Synthesis of N-(4-chlorophenyl) substituted pyrano[2,3-c]pyrazoles enabling PKBβ/AKT2 inhibitory and in vitro anti-glioma activity

Abstract

A series of N-(4-chlorophenyl) substituted pyrano[2,3-c]pyrazoles was synthesised and screened for their potential to inhibit kinases and exhibit anticancer activity against primary patient-derived glioblastoma 2D cells and 3D neurospheres. A collection of 10 compounds was evaluated against glioma cell lines, with compound 4j exhibiting promising glioma growth inhibitory properties. Compound 4j was screened against 139 purified kinases and exhibited low micromolar activity against kinase AKT2/PKBβ. AKT signalling is one of the main oncogenic pathways in glioma and is often targeted for novel therapeutics. Indeed, AKT2 levels correlated with glioma malignancy and poorer patient survival. Compound 4j inhibited the 3D neurosphere formation in primary patient-derived glioma stem cells and exhibited potent EC₅₀ against glioblastoma cell lines. Although exhibiting potency against glioma cells, 4j exhibited significantly less cytotoxicity against non-cancerous cells even at fourfold-fivefold the concentration. Herein we establish a novel biochemical kinase inhibitory function for N-(4-chlorophenyl) substituted pyrano[2,3-c]pyrazoles and further report their anti-glioma activity in vitro for the first time.KEY MESSAGEAnti-glioma pyrano[2,3-c]pyrazole 4j inhibited the 3D neurosphere formation in primary patient-derived glioma stem cells. 4j also displayed PKBβ/AKT2 inhibitory activity. 4j is nontoxic towards non-cancerous cells.

Keywords: Pyrano[2,3-c]pyrazole; anti-glioma; kinase inhibitor; neurosphere; stem cells.

Figure 1.

Cytotoxic activities of N-phenyl substituted pyrazole and pyran moieties against different glioma cell lines.

Scheme 1.

Two and multicomponent synthesis of pyrano[2,3-C]pyrazoles. Reaction type: (a) two-component, (b) three-component, and (c) four-component.

Scheme 2.

Two and multicomponent synthesis of pyrano[2,3-C]pyrazoles. Reaction type: (a) two-component, (b) three-component, and (c) four-component.

Scheme 3.

Synthesis of pyrano[2,3-c]pyrazoles by three-component reaction (with labelled atoms).

Figure 2.

4j exhibits biochemical inhibitory activity against AKT2 (A) Kinase profiling of 4j at 5 μM was carried out against the panel of 139 kinases at the International Centre for Protein Kinase Profiling (http://www.kinase-screen.mrc.ac.uk/). The IC₅₀ value of 4j was recorded in vitro using purified AKT2 (B) and AKT1 (C) over different 4j concentrations.

Figure 3.

AKT2 is overexpressed in glioma (A) High expression of AKT2 mRNA correlates with poorer survival in LGG patients. Individual values are sign-corrected log10 p-values of correlation. Non-abbreviated names of the cancer types: UVM: Uveal Melanoma; LIHC: Liver hepatocellular carcinoma; KICH: Kidney Chromophobe; ESCA: Oesophageal carcinoma; CHOL: Cholangiocarcinoma; UCS: Uterine Carcinosarcoma; KIRP: Kidney renal papillary cell carcinoma; BRCA: Breast invasive carcinoma; READ: Rectum adenocarcinoma; HNSC: Head and Neck squamous cell carcinoma; MESO: Mesothelioma; PAAD: Pancreatic adenocarcinoma; PRAD: Prostate adenocarcinoma; SARC: Sarcoma; DLBC: Lymphoid Neoplasm Diffuse Large B-cell Lymphoma; LUAD: Lung adenocarcinoma; PCPG: Pheochromocytoma and Paraganglioma; CESC: Cervical squamous cell carcinoma and endocervical adenocarcinoma; SKCM: Skin Cutaneous Melanoma; OV: Ovarian serous cystadenocarcinoma; THCA: Thyroid carcinoma; LUSC: Lung squamous cell carcinoma; KIRC: Kidney renal clear cell carcinoma; ACC: Adrenocortical carcinoma; UCEC: Uterine Corpus Endometrial Carcinoma; LGG: Brain Lower Grade Glioma. (B) AKT2 expression is highest in the tumour cell population of IDH1 WT GBM single-cell RNAseq dataset. (C) AKT2 expression is highest in the tumour cell population of IDH1 mutant astrocytoma single-cell RNAseq dataset.

Figure 4.

Compound 4j induces anti-glioma activity in 2D and 3D glioblastoma cell cultures. (A) Glioblastoma cells were treated with the indicated concentrations of 4j for 72 h, and fold viability was measured using MTS assay. (B) GBM12 cancer stem cells were treated with either DMSO or 20 μM 4j or 1 μM MK-2206 for 21 days and neurospheres were allowed to form. A representative image of the neurospheres is shown. Scale bar= 125 μm. (C) The diameter of the neurospheres were quantified using ImageJ. The significance of the differences was measured using one-way ANOVA with Tukey’s multiple comparisons. ****p < .0001; *p < .05 (D) As in (B) but GBM76 cancer stem cells were utilised. (E) The diameter of the neurospheres were quantified using ImageJ. The significance of the differences was measured using one-way ANOVA with Tukey’s multiple comparisons. ****p < .0001; ns: not-significant. Scale bar= 125 μm. (F) Non-cancerous RAW264.7, HEK293T, and BV2 cells were treated with the indicated concentrations of 4j for 72 h, and fold viability was measured using MTS assay. The significance of the differences was measured using two-way ANOVA with Tukey’s multiple comparisons. * = significant.