Cymantrenyl-Nucleobases: Synthesis, Anticancer, Antitrypanosomal and Antimicrobial Activity Studies

Abstract

The synthesis of four cymantrene-5-fluorouracil derivatives (1-4) and two cymantrene-adenine derivatives (5 and 6) is reported. All of the compounds were characterized by spectroscopic methods and the crystal structure of two derivatives (1 and 6), together with the previously described cymantrene-adenine compound C was determined by X-ray crystallography. While the compounds 1 and 6 crystallized in the triclinic P-1 space group, compound C crystallized in the monoclinic P2₁/m space group. The newly synthesized compounds 1-6 were tested together with the two previously described cymantrene derivatives B and C for their in vitro antiproliferative activity against seven cancer cell lines (MCF-7, MCF-7/DX, MDA-MB-231, SKOV-3, A549, HepG2m and U-87-MG), five bacterial strains Staphylococcus aureus (methicillin-sensitive, methicillin-resistant and vancomycin-intermediate strains), Staphylococcus epidermidis, and Escherichia coli, including clinical isolates of S. aureus and S. epidermidis, as well as against the protozoan parasite Trypanosoma brucei. The most cytotoxic compounds were derivatives 2 and C for A549 and SKOV-3 cancer cell lines, respectively, with 50% growth inhibition (IC₅₀) values of about 7 µM. The anticancer activity of the cymantrene compounds was determined to be due to their ability to induce oxidative stress and to trigger apoptosis and autophagy in cancer cells. Three derivatives (1, 4 and 5) displayed promising antitrypanosomal activity, with GI₅₀ values in the low micromolar range (3-4 µM). The introduction of the 5-fluorouracil moiety in 1 enhanced the trypanocidal activity when compared to the activity previously reported for the corresponding uracil derivative. The antibacterial activity of cymantrene compounds 1 and C was within the range of 8-64 µg/mL and seemed to be the result of induced cell shrinking.

Keywords: antibacterial activity; anticancer activity; antitrypanosomal activity; bioorganometallics; cymantrene; nucleobases.

Scheme 1

Synthesis of cymantrene-5-fluorouracil derivatives 1–4.

Scheme 2

Synthesis of cymantrene-adenine derivatives 5 and 6.

Figure 1

The molecular diagram of 1 with atomic displacement ellipsoids at the 50% probability level; Mp1 corresponds to the midpoint of the cyclopentadienyl ring. Hydrogen atoms have been omitted for clarity. Only molecule 1A is shown. Selected bond lengths [Å] and angles [°]: Mn1(A)-C1(A), 1.822(4); Mn1(A)-C2(A), 1.803(4); Mn1(A)-C3(A), 1.796(3); Mn1(A)-C4(A), 2.131(3); O1(A)-C1(A), 1.126(5); O2(A)-C2(A), 1.147(5); O3(A)-C3(A), 1.150(4); C4(A)-C9(A), 1.472(4); C9(A)-O4(A), 1.220(4); C10(A)-C11(A), 1.532(4); N1(A)-C12(A), 1.369(3); C13(A)-O6(A), 1.222(4); C13(A)-N2(A), 1.385(3); C14(A)-F1(A), 1.352(3); C5(A)-C4(A)-C9(A)-O4(A); −7.4(5); and, N1(A)-C12(A)-N2(A)-C13(A), 6.9(4).

Figure 2

The molecular diagram of 6 with atomic displacement ellipsoids at the 50% probability level; Mp1 corresponds to mid-point of the cyclopentadienyl ring. Hydrogen atoms have been omitted for clarity. Selected bond lengths [Å] and angles [°]: Mn1-C1, 1.795(2); Mn1-C2, 1.789(2); Mn1-C3, 1.797(2); O1-C1, 1.151(2); O2-C2, 1.152(3); O3-C3, 1.150(3); C4-C9, 1.503(2); C9-O4, 1.412(2); C19-O4, 1.423(2); C10-C11, 1.522(2); N1-C12, 1.368(2); N2-C13, 1.396(2); N5-C14, 1.353(2); C17-N5, 1.460(2); C5-C6-C7-C8; 0.3(2); N1-C12-N2-C13; −0.4(2); N3-C15-N4-C16; and, 1.1(3).

Figure 3

The molecular diagram of C with atomic displacement ellipsoids at the 50% probability level; Mp1 corresponds to mid-point of the cyclopentadienyl ring. Hydrogen atoms and solvent molecules have been omitted for clarity. Selected bond lengths [Å] and angles [°]: Mn1-C1, 1.760(3); Mn1-P1, 2.199(1); Mn1-P2, 2.193(1); O1-C1, 1.169(4); P2-C3, 1.862(3); P1-C2, 1.854(3); C2-C3, 1.540(4); C28-C33, 1.502(4); C33-O2, 1.417(4); C34-C35, 1.516(4); N1-C36, 1.356(4); N2-C37, 1.379(4); N5-C38, 1.327(4); P1-C2-C3-P2, −40.0(3); C29-C30-C31-C32, 0.3(4); N1-C36-N2-C37, 0.1(4); and, N3-C39-N4-C40, 0.0(7).

Figure 4

Structures of 7 and C.

Figure 5

Example enlarged photomicrographs illustrating induction of apoptosis, autophagy and mitotic catastrophe in human ovarian adenocarcinoma cells SKOV-3 by cymantrene derivatives 1, 2, 3, 7, B, and C and their core precursor CymH. The cells were stained with acridine orange (AO) and double-stained with fluorescence dyes Hoechst 33258 (Ho33258) and propidium iodide (PI) and then analyzed with an inverted fluorescence microscope (Olympus IX70, Tokyo, Japan) at 400× magnification, except cells stained with AO, which were photographed under 150× magnification. The images marked with arrows show the most typical morphological changes associated with different type of cell death induced by cymantrene compounds during 24 h-treatment and following culture of SKOV-3 cells in drug-free medium for 24 or 48 h (0 h—cells examined immediately after the treatment): (a) pale-blue live cells; (b) intense bright-blue early apoptotic shrunk cells with pycnotic nucleus; (c) violet late apoptotic cells; (d) cells with highly condensed and fragmented chromatin (karyorrhexis); (e) marginalization of chromatin; (f) cells with plasma membrane protrusions (“blebs”); (g) apoptotic bodies; (h) cytoplasmic bridges between the cells. Presence of giant cells (i), polyploid cells with two nuclei (j) and cells with AVOs (k) suggest concomitant mitotic catastrophe and autophagy, respectively.

Figure 6

Induction of apoptosis in human ovarian adenocarcinoma cells SKOV-3 by cymantrene derivatives 1–7, B, C, and their core precursor CymH. Changes in the fraction of live, early apoptotic, late apoptotic and necrotic fraction of SKOV-3 cells after 24 h treatment with IC₅₀ concentrations of cymantrenes and following post-treatment incubation in drug-free medium for 24 and 48 h.

Figure 7

Induction of AVOs in SKOV-3 cells by cymantrene derivatives 1–7, B, C and CymH. The cells were incubated with IC₅₀ concentrations of cymantrenes for 24 h under the same conditions as those applied in the cytotoxicity test and then examined immediately after the treatment (0 h time point) or cultured in drug-free medium for 24 or 48 h (24 h and 48 h time points, respectively). The time dependence of changes was statistically evaluated as described in Experimental Section (Section 4.5.2. Statistical Analysis). The fractions of AVOs containing cells at 24 h and 48 h time points were compared with corresponding fractions of AVOs containing cells at 0 h time point (* p < 0.05) and the fractions of AVOs containing cells at 48 h time point were compared with corresponding fractions of AVOs containing cells at 24 h time point (# p < 0.05). All of the changes in cells treated with cymantrenes were statistically significant in relation to untreated controls (for clarity not shown on the graph).

Figure 8

Changes in the relative level of ROS in SKOV-3 cells treated for 0.5 or 3 h with different concentrations (0.5 × IC₅₀, IC₅₀, and 2 × IC₅₀) of cymantrenes 1, 2, 3, 5, 6, 7, C, and CymH when compared with the ROS amount in untreated controls (100%). The kinetics of ROS formation was chased over 0–180 min time period after the treatment. The results are the means ± SD of 3 independent experiments, each performed in at least six repeats; * p < 0.05 as compared to untreated SKOV-3 cells (control).

Figure 8

Changes in the relative level of ROS in SKOV-3 cells treated for 0.5 or 3 h with different concentrations (0.5 × IC₅₀, IC₅₀, and 2 × IC₅₀) of cymantrenes 1, 2, 3, 5, 6, 7, C, and CymH when compared with the ROS amount in untreated controls (100%). The kinetics of ROS formation was chased over 0–180 min time period after the treatment. The results are the means ± SD of 3 independent experiments, each performed in at least six repeats; * p < 0.05 as compared to untreated SKOV-3 cells (control).

Figure 9

S. aureus morphology observed by SEM after incubation in growth medium in the absence (A1–A3, control) and presence of compound 1 (B1–B3, 48 µg/mL; C1–C3, 64 µg/mL) for 24 h.