Design, Synthesis, and Bioactivity Assessment of Modified Vemurafenib Analog

Abstract

Background: Metastatic melanoma is a highly aggressive malignancy with poor prognoses and frequent resistance to conventional chemotherapy. Approximately 40% of melanoma cases carry the BRAF^V600E mutation, for which vemurafenib, a selective BRAF^V600E inhibitor, is approved. Despite initial clinical benefits, vemurafenib often leads to drug resistance and relapse, highlighting the need for improved therapeutic strategies. Objectives, methods: In this study, we designed, synthesized, and characterized five novel vemurafenib analogs-RF-86A, RF-87A, RF-94A, RF-94B, and RF-96B-with the aim of enhancing anti-proliferative and anti-metastatic effects against human melanoma cells. Results: All compounds induced apoptosis in BRAF^V600E-mutated A375 cells, with RF-86A displaying the lowest IC₅₀ value among the series, comparable to that of vemurafenib. Moreover, RF-86A exhibited the highest selectivity index, as determined using HEK293T cells as a non-tumorigenic control. Additionally, migration assays and gelatin zymography demonstrated that the analogs, unlike vemurafenib, significantly inhibited matrix metalloproteinases MMP-2 and MMP-9, key enzymes involved in tumor invasion and metastasis. Conclusions: These findings suggest that structural modifications to the vemurafenib scaffold may improve therapeutic efficacy and offer a promising strategy to overcome acquired resistance.

Keywords: N-acylhydrazone; azaindole; cancer; melanoma.

Figure 1

Design concept of new pyrrolopyridine-N-acylhydrazone derivatives (2a–e).

Scheme 1

Synthesis of hydrazides (4a–e). Reagents and Conditions (a) MeOH, MePh, H₂SO₄ cat., 100 °C, reflux, 24 h, 42–82%; (b) NH₂NH₂ 1M in EtOH, 80 °C, reflux, 48 h, 50–63%.

Scheme 2

Synthesis of final products (2a–e). Reagents and Conditions: (a) HMTA, AcOH, and H₂O (2:1), reflux, 16 h, 66%; (b) hydrazides (5a–e), EtOH, HClcat., t.a, 6 h, 70–91%.

Figure 2

Vemurafenib analogs (2a–e) induce apoptosis in A375 cells. Apoptosis was detected by TUNEL assay. A double-staining technique was used. TUNEL staining by using an in situ cell death detection kit (fluorescein) for apoptotic cell nuclei and DAPI (blue) staining for all cell nuclei. A375 cells were treated for 24 h with or without (control) 5 μM of the test compounds. TUNEL-positive cells were visualized as indicated by green fluorescence staining. The images were obtained by EVOS M5000 microscope (Thermo Fisher, Waltham, MA, USA). Representative images from three independent experiments are shown. Magnificance: 20×. Scale bar: 150 μm.

Figure 3

Morphological appearance of A375 cells treated with vemurafenib and analogs (2a–e) (5 μM) for 24 h. The red arrows indicate apoptotic bodies, showing membrane blebs and fragmented nucleus. The images were obtained by an EVOS M5000 microscope (Thermo Fisher, Waltham, MA, USA). Representative images from three independent experiments are shown. Magnification of 20×.

Figure 4

Effects of vemurafenib and analogs (2a–e) on migration of A375 cells. After incubation with medium alone (controls), and the treatment with the compounds test (0.5 μM) for 24 h, cell migration was assayed through cell-based scratch method. (A) The images were obtained by EVOS M5000 microscope (Thermo Fisher, Waltham, MA, USA). Representative images from three independent experiments are shown. Magnification of 10×. Scale bar 50 μm. (B) The filled areas were analyzed and quantified using ImageJ software (version 1.54p). Statistical analyses were performed with GraphPad Prism 8.02 and included one-way ANOVA followed by Dunnett’s post hoc test. Statistical significance was defined as * p < 0.05 when compared with the control group.

Figure 5

Effects of vemurafenib and analogs (2a–e) on MMP-9 and MMP-2 expression and activity by A375 cells after incubation with medium alone (controls), and the treatment with the test compounds (1 and 5 μm) for 24 h. Gelatinase activity was assayed through zymography method. Representative images from three independent experiments are shown. The protein bands were analyzed using 6.1 software from Bio-Rad.