Synthesis and antitumor evaluation of some new substituted amidino-benzimidazolyl-furyl-phenyl-acrylates and naphtho[2,1-b]furan- carboxylates

Article abstract of DOI:10.1016/S0014-827X(03)00197-6

The multistep synthesis of a series of substituted amidino-benzimidazolyl- furyl-phenyl-acrylic acid's esters and substituted amidino-benzimidazolyl- naphtho[2,1-b]furan-carboxylic acid's esters is described starting from corresponding 3-(2-furyl)-2-phenyl-acrylic acids. The new compounds were tested on the cytostatic activities against malignant cell lines: pancreatic carcinoma (MiaPaCa2), breast carcinoma (MCF7), cervical carcinoma (HeLa), laryngeal carcinoma (Hep2), colon carcinoma (HT 29), melanoma (HBL), and human fibroblasts cell line (WI38). All compounds inhibited the proliferation of tumor cell lines. Inhibitory effect of examined compounds depended on concentration, but without significant difference among the type of tumor cells. The compounds 2 and 5 exerted very low inhibitory effect on the growth of human fibroblasts. Unsubstituted derivative 8 has not inhibited any tested cell lines.

Full text of DOI:10.1016/S0014-827X(03)00197-6

Il Farmaco 58 (2003) 1319ꢀ1324
/
www.elsevier.com/locate/farmac
Synthesis and antitumor evaluation of some new substituted amidino-
benzimidazolyl-furyl-phenyl-acrylates and naphtho[2,1-b]furan-
carboxylates
a
M. Hranjec , M. Grdi sˇ a , K. Pavelic , D.W. Boykin , G. Karminski-Zamola *
b
b
c
a,
a
Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, University of Zagreb, Marulicev trg 20, P.O. Box 177,
HR-10000 Zagreb, Croatia
b
Division of Molecular Medicine, Ruder Bo sˇ kovic Institute, Bijenicka cesta 54, P.O. Box 1016, HR-10000 Zagreb, Croatia
c
Department of Chemistry and Centre for Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30303-3083, USA
Received 29 March 2003; accepted 19 July 2003
Abstract
The multistep synthesis of a series of substituted amidino-benzimidazolyl-furyl-phenyl-acrylic acid’s esters and substituted
amidino-benzimidazolyl-naphtho[2,1-b]furan-carboxylic acid’s esters is described starting from corresponding 3-(2-furyl)-2-phenyl-
acrylic acids. The new compounds were tested on the cytostatic activities against malignant cell lines: pancreatic carcinoma
(
MiaPaCa2), breast carcinoma (MCF7), cervical carcinoma (HeLa), laryngeal carcinoma (Hep2), colon carcinoma (HT 29),
melanoma (HBL), and human fibroblasts cell line (WI38). All compounds inhibited the proliferation of tumor cell lines. Inhibitory
effect of examined compounds depended on concentration, but without significant difference among the type of tumor cells. The
compounds 2 and 5 exerted very low inhibitory effect on the growth of human fibroblasts. Unsubstituted derivative 8 has not
inhibited any tested cell lines.
´
#
2003 Editions scientifiques et m e´ dicales Elsevier SAS. All rights reserved.
Keywords: Pancreatic carcinoma; Laryngeal carcinoma; Breast carcinoma; Melanoma; Amidinobenzimidazolyl-furans
1
. Introduction
transcription factor Sp 1 were studied [15], while a new
derivatives of distamycine bearing one or more pyrazole
rings are synthesized and examined in vitro and in vivo
activities against L1210 leukaemia [16]. DNA-binding
properties and cytotoxic activity of novel aromatic
amidines in cultured human skin fibroblasts were
examined recently [17]. Antitumor activity of a series
of cyclic amidines of benzodiazepine series was investi-
gated too [18].
Amidino-substituted aromatic and heteroaromatic
compounds are widely investigated on their different
biological activity in the recent time [1ꢀ12] including
/
some phenyl substituted monoamidines which are
synthesized and their anti HIV activity examined [13].
On the other side, there is no much literature evidence
about the antitumor activity of amidines. A few papers
describe the synthesis of the natural oligopeptide anti-
viral antibiotics netropsyne and distamycine and their
synthetic derivatives. They have attracted considerable
attention on the part of synthetic chemists and pharma-
cists because some representatives exhibit anticancer
activity [14]. The effect of the synthetic derivative of
distamycine on molecular interaction between DNA and
2. Results and discussion
In this paper we describe the preparation of six new
substituted amidino-benzimidazolyl heterocyclic com-
pounds from furan series which could serve as potential
anticancer agent. The preparation of the target com-
pounds is outlined in the scheme. Amidino compounds
were prepared in the several steps from methyl (Z)-3-(5-
formyl-2-furyl)-2-phenyl-acrylic acrylate, 1 [19] with
* Corresponding author.
E-mail address: gzamola@pierre.fkit.hr (G. Karminski-Zamola).
´
0
014-827X/03/$ - see front matter # 2003 Editions scientifiques et m e´ dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0014-827X(03)00197-6

1320
M. Hranjec et al. / Il Farmaco 58 (2003) 1319ꢀ1324
/
earlier prepared 3,4-diamino-substituted-benzamidines
in the Pinner reaction [20,21]. In this way were prepared
methyl ester of 3-[5-(N-isopropylamidino-benzamidazo-
lyl)-2-furyl]-2-phenyl-acrylic acid 2, 3-[5-(N-2-imidazo-
linyl-benzimidazolyl-2-furyl]-2-phenyl-acrylic acid 3 and
cell lines HeLa ꢀ
carcinoma, HT29 ꢀ
creatic carcinoma, HBL ꢀ
cancer and normal human fibroblasts ꢀ
inhibitory effect varied between the compounds, and
depended on the concentration (as illustrated in Fig. 1).
All results are summarised in Table 1.
/
cervical carcinoma, Hep2 ꢀ
colon carcinoma, MiaPaCa2 ꢀ
melanoma, MCF7 ꢀ breast
WI38. The
/
laryngeal
/
/
pan-
/
/
/
3
2
-[5-(N-morpholinyl-amidino-benzimidazolyl)-2-furyl]-
-phenyl-acrylic acid 4 as hydrochloride salts.
The prepared compounds 2, 3 and 4 were photochemi-
cally dehydrogenated into methyl esters of 8-[5-
They are expressed as IC , a concentration at which
5
0
compound inhibit the cell proliferation for 50%. Very
strong inhibition was achieved on Hep2 (IC ꢁ1.66ꢀ
46.7 mM), MCF7
10 mM) and HBL (IC ꢁ1.78ꢀ39.8 mM)
cell lines. On the HeLa cells very good inhibition was
achieved with compound 2, 3, 4, 6 and 7 (IC ꢁ3.16ꢀ
5.07 mM) and slight inhibition with compound 5 (IC ꢁ
(
N-isopropylamidino-benzamidazolyl)]naphtho[2,1-b]-
/
/
5
0
furan-5-carboxylic acid 5, 8-[5-(N-2-imidazolinyl-benz-
imidazolyl)]naphtho[2,1-b]furan-5-carboxylic acid 6 and
39.8 mM), MiaPaCa2 (IC ꢁ
/
2.45ꢀ
/
5
0
(IC ꢁ
/
2.63ꢀ
/
/
/
5
0
50
8
-[5-(N -morpholinyl-amidino-benzimidazo-
lyl)]naphtho[2,1-b]furan-5-carboxylic acid 7 as hydro-
chloride salts. Compound 4 photolitically rearranged
into methyl ester of 8-(2-benzimidazolyl)naphtho[2,1-
b]furan-5-carboxylic acid 8 by the prolonged irradiation
in abs. ethanol (Scheme 1).
/
/
5
0
/
5
0
199 mM). Similar observation was on HT29 cell line. A
compound 4 inhibited very strong HT29 cells (IC ꢁ
/
5
0
1.38 mM), but other compounds exerted a wicker effect
(IC ꢁ42.7ꢀ1580 mM). The best inhibition was
achieved with compound 4, but it inhibits also the
growth of normal fibroblasts at the same extend (IC ꢁ
Structure identification of the synthetic products were
1
/
/
5
0
13
determined by H NMR, C NMR spectroscopy and
elemental analysis.
/
5
0
5.5 mM). Very strong inhibition of all examined tumor
cell lines was achieved with compound 2, and without
influence on proliferation of normal fibroblasts (WI38).
To compare an inhibitory effect among the compounds,
we can conclude that the basic structure of compounds
2
.1. Biological activity
All new prepared compounds 2, 3, 4, 5, 6, 7 and 8
were tested on their antitumor activity, using different
3
, 4, 6 and 7 and substituents (R) did not influence on
their inhibitory effect. But in compounds 2 and 5 the
substituent (R) has an influence on the inhibitory effect.
Unsubstituted derivative 8 has not inhibited any tested
cell lines. Thus, the inhibitory effect moderated with
substituent (R) and the concentration of substance,
rather than with type of tumor cell line.
3. Experimental
3.1. General
Melting points were determined on a Kofler block
apparatus and are uncorrected. IR spectra were deter-
mined with a Nicolet Magna 760 infrared spectro-
1
13
photometer in KBr pellets. H NMR and C NMR
spectral data were determined using Brucker Avance
DPX 300 MHz NMR or Varian-Gemini 300 MHz
spectrometers with tetramethylsilane as an internal
standard. Elemental analyses were carried out in the
Microanalitical laboratory at the ‘Rugjer Boskovic’
Institute.
3
(
.2. Methyl E-3-(5-formyl-2-furyl)-2-phenylacrylate
1)
Corresponding methyl-E-3-(2-furyl)-2-phenylacrylate
was formylated by Vilsmeier formilation.
Scheme 1.

M. Hranjec et al. / Il Farmaco 58 (2003) 1319ꢀ
/1324
1321
Fig. 1. The inhibitory effect of compound 2 and 4 on the growth of tumor cell lines (HT29, Hep2, HeLa, MCF7, MiaPaCa2, HBL) and normal
human fibroblasts (WI38).
Phosphorus oxychloride (22.8 ml, 250 mmol) was
added dropwise with cooling to DMF (20 ml, 250
mmol). The reaction mixture was stirred for half-an-
hour by cooling on ice, the apparatus being protected
with a calcium chloride tube. A solution of methyl-E-3-
temperature, and diethylether was added and the
resulting brown solid was filtered off. The crude product
was suspended in abs. ethanol and cooled to 0ꢀ5 8C.
/
Into the suspension was introduced HCl gas until the
suspension was saturated and the content was stirred
over night on the room temperature. Dry ether was
(
2-furyl)-2-phenylacrylate (19 g, 86 mmol) in DMF (10
ml) was added dropwise to the mixture. After the
addition was completed, the mixture was stirred for 1
h at room temperature, then heated at 90 8C 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. Orange crystals
added and the precipitated yellowꢀgreen powder was
filtered off and washed with dry ether. It was repeated
few times until the powder was analytically pure. It was
/
obtained 0.95 g (41%) slightly green powder. m.p. 235ꢀ
/
ꢂ1
1
2
37 8C. IR (cm ; KBr): 3300, 1675, 1590. H NMR (d
ppm) (DMSO-d ): 9.60 (s, 1H, NHamid.), 9.57 (s, 1H,
6
NHamid.), 9.49 (bs, 1H, NHamid.), 8.01 (s, 1H, NHbenzim.
H-13), 7.77 (d, 1H, Harom., H-10, Jꢁ8.64 Hz), 7.73 (s,
H, Harom., H-12), 7.61 (d, 1H, Harom., H-11, Jꢁ8.84
Hz), 7.60 (s, 1H, Hethen., H-7), 7.54ꢀ7.46 (m, 3H, Harom.
H-2, H-3, H-4), 7.38 (d, 1H, Hfur., H-9, Jꢁ3.83 Hz),
.31 (d, 2H, Harom., H-1,5, Jꢁ7.76 Hz), 5.71 (d,1H,
Hfur., H-8, Jꢁ3.78 Hz), 4.07 (m, 1H, CHi-pr), 3.90 (s,
H, H-6, OCH ), 1.34 (d, 6H, 2CH3i-Pr., Jꢁ6.21 Hz).
,
1
2.2 g (83.5%) are obtained, m.p. 103ꢀ
/
105 8C (literature
/
[
19] m.p. 103ꢀ105 8C.
/
1
/
/
,
3
.3. Methyl-E-3-[5-(5-N-isopropylamidino-2-
/
benzimidazolyl)-2-furyl]-2-phenyl-acrylate hydrochloride
2)
7
/
(
/
3
/
3
Compound 2 was prepared using the method de-
scribed earlier [20]. A mixture of 1 (1.01 g, 4.0 mmol),
,4-diamino-N-isopropylbenzamidine [21] (0.90 g, 4.0
1
3
C NMR (d ppm) (DMSO-d ): 166.9, 162.5, 152.9,
44.6, 144.4, 135.5, 132.9, 129.6, 129.3, 129.1, 127.1,
24.7, 124.2, 116.8, 116.6, 116.2, 115.3, 53.1, 45.7, 21.9.
6
1
1
3
mmol) and p-benzoquinone (0.43 g, 4.0 mmol) in abs.
EtOH (25 ml) was stirred at reflux for 4 h (under
nitrogen). The reaction mixture was cooled to room
Anal. Calc. for C25
H
N
O
Cl
N 9.43; Found: C 50.34, H 5.68, N 9.04.3%.
×
/
3H O: C 50.71, H 5.62,
27
4
3
3
2
Table 1
Inhibitory effect of compounds on tumor cell lines and normal fibroblasts
Comp.
IC50 (mM)
HT29
Hep2
HeLa
MCF7
MiaPaCa2
HBL
WI38
1 000 000
2
3
4
5
6
7
8
75.8
42.7
1.38
1580
398
12.6
5.01
1.66
39.8
6.02
4.17
3.24
5.62
3.16
199
2.63
5.12
4.37
6.3
3.09
35.5
2.45
21.4
3.72
46.7
2.57
41.7
1.78
6.3
ꢀ/
56
5.5
1 000 000
ꢀ
/
4.26
5.01
10
4.89
7.58
39.8
63
60.2
1 000 000
42.7
ꢀ/1 000 000
ꢀ/1 000 000
ꢀ/1 000 000
ꢀ/
1 000 000
ꢀ/1 000 000
ꢀ/1 000 000
ꢀ
/

1322
M. Hranjec et al. / Il Farmaco 58 (2003) 1319ꢀ1324
/
3
.4. Methyl E-3-{5-[5-N-(2-imidazolinyl)-
benzimidazolyl-2-furyl]}-2-phenylacrylate hydrochloride
3)
3.6. Methyl 2-[(5-N-
isopropylamidino)benzimidazolyl]naphtho[2,1-b]furan-
5-carboxylate hydrochloride (5)
(
Compound 3 was prepared using the method de-
scribed for the preparation of 2 from 1 (0.72 g, 2.8
mmol), 4-[N-(2-imidazolinyl)]-1,2-phenylenediamine
21] (0.50 g, 2.8 mmol) and p-benzoquinone (0.3 0 g,
A saturated solution of compound 2 (0.90 g, 2.0
mmol) in abs. ethanol (140 ml) was irradiated by pyrex
filtered light with UV light of 400 W high pressure
mercury arch lamp for 15 h at room temperature. Air
was bubbled through the solution. Irradiated solution
was concentrated under reduced pressure to 1/3 of the
volume. Diethylether was added to the solution and the
precipitated powder was filtered off and recrystallized
[
2
.8 mmol) in abs. ethanol (25 ml). It was obtained 0.80
246 8C. IR
cm ; KBr): 3350, 1700, 1610. H NMR (d ppm)
g, (54%) olive green powder m.p. 243ꢀ
(
/
ꢂ1
1
(
DMSO-d ): 10.76 (s, 2H, NHamid.), 8.38 (s, 1H,
6
NHbenzimid., H-13), 7.91 (d, 1H, Harom., H-10, Jꢁ
Hz), 7.78 (d, 1H, Harom., H-11, Jꢁ8.64 Hz), 7.70 (s, 1H,
Harom., H-12), 7.50 (s, 1H, Hethen., H-7), 7.47ꢀ7.44 (m,
H, Harom., H-2, H-3, H-4), 7.36 (d, 1H, Hfur., H-9, Jꢁ
.66 Hz), 7.30 (d, 1H, Harom., H-5, Jꢁ7.80 Hz), 7.29 (d,
H, Harom., H-1, Jꢁ7.64 Hz), 5.68 (d, 1H, Hfur., H-8,
3.66 Hz), 3.99 (bs, 4H, CH imid.), 3.41 (s, 3H, H-6,
/
8.64
from ethanol. It was obtained 0.37 g (35%) yellow
ꢂ1
/
powder m.p. 255ꢀ
/
257 8C. IR (cm ; KBr): 3300, 1640.
1
/
H NMR (d ppm) (DMSO-d ): 9.62 (bs, 2H, NHamid.),
6
3
3
1
/
9.52 (s, 1H, NHamid.), 9.07 (s, 1H, NHbenzimid., H-13),
8.88 (d, 1H, Harom., H-10, Jꢁ8.67 Hz), 8.63 (s, 1H,
Harom., H-12), 8.50 (d, 1H, Harom., H-11, Jꢁ8.30 Hz),
8.47 (d, 1H, Harom., H-5, Jꢁ7.60 Hz), 8.11 (s, 1H,
Hnaphthofur., H-7), 7.87ꢀ7.70 (m, 3H, Harom., H-2, H-3,
H-4), 7.63 (bs, 1H, Hfur., H-9), 4.46 (m, 1H, CHi-pr), 4.07
(s, 3H, H-6, OCH ), 1.24 (d, 6H, 2CH3 i-pr., Jꢁ6.21
/
/
/
/
Jꢁ
/
/
2
1
3
OCH3). C NMR (d ppm) (DMSO-d ): 166.3, 165.1,
6
/
1
1
51.9, 145.5, 145.0, 135.1, 131.8, 128.9, 128.7, 128.4,
26.6, 122.9, 115.9, 115.6, 114.9, 52.7, 44.2 (2C). Anal.
/
3
1
3
Calc. for C H N O Cl ×2H O: C 55.34, H 5.03, N
2 /
Hz). C NMR (d ppm) (DMSO-d ): 167.5, 162.7, 158.9,
151.2, 148.7, 145.6, 143.8, 141.7, 128.3, 128.1, 127.5,
2
4
22
4
3
2
6
1
0.76; Found: C 55.73, H 4.81, N 10.75%.
1
1
2
5
27.0, 126.3, 124.9, 124.3, 123.7, 117.3, 116.3, 115.7,
08.4, 53.1, 45.6, 21.9. Anal. Calc. for C H N O Cl ×
/
2
5
24
4
3
2
H O: C 56.12, H 5.27, N 10.47; Found: C 56.30, H
.16, N 10.72%.
2
3
.5. Methyl E-3-{5-[5-N-(N-morpholinylamidino)-
benzimidazolyl-2-furyl]}-2-phenylacrylate hydrochloride
4)
(
Compound 4 was prepared using the method de-
3.7. Methyl 2-{[(5-N-(2-
scribed for the preparation of 2 from 1 (0.54 g, 2.1
mmol), 4-(N-morpholynilylamidino)-1,2-phenylenedia-
mine [21] (0.50 g, 2.1 mmol) and p-benzoquinone (0.23
g, 2.1 mmol) in abs. ethanol (25 ml). It was obtained
imidazolinyl)]benzimidazolyl}naphtho[2,1-b]furan-5-
carboxylate hydrochloride (6)
Compound 6 was prepared on the way described for 5
by irradiation of saturated solution of 3 (0.50 g, 1.1
mmol) in ethanol: water (15 ml: 10 ml) during 35 h. It
0
.57 g, (54%) slightly green powder mp. 221ꢀ224 8C. IR
/
ꢂ1
1
cm ; KBr): 3300, 1720, 1650, 1620. H NMR (d ppm)
(
(
DMSO-d ): 11.17 (s, 1H, Hamid.), 9.81 (bs, 1H,
6
was obtained 0.075 g (16%) yellowꢀgreen powder, m.p.
/
ꢂ1
1
NHamid.), 9.11 (s, 1H, NHamid.), 8.10 (s, 1H, Hbenzimid.
H-13), 7.80 (d, 1H, Harom., H-10, Jꢁ8.45 Hz), 7.73 (s,
H, Harom., H-12), 7.65 (d, 1H, Harom., H-11, Jꢁ8.48
Hz), 7.53 (s, 1Hethen., H-7), 7.52ꢀ7.49 (m, 3H, Harom., H-
, H-3, H-4), 7.36 (d, 1H, Hfur., H-9, Jꢁ3.8 Hz), 7.32 (d,
H, Harom., H-1, H-5, Jꢁ7.81 Hz), 5.71 (d, 1H, Hfur.
3.6 Hz), 3.96 (s, 3H, H-6, OCH ), 3.77 (t, 4H,
,
ꢀ
/
300 8C. IR (cm ; KBr): 3350, 1690. H NMR (d
/
ppm) (DMSO-d ): 10.67 (bs, 2H, NHamid.), 8.87 (d, 1H,
6
1
/
Harom., H-10, Jꢁ
/
8.98 Hz ), 8.59 (s, 1H, NHbenzimid, H-
.
/
13 ), 8.53 (d, 1H, Harom., H-11, Jꢁ
/8.54 Hz), 8.46 (s, 1H,
.
2
2
/
Harom., H-12), 8.44 (d, 1H, Harom., H-5, Jꢁ7.38 Hz),
/
/
,
7.90 (bs, 1H, Harom., H-2), 7.84 (s, 1H, H-7), 7.80-7.70
(m, 2H, Harom., H-3, H-4), 7.65 (s, 1H, Hfuran., H-9),
H-7, Jꢁ
/
3
2
CH2 morph., Jꢁ
C NMR (d ppm) (DMSO-d ): 170.1, 166.3, 162.0,
6
/
7.20 Hz), 2.94 (bs, 4H, 2CH2 morph.).
4.25ꢀ
/
4.21 (bs, 4H, 2CH2 imid.), 4.16 (s, 3H, H-6, OCH3).
1
3
13
C NMR (d ppm) (DMSO-d ): 166.8, 165.0, 150.6,
6
1
1
52.2, 145.4, 144.5, 136.8, 132.1, 130.6, 130.1, 128.9,
28.4, 123.6, 120.5, 117.0, 116.4, 115.7, 115.6, 66.1, 53.8
148.1, 146.5, 142.2, 127.6, 127.5, 127.4, 127.3, 126.8,
126.3, 125.2, 124.3, 123.0, 116.1, 115.6, 115.4, 107.8,
(
C 50.61, H 5.24, N 11.35; Found: C 50.31, H 5.62, N
2C), 52.8 (2C). Anal. Calc. for C H N O Cl ×2H O:
3 /
107.3, 52.5, 44.2 (2C). Anal. Calc. for C H N O Cl×
/
2
6
28
5
4
2
24 20
4
3
3H O: C 53.67, H 4.88, N 10.43; Found: C 53.66, H
4.87, N 10.68%.
2
1
1.16%.

M. Hranjec et al. / Il Farmaco 58 (2003) 1319ꢀ
/1324
1323
3.8. Methyl 2-{5-[5-N-(N-
morpholinylamidino)]benzimidazolyl}naphtho[2,1-
b]furan-5-carboxylate hydrochloride (7)
PaCa2 (pancreatic carcinoma), HBL (melanoma),
MCF7 (breast cancer) and normal human fibroblasts
WI38. The cells were treated with different concentra-
tion of compounds (10 , 10 , 10 M) during 72 h.
All cell lines were grown in DMEM medium (supple-
ꢂ4
ꢂ5
ꢂ6
Compound 7 was prepared on the way described for 5
by irradiation of saturated solution of 4 ( 0.75 g, 1.5
mmol) in abs. ethanol (150 ml) during 10 h. It was
mented with 10% heat inactivated fetal bovine serum, 2
L
mM
-glutamine, 100 U/ml penicillin and 100 mg/ml
obtained 0.21 g (23%) yellow powder, mp. 270ꢀ
/
272 8C.
IR (cm ; KBr): 3300, 1650. H NMR (d ppm)
DMSO-d ): 11.32 (bs, 1H, NHamid.), 9.85 (bs, 1H,
streptomycin) at 37 8C in a humidified atmosphere with
ꢂ1
1
5% CO . For the purpose of the experiment, the cells
were plated in 96-microwell flat bottom plates at a
2
(
6
4
10 cells/ml (all tumor cell lines)
NHamid.), 9.20 (s, 1H, NHamid.), 8.23 (bs, 1H, NHbenzi-
mid., H-13), 7.90 (d, 1H, Harom., H-10, Jꢁ8.40 Hz), 7.85 (s,
H, Harom., H-12), 7.77 (d, 1H, Harom., H-11, Jꢁ
Hz), 7.70 (s, 1H, H-7), 7.68 (d, 1H, Harom., H-5, Jꢁ
concentration of 2ꢃ
/
4
10 cells/ml (WI38). The next day (24 h later)
/
and 3ꢃ
/
1
/
8.70
7.80
compounds were added to the cells at different concen-
ꢂ4
ꢂ5
ꢂ6
/
trations (10 , 10
and 10
dissolved in DMSO at a concentration of 10
M). Compounds were
ꢂ1
Hz), 7.48-7.37 (m, 3H, Harom., H-2, H-3, H-4), 7.28 (bs,
M and
1
6
H, Hfur., H-9), 3.76 (bs, 4H, 2CH2 morf.), 3.72 (s, 3H, H-
diluted with DMEM medium into working concentra-
tions. The concentration of DMSO was less than 0.1%
and at that concentration it does not affect the growth.
Control cells (without any compound) were growing
under the same conditions. Cell viability was measured
at day 0 and 72 h after addition of compounds, using
MTT assay, which detects dehydrogenase activity in
viable cell [22]. For this purpose the medium was
discarded and MTT was added to each well at a
concentration of 20 mg/40 ml. After 4 h of incubation
at 37 8C, the precipitates were dissolved in 160 ml of
DMSO. The absorbance was measured on an ELISA
reader at 570 nm, and the percentage of growth was
calculated. Each number was the mean of three indivi-
dual experiments done in quadruplicate. The results are
expressed as IC , what represent a concentration for
1
, OCH ), 2.96 (bs, 4H, 2CH2 morph.). C NMR (d
3
3
ppm) (DMSO-d ): 162.20, 162.12, 161.57, 150.66,
6
1
1
1
48.27, 145.41, 129.03, 128.49, 127.72, 127.61, 127.54,
27.50, 125.68, 125.62, 125.14, 124.41, 123.03, 122.75,
20.31, 116.17, 115.78, 115.05, 65.51 (2C), 62.02 (2C),
Anal. Calc. for C H N O Cl ×3H O: C 49.37, H 5.10,
N 11.07; Found: C 49.38, H 5.31, N 11.21%.
3 /
2
6
26
5
4
2
3.9. Methyl 2-(benzimidazolyl)naphtho[2,1-b]furan-5-
carboxylate (8)
When the saturated solution of compound 4 (0.25 g,
.5 mmol) in abs. ethanol (35 ml) was prolonged
0
irradiated (during 15 h and in the quantity of the 1/3
of the experiment 3.8) by pyrex filtered light with UV
light of 400 W high pressure mercury arch, only
photolitic product 8 (after recrystallization in ethanol)
5
0
50% inhibitory effect.
was isolated in the yield of 0.015 g (14%). Yellow
ꢂ1
powder m.p. 230ꢀ
/
232 8C. IR (cm ; KBr): 3310, 1650.
Acknowledgements
1
H NMR (d ppm) (DMSO-d ):, 8.87 (d, 1H, Harom., H-
6
1
1
1
0, Jꢁ
H, Harom., H-11, Jꢁ
2), 8.22 (s, 1H, H-7), 7.83(d, 1H, Harom., H-5, Jꢁ
/
8.87 Hz), 8.55 (s, 1H, NHbenzimid., H-13), 8.54 (d,
Support for this study by the Ministry of Science
(Project No 0125005 and 00981104) is gratefully ac-
knowledged.
/8.50 Hz), 8.45 (s, 1H, Harom., H-
/
7.60
Hz), 7.81 (s, 1H, Hfur., H-9), 7.76(d, 1H, Harom., H-2,
Jꢁ
J2ꢁ
J2ꢁ
ppm) (DMSO-d ): 167.5, 151.2, 148.6, 146.0, 128.2,
/
7.70 Hz), 7.72(dd, 1H, Harom., H-4, J ꢁ
/
8.40 Hz,
8.36 Hz,
1
/
8.38 Hz), 7.66(dd, 1H, Harom., H-3, J ꢁ
/
1
3
References
1
/8.40 Hz), 4.44 (s, 3H, H-6, OCH3). C NMR (d
6
[
[
[
[
1] M.A.H. de Zwart, H. van der Goot, H. Timmerman, Synthesis
and copper-dependent antimycoplasmal activity of 1-amino-3-(2-
pyridyl)isoquinoline derivatives. 2. Amidines, J. Med. Chem. 32
1
1
28.1, 128.0 (2C), 127.4, 126.9, 126.2, 125.0, 121.7,
20.3, 116.5, 116.2, 108.2, 105.3, 53.1. Anal. Calc. for
(
1989) 487ꢀ493.
/
C H N O : C 70.06.37, H 4.48, N 7.78; Found: C
3
2
1
14
2
2] M.M. Canto-Cavalheiro, A. Echevarria, M.A.S. de Souza, L.
Cysne-Finkelstein, M.A.D. Torres, L.L. Leon, Effect on amidine
derivatives on leishmania amazonensis axenic amastigotes, Arz-
7
0.35, H 4.24, N 8.03%.
3
.10. Biological assays
neimittel-Forschung 50 (2000) 925ꢀ
3] K. Okumura, T. Shimazaki, Y. Aoki, H. Yamashita, New platelet
fibrinogen recepzor glycoprotein II BꢀIII A antagonists-orallyac-
tive series of N-alkylated amidines with A 6,6-byciclic twemplate,
J. Med. Chem. 41 (1998) 4036ꢀ4052.
4] T.W. Ku, W.H. Miller, W.E. Bondinell, K.F. Erhard, R.M.
Keenan, A.J. Nicholas, C.E. Peishoff, J.M. Samanen, A.S. Wong,
W.F. Huffman, Potent non-peptide fibrinogen receptor antago-
/
928.
/
Biological activity of the compounds 2, 3, 4, 5, 6,7 and
was measured by their influence on growth of cell
8
/
cultures in vitro. The following human carcinoma cell
lines were used: HeLa (cervical carcinoma), Hep2
(
laryngeal carcinoma), HT29 (colon carcinoma), Mia-

1324
M. Hranjec et al. / Il Farmaco 58 (2003) 1319ꢀ1324
/
nists which present alternative pharmacophore, J. Med. Chem. 38
1987) 9ꢀ12.
5] E.S.H. El Ashry, N. Rashed, A.H.S. Shobier, Glycosidase
inhibitors and their chemotherapeutic value, part 3 (rewiew),
Pharmazie 55 (2000) 403ꢀ415.
6] T.M. Sielecki, J. Liu, S.A. Mousa, A.L. Racanelli, E.A. Hausner,
R.R. Wexler, R.E. Olson, Synthesis and pharmacology of
modified amidine isoxazoline glycoprotein IIb/IIIa receptor
N,N?-diphenyl-amidines, Bioorgan. Med. Chem. Lett. 6 (1996)
1901ꢀ1904.
[14] J.W. Lown, K. Krowicki, Efficient total syntheses of the
(
/
/
[
[
ologopeptide antibiotics netropsin and dystamicin, J. Org.
/
Chem. 50 (1985) 3774ꢀ3779.
/
[15] P.G. Baraldi, G. Cacciari, A. Guiotto, R. Romagnoli, et al., [2,1-
c] [1,4]Benzodiazepine (PBD)-dystamicin hybrid inhibits DNA
binding to transcription factor S
Acids 19 (2000) 1219ꢀ1229.
p
1, Nucleosides, Nucleot. Nucl.
antagonists, Bioorgan. Med. Chem. Lett. 11 (2001) 2201ꢀ
7] W.R. Ewing, M.R. Becker, V.E. Manetta, R.S. Davis, et al.,
Design and structureꢀactivity relationship of potent ans selective
inhibitors of blood coagulation factor Xa, J. Med. Chem. 42
1999) 3557ꢀ3571.
8] R. Ferroni, D. Simoni, S. Manfredini, M. Guarneri, P. Orlandini,
M. Barbieri, G.P. Franz e` , A. Mugelli, Synthesis of N -substituted
/
2204.
/
[
[
[16] P.G. Baraldi, P. Cozzi, C. Geroni, N. Mongelli, et al., Novel
benzoyl nitrogen mustard derivatives of pyrazole analogues of
distamicin a: synthesis and antileukemic activity, Bioorgan. Med.
/
(
/
Chem. 7 (1999) 251ꢀ262.
/
[17] A. Bielawska, K. Bielawski, S. Wolczynski, DNA-binding proper-
1
ties and cytotoxicity activity of novel aromatic amidines in
benzamidines. Effection blood coagulation, platelet aggregation
and antiarrhythic activity, Arzneimittel-Forschung 45 (1995)
cultured human skin fibroblasts, Pharmazie 56 (2001) 428ꢀ429.
/
[18] R. Janciene, L. Kosychova, V. Bukelskiene, V. Domkus, Z.
Strumbreviciute, V. Ragaleviciene, B.D. Puodziunaite, Synthesis
and antitumor activity of a series of cyclic amidines of 2,3-
dihydro-1H-1,5-benzodiazepines, Arzneimittel-Forschung 52
6
65ꢀ669.
/
[
9] J.M. Mellor, H. Rataj, A general route to cyclic amidines and
isothioureas based on formal Aza Diels Alder reactions of
aminoheterocycles, Tetrahedron Lett. 37 (1996) 2619ꢀ
/2622.
(2002) 475ꢀ481.
/
[
10] M.J. Rudolph, C.R. Illig, N.L. Subasinghe, et al., Design and
synthesis of 4,5-disustituted-thiophene-2-amidines as potent ur-
[19] M. Baji c´ , G. Karminski-Zamola, N. Blazevi c´ , Twofold photo-
chemical dehydrocyclization reaction of substituted 2,5-distyr-
ylthiophenes and 2,5-distyrylfuranes, Croat. Chem. Acta 65
okinase inhibitors, Bioorgan. Med. Chem. Lett. 12 (2002) 491ꢀ
/
4
95.
(1992) 835ꢀ
[20] R.R. Tidwell, Antipneumocystis activities of aromatic diamidox-
ime prodrugs, Antimicrob. Agents Chemother. (1998) 666ꢀ674.
[21] T.A. Farley, R.R. Tidwell, I. Donkor, N.A. Naiman, K.A.
Ohemeng, R.J. Lombardy, J.A. Bentley, M. Cory, Structure,
DNA minor groove binding, and base pair specificity of alkyl-
and aryl-linked bis(amidinobenzimidazoles) and bis(amidinoin-
/
846.
[
11] J.L. Collins, B.G. Shearer, J.A. Oplinger, S. Lee, E.P. Garvey, M.
Salter, C. Duffy, T.C. Burnette, E.S. Furfine, N-Phenylamidines
as selective inhibitors of human neuronal nitric oxide synthase:
/
structureꢀactivity studies and demonstration of in vivo activity, J.
Med. Chem. 41 (1998) 2858.
/
[
[
12] C.E. Stephens, F. Tanious, S. Kim, D.W. Wilson, W.A. Schell,
J.R. Perfect, S.G. Franzblau, D.W. Boykin, Diquanidino and
reversed diamidino-2,5-diarylfurans as antimicrobial agents, J.
Med. Chem. 44 (2001) 1741.
doles), J. Med. Chem. 36 (1993) 1746ꢀ
[22] J. Carmichael, W.G. DeGraff, A.F. Gazdar, J.D. Minna, J.B.
Mitchell, Evaluation of tetrazolium-based semiautomated
colorimetric assay: assessment of chemosensitivity testing, Cancer
Res. 47 (1987) 936ꢀ942.
/1753.
a
13] A. Echevarria, L.H. Santos, J. Miller, N. Mahmood, Synthesis,
characterization and anti-HIV activity of some 2-p-X-phenyl-1,3-
/