Brief Articles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17 5607
(2) Andreani, A.; Locatelli, A.; Leoni, A.; Rambaldi, M.; Morigi, R.;
Bossa, R.; Chiericozzi, M.; Fraccari, A.; Galatulas, I. Synthesis
and Potential Coanthracyclinic Activity of Substituted 3-(5-
Imidazo[2,1-b]thiazolylmethylene)-2-indolinones. Eur. J. Med.
Chem. 1997, 32, 919-924.
(3) Andreani, A.; Granaiola, M.; Leoni, A.; Locatelli, A.; Morigi, R.;
Rambaldi, M.; Giorgi, G.; Salvini, L.; Garaliene, V. Synthesis
and Antitumor Activity of Substituted 3-(5-Imidazo[2,1-b]thia-
zolylmethylene)-2-indolinones. Anticancer Drug Des. 2001, 16,
167-174.
(4) Comin-Anduix, B.; Agell, N.; Bachs, O.; Ovadi, J.; Cascante, M.
A New Bis-indole, KARs, Induces Selective M Arrest with
Specific Spindle Aberration in Neuroblastoma Cell Line SH-
SY5Y. Mol. Pharmacol. 2001, 60, 1235-1242.
(5) Mahboobi, S.; Pongratz, H.; Hufsky, H.; Hockmeyer, J.; Frieser,
M.; Lyssenko, A.; Paper D. H.; Burgermeister, J.; Bohmer F. D.;
Fiebig H. H.; Burger, A. M.; Baasner, S.; Beckers, T. Synthetic
2-Aroylindole Derivatives as a New Class of Potent Tubulin-
inhibitory, Antimitotic Agents. J. Med. Chem. 2001, 44, 4535-
4553.
(6) Fan, M.; Goodwin, M. E.; Birrer, M. J.; Chambers, T. C. The
c-Jun NH(2)-terminal Protein Kinase/AP-1 Pathway is Required
for Efficient Apoptosis Induced by Vinblastine. Cancer Res. 2001,
61, 4450-4458.
(7) Andreani, A.; Leoni, A.; Locatelli, A.; Morigi, R.; Rambaldi, M.;
Recanatini, M.; Garaliene, V. Potential Antitumor Agents. 29.
Synthesis and Potential Coanthracyclinic Activity of Imidazo-
[2,1-b]thiazole Guanylhydrazones. Bioorg. Med. Chem. 2000, 8,
2359-2366.
maintaining stirring and cooling. The reaction mixture was
kept for 3 h at room temperature and under reflux for 25 h.
Chloroform was removed under reduced pressure, and the
resulting oil was poured into ice. The crude aldehyde thus
obtained was collected by filtration and crystallized from
ethanol with a yield of 60%. C8H5F3N2OS (234.2) mp 115-
1
122 °C. IR: 3119, 1655, 1537, 1030, 861. H NMR: 2.55 (3H,
d, CH3, J ) 1.2), 8.31 (1H, q, th, J ) 1.2), 9.93 (1H, s, CHO).
General Procedure for the Synthesis of Compounds
16-28. The appropriate aldehyde 4-9 (10 mmol) was dissolved
in methanol (100 mL) and treated with the appropriate
indolinone 10-15 (10 mmol) and piperidine (1 mL). The
reaction mixture was refluxed for 3-5 h (according to a TLC
test). The precipitate formed on cooling was collected by
filtration and purified by crystallization from ethanol with a
yield of 20-30% (23, 26, 28), 40-50% (21, 22, 24, 27), 70-
80% (16-20, 25).
(B) Pharmacology. (a) In vitro Growth Inhibition and
Cytotoxicity. It was determined by the NCI according to
standard procedures.18
(b) Cell Cycle Analysis. The experiments were carried out
on HT-29 cells according to the procedures previously de-
scribed.19 Compound 20 and 21 were dissolved in dimethyl
sulfoxide (DMSO) at a concentration of 10 mg/mL, and a
solution 10-4 M was prepared in complete RPMI 1640. Ten-
fold serial dilutions were used to obtain the other concentra-
tions, and DMSO was added to control cells. Commercial
vincristine sulfate was diluted in the complete medium. Cell
cycle distribution was performed on nuclei isolated and stained
according to Nusse et al.20 Flow cytometric plots were analyzed
using the ModFit software (Verity). Nuclear morphology was
evaluated according to Comin-Anduix et al.4 All the data
presented are expressed as the mean ( SD of three indepen-
dent experiments.
(c) Effect on Tubulin Polymerization. The tubulin
polymerization assay was performed using CytoDYNAMIX
ScreenTM kit (Cytoskeleton Inc., CO) as per manufacturers’
protocol. Briefly, a 96-well microplate was prewarmed at 37
°C for 1 h. HTS-Tubulin (>97% pure) was reconstituted to 3
mg/mL in G-PEM buffer (80 mM PIPES pH 6.8, 0.5 mM
EGTA, 2.0 mM MgCl2, 1.0 mM GTP, and 5% glycerol).
Polymerization reaction was initiated by mixing 100 µL of
reconstituted tubulin with vehicle or test compound (10 µM
final concentration) into each well of the prewarmed plate.
Polymerization kinetics were then measured at 37 °C for 1 h
at 340 nm using a TECAN GENiospro microplate reader
(TECAN U.S. Inc., Research Triangle Park, NC).
(8) Beer, R. J. S.; Davenport, H. F.; Robertson, A. Extensions of the
Synthesis of Hydroxyindoles from p-Benzoquinones. J. Chem.
Soc. 1953, 1262-1264.
(9) Andreani, A.; Granaiola, M.; Leoni, A.; Locatelli, A.; Morigi, R.;
Rambaldi, M.; Garaliene, V.: Synthesis and Antitumor Activity
of 1,5,6-Substituted E-3-(2-chloro-3-indolylmethylene)1,3-dihy-
droindol-2-ones. J. Med. Chem. 2002, 45, 2666-2669.
(10) Andreani, A.; Rambaldi, M.; Bonazzi, D.; Greci, L.; Andreani,
F. Potential Antitumor Agents. III. Hydrazone Derivatives of
5-substituted 2-chloro-3-formyl-6-methylindole. Farmaco, Ed.
Sci. 1979, 34, 132-138.
(11) Porter, J. C.; Robinson, R.; Wyler, M. Monothiophthalimide and
Some Derivatives of Oxindole. J. Chem. Soc. 1941, 620-624.
(12) Koelsch C. F. A Synthesis of Ethyl Quininate from m-Cresol. J.
Am. Chem. Soc. 1944, 66, 2019-2020.
(13) Andreani, A.; Rambaldi, M.; Carloni, P.; Greci, L.; Stipa, P.
Imidazo[2,1-b]thiazole Carbamates and Acylureas as Potential
Insect Control Agents. J. Heterocycl. Chem. 1989, 26, 525-529.
(14) Andreani, A.; Rambaldi, M.; Mascellani, G.; Rugarli, P. Synthesis
and Diuretic Activity of Imidazo[2,1-b]thiazole Acetohydrazones.
Eur. J. Med. Chem. 1987, 22, 19-22.
(15) Andreani, A.; Rambaldi, M.; Leoni, A.; Locatelli, A.; Bossa, R.;
Fraccari, A.; Galatulas, I.; Salvatore, G.: Potential Antitumor
Agents. 24. Synthesis and Pharmacological Behavior of Imidazo-
[2,1-b]thiazole Guanylhydrazones Bearing at Least One Chlo-
rine. J. Med. Chem. 1996, 39, 2852-2855.
(d) Positive Inotropic Activity. The experiments were
carried out on the guinea-pig papillary muscles according to
a procedure previously described.7
(16) Andreani, A.; Granaiola, M.; Leoni, A.; Locatelli, A.; Morigi, R.;
Rambaldi, M.; Recanatini, M.; Lenaz, G.; Fato, R.; Bergamini,
C. Effects of New Ubiquinone-imidazo[2,1-b]thiazoles on Mito-
chondrial Complex 1 (NADH-ubiquinone reductase) and on
Mitochondrial Permeability Transition Pore. Bioorg. Med. Chem.
2004, 12, 5525-5532.
(17) Seki, T.; Tasaka, N.; Hoshino, R.; Kojima, J.; Fujii, K. Prepara-
tion of Imidazo[2,1-b]thiazoles for Improvement of Cerebral
Function and as Antiulcer Agents. Jpn. Kokai Tokkyo Koho JP
02178289, 1990. Chem. Abstr. 113, 191361.
(18) Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paull, K.;
Vistica, D.; Hose, C.; Langley, J.; Cronise, P.; Vaigro-Wolff, A.
Feasibility of a High-flux Anticancer Drug Screen Using a
Diverse Panel of Cultured Human Tumor Cell Lines. J. Natl.
Cancer Inst. 1991, 83, 757-766.
(19) Andreani, A.; Granaiola, M.; Leoni, A.; Locatelli, A.; Morigi, R.;
Rambaldi, M.; Garaliene, V.; Farruggia, G.; Masotti L. Substi-
tuted E-3-(2-chloro-3-indolylmethylene)1,3-dihydroindol-2-ones
with Antitumor Activity. Bioorg. Med. Chem. 2004, 12, 1121-
1128.
Acknowledgment. This work has been supported
by a grant from MIUR-COFIN 2002, contract number
2002033121_003. We are grateful to the National Can-
cer Institute (Bethesda, MD) for the antitumor tests.
Supporting Information Available: Additional data on
biological effects (Figure 1S: percentage of cell death. Figure
2S: time-dependent antiproliferative effect of compound 20
and 21. Figure 3S: cytograms of BrdU incorporation versus
PI fluorescence. Figure 4S: cell cycle phase distribution at
different times). C, H, N analytical data. IR and 1H NMR
spectroscopic data. This material is available free of charge
References
(1) Potential Antitumor Agents 38. For part 37 see Andreani, A.;
Granaiola, M.; Leoni, A.; Locatelli, A.; Morigi, R.; 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]quino-
line. J. Med. Chem. 2005, 48, 3085-3089.
(20) Nusse, M.; Beisker, W.; Hoffman, C.; Tarnok, A. Flow Cytometric
Analysis of G1- and G2/M-phase Subpopulations in Mammalian
Cell Nuclei using Side Scatter and DNA Content Measurements.
Cytometry 1990, 11, 813-821.
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