Synthesis and antitumor activity of guanylhydrazones from 6-(2,4-dichloro-5-nitrophenyl)imidazo[2,1-b]thiazoles and 6-pyridylimidazo[2,1- b]thiazoles

Article abstract of DOI:10.1021/jm061077m

The design and synthesis of antitumor imidazothiazole guanylhydrazones are reported. The compounds were submitted to NCI for testing. All but one were more active than methyl-GAG. A few compounds were selected for further studies in search of a possible mechanism of action. The results from these studies and a final search with the NCI COMPARE algorithm suggest that the guanylhydrazones described in this paper are acting through a novel mechanism of action.

Full text of DOI:10.1021/jm061077m

J. Med. Chem. 2006, 49, 7897-7901
7897
Brief Articles
Synthesis and Antitumor Activity of Guanylhydrazones from
6-(2,4-Dichloro-5-nitrophenyl)imidazo[2,1-b]thiazoles and 6-Pyridylimidazo[2,1-b]thiazoles1
Aldo Andreani,*,† Silvia Burnelli,† Massimiliano Granaiola,† Alberto Leoni,† Alessandra Locatelli,† Rita Morigi,†
Mirella Rambaldi,† Lucilla Varoli,† Giovanna Farruggia,‡ Claudio Stefanelli,‡ Lanfranco Masotti,‡ and Mark W. Kunkel§
Dipartimento di Scienze Farmaceutiche, UniVersita` di Bologna, Via Belmeloro 6, 40126 Bologna, Italy, Dipartimento di Biochimica “G.
Moruzzi”, UniVersita` di Bologna, Via Irnerio 48, 40126 Bologna, Italy, and DeVelopmental Therapeutics Program, Information Technology
Branch, DiVision of Cancer Treatment and Diagnosis, National Cancer Institute, 6130 ExecutiVe BouleVard, Room 8008, RockVille, Maryland,
20892-7444
ReceiVed September 13, 2006
The design and synthesis of antitumor imidazothiazole guanylhydrazones are reported. The compounds
were submitted to NCI for testing. All but one were more active than methyl-GAG. A few compounds were
selected for further studies in search of a possible mechanism of action. The results from these studies and
a final search with the NCI COMPARE algorithm suggest that the guanylhydrazones described in this paper
are acting through a novel mechanism of action.
Scheme 1
Introduction
In our third communication on the synthesis of antitumor
guanylhydrazones,2 we reported that in a series of 2,3-unsub-
stituted imidazo[2,1-b]thiazoles, 4-nitrophenyl and 4-chloro-3-
nitrophenyl at the 6 position are suitable pharmacophoric groups
giving rise to three potent antitumor agents. In a fourth
communication,3 we described the synthesis and antitumor
activity of new imidazothiazole guanylhydrazones bearing a
3-nitrophenyl, 4-nitrophenyl, or 4-chloro-3-nitrophenyl group
at the 6 position. The most active compounds were those bearing
chlorine and nitro groups on the phenyl ring. Encouraged by
the results with the compound unsubstituted at the 2,3-position,
we prepared the 2,3-dihydro analogue to evaluate the effect of
this change on the antitumor activity. We also performed the
synthesis of new analogues bearing an additional chlorine atom
on the phenyl ring, i.e., compounds with a 6-(2,4-dichloro-5-
nitrophenyl) group. An additional series of analogues bearing
a pyridyl group (i.e., a phenyl group bioisostere) at the 6 position
were also prepared.
Chemistry
The guanylhydrazones 6 reported in Scheme 1 and Table S1
(see Supporting Information) were prepared by reaction of
aminoguanidine with the appropriate aldehydes 5, obtained in
turn by means of the Vilsmeier reaction on the corresponding
imidazo[2,1-b]thiazoles 4, which were prepared from the
appropriate 2-aminothiazoles 1 and bromoketones 2. The
intermediate compounds 3 were isolated and used in the
Biology
All the guanylhydrazones 6 were submitted to the NCI cell
line screen for evaluation of their antitumor activity. For some
insights into the biological effects of these derivatives, a few
selected compounds were submitted to additional studies: 6Ha
(a member of the 2,4-dichloro-5-nitrophenyl derivatives and a
very potent cytotoxic agent on the leukemia panel); 6Ec and
6Fe (members of the pyridyl derivatives).
1
subsequent step without further purification. The IR and H
NMR spectra of the new compounds (Table S2) are in
agreement with the assigned structures. Analyses (C, H, N) were
within (0.4% of the theoretical values (Table S3).
* To whom correspondence should be addressed. Phone and fax: +39-
051-2099714. E-mail: aldo.andreani@unibo.it.
a. Antitumor Activity. In a preliminary test at a single
concentration against three human cell lines (see Supporting
Information) a compound is considered active when it reduces
the growth of any of the cell lines to 32% or less, and it will be
† Dipartimento di Scienze Farmaceutiche, Universita` di Bologna.
‡ Dipartimento di Biochimica “G. Moruzzi”, Universita` di Bologna.
§ National Cancer Institute.
10.1021/jm061077m CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/21/2006

7898 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26
Brief Articles
Table 1. Sixty-Cell Panel: Growth Inhibition and Cytostatic and Cytotoxic Activity of the Compounds That Passed the Three-Line Test
compda
6Aa
modes
leukemia
NSCLC
colon
CNS
melanoma
ovarian
renal
prostate
5.68
breast
MG-MIDb
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pTGI
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
5.84
5.37
4.22
6.47
5.50
5.75
5.46
5.20
5.94
5.59
5.99
5.67
4.99
6.35
5.66
5.81
5.46
5.80
5.52
5.29
6.12
5.60
5.73
5.45
5.20
5.88
5.55
5.35
5.68
4.71
5.70
5.35
4.62
5.93
5.54
5.28
5.68
4.93
4.22
4.77
4.48
4.19
5.06
4.44
4.07
4.53
4.09
4.01
5.82
5.52
5.19
5.78
5.51
5.27
5.00
4.55
4.19
5.10
4.71
4.27
5.18
4.50
4.24
5.10
4.82
4.54
5.02
4.56
4.21
4.74
4.45
4.16
4.63
4.37
4.14
4.40
4.05
5.18
4.80
4.35
4.70
3.30
2.70
5.55
5.22
5.76
5.44
4.64
6.19
5.45
4.38
5.67
4.48
4.03
4.87
4.48
4.17
5.20
4.49
4.04
4.91
4.23
6Ba
6Ca
6Da
6Ea
6Fa
6Ga
6Ha
6Bb
6Cc
6Ec
6Fc
6Cd
6Fd
6Ce
6.52
5.02
6.09
5.51
6.21
4.98
5.79
5.60
4.71
6.18
4.31
5.67
5.64
5.63
5.38
5.60
4.59
4.21
5.68
4.65
4.77
4.45
4.15
4.96
4.32
4.01
4.64
4.14
4.01
5.77
5.50
4.62
5.76
5.49
4.82
4.48
4.14
5.37
4.53
4.02
5.24
4.30
4.75
4.46
4.16
4.95
4.17
4.79
4.50
4.22
5.30
4.79
4.06
4.95
4.27
4.77
4.43
4.14
5.28
4.62
4.15
4.71
4.18
4.02
5.80
5.51
5.22
5.79
5.52
5.25
5.01
4.51
4.30
4.76
4.20
4.04
4.73
4.43
4.12
5.15
4.84
4.54
4.80
4.44
4.16
4.81
4.44
4.14
4.78
4.46
4.15
4.27
4.01
4.99
4.68
4.19
4.80
3.50
2.80
4.78
4.45
4.11
5.04
4.42
4.81
4.49
4.17
5.14
4.45
4.01
4.98
4.41
5.45
4.35
4.97
4.19
4.72
4.03
6.03
5.49
5.86
5.53
5.22
5.83
5.56
5.27
5.18
4.76
4.31
5.09
4.53
4.05
4.74
4.39
4.13
5.15
4.82
4.49
5.25
4.68
4.21
5.07
4.66
4.28
4.80
4.46
4.18
4.45
4.10
5.09
4.56
4.19
4.90
3.30
2.90
5.84
5.55
4.84
5.78
5.51
5.26
5.16
4.59
4.09
5.09
4.57
4.08
4.68
4.38
4.12
5.25
4.94
4.59
4.92
4.55
4.17
4.82
4.50
4.20
4.79
4.51
4.23
4.41
4.03
5.11
4.70
4.32
4.70
3.60
3.00
5.82
5.55
5.27
5.78
5.51
5.26
5.08
4.62
4.22
5.01
4.54
4.13
4.77
4.48
4.19
5.11
4.82
4.54
4.99
4.59
4.21
4.74
4.44
4.20
4.71
4.40
4.18
4.42
4.08
5.23
4.86
4.30
4.40
3.50
3.00
5.80
5.51
5.26
5.76
5.47
5.17
5.18
4.68
4.33
5.10
4.40
5.78
5.37
4.95
5.76
5.46
5.03
4.94
4.56
4.19
5.05
4.43
5.83
5.50
5.07
5.82
5.53
5.27
5.10
4.61
4.20
5.05
4.46
4.09
4.74
4.39
4.14
5.13
4.80
4.49
5.02
4.53
4.17
4.80
4.44
4.17
4.75
4.44
4.17
4.40
4.04
5.13
4.71
4.26
4.67
3.36
2.80
6.14
5.71
5.38
5.51
4.88
4.12
5.44
4.98
4.41
4.15
4.99
4.49
4.13
4.74
4.43
4.14
5.06
4.73
4.48
4.86
4.44
4.12
4.70
4.38
4.15
4.78
4.48
4.21
4.41
4.01
5.17
4.77
4.28
4.60
3.50
2.80
4.28
4.08
4.65
4.24
4.10
5.03
4.75
4.46
5.16
4.61
4.16
4.82
4.55
4.27
4.76
4.49
4.23
4.26
4.64
4.38
4.13
5.05
4.68
4.45
5.15
4.65
4.22
4.78
4.33
4.12
4.46
4.32
4.13
4.36
4.04
4.88
4.39
4.18
4.80
3.50
2.80
5.36
4.88
4.31
5.32
4.29
4.86
4.37
4.06
5.01
4.55
4.14
4.52
4.02
5.42
4.93
4.27
5.10
3.20
2.60
6Ee
6Fe
4.76
4.48
4.20
4.00
2.80
2.60
methyl-GAGc
a Highest concn ) 10-4 M unless otherwise reported; only modes showing a value of >4.00 are reported. b Mean graph midpoint, i.e., the calculated
mean panel. c Highest concn ) 10-2.6 M.
passed on for evaluation in the full panel of 60 cell lines. Table
1 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).
The dose response data from the NCI 60 cell line screen
showed a broader response range for 6Fe than for 6Ha and 6Ec;
the difference between the average concentration that caused
50% growth inhibition and the concentration that killed 50%
of the cells was almost 1 log (10-fold) for 6Fe and only 0.5 log
for 6Ha and 6Ec. Within each of the nine cell panels there was
a greater difference between the least-sensitive and the most-
sensitive cell lines in response to 6Fe than to the other two
compounds. For 6Fe about half of the cell lines were more
sensitive and half less sensitive than the average sensitivity of
all cell lines. For 6Ha and 6Ec all cell lines showed almost the
same sensitivity.
b. Effects on Cell Death of Leukemia HL60 Cells. The
leukemic cell line HL60 was used in these experiments. First,
we determined whether the guanylhydrazones induced apoptosis.
Figure 1A shows that none of the aforementioned compounds
(guanylhydrazones were tested at 1-100 µM) caused the
activation of caspase proteases acting on the substrate sequence
Asp-Glu-Val-Asp (DEVD), i.e., mainly effector caspases 3 and
7.4 Activation of this DEVDase activity represents a marker of
apoptotic cell death.4 On the other hand, etoposide, a classical
inducer of apoptosis5 that we used as a positive control,
substantially activated caspase activity. Also, cytofluorimetric
analysis of hypodiploid DNA content failed to detect any form
of apoptosis in guanylhydrazone-treated cells (data not shown).
Nevertheless, guanylhydrazones caused cell death, even if they

Brief Articles
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26 7899
Figure 3. Effects of 6Ec on cell cycle related events. (A) Cell cycle
distribution of HL60 cells after 24 h of treatments with different
amounts of 6Ec. Results are the mean ( SEM of three determinations.
(B) Morphological evaluation of nuclei stained with Hoechst 33258
obtained from untreated control cells or from cells treated for 72 h
with 25 µM 6Ec. (C) After 24 h in the absence (ctrl) or presence of
6Ec (25 µM), HL60 cells were collected and the cell cycle inhibitors
p21 and p27 were determined in cell extracts by Western blotting. All
the experiments shown in the figure were repeated at least twice with
similar results.
Figure 1. Induction of cell death by guanylhydrazones. (A) The activity
of caspase proteases acting on the peptide sequence DEVD (DEVDase
activity) was measured in HL60 cells treated for 24 h with 25 µM
guanylhydrazones or 10 µM etoposide. Guanylhydrazone concentrations
ranging from 1 to 100 µM were tested, with similar results. Data
represent the means ( SEM of triplicate measurements. (B) HL60 cells
were incubated for 24 h with 0.1-20 µM indicated guanylhydrazones.
Afterward cell death was evaluated by flow cytometry. The panel reports
the results obtained in one experiment repeated three times.
phase. Increasing the concentration of 6Ec resulted in a dose-
dependent increase in the percentage of cells in the G2/M
phases, while concomitantly the G0/G1 and S populations
decreased. At 25 µM 6Ec, 52% of HL60 cells were in the G2/M
phase. This block of the cell cycle in the G2/M phase was
maintained even after 48 and 72 h (data not shown). The
morphological evaluation of the nuclei, stained with Hoechst
33458 and examined by fluorescence microscopy, revealed that
in 6Ec-treated cells, the nuclei were bigger than in control cells,
but chromosomes were not detectable and the chromatin
appeared to be completely decondensed (Figure 3B). This
observation suggests that the cells were blocked in the G2 rather
than in the M phase.
At concentrations higher than 10 µM, 6Ec also caused an
increase in the content of the cell cycle inhibitors p21 and p27
(Figure 3C). Since these proteins are able to inhibit several
cyclin-dependent kinases,6 it appears likely that high concentra-
tions of 6Ec can also decrease the rate of cell cycle. The other
guanylhydrazones 6Ha and 6Fe did not cause any of these cell
cycle-related effects at sublethal concentrations (0.5 µM).
Some guanylhydrazones can block cell growth by inhibiting
the enzyme S-adenosylmethionine decarboxylase (SAMDC),
which is necessary for the synthesis of the polyamines spermi-
dine and spermine.7 The polyamines in turn are required for
cell cycle progression and cell proliferation. However, 6Ha, 6Ec,
and 6Fe did not inhibit the enzymatic activity of SAMDC in
extracts obtained from growing HL60 cells (data not shown),
in contrast to 4-amidinoindan-1-one-2′-amidinohydrazone
(CGP48664) and methylglyoxal bisguanylhydrazone (MGBG),
which are established inhibitors of the enzyme.8
d. Effects on the Mitochondrial Membrane Potential.
Previous studies have shown that the inhibition of mitochondrial
functions is a feature of many guanidino-containing drugs.9,10
Therefore, we investigated whether disruption of the mitochon-
drial transmembrane potential ∆Ψm was involved in the
antiproliferative action of these novel guanylhydrazones. HL60
cells were treated for 24 h with sublethal concentrations of the
drugs and then analyzed cytofluorimetrically by using the
potentiometric fluorescent dye DiOC6 (3,3′-dihexyloxacarbocya-
nine iodide) to measure mitochondrial membrane depolarization
in intact viable cells. Sublethal dosages of 6Ha and 6Fe (0.5
µM) did not affect the mitochondrial potential, whereas 6Ec
Figure 2. Effect of 6Ec on viability and growth of HL60 cells. Cells
were incubated for 24 h in the presence of the indicated concentrations
of 6Ec. Afterward the amount of cell survival (A) and the total cell
number (B) were determined by flow cytometry. The panels depict
the results obtained in one experiment repeated three times with
comparable results.
displayed different potencies. Figure 1B shows that 6Ha was
more toxic than 6Fe, whose cytotoxicity in turn was much higher
than that of 6Ec. Since 6Ec causes cell growth inhibition (see
the 60-cell panel), its effect must be mainly cytostatic. Actually,
sublethal concentrations of 6Ec were able to reduce cell
proliferation; a 25 µM concentration elicited only a 25% of cell
death after 48 h of treatment (Figure 2A), but cell proliferation
was reduced more than 80% (Figure 2B). Higher doses of 6Ec
were required to cause a marked cytotoxic effect; at 50 µM,
6Ec led to death about 45% of the cell populations within 3 h
and the whole culture after 24 h of incubation. Again, no sign
of apoptosis could be observed, indicating that all the tested
compounds exert their cytotoxic effect by inducing mainly
necrotic cell death.
c. Effects on Cell Cycle and on the Activity of S-
Adenosylmethionine Decarboxylase. To verify whether the
inhibition of cell growth by 6Ec might be attributable to
interference with the cell cycle program, HL60 cells were treated
with graded concentrations of the drugs for 24 h. Then the
analysis of DNA profiles was performed using propidium iodide
staining and flow cytometry. Figure 3A illustrates that the
majority of control cells were in the S phase or in the G0/G1
phase of the cell cycle and less than 10% were in the G2/M

7900 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26
Brief Articles
tested on the 60 cell line panel, showing pGI50 values between
4.74 and 5.13, were more active than methyl-GAG.
The introduction of a second chlorine atom on the phenyl
ring did not cause a significant change in the antitumor activity,
compared with the analogues with a single chlorine atom.3 The
substitution at the 2 position is more favorable if compared with
3-substitution and 2,3-disubstitution; the substituents leading to
higher growth inhibition were the ethyl (6Ga) and propyl group
(6Ha), but the corresponding methyl derivative (6Ca) showed
a more favorable ratio between growth inhibition and lethality
(pGI50/pLC50). Generally speaking, this class of 6-(2,4-dichloro-
5-nitrophenyl) derivatives is very interesting because all of
them entered the 60 cell line test and growth inhibition
(pGI50 ) 4.87-6.19) was more effective than that induced by
methyl-GAG.
Figure 4. Effect of 6Ec on the mitochondrial membrane potential ∆Ψm
(A) and ATP levels (B) in HL60 cells. Cells were cultured in the
presence of various concentrations of 6Ec for 24 h. (A) At the end of
the incubation the cells were stained with DiOC6 and analyzed
immediately by flow cytometry to measure ∆Ψm. (B) Acid extracts
were obtained, and the content of ATP was measured by HPLC. Results
are the mean ( SEM of triplicate determinations.
Eliminating the double bond at the 2,3 position (6Bb, mean
pGI50 ) 5.10) while maintaining the same substituent at the 6
position leads to a derivative that was a little less active than
the unsaturated parent compound previously published (mean
pGI50 ) 5.79 2), but the introduction of a second chlorine atom
on the 6-phenyl ring leads to a compound (6Ba) that was much
more active and less toxic (pGI50/pLC50 ) 1.41) than the
dose-dependently decreased the value of ∆Ψm, which was
reduced by about 50% when the drug concentration was 25 µM
(Figure 4A). In Figure 4B it is depicted that the collapse of
∆Ψm was accompanied by a decrease of cellular ATP content,
indicating an interference of 6Ec with cellular energetics. It is
worth noting that mitochondria depolarization often defines an
early but already irreversible stage of apoptosis,11 but this is
not the case because at toxic concentrations 6Ec appears to
trigger necrotic cell death, since no sign of apoptosis could be
detected.
e. COMPARE. We have used the NCI COMPARE algo-
rithm12 to search the NCI databases of public 60-cell screening
results and molecular target expression data to look for
additional effects of the guanylhydrazones that may occur
upstream of their effects on apoptosis and necrosis. COMPARE
can suggest compounds that might induce cytostasis and
cytotoxicity similar to our compounds and molecular targets
whose levels of expression in the cell lines are correlated with
the activity of our compounds. COMPARE identified com-
pounds with correlations to the GI50 data for the guanylhydra-
zones. These results included several other compounds that we
had previously submitted to the NCI 60-cell screen and a few
modestly correlated compounds whose mechanisms of action
are not known (see Supporting Information). There were no
strongly correlated molecular targets for any of the compounds
individually, but COMPARE did identify several members of
different classes of molecular targets that were common to the
results for nine or more of the compounds: receptor tyrosine
kinases and phosphatases, components of cell structure and
attachment, and components of the cell cycle machinery.
COMPARE cannot identify targets or mechanisms; it can only
make suggestions. The dearth of high correlations in the public
data set suggests that these guanylhydrazones may be acting
through a novel mechanism of action.
corresponding unsaturated derivative (6Aa, pGI50/pLC50
)
1.24). Compound 6Ba, with mean pGI50 of 6.19, was also the
most active derivative of the whole series presented in this paper.
The antiproliferative activity of these novel guanylhydrazones
can be mediated through cytotoxic or cytostatic effects. Among
the selected compounds that we tested against leukemic HL60
cells, 6Ha and 6Fe are mainly cytotoxic and trigger a nonapo-
ptotic cell death at relatively low concentrations (in the low
micromolar range). On the other hand, 6Ec is only cytotoxic at
high concentrations and its antiproliferative effect is largely due
to cytostasis. We found that the antiproliferative effect of 6Ec
was associated with a block in cell cycle progression, with cell
accumulation in the G2/M phase and with a marked reduction
in the mitochondrial transmembrane potential ∆Ψm and a
decrease in the intracellular ATP content. Other guanylhydra-
zones have been reported to disturb cell cycle progression and
mitochondrial function.2,9,10 We were not able to detect these
effects in cells treated with the powerful inducers of cell death
6Ha or 6Fe. However, we cannot exclude that cell cycle- and
mitochondria-related effects are common to many of these novel
guanylhydrazones but, being exerted at concentrations of about
10 µM, could be seen only in the case of compounds with low
toxicity. As regards the cytotoxic effect, the tested compounds
trigger cell death that does not appear to be apoptotic.
Experimental Section
1. Chemistry. Most of the starting compounds were prepared
according to the literature.13-20
Synthesis of the Guanylhydrazones 6. The appropriate aldehyde
5 (10 mmol) was dissolved 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 refluxed for
5-10 h according to a TLC test, and the resulting precipitate was
collected by filtration with a yield of 50-70% (6Ba, 6Ea, 6G-
Ha, 6Bb, 6Ac, 6Ec, 6C-Fd, 6De, 6Fe) and 80-95% (6Aa, 6C-
Da, 6Fa, 6B-Cc, 6Ec, 6A-Bd, 6Dd, 6Ae, 6Ce, 6Ee).
2. Biology. 2.a. Antitumor Activity. The antitumor tests (see
Supporting Information) were performed by the National Cancer
Institute (Bethesda, MD) as in our previous papers.21
Conclusion
The biological activity of the guanylhydrazones 6 is not
correlated to their clogP (see Table S4). When the 6 position
bears a pyridyl instead of a phenyl ring, a substituent at the 2
position is also necessary because the unsubstituted (6Ac-e,
6Bc-e) and the 3-substituted derivatives (6Dc-e) did not reach,
in the 3 cell line test, the minimum potency necessary to enter
the full 60 cell line panel. The methyl disubstitution at positions
2 and 3 leads to improvement of activity because compounds
6Ec,e were active on the 3 cell line test and therefore entered
the following step. The best results were obtained with chlorine
at the 2 position, but compounds with a methyl group in the
same position were also active. Seven of the eight derivatives
2.b. Effects on Cell Death of Leukemia HL60 Cells. Human
leukemia HL60 cells were routinely grown in RPMI 1640 medium
supplemented with 10% fetal calf serum and 2 mM glutamine at
37 °C in air/5% CO2.

Brief Articles
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26 7901
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Rambaldi, M.; Giorgi, G.; Garaliene, V. Potential Antitumor Agents.
34. Synthesis and Antitumor Activity of Guanylhydrazones from
Imidazo[2,1-b]thiazoles and from Diimidazo[1,2-a:1,2-c]pyrimidine.
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Compounds were dissolved in dimethylsulfoxide and diluted to
the required concentration in complete medium. Control cells
received the corresponding volume of dimethylsulphoxide. Cell
death was evaluated by flow cytometry with propidium iodide
staining as previously described10 and by conventional trypan blue
exclusion.
2.c. Effects on Cell Cycle and on the Activity of S-Adenos-
ylmethionine Decarboxylase. 2.c.1. Cell Cycle. At the indicated
time points, nuclei were isolated (ref 22 in Supporting Information)
and kept at 4 °C in the dark. Cells were stained with 50 µg/mL
propidium iodide immediately prior to analysis. Cell cycle profiles
were determined using an Epics Elite (Beckman Coulter) and
analyzed by Modfit software (Verity). Nuclear morphology was
evaluated according to Comin-Anduix et al. (ref 23 in Supporting
Information). To determine the content of p21 and p27 proteins, at
the end of the incubation the cellular proteins were extracted and
assayed by Western blotting as previously described (ref 24 in
Supporting Information). For immunodetection, specific antibodies
from Santa Cruz were used.
2.c.2. Enzyme Activity Assays. The activity of SAMDC was
assayed by measuring the release of radioactive CO2 from [14C-
carboxyl]S-adenosylmethionine.8 The activity of caspase enzymes
hydrolyzing the peptide sequence DEVD was measured in cell
extracts by a fluorimetric assay (ref 25 in Supporting Information).
One unit (U) of enzyme activity catalyzes the formation of 1 nmol
of product per minute.
2.d. Effects on the Mitochondrial Membrane Potential. To
assess the mitochondrial transmembrane potential ∆Ψm, 106 cells
were collected by centrifugation (200g for 10 min) and resuspended
in complete medium containing 4 nM DiOC6 (ref 26 in Supporting
Information). The cells were incubated for 40 min at 37 °C and
then analyzed by flow cytometry using a log scale as described.10
The cellular content of ATP was determined by HPLC after
extraction in perchloric acid (ref 27 in Supporting Information).
(7) Stanek, J.; Caravatti, G.; Capraro, H. G.; Furet, P.; Mett, H.;
Schneider, P.; Regenass, U. S-Adenosylmethionine Decarboxylase
Inhibitors: New Aryl and Heteroaryl Analogues of Methylglyoxal
Bis(guanylhydrazone). J. Med. Chem. 1993, 36, 46-54.
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C. W. CGP 48664, a New S-Adenosylmethionine Decarboxylase
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Rambaldi, M.; Lenaz, G.; Fato, R.; Bergamini, C.; Farruggia, G.
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. J.
Med. Chem. 2005, 48, 3085-3089.
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Potential (deltapsi(m)) in Apoptosis: An Update. Apoptosis 2003,
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A.; Rubinstein, L.; Plowman, J.; Boyd, M. R. Display and Analysis
of Patterns of Differential Activity of Drugs against Human Tumor
Cell Lines: Development of Mean Graph and COMPARE Algorithm.
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Chem. Abstr. 1947, 41, 15308.
(14) Erlenmeyer, H.; Jung, J. P. Derivatives of 2-Aminothiazoles. HelV.
Chim. Acta 1949, 32, 35-38.
Acknowledgment. This work was supported by a grant from
MIUR-COFIN 2002. We are grateful to NCI for the antitumor
tests and to Dr. Matteo Masetti (Dipartimento di Scienze
Farmaceutiche, Universita` di Bologna) for the helpful discus-
sions about clogP.
(15) Garg, H. G. Synthesis of Nitrophenacyl Bromides. J. Indian Chem.
Soc. 1961, 38, 59.
(16) Andreani, A.; Rambaldi, M.; Andreani, F.; Bossa, R.; Galatulas, I.
6-Pyridinylimidazo[2,1-b]thiazoles and Thiazolines as Potential Car-
diotonic Agents. Eur. J. Med. Chem. 1985, 20, 93-94.
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(19) Andreani, A.; Rambaldi, M.; Andreani, F.; Hrelia, P.; Cantelli Forti,
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Supporting Information Available: Detailed experimental
procedures with analytical and spectroscopic data for all the new
compounds. This material is available free of charge via the Internet
at http://pubs.acs.org.
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