Regio- and Stereospecific Synthesis of Oridonin D-Ring Aziridinated Analogues for the Treatment of Triple-Negative Breast Cancer via Mediated Irreversible Covalent Warheads

Abstract

Covalent drug discovery has undergone a resurgence in recent years due to comprehensive optimization of the structure-activity relationship (SAR) and the structure-reactivity relationship (SRR) for covalent drug candidates. The natural product oridonin maintains an impressive pharmacological profile through its covalent enone warhead on the D-ring and has attracted substantial SAR studies to characterize its potential in the development of new molecular entities for the treatment of various human cancers and inflammation. Herein, for the first time, we report the excessive reactivity of this covalent warhead and mediation of the covalent binding capability through a Rh₂(esp)₂-catalyzed mild and concise regio- and stereospecific aziridination approach. Importantly, aziridonin 44 (YD0514), with a more-druglike irreversible covalent warhead, has been identified to significantly induce apoptosis and inhibit colony formation against triple-negative breast cancer with enhanced antitumor effects in vitro and in vivo while displaying lower toxicity to normal human mammary epithelial cells in comparison to oridonin.

Figure 1.

(A) Representative molecules bearing covalent warheads enamide and ynamide (highlighted in red). (B) Representative molecules with covalent warheads epoxide and aziridine (highlighted in red).

Figure 2.

Reactivity of oridonin with GSH determined by ¹H NMR analysis.

Figure 3.

Plausible reaction mechanism for Rh₂(esp)₂ catalyzed regio- and stereospecific aziridination of oridonin-like substrates. (A) Analysis of the reactive substructure and substrate participating in the aziridination. (B) The structures of Rh₂(esp)₂. (C, D) The comparison of two different substrates (C) 33 vs (D) 21, assisted by the neighboring oxygen group participation in the Rh₂(esp)₂-promoted regio- and stereospecific aziridination.

Figure 4.

Reactivity of aziridinated oridonin analogue 25 with GSH determined by ¹H NMR.

Figure 5.

Reactivity of aziridinated oridonin analogue 44 with GSH determined by ¹H NMR.

Figure 6.

Growth inhibitory effects of 44 against MDA-MB-231, SK-BR3, MDA-MB-468, and MCF-7 breast cancer cells. (A) 44 significantly inhibits colony formation of MDA-MB-231, SK-BR3, MDA-MB-468, and MCF-7 cells. (B) 44 suppresses the proliferation of MDA-MB-231, SK-BR3, MDA-MB-468, and MCF-7 cells. Data are presented as the mean ± SD of at least three independent experiments. Significance between different groups was determined by using one-way ANOVA; p = 0.0003 (MDA-MB-231), p = 0.0002 (MDA-MB-468), p < 0.0001 (SK-BR3), and p = 0.0012 (MCF-7).

Figure 7.

Induction of apoptosis in MDA-MB-231 breast cancer cells by 44. (A) Flow-cytometry analysis of apoptotic MDA-MB-231 breast cancer cells induced by 44 at different concentrations. (B) Apoptotic ratio of different concentrations of 44 in MDA-MB-231 breast cancer cells. The values are means ± SD of at least three independent experiments. A single asterisk represents p < 0.05, two asterisks represent p < 0.01, and three asterisks represent p < 0.001 to 0 μM group (DMSO, vehicle control). (C) Western-blot analysis of biomarkers for apoptosis induced by 44 in MDA-MB-231 breast cancer cells at different concentrations (48 h).

Figure 8.

Effect of 44 and oridonin on normal mammary epithelial cells (MCF-10A) proliferation. MCF-10A cells were treated with varying concentrations of 44 and oridonin for 48 h. Values are mean ± SD of three independent experiments. Statistical significance was determined using a Student t-test, in comparison to the corresponding value of oridonin treatment at the same concentration. ns indicates not significant, a single asterisk indicates p < 0.05, triple asterisks indicate p < 0.001, and quadruple asterisks indicate p < 0.0001.

Figure 9.

In vivo efficacy of 44 in suppressing the xenograft tumor growth of triple-negative breast cancer. The breast cancer cell line MDA-MB-231 was used to generate xenograft tumors in nude mice for 10 and 15 mg/kg doses via intraperitoneal injection, respectively. Data are presented as the mean ± SEM of tumor volume at each time point. Significance between different groups was determined by using one-way ANOVA; p = 0.0023.

Scheme 1.. Synthesis of the Aziridinated Oridonin Analogues^a

a Reagents and conditions: (a) 5% HCl (aq), MeOH, CH₂Cl₂, 15 min, 28~62%; (b) Fmoc chloride, Na₂CO₃, dioxane, 0°C → rt, 12 h, 34%; (c) 5% HCl (aq), MeOH, CH₂Cl₂, 15 min, 75%; (d) PyHBr₃, THF, 0°C, 2 h, 82%; (e) N-allylthiourea, EtOH, reflux, 5 h, 44%; (f) piperidine, DMF, rt, 1 h, 82%; (g) acrylic acid, HBTU, DIPEA, CH₂Cl₂, 0°C → rt, 12 h, 33%; (h) propionic acid, HBTU, DIPEA, CH₂Cl₂, 0°C → rt, 12 h, 74%.