Synthesis and biological activity of novel series of heterocyclic compounds containing succinimide moiety

Chemistry

The preparation of 15 new N-substituted imides 3, 6, and 9 is described (Scheme 1). The starting imides 1, 4, and 7 were obtained by Diels-Alder reaction. Compounds 1 and 7 are known [15, 16] and compound 4 was prepared by the reaction of 2-methylcyclopent-1,3-dione with maleimide. Alkylation of imides 1, 4, and 7 with chloroalkylamines in the presence of catalytic amount of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) was attempted under conditions described by us previously [17–22]. Unfortunately, the final result of these reactions was not satisfactory. A mixture of products difficult to separate was obtained. Under these conditions, not only was self-condensation of alkylamines observed but also degradation of imides. The experiment was modified in that the starting imides 1, 4, and 7 were treated with DBU [23–29], and then the resultant products 2, 5, and 8 were condensed with chloroalkylamines. This modification resulted in a satisfactory yield in the range 73–95% of the desired products 3, 6, and 9 (Scheme 1).

Scheme 1

Synthesis of compounds 2, 5, 8 and 3a–e, 6a–e, 9a–e; (a) DBU, K₂CO₃, acetone; (b) ω-chloroalkylammonium chloride, DBU, K₂CO₃, acetone.

Single-crystal X-ray diffraction (XRD)

Ethyl 11-ethyl-7-methyl-3,5,10-trioxo-4-azatricyclo[5.2.2.0^2,6]undecane-8-carboxylate 2 crystallizes in orthorhombic P2₁2₁2₁with one molecule as an independent part of the unit cell. The molecules are lined by the hydrogen bond involving the NH ring fragment and one of the carbonyl group at the heterocyclic ring of the neighboring unit situated in (x+¹/₂,-y+¹/₂,-z) position. An infinite ribbon along the [100] direction is formed, with N(1)-H(1)…O(4) distance equal to 2.02 (4) Å [N…O distance is 2.817(4) Å] (see Figure 1 and Table 1). The other carbonyl group at the heterocyclic ring as well as the carbonyl groups at the carboxylate fragment or attached to the six-membered ring interact by a rather weak CH…O bond with methylene or methyl fragments of the neighboring molecules (the O…H contacts range from 2.234 Å to 2.716 Å). Similarly as in 2, the main structural motif in the crystal structure of 9-methyl-3,5,8-trioxo-4-azatricyclo[5.2.1.0^2,6]dec-1-yl acetate 5 is a ribbon that is formed along the [010] direction. Three molecules of 5 situated in (x, y, z), (³/₂-x, ¹/₂+y, -¹/₂-z) and (x, 1+y, z) positions are linked by hydrogen bonds involving NH and carbonyl fragments at the heterocyclic systems. The NH…O distances are rather strong and equal to 1.97(15) Å [N…O distances equal to 2.842(13) Å]. Noteworthy, the system crystallizes as a hydrate with the molecular ratio (compound/water) of 1:0.1. The water molecule expands the ribbon subunit by interacting with the carbonyl group of a molecule in the (x, y, z) position and by bifurcated hydrogen bond interaction with an ether or heterocyclic carbonyl fragment of the molecule situated in (x, 1+y, z). In both cases, water plays the role of a donor of the hydrogen bond. Owing to its small occupancy and rather long OH…O distances [2.884(11) Å or 3.036(11) Å and 2.958(11) Å, respectively], these interactions are much weaker, although very important for the architecture of the crystal (Figure 2).

Figure 1

Molecular structure and atomic numbering for 2, 5, and 8. Displacement ellipsoids are drawn at 50% probability level.

Figure 2

Hydrogen bond interactions in 2. View along the [001] direction.

In the case of 1,3-dioxo-4,5,6,7-tetraphenyl-2,3,3a,4,5,7a-hexahydro-1H-isoindole-4-carboxylic acid 8, the interaction pattern is different. This system crystallizes with 1.2 molecules of ethanol and 0.8 molecules of water. One molecule of ethanol is distorted over two positions in the ratio of 0.5:0.5. Another molecule of ethanol shares its position with water molecules in the ratio of 0.2:0.8. Unlikely to 2 and 8, the main structural motif is a cetrosymmetric dimer, which is formed due to hydrogen bond interactions between the NH fragment and the carbonyl group at the heterocyclic moiety of the neighboring units (Figure 3). This interaction seems to be the most effective in the set of analyzed compounds, as the NH…O distances are the shortest and equal to 1.93(2) Å [N…O distances equal to 2.795(17) Å]. The other carbonyl fragment at the heterocyclic moiety and the carboxylate group interact with the solvent molecules, which are disordered, as mentioned.

Figure 3

Hydrogen bond interactions in 5. View along the [100] direction.

Pharmacology

The cytotoxic properties of 12 compounds were tested in K562 (chronic myelogenous leukemia) and HeLa (cervix carcinoma) cells. As the control {100% viability in the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay}, cells which grew in the presence of 1% DMSO were used. The viability of cells was determined at four different drug concentrations: 1 mm, 1 × 10^-2 mm, 1 × 10^-4 mm, and 1 × 10^-6 mm. Based on dose-response curves, we calculated the IC₅₀ value for each compound, which corresponds to concentration that reduces cell viability by 50% (Table 2). Most compounds that were tested did not prove cytotoxic towards K562 or HeLa cells, which is demonstrated by IC₅₀ values which could not be determined under our experimental settings and are higher than 1 mm. By contrast, daunorubicin used as a reference drug exhibits potent anticancer activity after 48 h, which is evidenced by IC₅₀ values of 0.2 and 0.4 μm for K562 and HeLa cells, respectively. Interestingly, compound 3d shows toxicity specific for chronic myelogenous leukemia cells with IC₅₀ of 400 μm. This compound is not toxic for cervical carcinoma cells as evidenced by IC₅₀ >1 mm. By contrast, 48 h incubation with compound 9c induced similar toxicity towards both cancer cell lines, with IC₅₀ of 200 μm and 100 μm for K562 and HeLa cells, respectively (Table 2). Although compounds 3d and 9c show some measurable cytotoxicity against cancer cells, their activity is approximately three orders of magnitude lower than the activity of daunorubicin.

Conclusion

Fifteen new heterocyclic derivatives containing a succinimide moiety were synthesized. Twelve of them were screened for toxicity towards chronic myelogenous leukemia and cervix carcinoma cells. Only two compounds showed measurable cytotoxicity that allowed calculation of IC₅₀ values (Table 2). Other compounds did not show significant toxicity even at the concentration of 1 mm. Compound 3d is specifically toxic towards K562 leukemia cells while non-toxic for cervix carcinoma cells, as indicated by IC₅₀ values. Compound 9c seems to be more cytotoxic towards cancer cells than compound 3d, as evidenced by the lower IC₅₀ values.

General

Melting points were determined by the capillary method using Electrothermal 9100 apparatus and are uncorrected. The ¹H NMR spectra were recorded in DMSO-d₆ on a Bruker VMNRS 300 operating at 300 MHz. Mass spectral ESI (electrospray ionization) measurements were carried out on a Mariner Perspective-Biosystem instrument with a TOF detector. The spectra were obtained in the positive ion mode with a declustering potential of 140–300 V. Elemental analyses were obtained using 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 by thin layer chromatography (TLC) on silica gel (plates with 254 nm fluorescent indicator, layer thickness 0.2 mm, Kieselgel G., Merck), eluting with 9.8:0.2 or 9.5:0.5 chloroform/methanol.

Synthesis of 1,8-diacetoxy-9-methyl-4-azatricyclo[5.2.1.0^2,6]dec-8-ene-3,5-dione (4)

A mixture of 2-methylcyclopenta-1,3-dione (0.004 mol), maleimide (1H-pyrrole-2,5-dione) (0.0048 mol), and a catalytic amount of pTSA (p-toluenosulphonic acid) in isopropenyl acetate (15 mL) was heated under reflux for 14 h. The mixture was filtered hot and the solvent was evaporated. The residue was crystallized from hexanes. Compound 4 was obtained as a white powder; yield 95%; mp 154.7–156°C; ¹H NMR: δ 1.10 (3H, d, J = 7.2 Hz), 2.05 (3H, s), 2.15 (3H, s), 2.18 (1H, m), 2.28 (1H, m), 2.81 (1H, m), 3.66 (1H, dd, J = 16.2 Hz, J = 9.9 Hz), 4.00 (1H, d, J = 7.9 Hz), 11.26 (1H, s); MS: m/z 316.2 (100%) [M+Na]⁺. Anal. Calcd for C₁₄H₁₅NO₆: C, 57.34; H, 5.16; N, 4.78. Found: C, 57.35; H, 5.14; N, 4.84.

Synthesis of imides 1 and 7

Imides 1 and 7 were obtained as described previously [15, 16].

Synthesis of imides 2, 5, and 8

A mixture of imide 1, 4, or 7 (0.01 mol), excess of DBU and powdered anhydrous K₂CO₃ in acetone (15 mL) was heated under reflux for 8 h, then concentrated, and the residue was separated by column chromatography on silica gel using chloroform/methanol, from 100:0.2 to 100:5, as an eluent.

General procedure for synthesis of compounds 3a–e, 6a–e, and 9a–e

A solution of imide 2, 5, or 8 (0.001 mol) in acetone (10 mL) was treated with anhydrous K₂CO₃ (0.03 mol), a catalytic amount of DBU, and the ω-chloroalkylammonium chloride (0.03 mol). The mixture was heated under reflux for 8–10 h, then filtered and concentrated. The residue was purified by silica gel column chromatography eluting with chloroform or chloroform/methanol, 50:0.2. Hydrochlorides were obtained by treatment of free bases with a solution of hydrogen chloride in ethanol.

Single crystal X-ray diffraction (XRD)

Single crystals of 2, 5, and 8 suitable for XRD measurements were obtained by slow crystallization from ethanol. The XRD measurements were performed at 100 K on a Kuma KM4CCD κ-axis diffractometer with graphite-monochromated Mo Kα radiation. The crystals of 2, 5, and 8 were positioned at 50 mm from the KM4CCD camera, 216 frames were measured at 1° intervals with a counting time of 145 s 2, 1457 frames were measured at 1° intervals with a counting time of 6 s 5, 1240 frames were measured at 1° intervals with a counting time of 3 s 8, respectively. The data were corrected for Lorentz and polarization effects. Multiscan absorption correction was also applied [30]. Data reduction and analysis were carried out with the Kuma Diffraction (Wrocław, Poland) programs. The structures were solved by direct methods [31] and refined using SHELXL [32]. The refinement was based on F² for all reflections except for those with very negative F². The weighted R factor, wR and all goodness-of-fit S values are based on F². The non-hydrogen atoms were refined anisotropically. For 2 and 8, all hydrogen atoms (except those at nitrogen atoms) were located geometrically and their positions were refined using the riding model. The isotropic ADPs of hydrogen atoms were set to be either 1.2 or 1.5 times bigger than U_eq of the corresponding carbon atoms. The hydrogen atoms for 5 were located from a difference Fourier map, whereas their positions and thermal parameters were refined isotropically. Scattering factors were taken from Tables 6.1.1.4 and 4.2.4.2 in [33]. All figures presenting the results of XRD determination were made using the Diamond program [34]. The absolute configurations of 2, 5, and 8 were assigned based on the knowledge of stereochemistry of the synthetic precursors. The crystal data and structure refinement are specified in Table 3. Selected bond lengths, bond angles, major torsion angles, and hydrogen bonds are given in Tables 1 and 4. The configuration, conformation, and atom numbering are shown in Figure 4.

Figure 4

Dimeric interaction in 8.

Crystallographic data for the structures have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication no. 928940 (2), 928941 (5), and 928942 (8). Copies of the data can be obtained on application to CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (email: deposit@ccdc.cam.ac.uk).

Cells and cytotoxicity assay

The HeLa (human cervix carcinoma) and K562 (chronic myelogenous leukemia) cells were cultured in RPMI 1640 medium supplemented with antibiotics and 10% fetal calf serum, in a 5% CO₂-95% air atmosphere. The cells (7 × 10³) were seeded on each well on a 96-well plate (Nunc). After 24 h, the cells were exposed to the test compounds. Stock solutions (100 mm) of test compounds were freshly prepared in DMSO. The final concentrations of the compounds tested in the cell cultures were: 1 mm, 1 × 10^-2 mm, 1 × 10^-4 mm, and 1 × 10^-6 mm, except for compounds 9b and 9c which were used at single concentration of 100 μm. The concentration of DMSO in the cell culture medium was 1%. The cytotoxicity of all compounds was determined by the MTT assay, as described in [35]. Briefly, after 48 h of incubation with test compounds, cells were treated with MTT, and incubation was continued for 2 h. MTT-formazan crystals were dissolved in the buffer containing 20% SDS and 50% DMF (pH 4.7) and absorbance was read at 570 and 650 nm on an ELISA-PLATE READER (FLUOstar Omega). As a control (100% viability), cells grown in the presence of vehicle (1% DMSO) only were used.

The values of IC₅₀ (the concentration of test compound required to reduce the cell survival fraction to 50% of the control) were calculated from dose-response curves and used as a measure of cellular sensitivity to a given treatment.