Synthesis and spectroscopic properties of furyl-phenyl-acrylates and naphthofurans and their interaction with ct-DNA

Article abstract of DOI:10.1007/s00706-008-0859-7

A series of novel substituted derivatives related to furyl-phenyl-acrylates and naphthofurans, was synthesized and characterized by UV/Vis and fluorescence spectroscopy. Acyclic compounds can undergo photochemical dehydrocyclization by visible light irradiation in order to obtain their cyclic derivatives. The interactions of the prepared compounds with calf thymus DNA was investigated by means of electronic absorption and fluorescence spectra. It is intriguing that addition of ct-DNA induced a fluorescence increase of acyclic derivatives, exactly the opposite of the strong fluorescence quenching observed for cyclic derivatives 10 and 12. Compound 11 showed decreasing fluorescence intensity for lower concentrations of ct-DNA, while increasing of fluorescence is observed for high excess of added ct-DNA.

Full text of DOI:10.1007/s00706-008-0859-7

Monatsh Chem 139, 975–983 (2008)
DOI 10.1007/s00706-008-0859-7
Printed in The Netherlands
Synthesis and spectroscopic properties of furyl-phenyl-acrylates
and naphthofurans and their interaction with ct-DNA
1
1
2
1
ˇ ´ ´
Kristina Starcevic , Marijana Hranjec , Dejana Caric , Grace Karminski-Zamola
1
Department of Organic Chemistry, Faculty of Chemical Engineering and Technology,
University of Zagreb, Zagreb, Croatia
2
Department of Chemistry, Faculty of Agronomy, University of Zagreb, Zagreb, Croatia
Received 12 November 2007; Accepted 22 November 2007; Published online 12 May 2008
# Springer-Verlag 2008
Abstract A series of novel substituted derivatives re-
lated to furyl-phenyl-acrylates and naphthofurans, was
synthesized and characterized by UV=Vis and fluores-
cence spectroscopy. Acyclic compounds can undergo
photochemical dehydrocyclization by visible light ir-
radiation in order to obtain their cyclic derivatives.
The interactions of the prepared compounds with calf
thymus DNA was investigated by means of electronic
absorption and fluorescence spectra. It is intriguing
that addition of ct-DNA induced a fluorescence in-
crease of acyclic derivatives, exactly the opposite of
the strong fluorescence quenching observed for cyclic
derivatives 10 and 12. Compound 11 showed decreas-
ing fluorescence intensity for lower concentrations of
ct-DNA, while increasing of fluorescence is observed
for high excess of added ct-DNA.
with DNA involve three binding modes: namely
intercalative binding where molecules intercalate in-
to the base pairs of the nucleic acid, groove binding
in which the molecules bound on DNA are located in
the major or minor groove, and electrostatic binding,
which takes place along the external DNA double
helix and do not possess selectivity [2]. Understand-
ing the modes of the binding of small molecules to
DNA and the factors that can affect the binding is of
fundamental importance in understanding drug-DNA
interactions and in development of new efficient
drugs targeted DNA, such as antitumor agents [3].
The constant and growing interest in the develop-
ment of new efficient and general synthesis methods
for the preparation of fused heterocyclic systems in-
volving furan and thiophene subunits is justified by
their well-established valuable physiological and
pharmacological properties [4]; however, there is lit-
tle new literature data describing the synthesis and
biological activity of these systems [5, 6]. Recent
technical applications of polysubstituted benzofur-
ans and benzothiophenes, including numerous fluo-
rescent dyes used as retrograde tracers, and Ca2þ and
Mg2þ fluorescent indicator conjugates, etc. increase
their significance. The biological activity of many
arenofurane derivatives has been improved by addi-
tion of positively charged aliphatic substituents. For
example, amidino-substituted heterocycles based on
a furan moiety have shown a number of intriguing
Keywords Heterocycles; Photochemistry; UV=Vis spectros-
copy; Fluorescence spectroscopy; DNA.
Introduction
Over the past decades, there has been great interest
on the design of small molecules binding to DNA
[1]. Generally, the interactions of small molecules
Correspondence: Grace Karminski-Zamola, Department of
Organic Chemistry, Faculty of Chemical Engineering and
´
Technology, University of Zagreb, Marulicev trg 20, HR-
10000 Zagreb, Croatia. E-mail: gzamola@fkit.hr

ˇ ´
K. Starcevic et al.
976
modes of biological activity, like antimicrobial [7],
hallucinogenic agents [8], and some furamidines
were found to be active against diverse, highly infec-
tious parasites [9]; the best being selected for cur-
rently undergoing phase II clinical trials [10].
Present work is a continuation of researches
aimed on the search of novel acyclic and cyclic deriv-
atives of methyl (E)-3-(2-furyl)-2-phenyl-acrylate
with interesting spectral-luminescent properties for
biomedical application.
pared from aldehyde 2 in the reaction with hydrox-
ylamonium hydrochloride in a yield of 90% [17].
The novel N-isopropylamidino-substituted acyclic
compound 5 was prepared by a Pinner reaction [18]
from cyano derivative 4 and isopropylamine in the
second phase of the reaction. Acyclic cyano and
amidino 6–9 compounds were prepared by conden-
sation of corresponding heterocyclic aldehyde and
appropriate 4-substituted-1,2-phenylendiamines in a
yield of about 35–76% [16]. Compounds 7 and 11
have been prepared earlier [16] but their spectroscop-
ic properties and interaction with DNA have not been
examined. Attempts to isolate amidino derivatives of
6 and 9 by the Pinner procedure were not successful
because they polymerized during the reaction and the
isolation procedure owing to their instability and
sensitivity to air moisture. In the reaction of photo-
Results and discussion
Chemistry
All compounds 2–12 shown in Fig. 1 were prepared
according to the Scheme 1. Compound 4 was pre-
Scheme 1

Furyl-phenyl-acrylates and naphthofurans
977
chemical dehydrocyclization (20–70 h) acyclic com-
pounds 6, 7, and 9 were converted into cyclic products
10, 11, and 12. Methyl (E)-3-(5-N-isopropylamidino-
2-furyl)-2-(4-nitrophenyl)-acrylate hydrochloride (5)
and methyl (E)-3-[5-(5-N-isopropylamidino-2-benz-
imidazolyl)-2-furyl]-2-(4-nitrophenyl)-acrylate hydro-
chloride 8 did not give desired cyclic compounds
by UV irradiation under the oxidative conditions.
The structures of new compounds were confirmed
emission spectra. The obtained results are shown in
Table 1.
The UV=Vis spectra of all studied compounds did
not show any pH dependent changes in the physio-
logically relevant range (pH ¼ 3.5–8.0). Therefore,
we performed our studies at pH ¼ 7.0. Absorbancies
of aqueous solutions of compounds are proportiꢁon3 al to
their concentrations up to 3.69ꢀ10ꢁ5 mol dm (4),
4.07ꢀ10ꢁ5 mol dmꢁ3 (5), 4.3ꢀ10ꢁ5 mol dmꢁ3 (6),
1.27ꢀ10ꢁ5 mol dmꢁ3 (7), 3.7ꢀ10ꢁ5 mol dmꢁ3 (8),
3.3ꢀ10ꢁ5 mol dmꢁ3 (9), 2.72ꢀ10ꢁ5 mol dmꢁ3 (10),
5.42ꢀ10ꢁ5 mol dmꢁ3 (11), and 3.1ꢀ10ꢁ5 mol dmꢁ3
(12) indicating that there is no significant intermo-
lecular stacking which should give rise to hypochro-
micity effects.
All compounds showed one absorption band in the
region of 309–370 nm except 11 which showed two
absorption bands at 360 and 260 nm. Conversion
of cyano-substituted 4 into the amidino-substituted
5 resulted in bathochromic shifts (Ál ¼ 15 nm)
and decreased molar extinction coefficient values.
Cyano- and amidino-substituted benzimidazole nu-
clei attached directly on C-5 position of methyl
(E)-3-(2-furyl)-2-(4-nitro)-phenyl-acrylate (6 and 7)
induced a bathochromic shift (Ál ¼ 42 nm) and in-
creased the " value of a compound 7. Incorporation
of a nitro substituent on the phenyl ring in 8 did
not have any influence on the shift of absorption
maxima or molar extinction coefficient value. Di-
cyano substituted 9 showed a bathochromic shift
(Ál ¼ 4 nm) and decrease of the molar extinction
coefficient. Photochemical cyclization of 6, 7, and
9 into 10, 11, and 12 resulted in hypsochromic shifts
of the absorption maxima.
1
by elemental analysis, UV=Vis, MS, H, and 13C
NMR spectra.
In our previous paper [19] we have described ex-
periments that present the conceptual basis for de-
velopment of new photoinduced anticancer therapy
based on the photochemical transformation on a
DNA=RNA inactive into an active one. Additional
advantage of this concept lies in the fact the photo-
induced dehydrocyclization of the 1,2-arenyl ethenes
is the widely studied reaction offering thus the ac-
cess to a large number of already known or new
molecules with selected properties compatible with
physiologically relevant conditions. In order to ob-
tain new systems capable to undergo the photo-in-
duced dehydrocyclization by visible light irradiation,
we prepared some derivatives of methyl (E)-3-(2-
furyl)-2-phenyl-acrylate.
Spectroscopic characterization of 4–12 dissolved
in aqueous buffer
Since application of spectrophotometric methods in
the DNA interaction studies was necessary, we char-
acterized aqueous solutions of compounds by means
of electronic absorption (UV=Vis) and fluorescence
For compounds 6–12 fluorescence was excited
on the wavelength of absorption maxima. All studied
compounds exhibit characteristic fluorescence emis-
sion with maxima at about 416–432 nm and showed
one emission band. Cyclic compounds 10 and 11
showed a hypsochromic shift of the emission maxi-
ma, while 12 showed a bathochromic shift. Their
excitation spectra, except 4 and 5, being in good
agreement with the absorption spectra. Fluorescence
intensities were found to be linearly concentration
dependent up to 5ꢀ10ꢁ6 mol dmꢁ3.
Table 1 Electronic absorption and emission data of 4–12
Compound labs.max= "ꢀ103=
lem= lexc= ꢀ
nm
dm3 molꢁ1 cmꢁ1 nm nm
4
5
6
7
8
9
10
11
309
324
366
365
366
370
353
360
260
365
27.2
24.6
25.4
33.6
33.9
26.9
6.1
42.2
28.0
4.0
–
–
–
–
–
–
–
–
–
–
427 365
425 363
432 367
424 370
416 354 0.043
419 359 0.452
Under the same conditions cyclic compounds
10, 11, and 12 exhibit stronger emission (ꢀ (10) ¼
0.043, ꢀ (11) ¼ 0.452, ꢀ (12) ¼ 0.064) than their
acyclic analogues 6, 7, and 9 (ꢀ (6), ꢀ (7), and
ꢀ (9) <0.001).
12
429 363 0.064

ˇ ´
K. Starcevic et al.
978
Interactions of compounds 4–12 with
double-stranded ct-DNA
Addition of different concentrations of ct-DNA to 4
(1.71ꢀ10ꢁ5 mol dmꢁ3), 5 (1.89ꢀ10ꢁ5 mol dmꢁ3), 6
(1.71ꢀ10ꢁ5 mol dmꢁ3), 7 (2.04ꢀ10ꢁ5 mol dmꢁ3),
8 (1.47ꢀ10ꢁ5 mol dmꢁ3), 9 (1.71ꢀ10ꢁ5 mol dmꢁ3),
Interaction of all compounds with ct-DNAwas studied
by UV=Vis and fluorescence spectroscopic titrations.
Table 2 Electronic absorption data changes of compounds 6–11 upon the addition of ct-DNA at different ratio r((com-
pound)=(polynucleotide)) at pH ¼ 7.0 (Buffer Na-cacodylate, I ¼ 0.05 mol dmꢁ3
)
ra
l
abs=nm
Álabs=nm
Hb=%
Ál=nm
ra
l
abs=nm
Álabs=nm
Hb=%
Ál=nm
6
0
1
0.25
0
1
0.25
0
1
0.25
0.457
0.354
0.279
0.477
0.431
0.411
0.298
0.185
0.180
–
–
–
–
–
–
–
10
–
–
7
0
1
0.25
0
1
0.25
0
1
0.25
0.690
0.638
0.636
0.482
0.409
0.396
0.532
0.259
0.309
–
–
–
2
3
–
–
–
–
–
8
0.103
0.178
–
0.046
0.066
–
22.5
39.0
–
9.6
13.8
–
0.052
0.054
–
0.073
0.086
–
7.5
8.5
–
15.1
17.8
–
8
9
10
11
0.113
0.118
37.9
39.6
0.273
0.223
51.3
42.0
2
a
r ¼ (compound)=(polynucleotide)
b
Hypochromic effect, H ¼ (Abs(compound) ꢁ Abs(complex))=Abs(compound)ꢀ100
Fig. 1 Absorbance titrations of ct-DNA into samples of compounds 6, 8, 9, and 11. Mixing rations, compound=polynucleotide,
from bottom to top are 0.25 and 1. Spectrum of free compounds 6, 8, 9, and 11 in buffered aqueus solutions are indicated

Furyl-phenyl-acrylates and naphthofurans
979
10 (4.98ꢀ10ꢁ5 mol dmꢁ3), and 11 (1.26ꢀ
10ꢁ5 mol dmꢁ3) resulted in significant hypochromic
effects of some compounds (39% for 6, 8.5% for 7,
14% for 8, 18% for 9, 40% for 10, and 42% for 11)
which pointed to their strong interaction with the
DNA. Compounds 7, 8, 10, and 11 showed also bath-
ochromic shifts of absorption maxima (Álmax ¼ 2–
10 nm) in the range of l>300 nm at which ct-DNA
does not absorb light, according to Table 2. Addition
of ct-DNA to buffered solution of 12 (5.88ꢀ
10ꢁ5 mol dmꢁ3) at a ratio ((10)=(polynucleotide))
>0.1 induced precipitation.
Addition of ct-DNA into buffered aqueous solu-
tions of cyano-substituted methyl-E-3-(2-furyl)-2-(4-
nitrophenyl)acrylate 4 and its amidino-substituted
derivative 5 did not yield any measurable changes
in the UV=Vis spectra. As it is shown in Fig. 1,
compounds 6, 8, and 9 (ꢂ2ꢀ10ꢁ5 mol dmꢁ3) show-
ed decreasing of absorption maxima upon the ad-
dition of different concentration (ꢂ8.5ꢀ10ꢁ6–1ꢀ
10ꢁ4 mol dmꢁ3) of ct-DNA solution, while 7 and 11
showed increasing of absorption maxima and batho-
chromic shifts at large excess of ct-DNA.
fluorescence so we performed fluorimetric titrations
at 10 times lower concentration than used in the
UV=Vis experiments. Increasing the amount of
ct-DNA, F=F0 of 6, 7 and 8 reached ca. 2.29, 3.91,
and 9.72 at large excess of ct-DNA. F=F0 of cyclic
compounds 10, 11, and 12 reached ca. 0.2, 0.7, and
0.16, according to Table 3.
As shown in Fig. 2, compounds 6, 7, and 8
(ꢂ1.4ꢀ10ꢁ6 mol dmꢁ3) showed an increasing of
fluorescence intensity upon the addition of different
concentrations (ꢂ1.4ꢀ10ꢁ6–1.4ꢀ10ꢁ4 mol dmꢁ3)
of ct-DNA solutions, while 10 and 12 at the same
conditions showed decreasing of fluorescence inten-
sity. Compound 11 showed a decrease of the fluores-
cence intensity for r>0.25, while further addition of
ct-DNA (r ¼ 0.25) yielded an increase of fluores-
cence intensity. Increasing of fluorescence intensity
of 11 at r<0.25 resulted in appearance of two maxi-
ma. One of maxima appeared at lower wavelength
(406 nm) and the other one at higher wavelength
(426 nm) compared to emission maxima of 11.
Conclusion
Since aqueous solutions of cyclic compounds 10
and 12 exhibit strong fluorescence, it was possible to
perform fluorimetric titrations at 60 times lower con-
centration for 12 and 100 times lower concentration
for 10 than used in the UV=Vis experiments. Acyclic
compounds 6, 7, and 8 and cyclic 11 exhibit lower
The main aim of the present study was to prepare
small series of substituted methyl (E)-3-(2-furyl)-2-
phenylacrylates which can undergo photochemical
dehydrocyclization by visible light irradiation in or-
der to obtain its cyclic derivatives. Some changes in
Table 3 Fluorescence intensity changes of compounds 6–8 and 10–12 upon the addition of ct-DNA at different ratio
r((compound)=(polynucleotide)) at pH ¼ 7.0 (buffer Na-cacodylate, I ¼ 0.05 mol dmꢁ3
)
b
b
ra
Int=a.u.
ÁInt=a.u.
F=F0
ra
Int=a.u.
ÁInt=a.u.
F=F0
6
0
1
0.25
0.05
0.01
0
–
–
–
7
0
1
0.25
0.05
0.01
0
181.3
218.8
405.1
891.0
–
498.8
517.0
474.3
448.3
398.0
638.9
738.9
693.1
657.9
587.0
–
–
116.7
124.2
267.7
383.9
61.9
7.5
0.06
0.38
1.30
2.29
–
0.11
0.70
2.80
9.72
–
37.5
223.8
709.7
–
0.21
1.23
3.91
–
44.2
151.0
267.2
–
8
10
12
–
–
1
68.8
6.9
1
18.2
24.5
50.5
108.0
–
100.0
54.2
19.0
51.9
0.04
0.05
0.10
0.20
–
0.16
0.08
0.03
0.08
0.25
0.05
0.01
0
105.4
235.5
663.7
149.8
52.3
44.0
68.6
145.3
43.5
174.6
601.7
–
97.5
105.8
81.2
4.5
0.25
0.05
0.01
0
11
1
0.65
0.71
0.54
0.03
1
0.25
0.05
0.01
0.25
0.05
0.01
a
b
r ¼ (compound)=(polynucleotide); F ¼ fluorescence of compound-DNA complex, F0 ¼ fluorescence of compound without
ct-DNA addition

ˇ ´
K. Starcevic et al.
980
Fig. 2 Fluorescence titrations of ct-DNA into samples of compounds 7, 8, 10, 11, and 12. Mixing rations, compound=
polynucleotide, are 0.01, 0.05, 0.25, and 1. Spectrum of free compounds in buffered aqueus solutions is indicated
structure had an impact on reaction of photochemi-
cal dehydrocyclization. Compounds 4, 5, and 8 were
not capable of undergoing the aformentioned photo-
chemical dehydrocyclization, while acyclic com-
pounds 6, 7, and 9 were efficiently converted into
the corresponding naphthofurans 10, 11, and 12.
All prepared compounds had interesting spectro-
scopic properties and showed one absorption and
emission band (11 showed two absorption bands).
Cyano- and amidino substituents induced bathochro-
mic shift and mostly increasing of molar extinction
coefficient. We wanted to shift absorption maxima to
longer wavelengths, possibly in visible region, but
introducing of a nitro group did not yield any impor-
tant shift of absorption maxima. Compounds 6–12
showed moderate changes of their spectral-fluores-
cent properties in the presence of ct-DNA, except 4
and 5 which did not yield any measurable changes.
In the presence of different ratios of ct-DNA, acyclic
compounds 6, 7, and 8 increased fluorescence inten-

Furyl-phenyl-acrylates and naphthofurans
981
Methyl (E)-3-(5-cyano-2-furyl)-2-(4-nitro)-phenyl-acrylate
sity, while its cyclic derivatives 10 and 12 at the
same conditions showed exactly opposite quenching
of fluorescence intensity. The obtained results re-
vealed the possibility of two different types of inter-
actions with DNA, probably minor groove binding
and intercalation. Compounds 9 and 11 showed
both changes of fluorescence intensity, fluorescence
quenching at lower concentration of added ct-DNA,
and increase of fluorescence intensity at high excess
of ct-DNA. Since results obtained from spectroscop-
ic titrations of prepared compounds with ct-DNA are
interesting and might revealed the possibility for bio-
medical application, detailed binding studies using
some other spectroscopic methods will be further
investigated.
(4, C15H10N2O5)
Compound 2 (0.6g, 2.0 mmol) and hydroxyl ammonium hy-
drochloride (0.24g, 14.3mmol) were heated in 1.5 cm3 pyri-
dine at 60ꢃC for 30min, then 1 cm3 acetic anhydride was
successively added at a temperature not exceeding 95ꢃC,
which was being kept for additional 2 h. The mixture was
cooled to 20ꢃC, poured into 37.5cm3 water and the separated
nitrile was stirred for 1 h, filtered, washed with water, dried,
and recrystallized from petrolether. Yield 0.541g (90.2%) yel-
1
low powder; mp 113–114ꢃC; H NMR (300 MHz, DMSO):
ꢁ ¼ 8.30 (d, J ¼ 8.7 Hz, 2H), 7.70 (s, 1H), 7.60 (d, J ¼ 8.7 Hz,
2H), 7.55 (d, J ¼ 3.8 Hz, 1H), 6.62 (d, J ¼ 3.8 Hz, 1H), 3.73 (s,
CH3) ppm; 13C NMR (75 MHz, DMSO): ꢁ=165.58 (s), 153.02
(s), 148.50 (s), 141.85 (s), 131.40 (s), 130.91 (d, 2C), 126.11
(d), 125.92 (s), 125.84 (d), 123.41 (d, 2C), 117.32 (d), 111.75
(s), 52.81 (q) ppm; MS: m=z (%) ¼ 298.2 (M þ , 100).
Methyl (E)-3-(5-N-isopropylamidino-2-furyl)-2-(4-nitro)-phenyl-
acrylate hydrochloride (5, C18H20ClN3O5)
Experimental
A stirred suspension of 0.324 g 4 (1.1mmol) in 5 cm3 anhy-
drous EtOH was cooled in an ice-salt bath and was saturated
with HCl gas. The flask was then tightly stoppered and
the mixture was maintained at room temperature for 2 days,
until nitrile band disappeared (monitored by IR analysis at
2200cmꢁ1). The reaction mixture was purged with N2 gas
and diluted with ether (30 cm3). The crude imidate was filtered
off and was immediately suspended in 5 cm3 anhydrous etha-
nol. The 0.52cm3 isopropylamine (6.1mmol) was added and
the mixture was stirred for one day at room temperature. The
reaction mixture was diluted with 50 cm3 diethylether to give a
precipitate. The precipitate was collected and recrystallized
from ethanol=ether. Yield 0.120g (27.8%) yellow powder;
Chemistry
Melting points were obtained on an Original Kofler Mikro-
1
heitztisch apparatus (Reichert, Wien). The H and 13C NMR
spectra were recorded on a Varian Gemini 300 or a Bruker
Avance DPX 300 at 300 and 75MHz. All NMR spectra were
measured in DMSO-d6 solutions using TMS as an internal
standard. Elemental analysis for carbon, hydrogen and nitro-
gen were performed on a Perkin-Elmer 2400 elemental ana-
lyzer. Where analyses are indicated only as symbols of
elements, analytical results obtained are within 0.4% of the
theoretical value. All compounds were routinely checked by
TLC with Merck silica gel 60F-254 glass plates and HPLC-MS.
1
mp 225–226ꢃC; H NMR (300MHz, DMSO): ꢁ ¼ 9.51 (bs,
Preparation of compounds 2 and 4–12
3H), 8.31 (d, J ¼ 8.8 Hz, 2H), 7.76 (s, 1H), 7.64 (d, J ¼ 3.6 Hz,
1H), 7.61 (s, 1H), 6.20 (d, J ¼ 3.6 Hz, 1H), 4.05–3.95 (m,
CH), 3.75 (s, CH3), 1.20 (d, J ¼ 6.2 Hz, 6H, (CH3)2) ppm;
MS: m=z (%) ¼ 358.2 (Mþ 1, 100).
Methyl (E)-3-(5-formyl-2-furyl)-2-(4-nitro)-phenyl-acrylate
(2, C15H11NO6)
The corresponding methyl (E)-3-(2-furyl)-2-(4-nitro)-phenyl-
acrylate was formylated by Vilsmeier formylation. Phosphorus
oxychloride (1.58 cm3, 17.3mmol) was added drop-wise with
cooling to 1.3 cm3 DMF (16.3 mmol). The reaction mixture
was stirred for 0.5 h by cooling on ice, the apparatus being
protected with a CaCl2 tube. A solution of methyl (E)-3-(2-
furyl)-2-phenylacrylate (2g, 7.33 mmol) in 5 cm3 DMF was
added drop-wise to the mixture. After the addition was com-
pleted, the mixture was stirred for 1 h at room temperature,
then heated at 90ꢃC for 3 h, cooled and poured onto crushed
ice and made weakly alkaline with sodium carbonate solution,
and left over-night on ice. The solid was filtered off, washed
with water and recrystallized from methanol. Yield 0.602 g
(27.1%) yellow-orange crystals; mp 166–167ꢃC; 1H NMR
(300 MHz, DMSO): ꢁ ¼ 9.37 (s, 1H), 8.22 (d, J ¼ 8.5 Hz,
2H), 7.69 (s, 1H), 7.53 (d, J¼ 8.5 Hz, 2H), 7.35 (d, J¼ 3.7 Hz,
1H), 6.49 (d, J ¼ 3.7Hz, 1H), 3.38 (s, CH3) ppm; 13C NMR
(75 MHz, DMSO): ꢁ ¼ 175.31 (d), 154.40 (s), 149.42 (s),
141.53 (s), 132.11 (s), 131.36 (d, 2C), 126.57 (s), 125.21 (d),
124.40 (d, 2C), 118.42 (d), 116.73 (d), 111.28 (s), 59.79 (q)
ppm; MS: m=z (%) ¼ 301.2 (Mþ , 100).
Methyl (E)-3-[5-(5-cyano-2-benzimidazolyl)-2-furyl]-2-phenyl-
acrylate (6, C22H15N3O3)
A mixture of 0.5 g 1 (1.95 mmol), 0.256 g 4-cyano-1,2-phe-
nylenediamine (1.95 mmol), and 0.211 g p-benzoquinone
(1.95 mmol) in 10 cm3 absolute EtOH was stirred under nitro-
gen at reflux for 6 h. The reaction mixture was evaporated in
vacuum and the oily residue was dissolved in diethylether.
Petrolether was added and the precipitated product was filtered
1
off. Yield 0.550 g (76%) yellow powder; mp 169–170ꢃC; H
NMR (300MHz, DMSO): ꢁ ¼ 13.56 (s, NH, 1H), 8.11 (s, 1H),
7.71 (s, 1H), 7.69 (d, J ¼ 8.6 Hz, 1H), 7.60 (d, J ¼ 8.6Hz, 1H),
7.55–7.48 (m, 3H), 7.29 (d, J ¼ 7.6 Hz, 2H), 7.21 (d, J ¼
3.8 Hz, 1H), 5.66 (d, J ¼ 3.8Hz, 1H), 3.80 (s, CH3) ppm;
13C NMR (75 MHz, DMSO): ꢁ ¼ 168.22 (s), 166.36 (s),
151.75 (s), 145.32 (s), 144.71 (s), 135.11 (s), 134.77 (s),
131.72 (s), 128.99 (d, 2C), 128.76 (d, 3C), 128.44 (d),
126.65 (d), 125.67 (d), 119.84 (s), 119.77 (s), 115.61 (d),
115.15 (d), 114.75 (s), 52.4 (q) ppm; MS: m=z (%) ¼ 370.2
(Mþ 1, 100).

ˇ ´
K. Starcevic et al.
982
1
144ꢃC; H NMR (300MHz, DMSO): ꢁ ¼ 13.54 (s, NH, 1H),
8.15 (bs, 1H), 7.96 (d, J ¼ 8.2 Hz, 2H), 7.75 (s, 1H), 7.71–7.65
(m, 2H), 7.54 (d, J ¼ 8.2 Hz, 2H), 7.26 (d, J ¼ 3.4Hz, 1H),
6.06 (d, J ¼ 3.4 Hz, 1H), 4.15 (q, J ¼ 7.0 Hz, 2H, CH2CH3),
1.27 (t, J ¼ 7.1 Hz, 3H, CH2CH3) ppm; MS: m=z (%) ¼ 409.2
(Mþ 1, 100).
Methyl (E)-3-[5-(5-N-isopropylamidino-2-benzimidazolyl)-2-furyl]-
2-phenyl-acrylate hydrochloride (7, C25H27N4O3Cl3 ꢀ3H2O) [16]
A mixture of 1.01g 1 (4.0mmol), 0.90g 4-N-isopropylami-
dino-1,2-phenylenediamine (4.0mmol ), and 0.43 g p-benzo-
quinone (4.0mmol) in 25 cm3 absolute EtOH was stirred
under nitrogen at reflux for 4 h. The reaction mixture was
cooled to room temperature, diethylether was added and the
resulting brown solid was filtered off. The crude product was
suspended in absolute ethanol and cooled to 0–5ꢃC. Into the
suspension was introduced HCl gas until the suspension was
saturated and the content was stirred over night on the room
temperature. Dry diethylether was added, the precipitated
yellow-green powder was filtered off and washed with dry
diethyl-ether. Yield 0.95 g (41%) slightly green powder; mp
Methyl (E)-2-[(5-cyano)benzimidazolyl]naphtho[2,1-b]furan-5-
carboxylate (10, C22H13N3O3)
An ethanolic solution (35 cm3) of 0.10 g 6 (0.27 mmol), and
iodine (0.005g) was irradiated at room temperature with 400-
W high-pressure mercury lamp using a Pyrex filter for 50h.
The air was bubbled through the solution. The solution was
concentrated and resulting product was filtered off. Yield
0.050 g (50%) light yellow powder; mp 189–190ꢃC; 1H
NMR (300 MHz, DMSO): ꢁ ¼ 13.81 (bs, NH, 1H), 8.79 (d,
J ¼ 8.6Hz, 1H), 8.45 (d, J ¼ 8.3Hz, 1H), 8.43 (s, 1H), 8.37 (s,
1H), 7.72 (t, J ¼ 8.7 Hz, 2H), 7.63–7.61 (m, 3H), 3.74 (s, CH3)
ppm; MS: m=z (%) ¼ 368.3 (Mþ 1, 100).
1
236–238ꢃC; H NMR (300 MHz, DMSO): ꢁ ¼ 13.54 (s, NH,
1H), 9.60 (s, NH, 1H), 9.57 (s, NH, 1H), 9.49 (bs, NH, 1H),
8.01 (s, 1H), 7.77 (d, J ¼ 8.7Hz, 1H), 7.73 (s, 1H), 7.61 (d,
J ¼ 8.8 Hz, 1H), 7.60 (s, 1H), 7.54–7.46 (m, 3H), 7.38 (d,
J ¼ 3.8 Hz, 1H), 7.31 (d, J ¼ 7.8 Hz, 2H), 5.71 (d, J ¼
3.8 Hz, 1H), 4.07 (m, CH), 3.90 (s, CH3), 1.34 (d, J ¼
6.2 Hz, 6H, (CH3)2) ppm; 13C NMR (75 MHz, DMSO):
ꢁ ¼ 166.9 (s), 162.5 (s), 152.9 (s), 145.6 (s), 144.4 (s),
135.5 (s), 134.45 (s), 132.9 (s), 129.6 (d, 2C), 129.3 (d),
129.1 (d, 2C), 127.1 (d), 124.7 (d), 124.2(s), 119.87 (s),
116.8 (d), 116.6 (d), 116.2 (d), 115.3 (d), 53.1 (q), 45.7
(d), 21.9 (q, 2C) ppm; MS: m=z (%) ¼ 429.5 (M þ 1, 100).
Methyl (E)-2-[(5-N-isopropylamidino)benzimidazolyl]naphtho
[2,1-b]furan-5-carboxylate hydrochloride
(11, C25H24N4O3Cl2 ꢀ2H2O) [16]
An ethanolic solution 140 cm3 of 0.90 g 7 (2.0 mmol) was
irradiated at room temperature with 400-W high-pressure
mercury lamp using a Pyrex filter for 20 h. The air was
bubbled through the solution. The solution was concentrat-
ed, diethyl-ether was added to the solution and the preci-
pitated powder was filtered off and recrystallized from
ethanol. Yield 0.37 g (35%); yellow powder; mp 255–
257ꢃC; 1H NMR (300 MHz, DMSO): ꢁ ¼ 13.65 (s, NH,
1H), 9.62 (bs, NH, 2H), 9.52 (s, NH, 1H), 9.07 (s, NH,
1H), 8.88 (d, J ¼ 8.7 Hz, 1H), 8.63 (s, 1H), 8.50 (d, J ¼
8.4 Hz, 1H), 8.47 (d, J ¼ 7.6 Hz, 1H), 8.11 (s, 1H), 7.87–
7.70 (m, 3H), 7.63 (bs, 1H), 4.46 (m, CH), 4.07 (s, CH3),
1.24 (d, 6H, J ¼ 6.2 Hz, (CH3)2) ppm; 13C NMR (75 MHz,
DMSO): ꢁ ¼ 167.5 (s), 162.7 (s), 158.9 (s), 151.2 (s),
148.7 (s), 145.6 (s), 143.8 (s), 141.7 (s), 128.3 (d), 128.1
(s), 127.5 (d), 127.0 (d), 126.3 (d), 124.9 (s), 124.3 (d),
123.7 (d), 117.3 (s), 116.3 (d), 115.7 (d), 114.4 (d), 53.1
(q), 45.6 (d), 21.9 (q, 2C) ppm; MS: m=z (%) ¼ 427.4
(M þ 1, 100).
Methyl (E)-3-[5-(5-N-isopropylamidino-2-benzimidazolyl)-2-furyl]-
2-(4-nitrophenyl)-acrylate hydrochloride (8, C25H24ClN5O5)
A mixture of 0.2 g 2 (0.66 mmol), 0.150 g 4-N-isopropyl-
amidino-1,2-phenylenediamine (0.66 mmol), and 0.072 g
p-benzoquinone (0.66 mmol) in 10 cm3 absolute was stirred
under nitrogen at reflux for 3 h. The reaction mixture was
cooled to the room temperature, diethyl-ether was added
and the resulting product was filtered off and washed with
diethylether and recrystallized from ethanol=acetone. Yield
0.220 g (65%) green powder; mp 221–222ꢃC; 1H NMR
(300 MHz, DMSO): ꢁ ¼ 13.65 (bs, NH, 1H), 9.54 (bs, NH,
1H), 9.40 (bs, NH, 1H), 9.02 (bs, NH, 1H), 8.34 (d, J ¼
8.6 Hz, 2H), 7.96 (s, 1H), 7.79 (s, 1H), 7.70 (d, J ¼ 8.6 Hz,
2H), 7.63 (d, J ¼ 8.6 Hz, 2H), 7.35 (d, J ¼ 3.2 Hz, 1H), 6.26
(d, J ¼ 3.2 Hz, 1H), 4.10–4.06 (m, CH), 3.75 (s, CH3),
1.29 (d, J ¼ 6.3 Hz, 6H, (CH3)2) ppm; 13C NMR (75 MHz,
DMSO): ꢁ ¼ 168.76 (s), 166.28 (s), 162.77 (s), 151.39 (s),
147.89 (s), 147.32 (s), 146.73 (s), 146.490 (s), 142.92 (s),
141.66 (s), 131.56 (d, 2C), 129.32 (s), 127.91 (d), 127.17
(d), 124.74 (d), 124.48 (d, 2C), 118.38 (d), 116.09 (d),
114.95 (d), 53.86 (q), 45.48 (d), 21.70 (q, 2C) ppm; MS:
m=z (%) ¼ 474.3 (M þ 1, 100).
Ethyl (E)-2-[(5-cyano)benzimidazolyl-]-4-cyanonaphtho[2,1-
b]furan-5-carboxylate (12, C24H14N4O3)
An ethanolic solution (20 cm3) of 0.05 g 9 (0.13 mmol), and
iodine (0.002g) was irradiated at room temperature with
400-W high-pressure mercury lamp using a Pyrex filter for
70h. The air was bubbled through the solution. The solution
was concentrated and dark residue was treated with ethanol.
The resulting product was filtered off. Yield 0.020 g (40%)
Ethyl (E)-3-[5-(5-cyano-2-benzimidazolyl)-2-furyl]-2-(4-
cyanophenyl)-acrylate (9, C24H16N4O3)
1
A mixture of 0.2g 3 (0.68 mmol), 0.091 g 4-cyano-1,2-phenyl-
enediamine (0.68 mmol), and p-benzoquinone (0.073 g,
0.68mmol) 10 cm3 in absolute EtOH was stirred under nitro-
gen at reflux for 12h. The reaction mixture was evaporated
in vacuum and the crude product was recrystallizated from
petrolether. Yield 0.096 g (35%); yellow crystals; mp 142–
light brown powder; mp 167–168ꢃC; H NMR (300 MHz,
DMSO): ꢁ ¼ 13.60 (s, NH, 1H), 8.63 (s, 1H), 8.50–8.47 (m,
2H), 8.16 (s, 2H), 7.98 (d, J ¼ 8.5 Hz, 1H), 7.75 (d, J ¼
8.3 Hz, 1H), 7.60 (d, J ¼ 8.5 Hz, 1H), 4.20 (q, J ¼ 7.1 Hz,
2H, CH2CH3), 1.20 (t, J ¼ 7.1 Hz, 3H, CH2CH3) ppm; MS:
m=z (%) ¼ 407.2 (M þ 1, 100).

Furyl-phenyl-acrylates and naphthofurans
983
Spectroscopic measurements
References
UV absorption spectra were recorded against the solvent at
25 ꢄ 0.1ꢃC, using a Varian Cary 50 spectrophotometer oper-
ated in double-beam mode. The wavelength range covered
was 200–450 nm. Quartz cells of 1-cm path length were used
throughout and absorbancies were sampled at 0.1 nm intervals.
Samples were prepared by diluting the respective stock solu-
tions (in DMSO) with the appropriate aqueous buffer.
Fluorescence measurements were carried out on a Varian
Cary Eclipse fluorescence spectrophotometer at 25ꢃC using
1-cm path quartz cells. Excitation maxima were determined
from excitation spectra covering the range of 200–400 nm.
Emission spectra were recorded from 375 to 600 nm and
corrected for the effects of time- and wavelength-dependent
light-source fluctuations using a standard of rhodamine 101,
a diffuser provided with the fluorimeter and the software
supplied with the instrument. The measurements were per-
formed in aqueous buffer solution (pH ¼ 7.0, sodium caco-
dylate buffer, I ¼ 0.05 mol dmꢁ3). Relative fluorescence
quantum yields were determined according to Miller [13]
using Eq. (1):
1. a) Langer R (2001) Science 293:58; b) Rao KE, Lown JW
(1991) Chem Res Toxicol 4:661; c) Singh MP, Joseph T,
Kumar S, Lown JW (1992) Chem Res Toxicol 5:597; d)
Kelley SO, Orellana G, Barton JK (2000) J Photochem
Photobiol B 58:72
2. a) Erkkila KE, Odom RT, Barton JK (1999) Chem Rev
99:2777; b) Armitage B (1998) Chem Rev 98:1171
3. Demeunynck M, Bailly C, Wilson WD (2002) In DNA
and RNA Binders. Wiley-VCH, Weinheim
4. a) Cagniant P, Cagniant D (1975) In: Katritzky AR,
Boulton AJ (eds) Advances in Heterocyclic Chemistry,
vol. 18. Academic Press, New York, p 338; b) (1984) In:
Campaigne E, Bird CW, Cheeseman S (eds) Comprehen-
sive Heterocyclic Chemistry, vol. 4. G. W. H. Pergamon,
Oxford, p 910; c)Keay BA, DibblePW(1996)In: Katritzky
AR, Rees CW, Scriven EFV (eds) Comprehensive Hetero-
cyclic Chemistry II, vol. 2. Pergamon, Oxford, p 395;
d) Russel RK (1996) In: Katritzky AR, Rees CW, Scriven
EFV (eds) Comprehensive Heterocyclic Chemistry II, vol.
2. Pergamon, Oxford, p 679; e) Haugland RP (1996) In:
Spence MTZ (ed) Handbook of Fluorescent Probes and
Research Chemicals. Molecular Probes Inc., O. R. Eugene
5. Benameur L, Bouaziz Z, Nebois P, Bartoli MH, Boitard
M, Fillion H (1996) Chem Pharm Bull (Tokyo) 44:605
6. Bailly C (2000) Curr Med Chem 7:39
ꢀx ¼ ꢀs ꢀAsDxn2x=AxDsns2
ð1Þ
wherein ꢀ is the emission quantum yield, A is the absorbance
at the excitation wavelength, D is the area under the corrected
emission curve and n is the refractive index of the solvents
used. The subscripts s and x refer to the standard and to the
unknown. The standard we used was N-acetyl-l-tryptophan-
amide with a published fluorescence quantum yield of 0.14
[14, 15]. All samples were purged with argon to displace
oxygen. The reproducibility (difference between the largest
and the smallest value in a series of three independent mea-
surements, divided by their arithmetic mean) of quantum yield
measurements was better than 10%.
7. a) Wang S, Hall JE, Tanious FA, Wilson WD, Patrick DA,
McCurdy DR, Bender BC, Tidwell RR (1999) Eur J Med
Chem 34:215; b) Bilger C, Demerseman P, Buisson JP,
Royer R, Gayral P, Fourniat J (1987) Eur J Med Chem
Chim Ther 22:213
8. Monte AP, Marona-Lewicka D, Lewis MM, Mailman
RB, Wainscott DB, Nelson DL, Nichols DE (1998) J Med
Chem 41:2134
Calf thymus (ct-DNA) was purchased from Fluka, dissolved
in the sodium cacodylate buffer, I ¼ 0.05mol dmꢁ3, pH¼ 7.0
and additionally sonicated and filtered through the 0.45ꢂm
filter. The final concentration of the calf thymus (ct-DNA)
solution expressed as the concentration of phosphates was
determined spectroscopically [11, 12] by measuring the absor-
bance of buffered solution at l ¼ 260 nm for at least five in-
dependent additions of the DNA stock solution aliquots and
dividing the averaged absorbance value by molar extinction
9. a) Das BP, Boykin DW (1977) J Med Chem 20: 531;
b) Steck EA, Kinnamon KK, Davidson DE, Duxbury RE,
Johnson AJ, Masters RE (1982) Exp Parasitol 53:133
10. Boykin DW, Kumar A, Bender BK, Hall JE, Tidwell RR
(1996) Bioorg Med Chem Lett 6:3017
11. Chaires JB, Dattagupta N, Crothers DM (1982) Biochem-
istry 21:3933
ꢀ
12. Palm BS, Piantanida I, Zinic M, Schneider HJ (2000)
´
J Chem Soc Perkin Trans 2:385
coefficient "
¼ 6600mmolꢁ1 cmꢁ2. All reagents are of
ðct-DNAÞ
13. Miller JN (1981) Standards for Fluorescence Spectrome-
try. Chapman and Hall, London
14. Chen RF (1967) Anal Lett 1:35
analytical reagent grade and the doubly distilled water is used
all along.
Stock solutions were prepared by dissolving of com-
pounds 4–12 in DMSO. Before experiment respective ali-
quots were added to the aqueous buffer, partial volume of
DMSO in water not exceeding 0.1%. Stock solutions in
DMSO of all studied compounds were shown to be stable
over longer periods.
15. Eftink MR, Jia Y, Hu D, Ghiron CA (1995) J Phys Chem
99:5713
ꢀ
´
16. a) Hranjec M, Grdisa M, Pavelic K, Boykin DW,
Karminski-Zamola G (2003) IL Farmaco 58:1319; b)
ˇ
Starcevic K, Boykin DW, Karminski-Zamola G (2002)
Heterocycl Comm 8:221
´
ꢀ
ˇ
´
´
17. Starcevic K, Kralj M, Piantanida I, Suman L, Pavelic K,
Karminski-Zamola G (2006) Eur J Med Chem 41:925
18. Fairley TA, Tidwell RR, Donkor I, Naiman NA, Ohemeng
KA, Lombardy RJ, Bentley JA (1993) J Med Chem36:1746
Acknowledgement
This study was supported by grants (Nos.: 125-0982464-1356
and 178-0982929-2259) from the Ministry of Science and
Technology of the Republic of Croatia.
ꢀ
ˇ
´
19. Starcevic K, Karminski-Zamola G, Piantanida I, Suman
L, Kralj M (2005) J Am Chem Soc 127:1074