Thiazonaphthalimide derivatives: Synthesis and interaction with DNA

Article abstract of DOI:10.1016/j.tetlet.2018.05.052

The synthesis of several thiazonaphthalimide derivatives is described. The exclusive formation of angular rather than linear isomers was unequivocally demonstrated by NMR and single crystal X-ray diffraction. Their photophysical properties and ability to bind calf-Thymus DNA with affinities in the range of 10⁴ makes them interesting candidates to probe DNA by fluorescence.

Full text of DOI:10.1016/j.tetlet.2018.05.052

Accepted Manuscript
Thiazonaphthalimide Derivatives: Synthesis and Interaction with DNA
Alexandra Botz, Yves Chenavier, Jacques Pécaut, Pascale Delangle, Christelle
Gateau
PII:
S0040-4039(18)30666-X
https://doi.org/10.1016/j.tetlet.2018.05.052
TETL 49999
DOI:
Reference:
Tetrahedron Letters
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Received Date:
Revised Date:
Accepted Date:
8 March 2018
14 May 2018
18 May 2018
Please cite this article as: Botz, A., Chenavier, Y., Pécaut, J., Delangle, P., Gateau, C., Thiazonaphthalimide
Derivatives: Synthesis and Interaction with DNA, Tetrahedron Letters (2018), doi: https://doi.org/10.1016/j.tetlet.
2018.05.052
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Graphical Abstract
Thiazonaphthalimide derivatives: synthesis and
interaction with DNA
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Alexandra Botz, Yves Chenavier, Jacques Pécaut, Pascale Delangle, Christelle Gateau*

1
Tetrahedron Letters
jo u r n a l h o m e p a g e : w w w . e ls e v i e r . c o m
Thiazonaphthalimide Derivatives: Synthesis and Interaction with DNA
Alexandra Botz, Yves Chenavier, Jacques Pécaut, Pascale Delangle and Christelle Gateau
Univ. Grenoble Alpes, CEA, CNRS, INAC-SyMMES, 38000 Grenoble, France.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The synthesis of several thiazonaphthalimide derivatives is described. The exclusive formation
of angular rather than linear isomers was unequivocally demonstrated by NMR and single
crystal X-ray diffraction. Their photophysical properties and ability to bind calf-Thymus DNA
with affinities in the range of 104 makes them interesting candidates to probe DNA by
fluorescence.
Available online
Keywords:
DNA
2009 Elsevier Ltd. All rights reserved.
1,8-Naphthalimide
Thiazole formation
Fluorescence
———
 Corresponding author. Tel : +33 4 38 78 60 41
e-mail address: christelle.gateau@cea.fr

2
The design of polyaromatic molecules able to bind to DNA is
of significant importance for the development of anticancer and
fluorescent imaging agents. Among them, 1,8-naphthalimide
derivatives have received significant attention and have been
extensively investigated for their potent antitumor activity.1,2,3,4
Moreover their photophysical properties make them interesting
candidates to detect DNA by fluorescence.4
naphthalimide.10,11 Non-substituted thiazo compounds were
chosen (Fig. 1, R =H) for their large affinity for DNA and their
similar fluorescence properties to naphthalimide, which is known
to efficiently sensitize the lanthanide europium ion.11 An acidic
group was introduced by the reaction with glycine for efficient
coupling to the N-terminus of peptides. The two angular and
linear derivatives (6a and 6b respectively; Fig. 2) were targeted
in order to compare their behaviors with DNA. However, we
report herein that cyclisation occurs exclusively in positions 3
and 4 of the naphthalimide moiety affording the angular isomer
6a only, contrary to what was described in previous reports that
did not demonstrate unambiguous proof of the regioselectivity.7
DNA binding studies indicate that the aromatic moiety found in
the angular regioisomer 6a has promising DNA binding
properties with a large fluorescence quenching upon binding and
an affinity of 104 M-1.
Due to its tricyclic planar ring structure, 1,8-naphthalimide
(Fig. 1) interacts with DNA and its anticancer activity was
discovered 20 years ago.6 A large variety of 1,8-naphthalimide
derivatives has been designed and synthesized in order to
improve the interaction with DNA and has been studied as
anticancer and fluorescent imaging agents (Fig. 1).2,3,4 Synthetic
strategies such as functionalization of the imide nitrogen atom,
substitution at the 3 and 4 positions and ring expansion, have
provided access to a wide range of 1,8-naphthalimide derivatives
with various chemical and photophysical properties. The position
and nature of the substituents significantly influence the behavior
of these derivatives in terms of DNA binding and fluorescence
properties.2,3 Interestingly, the aromatic fused derivative
azonafide (Fig. 1) which contains an anthracene unit, displays a
larger affinity for DNA than its parent compound amonafide with
a naphthalene unit.6 This result can be explained by the increased
planar surface of the chromophore, thereby increasing the π-
stacking interactions between the aromatic rings and the base
pairs of DNA.
Figure 2. Angular and linear derivatives targeted
The angular thiazonaphthalimide derivative 6a was obtained
in five steps with an overall yield of 8%, starting from the
commercially available compound 3-nitro-1,8-naphthalic
anhydride 1 (Scheme 1). Detailed experimental conditions for
each step and compound characterization are given in the ESI.
The key step of this synthesis is the formation of the
aminothiazole ring starting from amino derivative 2. Compound
2 was obtained by reduction of the nitro group to the
corresponding amine, using tin chloride salts in acetic acid at
reflux, as described in the literature.8,12 The formation of the
aminothiazole moiety was then carried out by the treatment of 2
with potassium thiocyanate and bromine in acetic acid.8,13 This
synthesis involved a two-step reaction with the initial formation
of an aryl-thiocyanate. Indeed, it has been demonstrated that
aromatic hydrocarbons bearing strong electron-donating groups
such as -NH2 can react with thiocyanogen13 to afford aryl-
thiocyanates in ortho or in para positions. Thiocyanogen is
formed in situ from KSCN and Br2 according to the method
developed by Soderback.15 The thiocyanate moiety can then react
with the amine, leading to the formation of an aminothiazole
ring. Regardless of the number of equivalents of bromine and
potassium thiocyanate, the order or even the duration of addition
for each reagent, only one regioisomer was obtained and isolated,
with yields ranging from 60 to 95%. The experimental conditions
leading to the highest efficiency are described in the ESI and
gave compound 3 in 95% yield. Compound 3 displays limited
stability and was therefore quickly engaged in the following step,
which consists of deamination involving a diazonium ion. The
treatment of 3 with phosphoric acid, sodium nitrite and
hypophosphorous acid affords the crude product 4 in 36% yield.16
Compound 4 was then engaged in a condensation reaction with
glycine methyl ester hydrochloride, leading to stable compound 5
in 44% yield after purification on a silica gel column.17 Finally,
saponification with lithium hydroxide provides the desired
compound 6a in 52% yield. The full characterization of stable
compound 6a by 1H and 13C NMR, ESI-MS and microanalysis is
reported in the ESI.
Figure 1. 1,8-naphthalimide and selected derivatives
The heteroaromatic fused derivatives with furan or thiophene
developed by Brañas and co-workers demonstrate increased
antitumor activity.7 In fact, heteroaromatic rings likely contribute
to additional interactions such as Van der Waals forces.7 Qian
and co-workers have described a novel family of naphthalimide
derivatives, thiazonaphthalimides, in which the naphthalene ring
has been fused with a thiazole moiety (Fig. 1 – A and B).8
According to their study, the angular isomer A and the linear
isomer B of the aminothiazole derivative (R = NH2) were both
obtained and interact with calf-Thymus DNA (ct-DNA) with an
affinity in the range of 104.5 M-1. Following these promising early
results, new angular and linear thiazonaphthalimide derivatives
were reported by the same team and binding constants to DNA in
the range of 105-6 M-1 were achieved.9
The photophysical and DNA-binding properties of
thiazonaphthalimide derivatives make them ideal candidates for
the design of luminescent DNA probes. In particular their
insertion into lanthanide complexes would afford DNA-binding
long-lived luminescent probes, as previously reported for
lanthanide-binding peptides grafted with proflavine or

3
different molecules related by the symmetry center of the space
group (P21/n for 7 and P-1 for 8). These distances are short
enough to evidence π-stacking interactions in both structures.
Scheme 1. Reagents and conditions: a) SnCl2•2H2O (4 equiv.), HCl
(0.74 equiv.), AcOH, reflux, 22 h, quant.; b) KSCN (4 equiv.), Br2
(1.02 equiv.), AcOH, r.t., 48 h, 95%; c) i) NaNO2 (6 equiv.), H3PO4,
-10 °C, 1 h 50 min, ii) H3PO2 (23 equiv.), r. t., overnight, 36%; d)
H2NCH2COOCH3•HCl (1.1 equiv.), Et3N (1.1 equiv.), EtOH, reflux,
5 h, 44%; e) LiOH•H2O (1.99 equiv.), THF/MeOH, 50 °C, 52%.
Overall yield 8%
As the angular structure of compound 6a cannot be
differentiated from the linear structure 6b by 1H NMR
spectroscopy, a NOESY NMR experiment was performed to
unambiguously characterize the regioselectivity of the final
compound. In the linear compound 6b, a signature correlation
between the proton of the thiazole-fused ring (H4) and the H5
proton is expected. By comparison, there should be no
correlation between the proton of the thiazole-fused ring (H2) and
the H5 proton in angular compound 6a. Therefore the observed
correlation pattern (no correlation between H2 and H5; see ESI,
Fig. S7) evidences the formation of angular compound 6a with a
3,4-thiazolonaphthalimide moiety.
Scheme 2. Reagents and conditions: H2NCH2COOCH3•HCl (1.1
equiv.), Et3N (1.1 equiv.), EtOH, reflux, 5 h, 40%. Crystallographic
structures of compounds 7 and 8.
The literature reports of only 3- and 4-substitution on
naphthalic anhydride or naphthalimide may suggest the poor
reactivity of position 2 in the aromatic ring due to steric
hindrance or electronic effects. This would prevent the reaction
of the thiocyanate group at this position and the formation of 2,3-
thiazole fused linear compounds. In fact, the reported linear
compounds8 have been characterized by 1D NMR only and
1
therefore lack reliable structural assignment. Notably, the H
To unequivocally confirm the regioselectivity of the
cyclisation step, the unstable compound 3 was trapped with
glycine methyl ester hydrochloride, to afford stable compound 7
in 40% yield. The minor by-product 8 was also isolated in 3%
yield (Scheme 2). The two compounds 7 and 8 were fully
NMR signals of the aromatic moieties in compounds 7 and 8 are
very similar to those previously reported (see ESI, Table S1).
Therefore, our results based on unambiguous structural proof
strongly suggest that the thiazonaphthalimide major compounds
previously described by Qian and co-workers8 are in fact the
angular ones, and that their minor product is another angular
isomer with reversed positions of the nitrogen and sulfur atoms,
formed from an impurity present in the starting material.
1
characterized by H and 13C NMR and NOESY experiments.
NOE patterns similar to those observed for 6a confirm the
formation of 3,4-thiazonaphthalimide angular regioisomers.
Thiazonaphthalimide derivatives 5, 7 and 8 were studied for
their photophysical properties and their interactions with ct-
DNA. These ester derivatives are preferred over the acids, in
order to avoid electrostatic repulsions between the carboxylate
group and the negatively charged phosphates of DNA at
physiological pH. The comparison of these three derivatives
gives valuable information regarding the influence of the
nitrogen and sulfur atoms positions in the ring and the
substitution of the thiazole group by a hydrogen or an amine
group. The fluorescence of each compound was measured after
the addition of increasing amounts of ct-DNA (expressed in base
pairs (bp)) and excitation at its maximum absorption wavelength
at 298 K in a buffered solution. These results show a decrease of
the fluorescence of each aromatic compound after the addition of
ct-DNA in solution, reflecting an interaction between the
aromatic rings and the double-helix of DNA, as shown in Figure
3A for compound 5 and ESI Fig. S10 and S11 for compound 7
and 8, respectively.
The final proof for this regioselectivity was unambiguously
obtained by X-ray crystallography. Indeed, X-ray quality crystals
of 7 and 8 were obtained by the slow evaporation of
dichloromethane and their crystallographic structures solved by
X-Ray diffraction (see ESI, Tables S2 and S3 for details). The
solid-state structures confirm the formation of two angular
compounds. Compound 7 is the expected angular naphthalimide
derivative functionalized by an aminothiazole group with
nitrogen in position 3 and sulfur in position 4, whereas the minor
compound 8 is a similar derivative with the positions of nitrogen
and sulfur atoms inverted, i.e. sulfur in position 3 and nitrogen in
position 4 (Scheme 2). The formation of this by-product can be
explained by the presence of ~10% 4-nitro-1,8-naphthalic
anhydride in the commercially available 3-nitro-1,8-naphthalic
anhydride, as evidenced by the 1H NMR spectrum of this starting
material. The two X-ray structures show intermolecular hydrogen
bond networks that result in one dimensional arrangements along
the a axis for 7 and the a+b axis for 8: the protons of the amino
groups are H-bonded to one carbonyl ester and one nitrogen atom
of the thiazole ring for 7 or to one carbonyl ester and one oxygen
atom of the naphthalimide for 8. The aromatic rings are planar
with mean deviations of only 0.024 Å for 7 and 0.018 Å for 8.
The shortest distance between the two thiazonaphthalimide rings
were found at 3.412 Å for 7 and 3.438 Å for 8, between two
Two parameters were extracted from these experiments to
compare the interactions of the three compounds with ct-DNA.
The association constant Ka for the binding of the naphthalimide
derivatives to ct-DNA was calculated by fitting the evolution of
the fluorescence spectra with a 1:1 binding model using the
program Specfit (Fig. 3B). The corresponding Ka values are
reported in Table 1 and exemplifies differences in the binding of
the three compounds with a significantly larger affinity for the

4
non-substituted thiazonaphthalimide 5 (Ka = 104.0 M-1).18 The
substitution of the thiazole ring by the mesomeric electron-
donating amino group decreases the affinity for the double helix
by a factor of 2-3.5. Moreover, compound 5 also displays the
largest fluorescence quenching upon ct-DNA binding.
Acknowledgments
The authors thank Pierre-Alain Bayle for his contribution in
the recording of NMR data and Colette Lebrun for ESI-MS
spectrum. This research was supported by the Labex ARCANE
(Grant ANR-11-LABX-0003-01).
References
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5. Roldan, C. M.; Braña, M. F.; Berlango, J. M. 4-acylamino-n-
phenylbutyramides - useful as pharmaceuticals. Patent DE2318136 A1,
1973.
6. Sami, S. M.; Dorr, R. T.; Solyom, A. M.; Alberts, D.S.; Remers, W.A. J.
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Solyom, A. M.; Remers, W.A. J. Med. Chem. 1996, 39, 4978.
Figure 3. A) Titration of compound 5 (2.5 M) (HEPES buffer 10
mM, pH 7.0, NaCl 10 mM) at 298 K. Fluorescence upon excitation
at 356 nm as a function of ct-DNA (bp). B) Corresponding evolution
of the fluorescence at 418 nm; circles are experimental points and the
line represents calculated data with the program SPECFIT (logKa =
4.0).
7. Braña, M. F.; Cacho, M.; García, M. A.; de Pascual-Teresa, B.; Ramos,
A.; Domínguez, M. T.; Pozuelo, J. M.; Abradelo, C.; Rey-Stolle, M. F.;
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Table 1. Association constants of the naphthalimide derivatives with
ct-DNA at pH 7.
8. Li, Z.; Yang, Q.; Qian, X. Bioorg. Med. Chem. Lett. 2005, 15 (7), 1769.
9. Qian, X.; Li, Z.; Yang, Q. Bioorg. Med. Chem. 2007, 15 (21), 6846.
Compound
logKa
5
7
8
10. Ancel, L.; Gateau, C.; Lebrun, C.; Delangle, P. Inorg. Chem. 2013, 52,
552.
4.00(3)
3.45(7)
3.67(3)
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2012, 10, 126; Bonnet, C.S.; Devocelle, M.; Gunnlaugsson, T. Chem.
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23, 9974.
The melting temperature of double-stranded DNA can be used
to partially characterize the mode of interaction of aromatic
compounds with DNA.19 No significant alteration of the melting
temperature was observed in the presence of thiazonaphthalimide
compounds 5, 7 and 8, presumably indicating that none of the
three aromatic compounds interacts with DNA via an
intercalative process between the base pairs of DNA (see ESI,
Fig. S12). Indeed, although an intercalative process is mentioned
in the literature for the interaction between thiazonaphthalimide
derivatives and ct-DNA, little evidence has been provided.
12. Peters, A; T.; Bide, M.J. Dyes and Pigments 1985, 6, 267.
13. Prajapati, N.P., Vekariya, R. H.; Borad, A.; Patel, H.D. RSC Adv. 2014,
4, 60176; Deshmukh, R.; Thakur A. S.; Jha A. K.; Deshmukh R. Int. J. Res.
Pharm. Chem. 2011, 1, 329; Munirajasekhar, D.; HimajaM.; Mali sunil
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Pharm Res 2010, 3, 13; Malik, J. K.; Manvi, F.; Nanjwade B.; Singh S.
Drug Invent. Today 2009, 1, 32.
In summary, we have reported the synthesis and unambiguous
characterization of angular thiazonaphthalimide derivatives. The
formation of linear regioisomers was revealed to not be possible,
which is in accordance with the poor reactivity of position 2 in
the ring. Compounds 5, 7 and 8 strongly bind to ct-DNA, with no
significant evolution of the ct-DNA melting temperature upon
binding, which suggests a non-intercalative process. The largest
affinity and fluorescence quenching is observed for the non-
substituted ring. The next step will be to graft compound 6a to
amino groups found in peptides or proteins to obtain fluorescent
probes sensitive to DNA.
14. Prasad, J. V.; Panapoulous, A.; Rubin, J. R. Tetrahedron Lett. 2000,
41 (21), 4065.
15. Soderback, E Justus Liebig’s Annalen der Chemie, 1919, 419 (3),
217.
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Forood, B.; Flatt, B.; Nakai, H. J. Heterocycl. Chem. 2000, 37 (6), 1655.

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17. Yuan, D.; Brown, R.G.;, Hepworth, J.D.;, Alexiou, M. S.; Tyman,
J.H.P. J. Heterocyclic Chem. 2008, 45, 397; Kumpulainen, T.; Bakker,
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Kluwer Academic/Plenum Publishers: New York, NY, USA, 1999.
19. Horowitz, E. D.; Hud, N. V. J. Am. Chem. Soc. 2006, 128 (48), 15380.

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Highlights
 Thiazonaphtalimide derivatives were
synthesized
 Exclusive formation of linear isomer
was demonstrated by NMR and X-ray
diffraction.
 Photophysical
properties
and
interaction with ct-DNA were studied