Synthesis and preliminary studies of biological activity of amino derivatives of 4-azatricyclo- [5.2.1.0^2,6]dec-8-ene-3,5-dione with silicon in the structure

Chemistry

The synthesis was carried out by a three-step procedure for aryl/heteroarylpiperazine derivatives and by a two-step procedure for aminoalkyl derivatives (Scheme 1).

Scheme 1

Synthesis of the investigated compounds 1, 1a–1s, 2, and 2a–2s.

In the first step, the starting imides 1 and 2 were obtained in a Diels-Alder reaction. These compounds 1 and 2 were N-alkylated by treatment with 1,4-dichlorobutane or 1-bromo-3-chloropropane. The resultant N-(ω-haloalkyl)imides 1a,b and 2a,b were condensed with aryl/heteroarylopiperazines to yield the desired products 1c–n and 2c–n. The aminoalkyl derivatives 1o–s and 2o–s) were obtained by the reaction of starting imides 1 and 2 with an appropriate ω-haloalkylamine. The structures of all compounds were supported by ¹H NMR, MS, and elemental analysis. For the purpose of biological evaluations, the obtained compounds were converted into their hydrochlorides. Synthesis and characterization of selected compounds are given in Experimental, and the synthesis and characterization of all compounds can be found in the Supplementary Material.

Anticancer activity

Nine selected derivatives 1l,m, 1g, 1o,p, 2l,m, and 2o,p were screened for cytotoxic properties in human cervix carcinoma (HeLa), human chronic myelogenous leukemia (K562), and normal endothelial (HUVEC) cells, using MTT assay. The experimental conditions can be found in the Supplementary Material.

The basis for the design of the discussed compounds was the result of our previous study reported in a patent application [24]. This previous work indicated that aminoalkyl derivatives show the best activity. Accordingly, imide derivatives containing aliphatic amines 1o,p and 2o,p were included in this study in an attempt to determine the influence of the imide structure on the biological activity. The remaining compounds 1l,m and 2l,m are methoxyphenyl and fluorophenyl derivatives.

In the initial screening, human cancer cell viability after 48 h of incubation with the given compound at the concentration of 100 μm was measured. Next, for compounds that reduced cancer cell survival by more than 50%, the IC₅₀ values (concentration of compound that reduces cancer cell survival by 50%) were determined. To do that, cell viability was measured in the presence of different concentrations of the tested compound (1, 1×10^–2, 1×10^–4, and 1×10^–6 mm), and subsequently, based on dose-viability curves, the IC₅₀ values were calculated. Cells exposed to 1% DMSO (a vehicle) served as the control with 100% survival. Cells treated with 1 μm staurosporine served as the internal control of the cytotoxicity experiments [25, 26].

In the initial screening, seven compounds, 2o,p, 2l,m, 1l,m, and 1g (Table 1), that, at concentration of 100 μm, reduced viability of either K562 or HeLa cells (or both) by more than 50%, as compared with control cells, were identified. Interestingly, compound 1g and all compounds of 2 series show substantial toxicity toward both K562 and HeLa cell lines. Meanwhile, compounds 1l,m are selectively toxic to human leukemia cells (K562) and less toxic for human cervical carcinoma (HeLa) cells. Results presented in Table 1 indicate that compounds 2o,p, 2l,m, 1l,m, and 1g are also toxic against human normal endothelial cells. Therefore, these compounds do not show any selectivity between cancer and normal cells.

Next, for the seven compounds identified in the initial screening, IC₅₀ values were calculated (Table 2). As can be seen, these compounds show rather similar toxicity toward K562 and HeLa cancer cells, with IC₅₀ values in the range of 40–80 μm.

Based on cytotoxicity results, we would like to point out some structural features of the tested compounds that may influence their toxicity. First, among derivatives of 10-(trimethyl)silyl-4-azatrcicyclo[5.2.1.02,6]-dec-8-ene-3.5-dione, only aryl/heteroarylpiperazine derivatives (compounds 1g and 1l,m) are toxic for the tested cell lines, while aminoalkyl derivatives (1o and 1p) demonstrate no substantial toxicity. Interestingly, derivatives of 1 show rather enhanced selectivity toward leukemia cells (K562), except for compound 1g, which is toxic for both K562 and HeLa cells. Meanwhile, introduction of methyl groups into the imide system results in cytotoxic activity of both piperazine and aminoalkyl derivatives of 2 (compounds 2l,m and 2o,p, Table 1). These compounds are not selectively toxic toward K562 or HeLa cells (Table 1), which is also evidenced by similar IC₅₀ values (Table 2).

Microbiology

The selected derivatives (1a–n and 2a–n) were tested for their antimicrobial activity against a selection of Gram-positive cocci, Gram-negative rods, and yeasts. None of the tested compounds showed significant antimicrobial activity.

Radioligand binding

Compounds 1l and 2l were evaluated for in vitro affinity for 5-HT_1A, 5-HT₆, and 5-HT₇ receptors by means of radioligand binding assays [27–29] and the results are showed in Table 3. The experimental conditions can be found in the Supplementary Material.

The selected compounds belong to the long-chain arylpiperazine group, and they contain the necessary structural elements that determine affinity for 5 HT_1A receptors: 1-(2-methoxyphenyl)piperazine moiety, butyl chain, and amide/imide fragment. The influence of the piperazine fragment and alkyl chain on affinity is well known, but the importance of imide fragment has not been determined in detail. However, we can find literature reports that indicate that terminal imide plays an important role in the process of ligand recognition by serotonin reports. In addition, among derivatives that are 5-HT_1A receptor ligands, many demonstrate high affinity for 5-HT₇ receptors [10–13]. The aim of the study was to determine the impact of the imide structure on the affinity. Comparison of the results for these two derivatives allows for the formulation of the initial conclusions as follows. The K_i values show that compound 1l displays high affinity for 5-HT_1A receptors (K_i=1 nm) and moderate affinity for 5-HT₇ receptors (K_i=44 nm) and compound 2l has lower but still significant affinity for 5-HT_1A and 5-HT₇ receptors (K_i=76 and 53 nm, respectively) in comparison with reference compounds. Moreover, both compounds display low affinity for 5-HT₆ receptors. Generally, these results are consistent with literature reports.

Chemistry

All chemicals and solvents were purchased from Aldrich. Melting points were determined in a capillary in Electrothermal 9100 apparatus and are uncorrected. The ¹H NMR spectra were recorded in CDCl₃ on a Varian UNITY-plus 300 spectrometer operating at 300 MHz. Mass spectral (MS ESI) measurements were carried out on a Mariner PE Biosystems instrument with time of flight (TOF) detector. The spectra were obtained in the positive ion mode with a declustering potential of 140–300 V. Elemental analyses were recorded on a CHN model 2400 Perkin-Elmer instrument. Chromatographic columns were filled with Merck Kieselgel 0.05–0.2 mm (70–325 mesh ASTM) silica gel. Reactions were monitored, and the purity of the compounds was checked by thin layer chromatography using Merck silica gel GF254 aluminum sheets and eluting with chloroform-methanol (95:5, v/v). Spots were detected by their absorption under UV light.

General procedure of the preparation of imides 1 and 2

A mixture of 2,4-cyclopentadien-1-yl(trimethyl)silane (for imide 1, 0.02 mol) or 2,3,4,5-tetramethyl-2,4-cyclopentadien-1-yl)-trimethylsilane (for imide 2, 0.02 mol) and 1H-pyrrole-2,5-dione (0.022 mol) in benzene (15 mL) was heated under reflux for 8 h. When the reaction was completed, the mixture was cooled and the resultant precipitate was crystallized from benzene.

General procedure for preparation of alkyl derivatives 1a,b and 2a,b

The mixture of imide 1 or 2 (0.01 mol), anhydrous K₂CO₃ (0.01 mol), and 1-bromo-3-chloropropane (0.02 mol) or 1,4-dichlorobutane (0.02 mol) in acetonitrile (30 mL) was heated under reflux for 24–38 h. When the reaction was completed, the mixture was filtered and the solution concentrated. The residue was purified by column chromatography eluting with chloroform/methanol, 9:0.2, v/v.

General procedure for preparation of 4-arylpiperazino derivatives of N-substituted imides 1c–n and 2c–n

To the mixture of N-halogenoalkyl imide derivative (0.01 mol), a powdered anhydrous K₂CO₃ (0.01 mol) and a catalytic amount of KI in acetonitryle (30 mL), an appropriate amine was added, and the mixture was heated under reflux for 48–60 h. Then, an inorganic residue was filtered off and the solvent was evaporated. The residue was purified by column chromatography eluting with chloroform or chloroform/methanol, 90:0.5, v/v. Some compounds were converted into their hydrochlorides to improve their solubility for biological evaluations.

General procedure for preparation of N-alkylamino derivatives of imides 1o–s and 2o–s

A mixture of imide 1 or 2, powdered anhydrous K₂CO₃ (0.01 mol), a catalytic amount of 98% 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and an appropriate chloroalkylamine hydrochloride(0.01 mol) in acetone (30 mL) was heated for 8–14 h. After the reaction was completed, the inorganic residue was filtered off and the solvent was evaporated. The obtained compound was purified by column chromatography eluting with chloroform or chloroform/methanol, 50:0.2, v/v. All new derivatives were converted to into their hydrochlorides.

Characterization of selected products