Sulfur-substituted naphthalimides as photoactivatable anticancer agents: DNA interaction, fluorescence imaging, and phototoxic effects in cultured tumor cells

Article abstract of DOI:10.1016/j.bmc.2008.06.052

A series of sulfur-substituted naphthalimides (1-5) was prepared and investigated as antitumor drugs. Initial DNA interaction studies (by the fluorescence quenching method, UV/vis and CD spectroscopy, thermal denaturation, topoisomerase Western blot analysis, and DNA photocleavage experiments) expectedly suggested the DNA and topoisomerase as main targets of the agents. Fluorescence spectroscopic and microscopic experiments indicated a significant sensitivity of the emission intensities of 3 and 5 to the cellular environment and confirmed the cellular uptake and biodistribution into cell compartments for 1-3 and 5. A comparative evaluation of the antiproliferative effects under different experimental setups (concerning drug exposure period and an additional short-time UV irradiation) revealed significant phototoxic effects for the environmentally sensitive compounds 3 and 5 and strongly suggested the further development of sulfur-substituted naphthalimides for potential use in photodynamic tumor therapy.

Full text of DOI:10.1016/j.bmc.2008.06.052

Bioorganic & Medicinal Chemistry 16 (2008) 7107–7116
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Sulfur-substituted naphthalimides as photoactivatable anticancer agents: DNA
interaction, fluorescence imaging, and phototoxic effects in cultured
tumor cells
a
a
c
c
c
Ingo Ott a,b, , Yufang Xu , Jianwen Liu , Malte Kokoschka , Melanie Harlos , William S. Sheldrick ,
*
Xuhong Qian a,
*
a State Key Laboratory of Bioreactor Engineering and Shanghai Key Laboratory of Chemical Biology, East China University of Science and Technology, Meilong Road 130,
Shanghai 200237, China
b Institute of Pharmacy, Freie Universität Berlin, Königin-Luise-Str. 2+4, 14195 Berlin, Germany
c Lehrstuhl für Analytische Chemie, Ruhr-Universität Bochum, 44780 Bochum, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of sulfur-substituted naphthalimides (1–5) was prepared and investigated as antitumor drugs.
Initial DNA interaction studies (by the fluorescence quenching method, UV/vis and CD spectroscopy, ther-
mal denaturation, topoisomerase Western blot analysis, and DNA photocleavage experiments) expect-
edly suggested the DNA and topoisomerase as main targets of the agents. Fluorescence spectroscopic
and microscopic experiments indicated a significant sensitivity of the emission intensities of 3 and 5
to the cellular environment and confirmed the cellular uptake and biodistribution into cell compartments
for 1–3 and 5. A comparative evaluation of the antiproliferative effects under different experimental set-
ups (concerning drug exposure period and an additional short-time UV irradiation) revealed significant
phototoxic effects for the environmentally sensitive compounds 3 and 5 and strongly suggested the fur-
ther development of sulfur-substituted naphthalimides for potential use in photodynamic tumor therapy.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 20 May 2008
Revised 26 June 2008
Accepted 27 June 2008
Available online 2 July 2008
Keywords:
Antitumor drugs
Naphthalimides
Phototoxicity
Photodynamic therapy
Photoinduced electron transfer (PET)
1. Introduction
acetylation of the 50-primary-amino group on the aromatic ring
by N-acetyltransferase 2, which caused a high-variable, unpredict-
Despite tremendous efforts in the development of new drugs and
therapeutic procedures cancerous diseases are still difficult to treat
and the development of effective anticancer medicines remains one
of the main goals of modern medicinal chemistry research. As cancer
cells are highly proliferative tissues, one of the most promising bio-
logical targets to decrease tumor cell growth is the DNA.
Among the agents directly interacting with the DNA naphthali-
mide derivatives have gained considerable attention based on their
DNA-intercalating properties and topoisomerase-poisoning activi-
ties. The first drugs of this group that reached the clinical trial stage
were amonafide and mitonafide (see Fig. 1). However, due to unex-
pected central neurotoxicity and limited efficiency, the clinical
studies on these two drugs met some problems.
able toxicity because of the interindividual differences in N-acety-
lation and greatly obstructed its clinical development. Challenged
by such a toxic issue and for avoiding the release of N-acetylated
metabolites a broad variety of novel compounds (including bisin-
tercalators such as elinafide) has been reported.1–9
As the amino group of amonafide undergoes extensive metabo-
lism and the nitro-group of mitonafide was made responsible for
toxic side effects, our efforts in the development of novel naphthal-
N
N
Amonafide has so far failed to enter clinical phase III and has
long been challenged by its unpredictable side effects (e.g.,
dose-limiting bone marrow toxicity). In clinic, it was found that
amonafide was easily metabolized to N-acetyl-amonafide via N-
O
N
O
O
N
O
NH2
NO2
Abbreviations: CD, circular dichroism; PET, photoinduced electron transfer.
* Corresponding authors. Tel.: +86 21 64253589; fax: +86 21 65252603.
E-mail addresses: ottingo@zedat.fu-berlin.de (I. Ott), xhqian@ecust.edu.cn (X.
Qian).
amonafide
mitonafide
Figure 1. structural formulas of amonafide and mitonafide.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.06.052

7108
I. Ott et al. / Bioorg. Med. Chem. 16 (2008) 7107–7116
N
NH2
(1)
(2)
S
(4)
S
S
O
N
O
S
S
N
(3)
S
(5)
S
R16
aliphatic / aromatic residues
aromatic residues containing
additional heteroatoms (N or S)
O
HS
S
N
(7)
(6)
O
no substituent
N,N-dimethylamino group replaced with -SH
Figure 2. R16, novel target compounds and reference compounds.
imide antitumor drugs have focused so far on tetra- and pentacyclic
aromatic derivatives, which lack these supposedly therapeutically
problematic functions. This led to the discovery of several naphthal-
imides containing enlarged fused aromatic ring systems which dis-
played promising intercalating, DNA photocleaving, and tumor cell
growth-inhibiting properties. Among the compounds evaluated so
far we noted as an overall result that especially sulfur-containing
derivatives showed promising antiproliferative efficiency.10–12
One of the most active agents found within our studies is the
thio-heterocyclic naphthalimide R16 (see Fig. 2), which exhibited
superior cytotoxic effects in comparison to amonafide and miton-
afide in several tumor models, acted as a topoisomerase poison
intracellular distribution by fluorescence microscopy as well as
the in-vitro evaluation of their phototoxicity.
2. Results
2.1. Synthesis and fluorescence spectra
The synthesis of the target compounds was performed according
to established procedures as depicted in Scheme 1.10,12,16–19 Com-
mercially available 4-bromo-1,8-naphthalic anhydride was reacted
with the thiols to the corresponding 1,8-naphthalic anhydride thio-
ethers, which were then transformed into the target compounds by
refluxing with N,N-dimethlyaminoethylamine or cysteamine in
absolute ethanol. All target compounds used for the biochemical as-
says were purified by column chromatography over SiO2 and struc-
turally confirmed by 1H NMR, MS, and elemental analyses.
Fluorescence spectra were recorded in phosphate-buffered sal-
ine solution, pH 7.4 (PBS) under experimental conditions that were
also used for the study of the DNA binding by the fluorescence-
quenching method (see below).
The unsubstituted reference compound 7 showed an intense
maximum around Ex/Em 344/396 nm. For the ethyl-, butyl-, and
benzyl-substituted derivatives 1–3, this maximum was shifted to-
ward longer wavelengths (Ex/Em 396–403/506–523 nm; see Fig. 3
for representative examples) but no significant fluorescence inten-
sity was observed with the derivatives 4 and 5 containing addi-
tional heteroatoms (N and S) on the sulfur substituent. Absolute
fluorescence intensities at the respective Ex/Em maxima of 5 lM
of the compounds in PBS increased in the series 3 (649) < 7
(1896) < 1 (2968) < 2 (3539).
The weak or missing fluorescence emission of 3–5 can be attrib-
uted to changes in the photoinduced intramolecular electron trans-
fer (PET) caused by the aromatic substituents on the sulfur. PET
between the electron rich amino moiety and the relatively elec-
tron-deficient naphthalimide fluorophore component can be ex-
pected to be significantly influenced by these aromatic/
heteroaromatic moieties.25–27
and induced apoptosis.13
Although many naphthalimides have been prepared and
screened for cytotoxic effects, little attention has been paid to sim-
ple thio-substituted derivatives and no concrete structure–activity
relationship studies involving this substituent have been described
so far. Based on the structure of R16 and structurally closely re-
lated thio-heterocyclic compounds recently described by Brana et
al.,6 we developed a series of non-ring fused derivatives containing
sulfur in position 4 of the naphthalimide ring system (1–5, see Fig.
2). The target compounds contain the N,N-dimethylaminoethyl
side chain, which is considered to be an optimized pharmacophore
concerning the interaction with the DNA backbone and with the
topoisomerase enzyme.1 Compounds, each lacking one of the men-
tioned properties, were prepared as references (6 and 7).
Naphthalimides have also been described as strongly fluores-
cent agents14–15 and photoactivatable species that exhibit signifi-
cant DNA photocleaving10,11,16–20 activities. Interestingly, to the
best of our knowledge these properties have not been used so far
to evaluate the compounds as phototoxic antitumor drugs in vitro
or in vivo although there are few reports on the use as photoacti-
vatable antiviral agents.21–22 However, the presence of phototoxic
properties might widen the applicability of this class of cytostatics
concerning the use in photodynamic therapy.23 The investigation
of sulfur-substituted naphthalimides appears very promising in
this context based on recent observations showing that the DNA
photocleaving potency of thio or thiono naphthalimides was signif-
icantly higher than that of the corresponding oxygen derivatives.24
This report deals with the preparation and basic fluorescence
characteristics of a series of novel thiosubstituted naphthalimides,
the study of their DNA interaction properties, cellular uptake and
2.2. Interaction with the DNA
Based on their fluorescence-emitting properties, we studied the
binding of 1–3 and 7 to DNA by the fluorescence-quenching meth-

I. Ott et al. / Bioorg. Med. Chem. 16 (2008) 7107–7116
7109
R1
O
O
O
O
O
O
O
N
O
a)
b)
S-R2
Br
S-R2
O
(1): R1 = -N(CH3)2, R2 = ethyl
(2): R1 = -N(CH3)2, R2 = butyl
(3): R1 = -N(CH3)2, R2 = benzyl
N
(4): R1 = -N(CH3)2, R2 = 2-aminophenyl
(5): R1 = -N(CH3)2, R2 = 4,5-benzo-1,3-thiazol-2-yl
(6): R1 = SH, R2 = ethyl
N
(7)
O
Scheme 1. Synthesis of the target compounds. Reagents conditions: (a) thiol (R2-SH), DMF, K2CO3, 30–50 °C, 3–7 h; (b) N,N-dimethylethylamine or cysteamine, EtOH (abs),
reflux 5–7 h.
600
600
EX
(nm)
EX
(nm)
300
300
300
600
300
600
EM(nm)
EM(nm)
Figure 3. Contour plots of 3D fluorescence scans of 5.0
l
M of 7 (left) and 1 (right) in phosphate-buffered saline pH 7.4.
od. Figure 4 (top) shows exemplarily the quenching of the emission
curve of 5.0 M of 1 by increasing (5–400 M) amounts of DNA. In
Figure 4 (bottom), the relative fluorescence intensities at the max-
ima of 1–3 and 7 are plotted against the concentration of DNA.
Emissions for the stronger fluorescent target compounds 1 and
2 decreased exponentially and reached values under 50% of the ini-
The spectra recorded with 6 showed no bathochromic shift.
However, a slight hyperchromic shift was observed for this
compound.
l
l
In general, these results indicated intercalative properties for 1–
5 and 7 but not for 6. This can be attributed to the absence of the
nitrogen in the side chain of 6, which reduced the ability of the
compound to interact with the DNA (the protonable nitrogen in
the side chain is required to enable an initial electrostatic contact
with the negatively charged DNA phosphate backbone prior to
intercalation between the bases). It should be noted that in the
case of 5 a broadening of the absorption maximum was observed
(see Fig. 5, right), which indicates significant changes in the elec-
tron distribution upon interaction with the nucleobases.
Changes in the DNA melting temperature upon interaction with
an intercalating compound provide an effective means to estimate
tial fluorescence at incubation with 400 lM DNA. In contrast to
this, the emission of 3 could not be influenced strongly by the pres-
ence of DNA (>80% relative intensity at all concentrations) and for
7 the decrease in emission followed an almost linear trend. How-
ever, for all compounds fluorescence-quenching at least at the
highest DNA concentration (400 lM) could be observed, which is
in good agreement with a broad series of previous studies by dif-
ferent groups suggesting naphthalimides as agents with intercalat-
ing properties.1,7,8,10,12,17
UV/vis spectroscopy, CD (circular dichroism) spectroscopy, and
thermal denaturation studies offer useful tools to study the DNA
interaction independent of the presence of fluorescence
properties.28
the efficacy of intercalation. Melting temperature shifts DTm for 1–
7 are listed in Table 1. For 1–4 and 7 values between 6 and 9 °C
were measured and are indicative of an intercalative mode of inter-
action with the DNA. Small
DTm values were found for 5 and 6 cor-
The UV/vis spectra of 1–7 incubated with DNA at r = 0.1
(r = [compound]/[DNA]) for 60 min showed bathochromic and
hypochromic shifts in the case of 1, 2, 4, 5, and 7 (see Fig. 5 and
SI). For 3 a significant bathochromic and a slight hyperchromic
shift could be noted. This pattern could be caused by intramolecu-
relating to low intercalative efficacy.
Circulardichroism(CD) spectroscopy canbe used tomonitorcon-
formational changes of the DNA biopolymer as a consequence of
interaction with drugs. The CD spectrum of DNA shows a negative
peak at approximately 245 nm caused by the helical B conformation
and a positive peak at approximately 275 nm due to base stacking.
Incubation of DNA with 1–3 (see Fig. 6, left) led to a decrease of
the negative peak and an increase of the positive peak. Both effects
lar
p–p interactions between the aromatic ring of the side chain
and the naphthalimide core, which counteract an intercalative
interaction with the DNA.

7110
I. Ott et al. / Bioorg. Med. Chem. 16 (2008) 7107–7116
Table 1
Melting temperature shifts
DNA
DTm [°C] for the interaction of 1–7 with DNA at
3000
2000
1000
0
(0-400 µM)
r = [compound]/[DNA] = 0.1
Compound
D
Tm (°C)
1
2
3
4
5
6
7
8
8
7
9
3
2
6
440
480
520
nm
560
600
were slightly more pronounced than those of 1, 2, 4, 6, and 7
(see Fig. 7). This indicates that sulfur-substituted naphthalimides
could be suitable for triggering phototoxic effects, which is in line
with a series of previous findings.10,16–19
Besides DNA intercalation and photocleavage, the interaction
with the topoisomerase system has been made responsible for
the antiproliferative effects triggered by naphthalimides such as
R16.1,13. Accordingly, the influence of 1–5, 7 and R16 on topoiso-
(1)
(2)
(3)
(7)
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
merase II
a (Topo IIa) was evaluated by Western blot analysis
(see Fig. 8). This assay does not enable to determine an inhibitory
effect of the target compounds on topoisomerase but it reflects
changes in the expression of the enzyme, which may be the conse-
quence of a direct or indirect intracellular interaction with the en-
zyme itself or with related biochemical pathways.
0
50 100 150 200 250 300 350 400 450
DNA (µM)
In this assay 1, 4, and 5 and both reference compounds strongly
reduced Topo IIa protein levels, 2 and 3 were devoid of activity. As
Figure 4. Top: Quenching of the fluorescence emission curve of 5.0
increasing concentrations of DNA (37 °C, 1 h); bottom: fluorescence quenching of
the maxima of 5 M of 1–3 and 7 by increasing concentrations of DNA (37 °C, 1 h);
emissions were recorded at the respective Ex/Em maxima (Ex/Em 1, 402/521; 2, 403/
519; 3, 396/511; 7, 344/396 nm), I: fluorescence intensity in the presence of DNA,
I0: fluorescence intensity in the absence of DNA
lM of 1 by
the non-sulfur compound 7 displayed almost the same activity as
1, 4, 5, and R16, it can be concluded that the effect was indepen-
dent of the presence of the sulfur substituent on position 4 of the
ring system. The inactivity of 2 and 3 in this assay indicated that
long lipophilic side chains are not well tolerated.
l
2.3. Fluorescence-imaging: cellular uptake, intracellular
distribution, and photobleaching
areinagreementwithintercalativepropertiesofthecompounds. For
4 a strong decrease and deformation of the negative peak but no sig-
nificant change of the positive peak was observed. This pattern could
be the consequence of a –NH. . .O hydrogen-bonding interaction be-
tween the aniline nitrogen of 4 and a carbonyl group of the nucleo-
bases. Similar to the results obtained with 1–3 compounds 5 and 7
showed a decrease of the negative peak and an enhancement of
the positive peak (most marked for 7). No significant changes were
noted for 6, whichis in good agreement with theabove-described re-
sults concerning UV/vis spectroscopy and thermal denaturation.
As there are several reports on DNA photodamage and photoc-
leaving mechanisms of naphthalimides, we investigated if the tar-
get compounds 1–5 were able to photocleave pBR322 plasmid DNA
under UV irradiation conditions. Compounds 6 and 7 were used as
references. In this assay, the photocleaving efficacies of 3 and 5
The target compounds seemed suitable for fluorescence
microscopic studies concerning their cellular uptake and biodistri-
bution into the nuclei of tumor cells. For this purpose, MCF-7 hu-
man breast cancer cells were selected, incubated with the drugs,
and investigated using a fluorescence microscope equipped with
a Ex/Em 390 11/460 25 nm filter.
As expected, due to the fluorescence characteristics described
above, the cellular uptake of 1 and 2 was confirmed clearly by
the significant blue emission of the compounds (see Fig. 9). In con-
trast to this 4 and 7 could not be visualized, which can be attrib-
uted on the one hand to the weak intrinsic fluorescence noted
for 4 and on the other hand to the Ex/Em 344/396 maximum of 7,
1
0.3
0.2
0.1
0.0
0.3
0.2
0.1
0.0
5
1+ DNA
5+ DNA
300
350
400
nm
450
500
300
350
400
nm
450
500
Figure 5. UV/vis spectra of 20 lM of 1 and 5 incubated with DNA (200 lM) for 60 min (corresponding spectra of 2–4, 6, 7 are available in the SI).

I. Ott et al. / Bioorg. Med. Chem. 16 (2008) 7107–7116
7111
15
10
5
20
DNA + 1, DNA + 3
DNA + 2
DNA
DNA + 7
15
10
5
DNA + 5
DNA, DNA + 4, DNA + 6
0
0
DNA + 4
-5
-5
DNA, DNA + 6
-10
-10
250
300
350
400
250
300
350
400
nm
nm
Figure 6. CD spectra of DNA (200 lM) incubated with 20 lM of 1–3 (left) and 4–7 (right) for 60 min.
Figure 7. Gel electrophoresis of PBr322 plasmid DNA incubated with 50 lM of 1–7 for 2 h under irradiation at 365 nm. Lanes 1 and 9: control (C, no compound), lanes 2–8:
compounds 1–7; photocleaving properties are indicated by the appearance of a second DNA band (nicked or linear form) with lower electrophoretic mobility than the control
DNA (supercoiled closed circular form). The amount of cleaved DNA is indicated as mean percentage of two independent experiments. (One of the gels is depicted.)
their surrounding chemical environment and binding to mem-
control
1
2
3
4
5
7
R16
A
Topo IIα
branes or biological macromolecules as well as different pH values
within the cell will strongly affect the fluorescence properties of
the compounds.
As shown in Figure 9 the fluorescence intensity appeared to be
spread all over the cells but more intensive spots indicated an
accumulation of the target compounds in cell compartments such
as the nucleus (this effect was most marked for 5).
β-Actin
B
1200
During microscopy photobleaching (decrease of emission inten-
sity) was noted leading to an almost complete loss of blue emission
within a 2–4 min irradiation interval (see Fig. 9, middle left).
However, after keeping the cells for 4 min in the dark (no UV
irradiation) and repeated irradiation, the blue emission was recov-
ered but it remained stable only for a few seconds. In this case, the
blue fluorescence light appeared to be more diffusely spread over
the cells and no spots with increased emission intensity were ob-
served. It should be noted that for 5 almost no fluorescence emis-
sion was recovered after a ‘dark period’ whereas for 1 and 3 results
comparable with those exemplarily depicted for 2 were obtained
(compare Fig. 9 top right and middle pictures).
An important parameter for the fluorescence characteristics of
the presented naphthalimides is the protonable nitrogen of the
side chain. As shown previously for some structurally closely re-
lated compounds, protonation of the nitrogen caused an increase
in fluorescence emission.16 Therefore, we included 6, in which
the N,N-dimethylamino group is replaced by a mercapto group,
in the experiments. In contrast to the well-detectable 1, cells incu-
bated with its thiol derivative 6 did not show any significant blue
emission (data not shown).
1000
800
600
400
200
0
control
1
2
3
4
5
7
R16
Figure 8. (A) Western blot analysis of protein extracts obtained from HeLa cells
treated with drugs at their IC50 concentrations (see SI) for 24 h. (B) Quantification of
the bands was performed and normalized in relation to untreated cells. b-Actin was
used as a loading control. Data are presented as means SD of three independent
experiments.
which is outside the excitation and emission bandwidths of the
used filter.
Unexpectedly, 3 and 5 could be well monitored despite the low
or missing fluorescence emission in phosphate-buffered saline pH
7.4 (see Fig. 9, bottom). In fact, there are many reports on the
enhancement of naphthalimide fluorescence emission due to the
inhibition or switching-off of intramolecular PET processes upon
binding to metal ions,25,28–32 changing the pH value,14,16,34 or low-
ering the solvent polarity.26,34–36 Thus, the fluorescence properties
and quantum yields of naphthalimides are largely influenced by
2.4. Antiproliferative effects and phototoxicity
The antiproliferative effects of 1–5 were evaluated in MCF-7
breast cancer and HT-29 colon carcinoma cells. R16, 6, and 7 were

7112
I. Ott et al. / Bioorg. Med. Chem. 16 (2008) 7107–7116
Figure 9. Fluorescence microscopic studies of MCF-7 cells (40-fold enlargement) incubated for 6 h with 10 lM of 2, 3, and 5; top left: cells incubated with 2, picture taken
without using fluorescence equipment; top right: blue emission of 2 after a few seconds of fluorescence microscopy irradiation; middle left: no emission of 2 after 4 min
fluorescence microscopy irradiation; middle right: blue emission of 2 after a few seconds of fluorescence microscopy irradiation following a period of 4 min of fluorescence
microscopy irradiation and 4 min without irradiation; bottom left: blue emission of 3 after a few seconds of fluorescence microscopy irradiation; bottom right: blue emission
of 5 after a few seconds of fluorescence microscopy irradiation.
used as references. Experiments were performed under continuous
(72 h for HT-29 and 96 h for MCF-7) exposure to graded concentra-
tions of the compounds or under short-time exposure (24 h) to the
drugs followed by additional culture in drug-free media with or
without a 20-min 366-nm irradiation interval. According to this
experimental setup, only the drug taken up into the cells is irradi-
ated and no toxic photochemical products formed outside the cells
will influence the outcome of the results.
by far most active compound. However, 1–5 were also highly active
and displayed IC50 values between 1.9 and 4.6 M, which are well
in the range of established antitumor agents. Thus, cisplatin and 5-
fluorouracil showed IC50 values of 2.0 and 4.8 M in the same as-
say38 in MCF-7 cells and for amonafide comparable activity in
the used cell lines has been reported (IC50 value of 1.1 M in
MCF-7 cells5 and 4.7 M in HT-29 cells6,7). As expected compound
6, which does not contain the N,N-dimethylamino pharmacophore
required for proper interaction with the DNA or topoisomerase,
was devoid of any activity in all the experiments.
l
l
l
l
In the continuous exposure experiments (see ‘96 h’ and ‘72 h’
columns of Table 2) R16 was with IC50 values below 1.0 lM the
Table 2
Antiproliferative effects (IC50 value) in MCF-7 and HT-29 cells after continuous exposure to the drugs (MCF-7: 96 h, HT-29: 72 h), short exposure followed by culture in drug-free
media (MCF-7: 24/72 h, HT-29: 24/48 h) or short exposure followed by irradiation (20 min, 366 nm) and culture in drug-free media (MCF-7: 24 h/irrad./72 h, HT-29: 24 h/irrad./
48 h); experiments were performed in duplicate (each n = 6); n.d., not determined
IC50 MCF-7 (
l
M)
IC50 HT-29 (lM)
96 h
4.4
2.6
3.4
2.6
2.7
>50
4.5
0.20
24/72 h
24 h/irrad./72 h
72 h
24/48 h
24 h/irrad./48 h
1.6
0.6
0.5
0.9
0.5
0.7
0.5
0.1
0.3
0.4
1
2
3
4
5
6
7
R16
4.5
5.7
6.7
4.8
4.3
4.0
1.7
4.9
1.9
4.6
4.1
2.9
4.4
n.d.
3.5
2.2
4.3
4.8
2.8
2.5
3.0
1.3
2.8
1.0
0.3
0.2
0.1
0.9
1.4
0.0
0.5
1.1
0.4
0.4
0.0
1.6
1.8
0.7
0.3
0.4
0.6
1.1
0.4
7.3
2.4
5.5
1.8
>50
11.6
0.17
>50
11.3
0.18
>50
7.2
0.23
>50
5.3
0.21
0.3
3.8
3.3
0.9
0.8
0.3
0.03
0.01
0.02
0.18
0.03
0.06
0.62

I. Ott et al. / Bioorg. Med. Chem. 16 (2008) 7107–7116
7113
Interestingly, in the continuous exposure experiments no sig-
nificant effect of the sulfur substituents of 1–5 could be noted as
the unsubstituted counterpart 7 exhibited comparable activity.
This result was surprising as the ring-fused thio-heterocyclic R16
was the most active substance in the studied series.
irradiation. Although the IC50 values of the lead compound R16
could not be reached, the exclusive phototoxicity of 3 and 5 may
offer interesting therapeutic advantages concerning the applica-
tion in photodynamic tumor therapy.
In this context it should be noted that for therapeutic purposes
the use of advanced light dosimetry technology (e.g., fibers or
inflatable balloons as used for photodynamic treatment of brain tu-
mors40) should be envisaged due to the low tissue penetration of
low wavelength irradiation.41 Based on the fact that 3 and 5 dis-
played antiproliferative effects also under non-irradiation condi-
tions, they could be useful lead compounds for cytostatics that
are active under ‘normal’ therapeutic conditions and can be addi-
tionally activated in a controlled manner by the use of advanced
optical fiber technology. It can be assumed that under dark condi-
tions intercalation and most probably the interaction with topoiso-
merase system lead to cell growth-inhibiting effects. Under
photoirradiation supposedly the cleavage of DNA contributes addi-
tionally to the antiproliferative potency of the agents.
Short time exposure (24 h) to the agents followed by incubation
in drug-free media showed in most of the experiments an increase
of the IC50 values in MCF-7 cells but in HT-29 cells no strong effect
could be noted (compare ‘24 h/72 h’ and ‘24 h/48 h’ columns in Ta-
ble 2 with the corresponding ‘96 h’ and ‘72 h’ columns). This is
most probably the consequence of the longer incubation period
used for MCF-7 cells (which is based on the slower growth charac-
teristics of MCF-7 cells compared to HT-29 cells, see SI) allowing a
more effective recovery from the initial drug exposure period.
Analogous experiments including a 20 min irradiation interval
at 366 nm showed 3–5 times decreased IC50 values for 3 and 5 in
both investigated cell lines (for MCF-7 cells compare the ‘24 h/
72 h’ with the ‘24 h/irrad./72 h’ column in Table 2, for HT-29 com-
pare the corresponding ‘24 h/48 h’ and ‘24 h/irrad./48 h’ columns).
In contrast to this with 1, 4, 6, and R16 absolutely no changes in the
antiproliferative activity could be noted upon irradiation. For 7 in
HT-29 and for 2 in both cell lines a very weak (each 1.4-fold lower
IC50 values) effect of the irradiation procedure was observed.
The environmental sensitivity and phototoxic behavior of the
presented compounds can be seen as a consequence of the electron
distribution over the naphthalimide ring system, which is itself
influenced by the residues at the sulfur substituent in position 4.
Consequently, changes in electron distribution of the relatively
electron-deficient naphthalimide system will affect intramolecular
PET processes, which are themselves strongly dependent on the
chemical environment.14,16,25–36
The enhanced phototoxicity of 3 and 5 might be the conse-
quence of toxic radicals formed during the irradiation period and
seems to correlate with the sensitivity of their fluorescence emis-
sions to the surrounding chemical environment (as observed dur-
ing the microscopy studies) as well as their enhanced
photocleaving efficacy (see above).
According to the above-described results the introduction of
sulfur substituents containing non fused (hetero)aromatic rings lo-
cated close to the naphthalimide core can be recommended for fur-
ther drug development.
3. Discussion
4. Conclusion
A series of sulfur-substituted naphthalimides was prepared
and investigated for biological properties. Initial DNA interaction
studies (by fluorescence-quenching, UV/vis, thermal denatur-
A series of sulfur-substituted naphthalimides exhibiting the
established biological properties of this class of compounds (DNA
interaction and antiproliferative effects) was prepared. The present
study demonstrates that the development of phototoxic naphthal-
imides can be realized by introducing appropriate sulfur residues
on the 4 position of the naphthalimide core. To the best of our
knowledge, this is the first report proving the phototoxicity of
naphthalimides in tumor cell culture studies. The fluorescence
emission of the target compounds was significantly influenced by
the chemical (cellular) environment.
ation, plasmid DNA photocleaving, and Topo II
a Western blot
assays) indicated intercalation into the DNA and interaction
with the topoisomerase system as possible modes of drug ac-
tion of 1–5 and 7 and suggested a potential use of the target
compounds as photoactivatable antitumor agents. In contrast
the reference compound 6, which does not contain the N,N-
dimethylaminoethyl side chain, was devoid of significant activ-
ity in all the mentioned assays.
Fluorescence microscopic studies showed that the compounds
were taken up into the cells in significant amounts. Replacement
of the N,N-dimethylamino group with a mercapto group (6) pre-
vented fluorescence emission of the cells highlighting the impor-
tance of the side chain nitrogen for cellular uptake and/or
fluorescence properties. The detectability of 3 and 5, which exhib-
ited only weak or no fluorescence in buffer solution, was compara-
ble to that of 1 and 2, which showed strong fluorescence emission
in buffer solution. This effect may be attributed to sensitivity of the
fluorescence of the compounds to their chemical environment
(concerning pH value, polarity, membranes, and macromolecules).
Similar properties (meaning weak fluorescence in polar aqueous
solvents but intensive fluorescence emission in apolar solvents or
upon binding to hydrophobic regions of proteins or membranes)
have recently been applied for biological binding studies (e.g.,
opioide receptor binding) using an environment-sensitive naph-
thalimide fluorophore.37,39
5. Experimental
5.1. General
Chemicals and reagents were purchased from Sigma, Aldrich,
and Fluka. PBS: phosphate-buffered saline, pH 7.4; for biochemical
assays and in-vitro studies, the compounds were prepared as stock
solutions in dimethylsulfoxide (DMSO) and diluted 1:1000 with
the respective media. Human-reactive monoclonal antibodies,
anti-topoisomerase IIa, were from Thermo Scientific. NMR spectra
were recorded on a 400 MHz spectrometer or a 500 MHz NMR
spectrometer (Bruker); elemental analysis: Perkin-Elmer 240 C;
MS spectra: CH-/A- Varian MAT.
5.2. Cell culture
Interestingly, those target compounds (3 and 5) that were most
susceptible to fluorescence induction in cellular environment and
showed enhanced plasmid DNA photocleaving properties triggered
significant phototoxic effects in cultured tumor cells (MCF-7 and
HT-29) with 3–5 times lowered IC50 values after short-time UV
MCF-7 breast cancer, HT-29 colon carcinoma, and HeLa adeno-
carcinoma cells were maintained by standard procedures at 37 °C
in a humidified atmosphere of 95% air and 5% CO2; cell culture med-
ium for MCF-7 and HT-29 cells: minimum essential medium eagle
supplemented with 2.2 g NaHCO3, 110 mg/L sodium pyruvate, and

7114
I. Ott et al. / Bioorg. Med. Chem. 16 (2008) 7107–7116
50 mg/L gentamicin sulfate and adjusted to pH 7.4. Prior to use 10%
(V/V) fetal calf serum was added; cell culture medium for HeLa cells
RPMI 1640 medium supplemented with 10% heat-inactivated bo-
vine serum, penicillin (100 U/mL), and streptomycin (100 U/mL).
(DMSO-d6): d = 7.39 (m, 1H, ArH), 7.49 (ddd, 1H, 3J = 8.2 Hz,
3J = 7.2 Hz, 4J = 1.2 Hz, ArH), 7.88 (m, 1H, ArH), 7.97 (m, 1H, ArH),
8.02 (dd, 1H, 3J = 8.5 Hz, 4J = 1.2 Hz, ArH), 8.40 (d, 1H, 3J = 7.6 Hz,
ArH), 8.59 (d, 1 H, 3J = 7.6 Hz, ArH), 8.63 (dd, 1H, 3J = 7.3 Hz,
4J = 0.9 Hz), 8.81 (dd, 1H, 3J = 8.5 Hz, 4J = 0.9 Hz).
5.3. Synthesis of sulfur-substituted naphthalic anhydrides
5.9. Synthesis of 1–5 and 7
4-Bromo-1,8-naphthalic anhydride (1.75–5.77 mmol) was dis-
solved in 15 mL dimethylformamide and an excess of thiol and
K2CO3 were added. After stirring for 3–7 h at ambient temperature
(30–50 °C), the suspension was poured into 40 mL distilled water,
the precipitate was collected by filtration, washed with distilled
water, and dried. In case the 1H NMR spectra showed significant
amounts of the mercapto educts, the products were further puri-
fied by column chromatography over SiO2.
1,8-Naphthalic anhydrides were suspended in 15 mL EtOH abs
and an excess of amine was added. The mixture was stirred under
reflux conditions for 5–7.5 h. After evaporation of the solvent, the
naphthalimides were isolated by column chromatography (CC)
over SiO2. Some compounds were obtained as hydrates (the pres-
ence of water was confirmed by 1H NMR in CDCl3) due to water
impurities in the chromatographic solvents.
5.4. 4-Ethylthio-1,8-naphthalic anhydride
5.10. N-(N0,N0-Dimethyl-2-aminoethyl)-4-ethylthio-1,8-
naphthalimide (1)
4-Bromo-1,8-naphthalic anhydride: 1.60 g (5.77 mmol); etha-
nethiol: 0.44 mL (5.99 mmol), K2CO3: 0.51 g; 40 °C, 3 h; yield:
1.382 g (93%) yellow crystals; 1H NMR (DMSO-d6): d = 1.41 (t, 3H,
3J = 7.3 Hz, –CH3), 3.32 (q, 2H, 3J = 7.3 Hz, –CH2), 7.78 (d, 1H,
3J = 7.9 Hz, ArH), 7.92 (ddd, 1H, 3J = 8.2 Hz, 3J = 8.3 Hz, 4J = 0.8 Hz,
ArH), 8.40 (d, 1H, 3J = 7.9 Hz, ArH), 8.57 (m, 2H, 2 ArH).
4-Ethylthio-1,8-naphthalic anhydride: 106 mg, (0.41 mmol),
0.15 mL N,N-dimethylethylamine; 5 h, CC: CHCl3 followed by eth-
ylacetate/methanol 9:1 and 8:2; yield: 128 mg (0.39 mmol, 95%)
yellow crystals (mp 103 °C); 1H NMR (CDCL3): d = 1.50 (t, 3 H,
3J = 7.6 Hz), 2.39 (s, 6H, N(CH3)2), 2.68 (t, 2H, 3J = 7.2 Hz), 3.21 (q,
2H, 3J = 7.6 Hz), 4.34 (t, 2H, 3J = 7.2 Hz), 7.56 (d, 1H, 3J = 8.0 Hz,
ArH), 7.76 (m, 1H, ArH), 8.49 (d, 1H, 3J = 8.0 Hz, ArH), 8.58 (m,
1H, ArH), 8.64 (m, 1H, ArH); MS (EI): m/z = 328 (2.1%, M+), 298
(0.3%, M+ꢀ2 CH3), 58 (100%). Anal. (Calcd/Found): C (65.83/
65.70), H (6.14/6.14), N (8.53/8.53).
5.5. 4-Butylthio-1,8-naphthalic anhydride
4-Bromo-1,8-naphthalic anhydride: 485 mg (1.75 mmol),
0.32 mL butane-1-thiol; K2CO3: 0.54 g; 30 °C, 7 h; yield: 204 mg
(0.71 mmol, 41%) yellow crystals; 1H NMR (DMSO-d6): d = 0.94
(t, 3H, 3J = 7.3 Hz, CH3), 1.51 (m, 2H, CH2), 1.74 (m, 2H, CH2),
3.32 (t, 2H, 3J = 7.4 Hz, –S–CH2), 7.80 (d, 1H, 3J = 7.9 Hz, ArH),
7.92 (m, 1H, ArH), 8.40 (d, 1H, 3J = 7.9 Hz, ArH), 8.56 (m, 1H,
ArH), 8.62 (m, 1H, ArH).
5.11. N-(N0,N0-Dimethyl-2-aminoethyl)-4-butylthio-1,8-
naphthalimide (2)
4-Butylthio-1,8-naphthalic anhydride: 104 mg (0.36 mmol),
0.2 mL N,N-dimethylaminoethylamine; 6.5 h, CC: ethylacetate/
methanol 4:1 and CH2Cl2, CH2Cl2/MeOH 5:1; yield: 72 mg
(0.20 mmol, 56%) yellow crystals; 1H NMR (CDCl3): d = 0.99 (t,
3H, 3J = 7.3 Hz, –CH3), 1.56 (m, 2H, CH2), 1.80 (m, 2H, CH2), 2.36
(s, 6H, –N(CH3)2), 2.66 (t, 2H, 3J = 7.1 Hz, CH2), 3.16 (t, 2H,
3J = 7.4 Hz, CH2), 4.32 (t, 2H, 3J = 7.1 Hz, CH2), 7.54 (d, 1H,
3J = 7.9 Hz, ArH), 7.74 (dd, 1H, 3J = 8.5 Hz, 3J = 7.3 Hz, ArH), 8.47
(d, 1H, 3J = 7.9 Hz), 8.58 (dd, 1H, 3J = 8.5 Hz, 4J = 1.1 Hz, ArH), 8.62
(dd, 1H, 3J = 7.3 Hz, 4J = 1.1 Hz). Anal (Calcd/Found): C (67.38/
67.43), H (6.79/6.81), N (7.86/7.77).
5.6. 4-Benzylthio-1,8-naphthalic anhydride
4-Bromo-1,8-naphthalic anhydride: 703 mg (2.54 mmol), ben-
zylthiol: 0.4 mL, K2CO3: 0.55 g; 30–50 °C, 6 h; the crude product
was purified by column chromatography over SiO2 (eluent: CHCl3,
CHCl3/MeOH 10:1 and 10:2); yield: 317 mg (0.99 mmol, 39%) yel-
low crystals; 1H NMR (DMSO-d6): d = 4.62 (s, 2H, CH2), 7.29 (m, 1H,
ArH), 7.36 (m, 2H, 2 ArH), 7.52 (m, 2H, 2 ArH), 7.92 (m, 2H, 2 ArH),
8.39 (d, 1H, 3J = 7.9 Hz, ArH), 8.55 (dd, 1H, 3J = 7.3 Hz, 4J = 0.9 Hz),
8.62 (dd, 1H, 3J = 8.5 Hz, 4J = 0.9 Hz).
5.12. 4-Benzylthio-N-(N0,N0-dimethyl-2-aminoethyl)-1,8-
naphthalimideꢁ4H2O (3)
5.7. 4-(2-Aminophenyl)-thio-1,8-naphthalic anhydride
4-Bromo-1,8-naphthalicanhydride: 942 mg (3.40 mmol), o-ami-
nothiophenol: 0.4 mL; K2CO3: 0.61 g, 40 °C, 6 h; the crude product
was purified by column chromatography over SiO2 (eluent: CHCl3);
yield: 687 mg (2.14 mmol, 63%) yellow crystals; 1H NMR (DMSO-
d6): d = 5.60 (s, 2H, –NH2), 6.70 (m, 1H, ArH), 6.92 (dd, 1H,
3J = 8.2 Hz, 4J = 1.1 Hz, ArH), 7.00 (d, 1H, 3J = 8.0 Hz, ArH), 7.32 (m,
1H, ArH), 7.40 (dd, 1H, 3J = 7.7 Hz, 4J = 1.4 Hz, ArH), 7.99 (m, 1H,
ArH), 8.33 (d, 1H, 3J = 8.0 Hz, ArH), 8.60 (dd, 1H, 3J = 7.3 Hz,
4J = 0.8 Hz, ArH), 8.77 (dd, 1H, 3J = 8.5 Hz, 4J = 0.8 Hz, ArH), prepara-
tion of 4-(2-aminophenyl)-thio-1,8-naphthalic anhydride without
structural characterization data has been reported.19
4-Benzylthio-1,8-naphthalic anhydride: 118 mg (0.37 mmol),
0.20 mL N,N-dimethylaminoethylamine; 6.5 h, CC: ethylacetate/
methanol 4:1 and CH2Cl2/methanol 5:1; yield: 88 mg (0.19 mmol,
51%), yellow crystals (mp 252 °C); 1H NMR (DMSO-d6): d = 2.82 (br,
2H, CH2), 3.36 (s, 6H, –N(CH3)2), 4.22 (tbr, 2H, CH2), 4.57 (s, 2H,
CH2), 7.30 (m, 3H, 3ꢂ ArH), 7.49 (m, 2H, 2ꢂ ArH), 7.88 (m, 2H,
2ꢂ ArH), 8.37 (d, 1H, 3J = 8.0 Hz, ArH), 8.54 (m, 2H, 2ꢂ ArH); MS
(EI): m/z = 390 (3%, M+), 58 (100%). Anal. (Calcd/Found): C (59.72/
59.43), H (6.54/6.19), N (6.06/6.05).
5.13. 4-(2-Aminophenyl)-thio-N-(N0,N0-dimethyl-2-
aminoethyl)-1,8-naphthalimideꢁH2O (4)
5.8. 4-(4,5-Benzo-1,3-thiazol-2-yl)-thio-1,8-naphthalic
anhydride
4-(2-Aminophenyl)-thio-1,8-naphthalic anhydride: 106 mg
(0.33 mmol), 0.20 mL N,N-dimethylaminoethylamine; 7.5 h; CC:
4-Bromo-1,8-naphthalic anhydride: 977 mg (3.53 mmol), 2-
mercaptobenzothiazol: 668 mg (3.99 mmol); K2CO3: 0.52 g, 45 °C,
7 h; yield: 930 mg (2.56 mmol, 73%) yellow crystals; 1H NMR
ethylacetate/methanol
4:1;
yield:
115 mg
monohydrate
(0.28 mmol, 85%), orange crystals (mp 256 °C); 1H NMR (DMSO-
d6): d = 2.88 (br, 2H, CH2), 3.37 (s, 6H, –N(CH3)2), 4.23 (t, 2H,

I. Ott et al. / Bioorg. Med. Chem. 16 (2008) 7107–7116
7115
3J = 5.8 Hz), 5.57 (s, 2H, NH2), 6.70 (m, 1H, ArH), 6.91 (m, 1H, ArH),
7.0 (d, 1H, 3J = 7.9 Hz, ArH), 7.31 (m, 1H, ArH), 7.40 (m, 1H, ArH),
7.96 (m, 1H, ArH), 8.31 (d, 1H, 3J = 7.9 Hz, ArH), 8.58 (m, 1H,
ArH), 8.71 (m, 1H, ArH); MS (EI): m/z = 391 (4.6%, M+), 58 (100%).
Anal. (Calcd/Found): C (64.53/64.38), H (5.66/5.82), N (10.26/
10.16).
tion = M(nucleotide) = 200
l
M] were determined in a 10 mM phos-
phate buffer at pH = 7.2 in the presence of 1% DMSO. Melting
curves were recorded at 1 °C steps for the wavelength 260 nm with
an Analytik Jena SPECORD 200 spectrometer equipped with a Pel-
tier temperature controller. Tm values were calculated by deter-
mining the midpoints of melting curves from the first-order
derivatives. The experimental
DTm values are estimated to be accu-
5.14. 4-(4,5-Benzo-1,3-thiazol-2-yl)-thio-N-(N0,N0-dimethyl-2-
aminoethyl)-1,8-naphthalimideꢁ3H2O (5)
rate within 1 °C. Concentrations of CT DNA were determined
spectrophotometrically using the molar extinction coefficient e260
= 6600 Mꢀ1 cmꢀ1 42
All electronic absorption measurements were
.
4-(4,5-Benzo-1,3-thiazol-2-yl)-thio-1,8-naphthalic anhydride:
114 mg (0.31 mmol), 0.20 mL N,N-dimethylaminoethylamine;
6.5 h; SC: CH3Cl followed by ethylacetate/methanol 8:2 and 4:1;
yield: 88 mg (0.18 mmol, 58%), yellow crystals (mp 248 °C); 1H
NMR (CDCl3): d = 2.96 (d, 6H, 3J = 4.8 Hz, –NH+(CH3)2), 3.44 (m,
2H, CH2), 4.62 (m, 2H, CH2), 7.34 (m, 1H, ArH), 7.47 (m, 1H, ArH),
7.68 (m, 1H, ArH), 7.85 (m, 1H, ArH), 7.95 (m, 1H, ArH), 8.24 (d,
1H, 3J = 7.6 Hz, ArH), 8.64 (d, 1H, 3J = 7.6 Hz, ArH), 8.70 (m, 1H,
ArH), 8.81 (m, 1H, ArH); MS (EI): m/z = 433 (5.7%, M+), 58 (100%).
Anal. (Calcd/Found): C (56.66/56.99), H (5.17/4.92), N (8.62/8.88).
performed at 293 K. After sonication, buffered solutions of CT
DNA gave a UV absorbance ratio A260/A280 of approximately 1.90,
indicating that the DNA was sufficiently free of proton.43 Twenty
micromolar solutions of the compounds were treated with
200 lM DNA (nucleotide) for 60 min. All UV/vis and CD spectra
were measured after equilibration, that is, no further change in
the monitored absorbance or molar ellipticity. The UV/vis mea-
surements were performed on an Analytik Jena SPECORD 200 spec-
trometer and the CD measurements on a Jasco J-715 instrument.
5.19. DNA photocleavage
5.15. N-(N0,N0-Dimethylaminoethyl)-1,8-naphthalimide (7)
To 0.25
lg covalently closed pBR322 plasmid DNA (Takara Bio-
1,8-Naphthalic anhydride: 200 mg (1.01 mmol), 0.20 mL N,N-
dimethylaminoethylamine; 6.5 h; CC: dichloromethane/methanol
8:2; yield: 252 mg (0.94 mmol, 94%) white crystals, 1H NMR
(CDCl3): d = 2.37 (s, 6H, –N(CH3)2), 2.67 (t, 2H, 3J = 7.1 Hz CH2),
4.35 (t, 2H, 3J = 7.1 Hz CH2), 7.75 (dd, 2H, 3J = 7.4 Hz, 3J = 8.0 Hz,
2ꢂ ArH), 8.21 (m, 2H, 2ꢂ ArH), 8.61 (dd, 2H, 3J = 7.4 Hz,
technology, Dalian, China) in 9
l
L buffer (Tris–HCl, pH 7.5) 1.0 lL
of a 0.5 mM stock solution of 1–7 in DMSO or blank DMSO (control)
was added. The solutions were irradiated for 2 h at 365 nm (8 W,
distance: 10 cm). Gel electrophoresis of the samples was per-
formed in a 1% agarose gel. DNA was visualized by ethidium bro-
mide staining and photographed. Cleaved DNA was quantified
using Gel-Pro Analyzer software (Version 4.0) and expressed as
percentage of the total.
4J = 0.8 Hz, 2ꢂ ArH). Anal. (Calcd/Found):
C (71.62/71.84), H
(6.01/6.19), N (10.44/10.06); preparation of 7 without NMR struc-
tural characterization data has been reported.2,25
5.20. Western blot analysis of Topo II
treatment
a level after drugs
5.16. Synthesis of 4-ethylthio-N-(2-thiolethyl)-1,8-
naphthalimide (6)
HeLa cells (1 ꢂ 106/dish) were seeded in 10-cm dishes. After
12 h of incubation, cells were treated with drugs (1–7) and R16
at the concentration of their IC50 values (as determined in separate
experiments, see Supporting information) for 24 h, respectively.
Cells were lysed in a 0.1% NP40 buffer containing 10 mM NaCl,
5 mM EDTA, 50 mM NaF, 50 mM Tris–HCl (pH 7.5), 1 mM phenyl-
methylsulfonyl fluoride, 20 mg/mL leupeptin, 20 mg/mL aprotonin,
20 mg/mL antipaoen, 1 mM DTT, and 0.1% SDS and placed in ice for
30 min. After centrifugation for 15 min at 4 °C, the supernatant was
collected. Lysates were quantified using the BCA protein assay
(Pierce Biochemicals, Rockland, IL). For Western blot analysis,
4-Ethylthio-1,8-naphthalic anhydride (630 mg, 2.44 mmol) was
suspended in 50 mL absolute ethanol, 410 mg (3.61 mmol) cyste-
amine hydrochloride, and 0.5 mL triethylamine were added and
the mixture was stirred under reflux condition for 6 h. The formed
precipitate was isolated by filtration, washed with ethanol, and
dried. Yield 579 mg (1.76 mmol, 72%) yellow crystals; 1H NMR
(DMSO-d6): d = 1.37 (t, 3H, 3J = 7.3 Hz, CH3), 3.05 (t, 2H,
3J = 7.2 Hz, CH2), 3.25 (q, 2H, 3J = 7.3 Hz, CH2), 4.33 (t, 2H,
3J = 7.2 Hz, CH2), 7.72 (d, 1H, 3J = 8.0 Hz, ArH), 7.86 (dd, 1H,
3J = 7.9 Hz, 3J = 7.9 Hz, ArH), 8.33 (d, 1H, 3J = 8.0 Hz, ArH), 8.50
(m, 2H, 2ꢂ ArH); MS (EI): m/z = 317 (24.6%, M+), 284 (68.1%,
M+ꢀH2S), 258 (100%). Anal. (Calcd/Found): C (60.54/60.30), H
(4.76/4.75), N (4.41/4.66).
equal amounts of proteins (50 lg) were separated by SDS–poly-
acrylamide gel electrophoresis and transferred to a nitrocellulose
membrane. The blot was blocked in blocking buffer (5% non-fat
dry milk/1% Tween 20 in PBS) for 1 h at room temperature and
then incubated overnight with appropriate primary antibodies in
blocking buffer overnight at 4 °C. The blot was then incubated with
appropriate secondary antibody alkaline phosphatase (AP) conju-
5.17. Fluorescence scans and DNA fluorescence-quenching
Compounds were prepared as 5.0 mM stock solutions in DMSO.
For initial fluorescence-scanning investigations, the stock solutions
were diluted 1:1000 with PBS and measured. For DNA-binding
studies, the stock solutions were diluted 1:1000 with PBS contain-
ing DNA in graded concentrations (nucleoside concentration: 0, 5,
gate and detected in 10 mL AP buffer containing 33
lL BCIP and
66 l NBT (at room temperature for 10–20 min), and then photo-
l
graphed. b-Actin was used as a loading control.
25, 50, 100, 200, 400
lM), incubated for 60 min at 37 °C and mea-
5.21. Fluorescence microscopic studies
sured. Fluorescence measurements were performed using a Hitachi
F-4500 fluorescence spectrometer.
MCF-7 cells were grown in 6-well plates (Sarstedt) until at least
70% confluence. The cell culture medium was replaced with fresh
5.18. DNA binding studies (thermal denaturation, UV/vis, and
circular dichroism)
medium containing the compounds in a concentration of 10 lM
(0.1% V/V DMSO) and incubated for 6 h at 37 °C in a 5% CO2/95%
air atmosphere. The medium was removed, the cells were washed
The thermal denaturation temperature Tm of 1:10 compound/
with PBS, and finally 500
lL PBS was added to each well. Fluores-
DNA mixtures [compound concentration: 20 lM, DNA concentra-
cence microscopy was performed using a Axiovert 40 CFL micro-

7116
I. Ott et al. / Bioorg. Med. Chem. 16 (2008) 7107–7116
5. Sami, S. M.; Dorr, R. T.; Alberts, D. S.; Solyom, A. M.; Remers, W. A. J. Med. Chem.
2000, 43, 3067.
scope (Zeiss) equipped with a 50 W mercury vapor short arc lamp
and a Ex/Em 390 11/460 25 nm filter.
6. Brana, M. F.; Cacho, M.; Ramos, A.; Dominguez, M. T.; Pozuelo, J. M.; Abradelo, C.;
Rey-Stolle, M. F.; Yuste, M.; Carrasco, C.; Bailly, C. Org. Biomol. Chem. 2003, 1, 648.
7. Brana, M. F.; Cacho, M.; Garcia, M. A.; de Pascul-Teresa, B.; Ramos, A.;
Dominguez, M. T.; Pozuelo, J. M.; Abradelo, C.; Rey-Stolle, M. F.; Yuste, M.;
Banez-Coronel, M.; Lacal, J. C. J. Med. Chem. 2004, 47, 1391.
8. Antonini, I.; Santoni, G.; Lucciarini, R.; Amantini, C.; Sparapani, S.; Magnano, A.
J. Med. Chem. 2006, 49, 7198.
5.22. Antiproliferative effects and phototoxicity
The continuous exposure experiments were performed accord-
ing to a previously described assay procedure.38 In short each
9. Quaquebeke, E. V.; Mahieu, T.; Dumont, P.; Dewelle, J.; Ribaucour, F.; Simon, G.;
Sauvage, S.; Gaussin, J. F.; Tuti, J.; El Yazidi, M.; Van Vynckt, F.; Mijatovic, T.;
Lefranc, F.; Darro, F.; Kiss, R. J. Med. Chem. 2007, 50, 4122.
10. Li, Z.; Yang, Q.; Qian, X. Bioorg. Med. Chem. Lett. 2005, 15, 3143.
11. Li, Z.; Yang, Q.; Qian, X. Bioorg. Med. Chem. 2005, 13, 4864.
12. Li, F.; Cui, J.; Guo, L.; Qian, X.; Ren, W.; Wang, K.; Liu, F. Bioorg. Med. Chem. 2007,
15, 5114.
13. Zhu, H.; Huang, M.; Yang, F.; Chen, Y.; Miao, Z. H.; Qian, X.; Xu, Y.; Qin, Y.; Luo,
H.; Shen, X.; Geng, M.; Cai, Y.; Ding, J. Mol. Cancer Ther. 2007, 6, 484.
14. Pardo, A.; Poyato, J. M. L.; Martin, E. J. Photochem. 1987, 36, 323.
15. Cao, H.; Chang, V.; Hernandez, R.; Heagy, M. D. J. Org. Chem. 2005, 70, 4929.
16. Qian, X.; Li, Y.; Xu, Y.; Liu, Y.; Qu, B. Bioorg. Med. Chem. Lett. 2004, 14, 2665–
2668.
17. Li, Z.; Yang, Q.; Qian, X. Bioorg. Med. Chem. Lett. 2005, 15, 1769.
18. Xu, Y.; Qu, B.; Qian, X.; Li, Y. Bioorg. Med. Chem. Lett. 2005, 15, 1139.
19. Yang, Q.; Qian, X.; Xu, J.; Sun, Y.; Li, Y. Bioorg. Med. Chem. 2005, 13, 1615.
20. Rogers, J. E.; Abraham, B.; Rostkowski, A.; Kelly, L. A. Photochem. Photobiol.
2001, 74, 521.
21. Chanh, T. C.; Lewis, D. E.; Allan, J. S.; Sogandares-Bernal, F.; Judy, M. M.; Utecht,
R. E.; Matthews, J. L. AIDS Res. Hum. Retroviruses 1993, 9, 891.
22. Chanh, T. C.; Lewis, D. E.; Judy, M. M.; Sogandares-Bernal, F.; Michalek, G. R.;
Utecht, R. E.; Skiles, H.; Chang, S. C.; Matthews, J. L. Antiviral Res. 1994, 25, 133.
23. Pervaiz, S. FASEB J. 2001, 15, 612.
100 lL of 10,000 cells/mL (MCF-7) or 2850 cells/mL (HT-29) were
incubated in 96-well plates at 37 °C in 5% CO2/95% air atmosphere
for 48 h (HT-29) or 72 h (MCF-7). Then the medium was replaced
with medium containing the drugs in graded concentrations
(200 lL per well, six replicates). After further incubation for 72 h
(HT-29) or 96 h (MCF-7), the cell biomass was determined by crystal
violet staining. For the short-time incubation and phototoxicity
experiments this procedure was modified as follows: after 24 h of
incubation with the drug containing media, the wells were emptied
and new drug-free cell culture medium was added (200 lL per well).
The plates for evaluation of phototoxic effects were placed under a
4 W UV lamp and irradiated at 366 nm for 20 min at a distance of
5 cm. Then incubation at 37 °C in 5% CO2/95% air atmosphere was
continued for further 48 h (HT-29) or 72 h (MCF-7). The cell bio-
masses were determined by crystal violet staining. IC50 values were
determined as that concentration causing 50% inhibition of cell pro-
liferation compared to an untreated control and expressed as mean
value of at least two independent experiments.
24. Qian, X.; Huang, T. B.; Wei, D. Z.; Zhu, D. H.; Fan, M. C.; Yao, W. J. Chem. Soc.
Perkin. Trans. 2 2000, 715–718.
25. Ramachandram, B.; Saroja, G.; Sankaran, N. B.; Samanta, A. J. Phys. Chem. B
2000, 104, 11824.
Acknowledgment
26. Ramachandram, B. J. Fluoresc. 2005, 15, 71.
27. Cho, D. W.; Fujitsuka, M.; Sugimoto, A.; Yoon, U. C.; Mariano, P. S.; Majima, T. J.
Phys. Chem. B 2006, 110, 11062.
28. Schäfer, S.; Ott, I.; Gust, R.; Sheldrick, W. S. Eur. J. Inorg. Chem. 2007, 3034.
29. Grabchev, I.; Chovelon, J. M.; Qian, X. J. Photochem. Photobiol. A 2003, 158, 37.
30. Xu, Z.; Qian, X.; Cui, J. Org. Lett. 2005, 7, 3029.
31. Zhang, Z.; Wu, D.; Guo, X.; Qian, X.; Lu, Z.; Xu, Q.; Yang, Y.; Duan, L.; He, Y.;
Feng, Z. Chem. Res. Toxicol. 2005, 18, 1814.
Dr. Ingo Ott appreciates a postdoctoral fellowship from East
China University of Science and Technology.
Supplementary data
The supporting information contains results from the DNA
interaction studies (UV/vis spectra), cytotoxicity experiments
against Hela cells and evaluation of the cell growth characteristics
of HT-29 and MCF-7 cells. This information is available free of
charge in the internet. Supplementary data associated with this
article can be found, on the online version, at doi:10.1016/
j.bmc.2008.06.052.
32. Xu, Z.; Xiao, Y.; Qian, X.; Cui, J.; Cui, D. Org. Lett. 2005, 7, 889.
33. Parkesh, R.; Lee, T. C.; Gunssnlaugson, T. Org. Biomol. Chem. 2007, 5, 310.
34. Cui, D.; Qian, X.; Liu, F.; Zhang, R. Org. Lett. 2004, 6, 2757.
35. Martin, E.; Weigand, R.; Pardo, R. J. Lumin. 1996, 68, 157.
36. Berque-Bestel, I.; Soulier, J. L.; Giner, M.; Rivail, L.; Langlois, M.; Sicsic, S. J. Med.
Chem. 2003, 46, 2606.
37. Vazquez, M. E.; Blanco, J. B.; Imperiali, B. J. Am. Chem. Soc. 2005, 127, 1300.
38. Ott, I.; Schmidt, K.; Kircher, B.; Schumacher, P.; Wiglenda, T.; Gust, R. J. Med.
Chem. 2005, 48, 622.
39. Vazquez, M. E.; Blanco, J. B.; Salvadori, S.; Trapella, C.; Argazzi, R.; Bryant, S. D.;
Jinsmaa, Y.; Lazarus, L. H.; Negri, L.; Giannini, E.; Lattanzi, R.; Colucci, M.;
Balboni, G. J. Med. Chem. 2006, 49, 3653.
References
40. Muller, P. J.; Wilson, B. C. Lasers Surg. Med. 2006, 38, 384.
41. Dougherty, T. J.; Gomer, C. J.; Henderson, B. W.; Jori, G.; Kessel, D.; Korbelik, M.;
Moan, J.; Peng, Q. J. Nat. Cancer Inst. 1998, 90, 889.
1. Brana, M. F.; Ramos, A. Curr. Med. Chem. Anti-Cancer Agents 2001, 1, 237.
2. Idoipe, A.; Santillan, M. S.; Martinez-Larranaga, M. R.; Gonzalez, E.; de Jalon, P.
D. G. Arch. Farmacol. Toxicol. 1979, 5, 17.
42. Liu, H. Q.; Cheung, T. C.; Peng, S. M.; Chi, M. C. J. Chem. Soc., Chem. Commun.
1995, 1787.
3. Brana, M. F.; Castellano, J. M.; Jimenez, A. Cancer Chemother. Pharmacol. 1980, 4,
61.
43. Marmur, J. J. Mol. Biol. 1961, 3, 208.
4. Zee-Cheng, R. K.; Cheng, C. C. J. Med. Chem. 1985, 28, 1216.