Novel amidino substituted 2-phenylbenzothiazoles: Synthesis, antitumor evaluation in vitro and acute toxicity testing in vivo

Article abstract of DOI:10.1016/j.bmc.2009.12.054

The efficient synthesis of new bis-substituted nitro-amidino, amino-amidino (10a, 10b-13a, 13b) and previously prepared diamidino 2-phenyl-benzothiazoles (9a, 9b) is described. The compounds 11a and 11b were prepared by recently developed methodology of the key precursors in zwitterionic form 8a and 8b with 4-nitrobenzoylchloride in a very good yield (70%). All compounds except diamidino-substituted 2-phenylbenzothiazole 9a show exceptionally prominent tumor cell-growth inhibitory activity and cytotoxicity, whereby the special selectivity of amino-amidine 2-phenylbenzothiazole 12a towards MCF-7 and H 460 cells makes this compound a prospective lead compound that should be further evaluated in animal models. All in vivo tested compounds (12a, 12b, 13a and 13b) are absorbed from mice gastrointestinal system. LD₅₀ are between 67.33 and 696.2 mg/kg body weight (OECD/EPA toxicity categories 2-3).

Full text of DOI:10.1016/j.bmc.2009.12.054

Bioorganic & Medicinal Chemistry 18 (2010) 1038–1044
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel amidino substituted 2-phenylbenzothiazoles: Synthesis, antitumor
evaluation in vitro and acute toxicity testing in vivo
b
c
c
c
b
´
´
´
Livio Racané , Marijeta Kralj , Lidija Šuman , Ranko Stojkovic , Vesna Tralic-Kulenovic ,
Grace Karminski-Zamola a,
*
a Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, University of Zagreb, Maruli´cev trg 20, PO Box 177, HR-10000 Zagreb, Croatia
b Department of Applied Chemistry, Faculty of Textile Technology, University of Zagreb, baruna Filipovi´ca 10000 Zagreb, Croatia
c
-
ˇ
´
Division of Molecular Medicine, Ruder Boškovic Institute, Bijenicka cesta 54, PO Box 180, HR-10000 Zagreb, Croatia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 July 2009
Revised 17 December 2009
Accepted 21 December 2009
Available online 29 December 2009
The efficient synthesis of new bis-substituted nitro-amidino, amino-amidino (10a, 10b–13a, 13b) and
previously prepared diamidino 2-phenyl-benzothiazoles (9a, 9b) is described. The compounds 11a
and 11b were prepared by recently developed methodology of the key precursors in zwitterionic form
8a and 8b with 4-nitrobenzoylchloride in a very good yield (70%). All compounds except diamidino-
substituted 2-phenylbenzothiazole 9a show exceptionally prominent tumor cell-growth inhibitory
activity and cytotoxicity, whereby the special selectivity of amino-amidine 2-phenylbenzothiazole
12a towards MCF-7 and H 460 cells makes this compound a prospective lead compound that should
be further evaluated in animal models. All in vivo tested compounds (12a, 12b, 13a and 13b) are
absorbed from mice gastrointestinal system. LD50 are between 67.33 and 696.2 mg/kg body weight
(OECD/EPA toxicity categories 2–3).
Keywords:
Nitro-amidino-, amino-amidino- and
diamidino-substituted 2-
phenylbenzothiazoles
Antitumor activity ‘in vitro’
Toxicity testing ‘in vivo’
Ó 2010 Published by Elsevier Ltd.
1. Introduction
Our previously obtained results showed that antiproliferative
activity of cyano, amidino19 and amino20 substituted 2-phen-
In the last two decades, many heterocyclic compounds from the
benzothiazole series were synthesizedand theirbiological and phar-
macological activity investigated. They were studied extensively for
their antiallergic,1 anti-inflammatory,1,2 antitumor3–7 and analge-
sic8,9 activity. Considering the mechanism of action, it was shown
that benzothiazole derivatives act as tyrosine kinase10–13 and topo-
isomerase I and II inhibitors.14,15 Therefore, various benzothiazole
compounds are further of considerable interest for their diverse
pharmaceutical uses. It was reported recently about a novel series
of optically active 2-aminobenzothiazole derivatives and their
in vitro cytotoxicity against mouse Ehrlich Ascites Carcinoma
(EAC) and two human cancer cell lines MCF-7 and HeLa. In alkaline
comet assay some compounds showed dose-dependent DNA
damaging activity.16 Newly prepared 2-acetyl-3-(6-methoxyben-
zothiazo)-2-yl-amino-acrylonitrile showed significant antiprolifer-
ative activity and is a potent inducer of programed cell death in leu-
kemia cells.17 The most recent article describing the synthesis of
corresponding aminophenyl- substituted benzothiazoles with
different adenine mimic which resulted in the identification of
promising scaffolds, giving rise to the inhibition of different kinases
with the IC50 values in the nanomolar range.18
ylbenzothiazole derivatives strongly depends on the position of
the substituent on 2-phenylbenzothiazole skeleton, as well as on
the type of amidino substituent attached. We found that, in a series
of unsubstituted, N-isopropyl substituted, as well as 2-imidazolinyl
mono and bisamidino derivatives of 2-phenylbenzothiazole, N-iso-
propyl substituted amidine posses less pronounced antiprolifera-
tive activity on tested tumor cells.
In relation with the above considerations, we designed and effi-
ciently synthesized new nitro-amidino, amino-amidino and diami-
dino-substituted 2-phenylbenzothiazole derivatives and tested
their antitumor activity in vitro and determinate acute oral toxicity
in mice of the most active compounds.
2. Results and discussion
2.1. Chemistry
An efficient synthesis of different amidino substituted 2-phen-
ylbenzothiazole derivatives was carried out by reactions outlined
in Scheme 1.
Bis-nitrile and nitro-nitrile 5–7 derivatives were prepared by
condensation reaction of cyano or nitro-substituted 2-aminobenz-
othiole 1 or 221 with commercially available 4-cyano or 4-nitro-
benzoylchloride 3 and 4 (77–84%), respectively and were used as
* Corresponding author. Tel.: +385 14597297; fax: +385 14597224.
E-mail address: gzamola@fkit.hr (G. Karminski-Zamola).
0968-0896/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2009.12.054

L. Racané et al. / Bioorg. Med. Chem. 18 (2010) 1038–1044
1039
NH2
O
N
i)
+
R2
R2
S
Cl
R1
SH
R1
5; R1= CN R2=CN
6; R1= NO2 R2=CN
7; R1= CN R2=NO2
3
4
1; R1=CN
; R2=CN
; R2=NO2
2
; R1=NO2
N
ii) then
5
AmH
iii) or iv)
S
HAm
Cl
9a
9b
Cl
N
S
N
S
ii) then
v)
v)
AmH
NH2
6
7
AmH
NO2
iii) or iv)
H2N
O2N
Cl
Cl
10a
10b
12a
12b
N
S
N
S
ii) then
iii) or iv)
HAm
Cl
HAm
Cl
11a
11b
13a
13b
O
i)
NO2
Cl
NH2
S
HAm
NH
8a
8b
8a, 9a, 10a, 11a, 12a, 13a; Am=
8b, 9b, 10b, 11b, 12b, 13b; Am=
NH2
N
N
H
Scheme 1. Reagents and conditions: (i) HOAc, 4 h, reflux; (ii) 2-(2-ethoxyethoxy)ethanol/HCl(g), 5–7 days, rt; (iii) abs EtOH/NH3 (g), two days rt, then 1 h 60 °C; (iv) abs
EtOH/NH2(CH2)2NH2, 4 h, reflux; (v) SnCl2/MeOH/HCl, 15 min, reflux.
starting compounds for the preparation of targeted amidino substi-
tuted 2-phenylbenzothiazole by Pinner reaction. Compounds 5 and
9a were previously prepared by method different from ours, but
the authors did not report neither the yield of pure product nor
spectroscopic characteristics.22 For the conversion of nitrile 5–7
into corresponding imidoyl ether hydrochlorides, in the first step
of Pinner reaction, abs 2-(2-ethoxyethoxy)ethanol was used in-
stead of abs ethanol due to very low solubility of compounds.
The conversion of nitriles into imidoyl ether hydrochlorides was
quantitative followed by subsequent reaction with gaseous ammo-
nia or ethylenediamine affording amidine 9a, 9b–11a, 11b in
43–79% yield. Recently, we described an efficient method for prep-
aration of amidino substituted 2-aminobenzothiole23 thus opening
a possibility of another synthetic approach for preparation of
6-amidino substituted 2-phenyl or 2-heteroarylbenzothiazolyl
molecules. Condensation reaction of these key precursors in zwit-
terionic form 8a and 8b23 with 4-nitrobenzoylchloride in acetic
acid afforded compounds 11a and 11b in a very good yield (70%),
and this approach is much better in term of time consuming.
Finally, the reduction of nitro derivatives 10a, 10b–11a, 11b was
accomplished applying the reaction with tin(II) chloride as redu-
cens in hydrochloric acid–methanol mixture in a good yield (55–
80%). The amino-amidino derivatives 12a, 12b–13a, 13b were only
isolated from reaction as mono hydrochloride salt in spite of strong
acidic reaction conditions. The structure of compounds was deter-
mined by IR, 1H and 13C NMR spectroscopy as well as elemental
analysis. All amidino compounds 9a, 9b–13a, 13b were isolated
as hydrochloride salts to improve their aqueous solubility but ami-
dino-nitro compounds 10a, 10b–11a, 11b were sparingly water-
soluble and being the problem for in vitro testing.
2.2. Biological results
2.2.1. Antiproliferative activity
Compounds 9a, 9b–13a, 13b were screened for their potential
antiproliferative effects on a panel of five human cell lines, which
were derived from different cancer types including MCF-7 (breast
carcinoma), SW 620 and HCT 116 (colon carcinoma), H 460 (lung

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L. Racané et al. / Bioorg. Med. Chem. 18 (2010) 1038–1044
carcinoma) and MOLT-4 (acute lymphoblastic leukemia) (Table 1
and Fig. 1). All compounds except diamidino-substituted 2-phen-
ylbenzothiazole (9a) show exceptionally prominent tumor cell-
growth inhibitory activity and cytotoxicity. Interestingly, when
both phenyl and benzothiazole sides of the molecule are substi-
tuted with imidazoline (9b) the activity is dramatically stronger,
but not selective.
In general, nitro-amidino substituted 2-phenylbenzothiazole
(10 and 11) derivatives showed similar activity as their amino-
amidino substituted analogues (12 and 13). Still, nitro-imidazoli-
nyl substituted compounds (10b, 11b) exert somewhat lower
activity compared to nitro-amidino substituted analogues (10a,
11a) which are nonselective cytotoxic. On the contrary, amino-imi-
dazolinyl substituted compounds (12b and 13b) are to some extent
more potent compared to amino-amidino substituted 2-phen-
ylbenzothiazoles (12a and 13a). Moreover, although the differ-
ences are not marked, it appears that the position of the amidine
moiety on the phenyl ring results in a better activity compared
to its position on the benzothiazole ring.
In general, benzothiazoles were demonstrated to be potent aryl
hydrocarbon receptor (AhR) agonists, binding to AhR results in
induction of CYP1A1, causes generation of electrophilic reactive
species which forms DNA adduct, ultimately resulting in cell death
by activation of apoptotic machinery. Previously described 2-(4-
amino-3-methylphenyl)benzothiazole showed superb sensitivity
towards breast cancer MCF-7 cell line. This selectivity is assigned
to differences in metabolic activation and inactivation between cell
lines, whereby CYP1A1 and CYP1B1, activated via AhR (which was
shown to be highly expressed in breast cancer cells) are shown to
be the major determinants of this metabolic conversion.24,25
According to these data, we could speculate that the same
mechanism of action could be responsible for the here-presented
benzothiazole derivatives. Moreover, the cell line MCF-7 showed
superior sensitivity to 9a and 12a, compared to other cell lines.
However, compounds 13a and 13b, which are close analogues of
previously described 2-(4-amino-3-methylphenyl)benzothiazole
did not show any selectivity to MCF-7 cell line.
Still, prominent and selective activity of 12a towards MCF-7 and
H 460 cells makes this compound a prospective lead compound
that should be further evaluated in animal models. Therefore, pre-
liminary acute toxicity testing was performed.
2.2.2. Acute toxicity testing in vivo
The aim of the in vivo studies was to determine acute oral Tox-
icity LD50 dose of the test substances on murine model using OECD
Guideline for the testing of chemicals, Section 4: Health Effects
Test No. 425, Acute Oral Toxicity: Up-and-Down Procedure.26
Although all in vitro tested compounds except 9a showed similar
activities only the amino-amidinic derivative 12a, 12b, 13a, and
13b were sufficiently water soluble for in vivo testing. The esti-
mated LD50 for each of tested substances (based on maximum like-
lihood) of long term results is shown in Table 2.
From the obtained data it can be seen that all tested substances
absorbed in gastrointestinal tract of animals after intragastric ga-
vage caused biological/toxicological effect in animals. Substances
12a, 12b, 13a and 13b can be classified as category 2 and substance
13b can be classified as category 3 according to OECD/EPA toxicity
categories.27
Interestingly, compound 13b showed the lowest acute oral tox-
icity, although it exerted the most pronounced toxicity to tumor
cells. To check whether the differences in the toxicity could be a
consequence of different intestinal absorption, we utilized a web-
based application called PreADMET, which has been developed
for rapid prediction of drug-likeness and ADME/Tox data.28 How-
ever, according to these data the percentage of human intestinal
absorption prediction, defined as the sum of bioavailability and
absorption evaluated from ratio of excretion or cumulative excre-
tion in urine, bile and feces,29 for all compounds are very similar
(86% for 12a and 13a and 88% for 12b and 13b). Therefore, the
Table 1
In vitro inhibition of compounds 9a, 9b–13a, 13b on the growth of tumor cells
a
Compd
IC50
(
lM)
MOLT-4
>100b
HCT 116
64 35b
SW 620
MCF-7
11
H 460
9a
>100b
1
78 20b
9b
3
2
17
1
2
2
1
3
1
2
0.1
1
0.2
0.7
0.8
0.2
0.7
0.2
2
2
8
1
3
3
1
10
1
0.3
0.04
5
0.1
1
0.6
0.03
3
0.8 0.4
1
2
0.1
0.3
11 0.6
2
1
4
1
3
0.2
0.2
0.1
0.2
0.2
11a
11b
10a
10b
12a
12b
13a
13b
2
7
2
2
4
0.1
5
0.3
0.5
0.2
Table 2
1
3
0.3
0.4
Estimated LD50 (in mg/kg) in vivo
0.2 0.01
1
9
0.2 0.08
0.3 0.05
4
a
Compound
Estimated LD50 (mg/kg)
0.3 0.1
0.04
0.7
7
2
0.6
12a
12b
13a
13b
249.8
249.8
67.33
696.2
0.1
0.7 0.04
1.7 0.2
0.4 0.05
a
IC50; the concentration that causes a 50% reduction of the cell growth.
These IC50 measurements were not significantly smaller than 100 lM at p
b
a
<0.001 (one-tailed Z-test). For all other measurements, IC50 is significantly smaller
The LD50 is calculated with AOT425StatPgm based on maximum likelihood for
long term results at 95% confidence interval.
than 100 M, indicating that the compounds have cytostatic/cytotoxic activity.
l
Figure 1. Dose–response profiles for compounds 12a and 12b tested in vitro. PG = percentage of growth.

L. Racané et al. / Bioorg. Med. Chem. 18 (2010) 1038–1044
1041
differences in the toxicity data are probably consequences of the
compounds activity itself. The encouraging result pointing to lower
toxicity for the most active compound will be further evaluated in
in vivo antitumor studies, whereby the data attained herein will be
used to determine starting doses of tested substances.
crystals of imidoyl ether hydrochloride filtered off, washed with
abs ether, and dried under vacuum over KOH. The yield of corre-
sponding white hygroscopic crystalline solid of imidoyl ether
hydrochloride was quantitative and has been prepared prior to
use without purification in reaction with ammonia (iii) or ethy-
lenediamine (iv).
3. Conclusion
(iii) Reaction of imidoyl ether hydrochloride with ammonia: The
corresponding imidoyl ether hydrochloride (1 equiv) in abs etha-
nol was cooled to 5–10 °C and saturated with dry gaseous ammo-
nia. The flask was stoppered and content was stirred at room
temperature for three days. The reaction mixture was heated with
stirring at 60 °C for 1 h, cooled over night, and the resulting precip-
itate filtered off, washed with diethyl ether, and dried under vac-
uum. The crude product was crystallized from appropriate
solvent (charcoal) several times, until compound was analytically
pure (9a, 10a, and 11a).
(iv) Reaction of imidate hydrochloride with ethylenediamine: To
the suspension of corresponding imidoyl ether hydrochloride
(1 equiv) in abs EtOH freshly distilled ethylenediamine (5 equiv)
was added in nitrogen atmosphere. The reaction mixture was re-
fluxed for 4 h under nitrogen, cooled to rt and acidified with concd
HCl. After standing in refrigerator over night, the resulting precip-
itate was collected by filtration, washed with diethyl ether and
dried under vacuum. The crude product was purified by crystalliza-
tion giving pure compound (9b, 10b, and 11b).
The series of new amidino-nitro and amidino-amino substi-
tuted benzothiazoles was efficiently prepared by our recently pos-
tulated synthetic methodology, and the new convergent synthesis
of amidino-substituted benzothiazoles form zwitterions 8a and 8b
was developed.
All compounds except diamidino-substituted 2-phenylbenzo-
thiazole 9a show exceptionally prominent tumor cell-growth
inhibitory activity and cytotoxicity (IC50 were in the micromolar
range), whereby the special selectivity of amino-amidine 2-phen-
ylbenzothiazole 12a towards MCF-7 and H 460 cells makes this
compound a prospective lead compound that should be further
evaluated in animal models. All in vivo tested compounds (12a,
12b, 13a and 13b) are absorbed from mice gastrointestinal system.
LD50 are between 67.33 and 696.2 mg/kg body weight (OECD/EPA
toxicity categories 2–3). Compound 13b was least toxic, albeit very
active in antiproliferative study, which makes it exceptionally
interesting for further in vivo studies.
(v) Reduction of corresponding nitro-substituted compound: A
solution of tin(II) chloride dihydrate (10 equiv) in concd HCl and
methanol was added to the corresponding nitro compound
(1 equiv). The reaction mixture was stirred and refluxed for
15 min. The precipitate was filtered off, washed with methanol,
diethyl ether, and dried under vacuum. The crude product was
purified by crystallization giving pure compound (12a, 12b, 13a
and 13b).
4. Experimental
4.1. Chemistry
Melting points were determined on an Original Kofler Mikro-
heitztisch apparatus (Reichert, Wien). The 1H NMR and the 13C
NMR spectra were recorded with a Brucker Avance DPX-300 or
Brucker AV-600, the deuterated solvents indicated were used.
Chemical shifts are reported in parts per million (ppm) relative
to TMS. IR spectra were recorded with Bruker Vertex 70 FTIR spec-
trophotometer with an ATR sampling accessory and the signal are
given by wave numbers (cmꢀ1). Mass spectra were recorded with
an Agilent 1100 Series LC/MSD Trap SL spectrometer using electro-
spray ionization (ESI). Elemental analyses were performed at the
microanalytical laboratories of the ‘Rugjer Boskovic’ Institute. All
chemicals and solvents were purchased from Aldrich Chemical or
Acros Organics and dried by standard procedure. The 4-amino-3-
sulfanylbenzonitrile 1 and 2-amino-5-nitrobenzothiole 2 were
freshly prepared by alkaline hydrolysis of 6-cyano or 6-nitrobenzo-
thiazole according to the literature.21 The 5-amidinium-2-amin-
obenzothiolate 8a and 5-(imidazolinium-2-yl)-2-aminobenzothio-
late hydrate 8b were prepared according to the literature.23
4.1.1.1. 6-Cyano-2(4-cyanophenyl)benzothiazole (5). According
to (i), 4-amino-3-sulfanylbenzonitrile 1 (3.30 g, 22 mmol), 4-cya-
nobenzoylchloride 3 (3.31 g, 20 mmol), and 100 mL of glacial acetic
acid were used. Crystallization from toluene afforded 3.56 g
(77.7%) of pale yellow solid; mp 284–286 °C (lit.22, mp 288–
289 °C). IR (ATR):
m .
= 2227 (CN) cmꢀ1 1H NMR (300 MHz, DMSO-
d6): d = 7.93 (dd, 1H, J = 1.7 Hz, J = 8.5 Hz, H-Bt), 8.02 (d, 2H,
J = 8.4 Hz, H-Ph), 8.24 (d, 1H, J = 8.5 Hz, H-Bt), 8.28 (d, 2H,
J = 8.4 Hz, H-Ph), 8.76 (d, 1H, J = 1.6 Hz, H-Bt). MS (ESI) m/z:
262.2 (M+H+). Anal. Calcd for C15H7N3S (261.30): C, 68.95; H,
2.70; N, 16.08. Found: C, 69.03; H, 2.71; N, 16.01.
4.1.1.2. 6-Nitro-2-(4-cyanophenyl)benzothiazole (6). According
to (i), 2-amino-5-nitrobenzothiole 2 (3.78 g, 22 mmol), 4-cya-
nobenzoylchloride 3 (3.31 g, 20 mmol) and 100 mL of glacial acetic
acid were used. Crystallization from DMF afforded 4.44 g (79.0%) of
4.1.1. Synthesis; general procedures
(i) Condensation reaction of corresponding 6-substituted-2-amino-
thiophenole with 4-substituted benzoylchloride: To a solution of cor-
responding 6-substituted-2-aminothiophenole (1.1 equiv) in
glacial acetic acid 4-substituted-benzoylchloride (1.0 equiv) was
added in one portion and the reaction mixture was refluxed under
nitrogen for 4 h. After cooling to rt, the resulting precipitate was
collected by filtration, washed with diethyl ether and dried under
vacuum over KOH. The crude product was purified by crystalliza-
tion giving pure compound (5, 6, 7, 11a, and 11b).
yellow solid; mp 219–220 °C. IR (ATR):
m = 2229 (CN), 1523 (NO2)
cmꢀ1 1H NMR (600 MHz, CDCl3): d = 7.84 (d, 2H, J = 8.6 Hz, H-
.
Ph), 8.20 (d, 1H, J = 8.9 Hz, H-Bt), 8.25 (d, 2H, J = 8.5 Hz, H-Ph),
8.41 (dd, 1H, J = 2.2 Hz, J = 8.9 Hz, H-Bt), 8.89 (d, 1H, J = 2.1 Hz, H-
Bt). 13C NMR (75.5 MHz, CDCl3): d = 114.9 (s), 117.4 (s), 117.9 (d),
121.8 (d), 123.6 (d), 127.9 (d, 2C), 132.5 (d, 2C), 135.1 (s), 135.9
(s), 145.0 (s), 157.0 (s), 170.5 (s). MS (ESI) m/z: 282.0 (M+H+). Anal.
Calcd for C14H7N3O2S (281.29): C, 59.78; H, 2.51; N, 14.94. Found:
C, 59.83; H, 2.48; N, 15.03.
(ii) Conversion of corresponding nitrile into imidoyl ether hydro-
chloride: A suspension of corresponding nitrile (1 equiv) in 2-(2-
ethoxyethoxy)ethanol was saturated with dry gaseous HCl at
5 °C. The flask was stoppered, and stirred at rt until IR spectra indi-
cated the disappearance of the nitrile peak. The excess HCl was re-
moved from the suspension with a stream of nitrogen. The reaction
mixture was poured into dry ether (500 mL) and the resulting
4.1.1.3. 6-Cyano-2-(4-nitrophenyl)benzothiazole (7). According
to (i); 4-amino-3-sulfanylbenzonitrile 1 (3.30 g, 22 mmol), 4-nitro-
benzoylchloride 4 (3.71 g, 20 mmol) and 100 mL of glacial acetic
acid were used. Crystallization from xylene afforded 4.74 g
(84.3%) of yellow solid; mp 284–285 °C. IR (ATR):
m = 2229 (CN),
1512 (NO2) cmꢀ1 1H NMR (600 MHz, DMSO-d6): d = 7.92 (d, 1H,
.

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L. Racané et al. / Bioorg. Med. Chem. 18 (2010) 1038–1044
J = 7.7 Hz, H-Bt), 8.22 (d, 1H, J = 7.9 Hz, H-Bt), 8.33 (s, 4H, H-Ph),
8.77 (s, 1H, H-Bt). MS (ESI) m/z: 282.0 (M+H+). Anal. Calcd for
C14H7N3O2S (281.29): C, 59.78; H, 2.51; N, 14.94. Found: C,
59.69; H, 2.55; N, 14.91.
used according to (iv). Crystallization from 2 M HCl afforded
0.893 g (66.6%) of pale yellow solid; mp 292–294 °C (decomp.).
IR (ATR):
m .
= 3053 (NHCNH+), 2946 (NHCNH+), 1512 (NO2) cmꢀ1
1H NMR (300 MHz, DMSO-d6): d = 4.06 (s, 4H, H-CH2), 8.26 (d,
2H, J = 9.0 Hz, H-Ph), 8.34–8.38 (m, 4H, 2H-Ph and 2H-Bt), 9.17
(d, 1H, J = 2.2 Hz, H-Bt), 11.18 (br s, 2H, H-Am). 13C NMR
(75.5 MHz, DMSO-d6): d = 45.1 (t), 119.9 (d), 122.4 (d), 124.1 (d),
125.6 (s), 128.5 (d, 2C), 130.6 (d, 2C), 136.2 (s), 137.6 (s), 145.7
(s), 157.6 (s), 164.8 (s), 172.1 (s). MS (ESI) m/z: 324.7 [(M+H+) calcd
for free base C16H12N4O2S, 324.07]. Anal. Calcd for
C16H13ClN4O2SꢁH2O (378.83): C, 50.73; H, 3.99; N, 14.79. Found:
C, 50.70; H, 4.08; N, 14.81.
4.1.1.4. 2-(4-Amidinophenyl)-6-amidinobenzothiazole dihydr-
ochloride (9a). According to (ii), 6-cyano-2(4-cyanophenyl)ben-
zothiazole 5 (1.0 g, 3.83 mmol) and 50 mL of 2-(2-ethoxyeth-
oxy)ethanol were used, reaction time seven days. Afterwards, the
obtained diimidoyl ether dihydrochloride of 5 and 50 mL of abs
ethanol were used according to (iii). Crystallization from water–
acetone afforded 0.680 g (43.9%) of colorless solid; mp >300 °C
(lit.22 mp >300 °C). IR (ATR):
m
= 3030 (CðNH2ÞNH2þ), 1666
(CðNH2ÞNH2þ) cmꢀ1
.
1H NMR (300 MHz, DMSO-d6): d = 8.02 (dd,
4.1.1.8. 2-(4-Nitrophenyl)-6-amidinobenzothiazole hydrochlo-
ride (11a). According to (i), 5-amidinium-2-aminobenzothiolate
8a (0.50 g, 3 mmol), 4-nitrobenzoylchloride 4 (0.614 g, 3.3 mmol)
and 25 mL of glacial acetic acid were used. Crystallization from
2 M HCl afforded 0.747 g (70.6%) of colorless solid of 11a. According
to (ii), 6-cyano-2-(4-nitrophenyl)benzothiazole 7 (1.0 g, 3.55 mmol)
and 50 mL of 2-(2-ethoxyethoxy)ethanol were used, reaction time
seven days. Afterwards, the obtained imidoyl ether hydrochloride
of 7 and 50 mL of abs ethanol were used according to (iii). Crystalli-
zation from 2 M HCl afforded 0.924 g (73.8%) of colorless solid of
1H, J = 2.2 Hz, J = 8.4 Hz, H-Bt), 8.09 (d, 2H, J = 8.3 Hz, H-Ph), 8.32
(d, 1H, J = 8.7 Hz, H-Bt), 8.37 (d, 2H, J = 8.3 Hz, H-Ph), 8.82 (d, 1H,
J = 1.2 Hz, H-Bt), 9.58 (br s, 8H, H-Am). 13C NMR (75.5 MHz,
DMSO-d6): d = 123.9 (d), 124.2 (d), 125.9 (s), 127.1 (d), 128.3 (d,
2C), 129.9 (d, 2C), 131.3 (s), 135.5 (s), 136.9 (s), 156.8 (s), 165.4
(s), 165.9 (s), 170.3 (s). MS (ESI) m/z: 295.8 [(M+H+) calcd for free
base C15H13N5S, 295.09]. Anal. Calcd for C15H15Cl2N5Sꢁ2H2O
(404.31): C, 44.56; H, 4.74; N, 17.32. Found: C, 44.45; H, 4.82; N,
17.18.
11a; mp >300 °C. IR (AꢀT1R):
m
= 3023 (CðNH2ÞNH2þ), 1666 (CðNH2Þ
4.1.1.5. 2-[4-(Imidazolin-2-yl)phenyl]-6-(imidazolin-2-yl)ben-
zothiazole dihydrochloride (9b). According to (ii), 6-cyano-2(4-
cyanophenyl)benzothiazole 5 (1.0 g, 3.83 mmol) and 50 mL of
2-(2-ethoxyethoxy)ethanol were used, reaction time seven days.
Afterwards, the obtained diimidoyl ether dihydrochloride of 5, eth-
ylenediamine (2.57 mL, 38.3 mmol), and 50 mL of abs ethanol were
used according to (iv). Crystallization from water–acetone afforded
1.125 g (64.8%) of pale yellow solid; mp >300 °C (lit.19 mp >300 °C).
NH2þ), 1500 (NO2) cm 1H NMR (600 MHz, DMSO-d6): d = 7.90
.
(dd, 1H, J = 2.4 Hz, J = 8.6 Hz, H-Bt), 8.27 (d, 1H, J = 8.6 Hz, H-Bt),
8.36 (m, 4H, H-Ph), 8.70 (d, 1H, J = 2.4 Hz, H-Bt), 9.27 (br s, 2H, H-
Am), 9.51 (br s, 2H, H-Am). 13C NMR (150 MHz, DMSO-d6):
d = 124.0 (d), 124.3 (d), 125.1 (d, 2C), 126.2 (s), 127.2 (d), 129.3 (d,
2C), 135.7 (s), 138.1 (s), 149.7 (s), 156.8 (s), 165.9 (s), 169.5 (s). MS
(ESI) m/z: 298.7 [(M+H+) calcd for free base C14H10N4O2S, 298.05].
Anal. Calcd for C14H11ClN4O2SꢁH2O (352.80): C, 47.66; H, 3.71; N,
15.88. Found: C, 47.61; H, 3.78; N, 15.93.
IR (ATR):
m .
= 3081 (NHCNH+), 2962 (NHCNH+) cmꢀ1 1H NMR
(300 MHz, DMSO-d6): d = 4.06 (s, 8H, H-CH2), 8.22 (d, 1H,
J = 8.6 Hz, H-Bt), 8.29 (d, 2H, J = 8.4 Hz, H-Ph), 8.33 (d, 1H,
J = 8.6 Hz, H-Bt), 8.40 (d, 2H, J = 8.4 Hz, H-Ph), 9.02 (s, 1H, H-Bt),
10.98 (br s, 4H, H-Am). 13C NMR (75.5 MHz, DMSO-d6): d = 45.1
(t, 4C), 120.2 (d), 124.3 (d), 124.8 (s), 125.7 (s), 127.5 (d), 128.7
(d, 2C), 130.4 (d, 2C), 135.9 (s), 137.6 (s), 157.2 (s), 164.8 (s),
165.2 (s), 170.7 (s). MS (ESI) m/z: 347.8 [(M+H+) calcd for free base
C19H17N5S, 347.12]. Anal. Calcd for C19H19Cl2N5Sꢁ2H2O (456.39): C,
50.00; H, 5.08; N, 15.35. Found: C, 50.12; H, 4.92; N, 15.50.
4.1.1.9. 2-(4-Nitrophenyl)-6-(imidazolin-2-yl)benzothiazole hy-
drochloride (11b). According to (i), 5-(imidazolinium-2-yl)-2-
aminobenzothiolate hydrate 8b (0.634 g, 3 mmol), 4-nitrobenzoyl-
chloride 4 (0.614 g, 3.3 mmol) and 25 mL of glacial acetic acid were
used. Crystallization from 2 M HCl afforded 0.809 g (71.2%) of pale
yellow solid of 11b.
According to (ii), 6-cyano-2-(4-nitrophenyl)benzothiazole 7
(1.0 g, 3.55 mmol) and 50 mL of 2-(2-ethoxyethoxy)ethanol were
used, reaction time seven days. Afterwards, the obtained imidoyl
ether hydrochloride of 7, ethylenediamine (1.18 mL, 17.7 mmol)
and 50 mL of abs ethanol were used according to (iv). Crystalliza-
tion from 2 M HCl afforded 1.061 g (78.9%) of pale yellow solid of
4.1.1.6. 2-(4-Amidinophenyl)-6-nitrobenzothiazole hydrochlo-
ride (10a). According to (ii), 6-nitro-2-(4-cyanophenyl)benzothi-
azole 6 (1.0 g, 3.55 mmol) and 50 mL of 2-(2-ethoxyethoxy)-
ethanol were used, reaction time five days. Afterwards, the
obtained imidoyl ether hydrochloride of 6 and 50 mL of abs etha-
nol were used according to (iii). Crystallization from 2 M HCl affor-
ded 0.956 g (76.5%) of colorless solid; mp 292–295 °C (decomp.). IR
11b; mp >300 °C; IR (ATR):
1519 (NO2) cmꢀ1 1H NMR (300 MHz, DMSO-d6): d = 4.07 (s, 4H,
m
= 3058 (NHCNH+), 2964 (NHCNH+),
.
H-CH2), 8.17 (dd, 1H, J = 1.7 Hz, J = 8.7 Hz, H-Bt), 8.40 (d, 1H,
J = 8.7 Hz, H-Bt), 8.44 (s, 4H, H-Ph), 8.95 (d, 1H, J = 1.4 Hz, H-Bt),
10.89 (br s, 2H, H-Am). 13C NMR (75.5 MHz, DMSO-d6): 45.0 (t,
2C), 120.3 (s), 124.4 (d), 124.8 (d), 125.0 (d, 2C), 127.7 (d), 129.5
(d, 2C), 136.2 (s), 165.2 (s), 169.3 (s). MS (ESI) m/z: 324.7
[(M+H+) calcd for free base C16H12N4O2S, 324.07]. Anal. Calcd for
C16H13ClN4O2SꢁH2O (378.83): C, 50.73; H, 3.99; N, 14.79. Found:
C, 50.59; H, 3.83; N, 15.08.
(ATR):
m
= 2999 (CðNH2ÞNH2þ), 1663 (CðNH2ÞNH2þ), 1504 (NO2)
cmꢀ1
.
1H NMR (300 MHz, DMSO-d6): d = 8.06 (d, 2H, J = 8.4 Hz,
H-Ph), 8.25 (d, 1H, J = 9.0 Hz, H-Bt), 8.31–8.37 (m, 3H, 2H-Ph and
H-Bt), 9.25 (d, 1H, J = 2.2 Hz, H-Bt), 9.59 (br s, 4H, H-Am). 13C NMR
(75.5 MHz, DMSO-d6): d = 120.3 (d), 122.6 (d), 124.2 (d), 128.3 (d,
2C), 129.9 (d, 2C), 128.3 (s), 136.0 (s), 136.8 (s), 145.2 (s), 157.5 (s),
165.4 (s), 172.4 (s). MS (ESI) m/z: 298.7 [(M+H+) calcd for free base
C14H10N4O2S, 298.05]. Anal. Calcd for C14H11ClN4O2SꢁH2O (352.80):
C, 47.66; H, 3.71; N, 15.88. Found: C, 47.50; H, 3.88; N, 15.91.
4.1.1.10. 2-(4-Amidinophenyl)-6-aminobenzothiazole hydro-
chloride (12a). According to (v), 2-(4-amidinophenyl)-6-nitro-
benzothiazole hydrochloride hydrate 10a (1.0 g, 2.83 mmol),
tin(II) chloride dihydrate (6.4 g, 28 mmol), 12 mL of methanol
and 12 mL of concd HCl were used. Crystallization from water
4.1.1.7. 2-[4-(Imidazolin-2-yl)phenyl]-6-nitrobenzothiazole
hydrochloride (10b). According to (ii), 6-nitro-2-(4-cyano-
phenyl)benzothiazole 6 (1.0 g, 3.55 mmol) and 50 mL of 2-(2-eth-
oxyethoxy)ethanol were used, reaction time five days.
Afterwards, the obtained imidoyl ether hydrochloride of 6, ethy-
lenediamine (1.18 mL, 17.7 mmol) and 50 mL of abs ethanol were
afforded 0.628 g (68.7%) of yellow solid; mp 295–297 °C (decomp.).
þ
IR (ATR):
m
= 3193 (NH2), 3042 (CðNH2ÞNH2þ), 1667 (CðNH2ÞNH2
)
cmꢀ1
.
1H NMR (300 MHz, DMSO-d6): d = 5.68 (s, 2H, H-NH2), 6.86
(dd, 1H, J = 1.9 Hz, J = 8.8 Hz, H-Bt), 7.14 (d, 1H, J = 1.9 Hz, H-Bt),

L. Racané et al. / Bioorg. Med. Chem. 18 (2010) 1038–1044
1043
7.75 (d, 1H, J = 8.8 Hz, H-Bt), 7.97 (d, 2H, J = 8.4 Hz, H-Ph), 8.15 (d,
2H, J = 8.4 Hz, H-Ph), 9.43 (br s, 4H, H-Am). 13C NMR (75.5 MHz,
DMSO-d6): d = 103.9 (d), 116.2 (d), 124.2 (d), 127.0 (d, 2C), 129.3
(s), 129.6 (d, 2C), 137.5 (s), 138.4 (s), 145.6 (s), 148.7 (s), 158.8
(s), 165.5 (s). MS (ESI) m/z: 268.7 [(M+H+) calcd for free base
C14H12N4S, 268.08]. Anal. Calcd for C14H13ClN4SꢁH2O (322.81): C,
52.09; H, 4.68; N, 17.36. Found: C, 52.21; H, 4.69; N, 17.23.
MD, USA), which are derived from four cancer types. MCF-7 (breast
carcinoma), SW 620 (colon carcinoma), HCT 116 (colon carci-
noma), H 460 (lung carcinoma) were cultured as monolayers and
maintained in Dulbecco’s modified Eagle’s medium (DMEM), while
MOLT-4 (acute lymphoblastic leukemia) were cultured in suspen-
sion in RPMI medium, both supplemented with 10% fetal bovine
serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin and 100 lg/
mL streptomycin in a humidified atmosphere with 5% CO2 at
37 °C. The growth inhibition activity was assessed as described
previously, according to the slightly modified procedure of the Na-
tional Cancer Institute, Developmental Therapeutics Program. The
cells were inoculated onto a series of standard 96-well microtiter
plates on day 0, at 1–3 ꢂ 104 cells/mL, depending on the doubling
times of specific cell line. Test agents (dissolved in DMSO,
c = 4 ꢂ 10ꢀ2 M) were then diluted with the cell culture medium
in five serial 10-fold dilutions (10-8 to 10-4 M), added to cells and
incubated for a further 72 h. The cell growth rate was evaluated
by performing the MTT assay after 72 h of incubation, which de-
tects mitochondrial dehydrogenase activity in viable cells. Each
test was performed in quadruplicate in three individual experi-
ments. The results are expressed as IC50, which is the concentration
necessary for 50% of inhibition. The IC50 values for each compound
are calculated from concentration–response curves using linear
regression analysis by fitting the test concentrations that give PG
values above and below the reference value (i.e., 50%). If however,
for a given cell line all of the tested concentrations produce PGs
exceeding the respective reference level of effect (e.g., PG value
of 50), then the highest tested concentration is assigned as the de-
fault measurement value, which is preceded by a ‘>’ sign preceding
the number.
4.1.1.11. 2-[4-(Imidazolin-2-yl)phenyl]-6-aminobenzothiazole
hydrochloride (12b). According to (v), 2-[4-(imidazolin-2-yl)-
phenyl]-6-nitrobenzothiazole hydrochloride hydrate 10b (1.0 g,
2.64 mmol), tin(II) chloride dihydrate (6.0 g, 26 mmol), 12 mL of
methanol and 12 mL of concd HCl were used. Crystallization from
water–acetone afforded 0.505 g (54.8%) of orange solid; mp 292–
295 °C (decomp.). IR (ATR):
m
= 3360 (NH2), 3304 (NH2), 3198
.
(NHCNH+), 2963 (NHCNH+) cmꢀ1
1H NMR (300 MHz, DMSO-d6):
d = 4.03 (s, 4H, H-CH2), 5.80 (br s, 2H, H-NH2), 6.87 (dd, 1H,
J = 1.9 Hz, J = 8.8 Hz, H-Bt), 7.15 (d, 1H, J = 1.9 Hz, H-Bt), 7.76 (d,
1H, J = 8.8 Hz, H-Bt), 8.12 (d, 2H, J = 8.5 Hz, H-Ph), 8.21 (d, 2H,
J = 8.5 Hz, H-Ph), 10.8 (br s, 2H, H-Am). 13C NMR (75.5 MHz,
DMSO-d6): d = 44.9 (t, 2C), 104.1 (d), 116.4 (d), 123.3 (s), 124.4
(d), 127.3 (d, 2C), 130.0 (d, 2C), 137.6 (s), 139.0 (s), 145.7 (s),
148.5 (s), 158.6 (s), 164.7 (s). MS (ESI) m/z: 294.8 [(M+H+) calcd
for free base C16H14N4S, 294.09]. Anal. Calcd for C16H15ClN4SꢁH2O
(348.85): C, 55.09; H, 4.91; N, 16.06. Found: C, 55.18; H, 4.88; N,
16.12.
4.1.1.12. 2-(4-Aminophenyl)-6-amidinobenzothiazole hydro-
chloride (13a). According to (v), 2-(4-nitrophenyl)-6-amidinob-
enzothiazole hydrochloride hydrate 11a (1.0 g, 2.83 mmol), tin(II)
chloride dihydrate (6.4 g, 28 mmol), 12 mL of methanol and
12 mL of concd HCl were used. Crystallization from water afforded
4.2.2. Acute oral toxicity test in vivo
0.689 g (75.4%) of yellow solid; mp 260–262 °C (decomp.). IR
4.2.2.1. Animals. Healthy nulliparous and non-pregnant BALB/C
female mice, 12 weeks of age at the initiation of the experiment,
were used. Mice were obtained from Rudjer Boskovic Institute’s
breeding colony. During experimental period single animal was
kept per cage. Bottom of cage was covered with sawdust (AllspanÒ,
Germany). Standard food for laboratory mice (4RF 21 GLPÒ Muce-
dola srl, Italy) was used. All animals were fastening prior to dosing
by withholding food (not water) for 3–4 h and 1–2 h after dosing.
After that period access to food and water was ad libitum. Animals
were kept in conventional circumstances: light/dark rhythms 12/
12 h, temperature 22 °C, and humidity 55%. All experiments were
performed according to the OECD 425 Guideline for the testing of
chemicals—Acute oral Toxicity: Up-and-down procedure,26 ILAR
Guide for the Care and Use of Laboratory Animals, Council Direc-
tive (86/609/EEC) and Croatian animal protection law (NN 135/
2006).
Altogether 35 animals were used in experiments to determine
LD50 for all four tested substances. Animals are dosed, one at a
time, at 24 h intervals. The first animal receives a dose at the level
of the best estimate of the LD50. Depending on the outcome for the
previous animal, the dose for the next animal is adjusted up or
down. If an animal survives, the dose for the next animal is in-
creased; if it dies, the dose for the next animal is decreased. After
reaching the reversal of the initial outcome, that is, the point where
an increasing (or decreasing) dose pattern is reversed by giving a
smaller (or a higher) dose, four additional animals are dosed fol-
lowing the same UDP.26
þ
(ATR):
m
= 3180 (NH2), 3030 (CðNH2ÞNH2þ), 1662 (CðNH2ÞNH2
)
cmꢀ1
.
1H NMR (300 MHz, DMSO-d6): d = 6.11 (s, 2H, H-NH2), 6.69
(d, 2H, J = 8.7 Hz, H-Ph), 7.81–7.86 (m, 3H, H-Bt + 2H-Ph), 8.07
(d, 1H, J = 8.5 Hz, H-Bt), 8.53 (s, 1H, H-Bt), 9.00 (s, 2H, H-Am),
9.37 (s, 2H, H-Am). 13C NMR (75.5 MHz, DMSO-d6): d = 116.7 (d,
2C), 122.4 (d), 122.6 (s), 123.3 (d), 124.2 (s), 126.7 (d), 129.8 (d,
2C), 134.6 (s), 149.4 (s), 157.6 (s), 165.8 (s), 172.4 (s). MS (ESI)
m/z: 268.7 [(M+H+) calcd for free base C14H12N4S, 268.08]. Anal.
Calcd for C14H13ClN4SꢁH2O (322.81): C, 52.09; H, 4.68; N, 17.36.
Found: C, 52.31; H, 4.72; N, 17.13.
4.1.1.13. 2-(4-Aminophenyl)-6-(imidazolin-2-yl)benzothiazole
hydrochloride (13b). According to (v), 2-(4-nitrophenyl)-6-(imi-
dazolin-2-yl)benzothiazole hydrochloride hydrate 11b (1.0 g,
2.64 mmol), tin(II) chloride dihydrate (6.0 g, 26 mmol), 12 mL of
methanol and 12 mL of concd HCl were used. Crystallization from
water afforded 0.758 g (82.3%) of yellow solid; mp 278–282 °C (de-
comp.). IR (ATR):
m
= 3374 (NH2), 3310 (NH2), 3203 (NHCNH+),
1H NMR (300 MHz, DMSO-d6): d = 4.03 (s,
2980 (NHCNH+) cmꢀ1
.
4H, H-CH2), 6.17 (br s, 2H, H-NH2), 6.70 (d, 2H, J = 8.7 Hz, H-Ph),
7.83 (d, 2H, J = 8.7 Hz, H-Ph), 8.07 (m, 2H, H-Bt), 8.75 (s, 1H, H-
Bt), 10.77 (br s, 2H, H-Am); 13C NMR (75.5 MHz, DMSO-d6):
d = 44.6 (t, 2C), 113.9 (d, 2C), 117.5 (s), 119.4 (s), 122.1 (d), 123.2
(d), 126.7 (d), 129.7 (d, 2C), 134.5 (s), 153.4 (s), 157.8 (s), 164.8
(s), 173.2 (s). MS (ESI) m/z: 294.8 [(M+H+) calcd for free base
C16H14N4S, 294.09]. Anal. Calcd for C16H15ClN4SꢁH2O (348.85): C,
55.09; H, 4.91; N, 16.06. Found: C, 55.01; H, 4.95; N, 16.11.
4.2.2.2. Drugs. All test substances were stored at +4 °C. All test
substances were dissolved and diluted in physiological saline
immediately prior to injection by using ultrasonic water bath Bran-
sonicÒ 2510 for 15 min at the temperature of 69 °C. Following the
period of fasting the animals were weighed and the test substances
were administered. Doses of substances required by OECD 425
4.2. Biological evaluation
4.2.1. Antiproliferative activity
The experiments were carried out on five human cell lines (ob-
tained from American Type Culture Collection (ATCC, Rockville,

1044
L. Racané et al. / Bioorg. Med. Chem. 18 (2010) 1038–1044
Guideline26 were adjusted to be contained in 0.5 mL/25 g of mouse
body mass. The test substances were administered in a single dose
by gavage using a suitable intubation canula.
J. C.; Cryan, J. E.; Holland, V.; Winborn, K. Y.; Coleman, T.; Stevens, A. J.; Davis, J.
B.; Gunthorpe, M. J. Bioorg. Med. Chem. Lett. 2008, 18, 5609.
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DeFex, H. F.; Fang, Q.; Pang, S.; Pitt, S.; Ren Shen, D.; Schieven, G. L.; Barrish, J. C.
Bioorg. Med. Chem. Lett. 2003, 13, 2145.
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Med. Chem. Lett. 2003, 13, 2587.
4.2.2.3. Statistical analysis. Statistical analyses were conducted
by the ‘Acute Oral Toxicity (Guideline 425) Statistical Program’
(AOT425StatPgm) which is developed by Westat for the US EPA
and designed to be used with the acute oral toxicity testing proce-
dure presented in OECD Guideline for the testing of chemicals, Sec-
tion 4: Health Effects Test No. 425, Acute Oral Toxicity: Up-and-
Down Procedure.26
12. Gaillard, P.; Jeanclaude-Etter, I.; Ardissone, V.; Arkinstall, S.; Cambet, Y.;
Camps, M.; Chabert, C.; Church, D.; Cirillo, R.; Gretener, D.; Halazy, S.; Nichols,
A.; Szyndralewiez, C.; Vitte, P.-A.; Gotteland, J.-P. J. Med. Chem. 2005, 48, 4596.
13. Liu, C.; Lin, J.; Pitt, S.; Zhang, R. F.; Sack, J. S.; Kiefer, S. E.; Kish, K.; Doweyko, A.
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C.; Schieven, G. L.; Leftheris, K. Bioorg. Med. Chem. Lett. 2008, 18, 1874.
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Support for this study by the Ministry of Science, Education and
Sport of Croatia is gratefully acknowledged (Projects 0125-
0982464-1356, 098-0982464-2390, 098-0982464-2514, 117-
0000000-3283). The authors are very thankful to Fran Supek for
useful discussions and practical help.
19. Racanè, L.; Tralic´-Kulenovic´, V.; Kitson, R. P.; Karminski-Zamola, G. Monatsh.
Chem. 2006, 137, 1571.
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