Identification of pyrrolo[3',4':3,4]cyclohepta[1,2-d][1,2]oxazoles as promising new candidates for the treatment of lymphomas

Abstract

Unsatisfactory outcomes for relapsed/refractory lymphoma patients prompt continuing efforts to develop new therapeutic strategies. Our previous studies on pyrrole-based anti-lymphoma agents led us to synthesize a new series of twenty-six pyrrolo[3',4':3,4]cyclohepta[1,2-d] [1,2]oxazole derivatives and study their antiproliferative effects against a panel of four non-Hodgkin lymphoma cell lines. Several candidates showed significant anti-proliferative effects, with IC₅₀'s reaching the sub-micromolar range in at least one cell line, with compound 3z demonstrating sub-micromolar growth inhibitory effects towards the entire panel. The VL51 cell line was the most sensitive, with an IC₅₀ value of 0.10 μM for 3z. Our earlier studies had shown that tubulin was a prominent target of many of our oxazole derivatives. We therefore examined their effects on tubulin assembly and colchicine binding. While 3u and 3z did not appear to target tubulin, good activity was observed with 3d and 3p. Molecular docking and molecular dynamics simulations allowed us to rationalize the binding mode of the synthesized compounds toward tubulin. All ligands exhibited a better affinity for the colchicine site, confirming their specificity for this binding pocket. In particular, a better affinity and free energy of binding was observed for 3d and 3p. This result was confirmed by experimental data, indicating that, although both 3d and 3p significantly affected tubulin assembly, only 3d showed activity comparable to that of combretastatin A-4, while 3p was about 4-fold less active. Cell cycle analysis showed that compounds 3u and especially 3z induced a block in G2/M, a strong decrease in S phase even at low compound concentrations and apoptosis through the mitochondrial pathway. Thus, the mechanism of action of 3u and 3z remains to be elucidated. Very high selectivity toward cancer cells and low toxicity in human peripheral blood lymphocytes were observed, highlighting the good potential of these agents in cancer therapy and encouraging further exploration of this compound class to obtain new small molecules as effective lymphoma treatments.

Keywords: Antitumor agents; Hematological malignancies; Isoxazoles; Lymphoma; pyrrolo[3′,4’:3,4]cyclohepta[1,2-d][1,2]oxazoles.

Figure 1.

Best docked pose of A) 3d, B) 3p, C) 3u, and D) 3z with the 4O2B crystal structure of tubulin, depicting zones 1 and 2 of the colchicine site. Tubulin is shown in a faded yellow surface, while ligand and residues, involved in the most important interactions, are shown as sticks. H-bond and π-cation interactions are indicated as dashed yellow and green lines, respectively.

Figure 2.

Most representative MDs structure of tubulin (PDB code 4O2B) complexed with A) 3d, B) 3p, C) 3u, and D) 3z. Tubulin is depicted as a pale yellow surface, while ligand and residues, involved in the most important interactions, are shown as sticks. H-bond and π-cation interactions are indicated as dashed yellow and green lines, respectively.

Figure 3.

Cell cycle analysis of A549 (A), CCRF-CEM (B) and VL51 (C) cells treated for 24 h at the indicated concentrations with 3d, 3u and 3z. Cells were fixed and labeled with PI and analyzed by flow cytometry as described in the Experimental section. Data are presented as mean of two independent experiments ± SEM. (D) Percentage of p-histone H3 positive cells (mitotic cells) obtained from flow cytometric analysis of VL51 cells immunofluorescently labeled with an antibody to p-histone H3, following treatment with the indicated concentrations of compounds for 24 h.

Figure 4.

Compounds 3d, 3u and 3z induced apoptosis in A549 (A), CCRF-CEM (B) and VL51 (C) cells. Cells were treated with the compounds for 48 h at the indicated compound concentrations. The cells were then harvested and labeled with annexin-V-FITC and PI and analyzed by flow cytometry. Data are presented as mean ± S.E.M. for three independent experiments. The percentage of apoptotic cells refers to the sum of annexin-V positive and Annexin-V and PI double positive cells. (D) Assessment of mitochondrial membrane potential by flow cytometry with the fluorescent probe JC-1 after treatment for 24 h of VL51 cells with the indicated compounds at 0.5 and 1.0 μM.

Chart 1.

[1,2]Oxazolo[5,4-e]isoindoles (1), pyrrolo[2’,3’:3,4]cyclohepta[1,2-d][1,2]oxazoles (2), pyrrolo[3’,4’:3,4]cyclohepta[1,2-d][1,2]oxazoles (3).

Scheme 1.

Synthesis of 1-phenyl-5,6,7,8-tetrahydrocyclohepta[c]pyrrol-4(2H)-one (8a) and 1-(3,4,5-trimethoxyphenyl)-5,6,7,8-tetrahydrocyclohepta[c]pyrrol-4(2H)-one (8b). Reagents and conditions: (i) DMFDMA, reflux, 1 h, 99%; (ii) phenylglycine or 3,4,5-trimethoxy phenylglycine, AcONa^.3H₂O, ethanol, reflux, 90 min, 80 – 82%; (iii) Et₃N, Ac₂O, 30 min, reflux, 30 min, 53 – 73%; (iv) 80% acetic acid/37% HCl (1:12), 15 min, 60 °C, 75 – 81%.

Scheme 2.

Synthesis of 3-(4-methoxyphenyl)-8-oxo-2,4,5,6,7,8-hexahydrocyclohepta[c]pyrrole-1-carboxylate (8c) and 1-(4-methoxyphenyl)-5,6,7,8-tetrahydrocyclohepta[c]pyrrol-4(2H)-one (8d). Reagents and conditions: (i) a) ethyl azidoacetate, EtOK, ethanol, −20 °C, 4.5 h; b) toluene, reflux, 24 h, 74%; (ii) AlCl₃, glutaric anhydride, DCM, rt, 1 h then 10, rt, 24 h, 60%; (iii) triethylsilane, trifluoroacetic acid, rt, 24 h, 61%; (iv) trifluoroacetic anhydride, rt, 1 h, 60%; (v) a) 50% KOH, ethanol, reflux, 3 h; b) HCl 6 M, ethanol, reflux, 1 h, 60%.

Scheme 3.

Synthesis of pyrrolo[3’,4’:3,4]cyclohepta[1,2-d][1,2]oxazoles 3a-z. Reagents and conditions: (i) NaH, DMF, 0 °C to rt, 1 h then benzyl halides at 0 °C to rt, 3–12 h, 60 – 98%; (ii) ammonium formate, 10% Pd/C, ethyl acetate, rt, 12 h, 71 – 86%; (iii) TBDMAM, toluene, reflux, 12 h; (iv) NH₂OH·HCl, ethanol, reflux, 1 h, 60 – 93 %.