Potential antitumor agents. 37. Synthesis and antitumor activity of guanylhydrazones from imidazo[2,1-b]thiazoles and from the new heterocyclic system thiazolo[2′,3′:2,3]imidazo[4,5-c]quinoline

Article abstract of DOI:10.1021/jm040888s

This paper reports synthesis and antitumor activity of new guanylhydrazones from imidazo[2,1-b]thiazoles and from the new heterocyclic system thiazolo[2′,3′:2,3]imidazo[4,5-c]quinoline. The compounds were tested as potential antitumor agents at the National Cancer Institute. The effect of the guanylhydrazone of 2-chloro-6-(2,5-dimethoxy-4-nitrophenyl) imidazo[2,1-6]thiazole-5-carbaldehyde (41) was investigated, and it was found to be an inhibitor of Complex III of the mitochondrial respiratory chain and is able to induce apoptosis in the cell lines HT29 and HL60.

Full text of DOI:10.1021/jm040888s

J. Med. Chem. 2005, 48, 3085-3089
3085
Potential Antitumor Agents. 37.1 Synthesis and Antitumor Activity of
Guanylhydrazones from Imidazo[2,1-b]thiazoles and from the New Heterocyclic
System Thiazolo[2′,3′:2,3]imidazo[4,5-c]quinoline
Aldo Andreani,*,† Massimiliano Granaiola,† Alberto Leoni,† Alessandra Locatelli,† Rita Morigi,†
Mirella Rambaldi,† Giorgio Lenaz,‡ Romana Fato,‡ Christian Bergamini,‡ and Giovanna Farruggia‡
Dipartimento di Scienze Farmaceutiche, Universita` di Bologna, Via Belmeloro 6, 40126 Bologna, Italy, and
Dipartimento di Biochimica “G. Moruzzi”, Universita` di Bologna, Via Irnerio 48, 40126 Bologna, Italy
Received October 19, 2004
This paper reports synthesis and antitumor activity of new guanylhydrazones from imidazo-
[2,1-b]thiazoles and from the new heterocyclic system thiazolo[2′,3′:2,3]imidazo[4,5-c]quinoline.
The compounds were tested as potential antitumor agents at the National Cancer Institute.
The effect of the guanylhydrazone of 2-chloro-6-(2,5-dimethoxy-4-nitrophenyl)imidazo[2,1-b]-
thiazole-5-carbaldehyde (41) was investigated, and it was found to be an inhibitor of Complex
III of the mitochondrial respiratory chain and is able to induce apoptosis in the cell lines HT29
and HL60.
Introduction
distinctive biochemical properties, they all inhibit mito-
chondrial functions. MIBG showed to selectively inhibit
Complex I (NADH-Q1 reductase activity) in the respira-
tory chain,6,7 while methyl-GAG significantly inhibits
State 4 of respiration.8 Moreover it is known that
alkylguanidines such as octylguanidine are effective
inhibitors of State 3 of ADP-stimulated respiration.9
Since mitochondrial damage may be responsible, or
partly so, for the antiproliferative action,10-12 we studied
the effects on the mitochondrial respiratory chain of one
of the most interesting compounds synthesized, i.e., 41,
the guanylhydrazone of 2-chloro-6-(2,5-dimethoxy-4-
nitrophenyl)imidazo[2,1-b]thiazole-5-carbaldehyde. More-
over methyl-GAG is known to induce apoptosis in
different human cancer cell lines in a concentration- and
time-dependent way;13 therefore, we undertook a study
to elucidate if the mechanism of action of guanylhydra-
zone 41 was linked to a apoptotic or necrotic pathway.
Chemistry. The guanylhydrazones 29-41, 50, 51,
56, 58, 61, 66 (Schemes 1-4) were prepared from the
corresponding aldehydes, whereas 44 was from the
quinone 43. The aldehyde 60 (Scheme 3) was obtained
by reduction with Na2S2O4 of the quinone 59, prepared
in turn by a convenient method of oxidative demethyl-
ation of the hydroquinone dimethyl ether 57 with ceric
ammonium nitrate (CAN).14 A versatile approach was
used to afford the aldehyde 49 (Scheme 2) starting from
the 6-dimethoxyphenyl imidazothiazole 46: first it was
hydrolyzed by refluxing in aqueous HBr to give 47, then
the formyl group was introduced by means of the
Vilsmeier reaction. Moreover we attempted the reduc-
tion of the nitro group of the aldehydes 16, 23, 54, 65
which, under the experimental conditions employed
(Fe/acetic acid), did not bring to the corresponding
aminoaldehydes. When the nitro group was at the
2-position (16, 54) (Schemes 1, 2), the reduction was
accomplished with concomitant ring closure and forma-
tion of the new heterocyclic system thiazolo[2′,3′:2,3]-
imidazo[4,5-c]quinoline (42, 55). Due to the lack of
formyl group in compound 42, the reaction with ami-
In the previous papers2,3 we pointed out that in a
series of imidazo[2,1-b]thiazole guanylhydrazones, 2-,
3-, or 4-nitrophenyl at the 6 position is a suitable
pharmacophoric group giving rise to some potent anti-
tumor agents. To find congeners more active as potential
chemotherapeutic agents and to study in depth the
influence of the substituent in position 6, we designed
new imidazothiazole guanylhydrazones bearing differ-
ent dimethoxynitrophenyl groups in this position. We
chose as starting material for the synthesis some
dimethoxyacetophenones, which gave by nitration sev-
eral nitroaryl derivatives. From these intermediates we
could easily afford the guanylhydrazones bearing at the
6 position different nitroaryl groups, the feature sup-
posed to enhance the pharmacological activity. We also
prepared a few derivatives without the nitro group for
comparison purpose. The substituents in position 2 and
3 of the imidazothiazole ring were the same used in the
previous papers3,4 (2- and/or 3-methyl, 2-chloro, and
2-propyl), because they demonstrated an increase in the
cytotoxicity. In addition we have investigated the activ-
ity of a bis-guanylhydrazone, the correlated quinone,
and two new polycyclic compounds. The synthesized
compounds bear the guanidino group as other drugs:
m-iodobenzylguanidine (MIBG), the pyridyl cyanoguani-
dine CHS 828, and Mitoguazone (methyl-GAG, methyl-
glyoxal-bisguanylhydrazone, MGBG).5 MIBG and CHS
828 have shown promising antitumor activity in pre-
clinical tumor models, while methyl-GAG has useful
clinical activity as a single agent in malignant lymph-
oma, carcinoma of the head, neck, and esophageal and
non-small-cell lung cancer.5 Apart from having the
guanidino groups in common, there are many differ-
ences between these drugs in their structures and
mechanism of action. However, even if they have
* To whom correspondence should be addressed. Tel.:
+39-051-2099714, fax +39-051-2099734, e-mail: aldo.andreani@unibo.it.
† Dipartimento di Scienze Farmaceutiche.
‡ Dipartimento di Biochimica “G. Moruzzi”.
10.1021/jm040888s CCC: $30.25 © 2005 American Chemical Society
Published on Web 03/18/2005

3086 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Brief Articles
Scheme 1
noguanidine was performed at the carbonyl groups of
the corresponding p-quinone 43, prepared in turn by
oxidative demethylation with CAN. Under the same
experimental conditions, compound 55 did not furnish
the analogue o-quinone.
effect (TGI), and the cytotoxic effect (LC50). The values
of log GI50 range from -4.04 to -6.08, log TGI from
-4.01 to -5.65, and log LC50 from -4.01 to -5.01 (see
Supporting Information). The order of magnitude for the
activity of methyl-GAG16 and the tested compounds is
similar. Compound 41 is the most active of the series
showing the following log GI50 values (panel/cell line):
-5.81 (colon/COLO 205), -6.23 (ovarian/OVCAR-3),
-5.84 (renal/RXF 393), and -5.72 (leukemia/K-562) and
is under review by BEC (Biological Evaluation Com-
mittee of the NCI). The result obtained from all the
compounds tested shows that those bearing the nitro
group in ortho or meta position are weakly active
(29, 31, 33-35) or inactive (30, 32, 66).
An increase in activity is obtained with a para nitro
group (36-38, 40, 41) and in particular when a sub-
stituent is also present in position 2 of the imidazothia-
zole (38, 41). Moreover in the derivatives without the
nitro group (50, 61) it is evident a flattening of the
values (MG-MID ) 10-4 M) and a general decrease of
cytotoxic effect. These data confirm the importance of
Biological Results and Discussion
(a) In Vitro Growth Inhibition and Cytotoxic-
ity.15 The test was performed by the National Cancer
Institute (NCI, Bethesda, MD) as in our previous
papers.2,3 According to a primary screening, the guanyl-
hydrazones 29-41, 50, 51, 56, 58, 61, 66, the bisguanyl-
hydrazone 44, and the new heterocycles 42, 43, and 55
were evaluated for their cytotoxic activity on three
human cell lines. Compounds 29, 31, 33-38, 40, 41, 43,
44, 50, 55, 58, 61 were active on the three cell line test
and therefore were evaluated in the 60 cell lines, using
five concentrations at 10-fold dilutions, the highest
being 10-4M and the others 10-5, 10-6, 10-7, 10-8 M.
The results are expressed as log, taking into consider-
ation the growth inhibitory power (GI50), the cytostatic

Brief Articles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 3087
Scheme 2
Scheme 3
Scheme 4
Table 1. Effect of methyl-GAG and Compound 41 on
Respiration in Coupled Isolated RLMa (State 4 and State 3)
and on DNP-Uncoupled Respiration
the nitro group to increase the inhibition of cell prolif-
eration. SARs are not possible at present for compounds
43, 44, and 55 since they are derivatives of the new
heterocyclic system thiazolo[2′,3′:2,3]imidazo[4,5-c]quin-
oline. They did not show a potent antitumor activity,
but since 43 and 55 have an appreciable activity on
leukemia and renal cancer respectively, this new het-
erocyclic system will be considered as a possible new
lead.
(b) Effects on Respiration of Rat Liver Mito-
chondria. The experiments were conducted testing
methyl-GAG and 41 at the concentration of 35 µM and
measuring the inhibitory effect on mitochondrial res-
piration in coupled rat liver mitochondria. A polar-
ographyc apparatus (Clark electrode) detected the oxy-
gen consumption, and the reaction was started adding
a saturating amount of the appropriate substrate:
glutamate/malate for NADH-O2 activity, succinate for
succinate-O2 activity, and ascorbate/N,N,N′,N′-Tetram-
ethyl-p-phenylenediamine dihydrochloride (TMPD) to
test Complex IV (Cytochrome c oxidase enzyme) activity.
The respiratory rates were expressed as ngAoxigen/min/
mg of protein. As shown by data reported in Table 1,
methyl-GAG induces only a slight decrease in the
compd
(concn 35 µM)b
State 4
State 3
ICRc
DNP
Glutamate-Malate ngAoxigen/min/mg of RLM
control
17.4 ( 3.8 90.0 ( 5.5
5.3 ( 0.8
4.9 ( 1.2
110.5 ( 21.0
106.2 ( 14.2
methyl-GAG 17.9 ( 2.8 80.0 ( 4.0
41
23.1 ( 5.2 47.7 ( 23.6* 2.3 ( 1.2** 74.2 ( 19.3
Succinate ngAoxigen/min/mg of RLM
control
40.6 ( 2.8 134.5 ( 13.1 3.3 ( 0.3
191.0 ( 35.8
168.7 ( 19.8
methyl-GAG 39.2 ( 1.9 141.1 ( 26.1 3.7 ( 0.7
41
33.4 ( 4.3 54.9 ( 10.7** 1.7 ( 0.4** 81.7 ( 20.9
Ascorbate-TMPD ngAoxigen/min/mg of RLM
control
83.1 ( 14.6 125.9 ( 22.1 1.5 ( 0.2
141.9 ( 20.6
155.5 ( 9.9
158.3 ( 10.1
methyl-GAG 91.1 ( 15.1 128.2 ( 13.7 1.7 ( 0.4
41
98.0 ( 8.3 141.8 ( 15.9 1.5 ( 0.2
a RML: rat liver mitochondria. b Mean of five and eight inde-
pendent measures. c ICR: respiratory control index; * P < 0.05
** P < 0.001.
oxygen consumption rate when the substrate used is
ascorbate/TMPD. This is in disagreement with earlier
8,11
findings
suggesting that the significant inhibition
observed in the literature on State 4 respiration could
be an artifact due to the very high concentration of
methyl-GAG used in those experiments (6 mM). On the

3088 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Brief Articles
other hand, compound 41 has a more pronounced
inhibitory effect on the oxygen consumption induced by
both glutamate-malate/succinate, whereas it has no
effect on the respiration supported by ascorbate/TMPD
as described in Table 1. Beside an uncoupling effect,
compound 41 inhibits ADP-stimulated respiration (State
3) as well as uncoupled respiration, obtained by 2,4-
dinitrophenol (DNP) addition: it seems therefore rea-
sonable to assume a direct effect on one of the respira-
tory complexes. The lack of effect on ascorbate/TMPD
respiration excludes Complex IV as the target of com-
pound 41. Measurements of separate enzyme complexes
of the respiratory chain in the presence of 41 revealed
that it is not capable of inhibition on Complex I and on
Complex II (succinate-DCIP reductase activity) (data
not shown). These results suggest that Complex III
(ubiquinol-cytochrome c reductase) should be the target
of this compound. To test this hypothesis we have
measured the oxygen consumption induced by saturat-
ing amounts of CoQ1H2. The inhibition of 41 on
CoQ1H2-O2 activity is dose dependent (see Supporting
Information) but it was not possible to reach a 100%
inhibition (60 µM of 41 induces about 80% of inhibition).
It should be reminded that this compound has a very
low water-solubility, and it was impossible to test higher
concentrations. Moreover the low water-solubility of
compound 41 may induce aggregation that can prevent
its efficient partition in the lipid phase where the target
enzyme is located.
brane depolarization in intact viable cells,17,18 and
forward-angle light scattering (FS) signals to evaluate
variation in the cell size. In fact, in most cases, apoptosis
can be distinguished from necrosis on the basis of the
scatter parameters:19 in the initial stages of apoptosis
the cell shrinks, while the membrane remains intact;
during necrosis, cell swelling occurs as a result of early
failure of the membrane integrity. As a consequence of
these cellular changes, FS decreases during the initial
phases of apoptosis, whereas during necrosis, FS in-
creases immediately.20
Cytofluorimetric analysis showed that compound 41
was able to induce a rapid cell death in HT29 cells: the
percentage of dead cells increased from 7 ( 5% in
controls to 18 ( 8% after 5 h of treatments and to
32 ( 5% after 7 h. In parallel, an increase in cells
characterized by a low mitochondria potential and
reduced size was observed: this population represented
the 0.8% ( 0.6% in control cells and was respectively
the 6 ( 2%, 12 ( 4%, and 37 ( 8% after 5, 7, and 24 h.
Furthermore, a clear decrease in mitochondrial poten-
tial of viable cells with normal size was detectable after
7 h of treatment. The reduction in mitochondrial
potential preceded the cell shrinkage and obviously the
alteration in plasma membrane permeability, evidenced
by the PI uptake: this supported the hypothesis of
induction of apoptosis by mitochondrial perturbation,
which produces the characteristic morphological changes
of apoptosis culminating, at the end of the process, with
the loss of membrane integrity.
(c) Growth Inhibition and Effects on Cell Vi-
ability and Mitochondrial Potential. To investigate
the mechanism of action of these compounds, the
antiproliferative effects of compound 41 were studied
by monitoring them in two tumor cell lines: HT29 (colon
adenocarcinoma) and HL60 (promyelocytic leukemia).
These cell lines were chosen considering the interesting
activity reported by NCI’s assays on colon cancer and
leukemia panels. We have investigated the effects of
treatments on cell growth in the range of concentrations
between 10-8M and 10-4 M (see Supporting Informa-
tion). It is evident that no detrimental effect was
detectable after 24 h at doses up to 10-5 M; only after
48 h compound 41, just at the 10-6 M concentration,
affected the proliferation in HT29 cells. However, after
24 h in both the cell lines, the higher doses caused a
marked cytotoxic effect, leading to death about 75% of
the cell populations at the concentration 10-5 M and of
the whole cultures at 10-4 M. Moreover in HT29 cells a
growth inhibitory effect was observed after 48 h of
treatment at the 10-6 M concentration, but in this case
no increase in cell death was detectable. Taking into
account the activity shown by compound 41 on Complex
III and to assess if it could interfere with mitochondrial
function in intact viable cells as well as on isolated
mitochondria and in order to discriminate if the ob-
served cytotoxicity was due to an apoptotic or a necrotic
effect, the HT29 and HL60 cells were treated with
compound 41 at the10-5 M concentration. Five, seven,
and twenty-four hours after treatment, cells were
analyzed by flow cytometry for cell death, mitochondrial
membrane depolarization, and cell dimensions by using
propidium iodide (PI) exclusion for cell viability, the
potentiometric fluorescent dye DiOC6(3) (3,3′-dihexyl-
oxacarbocyanine iodide) to measure mitochondrial mem-
Interestingly, HL60 cells treated with this dose
(10-5 M) showed a very high cytotoxic effect even after
3 h, while 10-6 M did not show any effect. An interme-
diate dose (5 × 10-6 M) had to be used to induce effects
similar to those achieved in HT29 with the dose
10-5 M (data not shown). Methyl-GAG, at the same
concentrations, showed a faint effect on cell prolifera-
tion; only the 10-4 M dose is able to decrease HT29 and
HL60 growth respectively to 24 ( 6% and to 32 ( 4%
of the controls after 24 h of treatment. Moreover no
effect was detectable on mitochondrial membrane de-
polarization with methyl-GAG treatment.
Conclusion
In general the new derivatives did not show a very
high potency, but some of them had interesting activities
in selected cell lines. In particular compound 41 had
good features as potential antitumor agent; therefore,
its mechanism of action was studied. In the past decades
mitochondria have become the target of increased
interest because many studies have demonstrated that
they have an essential role on the regulation of cell
death.21-24 Moreover one mechanism by which tumor
cells survive in the presence of chemotherapeutic agents
is the increase of antiapoptotic activities.25 Development
of cytotoxic drugs that target mitochondria may provide
a new strategy to induce apoptosis in tumor cells. Since
drugs bearing the guanidino groups influence the mito-
chondrial functions at different levels, we studied
compound 41 by focusing our attention on these biologi-
cal organelles. Compound 41 was able to inhibit Com-
plex III and consequently produce a decrease in the
mitochondrial potential; moreover it was able to induce
at 10-5 M concentration, a time dependent increase of

Brief Articles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 3089
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through the respiratory chain may be responsible for
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Acknowledgment. This work has been supported
by a grant from MIUR-COFIN 2002, contract number
2002033121_003. We are grateful to NCI for the anti-
tumor tests and to CIRB of the University of Bologna
for flow cytometric measurements.
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Supporting Information Available: Additional experi-
1
mental procedures, C, H, N analytical data, H NMR and IR
spectroscopy, and tables and figures related to the biological
assays. This material is available free of charge via Internet
at http://pubs.acs.org.
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