Synthesis and antitumor evaluation of novel derivatives of 6-amino-2-phenylbenzothiazoles

Article abstract of DOI:10.3390/11050325

Novel derivatives of 6-amino-2-phenylbenzothiazole bearing different substituents (amino, dimethylamino or fluoro) on the phenyl ring were prepared as the corresponding hydrochloride salts. 6-Nitro-2-(substituted-phenyl) benzothiazoles (1-6) were synthesi

Full text of DOI:10.3390/11050325

Molecules 2006, 11, 325-333
molecules
ISSN 1420-3049
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Full Paper
Synthesis and Antitumor Evaluation of Novel Derivatives of 6-
Amino-2-phenylbenzothiazoles
Livio Racane 1, Ranko Stojkovic 2, Vesna Tralic-Kulenovic 1 and Grace Karminski-Zamola 3,*
1 Department of Applied Chemistry, Faculty of Textile Technology, University of Zagreb, Prilaz
baruna Filipovića 30, 10000 Zagreb, Croatia; e-mails: lracane@ttf.hr; vtralic@ttf.hr
2 Division of Molecular Medicine, Ruder Boskovic Institute, Bijenicka cesta 54, 10000 Zagreb,
Croatia; e-mail: stojkov@irb.hr
3 Department of Organic Chemistry, Faculty of Chem. Eng. and Technology, University of Zagreb,
Marulićev trg 19, 10000 Zagreb, Croatia
* Author to whom correspondence should be addressed; e-mail: gzamola@fkit.hr
Received: 4 April 2006; in revised form 20 April 2006 / Accepted: 24 April 2006 /
Published: 9 May 2006
Abstract: Novel derivatives of 6-amino-2-phenylbenzothiazole bearing different
substituents (amino, dimethylamino or fluoro) on the phenyl ring were prepared as the
corresponding hydrochloride salts. 6-Nitro-2-(substituted-phenyl)benzothiazoles (1-6) were
synthesized by condensation reactions of substituted benzaldehydes with 2-amino-5-
nitrothiophenol. Nitro derivatives were reduced to the amino derivatives with SnCl2/HCl.
Water soluble hydrochloride salts of 6-amino-2-(substituted-phenyl)benzothiazole (13-19)
were prepared using concentrated or gaseous HCl. Compounds 13-19 were found to exert
cytostatic activities against malignant human cell lines: cervical (HeLa), breast (MCF-7),
colon (CaCo-2), laryngeal carcinoma (Hep-2), and normal human fibroblast cell lines (WI-
38).
Keywords: Benzothiazole synthesis, antitumor activity.
Introduction
The study of benzothiazole derivates is of considerable current interest as a result of their
important biological and biophysical properties. 2-Aryl or 2-heteroaryl substituted benzothiazoles are
studied as antitumor [1], antimicrobial [2], antifungal [3] agents, as well as imaging agents for β-

Molecules 2006, 11
326
amyloid [4]. A series of potent and selective antitumor agents derived from 2-(4-
aminophenyl)benzothiazole was extensively examined and developed during recent years and the
fluorinated analogue, 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole is a novel agent with potent
and selective antitumor properties which in the form of its
phase clinical studies [5].
L-lysylamide prodrug, Phortress, is in early
In connection with our studies on the synthesis and biological properties of substituted
benzothiazoles [6-8] we turned our attention to the synthesis and antitumor evaluation of novel 2-
(substituted-phenyl)-6-aminobenzothiazole as analogous of 2-(4-aminophenyl)benzothiazole prepared
as hydrochloride or dihydrochloride salts.
Results and Discussion
Synthesis
An efficient synthesis of 2-phenylbenzothiazole derivatives was carried out by the reactions
outlined in Scheme 1.
Scheme 1.
1; R=2-NO2
H2N
N
S
O
H
R
R
2; R=3-NO2
3; R=4-NO2
4; R=4-NMe2
5; R=3-F
+
NO2
HS
NO2
6; R=4-F
SnCl2/HCl
7; R=2-NH2
8; R=3-NH2
9; R=4-NH2
10; R=4-NMe2
11; R=3-F
N
S
R
NH2
12; R=4-F
HCl (aq)
or HCl (gas)
13=7 x 2HCl
14=8 x 2HCl
15=9 x 2 HCl
16=10 x 2HCl
17=11 x HCl
18=11 x 2HCl
19=12 x HCl
N
S
R
x n HCl
NH2
Starting from 2-amino-5-nitrothiophenol (freshly prepared by basic hydrolysis of 6-
nitrobenzothiazole) and the corresponding aromatic aldehyde 6-nitro-2-(monosubstituted phenyl)-
benzothiazoles 1-6 were obtained in moderate yield (40-70%) by condensation in pyridine (procedure
A). The reduction of nitro compounds was accomplished with SnCl2 in methanol-HCl mixtures in a
short time at reflux giving the desired 6-amino-2-phenylbenzothiazole derivatives 7-12 in good yields
of 70-80% (procedure B). Treating the solutions of amines 10-11 or diamines 7-9, dissolved in a
mixture of abs. ethanol and xylene (1:1, v/v) with concd. hydrochloric acid (procedure C) the
corresponding amines were protonated and isolated as pure hydrochlorides 13-17, 19 in excellent

Molecules 2006, 11
327
yields (91-99%) without recrystallization. The same products can be obtained in similar yields by
treating the amines with gaseous HCl (procedure D). Only for 6-amino-2-(3-fluorophenyl)-
benzothiazole (11) was selective preparation of the monohydrochloride salt 17 (procedure C) and the
dihydrochloride salt 18 (procedure D), in which protonation occurs on both the amino and
benzothiazole nitrogens possible. The structures of new compounds 1-19 were confirmed by elemental
analysis and spectral data (IR, NMR). Due to a very low solubility of nitro compounds 2 and 3, only
elemental analysis, and IR spectra were recorded.
Biological Activity
The effects of the seven water soluble compounds 13-19 were tested on the human malignant cell
lines: cervical (HeLa), breast (MCF-7), colon (CaCo-2), laryngeal carcinoma (Hep-2) and normal
human fibroblast cell lines (WI-38). Inhibitory effects are shown in Table 1.
Table 1.
IC50 (M)
HEP-2
Comp.
No.
13
HeLa
6×10-4
3×10-3
9×10-5
4×10-3
8×10-5
9×10-5
5×10-5
CaCo-2
4×10-3
8×10-4
1×10-4
8×10-4
9×10-5
1×10-4
3×10-4
MCF7
6×10-4
7×10-5
6×10-5
2×10-4
4×10-5
3×10-5
4×10-5
WI-38
4×10-3
4×10-5
7×10-3
7×10-5
4×10-4
7×10-5
8×10-5
9×10-5
4×10-5
2×10-5
5×10-5
2×10-5
3×10-5
9×10-6
14
15
16
17
18
19
All benzothiazoles tested exhibited moderate antitumour activity with IC50 values ranging from
9×10-6 to 4×10-3 M. Most sensitive cell lines were HEP-2 and MCF-7. Our results showed up that
activity of the compounds investigated decreasing from ortho- meta- to para- position (p < 0.05) the
fluoro substituted benzothiazoles 17, 18 and 19 being more active.
Experimental
General
Melting points were determined on a Kofler hot stage microscope and are uncorrected. IR spectra
1
were recorded on a Nicolet Magna 760 spectrophotometer in KBr discs. H- and 13C-NMR spectra
were recorded on either a Varian Gemini 300 or a Brucker Avance DPX 300 spectrometer. Chemical
shifts (δ) are reported in parts per million (ppm) relative to tetramethylsilane (TMS) as an internal
standard, and coupling constants (J) are given in hertz (Hz). Elemental analyses were carried out in the
Microanalitical laboratory at the Rugjer Boskovic Institute. All compounds were routinely checked by

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TLC with Merck silica gel 60F-254 glass plates. 2-Amino-5-nitrothiophenole was prepared according
to the procedure described for preparation of 2-amino-5-cyanothiophenole [9] from 6-nitro-
benzothiazole.
Synthesis of 6-nitro-2-(substituted-phenyl)benzothiazoles 1-6
Procedure A: To a boiling solution of the appropriate substituted benzaldehyde (0.02 mol) in
pyridine (20 mL), a solution of 2-amino-5-nitrothiophenole (0.02 mol) in pyridine (20 mL) was added
dropwise, and the stirred reaction mixture was refluxed for 20 h. The mixture was then poured into 300
ml of 2 M hydrochloric acid and after cooling overnight the obtained crystalline product was filtered
off and crystallized from appropriate solvent. Using this procedure the following compounds were
prepared.
6-Nitro-2-(2-nitrophenyl)benzothiazole (1). Yield: 40.0% after crystallisation from xylene; mp 164-
165°C; IR cm-1: 1569, 1517, 1348, 842, 756, 749, 721; 1H-NMR (DMSO-d6) δ: 9.33 (d, 1H, J = 2.0
Hz), 8.40 (dd, 1H, J = 2.0 Hz, J = 8.9 Hz), 8.26 (d, 1H, J = 8.9 Hz), 8.16 (d, 1H, J = 8.3 Hz), 8.06 (d,
1H, J = 8.1 Hz), 7.96-7.87 (m, 2H); Anal. Calcd. for C13H7N3O4S: C, 51.83; H, 2.34; N, 13.95; Found:
C, 51.68; H, 2.40; N, 14.03.
6-Nitro-2-(3-nitrophenyl)benzothiazole (2). Yield: 70.9% (from xylene); mp 214-215 °C; IR cm-1:
1601, 1538, 1514, 1340, 829, 752, 739, 720; Anal. Calcd. for C13H7N3O4S: C, 51.83; H, 2.34; N, 13.95;
Found: C, 51.99; H, 2.11; N, 13.90.
6-Nitro-2-(4-nitrophenyl)benzothiazole (3) Yield: 67.1% (from xylene); mp 256-257 °C; IR cm-1: 1606,
1517, 1346, 854, 754, 719; Anal. Calcd. for C13H7N3O4S: C, 51.83; H, 2.34; N, 13.95; Found: C, 51.92;
H, 2.29; N, 13.88.
6-Nitro-2-(4-N,N-dimethylaminophenyl)benzothiazole (4). Yield: 66.1% (from xylene); mp 262-263
1
°C; IR cm-1: 1610, 1594, 1474, 1434, 1330, 810, 752; H-NMR (DMSO-d6) δ: 9.09 (s, 1H), 8.30 (d,
1H, J = 9.0 Hz), 8.05 (d, 1H, J = 9.0 Hz), 7.96 (d, 2H, J = 8.8 Hz), 6.85 (d, 2H, J = 8.8 Hz), 3.05 (s,
12H); Anal. Calcd for C15H13N3O2S: C, 60.18; H, 4.38; N, 14.04; Found: C, 60.41; H, 4.44; N, 14.30.
6-Nitro-2-(3-fluorophenyl)benzothiazole (5). Yield: 45.7% (from toluene); mp 199-200 °C; IR cm-1:
1588, 1515, 1448, 1343, 809, 785, 753; 1H-NMR (DMSO-d6) δ: 9.24 (d, 1H, J = 2.1 Hz), 8.35 (dd, 1H,
J = 8.9 Hz, J = 2.3 Hz), 8.24 (d, 1H, J = 8.9 Hz), 7.99-7.93 (m, 2H), 7.65 (m, 1H), 7.49 (m, 1H); Anal.
Calcd. for C13H7FN2O2S: C, 56.93; H, 2.57; N, 10.21; Found: C, 57.20; H, 2.63; N, 10.00.
6-Nitro-2-(4-fluorophenyl)benzothiazole (6). Yield: 58.2% (from xylene); mp 183-185 °C; IR cm-1:
1592, 1510, 1474, 1334, 839, 751; 1H-NMR (DMSO-d6) δ: 9.23 (d, 1H, J = 2.4 Hz), 8.36 (dd, 1H, J =
8.9 Hz, J = 2.4 Hz), 8.25-8.20 (m, 3H), 7.46 (dd, 2H, J = 8.8 Hz, J = 10.8 Hz); 13C-NMR (DMSO-d6)
δ: 172.6, 164.6 (d, J = 251 Hz), 157.4, 144.6, 135.4, 130.4 (d, J = 9.3 Hz, 2C), 129.0, 123.3, 122.1,

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119.7, 116.8 (d, J = 22.4 Hz, 2C); Anal. Calcd for C13H7FN2O2S: C, 56.93; H, 2.57; N, 10.21; Found:
C, 56.74; H, 2.65; N, 10.02.
Synthesis of 6-amino-2-(substituted-phenyl) benzothiazoles 7-12
Procedure B: A solution of tin(II) chloride dihydrate (9 g, 0.04 mol), concd. HCl (18 mL) and
methanol (18 mL) was added to the appropriate 6-nitro-2-(nitrophenyl)benzothiazole 1-3 (0.005 mol),
or 0.01 mol of the compounds 4-6. The mixture was stirred and refluxed for 15 min. Methanol was
removed by vacuum evaporation and the residue was dissolved in water. After basification to pH > 9
with 20% NaOH the product was extracted with ether and dried (MgSO4). The solvent was removed
under reduced pressure and the white solid obtained was crystallised from the appropriate solvent. The
following compounds were prepared by thus method:
6-Amino-2-(2-aminophenyl)benzothiazole (7). Yield: 74.5% (after crystallisation from toluene); mp
185-187 °C; IR cm-1: 3438, 3351, 3299, 1611, 1487, 817, 754; 1H-NMR (DMSO-d6) δ: 7.62 (d, 1H, J
= 8.6 Hz), 7.47 (d, 1H, J = 7.6 Hz), 7.13 (s, 2H), 7.10 (d, 1H, J = 8.0 Hz), 7.04 (s, 1H), 6.79 (d, 1H, J
= 8.3 Hz), 6.72 (m, 1H), 6.58 (m, 1H, J = 7.5 Hz), 5.42 (s, 2H); Anal. Calcd for C13H11N3S: C, 64.70;
H, 4.59; N, 17.41; Found: C, 64.83; H, 4.63; N, 17.29.
6-Amino-2-(3-aminophenyl)benzothiazole (8). Yield: 68.2% (from xylene); mp 202-205 °C; IR cm-1:
3406, 3300, 3200, 1621, 1602, 1485, 815, 788; 1H-NMR (DMSO-d6) δ: 7.62 (d, 1H, J = 8.6 Hz), 7.19
(s, 1H), 7.13-7.02 (m, 3H), 6.74 (d, 1H, J = 8.6 Hz), 6.62 (d, 1H, J = 7.6 Hz), 5.44 (s, 2H), 5.35 (s,
2H); 13C-NMR (DMSO-d6) δ: 161.4, 149.2, 147.2, 145.1, 136.0, 134.0, 129.5, 122.9, 115.7, 114.9,
114.0, 111.3, 103.7; Anal. Calcd for C13H11N3S: C, 64.70; H, 4.59; N, 17.41; Found: C, 64.75; H, 4.42;
N, 17.38.
6-Amino-2-(4-aminophenyl)benzothiazole (9). Yield: 68.8% (toluene); mp 263-265 °C; IR cm-1: 3456,
1
3365, 3304, 3193, 1621, 1606, 1488, 822; H-NMR (DMSO-d6) δ: 7.74 (d, 2H, J = 8.3 Hz), 7.67 (d,
1H, J = 8.6 Hz), 7.14 (s, 1H), 6.83 (d, 2H, J = 8.3 Hz), 5.84 (s, 2H), 5.43 (s, 2H); 13C-NMR (DMSO-
d6) δ: 161.6, 151.0, 146.4, 145.4, 135.3, 127.8 (2C), 122.0, 120.9, 114.4, 113.5 (2C), 104.0; Anal.
Calcd for C13H11N3S: C, 64.70; H, 4.59; N, 17.41; Found: C, 64.58; H, 4.50; N, 17.62.
6-Amino-2-(4-N,N-dimethylaminophenyl)benzothiazole (10). Yield: 68.7% (from xylene); mp 230-
233°C; IR cm-1: 3459, 3306, 3188, 1610, 1489, 813; 1H-NMR (DMSO-d6) δ: 7.78 (d, 2H, J = 8.9 Hz),
7.60 (d, 1H, J = 8.6 Hz), 7.07 (d, 1H, J = 2.1 Hz), 6.81 (d, 2H, J = 8.9 Hz), 6.75 (dd, 1H, J = 2.1 Hz, J
= 8.6 Hz), 5.36 (s, 2H), 3.02 (s, 6H); 13C-NMR (DMSO-d6) δ: 161.4, 151.4, 146.5, 145.4, 135.4, 127.5
(2C), 122.2, 120.9, 114.5, 111.8 (2C), 104.0, 39.7 (2C); Anal. Calcd for C15H15N3S: C, 66.88; H, 5.61;
N, 15.60; Found: C, 66.95; H, 5.72; N, 15.52.
6-Amino-2-(3-fluorophenyl)benzothiazole (11). Yield: 74.6% (toluene-petroleum ether, 1:1, v/v); mp
127-128 °C; IR cm-1: 3408, 3312, 3218, 1602, 1442, 852, 827, 793; 1H-NMR (DMSO-d6) δ: 7.76-7.68
(m, 3H), 7.54 (m, 1H), 7.31 (m, 1H), 7.08 (s, 1H), 6.79 (d, 1H, J = 8.6 Hz), 5.57 (s, 2H); 13C-NMR

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(DMSO-d6) δ: 162.4 (d, J = 244 Hz), 158.7, 147.8, 144.9, 136.5, 135.6 (d, J = 8.2 Hz), 131.3 (d, J =
8.4 Hz), 123.4, 122.6 (d, J = 2.6 Hz), 116.8 (d, J = 21.0 Hz), 115.3, 112.5 (d, J = 23.0 Hz), 103.5.
Anal. Calcd for C13H9FN2S: C, 63.92; H, 3.71; N, 11.47; Found: C, 63.73; H, 3.80; N, 11.52.
6-Amino-2-(4-fluorophenyl)benzothiazole (12). Yield: 79.3% (from toluene-petroleum ether, 1:1, v/v);
1
mp 156-157 °C; IR cm-1: 3407, 3314, 3218, 1606, 1487, 839, 827, 801; H-NMR (DMSO-d6) δ: 7.97
(dd, 2H, J = 8.6 Hz, J = 5.3 Hz), 7.66 (d, 1H, J = 8.6 Hz), 7.33 (dd, 2H, J = 8.6 Hz, J = 10.3 Hz), 7.07
(s, 1H), 6.77 (d, 1H, J = 8.6 Hz), 5.50 (s, 2H); 13C-NMR (DMSO-d6) δ: 163.2 (d, J = 247 Hz), 159.3,
147.6 145.2, 136.5, 130.2, 128.7 (d, J = 7.7 Hz), 123.3, 116.3 (d, J = 21.8 Hz), 115.3, 103.8; Anal.
Calcd for C13H9FN2S: C, 63.92; H, 3.71; N, 11.47; Found: C, 63.78; H, 3.85; N, 11.55.
Synthesis of amine hydrochloride salts 13-19.
Procedure C: The amines 7-12 (0.005 mol) were dissolved in a warm mixture of ethanol and
xylene (1:1, v/v) followed by the addition of concd. HCl (1.0 mL). The contents were stirred in a
stoppered flask for 4 h. The precipitated pure crystals were collected by filtration, washed with dry
ether, and dried in vacuo. The following compounds were prepared by this method:
6-Amino-2-(2-aminophenyl)benzothiazole dihydrochloride (13). Yield: 96.6%; mp 224-227 °C; IR
cm-1: 2816, 2662, 2524, 1486, 1463, 980, 767; 1H-NMR (DMSO-d6) δ: 8.11 (s, 1H), 8.07 (d, 1H, J =
8.6 Hz), 7.64 (d, 1H, J = 8.0 Hz), 7.49 (d, 1H, J = 8.6 Hz), 7.24 (dd, 1H, J = 8.0 Hz, J = 7.3 Hz), 6.29
13
(d, 1H, J = 8.3 Hz), 6.69 (dd, 1H, J = 7.7 Hz, J = 7.3), 4.78 (br s); C-NMR (DMSO-d6) δ: 169.6,
152.7, 144.2, 133.7, 132.3, 130.4, 129.2 (2C), 123.2, 122.3, 118.8, 117.1, 115.7; Anal. Calcd for
C13H13Cl2N3S: C, 49.69; H, 4.18; N, 13.37; Cl, 22.56; Found: C, 49.55; H, 4.15; N, 13.37; Cl, 22.38.
6-Amino-2-(3-aminophenyl)benzothiazole dihydrochloride (14). Yield: 97.4%; mp 253-255 °C; IR
cm-1: 2896, 2605, 1595, 1521, 1456, 818, 786; 1H-NMR (DMSO-d6) δ: 8.05 (d, 1H, J = 8.6 Hz), 7.97
(s, 1H), 7.88 (s, 1H), 7.80 (d, 1H, J = 7.7 Hz), 7.53 (dd, 1H, J = 8.0 Hz, J = 7.7 Hz), 7.40 (d, 1H, J =
8.8 Hz), 7.32 (d, 1H, J = 7.7 Hz); 13C-NMR (DMSO-d6) δ: 167.2, 152.4, 135.5, 134.3, 133.8, 131.0,
130.5, 126.1, 125.8, 124.0, 122.2, 121.1, 116.9; Anal. Calcd for C13H13Cl2N3S: C, 49.69; H, 4.18; N,
13.37; Cl, 22.56; Found: C, 49.70; H, 4.10; N, 13.35; Cl, 22.63.
6-Amino-2-(4-aminophenyl)benzothiazole dihydrochloride (15). Yield: 99.3%; mp 247-248 °C; IR
cm-1: 2852, 2554, 1605, 1507, 1485, 974, 822; 1H-NMR (DMSO-d6) δ: 7.95-7.92 (m, 2H), 7.79 (d, 2H,
J = 8.7 Hz), 7.36 (d, 1H, J = 8.3 Hz), 6.76 (d, 2H, J = 8.7 Hz), 4.41 (br s); 13C-NMR (DMSO-d6) δ:
168.5, 152.7, 143.5, 135.1 (2C), 129.4 (2C), 128.9, 126.1, 123.2, 122.0, 119.4, 116.9; Anal. Calcd for
C13H13Cl2N3S: C, 49.69; H, 4.18; N, 13.37; Cl, 22.56; Found: C, 49.68; H, 4.06; N, 13.37; Cl, 22.64.
6-Amino-2-(4-N,N-dimethylaminophenyl)benzothiazole dihydrochloride (16). Yield: 99.5%; mp 240-
242 °C; IR cm-1: 2796, 2568, 2453, 1607, 1519, 841; 1H-NMR (DMSO-d6) δ: 8.06 (s, 1H), 7.99 (d, 1H,
J = 8.6 Hz), 7.89 (d, 2H, J = 8.9 Hz), 7.44 (d, 1H, J = 8.6 Hz), 6.85 (d, 2H, J = 8.9 Hz), 3.01 (s, 6H);
13C-NMR (DMSO-d6) δ: 169.8, 152.0, 150.2, 134.5, 128.9 (2C), 127.6, 122.6, 122.4, 121.6, 116.5,

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114.7 (2C), 41.2 (2C); Anal. Calcd for C15H17Cl2N3S: C, 52.63; H, 5.01; N, 12.27; Cl, 20.71; Found: C,
52.60; H, 4.97; N, 12.23; Cl, 20.80.
6-Amino-2-(3-fluorophenyl)benzothiazole monohydrochloride (17). Yield: 96.1%; mp 206-207 °C; IR
cm-1: 2806, 2589, 1585, 1514, 1265, 848, 787; 1H-NMR (DMSO-d6) δ: 8.06 (d, 1H, J = 8.6 Hz), 7.99
(s, 1H), 7.89-7.83 (m, 2H), 7.60 (dd, 1H, J = 7.3 Hz, J = 7.0 Hz), 7.44-7.38 (m, 2H), 3.95 (br s, 3H);
13C-NMR (DMSO-d6) δ: 166.1, 162.4 (d, J = 245 Hz), 151.5, 135.5, 134.7 (d, J = 8.2 Hz), 132.0,
131.6 (d, J = 8.3 Hz), 123.8, 123.4 (d, J = 2.6 Hz), 121.4, 118.2 (d, J = 21.0 Hz), 115.4, 113.4 (d, J =
22.4 Hz); Anal. Calcd for C13H10ClFN2S: C, 55.62; H, 3.60; N, 9.98; Cl, 12.63; Found: C, 55.43; H,
3.56; N, 9.79; Cl, 12.81.
6-Amino-2-(4-fluorophenyl)benzothiazole monohydrochloride (19). Yield: 91.3%; mp 245-247 °C; IR
cm-1: 3058, 2854, 2586, 1602, 1488, 1237, 839, 816; 1H-NMR (DMSO-d6) δ: 8.13 (dd, 2H, J = 5.6 Hz,
J = 8.3 Hz), 8.06 (d, 1H, J = 8.6 Hz), 7.97 (s, 1H), 7.45-7.37 (m, 3H), 3.63 (br s); 13C-NMR (DMSO-
d6) δ: 166.7, 163.9 (d, J = 245 Hz), 151.8, 135.5, 131.0, 129.5 (d, J = 8.9 Hz, 2C), 129.1, 123.6, 121.1,
116.5 (d, J = 22.2 Hz, 2C), 115.3; Anal. Calcd for C13H10ClFN2S: C, 55.62; H, 3.60; N, 9.98; Cl, 12.63;
Found: C, 55.45; H, 3.56; N, 9.87; Cl, 12.78.
Procedure D: The amines 7-12 (0.005 mol) were dissolved in a warm mixture of ethanol and
xylene (1:1, v/v). A stream of dry hydrogen chloride was passed through the solution for 1 h while
cooling to room temperature. The contents were then stirred in a stoppered flask for 2 h. The
precipitated pure crystals were collected by filtration, washed with dry ether, and dried in vacuo. The
following compound was prepared by this method:
6-Amino-2-(3-fluorophenyl)benzothiazole dihydrochloride (18). Yield: 97.2%; mp 209-210 °C; IR
cm-1: 3045, 2798, 2590, 2541, 1588, 1486, 861, 795; 1H-NMR (DMSO-d6) δ: 8.17-8.14 (m, 2H), 7.94-
7.88 (m, 2H), 7.64 (dd, 1H, J = 7.6 Hz, J = 6.3 Hz), 7.53 (d, 1H, J = 8.6 Hz), 7.46 (m, 1H), 4.45 (br s);
13C-NMR (DMSO-d6) δ: 167.2, 162.4 (d, J = 244 Hz), 152.4, 135.3, 134.6 (d, J = 8.2 Hz), 131.6 (d, J
= 8.4 Hz), 129.7, 123.8, 123.6, 122.3, 118.4 (d, J = 20.9 Hz), 117.2, 113.5 (d, J = 21.4 Hz); Anal.
Calcd for C13H11Cl2FN2S: C, 49.22; H, 3.50; N, 8.83; Cl, 22.35; Found: C, 49.45; H, 3.50; N, 8.98; Cl,
22.30.
Cell culture
Five different human cell lines were used for cytotoxicity screening: colon adenocarcinoma (CaCo-
2), cervical adenocarcinoma (HeLa), laryngeal carcinoma (Hep-2), mammary adenocarcinoma
(MCF7), lung fibroblast (WI-38). Cells were seeded in tissue culture flasks (Sarstedt, Numbrecht,
Germany) in Dulbecco's Modified Eagle medium (DMEM) (Gibco, Paisley, Scotland) supplemented
with 10% foetal bovine serum (FBS; Gibco), 2 mM glutamine, 100 U/mL penicillin, and 100 µg/mL
streptomycin in a humidified atmosphere with 5% CO2 at 37 °C. The cultures were passaged at
preconfluent densities with the use of 0.025% trypsin (Institute of Immunology, Zagreb, Croatia).

Molecules 2006, 11
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Cytotoxicity assays
For the experiments the cells were seeded onto 96-well microtiter plates at a density of 2 x 103
cells per well and allowed to adhere for 24 h before the benzothiazoles were added over a
concentration range of 1-100 µM. Stock solutions of benzothiazoles were dissolved in DMSO (Sigma,
Deisenhofen, Germany) at the concentration of 0.1 M and were diluted with medium without bovine
calf serum. The DMSO concentration was adjusted to be the same in test and control plates (1%). All
drug solutions were freshly prepared on the day of testing. After incubation with benzothiazoles for 72
h, cell growth was established according to the results of MTT assay [10] - Sigma, Deisenhofen,
Germany. Each experiment was made in quadruplicate and repeated three times. The absorbency was
measured on a microplate reader at 570 nm. (Labsystems, Finland) The percentage of growth (PG) of
the cell lines was calculated according to the following expression:
[(Ti - Tz) / (C - Tz)] x 100 for concentrations for which Ti > / = Tz %
where mean Tz = the average of optical density measurements before exposure of cells to the test
compound, mean Ti = the average of optical density measurements after the desired period of time,
and mean C = the average of optical density measurements after the desired period of time with no
exposure of cells to the test compound. The results are expressed as IC50, which is the concentration
necessary for 50% inhibition. The IC50 values for each compound are calculated from dose response
curves using linear regression analysis by fitting the test concentrations that give PG values above and
below the reference value (i.e. 50%). Each result is a mean of IC50 ± SD value from three separate
experiments.
Acknowledgements
Support of this study by the Ministry of Science and Technology, Republic of Croatia (Projects No
0125005 and 0098145) is gratefully acknowledged.
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Sample availability: Available from the author.
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