Novel Cyano- And amidinobenzothiazole derivatives: Synthesis, antitumor evaluation, and X-ray and quantitative structure-activity relationship (QSAR) analysis

Article abstract of DOI:10.1021/jm801566q

Synthesis of a series of novel cyano-and amidinobenzothiazole derivatives 3-31 is described. All studied amidino derivatives showed noticeable antiproliferative effect on several tumor cell lines. Cyano derivatives 11-17 showed considerably less pronounced activity because of their poor solubility in aqueous cell culture medium, which was confirmed by the principal components (PC) analysis. Compounds 21, 22, 28, and 29 were tested for their effects on the cell cycle and apoptosis, whereby 22 and 29, having methyl group at the C-6 position in pyridine ring, showed drastic cell cycle perturbations that were both concentration- and time-dependent and induced apoptosis. The QSAR modeling, based on the physicochemical descriptors and on the measured biological activities, indicated the relevance of molecular polarizability and particular distribution of pharmacophores on the molecular surface for activity. In conclusion, benzothiazoles containing either isopropylamidino or imidazolyl groups will be considered as starting compounds for further investigation on lead identification.

Full text of DOI:10.1021/jm801566q

1744
J. Med. Chem. 2009, 52, 1744–1756
Novel Cyano- and Amidinobenzothiazole Derivatives: Synthesis, Antitumor Evaluation, and
X-ray and Quantitative Structure-Activity Relationship (QSAR) Analysis
Irena Caleta, Marijeta Kralj,*,‡ Marko Marjanovic´,‡ Branimir Bertosˇa,# Sanja Tomic´,# Gordana Pavlovic´, Kresˇimir Pavelic´,‡
†,§
´
and Grace Karminski-Zamola*,†
Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, UniVersity of Zagreb, Marulic´eV trg 20, P.O. Box 177,
HR-10000 Zagreb, Croatia, DiVision of Molecular Medicine and DiVision of Physical Chemistry, Ru{er BosˇkoVic´ Institute,
Bijenicˇka cesta 54, P.O. Box 1016, HR-10000 Zagreb, Croatia, and Faculty of Textile Technology, UniVersity of Zagreb,
Prilaz baruna FilipoVic´a 28a, HR-10000, Zagreb, Croatia
ReceiVed December 11, 2008
Synthesis of a series of novel cyano- and amidinobenzothiazole derivatives 3-31 is described. All studied
amidino derivatives showed noticeable antiproliferative effect on several tumor cell lines. Cyano derivatives
11-17 showed considerably less pronounced activity because of their poor solubility in aqueous cell culture
medium, which was confirmed by the principal components (PC) analysis. Compounds 21, 22, 28, and 29
were tested for their effects on the cell cycle and apoptosis, whereby 22 and 29, having methyl group at the
C-6 position in pyridine ring, showed drastic cell cycle perturbations that were both concentration- and
time-dependent and induced apoptosis. The QSAR modeling, based on the physicochemical descriptors and
on the measured biological activities, indicated the relevance of molecular polarizability and particular
distribution of pharmacophores on the molecular surface for activity. In conclusion, benzothiazoles containing
either isopropylamidino or imidazolyl groups will be considered as starting compounds for further investigation
on lead identification.
Introduction
acid metabolism23,24 inhibitors. Therefore, various benzothiazole
compounds are of considerable interest for their diverse
pharmaceutical uses and play a vital role in the synthesis of
fused heterocyclic systems.
The above considerations prompted us to design and syn-
thesize a new series of cyano- and amidino-substituted ben-
zothiazole derivatives (Figure 1) and to test their antiproliferative
activity of tumor cells in vitro. In order to find out which
chemical and physical descriptors are most relevant for inhibition
of the specific cell lines growth, we derived the Volsurf
parameters and, using PLSa statistical analysis, we determined
the relationship between these descriptors and molecular activi-
ties. In this way, QSAR models were derived for several tested
cell lines.
Despite major breakthroughs in diagnosis and treatment,
cancer is still the second leading cause of death in the Western
world. The discovery and development of new, more active,
more selective, and less toxic compounds for the treatment of
malignancy are one of the most important goals in medicinal
chemistry. The understanding of the fundamental biology of
cancer increased dramatically in recent years1 and has strongly
impacted both experimental and clinical tumor therapy. It is
believable that the future of tumor therapy is in the development
of molecularly targeted agents that specifically block key
mechanisms involved in development and progression of specific
types of cancer.2 Two potential targets for drug design in cancer
are DNA and different types of proteins like kinases or
topoisomerase I/II. The design of new intercalators3,4 and
groove-binders5,6 andthediscoveryofspecificproteininhibitors4,7,8
are two important approaches in the search of new chemotera-
peutic agents.
Since the 1990s, various pharmacological investigations of
newly synthesized benzothiazoles demonstrated interesting
pharmacological activities and led to development of new
medications for treating diseases. They were studied extensively
for their antiallergic,9 anti-inflammatory,9,10 antitumor11-14 and
analgesic activity.15,16 Apart from the above mentioned activi-
ties, benzothiazole derivatives also showed some interesting
efficacy as kinase,17-20 topoisomerase I/II21,22 and trans-retinoic
Chemistry
All compounds (3-31) shown in the Figure 1 were prepared
according to the Schemes 1 and 2, starting from commercially
available 4-aminobenzonitrile. 2-Amino-6-cyanobenzothiazole
1 was obtained using a slightly changed conventional procedure
with potassium thiocyanate and HCl(conc)/H2O in a two-step
synthesis.25,26 Cyano-substituted benzamides 3-6 were prepared
by a published method.27 Compound 1 was also treated with
a Abbreviations: 7-AAD, 7-amino-actinomycin D; DMEM, Dulbecco’s
modified Eagle medium; DMSO, dimethyl sulfoxide; FBS, fetal bovine
serum; H 460, lung carcinoma; HAS, hydrophobic surface area; HeLa,
cervical carcinoma; Hep-2, laryngeal carcinoma; LOO, Leave-One-Out; LV,
latent variables; MCF-7, breast carcinoma; MiaPaCa-2, pancreatic carci-
noma; MIF, molecular interaction fields; MTT, 3-(4,5-dimethylthiazole-2-
yl)-2,5-diphenyl tetrazolium bromide; OD, optical density; PBS, phosphate
buffered saline; PG, percentage of growth; PI, propidium iodide; PC,
Principal Components; PLS, Partial Least Square; QSAR, Quantitative
Structure Activity Relationship; SDEP, standard deviation of the error of
prediction; SW 620, colon carcinoma; VD, a measure of relative partition
of compounds between plasma and tissue; W, volume of water molecule;
WI 38, diploid fibroblasts; WN, volume of amide molecule; WO, volume
of the oxygen probe.
* To whom correspondence should be addressed. For M.K.: phone, +385
1 4571 235; fax, +3851 4561 010; e-mail, marijeta.kralj@irb.hr. For G.K.-
Z.: phone, ++38514597215; fax, ++38514597250; e-mail, gzamola@
fkit.hr.
†
Faculty of Chemical Engineering and Technology, University of Zagreb.
Current address: GlaxoSmithKline Research Center Zagreb Limited,
§
Prilaz baruna Filipovic´a 29, HR-10000 Zagreb, Croatia.
‡
Division of Molecular Medicine, Ru{er Bosˇkovic´ Institute.
#
Division of Physical Chemistry, Ru{er Bosˇkovic´ Institute.
Faculty of Textile Technology, University of Zagreb.
10.1021/jm801566q CCC: $40.75
2009 American Chemical Society
Published on Web 03/05/2009

Benzothiazole DeriVatiVes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1745
media. Since H-bonding is an important way of interaction of
biologicaly active molecules with a potential target, it could be
suggested that the ability of prepared molecules to form H-bonds
in aqueous biological media may play a role in their biological
activity.
Biological Results and Discussion
Antiproliferative Activity. The compounds 7-31 were tested
for their potential antiproliferative effect on a panel of human
cell lines, six of which were derived from cancer cell types:
HeLa (cervical carcinoma), Hep-2 (laryngeal carcinoma), MCF-7
(breast carcinoma), SW 620 (colon carcinoma), MiaPaCa-2
(pancreatic carcinoma), H 460 (lung carcinoma), and WI 38
(diploid fibroblasts). The results are expressed as IC50 (µM)
summarized in Tables 1 and 2. The cyano substituted ben-
zothiazoles (11-17) were poorly soluble in aqueous cell culture
medium; therefore, the highest concentration tested was 10-5
M, yet the precipitation still occurred during the test period (72
h). Nevertheless, all compounds except 11 showed antiprolif-
erative activity at least toward some cell types (e.g., MiaPaCa-
2), whereby 6-cyano-2-(N-(pyrimidin-2-yl)amino)benzothiazole
17 was the most active compound (Table 1). Cyano-substituted
benzothiazol-2-ylbenzamides 3-6 and amidino derivatives 25
and 27 were not tested for their antiproliferative effect because
of their poor solubility in aqueous cell culture medium.
These results (Table 1) point out that (i) two cyano groups
(11) strongly reduce the activity, (ii) replacing a pyridine with
the pyrimidine ring without any substituent significantly in-
creases the antiproliferative activity, (iii) the position of methyl
group at the pyridine ring (R2) does not significantly alter the
activity.
Figure 1. Substituted cyano- and amidinobenzothiazole derivatives
(3-31).
copper(II) bromide and tert-butyl nitrite in acetonitrile to form
the corresponding 2-bromo-6-cyanobenzothiazole 2. Compound
2 served as an intermediate for the synthesis of novel cyano
derivatives of 2-(hetero)arylaminobenzothiazole 11-17. Novel
amidinobenzothiazole derivatives 7-10 and 18-31 were pre-
pared by the classical Pinner reaction28 from cyano derivatives
(3-6, 11-17).
Molecular and Crystal Structure of Compound 15
On the other hand, all isopropylamidino- and imidazolinyl-
substituted benzothiazoles showed a strong antiproliferative
effect on the tested cell line panel (Table 2 and Figure 3),
while isopropylamidino-substituted benzothiazol-2-ylbenza-
mides 7-10 showed modest and mostly similar effects in
all cell lines tested.
The ORTEP-3 (version 1.08)29 drawing of the molecular
structure of compound 15 is depicted in Figure 2a and its crystal
structure in Figure 2b. The molecule is not essentially planar
in the crystalline state. The dihedral angle between planes
defined by the atoms of pyridine and benzothiazole ring amounts
to 5.68(8)°.
The rings bond lengths are normal. The thiazole ring C1-N1
bond length (1.313(2) Å) is shorter then N1-C2 (1.382(2) Å).
The bond lengths between the sulfur and the carbons of the
thiazole ring are 1.741(2) Å (S1-C7) and 1.751(2) Å (S1-C1).
The N2-C1 (1.359(3) Å) and N2-C8 (1.393(2) Å) bonds are
delocalized within the π-electron system of pyridine and the
benzothiazole ring.
The molecules are mutually linked into discrete centrosym-
metric dimmers by weak intermolecular hydrogen bond of the
N-H· · ·N type formed between NH group and thiazole nitrogen
atom (N2-H2N· · ·N1i; i ) x, y, 3/2 - z; N2-H2N, 0.84(3) Å;
H2N· · ·N1, 2.16(2) Å; N2 · · ·N1, 3.002(2) Å; N2-H2N· · ·N1,
178(1)°) (Figure 2b). No other types of hydrogen bonds are
found.
In our previous work we reported molecular and crystal
structures of compounds 2-chloro-N-(6-cyanobenzothiazol-2-
yl)benzamide 4 and 4-cyano-N-(benzothiazol-2-yl)benzamide
6.14,30 Both compounds also show similar crystal packing pattern
of mutually linked molecules into discrete centrosymmetric
dimers by weak intermolecular hydrogen bond of the N-H· · ·N
type. They are formed between the amide group and N atom of
benzothiazole moiety, while in compound 15 (present work)
they are formed between the amino group and N atom of
benzothiazole moiety.
Still, the imidazolinyl-substituted derivatives 26-31 show
more prominent, mostly nonselective cytotoxic activity, while
the isopropylamidino analogues 18-24 have more differential
activity showing less inhibitory effect in normal cells. Consider-
ing the isopropylamidino-substituted analogues, it was noticed
that the position of methyl group influences their antiproliferative
activity, whereby the C-5 position diminishes the activity.
Moreover, all isopropylamidino-substituted compounds showed
significant selectivity; they strongly inhibited the growth of
tumor compared with normal cells. In contrast, all imidazolinyl-
substituted benzothiazoles showed almost identical activity. In
order to shed more light on the mechanism of action of these
compounds, we tested the most interesting (active) and selective
isopropyl derivatives 21 and 22 and their direct imidazolinyl
analogues 28 and 29 for their effects on the cell cycle of the
most sensitive tumor cell line H 460, along with their potentials
to induce apoptosis in these cells.
Cell Cycle Perturbations. The benzothiazole compounds at
various concentrations result in a number of distinct perturba-
tions in cell cycle progression of H 460 cells (Figure 4),
confirming that the previously discussed structural differences
have strong impact on the activity. For example, isopropyla-
midino derivative 21, being less active by an order of magnitude
than other compounds, also showed notable effects on the cell
cycle only at significantly higher concentration than other
compounds (50 µM, which is 5 times higher than its IC50),
whereby it induced both G1 and G2 delay, along with the
Therefore, we assume that analogous compounds could also
form H-bonds not just in crystals but also in aqueous biological

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Caleta et al.
1746 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Scheme 1. Synthesis of Isopropylamidino-Substituted Benzothiazol-2-ylbenzamides 7-10
Scheme 2. Synthesis of Cyano- and Amidino-Substituted 2-(Hetero)arylaminobenzothiazoles 11-31
reduction of the percentage of cells in S-phase (Figure 4A,B).
Similarly, its imidazolinyl analogue 28 showed minor changes
at both time points.
On the other hand, both analogues bearing a methyl group at
position 6 of the pyridine ring (22 and 29) showed very
interesting and drastic cell cycle perturbations, whereby lower

Benzothiazole DeriVatiVes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1747
berberine induced different cell cycle responses at different
concentrations and time points.31
QSAR Analysis
A series of potentially relevant physical descriptors were
calculated using the program Volsurf.32 Principal components
(PC) analysis was accomplished in order to determine distribu-
tion of the compounds in the X-variable space, i.e., space of
physicochemical descriptors. Distribution of the compounds in
the descriptor space is shown in the score plot of the first two
principal components (Figure 6).
Most of the cyano-substituted compounds are grouped on the
left side of the plot. The exception is compound 17, pointing to
its significantly better solubility that correlates perfectly with
our experimental results, since as discussed previously, 17 was
the most active compound among cyano-substituted compounds,
which were in general poorly soluble. Such distribution is mostly
determined by the WO1 variable values, representing the volume
of the oxygen probe at an energy level of -0.837 kJ/mol (which
predominantly describes PC1 and which is for the cyano-
substituted compounds grouped on the left side of the graph
about 20% lower than for the other compounds), and WN1
variable values, representing the volume of amide molecule
probe at an energy level of -0.837 kJ/mol (which predominantly
describes PC2). These two descriptors describe most of the
variability in the X-variables space, i.e., space of the calculated
descriptors (Figure 7). Compound 31 is the only non-cyano-
substituted compound situated at the left half of the score plot.
Among properly soluble compounds it is the smallest one (i.e.,
it has the smallest V and S) with very high WN1 value (only
compound 9 has higher WN1 value). Although it has the
smallest WO1 in the group (about 370), this value is much above
WO1 values determined for cyano-substituted compounds (WO1
< 100).
Figure 2. (a) ORTEP-3 (version 1.08) drawing of the molecular
structure of 6-cyano-2-(N-(6-methylpyridin-2-yl)amino)benzothiazole
15 with the atomic numbering scheme. The thermal ellipsoids are drawn
at the 50% probability level at 293 K. (b) Crystal structure of 6-cyano-
2-(N-(6-methylpyridin-2-yl)amino)benzothiazole 15 viewed along the
y axis. Hydrogen bonds of the N-H· · ·N type are shown as dashed
lines.
concentrations lead to an increase in the percentage of cells in
the G1 phase. In contrast, 10 µM of both compounds did not
lead to a similar G1 increase but rather resulted in a marked
increase in the percentage of cells in G2/M. All concentrations
of tested compounds reduced the percentage of cells in the
S-phase. In addition, 22 and 29 increased the percentage of cells
in sub-G1, pointing to the induction of apoptosis. Moreover,
compound 22 at 50 µM induced apoptosis in about 40% of cells
after 24 h, while all cells died after 48 h of treatment (data not
shown).
In general, the oxygen and amide probe volumes are
proportional to compound hydrophilicity, while their shapes
correlate with the ability of the compound to fit into putative
receptor’s or transporter’s active site. Compounds with small
WO1 are grouped on the left side of the score plot, and those
with small WN1 are at its lower part. Compounds with small
WO1 are poorly soluble, and as a consequence, their activity
could not be precisely determined.
Further, we correlated the experimental results (IC50 for
growth inhibition) with the physical and chemical properties
of the compounds using partial least-squares (PLS) analysis.
For these calculations we used two data sets: (A) all compounds
that do not contain the cyano substitutent; (B) all compounds
on the right side of the score plot. Both groups contain 16
compounds, and they differ in one compound only; the first
group contains compound 17, and the second one contains
compound 31. The statistical parameters of the obtained PLS
models are given in Table 3. The models with the highest
predictive performances are derived for HeLa, Mia PaCa-2, and
SW 620 cell lines (see also Figure 8).
The optimal dimensionality depends on the cell line consid-
ered, but it is never above 3. The goodness of the model was
defined by fitting (R2 and standard deviation of the error of
calculation, SDEC) and cross-validation (Q2 and SDEP) pa-
rameters (see Table 3 for the definitions of these parameters).
From the obtained models we could elucidate which physi-
cochemical descriptors of the compounds, i.e., X-variables, are
most relevant for growth inhibition of different cell lines.
Variables with the highest absolute weights (either large positive
or negative values) are considered to be the most significant to
Annexin V Assay. To confirm the previously obtained results,
we assessed the annexin V assay, as a more sensitive method
for apoptosis detection and for quantification after 48 h of
treatment with compounds 21, 22, 28, and 29 at the same
concentrations as in the cell cycle perturbations assay. Indeed,
we confirmed that all compounds induced apoptosis in a dose-
dependent manner, whereby the effect of compounds 22 and
29 was the most prominent (Figure 5 and data not shown).
Our results demonstrate that different concentrations of
amidinobenzothiazoles 22 and 29 lead to distinct cellular
responses. These responses could be correlated with (i) specific
cellular localization patterns or (ii) different kinetics of inhibition
of specific cell-cycle-related protein. We propose that low
concentrations of compounds might selectively accumulate in
cell membrane and/or cytoplasm, resulting in a G1 checkpoint
activation and consequently arrest cell proliferation. Higher
concentrations of compounds accumulate in the cytoplasm and
nucleus where they directly interfere with DNA synthesis,
activate a G2 checkpoint, and consequently block cell cycle prior
to entry into M-phase. High concentrations resulted in sufficient
damage to trigger apoptosis. Similar results were described by
Serafim et al. who showed that plant isoquinoline alkaloid

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Caleta et al.
1748 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Table 1. In Vitro Inhibition of the Cyano-Substituted Compounds 11-17 on the Growth of Tumor Cells and Normal Human Fibroblasts (WI 38)
IC50 (µM)a
compd
WI 38
Hep-2
HeLa
MiaPaCa-2
SW 620
MCF-7
11
12
13
14
15
16
17
>10
2.3 ( 0.8
g10
4 ( 4
>10
>10
>10
7 ( 3
>10
>10
>10
5 ( 2
>10
>10
3 ( 2
9 ( 1
8 ( 2
>10
>10
>10
>10
>10
>10
>10
8 ( 2
10
3 ( 0.2
4 ( 0.04
5 ( 4
6 ( 3
6 ( 4
3 ( 2
3 ( 2
4 ( 2
>10
4 ( 1
g10
>10
g10
5 ( 2
9 ( 1
7 ( 1
a IC50: the concentration that causes a 50% reduction of the cell growth.
Table 2. In Vitro Inhibition of the Isopropylamidino- (7-10, 18-24) and Imidazolinyl- (26-31) Substituted Benzothiazole Derivatives on the Growth
of Tumor Cells and Normal Human Fibroblasts (WI 38)
IC50 (µM)a
compd
WI 38
>100
61 ( 31
>100
16 ( 3
92 ( 4
14 ( 1.6
12.5 ( 0.8
31 ( 1
19 ( 1
17 ( 0.5
75 ( 25
NTb
H 460
HeLa
MiaPaCa-2
SW 620
MCF-7
7
8
9
15 ( 1
59 ( 9
16 ( 5
23 ( 8
13 ( 1
9 ( 7
18 ( 2
5 ( 1.4
4 ( 1
12 ( 2
4 ( 3
9 ( 5
7 ( 3
3 ( 0.1
2 ( 1
2 ( 0.9
1 ( 0.01
1.4 ( 0.05
38 ( 11
45 ( 13
32 ( 3
17 ( 4
30 ( 18
3 ( 1
3 ( 0.2
14 ( 0.4
3 ( 1
12 ( 0.1
11 ( 8
3 ( 1
30 ( 0.4
45 ( 11
35 ( 5
17 ( 2
18 ( 6
5 ( 3
3 ( 0.9
12 ( 2
9 ( 1
13 ( 2
15 ( 3
2 ( 0.4
1 ( 0.2
2 ( 1
1.3 ( 0.1
1.7 ( 0.6
22 ( 5
16 ( 3
13 ( 2
13 ( 5
6 ( 8
22 ( 2
10
18
19
20
21
22
23
24
26
28
29
30
31
16 ( 2
16 ( 7
2 ( 0.4
2 ( 0.4
15 ( 0.9
3 ( 1
2 ( 0.7
2 ( 0.7
8 ( 0.04
3 ( 2
15 ( 0.7
5 ( 0.4
2 ( 0.2
2 ( 0.4
2 ( 0.2
1.5 ( 0.2
1.6 ( 0.08
5 ( 4
13 ( 3
2 ( 0.2
2 ( 1
6 ( 2
2 ( 1
1 ( 0.1
1.7 ( 0.06
2 ( 0.02
2 ( 0.3
1.6 ( 0.2
1.8 ( 0.1
2 ( 1
1.4 ( 0.2
1.7 ( 0.0
a IC50: the concentration that causes a 50% reduction of the cell growth. b NT: not tested.
Figure 3. Dose-response profiles for compounds 21 (A), 22 (B), 28 (C), and 29 (D). PG ) percentage of growth.
Overall, it could be concluded that the impact of descriptors
on the inhibition of growth of these cell lines is pretty even.
However, the WO descriptors are significantly more important
for SW 620 than other cell lines, pointing to the importance
of molecular polarizability, while molecular volume and
explain the compound efficiency on a certain cell line. The
descriptors specific for a certain cell line can be found by
comparing the X-variables with the highest weights in different
models. Weights for the most relevant descriptors obtained in
HeLa, MiaPaCa-2, and SW 620 models are given in Figure 9.

Benzothiazole DeriVatiVes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1749
Figure 4. Cell cycle analysis of H 460 cells treated with 5, 10, and 50 µM compounds 21, 22, 28, and 29 for 24 h (A, C, E, G, respectively) or
48 h (B, D, F, H, respectively). See results and discussion section for details.
surface are relatively important descriptors for MiaPaCa-2
and HeLa; i.e., benzothiazoles substituted with imidazolinyl
group, the molecules with the smallest surface and volume,
showed the strongest antiproliferative effect on these lines.
compounds are quite different, making the results inadequate
to obtain satisfactory models.
Conclusion
The QSAR model could not be derived for WI 38 cell line,
while for the MCF-7 cell line it is of a rather low quality,
with no predictive ability. The partial explanation lies in the
nature of WI 38 cells, which proliferate slowly compared to
other cell lines. Therefore, the influences of the tested
A series of novel cyano- and amidinobenzothiazole deriva-
tives (3-31) were prepared by multistep synthesis. All isopro-
pylamidino- and imidazolinyl-substituted benzothiazoles showed
strong antiproliferative effect on the tumor cell lines, whereby
imidazolinyl-substituted derivatives (25-31) showed more

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Caleta et al.
1750 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Figure 5. Annexin-V assay on H 460 cells treated with compounds 21 (c ) 50 µM) (B), 22 (c ) 5 µM (C) and c ) 10 µM (D)), 28 (c ) 10 µM)
(E), 29 (c ) 10 µM) (F) for 48 h. Control, vehicle-treated cells are shown in (A). The figure shows representative dot-plots of annexin V Alexa
Fluor 488 (x-axis)/7-AAD (y-axis) fluorescence. The percentages of population undergoing early and late apoptotic events are indicated in the
lower (R4) and upper (R2) right quadrant, respectively. Lower left quadrant (R3) represents the viable cells.

Benzothiazole DeriVatiVes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1751
higher concentrations (10 µM) of both compounds resulted in
a marked increase of cells in G2/M phase, while all concentra-
tions of tested compounds reduced the S-phase. In addition, 22
and 29 increased the percentage of cells in sub-G1, pointing to
the induction of apoptosis.
The principal components analysis and QSAR modeling
revealed that the cyano-substituted compounds 11-17 were
highly hydrophobic and consequently poorly soluble. The
exception was compound 17, thus correlating with its higher
biological activity. The best models were derived for the HeLa
cell line. The molecules with the smallest surface and volume
showed the strongest antiproliferative effect on this line.
Experimental Section
Chemistry. Melting points were determined on a Kofler hot stage
microscope and are uncorrected. IR spectra were recorded on a
1
Nicolet Magna 760 spectrophotometer in KBr disks. H and 13C
NMR spectra were recorded on either a Varian Gemini 300 or a
Bruker Avance DPX 300 spectrometer using TMS as an internal
standard in DMSO-d6. Elemental analysis for carbon, hydrogen,
and nitrogen were performed on a Perkin-Elmer 2400 elemental
analyzer. Where analyses are indicated only as symbols of ele-
ments, analytical results obtained are within (0.4% of the theoreti-
cal value. All compounds were routinely checked by TLC with
Merck silica gel 60F-254 glass plates. Experimental and spectro-
scopic data for cyano derivatives 1-6 and 11-17 with the reference
list are in Supporting Information.
Figure 6. Score plot of the first (PC1) and second (PC2) principal
components of the physicochemical descriptors (determined by the
program Volsurf, see Experimental Section) for 23 compounds. These
two principal components describe about 81% of variance among the
X-variables. Cyano-substituted compounds are encircled.
General Method for the Synthesis of Amidino Substituted
Benzothiazoles (7-10, 18-31). Amidino-substituted benzothiaz-
oles (7-10, 18-31) were prepared by modified Pinner reaction.27
The suspension of the corresponding cyano-substituted amides
(0.4 g) or cyano-substituted 2-(hetero)arylaminobenzothiazoles (0.2
g) in absolute carbitole (5 or 7 mL) at 0 °C was saturated with dry
HCl gas. After the mixture returned to room temperature, the flask
was stoppered and the contents were stirred until IR spectra
indicated the lack of the cyano peak (3 days). The corresponding
imido ester hydrochloride intermediate (90-95% yield) was
precipitated from the solution by the addition of dry diethyl ether,
filtered off, washed with dry ether, and dried under reduced pressure
over KOH.
Isopropylamidines. Isopropylamine (3 equiv) was added to a
suspension of the corresponding imido ester hydrochloride and
absolute ethanol (5 mL) under a nitrogen atmosphere. The reaction
mixture was left to stir for 24 h at room temperature. After 24 h
diethyl ether was added to the reaction mixture and the precipitate
was filtered off and dried. The crude product was dissolved in
absolute ethanol (5 mL) at 0 °C and saturated with dry HCl gas.
The corresponding isopropylamidine salt was precipitated from the
solution by addition of dry diethyl ether, filtered off, washed with
dry ether, and dried under reduced pressure over KOH.
Imidazolinylamidines. Ethylendiamine (3 equiv) was added to
a suspension of the corresponding imido ester hydrochloride and
absolute ethanol (5 mL) under a nitrogen atmosphere and left to
reflux for 4 h. After 4 h the reaction mixture was left to stir at
room temperature for the next 12 h. After 12 h, diethyl ether (20-40
mL) was added to the reaction mixture and the precipitate was
filtered off and dried. The crude product was recrystallized from
water (3-5 mL) to remove the remaining diethylamine hydrochlo-
ride salt, filtered off, and dried. Dry product was dissolved in
absolute ethanol (5 mL), and the suspension was saturated with
dry HCl gas at 0 °C. The corresponding imidazolinylamidine salt
was precipitated from the solution by the addition of dry diethyl
ether, filtered off, washed with dry ether, and dried under reduced
pressure over KOH.
Figure 7. Loading plot of the first (PC1) and second (PC2) principal
components of the physicochemical descriptors (determined by the
program Volsurf) for 23 compounds. Descriptors that are the most
important for differentiation of compounds are indicated. Their meaning
is the following: WO1 and WO2; W1 and W2; W1N and W2N are the
volumes of the oxygen probe, water molecule, and amide molecule,
respectively. Interaction fields are at two different energy levels: -0.837
and -1.674 kJ/mol. D1 describes the volume of the hydrophobic region
(interaction with the DRY probe); V represents molecular volume; S
describes molecular surface; HAS is the hydrophobic surface area.
prominent, mostly nonselective cytotoxic activity, while the
isopropylamidino analogues (18-24) had more differential
activity showing less inhibitory effect on normal cells. The
position of methyl group in pyridine ring of both amidino-
substituted groups of derivatives played a role in their antipro-
liferative activity, whereby the C-5 position diminished the
activity while its C-6 position led to very interesting and drastic
cell cycle perturbations. Lower concentrations of 22 and 29 (5
µM) led to an increase in the percentage of cells in G1, while
N-(6-(N-Isopropyl)amidinobenzothiazol-2-yl)benzamide Hydro-
chloride (7). White powder was isolated (0.184 g, 52.8%), mp )
296-298 °C. IR (KBr) (νmax/cm-1): 3417, 1682 (CdO), 1604
(CdN). 1H NMR (DMSO) (δ/ppm): 12.93 (brs, 1H, NH), 9.70 (d,
1H, J ) 7.83 Hz, Hamidine), 9.56 (s, 1H, Hamidine), 9.29 (s, 1H, Hamidine),

´
Caleta et al.
1752 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Table 3. Predictive Performances of the Best Volsurf Models Derived for Two Data Sets (A, Top, and B, Bottom, Both Containing 16 Compounds (for
Description, See Text)) Acting on Different Cell Line Types
b
c
model
A
cell line
HeLa
MiaPaCa-2
SW 620
MCF-7
HeLa
LVa
Q2
R2
SDEPd
SDECe
1
2
3
2
1
1
2
3
0.58
0.59
0.58
0.33
0.56
0.63
0.54
0.31
0.69
0.78
0.76
0.56
0.68
0.77
0.73
0.52
4.2
8.9
8.3
5.1
4.2
8.6
8.7
5.2
3.6
6.6
6.2
4.1
3.6
6.8
6.6
8.8
B
MiaPaCa-2
SW
620
MCF-7
b
a LV is the number of latent variables. Q2 is the cross-validated predictive performance and is given by Q2 ) 1 - (∑ni)1 (yexp(i) - ypred(i)))2)/(∑ni)1
(yexp (i) - yexp )2), where ypred corresponds to the value of IC50 predicted with the model for compound i, yexp is the experimentally determined inhibition, IC50,
c
of the compound. R2 is the equivalent of Q2 calculated for fitting. d SDEP is the standard deviation in cross-validated prediction and is given by SDEP )
[(∑in)1 (yexp(i) - ypred(i))2)/(n)]1/2 e SDEC is the equivalent of SDEP for fitting.
.
41.9%, mp ) 288-289 °C (lit.14 mp ) 288-289 °C). The 1H and
13C NMR spectra are in accordance with literature data.
6-(N-Isopropylamidino)-2-(N-(4-((N-isopropylamidino)pheny-
l))amino)benzothiazole Dihydrochloride (18). Beige powder was
isolated (0.11 g, 32.5%), mp ) 294-295 °C. IR (KBr) (νmax/cm-1):
1
3413, 3225, 1667, 1614. H NMR (DMSO) (δ/ppm): 12.10 (brs,
1H, NH), 9.60 (d, 1H, J ) 8.25 Hz, Hamidine), 9.53 (d, 1H, J ) 8.07
Hz, Hamidine), 9.46 (s, 1H, Hamidine), 9.42 (s, 1H, Hamidine), 9.15 (s,
1H, Hamidine), 9.07 (s, 1H, Hamidine), 8.31 (d, 1H, J ) 1.37 Hz), 8.09
(d, 2H, J ) 8.67 Hz), 7.82 (d, 3H, J ) 8.4 Hz), 7.71 (dd, 1H, J1
) 8.37 Hz, J2 ) 1.32 Hz), 4.17-4.07 (m, 2H, CH), 1.29 (d, 12H,
J ) 6.3 Hz, CH3). 13C NMR (DMSO) (δ/ppm): 163.99, 161.55,
161.06, 155.11, 144.33, 130.63, 129.58, 126.33, 123.04, 122.05,
121.98, 119.13, 117.40, 45.03, 44.93, 21.29, 21.27. Anal.
(C21H28Cl2N6S) C, H, N.
6-(N-Isopropyl)amidino-2-(N-(3-methylpyridin-2-yl)amino)ben-
zothiazole Dihydrochloride (19). Beige powder was isolated (0.2
g, 66.8%), mp ) 210-212 °C. IR (KBr) (νmax/cm-1): 3406, 3085,
1667, 1627. 1H NMR (DMSO) (δ/ppm): 9.60 (d, 1H, J ) 7.89 Hz,
Figure 8. Plot of experimental (X-axis) versus predicted (y-axis) growth
inhibition (IC50) for HeLa cell line with the outlier indicated (the
compound for which the discrepancy between the predicted and
experimental activity is larger than 2 times the standard deviation).
H
amidine), 9.47 (d, 1H, J ) 7.89 Hz, Hamidine), 9.14 (s, 1H, Hamidine),
8.36 (s, 1H), 8.30 (d, 1H, J ) 4.35 Hz), 7.80-7.72 (m, 3H), 7.09
(dd, 1H, J1 ) 6.80 Hz, J2 ) 5.44 Hz), 5.05 (brs, 2H, NH2+),
4.18-4.09 (m, 1H, CH), 2.41 (s, 3H, CH3), 1.29 (d, 6H, J ) 6.27
Hz, CH3). 13C NMR (DMSO) (δ/ppm): 163.37, 162.22, 152.32,
149.81, 143.09, 140.45, 131.95, 126.70, 123.14, 122.71, 121.79,
118.74, 118.50, 45.52, 21.80, 17.35. Anal. (C17H21Cl2N5S) C, H,
N.
8.50 (d, 1H, J ) 1.6 Hz), 8.16 (d, 2H, J ) 7.41 Hz), 7.94 (d, 1H,
J ) 8.47 Hz), 7.82 (dd, 1H, J1 ) 8.47 Hz, J2 ) 1.6 Hz), 7.71-7.56
(m, 3H), 4.24-4.11 (m, 1H, CH), 1.30 (d, 6H, J ) 6.3 Hz, CH3).
13C NMR (DMSO) (δ/ppm): 166.90, 162.53, 162.05, 152.11,
148.40, 133.65, 132.03, 129.19, 128.93, 126.89, 124.66, 123.36,
120.65, 45.62, 21.78. MS m/z: 339 (M+ - Cl, 100%). Anal.
(C18H19ClN4OS) C, H, N.
6-(N-Isopropyl)amidino-2-(N-(4-methylpyridin-2-yl)amino)ben-
zothiazole Dihydrochloride (20). Beige powder was isolated (0.15
g, 79.1%), mp ) 238-240 °C. IR (KBr) (νmax/cm-1): 3411, 3066,
1649, 1586. 1H NMR (DMSO) (δ/ppm): 9.56 (d, 1H, J ) 7.95 Hz,
2-Chloro-N-(6-(N-isopropyl)amidinobenzotiazol-2-yl)benza-
mide Hydrochloride (8). White powder was isolated (0.182 g,
69.9%), mp ) 291-292 °C. IR (KBr) (νmax/cm-1): 3414, 3053,
1
H
amidine), 9.42 (s, 1H, Hamidine), 9.07 (s, 1H, Hamidine), 8.34 (s, 1H),
1674 (CdO), 1622 (CdN). H NMR (DMSO) (δ/ppm): 13.27 (s,
8.27 (d, 1H, J ) 5.16 Hz), 7.77 (d, 1H, J ) 8.4 Hz), 7.71 (d, 1H,
J ) 8.4 Hz), 7.07 (s, 1H), 6.96 (d, 1H, J ) 5.1 Hz), 4.56 (brs, 2H,
NH2+), 4.15-4.04 (m, 1H, CH), 2.35 (s, 3H, CH3), 1.29 (d, 6H, J
) 6.24 Hz, CH3). 13C NMR (DMSO) (δ/ppm): 163.05, 162.30,
153.42, 151.44, 150.45, 146.09, 132.28, 126.57, 122.86, 122.56,
119.51, 119.18, 112.20, 45.49, 21.78, 21.28. Anal. (C17H21Cl2N5S)
C, H, N.
6-(N-Isopropyl)amidino-2-(N-(5-methylpyridin-2-yl)amino)ben-
zothiazole Dihydrochloride (21). White powder was isolated (0.28
g, 92.8%), mp ) 246-248 °C. IR (KBr) (νmax/cm-1): 3439, 3055,
1668, 1650. 1H NMR (DMSO) (δ/ppm): 9.61 (d, 1H, J ) 8.04 Hz,
1H, NH), 9.68 (d, 1H, J ) 7.11 Hz, Hamidine), 9.53 (s, 1H, Hamidine),
9.20 (s, 1H, Hamidine), 8.51 (s, 1H), 7.97 (d, 1H, J ) 8.46 Hz), 7.80
(d, 1H, J ) 8.31 Hz), 7.73 (d, 1H, J ) 7.56 Hz), 7.64-7.48 (m,
3H), 4.18-4.07 (m, 1H, CH), 1.30 (d, 6H, J ) 6.12 Hz, CH3). 13
C
NMR (DMSO) (δ/ppm): 166.52, 162.13, 161.38, 152.28, 134.21,
132.88, 132.13, 130.78, 130.41, 130.11, 127.84, 126.92, 124.89,
123.40, 121.15, 121.13, 45.59, 21.75. Anal. (C18H18Cl2N4OS) C,
H, N.
4-Fluoro-N-(6-(N-isopropyl)amidinobenzotiazol-2-yl)benza-
mid Hydrochloride (9). White powder was isolated (0.137 g,
52.0%), mp ) 296 - 298 °C. IR (KBr) (νmax/cm-1): 3428, 3052,
1675 (CdO), 1604 (CdN). 1H NMR (DMSO) (δ/ppm): 13.21 (brs,
1H, NH), 9.66 (d, 1H, J ) 7.5 Hz, Hamidine), 9.54 (s, 1H, Hamidine),
9.24 (s, 1H, Hamidine), 8.49 (s, 1H), 8.27-8.22 (m, 2H), 7.95 (d,
1H, J1 ) 8.46 Hz), 7.81 (dd, 1H, J1 ) 8.49 Hz, J2 ) 1.29 Hz),
7.46-7.40 (m, 2H), 4.17-4.08 (m, 1H, CH), 1.31 (d, 6H, J ) 6.3
Hz, CH3). 13C NMR (DMSO) (δ/ppm): 165.79, 165.53, 164.66,
164.13, 162.97, 131.98, 131.39, 128.16, 126.50, 123.17, 120.61,
116.90, 115.69, 45.64, 21.79. Anal. (C18H18ClFN4OS) C, H, N.
4-(N-Isopropylamidino)-N-(benzothiazol-2-yl)benzamide Hy-
drochloride (10). 10 was prepared by the above-described procedure
to give physical constants in accordance with literature values, yield
H
amidine), 9.48 (s, 1H, Hamidine), 9.19 (s, 1H, Hamidine), 8.37 (s, 1H),
8.25 (s, 1H), 7.78-7.71 (m, 3H), 7.26 (d, 1H, J ) 8.43 Hz), 5.60
(brs, 2H, NH2+), 4.20-4.09 (m, 1H, CH), 2.28 (s, 3H, CH3), 1.29
(d, 6H, J ) 6.36 Hz, CH3). 13C NMR (DMSO) (δ/ppm): 162.94,
162.10, 153.11, 149.17, 145.27, 140.69, 131.90, 127.29, 126.71,
122.87, 122.72, 118.99, 112.23, 45.53, 21.81, 17.70. Anal.
(C17H21Cl2N5S) C, H, N.
6-(N-Isopropyl)amidino-2-(N-(6-methylpyridin-2-yl)amino)ben-
zothiazole Dihydrochloride (22). White powder was isolated (0.24
g, 87.1%), mp > 300 °C. IR (KBr) (νmax/cm-1): 3413, 3052, 1668,
1620. 1H NMR (DMSO) (δ/ppm): 9.48 (d, 1H, J ) 7.92 Hz,

Benzothiazole DeriVatiVes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1753
Figure 9. Most important physicochemical descriptors in models: SW 620, black; MiaPaCa-2, white; HeLa, red. Explanation of descriptors are as follows:
V, volume; S, surface; W1-W6, molecular envelopes accessible by solvent water molecules: W1-W4, account for dispersion interaction and polarizability,
while higher W (W5 and higher), account for polar and hydrogen bond donor-acceptor regions; D1-D6, hydrophobic regions indicating interactions with
the hydrophobic probe at six different energy levels (-0.2, -0.4, -0.6, -0.8, -1.0, -1.2) × 4.186 kJ/mol; WO1-WO4, volumes of oxygen interaction
fields at four different energy levels (-0.2, -0.4, -0.6, -0.8) × 4.186 kJ/mol; WN1 and WN2, volume of nitrogen interaction fields at two different
energy levels (-0.2, -0.4) × 4.186 kJ/mol; MW, molecular weight; HAS, hydrophobic surface area; VD, a measure of relative partition of compounds
between plasma and tissue.
Hamidine), 9.37 (s, 1H, Hamidine), 9.07 (s, 1H, Hamidine), 8.29 (s, 1H),
7.69-7.61 (m, 3H), 6.99 (d, 1H, J ) 8.04 Hz), 6.87 (d, 1H, J )
7.32 Hz), 4.84 (brs, 2H, NH2+), 4.09-4.04 (m, 1H, CH), 2.46 (s,
3H, CH3), 1.22 (d, 6H, J ) 6.36 Hz, CH3). 13C NMR (DMSO)
(δ/ppm): 162.44, 161.62, 155.17, 152.71, 150.23, 138.99, 131.74,
126.06, 122.16, 122.11, 118.49, 116.71, 108.53, 45.03, 23.15, 21.31.
Anal. (C17H21Cl2N5S) C, H, N.
126.19, 122.86, 122.25, 119.07, 115.25, 45.07, 21.29. Anal.
(C15H18Cl2N6S) C, H, N.
6-Imidazolinyl-2-(N-(4-imidazolinylphenyl)amino)ben-
zothiazole Dihydrochloride (25). Yellow powder was isolated (0.105
g, 34%), mp ) 275-277 °C. IR (KBr) (νmax/cm-1): 3413, 3111,
2971, 1614. 1H NMR (DMSO) (δ/ppm): 12.18 (brs, 1H, NH), 10.67
(s, 4H, Hamidine), 8.55 (s, 1H), 8.03 (d, 2H, J ) 8.82 Hz), 8.04 (d,
2H, J ) 8.82 Hz), 7.95 (dd, 1H, J1 ) 8.46 Hz, J2 ) 1.2 Hz), 7.82
(d, 1H, J ) 8.46 Hz), 3.92 (s, 4H, CH2), 3.70 (s, 4H, CH2). 13C
NMR (DMSO) (δ/ppm): 164.53, 164.39, 163.95, 155.91, 145.16,
131.11, 130.15, 126.68, 122.30, 119.76, 117.72, 116.00, 115.21,
44.26, 44.09. Anal. (C19H20Cl2N6S) C, H, N.
6-(N-Isopropyl)amidino-2-(N-(5-chloropyridin-2-yl)amino)ben-
zothiazole Dihydrochloride (23). Beige powder was isolated (0.191
g, 65.4%), mp ) 220-223 °C. IR (KBr) (νmax/cm-1): 3412, 3060,
1675, 1617). 1H NMR (DMSO) (δ/ppm): 9.6 (d, 1H, J ) 7.98 Hz,
Hamidine), 9.47 (s, 1H, Hamidine), 9.15 (s, 1H, Hamidine), 8.45 (d, 1H, J
) 2.22 Hz), 8.38 (s, 1H), 7.93 (dd, 1H, J1 ) 8.84 Hz, J2 ) 2.44
Hz), 7.77 (d, 1H, J ) 8.61 Hz), 7.72 (d, 1H, J ) 8.61 Hz), 7.34 (d,
1H, J ) 8.84 Hz), 4.97(brs, 2H, NH2+), 4.18-4.07 (m, 1H, CH),
1.29 (d, 6H, J ) 6.27 Hz, CH3). 13C NMR (DMSO) (δ/ppm):
162.13, 161.67, 152.83, 149.93, 144.71, 138.40, 131.63, 126.16,
123.65, 122.48, 122.19, 118.80, 113.15, 45.03, 21.29. Anal.
(C16H18Cl3N5S) C, H, N.
6-Imidazolinyl-2-(N-(3-methylpyridin-2-yl)amino)benzothiaz-
ole Dihydrochloride (26). White powder was isolated (0.12 g,
40.5%), mp > 300 °C. IR (KBr) (νmax/cm-1): 3413, 3098, 2967,
1
1629, 1611. H NMR (DMSO) (δ/ppm): 10.76 (s, 2H, Hamidine),
8.70 (s, 1H), 8.29 (d, 1H, J ) 4.68 Hz), 8.06 (d, 1H, J ) 8.55 Hz),
7.78 (d, 1H, J ) 8.55 Hz), 7.68 (d, 1H, J ) 7.2 Hz), 7.07 (dd, 1H,
J1 ) 7.12 Hz, J2 ) 5.11 Hz), 4.00 (s, 4H, CH2), 3.76 (brs, 2H,
NH2+), 2.39 (s, 3H, CH3). 13C NMR (DMSO) (δ/ppm): 164.68,
163.40, 149.31, 143.08, 142.59, 139.55, 132.03, 126.41, 122.53,
120.98, 118.63, 118.08, 115.09, 44.20, 16.74. Anal. (C16H17Cl2N5S)
C, H, N.
6-(N-Isopropyl)amidino-2-(N-(pyrimidin-2-yl)amino)benzothia-
zole Dihydrochloride (24). Beige powder was isolated (0.125 g,
41.1%), mp > 300 °C. IR (KBr) (νmax/cm-1): 3363, 3138, 1669,
1611. 1H NMR (DMSO) (δ/ppm): 9.64 (d, 1H, J ) 8.07 Hz,
Hamidine), 9.50 (s, 1H, Hamidine), 9.20 (s, 1H, Hamidine), 8.76 (d, 2H, J
6-Imidazolinyl-2-(N-(4-methylpyridin-2-yl)amino)benzothiaz-
ole Dihydrochloride (27). White powder was isolated (0.12 g,
42.2%), mp > 300 °C. IR (KBr) (νmax/cm-1): 3423, 3051, 2941,
) 4.8 Hz), 8.41 (s, 1H), 7.81 (d, 1H, J ) 8.43 Hz), 7.74 (d, 1H, J
) 8.49 Hz), 7.19 (t, 1H, J ) 4.83 Hz), 5.89 (brs, 2H, NH2+),
4.19-4.09 (m, 1H, CH), 1.29 (d, 6H, J ) 6.24 Hz, CH3). 13C NMR
(DMSO) (δ/ppm): 162.70, 161.69, 158.20, 156.71, 152.53, 131.95,
1
2690, 1653. H NMR (DMSO) (δ/ppm): 10.71 (s, 2H, Hamidine),
8.64 (d, 1H, J2 ) 1.71 Hz), 8.21 (d, 1H, J ) 4.98 Hz), 7.97 (dd,

´
1754 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Caleta et al.
1H, J1 ) 8.52 Hz, J2 ) 1.71 Hz), 7.71 (d, 1H, J ) 8.52 Hz), 6.99
(s, 1H), 6.89 (d, 1H, J ) 4.98 Hz), 4.13 (brs, 2H, NH2+), 3.91 (s,
4H, CH2), 2.26 (s, 3H, CH3). 13C NMR (DMSO) (δ/ppm): 164.54,
163.14, 153.75, 150.63, 150.29, 145.36, 132.08, 126.46, 122.61,
119.19, 119.05, 115.09, 111.88, 44.17, 20.80. Anal. (C16H17Cl2N5S)
C, H, N.
linear regression analysis by fitting the test concentrations that give
PG values above and below the reference value (i.e. 50%). Each
result is the mean value from three separate experiments.
Cell Cycle Analysis. The 2 × 105 cells were seeded per well
in a six-well plate. After overnight incubation, tested compounds
were added. After the desired length of time, the attached cells
were trypsinized, combined with floating cells, washed with PBS,
and fixed with 70% ethanol. Immediately before analysis, the
cells were washed with PBS and incubated with 0.2 µg/µL RNase
A at 37 °C for 15 min. Subsequently, cells were stained with
50 µg/mL of propidium iodide (PI) and analyzed by Becton
Dickinson FACSCalibur flow cytometer. For each analysis,
20000 events were measured. Measurements were performed in
duplicate for two independent experiments. The percentage of
the cells in each cell cycle phase was based on the obtained
DNA histograms and determined using the ModFit LT software
(Verity Software House).
6-Imidazolinyl-2-(N-(5-methylpyridin-2-yl)amino)benzothiaz-
ole Dihydrochloride (28). White powder was isolated (0.19 g,
67.1%), mp > 300 °C. IR (KBr) (νmax/cm-1): 3370, 3082, 2961,
1
1654, 1590. H NMR (DMSO) (δ/ppm): 10.75 (s, 2H, Hamidine),
8.71 (d, 1H, J ) 1.38 Hz), 8.25 (s, 1H), 8.04 (dd, 1H, J1 ) 8.55
Hz, J2 ) 1.65 Hz), 7.18 (d, 1H, J ) 8.37 Hz), 4.23 (brs, 2H, NH2+),
3.99 (s, 4H, CH2), 2.27 (s, 3H, CH3). 13C NMR (DMSO) (δ/ppm):
164.68, 163.11, 154.02, 148.79, 145.68, 139.52, 132.11, 126.78,
126.39, 122.50, 118.90, 114.81, 111.41, 44.19, 17.22. Anal.
(C16H17Cl2N5S) C, H, N.
6-Imidazolinyl-2-(N-(6-methylpyridin-2-yl)amino)benzothiaz-
ole Dihydrochloride (29). White powder was isolated (0.12 g,
41.4%), mp > 300 °C. IR (KBr) (νmax/cm-1): 3385, 2958, 2545,
Detection of Apoptosis. Annexin-V Assay. Detection and
quantification of apoptotic cells at the single cell level were
performed by staining the cells with annexin-V Alexa Fluor 488
conjugate (Molecular Probes, Invitrogen) and 7-aminoactino-
mycin D (7-AAD, Invitrogen). The 2 × 105 cells were seeded
per well into a six-well plate and analyzed on a FACSCalibur
flow cytometer (Becton Dickinson, San Jose, CA). The tested
compounds were added to the attached cells at various concen-
trations (as shown in the results section). After the desired length
of time, both floating and attached cells were collected. The cells
were then washed with annexin-V binding buffer (10 mM
HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4), pelleted, and
incubated for 20 min in staining solution (annexin-V Alexa Fluor
488 conjugate and 1 µg/mL 7-AAD in annexin-V binding buffer).
The cells were then run on a flow cytometer and analyzed in
Summit software (Cytomation Inc., CO). Annexin-V positive
cells were determined to be apoptotic, and double positive cells
(both annexin-V and 7-AAD) were determined to be necrotic.
1
1648 (CdN). H NMR (DMSO) (δ/ppm): 10.67 (s, 2H, Hamidine),
8.62 (s, 1H), 7.97 (dd, 1H, J1 ) 8.43 Hz, J2 ) 1.23 Hz), 7.69 (d,
1H, J ) 8.52 Hz), 7.63 (dd, 1H, J1 ) 7.74 Hz, J2 ) 7.68 Hz), 6.96
(d, 1H, J ) 8.04 Hz), 6.86 (d, 1H, J ) 7.32 Hz), 3.92 (s, 4H,
CH2), 3.87 (brs, 2H, NH2+), 2.46 (s, 3H, CH3). 13C NMR (DMSO)
(δ/ppm): 164.58, 163.03, 155.36, 153.90, 150.09, 138.86, 132.26,
126.38, 122.45, 118.96, 116.80, 114.82, 108.50, 44.18, 23.25. Anal.
(C16H17Cl2N5S) C, H, N.
6-Imidazolinyl-2-(N-(5-chloropyridin-2-yl)amino)benzothiaz-
ole Dihydrochloride (30). Beige powder was isolated (0.14 g, 53.3%),
mp > 300 °C. IR (KBr) (νmax/cm-1): 3411, 3090, 2893, 1625. 1H NMR
(DMSO) (δ/ppm): 10.75 (brs, 2H, Hamidine), 8.68 (d, 1H, J2 ) 1.71
Hz), 8.37 (d, 1H, J ) 2.46 Hz), 7.98 (dd, 1H, J1 ) 8.58 Hz, J2 ) 1.71
Hz), 7.84 (dd, 1H, J1 ) 8.82 Hz, J2 ) 2.46 Hz), 7.70 (d, 1H, J ) 8.58
Hz), 7.22 (d, 1H, J ) 8.76 Hz), 4.64 (brs, 2H, NH2+), 3.91 (s, 4H,
CH2). 13C NMR (DMSO) (δ/ppm): 164.51, 162.75, 153.74, 149.71,
144.70, 138.42, 132.03, 126.48, 123.81, 122.70, 119.18, 115.18,
113.20, 44.18. Anal. (C15H14Cl3N5S) C, H, N.
6-Imidazolinyl-2-(N-(pyrimidin-2-yl)amino)benzothiazole Dihy-
drochloride (31). Beige powder was isolated (0.136 g, 48.9%), mp
> 300 °C. IR (KBr) (νmax/cm-1): 3415, 3084, 2972, 1611, 1591.
1H NMR (DMSO) (δ/ppm): 10.67 (s, 2H, Hamidine), 8.77-8.71 (m,
3H), 8.07 (dd, 1H, J1 ) 8.60 Hz, J2 ) 1.75 Hz), 7.84 (d, 1H, J )
8.55 Hz), 7.19 (t, 1H, J ) 4.83 Hz), 4.03 (s, 4H, CH2), 3.48 (brs,
2H, NH2+). 13C NMR (DMSO) (δ/ppm): 158.61, 150.99, 144.87,
134.48, 133.14, 130.67, 126.87, 123.09, 120.17, 116.16, 115.93,
44.87. Anal. (C14H14Cl2N6S) C, H, N.
Antitumor Activity Assays. The HeLa, MCF-7, SW 620,
MiaPaCa-2, Hep-2, H 460, and WI 38 cells were cultured as
monolayers and maintained in DMEM supplemented with 10%
FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/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
National Cancer Institute, Developmental Therapeutics Program.
The cells were inoculated onto standard 96-well microtiter plates
on day 0 at 3 × 103 to 6 × 103 cells/well, depending on the
doubling times of specific cell line. Test agents were then added
in five consecutive 10-fold dilutions (10-8-10-4 mol/L) and
incubated for a further 72 h. Working dilutions were freshly
prepared on the day of testing. The solvent (DMSO) was also
tested for eventual inhibitory activity by adjusting its concentra-
tion to be the same as the working concentrations (maximal
concentration of DMSO was 0.25%). After 72 h of incubation,
the cell growth rate was evaluated by performing the MTT
assay,33,34 which detects dehydrogenase activity in viable cells.
The absorbance (OD) was measured on a microplate reader at
570 nm.
Principal Components and Partial Least Square Analysis.
Smile codes of the molecules were used as input for the program
Volsurf.32,35 Volsurf utilizes information from the molecular
interaction fields (MIF) determined by GRID36 to calculate a series
of physicochemically relevant molecular descriptors.
For each MIF the interaction energy between a molecule of
known three-dimensional structure (“target”), generated by
Volsurf, and a small chemical group (“probe”) was computed
for a set of positions of the probe around the target. Probes that
mimic functional groups that may be present in a protein (e.g.,
receptor or transporter) active site (H2O, -NH2+, -CH3, O, and
DRY probe) were selected. For each probe thousands of
interaction energies were calculated at the nodes of the regular
three-dimensional grid around the target. Volsurf used calculated
MIFs to derive a series of 126 scalar descriptors that describe
molecular size, shape, solubility, distribution of hydrophilic and
hydrophobic regions, lipophilicity, flexibility, and presence/
distribution of pharmacophoric descriptors. Besides describing
pharmacokinetic processes,37 Volsurf has been used to also
describe ligand-protein interactions and antibacterial activity.38,39
In order to determine most variable descriptors (X-variables)
within the set of compounds and the compounds distribution in
the space of the descriptors, we conducted principal components
(PC) analysis. Further, to derive the relationship between several
most relevant X-variables and the measured inhibition, we per-
formed partial least-squares (PLS) statistical analysis. In this way,
we derived QSAR models that connect physicochemical molecular
descriptors with the specific response of certain cell line. The
predictivity of the models was evaluated by using “internal
validation”, by means of standard leave-one-out (LOO) analysis.
Each test point was performed in quadruplicate in three individual
experiments. The results are expressed as IC50, which is the
concentration necessary for 50% of inhibition. The IC50 values for
each compound are calculated from dose-response curves using
Acknowledgment. Support for this study by the Ministry
of Science, Education and Sport of Croatia is gratefully
acknowledged (Projects 125-0982464-1356, 098-0982464-

Benzothiazole DeriVatiVes
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2514, 098-0982464-2393, 098-1191344-2860, and 119-
119307-1332).
Supporting Information Available: Experimental and spec-
troscopic data for cyano derivatives 1-6 and 11-17 with the
reference list, elemental analysis results of novel compounds,
X-ray crystal structure analysis results, and selected crystal-
lographic data for compound 15. This material is available free
of charge via the Internet at http://pubs.acs.org. The CCDC
number 709670 for compound 15 contains the supplementary
crystallographic data for this paper. These data can be obtained
free of charge at www.ccdc.cam.ac.uk/conts/retrieving.html [or
from the Cambridge Crystallographic Data Centre (CCDC), 12
Union Road, Cambridge CB2 1EZ, U.K.; fax, +44(0)1223-
336033; e-mail, deposit@ccdc.cam.ac.uk]. A table of structure factors
is available from the authors.
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