Synthesis and biological evaluation of imidazolo[2,1-b]benzothiazole derivatives, as potential p53 inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.01.039

Since activation of p53 in response to cytotoxic stress may have proapoptotic or protective effects depending on the nature of the injury, inhibitors of p53 may have therapeutic interest as modulators of chemotherapy toxicity or efficacy. In an attempt to

Full text of DOI:10.1016/j.bmc.2011.01.039

Bioorganic & Medicinal Chemistry 19 (2011) 1649–1657
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of imidazolo[2,1-b]benzothiazole
derivatives, as potential p53 inhibitors
Michael S. Christodoulou b, Francesco Colombo a, Daniele Passarella a, , Gabriella Ieronimo a,
⇑
Valentina Zuco c, Michelandrea De Cesare c, Franco Zunino c,
⇑
a Dipartimento di Chimica Organica e Industriale, Università degli Studi di Milano, Via Venezian 21, 20133 Milano, Italy
b Chemistry Laboratory, Agricultural University of Athens, Iera Odos 75, Athens 11855, Greece
c Fondazione IRCCS Istituto Nazionale per lo Studio e la Cura dei Tumori, Via Venezian 1, 20133 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Since activation of p53 in response to cytotoxic stress may have proapoptotic or protective effects
depending on the nature of the injury, inhibitors of p53 may have therapeutic interest as modulators
of chemotherapy toxicity or efficacy. In an attempt to identify novel p53 inhibitors, a quality collection
of compounds structurally related to pifithrin-b were designed and synthesized as potential inhibitors
of p53. The biochemical and biological evaluations supported that compounds of the tetrahydrobenzo-
thiazole series were inhibitors of the p53 transcriptional activity and were effective in enhancing paclit-
axel-induced apoptosis. In contrast, in spite of the increased cytotoxic potency, selected compounds of
the benzothiazole series were not able to modulate the transcriptional activity of p53, as indicated by lack
of change of p21 expression. The therapeutic interest of the compounds of the former series in combina-
tion with taxanes was confirmed in a human tumor xenograft model.
Received 30 November 2010
Revised 14 January 2011
Accepted 19 January 2011
Available online 25 January 2011
Keywords:
p53 Inhibitors
Analogues of pifithrin-b
Benzothiazole derivatives
Tetrahydrobenzothiazole derivatives
Biochemical and biological evaluation
Combined treatment with taxanes
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
conditions, targeting p53 function may have therapeutic relevance
to improve the effects of mitotic spindle damage. In addition, when
The tumor suppressor protein p53 is a transcription factor,
which plays a critical role in cellular response to a variety of stres-
ses including DNA damage.1,2 The activation of p53 may induce
growth arrest or apoptosis depending on the extent or nature of
stress signals.1–3 Based on these functions, p53 can modulate tu-
mor cell sensitivity to chemotherapy or radiation. Since p53 has
been implicated as a determinant of apoptosis induced by DNA
damage, the loss of p53 function, as a consequence of mutation,
could result in a relative drug resistance of tumors carrying p53
mutations.4,5 However, the role of p53 in response to cytotoxic
treatment is dependent on the activation of distinct functions
and molecular context. For example, p53-mediated transcriptional
activation of p21 after genotoxic stress may result in cell cycle ar-
rest allowing DNA repair.6,7 In this case, p53 plays a role of survival
factor by allowing cells to reside in a growth arrested state, thereby
reducing the risk of accumulation of genotoxic lesions during cell
cycle progression. A protective role of the transcriptional activity
of p53 has been documented in response to antimicrotubule
agents.8 Indeed, cyclic pifithrin (PFT-b, Fig. 1)9 sensitizes wild-type
p53 tumor cells to paclitaxel (PTX) and vinca alkaloids. Under these
apoptosis is impaired as a consequence of alterations in the apop-
totic process, wild-type p53 may have a prosurvival function in re-
sponse to chemotherapy through prolonged growth arrest
allowing DNA repair. Therefore, although p53 inhibitors have been
developed as potential modulators of chemotherapy-induced tox-
icity,10–12 these agents may have potential applications to sensitize
tumor cells under particular treatment conditions. In the present
study, we report the synthesis of novel PFT-b analogues (Fig. 1)
and the biological evaluation of the obtained compounds.
2. Results and discussion
2.1. Chemistry
We considered as possible modifications (a) the replacing of the
tetrahydrobenzothiazole ring with a benzothiazole portion, (b) the
functionalization of the methyl group, and (c) the replacement of
the sulfur atom with alkylated nitrogen (Fig. 1). For the synthesis of
analogues 3a–f (Scheme 1) the commercially available 2-aminoben-
zothiazole 1a was used, while the starting 2-aminotetrahydrobenzo-
thiazole 1b was obtained following the procedure described by King
and Hlavacek13 in which cyclohexanone was reacted with thiourea
in the presence of iodine. Condensation of benzothiazoles 1a and 1b
⇑
Corresponding authors. Tel.: +39 02 50314081; fax: +39 02 50314078 (D.P.).
E-mail address: daniele.passarella@unimi.it (D. Passarella).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.01.039

1650
M. S. Christodoulou et al. / Bioorg. Med. Chem. 19 (2011) 1649–1657
S
N
Continuously, the reduction of nitriles 3a and 3e with DIBALH in
X
N
toluene provided the desired amines 5a and 5e in excellent yields
(90%). Nitro derivatives 3b and 3f were also transformed to the cor-
responding amines 6b and 6f using H2SO4 in the presence of Fe in
excellent yields (89%) (Scheme 2). The transformation of amines 5
and 6 in the corresponding hydrochloride salts was realized by a
treatment with a solution of satd HCl in MeOH .
N
N
R
PFT-β
PFT-β analogues
In order to investigate further the effects of substitution at the
para position of the right phenyl ring, several derivatives were
prepared. Acylation of amines 5a and 5e with acetylchloride in
DMF provided the corresponding amide analogues 7a and 7e,
respectively, in very high yields (85%). Analogue 8 was obtained
via an aza-Michael addition of N-isopropylacrylamide in H2O, while
analogue 9 was prepared in a two step synthesis, using the
Boc-protected b-alanine and subsequent removal of the terbutoxy-
carbonyl group (Scheme 3).
The obtainment of alcohol 12 and aldehyde 13 was planned in
two different approaches: (a) transformation of nitrile to the alde-
hyde and subsequent reduction and (b) reduction to alcohol and
oxidation to the desired aldehyde. Transformation of nitrile 3a to
aldehyde 13 using DIBALH in different solvents and temperatures
resulted unsuccessfully. Thus, nitrile 3a was first transformed into
acid 10 in basic conditions in almost quantitative yield (96%). The
subsequent reduction of acid 10 by LiAlH4 in THF afforded the de-
sired alcohol 12 in very low yield (10%). Therefore, acid 10 was
converted by trimethylsilyldiazomethane into the corresponding
Figure 1. Structure of pifithrin-b (PFT-b) and the general structure of designed
related analogues.
with a variety of a-bromoacetophenones 2a–d provided the corre-
sponding closed ring derivatives 3a–f in moderate yields.
The use of 2-aminobenzoimidazole 1c and 1-methyl-2-amin-
obenzoimidazole 1d permitted the obtainment of the compounds
that present the nitrogen atom replacing the sulfur atom. However,
the condensation of imidazoles 1c and 1d with 2-bromo-40-CN-
acetophenone 2a provided the open forms 4g (yield 60%) and 4h
(yield 75%), respectively, as the main products. Only in the case
of imidazole 1d a very low yield (5%) of the closed product 3h
was observed. Having this evidence, we considered to introduce
an aliphatic chain on the N-1 of the 2-aminobenzoimidazole. The
1-ethyl-2-aminobenzoimidazole 1e was easily prepared from 1c
using ethyliodide in the presence of KOH. Although the condensa-
tion of 1e with 2-bromo-40-CN-acetophenone 2a provided the de-
sired derivative 3i in higher yield (20%), still the open form 4i
remained the main product (60%) of the reaction (Scheme 1).
4''
3''
3' 'a
X
5''
6''
8
.
9
NH HBr
8a
4a
2''
X
7
9a
7''a N
1' '
O
1
7''
R
O
N
X
N
6
Br
N
4
2
a
3'
1
2
+
5
R
NH2
+
4'
1'
2'
2'
1'
3
R1
3'
R
R1
4'
R1
1
2
3
4
, R1 = NO2
, R1 = CN
, R1 = CN
3f, 4f X = S, R =
1a X = S, R =
2a R1 = CN
3a, 4a X = S, R =
2b
R
1 = NO2
2c R1 = Br
2d
, R1 = NO2
, R1 = Br
3b, 4b X = S, R =
3c, 4c X = S, R =
3g, 4g
3h, 4h
1b
1c
X= NH, R =
X = S, R =
R1 = CH3
, R1
CN
=
X = NCH3, R =
X = NH, R =
, R1 = CH3
, R1 = CN
3d, 4d
3e, 4e
X = S, R =
X = S, R =
,
3i, 4i X = NCH2CH3, R =
1d X = NCH3, R =
R1
=
CN
1e X = NCH2CH3, R =
Scheme 1. Reagents and condition: (a) EtOH, reflux, 90 min.
S
N
S
a
b
a
b
N
N
3a
3b
3e
3f
N
5a
CH2NH2
5e
CH2NH2
S
N
S
N
N
N
6b
NH2
6f
NH2
Scheme 2. Reagents and conditions: (a) DIBALH, toluene, reflux, 2 h; (b) Fe, H2SO4/H2O, 90 °C, 1 h.

M. S. Christodoulou et al. / Bioorg. Med. Chem. 19 (2011) 1649–1657
1651
S
a
N
N
R
H
N
7
7a
7e
R =
R =
O
S
N
S
b
N
N
N
R
H
N
H
N
CH2NH2
5
8
O
S
5a
R =
c
d
N
N
5e R =
H
N
NH2
9
O
Scheme 3. Reagents and conditions: (a) acetylchloride, Et3N, DMF, 24 h; (b) N-isopropylacrylamide, H2O, reflux, 16 h; (c) N-Boc-b-alanine, HATU, DIPEA, 20 h; (d) TMSCl,
MeOH, 1 h.
S
N
S
N
N
N
a
e
3a
3c
R
10
11
R = COOH
14
b
c
d
OCH3
R = COOCH3
12 R = CH2OH
13 R = CHO
Scheme 4. Reagents and conditions: (a) NaOH, EtOH, H2O, reflux, 4 h; (b) trimethylsilyldiazomethane, MeOH, 30 min; (c) LiAlH4, THF, 0 °C, 30 min; (d) Dess–Martin
periodinane, DCM, 20 min; (e) 4-methoxyphenylboronic acid, Na2CO3, Pd(PPh3)4, THF, EtOH, H2O, reflux, 12 h.
Table 1
ester 11 that by reaction with LiAlH4 provided alcohol 12 in good
yield. Oxidation of alcohol 12 using Dess–Martin periodinane in
DCM furnished aldehyde 13 in quantitative yield (Scheme 4). The
S
availability of the bromide derivative 3c permitted the application
of Suzuki coupling reaction with 4-methoxyphenyl boronic acid in
N
N
EtOH to obtain compound 14.
R
2.2. Biological activity
Compound
R
IC50
(
lV)
c log P**
*
The biological activity of all compounds described in the pres-
ent study was performed with an antiproliferative assay in the hu-
man ovarian carcinoma cell line, IGROV-1, following 72 h-
exposure. This cell system was chosen, because it is able to grow
in athymic nude mice. The results obtained in this cell system
show that the compounds with the tetrahydrobenzothiazole scaf-
fold of PFT-b (Table 1) retained a biological activity comparable
to that of the parent compound (i.e., IC50 values in the range of
PFT-b
3e
5e
CH3
CN
CH2NH2
CH2NH2ꢀHCl
NH2
23
8
12
31
4
4
3
2
3
2
4
2
4.397
3.703
3.133
0.588
3.024
0.691
2.868
5eꢀHCl
6f
6fꢀHCl
7e
NH2ꢀHCl
19
28
CH2NHCOCH3
*
IC50 = concentration of compound that reduces cell proliferation by 50%.
c log P = calculated octanol–water partition coefficient.
**
5–30 lM).
In contrast, the compounds of the benzothiazole series (Table 2)
exhibited a substantial increase of the antiproliferative potency
(i.e., IC50 values in the nanomolar range).
compounds 6f and 5eꢀHCl were able to reduce the up-regulation
of p21 induced by paclitaxel. In contrast 5aꢀHCl, benzothiazole
analogue of 5eꢀHCl, was not able to modulate the expression of
p21, thus suggesting that the biological activity of the benzothiaz-
oles was independent from the inhibition of p53. Thus the mecha-
nism of action of compounds of the latter series remains to be
determined.
The values of c log P14 for the compounds 3–14 are generally
lower than the one related to PFT-b, which indicates that new com-
pounds present a reduced lipophilicity.
Selected compounds of the two series were tested for their abil-
ity to modulate the expression of p53 and p21, a well-known p53
target, after paclitaxel (PTX) treatment.8 Figure 2 shows that the

1652
M. S. Christodoulou et al. / Bioorg. Med. Chem. 19 (2011) 1649–1657
Table 2
explored the ability of 6f, the most potent analogue of the tetra-
hydrobenzothiazole series, to modulate the tumor cell sensitivity
to paclitaxel. Using drug concentrations causing only antiprolifera-
tive effects (IC50), as indicated by the low level of apoptosis, the
combination of paclitaxel and 6f resulted in a substantial enhance-
ment of apoptosis induction (Fig. 3), thus suggesting that the cellu-
lar sensitization by the p53 inhibitor reflected an increased
susceptibility to apoptosis resulting in a cytotoxic, rather than
cytostatic, outcome.
X
N
N
R
c log P**
*
Compound
X
R
IC50
(
lV)
On the basis of this observation, the efficacy of this combination
was evaluated in vivo using the human osteosarcoma U2OS xeno-
graft, a model characterized by wild-type p53 (Fig. 4). Using well-
tolerated doses of each agent, the combination resulted in an in-
crease of efficacy as compared to paclitaxel in terms of tumor
growth-inhibition and complete response.
3a
3b
3c
3d
3h
3i
S
S
S
S
CN
NO2
Br
CH3
CN
0.03 0.01 3.967
2
1
4.171
5.021
4.661
3.290
3.666
0.3 0.1
0.3 0.2
7
NCH3
NCH2CH3 CN
3
4.5 0.5
5a
S
S
S
S
S
S
S
S
S
S
CH2NH2
0.03 0.01 3.396
5aꢀHCl
6b
6bꢀHCl
7a
8
9
12
13
14
CH2NH2ꢀHCl
0.3 0.1
0.852
3.288
0.955
3.132
3.821
2.167
3.550
4.002
6.064
NH2
1
5
0.8
2
NH2ꢀHCl
3. Discussion
CH2NHCOCH3
12
5
4
2
CH2NH(CH2)2CONHCH(CH3)2
CH2NHCO(CH2)2NH2
CH2OH
CHO
Ph (pOCH3)
In this report we described a novel series of compounds structur-
ally related to pifithrin-b as potential inhibitors of p53. These com-
pounds were prepared in an attempt to identify novel analogues
more suitable for in vivo use than the parent compound. We show
evidence that selected compounds of the tetrahydrobenzothiazole
series (i.e., 6f and 5e containing an amino group in the side chain),
characterized by improved physico-chemical properties in terms
of solubility and stability, also exhibited an increased potency as
antiproliferative agents. These changes are expected to influence
the pharmacokinetic behaviour. In particular, the changed mole-
cules may have a reduced uptake of an intracellular accumulation.
Indeed the changed forms of 5e (Table 1) and 5a (Table 2) exhibited
a reduced antiproliferative activity. This finding is consistent with a
modulation of the cellular pharmacokinetic behaviour. The water-
solubility of 5eꢀHCl and 5aꢀHCl allowed iv administration. At the
tested doses, both compounds, were well tolerated (Table 3). The
significant antitumor activity of 5aꢀHCl was consistent with its in-
creased antiproliferative activity as compared to 5eꢀHCl.
3
1
7
4
6
3
<1
⁄⁄
*
c log P = calculated octanol–water partition coefficient.
IC50 = concentration of compound that reduces cell proliferation by 50%.
Figure 2. Expression of p53 and p21waf1 in IGROV-1 cells treated for 24 h with
paclitaxel (dose corresponding to IC50: PTX 0.12 M) or selected compounds alone
(dose corresponding to IC50: 6f 5 M) or their
M; 5eꢀHCl 10
l
The tested compounds 5e and 6f were found to be able to re-
duce the up-regulation of p21 induced by paclitaxel. This effect
provides evidence that these agents are inhibitors of the transcrip-
tional activity of p53, which is activated in response to the mitotic
spindle damage induced by paclitaxel. In contrast, at equitoxic con-
centrations, a related compound of the benzothiazole series (i.e.,
5aꢀHCl) was not able to modify the expression of p21, thus indicat-
ing lack of inhibition of p53 in spite of its substantial antiprolifer-
ative potency. Consistent with this interpretation is lack of
potentiation of paclitaxel toxicity by compounds of the benzothia-
zole series (not shown).
l
M; 5aꢀHCl 0.3
l
l
combination. In the combined treatment, selected compounds were added 2 h
before treatment with PTX. Whole-cell extracts were prepared and analyzed by
Western Blot analysis. Actin is shown as a control of protein loading.
Table 3
Antitumor activity of compounds 5aꢀHCl and 5eꢀHCl in the treatment of the human
ovarian carcinoma IGROV-1 model
Drugs
Dose (mg/kg)
TVI%a
BWL%b
Toxc
5aꢀHCl
3
9
15
3
5
0/4
0/4
46*
In contrast, a representative compound of the tetrahydrobenzo-
thiazole series 6f was very effective in sensitizing ovarian carci-
noma cells to paclitaxel-induced apoptosis (Fig. 3). The ability of
6f to enhance the cytotoxic effects of paclitaxel may have pharma-
cological relevance as documented by a substantial increase of
antitumor activity of the combination of paclitaxel/6f in the treat-
ment of a human tumor xenograft (Fig. 4). The physical properties
allow the iv administration of 6f, which was well tolerated at the
dose and schedule used in this experiment. Future studies aimed
at optimizing the treatment schedule will better define the phar-
macological profile of the novel p53 inhibitors and the therapeutic
interest of the combination. Finally, selected compounds of the
benzothiazole series (e.g., 5aꢀHCl) exhibited a significant antitumor
activity at well tolerated doses. A better understanding of the
mechanism of action should provide the basis for optimization of
the pharmacological profile.
5eꢀHCl
9
18
21
27
6
4
0/4
0/4
Animals were treated iv every four days for a total of 4 administrations (q4dx4).
P <0.05 by Student’s test versus control mice.
*
a
Tumor volume inhibition in treated versus control mice.
Body weight loss induced by drug treatment.
Tox, that is, animals exhibiting lethal toxicity in each group.
b
c
The antitumor activity of 5eꢀHCl and 5aꢀHCl was compared in
the ovarian carcinoma xenograft, IGROV-1. The results indicate
that under comparable conditions only 5aꢀHCl exhibited a signifi-
cant tumor growth-inhibition (Table 3).
Since at the tested doses the compound was well tolerated, an
improvement of the efficacy is expected by increasing the dose le-
vel. On the basis of previous studies indicating the ability of pifith-
rin-b to sensitize wild-type p53 tumor cells to paclitaxel,8 we have
In conclusion, the obtained results are a further demonstration
of the importance of the chemical manipulation of small molecules

M. S. Christodoulou et al. / Bioorg. Med. Chem. 19 (2011) 1649–1657
1653
Figure 3. Induction of apoptosis in IGROV-1 cells exposed to an antiproliferative concentration of paclitaxel (PTX), 6f, or their combination. Drug treatment was performed
with IC50 concentrations for both agents, as indicated in legend to Figure 2. Cells were pretreated with 6f for 2 h and then exposed to the antimicrotubule agent. Apoptosis
was determined by TUNEL assay and FACScan analysis after 72 h of treatment. The percentages of TUNEL-positive cells are indicated in each panel. Representative of at least
three independent experiments is shown.
in ethanol (92 mL), 2-Br-40-CN-acetophenone 2a (1.5 g, 6.6 mmol)
was added and the solution was refluxed for 90 min. After the com-
pletion of the reaction, the solution was cooled to 0 °C. The result-
ing precipitate was filtered and washed with ethanol to provide 3a
as foam (yield 35%). 1H NMR (CDCl3 400 MHz): d = 8.09 (s, 1H, H-3),
7.98 (d, 2H, J = 8.2 Hz, H-30), 7.75 (d, 1H, J = 8.0 Hz, H-5), 7.70
(d, 2H, J = 8.2 Hz, H-20), 7.66 (d, 1H, J = 8.0 Hz, H-8), 7.51 (t, 1H,
J = 8.0 Hz, H-6), 7.41 (t, 1H, J = 8.0 Hz, H-7); 13C NMR (CDCl3,
400 MHz): d = 149.7 (C-9a), 146.4 (C-2), 138.9 (C-40), 133.2 (C-20),
132.6 (C-4a), 131.1 (C-8a), 127.1 (C-6), 126.2 (C-30), 126.1 (C-7),
125.2 (C-8), 119.6 (CN), 113.5 (C-5), 111.3 (C-10), 109.1 (C-3). Anal.
Calcd for C16H9N3S: C, 69.80; H, 3.29; N, 15.26. Found: C, 69.83; H,
3.24; N, 15.22; EI/MS m/z = 275.
4.2.1.2.
2-(40-Nitrophenyl)imidazo[2,1-b]benzothiazole
Figure 4. Antitumor activity of paclitaxel, compound 6f and their combination
against the osteosarcoma model U2OS. Animals were treated iv at the indicated
doses, every four days for 4 administrations. (s) Control (solvent-treated animals);
(3b). According to the general procedure analogue 3b was obtained
from 2-aminobenzothiazole 1a and 2-Br-40-NO2-acetophenone 2b
as yellow solid (yield 26%). 1H NMR (DMSO-d6, 300 MHz): d = 9.12
(1H, s, H-3), 8.32 (2H, d, J = 9.2 Hz, H-20), 8.13 (2H, d, J = 9.2 Hz, H-
30), 8.08 (1H, d, J = 8.1 Hz, H-5), 8.02 (1H, d, J = 8.1 Hz, H-8), 7.63
(1H, t, J = 8.1 Hz, H-6), 7.49 (1H, t, J = 8.1 Hz, H-7). Anal. Calcd for
(j) 6f (D) or PTX-treated animals; (N) combined treatment. Drug treatment started
when tumors were just measurable (around 80 mm3). CR: complete response (the
indicated ratio is referred to the ratio of the number of the complete tumor
regression/number of the treated tumors). No relevant manifestations of toxicity
were recorded.
C15H9N3O2S: C, 61.01; H, 3.07; N, 14.23. Found: C, 61.06; H, 3.11;
N, 14.25; EI/MS m/z = 295.
known as inhibitors to improve the performance or to modulate
the biological activity and throw light on the rule of p53.
4.2.1.3.
2-(40-Bromophenyl)imidazo[2,1-b]benzothiazole
(3c). According to the general procedure analogue 3c was ob-
tained from 2-aminobenzothiazole 1a and 2,40-dibromoacetophe-
none 2c as yellow solid (yield 23%). 1H NMR (DMSO-d6,
400 MHz): d = 8.86 (s, 1H, H-3), 8.06 (d, 1H, J = 8.1 Hz, H-5), 7.99
(d, 1H, J = 8.1 Hz, H-8), 7.83 (d, 2H, J = 8.4 Hz, H-20), 7.64 (d, 2H,
J = 8.4 Hz, H-30), 7.58 (t, 1H, J = 7.8 Hz, H-6), 7.45 (t, 1H, J = 7.8 Hz,
H-7); 13C NMR (DMSO-d6, 400 MHz): d = 148.7 (C-9a), 146.1
(C-2), 134.2 (C-40), 132.8 (C-20), 130.4 (C-4a), 129.6 (C-6), 127.9
(C-30), 127.5 (C-8a), 126.5 (C-7), 126.2 (C-8), 121.2 (C-10), 114.5
(C-5), 110.8 (C-3). Anal. Calcd for C15H9BrN2S: C, 54.72; H, 2.76;
N, 8.51. Found: C, 54.74; H, 2.71; N, 8.54 EI/MS m/z = 329 (M).
4. Experimental section
4.1. General experimental conditions
Thin-layer chromatography (TLC) was performed on Merck pre-
coated aluminium sheets of Silica Gel 60 F254 and visualization of
products being accomplished by UV absorbance at 254 nm. Flash
column chromatography was performed on Merck Silica gel
(240–400 mesh). 1H NMR and 13C NMR spectra were recorded at
300 and 400 MHz on Brucker spectrometers in the indicated sol-
vents. Chemical shifts (d) for proton and carbon resonances are re-
ported in parts per million (ppm) relative to tetramethylsilane
(TMS), which was used as an internal standard. EI mass spectra
were recorded at an ionizing voltage of 6 keV on a VG 70-70 EQ.
All reactions were carried out in dry solvents.
4.2.1.4.
2-(40-Methylphenyl)imidazo[2,1-b]benzothiazole
(3d). According to the general procedure analogue 3d was ob-
tained from 2-aminobenzothiazole 1a and 2-Br-40-methylaceto-
phenone 2d as yellow solid (yield 23%). 1H NMR (DMSO-d6,
400 MHz): d = 8.90 (s, 1H, H-3), 8.14 (d, 1H, J = 8.1 Hz, H-5),
8.09 (d, 1H, J = 8.1 Hz, H-8), 7.75 (d, 2H, J = 8.1 Hz, H-30), 7.64
(t, 1H, J = 7.8 Hz, H-6), 7.52 (t, 1H, J = 7.8 Hz, H-7), 7.30 (d, 2H,
J = 8.1 Hz, H-20), 2.34 (s, 3H, CH3); 13C NMR (DMSO-d6,
400 MHz): d = 148.7 (C-9a), 144.4 (C-2), 138.4 (C-10), 131.5
(C-40), 131.6 (C-4a), 130.6 (8a), 130.5 (C-20), 130.2 (C-6), 127.7
(C-30), 126.6 (C-7), 126.7 (C-8), 126.3 (C-5), 114.3 (C-3), 21.90
4.2. Synthesis
4.2.1. Preparation of compounds 3a–3i
4.2.1.1. 4-(Imidazo[2,1-b]benzothiazol-20-yl) benzonitrile
(3a). To a solution of 2-aminobenzothiazole 1a (0.93 g, 6.0 mmol)

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M. S. Christodoulou et al. / Bioorg. Med. Chem. 19 (2011) 1649–1657
(CH3). Anal. Calcd for C16H12N2S: C, 72.70; H, 4.58; N, 10.60.
Found: C, 72.73; H, 4.51; N, 10.64; EI/MS m/z = 264.
(DMSO-d6, 400 MHz): d = 8.75 (s, 1H, H-3), 8.03 (d, 1H, J = 8.0 Hz,
H-5), 7.98 (d, 1H, J = 8.0 Hz, H-8), 7.83 (d, 2H, J = 8.1 Hz, H-30),
7.57 (t, 1H, J = 8.0 Hz, H-6), 7.43 (t, 1H, J = 8.0 Hz, H-7), 7.41 (d,
2H, J = 8.1 Hz, H-20), 3.78 (s, 2H, CH2NH2); 13C NMR (DMSO-d6,
400 MHz): d = 147.9 (C-9a), 147.6 (C-2), 143.1 (C-10), 133.4 (C-40),
133.0 (C-4a), 130.3 (C-8a), 128.7 (C-20), 127.8 (C-6), 126.2 (C-8),
126.1 (C-7), 126.0 (C-30), 114.4 (C-5), 109.9 (C-3), 46.18 (CH2NH2).
Anal. Calcd for C16H13N3S: C, 68.79; H, 4.69; N, 15.04. Found: C,
68.73; H, 4.65; N, 15.09; EI/MS m/z = 279.
4.2.1.5. 4-(50,60,70,80-Tetrahydroimidazo[2,1-b]benzothiazol-20-yl)
benzonitrile (3e). According to the general procedure analogue
3e was obtained from 2-amino-4,5,6,7-tetrahydrobenzothiazole
1b and 2-Br-40-CN-acetophenone 2a as white solid (yield 30%).
1H NMR (DMSO-d6, 400 MHz): d = 8.42 (s, 1H, H-3), 8.00 (d, 2H,
J = 8.6 Hz, H-30), 7.83 (d, 2H, J = 8.6 Hz, H-20), 2.72–2.68 (m, 4H,
H-5 and H-8), 1.90–1.86 (m, 4H, H-6 and H-7); 13C NMR (DMSO-
d6, 400 MHz): d = 147.8 (C-9a), 143.4 (C-2), 139.1 (C-40), 133.1
(C-20), 126.9 (C-4a), 125.4 (C-30), 122.4 (C-8a), 119.6 (CN), 110.4
(C-10), 109.3 (C-3), 24.27 (C-5), 23.05 (C-7), 22.54 (C-8), 21.52
(C-6). Anal. Calcd for C16H13N3S: C, 68.79; H, 4.69; N, 15.04. Found:
C, 68.75; H, 4.72; N, 15.05; EI/MS m/z = 279.
4.2.2.2. 4-(50,60,70,80-Tetrahydroimidazo[2,1-b]benzothiazol-20-yl)
benzylamine (5e). According to the general procedure benzyl-
amine 5e was obtained from 4-(50,60,70,80-tetrahydroimidazo[2,1-
b]benzothiazol-20-yl) benzonitrile 3e as yellow solid (yield 90%).
1H NMR (DMSO-d6, 400 MHz): d = 8.14 (s, 1H, H-3), 7.76 (d, 2H,
J = 8.2 Hz, H-30), 7.72 (d, 2H, J = 8.2 Hz, H-20), 3.71 (s, 2H, CH2NH2),
2.68–2.66 (m, 4H, H-5 and H-8), 1.88–1.86 (m, 4H, H-6 and H-7);
13C NMR (DMSO-d6, 400 MHz): d = 146.9 (C-9a), 145.7 (C-2), 142.9
(C-10), 133.0 (C-40), 127.7 (C-20), 126.7 (C-4a), 124.8 (C-30), 120.9
(C-8a), 107.6 (C-3), 45.76 (CH2NH2), 24.20 (C-5), 23.12 (C-7), 22.58
(C-8), 21.60 (C-6). Anal. Calcd for C16H17N3S: C, 67.81; H, 6.05; N,
14.83. Found: C, 67.78; H, 6.10; N, 14.80; EI/MS m/z = 283.
4.2.1.6. 2-(40-Nitrophenyl)-5,6,7,8-tetrahydroimidazo[2,1-b]ben-
zothiazole (3f). According to the general procedure analogue 3f
was obtained from 2-amino-4,5,6,7-tetrahydrobenzothiazole 1b
and 2-Br-40-NO2-acetophenone 2b as white solid (yield 40%). 1H
NMR (DMSO-d6, 300 MHz): d = 8.53 (s, 1H, H-3), 8.27 (d, 2H,
J = 8.8 Hz, H-20), 8.09 (d, 2H, J = 8.8 Hz, H-30), 2.72–2.70 (m, 4H,
H-5 and H-8), 1.91–1.89 (m, 4H, H-6 and H-7). Anal. Calcd for
C
15H13N3O2S: C, 60.18; H, 4.38; N, 14.04. Found: C, 60.14; H,
4.2.3. Preparation of benzenamines 6b, 6f
4.33; N, 14.08.
4.2.3.1. 4-(Imidazo[2,1-b]benzothiazol-20-yl) benzenamine
(6b). To a solution of 2-(40-nitrophenyl)imidazo[2,1-b]benzothia-
zole 3b (2.0 g, 6.7 mmol) in ethanol (150 mL) were added iron
powder (7.5 g, 1.34 mol), H2O (30 mL) and H2SO4 12 N (750 mL)
and the mixture was stirred at 90 °C for 1 h. The hot solution
was filtered through Celite and washed with hot ethanol. The sol-
vent was removed in vacuum and the crude precipitate was trea-
ted with NaHCO3 (400 mL). The organic layer was extracted with
CH2Cl2 (3 ꢁ 150 mL), dried with Na2SO4 and evaporated in vacuum
to dryness to give 6b as white solid (yield 89%). 1H NMR (DMSO-d6,
300 MHz): d = 8.52 (1H, s, H-3), 8.05 (1H, d, J = 8.7 Hz, H-5), 7.95
(1H, d, J = 8.7 Hz, H-8), 7.61–7.51 (3H, m, H-30 and H-6), 7.40
(1H, t, J = 8.7 Hz, H-7), 6.65 (2H, d, J = 9.3 Hz, H-20), 5.25 (2H, s,
NH2). Anal. Calcd for C15H11N3S: C, 67.90; H, 4.18; N, 15.84. Found:
C, 67.87; H, 4.22; N, 15.87.
4.2.1.7. 4-(90-Methyl-9H-imidazo[1,2-a]benzimidazol-20-yl) ben-
zonitrile (3h). According to the general procedure analogue 3h
was obtained from 1-methyl-2-aminobenzimidazole 1d and 2-Br-
40-CN-acetophenone 2a as white solid (yield 5%). 1H NMR
(DMSO-d6, 400 MHz): d = 8.44 (s, 1H, H-3), 8.03 (d, 2H, J = 8.4 Hz,
H-30), 7.83 (d, 2H, J = 8.4 Hz, H-20), 7.78 (d, 1H, J = 7.8 Hz, H-5),
7.56 (d, 1H, J = 7.8 Hz, H-8), 7.37 (t, 1H, J = 7.8 Hz, H-7), 7.24
(t, 1H, J = 7.8 Hz, H-6), 3.79 (s, 3H, CH3N); 13C NMR (DMSO-d6,
400 MHz): d = 150.6 (C-9a), 141.9 (C-2), 140.1 (C-40), 136.6
(C-8a), 133.1 (C-20), 125.4 (C-30), 124.2 (C-7), 124.1 (C-4a), 120.7
(C-6), 119.7 (CN), 111.9 (C-5), 110.8 (C-8), 108.8 (C-10), 106.1
(C-3), 29.54 (CH3N). Anal. Calcd for C17H12N4: C, 74.98; H, 4.44;
N, 20.58. Found: C, 74.94; H, 4.47; N, 20.53; EI/MS m/z = 272.
4.2.1.8. 4-(90-Ethyl-9H-imidazo[1,2-a]benzimidazol-20-yl) ben-
zonitrile (3i). According to the general procedure analogue 3i
was obtained from 1-ethyl-2-aminobenzimidazole 1e and 2-Br-
40-CN-acetophenone 2a as yellow solid (yield 20%). 1H NMR
(DMSO-d6, 400 MHz): d = 8.80 (s, 1H, H-3), 8.05 (d, 2H, J = 8.4 Hz,
H-30), 7.99 (d, 2H, J = 8.4 Hz, H-20), 7.98 (d, 1H, J = 8.2 Hz, H-5),
7.84 (d, 1H, J = 8.2 Hz, H-8), 7.53 (t, 1H, J = 7.8 Hz, H-7), 7.43
(t, 1H, J = 7.8 Hz, H-6), 4.46 (q, 2H, J = 7.2 Hz, CH3CH2N), 1.47 (t,
3H, J = 7.2 Hz, CH3CH2N); 13C NMR (DMSO-d6, 400 MHz):
d = 148.1 (C-9a), 136.0 (C-2 and C-40), 135.7 (C-8a), 134.1 (C-20),
126.5 (C-30), 126.1 (C-7), 124.8 (C-4a), 123.0 (C-6), 119.9 (CN),
113.5 (C-5), 112.6 (C-8), 111.1 (C-10), 108.0 (C-3), 38.90 (CH3CH2N),
14.59 (CH3CH2N). Anal. Calcd for C18H14N4: C, 75.50; H, 4.93; N,
19.57. Found: C, 75.47; H, 4.98; N, 19.62; EI/MS m/z = 286.
4.2.3.2. 4-(50,60,70,80-Tetrahydroimidazo[2,1-b]benzothiazol-20-yl)
benzenamine (6f). According to the general procedure benzen-
amine 6f was obtained from 2-(40-nitrophenyl)-5,6,7,8-tetrahydro-
imidazo[2,1-b]benzothiazole 3f as white solid (yield 89%). 1H NMR
(DMSO-d6, 300 MHz): d = 7.87 (s, 1H, H-3), 7.50 (d, 2H, J = 8.3 Hz,
H-30), 6.58 (d, 2H, J = 8.3 Hz, H-20), 5.12 (s, 2H, NH2), 2.68–2.66
(m, 4H, H-5 and H-8), 1.89–1.87 (m, 4H, H-6 and H-7). Anal. Calcd
for C15H15N3S: C, 66.88; H, 5.61; N, 15.60. Found: C, 66.84; H, 5.65;
N, 15.55.
4.2.4. Preparation of hydrochloride salts 5aꢀHCl, 5eꢀHCl, 6bꢀHCl,
6fꢀHCl
4.2.4.1.
4-(Imidazo[2,1-b]benzothiazol-20-yl) benzylamine
hydrochloride salt (5aꢀHCl). A solution of 4-(imidazo[2,1-b]ben-
zothiazol-20-yl) benzylamine 5a (0.25 g, 0.89 mmol) and satd HCl
in methanol (50 mL) was stirred for 1 h and then evaporated till
dryness. The resulting precipitate was filtered and washed with
DEE to provide 5aꢀHCl as white solid (yield 95%). 1H NMR
(DMSO-d6, 400 MHz): d = 9.03 (s, 1H, H-3), 8.13 (d, 1H, J = 8.0 Hz,
H-5), 8.01 (d, 1H, J = 8.0 Hz, H-8), 7.94 (d, 2H, J = 8.1 Hz, H-30),
7.66–7.61 (m, 3H, H-20 and H-6), 7.51 (t, 1H, J = 8.0 Hz, H-7), 4.05
(q, 2H, J = 5.6 Hz, CH2NH2).
4.2.2. Preparation of benzylamines 5a, 5e
4.2.2.1. 4-(Imidazo[2,1-b]benzothiazol-20-yl) benzylamine
(5a). To
a cooled at 0 °C mixture of 4-(imidazo[2,1-b]ben-
zothiazol-20-yl) benzonitrile 3a (0.25 g, 0.90 mmol) in dry toluene
(5 mL) was added 1 M diisobutylaluminum hydride solution in
hexane (4.5 mL, 4.5 mmol) and the solution was refluxed for 2 h.
Then, the solution was poured into ice-water (50 mL), an aqueous
NaOH solution (1 N, 15 mL) was added and the organic layer was
extracted with Et2O (3 ꢁ 35 mL). The combined organic layers
were washed with brine, dried with Na2SO4, and evaporated in vac-
uum to dryness to provide 5a as yellow solid (yield 90%). 1H NMR
4.2.4.2. 4-(50,60,70,80-Tetrahydroimidazo[2,1-b]benzothiazol-20-yl)
benzylamine hydrochloride salt (5eꢀHCl). According to the
procedure described above 5eꢀHCl was obtained from 5e as a white

M. S. Christodoulou et al. / Bioorg. Med. Chem. 19 (2011) 1649–1657
1655
solid (yield 95%). 1H NMR (DMSO-d6, 400 MHz): d = 8.59 (s, 1H, H-3),
7.95 (d, 2H, J = 8.3 Hz, H-30), 7.62 (d, 2H, J = 8.3 Hz, H-20), 4.06
(q, 2H, J = 5.8 Hz, CH2NH2), 2.77–2.76 (m, 4H, H-5 and H-8),
1.92–1.87 (m, 4H, H-6 and H-7).
EtOAc dried with Na2SO4, filtered, and evaporated. The residue was
purified by flash column chromatography (EtOAc/MeOH/TEA
9:1:0.01) to provide 8 as yellow solid (yield 13%). 1H NMR (CDCl3,
300 MHz): d = 7.92 (s, 1H, H-3), 7.83 (d, 2H, J = 8.0 Hz, H-30), 7.68
(d, 1H, J = 8.0 Hz, H-5), 7.60 (d, 1H, J = 8.0 Hz, H-8), 7.48–7.45 (m,
3H, H-20 and H-6), 7.32 (t, 1H, J = 7.7 Hz, H-7), 4.06–3.90 (m, 3H,
CH(CH3)2, CH2NH), 3.06 (t, 2H, J = 6.0 Hz, CH2CH2CO), 2.61 (t, 2H,
J = 6.0 Hz, CH2CH2CO), 1.15 (d, 6H, J = 6.0 Hz, CH(CH3)2); 13C NMR
(CDCl3, 300 MHz): d = 173.4 (CO), 150.9 (C-9a), 143.0 (C-2), 138.7
(C-10), 136.1 (C-40), 135.8 (C-4a), 129.6 (C-8a), 127.8 (C-20), 126.8
(C-6), 125.3 (C-7), 124.4 (C-30), 123.8 (C-8), 113.5 (C-5), 108.8 (C-
3), 52.58 (CH2NH), 47.86 (CH2CH2CO), 42.90 (CH(CH3)2), 35,74
(CH2CH2CO), 22.65 (CH(CH3)2). Anal. Calcd for C22H24N4OS: C,
67.32; H, 6.16; N, 14.27. Found: C, 67.35; H, 6.11; N, 14.22; EI/
MS m/z = 392.
4.2.4.3.
4-(Imidazo[2,1-b]benzothiazol-20-yl) benzenamine
hydrochloride salt (6bꢀHCl). According to the general procedure
benzenamine hydrochloride salt 6bꢀHCl was obtained from 4-(imi-
dazo[2,1-b]benzothiazol-20-yl) benzenamine 6b as white solid
(yield 95%). 1H NMR (DMSO-d6, 400 MHz): d = 8.89 (1H, s, H-3),
8.08 (1H, d, J = 8.0 Hz, H-5), 8.04 (1H, d, J = 8.0 Hz, H-8), 7.96 (2H,
d, J = 8.4 Hz, H-30), 7.61 (1H, t, J = 7.8 Hz, H-6), 7.47 (1H, t,
J = 7.8 Hz, H-7), 7.44 (2H, d, J = 8.4 Hz, H-20).
4.2.4.4. 4-(50,60,70,80-Tetrahydroimidazo[2,1-b]benzothiazol-20-yl)
benzenamine hydrochloride salt (6fꢀHCl). According to the
general procedure benzenamine hydrochloride salt 6fꢀHCl was ob-
tained from 4-(50,60,70,80-tetrahydroimidazo[2,1-b]benzothiazol-20-
yl) benzenamine 6f as white solid (yield 95%). 1H NMR (DMSO-d6,
300 MHz): d = 8.61 (s, 1H, H-3), 7.94 (d, 2H, J = 8.2 Hz, H-30), 7.38
(d, 2H, J = 8.2 Hz, H-20), 2.78–2.76 (m, 4H, H-5 and H-8), 1.91–
1.89 (m, 4H, H-6 and H-7).
4.2.7. Preparation of N-(40-(imidazo[2,1-b]benzothiazol-200-
yl)benzyl)-3-aminopropanamide (9)
To a solution of tert-butyl (N-[3-oxo-3-(40-(imidazo[2,1-b]ben-
zothiazol-200-yl)
phenylmethylamino)propyl])
carbamate
(0.050 mg, 0.11 mmol) in MeOH (3 mL) was added slowly TMSCl
(0.077 mL, 0.50 mmol). After the completion of the reaction, NaH-
CO3 was added and the organic layer was extracted twice with
DCM. The combined organic layers were dried with Na2SO4, fil-
tered, and evaporated in vacuum to dryness to provide 9 as yellow
solid (yield 60%). 1H NMR (DMSO-d6, 400 MHz): d = 8.74 (s, 1H,
H-3), 8.38 (m, 1H, NH), 8.04 (d, 1H, J = 8.0 Hz, H-5), 8.00 (d, 1H,
J = 8.0 Hz, H-8), 7.84 (d, 2H, J = 8.2 Hz, H-30), 7.58 (t, 1H,
J = 7.3 Hz, H-6), 7.47 (t, 1H, J = 7.3 Hz, H-7), 7.34 (d, 2H, J = 8.2 Hz,
H-20), 4.32 (d, 2H, J = 5.6 Hz, CH2NH), 2.83 (t, 2H, J = 6.6 Hz, COCH2),
2.29 (t, 1H, J = 6.6 Hz, CH2NH2); 13C NMR (CD3OD-d4, 400 MHz):
d = 172.0 (CO), 147.5 (C-9a), 146.7 (C-2), 139.2 (C-10), 132.9
(C-40), 132.3 (C-4a), 129.7 (C-8a), 128.1 (C-20), 127.2 (C-6), 125.6
(C-7), 125.5 (C-30), 125.1 (C-8), 113.8 (C-5), 109.4 (C-3), 42.30
(CH2NH), 40.00 (CH2NH2), 38.90 (COCH2). Anal. Calcd for
4.2.5. Preparation of acetamides 7a, 7e
4.2.5.1. N-(4-(Imidazo[2,1-b]benzothiazol-20-yl)benzyl) acetam-
ide (7a). To a solution of 4-(imidazo[2,1-b]benzothiazol-20-yl)
benzylamine 5a (0.10 g, 0.36 mmol) in dry DMF (0.9 mL) dry
Et3N (0.33 mL, 2.4 mmol) was added and the solution was cooled
to 0 °C. Then, acetyl chloride was added (0.16 mL, 2.2 mmol) and
the solution was stirred overnight at rt. After the completion of
the reaction EtOAc was added and the organic layer was washed
with 5% NH4Cl, water and brine, dried with Na2SO4, filtered, and
evaporated. The residue was purified by flash column chromatog-
raphy (EtOAc) to provide 7a as white solid (85% yield). 1H NMR
(DMSO-d6, 400 MHz): d = 8.43 (s, 1H, H-3), 7.91 (d, 1H, J = 8.2 Hz,
H-5), 7.88 (d, 1H, J = 8.2 Hz, H-8), 7.83 (d, 2H, J = 8.2 Hz, H-30),
7.56 (t, 1H, J = 8.2 Hz, H-6), 7.44 (t, 1H, J = 8.2 Hz, H-7), 7.36
(d, 2H, J = 8.2 Hz, H-20), 4.40 (s, 2H, CH2NH), 2.03 (s, 3H, CH3CO);
13C NMR (CD3OD-d4, 400 MHz): d = 172.0 (CO), 147.5 (C-9a),
147.1 (C-2), 138.1 (C-10), 132.5 (C-40), 132.4 (C-4a), 129.8 (C-8a),
127.7 (C-20), 126.5 (C-6), 125.2 (C-7), 125.0 (C-30), 124.3 (C-8),
113.0 (C-5), 108.0 (C-3), 42.61 (CH2NH), 21.15 (CH3CO). Anal. Calcd
for C18H15N3OS: C, 67.27; H, 4.70; N, 13.07. Found: C, 67.23; H,
4.73; N, 13.12; EI/MS m/z = 321.
C19H18N4OS: C, 65.12; H, 5.18; N, 15.99. Found: C, 65.15; H, 5.14;
N, 15.95; EI/MS m/z = 350.
4.2.8. Preparation of 4-(imidazo[2,1-b]benzothiazol-20-yl)
benzoic acid (10)
A mixture of 4-(imidazo[2,1-b]benzothiazol-20-yl) benzonitrile
3a (0.50 g, 1.8 mmol) and NaOH (0.92 g, 23 mmol) in 1:1 EtOH–
H2O (20 mL) was heated under reflux for 4 h. The solvent was re-
moved in vacuum and the residue was triturated with water
(50 mL). The suspension was acidified with 10% aq HCl to pH 1
and stirred for 2 h at room temperature. The precipitate was fil-
tered off, washed with water (100 mL) and DEE (100 mL), and dried
over P2O5 atmosphere to afford 10 as white solid (yield 96%). 1H
NMR (DMSO-d6, 400 MHz): d = 8.94 (s, 1H, H-3), 8.05 (d, 1H,
J = 8.0 Hz, H-8), 8.02 (d, 2H, J = 8.6 Hz, H-20), 8.01–7.98 (m, 3H, H-30
and H-5), 7.59 (t, 1H, J = 7.8 Hz, H-6), 7.45 (t, 1H, J = 7.8 Hz, H-7);
13C NMR (DMSO-d6, 400 MHz): d = 168.2 (COOH),148.6 (C-9a),
146.4 (C-2), 139.1 (C-40), 132.8 (C-4a), 131.0 (C-20), 130.4 (C-10 and
C-8a), 127.9 (C-6), 126.5 (C-7), 126.2 (C-8), 125.7 (C-30), 114.6
(C-5), 111.7 (C-3). Anal. Calcd for C16H10N2O2S: C, 65.29; H, 3.42; N,
9.52. Found: C, 65.28; H, 3.40; N, 9.55; EI/MS m/z = 294.
4.2.5.2. N-(4-(50,60,70,80-Tetrahydroimidazo[2,1-b]benzothiazol-
20-yl)benzyl) acetamide (7e). According to the general procedure
acetamide 7e was obtained from 4-(50,60,70,80-tetrahydroimi-
dazo[2,1-b]benzothiazol-20-yl) benzylamine 5e after purification
by flash column chromatography (EtOAc) as yellow solid (85%
yield). 1H NMR (CD3OD-d4, 400 MHz): d = 7.87 (s, 1H, H-3), 7.75
(d, 2H, J = 8.2 Hz, H-30), 7.32 (d, 2H, J = 8.2 Hz, H-20), 4.38 (s, 2H,
CH2NH), 2.72–2.70 (m, 4H, H-5 and H-8), 2.02 (s, 3H, CH3CO),
1.96–1.94 (m, 4H, H-6 and H-7); 13C NMR (CD3OD-d4, 400 MHz):
d = 172.3 (CO), 148.8 (C-9a), 146.4 (C-2), 138.3 (C-10), 133.7 (C-
40), 128.2 (C-20), 127.1 (C-4a), 125.5 (C-30), 122.4 (C-8a), 107.1 (C-
3), 43.30 (CH2NH), 24.36 (C-5), 23.45 (C-6), 22.72 (C-8), 21.98 (C-
7), 21.81 (CH3CO). Anal. Calcd for C18H19N3OS: C, 66.43; H, 5.88;
N, 12.91. Found: C, 66.41; H, 5.84; N, 12.95; EI/MS m/z = 325.
4.2.9. Preparation of methyl 4-(imidazo[2,1-b]benzothiazol-20-
yl) benzoate (11)
To a mixture of 4-(imidazo[2,1-b]benzothiazol-20-yl) benzoic
acid 10 (0.50 g, 1.7 mmol) in MeOH (16 mL), a 2 M solution of trim-
ethylsilyldiazomethane in DEE (4 mL, 8.0 mmol) was added and
the solution was stirred for 30 min. Then, the solvent was removed
in vacuum, EtOAc was added and the organic layer was washed
with water and brine, dried with Na2SO4, filtered, and evaporated.
The residue was purified by flash column chromatography (Hex/
4.2.6. Preparation of 3-(40-(Imidazo[2,1-b]benzothiazol-200-yl)
benzylamino)-N-isopropylpropanamide (8)
A mixture of 4-(imidazo[2,1-b]benzothiazol-20-yl) benzylamine
5a (0.051 g, 0.18 mmol), and N-isopropylacrylamide (0.031 g,
0.27 mmol) in H2O (1 mL) was refluxed for 16 h. After the comple-
tion of the reaction the organic layer was extracted repeatedly with

1656
M. S. Christodoulou et al. / Bioorg. Med. Chem. 19 (2011) 1649–1657
EtOAc 4:6) to provide 11 as white solid (70% yield based on the
recovered starting material). 1H NMR (DMSO-d6, 400 MHz):
d = 8.94 (s, 1H, H-3), 8.04 (d, 1H, J = 8.0 Hz, H-8), 8.02–7.98
(m, 5H, H-20, H-30 and H-5), 7.58 (t, 1H, J = 7.8 Hz, H-6), 7.44
(t, 1H, J = 7.8 Hz, H-7), 3.86 (s, 3H, OCH3); 13C NMR (DMSO-d6,
400 MHz): d = 167.1 (COOCH3),146.4 (C-9a), 146.2 (C-2), 139.5
(C-40), 132.8 (C-4a), 131.0 (C-20), 130.6 (C-8a), 129.1 (C-10), 127.9
(C-6), 126.6 (C-7), 126.2 (C-8), 125.7 (C-30), 114.6 (C-5), 112.0
(C-3), 55.16 (COOCH3). Anal. Calcd for C17H12N2O2S: C, 66.22; H,
3.92; N, 9.08. Found: C, 66.17; H, 3.87; N, 9.04; EI/MS m/z = 308.
J = 8.8 Hz, PhHm–OCH3), 3.89 (s, 3H, OCH3); 13C NMR (CDCl3,
300 MHz): d = 160.1 (PhC–OCH3), 149.0 (C-9a), 147.5 (C-2), 141.5
(C10 and C-40), 134.4 (PhC-C10), 132.5 (C-4a), 130.7 (C-8a), 128.6
(PhCo–OCH3), 127.7 (C-20 and C-30), 127.3 (C-6), 126.4 (C-7),
125.2 (C-8), 114.9 (PhCm–OCH3), 113.7 (C-5), 108.7 (C-3), 56.00
(OCH3). Anal. Calcd for C22H16 N2OS: C, 74.13; H, 4.52; N, 7.86.
Found: C, 74.18; H, 4.57; N, 7.80; EI/MS m/z = 356.
4.3. Cellular sensitivity to drugs
In ovarian carcinoma IGROV-1 cells, cellular sensitivity to drugs
was evaluated by growth-inhibition assay after 72-h drug expo-
sure. Cells in the logarithmic phase of growth were seeded in
duplicate into 12-well plates. In the combined treatment with pac-
litaxel, cells were exposed to PFT 2 h before treatment with antim-
icrotubule agents. The highest final concentration of DMSO in
culture medium was 0.5%. After treatment, adherent cells were
trypsinized and counted by a cell counter (Beckam Coulter, Fuller-
ton, CA). IC50 is defined as the drug concentration causing a 50%
reduction of cell number compared with that of untreated control.
4.2.10. Preparation of 4-(imidazo[2,1-b]benzothiazol-20-yl)
benzyl alcohol (12)
To a cooled at 0 °C solution of benzoic acid 4-(imidazo[2,1-
b]benzothiazol-20-yl) methyl ester 11 (0.25 g, 0.82 mmol) in THF
(29 mL), LiAlH4 (0.021 g, 0.53 mmol) was added and the solution
was stirred for 30 min at 0 °C. Then, EtOAc was added and the or-
ganic layer was washed with HCl 1 N, water and brine, dried with
Na2SO4, filtered, and evaporated. The residue was purified by flash
column chromatography (Hex/EtOAc 4:6) to provide 12 as white
solid (70% yield based on the recovered starting material). 1H
NMR (DMSO-d6, 400 MHz): d = 8.77 (s, 1H, H-3), 8.04 (d, 1H,
J = 8.1 Hz, H-8), 7.99 (d, 1H, J = 8.1 Hz, H-5), 7.84 (d, 2H,
J = 8.2 Hz, H-30), 7.57 (t, 1H, J = 8.1 Hz, H-6), 7.43 (t, 1H, J = 8.1 Hz,
H-7), 7.39 (d, 2H, J = 8.2 Hz, H-20), 5.23 (t, 1H, J = 5.1 Hz, CH2OH)
4.53 (d, 2H, J = 5.1 Hz, CH2OH); 13C NMR (DMSO-d6, 400 MHz):
d = 147.9 (C-9a), 147.5 (C-2), 142.7 (C-10), 133.5 (C-40), 133.0
(C-4a), 130.3 (C-8a), 128.0 (C-20), 127.8 (C-6), 126.2 (C-7), 126.1
(C-8), 125.6 (C-30), 114.4 (C-5), 109.9 (C-3), 63.90 (CH2OH). Anal.
Calcd for C16H12N2OS: C, 68.55; H, 4.31; N, 9.99. Found: C, 68.58;
H, 4.35; N, 9.95; EI/MS m/z = 280.
4.4. Determination of apoptosis
Apoptosis was determined in ovarian carcinoma IGROV-1 cells
by TUNEL assay following 72 h-exposure to the selected com-
pounds, PTX alone or in combination. Treated cells were fixed in
4% paraformaldehyde, for 60 min, at room temperature, washed,
and resuspended in ice-cold PBS. The in situ cell death detection
kit fluorescein (Roche, Mannheim, Germany) was used according
to the manufacturers’ instructions and the samples were analyzed
by flow cytometry (Becton-Dickinson, Franklin Lakes, NJ).
4.2.11. Preparation of 4-(imidazo[2,1-b]benzothiazol-20-yl)
benzaldehyde (13)
4.5. Western blot analysis
To a solution of 4-(imidazo[2,1-b]benzothiazol-20-yl) benzyl
alcohol 12 (0.080 g, 0.28 mmol) in DCM (8 mL), Dess–Martin peri-
odinane (0.16 g, 0.36 mmol) was added and the mixture was stir-
red for 20 min. The solvent was evaporated and the residue was
purified by flash column chromatography (Hex/EtOAc 7:3) to pro-
vide 13 as white solid (98% yield). 1H NMR (DMSO-d6, 400 MHz):
d = 10.0 (s, 1H, CHO), 8.98 (s, 1H, H-3), 8.01 (d, 2H, J = 8.3 Hz,
H-30), 8.00 (d, 1H, J = 8.0 Hz, H-8), 7.99 (d, 1H, J = 8.0 Hz, H-5),
7.96 (d, 2H, J = 8.3 Hz, H-20), 7.59 (t, 1H, J = 8.0 Hz, H-6), 7.45
(t, 1H, J = 8.0 Hz, H-7); 13C NMR (DMSO-d6, 400 MHz): d = 193.4
(CHO), 148.8 (C-9a), 146.2 (C-2), 140.7 (C-40), 136.0 (C-10), 132.8
(C-4a), 131.3 (C-20), 130.5 (C-8a), 127.9 (C-6), 126.6 (C-7), 126.2
(C-8), 126.1 (C-30), 114.6 (C-5), 112.4 (C-3). Anal. Calcd for
Cells were treated as previously described for the indicated
times with paclitaxel (PTX) or selected compounds alone or in
combination at a cytotoxic concentration corresponding to IC50
.
Cells were collected and lysed as previously described.8
4.6. Tumor models and evaluation of antitumor activity
The experiments were performed using female athymic Swiss
nude mice. Mice were maintained in laminar flow rooms keeping
temperature and humidity constant. Mice had free access to food
and water. Experiments were approved by the Ethics Committee
for Animal Experimentation of the Istituto Nazionale Tumori of Mi-
lan according to institutional guidelines.
C16H10N2OS: C, 69.04; H, 3.62; N, 10.06. Found: C, 69.01; H, 3.67;
Exponentially growing tumor cells (107 cells/mouse) were sc in-
jected into the right flank of athymic nude mice. Tumor lines were
achieved by serial sc passages of fragments (about 3 ꢁ 3 ꢁ 3 mm)
from growing tumors. Animals were treated iv at the indicated
doses, every four days for 4 administrations starting when tumors
were measurable (around 80 mg). Compounds were dissolved in
DMSO and diluted in PBS containing 5% Cremophor to a final con-
centration of 10% DMSO.
N, 10.01; EI/MS m/z = 278.
4.2.12. Preparation of 2-(400-methoxybiphenyl-40-yl)
imidazo[2,1-b]benzothiazole (14)
To a solution of 2-(40-bromophenyl)imidazo[2,1-b]benzothia-
zole 3c (0.050 g, 0.15 mmol) in THF (6 mL) under nitrogen, a solu-
tion of 4-methoxyphenylboronic acid (0.20 g, 1.4 mmol) in EtOH
and a solution of Na2CO3 (0.20 g, 2.0 mmol) in H2O were added.
Then, Pd(PPh3)4 (cat. amount) was added and the mixture was
put under reflux for 12 h. After the completion of the reaction
EtOAc (10 mL) and H2O (10 mL) were added and the mixture was
filtered by Celite and washed with EtOAc. The organic layer was
dried with Na2SO4, filtered, and evaporated. The residue was puri-
fied by flash column chromatography (Hex/EtOAc 9:1) to provide
14 as yellow solid (18% yield). 1H NMR (CDCl3, 400 MHz):
d = 8.11 (s, 1H, H-3), 7.95 (d, 1H, J = 8.2 Hz, H-5), 7.77 (d, 1H,
J = 8.2 Hz, H-8), 7.62 (d, 2H, J = 8.2 Hz, H-30), 7.58–7.54 (m, 4H,
H-20, PhHo–OCH3), 7.51–7.40 (m, 2H, H-6 and H-7), 7.02 (d, 2H,
Acknowledgments
This research has been developed under the umbrella of
CM0602 COST Action ‘Inhibitors of Angiogenesis: design, synthesis,
and biological exploitation’. This study was supported by the Asso-
ciazione Ricerche sul Cancro and Ministero dell’Istruzione,
dell’Università e della Ricerca (MIUR) PRIN 2007—Program ‘Svilup-
po e caratterizzazione di nuovi inibitori di tirosine chinasi cellulari
con attività antiproliferativa e antiangiogenica nei confronti di dif-
ferenti tumori’.

M. S. Christodoulou et al. / Bioorg. Med. Chem. 19 (2011) 1649–1657
1657
McIldowie, M. J.; Piggott, M. J.; Grounds, M. D. Mol. Cell. Biochem. 2010, 337,
119; For previous synthesis of pifithrin and its analogs see: (e) Pietrancosta, N.;
Maina, F.; Dono, R.; Moumen, A.; Garino, C.; Laras, Y.; Burlet, S.; Quéléver, G.;
Kraus, J. L. Bioorg. Med. Chem. Lett. 2005, 15, 1561; (f) Carchéchath, S. D.;
Tawatao, R. I.; Corr, M.; Carson, D. A.; Cottam, H. B. J. Med. Chem. 2005, 48, 6409;
(g) Murru, S.; Singh, C. B.; Veerababurao, K.; Patel, B. K. Tetrahedron 2008, 64,
1931.
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