Potential antitumor agents. Part 29: Synthesis and potential coanthracyclinic activity of imidazo[2,1-b]thiazole guanylhydrazones

Article abstract of DOI:10.1016/S0968-0896(00)00165-6

This paper reports the synthesis of new imidazo[2,1-b]thiazole guanylhydrazones which were tested as potential antitumor agents. Three of these derivatives (those bearing a 3- or 4-nitrophenyl group) were the most potent and one of these showed a mild eff

Full text of DOI:10.1016/S0968-0896(00)00165-6

Bioorganic & Medicinal Chemistry 8 (2000) 2359±2366
Potential Antitumor Agents.
Part 291: Synthesis and Potential Coanthracyclinic Activity of
Imidazo[2,1-b]thiazole Guanylhydrazones
Aldo Andreani,a,* Alberto Leoni,a Alessandra Locatelli,a Rita Morigi,a
Mirella Rambaldi,a Maurizio Recanatinia and Vida Garalieneb
a
Á
Dipartimento di Scienze Farmaceutiche, Universita di Bologna, Via Belmeloro 6, 40126 Bologna, Italy
bLithuanian Institute of Cardiology, Kaunas, Lithuania LT-3007
Received 13 April 2000; accepted 21 June 2000
AbstractÐThis paper reports the synthesis of new imidazo[2,1-b]thiazole guanylhydrazones which were tested as potential anti-
tumor agents. Three of these derivatives (those bearing a 3- or 4-nitrophenyl group) were the most potent and one of these showed a
mild e€ect as CDK1 inhibitor. These same three derivatives were also tested as positive inotropic agents and two of them were more
5
potent than amrinone at 10 M. These two guanylhydrazones could be useful coanthracyclinic agents. # 2000 Elsevier Science
Ltd. All rights reserved.
Introduction
position. In compounds 7±8 the guanylhydrazone chain is
at the 6 position whereas a nitro group is present at
position 5. A di€erent electron attracting group was
considered at position 6 of compounds 9±10 whereas the
guanylhydrazone chain at position 5 bears an additional
methyl group. Compounds 11±14 belong to a series
where two of the previous molecules are connected by
means of a couple of amide (11±12) or ester (13±14)
bonds to a 2, 4 or 6 carbon spacer.
In 1998,1,2 we suggested the term `coanthracyclinic
activity' to indicate the pharmacological behavior of a
molecule endowed with both antitumor activity (in order
to reduce the anthracycline toxicity by reducing its
dosage) and positive inotropic activity (in order to coun-
teract the heart depression induced by anthracyclines).
We believe in this `bivalent' approach even though others
have been suggested, such as the possibility to prevent
anthracycline induced apoptosis of cardiac myocytes.3
Compounds 2±14 were subjected to the screening devel-
oped by the National Cancer Institute (NCI, Bethesda,
MD) and the most active compounds were also evaluated
for their positive inotropic activity, in search of poten-
tial coanthracyclinic agents. These same compounds
were tested as potential inhibitors of cyclin-dependent
kinase 1 (CDK1), in order to verify if the antitumor
activity was related to this mechanism of action.
In our ®rst search on imidazo[2,1-b]thiazole guanylhy-
drazones endowed with antitumor4 and cardiotonic5
activity, we described the synthesis and pharmacological
pro®le of compounds related to structure 1 (Chart 1).
The results showed that the active compounds had a
chlorine at the 6 position or at the phenyl ring of the 6
position. In the subsequent paper,6 the substitution at the
2 and 3 positions was also considered and two potential
coanthracyclinic agents were described.
Chemistry
In this paper we wish to report the synthesis and pharma-
cological activity of new analogues. The ®rst series bears a
nitro (2±5) or amino group (6) at the phenyl ring of the 6
The guanylhydrazones 2±8 (Scheme 1a) and 9±14 (Scheme
1b) were prepared by reaction of aminoguanidine with the
appropriate aldehydes 19±24, 27 and ketones 28, 29, 31±
34. Some of these compounds (19, 20, 24, 28) are
reported in the literature.7 9 The starting aldehydes 21±
22 which are necessary for the preparation of com-
pounds 4±5 were prepared by means of the Vilsmeier
*Corresponding author. Tel.:+39-051-2099714; fax:+39-051-2099734;
e-mail: aldoandr@alma.unibo.it
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00165-6

2360
A. Andreani et al. / Bioorg. Med. Chem. 8 (2000) 2359±2366
Chart 1. Design of the guanylhydrazones.
reaction from the corresponding imidazo[2,1-b]thiazoles
17±18. The aminoaldehyde 23 (starting material for 6)
was obtained by catalytic hydrogenation of the nitroder-
ivative 20. The aldehyde 27 (starting material for 8) was
prepared by oxidation with osmium tetroxide of 5-nitro-6-
(2-phenylethenyl)-2,3-dihydroimidazo[2,1-b]thiazole 26
obtained in turn from 6-methyl-5-nitro-2,3-dihydroimid-
azo[2,1-b]thiazole 25.7 The dimeric ketones 31±34 (for
the synthesis of 11±14) were prepared with two di€erent
approaches. The acid 29 (which is also the starting
material for 10) was obtained by hydrolysis of the ester
28: it was activated10 in the form of the N-succinimidyl
ester 30 and condensed with the suitable diamine to
a€ord the amides 31±32.
active when it reduces the growth of any of the cell lines
to 32% or less (negative numbers indicate cell kill) and it is
passed on for evaluation in the full panel of 60 cell lines.
As shown in Table 3, all the guanylhydrazones reported in
Scheme 1a (2±8, i.e. those arising from aldehydes) were
active, whereas among the guanylhydrazones arising from
ketones (Scheme 1b), only compound 14 was active.
The panel of 60 human tumor cell lines is organized into
subpanels representing leukemia, melanoma and can-
cers of lung, colon, kidney, ovary, breast, prostate and
central nervous system. The test compounds (2±8, 14)
were evaluated using ®ve concentrations at 10-fold
5
dilutions, the highest being 10 4 M and the others 10
,
8
10 6, 10 7, 10 M.
The acid 29 was also treated with 1,10-carbonyldiimida-
zole (CDI) to yield the acylimidazole, which in the pre-
sence of 1,4-butanediol or 1,6-hexanediol and 1,8-
diazobicyclo[5.4.0]undec-7-ene (DBU) as activator of
the alcoholic function11 gave the esters 33±34.
Compounds 3±5, i.e. those bearing a nitro group at the
position 3 or 4 of the phenyl ring in position 6 of the
imidazothiazole, were the most active. Table 4 reports
the results obtained (only for these compounds) expres-
sed as log10, taking into consideration the growth inhi-
bitory power (GI50), the cytostatic e€ect (TGI) and the
cytotoxic e€ect (LC50). For comparison purposes, the
results obtained by DTIC (4-dimethyltriazenoimidazole-
5-carboxamide) and Methyl-GAG (methylglyoxal bis-
guanylhydrazone dihydrochloride) are also reported.
The structure of the latter compound is related to that
Pharmacological Results and Discussion
In vitro growth inhibition and cytotoxicity12
As a primary screening, compounds 2±14 were eval-
uated for their cytotoxic potency on three human cell
lines, such as NCI-H460 lung cancer, MCF7 breast
cancer and SF-268 glioma. A compound is considered
of the guanylhydrazones described here but the highest
2.6
dose tested was 10
instead of 10 4. The antitumor

A. Andreani et al. / Bioorg. Med. Chem. 8 (2000) 2359±2366
2361
Scheme 1. (a) Synthesis of the guanylhydrazones 2±8. (b) Synthesis of the guanylhydrazones 9±14.
data from these and other reference drugs are available
at the address: http://dtp.nci.nih.gov/docs/cancer/sear-
ches/standard_agent_table.html
lines (leukemia, non-small cell lung cancer, colon, mel-
anoma, ovarian and breast cancer) it is possible to note
a signi®cant di€erence between cytostatic and cytotoxic
doses.
As shown in Table 4, compounds 3±5 are about equi-
potent with DTIC. Compound 4 showed cytostatic
e€ect at concentrations between 10 4 and 10 5 M. Both
compounds 3 and 5 were approximately 10 times more
potent, since they showed a good cytostatic activity in
CDK1 inhibitory activity13
As a ®rst attempt to investigate the mechanism of the
antitumor activity of compounds 3, 4 and 5, they were
tested in a CDK1/cyclin B kinase inhibition assay. This
kinase and other members of the CDK family play a
central role in the control of the cellular cycle, and it has
been demonstrated that the deregulation of their activity
may be involved in various human tumors.14 Compounds
3 and 4 did not result to be CDK1 inhibitors, whereas
compound 5 exhibited a slight inhibitory activity
(IC50=60 mM). Even if this is too weak an evidence to
assume a correlation between the antitumor activity of 5
and its intervention on the CDK-controlled mechanisms,
we will take it into account in order to further explore
the mechanism of action of the imidazo[2,1-b]thiazole
guanylhydrazones as possible CDK1 inhibitors.
the majority of the cell lines at concentrations in the
M. Both compounds showed a
5
range of 10 6±10
peculiar pro®le of cytotoxicity since in several cell lines
5
4
the increase of concentration from 10 to 10 deter-
mined a decrease of activity. This paradoxical e€ect does
not appear to be related to technical errors since it was
evident in both separate experiments (graphs not shown).
Moreover we wish to point out that, in most of the cel-
lular lines, the concentrations of compounds 3±4
endowed with cytostatic activity are very near to the
cytotoxic doses. By contrast, the pharmacological pro®le
of compound 5 was more promising since in six cellular

2362
A. Andreani et al. / Bioorg. Med. Chem. 8 (2000) 2359±2366
Positive inotropic activity
interesting concomitant cardiotonic e€ect was found in
two of these compounds (3, 5). We therefore suggest that
these guanylhydrazones could be endowed with the
coanthracyclinic activity we were looking for. It has been
recently reported15 the antitumor e€ect of vesnarinone,
an oral positive inotropic agent that has been used for
the treatment of patients with congestive heart failure.
There are no doubts that the mechanisms of action
involved in the pharmacological behavior of compounds
endowed with both cardiotonic and antitumor properties
require accurate investigations in order to determine
whether such agents can be introduced into practical
therapy. It is important to note that the concentrations
causing the useful cardiotonic and antitumor e€ects
should not be considerably di€erent, and this does not
seem to be the case with vesnarinone whereas this con-
dition was veri®ed in the compounds we tested.
The positive inotropic activity of the most potent anti-
tumor agents 3±5 is reported in Table 5 in comparison
with amrinone.
5
These compounds, at a concentration of 10 6±5Â10
M increased the contraction force of the guinea-pig
papillary muscle. Compound 4, in the range of above-
mentioned doses, showed only tendency to increase this
force. The inotropic action of amrinone accrued within
40±50 s and peak drug e€ect was within 4±5 min. By
contrast, the inotropic action of compounds 3, 5
accrued slowly (2±3 min) and the maximal response was
within 10±12 min.
In Table 5 it is possible to notice that compound 3
showed a dose-related e€ect and both the guanylhy-
drazones (3, 5) are more potent than amrinone.
Experimental
Chemistry
Conclusions
In the present work we pointed out that a 3- or 4-nitrophe-
nyl group is a suitable pharmacophoric group in the imi-
dazo[2,1-b]thiazole guanylhydrazone series, giving rise to
three potent antitumor agents. Even though the mechan-
ism of action of this activity has not been determined, an
The melting points are uncorrected. Analyses (C, H, N)
were within Æ0.4% of the theoretical values. Baker¯ex
plates (silica gel IB2-F) were used for TLC: the eluent
was petroleum ether:acetone 50:50 (with 0.1% of concd
NH4OH for the analysis of the guanylhydrazones as
Table 1. Compounds 2±34
Compound
x
R, R0 or Y, n
Formula
.
Mw
Mp, ꢀC or (lit.)
n
max (cm 1)a or (lit.)
.
2
3
4
5
6
7
8
9
±CHˆ
±CHˆ
±CH2
±CHˆ
±CHˆ
±CHˆ
±CH2
Ð
Ð
Ð
Ð
Ð
2-NO2, H
4-NO2, H
4-NO2, H
3-NO2, 4-Cl
4-NH2, H
Ð
Ð
C2H5
H
NH, 2
NH, 4
O, 4
C13H11N7O2S HCl H2O
383.8
383.8
422.3
436.7
390.3
399.1
328.2
348.8
320.8
683.6
675.6
695.6
705.6
245.3
245.3
247.3
279.7
273.3
273.3
275.3
307.7
243.3
197.2
185.2
273.3
199.2
238.3
210.2
307.3
444.5
472.5
474.5
502.6
280±284 dec
334±337 dec
296±298 dec
303±305 dec
258±263 dec
285±290 dec
275±280 dec
272±275 dec
275±280 dec
297±300 dec
288±292 dec
235±238 dec
300±305 dec
1675, 1660, 1515, 1155
1680, 1635, 1500, 1250
1670, 1620, 1510, 855
1675, 1640, 1525, 1150
1660, 1245, 1090, 820
1665, 1245, 1135, 1025
1675, 1575, 1180, 1140
1710, 1670, 1590, 1195
1720, 1675, 855, 720
1670, 1640, 1595, 715
1640, 1585, 1210, 860
1670, 1585, 1210, 1040
1670, 1590, 1260, 855
6
. .
C13H11N7O2S HCl H2O
.
C13H13N7O2S 2HCl H2O
.
.
C13H10ClN7O2S 2HCl
.
C13H13N7S 2HCl H2O
.
.
C7H7N7O2S 4HCl
.
C7H9N7O2S 2HCl
. .
C11H14N6O2S HCl H2O
. .
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
C9H10N6O2S HCl H2O
. .
C20H24N14O2S2 2HCl 3H2O
. .
C22H28N14O2S2 2HCl H2O
. .
C22H26N12O4S2 2HCl 2H2O
.
24H30N12O4S2 2HCl H2O
.
Ð
O, 6
C
±CHˆ
±CHˆ
±CH2±
±CHˆ
±CHˆ
±CHˆ
±CH2±
±CHˆ
Ð
Ð
Ð
Ð
Ð
2-NO2, H
4-NO2, H
4-NO2, H
3-NO2, 4-Cl
2-NO2, H
4-NO2, H
4-NO2, H
3-NO2, 4-Cl
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
NH, 2
NH, 4
O, 4
C11H7N3O2S
C11H7N3O2S
C11H9N3O2S
C11H6ClN3O2S
C12H7N3O3S
C12H7N3O3S
7
7
202±205
1535, 1510, 1195, 725
6
6
C12H9N3O3S
C12H6ClN3O3S
192±194
251±253 dec
178±180 dec
1650, 1500, 1250, 860
1645, 1095, 830, 715
3340-3060, 1610, 1250
6
C
12H9N3OS
C6H3N3O3S
C6H7N3O2S
C13H11N3O2S
C6H5N3O3S
C10H10N2O3S
C8H6N2O3S
12H9N3O5S
C18H16N6O4S2
20H20N6O4S2
C20H18N4O6S2
22H22N4O6S2
7
145±150
128±130 dec
1680, 1255, 1165, 710
1680, 1150, 870, 795
8
3140-2400, 1685, 1630
1800, 1770, 1650, 1310
3380, 1660, 1640, 970
b
Ð
Ð
Ð
177±178 dec
227±228 dec
275±278 dec
C
C
b
1705, 1640, 1185, 1020
O, 6
C
135±138
1
aIn all the guanylhydrazones the NH groups give broad bands in the range 3600±2300 cm
.

A. Andreani et al. / Bioorg. Med. Chem. 8 (2000) 2359±2366
Table 2. 1H NMR of compounds 2±14, 18, 21±23, 26, 27, 29±34
2363
Compound
d, ppm
2
7.58 (1H, d, th, J=4.5), 7.74 (2H, m, ar), 7.80 (3H, broad, NH+NH2), 7.83 (1H, t, ar, J=7.7), 8.05 (1H, d, ar, J=7.7), 8.29
(1H, s, -CHˆ), 8.74 (1H, d, th, J=4.5), 11.99 (1H, s, NH)
3
4
5
6
7,56 (1H, d, th, J=4.5), 7.80 (3H, broad, NH+NH2), 7.98 (2H, d, ar, J=7), 8.3 (2H, d, ar, J=7), 8.60 (1H, s, CHˆ), 8.68
(1H, d, th, J=4.5), 12.09 (1H, s, NH)
4.02 (2H, t, thn, J=7.2), 4.66 (2H, t, thn, J=7.2), 5.52 (2H, broad, NH2), 7.79 (1H, broad s, NH), 7.87 (2H, d, ar, J=8.8), 8.27
(2H, d, ar, J=8.8), 8.45 (1H, s, CHˆ), 12.22 (1H, s, NH)
7.57 (1H, d, th, J=4.4), 7.80 (3H, broad, NH+NH2), 7.91 (1H, d, ar-5, J=8.4), 8.03 (1H, dd, ar-6, J=2, J=8.4), 8.38
(1H, d, ar-2, J=2), 8.54 (1H, s, -CHˆ), 8.70 (1H, d, th, J=4,4), 11.84 (1H, s, NH)
7.18 (2H, s, C6H4NH2), 7.42 (2H, d, ar, J=8.5), 7.55 (1H, d, th, J=4.4), 7.77 (2H, d, ar, J=8.5), 7.80 (3H, broad, NH+NH2),
8.51 (1H, s, -CHˆ), 8.71 (1H, d, th, J=4.4), 12.00 (1H, s, NH)
7
8
9
7.76 (1H, d, th, J=4.4), 7.95 (3H, s, NH2+NH), 8.41 (1H, d, th, J=4.4), 8.81 (1H, s, -CH=), 12.61 (1H, s, NH)
4.06 (2H, t, thn, J=7.9), 4.63 (2H, t, thn, J=7.9), 7.91 (3H, s, NH2+NH), 8.62 (1H, s, -CHˆ), 12.68 (1H, s, NH)
1.28 (3H, t, CH2CH3, J=7), 2.37 (3H, s, CH3), 4.28 (2H, q, CH2CH3, J=7), 7.49 (1H, d, th, J=4.5), 7.91 (3H, s, NH2+NH),
7.97 (1H, d, th, J=4.5), 11.46 (1H, s, NH)
10
11
12
13
14
18
2.42 (3H, s, CH3), 5.30 (1H, broad, OH), 7.50 (1H, d, th, J=4.4), 8.00 (1H, d, th, J=4.4), 8.04 (3H, s, NH2+NH),
11.72 (1H, s, NH)
2.42 (6H, s, CH3), 3.47 (4H, broad, CH2), 7.46 (2H, d, th, J=4.5), 7.85 (6H, s, NH2+NH), 8.01 (2H, d, th, J=4.5), 8.58
(2H, s, CONH), 11.31 (2H, s, NH)
1.54 (4H, broad, CH2), 2.42 (6H, s, CH3), 3.28 (4H, broad, NHCH2), 7.45 (2H, d, th, J=4.5), 7.82 (6H, s, NH2+NH), 8.01
(2H, d, th, J=4.5), 8.47 (2H, t, CONH), 11.25 (2H, s, NH)
1.79 (4H, broad, CH2), 2.38 (6H, s, CH3), 4.28 (4H, broad, OCH2), 7.49 (2H, d, th, J=4.5), 7.89 (6H, s, NH2+NH), 7.98
(2H, d, th, J=4.5), 11.45 (2H, s, NH)
1.40 (4H, broad, CH2), 1.67 (4H, broad, CH2), 2.38 (6H, s, CH3), 4.25 (4H, t, OCH2, J=6), 7.49 (2H, d, th, J=4.5), 7.89
(6H, s, NH2+NH), 7.98 (2H, d, th, J=4.5), 11.45 (2H, s, NH)
7.33 (1H, d, th, J=4.5), 7.78 (1H, d, ar-5, J=8.5), 8.00 (1H, d, th, J=4.5), 8.13 (1H, dd, ar-6, J=2, J=8.5), 8.46
(2H: 1H, s, H-5+1H, d, ar-2, J=2)
21
22
4.05 (2H, t, thn, J=7.6), 4.53 (2H, t, thn, J=7.6), 8.08 (2H, d, ar, J=8.6), 8.30 (2H, d, ar, J=8.6), 9.79 (1H, s, -CHˆ)
7.65 (1H, d, th, J=4.3), 7.92 (1H, d, ar, J=8.5), 8.24 (1H, d, ar, J=8.5), 8.44 (1H, d, th, J=4.3), 8.57
(1H, s, ar), 9.96 (1H, s, -CHˆ)
23
6.95 (2H, d, ar, J=8.5), 7.54 (1H, d, th, J=4.4), 7.73 (2H, d, ar, J=8.5), 8.38 (1H, d, th, J=4.4), 8.60 (2H, broad, C6H4NH2),
9.87 (1H, s, CHO)
26
27
29
30
31
32
4.02 (2H, t, thn, J=7.5), 4.60 (2H, t, thn, J=7.5), 7.41 (3H, m: 2H, ar+1H, CH), 7.66 (4H, m: 3H, ar +1H, CH)
4.07 (2H, t, thn, J=7.8), 4.65 (2H, t, thn, J=7.8), 10.16 (1H, s, CHO)
2.62 (3H, s, CH3), 3.40 (1H, broad, OH), 7.63 (1H, d, th, J=4.4), 8.36 (1H, d, th, J=4.4)
2.64 (3H, s, CH3), 2.92 (4H, s, CH2), 7.75 (1H, d, th, J=4.5), 8.42 (1H, d, th, J=4.5)
2.66 (6H, s, CH3), 3.53 (4H, broad, CH2), 7.62 (2H, d, th, J=4.5), 8.39 (2H, d, th, J=4.5), 8.81 (2H, broad, NH)
1.58 (4H, broad, CH2), 2.63 (6H, s, CH3), 3.30 (4H, broad, NHCH2), 7.61 (2H, d, th, J=4.5), 8.38 (2H, d, th, J=4.5), 8.74
(2H, t, NH)
33
34
1.74 (4H, broad, CH2), 2.58 (6H, s, CH3), 4.32 (4H, t, OCH2, J=6.5), 7.61 (2H, d, th, J=4.5), 8.32 (2H, d, th, J=4.5)
1.45 (4H, broad, CH2), 1.74 (4H, broad, CH2), 2.59 (6H, s, CH3), 4.33 (4H, t, OCH2, J=6.4), 7.63 (2H, d, th, J=4.5),
8.34 (2H, d, th, J=4.5)
Table 3. Growth inhibition percentages of three human tumor cell
lines in the presence of compounds 2±14
shift (referenced to solvent signal) is expressed in d
(ppm) and J in Hz, with the following abbreviations:
th=thiazole, thn=thiazoline, ar=aromatic (Table 2).
Compound
4
NCI-H460
(Lung)
MCF7
(Breast)
SF-268
(CNS)
(10 M)
General procedure for the synthesis of the
guanylhydrazones 2±14
2
3
4
5
6
7
8
9
10
11
12
13
14
4
27
51
12
8
53
3
26
17
6
29
50
65
4
5
24
6
86
97
73
88
54
8
The appropriate aldehyde or ketone (10 mM) was dis-
solved in ethanol and treated with the equivalent of
aminoguanidine hydrochloride, prepared in turn from
an ethanol suspension of aminoguanidine bicarbonate
and excess of 37% hydrochloric acid. The reaction
mixture was re¯uxed for 30 min and the resulting pre-
cipitate was collected by ®ltration with a yield of 80±
90%. For the synthesis of guanylhydrazones 11±14 two
equivalents of aminoguanidine hydrochloride were
employed and the yield was 30±40%.
9
1
1
8
100
111
109
68
82
59
78
88
102
82
44
19
6-(4-Chloro-3-nitrophenyl)imidazo[2,1-b]thiazole (18). 2-
Aminothiazole (11 mM) was dissolved in acetone (50 mL)
and treated with 2-bromo-1-(4-chloro-3-nitrophenyl)-
ethanone8 (11 mM). The reaction mixture was re¯uxed for
1 h, the resulting salt was separated by ®ltration and
treated, without further puri®cation, with 20 mL of
free bases). Kieselgel 60 (Merck) was used for column
chromatography. The IR spectra were recorded in
1
Nujol on a Perkin±Elmer 683; nmax is expressed in cm
(Table 1). The 1H NMR spectra were recorded in
(CD3)2SO on a Varian Gemini (300 MHz); the chemical

2364
A. Andreani et al. / Bioorg. Med. Chem. 8 (2000) 2359±2366
Table 4. Growth inhibition, cytostatic and cytotoxic activity of compounds 3±5 on the 60 cell panela
3b
4b
5b
DTICb
TGI
Methyl-GAGc
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
LC50
GI50
TGI
LC50
Leukemia
NSCLC
Colon
5.9
5.7
5.9
5.8
5.7
5.7
5.7
5.8
5.5
5.2
5.5
5.5
5.5
5.5
5.3
5.4
5.5
5.3
4.3
5.1
5.1
5.5
5.0
4.8
4.9
5.3
4.9
5.3
4.8
5.2
5.2
4.9
4.9
4.9
4.9
4.9
4.7
4.4
4.8
4.8
4.5
4.5
4.6
4.6
4.6
4.1
4.2
4.3
4.4
4.2
4.1
4.2
4.3
4.1
5.9
5.7
5.9
5.8
5.7
5.8
5.8
5.8
5.7
5.4
5.5
5.6
5.5
5.4
5.5
5.5
5.5
5.4
4.0
4.9
5.0
5.2
4.6
4.5
5.2
5.2
4.4
4.8
4.4
4.2
4.2
4.5
4.3
4.3
4.1
4.1
4.1
4.2
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
5.1
4.6
4.9
4.7
4.4
4.8
4.7
4.0
4.8
3.2
3.5
3.3
3.6
3.5
3.5
3.3
2.8
3.5
2.6
2.8
2.9
3.0
3.0
2.8
2.7
2.6
2.8
CNS
Melanoma
Ovarian
Renal
Prostate
Breast
aAll values are log10
.
4
bHighest concn 10 M.
cHighest concn 10 2.6 M.
Table 5. Positive inotropic activity of compounds 3±5
®ltered through Celite and methanol was evaporated
under reduced pressure. The residue was puri®ed by col-
umn chromatography with a mixture of CH2Cl2:toluene:
MeOH (7.5:1:1.5) as the eluent. A yellow solid was
obtained which was crystallized from acetone/petroleum
ether with a yield of 25%.
Compound
Concn
(M)
Force of contraction
(P/P0ÆSEM,%)a
EC50 (M)
6
6
3
10
10
120.6Æ8.6
138.3Æ19.0
168.0Æ18.0
112.0Æ6.3
118.0Æ9.7
111.0Æ5.7
136.5Æ12.0
149.5Æ14.4
124.5Æ11.8
105.0Æ2.3
114.7Æ5.9
124.0Æ4.2
(4.0Æ0.56)Â10
5
5
5Â10
6
4
10
10
Ð
5
5-Nitro-6-(2-phenylethenyl)-2,3-dihydroimidazo[2,1-b]
thiazole (26). A mixture of 6-methyl-5-nitro-2,3-dihy-
droimidazo[2,1-b]thiazole 25 (3 g, 16 mM), benzalde-
hyde (10 mL) and piperidine (2.5 mL) was heated at
120±130 ꢀC for 6 h under stirring. After cooling, the
mixture was concentrated under reduced pressure and
the resulting yellow solid was puri®ed by column chro-
matography (eluent petroleum ether:acetone 70:30) and
crystallized from ethanol with a yield of 85%.
5
5
5
5Â10
6
6
5
10
10
(1Æ0.6)Â10
(2.5Æ0.4)Â104
5
5Â10
6
Amrinone
10
10
5
5Â10
aMean of ®ve experiments for 3 and amrinone, four for 4±5. P=force
of contraction at a given concentration; P0=control (100%).
5-Nitro-2,3-dihydroimidazo[2,1-b]thiazole-6-carbaldehyde
(27). A solution of sodium periodate (18 mM) in 15 mL
H2O was added to a stirred solution of compound 26
(9 mM) and osmium tetroxide (0.2 mM) in 50 mL THF.
The mixture was stirred at room temperature for 48 h.
The suspension was ®ltered and the ®ltrate was evapo-
rated under reduced pressure. The residue was puri®ed by
column chromatography with petroleum ether:acetone
70:30 as the eluent. Compound 27 was obtained as a
yellow solid with a yield of 65%.
ethanol and 400 mL of 2N HCl. After 1 h of re¯ux, the
solution was cautiously basi®ed by dropwise addition of
15% NH4OH. The resulting base was collected by ®l-
tration and crystallized from ethanol with a yield of
80%.
6-(4-Nitrophenyl)-2,3-dihydroimidazo[2,1-b]thiazole-5-car-
baldehyde (21) and 6-(4-chloro-3-nitrophenyl)imidazo[2,1-
b]thiazole - 5 - carbaldehyde (22). The Vilsmeier reagent
was prepared at 0±5 ꢀC by dropping POCl3 (54 mM)
into a stirred solution of DMF (65 mM) in CHCl3
(5mL). 6-(4-Nitrophenyl)-2,3-dihydroimidazo[2,1-b]thia-
zole 17 or 6-(4-chloro-3-nitrophenyl)imidazo[2,1-b]thia-
zole 18 (10 mM) in CHCl3 (60 mL) was added dropwise
to the Vilsmeier reagent while maintaining stirring and
cooling. The reaction mixture was kept for 3 h at room
temperature and under re¯ux for 20h. Chloroform was
removed under reduced pressure and the resulting oil
was poured onto ice. The crude aldehyde thus obtained
was collected by ®ltration and crystallized from ethanol
with a yield of 55% (21) and 90% (22).
5-Acetylimidazo[2,1-b]thiazole-6-carboxylic acid (29). A
solution of compound 289 (4.2 mM) in 0.1N NaOH
(20 mL) and MeOH (30 mL) was re¯uxed for 5 h. The
solvent was evaporated under reduced pressure and the
residue was dissolved in water (40 mL). The solution was
treated with 10% HCl until pH 2 was reached. The sus-
pension was extracted with CHCl3 which was evaporated
under reduced pressure. The residue, crystallized from
acetone/petroleum ether, gave a white solid with a yield
of 34%.
1-([(5-Acetylimidazo[2,1-b]thiazol-6-yl)carbonyl]oxy)pir-
rolidine-2,5-dione (30). A mixture of the carboxylic acid
29 (1.4 mM), diisopropylethylamine (DIEA, 1.4 mM) and
O-(N-succinimidyl)-N,N,N0,N0-tetramethyluronium tet-
ra¯uoroborate (STMU, 1.4 mM) in anhydrous DMF
was stirred at room temperature for 10 h. The mixture
was diluted with H2O (20 mL) and extracted with
6-(4-Aminophenyl)imidazo[2,1-b]thiazole-5-carbaldehyde
(23). The nitroderivative 20 (1.8 mM), dissolved in
30 mL of DMF:MeOH (0.1:10 v/v), was hydrogenated
in the presence of 10% Pd/C (10 mg) at atmospheric
pressure and room temperature for 8 h. The mixture was

A. Andreani et al. / Bioorg. Med. Chem. 8 (2000) 2359±2366
2365
CH2Cl2. The organic phase was dried and evaporated
under reduced pressure. The crude product was crystal-
lized from acetone/petroleum ether to a€ord a white
solid with a yield of 68%.
GI50 is the concentration of test drug where 100Â
(T T0)/(C T0)=50.
TGI is the concentration of test drug where 100Â
(T T0)/(C T0)=0.
N,N0-1,2-Ethanediylbis(5-acetylimidazo[2,1-b]thiazole-6-
carboxamide) (31) and N,N0-1,4-butanediylbis(5-acetyl-
imidazo[2,1-b]thiazole-6-carboxamide) (32). A mixture of
compound 30 (1 mM) and 0.5 mM of 1,2-diaminoethane
(or 1,4-diaminobutane) in 5 mL of DMF was re¯uxed
under stirring for 2 h. MeOH (10 mL) was added to the
cooled solution and the solid obtained was ®ltered and
used as such in the subsequent step. The yield was 9%
(31) and 37% (32).
LC50 is the concentration of test drug where 100Â
(T T0)/T0= 50 (the control optical density is not used
in the calculation of LC50).
Positive inotropic activity. Guinea-pig right ventricle
papillary muscle was mounted in an organ bath with a
circulation of physiological saline solution of following
composition (mM): NaCl 144; KCl 4; CaCl2 1.8; Tris.Cl
10; MgCl2 1; glucose 5; pH 7.3±7.4. The solution was aer-
ated with pure oxygen and the temperature was main-
tained at 36±37 ꢀC. The maximal perfusion value of the
papillary muscle was about 4.5 mL/min.
5-Acetylimidazo[2,1-b]thiazole-6-carboxylic acid 1,4-bu-
tanediyl ester (33) and 5-acetylimidazo[2,1-b]thiazole-6-
carboxylic acid 1,6-hexanediyl ester (34). 1,10-Carbonyl-
diimidazole (CDI, 2.4 mM) was added to a suspension
of compound 29 (2.4 mM) in anhydrous CH2Cl2 (35 mL)
and re¯uxed until e€ervescence ceased. Stirring at re¯ux
was maintained for 2 h. The cooled acylimidazole was
treated with 1,4-butanediol (or 1,6-hexanediol, 1.2 mM)
and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 2.4 mM).
The reaction mixture was stirred under re¯ux for 4 h,
cooled, washed with water, dried and concentrated under
reduced pressure to a€ord a crude residue which was used
as such in the subsequent step. The yield was 12% (33)
and 35% (34).
Electrostimulator ESU-2 (Russia) electrically stimulated
the muscle by square pulses with a voltage of 20% above
threshold, 10 ms duration at a frequency of 1.0 Hz. The
force of the isometric contraction was measured by the
6MX2B force displacement transducer (Russia) and
recorded by the H3030-4 recorder (Russia).
In order to obtain a concentration±response curve, each
papillary muscle, after 1 h equilibration period, was tested
in cumulative doses of the agents under test. Each com-
pound was dissolved in a minimum amount (0.1±0.3 mL)
of DMSO and diluted with physiological solution.
Quantitatively, the contraction force (P) was expressed
as respective ratio to the initial value of the contraction
force (P0) under normal conditions. From concentration±
response curves the negative logarithm of the concentra-
tion of the test agent required for a 50% increase in the
maximal force of contraction (EC50) was determined.
Results are presented as mean Æ SEM. Statistical analyses
were performed using Student's t-test.
Pharmacology
Antitumor activity
Three cell panel. Each cell line was inoculated and pre-
incubated on a microtiter plate. Test agents were then
added at a single concentration and the culture incubated
for 48 h. End-point determinations were made with sul-
forhodamine B (SRB), a protein-binding dye. Results
for each test compound are reported as the percent of
growth of the treated cells when compared to the
untreated control cells.
CDK1 inhibitory activity. CDK1-cyclin B was extracted
in homogenization bu€er from M-phase star®sh (Mar-
thasterias glacialis) oocytes and puri®ed by anity chro-
matography on p9CKShs1-Sepharose beads, from which
it was eluted by free p9CKShs1 as described.13 The
Sixty cell panel. Each cell line was inoculated onto
microtiter plates, then preincubated for 24±28 h at 37 ꢀC
for stabilization. Subsequently, test agents were added
in ®ve 10-fold dilutions and the culture was incubated
for an additional 48 h in 5% CO2 atmosphere and 100%
humidity. For each test agent, a dose±response pro®le
was generated. End-point determinations of cell growth
were performed by in situ ®xation of cells, followed by
staining with SRB, which binds to the basic amino acids
of cellular macromolecules. The solubilized stain was
measured spectrophotometrically in order to determine
relative cell growth in treated and untreated cells.
1
kinase activity was assayed with 1 mg mL histone H1
(Sigma type III-S), in the presence of 15 mM [g-32P]ATP
(3000 Ci mM 1; 1 mCi mL 1) in a ®nal volume of
30 mL. After a 10 min incubation at 30 ꢀC, 25 mL aliquots
of supernatant were spotted onto 2.5Â3 cm pieces of
Whatman P81 phosphocellulose paper and, 20 s later, the
®lters were washed ®ve times (for at least 5 min each
time) in a solution of 10 mL phosphoric acid per liter
water. The wet ®lters were counted in the presence of
1 mL scintillation ¯uid (Amersham). The kinase activity
was expressed as a percentage of maximal activity.
A plate reader was used to read the optical densities,
and a microcomputer processed the optical densities in
the special concentration parameters: GI50, TGI, LC50.
The optical density of the test well after 48 h period of
exposure to the test drugs is T, the optical density at
time zero is T0 and the control optical density is C.
Acknowledgements
We are grateful to the National Cancer Institute
(Bethesda, MD) for the antitumor tests and to Dr.
Laurent Meijer (CNRS, Station Biologique, Rosco€,

2366
A. Andreani et al. / Bioorg. Med. Chem. 8 (2000) 2359±2366
France) for the CDK1 test. We also wish to thank Dr.
Maurizio D'Incalci (Mario Negri Institute, Milan, Italy)
for helpful discussion. This work was supported by a
grant from MURST.
7. Andreani, A.; Leoni, A.; Rambaldi, M.; Locatelli, A.;
Bossa, R.; Galatulas, I.; Chiericozzi, M.; Cozzi, R. Eur. J.
Med. Chem. 1997, 32, 151.
8. Andreani, A.; Rambaldi, M.; Andreani, F.; Hrelia, P.;
Cantelli Forti, G. Arch. Pharm. Chem., Sci. Ed. 1987, 15,
41.
9. Canestrari S.; Sgarabotto P.; Andreani A.; Greci L. J.
Chem. Research (S) 1999, 412; (M) 1999, 1750.
10. Singh, M. A.; Plouvier, B.; Hill, G. C.; Gueck, J.; Pon, R.
T.; Lown, J. W. J. Am. Chem. Soc. 1994, 116, 7006.
11. Andreani, A.; Rambaldi, M.; Leoni, A.; Locatelli, A.;
Andreani, F.; Bossa, R.; Chiericozzi, M.; Cozzi, R.; Galatulas,
I. Eur. J. Med. Chem. 1996, 31, 741.
12. Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.;
Paull, K.; Vistica, D.; Hose, C.; Langley, J.; Cronise, P.; Vai-
gro-Wol€, A. J. Natl. Cancer Inst. 1991, 83, 757.
13. Meijer, L.; Borgne, A.; Mulner, O.; Chong, J. P.; Blow, J.
J.; Inagaki, N.; Inagaki, M.; Delcros, J. G.; Moulinoux, J. P.
Eur. J. Biochem. 1997, 243, 527.
References and Notes
1. For part 28 see Andreani, A.; Leoni, A.; Locatelli, A.;
Morigi, R.; Chiericozzi, M.; Fraccari, A.; Galatulas, I. Anti-
cancer Res. 1998, 18, 3407.
2. Andreani, A.; Locatelli, A.; Leoni, A.; Morigi, R.; Chier-
icozzi, M.; Fraccari, A.; Galatulas, I.; Salvatore, G. Eur. J.
Med. Chem. 1998, 33, 905.
3. Andrieu Abadie, N.; Ja€rezou, J. P.; Hatem, S.; Laurent,
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