Synthesis and antiproliferative activity of cyano and amidino substituted 2-phenylbenzothiazoles

Article abstract of DOI:10.1007/s00706-006-0546-5

Series of cyano, dicyano, amidino, and diamidino substituted 2-phenylbenzothiazoles were prepared. Mono- and dicyano substituted benzothiazoles were obtained by condensation of appropriate substituted benzaldehydes with 2-aminothiophenol or 4-amino-3-mercaptobenzonitrile. The appropriate amidines or diamidines were prepared by Pinner reaction. The compounds were tested against breast, prostate, and lung cancer cell lines in a 72∈h cytotoxicity assay. Many of the compounds had at 10∈μM activity equivalent to 2-(4-aminophenyl)benzothiazole, while four compounds had significantly better activity, particularly in the breast cancer model. Springer-Verlag 2006.

Full text of DOI:10.1007/s00706-006-0546-5

Monatshefte f€ur Chemie 137, 1571–1577 (2006)
DOI 10.1007/s00706-006-0546-5
Synthesis and Antiproliferative Activity
of Cyano and Amidino Substituted
2-Phenylbenzothiazoles
1
1;ꢀ
Livio Racane , Vesna Tralic-Kulenovic , Richard P. Kitson2,
`
´
´
and Grace Karminski-Zamola3
1
Department of Applied Chemistry, Faculty of Textile Technology, University of Zagreb,
Zagreb, Croatia
2
Department of Molecular Biology and Immunology, University of North Texas Health
Science Center, Fort Worth, TX, USA
3
Department of Organic Chemistry, Faculty of Chemical Engineering and Technology,
University of Zagreb, Zagreb, Croatia
Received April 20, 2006; accepted May 18, 2006
Published online November 6, 2006 # Springer-Verlag 2006
Summary. Series of cyano, dicyano, amidino, and diamidino substituted 2-phenylbenzothiazoles were
prepared. Mono- and dicyano substituted benzothiazoles were obtained by condensation of appropriate
substituted benzaldehydes with 2-aminothiophenol or 4-amino-3-mercaptobenzonitrile. The appro-
priate amidines or diamidines were prepared by Pinner reaction. The compounds were tested against
breast, prostate, and lung cancer cell lines in a 72h cytotoxicity assay. Many of the compounds had at
10ꢀM activity equivalent to 2-(4-aminophenyl)benzothiazole, while four compounds had significantly
better activity, particularly in the breast cancer model.
Keywords. Heterocycles; Benzothiazoles; Amidines; Antitumor agents; Structure-activity relationship.
Introduction
Study of benzothiazole derivatives is of considerable current interest as a result of
their important biological and biophysical properties. 2-Aryl or 2-heteroaryl sub-
stituted benzothiazoles have been studied as antitumor [1], antimicrobial [2], anti-
fungal agents [3], and as imaging agents for ꢁ-amyloid [4]. On the other hand, the
function of the amidinic group present in a variety of antimicrobial and antipara-
sitic agents is also well known [5].
Series of potent and selective antitumor agents derived from 2-(4-aminophenyl)
benzothiazole have been extensively examined and developed during recent years.
The fluorinated analogue, 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole is a
ꢀ
Corresponding author. E-mail: vtralic@ttf.hr

`
L. Racane et al.
1572
novel agent with potent and selective antitumor properties which in the form of its
L-lysylamide prodrug, Phortress, is in an early phase of clinical studies [6]. Besides,
two substituted 2-phenylbenzothiazole compounds enhanced reduction of B16 mel-
anoma pulmonary metastases mediated by A-NK cells adoptive immunotherapy
and can augment the anti-metastatic therapy [7].
In connection with our studies on synthesis and antitumor activity of amidino
substituted heterocyclic molecules [8], we describe the synthesis and biological
activity of a number of new cyano, dicyano, amidino, and diamidino substituted 2-
phenylbenzothiazoles, structurally similar to the previously described 2-(4-amino-
phenyl)benzothiazole [9].
Results and Discussion
Synthesis
The synthesis route to nitriles 1–3, monosubstituted amidines 4a–4c, 5a–5c, as
well as disubstituted amidines 6a and 6b of 2-phenylbenzothiazoles is outlined in
Scheme 1.
Scheme 1

Cyano and Amidino Substituted 2-Phenylbenzothiazoles
1573
The mononitriles 2-(4-cyanophenyl)benzothiazole (1) [10] and 6-cyano-2-phenyl-
benzothiazole (2) and the dinitrile, 6-cyano-2-(4-cyanophenyl)benzothiazole (3) [11],
were synthesized from benzaldehyde or 4-cyanobenzaldehyde by condensation with
4-amino-3-mercaptobenzonitrile [12] or 2-aminothiophenol in pyridine followed by
oxidation with alcoholic ferric chloride solution [13]. As previously developed [8] a
Pinner reaction was used to transform the cyano into an amidino group. The imidate
ester hydrochloride generated as an intermediate product was very hygroscopic and
tended to hydrolyse fairly rapidly if left exposed to the atmosphere. Thus, it was im-
mediately converted into imidazolinyl and isopropyl substituted amidines.
For preparing 2-(4-amidinophenyl)benzothiazole hydrochloride (4a) [14] and
6-amidino-2-phenylbenzothiazole hydrochloride (5a) the corresponding imidate
ester hydrochloride was converted into the free base. Treating the solution of im-
idate ester with NH4Cl in 75% ethanol the corresponding amidine was obtained.
The hydrochloride salt was obtained from the amidine by treatment of the ethanol-
xylene solution of the corresponding free base with HCl. In spite of an over-
all yield for the monoamidines 4a and 5a of about 60%, the same approach to
prepare 6-amidino(4-amidinophenyl)benzothiazole [11] was unsuccessful. The at-
tempted reaction of the corresponding imidate ester hydrochloride with ethylene-
diamine in ethanol gave the free bases 2-[(4-(imidazolin-2-yl)phenyl)]benzothiazole,
6-(imidazolin-2-yl)-2-phenylbenzothiazole, and 2-[(4-(imidazolin-2-yl)phenyl)]-6-
(imidazolin-2-yl)benzothiazole. They were converted into their water-soluble hydro-
chlorides 4b, 5b, and 6a with HCl in overall moderate to good yield. Analogous
treatment of the corresponding imidate ester hydrochloride with isopropylamine
gave 2-[4-(N-isopropylamidino)phenyl]benzothiazole hydrochloride (4c), 6-(N-iso-
propylamidino)-2-phenylbenzothiazole hydrochloride (5c), and 6-(N-isopropyl-
amidino)-2-[4-(N-isopropylamidino)phenyl]benzothiazole dihydrochloride (6b). In
spite of the excess of isopropylamine the hydrochlorides were isolated in a yield of
about 50%.
The structures of the new compounds were confirmed by elemental analysis,
1
IR, H NMR, and 13C NMR spectra.
Cytotoxicity
The compounds were tested against DU-145 (human metastatic prostate carcinoma),
HT-1080 (human fibrosarcoma), MDA-MB-231 (human metastatic breast adenocar-
cinoma, and A549-Met2 (a metastatic variant of the human A549 lung carcinoma
[Brunson et al., manuscript in preparation]) at 10 ꢀM for 72 h and analyzed for their
effect on cell proliferation using an MTT assay. The results are shown in Table 1. A
two-way ANOVA using a Bonferroni post-test was used to compare each drug to 2-(4-
aminophenyl)benzothiazole (7), which was prepared as described by Shi et al. [9].
Most of the compounds inhibited cell proliferation to a similar extent as 2-(4-
aminophenyl)benzothiazole. However, 4a, 4b, 5b, and 6a were more effective
against the HT-1080 and MDA-MB-231 cell lines than 2-(4-aminophenyl)ben-
zothiazole (p<0.001). It can be seen that the antitumor activity depends on the
kind and position of an amidino substituent on the 2-phenylbenzothiazole skeleton.
It is interesting that the activity of imidazoline derivatives is fairly better than that
of isopropylamidino derivatives, the best being the activity of 6a. Further analysis

`
L. Racane et al.
1574
Table 1. Effect of novel benzothiazoles on cell number (Percent of Control Cell Number ꢆ SD) after
72 h incubation
Comp.
Cell Line
A549-Met2
DU-145
HT-1080
MDA-MB-231
1
47 ꢆ 6
49 ꢆ 5
43 ꢆ 8
42 ꢆ 8
43 ꢆ 5
50 ꢆ 6
44 ꢆ 4
39 ꢆ 5
49 ꢆ 4
4 ꢆ 3a
64 ꢆ 17a
42 ꢆ 5
47 ꢆ 7
48 ꢆ 5
48 ꢆ 7
42 ꢆ 9
18 ꢆ 18a
12 ꢆ 9a
43 ꢆ 6
64 ꢆ 22
16 ꢆ 16a
36 ꢆ 14
4 ꢆ 2a
36 ꢆ 3
2
45 ꢆ 11
33 ꢆ 4
39 ꢆ 6
3
35 ꢆ 14
8 ꢆ 6a
4a
4b
4c
5a
5b
5c
6a
6b
7
61 ꢆ 10a
57 ꢆ 6a
68 ꢆ 6a
98 ꢆ 15a
64 ꢆ 5a
67 ꢆ 8a
56 ꢆ 14a
58 ꢆ 10a
40 ꢆ 4
15 ꢆ 6a
28 ꢆ 21
23 ꢆ 11a
11 ꢆ 8a
53 ꢆ 10a
2 ꢆ 2a
68 ꢆ 4a
44 ꢆ 2
42 ꢆ 10
35 ꢆ 4
a
Significantly different (p<0.001) than treatment of the cell line with 2-(4-aminophenyl)benzothia-
zole (7)
of other cell lines at various concentrations will provide a more complete picture of
the possible anti-cancer activity of these novel compounds.
Experimental
Melting points were determined on a Kofler block apparatus. 1H and 13C NMR spectral data were de-
termined with a Brucker Avance DPX 300MHz NMR spectrometer with TMS as an internal standard. IR
spectra were determined with a Nicolet Magna 760 infrared spectrophotometer in KBr pellets. Elemental
analyses (C, H, N) were carried out in the Microanalitical Laboratory at the ꢁRudjer Boskovic
Instituteꢂ, Zagreb, and the results agreed with the calculated values within experimental errors.
2-(4-Cyanophenyl)benzothiazole (1) [10], 6-cyano-2-(4-cyanophenyl)benzothiazole (3) [11], and
2-(4-aminophenyl)benzothiazole (7) [9] were prepared according to established procedures.
Cell lines (HT-1080, DU-145, and MDA-MB-231) were obtained from the American Type Culture
Collection (Manassas, VA). The A549-Met2 cell line was a generous gift of Dr. K.W. Brunson (UNT
Health Science Center). Cell lines were cultured in RPMI 1640 (HT-1080), L-15 (MDA-MB-231),
F-12K (A549-Met2), or DMEM (DU-145) medium containing 10% fetal bovine serum, 100 U=cm3
penicillin, and 100ꢀg=cm3 streptomycin sulfate (complete medium). All cell culture reagents were
from Gibco BRL (Rockville, MD).
6-Cyano-2-phenylbenzothiazole (2, C14H8N2S)
To 0.940 g benzaldehyde (10.0 mmol) dissolved in 25 cm3 pyridine 1.65 g 4-amino-3-mercaptobenzo-
nitrile (11.0mmol) were added, and the stirred reaction mixture was refluxed for 4 h. The mixture was
then poured into 200 cm3 2 M HCl, and after cooling overnight the obtained crystalline product was
filtered off, washed with water and dried. The crude product was dissolved in EtOH, gently boiled for
5 min with an EtOH solution of 2.7 g FeCl3 ꢃ 6H2O, and poured in 200 cm3 H2O, and filtered off.
1
Recrystallization from EtOH afforded 1.13 g (48%) 2. Mp 240–242ꢄC; H NMR (300MHz, DMSO-
d6): ꢂ ¼ 8.79 (d, J ¼ 1.4 Hz, 1H, H-Bt), 8.23 (d, J ¼ 8.5 Hz, 1H, H-Bt), 8.16 (dd, J ¼ 1.8, 7.9 Hz, 2H,
H-Ph), 7.97 (dd, J ¼ 1.6, 8.5 Hz, 1H, H-Ph), 7.66–6.61 (m, 3H, H-Ph) ppm; 13C NMR (75 MHz, DMSO-
d6): ꢂ ¼ 107.5 (s), 118.7 (s), 123.6 (d), 127.5 (d, 2C), 127.7 (d), 128.5 (s), 129.5 (d, 2C), 129.8 (d), 132.1
(s), 132.3 (d), 155.8 (s), 171.9 (s) ppm; IR (KBr): ꢃꢀ¼ 2228 (CN), 1508 (C¼N) cmꢅ1
.

Cyano and Amidino Substituted 2-Phenylbenzothiazoles
1575
2-(4-Amidinophenyl)benzothiazole hydrochloride hydrate (4a)
A suspension of 2.36g 1 (10 mmol) in 100 cm3 2-(2-ethoxyethoxy)ethanol was saturated with HCl gas
at 5ꢄC. The flask was then stoppered, and the content was stirred at room temp. for 5 d (until IR spectra
indicated the disappearance of the cyano peak). The excess HCl was removed from the suspension by a
stream of N2. The reaction mixture was poured into 500 cm3 dry ether and the crystals of imidate ester
dihydrochloride were filtered off, washed with dry ether, and dried under reduced pressure over KOH.
The corresponding crude imidate ester hydrochloride was poured into 200 cm3 cold H2O containing
30cm3 20% K2CO3 and the free base was extracted with CHCl3. The solvent was evaporated and the
residual oil was dissolved in 70cm3 EtOH and 0.588 g NH4Cl (11 mmol) dissolved in 10cm3 H2O
were added. The mixture was heated under reflux for 5 h. After filtration (charcoal) the solvent was
evaporated and the residue was dissolved in H2O. The cold solution was made alkaline with 2 M NaOH
and the crystals were filtered off, washed with H2O, and dried. Free base was dissolved in 100 cm3 EtOH-
xylene mixture (v=v, 1=1) and to the obtained solution 1 cm3 HCl was added by stirring. The stirring was
continued overnight and after cooling for 2 d at 5ꢄC the product was collected. Recrystallization from
1
EtOH afforded 1.85g (60%) 4a. Mp >300ꢄC (Ref. [14] 305ꢄC); H NMR (300MHz, DMSO-d6):
ꢂ ¼ 9.67 (brs, 2H, H-Am disappeared with D2O), 9.47 (brs, 2H, H-Am disappeared with D2O), 8.32
(d, J ¼ 8.5 Hz, 2H, H-Ph), 8.23 (d, J ¼ 8.0 Hz, 1H, H-Bt), 8.14 (d, J ¼ 8.0 Hz, 1H, H-Bt), 8.06 (d,
J ¼ 8.5 Hz, 2H, H-Ph), 7.64–7.51 (m, 2H, H-Bt) ppm; 13C NMR (75 MHz, DMSO-d6): ꢂ ¼ 122.5 (d),
123.2 (d), 126.1 (d), 126.9 (d), 127.3 (d, 2C), 129.2 (d, 2C), 130.1 (s), 134.8 (s), 137.1 (s), 153.4 (s), 164.9
(s), 165.5 (s) ppm; IR (KBr): ꢃꢀ¼ 3358 and 3056 (NH), 1679, 1657, and 1611 (C¼N) cmꢅ1
.
2-[4-(Imidazolin-2-yl)phenyl]benzothiazole hydrochloride hydrate (4b, C16H13N3S ꢃ HCl ꢃ H2O)
A suspension of 2.36g 1 (10 mmol) in 100 cm3 2-(2-ethoxyethoxy)ethanol was saturated with HCl gas
at 5ꢄC. The flask was then stoppered, and the content was stirred at room temp. for 5 d (until IR spectra
indicated the disappearance of the cyano peak). The excess HCl was removed from the suspension by a
stream of N2. The reaction mixture was poured into 500 cm3 dry ether and the crystals of imidate ester
dihydrochloride were filtered off, washed with dry ether, and dried under reduced pressure over KOH.
To the suspension of crude imidate ester hydrochloride in 40cm3 dry EtOH, a solution of 1.2g freshly
distilled ethylenediamine (20.0 mmol) dissolved in 10 cm3 dry EtOH was added. The reaction mixture
was refluxed for 20 h. After evaporation of solvent the residue was suspended in H2O, and basified with
2 M NaOH. The crude product was filtered off, washed with H2O, and dried in vacuum at 80ꢄC. The
free base was dissolved in 100 cm3 EtOH-xylene mixture (v=v, 1=1) and to the hot solution 1 cm3 HCl
was added by stirring. The stirring was continued overnight and after cooling for 2 d at 5ꢄC the prod-
1
uct was collected. Recrystallization from EtOH afforded 0.85g (64%) 4b. Mp >300ꢄC; H NMR
(300 MHz, DMSO-d6): ꢂ ¼ 11.01 (brs, 2H, H-Am disappeared with D2O), 8.36 (d, J ¼ 8.3Hz, 2H,
H-Ph), 8.26–8.22 (m, 3H, 1H-Bt þ 2H-Ph), 8.14 (d, J ¼ 8.1Hz, 1H, H-Bt), 7.64–7.51 (m, 2H, H-Bt),
4.04 (s, 4H, H-CH2) ppm; 13C NMR (DMSO-d6): ꢂ ¼ 45.0 (t, 2C), 123.2 (d), 123.9 (d), 124.8 (s), 126.8
(d), 127.6 (d), 128.2 (d, 2C), 130.2 (d, 2C), 135.5 (s), 138.2 (s), 154.0 (s), 164.5 (s), 166.0 (s) ppm; IR
(KBr): ꢃꢀ¼ 3065 and 2965 (NH), 1623 and 1606 (C¼N) cmꢅ1
.
2-[4-(N-Isopropylamidino)phenyl]benzothiazole hydrochloride hydrate
(4c, C17H17N3S ꢃ HCl ꢃ H2O)
A suspension of 0.94 g 1 (4mmol) in 40cm3 2-(2-ethoxyethoxy)ethanol was saturated with HCl gas at
5ꢄC. The flask was then stoppered, and the content was stirred at room temp for 5 d (until IR spectra
indicated the disappearance of the cyano peak). The excess HCl was removed from the suspension by
a stream of N2. The reaction mixture was poured into 300 cm3 dry ether and the crystals of imidate
ester dihydrochloride were filtered off, washed with dry ether, and dried under reduced pressure over
KOH. To the suspension of crude imidate ester hydrochloride in 20cm3 dry EtOH 0.59 g freshly
distilled isopropylamine (10.0 mmol) dissoved in 5 cm3 dry EtOH were added. The reaction mixture
was refluxed for 24 h, solvent was evaporated, and the obtained crude product filtered off. Recrystalli-
1
zation from H2O-acetone afforded 0.670 g (48%) 4c. Mp >300ꢄC; H NMR (300MHz, DMSO-d6):
ꢂ ¼ 9.70 (brs, 3H, H-Am disappeared with D2O), 8.29 (d, J ¼ 7.5 Hz, 2H, H-Ph), 8.23 (d, J ¼ 7.9 Hz,

`
L. Racane et al.
1576
1H, H-Bt), 8.14 (d, J ¼ 8.0Hz, 1H, H-Bt), 7.94 (d, J ¼ 7.4 Hz, 2H, H-Ph), 7.64–7.51 (m, 2H, H-Bt),
4.22 (m, 1H, H-CH), 1.31 (d, J ¼ 5.7 Hz, 6H, H-CH3) ppm; 13C NMR (75 MHz, DMSO-d6): ꢂ ¼ 21.8
(q, 2C), 45.8 (d), 123.1 (d), 123.8 (d), 126.7 (d), 127.5 (d), 127.7 (d, 2C), 130.2 (d, 2C), 131.9 (s), 135.3
(s), 137.1 (s), 154.0 (s), 161.5 (s), 166.6 (s) ppm; IR (KBr): ꢃꢀ¼ 3204 and 3046 (NH), 1671 and 1618
(C¼N) cmꢅ1
.
6-Amidino-2-phenylbenzothiazole hydrochloride hydrate (5a, C14H11N3S ꢃ HCl ꢃ H2O)
Compound 5a was prepared by the method described for 4a from 2.36 g 2 (10 mmol) and 0.588g
NH4Cl (11 mmol). Recrystallization from EtOH afforded 1.94 g (63%) 5a. Mp 274–278ꢄC; 1H NMR
(300 MHz, DMSO-d6): ꢂ ¼ 9.68 (brs, 2H, H-Am disappeared with D2O), 9.49 (brs, 2H, H-Am dis-
appeared with D2O), 8.77 (d, J ¼ 1.6 Hz, 1H, H-Bt), 8.27 (d, J ¼ 8.7Hz, 1H, H-Bt), 8.18 (dd, J ¼ 1.5,
7.3 Hz, 2H, H-Ph), 8.00 (dd, J ¼ 1.8, 8.6 Hz, 1H, H-Bt), 7.66–7.60 (m, 3H, H-Ph) ppm; 13C NMR
(75 MHz, DMSO-d6): ꢂ ¼ 122.9 (d), 123.4 (d), 124.7 (s), 126.3 (d), 127.5 (d, 2C), 129.5 (d, 2C), 132.1
(s), 132.2 (d), 134.6 (s), 156.5 (s), 165.4 (s), 171.4 (s) ppm; IR (KBr): ꢃꢀ¼ 3237 and 2962 (NH), 1672
and 1594 (C¼N) cmꢅ1
.
6-(Imidazolin-2-yl)-2-phenylbenzothiazole hydrochloride hydrate (5b, C16H13N3S ꢃ HCl ꢃ H2O)
Compound 5b was prepared by the method described for 4b from 0.94g 2 (4mmol) and 1.2 g
ethylenediamine (20.0 mmol). Recrystallization from H2O afforded 0.867g (65%) 5b. Mp >300ꢄC;
1H NMR (300 MHz, DMSO-d6): ꢂ ¼ 11.09 (brs, 2H, H-Am disappeared with D2O), 8.99 (s, 1H, H-Bt),
8.28 (d, J ¼ 8.6 Hz, 1H, H-Bt), 8.21 (d, J ¼ 8.7 Hz, 1H, H-Bt), 8.15 (d, J ¼ 8.0 Hz, 2H, H-Ph), 7.69–7.59
(m, 3H, H-Ph), 4.05 (s, 4H, H-CH2) ppm; 13C NMR (75 MHz, DMSO-d6): ꢂ ¼ 44.3 (t, 2C), 118.8 (s),
123.2 (d), 123.8 (d), 126.7 (d), 127.6 (d, 2C), 129.5 (d, 2C), 132.0 (s), 132.3 (d), 134.7 (s), 156.7 (s),
164.2 (s), 171.9 (s) ppm; IR (KBr): ꢃꢀ¼ 3068 and 2969 (NH), 1613 (C¼N) cmꢅ1
.
6-(N-Isopropylamidino)-2-phenylbenzothiazole hydrochloride hydrate (5c, C17H17N3S ꢃ HCl ꢃ H2O)
Compound 5c was prepared by the method described for 4c from 0.94 g 2 (4mmol) and 1.18g iso-
propylamine (20.0 mmol). Recrystallization from H2O afforded 0.812 g (58%) 5c. Mp 292–294ꢄC;
1H NMR (300MHz, DMSO-d6): ꢂ ¼ 9.63 (brs, 3H, H-Am disappeared with D2O), 8.65 (s, 1H, H-Bt),
8.26 (d, J ¼ 8.5 Hz, 1H, H-Bt), 8.17 (d, J ¼ 7.8 Hz, 2H, H-Ph), 7.88 (d, J ¼ 8.5Hz, 1H, H-Bt), 7.64–7.60
(m, 3H, H-Ph), 4.19 (m, 1H, H-CH), 1.31 (d, J ¼ 6.3 Hz, 6H, H-CH3) ppm; 13C NMR (75 MHz, DMSO-
d6): ꢂ ¼ 21.2 (q, 2C), 45.2 (d), 122.7 (d), 123.5 (d), 126.0 (s), 126.6 (d), 127.5 (d, 2C), 129.5 (d, 2C),
132.1 (d), 132.2 (s), 134.4 (s), 156.1 (s), 161.3 (s), 171.1 (s) ppm; IR (KBr): ꢃꢀ¼ 3192 and 3056 (NH),
1665 and 1617 (C¼N) cmꢅ1
.
2-[4-(Imidazolin-2-yl)phenyl]-6-(imidazolin-2-yl)benzothiazole dihydrochloride dihydrate
(6a, C19H17N5S ꢃ 2HCl ꢃ 2H2O)
Compound 6a was prepared by the method described for 4b from 1.045g 3 (4mmol) and 2.4 g ethy-
lenediamine (40.0mmol). Recrystallization from EtOH afforded 0.754 g (41%) 6a. Mp >300ꢄC;
1H NMR (300MHz, DMSO-d6): ꢂ ¼ 11.98 (brs, 4H, H-Am disappeared with D2O), 9.02 (s, 1H, H-
Bt), 8.40 (d, J ¼ 8.4 Hz, 2H, H-Ph), 8.33 (d, J ¼ 8.6 Hz, 1H, H-Bt), 8.29 (d, J ¼ 8.4 Hz, 2H, H-Ph), 8.22
(d, J ¼ 8.6Hz, 1H, H-Bt), 4.06 (s, 4H, H-CH2), 4.05 (s, 4H, H-CH2) ppm; 13C NMR (75 MHz, DMSO-
d6): ꢂ ¼ 45.1 (t, 4C), 120.2 (s), 124.3 (d), 124.8 (d), 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) ppm; IR (KBr): ꢃꢀ¼ 3081 and 2962 (NH),
1618 (C¼N) cmꢅ1
.
6-(N-Isopropylamidino)-2-[4-(N-isopropylamidino)phenyl]benzothiazole dihydrochloride
dihydrate (6b, C21H25N5S ꢃ 2HCl ꢃ 2H2O)
Compound 6b was prepared by the method described for 4c from 1.045g 3 (4 mmol) and 2.36g
isopropylamine (40.0 mmol). Recrystallization from H2O-acetone afforded 0.742 g (38%) 6b. Mp
1
>300ꢄC; H NMR (300MHz, DMSO-d6): ꢂ ¼ 9.62 (brs, 6H, H-Am disappeared with D2O), 8.71 (d,

Cyano and Amidino Substituted 2-Phenylbenzothiazoles
1577
J ¼ 1.4 Hz, 1H, H-Bt), 8.36 (d, J ¼ 8.4 Hz, 2H, H-Ph), 8.31 (d, J ¼ 8.5 Hz, 1H, H-Bt), 7.98 (d, J ¼ 8.4 Hz,
2H, H-Ph), 7.91 (dd, J ¼ 1.4, 8.6 Hz, 1H, H-Bt), 4.16 (m, 2H, H-CH), 1.31 (d, J ¼ 6.2 Hz, 12H, H-CH3)
ppm; 13C NMR (75 MHz, DMSO-d6): ꢂ ¼ 21.1 (q, 2C), 21.2 (q, 2C), 45.2 (d, 2C), 123.1 (d), 123.7 (d),
126.5 (s), 126.9 (d), 127.5 (d, 2C), 129.6 (d, 2C), 132.0 (s), 134.7 (s), 135.9 (s), 155.9 (s), 160.9 (s), 161.3
(s), 169.5 (s) ppm; IR (KBr): ꢃꢀ¼ 3222 and 3081 (NH), 1668 and 1619 (C¼N) cmꢅ1
.
Cytotoxicity Assay
A 10mM stock solution of each compound was prepared in DMSO. A single cell suspension was pre-
pared from each cell line and diluted to a concentration of 25000 cells=cm3 in their respective complete
medium. To aliquots of this suspension, compounds were added to a final concentration of 10 ꢀM.
Samples (200 mm3) of each aliquot were placed in 96 well plates in replicates of five. Cells with no
drug, but containing solvent (DMSO) were run as a control. The plates were incubated for 72h at 37ꢄC.
Cell viability after incubation with the compounds was measured using MTT (3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl tetrazolium bromide) (Sigma, St. Louis, MO). Results were expressed as percent of
control cell number and represent an average of 10 readings (2 sets of 5 replicates).
Acknowledgements
The support of the Ministry of Science and Technology, Republic of Croatia (Project No. 125005) is
gratefully acknowledged.
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