Antitumor Activity of New Substituted 3-(5-Imidazo[2,1-b

Nov 12, 2008 - Mirella Rambaldi,† Lucilla Varoli,† Natalia Calonghi,‡ Concettina Cappadone,‡ Manuela Voltattorni,§ Maddalena Zini,‡. Claudi...
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J. Med. Chem. 2008, 51, 7508–7513

Antitumor Activity of New Substituted 3-(5-Imidazo[2,1-b]thiazolylmethylene)-2-indolinones and 3-(5-Imidazo[2,1-b]thiadiazolylmethylene)-2-indolinones: Selectivity against Colon Tumor Cells and Effect on Cell Cycle-Related Events1 Aldo Andreani,*,† Silvia Burnelli,† Massimiliano Granaiola,† Alberto Leoni,† Alessandra Locatelli,† Rita Morigi,† Mirella Rambaldi,† Lucilla Varoli,† Natalia Calonghi,‡ Concettina Cappadone,‡ Manuela Voltattorni,§ Maddalena Zini,‡ Claudio Stefanelli,‡ Lanfranco Masotti,‡ and Robert H. Shoemaker| 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, Centro Interdipartimentale di Ricerche Biotecnologiche (CIRB), UniVersita` di Bologna, Via S. Donato, 15, 40127 Bologna, Italy, and Screening Technologies Branch, DeVelopmental Therapeutics Program, DiVision of Cancer Treatment and Diagnosis, National Cancer Institute at Frederick, Frederick, Maryland 21702 ReceiVed July 8, 2008

The synthesis of new 3-(5-imidazo[2,1-b]thiazolylmethylene)-2-indolinones and 3-(5-imidazo[2,1-b]thiadiazolylmethylene)-2-indolinones is reported. The antitumor activity was evaluated according to the protocols available at the National Cancer Institute (NCI), Bethesda, MD. To investigate the mechanism of action of the most potent antitumor agent of this series, its effect on growth of HT-29 colon carcinoma cells was studied. Its ability to inhibit cellular proliferation was mediated by cell cycle arrest at the G2/M phase, accompanied by inhibition of ornithine decarboxylase (ODC), the limiting enzyme of polyamine synthesis, and followed by induction of apoptosis. Chart 1

Introduction This is our fourth paper devoted to the synthesis of antitumor 3-(5-imidazo[2,1-b]thiazolylmethylene)-2-indolinones.2-4 Chart 1 reports the most active derivatives so far obtained: A (mean pGI50 ) 5.82)2 and B (mean pGI50 ) 6.42).4 We describe here new analogues with different substituents in the indole as well in the imidazothiazole portion. The synthesis of analogues bearing the imidazothiadiazole in place of the imidazothiazole system was also performed. The antitumor activity of all the new compounds was evaluated in a preliminary test according to the protocols available at the National Cancer Institute (NCIa), Bethesda, MD, and the active compounds were further tested in the full five-dose assay. To investigate the mechanism of action of the most potent antitumor agent, its effect on growth of HT-29 colon carcinoma cells was studied.

Scheme 1a

Chemistry In the design of new derivatives (Scheme 1 and Table 1), we took into account five approaches: (1) With the same imidazothiazole portion (2,6-dimethyl substituted) that gave the best results in the last two papers of this series,3,4 different substituents have been considered at the 5-position of the indolinone portion (compounds 3-7). This set of compounds includes N-methyl derivatives too (see point 4). * To whom correspondence should be addressed. Phone: +39-0512099714. Fax: +39-051-2099734. E-mail: [email protected]. † Dipartimento di Scienze Farmaceutiche, Universita` di Bologna. ‡ Dipartimento di Biochimica “G. Moruzzi”, Universita` di Bologna. § Centro Interdipartimentale di Ricerche Biotecnologiche (CIRB), Universita` di Bologna. | National Cancer Institute at Frederick. a Abbreviations: DTP, Developmental Therapeutics Program; NCI, National Cancer Institute; GI, growth inhibition; TGI, total growth inhibition; LC, lethal concentration; BEC, Biological Evaluation Committee; ODC, ornithine decarboxylase; DMSO, dimethyl sulfoxide; DAPI, 4′,6-diamidino2-phenylindole; EDTA, ethylenediaminetetraacetic acid; PBS, phosphatebuffered saline; PI, propidium iodide.

a

For x-y, R, R1, R2, see Table 1.

(2) In additional tests performed by NCI on the compounds published in the first paper of this series,2 a significant antitumor activity was found in two compounds bearing a 2,5-dimethoxyphenyl group at the 6-position of the imidazothiazole portion, and one of them (compound A in Chart 1) was submitted to the Biological Evaluation Committee (BEC) of the NCI (unpublished results). On this basis, we planned new derivatives with the same imidazothiazole portion (and its 2-methyl derivative) but different indolinones, in particular those considered in the previous paper4 (compounds 8-11 and 19-22). (3) With the same indolinone portion as above and with the same 2-methyl group in the ring condensed with imidazole, the effect of the introduction of an additional nitrogen was considered (imidazothiadiazole derivatives 23-27).

10.1021/jm800827q CCC: $40.75  2008 American Chemical Society Published on Web 11/12/2008

Antitumor ActiVity of Indolinones

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7509

Table 1. Compounds 3-28a

a

compd

x-y

R

R1

R2

formula

MW

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

CH3CdCH CH3CdCH CH3CdCH CH3CdCH CH3CdCH CH3CdCH CH3CdCH CH3CdCH CH3CdCH CH3CdCH CH3CdCH CH3CdCH CH3CdCH CH3CdCH CH3CdCH CH3CdCH CHdCH CHdCH CHdCH CHdCH CH3CdN CH3CdN CH3CdN CH3CdN CH3CdN CH3CdN

CH3 CH3 CH3 CH3 CH3 2,5-DMPh 2,5-DMPh 2,5-DMPh 2,5-DMPh 4-Tol 4-Tol 4-Tol 4-CPh 4-CPh 4-CPh 4-CPh 2,5-DMPh 2,5-DMPh 2,5-DMPh 2,5-DMPh CH3 CH3 CH3 CH3 CH3 4-CPh

H H CH3 CH3 CH3 H H H CH3 H H H H H H H H H CH3 CH3 H H H H CH3 H

F COOH H Cl OH OCH3 Cl OH OCH3 OCH3 Cl OH H OCH3 Cl OH Cl OH H OCH3 H OCH3 Cl OH OCH3 H

C16H12FN3OS C17H13N3O3S C17H15N3OS C17H14ClN3OS C17H15N3O2S C24H21N3O4S C23H18ClN3O3S C23H19N3O4S C25H23N3O4S C23H19N3O2S C22H16ClN3OS C22H17N3O2S C21H14ClN3OS C22H16ClN3O2S C21H13Cl2N3OS C21H14ClN3O2S C22H16ClN3O3S C22H17N3O4S C23H19N3O3S C24H21N3O4S C15H12N4OS C16H14N4O2S C15H11ClN4OS C15H12N4O2S C17H16N4O2S C20H13ClN4OS

313.35 339.37 309.39 343.83 325.39 447.51 451.93 433.48 461.54 401.48 405.90 387.46 391.88 421.90 426.32 407.88 437.90 419.46 417.48 447.51 296.35 326.38 330.79 312.35 340.40 392.86

mp, °C 314-315 314-316 180-182 210-211 275-277 224-226 230-232 317-319 257-260 270-272 285-287 340-342 314-316 264-266 342-344 338-340 262-264 298-300 100-105 160-162 288-290 270-272 315-320 285-287 100-103 316-318

dec dec dec

dec dec dec dec

dec dec dec

DMPh ) dimethoxyphenyl. Tol ) methylphenyl. CPh ) chlorophenyl.

Figure 1. Effects of compound 24 on cell cycle-related events in HT29 cells. (A) Cell cycle distribution after 24 h of treatments with the indicated concentration of compound 24 or vincristine. Results are mean values ( SEM of three determinations. (B) Influence of compound 24 on ODC activity. Extracts were prepared from cells incubated for 24 h in the presence of the indicated concentration of compound 24 or vincristine. Results are mean values ( SEM of three determinations.

(4) As was successfully done for one compound in the previous paper,4 the N-methyl homologues of some derivatives were also considered. These compounds (5-7, 11, 21, 22, 27) show different features and therefore are reported in the three groups described above. (5) A number of derivatives were prepared to evaluate the effect of different substituents on the phenyl ring at the 6-position of the 2-methylimidazothiazole/2-methylimidazothiadiazole (compounds 12-18, 28). Compounds 3-28 were prepared by means of the single step Knoevenagel reaction between the aldehydes 1 and the indolinones 2 in methanol/piperidine or acetic acid/hydrochloric acid. The structures of the final compounds were confirmed by means of IR and 1H NMR spectra (Table S1 in Supporting Informa-

Figure 2. Effects of compound 24 on microtubule networks in HT29 cells. Cells were treated for 24 h with compound 24 (1 µM) or vincristine (30 nM). Afterward, the cells were fixed and processed for double immunofluorescence labeling with antibody against R-tubulin (upper panels) to assess the level of tubulin polymerization, and DAPI (lower panels) to detect nuclear morphology. Staining was analyzed by laser scanning confocal microscopy. All the images have been taken at the same magnification and depict microscopic fields representative of the whole cell population.

tion). Most of them were obtained as almost pure geometrical isomers that, according to the usual NOE experiments described in the previous papers,2,3 were assigned to the E configuration. Compounds 3, 6, 13, 17, 21 were obtained as evident E/Z mixtures and submitted as such to the biological tests. The E/Z ratio in solution is time dependent and tends to 50/50. In our experience with similar derivatives, we described the separations of the two isomers by fractional crystallization2 but no significant difference in the pharmacological behavior was noticed. Biology a. Antitumor Activity. Compounds 3-28 were preliminary tested at a single high dose (10-5 M) in the full NCI 60 cell panel. This panel is organized into subpanels representing

7510 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23

Andreani et al.

Table 2. Sixty Cell Panel: Growth Inhibition and Cytostatic and Cytotoxic Activity of the Compounds That Passed the Preliminary Test compda 3 5 6

7 11 12 13

15

18

19

21

23 24c 25 28 vincristine sulfated

modes

leukemia

NSCLC

colon

CNS

melanoma

ovarian

renal

prostate

breast

MG-MIDb

pGI50 pTGI pGI50 pTGI pGI50 pTGI pLC50 pGI50 pTGI pGI50 pTGI pGI50 pTGI pGI50 pTGI pLC50 pGI50 pTGI pLC50 pGI50 pTGI pLC50 pGI50 pTGI pLC50 pGI50 pTGI pLC50 pGI50 pGI50 pTGI pGI50 pTGI pGI50 pTGI pLC50 pGI50 pTGI pLC50

5.34 4.63 5.30 4.68 5.84 4.93

5.09 4.37 4.75 4.12 5.10 4.38

5.14 4.35 4.81 4.13 5.42 4.35

4.94 4.39 4.65 4.01 5.22 4.21

5.02 4.39 4.67

5.35 4.63 5.26 4.26 5.17 4.48

4.89 4.17 4.54 4.05 4.84 4.11 4.87 4.43 4.16 5.09 4.75 4.42 4.78 4.26 4.04 4.56 4.13

5.22 4.48 5.06 4.29 5.50 4.54 4.10 5.34 4.43 4.62 4.13 4.84

5.20 4.45 4.88 4.14 5.26 4.20

5.47 4.56 4.80 4.20 5.39 4.29 4.64 4.14

5.62 4.39 5.34 4.16 5.57 4.42 4.08 5.41 4.25 4.48 4.09 4.89 4.18 4.73 4.36 4.10 4.99 4.67 4.41 4.61 4.12

5.13 4.20 4.60 4.25 4.79 4.26 4.83 4.50 4.18 5.02 4.72 4.49 4.82 4.24 4.05 4.71 4.37 4.10 4.71 4.32 4.06 4.34 5.20 4.16 4.38 4.01 4.70 4.45 4.32 6.80 5.10 3.60

5.04 4.28 4.59 4.04 4.74 4.01 4.84 4.46 4.13 5.00 4.69 4.40 4.86 4.35 4.05 4.59 4.13

4.90

4.53 4.09

4.88 4.07 4.51 4.01 4.88 4.17 4.90 4.49 4.19 5.48 4.73 4.48 4.82 4.30 4.06 4.53 4.06 4.02 4.56 4.14

4.30 5.30 4.14 4.28 4.59

5.22 4.46 4.98 4.20 5.37 4.43 4.02 5.16 4.27 4.58 4.09 4.90 4.12 4.86 4.43 4.14 5.11 4.6 4.44 4.81 4.29 4.04 4.60 4.16 4.02 4.69 4.21 4.01 4.49 5.51 4.23 4.47 4.03 4.81 4.43 4.32 6.70 5.00 3.60

4.74 4.35 4.90 4.41 4.02 4.58 4.09 5.13 4.35 4.01 4.79 5.94 4.42 4.85 4.09 4.75

4.57 4.13

7.00 4.80 3.20

6.60 4.80 3.60

4.41 5.27 4.25 4.27 4.05 4.72 4.34

4.60 4.18 4.04 4.63 4.17 4.02 5.03 6.16 4.38 4.85 4.73 4.35 7.00 5.40 4.10

5.18 4.69 4.26 5.17 4.82 4.52 4.88 4.45 4.09 4.60 4.16 4.67 4.24 4.01 4.31 5.60 4.42 4.53 4.01 5.49 4.78 4.41 6.90 5.20 3.70

6.50 4.70 3.50

6.00 5.36

4.51 4.01 5.33 4.65 4.26 5.01 4.77 4.54 4.82 4.30 4.10 4.47

5.34 4.42 4.54 4.02 4.89 4.90 4.41 4.12 5.19 4.75 4.45 4.80 4.25 4.02 4.66 4.18

4.60 4.04

4.80 4.27

4.21 5.21 4.04 4.25

4.32 5.41

4.75 4.48 4.33 6.50 4.70 3.60

4.91 4.49

4.64 5.58 4.12 4.55 4.08 4.77 4.37

6.90 5.20 3.50

6.50 5.10 3.50

4.36

a Highest concentration ) 10-4 M. Only modes showing a value >4.00 are reported. b Mean graph midpoint, i.e., the mean concentration for all cell lines. c Mean of two different experiments. d Highest concentration ) 10-3 M.

leukemia, melanoma, and cancers of lung, colon, kidney, ovary, breast, prostate, and central nervous system. Only compounds that satisfy predetermined threshold inhibition criteria in a minimum number of cell lines entered the full five-dose assay. The one-dose data are reported as a mean graph of the percent growth of treated cells (data not shown). Fifteen of twenty-six compounds reported in Table 1 were active in this preliminary test and passed on for evaluation in the full five-dose assay. The test compounds were dissolved in dimethyl sulfoxide (DMSO) and evaluated using five concentrations at 10-fold dilutions, the highest being 10-4 M. Table 2 reports the results obtained, expressed as the negative log of the molar concentration at three assay end points: the 50% growth inhibitory power (pGI50), the cytostatic effect (pTGI ) total growth inhibition), and the cytotoxic effect (pLC50). Vincristine sulfate is reported as the reference drug. In the Supporting Information a table is available where the growth inhibition is expressed as micromolar concentration (see Table S2). b. Effect of Compound 24 on Growth of HT-29 Colon Carcinoma Cells. Compound 24is the most active of this series and is particularly active against colon carcinoma cell lines (Table 2). To investigate the mechanisms of its antiproliferative action, we first examined its effect on the cell cycle profile of HT-29 colon carcinoma cells. The cells were incubated for 24 h in the presence of compound 24 at concentrations ranging from 0.5 to 2 µM. Afterward the analysis of DNA profiles was performed

by flow cytometry. Figure 1A shows that compound 24 caused a dose-dependent accumulation of HT-29 cells in the G2/M phase, whereas most of the control cells were in the G0/G1 phase of the cell cycle. Actually, the G2/M cell population increased from 18% in the control to 58% in HT-29 cells treated with 2 µM compound 24 for 24 h. A similar effect was obtained with vincristine, used as a reference compound, but at lower concentration (30 nM). A cellular event strictly correlated with cell cycle progression is represented by the induction of ornithine decarboxylase (ODC), the limiting enzyme for the biosynthesis of polyamines, which are absolutely required for cell proliferation.5 Figure 1B shows that, together with HT-29 cell arrest in the G2/M phase, compound 24 also caused the dose-dependent inhibition of ODC activity. Again, vincristine had a similar, but more pronounced effect. Next we examined the effect of compound 24 on cellular microtubule networks by using immunofluorescence techniques. As shown in Figure 2, the microtubules exhibit normal arrangement and organization in control HT-29. However, after treatment for 24 h with compound 24 (1 µM), we observed an increase of cellular tubulin condensation, which represents a marker of cytoskeleton reorganization, together with a great number of mitotic spindles. These observations suggested that compound 24 arrested the cells in mitosis, similar to vincristine. Morphological evaluation using 4′,6-diamidino-2-phenylindole (DAPI) staining revealed condensed chromatin along with fragmented nuclei, suggestive of activation of apoptosis.

Antitumor ActiVity of Indolinones

Figure 3. Effects of compound 24 on growth and death of HT-29 cells. (A) Rate of cell growth determined as total cell number. The cells were incubated in the presence of the indicated concentration of compound 24 and counted daily. (B) Activation of apoptotic caspase proteases. Caspase activity acting on the peptide sequence Asp-GluVal-Asp (DEVD), indicated as DEVDase activity, was measured in extracts obtained from cells treated for 24 or 48 h with the indicated concentration of compound 24. Each panel represents the results obtained in one typical experiment repeated at least twice with comparable results.

As a consequence of its effects on the cell-division machinery, depicted in Figures 1 and 2, compound 24 inhibited cell growth and, at 2 µM, completely and irreversibly blocked HT-29 tumor cell proliferation (Figure 3A). Following 48 h of cycle arrest, the activity of caspase proteases acting on the amino acid sequence Asp-Glu-Val-Asp (DEVD) began to increase (Figure 3B), confirming the onset of apoptosis. Conclusion From the biological results, it is possible to schematize the following conclusions. The introduction of a carboxyl group at the 5 position of the indolinone system led to loss of activity (compound 4 did not pass the preliminary test). Good results were instead obtained with the introduction, at the same position, of fluorine, since compound 3 showed a mean growth inhibition of 5.22 and was particularly effective against colon tumors (pGI50 ) 5.62). Compounds bearing a methyl group at the indole nitrogen (5-7) were a little less active than the corresponding unsubstituted analogues (mean pGI50 of 5.80, 6.42, and 5.48, respectively3,4), but compound 6 was especially active against prostate tumors (pGI50 ) 6.00) and leukemias (pGI50 ) 5.84). Compounds bearing a 2,5-dimethoxyphenyl group at the 6-position of the imidazothiazole (19-22) and 2-methylimidazothiazole system (8-11) did not lead to improvement of the result obtained with previously published analogues such as compound A (Chart 1). It is interesting that compound 21 (a N-methylindole derivative) was mainly active against leukemias (pGI50 ) 5.13) with relatively low cytotoxicity (pLC50 ) 4.01). Better results were obtained with the 2-methylimidazothiazole system bearing, at position 6, a 4-methyphenyl (12-14) or a 4-chlorophenyl group (15-18). Some of these compounds (12, 13, 15, 18) were more active than two analogues where the phenyl ring was unsubstituted: the analogue of 12 did not pass the preliminary test and the analogue of 15 showed a mean pGI50 of 4.2.3 In particular compound 15 showed a mean pGI50 of 5.11 and was most active toward kidney tumors. Compound 12 was most active toward prostate cancers and leukemias, and compound 13 was most active toward CNS tumors. The results from the imidazothiadiazole derivatives (23-28) are very interesting. Compound 24 was the most active of the whole series presented in this paper, with a mean pGI50 of 5.51,

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7511

a relatively low toxicity (mean pLC50 < 4.00, not shown), and a pGI50 of 6.16 against colon tumors. Even compound 23 showed a certain degree of selectivity against these tumor cells, whereas compound 28 was most active against CNS tumors. The antiproliferative action of the tested compounds can be mediated through either cytotoxic or cytostatic effects. We found that the effect of the very active compound 24 against HT-29 colon carcinoma cells was associated with a block in cell cycle progression, with cell arrest in the G2/M phase. The ability of 24 to interfere with cell cycle in tumor cells was also evidenced by inhibition of ODC, the first and rate limiting enzyme for polyamine synthesis. It is known that ODC inhibitors can interfere with the cell cycle at the G1-S and G2-M transitions.6 For this reason, ODC represents a potential target for anticancer drug development.7 As expected, the arrest of the cell cycle is then followed by a late induction of apoptosis, with activation of caspase proteases. These observations show that compound 24 can interfere with proliferation of colon cancer cells by multiple mechanisms. From a quantitative standpoint, compounds 3, 5, 7, 23, 24, and 25 showed the most similar profiles of activity in the NCI60 testing with Pearson correlation coefficients of 0.8 or greater with each other (Table S3 of Supporting Information). Compound 24 showed the strongest pattern of colon panel selectivity and was tested twice in the NCI60 to confirm this pattern. Compounds 5, 7, and 25 demonstrated very similar patterns of activity against colon tumor panel. While not unprecedented in the NCI60 screening experience, observation of this pattern within an analogue series is quite striking and suggests a common mechanism of action, targeting events particularly important in colon tumors. As stated above for compound 24, we believe that all the compounds of this series act by multiple mechanisms. This theory was widely discussed and documented for another series of compounds we recently published.8 Experimental Section 1. Chemistry. The melting points are uncorrected. Elemental analyses were performed with a Fisons Carlo Erba EA1108 instrument, and results were within 0.4% of the theoretical values (Table S4 of Supporting Information). TLC was performed on Bakerflex plates (silica gel IB2-F) and column chromatography on Kieselgel 60 (Merck). The eluent was a mixture of petroleum ether/ acetone in various proportions. The IR spectra were recorded in Nujol on a Nicolet Avatar 320 E.S.P.; νmax is expressed in cm-1. The 1H NMR spectra were recorded on a Varian Gemini (300 MHz); the chemical shift (referenced to solvent signal) is expressed in δ (ppm) and J in Hz. For the spectra that are not reported here, see Table S1 of Supporting Information. 5-Chloro-2-indolinone and 2-oxindole are commercially available, whereas the other substituted 2-indolinones (5-hydroxy,9 1-methyl-5-hydroxy,10 1-methyl-5methoxy,10 5-methoxy,11 5-fluoro,12 5-carboxy,13 1-methyl14) and the starting aldehydes [(2,6-dimethyl-imidazo[2,1-b]thiazole-5-carboxaldehyde,15 6-(2,5-dimethoxyphenyl)imidazo[2,1-b]thiazole-5carboxaldehyde,16 2-methyl-6-(2,5-dimethoxyphenyl)imidazo[2,1b]thiazole-5-carboxaldehyde,16 2-methyl-6-p-methylphenylimidazo[2,1-b]thiazole-5-carboxaldehyde,17 2-methyl-6-p-chlorophenylimidazo[2,1-b]thiazole-5-carboxaldehyde,18 2,6-dimethylimidazo[2,1-b][1,3,4]thiadiazole-5-carboxaldehyde,19 2-methyl-6-p-chlorophenylimidazo[2,1-b][1,3,4]thiadiazole-5-carboxaldehyde20] were prepared according to the literature. General Procedure for the Synthesis of Compounds 3 and 5-28. The appropriate aldehyde 1 (10 mmol) was dissolved in methanol (100 mL) and treated with an equivalent of the indolinone 2 and piperidine (1 mL). The reaction mixture was refluxed for 3-5 h, according to a TLC test. The precipitate that formed on cooling was collected by filtration with a yield of 45-55%. Compound 21 was purified by column chromatography.

7512 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23

Data for 3. IR: 3068, 1713, 1183, 1001, 765. 1H NMR: 2.21 (3H, s, CH3), 2.40 (3H, d, CH3, J)1.4), 6.48 (1H, dd, ind, J ) 9.1, J ) 2.5), 6.84 (1H, m, ind), 7.06 (1H, m, ind), 7.56 (1H, q, th, J ) 1.4), 7.87 (1H, s, CH), 10.64 (1H, s, NH). Anal. (C16H12FN3OS) C, H, N. Data for 6. IR: 1685, 1618, 1104, 881, 666. 1H NMR: 2.20 (3H, s, CH3), 2.41 (3H, d, CH3, J ) 1.5), 3.23 (3H, s, NCH3), 6.69 (1H, d, ind-4, J ) 1.9), 7.08 (1H, d, ind-7, J ) 9.6), 7.37 (1H, dd, ind6, J ) 9.6, J ) 1.9), 7.57 (1H, q, th, J ) 1.5), 7.75 (1H, s, CH). Anal. (C17H14ClN3OS) C, H, N. Data for 7. IR: 3104, 1698, 1117, 810, 661. 1H NMR: 2.19 (3H, s, CH3), 2.39 (3H, d, CH3, J ) 1.4), 3.17 (3H, s, NCH3), 6.39 (1H, d, ind-4, J ) 2.6), 6.69 (1H, dd, ind-7, J ) 8.4, J ) 2.6), 6.84 (1H, d, ind-6, J ) 8.4), 7.56 (1H, q, th, J ) 1.4), 7.59 (1H, s, CH), 9.08 (1H, s, OH). Anal. (C17H15N3O2S) C, H, N. Data for 15. IR: 3204, 1702, 1197, 833, 727. 1H NMR: 2.44 (3H, s, CH3), 6.88 (1H, d, ind, J ) 7.5), 6.98 (1H, t, ind, J ) 7.5), 7.24 (1H, t, ind, J ) 7.5), 7.41 (1H, s, th), 7.54 (2H, d, ph, J ) 8.5), 7.73 (4H, m, 1H-ind + 2H-ph + CH), 10.58 (1H, s, NH). Anal. (C21H14ClN3OS) C, H, N. Data for 24. IR: 3200, 1703, 1033, 876, 768. 1H NMR: 2.25 (3H, s, CH3), 2.72 (3H, s, CH3), 3.60 (3H, s, OCH3), 6.54 (1H, d, ind-4, J ) 2.2), 6.77 (1H, d, ind-7, J ) 8.4), 6.83 (1H, dd, ind-6, J ) 8.4, J ) 2.2), 7.50 (1H, s, CH), 10.45 (1H, s, NH). Anal. (C16H14N4O2S) C, H, N. Procedure for the Synthesis of Compound 4. 2,6-Dimethylimidazo[2,1-b]thiazole-5-carbaldehyde (10 mmol) was dissolved in acetic acid (50 mL) and treated with an equivalent of 2-oxoindoline5-carboxylic acid and 37% hydrochloric acid (1 mL). The reaction mixture was refluxed for 15-20 h, according to a TLC test, and the precipitate that formed on cooling (yield 65%) was collected by filtration. IR: 3310, 1709, 1163, 892, 765. 1H NMR: 2.19 (3H, s, CH3), 2.39 (3H, d, CH3, J ) 1.2), 6.98 (1H, d, ind-7, J ) 8.2), 7.41 (1H, q, th, J ) 1.2), 7.62 (1H, d, ind-4, J ) 1.5), 7.65 (1H, s, CH), 7.85 (1H, dd, ind-6, J ) 8.2, J ) 1.5), 11.07 (1H, s, NH). Anal. (C17H13N3O3S) C, H, N. 2. Biology. 2.a. Antitumor Activity. The screening is a twostage process,21 beginning with the evaluation of all compounds against the 60 cell lines at a single dose of 10-5 M. Compounds that exhibit significant growth inhibition are evaluated against the 60 cell panel at five concentration levels by the NCI according to standard procedures (http://dtp.nci.nih.gov/branches/btb/ivclsp.html). 2.b. Effects on Growth of HT-29 Colon Carcinoma Cells. Cell Culture and Treatment. HT-29 cells were maintained in RPMI 1640 medium (Labtek Eurobio, Milan, Italy) supplemented with 10% fetal bovine serum (Euroclone, Milan, Italy) and 2 mM L-glutamine (Sigma-Aldrich, St. Louis, MO) at 37 °C and 5% CO2. The molecules under test were dissolved in DMSO (Sigma) at 10 mmol/L immediately before use. Cells were plated at 2 × 104 cells/ cm2 in a plastic well (60 cm2), and after 24 h the medium was removed and fresh medium, containing the studied compound at different concentrations, was added. In control cells only DMSO was added. Cell viability was determined by Trypan blue exclusion. Cell Cycle Analysis. Control and treated cells were detached with 0.11% trypsin (Sigma-Aldrich, St. Louis, MO) and 0.02% EDTA (ethylenediaminetetraacetic acid), washed twice in PBS (phosphate buffered saline), and centrifuged. The pellet was suspended in 0.01% nonidet P-40 (Sigma), 10 µg/mL RNase, 0.1% sodium citrate (Sigma), and 50 µg/mL propidium iodide (PI) (Sigma) for 30 min at room temperature in the dark. PI fluorescence was analyzed by using a Beckman Coulter Epics XL MCL cytometer. Cell cycle analysis was performed using Modfit 5.0 software. ODC Activity. ODC activity in cell extracts was assayed by measuring the release of radioactive CO2 from [14C-carboxyl]ornithine.22 Caspase Activity. The activity of caspase enzymes hydrolyzing the peptide sequence Asp-Glu-Val-Asp (DEVD), indicated as DEVDase activity, was measured in cell extracts by a fluorometric assay.23 Confocal Microscopy. Cells were seeded at 1 × 104 cells/cm2 on glass coverslips and treated for 24 h with compound 24 (1 µM).

Andreani et al.

At the end of the incubation, the cells were washed twice with PBS, fixed with 3% paraformaldehyde, subsequently washed with 0.1 M glycine in PBS, and permeabilized in 70% ice-cold ethanol. Microtubules staining was performed, incubating overnight at 4 °C with a mouse monoclonal anti-R-tubulin primary antibody (Upstate). Cells were then washed, incubated with fluorescein isothiocyanate conjugated antimouse IgG antibody (Sigma) for 1 h at room temperature, and finally stained with DAPI. All preparations were embedded in Mowiol and analyzed using a laser scanning confocal microscope (Nikon C1s) equipped with Nikon eclipse TE300.

Acknowledgment. This work was supported by a grant from MIUR-COFIN 2006. We are grateful to NCI for the antitumor tests and to CIRB for the use of confocal microscope and flow cytometer. Supporting Information Available: IR and 1H NMR of the new compounds (Table S1), antitumor activity expressed as micromolar concentrations (Table S2), matrix compare analysis results for compounds with correlation coefficient g0.8 (Table S3), elemental analysis results (Table S4), and NSC numbers (Table S5). This material is available free of charge via the Internet at http://pubs.acs.org.

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