Discovery and Optimization of a Series of 2-Aryl-4-Amino-5-(3′,4′,5′-trimethoxybenzoyl)Thiazoles as Novel Anticancer Agents

Abstract

A new series of tubulin polymerization inhibitors based on the 2-aryl/heteroaryl-4-amino-5-(3′,4′,5′-trimethoxybenzoyl)thiazole scaffold was synthesized and evaluated for growth inhibition activity on a panel of cancer cell lines, cell cycle effects, and in vivo potency. Structure–activity relationships were elucidated with various substitutions at the 2-position of the thiazole skeleton. Hydrophobic moieties, such as phenyl and 3-thienyl, were well tolerated at this position, and variation of the phenyl substituents had remarkable effects on potency. The most active compound (3b) induced apoptosis through the mitochondrial pathway with activation of caspase-3. We also showed that it has potential antivascular activity since it reduced in vitro endothelial cell migration and disrupted capillary-like tube formation at noncytotoxic concentrations. Furthermore, compound 3b significantly reduced the growth of the HT-29 xenograft in a nude mouse model, suggesting that 3b is a promising new antimitotic agent with clinical potential.

INTRODUCTION

Besides being critical for cell architecture, the microtubule system of eukaryotic cells is essential for cell division since microtubules are key components of the mitotic spindle.¹ Microtubules are a dynamic cellular compartment in both neoplastic and normal cells. This dynamicity is characterized by the continuous turnover of αβ-tubulin heterodimers in the polymeric microtubules. The microtubule system is also important in other fundamental cellular processes, such as regulation of motility, cell signaling, formation and maintenance of cell shape, secretion, and intracellular transport.²

In the last decades, there has been a continuing interest in the discovery and development of novel small molecules able to inhibit tubulin polymerization.³ Numerous chemically diverse antimitotic agents, many of which are natural products, interact specifically with tubulin.⁴ Among the naturally occurring derivatives, combretastatin A-4 (CA-4, 1, Chart 1), isolated from the bark of the South African tree Combretum caf frum,⁵ is one of the well-known tubulin-binding molecules affecting microtubule dynamics, and compound 1 strongly inhibits the polymerization of tubulin by binding to the colchicine site.⁶ Compound 1 shows potent cytotoxicity against a wide variety of human cancer cells, including those that are multidrug resistant.⁷

Chart 1.

Chemical Structures of CA-4 and 2-Substituted-4-amino-5-aroylthiazoles 2a and 3a–o

In the course of our search for new synthetic tubulin inhibitors, we recently reported the synthesis and biological characterization of a series of 2-alkylamino-4-amino-5-aroylthiazoles with general structure 2, prepared by an easy one-pot four step procedure.⁸ Among the synthesized compounds, the 2-(pyrrolidin-1-yl)-4-amino-5-(3′,4′,5′-trimethoxybenzoyl)-thiazole derivative 2a was the only compound of this series active at submicromolar concentrations against a panel of five cancer cell lines, with IC₅₀ values ranging from 0.2 to 0.4 µM. Compound 2a was 10- to 100-fold less active than the reference compound 1 as an antiproliferative agent, while it was comparable to 1 as an inhibitor of tubulin polymerization. Structure–activity relationship studies indicated that the pyrrolidine is the only substituent tolerated at the 2-position of the thiazole scaffold. This finding led us to assume that the tubulin binding pocket for this portion of the molecule is quite small and that tubulin only tolerated the presence of hydrophobic substituents, indicating the possibility for further improvement in activity. Thus, once the 4-amino-5-(3′,4′,5′-trimethoxybenzoyl) thiazole scaffold was identified as the minimum structural requirement for activity in this new series of compounds, our strategy for further development of active antimitotic agents was to perform modifications at the 2-position of the thiazole ring. The first round of optimization included replacement of the pyrrolidine ring of compound 2a with a phenyl or bioisosteric 3-thienyl ring, to furnish derivatives 3a and 3b, respectively. As shown in Table 1, phenyl or 3′-thienyl substitutions at the C-2 position of the thiazole ring significantly enhanced antiproliferative activity. Encouraged by the increased potency obtained with 3a, we then synthesized compounds 3c–o to determine whether various electron-releasing (Me, Et, C(CH₃)₃, OMe, OEt, and OCF₃) or electron-withdrawing (F, Cl, CF₃, CN, and NO₂) substituents on the para- or meta-positions of the phenyl ring would further enhance activity. To compare the effects of para- and meta-substitution, the chloro atom and the methoxy-group were also introduced at the meta-position of the phenyl ring. We are including 3b in this article because it had the best antiproliferative activity (see below) of all the compounds prepared in this study.

Table 1.

In Vitro Cell Growth Inhibitory Effects of Compounds 1 and 3a−o

	IC50a (nM)
compd	HeLa	A549	HL-60	Jurkat	MCF-7	HT-29
3a	7.6 ± 1.7	367 ± 11.0	80.8 ± 7.1	58.0 ± 5.6	2.2 ± 0.8	13.2 ± 5.4
3b	2.4 ± 0.9	210 ± 38.1	77.9 ± 2.6	26.4 ± 2.3	51.2 ± 9.3	11.4 ± 2.8
3c	24.1 ± 4.3	1082 ± 36.7	384 ± 25.0	582 ± 15.4	29.4 ± 1.5	115 ± 3.5
3d	188 ± 46.0	6730 ± 202	4445 ± 68.2	1749 ± 58.8	845 ± 18.5	2083 ± 50.5
3e	2.8 ± 1.0	326 ± 14.8	137 ± 27.2	35.9 ± 4.4	41.7 ± 3.5	24.0 ± 5.8
3f	22.7 ± 7.8	1223 ± 97.1	295 ± 27.0	201 ± 57.1	3.2 ± 0.7	53.1 ± 14.8
3g	191 ± 44.8	5175 ± 135	1813 ± 244	2328 ± 577	1583 ± 267	1018 ± 135
3h	4148 ± 75.4	>10,000	>10,000	>10,000	508 ± 123	9067 ± 158
3i	1799 ± 27.6	>10,000	>10,000	>10,000	>10,000	>10,000
3j	244 ± 41.3	>10,000	>10,000	2023 ± 75.9	1255 ± 25.7	2806 ± 72.2
3k	13.7 ± 4.5	720 ± 28.3	41.2 ± 5.5	66.6 ± 8.9	11.3 ± 5.1	22.3 ± 4.4
3l	191 ± 35.9	>10,000	1136 ± 74.4	606 ± 45.7	257 ± 65.4	220 ± 18.6
3m	1981 ± 36.0	6253 ± 318	>10,000	4829 ± 64.1	>10,000	>10,000
3n	>10,000	>10,000	>10,000	>10,000	>10,000	>10,000
3o	570 ± 10.2	6106 ± 169.9	>10,000	>10,000	>10,000	4785 ± 83.2
1	4 ± 1	180 ± 50	1 ± 0.2	5 ± 0.6	370 ± 100	3100 ± 100

^aIC₅₀ = compound concentration required to inhibit tumor cell proliferation by 50%. Data are expressed as the mean ± SEM from the dose–response curves of at least three independent experiments.

We examined the efficacy of the newly synthesized compounds on a panel of human cancer cell lines, including multidrug resistant lines overexpressing the 170-kDa P-glycoprotein drug efflux pump, and we investigated in detail the modalities of cell death induced by these derivatives. Since many antimitotic drugs, such as compound 1, have been shown to possess antiangiogenic and antivascular activities,^9,10 we also investigated the effects of these compounds on in vitro assays with HUVECs.

CHEMISTRY

The general synthesis of 2-aryl/heteroaryl-4-amino-5-(3′,4′,5′-trimethoxybenzoyl)thiazoles 3a–o is outlined in Scheme 1. These molecules were prepared by a one-step efficient synthetic procedure, starting from an easily accessible common intermediate 5.⁸ This latter compound was obtained in good yield by a one-pot three-step sequential procedure, starting from dimethyl cyanodithiocarbonate 4, which was reacted successively with sodium sulfide, 2-bromo-1-(3,4,5-trimethoxyphenyl)ethanone and potassium carbonate.¹¹ The Pd₂dba₃-catalyzed, Cu(I)Tc-mediated coupling of thiazole-2-thiomethyl ether 5 with the appropriate aryl/heteroaryl boronic acid, in the presence of TFP, furnished the 2-aryl/heteroaryl substituted thiazoles 3a–o in high yields, avoiding the protection/deprotection sequence of the amino group at the 4-position of the thiazole ring.¹²

Scheme 1^a.

^aReagents: a, Na₂S·9H₂O, DMF, 70 °C, 2 h; b, 1-(3′,4′,5′-trimethoxyphenyl)-2-bromoethanone, 50 °C, 2 h; c, K₂CO₃, 1 h; d, Pd₂dba₃, Cu(I)Tc, TFP, ArB(OH)₂, THF, 60 °C, 18 h.

BIOLOGICAL RESULTS AND DISCUSSION

In Vitro Antiproliferative Activities

The 2-aryl/heteroaryl-4-amino-5-(3′,4′,5′-trimethoxybenzoyl)thiazoles 3a–o were evaluated for their antiproliferative activity against a panel of six human tumor cell lines and compared with reference compound 1. As shown in Table 1, the antiproliferative activities of the tested compounds were generally more pronounced against HeLa and MCF-7 cells as compared with the other cell lines. With the exception of MCF-7 cells, the 3-thienyl derivative 3b was the most active compound in this series, exhibiting IC₅₀ values ranging from 2.4 to 78 nM against five of the six cancer cell lines and an IC₅₀ of 210 nM against the A549 cells. Moreover, with the MCF-7 and HT-29 cells, compounds 3a–c, 3e–f, and 3k were more potent than 1, with IC₅₀ values in the single- or double-digit nanomolar range. Compounds 3b and 3e showed comparable potency to 1 against the HeLa cells. Of the 15 tested compounds, 3a–b, 3e, and 3k possessed the highest overall potency, with IC₅₀ values of 2.4–140 nM against five of the six cancer cell lines and IC₅₀ values of 200–700 nM against the A549 cells. With the exception of MCF-7 and HT-29 cells, reference compound 1 possessed the highest potency in four of the six cell lines tested.

The bioisosteric replacement of the phenyl ring of compound 3a with the 3-thienyl group (3b) produced a 1.5- to 3-fold increase of potency against A549, Jurkat, and HeLa cells, while the differences between 3a and 3b were minimal in HL-60 and HT-29 cells. Only in MCF-7 cells was 3b less active than 3a (IC₅₀ values of 51 and 2.2 nM, respectively). Excluding the A549 cells, compounds 3a and 3b had IC₅₀ values ranging from 24 to 80 nM against the cell lines, compared with a range of 1–3100 nM obtained with 1.

The data shown in Table 1 demonstrated the importance of substituents on the phenyl ring at the 2-position of the thiazole system for activity and selectivity against the different cancer cell lines. In general, all substituents caused a reduction in antiproliferative activity relative to 3a, except in four cases. The meta-chloro substituent of 3e resulted in enhanced antiproliferative activity against three cell lines (HeLa, A549, and Jurkat cells), while the meta-methoxy substituent of 3k resulted in enhanced activity in one cell line (HL-60 cells). Otherwise, all substituents led to reduced activity, sometimes mild to moderate (e.g., 3c, 3e, and 3f) and sometimes profound (e.g., 3h–j and 3l–o).

Turning specifically to the para-substituted phenyl derivatives, these showed highly variable potencies. Generally, it was found that most substituents in the para-position resulted in lower activity as compared to the unsubstituted parent compound 3a, with the least deleterious being fluorine and methyl (compounds 3c and 3f, respectively). Comparing the fluorine derivative (3c) with the methyl derivative (3f), the latter was more active in five of the six cell lines we examined, with the greatest difference observed in the MCF-7 cells. Comparing the fluorine (3c) and the chlorine (3d) derivatives, increasing the size of the halide led to a large loss of activity with all six cell lines. In contrast, as noted above, the meta-chloro derivative (3e) was much more active than its para-congener 3d, including enhanced activity relative to 3a in three cell lines. Indeed, the activity of 3e did not differ greatly from the 3-thienyl analogue 3b in all cell lines.

Turning to the electronic characteristics of the para-substituents, the introduction of a weak electron-releasing methyl group (3f) caused a 1.5–4-fold loss in antiproliferative activity in the six cell lines relative to the unsubstituted 3a. A para-ethyl group (compound 3g) caused a further, major decrease in antiproliferative activity relative to 3a, and 3f was 14–700-fold less active in the six cell lines. Little antiproliferative activity was observed with still larger para-substituents (3h–j, 3m–o), regardless of their electronic characteristics.

The antiproliferative activities of compounds 3j, 3k, and 3l were influenced by the number and position of methoxy substituents on the phenyl ring. The meta-methoxy derivative 3k, as noted above, was relatively active. Relative to the unsubstituted 3a, 3k was 1.1–5-fold less active in five cell lines and twice as active in HL-60 cells. In contrast, the para-methoxy derivative 3j was 32–570-fold less active than 3a when IC₅₀ values could be determined. The 3,4-dimethoxy substituted analogue 3l was generally intermediate in activity between 3j and 3k.

In summary, comparing the effects of ERGs and EWGs on the phenyl at the C2-thiazole position, no consistent difference in effects on antiproliferative activity occurred. Overall, all substituents at the para-position led to a loss of activity, and profound loss occurred with larger substituents. The effects of the two small meta-substitents examined were not dramatic, leading, perhaps, to some enhancement of antiproliferative activity. In different cell lines, moreover, different effects were observed. For example, replacement of the electron-donating methoxy group with the electron-withdrawing chlorine atom (compounds 3k and 3e, respectively), resulted in a 3- and 4-fold reduction in activity against HL-60 and MCF-7 cells, while 3k and 3e showed comparable potencies against HT-29, and 3e was 5- and 2-fold more active than 3k against HeLa and Jurkat cells.

Effect of Compound 3b on Multidrug Resistant Cells

To investigate whether these derivatives are substrates of drug efflux pumps, one of the most active compounds (3b) was tested against a panel of drug resistant cell lines which either overexpress P-glycoprotein (Lovo^Doxo and Cem^Vbl100)^13,14 or are associated with tubulin gene mutations (A549-T12)¹⁵ that result in modified tubulin with impaired polymerization properties. As shown in Table 2, 3b exhibited cytotoxic activity in all three of the drug resistant cell lines. The activity in the two P-glycopprotein overexpressing cell lines demonstrated that 3b is not a substrate for this important drug pump.

Table 2.

In Vitro Cell Growth Inhibitory Effects of Compound 3b on Drug Resistant Cell Lines

	IC50a (nM)
compd	LoVo	LoVoDoxo	resistance ratiob
3b	18.0 ± 4.5	24.8 ± 5.3	1.4
doxorubicinc	120 ± 30	13150 ± 210	109.6
	CEM	CEMVbl100	resistance ratiob
3b	273.3 ± 20.3	666.7 ± 12.0	2.4
vinblastine	0.8 ± 0.1	205 ± 46	256.2
	A549	A549-T12	resistance ratiob
3b	209.6 ± 38.1	201.5 ± 46.7	0.96
taxolc	7.2 ± 0.1	75.2 ± 12.5	10.4

^aIC₅₀= compound concentration required to inhibit tumor cell proliferation by 50%. Data are expressed as the mean ± SEM from the dose–response curves of at least three independent experiments.

^bThe values express the ratio between IC₅₀ determined in resistant and nonresistant cell lines.

^cData are from ref 8.

Inhibition of Tubulin Polymerization and Colchicine Binding

To investigate whether the antiproliferative activities of compounds 3a–c, 3e–f, and 3k derived from an interaction with tubulin, these agents were evaluated for their inhibition of tubulin polymerization and for effects on the binding of [³H]colchicine to tubulin (Table 3).^16a,b For comparison, compound 1 was examined in contemporaneous experiments. In the assembly assay, compounds 3a–b and 3e–f were found to be the most active compounds (IC₅₀ values, 1.5–1.7 µM), with activity comparable with that of 1 (IC₅₀, 1.2 µM). Compounds 3c and 3k were half as active as 1, although they were more potent than 1 as antiproliferative agents against MCF-7 and HT-29 cells.

Table 3.

Inhibition of Tubulin Polymerization and Colchicine Binding by Compounds 1, 3a–c, 3e–f and 3k

compd	tubulin assemblya IC50 ± SD (µM)	colchicine bindingb % ± SD
3a	1.6 ± 0.2	78 ± 6
3b	1.5 ± 0.2	77 ± 4
3c	2.7 ± 0.2	45 ± 6
3e	1.7 ± 0.2	63 ± 3
3f	1.5 ± 0.1	59 ± 4
3k	2.2 ± 0.3	51 ± 6
1	1.2 ± 0.1	98 ± 0.6

^aInhibition of tubulin polymerization. Tubulin was at 10 µM.

^bInhibition of [³H]colchicine binding. Tubulin, colchicine, and the tested compound were at 1, 5, and 5 µM, respectively.

When comparing the inhibition of tubulin polymerization versus the growth inhibitory effects, we found a positive correlation for most, but not all, of the active compounds. While 3f was generally less potent than 3b as an antiproliferative agent, 3f and 3b showed equal potency as inhibitors of tubulin assembly. Similarly, 3k is 1.5-fold less active than 3f in the tubulin assay, but 3k is more active than 3f in five of the cell lines. Such discrepancies in antitubulin versus antiproliferative activity are not infrequently observed, but the reasons are usually uncertain, as in our case. Among possible explanations is that we used bovine brain tubulin in the former studies, and its composition in terms of tubulin isotypes differs significantly from that of different human cancer cell lines.^16c

In the colchicine binding studies, derivatives 3a and 3b potently inhibited the binding of [³H]colchicine to tubulin, with 79% and 77% inhibition occurring with these agents. Inhibition of colchicine binding by compounds 3c, 3e–f, and 3j was lower, ranging from 45 to 63%. None, however, was quite as potent as 1, which in these experiments inhibited colchicine binding by 98%.

For the most active compounds 3a and 3b, a good correlation was observed between antiproliferative activities, inhibition of tubulin polymerization, and inhibition of colchicine binding. In general, inhibition of [³H]colchicine binding to tubulin correlated more closely with antiproliferative activity than did inhibition of tubulin assembly.

Molecular Modeling

A series of molecular docking simulations were performed to rationalize the observed SARs for this series of compounds. The proposed binding mode that emerged from these studies is virtually identical to the pyrrolidine analogue 2a reported previously.⁸ Figure 1 shows how the thienyl analogue 3b sits in a tight hydrophobic pocket defined by β-tubulin residues Val181, Asn258, Met259, and Lys352. It is interesting to note that this pocket can only accommodate an aromatic ring with relatively small substituents, like the ones present in 3a, 3c–f, and 3k (Figure 2), while compounds with bulkier substituents (e.g., the t-butyl of 3h) do not dock well in the binding site because of steric hindrance. In addition to these nonpolar interactions, the trimethoxyphenyl ring of 3b is also in contact with βCys241, while the amino group establishes a hydrogen bond with βThr179. These latter two interactions are often observed in models of the binding of tubulin inhibitors in the colchicine site.¹⁷

Figure 1.

Proposed binding mode of 3b in the colchicine site of β-tubulin.

Figure 2.

Putative binding mode of 3c (in cyan), 3e (in magenta), and 3f (in orange) in the colchicine site of β-tubulin.

Analysis of Cell Cycle Effects

The effect of 3a and 3b on cell cycle progression was examined by flow cytometry in HeLa and Jurkat cells. Treatment with the two compounds in HeLa cells, resulted in the accumulation of cells in the G2/M phase, with a concomitant reduction in cells in both the S and G1 phases. These changes occurred in a concentration-dependent manner (Figure 3, panel A), but changes were observed even at the lowest concentration (62 nM) used. A similar behavior was observed also for Jurkat cells (Figure 3, panel B), except that for compound 3a we detected a less pronounced G2/M arrest in comparison with 3b in the same cell lines accompanied with a reduction of cells in the G1 phase while cells in the S phase remained constant.

Figure 3.

Effect of compounds 3a and 3b on cell cycle phase arrest in HeLa (panel A) and Jurkat cells (panel B). Cells were treated with different concentrations of 3a or 3b, as indicated, ranging from 62.5 to 500 nM for 24 h. Then, the cells were fixed and stained with PI to analyze DNA content by flow cytometry. Data are presented as the mean ± SEM of three independent experiments. Panel C: effect of 3b on some G2/M regulatory proteins. HeLa cells were treated for 24 or 48 h with the indicated concentration of the compound. The cells were harvested and lysed for the detection of cyclin B, p-Cdc2^Y15, and Cdc25c expression by Western blot analysis. To ensure equal protein loading, each membrane was stripped and reprobed with anti-β-actin antibody.

Next, we investigated the association between 3b-induced G2/M arrest and alterations in G2/M regulatory protein expression in HeLa cells. As shown in Figure 3 (panel C), 3b caused an increase in cyclin B1 expression after 24 and 48 h, indicating an activation of the mitotic checkpoint following drug exposure. This effect was confirmed by the appearance of slower migrating forms of phosphatase cdc25c at 24 h, followed by a strong reduction at 48 h. The phosphorylation of cdc25c directly stimulates its phosphatase activity, and this is necessary to activate cdc2/cyclin B on entry into mitosis.¹⁸ In good agreement, we also observed at 24 h of incubation, at 100 nM, a remarkable dephosphorylation at Tyr15 of cdc2 kinase, and this effect, although less pronounced, was also present after 48 h of incubation.

Compound 3b Induced Apoptosis

To evaluate the mode of cell death induced by compound 3b, we performed a biparametric cytofluorimetric analysis using propidium iodide (PI) and annexin-V-FITC, which stain DNA and phosphatidylserine (PS) residues, respectively.

After treatment with 3b at 50 or 100 nM for 24 or 48 h, HeLa cells were labeled with the two dyes, and the resulting red (PI) and green (FITC) fluorescence was monitored by flow cytometry. As shown in Figure 4 (panel A), 3b caused a significant induction of apoptotic cells after 24 h. The percentage of annexin-V positive cells then further increased at 48 h (Figure 4, panel B). These findings prompted us to further investigate the apoptotic process after treatment of cells with 3b.

Figure 4.

Panel A: representative flow cytometric histograms of apoptotic HeLa cells after 48 h treatment with 3b. The cells were harvested and labeled with annexin-V-FITC and PI and analyzed by flow cytometry. Panel B: percentage of cells found in the different regions of the biparametric histograms after incubation with 3b for 24 or 48 h. Data are expressed as the mean ± SEM for five independent experiments.

Compound 3b Induced Mitochondrial Depolarization and ROS Production

Mitochondria play an essential role in the propagation of apoptosis.¹⁹ It is well established that, at an early stage, apoptotic stimuli alter the mitochondrial transmembrane potential (Δψ_mt). Δψ_mt was monitored by the fluorescence of the dye JC-1.²⁰ Treated HeLa cells in the presence of 3b (50 and 100 nM) exhibited a marked shift in fluorescence compared with control cells, indicating depolarization of the mitochondrial membrane potential (data not shown). Following treatment, the percentage of cells with low Δψ_mt increased in a time-dependent fashion (Figure 5, panel A). The disruption of Δψ_mt is associated with the appearance of annexin-V positivity in the treated cells when they are in an early apoptotic stage. In fact, the dissipation of Δψ_mt is characteristic of apoptosis and has been observed with both microtubule stabilizing and destabilizing agents, including 1, in different cell types.¹⁸

Figure 5.

Assessment of HeLa mitochondrial dysfunction after treatment with compound 3b. Panel A: induction of the loss of mitochondrial membrane potential after 24 or 48 h incubations with compound 3b. Cells were stained with the fluorescent probe JC-1 and analyzed by flow cytometry. Data are expressed as the mean ± SEM for three independent experiments. Panels B and C: mitochondrial production of ROS in HeLa cells. After 24 or 48 h incubations with 3b, cells were stained with H₂-DCFDA (panel B) or HE (panel C) and analyzed by flow cytometry. Data are expressed as the mean ± SEM of three independent experiments.

Mitochondrial membrane depolarization is associated with mitochondrial production of ROS.²¹ Therefore, we investigated whether ROS production increased after treatment with the test compounds. We utilized the two fluorescence indicators hydroethidine (HE) and 2,7-dichlorodihydrofluorescein diacetate (H₂-DCFDA).²² The results are presented in Figure 5, panels B and C, which show that 3b induced the production of significant amounts of ROS in comparison with control cells, in agreement with the dissipation of Δψ_mt described above.

Effect of 3b on Bcl-2 and Bax Expression and Caspase-3 Activation

We evaluated the activity of caspase-3 after treatment of HeLa cells with 3b since this enzyme is essential for the propagation of the apoptotic signal after exposure to many antimitotic compounds.²³ We observed a clear activation of caspase-3, as well as cleavage of the caspase-3 substrate PARP after 24 and 48 h of 3b exposure (Figure 6). In addition, after a 48 h exposure to 100 nM, we also found an increase in the expression of phospho-γH2AX, a well-known marker for cellular DNA double strand breaks.²⁴ This suggests that DNA damage probably occurred in the unsegregated chromosomes resulting from the stalled replication caused by compound 3b. There is increasing evidence that regulation of the Bcl-2 family of protein shares the signaling pathways induced by antimicrotubule compounds.^18,25 The proteins of the Bcl family play a major role in controlling apoptosis through the regulation of mitochondrial processes and the release of mitochondrial proapoptotic molecules that are important for the cell death pathway.¹⁸ Our results (Figure 6) showed that the expression of the antiapoptotic protein Bcl-2 was strongly reduced after 24 h of treatment with 100 nM 3b and after 48 h with either 50 or 100 nM 3b. In contrast, expression of Bax, a proapoptotic protein of the Bcl-2 family, was unchanged.

Figure 6.

Western blot analysis for caspase-3 activation, PARP cleavage, and the expression of Bcl-2, Bax, and histone γH2AX in HeLa cells. The cells were treated with the indicated concentration of 3b for 24 or 48 h. Whole cell lysates were subjected to SDS–PAGE, followed by blotting with the appropriate antibody or an antiactin antibody.

In Vitro Evaluation of Antivascular Activity of 3b

Many studies have shown that most microtubule binding drugs possess vascular disrupting activity, and such data suggest that this class of drugs could be useful as antiangiogenic compounds.^9,10 In this context, CA-4 and its analogues in clinical development have been shown to quickly and selectively shut down the blood flow of tumors.^26,27 The effect is thought to be mediated by inducing endothelial cell shape change, possibly through disruption of microtubule dynamics.⁹ We tested 3b for its ability to induce rapid endothelial cell shape changes using a human umbilical vein endothelial cell (HUVEC) culture assay. We first investigated the effects of 3b on endothelial cell migration using an in vitro scratch assay.²⁸ We scraped confluent monolayers of HUVECs to clear space for motile cells to move into. As shown in Figure 7 (panel A), we observed that after 24 h nontreated cells migrated to completely fill the area that was initially scraped. In contrast, 3b significantly inhibited HUVEC migration in a concentration and time dependent manner (Figure 7, panels A and B).

Figure 7.

Antivascular activity of compound 3b. Panel A: 3b inhibits HUVEC migration. Confluent HUVEC monolayers were scratch wounded. The cells were treated with various concentrations of 3b and at different times cells were photographed (40× magnification). The dotted lines define the areas lacking cells. Panel B: the graph shows the quantitative effect of 3b. Data are presented as the mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 vs control. Panel C: inhibition of endothelial cell capillary-like tubule formation by 3b. Tubule formation on Matrigel was performed as described in Materials and Methods. Representative pictures (40× magnification) of HUVECs treated with the indicated concentrations of 3b for 3 h. Panel D: the graph shows the quantitative effects of 3b on the dimensional and topological parameters of the HUVEC network.

We also tested the effects of 3b in a tube formation assay. After being seeded on Matrigel, HUVECs form a rich meshwork of branching capillary-like tubules with multicentric junctions. After just 1 h in different concentrations (100–500 nM) of 3b, the capillary-like tubes were interrupted. At the two higher concentrations of 3b, most cells were spherical and either isolated or aggregated in small clumps (Figure 7, panel C). Quantitative image analysis (Figure 7, panel D) showed that 3b markedly decreased in a concentration dependent manner both dimensional (percent of area covered by HUVECs, total length per field) and topological parameters (number of mesh per field, number of branching points) of the capillary-like network.

To evaluate if the inhibition of cell migration and tube formation was due to a cytotoxic action of 3b, we analyzed cell proliferation of the HUVECs by the MTT assay. The calculated GI₅₀ after 72 h was 4.4 ± 0.9 µM, indicating that this compound inhibited proliferation of these cells only at concentrations higher than that required for the inhibition of cell migration and tube formation. Altogether, these preliminary observations suggest that 3b, like CA-4, would most likely cause severe vascular disruption in vitro, although at a concentration higher than that of CA-4,^26,27 and warrant further studies to evaluate in depth its vascular disrupting properties.

In Vivo Antitumor Activity of Compound 3b

To evaluate the in vivo antitumor activity of 3b, human colon adenocarcinoma xenografts were established by subcutaneous injection of HT29 cells into the backs of nude mice. Once the HT-29 xenografts reached a size of ~300 mm³, 12 mice were randomly assigned to one of two groups. In one of the groups, compound 3b dissolved in DMSO was injected intraperitoneally at doses of 100 mg/kg. The drug, as well as the vehicle control, were administered three times a week for one week. As shown in Figure 8 (panel A), compound 3b caused a significant reduction in tumor growth (58%, p < 0.01) as compared with the administration of vehicle only.

Figure 8.

Inhibition of human xenograft growth in vivo by compound 3b. Panel A: HT29 tumor-bearing nude mice were administered either vehicle control or 100 mg/kg of 3b in vehicle intraperitoneally on days 0, 2, and 4 (indicated by arrows). The figure shows the tumor volume (panel A) and body weight (panel B) recorded at the indicated days after treatments. Data are expressed as the mean ± SEM of tumor volume and body weight at each time point for six animals per group. *p < 0.01 vs the control.

During the whole treatment period, no significant weight changes or macroscopic signs of toxicity occurred in the treated animals (Figure 8, panel B) suggesting that the administration of 3b was well tolerated.

CONCLUSIONS

The structural refinement of compound 2a led to the discovery of a novel series of synthetic inhibitors of tubulin polymerization with general structure 3, based on the 2-aryl/heteroaryl-4-amino-5-(3′,4′,5′-trimethoxybenzoyl)thiazole molecular skeleton, prepared in a one-step procedure by Liebeskind–Srogl conditions starting from an easily accessible building block 5 bearing an unprotected amino group function. The antiproliferative activity of these compounds was highly dependent on the structure modifications on the 2-position of the thiazole ring. The substituents examined included phenyl, 3-thienyl, and substituted phenyls bearing EWGs or ERGs.

The study revealed that phenyl (3a) and 3-thienyl (3b) thiazole derivatives exhibited improved growth inhibition activity as compared with the activity of the corresponding pyrrolidine analogue 2a. Moreover, compounds 3a and 3b exhibited the best antiproliferative activity among the compounds synthesized in this study.

Overall, these compounds had greater potency than 1 against MCF-7 and HT-29 cells, comparable activity against HeLa and A549 cells, and less activity against HL-60 and Jurkat cells. Comparing 3a with the bioisosteric 3-thienyl derivative 3b, the latter was less active than 3a only in MCF-7 cells. In general, the introduction of EWGs and ERGs reduced activity compared with the unsubstituted phenyl derivative 3a, with no clear difference in effect on potency between EWGs and ERGs. At the para-position, only fluorine and methyl were tolerated, while the introduction of all other EWGs (Cl, NO₂, and CF₃) and ERGs (Et, C(CH₃)₃; OCF₃ and OEt) significantly reduced antiproliferative activity. For methoxy and chloro substituents, their position on the phenyl ring had a profound influence on potency. Moving the chloro group from the para- to the meta-position (compounds 3d and 3e, respectively) led to a dramatic increase in antiproliferative activity. The same effect was observed for the methoxy substituent (3j vs 3k). In contrast, the insertion of an additional methoxy group, to yield the m,p-dimethoxy derivative 3l, substantially reduced antiproliferative activity. These substituent effects are probably caused by increased steric hindrance in the colchicine site, preventing efficient binding, as observed in the molecular modeling studies. Thus, a selected, single, and small EWG or ERG could be placed on the phenyl ring with relatively minor effects on antiproliferative activity, but no modification improved activity relative to the unsubstuted phenyl or 3-thienyl derivatives 3a and 3b, respectively. Moreover, several compounds, such as 3c and 3f, characterized by the presence of substituents with opposite electronic effects showed approximately the same potency.

We identified tubulin as the molecular target of the compounds since derivatives 3a and 3b, with the greatest inhibitory effects on cell growth, strongly inhibited tubulin assembly and the binding of colchicine to tubulin. Their potency for inhibition of tubulin polymerization was comparable with that of the reference compound 1.

We also showed that 3b had cellular effects typical for microtubule interacting agents, causing accumulation of cells in the G2/M phase of the cell cycle. Further studies showed that 3b was a potent inducer of apoptosis in HeLa cells. Apoptosis induced by antimitotic agents has been associated with alterations in a variety of cellular signaling pathways. As with many antimitotic drugs, compound 3b induced Bcl-2 down-regulation after a 24 h treatment. Bcl-2 prevents the initiation of the cellular apoptotic program by stabilizing mitochondrial permeability. Our results confirmed that the induction of apoptosis by 3b was associated with down-regulation of Bcl-2, dissipation of the mitochondrial transmembrane potential, and activation of caspase-3, which is coupled with terminal events of apoptosis, such as PARP cleavage. These effects were in good agreement with the cytotoxic potency of this compound and occur at lower concentrations than was observed with compound 2a.⁸ Compound 3b was also active in suppressing the growth of drug resistant cells, and even more importantly, it had significant in vivo activity in a colon cancer xenograft model. Finally, preliminary experiments were performed to assess the potential antivascular activity of compound 3b. The ability of this compound to inhibit HUVEC migration and to destroy pre-established vessels formed by HUVECs is consistent with antivascular activity and warrants further testing in in vivo cancer models.

EXPERIMENTAL SECTION

Chemistry

Materials and Methods

¹H and ¹³C NMR spectra were recorded on a Bruker AC 200 and Varian 400 Mercury Plus spectrometer, respectively. Chemical shifts (δ) are given in ppm, and the spectra were recorded in appropriate deuterated solvents, as indicated. Infrared spectra were recorded on a Perkin-Elmer Spectrum 100 FTIR spectrometer using the ATR technique. The main bands are given in cm⁻¹. Positive-ion electrospray ionization (ESI) mass spectra were recorded on a double-focusing Finnigan MAT 95 instrument with BE geometry. Melting points (mp) were determined on a Buchi-Tottoli apparatus and are uncorrected. All products reported showed ¹H NMR spectra in agreement with the assigned structures. The purity of tested compounds was determined by combustion elemental analyses conducted by the Microanalytical Laboratory of the Chemistry Department of the University of Ferrara with a Yanagimoto MT-5 CHN recorder elemental analyzer. All tested compounds yielded data consistent with a purity of at least 95% as compared with the theoretical values. All reactions were carried out under an inert atmosphere of dry nitrogen, unless otherwise indicated. Standard syringe techniques were used for transferring dry solvents. Reaction courses and product mixtures were routinely monitored by TLC on silica gel (precoated F₂₅₄ Merck plates), and compounds were visualized with aqueous KMnO₄. Flash chromatography was performed using 230–400 mesh silica gel and the indicated solvent system. Organic solutions were dried over anhydrous Na₂SO₄. Arylboronic acids are commercially available and used as received. All chemicals and reagents were purchased from Aldrich (Sigma-Aldrich) or Lancaster (Alfa Aesar, Johnson Matthey Company).

Synthesis of (4-Amino-2-(methylthio)thiazol-5-yl)(3,4,5-trimethoxyphenyl)methanone (5)

To a stirred suspension of Na₂S·9H₂O (1.2 g., 5 mmol) in DMF (7 mL), dimethyl cyanodithioimidocarbonate 4 (730 mg, 5 mmol) dissolved in DMF (5 mL) was added, and the mixture was heated at 70 °C for 2 h. Then, 2-bromo-1-(3,4,5-trimethoxyphenyl)ethanone (2.89 g., 10 mmol) dissolved in DMF (5 mL) was slowly added dropwise at 50 °C. The mixture was heated at 50 °C for 2 h, and potassium carbonate was added (690 mg, 10 mmol). The reaction was stirred at 50 °C for an additional hour. The mixture was poured onto water (100 mL) with stirring. When a yellow precipitate appeared, it was filtered, washed with water, and dried at room temperature until a constant weight was reached. The isolated solid was purified by stirring in petroleum ether for 1 h and filtered to afford (5) as a yellow solid. Yield 52%, mp 130–132 °C. ¹H NMR (CDCl₃) δ: 2.67 (s, 3H), 3.90 (s, 3H), 3.91 (s, 6H), 6.82 (bs, 2H), 7.03 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ: 13.6, 56.3 (2C), 61.0, 104.7 (2C), 105.2, 137.1, 139.9, 153.0 (2C), 165.2, 172.2, 183.3. IR (ATR) ν: 3293, 1594, 1532, 1455, 1407, 1337, 1116, 998, 736.

General Procedure for the Synthesis of (4-Amino-2-arylthiazol-5-yl)(3,4,5-trimethoxyphenyl)methanone (3a–o)

To a stirred solution of (4-amino-2-(methylthio)thiazol-5-yl)(3,4,5-trimethoxyphenyl)-methanone 5 (170 mg, 0.5 mmol) in THF (10 mL) were subsequently added the appropriate arylboronic acid (1 mmol), TFP (19 mg, 0.08 mmol), and CuTC (0.125 g, 0.65 mmol). The reaction vessel was flushed with argon, then Pd₂-dba₃·CHCl₃ (20 mg, 0.02 mmol) was added, and the reaction mixture was stirred at 70 °C for 18 h. After cooling, the mixture was diluted with dichloromethane (5 mL) and filtered through Celite, and the filtrate was evaporated. The residue was dissolved in dichloromethane (15 mL), and the mixture was washed with 25% aqueous ammonia (2 × 5 mL). The organic layer was dried and the residue after evaporation purified by silica gel column chromatography (gradient of petroleum ether and ethyl acetate), giving the desired product.

(4-Amino-2-phenylthiazol-5-yl)(3,4,5-trimethoxyphenyl)-methanone (3a)

Following the general procedure, the crude residue, purified by flash chromatography using ethyl acetate/petroleum ether 4:6 (v/v) as eluent, furnished 3a as a yellow solid (52% yield); mp 156–158 °C. ¹H NMR (CDCl₃) δ: 3.93 (s, 6H), 3.94 (s, 3H), 6.94 (bs, 2H), 7.11 (s, 2H), 7.48 (m, 3H), 7.95 (dd, J = 8.0 and 2.0 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃) δ: 56.3 (2C), 60.9, 105.2 (2C), 108.9, 127.0 (2C), 129.3 (2C), 131.2, 132.4, 133.4, 135.7, 141.1, 153.2 (2C), 163.7, 186.4. IR (ATR) ν: 3384, 1620, 1578, 1450, 1411, 1337, 1127, 999, 817, 760. MS (ESI): [M]⁺ = 370.7. Anal. (C₁₉H₁₈N₂O₄S) C, H, N.

(4-Amino-2-(thiophen-3-yl)thiazol-5-yl)(3,4,5-trimethoxyphenyl)-methanone (3b)

Following the general procedure, the crude residue, purified by flash chromatography using ethyl acetate/petroleum ether 1:1 (v/v) as eluent, furnished 3b as a yellow solid (58% yield); mp 198–200 °C. ¹H NMR (CDCl₃) δ: 3.92 (s, 6H), 3.93 (s, 3H), 6.98 (bs, 2H), 7.09 (s, 2H), 7.42 (d, J = 8.4 Hz, 1H), H), 7.51 (d, J = 8.4 Hz, 1H), 8.03 (s, 1H). ¹³C NMR (100 MHz, CDCl₃) δ: 56.3 (2C), 60.9, 101.4, 105.2 (2C), 125.9, 127.6, 133.9, 134.2, 135.8, 141.0, 153.1 (2C), 164.0, 166.4, 186.3. IR (ATR) ν: 3377, 1614, 1577, 1445, 1408, 1338, 1234, 1125, 989, 819, 763. MS (ESI): [M]⁺ = 376.8. Anal. (C₁₇H₁₆N₂O₄S₂) C, H, N.

(4-Amino-2-(4-fluorophenyl)thiazol-5-yl)(3,4,5-trimethoxyphenyl)methanone (3c)

Following the general procedure, the crude residue, purified by flash chromatography using ethyl acetate/petroleum ether 6:4 (v/v) as eluent, furnished 3c as a yellow solid (49% yield); mp 196–197 °C. ¹H NMR (CDCl₃) δ: 3.92 (s, 6H), 3.93 (s, 3H), 7.02 (bs, 2H), 7.12 (s, 2H), 7.19 (m, 2H), 7.94 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ: 56.4 (2C), 61.0, 102.4, 105.2 (2C), 116.3, 116.6, 128.5, 129.1, 129.2, 135.9, 141.0, 153.2 (2C), 163.8, 165.3 (d, J = 253.3 Hz), 170.8, 186.5. IR (ATR) ν: 3262, 1620, 1578, 1450, 1408, 1337, 1236, 1129, 994, 813, 759. MS (ESI): [M]⁺ = 388.9. Anal. (C₁₉H₁₇FN₂O₄S) C, H, N.

(4-Amino-2-(4-chlorophenyl)thiazol-5-yl)(3,4,5-trimethoxyphenyl)methanone (3d)

Following the general procedure, the crude residue, purified by flash chromatography using ethyl acetate/petroleum ether 6:4 (v/v) as eluent, furnished 3d as a yellow solid (48% yield); mp 146–148 °C. ¹H NMR (CDCl₃) δ: 3.92 (s, 6H), 3.93 (s, 3H), 6.82 (bs, 2H), 7.10 (s, 2H), 7.42 (d, J = 8.8 Hz, 2H), 7.88 (d, J = 8.8 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃) δ: 56.4 (2C), 61.1, 102.6, 105.3 (2C), 110.2, 128.1 (2C), 129.5 (2C), 130.7, 135.9, 138.2, 153.2 (2C), 164.8, 170.7, 186.6. IR (ATR) ν: 3313, 2930, 1614, 1572, 1414, 1335, 1129, 818, 758. MS (ESI): [M]⁺ = 404.9. Anal. (C₁₉H₁₇ClN₂O₄S) C, H, N.

(4-Amino-2-(3-chlorophenyl)thiazol-5-yl)(3,4,5-trimethoxyphenyl)methanone (3e)

Following the general procedure, the crude residue, purified by flash chromatography using ethyl acetate/petroleum ether 6:4 (v/v) as eluent, furnished 3e as a yellow solid (58% yield); mp 185–186 °C. ¹H NMR (CDCl₃) δ: 3.93 (s, 6H), 3.94 (s, 3H), 6.84 (bs, 2H), 7.11 (s, 2H), 7.35 (d, J = 7.8 Hz, 2H), 7.46 (dt, J = 7.2 and 1.6 Hz, 1H), 7.83 (dt, J = 7.2 and 1.6 Hz, 1H), 7.96 (t, J = 1.4 Hz, 1H). ¹³C NMR (100 MHz, CDCl₃) δ: 56.3 (2C), 60.9, 102.7, 105.2 (2C), 124.9, 126.7, 130.4, 131.7, 133.9, 135.3, 135.8, 141.0, 153.1 (2C), 164.7, 170.2, 186.6. IR (ATR) ν: 3307, 1614, 1577, 1339, 1236, 1133, 992, 820, 761. MS (ESI): [M]⁺ = 404.9. Anal. (C₁₉H₁₇ClN₂O₄S) C, H, N.

(4-Amino-2-p-tolylthiazol-5-yl)(3,4,5-trimethoxyphenyl)-methanone (3f)

Following the general procedure, the crude residue, purified by flash chromatography using ethyl acetate/petroleum ether 1:1 (v/v) as eluent, furnished 3f as a yellow solid (45% yield); mp 130–132 °C. ¹H NMR (CDCl₃) δ: 2.41 (s, 3H), 3.92 (s, 6H), 3.93 (s, 3H), 6.84 (bs, 2H), 7.11 (s, 2H), 7.26 (d, J = 8.2 Hz, 2H), 7.84 (d, J = 8.2 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃) δ: 21.7, 56.3 (2C), 60.9, 101.9, 105.2 (2C), 126.9 (2C), 129.2, 129.9 (2C), 135.9, 140.9, 143.0, 153.1 (2C), 164.4, 172.2, 186.3. IR (ATR) ν: 3314, 2928, 1612, 1574, 1412, 1335, 1235, 1128, 991, 816, 759. MS (ESI): [M]⁺ = 384.9. Anal. (C₂₀H₂₀N₂O₄S) C, H, N.

(4-Amino-2-(4-ethylphenyl)thiazol-5-yl)(3,4,5-trimethoxyphenyl)-methanone (3g)

Following the general procedure, the crude residue, purified by flash chromatography using ethyl acetate/petroleum ether 1:1 (v/v) as eluent, furnished 3g as a yellow solid (56% yield); mp 86–87 °C. ¹H NMR (CDCl₃) δ: 1.26 (t, J = 7.2 Hz, 3H), 2.69 (q, J = 7.2 Hz, 2H), 3.92 (s, 6H), 3.93 (s, 3H), 6.94 (bs, 2H), 7.12 (s, 2H), 7.29 (d, J = 8.2 Hz, 2H), 7.88 (d, J = 8.2 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃) δ: 15.3, 29.0, 56.4 (2C), 61.0, 102.2, 105.2 (2C), 127.6 (2C), 133.9, 135.9 (2C), 136.1, 140.9, 149.0, 153.1 (2C), 165.1, 172.6, 186.5. IR (ATR) ν: 3273, 2931, 1621, 1574, 1453, 1412, 1334, 1237, 1128, 1004, 820, 759. MS (ESI): [M]⁺ = 398.8. Anal. (C₂₁H₂₂N₂O₄S) C, H, N.

(4-Amino-2-(4-tert-butylphenyl)thiazol-5-yl)(3,4,5-trimethoxyphenyl)methanone (3h)

Following the general procedure, the crude residue, purified by flash chromatography using ethyl acetate/petroleum ether 4:6 (v/v) as eluent, furnished 3h as a yellow solid (54% yield); mp 66–68 °C. ¹H NMR (CDCl₃) δ: 1.35 (s, 9H), 3.93 (s, 6H), 3.94 (s, 3H), 6.91 (bs, 2H), 7.12 (s, 2H), 7.47 (d, J = 8.4 Hz, 2H), 7.89 (d, J = 8.4 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃) δ: 31.3 (3C), 35.2, 56.3 (2C), 61.1, 105.3 (2C), 110.9, 125.0, 126.2 (2C), 126.8 (2C), 128.5, 136.1, 142.1, 153.2 (2C), 155.9, 164.9, 186.5. IR (ATR) ν: 2958, 1574, 1402, 1334, 1233, 1124, 1000, 818, 763. MS (ESI): [M]⁺ = 426.7. Anal. (C₂₃H₂₆N₂O₄S) C, H, N.

(4-Amino-2-(4-trifluoromethylphenyl)thiazol-5-yl)(3,4,5-trimethoxyphenyl)methanone (3i)

Following the general procedure, the crude residue, purified by flash chromatography using ethyl acetate/petroleum ether 4:6 (v/v) as eluent, furnished 3i as a yellow solid (56% yield); mp 166–167 °C. ¹H NMR (CDCl₃) δ: 3.93 (s, 6H), 3.94 (s, 3H), 6.86 (bs, 2H), 7.11 (s, 2H), 7.71 (d, J = 8.2 Hz, 2H), 8.05 (d, J = 8.2 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃) δ: 56.4 (2C), 61.0, 103.2, 105.3 (2C), 123.7 (q, J = 271.5 Hz), 126.2 (2C), 126.4, 127.1 (2C), 133.2 (q, J = 32.7 Hz), 135.5, 135.8, 141.2, 153.2 (2C), 164.9, 169.9, 186.8. IR (ATR) ν: 3314, 2930, 1613, 1575, 1322, 1237, 1127, 1104, 1067, 845, 817, 760. MS (ESI): [M]⁺ = 438.7. Anal. (C₂₀H₁₇F₃N₂O₄S) C, H, N.

(4-Amino-2-(4-methoxyphenyl)thiazol-5-yl)(3,4,5-trimethoxyphenyl)methanone (3j)

Following the general procedure, the crude residue, purified by flash chromatography using ethyl acetate/petroleum ether 6:4 (v/v) as eluent, furnished 3j as a yellow solid (46% yield); mp 196–198 °C. ¹H NMR (CDCl₃) δ: 3.87 (s, 3H), 3.92 (s, 6H), 3.93 (s, 3H), 6.82 (bs, 2H), 6.93 (d, J = 8.0 Hz, 2H), 7.11 (s, 2H), 7.90 (d, J = 8.0 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃) δ: 55.8, 56.5 (2C), 61.2, 101.8, 105.3 (2C), 114.8 (2C), 124.9, 129.0 (2C), 136.3, 141.1, 153.2 (2C), 163.1, 164.8, 172.3, 186.4. IR (ATR) ν: 3298, 1573, 1397, 1259, 1124, 1004, 825, 756. MS (ESI): [M]⁺ = 400.8. Anal. (C₂₀H₂₀N₂O₅S) C, H, N.

(4-Amino-2-(3-methoxyphenyl)thiazol-5-yl)(3,4,5-trimethoxyphenyl)methanone (3k)

Following the general procedure, the crude residue, purified by flash chromatography using ethyl acetate/petroleum ether 1:1 (v/v) as eluent, furnished 3k as an orange solid (47% yield); mp 167–168 °C. ¹H NMR (CDCl₃) δ: 3.87 (s, 3H), 3.92 (s, 6H), 3.93 (s, 3H), 6.84 (bs, 2H), 7.07 (m, 1H), 7.11 (s, 2H), 7.36 (t, J = 7.2 Hz, 1H), 7.54 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ: 55.7, 56.4 (2C), 61.1, 105.3 (2C), 111.7, 118.7, 119.5, 130.5, 132.9. 135.8, 141.1, 153.2 (2C), 160.2, 162.8, 170.5, 171.8, 186.5. IR (ATR) ν: 3275, 1615, 1579, 1503, 1409, 1337, 1235, 1129, 994, 826, 769, 674. MS (ESI): [M]⁺ = 400.8. Anal. (C₂₀H₂₀N₂O₅S) C, H, N.

(4-Amino-2-(3,4-dimethoxyphenyl)thiazol-5-yl)(3,4,5-trimethoxyphenyl)methanone (3l)

Following the general procedure, the crude residue, purified by flash chromatography using ethyl acetate/petroleum ether 2:8 (v/v) as eluent, furnished 3l as a red solid (45% yield); mp 176–177 °C. ¹H NMR (CDCl₃) δ: 3.92 (s, 6H), 3.93 (s, 3H), 3.95 (s, 3H), 3.98 (s, 3H), 6.80 (bs, 2H), 6.88 (d, J = 8.8 Hz, 1H), 7.10 (s, 2H), 7.51 (d, J = 8.8 Hz, 1H), 7.54 (s, 1H). ¹³C NMR (100 MHz, CDCl₃) δ: 56.3, 56.5 (2C), 61.1, 62.3, 100.4, 105.2, 107.2 (2C), 107.6, 109.8, 111.4, 121.5, 135.3, 141.3, 149.7, 153.2 (2C), 169.2, 170.9, 186.0. IR (ATR) ν: 2933, 1579, 1454, 1411, 1333, 1261, 1121, 1101, 1006, 799. MS (ESI): [M]⁺ = 431.0. Anal. (C₂₁H₂₂N₂O₆S) C, H, N.

(4-Amino-2-(4-ethoxyphenyl)thiazol-5-yl)(3,4,5-trimethoxyphenyl)methanone (3m)

Following the general procedure, the crude residue, purified by flash chromatography using ethyl acetate/petroleum ether 6:4 (v/v) as eluent, furnished 3m as an orange solid (44% yield); mp 185–186 °C. ¹H NMR (CDCl₃) δ: 1.44 (t, J = 7.2 Hz, 3H), 3.92 (s, 6H), 3.93 (s, 3H), 4.11 (q, J=7.2 Hz, 2H), 6.80 (bs, 2H), 6.94 (d, J = 8.6 Hz, 2H), 7.11 (s, 2H), 7.90 (d, J = 8.6 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃) δ: 14.8, 56.4 (2C), 61.1, 63.9, 102.0, 105.6 (2C), 115.2 (2C), 124.6, 128.9 (2C), 136.1, 141.3, 153.2 (2C), 163.1, 164.6, 172.2, 186.0. IR (ATR) ν: 2996, 1597, 1573, 1406, 1337, 1235, 1119, 104, 1006, 820, 763. MS (ESI): [M]⁺ = 414.9. Anal. (C₂₁H₂₂N₂O₅S) C, H, N.

(4-Amino-2-(4-trifluoromethoxyphenyl)thiazol-5-yl)(3,4,5-trimethoxyphenyl)methanone (3n)

Following the general procedure, the crude residue, purified by flash chromatography using ethyl acetate/petroleum ether 1:1 (v/v) as eluent, furnished 3n as a yellow solid (49% yield); mp 174–176 °C. ¹H NMR (CDCl₃) δ: 3.92 (s, 6H), 3.93 (s, 3H), 6.90 (bs, 2H), 7.11 (s, 2H), 7.28 (d, J = 9.0 Hz, 2H), 7.98 (d, J = 9.0 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃) δ: 56.4 (2C), 61.1, 102.6, 105.3 (2C), 114.4, 120.4 (q, J = 257.8 Hz), 121.3 (2C), 128.7 (2C), 135.8, 151.9, 153.2 (2C), 162.6, 163.6, 170.1, 186.6. IR (ATR) ν: 3315, 1614, 1576, 1337, 1236, 1127, 997, 816, 759. MS (ESI): [M]⁺ = 454.7. Anal. (C₂₀H₁₇F₃N₂O₅S) C, H, N.

(4-Amino-2-(4-nitrophenyl)thiazol-5-yl)(3,4,5-trimethoxyphenyl)-methanone (3o)

Following the general procedure, the crude residue, purified by flash chromatography using ethyl acetate/petroleum ether 6:4 (v/v) as eluent, furnished 3o as a red solid (42% yield); mp 234–236 °C. ¹H NMR (CDCl₃) δ: 3.93 (s, 6H), 3.94 (s, 3H), 6.92 (bs, 2H), 7.11 (s, 2H), 8.15 (d, J = 9.0 Hz, 2H), 8.31 (d, J = 9.0 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃) δ: 56.4 (2C), 61.1, 105.3 (2C), 106.5, 124.5 (2C), 127.6 (2C), 124.8, 135.7, 137.9, 149.4, 153.3 (2C), 160.2, 168.4, 186.9. IR (ATR) ν: 3337, 1593, 1577, 1519, 1415, 1331, 1234, 1130, 850, 816. MS (ESI): [M]⁺ = 415.8. Anal. (C₁₉H₁₇N₃O₆S) C, H, N.

Antiproliferative Assays

Human T-leukemia (Jurkat) and human promyelocytic leukemia (HL-60) cells were grown in RPMI-1640 medium, (Gibco, Milano, Italy). Breast adenocarcinoma (MCF-7), human nonsmall cell lung carcinoma (A549), human cervix carcinoma (HeLa), and human colon adenocarcinoma (HT-29) cells were grown in DMEM medium (Gibco, Milano, Italy). Both media were supplemented with 115 units/mL of penicillin G (Gibco, Milano, Italy), 115 µg/mL of streptomycin (Invitrogen, Milano, Italy), and 10% fetal bovine serum (Invitrogen, Milano, Italy). LoVo^Doxo cells are a doxorubicin resistant subclone of LoVo cells¹³ and were grown in complete Ham’s F12 medium supplemented with doxorubicin (0.1 µg/mL). CEM^Vbl-100 cells are a multidrug-resistant line selected against vinblastine.¹⁴ A549-T12 cells are a nonsmall cell lung carcinoma line exhibiting resistance to taxol.¹⁵ They were grown in complete DMEM medium supplemented with taxol (12 nM). Stock solutions (10 mM) of the different compounds were obtained by dissolving them in DMSO. Individual wells of a 96-well tissue culture microtiter plate were inoculated with 100 µL of complete medium containing 8 × 10³ cells. The plates were incubated at 37 °C in a humidified 5% CO₂ incubator for 18 h prior to the experiments. After medium removal, 100 µL of fresh medium containing the test compound at different concentrations was added to each well and incubated at 37 °C for 72 h. The percentage of DMSO in the medium never exceeded 0.25%. This was also the maximum DMSO concentration in all cell-based assays described below. Cell viability was assayed by the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test as previously described.²⁰ The IC₅₀ was defined as the compound concentration required to inhibit cell proliferation by 50%, in comparison with cells treated with the maximum amount of DMSO (0.25%) and considered as 100% viability.

Effects on Tubulin Polymerization and on Colchicine Binding to Tubulin

To evaluate the effect of the compounds on tubulin assembly in vitro,^16a varying concentrations of compounds were preincubated with 10 µM bovine brain tubulin in glutamate buffer at 30 °C and then cooled to 0 °C. After the addition of 0.4 mM GTP, the mixtures were transferred to 0 °C cuvettes in a recording spectrophotometer and warmed to 30 °C. Tubulin assembly was followed turbidimetrically at 350 nm. The IC₅₀ was defined as the compound concentration that inhibited the extent of assembly by 50% after a 20 min incubation. The capacity of the test compounds to inhibit colchicine binding to tubulin was measured as described,^16b except that the reaction mixtures contained 1 µM tubulin, 5 µM [³H]colchicine, and 5 µM test compound.

Molecular Modeling

All molecular modeling studies were performed on a MacPro dual 2.66 GHz Xeon running Ubuntu 10. The tubulin structure was downloaded from the Protein Data Bank (http://www.rcsb.org/; PDB code 3HKC).²⁹ Hydrogen atoms were added to the protein, using the Protonate3D function of Molecular Operating Environment (MOE).³⁰ Ligand structures were built with MOE and minimized using the MMFF94x forcefield until a rmsd gradient of 0.05 kcal mol⁻¹ Å⁻¹ was reached. The docking simulations were performed using PLANTS.³¹

Flow Cytometric Analysis of Cell Cycle Distribution

For flow cytometric analysis of DNA content, 5 × 10⁵ HeLa or Jurkat cells in exponential growth were treated with different concentrations of the test compounds for 24 or 48 h. After the incubation, the cells were collected, centrifuged, and fixed with ice-cold ethanol (70%). The cells were treated with lysis buffer containing RNase A and 0.1% Triton X-100 and stained with PI. Samples were analyzed on a Cytomic FC500 flow cytometer (Beckman Coulter). DNA histograms were analyzed using MultiCycl for Windows (Phoenix Flow Systems).

Annexin-V Assay

Surface exposure of PS on apoptotic cells was measured by flow cytometry with a Coulter Cytomics FC500 (Beckman Coulter) by adding annexin-V-FITC to cells according to the manufacturer’s instructions (Annexin-V Fluos, Roche Diagnostic). Simultaneously, the cells were stained with PI.

Assessment of Mitochondrial Changes

The mitochondrial membrane potential was measured with the fluorescent dye JC-1 (Molecular Probes, USA) by flow cytometry, as described previously.²⁰ The production of ROS was measured by flow cytometry using either HE (Molecular Probes) or H₂DCFDA (Molecular Probes). After different times of treatment, cells were collected by centrifugation and resuspended in HBSS containing the fluorescence probes HE or H₂DCFDA at 2.5 and 0.1 µM, respectively. The cells were incubated for 30 min at 37 °C, centrifuged and resuspended in HBSS. The fluorescence was directly recorded with the flow cytometer, using as excitation wavelength 488 nm and emission at 585 and 530 nm for HE and H₂DCFDA, respectively.

Western Blot Analysis

HeLa cells were incubated in the presence of test compounds and, after different times, were collected, centrifuged, and washed two times with ice cold phosphate-buffered saline (PBS). The pellet was resuspended in lysis buffer. After the cells were lysed on ice for 30 min, lysates were centrifuged at 15000g at 4 °C for 10 min. The protein concentration in the supernatant was determined using BCA protein assay reagents (Pierce, Italy). Equal amounts of protein (20 µg) were resolved using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) (7.5–15% acrylamide gels) and transferred to a PVDF Hybond-p membrane (GE Healthcare). Membranes were blocked with I-block (Tropix), the membrane being gently rotated overnight at 4 °C. Membranes were incubated with primary antibodies against Bcl-2, cleaved PARP, cdc25C, Bax, phospho-histone γH2AX and cdc2 (all rabbit, 1:1000, Cell Signaling, Milano, Italy), cyclin B1 (mouse, 1:1000, BD, Milano, Italy), or β-actin (mouse, 1:10,000, Sigma-Aldrich, Milano, Italy) for 2 h at room temperature. Membranes were next incubated with peroxidase-labeled goat antirabbit IgG (1:100,000, Sigma-Aldrich) or peroxidase-labeled goat antimouse IgG (1:100,000, Sigma-Aldrich) for 60 min. All membranes were visualized using ECL Advance (GE Healthcare) and exposed to Hyperfilm MP (GE Healthcare). To ensure equal protein loading, each membrane was stripped and reprobed with anti-β-actin antibody.

Antivascular Activity

HUVECs were prepared from a primary culture of human umbilical cord veins. The adherent cells were maintained in M200 medium supplemented with LSGS (Low Serum Growth Supplement), which contains FBS, hydrocortisone, hEGF, bFGF, heparin, and gentamycin/amphotericin B (Invitrogen, Milano, Italy), and seeded on collagen I (BD Biosciences). Once confluent, the cells were detached by treatment with a trypsin–EDTA solution and used in experiments from the third to seventh passages.

Motility Assay

Motility assay for HUVECs was based on “scratch” wounding of a confluent monolayer.²⁶ Briefly, HUVECs (1 × 10⁵) were seeded onto 0.3% collagen I-coated six well plates in complete medium until a confluent monolayer was formed. The cells were scratch wounded using a pipet tip. Then the cells were treated with the test compounds, and at different times after the scratch, the cells were photographed under a light microscope (40× magnification). Wound closure was evaluated by Image-J software.

Endothelial Cell Vessel Formation on a Matrigel Matrix

A Matrigel matrix was kept at 4 °C for 16 h. Two hundred microliters of Matrigel (BD Biosciences) was added to each well of a 24-well plate. After gelling at 37 °C for 30 min, gels were overlaid with 500 µL of medium containing 6 × 10⁴ HUVECs. Different concentrations of test compound were added to the cultures, which were incubated for different times. To explore the effect of test compounds on pre-established vasculature, HUVECs were seeded and incubated over Matrigel for 6 h to allow the capillary tubes to form. Then the compound was added and continuously incubated for additional times, and the disappearance of existing vasculature was monitored by recording phase contrast images (five fields for each well: the four quadrants and the center) using a digital camera. The images were saved as TIFF files. Image analysis was carried out using the ImageJ image analysis software and evaluating both dimensional parameters (percent area covered by HUVECs and total length of HUVECs network per field) and topological parameters (number of meshes and branching points per fields).³² Values were expressed as percent change from control cultures grown with complete medium.

Antitumor Activity in Vivo

Four week old female BALB/c-nu nude mice (15–18 g) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). The animals were maintained under specific pathogen-free conditions with food and water supplied ad libitum in Zhejiang University of Traditional Chinese Medicine Laboratory Animal Center. Human colon adenocarcinoma HT-29 cells in logarithmic growth phase were resuspended in RPMI 1640 without fetal bovine serum at 1 × 10⁷ cells/mL and inoculated (0.2 mL) in the hypodermis of the pars dorsalis of each mouse. Once the HT-29 xenografts reached a size of ~300 mm³, 12 mice were randomly assigned to two groups. For the first group, compound 3b was prepared in DMSO and injected intraperitoneally at volumes of 0.01 mL/g body weight to give a dose of 100 mg/kg to each mouse. The compound was administered three times a week for one week. After completing the treatment schedule and the evaluation period, tumor-bearing mice were euthanized. Tumor volume was calculated by the following formula: V = (L × W²)/2 where L is the length, and W is the width of the tumor nodules measured by Vernier calipers. The study was approved by the Institutional Animal Ethical Committee of the Second Affiliated Hospital, School of Medicine, Zhejiang University (PRC).

Statistical Analysis

Unless indicated otherwise, results are presented as the mean ± SEM. The differences between different treatments were analyzed using the two-sided Student’s t test. P values lower than 0.05 were considered significant.

ABBREVIATIONS USED

CA-4

combretastatin A-4

HUVEC

human umbilical vein endothelial cells

EWG

electron-withdrawing group

ERG

electron-releasing group

SAR

structure–activity relationships

CuTc

copper(I)thiophene-2-carboxylate

Pd₂dba₃·CHCl₃

tris-(dibenzylideneacetone)dipalladium complex with chloroform

TFP

tris(2-furyl)phosphine

FITC

fluorescein isothiocyanate

PI

propidium iodide

PS

phospholipid phosphatidylserine

ROS

reactive oxygen species

HE

hydroxyethidine

H₂DCFDA

2,7-dichlorodihydrofluorescein

PARP

poly-ADP-ribose polymerase

HBSS

Hank’s Balanced Salt Solution

PBS

phosphate-buffered saline

SDS–PAGE

sodium dodecyl sulfate–polyacrylamide gel electrophoresis