Symmetrical α-bromoacryloylamido diaryldienone derivatives as a novel series of antiproliferative agents. Design, synthesis and biological evaluation

Abstract

In a continuing study of hybrid compounds containing the α-bromoacryloyl moiety as potential anticancer drugs, we synthesized a novel series of hybrids 4a–h, in which this moiety was linked to a 1,5-diaryl-1,4-pentadien-3-one system. Many of the conjugates prepared (4b, 4c, 4e and 4g) demonstrated pronounced, submicromolar antiproliferative activity against four cancer cell lines. Moreover, compound 4b induced apoptosis through the mitochondrial pathway and activated caspase-3 in a concentration-dependent manner.

Because of their ability to interact with cellular nucleophiles, Michael acceptors are often employed as a powerful tool in the design of anticancer agents.¹ The α,β-unsaturated carbonyl group is capable of undergoing Michael addition, and this moiety may be highly deleterious for malignant cells because it could interfere at multiple points in biological cascades through successive attacks on cellular nucleophiles.² Among the α,β-unsaturated ketones, enones and dienones derivatives, the chalcones³ and the α,α′-bis(arylidene)ketones,^4–7 are versatile pharmacophores belonging to the class of Michael acceptors. In previous studies, the 1,5-diaryl-3-oxo-1,4-pentadienyl system was incorporated into various cyclic and acyclic scaffolds with general formula 1a and 1b, and these compounds included derivatives that demonstrated promising antiproliferative activities against cancer cell lines.^4,8 The aromatic symmetrical dienones 1a and 1b act as Michael acceptors, suggesting that two successive alkylations of the β-positions of the reactive dienone system by cellular thiols could be one mechanism by which these compounds exert their antiproliferative activity in vitro.^9–12

The α-bromoacryloyl alkylating moiety is present in a series of potent anticancer distamycin-like minor groove binders, including PNU-166196 (brostallicin), which is currently undergoing Phase II clinical trials as a first-line single agent chemotherapy in patients with advanced or metastatic soft tissue sarcoma (Chart 1).¹³ It has been hypothesized that the reactivity of the α-bromoacryoyl moiety results from a first-step Michael-type nucleophilic attack, followed by a further reaction of the former vinylic bromo substituent alpha to the carbonyl, leading successively either to a second nucleophilic substitution or to a beta elimination.¹⁴

Chart 1.

In a recent publication, we reported a series of α-bromoacryloylamido chalcones with general structure 2, containing a pair of Michael acceptors in their structure, corresponding to the α-bromoacryloyl moiety and the α,β-unsaturated ketone system of the chalcone framework.¹⁵ Electron-releasing and electron-withdrawing groups on the phenyl linked to the carbonyl significantly influenced the antiproliferative activity. Of the tested compounds, derivative 2a (4-Br) and 2b (4-OMe) displayed IC₅₀ values of 0.62–1.1 and 0.98–1.4 µM, respectively, against a panel of four cancer cell lines (Table 1).

Table 1.

In vitro inhibitory effects of compounds 2ab, 3ab, 4a–h and 6a–i on the proliferation of murine leukemia (L1210), murine mammary carcinoma (FM3A), human T-leukemia (CEM) and human cervix carcinoma (HeLa) cells

Compound	IC50a (µM)
	L1210	FM3A	CEM	HeLa
2a	0.62 ± 0.20	1.1 ± 0.8	0.99 ± 0.31	1.0 ± 0.0
2b	0.98 ± 0.96	1.2 ± 0.5	1.4 ± 1.2	1.4 ± 0.2
3a	9.3 ± 6.9	40 ± 8	11 ± 4	8.3 ± 0.2
3b	2.4 ± 1.6	2.2 ± 0.5	6.5 ± 0.5	4.4 ± 4.3
6a	7.8 ± 0.7	9.2 ± 0.7	6.1 ± 0.9	4.8 ± 4.4
6b	6.9 ± 0.5	9.1 ± 0.7	1.7 ± 0.1	5.2 ± 3.4
6c	4.8 ± 1.0	8.9 ± 0.8	1.6 ± 0.1	1.8 ± 0.5
6d	35 ± 2	42 ± 0	11 ± 2	30 ± 17
6e	18 ± 7	29 ± 9	8.5 ± 1.9	10 ± 5
6f	6.8 ± 1.0	9.5 ± 1.4	1.9 ± 0.2	5.2 ± 4.7
6g	9.7 ± 0.6	18 ± 6	1.7 ± 0.1	7.6 ± 2.0
6h	9.0 ± 0.0	17 ± 5	1.7 ± 0.05	8.5 ± 0.1
6i	60 ± 14	207 ± 6	168 ± 16	107 ± 91
4a	4.1 ± 3.4	6.5 ± 4.1	2.5 ± 0.9	4.6 ± 4.4
4b	0.32 ± 0.01	0.47 ± 0.10	0.30 ± 0.01	0.70 ± 0.60
4c	0.32 ± 0.03	0.48 ± 0.02	0.41 ± 0.09	0.30 ± 0.02
4d	1.7 ± 1.0	1.7 ± 1.1	1.0 ± 2.0	4.3 ± 3.7
4e	0.38 ± 0.06	0.73 ± 0.00	0.34 ± 0.04	0.35 ± 0.02
4f	1.6 ± 0.7	5.4 ± 1.2	1.6 ± 0.0	0.73 ± 0.63
4g	0.39 ± 0.02	0.79 ± 0.30	0.47 ± 0.17	0.84 ± 0.74
4h	0.62 ± 0.42	1.1 ± 0.9	1.4 ± 0.2	1.6 ± 0.0
Melphalanb	2.13 ± 0.03	n.d	2.47 ± 0.30	n.d

n.d. not determined.

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

^bThese data were previously reported in Ref. 7.

The conversion of α-bromoacryloylamido chalcones 2ab into the corresponding unsymmetrical dienones 3ab led to a reduction in activity (0.62–1.1 µM for 2a vs 8.3–40 µM for 3a, 0.98–1.4 µM for 2b vs 2.2–6.5 µM for 3a).¹⁶

The observations that both the 1,5-diaryl-3-oxo-1,4-pentadienyl and the α-bromoacryloyl groups can act as trapping agents of cellular nucleophiles led us to prepare and evaluate a novel class of synthetic conjugates with general formulae 4, incorporating these two moieties within their structures (Chart 1). If such processes occur, the bis-(α-bromoacryloylamidoarylidene) ketone derivatives 4a–h, characterized by the presence of four potential sites for electrophilic attack on cellular constituents, should be more active than the corresponding 1,5-diaryl-1,4-pentadien-3-one system, containing only two nucleophilic centers.

In particular, we have synthesized three different small series of bis-(α-bromoacryloylamidoarylidene) ketone derivatives, corresponding to the acyclic analogue 4a, the cyclic conjugates 4c–e, and finally the 4-piperidone derivatives 4f–h.

The three different series of cyclic ketones 4b and 4de were prepared with the aim of evaluating how the size of the ring influenced antiproliferative activity. By the synthesis of compound 4c, we evaluated the effect of the introduction of a small hydrophobic substituent, corresponding to a methyl group, at the 4-position of the cyclohexanone ring.

Hybrid compounds 4a–h were prepared by the procedure described in Scheme 1. The Claisen–Schmidt aldol condensation¹⁷ between 3-nitrobenzaldehyde or 4-nitrobenzaldehyde with the appropriate commercially available acetone, cyclic ketone or N-substituted 4-piperidone in the presence of lithium hydroxide monohydrate as base in ethanol led to the formation of the corresponding α,α-bis (3-nitrobenzylidene)ketones 5a–h or (1E,4E)-1,5-bis(4-nitrophenyl)penta-1,4-dien-3-one 5i in 65–73% yields. α,α-Bis-(aminobenzylidene) ketones 6a–i were generated from the corresponding nitro derivatives 5a–i by reduction with iron and ammonium chloride in a refluxing mixture of water and ethanol (1:4 v/v).

Scheme 1.

Reagents: (a) LiOH·H₂O, cyclic/acyclic ketone, m-nitrobenzaldehyde for the synthesis of 5a–b, p-nitrobenzaldehyde for the synthesis of 5i, EtOH, rt, 18 h; (b) Fe, NH₄Cl, H₂O–EtOH, reflux, 2 h; (c) α-bromoacrylic acid, EDCI, HOBt, DMF, 18 h, rt.

Finally, the hybrid compounds 4a–k were prepared in 50–62% yields by the condensation of α-bromoacrylic acid with bis-(3-anilinomethylidene) ketones 6a–h, respectively. This condensation was performed using an excess (4 equiv) of both 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDCl) and 1-hydroxy-1,2,3-benzotriazole (1-HOBt) in dry DMF as solvent at room temperature and with identical reaction times (18 h).

Table 1 summarizes the inhibitory effects of bis-(α-bromoacryloylamido arylidene) ketone derivatives 4a–h on the proliferation of murine leukemia (L1210), murine mammary carcinoma (FM3A), human T-leukemia (CEM) and human cervix carcinoma (HeLa) cells, with bis-(aminobenzylidene)ketones 6a–i, α-bromoacryloylamido chalcones 2ab and α-bromoacryloylamido bis-(arylidene)ketones 3ab as reference compounds. The alkylating agent melphalan was used as a reference drug.

Compounds 6a and 6i were synthesized in order to evaluate the influence of the position of amino group on the benzylidene moiety on antiproliferative activity. Changing the location of the amino group from the para- in 6i to the meta-position as in 6a, led to a 10–25-fold increase in antiproliferative activity against all four cancer cell lines. The higher inhibition of cell growth showed by 6a led us to synthesize α,α′-bis-(meta-aminobenzylidene) ketone derivatives 6b–h.

Comparing the activities of 4a–h with those of 6a–h demonstrates that insertion of the two α-bromoacryloyl moieties was an important molecular change leading to a significant increase in antiproliferative activity. The bis-(α-bromoacryloylamido benzylidene) ketones 4a–h were from 2- to 50-fold more active than the corresponding bis-(anilinomethylidene) precursors 6a–h. These values are greater or equal to those obtained with the α-bromoacryloylamido enones 2ab. The greater activity of 4bc, 4e and 4gh compared to that of 3ad indicated the contribution of a second α-bromoacryloyl moiety to antiproliferative activity.

The compounds displaying the greatest potency were 4b, 4c and 4g, with IC₅₀ values of 0.32–0.38, 0.47–0.79, 0.30–0.47 and 0.30–0.84 µM against the L1210, FM3A, CEM and HeLa cell lines, respectively. Notably, the acyclic derivative 4a was significantly less active than its cyclic counterparts 4b–h. Derivatives 4b–h were found to have greatest potency to melphalan against tested cancer cell lines.

In the series of compounds 4b–e, activity decreased when the size of the cyclic ketone was increased from six to seven carbon units, while it increased greatly when the size of the ring increased from seven to eight carbons (compounds 4b, 4d and 4e, respectively). Starting from 4b, the 4-methyl substitution on the cyclohexanone ring, to furnish 4c, did not improve antiproliferative activity against L1210, FM3A and CEM cells, but the methyl group doubled potency with respect to HeLa cells.

Comparing the 4-piperidone derivatives 4f–h, lengthening the alkoxy carbonyl group from methyl to ethyl (compounds 4g and 4h, respectively) halved antiproliferative activity.

The more basic N-benzyl derivative 4f showed reduced potency relative to 4g and 4h against L1210, FM3A and CEM cells, but 4g and 4h were equipotent against HeLa cells.

Next, we examined the effects of increasing concentrations of compounds 4bc, 4e and 4g on cell-cycle progression by performing a flow cytometric analysis on HeLa cells (Table 2). Cells were cultured for 24 h in the absence or presence of each compound at 0.25, 0.5 or 1 µM, and the cells were stained with propidium iodide (PI). Treatment with 4b, 4c or 4e at 1 µM for 24 h induced a major shift from the G1 to the G2-M phase compared with control cells, but no concentration, of those tested, of compound 4g altered cell cycle progression. In particular, as shown in Table 2, treatment with 0.25, 0.5 and 1 µM of compound 4b for 24 h led to a dose-dependent decrease of the G1 peak and a proportional accumulation of cells in the G2-M phase, accompanied by a concomitant increase in the population of apoptotic cells, as demonstrated by the increase of the sub-G1 peak. Similar effects on the cell cycle progression were observed with 4bc, 4e or 4g after 48 h of treatment (see Table 1 in the Supplementary data section).

Table 2.

Cell-cycle distribution of HeLa cells after 24 h of treatment with compounds 4b, 4c, 4e or 4g

Compound	Concentration (µM)	Cell cycle percentage (%)
		Sub-G1a	G1b	Sb	G2-Mb
Control	NM	1.9 ± 0.3	52.9 ± 3.3	29.0 ± 3.0	18.1 ± 0.9
4b	0.25	4.7 ± 2.1	44.7 ± 1.1	35.8 ± 4.0	16.1 ± 0.5
4b	0.5	10.5 ± 4.6	50.9 ± 10.1	21.6 ± 6.3	27.8 ± 6.8
4b	1.0	27.2 ± 1.2	28.8 ± 6.0	22.7 ± 7.1	51.9 ± 10.4
4c	0.25	3.2 ± 0.6	41.6 ± 5.5	43.5 ± 0.8	14.8 ± 4.7
4c	0.5	4.0 ± 1.1	59.2 ± 9.0	16.1 ± 2.6	27.5 ± 3.8
4c	1.0	4.5 ± 0.9	26.5 ± 1.5	31.3 ± 6.5	41.9 ± 8.0
4e	0.25	2.5 ± 0.9	37.2 ± 8.6	46.2 ± 7.6	16.5 ± 1.0
4e	0.5	6.0 ± 3.0	64.4 ± 1.4	25.0 ± 5.0	10.5 ± 2.5
4e	1.0	15.6 ± 7.5	32.6 ± 10.6	12.9 ± 2.5	54.5 ± 7.8
4g	0.25	8.0 ± 3	49.2 ± 4.0	36.8 ± 6.8	14.5 ± 3.4
4g	0.5	7.2 ± 1.0	51.9 ± 3.9	34.6 ± 11.7	13.4 ± 8.2
4g	1.0	8.2 ± 2.1	57.0 ± 2.5	17.5 ± 2.0	24.9 ± 4.1

Data are expressed as the mean ± SE from the dose–response curves of at least three independent experiments.

NM: not meaningful.

^aPercentage of the cell population with hypodiploid DNA content peak (apoptotic cells).

^bThe percentage of cells in each phase of the cell cycle was calculated using living cells.

The induction of apoptosis in HeLa cells was confirmed by the annexin-V test¹⁸ with compounds 4bc, 4e and 4g at various concentrations for 24 or 48 h. Figure 1 shows a time and concentration-dependent increase of the percentage of annexin-V positive cells for all the tested compounds. Only a small percentage (2–5%) of necrotic cells was observed with all compounds and only at the highest concentration employed (data not shown).

Figure 1.

Flow cytometric analysis of apoptotic cells after treatment of HeLa cells with compound 4bc, 4e or 4g. After 24 and 48 h of treatment, cells were harvested and labeled with Annexin-V-FITC and PI and then analyzed by flow cytometry. The data are expressed as mean of percentage of Annexin-V positive cells ± S.E.M. for three independent experiments.

Since many compounds that arrest the cell cycle at the G2-M phase are tubulin inhibitors,¹⁹ compounds 4bc, 4e and 4g were evaluated for their inhibitory effects on tubulin polymerization. None of tested compounds were effective as inhibitors of tubulin assembly (IC₅₀ >40 µM). These data make it unlikely that the antiproliferative activity of these hybrid compounds results from a direct interaction with tubulin and that they act as microtubule depolymerizing agents.

Impairment of mitochondrial function is an early event in the executory phase of programmed cell death in different cell types, and it occurs as a consequence of a preliminary reduction of the mitochondrial transmembrane potential (Δψ_mt).^20,21 Early Δψ_mt disruption results from an opening of mitochondrial permeability transition pores, and this permeability transition triggers the release of apoptogenic factors, such as apoptosis inducing factor and cytochrome c, which in turn lead to later apoptotic events.^20,21

We used the lipophilic cation 5,5′,6,6′-tetrachlo-1,1′,3,3′-tetraethylbenzimidazol-carbocyanine (JC-1) to monitor changes in Δψ_mt induced by 4b. The method is based on the ability of this fluorescent probe to enter selectively into the mitochondria, and its color changes reversibly from green to orange as membrane potential increases.²² This property is due to the reversible formation of JC-1 aggregates upon membrane polarization. Aggregation causes a shift in the emitted light from 530 nm (i.e., emission by JC-1 monomers) to 590 nm (emission by JC-1 aggregates) following excitation at 490 nm.

HeLa cells were treated with compound 4b for 24, 48 and 72 h at 0.5 and 1 µM. As shown in Figure 2, compound 4b induces substantial mitochondrial depolarization in a time- and concentration-dependent manner. The disruption of the Δψ_mt is associated with the appearance of sub-G1 cells and with the marked increase in the percentage of annexin-V positive cells.

Figure 2.

Assessment of mitochondrial potential after treatment with compound 4b. Upper panel: induction of loss of mitochondrial membrane potential after 24, 48 and 72 h of incubation of HeLa cells with 4b at 0.5 or 1.0 µM. Cells were stained with the fluorescent probe JC-1 and analyzed by flow cytometry.

Mitochondrial membrane depolarization is associated with mitochondrial production of reactive oxygen species (ROS).^23,24 ROS are highly reactive molecules with both free radical and non-radical structures, including superoxide anion, hydrogen peroxide, hydroxyl radical and single oxygen. ROS play a central role as second messengers in a number of signal transduction pathways and cause a wide range of cellular responses ranging from transient to permanent growth arrest, and from apoptosis to necrosis, dependingon the level of ROS.

To investigate the effects of 4b on the production of oxygen species during apoptosis, we utilized two fluorescence probes, hydroethidine (HE) and 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA). The fluorescence of these compounds occurs if ROS are generated.²⁵ As shown in Figure 3 (upper and medium panels), there was an increase in cells producing ROS that closely paralleled the increase in cells with low Δψ_mt, as a function of both the concentration of 4b and treatment time.

Figure 3.

Mitochondrial production of ROS in HeLa cells. After 24 and 48 h of incubation with 4b, cells were stained with HE (upper panel) or H₂DCFDA (middle panel) and analyzed by flow cytometry. Increase of mitochondrial mass in HeLa cells treated with compound 4b (lower panel). Cells were treated as above and the relative NAO intensity was analyzed by flow cytometry. The results are expressed as percentage of the fluorescence intensity with respect to untreated control cells. Data are expressed as mean ± S.E.M. of three independent experiments.

We also evaluated the damage caused by ROS in mitochondria by assessing the oxidation state of cardiolipin, a phospholipid restricted to the inner mitochondrial membrane. We used 10 N-non-yl acridine orange (NAO), a fluorescent probe which is independent of the mitochondrial permeability transition.²⁶ The dye interacts stoichiometrically with intact, non-oxidized cardiolipin.²⁷ Some-what unexpectedly, the cells did not show reduction in NAO fluorescence, but, rather, a marked increase in NAO, especially after 48 h of treatment with 4b (Fig. 3, lower panel). This effect suggests an increase in cardiolipin content as a consequence of increased mitochondrial mass. This has been recently observed by our group in K562 cells following treatment with hybrid α-bromoacryloylamido chalcones¹⁵ but also in other tumor cell lines after treatment with herbimycin A,²⁸ genistein,²⁹ and the acronicyne derivative S23906-1³⁰ and following oxidative stress.³¹

Altogether these results indicated that the cell cycle arrest and apoptosis induced by 4b in HeLa cells were correlated with a dose-and time-dependent appearance of mitochondrial dysfunction.

The mechanism of apoptosis depends on a cascade of proteolytic enzymes called caspases.³² They exist in most cells as inactive precursors (zymogens) that, once activated sequentially, lead to irreversible cell death. Within the caspase family, caspase-3 is essential for propagation of the apoptotic signal after exposure of cells to many DNA-damaging agents and other anticancer drugs.^31,33 We therefore determined whether the activation of caspase-3 was involved in the apoptosis induced by compound 4b. We used a monoclonal antibody specific for the active fragment of caspase-3. As shown in Figure 4, with 0.5 and especially 1 µM 4b there was a marked activation of caspase-3 after 24 h of treatment, and no further increase of caspase-3 activity occurred after 48 h.

Figure 4.

Caspase-3 induced activity by compound 4b. HeLa cells were incubated in the presence of 4b at 0.5 or 1.0 µM. After 24 or 48 h of treatment, cells were harvested and stained with an anti-human active Caspase-3 fragment monoclonal antibody conjugated with FITC. Data obtained by flow cytometric analysis are expressed as percentage of caspase-3 active fragment positive cells. Data are expressed as mean ± S.E.M. of three independent experiments.

In summary, our plan to optimize the potency of a double Michael acceptor by providing multiple sites of reactivity towards cellular nucleophiles led us to synthesize a series of bis-(α-bromo-acryloylamido benzylidene) ketones 4a–h. Compared to melphalan, an established anticancer agent, derivatives 4b–h displayed significantly greater potency. These compounds were derived from the hybridization of two kinds of Michael acceptors, corresponding to the 1,5-diaryl-1,4-pentadien-3-one system of α,α′-bis-(aminobenzylidene) ketones 6a–i and the α-bromoacryloyl moiety. While compounds 6a–i showed weak or no antiproliferative activity against five cancer cell lines, the attachment of two α-bromoacryloyl moieties led to significantly increased activity. The greater potency of hybrid compounds 4a–h than bis-(aminoarylidene) ketones 6a–i was attributed to the additional electrophilic groups in 4a–h, enabling these compounds to act potentially as multifunctional Michael acceptors. In the series of cyclic ketones 4b–e, the six- and the eight-membered cyclic ring was preferred to the seven-membered ring. Compound 4g had no effect on cycle progression of HeLa cells even at higher concentrations, while compounds 4bc and 4e induced cell cycle arrest in a concentration-dependent manner. In particular, treatment with increasing concentrations of 4b led to cell cycle arrest in the G2-M phase accompanied by an increase in the percentage of apoptotic cells after 24 and 48 h.

As demonstrated with 4b, the mechanism of action of these compounds appears to be induction of apoptosis mediated by the activation of caspase-3 and by important changes in mitochondrial function, such as dissipation of the transmembrane potential and generation of ROS. Further studies to clarify additional details of the molecular mechanism of action of these compounds and their selectivity in inhibiting the growth of cancer cells are underway.