New antitumor imidazo[2,1-b]thiazole guanylhydrazones and analogues

Article abstract of DOI:10.1021/jm701246g

The synthesis of new antitumor 6-substituted imidazothiazole guanylhydrazones is described. Moreover, a series of compounds with a different basic chain at the 5 position were prepared. Finally, the replacement of the thiazole ring in the imidazothiazole system was also considered. All the new compounds prepared were submitted to the NCI cell line screen for evaluation of their antitumor activity. A few selected compounds were submitted to additional biological studies concerning effects on the cell cycle, apoptosis, and mitochondria.

Full text of DOI:10.1021/jm701246g

J. Med. Chem. 2008, 51, 809–816
809
New Antitumor Imidazo[2,1-b]thiazole Guanylhydrazones and Analogues1
Aldo Andreani,*,† Silvia Burnelli,† Massimiliano Granaiola,† Alberto Leoni,† Alessandra Locatelli,† Rita Morigi,†
Mirella Rambaldi,† Lucilla Varoli,† Natalia Calonghi,‡ Concettina Cappadone,‡ Giovanna Farruggia,‡ Maddalena Zini,‡
Claudio Stefanelli,‡ Lanfranco Masotti,‡ Norman S. Radin,§ and Robert H. Shoemaker4
Dipartimento di Scienze Farmaceutiche, UniVersità di Bologna, Via Belmeloro 6, 40126 Bologna, Italy, Dipartimento di Biochimica “G.
Moruzzi”, UniVersità di Bologna, Via Irnerio 48, 40126 Bologna, Italy, Molecular & BehaVioral Neuroscience Institute, UniVersity of
Michigan, Ann Arbor, Michigan 48109, and Screening Technologies Branch, DeVelopmental Therapeutics Program, DiVision of Cancer
Treatment and Diagnosis, National Cancer Institute at Frederick, Frederick, Maryland 21702
ReceiVed October 2, 2007
The synthesis of new antitumor 6-substituted imidazothiazole guanylhydrazones is described. Moreover, a
series of compounds with a different basic chain at the 5 position were prepared. Finally, the replacement
of the thiazole ring in the imidazothiazole system was also considered. All the new compounds prepared
were submitted to the NCI cell line screen for evaluation of their antitumor activity. A few selected compounds
were submitted to additional biological studies concerning effects on the cell cycle, apoptosis, and
mitochondria.
Introduction
the 5 position, both the introduction of a diaminoguanidine/
dimethyldiaminoguanidine chain (20Gb, 20Ic) and the above-
described introduction of a heterocycle-containing chain (20Gd-
Ge). According to the literature, the reaction of an aldehyde
with diaminoguanidine may give bishydrazones.5–7 We obtained
compounds 22J and 23H which were submitted to antitumor
tests since analogous derivatives have been reported as possible
antitumor agents.8
(3) Replacement of the thiazole ring in the imidazothiazole
system. Taking the 4-chloro-3-nitrophenyl derivatives as the lead
compounds,9,3 the thiazole ring was replaced by the following
heterocycles: thiadiazole (36), benzothiazole (37), pyridine (38),
and pyrimidine (39).
Methyl-gag (Mitoguazone) is still present in the “Cumulative
List of All Orphan Designated Products” published by the FDA
in October 2007. It is approved only for the treatment of diffuse
non-Hodgkin’s lymphoma, including AIDS-related diffuse non-
Hodgkin’s lymphoma. We believe that the search for new
antitumor guanylhydrazones could lead to safer drugs possibly
endowed with a broader range of activity. In our last paper in
this field2 and in the previous ones, the starting materials were
mainly imidazothiazoles with substituents at the positions 2, 3,
and 6. In this paper, we describe the search for new leads with
the following rationale:
(1) Additional 6-substituted imidazothiazole guanylhydra-
zones. We took into consideration the thiophene ring (and its
dimethyl and dichloro derivatives) connected to the 6 position
of the imidazothiazole system which may be unsubstituted (18,
19 Aa-Da, see Scheme 1) or may bear a methyl group at the 2
position (20 Aa-Da), thus being the same system present in
one of the most interesting derivatives of this series, the
guanylhydrazone of 6-(4-chloro-3-nitrophenyl)-2-methylimi-
dazo[2,1-b]thiazole.3
Chemistry
The hydrazones 18–21 (Scheme 1, Table 1) and 36–39 (Scheme
2, Table 1) were prepared by reaction between an aldehyde and
the appropriate hydrazine: aminoguanidine (a), 1,3-diaminoguani-
dine (b, c), 2-hydrazino-2-imidazoline (d), 2-hydrazinopyridine (e),
and 2-hydrazino-4-(trifluoromethyl)pyrimidine (f).
When the reaction was performed with 1,3-diaminoguanidine,
compound 20Gb, and the bishydrazones 22J, 23H were obtained,
whereas compound 20Ic was isolated in the presence of acetone.
The starting aldehydes 14–17 and 32–35 were obtained by
means of the Vilsmeier reaction on compounds 10–13 and 28–31
prepared in turn from the appropriate 2-aminothiazoles 1–4 and
the bromoketones 5 (to obtain the imidazo[2,1-b]thiazoles
10–13) or from compounds 24–27 treated with 2-bromo-4′-
chloro-3′-nitroacetophenone 5G (for the derivatives 28–31).
The IR and 1H NMR spectra of the new compounds
(Supporting Information, Table S1) are in agreement with the
assigned structures.
Two additional substituents for the 6 position have been
considered: the 3,5-dimethoxyphenyl group (19Fa) and the
carboxyethyl group (21Ea).
(2) Variations in the basic chain at the 5 position. These have
been performed in the imidazothiazoles bearing, at the 6
position, the most favorable substituents: 4-chloro-3-nitrophenyl3
and 2,5-dimethoxy-4(or 6)-nitrophenyl.4 In detail, while main-
taining chlorine at the 2 position and the 2,5-dimethoxy-4-
nitrophenyl group at the 6 position, the linear basic chain was
replaced by a chain including an heterocycle such as imidazo-
line, pyridine, and pyrimidine (21Hd-Hf). Moreover, while
maintaining the methyl group at the 2 position and the above-
mentioned phenyl groups (4-chloro-3-nitrophenyl and 2,5-
dimethoxy-6-nitrophenyl) at the 6 position, we considered, for
Biology
As a primary screening, all the new hydrazones were
submitted to the Developmental Therapeutics Program (DTP)a
* To whom correspondence should be addressed. Tel.: +39-51-2099714.
Fax: +39-51-2099734. E-mail: aldo.andreani@unibo.it.
a Abbreviations: DTP, Developmental Therapeutics Program; NCI,
National Cancer Institute; GI, growth inhibition; TGI, total growth inhibition;
LC, letal concentration; LDH, lactate dehydrogenase; DiOC6, 3,3′-dihexy-
loxacarbocyanine iodide; DClF-DA, dichlorofluorescin diacetate; PBS,
phosphate-buffered saline.
†
Dipartimento di Scienze Farmaceutiche, Università di Bologna.
‡
Dipartimento di Biochimica “G. Moruzzi”, Università di Bologna.
University of Michigan.
§
4
National Cancer Institute at Frederick.
10.1021/jm701246g CCC: $40.75
2008 American Chemical Society
Published on Web 02/06/2008

810 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Andreani et al.
Scheme 1a
a (x-y see Table 1).
at the National Cancer Institute (NCI) for evaluation of antitumor
activity in the human cell line screen. For some insights into
the biological effects of these derivatives, a few selected
compounds were submitted to additional studies: 20 Da (the
most active 6-thienyl derivative), 20Gb, 20Gd, 21Hd (not
aminoguanidine derivatives), 36 and 37 (not imidazothiazole
derivatives and in particular compound 36 showed a very potent
cytotoxic activity on HL-60 leukemia, subpanel not shown).
a. Antitumor Activity. In a preliminary test at a single
concentration (100 µM) against three human cell lines (NCI-
H460 lung cancer, MCF7 breast cancer, and SF-268 glioma), a
compound is considered active when it reduces the growth of
any of the cell lines to 32% or less, and it will be passed on for
evaluation in the full panel of 60 cell lines. This panel is
organized into subpanels representing leukemia, melanoma, and
cancers of the lung, colon, kidney, ovary, breast, prostate, and
central nervous system.
The test compounds were dissolved in DMSO and evaluated
using five concentrations at 10-fold dilutions, the highest being
10-4 M. Table 2 reports the results obtained with this test
(methyl-GAG is reported for comparison purposes) expressed
as -log of the molar concentration that inhibited cell growth
by 50% (pGI50), caused total cytostasis (pTGI ) Total Growth
Inhibition), or killed half of the cells (pLC50). These are averages
of data for nine different broad categories of the 60-cell panel:
the detailed results from each cell line are reported under the
Supporting Information (Table S5) for the most active com-
pounds as well as a correlation analysis (Table S6) which
provides a statistical comparison of the profiles of activity of
these compounds in this screen (the highest correlation coef-
ficients support the closest mechanistic connections between the
compounds).
or even lower than methyl-GAG. The most interesting results
obtained may be summarized as follows.
(1) 6-Substituted imidazothiazole guanylhydrazones. The
variations at the 6 position of the imidazothiazole system showed
that the carboxyethyl group (21Ea) is not suitable for this
position, whereas better results were obtained with the 3,5-
dimethoxyphenyl group (19Fa) even though the activity was
not higher than that of the compounds considered in our previous
papers.9,3,4,2 The same happens with the thiophene ring and its
dimethyl derivative in particular when connected to unsubsti-
tuted imidazothiazoles (18Aa-Ca; 19Aa-Ca).
By contrast, in the 2-methyl-imidazothiazoles, the antitumor
activity was higher (20Aa-20Ca) with mean pGI50 values of
5.03–5.40. For those derivatives, the pattern of selectivity is
also interesting, since about half of the cell lines were more
sensitive and half less sensitive than the average sensitivity of
all cell lines. Very good results were obtained when the
substituent at the 6 position was a dichlorothiophene: the
resulting compounds (18 Da, 19 Da, and 20 Da) showed mean
pGI50 values of 5.06, 5.79, and 5.96, respectively. This result
and the previous one provide evidence3 that a methyl group at
the 2 position of the imidazothiazole system leads to enhance-
ment of the antitumor activity, and a further confirmation is
the behavior of the guanylhydrazone 20 Da which is the most
active compound of this group with a low toxicity; in fact, it
showed a difference of 1.11 log units between the average
concentration which caused 50% growth inhibition (pGI50) and
the concentration which killed 50% of the cells (pLC50).
Moreover, for this molecule, six of the nine subpanels were
more sensitive than the average sensitivity of all cell lines.
(2) Variations in the basic chain at the 5 position. The
introduction of a basic chain different from that so far considered
showed that the dimethyldiaminoguanidine chain and the
pyrimidine ring lead to inactive compounds (20Ic and 21Hf).
Better results were obtained with the pyridine ring (20Ge and
Among the 27 derivatives tested by the NCI, 23 were active
in the primary test and were evaluated in the full panel of 60
cell lines showing growth inhibition concentrations similar to

Antitumor Guanylhydrazones and Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 811
Table 1. New Compounds
significant antitumor activity: this compound gave a mean pGI50
value of 5.67 and a difference of 1.11 log units between mean
pGI50 and mean pLC50.
compda
x-y
formula
MW
Mp, °C
10B
10C
10D
11C
11D
12C
12D
28
CH2-CH2 C9H8N2S2
CH2-CH2 C11H12N2S2
CH2-CH2 C9H6Cl2N2S2
208.29 146–151
236.36 86–87
277.20 85–86
(3) Replacement of the thiazole ring in the imidazothiazole
system. Replacement by a pyrimidine ring leads to an inactive
compound (39), whereas replacement by thiadiazole (36),
benzothiazole (37), and pyridine (38) gave interesting results
with mean pGI50 values between 5.74 and 6.23. Noteworthy is
the difference shown by these three compounds (1.10–1.59 log
units) between mean pGI50 and mean pLC50. In particular,
compound 37 gave the best result with an evident selectivity
toward the leukemia subpanel (mean pGI50 ) 6.86); therefore,
it will be considered a new lead in the design of the next series
of compounds.
b. Effects on Growth and Death of HL-60 Leukemia
Cells. A few compounds selected as described above were used
for further assays in a biological model represented by HL-60
leukemia cells. First, we examined whether the antiproliferative
effect was associated to interference in the cell cycle progression.
The cells were incubated for 24 h in the presence of a relatively
low concentration of the hydrazones (2 µM), and the analysis
of DNA profiles was performed by flow cytometry. No
significant differences were detected on cell cycle distribution
(Table 3), but a decrease in cell number associated to increased
cell death was observed. At the same concentration, methyl-
GAG, used as a positive control, did not cause any significant
effect. Only at 10 µM concentration, methyl-GAG began to
cause a decrease in the cell number, accompanied with a slight
change in cell cycle distribution, probably associated to the well-
known inhibition of polyamine synthesis.8 On the other hand,
none of new hydrazones inhibited the activity of S-adenosyl-
methionine decarboxylase in extracts from HL60 cells in the
range 1–100 µM (data not shown), and their effect was related
to cytotoxicity, which was particularly evident with compounds
21Hd and 36.
On the other hand, a decrease in cell number associated with
increased cell death was observed, being particularly evident
with compounds 21Hd and 36. The effects of these latter
derivatives, which appear to be cytotoxic at low concentrations
against HL-60 cells, were further studied. First, we determined
whether these derivatives induced apoptosis. Cells were treated
with 5 µM of the compounds under test, and the activation of
effector caspases acting on the substrate sequence Asp-Glu-
Val-Asp (DEVD) was determined. The activity of these caspase
proteases, a marker of apoptotic cell death, rapidly increased
in a dose-dependent mode (Figure 1A), reaching a maximum
after 4 h, and thereafter declined (Figure 1B). When the assay
was performed after 24 h of treatment, no caspase activation
could be detected, even if cell death was largely increased, as
determined by lactate dehydrogenase (LDH) release into the
medium (Figure 1C). The rapid onset of apoptosis was
confirmed by fluorescence microscopy analysis of treated cells.
In Figure 1D, HL-60 cells stained with Hoechst 33258 after
3 h of treatment are depicted. Nuclear changes associated with
hydrazones in treated cells were evident, in particular, the
characteristic chromatin condensation and the presence of
apoptotic bodies.
CHdCH
CHdCH
C11H10N2S2
234.34 58–60
C9H4Cl2N2S2
H3CCdCH C12H12N2S2
H3CCdCH C10H6Cl2N2S2
275.18 110–112
248.37 111–112
289.21 144–145
294.72 194–196
329.76 258–260
273.68 192–194
274.66 235–237
236.32 157–158
236.32 105–107
264.37 114–115
305.21 118–120
234.30 89–91
-
-
-
-
C11H7ClN4O2S
C15H8ClN3O2S
C13H8ClN3O2
C12H7ClN4O2
29
30
31
14A
14B
14C
14D
15A
15B
15C
15D
16A
16B
16C
16D
17E
32
CH2-CH2 C10H8N2OS2
CH2-CH2 C10H8N2OS2
CH2-CH2 C12H12N2OS2
CH2-CH2 C10H6Cl2N2OS2
CHdCH
CHdCH
CHdCH
CHdCH
C10H6N2OS2
C10H6N2OS2
C12H10N2OS2
C10H4Cl2N2OS2
234.30 150–152
262.35 137–140
303.19 188–190
248.33 115–117
248.33 158–160
276.38 145–147
317.22 165–167
258.68 150–152
322.73 205–207 dec
357.77 242–244
301.69 257–259
302.67 268–270
328.85 258–260 dec
328.85 268–270
356.90 232–234dec
397.74 215–217 dec
315.83 190–192
315.83 211–212
354.89 254–256
395.72 285–287 dec
380.86 297–300 dec
340.86 277–280 dec
340.86 314–316 dec
368.91 280–282 dec
397.74 290–291 dec
429.29 257–259 dec
484.76 324–326 dec
449.32 280–282 dec
494.96 195–198 dec
351.21 190–192 dec
486.34 250–252
495.34 225–227dec
H3CCdCH C11H8N2OS2
H3CCdCH C11H8N2OS2
H3CCdCH C13H12N2OS2
H3CCdCH C11H6Cl2N2OS2
ClCdCH
C9H7ClN2O3S
C12H7ClN4O3S
C16H8ClN3O3S
C14H8ClN3O3
C13H7ClN4O3
-
-
-
-
33
34
35
18Aa CH2-CH2 C11H12N6S2 ·HCl
18Ba CH2-CH2 C11H12N6S2 ·HCl
18Ca CH2-CH2 C13H16N6S2 ·HCl
18 Da CH2-CH2 C11H10Cl2N6S2 ·HCl
19Aa CHdCH
19Ba CHdCH
19Ca CHdCH
19 Da CHdCH
C10H11N6S2 ·HCl
C10H11N6S2 ·HCl
C13H14N6S2 ·HCl
C11H8Cl2N6S2 ·HCl
C15H16N6O2S·HCl
19Fa
CHdCH
20Aa H3CCdCH C12H12N6S2 ·HCl
20Ba H3CCdCH C12H12N6S2 ·HCl
20Ca H3CCdCH C14H16N6S2 ·HCl
20 Da H3CCdCH C11H10Cl2N6S2 ·HCl
20Gb H3CCdCH C14H13ClN8O2S·HCl
20Gd H3CCdCH C16H14ClN7O2S·HBr
20Ge H3CCdCH C18H13ClN6O2S·HCl
20Ic
H3CCdCH C19H22N8O4S·HCl
21Ea ClCdCH
21Hd ClCdCH
21He ClCdCH
21Hf
22J
23H
36
37
38
39
C10H11ClN6O2S·HCl
C17H16ClN7O4S·HCl
C19H15ClN6O4S·HCl
C19H13ClF3N7O4S·HCl 564.33 262–264 dec
C29H25N11O8S2 ·HCl 756.17 231–233dec
C29H23Cl2N11O8S2 ·HCl 825.06 260–262 dec
ClCdCH
CHdCH
ClCdCH
-
-
-
-
C13H11ClN8O2S·HCl
C17H12ClN7O2S·HCl
C15H12ClN7O2 ·HCl
C14H11ClN8O2 ·HCl
415.26 318–320dec
450.30 288–290 dec
394.22 310–312 dec
395.20 268–270dec
a According to Scheme 1, the uppercase letter is related to the substituent
at the 6 position (R) and the lowercase letter to the substituent at the side
chain (R′).
21He), whereas the best results were obtained with the unsub-
stituted diaminoguanidine chain (compound 20Gb showed mean
pGI50 ) 5.72) and with the imidazoline ring which is present
in compounds 20Gd and 21Hd giving mean pGI50 values of
5.90 and 5.99, respectively. It is interesting to point out that
21Hd (showing a difference of 1.36 log units between mean
pGI50 and mean pLC50) was more active than the corresponding
guanylhydrazone which showed a pGI50 value of 5.35.4 More-
over, compound 21Hd is also selective toward the colon
subpanel (pGI50 ) 6.30).
c. Effects on Mitochondria. Many guanidine-containing
drugs have been reported to interfere with mitochondrial
functions. Therefore, we investigated the effect of compounds
21Hd and 36 on the mitochondrial potential (∆Ψm) by using
the fluorescent dye DiOC6 (3,3′-dihexyloxacarbocyanine iodide)
to measure mitochondria depolarization in intact cells. Figure
2A shows that 1 h of treatment with either 21Hd or 36 resulted
The bishydrazones are only two compounds; nevertheless,
they provide a useful suggestion for the synthesis of the next
analogues since only the 2-chloro derivative 23H showed

812 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Andreani et al.
Scheme 2a
a i ) POCl3/DMF; ii ) aminoguanidine.
in a marked disruption of ∆Ψm. Mitochondrial membrane
depolarization has been associated with mitochondrial produc-
tion of reactive oxygen species.10 We therefore investigated
whether oxidative stress increased after treatment with the
hydrazones by using dichlorofluoresceine diacetate (DClF-DA),
a probe for peroxide species. As shown in Figure 2B, treatment
with compounds 21Hd and 36 caused an evident increase in
peroxide formation. These mitochondrial effects of the hydra-
zones were associated with loss of mitochondrial function and
a reduction in ATP synthesis. In fact, HPLC analysis of adenine
nucleotides showed a sharp decrease of cellular ATP, evidenced
by the large increase in the AMP/ATP ratio (Figure 2C) that
revealed a profound drop in the cellular energetic potential.
The biological studies of a few selected hydrazones have
shown that the efficient antiproliferative power of compounds
21Hd and 36 was associated with early damage to mitochondria.
Actually, a marked decrease in the mitochondrial transmem-
brane potential was detectable as soon as 1 h after the
treatment and was accompanied, as expected, with a large
increase in cellular peroxides and inhibition of ATP synthesis.
It appears reasonable to think that the oxidative stress and
the interference with cellular energetics are major molecular
mechanisms responsible for the cytotoxicity of the com-
pounds. Mitochondrial effects of compounds 21Hd and 36
(the effects of compound 37 were very similar to those of
36, data not shown) were associated with an early onset of
apoptosis, revealed by typical nuclear changes and by the
rapid activation of caspase proteases. It is worth noting that
the molecular characteristics of apoptosis are no longer
evident after 24 h of treatment, suggesting an abortive form
of apoptosis, possibly as a consequence of the fall in ATP,
that is required to maintain the active process of apoptosis.11
Some of the compounds reported in this series may owe a
significant part of their antitumor activity to the allylic imino
nitrogen atom moiety, in which the N is part of the hydrazone
link and the allylic CdC link is in the imidazole ring. This kind
of allylic moiety is seen in many compounds or their metabolites
that exhibit antitumor activity or a specific biological activity
that can produce cancer cell death, such as angiogenesis or
protein phosphorylation.12,13 The typical allylic moiety consists
of a propene sequence [-CRdCR-CH(X)-], with X signifying
an imino N, amino N, or oxygen that can be an OH, a carbonyl
O, or an O in ester, ether, or amide linkage. Many drugs with
this structural feature also exhibit an oxygen or nitrogen bound
to one or both carbon atoms of the double bond.13 Moreover,
in some of the compounds here described, metabolic oxidation
of the aromatic methoxy groups may result in demethylation,
followed by oxidation of the hydroxyls. This would yield a
quinone, typically formed by metabolism of the catechol and
hydroquinone moieties in drugs. Quinone groups are also present
in unmetabolized antitumor drugs, such as the anthraquinones.
Since quinones are double allylic ketones, they can be expected
to contribute further to the anticancer activity. Also adding to
the antitumor effect is the conjugation of the allylic double bond
produced when the quinone structure is formed.13 The effect
of the nitro group in the hypothetical quinone is not known.

Antitumor Guanylhydrazones and Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 813
Table 2. Sixty Cell Panel (Growth Inhibition and Cytostatic and Cytotoxic Activity of the Compounds which Passed the Three Line Test)
compda
18Aa
modes
leukemia
NSCLC
colon
CNS
melanoma
ovarian
renal
prostate
breast
MG-MIDb
pGI50
pTGI
pGI50
pGI50
pTGI
pGI50
pTGI
pLC50
pGI50
pTGI
pGI50
pTGI
pGI50
pTGI
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pTGI
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pTGI
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
4.73
4.41
4.72
4.68
-
4.99
4.61
4.32
5.17
4.42
5.16
4.62
4.81
-
6.00
5.67
5.21
4.99
4.72
4.45
5.74
5.39
4.98
5.11
4.33
4.32
-
4.54
-
4.76
4.41
4.64
4.86
4.53
5.74
4.79
4.52
5.01
4.62
4.85
4.53
4.81
4.49
6.01
5.63
4.95
5.22
4.83
4.50
5.59
5.09
4.64
5.27
4.69
5.43
4.93
4.41
5.99
5.40
4.70
6.02
5.68
4.83
5.92
5.00
4.68
4.58
4.37
4.32
4.68
4.32
6.30
5.53
5.07
4.35
-
4.72
4.40
4.60
4.73
4.46
5.06
4.76
4.47
4.92
4.59
4.82
4.54
4.81
4.52
5.95
5.55
4.93
5.09
4.76
4.46
5.37
4.93
4.48
5.16
4.71
5.08
4.74
4.42
5.95
5.36
-
5.95
5.53
5.01
5.97
5.37
4.88
5.11
4.71
4.45
4.71
-
6.19
5.68
4.69
4.81
4.52
4.40
5.79
5.59
4.43
5.91
5.01
-
4.67
4.42
4.66
4.81
4.53
5.00
4.69
4.45
4.82
4.54
4.77
4.50
4.79
4.51
5.55
5.14
4.81
5.07
4.75
4.51
5.65
5.23
4.86
5.09
4.71
5.13
4.82
4.56
5.98
5.51
5.11
5.74
5.34
4.62
6.01
5.62
5.03
4.74
4.42
4.33
4.71
4.34
5.90
5.41
4.94
4.48
4.33
-
4.40
-
4.64
-
4.71
4.44
4.76
4.81
4.51
5.09
4.82
4.55
4.91
4.61
4.83
4.54
4.86
4.57
5.81
5.21
4.76
5.03
4.79
4.55
5.52
4.91
4.40
5.12
4.70
5.32
4.72
-
6.10
5.83
5.56
5.47
5.02
4.56
5.94
5.53
4.82
4.93
4.41
-
4.76
4.39
4.49
4.62
4.38
4.91
4.64
4.38
4.83
4.55
4.76
4.50
4.79
4.52
5.78
5.37
4.63
5.00
4.69
4.43
5.24
4.73
-
5.11
4.65
5.35
4.87
4.41
5.91
5.58
4.40
5.81
5.27
4.60
5.74
5.12
4.69
4.86
4.41
-
4.64
-
5.74
4.37
-
4.76
4.38
-
5.48
5.17
-
5.99
5.16
-
6.13
5.02
-
5.64
5.27
4.64
4.80
3.50
2.80
4.66
4.32
4.62
4.74
4.43
5.06
4.68
4.43
4.96
4.56
4.85
4.53
4.79
4.48
5.79
5.32
4.81
5.07
4.74
4.47
5.40
4.93
4.46
5.06
4.57
5.03
4.65
4.32
5.96
5.53
4.85
5.72
5.23
4.63
5.90
5.32
4.71
4.86
4.48
4.33
4.70
4.31
5.99
5.35
4.63
4.70
4.41
4.33
5.67
5.30
4.56
5.90
5.32
4.37
6.23
5.40
4.64
5.74
5.36
4.64
4.67
3.36
2.80
18Ba
18Ca
4.54
4.77
4.45
4.95
4.65
4.43
4.89
4.53
4.82
4.52
4.78
4.48
5.57
4.97
4.49
5.03
4.69
4.46
5.11
4.60
-
4.86
4.38
5.01
4.60
-
5.96
5.44
4.71
5.62
5.01
-
5.81
5.22
4.55
4.94
4.51
4.32
4.74
4.38
6.03
5.32
4.64
4.59
-
4.84
4.62
4.32
4.98
4.64
4.41
4.99
4.57
4.81
4.50
4.76
4.47
5.96
5.45
4.79
5.10
4.77
4.46
5.19
4.76
-
4.55
4.76
4.42
5.00
4.70
4.46
5.08
4.66
4.83
4.53
4.75
4.49
5.79
5.24
4.88
5.07
4.75
4.50
5.37
4.85
4.36
4.99
4.55
4.85
4.49
-
5.91
5.52
4.85
5.49
5.07
4.72
5.84
5.43
4.83
4.81
4.40
4.33
4.67
-
5.87
5.47
4.84
4.87
4.51
4.34
5.66
5.31
4.60
5.91
5.44
4.39
6.08
5.41
4.95
5.66
5.31
4.64
4.70
3.30
2.70
18Da
19Aa
19Ba
19Ca
19Da
19Fa
20Aa
20Ba
20Ca
4.96
4.56
4.92
4.48
-
-
20Da
20Gb
20Gd
20Ge
6.05
5.75
5.45
5.20
-
5.99
5.63
4.71
5.89
5.43
4.85
5.85
5.47
4.32
5.04
4.59
4.31
4.68
-
6.13
5.73
4.64
4.70
4.48
4.38
5.74
5.42
4.61
5.83
5.37
4.37
5.96
5.71
4.72
5.73
5.44
5.20
4.80
3.50
2.80
-
6.17
5.21
-
4.78
4.47
-
4.85
4.41
5.58
-
21Ea
21Hd
4.77
4.41
6.12
5.94
4.73
4.79
-
-
21He
5.04
4.45
4.33
5.63
-
-
-
-
23H
5.69
5.40
-
5.48
4.94
-
6.17
5.42
4.62
5.77
5.42
5.20
4.60
3.50
2.80
5.65
5.15
4.85
6.19
5.55
4.79
6.47
5.44
4.72
5.73
5.10
4.57
4.90
3.30
2.90
5.72
5.29
4.91
5.84
5.53
4.45
6.15
5.56
4.70
5.69
5.44
-
5.71
5.42
5.13
5.96
5.58
5.23
5.64
4.83
-
5.82
5.51
5.24
4.00
2.80
2.60
-
36
6.24
5.44
-
6.86
-
37
6.35
5.52
4.84
5.92
5.52
4.84
4.70
3.60
3.00
-
38
5.77
5.42
-
5.10
3.20
2.60
methyl-GAGc
4.40
3.50
3.00
a Highest conc. ) 10-4 M. Only modes showing a value >4.30 are reported. b Calculated mean panel. c Highest conc. ) 10–2.6 M.
recorded in (CD3)2SO on a Varian Gemini (300 MHz); the chemical
shift (referenced to solvent signal) is expressed in δ (ppm) and J
in Hz (Table S1). The EI-MS were recorded at 70 eV on a VG
7070E. Elemental analyses were performed with a Fisons Carlo
Erba Instrument EA1108 and resulted within ( 0.4% of the
theoretical values (Table S3).
Experimental Section (See Also Supporting Information)
1. Chemistry. The melting points are uncorrected. Bakerflex
plates (silica gel IB2-F) were used for TLC. The eluent was
petroleum ether/acetone in various proportions (with 0.1% of conc.
NH4OH for the analysis of the guanylhydrazones as free bases).
Kieselgel 60 (Merck) was used for column chromatography. The
IR spectra were recorded in nujol on a Nicolet Avatar 320 ESP
spectrometer; νmax is expressed in cm-1. The 1H NMR spectra were
The starting compounds 1–5 and 24–27 are commercially
available. The imidazothiazoles 10A; 11A,B,F,J; 12A,B,G,I; and

814 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Andreani et al.
absorption was confirmed around 1700 cm-1) in the subsequent
step. It was refluxed for 1 h with 200 mL of 2 N HCl, and before
complete cooling, the solution was cautiously basified by dropwise
addition of 15% NH4OH. The resulting base was collected by
filtration and crystallized from petroleum ether (10C,D, 11C,D,
12C,D) or from acetone/petroleum ether (10B), with a yield of
70–80% (10C; 11C,D; 12C,D) and 45% (10B,D).
2a. Synthesis of Compounds 28-31 (Scheme 2). Compounds
28-31 were prepared with the same procedure described for the
synthesis of the imidazo[2,1-b]thiazoles, using the appropriate
2-amino derivative (24-27) which was treated with the 2-bro-
moacetophenone 5G. The crude products were purified by crystal-
lization from ethanol with a yield of 60–70%.
Table 3. Effects of Some Derivatives on Cell Cycle, Growth, and Death
of HL-60 Leukemia Cells
cell cycle phaseb
propidium iodide
G0/G1 ) positive cells (%)
G2/M cells/mL (x10-3
cell no.
Cell Death
compda
Ctrl
20 Da
20Gb
20Gd
21Hd
36
S
34.7 51.0 14.3
35.3 50.0 14.7
35.0 51.9 13.1
31.3 53.7 15.0
33.1 51.9 15.1
29.9 53.6 16.5
850 ( 72
750 ( 23
760 ( 11
780 ( 12
485 ( 10
410 ( 64
702 ( 26
4.4 ( 1.8
7.4 ( 1.6
7.8 ( 4.3
11.9 ( 2.7
22.0 ( 1.5
31.0 ( 1.7
5.2 ( 2.0
methyl-GAG 43.6 40.9 15.5
a 2 µM concentration except methyl-GAG which was 10 µM. b Percentage
of cells in the different phases of the cell cycle.
3a. Synthesis of the Aldehydes 14A-D, 15A-D, 16A-D,
17E (Scheme 1), and 32–35 (Scheme 2). The Vilsmeier reagent
was prepared at 0–5 °C by dropping POCl3 (54 mmol) into a stirred
solution of DMF (65 mmol) in CHCl3 (5 mL). The appropriate
condensed imidazole system (10–13, 28–31, 5 mmol) was sus-
pended in CHCl3 (20 mL). The mixture thus obtained was dropped
into the Vilsmeier reagent while maintaining stirring and cooling.
The reaction mixture was kept for 3 h at room temperature and
under reflux for 1–24 h (according to a TLC test). Chloroform was
removed under reduced pressure, and the resulting oil was poured
onto ice.
13E,H and the aldehydes 15F,J; 16G,I; and 17H were prepared
according to the literature.14–16,3,4,17
1a. Synthesis of the Imidazo[2,1-b]thiazoles 10B-D;
11C,D; and 12C,D (Scheme 1). The appropriate 2-aminothiazole
or 2-aminothiazoline 1–4 (20 mmol) was dissolved in 100 mL of
acetone and treated with the equivalent of the appropriate 2-bro-
moacetylthiophene 5B-D. The reaction mixture was refluxed for
2–3 h (according to a TLC test), and the resulting salt (6–9) was
separated by filtration and used without further purification (νC)O
Figure 1. Induction of apoptosis in HL-60 cells by derivatives 21Hd and 36. (A) The activity of caspase proteases cutting the peptide sequence
DEVD (DEVDase activity) was measured in cells treated for 4 h with the indicated concentration of the compounds. (B) DEVDase activity in cells
treated with 5 µM of compounds 21Hd and 36 was measured at the indicated time. (C) LDH release into the medium following 24 h in the presence
of 5 µM of 21Hd and 36. (D) Morphological evaluation of nuclei stained with Hoechst 33258 obtained from control cells (left), cells treated for
3 h with 5 µM of compounds 21Hd (central panel) or 36 (right). Each panel represents the results obtained in one typical experiment repeated at
least twice with comparable results.
Figure 2. Effects of compounds 21Hd and 36 on mitochondria. HL-60 cells were treated with 5 µM of the derivatives. (A) The mitochondrial
membrane potential ∆Ψm was determined by flow cytometry after 1 h. (B) Intracellular peroxides were detected by flow cytometry following a 3 h
incubation. (C) The AMP/ATP ratio was calculated following the measurement of adenine nucleotide levels in cells treated for 3 h. All the results
are mean ( SEM of triplicate determinations.

Antitumor Guanylhydrazones and Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 815
The crude aldehydes 14A,D; 15A-D; 16A-D; 17E; and 32-35
were collected by filtration, whereas compounds 14B,C precipitated
when the solution was basified by addition of NH4OH.
The crude aldehydes were crystallized from ethanol with a yield
of 70–90%.
4a. Synthesis of the Hydrazones 18–23 (Scheme 1) and 36–
39 (Scheme 2). The appropriate aldehyde (10 mmol) was dissolved
in ethanol and treated with the equivalent of the appropriate
hydrazine hydrochloride: aminoguanidine (for compounds 18Aa-
Da, 19Aa-Da,Fa, 20Aa-Da, 21Ea, and 36–39), 1,3-diaminoguani-
dine (for compounds 20Gb, 20Ic, 22J, and 23H), 2-hydrazinopy-
ridine (for compounds 20Ge and 21He), 2-hydrazino-4-
(trifluoromethyl)pyrimidine (for compound 21Hf).
In the case of compounds 20Gd and 21Hd, the aldehyde was
treatedwith2-hydrazino-2-imidazolinehydrobromideandhydrochloride.
The reaction mixture was refluxed for 5–30 h according to a
TLC test, and the resulting precipitate was collected by filtration
with a yield of 25–30% (21Hd, 22J, and 23H), 50–60% (19Ca,
19 Da, 20Gb, 20Gd, 20Ic, 21Hf, and 37), and 80–90% (18Aa-
Da, 19Aa, 19Ba, 19Fa, 20Aa, 20Ba, 20Ca, 20 Da, 20Ge, 21Ea,
21He, 36, 38, and 39).
aliquots of medium from each flask; these aliquots were then
analyzed spectrophotometrically for LDH activity by measuring
NADH levels at 340 nm.21
Caspase Activity. The activity of caspase enzymes hydrolyzing
the peptide sequence DEVD (Asp-Glu-Val-Asp), indicated as
DEVDase activity, was measured in cell extracts by a fluorimetric
assay.2
Adenine Nucleotide Level. The cellular content of adenine
nucleotides was determined by HPLC after extraction in perchloric
acid and conversion into fluorescent etheno-derivatives.22
Acknowledgment. This work has been supported by a grant
from MiUR-Prin 2006. We are grateful to NCI for the antitumor
tests.
Supporting Information Available: IR and 1H NMR of the
new compounds (Table S1), NSC numbers (Table S2), elemental
analyses (Table S3), mass spectra (Table S4), detailed results from
NCI for the most active compounds (Table S5), and correlation
analysis of the profiles of activity of these compounds (Table S6).
This material is available free of charge via the Internet at http://
pubs.acs.org.
Compound 20Ic was obtained by refluxing the reaction mixture
in the presence of acetone.
2. Biology. 2a. Antitumor Activity. The antitumor tests were
performed by the National Cancer Institute (NCI, Bethesda, Md)
as in our previous papers.18 The test compounds were dissolved in
DMSO and diluted 1:400 in complete culture medium resulting in
a final DMSO concentration of 0.25%.
2b. Effects on HL-60 Leukemia Cells. Cell Culture. HL-60
(human promyelocytic leukemic) cells were maintained in expo-
nential growth at 37 °C in a humidified 5% CO2 atmosphere in
RPMI 1640 medium supplemented with 10% Foetal Bovine Serum
and 2 mM glutamine. Under these growth conditions, cell doubling
time was about 24 h. Cell growth was assessed by cell counting
using Coulter Counter model Z1 (Florida, USA). All the compounds
under test were dissolved in dimethylsulfoxide and diluted to the
required concentration in medium.
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and 605 nm, respectively. At least 10 000 cells were collected, and
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