxide
derivatives. The single crystal X-ray structure of derivative 5a was determined. Moreover, the DNA-binding properties
of these compounds were investigated by electronic absorption, fluorescence and CD spectroscopy. The experimental
results showed that compounds 5ah bind with DNA via intercalation. The highest values of the KSV constant were
established for derivatives 5f, h which containing electron acceptor substituents (NO2, Br) at the para position (R3) of
the aromatic ring. This was in contrast to derivative 5e with an electron donating substituent (OCH3) at the para
position. Derivatives 5ad, g did not significantly differ in their KSV values. Compounds 5a, 5fh showed inhibition
activity against topoisomerase I, suggesting that these spiro-acridine derivatives may act as potential anticancer agents.
Acknowledgements
This work was supported by the VEGA grants Nos. 1/0001/13 and 1/0131/16.
Supplementary data
CCDC 1497242 contains the supplementary crystallographic data for this paper. These data can be obtained free of
charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK;
fax: +44 1223 336033; e-mail: deposit@ccdc.cam.ac.uk).
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Highlights:
New spiro acridin-isoxazolines were synthesized by 1,3-dipolar cycloaddition
Single crystal X-Ray structure of spiro-acridine derivative 5a was solved
Evaluation of DNA-binding properties by spectroscopic methods
Topoisomerase I inhibition assay